Pharmaceutical combination of wnt signaling and MACC1 inhibitors

Abstract

To provide a combination or composition in the treatment of a tumor disease, such as a solid tumor, and / or for the treatment and / or prophylaxis of tumor metastasis.SOLUTION: The present invention provides a pharmaceutical combination comprising a. niclosamide, and b. an inhibitor of MACC1 selected from a MEK1 inhibitor and / or a statin. The niclosamide and the inhibitor of MACC1 are present in a kit, in spatial proximity but in separate containers and / or compositions, or are combined in a single pharmaceutical composition in admixture with a pharmaceutically acceptable carrier.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical combination comprising an inhibitor of the Wnt / β-catenin signaling pathway and an inhibitor of MACC1. In a preferred embodiment, the present invention relates to a combination of an inhibitor of S100A4, preferably niclosamide, as a Wnt signaling inhibitor, and a statin or a MEK1 inhibitor as an inhibitor of MACC1. The present invention further relates to a pharmaceutical composition comprising said combination, as well as to the use of said combination or composition in the treatment of neoplastic diseases, such as solid tumors, and / or for the treatment and / or prevention of tumor metastasis. [Background technology]

[0002] Metastatic dissemination of primary tumors is directly related to patient survival and represents the most fatal attribute of cancer. It critically limits successful treatment in many tumor entities. Biomarkers that identify cancer patients at high risk of metastasis and at the same time act as key drivers of metastasis progression are highly desired. Clinical interventions targeting these molecules are of paramount importance.

[0003] To date, approximately 25%–30% of all patients with colorectal cancer (CRC) already have distant metastases at first presentation (stage IV). 40%–50% of all patients newly diagnosed with CRC without distant metastases (stage I–stage III) develop distant metastases at a later stage (metachronous) after the initial surgery, which is highly associated with a shorter survival time. The 5-year survival rate of patients is approximately 80% in early stage disease, but less than 10% when distant metastases occur. Novel therapeutic options are therefore needed. One of the important therapeutic goals is the limitation of solid tumor metastasis by novel combinatorial molecular targeted therapies.

[0004] The present inventors previously identified a novel gene, MACC1 (Metastasis-Associated in Colon Cancer), in human colorectal cancer (CRC) (Non-Patent Document 1). MACC1 induces fundamental processes such as proliferation, migration, invasion, and metastasis in xenograft and transgenic mice. MACC1 has now been established as an important factor for tumor progression and metastasis in various solid cancers (CRC, gastric cancer, esophageal cancer, pancreatic cancer, hepatocellular / biliary cancer, lung cancer, nasopharyngeal cancer, renal cancer, bladder cancer, ovarian cancer, breast cancer, glioblastoma, osteosarcoma), as well as a prognostic and predictive biomarker. High MACC1 levels in primary tumors or in the patient's blood predict metastasis formation, which is associated with shortened patient survival.

[0005] The present inventors previously identified the first MACC1 transcription inhibitor (Non-Patent Document 2). By performing a human MACC1 promoter-based high-throughput screen (HTS), we identified FDA-approved small molecule drugs such as lovastatin as transcription inhibitors of MACC1 (Figure 1). These compounds inhibited cell motility in cell cultures and, more importantly, limited the development of metastasis in xenograft mice. Following this finding, additional statins were investigated and identified as inhibitors of MACC1 transcription.

[0006] We previously characterized the interactome and phospho-interactome of MACC1. MEK1 was identified as an essential kinase that can phosphorylate MACC1. MEK inhibitors significantly reduced cell motility in vitro. The PoC for MACC1-induced metastasis restriction in mice was demonstrated by deleting MACC1 pY sites or inhibiting pY-MACC1 with FDA-approved MEK1 inhibitors such as AZD6244 (selumetinib) and GSK 1120212 (trametinib).

[0007] By using an isogenic cell line model, the present inventors first identified the now established metastasis inducer S100A4 as a transcriptional target of the Wnt / β-catenin signaling pathway (Non-Patent Document 3). Thus, interventions targeting the Wnt pathway reduced S100A4 expression and significantly reduced metastasis formation in mice.

[0008] The present inventors have previously identified the first transcription inhibitors of the metastatic gene S100A4 using human S100A4 promoter-based HTS (Non-Patent Document 4, Non-Patent Document 5, Non-Patent Document 6). Among the best candidates, the FDA-approved nonsteroidal anti-inflammatory drug sulindac, the antibiotic calcimycin, and the anthelmintic drug niclosamide (Patent Document 1) were identified. Interestingly, both of these drugs interfere with the Wnt / β-catenin signaling pathway, reduce cell motility in vitro, and more importantly, significantly limit metastasis formation in mice.

[0009] Translation of these preclinical findings regarding inhibition of metastasis by repositioning small molecule drugs is currently underway with the FDA-approved anthelmintic drug niclosamide in a clinical Phase II trial: Treatment of patients with stage IV colorectal cancer with niclosamide to limit tumor progression and metastasis (EudraCT 2014-005151-20, NCT02519582).

[0010] The prior art describes the use of niclosamide to enhance the bioavailability of peptide active substances in the treatment of atherosclerosis and also mentions the administration of pravastatin (Patent Document 2 and Non-Patent Document 7), but does not mention cancer treatment or reducing cell motility / metastasis.

[0011] Despite the substantial role of MACC1 and the Wnt / β-catenin signaling pathway, especially S100A4, as prognostic biomarkers and drivers of tumor metastasis progression, their combined targeting as therapeutic targets to inhibit tumor and metastasis progression has not been previously explored in cancer patients. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] International Publication No. 2012143377 [Patent Document 2] International Publication No. 2008 / 021088 [Non-patent literature]

[0013] [Non-Patent Document 1] Stein et al., Nat Med. 2009 Jan;15(1):59-67 [Non-Patent Document 2] Juneja, Kobelt et al. PLoS Biol. 2017 Jun 1;15(6) [Non-Patent Document 3] Stein et al., Gastroenterology, 2006 Nov;131(5):1486-500 [Non-Patent Document 4] Stein et al., Neoplasia. 2011 Feb;13(2):131-44 [Non-Patent Document 5] Sack et al. Mol Biol Cell. 2011 Sep;22(18):3344-54 [Non-Patent Document 6] Sack et al., J Natl Cancer Inst. 2011 Jul 6;103(13):1018-36 [Non-Patent Document 7] Navab et al, J. Lipid Research, vol. 50, 8, 1538-154, 2009 Summary of the Invention [Problem to be solved by the invention]

[0014] In light of the prior art, the technical problem underlying the present invention is to provide an alternative or improved means for the treatment of cancer.The technical problem can also be seen as providing a means for the treatment and / or reduction of the risk of cancer progression in patients with early stage cancer.The technical problem can also be seen as providing a means for the treatment and / or reduction of the risk of metastasis in patients with solid tumors, more particularly in patients with elevated MACC1 and / or S100A4 levels. [Means for solving the problem]

[0015] The object is achieved by the features of the independent claims. Preferred embodiments of the invention are provided by the dependent claims.

[0016] Thus, the present invention provides a.An inhibitor of the Wnt / β-catenin signaling pathway; b. an inhibitor of MACC1; The present invention relates to a pharmaceutical combination comprising:

[0017] The present invention also relates to combinations for use in the treatment of neoplastic diseases, such as solid tumors, and / or for the treatment and / or prevention of tumor metastases, as well as corresponding therapeutic methods. The present invention also relates to the combined administration of inhibitors of the Wnt / β-catenin signaling pathway and inhibitors of MACC1 in such treatments.

[0018] Thus, the present invention represents a personalized intervention for limiting cancer progression and metastasis, combining and targeting the recently discovered key progression factor and prognostic / predictive biomarker MACC1 with the known inducer of metastasis, the Wnt / β-catenin signaling pathway.

[0019] The combined effect of inhibiting both the Wnt / β-catenin signaling pathway and MACC1 results in an unexpected synergistic effect in reducing cell motility and tumor metastasis.

[0020] The additional beneficial effect of reducing tumor cell proliferation is also achieved by MACC1 inhibitors. The synergistic effect evident in this combined treatment with respect to the reduction of cell motility and metastasis is quantitatively reproducible. By treating tumor cells with a combination of both an inhibitor of the Wnt / β-catenin signaling pathway and an inhibitor of MACC1, cell motility is reduced to a greater extent than the sum of the effects induced by each inhibitor when administered alone.

[0021] Moreover, this quantitative synergy is evident in both in vitro and in vivo experimental systems, and therefore the observed synergy is likely to translate to the clinical setting and provide an effective means for tumor treatment in mammalian, preferably human, subjects.

[0022] Quantitative synergy is also evident with multiple inhibitors of the Wnt / β-catenin signaling pathway and multiple inhibitors of MACC1. This supports the synergy of the present invention for the classes of active agents described herein, without being limited to the specific agents tested. The inventors have established that by interfering with the Wnt / β-catenin signaling pathway, beneficial effects in reducing cancer cell motility can be achieved. By combining these effects with inhibition of MACC1, a synergistic therapeutic effect is achieved that is greater than the sum of the individual effects, regardless of the specific pharmaceutical agent used. A more detailed description of the quantitative synergy achieved by the combination is evident in the Examples, particularly in Figures 8 to 10.

[0023] Moreover, this quantitative synergy is evident at multiple concentrations of various combinations of inhibitors of the Wnt / β-catenin signaling pathway and inhibitors of MACC1. Varying the relative concentrations of the two agents does not appear to result in a significant loss of quantitative synergy.

[0024] In some embodiments, the respective doses of the inhibitor of the Wnt / β-catenin signaling pathway (preferably niclosamide or a derivative thereof) and the inhibitor of MACC1 (preferably a statin) can be reduced compared to the doses normally administered. As shown in the examples below, the synergistic effect of the combination of active agents allows for lower doses to be administered, e.g., doses that would be ineffective when administered alone show efficacy when administered in the combination of the present invention. A person skilled in the art could not derive from general knowledge or the prior art that the combination of the present invention allows for more effective and lower dosing of the active agents, thereby potentially maintaining or enhancing efficacy while potentially reducing side effects. As is evident from the experimental support provided herein, even low doses of the active agents (e.g., between 10% and 50% of the maximum dose established in humans for some active agents) can be employed. Even when administered at such reduced doses, the desired effect of inhibition of cancer metastasis remains greater than the sum of the effects of the components administered individually, thereby supporting the synergistic effect.

[0025] Moreover, MACC1 induces the expression and secretion of S100A4 (Figures 2-4). Thus, the inventors have identified a novel link between these two molecular targets. The observed synergy obtained by the combination of the present invention therefore represents an unexpected synergy. A functional interaction between MACC1 and S100A4 has not been described before in the context of cancer growth or metastasis.

[0026] Moreover, a person skilled in the art would not have expected that these two molecular targets would interact beyond a cumulative effect. Even if a relationship between MACC1 and the Wnt / β-catenin signaling pathway could be derived from the art (this is not evident to the inventors' knowledge), the synergistic effect of targeting these factors could not be predicted based on the expectations of a person skilled in the art. Typically, targeting two factors in a related pathway does not usually lead to a synergistic effect, but to an additional or negligible additive effect. Given that MACC1 and S100A4 are mechanistically related, a person skilled in the art would expect the opposite of what has been observed in the present invention. Without being bound by theory, targeting two locations in the same or related pathways typically does not provide an additional quantitative effect since the pathway is already inhibited by the first agent, so the second (combined) agent typically provides no additional effect or at most leads to a small cumulative effect, so that targeting another location in the same or related pathway by a second agent does not provide an additional effect, but merely enhances the effect already achieved by the first agent. Contrary to expectations, a surprising quantitative synergy was achieved by inhibiting both MACC1 and the Wnt / β-catenin signaling pathway.

[0027] In one embodiment, the present invention relates to a pharmaceutical combination comprising: a. an inhibitor of the Wnt / β-catenin signaling pathway and b. an inhibitor of MACC1, wherein inhibitors a. and b. achieve a synergistic effect in reducing cell motility and / or tumor metastasis.

[0028] In one embodiment, inhibitors a. and b. preferably achieve a synergistic effect in reducing cell motility in an in vitro wound healing assay, as described in the examples below.

[0029] In one embodiment, inhibitors a. and b. achieve a synergistic effect in reducing cell motility in an in vitro wound healing assay using HCT116 cells.

[0030] In one embodiment, synergy is determined using the Loewe or HSA model, hi another embodiment, synergy is determined using the Bliss model and / or the ZIP model.

[0031] In one embodiment, the inhibitor of the Wnt / β-catenin signaling pathway is an inhibitor of S100A4.

[0032] S100A4, a target gene of the Wnt / β-catenin signaling pathway, is overexpressed in many different types of cancer, including gallbladder cancer, bladder cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, hepatocellular carcinoma, non-small cell lung cancer, and especially colorectal cancer. Increased expression of S100A4 is strongly associated with tumor aggressiveness, metastatic ability, and reduced patient survival. Inhibition of S100A4 has also been shown to lead to inhibition of S100A4-induced cell migration and invasion, as well as cell proliferation and colony formation in vitro.

[0033] In one embodiment, the inhibitor of the Wnt / β-catenin signaling pathway is niclosamide or a derivative thereof, sulindac, calcimycin, ICG001, FH535, LF3, a phenothiazine such as trifluoperazine (TFP), or a functionally similar derivative thereof.

[0034] Niclosamide, sold under the trade name Yomesan among others, is a drug originally used to treat tapeworm infestations. Niclosamide is also known as 5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide. [ka]

[0035] Niclosamide has also been shown to exhibit a strong inhibitory effect in suppressing the motility and metastasis of cancerous cells expressing S100A4. Niclosamide effectively inhibits the expression of S100A4 and regulates the TCF / beta-catenin protein complex. The S100A4 gene is a target of the Wnt pathway, and its expression is activated by the transcription complex TCF / beta-catenin. Regulation of the transcription complex TCF / beta-catenin via niclosamide treatment results in the cessation or inhibition of transcription of the S400A4 gene. The effect of niclosamide in inhibiting S100A4 was discovered through a high-throughput screening of small molecules to identify inhibitors of S100A4 promoter-driven reporter gene expression with potential clinical antimetastatic activity.

[0036] Various derivatives of niclosamide have been shown to be effective in S100A4-driven cell migration, invasion, and metastasis, and are included in the present application. Those skilled in the art are aware of various chemical methods and techniques for providing chemicals to generate derivatives, which still include chemical bases such as addition, deletion, or substitution of groups or functional groups. Derivatives include, for example, those described in Patent Document 1.

[0037] Examples of niclosamide derivatives include, but are not limited to, the following: [ka]

[0038] Further examples of niclosamide derivatives include, but are not limited to, the following:

[0039] DK419 as disclosed in Wang et al (Bioorg Med Chem. 2018 Nov 1;26(20):5435-5442): [ka]

[0040] A17 or B17 as disclosed in Wu et al. (Cancer Med. 2018 Aug;7(8):3945-3954): [ka]

[0041] Analog 11 or Analog 32 as disclosed in Arend et al. (Oncotarget. 2016 Dec 27;7(52):86803-86815): [ka]

[0042] Sulindac is a nonsteroidal anti-inflammatory drug (NSAID) of the arylalkanoic acid class, marketed as Clinoril by Merck in the UK and the US. Like other NSAIDs, Sulindac is useful in the treatment of acute or chronic inflammatory conditions. Sulindac was identified as a transcriptional inhibitor of the metastatic gene S100A4 using a human S100A4 promoter-based high-throughput screen. [ka]

[0043] Calcimycin is an ionophoric polyether antibiotic derived from Streptomyces chartreusensis. It binds and transports calcium and other divalent cations across membranes, uncouples oxidative phosphorylation, and inhibits rat liver mitochondrial ATPase. Calcimycin is also known as A23187, calcium ionophore, antibiotic A23187, and calcium ionophore A23187. Calcimycin is produced during fermentation of Streptomyces chartreusensis. We have demonstrated that calcimycin also inhibits S100A4. [ka]

[0044] Further inhibitors of S100A4 are known in the art. For example, 6-B12 is under investigation for the treatment of metastatic cancer and breast tumors with lung metastasis. 6-B12 is a monoclonal antibody acting against S100A4 (Cancer Research UK). 6-B12 has been shown to neutralize S100A4, inhibit spontaneous tumor progression and pre-metastatic niche formation, and alter the T cell polarization balance. The effect of spontaneous breast cancer on tumor growth and metastasis dynamics was assessed in a mouse model. In a model of metastatic niche formation, the expression of metastatic niche markers was determined. The S100A4 blocking antibody 6-B12 reduces tumor growth and metastasis in a model of spontaneous breast cancer. The 6B12 antibody inhibits the accumulation of T cells at primary and pre-metastatic tumor sites. The 6B12 antibody also acts as an immunomodulatory agent (Grum-Schwensen et al, BMC Cancer. 2015; 15:44).

[0045] LK-1 is in development for the treatment of solid tumors and pancreatic cancer. LK-1 is a humanized monoclonal antibody that acts by targeting S100A4 (Lykera Biomed).

