Pharmaceutical composition comprising mitogens and stress control pathway inhibitors for the treatment of cancer

Abstract

The present invention relates to a pharmaceutical composition comprising mitogens and stress control pathway inhibitors for the treatment of cancer. The present invention more specifically relates to a pharmaceutical composition comprising FGF-2 and bortezomib or LB-100 and bortezomib for the treatment of cancer cells without compromising the survival of normal cells.

Description

PHARMACEUTICAL COMPOSITION COMPRISING MITOGENS AND INHIBITORS OF STRESS CONTROL PATHWAYS FOR THE TREATMENT OF CANCER FIELD OF THE INVENTION

[0001] The present invention relates to a pharmaceutical composition comprising mitogens and stress control pathway inhibitors for the treatment of cancer. More specifically, the present invention relates to mitogenic activation using the growth factor FGF-2 or LB-100 combined with bortezomib (a proteotoxic stress control pathway inhibitor). The combination of mitogenic stimulation with cellular stress control pathway inhibitors leads to the death of triple-negative breast cancer cells without compromising the survival of normal breast cells. BACKGROUND AND BACKGROUND OF THE INVENTION: Normal and Malignant Cell Proliferation

[0002] The present invention proposes an approach to cancer therapy that at first glance seems counterintuitive: instead of inhibiting tumor cell proliferation, it stimulates it. By increasing the rate of cell proliferation, the tumor becomes more dependent on stress control pathways. The key to this strategy is the appropriate modulation of cellular stress control pathways, which can induce programmed cell death only in tumor cells. This counterintuitive approach offers the potential for a new way to treat cancer, harnessing the very mechanisms that drive malignant proliferation.

[0003] The control of cell proliferation, of both normal and malignant cells, is subject to two main types of molecular regulatory pathways: mitogenic pathways and cellular stress control pathways. These pathways, which include cell proliferation, cell death evasion, angiogenesis, and invasion and metastasis, have been identified as the "principles" of of cancer.” Normal cells have mechanisms to deal with mitogenic overstimulation, while malignant cells, faced with mitogenic overstimulation, experience a wide range of stresses, becoming acutely dependent on stress control pathways for survival.

[0004] The molecular mechanisms that lead to malignant cell transformation are complex, highly diverse, and still poorly understood. On the other hand, it is possible to show that malignant cells, when stimulated by the combination of a mitogen and an inhibitor of a defined stress-controlling pathway, die, while normal cells survive this treatment well.

[0005] Thus, the aim of the present invention is to present this phenomenon as the basis for a promising therapeutic treatment for cancer. It should be emphasized, from the outset, that this treatment may be useful in the treatment of any type of cancer. Fibroblast growth factors (FGFs)

[0006] Among the factors that play an important role in the cell cycle are fibroblast growth factors (FGFs), whose expression and biological function span a variety of cells and tissues, including the adrenal cortex. The first FGFs discovered were characterized based on their mitogenic activity. The existence of peptides in brain and pituitary extracts capable of promoting the growth of cultured fibroblast lines was initially described by Armelin in 1973 and in adrenal cells by Gospodarowicz in 1975 (ARMELIN, 1973; GOSPODAROWICZ, 1975). Thus, the first components of the FGF family were identified: FGF-a (acidic) or FGF-1 and FGF-b (basic) or FGF-2. To date, 23 types of FGFs have been described.

[0007] The interaction of FGF-2 with FGF receptors (FGFRs) requires the presence of heparan sulfate, which is a component of membrane or extracellular matrix proteoglycans. FGFRs are receptors with tyrosine kinase activity to which five genes correspond: flg (FGFR1), bek (FGFR2), flg-2 (FGFR3), and FGFR4. These genes undergo alternative splicing involving exons 8 and 9. These exons encode part of the third immunoglobulin domain of FGFR1-3, generating isoforms IIIb (more expressed in epithelial tissues) and IIIc (more expressed in mesenchymal tissues), which bind to different FGFs. FGFR4 only has exon 8 (ESWARAKUMAR; LAX; SCHLESSINGER, 2005).

[0008] FGF-2 is a growth factor that binds to tyrosine kinase receptors (FGFRs) and activates several intracellular signaling pathways, such as RAS-MAPK, PI3K-AKT, PLCγ, and STAT. These pathways can regulate cellular processes such as proliferation, survival, migration, differentiation, angiogenesis, wound repair, tissue regeneration, and cell differentiation, depending on the cell type and microenvironmental context. FGFs are important for embryonic development and adult homeostasis. Mutations in FGFRs or FGF signaling pathways can cause metabolic diseases, cancer, and serious congenital syndromes.

