iNOS INHIBITORY COMPOSITIONS AND THEIR USE AS BREAST CANCER THERAPEUTICS

Abstract

To provide improved chemotherapeutic compositions for the treatment and / or remission of one or plural symptoms of human cancer.SOLUTION: Disclosed is a method for treating one or more mammalian cancers by using one or more iNOS pathway inhibitory compounds in combination with one or more selected hypotensive agents including calcium channel antagonists, or in combination with one or more conventional chemotherapies or anti-cancer regimen, in particular a method for treating human breast cancers. Also disclosed are specific therapeutic formulations comprising these compositions, and use methods thereof for managing or reversing treatment-resistance in the treatment of refractory, metastatic and relapsed cancers, especially treatment resistance in human triple negative breast cancer.SELECTED DRAWING: None

Description

[Technical field]

[0001] 2. Background of the Invention CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 61 / 976,956, filed April 8, 2014 (pending; Attorney Docket No. 37182.170), the contents of which are specifically incorporated herein in their entirety by express reference thereto.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant R01-CA138197 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Names of parties involved in the joint research agreement Not applicable.

[0004] FIELD OF THEINVENTION The present invention relates generally to the fields of medicine and oncology. In particular, the present invention provides improved chemotherapeutic compositions for the treatment and / or amelioration of one or more symptoms of human cancer. In an illustrative embodiment, a method for treating human breast cancer is provided using administration of one or more effectors of the iNOS pathway. In an exemplary embodiment, for example, administration of N-acetylglucosamine, alone or in combination with one or more antihypertensive agents, including calcium channel antagonists, is provided. G A formulation of an iNOS inhibitor comprising -monomethyl-L-arginine (L-NMMA; C9H2ON4O4; MW248.28) is provided as a therapeutic formulation for the treatment of mammalian breast cancer, in particular for the treatment of triple-negative breast cancer (TNBC) in humans, which is a refractory form of the disease that is resistant to conventional chemotherapy drugs and has a poor prognosis. [Background technology]

[0005] Despite significant advances in breast cancer biology, progress in the treatment of advanced breast cancer has been limited, with little change in overall survival for women with treatment-resistant metastatic breast cancer over the past few decades. It is noted that approximately 40,000 women with metastatic breast cancer die each year due to treatment resistance and failure of current therapies. The classical model of carcinogenesis can be described as random or "stochastic," where any cell can be transformed by accumulating the right combination of mutations. An alternative model is that subpopulations or clones of cells retain key stem-like properties, including the capacity for self-renewal and differentiation that contribute to the cellular heterogeneity that drives carcinogenesis. Experimental evidence supporting such intratumoral clonal heterogeneity was first reported in human leukemias by Dick et al. These concepts were subsequently supported by the development of a new model in which human breast cancers express CD44 + / CD24 - / low3 This has been extended to solid tumors by several groups demonstrating that tumors are driven by stem-like cells characterized by cell surface expression of ENREF13. Large-scale sequencing analysis of solid tumors has provided further evidence of extensive heterogeneity within individual tumors. This intratumor heterogeneity may be a major contributor to treatment resistance and treatment failure. Different subpopulations may be associated with heterogeneous protein functions that may foster tumor fitness and lead to treatment failure by Darwinian selection. Thus, subpopulations of cells with stem-like properties within heterogeneous bulk tumors have been shown to be responsible for tumor initiation and recurrence.

[0006] Three groups have recently independently provided direct functional evidence for the presence of cells with stem-like properties by lineage tracing experiments in glioblastoma (GBM), squamous cell skin tumors, and intestinal adenomas, further demonstrating the hierarchical nature of cancer. These independent groups confirm that a small fraction of cells within bulky tumors possesses clonogenic potential, and that this fraction is intrinsically resistant to chemotherapy.

[0007] Triple-negative breast cancer Triple-negative breast cancer (TNBC) is an aggressive and lethal form of cancer lacking estrogen (ERα), progesterone (PR) and human epidermal growth factor (HER-2) receptors with no approved targeted therapeutic options. Despite numerous advances, treatment resistance and metastasis are the main causes of death in TNBC patients. The development of resistance to conventional treatments and metastasis may result from a subpopulation of cells with tumor-initiating capabilities. Residual tumors after chemotherapy are characterized by CD44 expression, which exhibits self-renewal capacity and mesenchymal features. + / CD24 - / low These cancer stem cells (CSCs) are enriched in tumor cells. These cancer stem cells (CSCs) can be useful for reinitiating tumor growth and seeding metastasis. Therefore, combinatorial treatment with conventional chemotherapy and anti-CSC compounds will be required to reduce tumor burden, recurrence and metastasis to distant organs. Unfortunately, such combinations are not currently available for routine use in clinics.

[0008] Currently, there is no targeted treatment for TNBC. Inducible nitric oxide synthase (iNOS) has been found to promote the invasiveness of breast tumors. Previous studies have demonstrated that high endogenous iNOS expression correlates with and predicts poor TNBC patient survival. However, despite the progress made to date in the treatment of breast cancer, clinicians still agree that there is still a significant need for the development of novel agents active in its treatment, particularly chemotherapy for use in the treatment of TNBC.

[0009] Indeed, there remains a significant unmet medical need for new agents that are effective in the treatment of hyperproliferative disorders, particularly breast cancer that has become resistant to conventional chemotherapeutic agents.

[0010] Hypertensive comorbidity in breast cancer patients Hypertension is one of the most common disorders enhancing morbidity in women with breast cancer (Sarfati et al., 2013; Gampenrieder et al., 2014). Chemotherapy-induced hypertension is a common effect that increases patient mortality in metastatic and TNBC (Cameron et al., 2013; Fan et al., 2014). Thus, the co-administration of one or more antihypertensive drugs that can counteract the deleterious hypertensive side effects of chemotherapy administration represents an important consideration to improve patient health and increase survival.

[0011] The present invention demonstrated the synergistic effect of co-administration of calcium channel blockers (currently used in the clinic as traditional antihypertensive drugs) in both in vitro and in vivo models to treat TNBC. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] CAMERON, D. et al., Lancet Oncol. (September 2013) 14(10):933-942 [Non-Patent Document 2] GAMPENRIEDER, SP et al., Anticancer Res. (January 2014) 34(1):227-233 Summary of the Invention [Means for solving the problem]

[0013] The present invention addresses the above and other unmet deficiencies inherent in the relevant oncology and pharmaceutical fields by providing formulations of iNOS inhibitors, alone or in combination with one or more calcium ion antagonists (slow channel type blockers), as chemotherapeutic agents for treating mammalian cancers, particularly human breast cancers, such as metastatic or TNBC. The present invention also provides methods of using and repurpose iNOS inhibitor compounds in new cancer treatment modalities that advantageously provide unexpected benefits to patients in need of such treatment.

[0014] In a general and general sense, the present invention first provides a pharmaceutical composition for treating and / or ameliorating one or more symptoms of cancer in a mammal in need thereof.In an exemplary embodiment, such a chemotherapeutic formulation comprises a therapeutically effective amount of at least a first iNOS inhibitor compound, 1) alone, 2) a) a therapeutically effective amount of one or more antihypertensive agents, such as calcium channel antagonists; c) one or more conventional chemotherapeutic, therapeutic, diagnostic or symptomatic compounds; b) in combination with a therapeutically effective amount of one or more antihypertensive agents, such as calcium channel antagonists, or 3) together with the antihypertensive agents from b) and one or more conventional compounds from c).

[0015] In the practice of the present invention, an exemplary iNOS inhibitor compound is N G -Monomethyl-L-arginine [L-NMMA], (N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide) [1400W], (N 5 -[imino(nitroamino)methyl]-L-ornithine methyl ester) [L-NAME], and salts, derivatives and combinations thereof.

[0016] Similarly, in the practice of the present invention, exemplary calcium channel antagonists include, without limitation, one or more antihypertensive agents selected from the group consisting of amlodipine, felodipine, lacidipine, nicardipine, nivaldipine, azelnidipine, and combinations thereof.

[0017] In illustrative embodiments, the inventors have demonstrated that synergistic therapeutic outcomes can be achieved when one or more iNOS inhibitors and one or more calcium channel antagonists are co-administered. In one such embodiment, the iNOS inhibitor, L-NMMA, and the calcium channel antagonist, amlodipine besylate (NORVASC®, 3-ethyl-5-methyl(±)-2-[(2-aminoethoxy)methyl]4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridinedicarboxylate, monobenzenesulfonate; C 20 H 25 Notably, when CIN2O5·C6H6O3S; MW = 567.1) was co-administered to TNBC cell lines, a surprising and unexpected synergistic effect was obtained.

[0018] Optionally, the compositions of the invention may further comprise one or more additional separate iNOS inhibitors and / or one or more additional separate antihypertensive agents and / or one or more additional separate conventional treatments.

[0019] In certain embodiments, the inventors contemplate co-therapy formulations comprising one or more iNOS inhibitory compounds formulated with one or more additional active ingredients, including, without limitation, one or more antihypertensive agents, anti-neoplastic agents, cytotoxic agents, cytostatic agents or chemotherapeutic agents, or any combination thereof.

[0020] Exemplary chemotherapeutic agents include, without limitation, anti-cancer compounds such as cyclophosphamide, doxorubicin, 5-fluorouracil, docetaxel, paclitaxel, trastuzumab, methotrexate, epirubicin, cisplatin, carboplatin, vinorelbine, capecitabine, gemcitabine, mitoxantrone, isabepilone, eribulin, lapatinib, carmustine, nitrogen mustard, sulfur mustard, platin tetranitrate, vinblastine, etoposide, camptothecin, topoisomerase inhibitors (including topoisomerase I and II inhibitors), and derivatives, analogs, salts, active metabolites, or one or more combinations thereof.

[0021] Exemplary therapeutic agents include, without limitation, one or more of an immunomodulatory agent, a neuroactive agent, an anti-inflammatory agent, an anti-hyperlipidemic agent, a hormone, a receptor agonist or antagonist or an anti-infective agent, or a compound selected from a protein, a peptide, an antibody, an enzyme, RNA, DNA, siRNA, mRNA, a ribozyme, a hormone, a cofactor, a steroid, an antisense molecule, and combinations thereof.

[0022] Similarly, the administration of the chemotherapeutic formulations disclosed herein can be further augmented by one or more additional cancer therapies, including, without limitation, the administration of a therapeutically effective amount of radiation to the mammal undergoing treatment.

[0023] In the practice of the present invention, the disclosed chemotherapeutic compositions can be administered systemically to an animal in a single dose or in multiple doses over a period ranging from one or more weeks to one or more months, as deemed necessary by the medical provider in charge of the treatment regimen.

[0024] Preferably, the iNOS inhibitory anti-cancer compositions disclosed herein further comprise one or more pharma- ceutically acceptable carriers, buffers, diluents, vehicles, excipients, or any combination thereof, suitable for administration to mammalian host cells, particularly human host cells.

[0025] In another embodiment, the present invention provides a method for treating or ameliorating one or more symptoms of cancer in an animal in need thereof, comprising at least the step of administering to an animal in need thereof an effective amount of one or more of the chemotherapeutic compositions disclosed herein for a time sufficient to treat or ameliorate, in a general and general sense, one or more symptoms of cancer in the animal.

[0026] In another embodiment, the present invention provides a method for treating or ameliorating one or more symptoms of cancer in a mammalian subject. In a general and general sense, the method at least comprises administering to a mammalian subject in need thereof a therapeutically effective amount of one or more of the chemotherapeutic compositions disclosed herein for a time effective to treat or ameliorate one or more symptoms of cancer in the subject.

[0027] In certain embodiments, the inventors believe that the disclosed chemotherapeutic formulations are particularly useful in situations where the cancer is diagnosed or identified as a refractory, metastatic, relapsing or treatment-resistant cancer, including, for example, situations where the cancer is diagnosed or identified as a treatment-resistant triple-negative breast cancer.

[0028] The present invention also provides a method for treating or ameliorating one or more symptoms of cancer in an animal in need thereof. Such a method generally comprises at least administering to the animal (systemically or locally to one or more regions or sites in or around the body of the animal) an effective amount of at least a first chemotherapeutic iNOS inhibitor formulation or its analog, agonist, antagonist or derivative or salt disclosed herein, alone or in combination with one or more calcium channel antagonists, for a time sufficient to treat or ameliorate one or more symptoms of cancer in the animal.

[0029] In a further aspect, the present invention also provides a method for inhibiting the growth of cancer cells or tumors in an animal, comprising providing to one or more cells or tissues of the body of an animal in need thereof an amount of one or more of the chemotherapeutic iNOS inhibitor formulations disclosed herein in an amount and for a time effective to inhibit the growth of cancer cells or tumors in a global and general sense.

[0030] In another aspect, the present invention provides a method for treating cancer in a subject, preferably a human. In an overall and general sense, the method generally comprises administering to a subject in need thereof a therapeutically effective amount of one or more of the iNOS inhibitory chemotherapy formulations disclosed herein, alone or in combination with one or more calcium channel antagonists, such as one or more antihypertensive calcium channel antagonist compounds disclosed herein, one or more additional chemotherapeutic agents, a therapeutically effective amount of ionizing radiation, or any combination thereof. Exemplary additional compositions that can be co-administered to a subject include, without limitation, one or more conventional anticancer drugs. Alternatively, the method of the present invention can also comprise one or more surgical interventions, such as tumor resection, or can further optionally comprise one or more courses of therapeutically effective ionizing radiation (i.e., radiation therapy).

[0031] The present invention also provides a method for treating or ameliorating one or more symptoms of cancer in a mammal. Such a method generally comprises administering to a mammal an effective amount of an iNOS inhibitory chemotherapeutic formulation disclosed herein, alone or in combination with one or more antihypertensive drugs, particularly calcium channel antagonists, alone or in further combination with one or more conventional chemotherapeutic agents, for a time sufficient to treat or ameliorate one or more symptoms of cancer in the mammal.

[0032] The present invention also provides pharmaceutical compositions for use in the treatment of cancer in an animal subject, comprising one or more of the iNOS inhibitory chemotherapeutic formulations disclosed herein, alone or in combination with one or more calcium channel antagonists, and can include such use to treat or ameliorate one or more symptoms of malignant breast cancer in a human subject.

[0033] The present invention also provides methods of altering, affecting, destroying or killing one or more mammalian cells in or around the body of an animal having, suspected of having, or diagnosed with one or more forms of mammalian cancer, including, but not limited to, breast cancer, lung cancer, prostate cancer, fibrosarcoma, synovial sarcoma, pancreatic cancer and other forms of the disease. Such methods generally include providing to one or more animal cells a therapeutically effective amount of one or more of the disclosed iNOS inhibitory chemotherapeutic compositions, alone or in combination with one or more antihypeprtensive agents, including, inter alia, calcium channel antagonists, for a time sufficient to treat and / or ameliorate one or more symptoms of cancer in the animal.

[0034] Also provided herein are methods of altering, modulating, regulating, increasing and / or attenuating at least one component, pathway, enzyme or step involved in the process of hyperproliferative cells growing in or around the body of an animal by providing an effective amount of one of more of the disclosed iNOS inhibitory chemotherapeutic compositions, alone or in combination with one or more calcium channel antagonists, to one or more cells, tissues and / or organs of a subject in need thereof for a time effective to alter, modulate, control, increase and / or attenuate at least one component, pathway, enzyme or step involved in the process of hyperproliferative cell growth in such cells, tissues, organs and / or body.

[0035] Further provided herein are methods of treating and / or ameliorating at least one symptom of cancer in a mammal, including, without limitation, human breast cancer and human therapy-resistant triple-negative breast cancer.

[0036] iNOS inhibitor compounds and preparations thereof As noted herein, the iNOS inhibitory chemotherapeutic formulations of the present invention may be used as a single cancer treatment modality or may be combined with one or more additional chemotherapeutic agents, diagnostic reagents and / or the like, including, without limitation, one or more proteins, peptides, polypeptides (including, without limitation, enzymes, antibodies, antigens, antigen-binding fragments, etc.); RNA molecules (including, without limitation, catalytic RNAs such as siRNA, iRNA, mRNA, tRNA and ribozymes, etc.), DNA molecules (including, without limitation, oligonucleotides, polynucleotides, genes, coding sequences (CDS), introns, exons, plasmids, cosmids, phagemids, baculoviruses, vectors (including, without limitation, viral vectors, virions, viral particles, etc.)); peptide nucleic acids, detection agents, imaging agents, contrast agents, detectable gases, radionuclides, etc., and one or more additional chemotherapeutic agents, surgical intervention (e.g., tumor resection), radiation therapy, etc., or any combination thereof as part of a multifactorial or multifocal treatment plan for an affected patient.

[0037] The chemotherapeutic formulations of the present invention may optionally further comprise one or more additional components to aid, facilitate or improve delivery of the iNOS inhibitory chemotherapeutic formulation, including, without limitation, one or more liposomes, particles, lipid complexes, may further optionally comprise one or more binding agents, cell surface active agents, surfactants, lipid complexes, niosomes, ethosomes, transferosomes, phospholipids, sphingolipids, sphingosomes or any combination thereof, and may optionally be provided within a pharmaceutical formulation comprising one or more nanoparticles, microparticles, nanocapsules, microcapsules, nanospheres, microspheres or any combination thereof.

[0038] The pharmaceutical compositions may also be mixed with one or more pharma- ceutically acceptable carriers, diluents, excipients, or any combination thereof, and may be further optionally formulated to include liposomes, surfactants, niosomes, ethosomes, transferosomes, phospholipids, sphingosomes, nanoparticles, microparticles, or any combination thereof.

[0039] Preferably, the chemotherapeutic formulations disclosed herein will be at least substantially stable at a pH of about 4.2 to about 8.2, and more preferably, substantially stable at a pH of about 5 to about 7.5. Preferably, the active ingredients will be substantially active at the physiological conditions of the animal to which they are administered.

[0040] Chemotherapy Methods and Uses Another important aspect of the present invention pertains to methods for using the disclosed iNOS inhibitory chemotherapeutic formulations to treat or ameliorate one or more symptoms of one or more forms of breast cancer, including, for example, treatment-resistant breast cancer such as triple-negative breast cancer. Such methods generally include administering to a mammal (particularly a human in need thereof) one or more of the disclosed anti-cancer compositions in an amount and for a time sufficient to treat (or ameliorate one or more symptoms of) breast cancer in the affected mammal.

[0041] In certain embodiments, the chemotherapy formulations described herein can be provided to animals in a single treatment modality (as a single dose, or multiple doses over a period of hours to days or weeks) as needed for cancer treatment.Alternatively, in some embodiments, it may be desirable to continue treatment for a period of weeks to months or longer, or to include it in combination with one or more additional modes of treatment.In other embodiments, it may be desirable to provide treatment in combination with one or more existing or conventional treatment regimens.

[0042] The invention also provides the use of one or more of the disclosed chemotherapeutic compositions in the manufacture of a medicament for the treatment and / or amelioration of one or more symptoms of cancer, particularly in the manufacture of a medicament for the treatment and / or amelioration of one or more symptoms of cancer in a mammal, such as human breast cancer.

[0043] The invention also provides the use of one or more of the disclosed iNOS inhibitor / calcium channel antagonist formulations in the manufacture of a medicament for the treatment of cancer, in particular for the treatment of treatment-resistant triple-negative breast cancer in humans.