[0046] ICG001 antagonizes Wnt / β-catenin / TCF-mediated transcription and specifically binds to CREB-binding protein (CBP). By binding to CBP and blocking its interaction with β-catenin, ICG001 selectively induces apoptosis in transformed colon cells, but not in normal cells, and prevents the proliferation of colon cancerous cells. [ka]

[0047] FH535 is a reversible dual inhibitor of Wnt / β-catenin, a compound that suppresses both Wnt / β-catenin and peroxisome proliferator-activated receptor (PPAR) signaling. FH535 is unique in its ability to inhibit the Wnt / β-catenin pathway. This compound is selectively toxic to several carcinomas that express the Wnt / β-catenin pathway. [ka]

[0048] LF3 is a specific inhibitor of canonical Wnt signaling by interfering with the interaction between β-catenin and TCF4. LF3 does not cause cell death or interfere with cadherin-mediated cell-cell adhesion. The self-renewal ability of cancer stem cells is blocked by LF3 in a concentration-dependent manner. LF3 blocks the expression of a set of Wnt target genes in Wnt-dependent colon cancer cells. LF3 inhibits the proliferation of Wnt-dependent colon cancer cells through induction of cell cycle arrest and also inhibits the self-renewal ability of CSCs. [ka]

[0049] Phenothiazines inhibit the function of S100A4 by inducing protein oligomerization. Phenothiazines such as trifluoperazine (TFP) have been shown to inhibit S100A4 (Malashkevich et al, PNAS, 2010, vol. 107, no. 19, 8605-8610). Trifluoperazine (TFP) was identified as an inhibitor that disrupts the S100A4 / myosin-IIA interaction. [ka]

[0050] In one embodiment, the inhibitor of MACC1 is a statin.

[0051] Statins, also known as HMG-CoA reductase inhibitors, are a type of lipid-lowering drug. Statins reduce cardiovascular disease and mortality in people at high risk for cardiovascular disease. Statins are effective in lowering LDL cholesterol and are therefore widely used in primary prevention in people at high risk for cardiovascular disease as well as secondary prevention in people who have developed cardiovascular disease. The present inventors have shown herein that statins result in inhibition of MACC1. As shown in more detail in the examples below, all statins tested were able to reduce MACC1 mRNA expression and MACC1 protein. The present inventors have confirmed that all statins tested are able to reduce cell motility when applied as monotherapy in wound healing assays. A dose-dependent decrease in wound closure over time was observed (Figure 7).

[0052] In one embodiment, the inhibitor of MACC1 is a statin selected from atorvastatin, lovastatin, fluvastatin, pitarvastatin, pravastatin, rosuvastatin and / or simvastatin, preferably atorvastatin, lovastatin and / or fluvastatin.

[0053] It was quite surprising that statins show the effects described herein, i.e., they reduce MACC1 mRNA expression and MACC1 protein, and furthermore, they can reduce cell motility when applied as monotherapy in wound healing assays. The synergistic effect of combination with Wnt signaling inhibitors (such as niclosamide) also represents an unexpected finding, and the combined beneficial effect could not be derived from any suggestion in the art.

[0054] Atorvastatin, sold under the brand name Lipitor among others, is a statin drug used to prevent cardiovascular disease and treat abnormal lipid levels in high-risk individuals. Atorvastatin has been shown to reduce MACC1 mRNA expression and MACC1 protein levels. [ka]

[0055] Lovastatin (Mevacor) is a statin drug used to lower cholesterol and reduce the risk of cardiovascular disease in people with hypercholesterolemia. Lovastatin has been shown to reduce MACC1 mRNA expression and MACC1 protein levels. [ka]

[0056] Fluvastatin (Lescol, Canef, Vastin) is a member of the statin drug class used to treat hypercholesterolemia and prevent cardiovascular disease. Fluvastatin has been shown to reduce MACC1 mRNA expression and MACC1 protein levels. [ka]

[0057] Pitavastatin is a member of the cholesterol-lowering drug class of statins and is marketed in the United States under the trade name Livalo. Like other statins, pitavastatin is an inhibitor of HMG-CoA reductase, the enzyme that catalyzes the first step in cholesterol synthesis. Pitavastatin has been shown to reduce the expression of MACC1 mRNA and the levels of MACC1 protein. [ka]

[0058] Pravastatin (Pravachol) is a statin drug used to prevent cardiovascular disease and treat abnormal lipids in high-risk individuals. Pravastatin can reduce MACC1 mRNA expression and / or MACC1 protein levels. [ka]

[0059] Rosuvastatin (Crestor) is a statin drug used to prevent cardiovascular disease and treat abnormal lipids in high-risk individuals. Rosuvastatin can reduce MACC1 mRNA expression and / or MACC1 protein levels. [ka]

[0060] Simvastatin (Zocor) is a lipid-lowering drug. Simvastatin is used in conjunction with exercise, diet, and weight loss to prevent elevated lipid (fat) levels. Simvastatin can reduce MACC1 mRNA expression and / or MACC1 protein levels. [ka]

[0061] The above statins are provided as non-limiting preferred embodiments of the statin class of compounds. To date, several statins tested by the present inventors achieved the desired MACC1 inhibition in addition to the desired synergistic effect in reducing cell motility when used in combination with an inhibitor of the Wnt / β-catenin signaling pathway, preferably niclosamide.

[0062] In one embodiment, the inhibitor of MACC1 is a MEK1 inhibitor.

[0063] As shown below, the MEK inhibitors tested herein, preferably the MEK1 inhibitors AZD6244 (selumetinib), GSK1120212 (trametinib) or cobimetinib, result in the desired MACC1 inhibition and synergism with an inhibitor of the Wnt / β-catenin signaling pathway, preferably niclosamide. MACC1 is phosphorylated by MEK1, leading to the induction of MACC1-mediated effects. Thus, MEK1 inhibitors such as GSK1120212 (trametinib, approved for the treatment of melanoma) and AZD6244 (selumetinib) act to reduce MACC1 activity and, when combined with an inhibitor of the Wnt / β-catenin signaling pathway, preferably niclosamide, act synergistically on cell motility.

[0064] MEK inhibitors are chemicals or drugs that inhibit the mitogen-activated protein kinase enzymes MEK1 and / or MEK2. Inhibitors of MEK1 and MEK2 are included in the present invention. These inhibitors can be used to affect the MAPK / ERK pathway, which is overactive in some cancers.

[0065] As a non-limiting example of a MEK inhibitor, selumetinib (AZD6244) is a drug being investigated for the treatment of various types of cancer, such as non-small cell lung cancer (NSCLC) and thyroid cancer. MACC1 is phosphorylated by MEK1, and selumetinib inhibits MEK1, reducing MACC1-mediated effects and decreasing cell proliferation and / or motility. [ka]

[0066] Trametinib (GSK1120212) is FDA approved for the treatment of BRAF-mutated melanoma. Trametinib (Mekinist) is an MEK inhibitor with anti-cancer activity that inhibits MEK1 and MEK2. MACC1 is phosphorylated by MEK1, and trametinib inhibits MEK1, reducing MACC1-mediated effects and decreasing cell proliferation and / or motility. [ka]

[0067] Cobimetinib or XL518 is approved by the US FDA for use in combination with vemurafenib (Zelboraf™) for the treatment of advanced melanoma with BRAF V600E or V600K mutations. MACC1 is phosphorylated by MEK1, and cobimetinib inhibits MEK1, reducing MACC1-mediated effects and decreasing cell proliferation and / or motility. [ka]

[0068] Binimetinib (MEK162) was approved by the FDA in 2018 in combination with encorafenib for the treatment of patients with unresectable or metastatic BRAF V600E or V600K mutation-positive melanoma. [ka]

[0069] In some embodiments, the combinations described herein comprise two or more (or at least two) separate compounds. In some embodiments, the inhibitor of the Wnt / β-catenin signaling pathway may also be an inhibitor of MACC1 (in some embodiments, to some residual extent). In some embodiments, the inhibitor of MACC1 may also be an inhibitor of the Wnt / β-catenin signaling pathway (in some embodiments, to some residual extent).

[0070] Preferred MACC1 inhibitors have an inhibitory effect on MACC1 protein function (post-translational inhibitors), preferably by inhibiting MACC1 phosphorylation (in some embodiments by inhibiting the mitogen-activated protein kinase enzyme (MEK)). Preferred Wnt signaling inhibitors have an inhibitory effect on S100A4 transcription, preferably by disrupting the transcription complex TCF / beta-catenin (e.g., via niclosamide or a derivative thereof). In some embodiments, inhibitors of MACC1 have a smaller inhibitory effect on S100A4 transcription than niclosamide, i.e., MACC1 inhibitors act primarily on MACC1 protein function with a smaller effect on S100A4 transcription than the preferred Wnt signaling inhibitors described herein.

[0071] In one embodiment, the present invention provides a method for producing a method for treating a cancer cell comprising: a. niclosamide or a derivative thereof, preferably as disclosed herein; b. statins and / or MEK1 inhibitors; The present invention relates to a pharmaceutical combination as described herein, comprising:

[0072] Combinations of niclosamide with either statins and / or MEK1 inhibitors represent preferred embodiments, the synergistic effects of which are demonstrated in the examples below. It was quite surprising that quantitative synergy in reducing cancerous cell motility and metastasis could be achieved with these combinations.

[0073] In preferred embodiments, the combination is selected from the group consisting of niclosamide and atorvastatin, niclosamide and lovastatin, niclosamide and fluvastatin, niclosamide and AZD6244 (selumetinib), or niclosamide and GSK1120212 (trametinib).

[0074] In some embodiments, the inhibitor of the Wnt / β-catenin signaling pathway (a.) and the inhibitor of MACC1 (b.) have a relative amount of 10000:1 to 1:10000 by weight, preferably 5000:1 to 1:1 by weight.

[0075] As demonstrated in the Examples below, the relative concentrations of the combination agents tested indicate that no loss of synergy occurs when the agents are tested at various relative concentrations, and thus the invention encompasses any relative concentrations and / or amounts of the two classes of agents disclosed herein.

[0076] In one embodiment, (a.) the inhibitor of the Wnt / β-catenin signaling pathway is niclosamide and (b.) the inhibitor of MACC1 is a statin, preferably atorvastatin, lovastatin, fluvastatin, pitarvastatin, pravastatin, rosuvastatin and / or simvastatin, and (a.) and (b.) have a relative amount of 1000:1 to 1:1, preferably 500:1 to 1:1, more preferably 50:1 to 10:1, more preferably about 25:1.

[0077] In some embodiments, (a.) the inhibitor of the Wnt / β-catenin signaling pathway is niclosamide and (b.) the inhibitor of MACC1 is a MEK1 inhibitor, and (a.) and (b.) have a relative amount of from 5000:1 to 1:1, more preferably from 2000:1 to 500:1.

[0078] In some embodiments, (a.) the inhibitor of the Wnt / β-catenin signaling pathway is niclosamide, and (b.) the MEK1 inhibitor is GSK1120212 (trametinib), and (a.) and (b.) have a relative amount of 2000:1 to 500:1, preferably about 1000:1.

[0079] In some embodiments, (a.) the inhibitor of the Wnt / β-catenin signaling pathway is niclosamide, and (b.) the MEK1 inhibitor is AZD6244 (selumetinib) or cobimetinib, and (a.) and (b.) have a relative amount of 1000:1 to 1:1, preferably 500:1 to 1:1, more preferably 50:1 to 10:1, more preferably about 35:1 to 25:1.

[0080] The above embodiments regarding specific relative amounts are based on clinically tolerated or currently tested doses of the various compounds described herein and combined in pharmaceutical combinations described herein.

[0081] In a preferred embodiment, the agents a. and b. (inhibitors of the Wnt / β-catenin signaling pathway (a.) and inhibitors of MACC1 (b.)) are administered in a concentration or amount sufficient to provide a therapeutic effect. Such an effect can be determined by one skilled in the art without undue effort. This amount relates to a therapeutically effective amount when the agent is used alone or when the agent is used in combination with a second agent. In a preferred embodiment, the agent is administered at a concentration or amount or according to a dosing regimen already established in the art, such as for which regulatory approval has been issued (e.g. by the FDA or EMA), or at a dose currently being evaluated during phase 2 or 3 clinical trials and / or according to the maximum tolerated dose from phase I trials.

[0082] In such a treatment, the combination of agent (a.) and agent (b.) can be administered to the patient simultaneously or sequentially, agent (a.) being administered indifferently before or after agent (b.). Agent (a.) and agent (b.) can also be administered by the same or different routes of administration.

[0083] In one embodiment, niclosamide is administered to a human subject in an amount of 100 mg to 5000 mg, preferably 500 mg to 3000 mg, more preferably 500 mg to 2000 mg, or 1000 mg to 2000 mg, or 1500 mg to 2500 mg, preferably about 1000 mg, 1500 mg or 2000 mg per day. In a preferred embodiment, the dose of niclosamide is an oral daily dose of 1000 mg to 3000 mg, preferably 1500 mg to 2500 mg, more preferably 2000 mg.

[0084] In one embodiment, one or more statins are administered to a human subject in an amount of 5 mg to 500 mg, preferably 20 mg to 200 mg, more preferably 50 mg to 150 mg, more preferably about 80 mg per day. These embodiments relate individually to one or more of atorvastatin, lovastatin, fluvastatin, pitarvastatin, pravastatin, rosuvastatin, and / or simvastatin.

[0085] In one embodiment, one or more statins, preferably atorvastatin, lovastatin and / or fluvastatin, are administered to a human subject in an amount of 1 mg to 500 mg per daily oral dose, or 5 mg to 500 mg, preferably 20 mg to 200 mg, more preferably 50 mg to 150 mg, more preferably about 80 mg per daily oral dose, in combination with niclosamide in an amount of 500 mg to 3000 mg, preferably 800 mg to 2500 mg, more preferably 1000 mg to 2000 mg per daily oral dose.

[0086] In one embodiment, the one or more MEK inhibitors (inhibitors of MEK1 or MEK2) are administered to a human subject in an amount of 0.1 mg to 500 mg per day, preferably 1 mg to 200 mg, more preferably 20 mg to 150 mg, more preferably about 2 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, or 90 mg per day.

[0087] In a preferred embodiment, trametinib is administered to a human subject in an amount of 0.1 mg to 20 mg, preferably 1 mg to 10 mg, more preferably 1 mg to 5 mg, more preferably about 2 mg per day.

[0088] In a preferred embodiment, selumetinib is administered to a human subject in an amount of 1 mg to 500 mg, preferably 10 mg to 200 mg, more preferably 20 mg to 150 mg, more preferably about 75 mg per day.

[0089] In a preferred embodiment, cobimetinib is administered to a human subject in an amount between 1 mg and 500 mg, preferably between 10 mg and 200 mg, more preferably between 20 mg and 150 mg, more preferably about 60 mg per day.

[0090] A further aspect of the present invention relates to a pharmaceutical combination as described herein for use in the treatment of a neoplastic disease.

[0091] Thus, the present invention relates to a method for treating a neoplastic disease in a subject in need thereof or in a patient at risk of suffering from a neoplastic disease, the method of treatment preferably comprising administering to said subject a therapeutically effective amount of a pharmaceutical combination or a combination of two active substances to obtain a therapeutic effect.

[0092] Thus, a further aspect of the present invention relates to an inhibitor of the Wnt / β-catenin signaling pathway, preferably niclosamide, sulindac, calcimycin, ICG001, FH535, LF3 and / or a phenothiazine such as trifluoperazine (TFP), more preferably niclosamide, for use as a medicament in the treatment of a neoplastic disease, preferably for the treatment of tumor metastasis, solid tumors or a specific neoplastic disease as disclosed herein, said treatment comprising the concomitant administration of an inhibitor of MACC1, preferably a statin or a MEK1 inhibitor, more preferably atorvastatin, lovastatin, fluvastatin, pitarvastatin, pravastatin, rosuvastatin and / or simvastatin.

[0093] Thus, a further aspect of the present invention relates to an inhibitor of MACC1, preferably a statin or MEK1 inhibitor, more preferably atorvastatin, lovastatin, fluvastatin, pitarvastatin, pravastatin, rosuvastatin and / or simvastatin, or more preferably AZD6244 (selumetinib), GSK1120212 (trametinib) or cobimetinib, for use as a medicament in the treatment of a neoplastic disease, preferably for use in the treatment of tumor metastasis, a solid tumor or a specific neoplastic disease as described herein, said treatment comprising the concomitant administration of an inhibitor of the Wnt / β-catenin signaling pathway, preferably niclosamide, sulindac, calcimycin, ICG001, FH535, LF3 or a phenothiazine such as trifluoperazine (TFP), more preferably niclosamide.

[0094] In one embodiment, the invention relates to a pharmaceutical combination or a combination of two agents for use in the treatment and / or prevention of tumor metastasis, preferably by reducing cell motility of cancerous cells.