[0009] The FGF-2 / FGFR axis has been implicated in the origin and maintenance of multiple cancer processes, as it stimulates cell survival, migration, and proliferation. For example, the FGF-2 / FGFR-ERK pathway maintains pluripotent tumor cells in esophageal squamous cell carcinoma; FGF-2 induces the proliferation and invasion of intraocular melanoma cells; amplification or gain-of-function mutations in FGFRs have been reported in tumors originating from various organ types; and FGF-2 is overexpressed in several types of human tumors.

[0010] However, some studies have also reported antiproliferative activity of FGF-2 in malignantly transformed cells. For example, FGF-2 inhibits proliferation and promotes apoptosis in MDA-MB-134 and MCF-7 breast tumor cells (MCLESKEY; DING; LIPPMAN; KERN, 1994; WANG; MALOOF; WANG; FENIG et al., 1998); blocks the proliferation of neuroepithelioma cells (HIGGINS; WONG; RICHNER; ROWE et al., 2009; SMITS; VAN PEER; ESSERS; KLOMPMAKER et al., 2000) and induces cell death in Ewing's sarcomas (Ewing's Sarcoma Family Tumors, or ESFTs) (STURLA; WESTWOOD; SELBY; LEWIS et al. al., 2000; WESTWOOD; CUTHBERT-HEAVENS;

[0011] ESFT cell lines are very sensitive to the toxic effects of FGF-2. These cell lines are poorly differentiated tumors that occur mainly in childhood and adolescence and derive from bone and soft tissues. The cytostatic and cytotoxic effects of FGF-2 on ESFT cells have been reported by several researchers in recent decades, with aspects of the specific molecular mechanisms of this toxicity varying between these studies (PASSIATORE; GENTILELLA; ROM; PACIFICI et al., 2011; SCHWEIGERER; NEUFELD; GOSPODAROWICZ, 1987; WILLIAMSON; DIBLING; BOYNE; SELBY et al., 2004). Proliferative effects on ESFT cells have also been attributed to FGF-2 signaling, often under low-serum growth conditions (CIDRE-ARANAZ; GRÜNEWALD; SURDEZ; GARCÍA-GARCÍA et al., 2017; GRÜNEWALD; BERNARD; GILARDI-HEBENSTREIT; RAYNAL et al., 2015). Protein phosphatases

[0012] Reversible protein phosphorylation, crucial for regulating cellular signal transduction, is controlled by phosphatases and kinases. Phosphatases are enzymes that remove a phosphate group from their substrates, a process known as dephosphorylation. They are classified into four classes: serine / threonine phosphoprotein phosphatases (PPPs), protein tyrosine phosphatases (PTPs), dual-specificity protein phosphatases (DUSPs), and protein histidine phosphatases. The PPP class is the largest of the serine / threonine family and includes PP1, PP2A, PP2B, PP4, PP5, PP6, and PP7.

[0013] PP2A, a serine / threonine phosphatase, is crucial for several cellular functions, including the regulation of signal transduction pathways and cellular processes such as cell cycle progression, DNA replication, gene transcription, and protein synthesis (THOMPSON; WILLIAMS, 2018). This heterotrimeric enzyme is composed of a scaffolding subunit "A" (PP2A-A), a regulatory subunit "B" (PP2A-B), and a catalytic subunit "C" (PP2A-C). Among its actions is the dephosphorylation of MEK, ERK1, and ERK2.

[0014] The inhibitor LB-100, a small molecule that inhibits PP2A, has demonstrated antineoplastic effects, especially when combined with radiotherapy or specific chemotherapy. PP2A inhibition is believed to affect DNA repair mechanisms, making cells more vulnerable to DNA-damaging agents. Furthermore, recent data indicate that PP2A inhibition may increase oncogenic signaling through the MAPKinase pathway, causing stress on DNA replication. This may contribute to greater sensitivity to DNA-damaging agents, potentiating the antineoplastic effect of LB-100, a characteristic that demonstrates that LB-100 acts as a mitogen, with an effect similar to that of FGF-2.

[0015] LB-100 also enhances the efficacy of immune checkpoint blockade (ICB) in several cancer models by stimulating T-cell activation and cGASSTING. Furthermore, PP2A inhibition can transform immunologically "cold" tumors into "hot" tumors by inhibiting DNA repair.

[0016] Therefore, PP2A inhibition in tumor cells, achieved through LB-100, results in the continuous activation of the MAPKinase pathway, leading to an overstimulation of cell proliferation. This mitogenic activation, similar to that of FGF-2, can trigger ER stress. endoplasmic, eventually culminating in cell death. The strategy of stimulating tumor proliferation to subject the tumor to control of stress pathways proves particularly effective in tumor cells that exhibit PP2A inhibition and sustained ERK activation.