[0044] Treatment kit Therapeutic kits comprising one or more of the disclosed iNOS inhibitor formulations and instructions for using the kit in a particular cancer treatment modality also represent preferred embodiments of the present invention. These kits may further optionally comprise one or more additional anti-cancer compounds, one or more diagnostic reagents or one or more additional therapeutic compounds, pharmaceuticals, etc.

[0045] The kit of the present invention can be packaged for commercial distribution and can further optionally include one or more delivery devices (e.g., syringes, injections, etc.) adapted for delivery of the chemotherapeutic composition(s) to animals. Such kits typically include at least one vial, test tube, flask, bottle, syringe or other container into which the pharmaceutical composition(s) can be placed and preferably dispensed accordingly. If a second pharmaceutical product is also provided, the kit can also contain a second separate container into which the second composition can be placed. Alternatively, multiple pharmaceutical compositions disclosed herein can be prepared in a single mixture, such as a suspension or solution, and packaged in a single container, such as a vial, flask, syringe, catheter, cannula, bottle or other suitable single container.

[0046] The kits of the present invention typically may also include a retention mechanism adapted to contain or hold the vial(s) or other container(s) in close confinement for commercial sale, such as, for example, an injection or blow molded plastic container that can hold the desired vial(s) or other container(s) to minimize or prevent breakage, exposure to sunlight, or other undesirable factors, or to allow for immediate use of the composition(s) included within the kit.

[0047] Pharmaceutical Formulations In certain embodiments, the present invention relates to the formulation of one or more chemotherapeutic and / or diagnostic compounds in a pharma- ceutically acceptable formulation for delivery to one or more cells or tissues of an animal, either alone or in combination with one or more other modalities of diagnosis, prophylaxis and / or treatment.The formulation of pharma-ceutically acceptable excipients and carrier solutions is well known to those skilled in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in various treatment regimens.

[0048] In certain circumstances, it may be desirable to deliver the disclosed chemotherapeutic compositions in an appropriately formulated pharmaceutical vehicle by one or more standard delivery devices, including, but not limited to, subcutaneous, parenteral, intravenous, intramuscular, intrathecal, oral, intraperitoneal, transdermal, topical, oral or nasal inhalation, or direct injection to one or more cells, tissues, or organs in or around the body of an animal.

[0049] Methods of administration may also include modalities described in U.S. Patent Nos. 5,543,158, 5,641,515, and 5,399,363, each of which is specifically incorporated herein by express reference in its entirety. Solutions of the active compounds as free bases or pharmacologically acceptable salts may be prepared in sterile water, suitably mixed with one or more surfactants, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, oils, or mixtures thereof. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0050] For administration of an injectable aqueous solution, without limitation, the solution can be appropriately buffered if necessary, and the liquid diluent is first made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, transdermal, subdermal and / or intraperitoneal administration. In this regard, the compositions of the present invention can be formulated in one or more pharma-ceutically acceptable vehicles, including, for example, sterile aqueous media, buffers, diluents, and the like. For example, a given dosage of active ingredient(s) can be dissolved in a particular volume of an isotonic solution (e.g., an isotonic NaCl-based solution) and then injected at the proposed site of administration, or further diluted in a vehicle suitable for intravenous infusion (see, for example, "Remington's Pharmaceutical Sciences", 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated, the extent of treatment, and the site of administration; nevertheless, the person responsible for administration will be able to determine the correct dosing regimen appropriate for the individual subject, using ordinary knowledge in the medical and pharmaceutical arts.

[0051] Sterile injectable compositions can be prepared by incorporating the disclosed compositions in the required amount in a suitable solvent, together with some of the other ingredients listed above, as needed, followed by sterile filtration. In general, dispersions can be prepared by incorporating the selected sterilized active ingredient(s) into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those listed above. The compositions disclosed herein can also be formulated in neutral or salt form.

[0052] Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) formed with inorganic acids such as, but not limited to, hydrochloric or phosphoric acids, or organic acids such as, but not limited to, acetic, oxalic, tartaric, mandelic, and others. Salts formed with free carboxyl groups can also be derived from inorganic bases such as, but not limited to, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and others. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in an amount effective for the intended application. The formulations are easily administered in a variety of dosage forms, such as injectable solutions, topical preparations, oral formulations, including time-release capsules, hydrogels, colloids, viscous gels, transdermal agents, intranasal and inhalation formulations, and others.

[0053] The amount, dosage regimen, formulation and administration of the chemotherapeutic agent disclosed herein will be within the skill of a person skilled in the art who has the benefit of the present teachings.However, administration of a therapeutically effective (i.e., pharmacologic effective) amount of the disclosed compositions may be achieved by a single administration, such as, but not limited to, a single injection of the agent delivered in an amount sufficient to bring about the desired benefit to the patient undergoing such procedure.Alternatively, in some circumstances, it may be desirable to give multiple or sequential administrations of the composition over a relatively short or even relatively extended period of time, as may be determined by the physician supervising the administration of such compositions to selected individuals.

[0054] Typically, one or more formulations of the compositions described herein will contain at least a chemotherapeutic effective amount of the first active agent. Preferably, the formulations will contain at least about 0.001% of each active ingredient, preferably at least about 0.01%, although the percentage of the active ingredient(s) can, of course, vary and can conveniently be present in an amount of about 0.01 to about 90% by weight or volume, or about 0.1 to about 80% by weight or volume, or more preferably about 0.2 to about 60% by weight or volume, based on the total formulation. Naturally, the amount of active compound(s) in each composition can be prepared in such a way that a suitable dosage is obtained in any given unit dose of the compound. Solubility, bioavailability, biological activity, and the like can be considered. 1 / 2 Factors such as route of administration, product shelf life and other pharmacological considerations will be taken into account by those skilled in the art of preparing such pharmaceutical formulations, and therefore various dosages and treatment regimens may be desirable.

[0055] The chemotherapeutic compositions disclosed herein can be administered by any effective method, including, but not limited to, parenteral, intravenous, intramuscular, or even intraperitoneal administration, for example, as described in U.S. Patent Nos. 5,543,158, 5,641,515, and 5,399,363, each of which is specifically incorporated herein by express reference in its entirety. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water, suitably mixed with a surfactant, such as hydroxypropylcellulose, or in other similar manner. Pharmaceutical forms adapted for injectable administration include sterile aqueous solutions or dispersions, including, but not limited to, those described in U.S. Patent No. 5,466,468, which is specifically incorporated herein by express reference in its entirety, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be at least sufficiently stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as viruses, bacteria, fungi, and the like.

[0056] The carrier(s) can be a solvent or dispersion medium including, without limitation, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like, or combinations thereof), one or more vegetable oils, or any combination thereof, but may contain additional pharma- ceutically acceptable components.

[0057] The proper fluidity of the pharmaceutical preparations disclosed herein can be maintained by, for example, the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion, by using surfactants, or by any combination of these techniques. The inhibition or prevention of the action of microorganisms can be brought about by one or more antibacterial or antifungal agents, such as, but not limited to, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include an isotonic agent, such as, but not limited to, one or more sugars or sodium chloride, or any combination thereof. Prolonged absorption of an injectable composition can be brought about by the use of an agent that delays absorption in the composition, such as, but not limited to, aluminum monostearate, gelatin, or a combination thereof.

[0058] While systemic administration is believed to be effective in many embodiments of the present invention, it is also believed that the formulations disclosed herein are suitable for direct injection into one or more organs, tissues or cell types in the body. Administration of the disclosed compositions can be carried out using any suitable means, including those known to those skilled in the relevant medical arts.

[0059] The pharmaceutical formulations disclosed herein are by no means limited to use only in humans, or even primates or mammals. In certain embodiments, the methods and compositions disclosed herein can be used with birds, amphibians, reptiles or other animal species. However, in preferred embodiments, the compositions of the present invention are preferably formulated for administration to mammals, particularly humans, in various diagnostic and / or therapeutic regimens. The compositions disclosed herein can also be provided in formulations acceptable for veterinary administration, including, but not limited to, selected livestock, exotic or farm animals, companion animals (including pets and the like), non-human primates and zoological or other captive specimens and the like. The present specification provides, for example, the following items: (Item 1) 1) a chemotherapeutic effective amount of a first iNOS inhibitor; 2) a therapeutically effective amount of: a) a first antihypertensive agent; b) a first chemotherapeutic agent, or c) a combination of a) and b) 13. A pharmaceutical composition comprising: (Item 2) The first iNOS inhibitor is G -Monomethyl-L-arginine [L-NMMA], (N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide) [1400W] or (N 5 2. The pharmaceutical composition according to item 1, comprising -[imino(nitroamino)methyl]-L-ornithine methyl ester) [L-NAME]. (Item 3) 3. The pharmaceutical composition of claim 1, wherein the first antihypertensive agent comprises a first calcium channel antagonist. (Item 4) The pharmaceutical composition of any of the preceding items, wherein the first antihypertensive agent comprises a first calcium channel antagonist selected from the group consisting of amlodipine, aranidipine, azelnidipine, valnidipine, benidipine, clinidipine, clevidipine, diltiazem, efonidipine, fendiline, felodipine, gallopamil, isradipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nimodipine, nisoldipine, nitrendipine, nivaldipine, pranidipine, and verapamil. (Item 5) 3) The pharmaceutical composition of any of the preceding items, further comprising a second, separate iNOS inhibitor. (Item 6) d) The pharmaceutical composition according to any of the preceding items, further comprising one or more of an immunomodulatory agent, a neuroactive agent, an anti-inflammatory agent, an antihyperlipidemic agent, a hormone, a receptor agonist, a receptor antagonist, an anti-infective agent, a protein, a peptide, an antibody, an antigen-binding fragment, an enzyme, RNA, DNA, siRNA, mRNA, a ribozyme, a hormone, a cofactor, a steroid, an antisense molecule, a second separate antihypertensive agent, a second separate chemotherapeutic agent, or any combination thereof. (Item 7) The pharmaceutical composition of any of the preceding items, wherein the first chemotherapeutic agent comprises one or more antineoplastic compounds, one or more cytotoxic compounds, one or more cytostatic compounds, or any combination thereof. (Item 8) The pharmaceutical composition of any of the preceding items, wherein the first chemotherapeutic agent is selected from the group consisting of cyclophosphamide, doxorubicin, 5-fluorouracil, docetaxel, paclitaxel, trastuzumab, methotrexate, epirubicin, cisplatin, carboplatin, vinorelbine, capecitabine, gemcitabine, mitoxantrone, isabepilone, eribulin, lapatinib, carmustine, nitrogen mustard, sulfur mustard, platin tetranitrate, vinblastine, etoposide, camptothecin, and any combination thereof. (Item 9) The pharmaceutical composition of any of the preceding items, wherein i) the first iNOS inhibitor comprises L-NMMA, and ii) the first antihypertensive agent comprises amlodipine or the first chemotherapeutic agent comprises docetaxel. (Item 10) The pharmaceutical composition of any of the preceding items, wherein i) the first iNOS inhibitor comprises L-NMMA, ii) the first antihypertensive agent comprises amlodipine, and iii) the first chemotherapeutic agent comprises docetaxel. (Item 11) The pharmaceutical composition according to any of the preceding items, further comprising a liposome, a surfactant, a niosome, an ethosome, a transferosome, a phospholipid, a sphingosome, a nanoparticle, a microparticle, or any combination thereof. (Item 12) The pharmaceutical composition according to any of the preceding items, further comprising a pharma- ceutically acceptable carrier, buffer, diluent, vehicle, excipient, or any combination thereof. (Item 13) A pharmaceutical composition according to any of the preceding items, formulated for mammalian administration, preferably for human administration. (Item 14) The pharmaceutical composition of any of the preceding items, adapted and configured as part of a therapeutic kit comprising the composition and at least a first set of instructions for administration of the composition to a human in need thereof. (Item 15) A pharmaceutical composition according to any of the preceding items for use in the treatment, prevention or amelioration of one or more symptoms of cancer in a mammal. (Item 16) A pharmaceutical composition according to any of the preceding items for use in the treatment, prevention or amelioration of one or more symptoms of human breast cancer, in particular resistant, metastatic or triple-negative human breast cancer. (Item 17) 17. A pharmaceutical composition according to any one of items 1 to 16 for use in therapy. (Item 18) 17. The pharmaceutical composition according to any one of items 1 to 16, for use in the treatment of cancer in a mammalian subject. (Item 19) 14. Use of a pharmaceutical composition according to any one of items 1 to 13 in the manufacture of a medicament for treating or ameliorating one or more symptoms of cancer in a mammal. (Item 20) 20. The use according to item 19 in the manufacture of a medicament for treating human breast cancer, in particular refractory, treatment-resistant, relapsed, metastatic or triple-negative breast cancer. (Item 21) 17. A method of treating or ameliorating one or more symptoms of cancer in an animal in need thereof, comprising administering to said animal an effective amount of a pharmaceutical composition according to any one of items 1 to 16 for a time sufficient to treat or ameliorate said one or more symptoms of said cancer in said animal. (Item 22) 22. The method of claim 21, wherein the cancer is diagnosed or identified as a refractory, metastatic, relapsing or treatment-resistant cancer. (Item 23) The method of claim 21 or claim 22, wherein the cancer is diagnosed or identified as a treatment-resistant or metastatic cancer, in particular a treatment-resistant, triple-negative human breast cancer. (Item 24) 24. The method according to any one of items 21 to 23, further comprising administering to the animal a therapeutically effective amount of radiation. (Item 25) 25. The method of any one of items 19 to 24, wherein the pharmaceutical composition is administered systemically to the animal in a single dose or in a series of multiple doses over a period of one or more days, over a period of one or more weeks, or over a period of one or more months or longer. (Item 26) 26. The method of any one of items 19 to 25, wherein the pharmaceutical composition further comprises a second distinct chemotherapeutic agent or a second distinct iNOS inhibitor according to any one of items 1 to 16.

[0060] To promote an understanding of the principles of the invention, reference will now be made to the embodiments or examples illustrated in the drawings, and specific terminology will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. Any changes and further modifications in the described embodiments, and any further applications of the principles of the invention as described herein, are contemplated as would normally occur to one skilled in the art to which the invention pertains.

[0061] The following drawings form part of the present specification and are included to demonstrate certain aspects of the present invention. This application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee. The present invention can be more fully understood by reference to the following description taken in conjunction with the accompanying drawings, in which like reference numerals identify like elements. The accompanying drawings are shown below. [Brief description of the drawings]