[0095] As shown in the following examples, synergy has been demonstrated in both in vitro and in vivo models. The in vitro approach employed uses a wound healing assay with the HCT116 human colon cancer cell line. HCT116 cells are endogenously positive for MACC1 and S100A4 expression at the mRNA and protein levels. Synergy is quantified based on the direct effect on the reduction of cell motility of HCT116 cells observed in the wound healing assay. Cell motility is a fundamental and ancient cellular behavior that directly contributes to the pathology of metastasis. Demonstrating an effect on cell motility, as evidenced by the wound healing assay, supports a therapeutic setting to treat and / or prevent metastasis. Synergy can also be observed on the reduction of cancer cell migration and / or invasion.

[0096] In one embodiment, the tumor is a solid tumor, preferably gastrointestinal cancer, colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, hepatocellular carcinoma, bile duct cancer, lung cancer, nasopharyngeal cancer, renal cancer, bladder cancer, ovarian cancer, brain cancer, bone cancer or breast cancer.

[0097] Solid tumors have been previously analyzed by the present inventors and characterized for their responsiveness to inhibitors of the Wnt / β-catenin signaling pathway and / or inhibitors of MACC1. The solid tumors listed above are known to be responsive to, for example, niclosamide. The solid tumors listed above are known to exhibit expression of MACC1 and / or exhibit active Wnt / β-catenin signaling, preferably as manifested through expression of S100A4.

[0098] In one embodiment, tumor cells exhibit increased expression and / or activity of MACC1 and S100A4 compared to a control, such as a healthy control.

[0099] The inventors herein demonstrate the beneficial role of combining both biomarkers to improve the prognosis of CRC patients (Stein et al. 2012, PLoS One. 2012;7(11)) and gastric cancer patients (Burock et al. World J Gastroenterol 2015 January 7; 21(1):333-341) in the blood of patients as well as in the tumor. A significant advantage for patients showing increased expression and / or activity of MACC1 and S100A4 is evident when treated with the pharmaceutical combination described herein, i.e. a combination of drugs acting on both molecules (Figure 4 shows the additional survival rate of patients based on the expression levels of MACC1 and S100A4).

[0100] In some embodiments, drug-induced S100A4 and / or MACC1 modulation and therapeutic efficacy can also be monitored for therapeutic response using blood-based S100A4 and / or MACC1 assays in combination with HPLC measurement of niclosamide in the patient's blood.

[0101] In a preferred embodiment, the pharmaceutical combination for use as a medicament described herein is characterized in that the treatment of cancer comprises the treatment of a subject having elevated levels of MACC1 and S100A4 compared to a control, such as a healthy control.

[0102] Those skilled in the art can use standard techniques to determine elevated levels of MACC1 and S100A4. For example, a method selected from the group consisting of nucleic acid amplification methods such as PCR, qPCR, RT-PCR, qRT-PCR or isothermal amplification, mass spectrometry (MS), luminescence immunoassays (LIA), radioimmunoassays (RIA), chemiluminescence and fluorescence immunoassays, enzyme immunoassays (EIA), enzyme-linked immunoassays (ELISA), luminescence-based bead arrays, magnetic bead-based arrays, protein microarray assays, rapid test formats (e.g., immunochromatographic strip tests), rare cryptate assays, and automated systems / analyzers can be used.

[0103] The increase in the levels of MACC1 and S100A4 can be determined by comparison with a suitable control. The control sample or patient can relate to a healthy control subject or a sample derived from a control subject, such as a subject with no detectable cancer or history of cancer. When considering the risk of metastasis, the control can also include the use of a sample from a patient with cancer (e.g., those specific cancers described herein) but without metastasis. As a positive control, a sample from a patient with elevated expression of MACC1 and S100A4 and / or a patient with widespread metastasis can be used.

[0104] The elevated levels of MACC1 and / or S100A4 can be determined via nucleic acid and / or protein levels. Increased amounts of MACC1 and / or S100A4 protein and / or mRNA transcripts can be used to detect the levels of MACC1 and / or S100A4 in patients or control subjects. MACC1 and S100A4 are expressed in tumor tissue. Biopsy samples or removed tumors can be used to measure MACC1 and / or S100A4 levels as described herein. Furthermore, MACC1 and / or S100A4 protein and / or nucleic acid encoding MACC1 and / or S100A4 can be detected in a patient's body fluids, such as the patient's urine or blood, or in a sample derived from the blood, such as plasma or serum. Specific methods for identifying elevated levels of MACC1 and / or S100A4 that are useful in the present invention but are not limited to the present invention, are described in more detail below.

[0105] Treatment of patients with elevated levels of MACC1 and / or S100A4 may, in some embodiments, include a combination method incorporating a diagnosis of cancer based on elevated levels of MACC1 and / or S100A4, e.g., elevated MACC1 and / or S100A4 transcript levels, and subsequent treatment with a combination as described herein.

[0106] Treatment of patients with elevated levels of MACC1 and / or S100A4 represents a novel clinical situation resulting from the inhibition of MACC1 and Wnt signaling as described herein. Although MACC1 and S100A4 are known to be prognostic markers for cancer metastasis, no treatment based on providing a MACC1 and Wnt signaling (S100A4) inhibitor has been proposed so far.

[0107] Patients diagnosed with cancer or at high risk of developing cancer or metastasis based on their levels of MACC1 and / or S100A4 can now be directly treated by disrupting the function of MACC1 and S100A4. Thus, the present invention allows an efficient treatment approach for patients with elevated levels of MACC1 and / or S100A4. The inhibitors of the present invention allow effective treatment with novel patient stratification strategies. Those patients who do not have elevated levels of MACC1 and / or S100A4 are typically not selected for treatment with the compounds disclosed herein, thereby targeting the administration of the combinations described herein to a population of patients with elevated levels of MACC1 and / or S100A4 who are more likely to respond to treatment, thereby saving the time and economic costs of treating non-responders who do not have elevated levels of MACC1 and / or S100A4.

[0108] The patient group defined by elevated levels of MACC1 and / or S100A4 compared to appropriate control groups represents a patient group defined by distinct pathological and physiological criteria directly related to the development and progression of disease.Furthermore, there is no suggestion in the art that this patient group can be treated by a combination of inhibitors directed specifically at the disease-causing and disease-indicating molecules MACC1 and S100A4 themselves.It was completely unexpected that an effective treatment for this patient group could be developed based on the synergistic effect of the combined targeting of MACC1 and S100A4.

[0109] In a preferred embodiment, the MACC1 inhibitors described herein are post-translational MACC1 inhibitors. For example, MEK inhibitors cause inhibition of MACC1 phosphorylation, thereby resulting in reduced activity. Reduced activity can be determined by comparison with other subjects with elevated MACC1 activity and / or appropriate controls, such as subjects not receiving the treatment. In other embodiments, transcriptional repressors of MACC1, such as Rottlerin, can be used.

[0110] In a preferred embodiment, the inhibitor of the Wnt / β-catenin signaling pathway is a transcriptional repressor of S100A4, preferably causing inhibition or suppression of transcription of the nucleic acid encoding S100A4. The reduction in transcription may be determined by comparison with other subjects with elevated S100A4 levels and / or appropriate controls, such as subjects not receiving the treatment.

[0111] MACC1 inhibitors and inhibitors of the Wnt / β-catenin signaling pathway can be characterized as anti-metastatic agents. The anti-metastatic effect can be characterized in that the cancer treatment of the present invention comprises preventing and / or reducing cancer cell metastasis and / or cell migration or motility in the subject undergoing treatment.

[0112] In particular, the preventive effect on reducing the risk of metastasis is a unique feature of the present invention. Patients previously diagnosed with elevated levels of MACC1 and / or S100A4 without any appropriate "preventive" treatment were associated with a high risk of metastasis. Herein, the identification of MACC1 and S100A4 inhibition by the combinations described herein allows these patients to be effectively directly treated before cancer metastasis.

[0113] In some embodiments, the MACC1 inhibitors used as medicaments described herein can be characterized as anti-proliferative agents. The anti-proliferative effect can be characterized in that the cancer treatment of the invention includes preventing and / or reducing cancer cell proliferation in the subject undergoing treatment.

[0114] In a preferred embodiment, the cancer being treated is colorectal cancer or a metastasis of colorectal cancer.In one embodiment, the cancer being treated is pancreatic cancer or a metastasis of pancreatic cancer.

[0115] A further embodiment of the present invention relates to the use of the combinations described herein for the treatment of a subject at risk for and / or having glioma, lung cancer, hepatocellular carcinoma, gastric cancer, ovarian cancer, esophageal cancer, gallbladder cancer, renal cancer, nasopharyngeal cancer and / or breast cancer, or osteosarcoma.

[0116] The anti-cancer effects of niclosamide have been demonstrated in various human cancers. In further embodiments, the following cancers for which niclosamide has been shown to be effective can be treated: human breast cancer, prostate cancer, colon cancer, ovarian cancer, multiple myeloma, melanoma, acute myeloid leukemia, glioblastoma, head and neck cancer, and lung cancer (reviewed in Li et al, Cancer Lett. 2014 Jul 10; 349(1):8-14). As summarized herein, niclosamide can block the Wnt signaling pathway that governs cancer initiation, progression, and metastasis, and thus niclosamide has potent activity in inducing cancer cell cycle arrest, growth inhibition, reduced motility, and / or apoptotic death across multiple cancer types.

[0117] In a preferred embodiment, the combinations used as pharmaceuticals described herein are characterized in that the cancer treatment comprises the treatment of subjects with solid tumors and no detectable metastases in one or more lymph nodes.

[0118] The treatment of patients without lymph node metastases is relevant for treating subjects with relatively early stages of cancer, where said patients are still at risk of metastasis.

[0119] In addition to being an effective preventative (or risk-reducing) treatment for future metastases, especially in patients with early stage (such as stage 0, I or II) cancer, preferably colorectal or pancreatic cancer, the present invention provides an effective anti-tumor treatment.

[0120] Treatment of patients with early stage cancer with disruption of MACC1 and S100A4 function by the combination described herein represents a novel clinical situation. Many early stage cancer patients are not initially treated with chemotherapy. Tumor removal is generally performed at an early stage before chemotherapy begins. The therapeutic approach described herein, particularly for patients with early stage cancer with elevated MACC1 and / or S100A4 levels, represents a therapeutic approach that was previously thought to be impossible. For example, many treatment guidelines recommend that antiproliferative drug therapy be initiated only at later cancer stages, such as stage III colorectal cancer. Until now, there has been a need in the art for a means to (potentially prophylactically) treat patients with tumors that have not yet metastasized. Few products are available that can actively prevent or reduce the risk of cancer metastasis, except for surgery.

[0121] The selection of patient populations and cancer stages described herein allows for an effective preventative or risk-reducing treatment approach to avoid or minimize the risk of tumor metastasis in patients at increased risk of metastasis. Traditional protocols require clinicians to wait and observe whether tumor metastasis occurs after the tumor is surgically removed before initiating further treatment. The present invention herein provides a means to alleviate the challenges inherent in previous treatment regimes and reduce the risk of metastasis in patients diagnosed with early stage cancer, preferably solid tumors.

[0122] To date, early diagnosis of increased risk of metastasis (e.g., by detection of elevated levels of MACC1 and / or S100A4) has not been able to be effectively followed up with appropriate treatment, since no agents have been identified that can adequately address early stage cancers with elevated levels of MACC1 and / or S100A4. Clinical practice typically removes tumors as quickly as possible and does not initiate additional treatments until later stages. Even when elevated levels of MACC1 and / or S100A4 are detected, no direct and effective therapeutic options have been available for treating early stage cancer patients, for example to prevent or reduce the risk of developing metastases following removal of the tumor by surgery. The present invention solves this problem of the prior art by providing MACC1 inhibitors as described herein.

[0123] In a preferred embodiment, the present invention is characterized in that the cancer treatment according to the present invention comprises the treatment of a subject with stage 0, I or II colorectal cancer. In another embodiment, the present invention encompasses the treatment of a patient with stage 0, I, IIA, IIB or IIC colorectal cancer. Based on the information provided herein and known guidelines, a person skilled in the art can clearly determine which patients fall into these particular stages.

[0124] As described in more detail below, staging of colorectal cancer is an accepted method to assess the progression of cancer in patients and determine appropriate treatment approaches. Stage 0, I, and II colorectal cancer patients typically do not exhibit metastasis to the subject's lymph nodes, but may still be susceptible to developing life-threatening metastases in the future, depending on MACC1 and / or S100A4 levels. Treatment of these patients represents a new patient collective that is made possible by the inhibition of MACC1 and S100A4 as described herein.

[0125] In a preferred embodiment, the invention is characterized in that the cancer treatment according to the invention comprises the treatment of a subject with stage 0, I or II pancreatic cancer. In particular, the invention contemplates the treatment of stages 0, IA, IB and IIA pancreatic cancer, which are characterized by the absence of lymph node involvement.

[0126] Treatment of stage III and IV colorectal and pancreatic cancer is also envisaged by the present invention, due to the combined anti-metastatic and anti-proliferative effects of the combinations described herein.

[0127] In one embodiment, the subject of the treatment is undergoing and / or has undergone surgery to remove a solid tumor.

[0128] In one embodiment, the treatments and combinations described herein are used in a method of neoadjuvant treatment of a disease state. Neoadjuvant therapy refers to the administration of a therapeutic agent prior to another treatment. Neoadjuvant therapy aims to reduce the size or extent of a cancer before using a definitive therapeutic intervention such as surgery, making the procedure simpler and more likely to be successful, reducing the importance of more extensive treatment procedures that would be required if the size or extent of the tumor had not been reduced. In one embodiment, the combination or use thereof, or the treatment described herein is employed as a neoadjuvant prior to surgery to remove the tumor. In another embodiment, the combination or use thereof, or the treatment described herein is employed to treat and / or prevent metastatic disease prior to and / or during surgery to remove the tumor.

[0129] As described in more detail below, staging of pancreatic cancer is an accepted method for assessing the progression of cancer in a patient and determining an appropriate treatment approach. Based on the information provided herein and known guidelines, one skilled in the art can clearly determine which patients fall into these particular stages.

[0130] In one embodiment of the invention, the combinations described herein can be used to treat or reduce the risk of developing (metastatic) cancer, including the treatment of a subject at risk of developing cancer, said subject comprising: a. has been previously treated for cancer, preferably including a reduction in the number of cancerous cells and / or a reduction in cancer symptoms compared to before the previous treatment; b. Suitable controls, such as elevated levels of MACC1 and S100A4 compared to healthy control subjects, preferably subjects with no history of cancer.

[0131] In one embodiment of the invention, the combinations described herein can be used to treat or reduce the risk of developing (metastatic) cancer, including the prophylactic treatment of cancer cell metastasis and / or cell migration in a subject at risk of developing metastasis, said subject being a. has been previously treated after suffering from colorectal cancer, preferably including a reduction in the number of cancerous cells and / or a reduction in cancer symptoms compared to before the previous treatment; b. Suitable controls, such as elevated levels of MACC1 and S100A4 compared to healthy control subjects, preferably subjects with no history of cancer.

[0132] In some embodiments of the present invention, a patient identified as having a tumor typically has the tumor removed by surgery. The removed tumor or the patient's blood or a suitable sample thereof can be tested for levels of MACC1 and / or S100A4. If the patient is identified as having elevated levels of MACC1 and / or S100A4 compared to relevant controls, the subject is a candidate for treatment with the compounds described herein. After surgery, physicians cannot guarantee that all cancerous cells have been removed in all cases. Elevated levels of MACC1 and / or S100A4 in the removed tumor or in the subject's blood indicate a high risk of metastasis, even after surgical removal of the tumor. Administration of the combinations described herein can be performed to reduce the risk of metastasis and subsequent development of cancer recurrence.

[0133] The present invention further relates to the co-administration of the compounds described herein with additional cytostatic agents. Such agents may relate to any anti-proliferative compound known in the art.

[0134] In a preferred embodiment, the MACC1 inhibitors described herein are administered to early stage cancer patients in combination with 5-FU. Fluorouracil or 5-FU (trademarked as Adrucil(iv), Carac or Efudex) is a drug that is a pyrimidine analog used in the treatment of cancer. This drug is a suicide inhibitor and functions through irreversible inhibition of thymidylate synthase. Coadministration with the combinations described herein is associated with improved anti-proliferative and anti-metastatic effects.

[0135] Further candidates for combination treatment regimens involve platinum-based antitumor agents such as oxaliplatin, marketed as Eloxatin by Sanofi, which are used in cancer chemotherapy. Oxaliplatin is used in the treatment of colorectal cancer, typically with folinic acid and 5-fluorouracil, sometimes in combination. Other platinum compounds used in advanced cancers, such as cisplatin and carboplatin, may also be considered.

[0136] The present invention therefore relates to methods for the treatment of the above mentioned cancers and patient groups, and also to combined diagnostic, prognostic and therapeutic methods, respectively, based on the detection and subsequent inhibition of MACC1 expression.

[0137] The present invention further provides a method of treating a cancer as described herein, or a particular cancer or patient population, in a human subject, such as a subject having an early stage cancer as described herein, comprising the steps of: i. having a sample, such as a biological fluid, obtained from a subject; ii. performing an assay on the sample, for example as described for the diagnostic assays above, comprising determining the level of MACC1 and / or S100A4 expression in the sample and comparing the level of MACC1 and / or S100A4 expression with a control sample; iii. if elevated levels of MACC1 and / or S100A4 are detected, administering to the subject a treatment comprising administering a pharmaceutical combination as described herein to reduce expression and / or activity of MACC1 and / or S100A4; The present invention relates to a method comprising the steps of:

[0138] Features of the invention relating to methods of treatment and diagnosis, as well as descriptions of pharmaceutical formulations used to treat various medical conditions, apply to the pharmaceutical formulations themselves, and vice versa.