[0017] This approach, explored in the present invention, represents a paradigm shift in cancer treatment, with the potential to significantly improve patient survival. Proteotoxic Stress

[0018] Proteotoxic stress, which occurs when there is an imbalance in protein homeostasis, is associated with several diseases, including cancer. This imbalance can lead to the accumulation of misfolded or aggregated proteins in cells, triggering a series of cellular responses, including the activation of signaling pathways that promote cell survival, adaptation, and, in some cases, cell death.

[0019] These responses occur at various levels, from the activation of unconventional chaperones to the remodeling of proteolytic systems. When mutations make a protein more susceptible to misfolding, aggregation, and degradation, diseases can develop.

[0020] In the context of cancer, proteotoxic stress can contribute to disease progression. Studies suggest that physical or psychological stress may be linked to increased cancer progression and even an increased incidence.

[0021] Since cancer is a complex disease, one of the possible treatments to inhibit tumor growth is through inhibition of the proteotoxic stress control pathway. The proteasome, a protein complex that degrades proteins modified primarily by polyubiquitin chains, is formed by a hollow cylinder of several protein subunits. In addition to the central cylinder, the proteasome has a cap at each end that selectively binds to proteins marked for degradation. Because it is responsible for by degrading damaged and / or harmful proteins, the proteasome is the target of studies for the treatment of cancer and tumors. BORTEZOMIB

[0022] Bortezomib is an effective drug in the treatment of multiple myeloma, acting by inhibiting the 26S proteasome. By blocking the activity of this complex, bortezomib prevents the normal degradation of proteins within cancer cells, increasing proteotoxic stress and potentially leading to cell death.

[0023] Therefore, by inhibiting the proteasome, bortezomib contributes to reducing cancer growth and spread. In this sense, since it can increase tumor cell sensitivity to the proteotoxic stress control pathway by stimulating proliferation, the effect of bortezomib can be potentiated.

[0024] Figure 20 shows the action of Bortezomib on the proteasome, demonstrating that inhibition of the 26S proteasome leads to increased JNK, stabilization of p53, and decreased NF-κβ, which increase apoptosis. In addition, the stabilization of p21 and p27 decreases proliferation. The stabilization of Caveolin-1 decreases cell migration, and the decrease in NF-κβ decreases proliferation and angiogenesis (RUSSO; FRATTO; BAZAN; SCHIRÓ et al., 2007). Triple Negative Breast Cancer

[0025] Breast cancer is a complex and heterogeneous disease, with different histological and dissemination patterns, as well as varied therapeutic responses and patient outcomes (JOHNSON; CONANT; SOO, 2021). It is one of the leading causes of death among women globally (IARC; WHO; GCO, 2020; IBRAHIM; AL-GAHMI; ZEENELIN; ZEKRI et al., 2009; JOHNSON; CONANT; SOO, 2021; MCDONALD; KAWAGUCHI; QI; PENG et al., 2019). This heterogeneity is due to differences in genomic characteristics, epigenetic, transcriptomic and proteomic characteristics of cancer cells, which influence properties such as proliferation, apoptosis, metastasis and response to treatment (GUO; KONG; LIU; ZHAN et al., 2023).

[0026] In Brazil, breast cancer is the leading cause of cancer death among women, except in the North region, where cervical cancer is more prevalent. In 2020, the global population-adjusted mortality rate was 11.84 deaths per 100,000 women, being highest in the Southeast and South regions.

[0027] Breast cancer can be classified into four subtypes based on the expression of certain hormone receptors: Luminal A, Luminal B, HER2, and Basal-like. However, the genetic heterogeneity of breast cancer is likely more complex than currently understood.

[0028] The basal-like subtype of breast cancer, often associated with triple-negative breast cancer (TNBC), is high-grade and has high recurrence rates. Imaging studies often reveal irregular masses, but some basal-like tumors can be mistaken for benign lesions. Studies indicate a high correlation between the basal-like subtypes PAM50, Lehmann BL1 / BL2, and Baylor BLIA / BLIS, highlighting the stability of the basal-like subtype in TNBC.

[0029] TNBC is more common in young, Black, and Latina women, and in women with mutations in the BRCA1 and / or BRCA2 genes (CANCER, 2022a). Representing 15–20% of all breast carcinomas, TNBC has an aggressive clinical course, with early onset, high metastatic potential, and worse clinical outcomes.

[0030] Treatments for TNBC include surgery, radiation therapy, chemotherapy, and targeted therapy, depending on the stage of the cancer and the patient's overall health. It's important to emphasize that the treatment plan is individualized for each patient, taking into account their specific circumstances.