[0062] [Figure 1A-D]Figure 1A, Figure 1B, Figure 1C, Figure 1D, Figure 1E, and Figure 1F illustrate that enhanced NOS2 expression correlates with poor patient survival in invasive TNBC. Oncomine cancer microarray analysis of The Cancer Genome Atlas (TCGA) database (Figure 1A and Figure 1B). Figure 1A: Higher NOS2 mRNA expression in invasive TNBC versus non-TNBC. P=3.85E-5, t-test. Figure 1B: High NOS2 expression correlates with 5-year death in invasive breast cancer. P=0.037, t-test. Kaplan-Meier survival analysis in the Van de Vijver (n=69; p=0.04) (Figure 1C) and Curtis (n=260; p=0.01) (Figure 1D) (Wilcoxon test) breast databases show that high NOS2 expression correlates with worse overall survival in TNBC patients. (Figure 1E) Immunohistochemical analysis of TNBC human samples for iNOS protein expression. Weak-moderate (3-4), moderate-strong (5-6) and strong (7) were the established cut-offs for further analysis of survival. Several samples showed expression in both tumor (T) and stromal (S) cells (original optical objective: 20x). MDA-MB-231 cells transfected with either NOS2-directed shRNA (shRNA1) or empty vector (EV) were used as negative and positive controls for iNOS staining, respectively (original optical objective: 10x). Counterstain: hematoxylin. Figure 1F: Increased iNOS expression is associated with poorer patient survival compared to low iNOS expression. Kaplan-Meier survival analysis of TNBC human patient samples (n=83). P=0.05, log-rank test. [Figure 1E-F]Figure 1A, Figure 1B, Figure 1C, Figure 1D, Figure 1E, and Figure 1F illustrate that enhanced NOS2 expression correlates with poor patient survival in invasive TNBC. Oncomine cancer microarray analysis of The Cancer Genome Atlas (TCGA) database (Figure 1A and Figure 1B). Figure 1A: Higher NOS2 mRNA expression in invasive TNBC versus non-TNBC. P=3.85E-5, t-test. Figure 1B: High NOS2 expression correlates with 5-year death in invasive breast cancer. P=0.037, t-test. Kaplan-Meier survival analysis in the Van de Vijver (n=69; p=0.04) (Figure 1C) and Curtis (n=260; p=0.01) (Figure 1D) (Wilcoxon test) breast databases show that high NOS2 expression correlates with worse overall survival in TNBC patients. (Figure 1E) Immunohistochemical analysis of TNBC human samples for iNOS protein expression. Weak-moderate (3-4), moderate-strong (5-6) and strong (7) were the established cut-offs for further analysis of survival. Several samples showed expression in both tumor (T) and stromal (S) cells (original optical objective: 20x). MDA-MB-231 cells transfected with either NOS2-directed shRNA (shRNA1) or empty vector (EV) were used as negative and positive controls for iNOS staining, respectively (original optical objective: 10x). Counterstain: hematoxylin. Figure 1F: Increased iNOS expression is associated with poorer patient survival compared to low iNOS expression. Kaplan-Meier survival analysis of TNBC human patient samples (n=83). P=0.05, log-rank test. [Figure 2A-C]Figures 2A, 2B, 2C, 2D, 2E and 2F illustrate the effect of iNOS inhibitors on the tumorigenicity of TNBC cell lines. Proliferation (Figures 2A and 2B), primary (Figure 2C) and secondary (Figure 2D) mammospheres and migration index (Figures 2E and 2F) of MDA-MB-231 and SUM159 cell lines treated with 1400W and L-NMMA for 96 hours. Results were normalized to vehicle. Data are presented as mean ± SEM. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, one-way ANOVA and Bonferroni post-hoc test. [Fig. 2D-F] Figures 2A, 2B, 2C, 2D, 2E and 2F illustrate the effect of iNOS inhibitors on the tumorigenicity of TNBC cell lines. Proliferation (Figures 2A and 2B), primary (Figure 2C) and secondary (Figure 2D) mammospheres and migration index (Figures 2E and 2F) of MDA-MB-231 and SUM159 cell lines treated with 1400W and L-NMMA for 96 hours. Results were normalized to vehicle. Data are presented as mean ± SEM. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, one-way ANOVA and Bonferroni post-hoc test. [Figure 3A-D]Figure 3A, Figure 3B, Figure 3C, Figure 3D, Figure 3E, Figure 3F, Figure 3G, Figure 3H, Figure 3I and Figure 3J show that iNOS knockdown impairs tumorigenicity and EMT through dual effects on HIF1α and endoplasmic reticulum (ER) stress / TGFβ / AFT4 / ATF3 crosstalk. Proliferation (Figure 3A), migration (Figure 3B) and self-renewal capacity (primary and secondary mammospheres) (Figure 3C) in MDA-MB-231 cells transfected with two different NOS2-directed shRNAs (shRNA1, shRNA2) compared to empty vector (shRNA-EV). Western blot analysis of NOS isoforms (iNOS, eNOS, nNOS) and EMT transcription factors in MDA-MB-231 and SUM159 cell lines treated with 1400W (Figure 3D) and shRNA-mediated NOS2 knockdown (Figure 3E). Selective iNOS inhibition reduced hypoxia (HIF1α), ER stress markers (IRE1α, ATF4) (Figure 3F), phospho-Smad2 / 3, Smad2 / 3 and mature TGFβ protein levels (Figure 3G) in MDA-MB-231 and SUM159 cells. Figure 3H: Recombinant TGFβ1 (10 ng / mL, 24 h) activates the PERK / eIF2α / ATF4 / ATF3 axis in MCF10A. Figure 3I: Effect of co-treatment of recombinant TGFβ1 (10 ng / mL) and 1400W (4 mM) for 24 h on the PERK / eIF2α / ATF4 / ATF3 axis in MCF10A cells. iNOS, ATF4, ATF3 and mature TGFβ protein levels in siRNA-mediated NOS2 knockdown (siRNA20) MCF10A cells for 96 h. FIG. 3J: Selective iNOS inhibition is hypothesized to impair EMT and tumor cell migration through effects on HIF1α, ER stress (IRE1α / XBP1), and crosstalk between ATF4, ATF3, and TGFβ. Results were normalized to empty vector. Data are presented as mean ± SEM. ****p<0.0001, ***p<0.001, **p<0.01. One-way ANOVA and Bonferroni post-hoc test. [Figure 3E-G]Figure 3A, Figure 3B, Figure 3C, Figure 3D, Figure 3E, Figure 3F, Figure 3G, Figure 3H, Figure 3I and Figure 3J show that iNOS knockdown impairs tumorigenicity and EMT through dual effects on HIF1α and endoplasmic reticulum (ER) stress / TGFβ / AFT4 / ATF3 crosstalk. Proliferation (Figure 3A), migration (Figure 3B) and self-renewal capacity (primary and secondary mammospheres) (Figure 3C) in MDA-MB-231 cells transfected with two different NOS2-directed shRNAs (shRNA1, shRNA2) compared to empty vector (shRNA-EV). Western blot analysis of NOS isoforms (iNOS, eNOS, nNOS) and EMT transcription factors in MDA-MB-231 and SUM159 cell lines treated with 1400W (Figure 3D) and shRNA-mediated NOS2 knockdown (Figure 3E). Selective iNOS inhibition reduced hypoxia (HIF1α), ER stress markers (IRE1α, ATF4) (Figure 3F), phospho-Smad2 / 3, Smad2 / 3 and mature TGFβ protein levels (Figure 3G) in MDA-MB-231 and SUM159 cells. Figure 3H: Recombinant TGFβ1 (10 ng / mL, 24 h) activates the PERK / eIF2α / ATF4 / ATF3 axis in MCF10A. Figure 3I: Effect of co-treatment of recombinant TGFβ1 (10 ng / mL) and 1400W (4 mM) for 24 h on the PERK / eIF2α / ATF4 / ATF3 axis in MCF10A cells. iNOS, ATF4, ATF3 and mature TGFβ protein levels in siRNA-mediated NOS2 knockdown (siRNA20) MCF10A cells for 96 h. FIG. 3J: Selective iNOS inhibition is hypothesized to impair EMT and tumor cell migration through effects on HIF1α, ER stress (IRE1α / XBP1), and crosstalk between ATF4, ATF3, and TGFβ. Results were normalized to empty vector. Data are presented as mean ± SEM. ****p<0.0001, ***p<0.001, **p<0.01. One-way ANOVA and Bonferroni post-hoc test. [Figure 3H-J]Figure 3A, Figure 3B, Figure 3C, Figure 3D, Figure 3E, Figure 3F, Figure 3G, Figure 3H, Figure 3I and Figure 3J show that iNOS knockdown impairs tumorigenicity and EMT through dual effects on HIF1α and endoplasmic reticulum (ER) stress / TGFβ / AFT4 / ATF3 crosstalk. Proliferation (Figure 3A), migration (Figure 3B) and self-renewal capacity (primary and secondary mammospheres) (Figure 3C) in MDA-MB-231 cells transfected with two different NOS2-directed shRNAs (shRNA1, shRNA2) compared to empty vector (shRNA-EV). Western blot analysis of NOS isoforms (iNOS, eNOS, nNOS) and EMT transcription factors in MDA-MB-231 and SUM159 cell lines treated with 1400W (Figure 3D) and shRNA-mediated NOS2 knockdown (Figure 3E). Selective iNOS inhibition reduced hypoxia (HIF1α), ER stress markers (IRE1α, ATF4) (Figure 3F), phospho-Smad2 / 3, Smad2 / 3 and mature TGFβ protein levels (Figure 3G) in MDA-MB-231 and SUM159 cells. Figure 3H: Recombinant TGFβ1 (10 ng / mL, 24 h) activates the PERK / eIF2α / ATF4 / ATF3 axis in MCF10A. Figure 3I: Effect of co-treatment of recombinant TGFβ1 (10 ng / mL) and 1400W (4 mM) for 24 h on the PERK / eIF2α / ATF4 / ATF3 axis in MCF10A cells. iNOS, ATF4, ATF3 and mature TGFβ protein levels in siRNA-mediated NOS2 knockdown (siRNA20) MCF10A cells for 96 h. FIG. 3J: Selective iNOS inhibition is hypothesized to impair EMT and tumor cell migration through effects on HIF1α, ER stress (IRE1α / XBP1), and crosstalk between ATF4, ATF3, and TGFβ. Results were normalized to empty vector. Data are presented as mean ± SEM. ****p<0.0001, ***p<0.001, **p<0.01. One-way ANOVA and Bonferroni post-hoc test. [Figure 4A-B]Figure 4A, Figure 4B, Figure 4C and Figure 4D show the reduction of tumor initiation and lung metastasis in MDA-MB-231 xenografts. Figure 4A: Tumor volume of MDA-MB-231 breast xenografts (n=5 / group) after daily injection of L-NAME (80 mg / kg, ip). Two-way ANOVA and Bonferroni post-hoc test. Figure 4B: Primary and secondary MSFE of cancer cells isolated from tumor tissue. Student's t-test. Figure 4C: Tumor initiation ability of tumor cells assayed by limiting dilution method. Fisher's exact test. Figure 4D: Luminescence of MDA-MB-231 L / G tumor cells in the lungs of vehicle and L-NAME treated mice. Student's t-test. Results were normalized to vehicle. Data are shown as mean ± SEM. ***p<0.001, **p<0.01, *p<0.05. [Figure 4C-D] Figure 4A, Figure 4B, Figure 4C and Figure 4D show the reduction of tumor initiation and lung metastasis in MDA-MB-231 xenografts. Figure 4A: Tumor volume of MDA-MB-231 breast xenografts (n=5 / group) after daily injection of L-NAME (80 mg / kg, ip). Two-way ANOVA and Bonferroni post-hoc test. Figure 4B: Primary and secondary MSFE of cancer cells isolated from tumor tissue. Student's t-test. Figure 4C: Tumor initiation ability of tumor cells assayed by limiting dilution method. Fisher's exact test. Figure 4D: Luminescence of MDA-MB-231 L / G tumor cells in the lungs of vehicle and L-NAME treated mice. Student's t-test. Results were normalized to vehicle. Data are shown as mean ± SEM. ***p<0.001, **p<0.01, *p<0.05. [Figure 5A-C]Figure 5A, Figure 5B, Figure 5C, Figure 5D, Figure 5E and Figure 5F illustrate the in vivo effect of L-NMMA in MDA-MB-231 xenografts. Figure 5A: Tumor volume of MDA-MB-231 breast xenografts (n=10 / group) treated with vehicle, L-NMMA, chemotherapy and combination. Two-way ANOVA and Bonferroni's post-hoc test. Figure 5B: Illustrative images of Ki67 staining in vehicle, L-NMMA, docetaxel and combination groups. Original optical objective: 10x. Counterstain: hematoxylin. Figure 5C: Cell proliferation of tumor xenografts is depicted as Ki67 positive cells. 1,000 cells were counted from 10 different fields and the percentage was determined. Figure 5D: Nuclear cleaved caspase-3 staining in Chemo and Combo groups; 1,000 cells were counted from 10 different fields and the percentages were determined. Figure 5E: Primary and secondary MSFE of breast cancer cells isolated from tumor tissues. One-way ANOVA and Bonferroni's post-hoc test. Figure 5F: Tumor-initiating ability of tumor cells assayed by limiting dilution method. Fisher's exact test. Results were normalized to vehicle. Data are presented as mean ± SEM. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. [Figure 5D-E]Figure 5A, Figure 5B, Figure 5C, Figure 5D, Figure 5E and Figure 5F illustrate the in vivo effect of L-NMMA in MDA-MB-231 xenografts. Figure 5A: Tumor volume of MDA-MB-231 breast xenografts (n=10 / group) treated with vehicle, L-NMMA, chemotherapy and combination. Two-way ANOVA and Bonferroni's post-hoc test. Figure 5B: Illustrative images of Ki67 staining in vehicle, L-NMMA, docetaxel and combination groups. Original optical objective: 10x. Counterstain: hematoxylin. Figure 5C: Cell proliferation of tumor xenografts is depicted as Ki67 positive cells. 1,000 cells were counted from 10 different fields and the percentage was determined. Figure 5D: Nuclear cleaved caspase-3 staining in Chemo and Combo groups; 1,000 cells were counted from 10 different fields and the percentages were determined. Figure 5E: Primary and secondary MSFE of breast cancer cells isolated from tumor tissues. One-way ANOVA and Bonferroni's post-hoc test. Figure 5F: Tumor-initiating ability of tumor cells assayed by limiting dilution method. Fisher's exact test. Results were normalized to vehicle. Data are presented as mean ± SEM. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. [Figure 5F]Figure 5A, Figure 5B, Figure 5C, Figure 5D, Figure 5E and Figure 5F illustrate the in vivo effect of L-NMMA in MDA-MB-231 xenografts. Figure 5A: Tumor volume of MDA-MB-231 breast xenografts (n=10 / group) treated with vehicle, L-NMMA, chemotherapy and combination. Two-way ANOVA and Bonferroni's post-hoc test. Figure 5B: Illustrative images of Ki67 staining in vehicle, L-NMMA, docetaxel and combination groups. Original optical objective: 10x. Counterstain: hematoxylin. Figure 5C: Cell proliferation of tumor xenografts is depicted as Ki67 positive cells. 1,000 cells were counted from 10 different fields and the percentage was determined. Figure 5D: Nuclear cleaved caspase-3 staining in Chemo and Combo groups; 1,000 cells were counted from 10 different fields and the percentages were determined. Figure 5E: Primary and secondary MSFE of breast cancer cells isolated from tumor tissues. One-way ANOVA and Bonferroni's post-hoc test. Figure 5F: Tumor-initiating ability of tumor cells assayed by limiting dilution method. Fisher's exact test. Results were normalized to vehicle. Data are presented as mean ± SEM. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. [Figure 6A-C]Figure 6A, Figure 6B, Figure 6C, Figure 6D and Figure 6E show the clinically relevant dose regimen of L-NMMA in an orthotopic mouse model of TNBC. Figure 6A: Mean systolic pressure of mice (n=5) given the dose rate proposed in this study for one cycle. One-way ANOVA and Bonferroni post-hoc test. Figure 6B: Mean systolic pressure of mice (n=5) 30 min and 24 h after the last injection of one cycle treatment. One-way ANOVA and Bonferroni post-hoc test. Figure 6C: Tumor volume of MDA-MB-231 breast xenografts (n=10 / group) treated with vehicle, amlodipine, docetaxel and combination (docetaxel+L-NMMA). Two-way ANOVA and Bonferroni post-hoc test. Figure 6D: Kaplan-Meier survival curves of mice bearing vehicle, chemotherapy and combo treated MDA-MB-231 xenografts. Wilcoxon test. Figure 6E: Tumor volumes of SUM159 breast xenografts treated with vehicle, amlodipine, docetaxel and combination (docetaxel + L-NMMA). Two-way ANOVA and Bonferroni post-hoc test. Data are presented as mean ± SEM. ****p<0.0001, ***p<0.001. [Figure 6D-E]Figure 6A, Figure 6B, Figure 6C, Figure 6D and Figure 6E show the clinically relevant dose regimen of L-NMMA in an orthotopic mouse model of TNBC. Figure 6A: Mean systolic pressure of mice (n=5) given the dose rate proposed in this study for one cycle. One-way ANOVA and Bonferroni post-hoc test. Figure 6B: Mean systolic pressure of mice (n=5) 30 min and 24 h after the last injection of one cycle treatment. One-way ANOVA and Bonferroni post-hoc test. Figure 6C: Tumor volume of MDA-MB-231 breast xenografts (n=10 / group) treated with vehicle, amlodipine, docetaxel and combination (docetaxel+L-NMMA). Two-way ANOVA and Bonferroni post-hoc test. Figure 6D: Kaplan-Meier survival curves of mice bearing vehicle, chemotherapy and combo treated MDA-MB-231 xenografts. Wilcoxon test. Figure 6E: Tumor volumes of SUM159 breast xenografts treated with vehicle, amlodipine, docetaxel and combination (docetaxel + L-NMMA). Two-way ANOVA and Bonferroni post-hoc test. Data are presented as mean ± SEM. ****p<0.0001, ***p<0.001. [Figure 7A-B] Figures 7A and 7B show representative images of mammospheres in MDA-MB-231 cells treated with iNOS inhibitors. Illustrative images of primary (Figure 7A) and secondary (Figure 7B) mammospheres after 96 hours of treatment with 1400W, L-NMMA (vehicle, 1, 2, 4 mM) and L-NAME (vehicle, 1, 2, 5 mM). [Figure 8A-B] Figures 8A and 8B show representative images of mammospheres in SUM159 cells treated with iNOS inhibitors. Illustrative images of primary (Figure 8A) and secondary (Figure 8B) mammospheres after 96 hours of treatment with 1400W, L-NMMA (vehicle, 1, 2, 4 mM) and L-NAME (vehicle, 1, 2, 5 mM). [Figure 9A-B]Figure 9A, Figure 9B, Figure 9C, Figure 9D and Figure 9E show the effect of L-NAME and micromolar concentrations of 1400W and L-NMMA on the tumorigenicity of TNBC cell lines. Growth (Figure 9A), primary (Figure 9B) and secondary (Figure 9C) mammospheres of MDA-MB-231 and SUM159 cell lines treated with L-NAME. Effect of micromolar concentrations of 1400W (Figure 9D) and L-NMMA (Figure 9E) on the migration index of MDA-MB-231 and SUM159 cells. Results were normalized to vehicle. Data are presented as mean ± SEM. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, one-way ANOVA and Bonferroni post-hoc test. [Figure 9C-D] Figure 9A, Figure 9B, Figure 9C, Figure 9D and Figure 9E show the effect of L-NAME and micromolar concentrations of 1400W and L-NMMA on the tumorigenicity of TNBC cell lines. Growth (Figure 9A), primary (Figure 9B) and secondary (Figure 9C) mammospheres of MDA-MB-231 and SUM159 cell lines treated with L-NAME. Effect of micromolar concentrations of 1400W (Figure 9D) and L-NMMA (Figure 9E) on the migration index of MDA-MB-231 and SUM159 cells. Results were normalized to vehicle. Data are presented as mean ± SEM. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, one-way ANOVA and Bonferroni post-hoc test. [Figure 9E] Figure 9A, Figure 9B, Figure 9C, Figure 9D and Figure 9E show the effect of L-NAME and micromolar concentrations of 1400W and L-NMMA on the tumorigenicity of TNBC cell lines. Growth (Figure 9A), primary (Figure 9B) and secondary (Figure 9C) mammospheres of MDA-MB-231 and SUM159 cell lines treated with L-NAME. Effect of micromolar concentrations of 1400W (Figure 9D) and L-NMMA (Figure 9E) on the migration index of MDA-MB-231 and SUM159 cells. Results were normalized to vehicle. Data are presented as mean ± SEM. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, one-way ANOVA and Bonferroni post-hoc test. [Figure 10A-C] Figure 10A, Figure 10B, Figure 10C, Figure 10D, Figure 10E, Figure 10F and Figure 10G show migration in TNBC cell lines treated with iNOS inhibitors, and Western blots of NOS isoforms, EMT transcription factors and hypoxia. (Figure 10A) Tumor cell migration after treatment with L-NAME in MDA-MB-231 and SUM159 cell lines. Western blot analysis of NOS isoforms (iNOS, eNOS and nNOS) in MDA-MB-231 and SUM159 cells treated with 1400W (Figure 10B) and L-NMMA (Figure 10C). EMT marker protein levels in MDA-MB-231 and SUM159 cells after treatment with micromolar concentrations of 1400W (Figure 10D) or L-NMMA (Figure 10E). (FIG. 10F) Western blot analysis of NOS isoforms and EMT transcription factors in MDA-MB-231 and SUM159 cell lines treated with L-NAME. (FIG. 10G) Quantification of HIF1α protein levels relative to β-actin in MDA-MB-231 and SUM159 cells treated with 1400W. Results were normalized to vehicle. Data are presented as mean ± SEM. **p<0.01, *p<0.05, one-way ANOVA and Bonferroni post-hoc test. [Fig. 10D-F]Figure 10A, Figure 10B, Figure 10C, Figure 10D, Figure 10E, Figure 10F and Figure 10G show migration in TNBC cell lines treated with iNOS inhibitors, and Western blots of NOS isoforms, EMT transcription factors and hypoxia. (Figure 10A) Tumor cell migration after treatment with L-NAME in MDA-MB-231 and SUM159 cell lines. Western blot analysis of NOS isoforms (iNOS, eNOS and nNOS) in MDA-MB-231 and SUM159 cells treated with 1400W (Figure 10B) and L-NMMA (Figure 10C). EMT marker protein levels in MDA-MB-231 and SUM159 cells after treatment with micromolar concentrations of 1400W (Figure 10D) or L-NMMA (Figure 10E). (FIG. 10F) Western blot analysis of NOS isoforms and EMT transcription factors in MDA-MB-231 and SUM159 cell lines treated with L-NAME. (FIG. 10G) Quantification of HIF1α protein levels relative to β-actin in MDA-MB-231 and SUM159 cells treated with 1400W. Results were normalized to vehicle. Data are presented as mean ± SEM. **p<0.01, *p<0.05, one-way ANOVA and Bonferroni post-hoc test. [Figure 10G]Figure 10A, Figure 10B, Figure 10C, Figure 10D, Figure 10E, Figure 10F and Figure 10G show migration in TNBC cell lines treated with iNOS inhibitors, and Western blots of NOS isoforms, EMT transcription factors and hypoxia. (Figure 10A) Tumor cell migration after treatment with L-NAME in MDA-MB-231 and SUM159 cell lines. Western blot analysis of NOS isoforms (iNOS, eNOS and nNOS) in MDA-MB-231 and SUM159 cells treated with 1400W (Figure 10B) and L-NMMA (Figure 10C). EMT marker protein levels in MDA-MB-231 and SUM159 cells after treatment with micromolar concentrations of 1400W (Figure 10D) or L-NMMA (Figure 10E). (FIG. 10F) Western blot analysis of NOS isoforms and EMT transcription factors in MDA-MB-231 and SUM159 cell lines treated with L-NAME. (FIG. 10G) Quantification of HIF1α protein levels relative to β-actin in MDA-MB-231 and SUM159 cells treated with 1400W. Results were normalized to vehicle. Data are presented as mean ± SEM. **p<0.01, *p<0.05, one-way ANOVA and Bonferroni post-hoc test. [Figure 11A-E]Figure 11A, Figure 11B, Figure 11C, Figure 11D, Figure 11E, Figure 11F, Figure 11G, Figure 11H and Figure 11I show that NOS2 knockdown reduces cell tumorigenicity, EMT transcription factors, spliced ​​XBP1 and Smad2 / 3 signaling. Crosstalk between ER stress and TGFβ. Proliferation (Figure 11A), migration (Figure 11B) and primary and secondary mammospheres (Figure 11C) of SUM159 cells transfected with two different NOS2-directed shRNAs (shRNA1, shRNA2) compared to empty vector (shRNA-EV). Figure 11D: EMT transcription factors Snail and Slug in shRNA-mediated NOS2 knockdown MDA-MB-231 and SUM159 cells. Figure 11E: Changes in Zeb1 and Twist1 protein levels were confirmed in SUM159 cells transfected with two different NOS2-directed siRNAs (siRNA18, siRNA20; 100 nM siRNA) for 96 h. Figure 11F: Unspliced ​​XBP1 (uXBP1), spliced ​​XBP1 (sXBP1) and β-actin RT-PCR cDNA amplicons from MDA-MB-231 cells treated with 1400W for 96 h. Figure 11G: Protein-protein interaction analysis (STRING 9.1) deciphered the link between NOS2, TGFβ1 and ATF4 / ATF3 axis. Figure 11H: iNOS inhibitor 1400W can reduce Smad2 / 3 signaling in MDA-MB-231 cells under treatment with recombinant TGFβ1 (10 ng / mL) for 72 h. Figure 11I: Tunicamicyn (5 μM) confirmed the crosstalk between ER stress and TGFβ via ATF4 / ATF3 transcription factors. Results were normalized to empty vector. Data are shown as mean ± SEM. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, One-way ANOVA and Bonferroni post-hoc test. [Fig. 11F-I]Figure 11A, Figure 11B, Figure 11C, Figure 11D, Figure 11E, Figure 11F, Figure 11G, Figure 11H and Figure 11I show that NOS2 knockdown reduces cell tumorigenicity, EMT transcription factors, spliced ​​XBP1 and Smad2 / 3 signaling. Crosstalk between ER stress and TGFβ. Proliferation (Figure 11A), migration (Figure 11B) and primary and secondary mammospheres (Figure 11C) of SUM159 cells transfected with two different NOS2-directed shRNAs (shRNA1, shRNA2) compared to empty vector (shRNA-EV). Figure 11D: EMT transcription factors Snail and Slug in shRNA-mediated NOS2 knockdown MDA-MB-231 and SUM159 cells. Figure 11E: Changes in Zeb1 and Twist1 protein levels were confirmed in SUM159 cells transfected with two different NOS2-directed siRNAs (siRNA18, siRNA20; 100 nM siRNA) for 96 h. Figure 11F: Unspliced ​​XBP1 (uXBP1), spliced ​​XBP1 (sXBP1) and β-actin RT-PCR cDNA amplicons from MDA-MB-231 cells treated with 1400W for 96 h. Figure 11G: Protein-protein interaction analysis (STRING 9.1) deciphered the link between NOS2, TGFβ1 and ATF4 / ATF3 axis. Figure 11H: iNOS inhibitor 1400W can reduce Smad2 / 3 signaling in MDA-MB-231 cells under treatment with recombinant TGFβ1 (10 ng / mL) for 72 h. Figure 11I: Tunicamycin (5 μM) confirmed the crosstalk between ER stress and TGFβ through ATF4 / ATF3 transcription factors. Results were normalized to empty vector. Data are shown as mean ± SEM. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, One-way ANOVA and Bonferroni post-hoc test. [Figure 12A-B]Figure 12A, Figure 12B, Figure 12C and Figure 12D show representative images of mammospheres and wound healing assays in shRNA-mediated NOS2 knockdown cells. Illustrative images of primary and secondary mammospheres (Figure 12A and Figure 12B) and migration (wound healing assay) (Figure 12C and Figure 12D) in SUM159 and MDA-MB-231 cells transfected with two different NOS2-directed shRNAs (shRNA1, shRNA2) or empty vector (sRNA-EV). [Fig. 12C-D] Figure 12A, Figure 12B, Figure 12C and Figure 12D show representative images of mammospheres and wound healing assays in shRNA-mediated NOS2 knockdown cells. Illustrative images of primary and secondary mammospheres (Figure 12A and Figure 12B) and migration (wound healing assay) (Figure 12C and Figure 12D) in SUM159 and MDA-MB-231 cells transfected with two different NOS2-directed shRNAs (shRNA1, shRNA2) or empty vector (sRNA-EV). [Figure 13A-B]Figure 13A, Figure 13B, Figure 13C, Figure 13D, Figure 13E and Figure 13F show the in vivo effect of L-NMMA in SUM159 xenografts. Figure 13A: Tumor volume of SUM159 breast xenografts (n=10 / group) treated with vehicle, L-NMMA, chemotherapy and combination. Two-way ANOVA and Bonferroni post-hoc test. Figure 13B: Illustrative images of Ki67 staining in vehicle, L-NMMA, chemotherapy (docetaxel) and combination groups. Original optical objective: 10x. Counterstain: hematoxylin. (Figure 13C) Cell proliferation of tumor xenografts is depicted as Ki67 positive cells. 1,000 cells were counted from 10 different fields and the percentage was determined. One-way ANOVA and Bonferroni post-hoc test. Primary and secondary MSFE of breast cancer cells isolated from tumor tissues (Figure 13D and Figure 13E). One-way ANOVA and Bonferroni post-hoc test. Figure 13F: Tumor-initiating ability of tumor cells assayed by limiting dilution method. Fisher's exact test. Results were normalized to vehicle. Data are presented as mean ± SEM. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. [Figure 13C-E]Figure 13A, Figure 13B, Figure 13C, Figure 13D, Figure 13E and Figure 13F show the in vivo effect of L-NMMA in SUM159 xenografts. Figure 13A: Tumor volume of SUM159 breast xenografts (n=10 / group) treated with vehicle, L-NMMA, chemotherapy and combination. Two-way ANOVA and Bonferroni post-hoc test. Figure 13B: Illustrative images of Ki67 staining in vehicle, L-NMMA, chemotherapy (docetaxel) and combination groups. Original optical objective: 10x. Counterstain: hematoxylin. (Figure 13C) Cell proliferation of tumor xenografts is depicted as Ki67 positive cells. 1,000 cells were counted from 10 different fields and the percentage was determined. One-way ANOVA and Bonferroni post-hoc test. Primary and secondary MSFE of breast cancer cells isolated from tumor tissues (Figure 13D and Figure 13E). One-way ANOVA and Bonferroni post-hoc test. Figure 13F: Tumor-initiating ability of tumor cells assayed by limiting dilution method. Fisher's exact test. Results were normalized to vehicle. Data are presented as mean ± SEM. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. [Figure 14A]Figure 14A, Figure 14B, Figure 14C, Figure 14D, Figure 14E and Figure 14F show NMMA levels in plasma and tumor tissues. CD44+ / CD24- / low population in L-NMMA treated xenografts. Flow cytometry analysis (% parent) of CD44+ / CD24- / low cells isolated from SUM159 (Figure 14A) and MDA-MB-231 (Figure 14B) xenograft tumor tissues of mice treated with vehicle, L-NMMA, docetaxel and combination (docetaxel + L-NMMA). Figure 14C and Figure 14D: Ratiometric quantification of methylarginine in plasma and tumor tissues (MDA-MB-231 and SUM159 xenografts) by LC-MS / MS (Student's t-test). Figure 14E: iNOS catalyzes the reaction of L-arginine to L-citrulline + nitric oxide (NO). Ratiometric quantification of citrulline SUM159 xenograft tissue LC-MS / MS (Student's t-test). Figure 14F: Total nitric oxide production in SUM159 cells treated with L-NMMA and 1400W (4 mM) for 0.5, 2, 6 and 24 hours. Results were normalized to vehicle. Data are presented as mean ± SEM. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, one-way ANOVA and Bonferroni's post-hoc test. [Figure 14B]Figure 14A, Figure 14B, Figure 14C, Figure 14D, Figure 14E and Figure 14F show NMMA levels in plasma and tumor tissues. CD44+ / CD24- / low population in L-NMMA treated xenografts. Flow cytometry analysis (% parent) of CD44+ / CD24- / low cells isolated from SUM159 (Figure 14A) and MDA-MB-231 (Figure 14B) xenograft tumor tissues of mice treated with vehicle, L-NMMA, docetaxel and combination (docetaxel + L-NMMA). Figure 14C and Figure 14D: Ratiometric quantification of methylarginine in plasma and tumor tissues (MDA-MB-231 and SUM159 xenografts) by LC-MS / MS (Student's t-test). Figure 14E: iNOS catalyzes the reaction of L-arginine to L-citrulline + nitric oxide (NO). Ratiometric quantification of citrulline SUM159 xenograft tissue LC-MS / MS (Student's t-test). Figure 14F: Total nitric oxide production in SUM159 cells treated with L-NMMA and 1400W (4 mM) for 0.5, 2, 6 and 24 hours. Results were normalized to vehicle. Data are presented as mean ± SEM. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, one-way ANOVA and Bonferroni's post-hoc test. [Fig. 14C-D]Figure 14A, Figure 14B, Figure 14C, Figure 14D, Figure 14E and Figure 14F show NMMA levels in plasma and tumor tissues. CD44+ / CD24- / low population in L-NMMA treated xenografts. Flow cytometry analysis (% parent) of CD44+ / CD24- / low cells isolated from SUM159 (Figure 14A) and MDA-MB-231 (Figure 14B) xenograft tumor tissues of mice treated with vehicle, L-NMMA, docetaxel and combination (docetaxel + L-NMMA). Figure 14C and Figure 14D: Ratiometric quantification of methylarginine in plasma and tumor tissues (MDA-MB-231 and SUM159 xenografts) by LC-MS / MS (Student's t-test). Figure 14E: iNOS catalyzes the reaction of L-arginine to L-citrulline + nitric oxide (NO). Ratiometric quantification of citrulline SUM159 xenograft tissue LC-MS / MS (Student's t-test). Figure 14F: Total nitric oxide production in SUM159 cells treated with L-NMMA and 1400W (4 mM) for 0.5, 2, 6 and 24 hours. Results were normalized to vehicle. Data are presented as mean ± SEM. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, one-way ANOVA and Bonferroni's post-hoc test. [Fig. 14E-F]Figure 14A, Figure 14B, Figure 14C, Figure 14D, Figure 14E and Figure 14F show NMMA levels in plasma and tumor tissues. CD44+ / CD24- / low population in L-NMMA treated xenografts. Flow cytometry analysis (% parent) of CD44+ / CD24- / low cells isolated from SUM159 (Figure 14A) and MDA-MB-231 (Figure 14B) xenograft tumor tissues of mice treated with vehicle, L-NMMA, docetaxel and combination (docetaxel + L-NMMA). Figure 14C and Figure 14D: Ratiometric quantification of methylarginine in plasma and tumor tissues (MDA-MB-231 and SUM159 xenografts) by LC-MS / MS (Student's t-test). Figure 14E: iNOS catalyzes the reaction of L-arginine to L-citrulline + nitric oxide (NO). Ratiometric quantification of citrulline SUM159 xenograft tissue LC-MS / MS (Student's t-test). Figure 14F: Total nitric oxide production in SUM159 cells treated with L-NMMA and 1400W (4 mM) for 0.5, 2, 6 and 24 hours. Results were normalized to vehicle. Data are presented as mean ± SEM. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, one-way ANOVA and Bonferroni's post-hoc test. [Figure 15A-F] Figures 15A, 15B, 15C, 15D, 15E and 15F show the effect of calcium channel antagonists on the proliferation of MDA-MB-231 cells (percentage of proliferation reduction is shown above the bars). [Figure 16A-F] Figures 16A, 16B, 16C, 16D, 16E and 16F show the effect of calcium channel antagonists on cell proliferation of SUM159 cells (percentage of proliferation reduction is shown above the bars). [Figure 17A-B] FIG. 17A and FIG. 17B illustrate the antitumor activity of amlodipine in a mouse model of triple-negative breast cancer. [Figure 18A-B]Figure 18A and Figure 18B show that the migration of MDA-MB-231 and SUM159 cells was evaluated by "wound healing" assay. Briefly, 3x105 cells were seeded in growth medium to confluence in 6-well plates. Cell monolayers were treated with 1400W (0, 0.0001, 0.001, 0.01, 0.1, 1, 2, 4mM) for 72 hours in low serum conditions (1%) and 24 hours in normal growth medium in the presence of inhibitors (total 96 hours). Subsequently, a "wound" was created in the cell monolayer. Images were taken at 0 and 12 hours. Wound healing capacity was determined by the software Image J. Data were replicated in three independent experiments. Results were normalized to vehicle. [Figure 19A-B] Figure 19A and Figure 19B show that the effect of selective iNOS inhibition on epithelial-mesenchymal (EMT) inducers was examined by Western blot in MDA-MB-231 and SUM159 cell lines. Cells were treated with 1400W (0.1, 1, 10, 100 μM; 1, 2, 4 mM) for 96 hours. Protein levels of iNOS and EMT inducers were determined by Western blot using antibodies against iNOS (N-20) and Twist1 (L-21) (Santa Cruz Biotechnology), Snail (C15D3), Slug (C19G7) and TCF8 / Zeb1 (D80D3) (Cell Signaling) (1:1000 dilution). β-actin (Cell Signaling; 1:2000) was used as a loading control. [Figure 20A-B]Figure 20A, Figure 20B, Figure 20C and Figure 20D show that MDA-MB-231 and SUM159 cells (3x106) were injected into the right mammary fat pad of female SCID beige mice (n=10 / group). The clinically relevant dose regimen consisted of two cycles of docetaxel (20mg / kg, ip, day 0) 12 hours prior to L-NMMA (400mg / kg on day 1 and 200mg / kg for an additional 4 days, by oral gavage) and amlodipine (10mg / kg, ip, daily, for 6 days) on day 0. Docetaxel alone and saline (ip) + sterile water (oral gavage) were used as controls. The combination of L-NMMA and docetaxel was able to reduce tumor growth in MDA-MB-231 and SUM159 xenografts (Figure 20A and Figure 20D). Amlodipine prevented the L-NMMA-induced increase in blood pressure (Figure 20B). This dose regimen also improved survival compared to docetaxel alone in MDA-MB-231 xenografts (Figure 20C). ****p<0.0001, ***p<0.001. [Figure 20C-D]Figure 20A, Figure 20B, Figure 20C and Figure 20D show that MDA-MB-231 and SUM159 cells (3x106) were injected into the right mammary fat pad of female SCID beige mice (n=10 / group). The clinically relevant dose regimen consisted of two cycles of docetaxel (20mg / kg, ip, day 0) 12 hours prior to L-NMMA (400mg / kg on day 1 and 200mg / kg for an additional 4 days, by oral gavage) and amlodipine (10mg / kg, ip, daily, for 6 days) on day 0. Docetaxel alone and saline (ip) + sterile water (oral gavage) were used as controls. The combination of L-NMMA and docetaxel was able to reduce tumor growth in MDA-MB-231 and SUM159 xenografts (Figure 20A and Figure 20D). Amlodipine prevented the L-NMMA-induced increase in blood pressure (Figure 20B). This dose regimen also improved survival compared to docetaxel alone in MDA-MB-231 xenografts (Figure 20C). ****p<0.0001, ***p<0.001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0063] A quick explanation of the sequence: SEQ ID NO:1 is an exemplary DNA oligonucleotide forward primer for use according to one embodiment of the present invention.