[0139] drawing The invention is further illustrated by the drawings, which are not intended to limit the scope of the invention. [Brief description of the drawings]

[0140] [Figure 1-1] Figure 1: Statins inhibit MACC1 expression. HCT116 cells were treated with increasing doses (1 μM-10 μM) of different clinically relevant statins dissolved in DMSO. After 24 h of treatment, cells were harvested for RNA and protein isolation. MACC1 mRNA expression was analyzed by qRT-PCR and is expressed relative to DMSO-treated control samples. Protein amounts were analyzed by Western blot. All tested statins were able to significantly decrease MACC1 mRNA expression and MACC1 protein in a dose-dependent manner. [Figure 1-2] Same as above [Figure 2-1]Figure 1. Migration induced by supernatants of MACC1-overexpressing cells. Cell culture supernatants from SW480 / vector (low MACC1 expression), SW480 / MACC1 (different clones, ectopically overexpressing MACC1) and SW620 (endogenously high MACC1 expression) were collected to treat different tumor cell lines in vitro. SW480 cells (low MACC1 expression) show increased migration in Boyden chamber assays when treated with cell culture supernatants of MACC1-positive cells compared to controls treated with supernatants of MACC1-low SW480 cells. SW620 cells (high endogenous MACC1 expression) do not show increased migration under these conditions. When MACC1 is depleted by shRNA in these cells, motility is reduced and can be rescued by cell culture supernatants of MACC1-positive cells. These results were confirmed in another human tumor cell line from another entity (MiaPaCa, pancreatic cancer). Thus, supernatants collected from MACC1-overexpressing cells induce migration towards a variety of wild-type cells without ectopic MACC1 overexpression. [Figure 2-2] Same as above [Diagram 3] Figure 1. SILAC-mass spectrometry. SW480 / vector (low MACC1 expression) and SW480 / MACC1 (ectopically high MACC1 expression) cell cultures were treated with light and heavy media to label newly synthesized proteins secreted into cell culture supernatants. After protein isolation and LC-MS / MS analysis, the MACC1-induced secretome was identified. MACC1-specific supernatants contain newly secreted S100A4. SW480 / vector vs. SW480 / MACC1: MACC1 overexpression induces S100 protein, especially S100A4, secretion. [Figure 4]FIG. 1 shows that the combination of MACC1 and S100A4 biomarkers identifies the best high-risk colorectal cancer patients. Tumor (A) and plasma (B) samples were collected from colorectal cancer patients and analyzed for MACC1 and S100A4 gene expression by qRT-PCR. Patients with low gene expression of both biomarkers show the best metastasis-free overall survival. Patients with high gene expression of one biomarker show a shortened metastasis-free overall survival. Patients with high gene expression of both biomarkers showed the worst metastasis-free overall survival. Thus, MACC1 and S100A4 can identify patients at increased risk of metastasis formation compared to patients with low expression. This prognosis is further improved when both biomarkers are highly expressed in (A) primary CRC tumors and (B) plasma of CRC patients. [Diagram 5] MACC1 enhances Wnt signaling. Overexpression of MACC1 in SW480 (A) and HCT116 (B) cells increases, whereas depletion of MACC1 in SW620 (C) cells decreases, TCF signaling activity, indicating positive regulation of Wnt signaling by MACC1. Immunoblots of MACC1 overexpression in SW480 and knockdown in SW620 (D). β-catenin knockdown reduces MACC1-induced TCF promoter activity (E) and migration induced by MACC1 (F), indicating that MACC1 induces Wnt signaling via β-catenin in colorectal cancer cells. [Figure 6-1]MACC1 induces the expression of S100A4 through Wnt signaling. Ectopic overexpression of MACC1 in HCT116 cells (A) increases TCF promoter activity (B), subsequently upregulating S100A4 promoter activity (C) and S100A4 gene expression at the mRNA (D) and protein (E) levels. Knockout of MACC1 in SW620 cells (F) decreases TCF promoter activity (G), subsequently downregulating S100A4 promoter activity (H) and S100A4 gene expression at the mRNA (I) and protein (J) levels. In a 54-patient colorectal cancer cohort, the mRNA expression levels of MACC1 and S100A4 were positively correlated (K). [Figure 6-2] Same as above [Figure 7] Figure 1 shows the effect of statins and niclosamide applied as single agents on cell motility and proliferation in vitro. HCT116 cells were treated with three concentrations of atorvastatin, lovastatin, fluvastatin, and niclosamide for 48 hours. Wound closure was monitored every 2 hours using an IncuCyte real-time system. Wound closure was measured relative to the initial wound and expressed as fold change compared to DMSO-treated samples. A pre-established image collection of HCT116 was used to teach the software detection of cells and wounds. The experiment shows that for all drugs tested, there is a dose-dependent decrease in wound closure ability. Untreated and DMSO-treated cells were used as controls. Data represent the mean ± SEM of at least three independent triplicate experiments. [Figure 8-1]Figure 1 shows the effect of applying statins and niclosamide as combination agents on cell motility and proliferation in vitro. HCT116 cells were treated with three concentrations of atorvastatin, lovastatin, and fluvastatin in combination with three concentrations of niclosamide for 48 hours. A representative example shows a combination of doses reduced by 50%. For each drug combination, a synergy matrix of all concentrations is shown. Positive values ​​demonstrate the presence of synergy between the two agents. Negative values ​​may indicate antagonism between the two agents. A value of 0 indicates an additive effect of the two agents. Wound closure was monitored every 2 hours using an IncuCyte real-time system. Wound closure was measured relative to the initial wound and expressed as a fold change compared to the DMSO-treated sample. A pre-established image collection of HCT116 was used to teach the software detection of cells and wounds. The experiment shows that when statins are combined with niclosamide, there is a synergistic decrease in wound closure. Untreated and DMSO-treated cells were used as controls. Data represent the mean ± SEM of at least three independent triplicate experiments. [Figure 8-2] Same as above [Figure 8-3] Same as above [Figure 9-1]Figure 1 shows the effect of MEK1 inhibitors AZD6244 and GSK1120212, and niclosamide applied as combination agents on cell motility and proliferation in vitro. HCT116 cells were treated with a combination of three concentrations of GSK1120212 and three concentrations of niclosamide. For each drug combination, a synergy matrix of all concentrations is shown. Positive values ​​demonstrate the presence of synergy between the two agents. Negative values ​​may indicate antagonism between the two agents. A value of 0 indicates an additive effect of the two agents. A synergistic example of a combination of 0.1 μM GSK1120212 and 0.25 μM niclosamide is shown. A synergy matrix of all concentrations for the treatment is shown. Wound closure was monitored every 2 hours using an IncuCyte real-time system. Wound closure was measured relative to the initial wound and expressed as a fold change compared to the DMSO-treated sample. A pre-established image collection of HCT116 was used to teach the software detection of cells and wounds. Experiments show that when the MEK1 inhibitor GSK1120212 (trametinib) is combined with niclosamide, there is a synergistic reduction in wound closure. Untreated and DMSO-treated cells were used as controls. [Figure 9-2] Same as above [Figure 10]Figure 1 shows the effect of atorvastatin, fluvastatin, and niclosamide in an in vivo mouse model that corresponds to the maximum human dose. Severe combined immunodeficiency (SCID)-beige mice (n=10 per group) were intrasplenic implanted with HCT116-CMVp-Luc cells and treated daily with vehicle, 13 mg / kg atorvastatin or fluvastatin (po), 328 mg / kg niclosamide (po), or a combination of niclosamide and one of the statins at the indicated doses. These doses correspond to the maximum human doses established for statins and niclosamide. Liver metastases were analyzed by bioluminescence imaging after intraperitoneal application of 150 mg / kg D-luciferin at the experimental endpoint. Following whole animal imaging, the liver was removed and analyzed as an isolated organ. Metastasis formation was significantly reduced when atorvastatin, fluvastatin, and niclosamide were applied as single agents. This metastasis inhibition was also evident in the combination treatments. [Figure 11] Figure 1 shows the effect of reduced doses of atorvastatin, fluvastatin, and niclosamide in an in vivo mouse model. Severe combined immunodeficiency (SCID)-beige mice (n=10 per group) were intrasplenic implanted with HCT116-CMVp-Luc cells and treated daily with vehicle, 1.5 mg / kg atorvastatin or fluvastatin (po), 164 mg / kg niclosamide (po), or a combination of niclosamide and one of the statins at the indicated doses. These doses therefore represent 12.5% ​​of the maximum human dose of a statin and 50% of the maximum human dose of niclosamide. Liver metastases were analyzed by bioluminescence imaging after intraperitoneal application of 150 mg / kg D-luciferin at the experimental endpoint. Following whole animal imaging, the liver was removed and analyzed as an isolated organ. No reduction in metastasis formation was observed when atorvastatin, fluvastatin, and niclosamide were applied as single agents. However, a reduction in metastasis was observed with combined treatment, further supporting the synergistic effect of the combination of active agents. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0141] Pharmaceutical combinations: According to the present invention, a "pharmaceutical combination" is the coexistence, i.e. close proximity, of an inhibitor of the Wnt / β-catenin signaling pathway and an inhibitor of MACC1. In one embodiment, the combination is suitable for co-administration.

[0142] In one embodiment, the pharmaceutical combination described herein is characterized in that the inhibitor of the Wnt / β-catenin signaling pathway is in a pharmaceutical composition mixed with a pharma- ceutically acceptable carrier, and the inhibitor of MACC1 is in a separate pharmaceutical composition mixed with a pharma- ceutically acceptable carrier. Thus, the pharmaceutical combination of the present invention may in some embodiments involve the presence of two separate compositions or dosage forms in close proximity to each other. The combined agents need not be present in a single composition.

[0143] In one embodiment, the pharmaceutical combination described herein is characterized in that the inhibitor of the Wnt / β-catenin signaling pathway and the inhibitor of MACC1 as described in any one of the appended claims are present in a kit in spatially adjacent but separate containers and / or compositions. The manufacture of the kit is within the capabilities of a person skilled in the art. In one embodiment, separate compositions containing two separate agents can be packaged and sold together as a combination. In another embodiment, the provision of two agents in combination in a single catalogue or the like but in separate packages is understood as a combination.

[0144] In one embodiment, the pharmaceutical combination described herein is characterized in that an inhibitor of the Wnt / β-catenin signaling pathway and an inhibitor of MACC1 as described in any one of the appended claims are combined in a single pharmaceutical composition mixed with a pharma- ceutically acceptable carrier. The preparation or composition of the combination is known to the skilled artisan, who can assess which carrier materials are compatible with both active substances in the combination and their suitable formulations.

[0145] Inhibitors: According to the present invention, an "inhibitor" in the context of an "inhibitor of the Wnt / β-catenin signaling pathway" or an "inhibitor of MACC1" is considered to be any agent, substance, compound, molecule or other means that slows down, suppresses, blocks or otherwise interferes with or negatively affects the activity, function, expression or signaling caused by a specified target.

[0146] For example, an inhibitor of MACC1 may directly or indirectly affect the function of MACC1 protein, the expression (transcription or translation) of MACC1, and / or the signal transduction mediated by MACC1. For example, an inhibitor of Wnt / β-catenin signaling pathway may affect Wnt / β-catenin signaling or any factor directly or indirectly involved in Wnt / β-catenin signaling. The inhibitors described herein may also be referred to as "agents". Reference to "agents" in the context of the combinations and methods described herein is to be understood as inhibitors of Wnt / β-catenin signaling pathway and inhibitors of MACC1. Preferred inhibitors are those described herein.

[0147] Synergy: Several models can be used to determine or quantify the degree of synergy or antagonism obtained by any given combination. Typically, synergy is considered to be an effect whose magnitude exceeds the sum of two known effects. In some embodiments, the combination response is compared to the expected combination response under the assumption of no interaction calculated using a reference model (see Tang J. et al. (2015) What is synergy? The saariselkae agreement revisited. Front. Pharmacol., 6, 181).

[0148] Commonly used reference models include the Highest single agent (HSA) model (Berenbaum MC(1989) What is synergy. Pharmacol. Rev., 41, 93-141), the Loewe additivity model (Loewe S.(1953) The problem of synergism and antagonism of combined drugs. Arzneimiettel Forschung, 3, 286-290), the Bliss independence model (Bliss CI(1939) The toxicity of poisons applied jointly. Ann. Appl. Biol., 26, 585-615.), and more recently the Zero interaction potency (ZIP) model (Yadav B. et al.(2015) Searching for drug synergy in complex dose-response landscapes using an interaction potency model. Comput. Struct. Biotechnol. J., 13, 504-505). The assumptions made in these reference models differ from one another and may produce somewhat inconsistent conclusions about the degree of synergy. Nevertheless, according to the present invention, if any of these models show synergy between the agents in the combinations described herein, synergy can be considered to have been achieved. Preferably, two, three or all four of these models will reveal synergy between any two agents in the combinations described herein.

[0149] Without limitation, four reference models that can produce reliable results are preferred: (i) the HSA model, where the synergy score quantifies the excess over the best single drug response; (ii) the Loewe model, where the synergy score quantifies the excess over the expected response when the two drugs are the same compound; (iii) the Bliss model, where the expected response is a multiplicative effect as if the two drugs acted independently; and (iv) the ZIP model, where the expected response corresponds to an additive effect as if the two drugs did not affect each other's efficacy.

[0150] The most widely used combination criterion and the preferred model for determining synergy is "Loewe additivity" or "Loewe model" (Loewe (1928), Ergebn. Physiol. 27:47-187; Loewe and Muischnek. "Effect of combinations: mathematical basis of the problem" Arch. Exp. Pathol. Pharmakol. 114:313-326, 1926; Loewe S.(1953) The problem of synergism and antagonism of combined drugs. Arzneimittel Forschung, 3, 286-290), or "dose additivity," which describes the trade-off in potency between two agents when the same compound is included on both sides of a dose matrix. For example, if 50% inhibition is achieved by 1 μM drug A or 1 μM drug B separately, then the combination of 0.5 μM A with 0.5 μM B should also inhibit 50%. Synergy above this level is particularly important in justifying the clinical use of a proposed combination therapy, as it defines the point at which the combination may provide additional benefit over simply increasing the dose of either agent.

[0151] As a further example of determining Loewe additivity (or dose additivity), let d1 and d2 be the doses of compound 1 and compound 2 that, in combination, produce effect e. We define the doses of compound 1 and compound 2 required to produce effect e alone as D e1 and D. e2 (Assume that these conditions uniquely define the doses of the compounds, i.e., the individual dose-response functions are bijective.) d e1 / D e2 quantifies the potency of Compound 1 relative to that of Compound 2. e1 / D e2 can be interpreted as converting the dose of compound 2 to the corresponding dose of compound 1 after taking into account the difference in potency. Loewe additivity is e1 / D e2 =D e1 or d1 / D e1 +d2 / D e2 = 1. Geometrically, Loewe additivity is defined as the situation where an isoball is a point (D e1 , 0) and (0, D e2 ) is a line segment connecting f1(d1), f2(d2), and the dose-response functions of compound 1, compound 2, and the mixture, respectively, as follows: d1 / f1 -1 (f 12 (d1,d2))+d2 / f2 -1 (f 12 Dose additivity holds when (d1,d2) = 1.

[0152] Coadministration: According to the present invention, the term "combined administration" (otherwise known as simultaneous administration or joint treatment) in some embodiments includes administration of separate formulations of the compounds described herein, whereby treatments can occur within minutes of each other, at the same time of each other, on the same day, the same week, or the same month. Alternating administration of two agents is considered an embodiment of combined administration. Staggered administration is encompassed by the term combined administration, whereby one agent can be administered, followed by administration of a second agent, optionally followed by administration of the first agent again, and so on. Simultaneous administration of multiple agents is considered an embodiment of combined administration. Simultaneous administration in some embodiments includes, for example, oral ingestion of multiple compositions containing multiple agents at the same time, for example, by taking separate tablets at the same time. Combination drugs, such as a single formulation containing multiple agents disclosed herein, and optionally an additional anticancer drug, can also be used to simultaneously administer various components in a single administration or dosage.

[0153] The combination therapy or administration of one agent may precede or follow treatment with the other agent in combination, with intervals ranging from minutes to weeks. In embodiments in which the second and first agents are administered separately, it is generally ensured that no significant period expires between the time of each delivery, so that the first and second agents can still be advantageously combined to exert a synergistic effect on the treatment site. In such cases, it is contemplated to contact the subject with both modalities within about 12 to 24 hours of each other, more preferably within about 6 to 12 hours of each other, and most preferably with a delay of only about 12 hours. In some circumstances, it may be desirable to significantly extend the treatment period, but several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) pass between the respective administrations.