[0031] The heterogeneity of breast cancer makes early diagnosis, treatment selection, and prognosis prediction difficult. Understanding the gene expression profile of each subtype can help explain the clinical behavior of different breast cancer tumors and offer hope for targeted therapies. However, this type of therapy is very expensive and still restricted to a small portion of the eligible women population.

[0032] TNBC-derived cell lines, such as MDA-MB-231, which has mutations in several genes, are frequently used to study new therapeutic approaches for TNBC (SATO; SAGARA; TAJIMA; MIURA et al., 2022; YIN; DUAN; BIAN; YU, 2020).

[0033] Oncogenic RAS signaling is an important genetic element for the transformation of primary human cells and is present in the MDA-MB-231 cell line. However, TNBC, which does not express hormone receptors or HER2, does not respond to hormonal or HER2-targeted therapies, making recurrence and metastasis more common. This highlights the urgent need for new therapeutic strategies.

[0034] In this context, TNBC, in the form of the MDA-MB-231 cell line, serves as an excellent model for testing new therapeutic approaches. By stimulating tumor proliferation and subjecting it to the control of stress pathways, the present invention explored a new approach to combating TNBC, potentially overcoming some of the limitations of current treatments.

[0035] It's worth noting that all currently available cancer treatments aim to inhibit mitogenic stimulation in order to stop or slow the high levels of tumor cell proliferation. Thus, radiotherapy and chemotherapy are treatments that follow this principle. However, in addition to reducing tumor cell proliferation, these treatments also affect normal cells, especially those that have high growth rates.

[0036] Other approaches used in cancer treatment involve antibodies that specifically recognize tumor cells, reducing side effects on normal cells. However, this approach, in addition to requiring the discovery of specific antibodies to recognize each type of tumor, requires molecular engineering to produce the antibodies, significantly increasing the cost of these treatments. SUMMARY OF THE INVENTION

[0037] The present invention will provide significant advantages in relation to a pharmaceutical composition comprising mitogens and inhibitors of stress control pathways for the treatment of cancer, enabling an increase in its performance and presenting a more favorable cost / benefit ratio.

[0038] More specifically, the present invention relates to the growth factor FGF-2, more specifically, the present invention relates to FGF-2 inhibiting the proliferation of malignant adrenocortical cells of the Y1 lineage. The combination of mitogenic stimulation with inhibitors of cellular stress control pathways leads to the death of triple-negative breast cancer cells, without compromising the survival of normal breast cells.

[0039] The present application demonstrated that FGF-2 has antagonistic effects on the proliferation of the Y1 line of murine adrenocortical tumorigenic cells dependent on amplification of the K-ras oncogene. In this case, FGF-2 stimulates the G0 transition. G1, but irreversibly blocks the G2 → M transition of the cell cycle. In particular, the present application presents a novel approach to the effects of FGF-2 on malignant cells. Contrary to the indications of the prior art, the present invention reveals the mitogenic effect and the blocking effect of cell multiplication. performed by FGF-2. In fact, this phenomenon stems from the mitogenic effect that, in malignant cells, causes stress that can lead to cell death. This dual antagonistic effect of FGF-2 is exploited in the present application to develop a new pharmaceutical composition for cancer treatment.

[0040] The present invention demonstrates that the growth factor FGF-2 (from the English fibroblast growth factor 2) inhibits the proliferation of Y1 cells, a murine cancer cell model induced by K-ras amplification. In recent years, it has been demonstrated that these cells, when stimulated by FGF-2, are highly dependent on stress control pathways, such as replication stress (RTS) and proteotoxic stress (ubiquitin proteasome system). Based on these observations, the inventors of the present application, contrary to the indications of the prior art, hypothesized that tumor cells would be hypersensitive to combinations of mitogens plus inhibitors of stress control pathways, while normal cells are tolerant to these combinations.

[0041] Therefore, the present application demonstrates that these combinations are promising therapeutic protocols for cancer treatment, as they trigger the death of tumor cells, sparing normal tissue.

[0042] Therefore, the main objective of this application is to provide pharmaceutical compositions containing a combination of mitogens and stress control pathway inhibitors for the treatment of cancer, preferably triple-negative breast tumors (TNBC). In vivo analysis of the composition was demonstrated in a model of MDA-MB-231 tumors, for which there is no available therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The structure and operation of the present invention, together with additional advantages thereof, may be better understood by reference to the accompanying drawings and the following descriptions:

[0044] Figure 1 shows mitogenic stimulation via FGF-2 or by inhibition of PP2A by LB-100, elevating pERK1 / 2 increases intracellular stress, triggering the need for activation of stress control pathways, making the tumor cell more sensitive to treatments to inhibit these stress control pathways, such as the proteasome pathway, using Bortezomib.