[0064] SEQ ID NO:2 is an exemplary DNA oligonucleotide reverse primer for use in accordance with one embodiment of the present invention.

[0065] SEQ ID NO:3 is an exemplary DNA oligonucleotide forward primer for use in accordance with one embodiment of the present invention.

[0066] SEQ ID NO:4 is an exemplary DNA oligonucleotide reverse primer for use in accordance with one embodiment of the present invention.

[0067] Description of Illustrative Embodiments Illustrative embodiments of the present invention are described below. In the interest of clarity, not all features of an actual implementation are described herein. It will, of course, be appreciated that in the development of any such actual implementation, numerous implementation-specific decisions must be made to achieve the developer's specific goals, including compliance with system-related and business-related constraints that vary from implementation to implementation. It will further be appreciated that such a development effort may be complex and time-consuming, but would be a routine endeavor for those of ordinary skill in the art having the benefit of this disclosure.

[0068] The present inventors and collaborators were among the first groups to demonstrate that breast cancer cells with stem-like properties were intrinsically resistant to conventional therapies. Since these early observations, other groups have confirmed this view by establishing the resistance of these cells to conventional chemotherapy and radiotherapy. These and other studies have also supported our findings that an increase in the stem-like cell population is associated with a worse prognosis. These findings have fundamental clinical implications. Current development of cancer therapeutics is based in large part on the identification of agents capable of causing bulk tumor regression in animal models or clinical trials, but an exclusive focus on drugs that induce tumor regression by killing actively circulating or fully differentiated cells may spare a critical population of therapy-resistant cells. These observations have recently been extended to breast cancer, where the present inventors have shown that a subpopulation of chemotherapy-resistant cells within bulk primary tumors has a propensity to metastasize by a series of different adaptive mechanisms.

[0069] The inventors also identified a tumorigenic signature from patient breast cancer biopsies and then used a functional approach to identify novel targets of treatment resistance from this gene set. A high-throughput mammosphere formation efficiency (MSFE) screen was performed using shRNA knockdown of 477 genes in the tumorigenic signature. This approach identified two target proteins, RPL39 and MLF2. RPL39 was previously recognized as a component of the 60S ribosomal complex located on the X chromosome (XQ24) with proposed roles in spermatogenesis and protein translation. MLF2 is located on chromosome 12 and may be involved in chromosomal abnormalities and cellular defense responses. Little is known about the role of RPL39 in cancer, and even more limited knowledge is available for MLF2. A series of amino acid modifications of MLF2 at Ser144, 152 and 238 as well as a somatic mutation (Phe80Cys) have been associated with colorectal cancer. Notably, RPL39 and MLF2 overexpression increased cell migration, proliferation and mammosphere formation, suggesting the potentially important functions of these two genes in cancer. A comprehensive understanding of the mechanisms of RPL39 and MLF2 is crucially essential for the validation of these two genes as novel cancer targets. By mutual exclusivity analysis of RPL39 and MLF2 using The Cancer Genome Atlas (TCGA) database, RPL39 and MLF2 were found to be exclusively co-occurring (p<0.00001), suggesting a mechanistic pathway shared by both genes. Using microarray analysis, we identified the "cellular effect of sildenafil (Viagra)", i.e., nitric oxide (NO) signaling, as the major pathway that linked both RPL39 and MLF2. We then confirmed the role of NO signaling by inducing iNOS (inducible nitric oxide synthase) protein by overexpression of RPL39 and MLF2 and reducing iNOS protein levels by siRNA (small interfering ribonucleic acid) silencing of RPL39 and MLF2.The role of NOS signaling in breast cancer biology has not been extensively studied in the literature.Reports to date suggest that high NO concentrations are cytotoxic to cancer cells, while lower NO concentrations may enhance tumor growth.