[0154] In the sense of the present invention, any form of administration of the agents described herein is encompassed by combined administration, since a beneficial additive therapeutic effect, preferably a synergistic effect, is achieved by combined administration of two agents.

[0155] MACC1: Metastasis remains a major challenge in colorectal cancer (CRC) management. One of the recent key findings in understanding the molecular pathogenesis of CRC metastasis is the identification of the gene MACC1. MACC1 was discovered as a prognostic biomarker for metastasis and metastasis-free survival in CRC. Meanwhile, MACC1 has been identified as a critical progression factor for tumorigenesis and metastasis in various other solid cancers.

[0156] As used herein, "MACC1" refers to the metastasis associated in colon cancer 1 gene in Homo sapiens according to NCBI database gene ID: 346389. This gene encodes a protein of 852 amino acids recorded under GenBank ID: AAI37091. Suitable means for detecting MACC1 encoding nucleic acids or proteins are provided in the Examples below.

[0157] Inhibitors of MACC1 can be determined using established routine techniques. For example, any given assay can be employed to assess substances suspected to be MACC1 inhibitors and compare with appropriate controls using commercially available techniques described herein (e.g., ELISA kits available from Aviva Systems Biology OKCD09378 or Biomatik EKU05926) or cited prior art techniques (e.g., Non-Patent Document 2). To determine the reduction in activity or amount of MACC1 induced by treatment of any given substance (candidate inhibitor), MACC1 protein or activity can be assessed or MACC1 transcript levels can be determined from cell assays or biological samples.

[0158] MEK inhibitors: In some embodiments, the MACC1 inhibitor is a MEK inhibitor, preferably a MEK1 inhibitor. A MEK inhibitor is any substance that inhibits mitogen-activated protein kinase enzymes, preferably MEK1 and / or MEK2. These inhibitors affect the MAPK / ERK pathway, which is overactive in some cancers.

[0159] The mitogen-activated protein kinase (MAPK) signaling pathway involves a family of protein kinases that play a key role in regulating diverse cellular activities, including cell proliferation, survival, differentiation, motility, and angiogenesis. MAPK pathways transduce signals from a variety of extracellular stimuli (growth factors, hormones, cytokines, and environmental stresses) resulting in distinct intracellular responses through a series of phosphorylation events and protein-protein interactions. MEK proteins belong to a family of enzymes that are upstream of specific MAPK targets in each of the four MAP kinase signaling pathways. Several MEK enzymes have been identified. These MEK enzymes selectively phosphorylate serine / threonine and tyrosine residues within the activation loop of specific MAP kinase substrates. MEK1 and MEK2 are closely related. They are involved in the Ras / Raf / MEK / ERK signaling cascade. MEK1, also called MAPKK-1, is the prototypic member of the MEK family of proteins. MEK1 is encoded by the gene MAP2K1 located on chromosome 15q22.31. The gene MAP2K2, which encodes the MEK2 protein, is located on chromosome 19p13.3. The MEK 1 / 2 proteins consist of an N-terminal sequence, a protein kinase domain, and a C-terminal sequence (reviewed in Akinleye et al, J Hematol Oncol. 2013; 6:27).

[0160] Many MEK inhibitors are known in the art, and one of skill in the art can identify agents from this established class using standard methods or literature resources. MEK inhibitors currently in clinical development include, for example, CI-1040 (PD184352), PD0325901, selumetinib (AZD6244), MEK162, AZD8330, TAK-733, GDC-0623, refametinib (RDEA119; BAY 869766), pimasertib (AS703026), RO4987655 (CH4987655), RO5126766, WX-554, RO4987655 (CH4987655), GDC-0973 (XL518), AZD8330, and HL-085 (Cheng and Tian, ​​Molecules 2017, 22, 1551, and in Akinleye et al, J Hematol Oncol.2013; 6:27).

[0161] MEK inhibitors are commercially available and can be procured as needed, and include, but are not limited to, selumetinib (AZD6244), trametinib (GSK1120212), PD0325901, U0126, PD184352 (CI-1040), PD98059, BIX 02189, pimasertib (AS-703026), BIX 02188, TAK-733, AZD8330, binimetinib (MEK162, ARRY-162, ARRY-438162), PD318088, honokiol, SL-327, refametinib (RDEA119, Bayer, 86-9766), myricetin, BI-847325, cobimetinib (GDC-0973, RG7420), GDC-0623 and APS-2-79HCl. Additional MEK inhibitors in development are related to LNP-3794, SHR-7390, CKI-27, CS-3006, and E-6201. In one embodiment, the MEK inhibitor is not U0126.

[0162] Statins: Statins, also known as HMG-CoA reductase inhibitors, are a type of lipid-lowering drug. Statins reduce cardiovascular disease and mortality in people at high risk for cardiovascular disease. Statins are effective in lowering LDL cholesterol, so statins are widely used in primary prevention in people at high risk for cardiovascular disease as well as secondary prevention in people who have developed cardiovascular disease. Statins are commercially available and include, but are not limited to, atorvastatin (Lipitor), fluvastatin (Lescol, Lescol XL), lovastatin (Mevacor, Altoprev), pravastatin (Pravachol), rosuvastatin (Crestor), simvastatin (Zocor), and pitavastatin (Livalo).

[0163] Determining the patient's MACC1 level: The determination of elevated MACC1 levels in patient samples has been described in the art. These methods can be applied to the present invention.

[0164] An example of detecting MACC1 levels from plasma is disclosed in Burock S, Herrmann P, Wendler I, Niederstrasser M, Wernecke KD, Stein U. "Circulating MACC1 transcripts in gastric cancer patient plasma as diagnostic and prognostic biomarker", World J Gastroenterol (accepted July 22, 2014). Briefly, the authors tested for differences between groups regarding MACC1 transcript levels in plasma by using the non-parametric (exact) Wilcoxon-Mann-Whitney test (for deviations from normal distribution and distribution from small samples). Samples obtained from tumor-free volunteers were compared with samples from patients with primary tumors without or with synchronous organ metastases, patients with metachronous metastases, and follow-up patients. Furthermore, samples obtained from patients with tumors were compared with samples obtained from patients with tumors and metastases. Results are expressed as median (range) or frequency (%). p<0.05 was considered significant. To evaluate the diagnostic value of circulating MACC1 transcripts in the plasma of cancer patients, the authors calculated the sensitivity and specificity in quartiles of newly diagnosed cancer patients with primary tumors with or without synchronous metastases compared with blood samples from tumor-free volunteers. For the survival analysis of all newly diagnosed gastric patients, the authors used Kaplan-Meier curves combined with the log-rank test. The cut-off value for each of the biomarkers was the median value of the studied group (primary diagnosis or all patients).

[0165] Further examples of methods for detecting elevated MACC1 levels are described in Stein U, Burock S, Herrmann P, Wendler I, Niederstrasser M, Wernecke KW, Schlag PM, "Circulating MACC1 transcripts in colorectal cancer patient plasma predict metastasis and prognosis", PLoS ONE, 7:e49249, 2012. Differences between groups regarding MACC1 transcript levels in plasma were tested using the non-parametric Wilcoxon-Mann-Whitney test (depending on normal distribution): tumor-free volunteers versus patients with primary tumors with or without synchronous metastases, patients with metachronous metastases, and follow-up patients; patients with tumors versus patients with tumors and metastases. In case of small samples, large differences in sample sizes, large but unbalanced groups, datasets with ties, or sparse data, the test was performed in the exact version. P<0.05 was considered significant. To define the diagnostic value of circulating MACC1 transcripts in plasma, sensitivity and specificity were calculated in quartiles of patients with colorectal cancer (test and validation sets), colon and rectal cancer, newly diagnosed with primary tumors with or without synchronous metastases, compared with blood samples from tumor-free volunteers. Patients were grouped into test and validation sets according to the time course of examinations and first blood draw. The test set of newly diagnosed CRC patients was taken to determine the optimal cut-off value of MACC1 (sensitivity 75%, specificity 76%) and this cut-off value was applied to the validation set of CRC patients. Kaplan-Meier curves combined with the log-rank test were used for the survival of all newly diagnosed cancer patients. The cut-off value of MACC1 was the median value of the studied group (primary diagnosis or all patients).

[0166] An example of testing and determining elevated levels in tumor tissue is disclosed in Stein U, Walther W, Arlt F, Schwabe H, Smith J, Fichtner I, Birchmeier W, Schlag PM, "MACC1, a newly identified key regulator of HGF / Met signaling, predicts colon cancer metastasis", Nature Med 15:59-67, 2009. The authors used a nonparametric two-sided Mann-Whitney rank sum test to assess statistical significance. The authors used ROC analysis and Mann-Whitney tests to compare non-metastatic and metastatic cases. The authors used a nonparametric Spearman-Rho test to assess the correlation between MACC1 and MET in subjects. The authors used the log-rank test to assess Kaplan-Meier curves. The authors used logistic and Cox regressions to evaluate MACC1 as an independent metastatic marker. Data represent mean ± sd.

[0167] Further examples of testing and determining elevated MACC1 levels in tumor tissues are disclosed in Nitsche U, Rosenberg R, Balmert A, Schuster T, Slotta-Huspenina J, Herrmann P, Bader FG, Friess H, Schlag PM, Stein U, Janssen KP, "Integrative marker analysis allows risk assessment for metastasis in stage II colon cancer", Ann Surgery, 256:763-771, 2012. Metastasis-free survival (i.e., survival without distant metastasis) was considered as the primary endpoint. Statistical evaluation was performed using IBM™ SPSS™ Statistics Version 19 (SPSS Inc., IBM Corporation, Somers, NY, USA). To derive optimal cut-off values ​​for gene expression levels, the maximally selected log-rank statistic was used, implemented by R Software version 2.13.0 (R Foundation for Statistical Computing, Vienna, Austria). To address multiple study questions within these analyses, the R-function maxstat.test was employed. Time-dependent survival probabilities were estimated by the Kaplan-Meier method and independent subgroups were compared using the log-rank test. Cox proportional hazards models were used to investigate the impact of multivariate relationships between covariates on survival. Multivariate analysis of binary outcome data was assessed by logistic regression. Area under the curve (AUC) values ​​were calculated by time-dependent receiver operating characteristic (ROC) analysis for censored survival data. Recurrence-free survival times as well as estimated hazard ratios (HRs) were calculated and reported with 95% confidence intervals (95% CI). Clustering of patients into different groups was performed by the SPSS™ Two Step Cluster analysis function.A post hoc power analysis using N-Query software revealed that in the total number of distant recurrence cases, a hazard ratio of 3.2 or greater could be detected using the log-rank test with a type 2 error ≤ 20% (power 80%) at a two-sided level of significance of 5%. All statistical tests were performed two-sided, and a p value of < 0.05 was considered statistically significant. No p value correction was applied to adjust for the problem of multiple testing.

[0168] Wnt / β-catenin signaling pathway: As used herein, the terms "Wnt / β-catenin signaling pathway" and "Wnt signaling" or "Wnt signaling pathway" may be used interchangeably.

[0169] Colorectal cancer is often associated with activation of the Wnt / β-catenin signaling pathway and high expression of the prometastatic gene S100A4. In addition to its function in cell-cell adhesion, β-catenin is also a key mediator of the canonical Wnt signaling pathway. In the absence of Wnt signaling, two scaffolding proteins, the tumor suppressors APC and AXIN, form a so-called destruction complex with β-catenin, which promotes the sequential phosphorylation of β-catenin by CKI and GSK3-β at the amino terminus. Phosphorylation recruits E3 ubiquitin ligases, including the F-box / WD repeat protein β-TrCP, which marks the proteasomal degradation of β-catenin. The Wnt signaling pathway is activated by binding of Wnt ligands to Frizzled transmembrane receptors, which are serpentine receptors with amino-terminal cysteine-rich domains. This complex then interacts with single-pass transmembrane proteins of the LDL receptor family (LRP5 / 6). It is not clear how the FRZ / LRP complex regulates the kinase activity of the destruction complex. However, it has been suggested that the activity of Axin / GSK3-β is inhibited by a mechanism involving the interaction of Axin with LRP5 / 6 or the action of the Axin-binding molecule Dishevelled (DSH). Unphosphorylated β-catenin translocates to the nucleus, where it binds to the amino terminus of the Tcf / Lef (T cell factor / lymphoid enhancer factor) family of DNA-binding proteins and activates the transcription of target genes. Tcf / Lef proteins repress target genes in the absence of β-catenin, but upon binding to β-catenin, they convert them into transcriptional activators.

[0170] The relevance of the Wnt pathway to cancer cells is indicated by the high rate of mutations occurring in Wnt pathway genes. For example, over 90% of colorectal cancers harbor mutations that lead to activation of the Wnt pathway. These mutations generally affect the phosphorylation and stability of β-catenin, preventing its degradation via the ubiquitin pathway. Unphosphorylated β-catenin accumulates in the cytoplasm and is transported to the nucleus, where it interacts with TCF family transcription factors to regulate target genes. Accumulation of nuclear β-catenin is associated with later stages of tumor progression and the development of metastasis, and the presence of mutated β-catenin is associated with aggressive tumor growth and poor prognosis.

[0171] As an example, S100A4 is considered a marker molecule or readout of Wnt / β-catenin signaling. Other readout molecules whose levels indicate Wnt / β-catenin signaling inhibition include, but are not limited to, c-myc, MMP-7, MMP-9 or MMP-13.

[0172] Wnt / beta-catenin signaling and small molecule inhibitors are described in detail in Voronkov et al (Current Pharmaceutical Design, 2013, 19, 634-664).

[0173] Additional inhibitors of the Wnt / β-catenin signaling pathway include XAV939, IWR1, IWP-1, IWP-2, JW74, JW55, okadaic acid, tautomycin, SB239063, SB203580, ADP-HPD, 2-[4-(4-fluorophenyl)piperazin-1-yl]-6-methylpyrimidin 4(3H)-one, PJ34, Cambinol, Sulindac, 3289-86 25, J01-017a, NSC668036, filipin, IC261, PF670462, bosutinib, PHA665752, imatinib, ICG-001, ethacrynic acid, PKF115-584, PNU-74654, PKF118-744, CGP049090, PKF118-310, ZTM000990, BC21, GDC-0941, Rp-8-Br-cAMP.

[0174] Inhibitors of Wnt / β-catenin signaling pathway can be determined using established routine techniques. For example, QIAGEN provides a wide range of assay techniques for evaluating WNT signaling studies, which allow the analysis of gene expression and regulation, epigenetic modification, genotyping, and activation of signaling pathways. Any given assay can be employed to evaluate suspected Wnt / β-catenin signaling pathway inhibitors using commercially available techniques described herein or cited prior art techniques (e.g., Sack et al. Mol Biol Cell. 2011 Sep; 22(18): 3344-54; Sack et al., J Natl Cancer Inst. 2011 Jul 6; 103(13): 1018-36) and compare with appropriate controls.

[0175] S100A4: One major target associated with metastasis formation is S100 calcium binding protein A4 (S100A4), an 11 kDa protein originally identified as metastasin 1 (MTS1). S100A4 is also known as 18A2, 42A, CAPL, FSP1, MTS1, P9KA, PEL98. As used herein, "S100A4" refers to S100 calcium binding protein A4 in Homo sapiens according to the protein ID: NP_002952 in the NCBI database. The S100A4 gene encodes a 101 amino acid protein recorded under gene ID: 6275. Suitable means for detecting nucleic acids encoding S100A4 or S100A4 protein are provided herein.

[0176] S100A4 is overexpressed in various types of cancer, including gallbladder, bladder, breast, esophageal, gastric, pancreatic, hepatocellular, non-small cell lung, and especially colorectal cancer. Increased expression of S100A4 is strongly associated with tumor aggressiveness, ability to metastasize, and poor patient survival. However, S100A4 itself is not tumorigenic, as transgenic mice overexpressing S100A4 do not develop tumors by themselves. However, crossing S100A4 transgenic mice with mice showing spontaneous tumor formation leads to aggressive tumor growth and metastasis. Furthermore, S100A4 null mice injected with highly metastatic mouse breast cancer cells do not show metastasis. These observations suggest that S100A4 is essential for the process of metastasis formation.

[0177] S100A4 plays a key role in cellular processes such as migration, invasion, adhesion, and angiogenesis, which form the basis of metastasis formation. For example, S100A4 enhances cell motility by interacting with proteins from the cytoskeleton, such as the heavy chain of nonmuscle myosin II (MYH9). In addition, S100A4 is involved in cell adhesion by interacting with protein tyrosine phosphatase receptor type F (PTPRF)-interacting protein, binding protein 1 (PPFIBP1; also known as liprin β-1), and promotes cell invasion and angiogenesis via upregulation of metallomatrix peptidases (MMPs).

[0178] Inhibitors of S100A4 can be determined using established routine techniques. For example, any given assay can be employed to assess suspected S100A4 inhibitors and compare with appropriate controls using commercially available techniques described herein (e.g., ELISA kits available from Aviva Systems Biology OKCA02438 or LifeSpan Biolsciences LS-F9180-1) or cited prior art techniques (e.g., Sack et al., J Natl Cancer Inst.2011 Jul 6;103(13):1018-36; Garrett et al, Biochemistry.2008 Jan 22;47(3):986-996.). To determine the reduction in activity or amount of S100A4 induced by the treatment of any given substance (candidate inhibitor), S100A4 protein or activity can be assessed or S100A4 transcript level can be determined from cell assay or biological sample.