[0045] Figure 2 is a proof of concept of the hypothesis with the B61 cell line. A and B). Representative figures of the viability assay stained with Crystal Violet demonstrating the decrease in cell viability as the concentration of bortezomib and FGF-2 increases; A) Tumor cell line B61; B) Normal cell line Balb / 3T3 clone A31; C and D) Dose-response matrix demonstrating the synergistic inhibition of different concentrations of FGF-2 and bortezomib and the ZIP Score, n = 3; C) Tumor cell line B61; D) Normal cell line Balb / 3T3 clone A31; E and F) Representative figure of the protein expression kinetics by Western blot for pERK, total ERK, and BIP, treated with FGF-2 (10 ng / ml), at the top, and graph of the expression ratio of pERK and total ERK, at the bottom.

[0046] Figure 3 shows an assay demonstrating the sensitivity of the B61 tumor cell line to FGF-2 and combined treatment: A and B) Growth curve of the murine cell lines B61 (A) and Balb / 3T3 clone A31 (B) for treatments with: Saline (control), FGF-2 (10 ng / ml), with Bortezomib (5 nM) and joint treatment with FGF-2 plus Bortezomib; C and D) Representative figure of the clonogenic assay of the murine cell lines B61 (C) and Balb / 3T3 clone A31 (D) treated with: Saline (control), FGF-2 (10 ng / ml), Bortezomib (3 nM) and joint treatment with FGF-2 plus Bortezomib. All graphs were made using Prism 9.4 (GraphPad Software), n = 3, bar = mean error. * p < 0.0001.

[0047] Figure 4 shows an in vivo assay for B61 cell line. A) Treatment scheme for B61 tumor cell line. 1 x 10 6B61 tumor cell line cells were inoculated into the right back. After inoculation, the males were divided into 4 groups: Control (50 ml of PBS); FGF-2 (200 ng / animal); Bortezomib (1 mg / kg / animal); Combined treatment (FGF-2 + Bortezomib). B) Growth curve graph of the mean volume in cubic millimeters of B61 tumors (day 0). C) Graph showing the percentage of weight gained or lost after the start of the experiment in mice weighed before treatments. The first weighing before inoculation of B61 cells is considered 100%. D) Representative photo of the tumors formed in male Balb / c mice. E) Graph showing the mass in milligrams of the weighed tumors after extraction. All graphs were created using Prism 9.4 (GraphPad Software), and we used the same program for statistical analyses; the means represent the sum of 2 distinct groups. We used the Two-way ANOVA test and the Tukey multiple comparison test as post-test.**** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05, n = 13 (n = 5 in group 1 and n = 8 in group 2). Bars = mean error. .

[0048] Figure 5 shows evidence of drug diffusion and its effect. Mice were inoculated on both flanks with 1 x 10 6cells of the B61 tumor line each, and the treatments were injected only next to the tumor on the right flank. After inoculation, the males were divided into 4 groups: Control (50 ml of PBS); FGF-2 (200 ng / animal); Bortezomib (1 mg / kg / animal); Combined treatment (FGF-2 + Bortezomib). A) Growth curve graph of the mean volume in cubic millimeters of the B61 tumors (day 0). B) Graph showing the mass in milligrams of the tumors weighed after extraction. C) Graph showing the percentage of weight gained or lost after the start of the experiment in the mice weighed before treatments. The first weighing before inoculation of the B61 cells is considered 100%. All graphs were made using Prism 9.4 (GraphPad Software) and we used the same program for statistical analysis. We used the Two-way ANOVA test and the Tukey multiple comparison test as post-test. NS = not significant, n = 9. Bars = standard deviation.

[0049] Figure 6 shows in vitro assays of the 4T1 cell line. A) Viability assay stained with Crystal Violet to verify the synergism of the treatment of LB-100 plus Bortezomib at different concentrations, upper part, representation of a plate, lower figure, table showing the cell density in relation to the control; B) Graph of the Viability Assay with XTT using one dose of LB-100 (10 µM) and 3 doses of Bortezomib (2.5 nM, 5 nM and 10 nM); C) Kinetics of pERK, total ERK and BIP protein by Western Blotting with doses of FGF-2 (20 ng / ml) and EGF (200 ng / ml); D) Clonogenic assay of the 4T1 lineage treated with LB-100, Bortezomib and combined treatment, upper part of the figure, representative photograph of the plates showing the visible colonies marked with Crystal Violet, lower part of the figure, Graphs of the average colony size, Percentage of the sum of colonies that fill the plate and Total number of colonies per plate and treatment.The 3 graphs are relative to the mean count of the control (considered 1); E) Graph showing the growth curve of the 4T1 cell line treated with LB-100, Bortezomib and combined treatment; F) Graph showing the growth curve of the 4T1 cell line treated with FGF-2, Bortezomib and combined treatment. All graphs were made using Prisma 9.4 (GraphPad Software) and we used the same program for statistical analyses, the means represent the sum of 3 distinct groups. We used the Two-way ANOVA test and the Tukey multiple comparison test as post-test. **** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05. Bars = standard deviation. .