[0070] Two novel oncogenes (RPL39 and MLF2) were previously identified that play a role in treatment resistance and lung metastasis. Upregulation of NO signaling was shown to be a common mechanistic pathway for both genes. Inhibition of NO signaling by LNMMA was shown to reduce the number of treatment-resistant cells and lung metastasis in human TNBC cell lines.

[0071] Pharmaceutical Formulations The pharmaceutical formulation of the present invention may further comprise one or more excipients, buffers or diluents, which are specifically formulated for administration to a human patient. The composition may further optionally comprise one or more microspheres, microparticles, nanospheres or nanoparticles, and may be formulated for administration to one or more cells, tissues, organs or bodies of a human undergoing treatment for cancer, particularly breast cancer.

[0072] The formulation of pharma- ceutically acceptable excipient and carrier solutions is well known to those of skill in the art, as is the development of appropriate dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including, for example, but not limited to, oral, parenteral, intravenous, intranasal, intratumoral, and intramuscular routes of administration.

[0073] Typically, the iNOS inhibitory chemotherapeutic formulations of the present invention can be formulated to contain at least about 0.1% or more of active compound, although the percentage of active ingredient(s) can of course vary and can conveniently be between about 1 or 2% to about 70% or 80% or more by weight or volume of the total formulation. Of course, the amount of active compound(s) in each diagnostic or therapeutically useful composition can be prepared in such a way that a suitable dosage of diagnostic or therapeutic agent is obtained in any given unit dose of the chemotherapeutic formulation disclosed herein. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life and other pharmacological considerations will be considered by those skilled in the art of preparing such pharmaceutical formulations, and therefore various dosages and treatment regimens may be desirable.

[0074] The specific amount of the composition used and the specific administration time or dosage regimen of the composition using the disclosed iNOS inhibitory chemotherapy formulation will be within the skill of the artisan who has the benefit of the present teachings.However, the administration of the diagnostically or therapeutically effective amount of the disclosed formulation may be achieved by administering one or more doses of the formulation for a time effective to bring about the desired chemotherapy benefit to the patient undergoing such treatment.Such dosage regimen can be determined by the physician supervising the administration of the chemotherapy drug depending on the specific condition or patient, the degree of cancer, etc.

[0075] Typically, the formulation of active ingredients in the disclosed compositions will contain an effective amount for a given patient's particular treatment regimen. Preferably, the formulation will contain at least about 0.1% of each active ingredient, although the percentage of the active ingredient(s) may, of course, vary and may conveniently be present in an amount of about 0.5 to about 80% by weight or volume, or about 1 to about 70% by weight or volume, or more preferably, about 2 to about 50% by weight or volume, based on the total formulation. Naturally, the amount of active compound(s) can be prepared in such a way that a suitable dosage is obtained in any given unit dose of the compound. Solubility, bioavailability, biological activity, and the like can be considered.1 / 2 Factors such as route of administration, product shelf life and other pharmacological considerations will be taken into account by those skilled in the art of preparing such pharmaceutical formulations, and therefore various dosages and treatment regimens may be desirable.

[0076] Compositions for the preparation of medicines Another important aspect of the present invention relates to methods for using the disclosed compositions (and formulations containing them) in the preparation of medicaments for treating or ameliorating various disease, dysfunction or deficiency symptoms in animals, such as vertebrate mammals. The use of the disclosed compositions is particularly contemplated in the chemotherapy treatment of one or more types of cancer in humans, particularly in the treatment of TNBC in human females.

[0077] Such uses generally involve administration of one or more of the disclosed iNOS inhibitory chemotherapeutic compositions to a mammal in need thereof in an amount and for a time sufficient to treat, alleviate or ameliorate one or more symptoms of cancer in the affected mammal.

[0078] Pharmaceutical formulations comprising one or more of the disclosed chemotherapeutic agents also form part of the present invention, particularly compositions further comprising at least a first pharma- ceutically acceptable excipient for use in the treatment or amelioration of one or more symptoms of mammalian breast cancer, and particularly for use in the treatment or amelioration of one or more symptoms of TNBC in human females.

[0079] Chemotherapeutic agents and their preparations Nitric oxide (NO) is a bioactive molecule that exhibits pleiotropic effects in cancer cells and tumors, with concentration-dependent tumor-promoting and antitumor effects. NO is produced by three different nitric oxide synthase (NOS) isoforms: neuronal (nNOS / NOS1), inducible (iNOS / NOS2) and endothelial (eNOS / NOS3). Increased iNOS expression has been found in breast cancer and other different cancers such as lung, colon, melanoma and glioblastoma. Previous reports have demonstrated a correlation between high iNOS expression, invasiveness and poor prognosis in breast cancer patients. Increased iNOS expression has recently been hypothesized as a prognostic factor for reduced survival in patients with basal-like estrogen receptor-negative breast cancer, through induction of interleukin-8 (IL-8), CD44, c-Myc (7), and in part through activation of the transcription factor Ets-1. In the present invention, it is hypothesized that enhancing endogenous iNOS expression promotes tumor recurrence and metastasis by modulating CSC self-renewal properties and tumor cell migration, thereby driving poor patient survival. It is further hypothesized that inhibition of endogenous iNOS, in combination with conventional chemotherapy, reduces the invasiveness and mesenchymal characteristics of residual TNBC cells and the number of metastases to distant organs, thereby improving the survival of TNBC patients.

[0080] In the following examples, the selective iNOS inhibitor 1400W (N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide) and two pan-NOS inhibitors, L-NMMA (N G -monomethyl-L-arginine) and L-NAME (N 5 Inhibition of iNOS by different small molecule inhibitors has been demonstrated, including L-NMMA (imino(nitroamino)methyl]-L-ornithine methyl ester). L-NMMA has been extensively studied in hundreds of patients for cardiogenic shock, facilitating its immediate translation into human clinical trials without the need for extensive preclinical testing.

[0081] Amlodipine Amlodipine is a dihydropyridine calcium antagonist that inhibits transmembrane influx of calcium ions into vascular smooth muscle and cardiac muscle. Experimental data suggest that amlodipine binds to both dihydropyridine and non-dihydropyridine binding sites. The contractile process of cardiac and vascular smooth muscle depends on the movement of extracellular calcium ions into these cells through specific ion channels. Amlodipine selectively inhibits calcium ion influx across cell membranes, and superior effects in blood vessels were seen in intact animals at therapeutic doses. Serum calcium concentrations are not affected by amlodipine. Within the physiological pH range, amlodipine is an ionized compound (pKa=8.6) and its kinetic interaction with calcium channel receptors is characterized by gradual association and dissociation rates with the receptor binding site, resulting in gradual onset of effect.

[0082] Amlodipine is a peripheral arterial vasodilator that acts directly on vascular smooth muscle, causing a decrease in peripheral vascular resistance and a decrease in blood pressure. The exact mechanism by which amlodipine relieves angina has not been fully delineated but is thought to include the following:

[0083] Exertional Angina: In patients with exertional angina, amlodipine reduces the total peripheral resistance (afterload) against which the heart must work, reducing myocardial work and hence myocardial oxygen demand at any given level of exercise.

[0084] Vasospastic angina: Amlodipine has been demonstrated to block stenosis and restore blood flow in coronary arteries and arterioles in response to calcium, potassium epinephrine, serotonin, and thromboxane A2 analogs in experimental animal models and in human coronary vessels in vitro. This inhibition of coronary spasm accounts for the efficacy of amlodipine in vasospastic (Prinzmetal or variant) angina.

[0085] Illustrative Definitions In accordance with the present invention, polynucleotides, nucleic acid segments, nucleic acid sequences, and the like, include, but are not limited to, DNA (including, but not limited to, genomic or extragenomic DNA), genes, peptide nucleic acids (PNAs), RNA (including, but not limited to, rRNA, mRNA, and tRNA), nucleosides, and suitable nucleic acid segments, obtained in whole or in part from natural sources, chemically synthesized, modified, or otherwise prepared or synthesized by the hand of man.

[0086] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and compositions similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and compositions are described herein. For purposes of the present invention, the following terms are defined below for clarity and ease of reference:

[0087] Following long-standing patent law convention, the words "a" and "an" refer to "one or more" when used in this application, including the claims.

[0088] The terms "about" and "approximately" are herein understood to be interchangeable and generally refer to a range of numbers around a given number and any number within the range of numbers recited (e.g., "about 5 to 15" means "about 5 to about 15" unless otherwise specified). Additionally, any numerical range herein is understood to include each and every whole integer within the range.

[0089] "Biocompatible" refers to a material that, when exposed to living cells, will support proper cellular activity of the cells without causing undesirable effects in the cells, such as alterations in the cell's living cycle, alterations in the cell's proliferation rate, or cytotoxic effects.

[0090] As used herein, the term "buffer" includes one or more compositions or aqueous solutions thereof that resist pH fluctuations when an acid or alkali is added to a solution or composition containing the buffer. This resistance to pH change is due to the buffering properties of such a solution and may be a function of one or more specific compounds contained in the composition. Thus, a solution or other composition that exhibits buffering activity is referred to as a buffer or buffer solution. Buffers generally do not have an unlimited capacity to maintain the pH of a solution or composition; rather, they are typically capable of maintaining a pH within a certain range, e.g., a pH of about 5-7.

[0091] As used herein, the term "carrier" is intended to include any solvent(s), dispersion medium, coating(s), diluent(s), buffer(s), isotonic agent(s), solution(s), suspension(s), colloid(s), inert(s) etc., or combinations thereof, that are pharma- ceutically acceptable for administration to the relevant animal or, where applicable, acceptable for therapeutic or diagnostic purposes.

[0092] The term "effective amount" as used herein refers to an amount capable of treating or ameliorating a disease or condition, or otherwise producing the intended therapeutic effect.

[0093] The term "for example" or "eg" is used herein merely as an example, without any sense of limitation, and should not be construed as referring only to items specifically recited herein.

[0094] As used herein, the phrase "in need of treatment" refers to a judgment made by a caregiver, such as a physician or veterinarian, that a patient requires treatment (or will benefit in one or more ways from treatment). Such a judgment can be made based on a variety of factors within the caregiver's area of ​​expertise, and can include knowledge that the patient is ill as a result of a disease condition that can be treated with one or more compounds or pharmaceutical compositions, such as those described herein.

[0095] As used herein, the term "kit" can be used to describe a variety of portable, self-contained enclosures that include at least one set of reagents, components, or pharma- ceutical formulated compositions of the present invention. Optionally, such kits can include one or more sets of instructions for use of the enclosed compositions, for example, in laboratory or clinical applications.

[0096] The term "naturally occurring" as used herein refers to the fact that the object can be found in nature.For example, the polypeptide or polynucleotide sequence that exists in an organism (including viruses) that can be isolated from a source in nature and has not been intentionally modified by man in a laboratory is naturally occurring.In this specification, a laboratory rodent line that may have been selectively bred according to classical genetics is considered to be a naturally occurring animal.

[0097] As used herein, the term "nucleic acid" includes one or more of polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, or a modified purine or pyrimidine base (including abasic sites). As used herein, the term "nucleic acid" also includes polymers of ribonucleosides or deoxyribonucleosides, typically covalently linked by phosphodiester bonds between the subunits, and sometimes by phosphorothioates, methylphosphonates, etc. "Nucleic acid" includes single- and double-stranded DNA, as well as single- and double-stranded RNA. Exemplary nucleic acids include, without limitation, gDNA; hnRNA; mRNA; rRNA, tRNA, microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snORNA), small nuclear RNA (snRNA) and small temporal RNA (stRNA), among others, and any combination thereof.

[0098] As used herein, the term "patient" (also interchangeably referred to as "recipient", "host" or "subject") refers to any host capable of serving as a recipient for one or more of the vascular access devices described herein. In certain aspects, the recipient will be a vertebrate intended to represent any animal species, preferably a mammalian species such as mankind. In certain embodiments, "patient" refers to any animal host, including but not limited to human and non-human primates, birds, reptiles, amphibians, cattle, dogs, goats, cabins, crows, epines, horses, cats, goats, lapines, leporines, wolves, mice, sheep, pigs, racines, foxes, and others, including, without limitation, domestic livestock, herding or migratory animals or birds, exotic or zoological specimens and companion animals, pets, and any animals under the care of a veterinary professional.

[0099] The phrase "pharmaceutical acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to humans, particularly when administered to the human eye. The preparation of aqueous compositions containing proteins as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as solutions or suspensions in liquid. Alternatively, they can be prepared in solid forms suitable for solution or suspension in liquid prior to injection.

[0100] As used herein, "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects.Examples of such salts include, but are not limited to, the acid addition salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc.; and the salts formed with organic acids, such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic (embonic) acid, alginic acid, naphthoic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, etc.; salts with polyvalent metal cations, such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, etc.; salts formed with organic cations formed from N,N'-dibenzylethylenediamine or ethylenediamine; and combinations thereof.

[0101] As used herein, the term "polypeptide" is intended to encompass both "polypeptide" and "polypeptides", and includes any chain or chains of two or more amino acids. Thus, as used herein, terms including, but not limited to, "peptide", "dipeptide", "tripeptide", "protein", "enzyme", "amino acid chain" and "contiguous amino acid sequence" are all included within the definition of "polypeptide", and the term "polypeptide" can be used in place of or interchangeably with any of these terms. The term further includes polypeptides that have undergone one or more post-translational modifications, including, but not limited to, modifications such as glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, post-translational processing, or inclusion of one or more non-naturally occurring amino acids. Conventional nomenclature exists in the art of polynucleotide and polypeptide structure. For example, one-letter and three-letter abbreviations are commonly used to describe amino acids: alanine (A; Ala), arginine (R; Arg), asparagine (N; Asn), aspartic acid (D; Asp), cysteine ​​(C; Cys), glutamine (Q; Gln), glutamic acid (E; Glu), glycine (G; Gly), histidine (H; His), isoleucine (I; Ile), leucine (L; Leu), methionine (M; Met), phenylalanine (F; Phe), proline (P; Pro), serine (S; Ser), threonine (T; Thr), tryptophan (W; Trp), tyrosine (Y; Tyr), valine (V; Val) and lysine (K; Lys). The amino acid residues described herein are preferably in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, provided the desired properties of the polypeptide are retained.

[0102] As used herein, the terms "prevent", "preventing", "prevention", "inhibit", "inhibiting" and "inhibition" refer to the administration of a compound, alone or contained in a pharmaceutical composition, prior to the onset of clinical symptoms of a disease state, to prevent any symptom, aspect or feature of the disease state. Such prevention and inhibition need not be absolute to be considered medically useful.

[0103] "Protein" is used interchangeably herein with "peptide" and "polypeptide" and includes both synthetically, recombinantly, or in vitro produced peptides and polypeptides, as well as peptides and polypeptides expressed in vivo after a nucleic acid sequence is administered to a host animal or human subject. The term "polypeptide" is intended to refer to any amino acid chain length, including short peptides, preferably from about 2 to about 20 amino acid residues in length, oligopeptides, preferably from about 10 to about 100 amino acid residues in length, and longer polypeptides, including about 100 amino acid residues or more in length. Additionally, the term is also intended to include enzymes, i.e., functional biomolecules that include at least one amino acid polymer. The polypeptides and proteins of the present invention also include polypeptides and proteins that are or have been post-translationally modified, including any sugar or other derivative(s) or conjugate(s) added to the backbone amino acid chain.

[0104] "Purified" as used herein means separated from many other compounds or entities. A compound or entity may be partially purified, substantially purified, or pure. A compound or entity is considered pure when it is substantially removed from all other compounds or entities, i.e., preferably at least about 90%, more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99% pure. A partially or substantially purified compound or entity may be removed from at least 50%, at least 60%, at least 70% or at least 80% of the material in which it is naturally found, e.g., cellular material such as cellular proteins and / or nucleic acids.

[0105] The term "subject" as used herein describes an organism, including mammals such as primates, that can be provided with treatment with the compositions of the present invention. Mammalian species that can benefit from the disclosed treatment methods include, but are not limited to, apes; chimpanzees; orangutans; humans; monkeys; domestic animals, such as dogs and cats; livestock, such as horses, cows, pigs, sheep, goats, and chickens; and other animals, such as mice, rats, guinea pigs, and hamsters.

[0106] As used herein, the terms "substantially free" or "essentially free" in relation to the amount of a component preferably refer to a composition containing less than about 10 weight percent, preferably less than about 5 weight percent, more preferably less than about 1 weight percent of the compound. In preferred embodiments, these terms refer to less than about 0.5 weight percent, less than about 0.1 weight percent, or less than about 0.01 weight percent.

[0107] As used herein, the term "plasmid" or "vector" refers to a genetic construct made up of genetic material (i.e., nucleic acid). Typically, a plasmid or vector contains an origin of replication that is functional in a bacterial host cell, e.g., Escherichia coli, and a selectable marker for detecting bacterial host cells containing the plasmid. The plasmids and vectors of the present invention can include one or more genetic elements as described herein, arranged such that the inserted coding sequence can be transcribed and translated in a suitable expression cell. In addition, a plasmid or vector can include one or more nucleic acid segments, genes, promoters, enhancers, activators, multiple cloning regions, or any combination thereof, including segments obtained or derived from one or more natural and / or artificial sources.

[0108] The term "a sequence essentially as set forth in SEQ ID NO:X" means that the sequence substantially corresponds to a portion of SEQ ID NO:X, but has a relatively small number of nucleotides (or amino acids, in the case of a polypeptide sequence) that are not identical to or biologically functional equivalent to the nucleotides (or amino acids) of SEQ ID NO:X. The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein. Thus, sequences having about 85% to about 90%; or more preferably about 91% to about 95%; or even more preferably about 96% to about 99% of the nucleotides that are identical or functionally equivalent to one or more of the nucleotide sequences provided herein are particularly contemplated as useful in the practice of the invention.

[0109] Standard hybridization conditions suitable for the present invention include, for example, hybridization in 50% formamide, 5x Denhardt's solution, 5x SSC, 25 mM sodium phosphate, 0.1% SDS and 100 μg / ml denatured salmon sperm DNA at 42°C for 16 hours, followed by sequential washing with 0.1x SSC, 0.1% SDS solution at 60°C for 1 hour to remove a desired amount of background signal. Lower stringency hybridization conditions for the present invention include, for example, hybridization in 35% formamide, 5x Denhardt's solution, 5x SSC, 25 mM sodium phosphate, 0.1% SDS and 100 μg / ml denatured salmon sperm DNA or E. coli DNA at 42°C for 16 hours, followed by sequential washing with 0.8x SSC, 0.1% SDS at 55°C. Those skilled in the art will recognize that conditions can be easily adjusted to obtain a desired level of stringency.

[0110] Of course, the present invention also encompasses nucleic acid segments that are complementary, essentially complementary and / or substantially complementary to at least one or more of the specific nucleotide sequences specifically depicted herein. A "complementary" nucleic acid sequence is one that can base pair according to the standard Watson-Crick rules of complementarity. As used herein, the term "complementary sequence" refers to a substantially complementary nucleic acid sequence, as defined by the same nucleotide comparison depicted above, or capable of hybridizing to one or more of the specific nucleic acid segments disclosed herein under relatively stringent conditions, such as those described immediately above.