[0179] Determination of S100A4 levels in patients: Determining elevated levels of S100A4 in patient samples has been described in the art, and these methods can be applied to the present invention.

[0180] In a preferred embodiment, the present invention relates to the treatment of a patient population identified by increased expression of S100A4, preferably the cancerous cells show increased expression of S100A4. S100A4 expression can be determined using any method commonly known in the art, such as RT-PCR to analyze increased expression of S100A4 transcripts, or immunologically based methods such as Western Blot or ELISA to detect increased expression of S100A4 protein, or any other diagnostic tool capable of providing a relative measure of S100A4 expression compared to "normal" or "healthy" or "low risk" cells.

[0181] As disclosed in US Patent Publication No. 20140294957, 4 × 10 cells were seeded in 6-well plates 24 hours before cells were lysed with Trizol Reagent (Invitrogen). 5 Total RNA can be isolated from cells. Alternatively, RNA can be isolated from blood samples or tumor tissue samples and processed accordingly. For example, RNA is extracted using Trizol RNA extraction reagent (Invitrogen) according to the manufacturer's instructions. Quantification of RNA concentration is performed using Nanodrop (Peqlab), and 50 ng of total RNA is reverse transcribed with random hexamers in 10 mM MgCl2, 1×RT buffer, 250 μM pooled dNTPs, 1 U / μL RNase inhibitor, and 2.5 U / μL MuLV reverse transcriptase (all Applied Biosystems). The reaction occurs at 42° C. for 15 min, 99° C. for 5 min, followed by cooling at 5° C. for 5 min. The cDNA products are amplified in a total volume of 10 μL in a 96-well plate in a LightCycler 480 (Roche) using the following conditions: 95° C. for 10 min, followed by 45 cycles of 95° C. for 10 s, 61° C. for 30 s, and 72° C. for 4 s, using appropriate primers such as those disclosed in US Patent Application Publication No. 20140294957, which is incorporated herein by reference in its entirety. For cDNA quantification of the housekeeping gene glucose-6-phosphate dehydrogenase (G6PD), the hG6PDH Roche Kit (Roche Diagnostics, Mannheim, Germany) is used according to standard instructions. Data analysis is performed, for example, using the LightCycler™ 480 software release 1.5.0 SP3. For each qRT-PCR reaction, the average of the duplicates is calculated. Each average value of the expressed genes is normalized to the respective average amount of G6PD cDNA.

[0182] Additional diagnostic approaches for determining S100A4 and / or MACC1 levels: The present invention further relates to a kit, the use of said kit and a method for determining S100A4 and / or MACC1 levels. Such means can also be used for determining the levels of S100A4 and / or MACC1 during functional testing of inhibitors. The definitions provided herein (e.g. provided in relation to the method) also apply to the kit of the present invention. In particular, the present invention relates to the use of a kit for determining S100A4 and / or MACC1 levels, which kit comprises: A detection reagent for determining the level of S100A4 and / or MACC1; Reference data, in particular reference data regarding threshold or cut-off value(s) for determining elevated levels of S100A4 and / or MACC1; wherein said reference data is preferably stored on a computer readable medium and / or used in the form of a computer executable code configured to compare the determined levels of S100A4 and / or MACC1 with a threshold or cut-off value(s).

[0183] As used herein, "reference data" preferably comprises reference level(s) of S100A4 and / or MACC1 from healthy patients. The level of S100A4 and / or MACC1 in a sample of a subject can be compared to the reference levels contained in the reference data of the kit. The reference data can also comprise a reference sample against which the levels of S100A4 and / or MACC1 are compared. The reference data can also comprise instructions on how to use the kit of the invention.

[0184] The kit may further comprise items useful for obtaining a sample, such as a blood sample, for example the kit may comprise a container comprising a device for attaching the container to a cannula or syringe, a syringe suitable for isolating blood, exhibiting an internal pressure below atmospheric pressure suitable for drawing a predetermined amount of sample into said container, and / or comprising additional detergents, chaotropic salts, ribonuclease inhibitors, chelating agents, RNase inhibitor proteins, and mixtures thereof.

[0185] As used herein, a "detection reagent" or the like is a reagent suitable for determining the marker(s) described herein, e.g., S100A4 and / or MACC1. Such an exemplary detection reagent is, for example, a nucleic acid primer for amplifying an S100A4 and / or MACC1 encoding nucleic acid sequence. Such a primer can be used in a nucleic acid amplification reaction, e.g., a PCR method. Such an exemplary detection reagent is, for example, a ligand, e.g., an antibody or a fragment thereof, that specifically binds to an S100A4 and / or MACC1 peptide or epitope of the marker(s) described herein. Such a ligand may be used in an immunoassay.

[0186] The term "nucleic acid amplification reaction" refers to any method that includes an enzymatic reaction that allows the amplification of nucleic acids. A preferred embodiment of the invention relates to polymerase chain reaction (PCR). Another preferred embodiment relates to real-time PCR (RT-PCR) or quantitative RT-PCR (qRT-PCR), since it allows the quantification of the amplified target in real time. The term "real-time PCR" is intended to mean any amplification technique that allows the progress of the amplification reaction to be monitored (i.e. in real time) as it occurs. Data is therefore collected during the exponential amplification phase of the PCR reaction, rather than at the end point as in conventional PCR. Measuring the reaction rate at the early stage of PCR offers a distinct advantage over traditional PCR detection. In real-time PCR, the reaction is characterized by the time point during cycling at which amplification of the target is first detected, rather than the amount of target accumulated after a certain number of cycles. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed. Traditional PCR methods can also be applied, using separation methods such as agarose gels for detection of PCR amplification at the final stage or end point of the PCR reaction. In the case of qRT-PCR, no post-PCR processing of the unknown DNA sample is required, since quantification occurs in real time during the reaction. Furthermore, the increase in reporter fluorescent signal is directly proportional to the number of amplicons produced. Those skilled in the art are aware of the reagents and methods necessary to perform such detection.

[0187] cancer: The present invention relates to a pharmaceutical combination for inhibition of MACC1 and inhibition of Wnt / β-catenin signaling for the treatment of cancer or cancer-like disorders such as cell proliferation disorders or cancer metastasis. The terms "cancer", "proliferative disorder" or "cell proliferation disorder" can be understood interchangeably and refer to any disorder in which the proliferation capacity of affected cells differs from the normal proliferation capacity of unaffected cells. An example of a cell proliferation disorder is a neoplasm. Malignant cells develop as a result of a multi-step process. The terms "cancer", "neoplastic disease" or "malignant tumor" may refer to tumors or hematopoietic disorders that are no longer under normal cell proliferation control. The term "cancerous cell" includes cells afflicted by any one of the cancerous conditions provided herein. The term "carcinoma" refers to a malignant new growth composed of epithelial cells that tend to invade surrounding tissues and cause metastases.

[0188] The cell proliferative disorder described herein may be a neoplasm, commonly referred to as a tumor or tumour. Such neoplasms are either benign or malignant. The terms "neoplasm", "tumour" or "tumour" refer to new abnormal growth of cells or abnormal cell growth that reproduces faster than normal. A neoplasm creates an unstructured mass (tumour) that can be either benign or malignant. The term "benign" refers to a non-cancerous tumour, e.g., whose cells do not proliferate or invade surrounding tissues.

[0189] In another aspect, the invention provides a method of preventing, treating, and / or managing a solid tumor in a patient, the method comprising administering to a patient in need thereof a prophylactically effective regimen or a therapeutically effective regimen, the regimen comprising administering to the patient a compound of the invention or a pharma- ceutically acceptable salt thereof, and the patient has been diagnosed with a solid tumor. In certain embodiments of this aspect, the solid tumor is selected from the group consisting of fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelioma, lymphangiosarcoma, lymphangioendothelioma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, gastric cancer, oral cancer, nasal cancer, pharyngeal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, and papillary carcinoma. , papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung cancer, bladder cancer, lung cancer, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma, or retinoblastoma.

[0190] Transference: In one embodiment, the present invention relates to a pharmaceutical combination or a combination of two agents used for the treatment and / or prevention of tumor metastasis, preferably by reducing the cell motility of cancerous cells. As used herein, the term "metastasis" refers to the spread of cancer cells from where they were originally formed to another part of the body.

[0191] Metastasis can be considered as a multi-step process in which tumor cells disseminate from a primary tumor and colonize distant organs. The steps include, but are not limited to, tumor cell invasion into basement membranes and surrounding tissues, intravasation into blood vessels, survival therein, extravasation and / or growth at different organ sites. To achieve these steps, precise coordination of cell movement and matrix remodeling is required and can be interrupted by anti-metastatic agents. Thus, determining the effect on cancer metastasis may employ determining the inhibitory effect on one or more of cell invasion, i.e. invasion into basement membranes and surrounding tissues, cell motility or migration, invasion into blood or lymphatic vessels (intravasation), and / or leakage of cancer cells from blood or lymphatic vessels (extravasation). According to the present invention, preferably a reduction in cell motility is assessed.

[0192] Models for assessing the effect on metastasis are disclosed herein and known to those skilled in the art.The in vitro approach adopted preferably uses wound healing assay with HCT116 human colon cancer cell line.The effect on metastasis is quantified based on the direct effect on the reduction of cell motility of HCT116 cells observed in wound healing assay.

[0193] The effect of reducing cancer cell motility, migration and / or invasion can also be observed. In some embodiments, cancer metastasis can be distinguished from cancer proliferation, and thus represents a separate medical application. In some embodiments, both cell proliferation and cell metastasis are reduced.

[0194] As used herein, "cell migration" or "motility" is intended to mean the movement of cells toward a target, such as a chemical or physical signal. Cells often migrate in response to external signals, including chemical and mechanical signals. Cancer cells can migrate and exhibit cell motility, which is a hallmark of metastatic cancer, and can be targeted to treat and / or reduce metastasis or reduce the risk of metastasis. Chemotaxis is one example of cell migration or motility in response to a stimulus. In vitro chemotaxis assays, such as Boyden chamber assays, can be used to determine whether cell migration occurs in any given cell.

[0195] For example, cancer cells can be purified and analyzed. Chemotaxis assays (e.g., by Falk et al., 1980 J. Immuno. Methods 33:239-247) can be performed on cells of interest using plates on which specific chemical signals are arranged, and the transmigrated cells are then collected and analyzed. For example, the Boyden chamber assay requires the use of chambers separated by filters and is used as a tool for accurate determination of chemotactic behavior. The pioneering type of these chambers was created by Boyden (Boyden (1962) "The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes". J Exp Med 115 (3): 453). Motile cells are placed in the upper chamber, while the fluid containing the test substance is filled in the lower chamber. The size of the motile cells to be investigated determines the pore size of the filter. It is essential to choose a diameter that allows active transmigration. To mimic in vivo conditions, in some protocols it is preferred to coat the filter with molecules of the extracellular matrix (collagen, elastin, etc.). The efficiency of the measurement can be increased by the development of multi-well chambers (e.g. NeuroProbe) in which 24, 96, 384 samples are evaluated in parallel. The advantage of this variant is that several counterparts are assayed under the same conditions. Such a method can be used to assess whether any given cancer cell type exhibits cell motility or migration, i.e. whether a tumor sample can be obtained and the cells can be cultured and assayed in such a way. Furthermore, any given inhibitor can be assessed for its effect on cell motility using such an assay. The effect of any given combination described herein can be assessed to determine synergistic effects in affected migration, which is a readout of efficacy in treating metastasis.

[0196] Cancer staging: A staging system is a standardized way that clinicians describe the extent of cancer. The most commonly used staging system for colorectal cancer is the American Joint Committee on Cancer (AJCC) staging system, sometimes known as the TNM system (see the American Cancer Society for more information).

[0197] The TNM system describes three important pieces of information: T, which describes how far the main (primary) tumor has grown into the intestinal wall and whether it has grown into nearby areas; N, which describes the extent of spread to nearby (regional) lymph nodes, which are small, bean-shaped collections of immune system cells that are important in fighting infection; and M, which indicates whether the cancer has spread (metastasized) to other organs in the body.

[0198] Staging of colorectal cancer: Colorectal cancer can spread almost anywhere in the body, but the most common sites of spread are the liver and lungs. Numbers or letters appear after the T, N, and M to provide further detail about each of these factors. Numbers 0 through 4 indicate increasing severity. The letter X means "unable to rate, information not available."

[0199] The T categories of colorectal cancer describe the extent of spread through the layers that make up the walls of the colon and rectum. Tx: information is incomplete and the extent of the tumor cannot be described;Tis: cancer is in the earliest stage (intraepithelial). Cancer involves only the mucosa. Cancer has not grown beyond the muscularis mucosa (inner muscularis layer);T1: cancer has grown through the muscularis mucosa and spread to the submucosa;T2: cancer has grown through the submucosa and spread to the muscularis propria (thick outer muscularis layer);T3: cancer has grown through the muscularis propria to the outermost layer of the colon or rectum but not through it. Cancer has not reached nearby organs or tissues;T4a: cancer has grown through the serosa (also known as the visceral peritoneum), which is the outermost layer of the intestine;T4b: cancer has grown through the wall of the colon or rectum and is attaching to or invading nearby tissues or organs.

[0200] The N category indicates whether the cancer has spread to nearby lymph nodes and, if so, how many are involved. To get an accurate idea of ​​lymph node involvement, most doctors recommend removing at least 12 lymph nodes during surgery and examining them under a microscope. Nx: No description of lymph node involvement possible due to incomplete information;N0: No cancer in nearby lymph nodes;N1: Cancer cells are found in or near 1-3 nearby lymph nodes;N1a: Cancer cells are found in 1 nearby lymph node;N1b: Cancer cells are found in 2-3 nearby lymph nodes;N1c: Small deposits of cancer cells are found in an area of ​​fat near the lymph node, but not in the lymph node itself;N2: Cancer cells are found in 4 or more nearby lymph nodes;N2a: Cancer cells are found in 4-6 nearby lymph nodes;N2b: Cancer cells are found in 7 or more nearby lymph nodes.

[0201] The M category indicates whether the cancer has spread (metastasized) to distant organs such as the liver, lungs, or distant lymph nodes: M0: no spread to distant organs; M1a: cancer has spread to one distant organ or to a set of distant lymph nodes; M1b: cancer has spread to two or more distant organs or to a set of distant lymph nodes, or to distant parts of the peritoneum (lining of the abdominal cavity).

[0202] Once a person's T, N, and M categories have been determined, usually after surgery, this information is combined in a process called stage grouping. Stages are designated by Roman numerals, ranging from Stage I (least advanced) to Stage IV (most advanced). Some stages are further subdivided by letters.

[0203] Stage 0: Tis, N0, M0: Cancer is in the earliest stage. It has not grown beyond the inner lining (mucosa) of the colon or rectum. This stage is also known as carcinoma in situ or intramucosal carcinoma.

[0204] Stage I: T1-T2, N0, M0: Cancer has grown through the muscularis mucosae into the submucosa (T1) or may have grown into the muscularis propria (T2). Cancer has not spread to nearby lymph nodes or distant sites.

[0205] Stage IIA: T3, N0, M0: Cancer has grown into the outermost layers of the colon or rectum but not through them (T3). Cancer has not reached nearby organs. Cancer has not yet spread to nearby lymph nodes or distant sites.

[0206] Stage IIB: T4a, N0, M0: Cancer has grown through the wall of the colon or rectum but has not grown into other nearby tissues or organs (T4a). Cancer has not yet spread to nearby lymph nodes or distant sites.

[0207] Stage IIC: T4b, N0, M0: Cancer has grown through the wall of the colon or rectum and has attached to or grown into other nearby tissues or organs (T4b). Cancer has not yet spread to nearby lymph nodes or distant sites.

[0208] Stage IIIA: One of the following applies: T1-T2, N1, M0: Cancer has grown through the mucosa into the submucosa (T1) and may have grown into the muscularis propria (T2). Cancer has spread to 1-3 nearby lymph nodes (N1a / N1b) or areas of fat near the lymph nodes, but not into the lymph nodes themselves (N1c). Cancer has not spread to distant sites; or T1, N2a, M0: Cancer has grown through the mucosa into the submucosa (T1). Cancer has spread to 4-6 nearby lymph nodes (N2a). Cancer has not spread to distant sites.

[0209] Stage IIIB: One of the following applies: T3-T4a, N1, M0: Cancer has grown through the outermost layer of the colon or rectum (T3) or the visceral peritoneum (T4a) but has not reached nearby organs. Cancer has spread to one to three nearby lymph nodes (N1a / N1b) or areas of fat near the lymph nodes but not to the lymph nodes themselves (N1c). Cancer has not spread to distant sites; T2-T3, N2a, M0: Cancer has grown into the muscularis propria (T2) or the outermost layer of the colon or rectum (T3). Cancer has spread to four to six nearby lymph nodes (N2a). Cancer has not spread to distant sites; or T1-T2, N2b, M0: Cancer has grown through the mucosa into the submucosa (T1) or may have also grown into the muscularis propria (T2). Cancer has spread to seven or more nearby lymph nodes (N2b). Cancer has not spread to distant sites.