[0050] Figure 7 shows in vivo assays of the 4T1 cell line. A) Treatment scheme for the triple-negative murine breast tumor cell line 4T1. 1 x 10 5 4T1 lineage cells in the breast Right abdominal area of ​​Balb / c females. After inoculation, the females were divided into 4 groups: Control (50 ml of PBS); LB-100 (1 mg / kg / animal); Bortezomib (1 mg / kg / animal); Combined treatment (LB-100 + Bortezomib). B) Growth curve graph of the mean volume, in cubic millimeters, of 4T1 tumors (day 0). C) Graph showing the percentage of weight gained or lost after the start of the experiment in female mice weighed before treatments. The first weighing before inoculation of 4T1 cells is considered 100%. D) Representative photo of the tumors formed in female Balb / c mice after euthanasia and mammary dissection. E) Graph showing the mass, in milligrams, of the tumors weighed after extraction. All graphs were made using Prism 9.4 (GraphPad Software) and we used the same program for statistical analyses, the means represent the sum of 2 distinct groups.We used Two-way ANOVA and Tukey's multiple comparison test as post-test. **** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05, n = 8 Bars = standard deviation. .

[0051] Figure 8 shows representative photographs of histological sections of 4T1 tumors stained with hematoxylin and eosin: A) Control; B) LB-100; C) Bortezomib; D) LB-100 + Bortezomib. Black arrows pointing to the necrotic region, red arrows pointing to the capsule. 10x objective magnification.

[0052] Figure 9 shows the pulmonary metastasis and pneumonitis caused by the 4T1 line. A) representative figure of the only female in the control group that presented pulmonary metastasis; B) graph representing the presence (to different degrees) or absence of signs of inflammation in the pulmonary alveoli.

[0053] Figure 10 shows the quantification of distant sites of metastases. A) p100 plates of 4T1 colonies after extraction and digestion of the lung and lymph node after euthanasia. B) Representative figure of the colonies formed from 4T1 cells present in the lung or inguinal lymph node. C) Graph of mean colony counts in the lung and inguinal lymph node in relation to the number of positive plaques within and across treatments. Green border, negative for the presence of micrometastasis; Red border, positive for the presence of micrometastasis; Circle with a diagonal line and yellow borders, plaques that were discarded due to contamination.

[0054] Figure 11 shows in vitro assays of the MDA-MB-231 cell line. A and B) Viability assay stained with Crystal Violet to verify the synergism of treatments with FGF-2 plus Bortezomib or with LB-100 plus Bortezomib at different concentrations, upper part, representation of a plate, lower figure, table showing the cell density in relation to the control; C and D) Graphical representations of the synergistic activity of the different concentrations of FGF-2 and Bortezomib or LB-100 and Bortezomib for tumor cell line MDA-MB-231, respectively, n = 3; E and F) Dose-response curve graph showing the IC50 of Bortezomib; G and H) Dose-response curve graph showing the IC50 of FGF-2 and LB-100, respectively.

[0055] Figure 12 shows a representative photo of the ERK activation kinetics after FGF-2 stimulation (10 ng / ml) for the MCF-7 and MDA-MB-231 cell lines and stimulation with 17β-estradiol (E-21 µM).

[0056] Figure 13 shows an in vitro survival assay and growth curve of MDA-MB-231. A and B) XTT survival assay of the triple-negative breast tumor cell line MDA-MB-231, treated with FGF-2 alone or combined with bortezomib and treated with LB-100 alone or combined with bortezomib, respectively; C and D) Growth curve of the triple-negative breast tumor cell line MDA-MB-231, treated with FGF-2 alone or combined with bortezomib and treated with LB-100 alone or combined with Bortezomib, respectively. All graphs were made using Prism 9.4 (GraphPad Software) and we used the same program for statistical analyses, the means represent the sum of 2 distinct groups. We used the Two-way ANOVA test and the Tukey multiple comparison test as post-test. **** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05. Bars = standard deviation.

[0057] Figure 14 shows an in vitro demonstration of the non-toxicity of our treatment in normal breast cell lines. A and B) XTT viability assay of normal breast cell lines treated with FGF-2 (2 ng / ml) individually or combined with Bortezomib (5, 10, and 20 nM) (A) MCF10A and (B) MCF-12A. All graphs were made using Prism 9.4 (GraphPad Software), and we used the same program for statistical analyses. The means represent the sum of two distinct groups. We used the Two-way ANOVA test and the Tukey multiple comparison test as post-test. Bars = standard deviation.