[0111] As mentioned above, the probes and primers of the present invention can be of any length. An algorithm can be proposed to define every probe or primer contained within a given sequence by assigning a numerical value to the sequence, for example, the first residue is 1, the second residue is 2, etc.: n to n+y, where n is an integer from 1 to the last number of the sequence, y is the length of the probe or primer minus 1, and n+y does not exceed the last number of the sequence. Thus, for a 25 base pair probe or primer (i.e., a "25-mer"), the collection of probes or primers corresponds to bases 1-25, bases 2-26, bases 3-27, bases 4-28, etc., throughout the length of the sequence. Similarly, for a 35 base pair probe or primer (i.e., a "35-mer"), exemplary primer or probe sequences include, without limitation, sequences corresponding to bases 1-35, bases 2-36, bases 3-37, bases 4-38, etc., throughout the length of the sequence. Similarly, for a 40-mer, such a probe or primer may correspond to the nucleotides of the first base pair to bp40, the second bp to bp41, the third bp to bp42, etc., of the sequence, while for a 50-mer, such a probe or primer may correspond to a nucleotide sequence extending from bp1 to bp50, bp2 to bp51, bp3 to bp52, bp4 to bp53, etc. "Treating" or "treatment of" as used herein refers to the provision of any type of medical or surgical management to a subject. Treating can include, but is not limited to, the administration of a composition comprising a therapeutic agent to a subject. "Treating" includes the administration or application of any of the compounds or compositions of the present invention to a subject for purposes such as curing, reversing, alleviating, reducing the severity of, inhibiting the progression of, or reducing the likelihood of, a disease, disorder, or condition, or one or more symptoms or manifestations of a disease, disorder, or condition. In certain embodiments, the compositions of the present invention can also be administered prophylactically, i.e., prior to the onset of any symptoms or manifestations of a condition, and such prevention is warranted. Typically, in such cases, the subject will be one who has been diagnosed as being "at risk" for developing such a disease or disorder, either as a result of family history, medical records, or the completion of one or more diagnostic or prognostic tests that are indicative of a propensity to subsequently develop such a disease or disorder.

[0112] The term "therapeutically effective period" refers to the period of time necessary for an active agent to be therapeutically effective. The term "therapeutically effective" refers to the reduction in severity and / or frequency of symptoms, the elimination of symptoms and / or their underlying causes, the prevention of the appearance of symptoms and / or their underlying causes, and the amelioration or correction of damage.

[0113] A "therapeutic agent" may be any physiologically or pharmacologically active substance capable of producing a desired biological effect at a targeted site in a subject. Therapeutic agents may be chemotherapeutic agents, immunosuppressants, cytokines, cytotoxic agents, nucleolytic compounds, radioisotopes, receptors and prodrug activating enzymes, which may be naturally occurring or produced by synthetic or recombinant methods, or any combination thereof. Drugs affected by classical multidrug resistance, such as vinca alkaloids (e.g., vinblastine and vincristine), anthracyclines (e.g., doxorubicin and daunorubicin), RNA transcription inhibitors (e.g., actinomycin-D) and microtubule stabilizing drugs (e.g., paclitaxel), may have particular utility as therapeutic agents. Cytokines may also be used as therapeutic agents. Examples of such cytokines are lymphokines, monokines and traditional polypeptide hormones. Cancer chemotherapeutic agents may be preferred therapeutic agents. For a more detailed description of anti-cancer and other therapeutic agents, those skilled in the art can refer to the Physician's Desk Reference and Goodman and Gilman's "Pharmacological For purposes of illustration, reference may be made to a number of instructional manuals, including, but not limited to, Basis of Therapeutics, 10th Edition, Hardman et al., eds., 2001.

[0114] "Transcriptional regulatory element" refers to a polynucleotide sequence that activates transcription alone or in combination with one or more other nucleic acid sequences. Transcriptional regulatory elements can include, for example, one or more promoters, one or more response elements, one or more negative regulatory elements, and / or one or more enhancers.

[0115] As used herein, "transcription factor recognition site" and "transcription factor binding site" refer to a polynucleotide sequence(s) or sequence motif(s) that are identified as sites for sequence-specific interaction of one or more transcription factors, often in the form of direct protein-DNA binding.Typically, transcription factor binding sites can be identified by DNA footprinting, gel mobility shift assays, etc., and / or predicted based on known consensus sequence motifs or by other methods known to those skilled in the art.

[0116] A "transcription unit" refers to a polynucleotide sequence comprising at least a first structural gene operably linked to at least a first cis-acting promoter sequence, and optionally operably linked to one or more other cis-acting nucleic acid sequences necessary for efficient transcription of the structural gene sequence, at least a first distal regulatory element that may be required for proper tissue-specific and developmental transcription of the structural gene sequence operably positioned under the control of the promoter and / or enhancer element, and any additional cis sequences necessary for efficient transcription and translation (e.g., polyadenylation site(s), mRNA stability control sequence(s)), etc.).

[0117] The term "substantially complementary", when used to define either amino acid or nucleic acid sequences, means that a particular target sequence, e.g., an oligonucleotide sequence, will be substantially complementary to the whole or part of the selected sequence and thus will specifically bind to the part of the mRNA that codes for the selected sequence.Thus, typically, the sequence will be highly complementary to the mRNA "target" sequence and will have no more than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10 bases mismatch through the complementary part of the sequence.In many cases, it may be desirable for the sequence to be an exact match, i.e., completely complementary to the sequence to which the oligonucleotide specifically binds, and thus no mismatch along the complementary stretch.Thus, a highly complementary sequence will typically bind very specifically to the target sequence region of the mRNA and thus be highly efficient in reducing and / or even inhibiting the translation of the target mRNA sequence into a polypeptide product.

[0118] A substantially complementary nucleic acid sequence will be more than about 80 percent complementary (or "percent exact match") to the corresponding nucleic acid target sequence to which the nucleic acid specifically binds, more preferably more than about 85 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds. In certain embodiments, as described above, it will be desirable to have even more substantially complementary nucleic acid sequences for use in the practice of the present invention, in which case the nucleic acid sequence will be more than about 90 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds, and in certain embodiments, may be more than about 95 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds, and may even be up to about 96%, about 97%, about 98%, about 99% or even about 100% exact match complementary to the entire or part of the target sequence to which the designed nucleic acid specifically binds.

[0119] The percent similarity or percent complementarity of any of the disclosed nucleic acid sequences can be determined by comparing sequence information using, for example, the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (1970). Briefly, the GAP program defines similarity as the number of aligned symbols that are similar divided by the total number of symbols (i.e., nucleotides or amino acids) in the shorter of the two sequences. Preferred default parameters of the GAP program include: (1) a unary comparison matrix for nucleotides (containing a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov and Burgess (1986); (2) a penalty of 3.0 per gap and an additional penalty of 0.10 per symbol in each gap; and (3) no penalty for terminal gaps.

[0120] As used herein, the term "transformed cell" is intended to mean a host cell whose nucleic acid complement has been altered by the introduction of one or more exogenous polynucleotides into said cell.

[0121] As used herein, the term "transformation" is intended to generally describe the process of introducing an exogenous polynucleotide sequence (e.g., a viral vector, a plasmid, or a recombinant DNA or RNA molecule) into a host cell or protoplast, where the exogenous polynucleotide is integrated into at least a first chromosome or is capable of autonomous replication within the transformed host cell. Transfection, electroporation, and "naked" nucleic acid uptake all represent examples of techniques used to transform a host cell with one or more polynucleotides.

[0122] As used herein, the terms "treat", "treating" and "treatment" refer to the administration of one or more compounds (alone or contained in one or more pharmaceutical compositions) after the onset of clinical symptoms of a disease state to reduce or eliminate any symptom, aspect or characteristic of the disease state. Such treatment need not be absolute to be considered medically useful. Thus, the terms "treat", "treat", "treated" or "treating" can refer to the treatment of one or more symptoms, or the amelioration or reduction in the extent or severity of a disease, whether its onset is before or after it afflicts the patient.

[0123] In certain embodiments, it will be advantageous to use one or more nucleic acid segments of the present invention in combination with a suitable detectable marker (i.e., "label"), such as when using a labeled polynucleotide probe in determining the presence of a given target sequence in a hybridization assay. A wide variety of suitable indicator compounds and compositions are known in the art for labeling oligonucleotide probes, including, but not limited to, fluorescent, radioactive, enzymatic or other ligands, such as avidin / biotin, that can be detected in a suitable assay. In certain embodiments, one or more fluorescent labels or enzyme tags, such as urease, alkaline phosphatase or peroxidase, can also be used in place of radioactive or other environmentally undesirable reagents. In the case of enzyme tags, colorimetric, chromogenic or fluorogenic indicator substrates are known that can be used to provide methods for detecting samples visible to the human eye, or analytical methods such as scintigraphy, fluorimetry, spectrophotometry, etc., for identifying specific hybridization with samples containing one or more complementary or substantially complementary nucleic acid sequences. In the case of so-called "multiplexed" assays, in which two or more labeled probes are detected simultaneously or sequentially, it may be desirable to label a first oligonucleotide probe with a first label having a first detection property or parameter (e.g., emission and / or excitation spectral maximum), which also labels a second oligonucleotide probe with a second label having a second detection property or parameter that is different (i.e., separate or distinguishable) from the first label. The use of multiplexed assays, particularly in the context of genetic amplification / detection protocols, is well known to those skilled in the art of molecular genetics.

[0124] The section headings used throughout are for organizational purposes only and should not be construed as limiting the subject matter described. Any documents or portions of documents cited in this application, including but not limited to patents, patent applications, papers, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for all purposes. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that is inconsistent with the definition of that term in this application, this application will control. EXAMPLES

[0125] The following examples are included to demonstrate preferred embodiments of the present invention. Those skilled in the art should appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the present invention, and thus can be considered to constitute preferred modes of its practice. However, those skilled in the art should appreciate in light of this disclosure that many changes may be made in the specific embodiments disclosed and still obtain similar or similar results without departing from the spirit and scope of the present invention.

[0126] Example 1 iNOS inhibition as an effective targeted therapy for TNBC

[0127] As noted above, TNBC is an aggressive form of breast cancer for which there is no effective targeted therapy. iNOS is associated with poor survival in breast cancer patients by increasing tumor aggressiveness. It was hypothesized that inhibition of endogenous iNOS would reduce the aggressiveness of TNBC by decreasing tumor initiation and metastasis through modulation of epithelial-mesenchymal transition (EMT) inducers.

[0128] This example describes the use of iNOS inhibitors as a targeted therapy for TNBC. iNOS protein levels were determined in 83 human TNBC tissues and correlated with clinical outcomes. Proliferation, mammosphere formation efficiency, migration, and EMT transcription factors were evaluated in vitro after iNOS inhibition. Endogenous iNOS targeting was evaluated as a potential treatment in a TNBC mouse model.

[0129] Gene expression and immunohistochemical analyses showed that high endogenous iNOS expression was associated with a worse prognosis in TNBC patients. Selective iNOS (1400W) and pan-NOS (L-NMMA and L-NAME) inhibitors, together with inhibition of EMT transcription factors (Snail, Slug, Twist1 and Zeb1), reduced cell proliferation, CSC self-renewal and cell migration in vitro. Impairment of crosstalk between HIF1α, endoplasmic reticulum stress (IRE1α / XBP1) and ATF4 / ATF3 and TGFβ was observed. iNOS inhibition significantly reduced tumor growth, decreased cell proliferation, and reduced the number of lung metastases as well as tumor initiation and self-renewal capacity. Based on the success of L-NMMA in reducing tumor growth and enhancing survival in TNBC, the inventors propose an effective targeted therapeutic regimen by retargeting iNOS inhibitors in general, and the pan-NOS inhibitor L-NMMA in particular (which has already been extensively investigated for cardiogenic shock), as anti-cancer therapeutics.

[0130] Materials and Methods

[0131] Oncomine gene expression data analysis. Relative levels of NOS2 mRNA expression in human triple-negative breast cancers were investigated by analysis of the Oncomine cancer microarray database from The Cancer Genome Atlas (TCGA) database (n=593). Patient survival analyses of two different gene expression datasets were obtained.

[0132] Cell culture. Mesenchymal-like triple-negative breast cancer cell lines, MDA-MB-231 and SUM159, were purchased from American Type Culture Collection and Asterand, respectively. Cells were treated daily for 96 h with either 1400W (0.1, 1, 10, 100 μM; 1, 2, 4 mM), L-NMMA (0.1, 1, 10, 100 μM; 1, 2, 4 mM) or L-NAME (0.1, 1, 10, 100 μM; 1, 2, 5 mM) unless otherwise specified. Mammosphere formation efficiency (MSFE), cell proliferation and migration assays are described in detail below.

[0133] Immunohistochemistry. Paraffin-embedded sections of human patient, MDA-MB-231 and SUM159 orthotopic tumor tissues were incubated with either anti-iNOS (1:50 dilution) or anti-Ki67 (1:100 dilution) antibodies. Slides were counterstained with hematoxylin. Additional information is included below.

[0134] Animal studies. Female SCID beige mice (4-5 weeks old) were housed under standard laboratory conditions (22°C; 12 h / 12 ​​h light / dark cycle and free access to food and water). All animal procedures and experimental protocols were performed using institutional and federally approved Animal Care and Use guidelines. Detailed information is provided below.

[0135] Statistical analysis. Data are presented as mean ± SEM. A p value of <0.05 was considered significant.

[0136] Reagents: N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide (1400W) and N 5 -[Imino(nitroamino)methyl]-L-ornithine methyl ester (L-NAME) was purchased from Cayman Chemical. G-monomethyl-L-arginine (L-NMMA) was from Enzo Life Sciences and kindly provided by Arginox Pharmaceuticals. Tunicamycin and recombinant human TGF-β1 (CHO cell-derived) were from Abcam and Peprotech, respectively. Anti-iNOS (N-20), anti-eNOS (C-20), anti-nNOS (R-20), anti-Twist1 (L-21), anti-Twist1 (2C1a), anti-ATF3 (C-19) and anti-CREB-2 (C-20) (ATF4) antibodies were from Santa Cruz Biotechnology, Inc. Antibodies, anti-Snail (C15D3), anti-Slug (C19G7), anti-TCF8 / Zeb1 (D80D3), anti-PERK (C33E10), anti-TGFβ, anti-phospho-Smad2 (Ser465 / 467) / Smad3 (Ser423 / 425) (D6G10), anti-Smad2 / 3, anti-IRE1α (14C10), anti-phospho-PERK (Thr980) (16F8), anti-PERK (C33E10), anti-phospho-eIF2α (Ser51) (119A11), anti-eIF2α, anti-β-actin (13E5), anti-rabbit and anti-mouse IgG (linked to HRP) were obtained from Cell Signaling Technology Inc. Anti-HIF1α (EP1215Y) was from Abcam. For immunohistochemistry, anti-Ki67 (SP6) was from Abcam, anti-iNOS (K13-A) was purchased from Novus Biologicals, and anti-cleaved caspase-3 (Asp175) was from Cell Signaling. PCR primers for XBP1 and β-actin were from Invitrogen. Mouse anti-human CD24-FITC (clone ML5) and mouse anti-human CD44-APC (clone G44-26) were from BD Biosciences. Anti-mouse MHC class I (H-2Kd)-PE (clone SF1-1.1.1) was from eBioscience.

[0137] Cell culture and mammosphere formation efficiency assay. Mesenchymal-like triple-negative breast cancer cell lines, MDA-MB-231 and SUM159 (purchased from American Type Culture Collection and Asterand, respectively), were selected based on their high expression of EMT markers, metastatic properties, percentage of CD44+ / CD24- cells (MDA-MB-231: approximately 80-90%; SUM159: approximately 40-50%) and iNOS protein levels. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (Thermo Scientific) and 1% antibiotic-antimycotic (Gibco). Stock solutions of iNOS inhibitors (1400W, L-NMMA and L-NAME) were made in 1x PBS. Prior to addition to cells, inhibitors were further diluted in cell culture medium. Cells were treated daily for 96 h with either 1400W (0.1, 1, 10, 100 μM; 1, 2, 4 mM), L-NMMA (0.1, 1, 10, 100 μM; 1, 2, 4 mM), or L-NAME (0.1, 1, 10, 100 μM; 1, 2, 5 mM) unless otherwise stated. For mammosphere formation efficiency (MSFE) assays, 2,000 (SUM159) and 5,000 (MDA-MB-231) cells / well were plated in 0.5% methylcellulose (MethoCult MammoCult basal medium supplemented with 10% MammoCult proliferation supplement, 4 μg / mL heparin and 0.48 μg / mL hydrocortisone (StemCell Technologies) was used. After 96 h of treatment with either 1, 2 and 4 mM (1400W and L-NMMA) or 1, 2 and 5 mM L-NAME, primary mammospheres (MS) were scanned and counted using a colony counter (GelCount, Oxford Optronicx). Primary MSFE was assessed by dividing the mammosphere number by the cell number. After trypsinization of primary MS, single cells were grown in 0.5% methylcellulose and mammosphere medium (as described above) without treatment. Secondary MS were scanned and counted, and secondary MSFE was assessed. For a mouse model of lung metastasis, MDA-MB-231 cells were transfected with a luciferase / GFP-based dual reporter plasmid and stable clones (MDA-MB-231 L / G) were selected with 1 mg / mL blasticidin (InvivoGen).

[0138] Cell proliferation assay. The effect of iNOS inhibition on cell proliferation was assayed by the WST-1 method. Briefly, 500 (SUM159) and 1,000 (MDA-MB-231) cells / well were plated in 96-well plates and treated with either 1, 2 and 4 mM (1400W and L-NMMA) or 1, 2 and 5 mM L-NAME for 96 h. Proliferation rates were determined by adding premixed WST-1 reagent (Clontech). After 3 h of incubation at 37° C., absorbance was read at 450 nm (reference wavelength 690 nm).

[0139] Cell migration ability. Cell migration was determined by the "wound healing assay". Briefly, 3 × 10 5Cells / well were grown in 6-well plates until confluence. Cells in monolayer were treated with different concentrations of 1400W, L-NMMA and L-NAME under starvation conditions (1% serum) for 72 hours. To avoid any effect on cell proliferation, the low serum medium was replaced by normal growth medium in the presence of inhibitors for 24 hours (total 96 hours). Then, a "wound" was created in the cell monolayer using a 100 μL pipette tip. Images were taken at 0 hours and cells were allowed to heal the wound for 12 hours. Wound healing capacity was determined by the software Image J. Data were replicated in three independent experiments.

[0140] Lentivirus-mediated shRNA knockdown. GIPZ NOS2 lentiviral shRNA clones (shRNA1-V3LHS_360691; shRNA2-V2LHS_111769) and GIPZ lentiviral empty vector shRNA control were purchased from Thermo Scientific. MDA-MB-231 and SUM159 cells were treated with lentiviral particles and polybrene (6 μg / mL) (Sigma-Aldrich) for 48 h. Cell clones carrying shRNA were selected by puromycin (2 μg / mL) (Sigma-Aldrich) for 1 week. Then, cells were harvested and plated for proliferation, mammospheres, wound healing and Western blot assays.