[0210] Stage IIIC: One of the following applies: T4a, N2a, M0: Cancer has grown through the wall of the colon or rectum (including the visceral peritoneum) but has not reached nearby organs (T4a). Cancer has spread to 4-6 nearby lymph nodes (N2a). Cancer has not spread to distant sites; T3-T4a, N2b, M0: Cancer has grown through the outermost layer of the colon or rectum (T3), or the visceral peritoneum (T4a), but has not reached nearby organs. Cancer has spread to 7 or more nearby lymph nodes (N2b). Cancer has not spread to distant sites; or T4b, N1-N2, M0: Cancer has grown through the wall of the colon or rectum and has attached to or grown into other nearby tissues or organs (T4b). Cancer has spread to at least one nearby lymph node or to an area of ​​fat near lymph nodes (N1 or N2). The cancer has not spread to distant sites.

[0211] Stage IVA: Any T, Any N, M1a: Cancer may or may not have grown through the wall of the colon or rectum and may or may not have spread to nearby lymph nodes. Cancer has spread to one distant organ (such as the liver or lung) or group of lymph nodes (M1a).

[0212] Stage IVB: Any T, Any N, M1b: Cancer may or may not have grown through the wall of the colon or rectum and may or may not have spread to nearby lymph nodes. Cancer has spread to two or more distant organs (such as the liver or lungs) or lymph node groups, or to distant parts of the peritoneum (the lining of the abdominal cavity) (M1b).

[0213] Staging of pancreatic cancer: T Categories:TX: Primary tumor cannot be assessed;T0: No evidence of primary tumor;Tis: Carcinoma in situ (tumor is confined to the top layer of pancreatic duct cells). At this stage, there is little to no pancreatic tumor;T1: Cancer is still within the pancreas and is 2 centimeters (cm) (about 3 / 4 inch) or less in diameter;T2: Cancer is still within the pancreas, but is more than 2 cm in diameter;T3: Cancer has grown outside the pancreas into nearby surrounding tissue, but has not grown into major blood vessels or nerves;T4: Cancer has grown beyond the pancreas into nearby large blood vessels or nerves.

[0214] N Categories: NX: Nearby (regional) lymph nodes cannot be assessed; N0: Cancer has not spread to nearby lymph nodes; N1: Cancer has spread to nearby lymph nodes.

[0215] M Categories: M0: Cancer has not spread to distant lymph nodes (except those near the pancreas) or distant organs such as the liver, lungs, or brain; M1: Cancer has spread to distant lymph nodes or distant organs.

[0216] Pancreatic Cancer Stage Grouping: Once the T, N, and M categories have been determined, this information is combined to assign an overall stage of 0, I, II, III, or IV (sometimes followed by a letter), a process called stage grouping.

[0217] Stage 0 (Tis, N0, M0): The tumor is confined to the top layer of pancreatic duct cells and has not invaded deeper tissues. The tumor has not spread outside the pancreas. These tumors are sometimes called pancreatic intraepithelial carcinoma or pancreatic intraepithelial neoplasia III (PanIn III).

[0218] Stage IA (T1, N0, M0): The tumor is confined to the pancreas and is 2 cm or less in diameter (T1). The tumor has not spread to nearby lymph nodes (N0) or distant sites (M0).

[0219] Stage IB (T2, N0, M0): The tumor is confined to the pancreas and is larger than 2 cm in diameter (T2). The tumor has not spread to nearby lymph nodes (N0) or distant sites (M0).

[0220] Stage IIA (T3, N0, M0): The tumor has grown outside the pancreas but has not grown into major blood vessels or nerves (T3). The tumor has not spread to nearby lymph nodes (N0) or distant sites (M0).

[0221] Stage IIB (T1-T3, N1, M0): The tumor is confined to the pancreas or has grown outside the pancreas but not into major blood vessels or nerves (T1-T3). The tumor has spread to nearby lymph nodes (N1) but has not spread to distant sites (M0).

[0222] Stage III (T4, Any N, M0): The tumor has grown outside the pancreas near major blood vessels or nerves (T4). The tumor may or may not have spread to nearby lymph nodes (Any N). The tumor has not spread to distant sites (M0).

[0223] Stage IV (Any T, Any N, M1): Cancer has spread to distant sites (M1).

[0224] Treatment: In the present invention, "treatment" or "therapy" generally refers to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, considering that the disease and / or condition is completely or partially prevented, for example by reducing the risk of the subject having a particular disease or condition, or may be therapeutic, considering that the disease and / or adverse effects of the disease are partially or completely cured.

[0225] In the present invention, "therapy" includes any treatment of a disease or condition in a mammal, particularly a human, such as the following treatments (a) to (c): (a) preventing the onset of the disease, condition or symptom in a patient; (b) inhibiting the symptoms of the condition, i.e. preventing the progression of the symptom; (c) ameliorating the symptoms of the condition, i.e. inducing relief of the disease or condition.

[0226] In particular, the treatments described herein relate to reducing or inhibiting tumor growth by reducing or inhibiting the proliferation of cancerous cells or reducing the metastatic properties of cancerous cells. The prophylactic therapies described herein are intended to encompass prevention of metastatic cancer or reducing the risk of developing metastatic cancer, as the likelihood of cancerous cells metastasizing following treatment with the compounds described herein is reduced.

[0227] Pharmaceutical Compositions and Methods of Administration: The present invention also relates to pharmaceutical compositions comprising the compounds described herein.

[0228] The present invention also relates to pharma- ceutically acceptable salts of the compounds described herein, in addition to the enantiomers and / or tautomers of the compounds described.

[0229] The term "pharmaceutical composition" refers to a combination of an agent described herein and a pharma- ceutically acceptable carrier. The phrase "pharmaceutical acceptable" refers to molecular entities and compositions that do not produce severe allergic or similar adverse reactions when administered to humans. As used herein, "carrier" or "carrier material" includes any and all solvents, dispersion media, vehicles, coating agents, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is known in the art. Supplementary active ingredients may also be incorporated into the composition.

[0230] The pharmaceutical composition containing the active ingredient may be in a form suitable for oral use, such as, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions. Tablets contain the active ingredient in admixture with non-toxic pharma- ceutically acceptable excipients suitable for the manufacture of tablets. Tablets may be uncoated or may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over a longer period.

[0231] Dosage levels on the order of about 0.01 mg to about 500 mg per kilogram of body weight per day are useful in treating a given condition. For example, cancer can be effectively treated by administration of the molecules of the invention at about 0.01 mg to 50 mg per kilogram of body weight per day (about 0.5 mg to about 5 g per patient per day). The amount of active ingredient which can be combined with the carrier materials to produce a single dosage form will vary depending on the host treated and the particular mode of administration. For example, formulations intended for oral administration to humans may vary from about 5% to about 95% of the total composition. Dosage unit forms generally contain from about 1 mg to about 5000 mg of active ingredient. However, it should be understood that the specific dosage level for any particular patient will depend on a variety of factors, including the activity of the particular compound employed, age, body weight, general health, sex, diet, time of administration, route of administration, excretion rate, drug combination, and the severity of the particular disease undergoing therapy. The effective amount of a compound according to the invention to be administered will vary depending on factors including the particular compound, its toxicity and inhibitory activity, the condition being treated, and whether the compound is administered alone or in combination with other therapeutic agents.

[0232] The present invention also relates to a process or method for treating the mentioned pathological conditions. The compounds of the present invention can be administered prophylactically or therapeutically to a warm-blooded animal, such as a human, in need of such treatment, preferably in an amount effective against the mentioned disorders, and the compounds are preferably used in the form of a pharmaceutical composition.

[0233] In some embodiments, before, during, or after administering a therapeutically effective regimen of the compound of the present invention or a pharma- ceutically acceptable salt thereof, the patient may receive a therapy for the treatment and / or management of cancer. Non-limiting examples of such therapies include chemotherapy, radioimmunotherapy, toxin therapy, prodrug-activated enzyme therapy, antibody therapy, surgical therapy, immunotherapy, radiation therapy, targeted therapy (i.e., therapy directed at a specific target or pathway, such as tyrosine kinase), and any combination thereof. In some embodiments, the patient has not previously received a therapy for the treatment and / or management of cancer. EXAMPLES

[0234] The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.

[0235] Materials and Methods Cell lines and drug treatments HCT116, SW620 and SW480 colorectal cancer cells were purchased from ATCC. HCT116 cells were stably transduced with GFP / MACC1-GFP plasmid to generate MACC1 overexpressing cell lines, and SW620 cells were stably transduced with sh-control / sh-MACC1 plasmid to generate MACC1 knockdown cell lines. SW480 stable MACC1 overexpressing cell lines were generated using pCDNA3.1 MACC1 plasmid, and control cells were generated using pCDNA3.1 empty plasmid, followed by clonality selection using gentamicin (Gibco). Knockout of MACC1 in SW620 cells was achieved by CRISPR-Cas9-based gene editing followed by puromycin (Gibco)-based selection, after which clones were selected using single cell sorting. Cells were cultured in RPMI supplemented with 10% FCS in a 5% CO2 humidified atmosphere and passaged 2-3 times a week to keep the cultures subconfluent. Drugs were solubilized in DMSO with stock concentrations of 2 mM for niclosamide (Sigma-Aldrich, St. Louis, MO, USA), 2 mM for GSK1120212 (trametinib, Selleck Chemicals, Munich, Germany) and selumetinib (AZD6244, Selleck Chemicals, Munich, Germany), and 10 mM for statins (atorvastatin, lovastatin, fluvastatin, TCI Deutschland GmbH, Eschborn, Germany). All drugs were pre-diluted in DMSO to 1000-fold the final concentration. Control cells were treated with an equal volume of DMSO.

[0236] Reporter assays Luciferase reporter assays were used to assess Wnt signaling and S100A4 promoter activity. Wnt signaling activity was measured using a TOP-flash reporter construct containing the 6xTCF promoter consensus sequence cloned in front of firefly luciferase in the pGL4.23 reporter plasmid (Promega) (kindly provided by Dr. Giridhar Mudduluru). S100A4 promoter activity was measured using the S100A4 promoter luciferase system with the core S100A4 promoter sequence cloned in front of the firefly luciferase reporter in the pGL1.4 reporter plasmid (Invitrogen). Briefly, 2x10 5 Cells were seeded in 24-well plates. After 24 h of incubation, cells were transfected with 500 ng of reporter luciferase construct along with 25 ng of Renilla luciferase to normalize transfection efficiency. Transfection was performed using Lipofectamine 2000 (Life Technologies) transfection reagent according to the manufacturer's instructions. Forty-eight hours after transfection, luciferase activity was measured using the Dual Luciferase Assay Kit (Promega). Firefly luciferase activity values ​​were normalized using Renilla luciferase values, and data are expressed as the mean ± SEM of three independent experiments.

[0237] RNA interference Pre-designed Silencer Select siRNAs against β-catenin (#16704) and negative control (#4390844) were purchased from Ambion. Cells were transfected using Lipofectamine RNAiMAX (Life Technologies) transfection reagent according to the manufacturer's instructions. After 48 hours, cells were replated for further treatment and / or analysis.

[0238] Boyden chamber migration assay Cells were starved overnight in FCS-free medium. Transwell Boyden chamber inserts (Corning) were equilibrated in medium containing 10% FCS for 4 h at 37°C before seeding the cells. 1 × 10 5 Serum-starved cells were seeded onto each insert in medium containing 2% FCS. Medium containing 10% FCS was added to the bottom well as the chemoattractant, and the chambers were incubated at 37°C in a humidified incubator with 5% CO2. After 16 h, cells at the bottom edge of the transwell membrane were harvested by trypsinization. Migrated cells were quantified using Cell-Titer-Glo reagent (Promega) according to the manufacturer's instructions.

[0239] RNA isolation and qRT-PCR RNA was isolated using the Gene Matrix Universal RNA Purification Kit (EURx, Poland) according to the manufacturer's instructions. RNA was quantified and 50 ng of RNA was reverse transcribed using a reaction mix containing MuLV reverse transcriptase, 10 mM MgCl2, 1x PCR-buffer, 250 μM pooled dNTPs, 1U RNase inhibitor, and random hexamers (all from Applied Biosystems). The reaction was carried out at 42°C for 15 min, 99°C for 5 min, and then cooled at 4°C for 5 min. cDNA was amplified using SYBR Green chemistry (Promega) using a LightCycler 480 II system (Roche Diagnostics) with the following PCR conditions: 95°C for 2 min, followed by 45 cycles of 95°C for 7 s, 60°C for 10 s, and 72°C for 20 s. Primers used for quantification are: MACC1 Fwd 5'-TTCTTTTGATTCCTCCGGTGA-3' and Rev 5'-ACTCTGATGGGCATGTGCTG-3'; S100A4 Fwd 5'-TGTGATGGTGTCCACCTTCC-3' and Rev 5'-CCTGTTGCTGTCCAAGTTGC-3'. Data analysis was performed using LightCycler 480 software release 1.5.0 SP3 (Roche Diagnostics). Mean values ​​were calculated from RT-qPCR performed in duplicates. Each mean value of expressed genes was normalized to the respective RP-II cDNA. Data are presented as mean ± SEM of three independent experiments.

[0240] Protein extraction and immunoblotting Whole cell protein extract, 5 x 10 5The assay was performed on HCT116 and SW620 cells. Cells were lysed for 30 min at 4°C using RIPA buffer (50 mM Tris-HCT pH 7.5, 150 mM NaCl, 1% Nonidet P-40) supplemented with cOmplete protease inhibitors (Roche). Lysates were clarified and protein content was measured using a BCA kit (Pierce). 20 μg protein lysates were denatured with NuPage loading buffer containing LDS (invitrogen) and DTT for 10 min. Proteins were then separated by 10% SDS-PAGE before transferring proteins to a PVDF membrane (BioRAD). Membranes were blocked for 60 min with 5% wt / vol skim milk powder and 1% wt / vol BSA in TBST (10 mM Tris-HCL pH 8, 150 mM NaCl and 0.1% Tween 20). Membranes were incubated overnight with the respective primary antibodies (anti-MACC1, #HPA020081, Sigma Aldrich; S100A4, #5114, Dako and β-actin, #31430, Invitrogen; Lamin B1, #12586s, Cell Signalling). Membranes were washed and further incubated for 60 min with the corresponding horseradish peroxidase tagged secondary antibodies (anti-mouse IgG HRP conjugated, #31430, Invitrogen and anti-rabbit IgG HRP conjugated, #W4018, Promega). Membranes were further washed and developed using the chemiluminescence reagent WesternBright ECL kit (Advansta) and then exposed to Fuji medical X-ray film SuperRX (Fujifilm).

[0241] Wound healing assay and synergy calculation For the wound healing assay, the HCT116 human colon cancer cell line was used. HCT116 cells are endogenously positive for the expression of MACC1 and S100A4 at the mRNA and protein levels. Furthermore, inhibition of either marker reduces the motility of these cells.

[0242] For wound healing (scratch) experiments, cells were cultured at 6 × 10 per ml. 5 The cells were passaged to a density of 1.2 × 10 and cultured for 48 h. Cells were then harvested and counted. For wound healing assay, 1.2 × 10 5 Cells were seeded in 100 μl RPMI supplemented with 10% FCS in 96-well Image Lock plates (Essen Bioscience, Hertfordshire, UK). Cells were allowed to attach for 6 hours. Monolayer wounds were applied using an IncuCyte WoundMaker (Essen Bioscience). After wound application, 100 μl of 2x concentrated drug solution was added. DMSO-treated and untreated samples were used as controls. For each drug, three different concentrations (niclosamide 1 μM, 0.5 μM, and 0.25 μM, each statin 5 μM, 2.5 μM, and 1.25 μM, MEK1 inhibitor 1 μM, 0.1 μM, and 0.01 μM) and their combinations were applied, each in triplicate. Of all statins, we tested lovastatin, fluvastatin, and atorvastatin. Wound closure was monitored every 2 hours with an IncuCyte system (Essen Bioscience). Pre-established image collection of HCT116 was used to teach the software detection of cells and wounds. Wound confluency was expressed relative to DMSO-treated controls.

[0243] Synergy was analyzed using combinefit 2.021. Samples treated with DMSO were set to 100%. To calculate synergy, we used Loewe's isobole equation model. The dose-response curves of three single concentrations and their nine combinations were used for the calculation. The Loewe model was applied because a certain degree of target interaction has already been shown: first, active Wnt signaling is important for MACC1 to promote tumor progression and invasiveness (Lemos C, Clin Cancer Res, 2016); second, S100A4 is a Wnt signaling target gene and is found in the MACC1-induced secretome. Three single concentrations and their nine combinations were expressed relative to the control.