[0058] Figure 15 shows a schematic of the protocol for induction and treatment of the MDA-MB-231 strain. Female Balb / c-Nude mice were inoculated with 3x10 6cells of the breast tumor line MDA-MB-231 in the right abdominal breast.: A) After inoculation, the females were divided into 4 groups: Control (50ml of PBS); FGF-2 (200ng / animal); Bortezomib (0.3mg / kg / animal) the treatment followed as per the scheme; B) After inoculation, the females were divided into 4 groups: Control (50ml of PBS); LB-100 (1mg / kg / animal); Bortezomib (0.3mg / kg / animal); Combined treatment (LB-100 + Bortezomib) the treatment followed as per the scheme.

[0059] Figure 16 shows the in vivo data of tumors formed by the MDA-MB-231 cell line. A and B) Growth curve graphs of the mean volume, in cubic millimeters, of the tumors formed measured every 3 days from day 2 after inoculation of the MDA-MB-231 cell line; C and B) Representative figures of tumors after breast extraction and dissection using a magnifying glass; E and F) Graphs showing the mass, in milligrams, of the tumors formed after extraction and dissection using a magnifying glass. G and H). Graphs showing the percentage of weight gained or lost after the start of the experiment in female mice weighed before treatments. The first weighing before inoculation of MDA-MB-231 cells is considered 100%. The data on the left side of Figure 11 (A, C, E, G) are from the groups treated with FGF-2, Bortezomib, and FGF-2 + Bortezomib; the data on the right side of Figure 11 (B, D, F, and G) are from the groups treated with LB-100, Bortezomib, and LB-100 + Bortezomib. All graphs were created using Prism 9.4 (GraphPad Software), and we used the same program for statistical analysis. The means represent the sum of two distinct groups. We used the two-way ANOVA test and the Tukey multiple comparison test as post-test.**** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05, n = 13. For the experiments using. Bars = mean error. .

[0060] Figure 17 shows representative photographs of histological sections of MDA-MB-231 tumors stained with hematoxylin and eosin: A) Control; B) FGF-2; C) Bortezomib; D) FGF-2 + Bortezomib. 10x objective magnification.

[0061] Figure 18 shows a correlation graph between apoptotic cells and cell proliferation in relation to treatments. Graph generated in RStudio using Pearson's correlation method.

[0062] Figure 19 shows confirmation of the low toxicity of the combined treatment: A and B) Graphs showing the percentage of weight gained or lost after the start of the experiment in mice weighed before treatments. The first day the animals were treated (day 0) is considered 100%, (A) Animals treated every 3 days until day 12 and sacrificed on day 13, (B) Animals treated every 3 days, 4 times, after a 12-day rest and new treatment every 3 days, 3 times, the last treatment was on day 33 and they were sacrificed on day 35. C – F) Graphs in mg / dL of serum quantification for markers of liver (ALT and AST) and kidney (urea and creatinine) toxicity, (C) ALT, (D) AST, (E) Urea and (F) Creatinine. All graphs were made using Prism 9.4 (GraphPad Software) and we used the same program for statistical analyses, the means represent the sum of 1 group composed of 5 mice per group. We used the Two-way ANOVA test and the Tukey multiple comparison test as a post-test. ** p < 0.01 and * p < 0.05. Bars = standard deviation.

[0063] Figure 20 is a representative figure of the action of Bortezomib on the proteasome demonstrating that inhibition of the 26S proteasome leads to increased JNK, stabilization of p53 and decreased NF-κβ which increase apoptosis, in addition, the stabilization of p21 and p27 decreases proliferation. The stabilization of Caveolin-1 decreases cell migration and ending the decrease in NF-κβ decreases proliferation and angiogenesis, figure adapted and translated (RUSSO; FRATTO; BAZAN; SCHIRÓ et al., 2007).

[0064] Figure 21 shows combined treatment with mitogenic stress inhibitors in human glioblastoma U251-MG cells. 4 x 10 4U251-MG cells were plated in 1.93 cm² wells (upper panel). After 24 h, they were treated in the presence or absence of a combination of the mitogen FGF-2 (Fig 21.1), PMA (Fig 21.2), LB-100 (Fig 21.3) or GATPT (Fig 21.4) and specific inhibitors of: proteasome (Bortezomib - A), Histone deacetylase (SAHA - B), ATR (VE-822 - C) and autophagy (Chloroquine - D) at the concentrations described in each figure and maintained in a humidified atmosphere at 37°C and 5% CO2, for 72 h. The cells were then washed in PBSA (1x), fixed, and stained in a solution of Crystal Violet (0.5g / L), 37% formaldehyde (2.7% v / v), and 100% methanol (1% v / v) at room temperature for 20 minutes. Afterwards, the plates were washed 3x in distilled water. The plates were photographed in UVITEC, and the area quantified The demarcation was made in the ImageJ-FIJI program (bottom panel). The combinations that led to synthetic lethality are marked with a red circle or black rectangle (n≥2).