[0141] siRNA-mediated NOS2 knockdown. SUM159 and MDA-MB-231 cells were transfected with scrambled siRNA, siRNA18 (s9618) or siRNA20 (s9620) (Silencer Select, Ambion) for 96 hours. Briefly, cells grown in 6-well plates (100,000 cells / well) in serum-, antibiotic / antimycotic-free DMEM medium were transfected with NOS2 siRNA or scrambled siRNA (100 nM) packaged in Lipofectamine RNAiMAX (Invitrogen) for 6 hours. Complete DMEM medium was added and cells were grown for 96 hours.

[0142] Nitric oxide production in SUM159 cells. Cells were treated with L-NMMA or 1400W for 24 hours in phenol red and serum free DMEM medium. Aliquots of cell culture supernatants were taken at 0, 0.5, 2, 6 and 24 hours for nitrate + nitrite (total nitric oxide) production by Nitrate / Nitrite Fluorometric Assay Kit (Cayman Chemical) according to the manufacturer's instructions.

[0143] Western blot. 2.5 × 10 5 Cells were cultured for 96 h at a density of 1000 cells / well. Cells were resuspended in 1× lysis buffer (Cell Signaling Technology, Inc.) and 1× protease / phosphatase inhibitor cocktail (Thermo Scientific). Samples (30 μg protein) were boiled in 4× LDS sample buffer (Thermo Scientific) containing β-mercaptoethanol (Sigma Aldrich) and subjected to SDS-PAGE electrophoresis in 4–20% polyacrylamide gels (Bio-Rad). Proteins were transferred to nitrocellulose membranes (Bio-Rad) and incubated with 5% nonfat dry milk in 1× Tris-buffered saline (TBS) for 1 h to avoid nonspecific binding. Membranes were incubated with primary antibodies (1:1,000 dilution; anti-β-actin, 1:2,000 dilution) overnight at 4°C. After washing and 1 h incubation with the appropriate secondary antibodies (1:2,000 dilution), the membranes were washed and incubated with enhanced chemiluminescence substrate. Protein bands were developed onto autoradiography film (Denville Scientific, Inc.).

[0144] RT-PCR analysis of spliced ​​XBP1. Total RNA was extracted from MDA-MB-231 and SUM159 cells using the RNeasy micro kit (Qiagen) and cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer's instructions. PCR amplification (50 ng cDNA) was performed using 2.5 U / μL Taq DNA polymerase (native, 5 U / μL), 0.2 mM dNTPs, 1.5 mM MgCl2 (50 mM) and 0.5 μM of each primer. The primers were: XBP1-forward 5'-GGGTCCAAGTTGTCCAGAATGC-3' (SEQ ID NO: 1) XBP1-reverse 5'-TTACGAGAGAAAACTCATGGC-3' (SEQ ID NO: 2) β-actin-forward 5'-CTGGAACGGTGAAGGTGACA-3' (SEQ ID NO: 3) β-actin-reverse 5'-AAGGGACTTCCTGTAACAATGCA-3' (SEQ ID NO: 4). PCR conditions were 1 cycle of 95° C. for 5 min, 25 cycles of 95° C. for 30 s, 50° C. for 1 min, and 68° C. for 1 min, followed by 1 cycle of 68° C. for 5 min. cDNA amplicons were separated in 2% agarose.

[0145] Immunohistochemistry. Paraffin-embedded sections of human patient, MDA-MB-231 and SUM159 orthotopic tumor tissues were subjected to antigen retrieval using Tris-HCl buffer (pH=9.0) and blocked using hydrogen peroxide for 5 min. Human patient samples and xenograft tumors were then incubated with anti-iNOS (1:50 dilution), anti-Ki67 (1:100 dilution) and anti-cleaved caspase-3 (1:50) antibodies for 1 h at room temperature. Samples were developed with a peroxidase-based EnVision kit (Dako) and compared with negative controls to exclude false positives. Slides were counterstained with hematoxylin. iNOS score method: intensity (0-3): negative, weak, moderate, strong; distribution (0-4): <10%, 10-30%, >30-50%, >50-80%, >80%. The total score can be divided into four groups: negative (0–1), weak (2–3), moderate (4–5) and strong (6–7). MDA-MB-231 cells transfected with either NOS2-directed shRNA (shRNA1) or empty vector (EV) were used as negative and positive controls for iNOS staining, respectively.

[0146] Animal studies. Female SCID beige mice (4-5 weeks old) (Harlan Laboratories) were housed under standard laboratory conditions (22°C; 12 h / 12 ​​h light / dark cycle and free access to food and water). MDA-MB-231 or SUM159 cells (3 × 10 6 ) was injected into the right mammary fat pad. 3 Once this was reached, mice were randomized into different groups (n=10 / group) as follows: 1) vehicle (saline, ip), 2) L-NMMA (either 80 mg / kg or 200 mg / kg, ip, daily), 3) docetaxel (20 mg / kg), 4) combo (L-NMMA and docetaxel).

[0147] For lung metastasis prevention studies, MDA-MB-231 L / G cells were implanted as described above. Mice were randomized and treatments were initiated 48 hours after cell injection (n=5 / group): 1) vehicle (saline, ip), 2) L-NAME (80 mg / kg, ip, daily for 35 days). Prior to injection of luciferin, lungs were removed and washed in cold DMEM+10% FBS+1% antibiotic / antimycotic. Lungs were then incubated for 10 minutes in cold DMEM medium containing 50 μM luciferin. This protocol avoids a time lapse between lung removal and exposure to luciferin. Fluorescent cancer cells were detected by an IVIS-200 in vivo imaging system (Perkin Elmer, Inc.).

[0148] The clinically relevant dose regimen consisted of two cycles of docetaxel (20 mg / kg, ip, day 0) 12 hours prior to L-NMMA (400 mg / kg on day 1 and 200 mg / kg for four additional days by oral gavage) and combined with amlodipine (10 mg / kg, ip, daily for 6 days) on day 0. Docetaxel alone and saline (ip) plus sterile water (oral gavage) were used as controls.

[0149] Self-renewal and tumor-initiating abilities were determined in single cells isolated from tumor tissue by MSFE and limiting dilution assays, respectively. Briefly, mammary tumor tissue was minced and digested with 100 U / mL collagenase type 3 (Worthington) and 0.8 U / mL dispase (Gibco) in DMEM:F12 medium for 45 min at 37°C. MSFE was assayed in single cells isolated as described above. 5 × 10 6 cells derived from tumor tissue in the mammary fat pad of SCID beige mice (n = 12 / group) were cultured at 37°C for 45 min. 4 or 2×10 4 A limiting dilution assay (LDA) was performed by injecting either one of the isolated cells. CD44 + / CD24 - / low Flow cytometric analysis of cell populations was assayed.

[0150] Metabolite profiling by liquid chromatography-tandem mass spectrometry (LC-MS / MS). MDA-MB-231 and SUM159 xenograft tissues and plasma samples from animal studies (L-NMMA daily administration) were prepared as previously described (2). L-NMMA (200 mg / kg) was administered orally by gavage to female SCID beige mice (n=5). Blood was collected before (baseline, 0 h) and after (0.5, 2, 12, 24 h) L-NMMA administration. The LC-MS / MS platform used for metabolite profiling has been described previously. Ratiometric quantification of methylarginine (L-NMMA) and citrulline was determined as ion abundance levels in plasma and tumor tissue.

[0151] Blood pressure measurements. Blood pressure (BP) of 15 female SCID beige mice was measured for 3 days (basal BP) and then treated with one cycle of clinically relevant dose regimens (n=5 / group) as follows: amlodipine (10 mg / kg, ip) for 6 days (starting on day 0), L-NMMA (200 mg / kg, gavage) for 5 days (starting on day 1) and combination (L-NMMA+amlodipine). Average daily BP was determined by averaging the last 10 of 20 BP measurements during the last 3 consecutive days of cycle treatment using a computerized tail cuff monitor (BP-2000 Series II, Visitech).

[0152] Statistical analysis. All data were analyzed using GraphPad™ Prism software (GraphPad Sofware, Inc.). Data are presented as mean ± SEM. Statistical significance between two groups was analyzed by two-tailed Student's t-test. Experiments with more than three groups were analyzed by one-way ANOVA (analysis of variance) followed by Bonferroni's post-hoc test. Statistical analysis of tumor volume was evaluated by two-way ANOVA and Bonferroni's post-hoc test. Fisher's exact test was used to determine significant differences in limiting dilution assays. Survival fraction was evaluated using the Kaplan-Meier method and further analyzed by either Wilcoxon or log-rank test. Proliferation, MSFE, migration index and Ki67 staining are normalized to the vehicle group (100%). A p-value of less than 0.05 was considered significant.

[0153] result

[0154] Enhanced iNOS expression correlates with poor patient survival in invasive TNBC. iNOS has been described as a mediator of metastasis in different cancer types. Elevated iNOS expression has been associated with poor survival in ERα-negative breast cancer patients. The inventor hypothesized that enhanced iNOS expression in TNBC correlates with poor patient survival and metastasis.

[0155] An Oncomine cancer microarray database analysis of NOS2 (iNOS mRNA expression) expression in breast cancer was performed. Analysis of The Cancer Genome Atlas (TCGA) database showed that NOS2 mRNA expression was significantly higher in invasive TNBC patient samples (n=46) versus non-TNBC (n=250) (fold change 1.425, p=3.85×10 -5, Student's t-test) (Figure 1A). Patient survival analysis demonstrated a correlation between increased NOS2 expression and worse survival at 5 years in invasive ductal carcinoma patients (n=79) (fold change 1.275, p=0.037, Student's t-test). Of these, 46 samples were TNBC (n=37, high NOS2 expression; n=9, low iNOS expression) (Figure 1B). The inventors and colleagues further tested whether NOS2 expression correlated with worse survival in two additional databases of TNBC patients. Analysis of the Van de Vijver (n=69 samples) and Curtis (n=260 samples) databases confirmed that high NOS2 expression was associated with survival in TNBC patients (Figures 1C and 1D).

[0156] Next, the inventors and colleagues tested iNOS protein expression by immunohistochemistry in 83 surgically resected TNBC primary breast cancer samples and correlated expression with known patient outcomes. iNOS was predominantly cytoplasmic, but some cells showed both cytoplasmic and nuclear localization (Figure 1E). The overall scores showed that iNOS levels were weak to moderate (score 3-4) in 14 samples (16.9%) (Figure 1E, Figure 3, and Figure 4), moderate to strong (score 5-6) in 50 samples (60.2%) (Figure 1E), and strong (score 7) in 19 specimens (22.9%) (Figure 1E). Using this stratification, the correlation of iNOS expression and patient survival was analyzed using Kaplan-Meier analysis. Consistent with the mRNA mining analysis (FIGS. 1C and 1D), we confirmed that enhanced iNOS protein levels were associated with poorer patient survival compared to low iNOS expression (p=0.05, chi-square test) (FIG. 1F). These results demonstrate that increased iNOS by mRNA and protein expression in invasive TNBC is associated with poor patient survival.

[0157] Inhibition of iNOS reduces the tumorigenic potential of TNBC cells. We and colleagues evaluated the effect of iNOS inhibition on the proliferation of SUM159 and MDA-MB-231 cell lines after 96 h of treatment with a selective iNOS inhibitor, 1400W, and pan-NOS inhibitors, L-NMMA and L-NAME (Figure 2A). High concentrations of 1400W (1, 2 and 4 mM) were able to significantly reduce proliferation in both cell lines (Figure 2A). Similar results were observed after treatment with L-NAME (Figure 9A). The highest concentration (4 mM) of L-NMMA showed antiproliferative activity in both cell lines (Figure 2B).

[0158] Resistance to treatment and metastasis may arise from a subpopulation of cancer stem cells (CSCs) within heterogeneous primary cancers that may serve to re-initite tumor growth and seed metastases. Thus, we and colleagues investigated the effect of iNOS inhibition in cancer stem cell self-renewal by using a mammosphere formation efficiency (MSFE) assay. iNOS inhibition reduced MSFE of primary mammospheres (MS) in both cell lines (Figure 2C). A similar effect was found for L-NAME (Figure 9B). We identified a secondary MSFE reduction in both cell lines for all inhibitors tested (Figure 2D; Figure 9C). As findings indicate enhanced iNOS expression in invasive TNBC (Figure 1A), we further investigated the role of iNOS in cell migration using a wound-healing assay. Selective iNOS inhibition with 1400W caused a significant dose-dependent decrease in migration of both cell lines at millimolar (Figure 2E) and micromolar (Figure 9D) concentration ranges. Cells treated with L-NMMA showed a reduced migration ability (Figure 2F). Lower concentrations in the micromolar range were inconsistent and less effective (Figure 9E). Similar results were found for L-NAME (Figure 10A). These results were further confirmed in shRNA-mediated iNOS (NOS2) knockdown MDA-MB-231 (Figures 3A, 3B, and 3C) and SUM159 cells (Figures 11A, 11B, and 11C). Taken together, the results indicate that basal levels of iNOS have a major role in the CSC self-renewal and migration properties of TNBC cell lines, with a more unclear effect on proliferation.

[0159] Suppression of endogenous iNOS can impair EMT and cell migration by impairing HIF1α and endoplasmic reticulum (ER) stress / TGFβ / AFT4 / ATF3 crosstalk. Transdifferentiation (EMT) of polarized epithelial cells into mesenchymal cells is induced during tumor invasion and metastasis. Next, we considered the effect of iNOS inhibition on EMT-inducing transcription factors in mesenchymal-like TNBC MDA-MB-231 and SUM159 cells by Western blot. We first tested the effect on NOS isoforms (iNOS, eNOS and nNOS) after either selective or pan-inhibition (Figure 3D; Figure 10B, Figure 10C and Figure 10F). The findings revealed that selective iNOS blockade with 1400W caused a decrease in the protein levels of EMT transcription factors Snail, Slug and Twist1 at millimolar (Fig. 3D) and micromolar (Fig. 10D) concentrations in both cell lines. Zeb1 protein levels were decreased at millimolar concentrations (Fig. 3D). Similar, albeit less consistent, results were found for pan-NOS inhibitors (Fig. 10E and Fig. 10F). iNOS knockdown with shRNA correlated with a decrease in Zeb1 and Twist1 protein levels (Fig. 3E). Snail and Slug were blocked only in SUM159 (Fig. 11D); we found similar results in MDA-MB-231 after 2 weeks of clonal selection. The decrease in Zeb1 and Twist1 was confirmed by transient iNOS knockdown in SUM159 cells (Fig. 11E). Overall, these data suggest that selective iNOS inhibition efficiently reduces migration of TNBC cell lines, which consistently correlates with a reduction in EMT transcription factors.

[0160] Different pathways are responsible for EMT induction and tumor cell metastasis, among which nitric oxide is the common denominator of HIF1α and endoplasmic reticulum (ER) stress. The findings show that selective iNOS inhibition led to a dose-dependent decrease in hypoxia (HIF1α) in both cell lines (Figure 3F; Figure 10G) as well as the ER stress markers IRE1α / spliced ​​XBP1 (Figure 3F; Figure 11F) and ATF4 (spliced ​​XBP1 was not detected in SUM159 cells, data not shown) (Figure 3F). Functional protein-protein interaction (STRING 9.1) analysis revealed a link between iNOS and TGFβ1 (Figure 11G). The study confirmed that 1400W could inhibit TGFβ signaling (phospho-Smad2 / 3, Smad2 / 3 and mature TGFβ) in the absence (Figure 3G) and presence (Figure 11H) of recombinant TGFβ1 (10 ng / mL, 72 h) by an undetermined mechanism. Additional protein-protein interaction analysis showed an interaction between ATF4 and ATF3, both of which activate transcription factors that interact with TGFβ (Figure 11G). The experiment confirmed the crosstalk between ER stress and TGFβ by ATF4 / ATF3 (Figure 11I); similarly, recombinant TGFβ1 (10 ng / mL, 24 h) induced the PERK / eIF2α / ATF4 / ATF3 axis (Figure 3H). The results showed that co-treatment of iNOS inhibitor 1400W (4 mM) and recombinant TGFβ1 for 24 h could inhibit the stimulation of ATF4 and ATF3 protein levels by TGFβ1 independently of the PERK / eIF2α pathway. This result was further confirmed in siRNA-mediated iNOS (NOS2) knockdown cells (Figure 3I). Overall, these data demonstrated that iNOS inhibition could impair EMT and tumor cell migration by impairing ER stress (IRE1α / XBP1) and the crosstalk between ATF4, ATF3 and TGFβ.

[0161] iNOS inhibition reduces tumor growth, tumor initiation ability, and prevents lung metastasis in a mouse model of triple-negative breast cancer. Based on this in vitro data, we next investigated whether iNOS inhibition could prevent tumor initiation and metastasis of mammary tumor cells in a mouse model of lung metastasis. Daily ip injections of 80 mg / kg L-NAME were given to mice bearing MDA-MB-231 xenografts for 35 days. L-NAME significantly reduced tumor growth (p=0.001) (Figure 4A) and MSFE of primary MS (Figure 4B). Secondary MSFE was also reduced, but not significantly, compared to the vehicle group (Figure 4B). Moreover, single cells (5×10 5 or 1×10 5 The tumor-initiating ability of CSCs was evaluated by limiting dilution assay (LDA) by injecting 5 × 10 cells into the right mammary fat pad. All animals (n = 5) in the vehicle group developed tumors, whereas treatment with L-NAME led to the development of tumors up to 5 × 10 5 The cells produced 3 / 5 tumors at 1.5 weeks. Compared to the L-NAME-treated group (0 / 5 tumors), 1×10 5 The same results were observed in the vehicle group at 2.5 weeks with cells (p<0.05, Fisher's exact test) (Figure 4D).

[0162] Next, we tested whether iNOS inhibition could suppress lung metastasis in a TNBC xenograft model. The luciferase / GFP-based MDA-MB-231 (MDA-MB-231 L / G) xenograft mouse model mimics the lung metastasis process in patients. In this lung metastasis model, cells metastasize from the primary tumor to the lungs approximately 35 days after implantation. MDA-MB-231 L / G cells were injected into the right mammary fat pad of SCID mice and given 80 mg / kg L-NAME daily for 35 days. Ex vivo imaging of the lungs in the presence of luciferin showed higher fluorescence in the vehicle group compared to the L-NAME group (Figure 4C). These results suggested that iNOS inhibition with daily L-NAME could also prevent lung metastasis in a TNBC mouse model.

[0163] In order to translate these results into future clinical trials, the pan-NOS inhibitor, L-NMMA, was selected for additional intensive study. Although L-NMMA has previously been investigated in cardiogenic shock and has been administered to thousands of patients for that indication, the present invention provides the first reported repurposing of this compound for an anti-cancer indication, and little data exists prior to these results to suggest preclinical doses that may be effective as an anti-cancer therapeutic.