[0244] Animal and in vivo drug treatment All experiments were performed according to the United Kingdom Coordinated Committee on Cancer Research (UKCCCR) guidelines and approved by the responsible local authority (State Department of Health and Social Services, Berlin, Germany). For in vivo drug testing, xenograft mouse models were used as previously described (Stein 2009 Nat Med, Sack 2011 J Natl Cancer Inst, Juneja & Kobelt 2017 Plos Biol). Briefly, HCT116-CMVp-LUC cells (5 × 10 per mouse) were cultured in 1000 x 1000 cells / mL. 5 Cells (resuspended in 50 μL of PBS) were implanted intrasplenically into 6-week-old female SCID beige (SCID bg / bg) mice. Animals were randomly assigned to treatment groups.

[0245] Drugs were administered as suspensions using a gavage tube. Both niclosamide (328 mg / kg or 164 mg / kg) and statins (atorvastatin or fluvastatin, 13 mg / kg or 1.5 mg / kg) were orally administered daily. Control mice were treated with an appropriate volume of vehicle solution. In vivo experiments and luminescence imaging were performed as described below.

[0246] In vivo experiments were terminated when control animals showed signs of increasing tumor / metastasis burden and liver damage such as abdominal distension (ascites formation), decreased activity, and distress due to reduced food intake (ethical / humane endpoints).

[0247] For luminescence imaging, mice were anesthetized with 5% isoflurane and intraperitoneally administered 150 mg / kg D-luciferin (Biosynth, Stadt, Switzerland) dissolved in sterile PBS. Anesthesia was maintained with 2% isoflurane. Imaging was performed using a NightOWL LB 981 system (Berthold Technologies, Bad Wildbad, Germany). ImageJ version 1.48v (NIH, Bethesda, MD) was used for color coding of signal intensity (displaying 256 gray scale) and overlaying photographs.

[0248] result MACC1 has been reported to enhance WNT signaling and its target gene expression, leading to increased cancer cell migration and invasion in vitro, and tumor formation and metastasis in vivo (Stein et al. 2009 Nat Med. Jan; 15(1):59-67; Zhen et al. 2014, Oncotarget. Jun 15; 5(11):3756-69; Lemos et al. 2016, Clin Cancer Res. Jun 1; 22(11):2812-24). Ectopic overexpression of MACC1 increased β-catenin nuclear localization, thereby increasing WNT target genes. Conversely, MACC1 knockdown decreased β-catenin nuclear localization and reduced their expression. To inhibit MACC1 gene expression, various clinically relevant statins were tested for their ability to reduce MACC1 mRNA and protein expression. All statins tested significantly reduced MACC1 gene expression (Figure 1).

[0249] Moreover, ectopic overexpression of MACC1 induces the release of factors into the medium in vitro that mediate increased cell motility and invasion in cells of different origins (Figure 2).

[0250] These MACC1-specific supernatants contain newly secreted S100A4, as determined by SILAC analysis (Figure 3).

[0251] This was further substantiated by expression correlation analysis of MACC1 and S100A4 in a publicly available microarray dataset of CRC patients from the NCBI GEO database (Tsuji et al. 2012). Gene expression of MACC1 and S100A4 was positively and significantly correlated with metastasis-free and overall survival in the CRC patient cohort (ρ = 0.4115, p = 0.002, n = 54) (Figure 4).

[0252] We determined that overexpression of MACC1 increased WNT signaling (TCF promoter) activity and subsequently induced S100A4 promoter activity in colorectal cancer cells (Figure 5). This increase in S100A4 promoter activity was further evident in the expression of S100A4 gene and protein in these cells.

[0253] We also determined that increased S100A4 expression in MACC1 ectopically overexpressing cells was due to increased WNT signaling activity in these cells. Conversely, MACC1 knockdown reduced S100A4 promoter activity and gene expression (Figure 6).

[0254] Both MACC1 and S100A4 have been shown to independently induce migration and metastasis. Our recent study uncovered a novel mechanism by which MACC1 induces migration and metastasis via the Wnt / S100A4 axis. Therefore, direct inhibition of Wnt / β-catenin signaling together with MACC1 inhibition represents a viable therapeutic strategy.

[0255] Consistent with this, we tested three different statins in combination with niclosamide, which target two separate pathways. All statins tested were able to reduce MACC1 mRNA expression and MACC1 protein (Figure 1). MACC1 expression is inhibited by statins, thus reducing MACC1-induced effects, and niclosamide interferes with Wnt / β-catenin signaling, reducing S100A4 expression. First, we confirmed that all compounds were able to reduce cell motility when applied as monotherapy in wound healing assays. We found a dose-dependent decrease in wound closure over time. Either untreated or DMSO-treated cells closed the monolayer wound within 48 hours. In comparison, wound closure in cells treated with monotherapy was delayed with all drugs (Figure 7).

[0256] Next, we tested whether statins could act synergistically to reduce cell motility when applied together with niclosamide (Figures 8A-8C). Here, we aim to reduce the amount of drug required to inhibit motility. Compared to single treatments, there was increased inhibition of wound closure when combining two drugs, niclosamide and one statin. In our example, with lower concentrations (2.5 μM for statins and 0.5 μM for niclosamide) compared to our original description of HCT116 (5 μM for statins and 1 μM for niclosamide), we found a synergistic decrease in the ability to close wounds applied to HCT116 monolayers.

[0257] We have shown that MACC1 is phosphorylated by MEK1, leading to the induction of MACC1-mediated effects. Therefore, we tested whether the MEK1 inhibitors GSK1120212 (trametinib, approved for melanoma treatment) and AZD6244 (selumetinib) act synergistically on cell motility when combined with niclosamide. Similarly, we applied three different concentrations of trametinib, selumetinib, and niclosamide to HCT116 cells and monitored wound closure over time. In this case, synergistic activity is detectable, but at a lower level compared to the combination of statins and niclosamide (Figures 9A-B).

[0258] To translate our results into clinical applications, drug combinations consisting of niclosamide and statins were tested in vivo. The combinations of niclosamide and fluvastatin, and niclosamide and atorvastatin were evaluated in an in vivo model (mice intrasplenic implanted with HCT116-CMVp-Luc cells) using oral administration, as described in the Methods section above and in Figures 10-11.

[0259] In the first series of experiments, niclosamide, atorvastatin, and fluvastatin were administered individually and in combination: statins were administered at 13 mg / kg and niclosamide was administered at 328 mg / kg, which corresponds to the maximum established human doses of these agents (80 mg daily for statins and 2 g daily for niclosamide).

[0260] As can be seen in Figure 10, atorvastatin, fluvastatin, and niclosamide reduced metastasis formation (as determined by luminescence measurements) when applied as single agents at these high doses. This inhibition of metastasis was also evident in the combination treatment.

[0261] This experiment was repeated using reduced doses of niclosamide, atorvastatin, and fluvastatin. Statins were administered at 1.5 mg / kg, and niclosamide was administered at 164 mg / kg. These doses represent 12.5% ​​of the maximum human dose of statins and 50% of the maximum human dose of niclosamide (9.2 mg daily for statins and 1 g daily for niclosamide).

[0262] As can be seen from Figure 11, no reduction in metastasis formation as determined by luminescence measurements was observed when atorvastatin, fluvastatin, and niclosamide were applied as single agents. However, a reduction in metastasis was observed with the combination treatment, further supporting that the combination of active agents results in an unexpected synergistic effect in reducing metastases.

[0263] Experiments using a combination of lovastatin and niclosamide have also been performed and show similar results.

[0264] In summary, the combination of niclosamide with statins and the combination of niclosamide with the MEK1 inhibitor GSK1120212 (trametinib) can synergistically reduce cell motility in an in vitro wound-healing assay. Furthermore, the combination of niclosamide with lovastatin, fluvastatin, or atorvastatin is superior and synergistic to monotherapies in reducing metastasis formation in xenograft mouse models.

[0265] Conclusion of the Example We show that the close association of S100A4 and MACC1 and their overexpression are associated with poor prognosis in affected CRC patients. We also show the association of these unexplored biomarkers with Wnt signaling. These findings prompted us to exploit these targets by combined intervention of tumor progression and metastasis formation using repositioned small molecule inhibitors such as niclosamide, statins, and MEK1 tyrosine kinase inhibitors. This intervention strategy using drug combinations showed unexpected synergistic efficacy for metastasis inhibition. This strongly indicates that targeting these important pathways and molecules causally related to cancer metastasis may lead to efficient intervention, especially for patients at high risk of metastasis due to overexpression of S100A4 and MACC1. Thus, treatment of these patients stratified for overexpression of S100A4 and MACC1 will be beneficial for them and holds great promise for clinical use. [Explanation of symbols]

[0266] Drawing translation Figure 1 Lovastatin Relative mRNA Concentration β-Actin Concentration [μM] Fluvastatin Pitarvastatin Simbastatin Atorvastatin Figure 2 SW480+ supernatants SW480+ supernatant Migrated cells (fold vector) 24h 24 hours 48h 48 hours SW460 / vector SW460 / vector SW620+ supernatants SW620+ supernatant SW480 / vector SW480 / vector MACC1 specific supernatants MACC1 specific supernatant vector Figure 3 Vector Vector SILAC Light SILAC Light SILAC Heavy Mix supernatant 1:1 Mix the supernatant 1:1 Protein precipitation and concentration Protein digestion LC-MS / MS analysis LC-MS / MS analysis Bioinformatics analysis MACC1 Secretome MACC1 Secretome Figure 4 Metastasis-free survival MACC1 and S100A4 low S100A4 high S100A4 high MACC1 high MACC1 high MACC1 and S100A4 high Months Survival Survival Figure 5 TCF promoter activity (TOP-flash luciferase, RLU) vector Lamin B1 si-β-catenin si-β-catenin Migration CellTiter Glo Migration CellTiter Glo Migrated cells (fold vector) Figure 6 endogenously low Rel. mRNA expression TCF promoter activity (TOP-flash luciferase, RLU) S100A4 promoter S100A4 promoter S100A4 promoter activity (relative luciferase activity, RLU) S100A4 promoter activity (relative luciferase activity, RLU) β-actin endogenously high Figure 7 wound confluence [%] Wound confluence [%] time [h] time [hour] Medium Atorvastatin Lovastatin Fluvastatin Figure 8 Medium Atorvastatin Niclosamide Combination wound confluence [%] Wound confluence [%] time [h] time [hour] Fluvastatin Lovastatin Figure 9 wound confluence [%] Wound confluence [%] time [h] time [hour] Medium Niclosamide combination Figure 10 luminescence Solvent Atorvastatin Fluvastatin Niclosamide Combination Niclosamide / Atorvastatin Combination Niclosamide / Fluvastatin Figure 11 luminescence Solvent Atorvastatin Fluvastatin Niclosamide Combination Niclosamide / Atorvastatin Combination Niclosamide / Fluvastatin

Claims

1. a. an inhibitor of the Wnt / β-catenin signaling pathway; b. an inhibitor of MACC1; A pharmaceutical combination comprising:

2. The pharmaceutical combination according to claim 1, wherein the inhibitor of the Wnt / β-catenin signaling pathway is an inhibitor of S100A4.

3. 3. The pharmaceutical combination according to claim 2, wherein the S100A4 inhibitor is a phenothiazine such as niclosamide or a derivative thereof, sulindac, calcimycin, ICG001, FH535, LF3, or trifluoperazine (TFP).

4. The pharmaceutical combination according to any one of claims 1 to 3, wherein the inhibitor of MACC1 is a statin.

5. The pharmaceutical combination according to any one of claims 1 to 4, wherein the MACC1 inhibitor is a MEK1 inhibitor.

6. Niclosamide and b. statins and / or MEK1 inhibitors; The pharmaceutical combination according to any one of claims 1 to 5, comprising:

7. 7. The pharmaceutical combination according to any one of claims 1 to 6, wherein the inhibitor of MACC1 is a statin selected from atorvastatin, lovastatin, fluvastatin, pitarvastatin, pravastatin, rosuvastatin and / or simvastatin, preferably atorvastatin, lovastatin and / or fluvastatin.

8. 8. The pharmaceutical combination according to any one of claims 1 to 7, wherein the inhibitor of MACC1 is a statin selected from atorvastatin, lovastatin, fluvastatin, pitarvastatin, pravastatin, rosuvastatin and / or simvastatin, preferably atorvastatin, lovastatin and / or fluvastatin, and the inhibitor of the Wnt / β-catenin signaling pathway is niclosamide or a derivative thereof.

9. The pharmaceutical combination according to any one of claims 1 to 6, wherein the MACC1 inhibitor is a MEK1 inhibitor selected from AZD6244 (selumetinib), GSK1120212 (trametinib) and / or cobimetinib.

10. 10. The pharmaceutical combination according to any one of claims 1 to 9, wherein the combination is selected from the group consisting of niclosamide and atorvastatin, niclosamide and lovastatin, niclosamide and fluvastatin, niclosamide and AZD6244 (selumetinib), or niclosamide and GSK1120212 (trametinib).

11. (a.) the inhibitor of the Wnt / β-catenin signaling pathway and (b.) the inhibitor of MACC1 in relative amounts of 10000:1 to 1:10000 by weight, preferably 5000:1 to 1:1 by weight; Preferably, (a.) the inhibitor of the Wnt / β-catenin signaling pathway is niclosamide and (b.) the inhibitor of MACC1 is a statin, preferably as claimed in claim 7, wherein (a.) and (b.) have a relative amount of 1000:1 to 1:1, preferably 500:1 to 1:1, more preferably 50:1 to 10:1, more preferably about 25:1, Preferably, (a.) the inhibitor of the Wnt / β-catenin signaling pathway is niclosamide and (b.) the inhibitor of MACC1 is a MEK1 inhibitor, and (a.) and (b.) have a relative amount of 5000:1 to 1:1, more preferably 2000:1 to 500:1; Preferably, (a.) the inhibitor of the Wnt / β-catenin signaling pathway is niclosamide, and (b.) the MEK1 inhibitor is GSK1120212 (trametinib), and (a.) and (b.) have a relative amount of 2000:1 to 500:1, preferably about 1000:1; 11. The pharmaceutical combination according to any one of claims 1 to 10, preferably, (a.) the inhibitor of the Wnt / β-catenin signalling pathway is niclosamide, and (b.) the MEK1 inhibitor is AZD6244 (selumetinib) or cobimetinib, and (a.) and (b.) have a relative amount of 1000:1 to 1:1, preferably 500:1 to 1:1, more preferably 50:1 to 10:1, more preferably about 35:1 to 25:

1.

12. The inhibitor of the Wnt / β-catenin signaling pathway is in a pharmaceutical composition admixed with a pharma- ceutically acceptable carrier, and the inhibitor of MACC1 is in a separate pharmaceutical composition admixed with a pharma- ceutically acceptable carrier; or The inhibitor of the Wnt / β-catenin signaling pathway according to any one of claims 1 to 11 and the inhibitor of MACC1 are in spatial proximity in the kit but in separate containers and / or compositions, or 12. The pharmaceutical combination according to any one of claims 1 to 11, wherein the inhibitor of the Wnt / β-catenin signalling pathway according to any one of claims 1 to 11 and the inhibitor of MACC1 are combined in a single pharmaceutical composition mixed with a pharma- ceutical acceptable carrier.

13. The pharmaceutical combination according to any one of claims 1 to 12, for use in the treatment of a neoplastic disease.

14. 14. The pharmaceutical combination for use according to claim 13, wherein the neoplastic disease is a solid tumor or is selected from the group consisting of gastrointestinal cancer, colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, hepatocellular carcinoma, bile duct cancer, lung cancer, nasopharyngeal cancer, renal cancer, bladder cancer, ovarian cancer, brain cancer, bone cancer, head and neck cancer, prostate cancer, melanoma or breast cancer.

15. 15. The pharmaceutical combination according to claim 13 or 14, for use in the treatment and / or prevention of tumor metastasis, preferably by reducing cell motility of cancerous cells.

16. The pharmaceutical combination for use according to any one of claims 13 to 15, wherein the tumour cells show increased expression and / or activity of MACC1 and S100A4 compared to a control, such as a healthy control.

17. 17. The pharmaceutical combination for use according to any one of claims 13 to 16, wherein the subject of treatment presents with stage 0, I, II, III or IV colorectal cancer and / or the subject of treatment is undergoing and / or has undergone surgery to remove a solid tumor.

18. An inhibitor of the Wnt / β-catenin signaling pathway, preferably an inhibitor of the Wnt / β-catenin signaling pathway according to claim 2 or 3, more preferably niclosamide, for use as a medicament in the treatment of a neoplastic disease, preferably for the treatment of tumor metastasis, solid tumors or a neoplastic disease according to any one of claims 13 to 17, said treatment comprising the co-administration of an inhibitor of MACC1, preferably a statin or a MEK1 inhibitor, more preferably an inhibitor of MACC1 according to claim 7.

19. An inhibitor of MACC1, preferably a statin or a MEK1 inhibitor, more preferably an inhibitor of MACC1 according to claim 7, for use as a drug in the treatment of a neoplastic disease, preferably for the treatment of tumor metastasis, a solid tumor or a neoplastic disease according to any one of claims 13 to 17, wherein said treatment comprises the co-administration of an inhibitor of the Wnt / β-catenin signalling pathway, preferably an inhibitor of the Wnt / β-catenin signalling pathway according to claim 2 or 3, more preferably an inhibitor of MACC1.

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