[0164] To address this issue, we first gave daily injections of 80 mg / kg L-NMMA to mice bearing SUM159 xenografts, either alone or in combination with docetaxel (20 mg / kg). After 10 days, no differential effects were observed between the groups, and the daily dose was increased to 200 mg / kg. Tumor growth was efficiently blocked by L-NMMA administered alone or in combination with docetaxel (Figure 13A). We correlated these results with tumor cell proliferation by immunohistochemistry (Ki67). Higher proliferation rates were observed in the vehicle and chemotherapy groups compared to the L-NMMA and combination groups (Figures 13B and 13C). We then analyzed the effect on CSC self-renewal by MSFE assay. Docetaxel showed a dramatic increase in primary and secondary MSFE of single tumor cells isolated from breast tumor tissue. This increase was effectively blocked by the addition of L-NMMA (combination group) (Figure 13D). Flow cytometry analysis showed that CD44 + / CD24 - / low LDA showed a slight increase in the population of 5×10 4 At week 7, 12 / 12, 4 / 12, 12 / 12, and 6 / 12 tumors were observed, respectively. At week 9, 2 × 10 4 A significant reduction in tumor initiation ability was observed as the cells in different groups gave rise to 7 / 12, 0 / 12, 3 / 12, and 0 / 12 tumors in vehicle, L-NMMA, docetaxel, and the combination, respectively (p<0.05, Fisher's exact test) (FIG. 14E).

[0165] Next, the role of L-NMMA was investigated in different TNBC mouse models, alone or in combination with docetaxel. Docetaxel (20 mg / kg, days 0 and 21) and 200 mg / kg L-NMMA (daily) were given to mice bearing MDA-MB-231 xenografts for 31 days. First, tumor growth reduction was found in the L-NMMA group compared to vehicle, but there was no change between the docetaxel and combination groups (Figure 5A). These results were further correlated with the lower proliferation rate in the L-NMMA and combination groups, seen by immunohistochemistry (Figure 5B and Figure 5C). Moreover, higher apoptosis levels were found in docetaxel-treated xenografts; these results can offset the higher proliferation rate compared to the combination group (Figure 5D). There was less primary MS in both L-NMMA and combination compared to vehicle and chemotherapy alone groups. L-NMMA treatment was able to reduce secondary MS, but no changes were observed in the combination group (Figure 5E). Flow cytometry analysis showed that CD44 + / CD24 - / low No change in population was noted (FIG. 14B). LDA showed that the vehicle- and docetaxel-treated groups showed 12 / 12 and 8 / 12 tumors, respectively, with 5×10 4The cells showed a significant reduction in L-NMMA-treated (1 / 12) and combination-treated (4 / 12) xenografts at week 5. After 6 weeks, a reduction was observed in both the L-NMMA and combination groups (3 / 12 and 5 / 12, respectively) compared to the vehicle and docetaxel groups (6 / 12 and 8 / 12, respectively) (p<0.05, Fisher's exact test) (Figure 5F). The study demonstrates that L-NMMA plasma levels are rapidly cleared (Figure 14C), while 24 hours after completion of treatment, it accumulates in tumor tissue (Figure 14D) and inhibits the conversion of L-arginine to L-citrulline and NO by iNOS (Figure 14E). This inhibition led to a reduction in total NO production, as seen in SUM159 cells (Figure 14F). Overall, these results demonstrate that in vivo iNOS inhibition by L-NMMA reduced tumor growth, cell proliferation and tumor-initiating capacity of CSCs, with a significant reduction in lung metastasis.

[0166] Efficient dose regimen of L-NMMA and docetaxel with potential clinical application. Clinically, L-NMMA causes acute blood pressure (BP) elevation due to inhibition of constitutive eNOS. Here, we describe a regimen with attenuated duration of iNOS inhibitor (in this case L-NMMA) together with antihypertensive drug (in this example amlodipine) for two cycles in two different TNBC mouse models (MDA-MB-231 and SUM159 xenografts). Standard docetaxel was administered at 20 mg / kg every 2 weeks. L-NMMA was given 24 hours after chemotherapy at 200 mg / kg for 5 days, a dosage comparable to previous clinical reports. Calcium channel blocker amlodipine (10 mg / kg, daily for 6 days, ip) was used to counteract the BP increment. Oral L-NMMA significantly increased mean systolic pressure (147 mmHg) compared to basal levels (120 mmHg) in mice, and this increase was efficiently reversed by amlodipine (10 mg / kg) (Figure 6A). This BP increase was transient and disappeared 24 hours after the last injection of L-NMMA (Figure 6B).

[0167] The combination of L-NMMA and docetaxel was able to reduce tumor growth in the MDA-MB-231 orthotopic model (Figure 6C). This dose regimen also improved survival compared to the docetaxel-treated group (p=0.0001, Wilcoxon test) (Figure 6D). Similar results were found in SUM159 xenografts (Figure 6E). Overall, the data show that the dose regimen proposed herein is effective in reducing tumor growth and results in better survival. By combining administration of an iNOS inhibitor with an antihypertensive drug for at least part of the protocol, the troublesome adverse side effects of iNOS inhibitors in transiently increasing BP can be minimized and / or avoided.

[0168] Further combination of iNOS inhibitor therapy with one or more conventional chemotherapeutic agents, such as docetaxel, provides additional synergistic treatment benefits and represents a new strategy to overcome treatment resistance in refractory, metastatic and TNBC patients.

[0169] As described herein, TNBC is a highly aggressive and lethal form of cancer that lacks effective targeted therapies. TNBC patients exhibit a higher risk of metastasis and tumor relapse. iNOS levels have been suggested to predict worse survival in basal-like estrogen receptor-negative breast cancer patients and increase tumor invasiveness and metastatic propensity of cells by modulating cancer stem cells (CSCs). The present invention is the first report to demonstrate that inhibition of the iNOS pathway can reduce the tumorigenic potential of TNBC cells by affecting cell proliferation, CSC self-renewal and / or cell migration. The in vivo studies presented herein demonstrate the efficacy of several exemplary small molecule iNOS inhibitors, such as L-NMMA, as novel targeted therapies for cancer patients, including those with refractory cancers, such as TNBC, and provide the need for immediate translation of these results into human clinical trials.

[0170] These examples demonstrated that NOS2 is commonly increased in invasive TNBC and associated with poor survival in invasive breast cancer patients. Additional data demonstrated that high iNOS protein levels by immunohistochemistry in 83 human TNBC patient samples also correlated with poor patient outcome, consistent with earlier reports in ERα-negative and invasive breast cancer. Kaplan-Meier analysis of the Van de Vijver and Curtis database as well as human TNBC patient samples strongly indicates that high iNOS expression was associated with poor overall survival in TNBC patients. These observations also establish that increased iNOS expression may predict poor prognosis in certain subsets of cancer patients.

[0171] iNOS expression correlates with increased tumor grade and invasiveness of breast cancer cells. The present invention describes the effect of iNOS on CSC self-renewal, tumor initiation and migration ability of TNBC cells. The antitumor activity of iNOS inhibitors has been previously reported in epidermoid carcinoma, oral, glioblastoma and breast cancer, consistent with in vitro and in vivo findings. Increased iNOS expression has been described to contribute to resistance to conventional treatments by promoting tumor initiation in glioblastoma cells. Moreover, iNOS may affect CSC self-renewal by modulating CD44 and c-Myc in ERα-negative breast cancer. For the first time, the inventors demonstrated that iNOS inhibition reduces CSC self-renewal and tumor initiation in both in vitro and in vivo models of TNBC.

[0172] Nitric oxide can either promote or inhibit metastatic events depending on endogenous levels. The role of NOS inhibitors in metastasis has been previously studied, but the underlying mechanisms remain unclear. Initial studies demonstrated that the pan-NOS inhibitor L-NAME can reduce tumor growth and lung metastasis in a mouse breast cancer model (EMT-6 cells). Similarly, L-NAME inhibited the invasive and migratory potential of two metastatic breast cell lines (C3L5 and C10). In another study with metastatic human adenocarcinoma HRT-18 cells, invasiveness was substantially reduced by daily treatment with 500 μM of the selective iNOS inhibitor 1400W. More recently, 1400W was shown to significantly inhibit spontaneous lung metastasis in a mouse model of oral adenoid cystic carcinoma.

[0173] This example demonstrated that iNOS inhibition reduced cell migration and lung metastasis in an in vivo model of TNBC. It was suggested that NO and iNOS may lead to early metastasis by inducing IL-8 and CXC chemokine receptor 4. CSCs exhibit mesenchymal features that lead to increased cell migration and metastasis. iNOS inhibition reduced CSC self-renewal and tumor initiation, thus indicating that inhibitors against this pathway may reverse the transition of tumor cells to a more mesenchymal-like phenotype. Consistent with the effects on cell migration, selective iNOS inhibition and NOS2 knockdown reduced transcription factors that drive EMT in all TNBC cell lines examined.

[0174] To better understand the mechanism of effect of endogenous iNOS inhibition in reducing EMT transcription factors, we analyzed the impact of an iNOS selective inhibitor, 1400W. EMT can be promoted by different signaling pathways, such as TGFβ, Wnt / β-catenin, Notch, Hedgehog and multiple growth factors. EMT transcription factors (Snail, Slug, Twist1 or Zeb1) are activated by diverse intermediate effectors, such as c-Myc, Ets, HIF1α or NFκB. Moreover, ER stress has been linked to EMT in thyroid, alveolar epithelial and human renal proximal tubule cells by activation of PERK, XBP1 or Grp78. Interestingly, among these disparate signaling networks, iNOS is the common denominator between HIF1α and ER stress. Inhibition of endogenous iNOS-derived NO production could reduce HIF1α stabilization and protein levels in colon cancer cells. In particular, the transcription factors Twist1, Snail, Slug, and Zeb1 are directly or indirectly affected by HIF1α.

[0175] Moreover, hypoxia induces ER stress and unfolded protein response (UPR), which has recently been linked to migration and sphere formation in breast cancer cells through activation of the PERK / ATF4 / LAMP3 arm under hypoxic conditions. The results suggest that iNOS inhibition correlates with impaired TGFβ signaling via the ER stress ATF4 / ATF3 axis. It is known that TGFβ stimulates ATF4 protein levels to suppress differentiation in calvarial osteoblasts. Certain conditions, such as ER stress via the PERK / eIF2α axis, can activate ATF4, which in turn induces ATF3 transcription, while ATF3 itself is an activating transcription factor that cooperates with Twist-1 to enhance TGFβ, mammosphere formation, and EMT.

[0176] The translation of these results into clinical implementation represents the next step in this new knowledge. L-NMMA is a pan-NOS inhibitor that has been studied extensively in several clinical trials of circulatory shock. In cardiogenic shock trials, L-NMMA was found to be safe and with few adverse events other than transient reversible hypertension. In normotensive patients, L-NMMA was administered to patients with metastatic renal cell carcinoma prior to infusion of interleukin-2. Doses of 3 and 6 mg / kg did not induce clinically evident side effects and BP remained unchanged. At the 12 mg / kg dose level, patients experienced an increase in systolic BP of up to 25 mmHg without any clinical symptoms, which rapidly normalized upon cessation of L-NMMA infusion. Determining a safe and effective regimen with clinical applicability was the major challenge of these preclinical studies. The dose rates in this study were selected based on previous clinical trials in patients with septic shock, with modifications. These results demonstrated that tumor growth could be limited by an attenuated regimen of L-NMMA given with amlodipine for 5 days after chemotherapy. Randomized, placebo-controlled, double-blind studies of L-NMMA have been previously reported in septic shock patients for up to 14 days. The regimen followed consisted of an initial dose of 2.5 mg / kg / hr, followed by adjustments to different rates (0.5, 1, 2.5, 5, 7.5, 10, 15 and 20 mg / kg / hr). The current dosing regimen for the use of L-NMMA as an anticancer therapeutic is much lower than that previously reported in the literature on septic shock.

[0177] In conclusion, this example provides new evidence for a correlation between enhanced endogenous iNOS expression and poor survival in TNBC patients. Targeted treatment with iNOS inhibitors was demonstrated to inhibit not only tumor cell proliferation but also CSC self-renewal and migration, reducing tumor growth, tumor initiation and the number of lung metastases. Inhibition of metastatic events may be due to the reduction of EMT transcription factors through inhibition of HIF1α, ER stress (IRE1α / spliced ​​XBP1) and the TGFβ / ATF4 / ATF3 axis. These results define a targeted therapeutic regimen using the compounds described herein to reduce tumor growth and enhance survival in vivo, establishing the importance of clinical trials targeting this pathway in TNBC patients.

[0178] Example 2 Effects of calcium channel antagonists Materials and Methods

[0179] In vitro cell proliferation assay. Mesenchymal-like TNBC cell lines, MDA-MB-231 and SUM159, were grown in DMEM supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic. Stock solutions of various calcium channel antagonists (amlodipine, nicardipine, nifedipine, felodipine, isradipine, diltiazem, verapamil, lacidipine, nisoldipine, nitrendipine, nivaldipine, azelnidipine, valnidipine, benidipine, efonidipine, lercanidipine, pranidipine, manidipine) were prepared in DMSO. The effect on cell proliferation was assayed by the WST-1 method. Briefly, 1,000 (SUM159) and 2,000 (MDA-MB-231) cells / well were plated in 96-well plates and treated with different concentrations (0, 1, 5 and 10 μM) of calcium channel antagonists for 72 h. Proliferation rates were determined by adding premixed WST-1 reagent. After 3 h of incubation at 37° C., absorbance was read at 450 nm (reference wavelength 690 nm). Results were normalized to vehicle (100%). Antagonists were considered active if they reduced proliferation by ≥30%.

[0180] Animal studies. Female SCID beige mice (4-5 weeks old) were housed under standard laboratory conditions (22 °C; 12 h / 12 ​​h light / dark cycle and free access to food and water). MDA-MB-231 or SUM159 cells (3 × 10 6 ) was injected into the right mammary fat pad. 3 Once the mice reached maturity, they were randomized into different groups (n=5 / group): 1) vehicle (saline, ip), 2) amlodipine (10 mg / kg, ip) on days 0 and 14 for 2 cycles (daily for 6 days each cycle). All animal procedures and experimental protocols were approved by the institution and in full strict adherence to the federal guidelines for the use and care of laboratory animals.

[0181] result

[0182] Cell proliferation in vitro. We demonstrated the anti-proliferative efficacy of several calcium channel antagonists in two different TNBC cell lines (MDA-MB-231 and SUM159). Results showed a dose-dependent decrease in proliferation after treatment with amlodipine, felodipine, lacidipine, nicardipine, nivaldipine and azelnidipine in both MDA-MB-231 (Figure 15A, Figure 15B, Figure 15C, Figure 15D, Figure 15E and Figure 15F) and SUM159 (Figure 16A, Figure 16B, Figure 16C, Figure 16D, Figure 16E and Figure 16F). In these figures, the percentage of proliferation decrease is shown above the bars.

[0183] Animal studies. Data obtained from in vivo models of TNBC showed that administration of amlodipine (10 mg / kg) can limit the growth of MDA-MB-231 and SUM159 orthotopic tumors (Figures 17A and 17B, respectively). The most effective antagonists identified from the in vitro results (amlodipine, felodipine, lacidipine, nivaldipine, and azelnidipine) can also be tested for in vivo antitumor activity in suitable animal models of TNBC (including, for example, MDA-MB-231 and SUM159). A dose of approximately 20 mg / kg ip was considered to be within the effective range.

[0184] References: The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference in their entireties: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

[0185] It should be understood that the examples and embodiments described herein are merely illustrative, and various modifications or changes therein may be suggested to those skilled in the art and should be included within the spirit and scope of the present application and the appended claims.All references cited herein (including publications, patent applications and patents) are incorporated herein by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and set forth in its entirety herein.The recitation of ranges of values ​​herein is intended to serve as a simple way of individually referring to each separate value falling within the range, unless otherwise stated herein, and each separate value is incorporated herein as if it were individually recited herein.

[0186] Description herein of any aspect or embodiment of the invention using terms such as "comprising," "having," "including," or "containing" with reference to an element or elements is intended to provide support for similar aspects or embodiments of the invention that "consist," "consist essentially of," or "substantially comprise" the particular element or elements, unless otherwise indicated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element will be understood to also describe a composition consisting of that element, unless otherwise indicated or clearly contradicted by context).

[0187] All compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described herein in terms of illustrative embodiments, it will be apparent to one of ordinary skill in the art that variations may be applied to the compositions, methods, and / or steps or order of steps of the methods without departing from the spirit, scope and concept of the invention. More specifically, it will be apparent that certain compounds that are chemically and / or physiologically related may be substituted for one or more of the compounds described herein while still achieving the same or similar results. Any such substitutions and / or modifications that are apparent to one or more of those skilled in the relevant art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

1. A product for the treatment of breast cancer comprising: (i) a chemotherapy-effective amount of N-G-monomethyl-L-arginine [L-NMMA] and (ii) a therapeutically effective amount of a first chemotherapeutic agent and (iii) a therapeutically effective amount of a first antihypertensive agent as a combination formulation for separate administration in the treatment of breast cancer.

2. The product described in claim 1, wherein the breast cancer is triple-negative breast cancer.

3. The product of claim 1, wherein the first chemotherapeutic agent comprises one or more antineoplastic compounds, one or more cytotoxic compounds, one or more cytostatic compounds, or any combination thereof, and / or is selected from the group consisting of cyclophosphamide, doxorubicin, 5-fluorouracil, docetaxel, paclitaxel, trastuzumab, methotrexate, epirubicin, cisplatin, carboplatin, vinorelbine, capecitabine, gemcitabine, mitoxantrone, ixabepilone, eribulin, lapatinib, carmustine, nitrogen mustard, sulfur mustard, platin tetranitrate, vinblastine, etoposide, camptothecin, and any combination thereof. (a) the first antihypertensive agent comprises a first calcium channel antagonist; and / or (b) The product of any one of claims 1 to 3, wherein the first antihypertensive agent comprises a first calcium channel antagonist selected from the group consisting of amlodipine, aranidipine, azelnidipine, valnidipine, benidipine, clinidipine, clevidipine, diltiazem, efonidipine, fendiline, felodipine, gallopamil, isradipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nimodipine, nisoldipine, nitrendipine, nivaldipine, pranidipine, and verapamil. (a) a second, separate iNOS inhibitor, and / or (b) one or more of an immunomodulatory agent, a neuroactive agent, an anti-inflammatory agent, an antihyperlipidemic agent, a hormone, a receptor agonist, a receptor antagonist, an anti-infective agent, a protein, a peptide, an antibody, an antigen-binding fragment, an enzyme, RNA, DNA, siRNA, mRNA, a ribozyme, a hormone, a cofactor, a steroid, an antisense molecule, a second distinct antihypertensive agent, a second distinct chemotherapeutic agent, or any combination thereof. The article of manufacture of claim 1 further comprising:

6. The product of claim 3, wherein the first chemotherapeutic agent comprises docetaxel.

7. The product of claim 3, wherein the first chemotherapeutic agent comprises trastuzumab.

8. The product of claim 1, wherein the chemotherapeutic effective amount of N G -monomethyl-L-arginine [L-NMMA] further comprises liposomes, surfactants, niosomes, ethosomes, transferosomes, phospholipids, sphingosomes, nanoparticles, microparticles, or any combination thereof, and / or further comprises a pharma-ceutically acceptable carrier, buffer, diluent, vehicle, excipient, or any combination thereof.

9. The product of claim 1, adapted and configured as part of a therapeutic kit comprising the product and at least a first set of instructions for administration of the product to a human in need thereof.

10. The product of claim 1, wherein the product is administered in conjunction with a therapeutically effective amount of radiation.

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