[0065] Figure 22 shows that mitogens promote sustained MAPK-ERK1 / 2 overactivation and lethally sensitize human glioblastoma U251-MG cells to oxidative stress and autophagy inhibition. (AJ) Representative clonogenic assays with U251-MG cells. 2.0 × 10 2 cells / cm 2were plated in complete medium in the presence or absence of LB-100 (5µM - AC and J), PMA (5ng / ml - DF and J) or FGF-2 (30ng / ml - GJ), combined or not with Bortezomib (5nM), SAHA (2µM) or Chloroquine (15µM). cultured for 10 (A_I) or 15 days (J), and then fixed / stained. Culture media were renewed every 2 or 3 days. Data are presented as mean ± SD of triplicate values ​​and statistical significance was determined using one-way ANOVA followed by Tukey's test for multiple comparisons (*p<0.05, **p<0.001, ***p<0.0001). (K) Western blots comparing the pERK / ERK ratio in the U251-MG line. Lysates were prepared from cells grown in complete medium in the presence or absence of PMA (10 ng / ml), GATPT (200 nM), or LB-100 (10 µM) for 10 min, 4 h, and 24 h. HPRT was used as a loading control (n = 1).

[0066] Figure 23 shows combined treatment with mitogenic stress inhibitors in human hepatocarcinoma HEPG2 cells. 4 x 10 4 HEPG2 cells were plated in 1.93 cm² wells (upper panel). After 24 h, they were treated in the presence or absence of a combination of the mitogens FGF-2 (23.1), PMA (23.2), LB-100 (23.3), GATPT (23.4), HGF (23.5) and EGF (23.6) and specific inhibitors of: proteasome (Bortezomib - A), Histone deacetylase (SAHA - B), ATR (VE-822 - C) and autophagy (Chloroquine - D) at the concentrations described in each figure and maintained in a humidified atmosphere at 37°C and 5% CO2, for 72 h. Then, the cells were washed in PBSA (1x), fixed and stained in Crystal Violet (0.5g / L), 37% formaldehyde (2.7% v / v) and 100% methanol (1% v / v) solution at room temperature for 20 minutes. Afterwards, the plates were washed 3x in distilled water. The plates were photographed in UVITEC and the quantification of the demarcated area was done in the ImageJ-FIJI program (lower panel). The combinations that led to synthetic lethality are marked with a red circle or black rectangle (n≥2).

[0067] Figure 24 shows that mitogens lethally sensitize human cancer HEPG2 cells to stress inhibition. Representative clonogenic assays with HEPG2 cells. 1.0 × 10 3Cells / cm2 were plated in complete medium in the presence or absence of FGF-2 (10ng / ml), HGF (100ng / ml) or PMA (10ng / ml), combined or not with (A-C) Bortezomib (2.5nM) or SAHA (0.5µM); (DF) VE-822 (200nM) or Chloroquine (20µM) and cultured for 15 days and then fixed / stained. Culture media were renewed every 2 or 3 days. Data are presented as mean ± sd. of values ​​from triplicates and statistical significance was determined using one-way ANOVA followed by Tukey's test for multiple comparisons.

[0068] Figure 25 shows combined treatment with mitogenic stress inhibitors in human colorectal cancer HCT15 cells. 4 x 10 4HCT15 cells were plated in 1.93 cm² wells (upper panel). After 24 h, they were treated in the presence or absence of a combination of the mitogen FGF-2 (25.1), PMA (25.2), LB-100 (25.3), GATPT (25.4) or EGF (5.5) and specific inhibitors of: proteasome (Bortezomib - A), Histone deacetylase (SAHA - B), ATR (VE-822 - C) and autophagy (Chloroquine - D) at the concentrations described in each figure and maintained in a humidified atmosphere at 37°C and 5% CO2, for 72 h. Then, the cells were washed in PBSA (1x), fixed and stained in Crystal Violet solution (0.5g / L), 37% formaldehyde (2.7% v / v) and 100% methanol (1% v / v) at room temperature. at room temperature for 20 minutes. Afterwards, the plates were washed 3 times in distilled water. The plates were photographed on the UVITEC, and the demarcated area was quantified using ImageJ-FIJI (bottom panel). Combinations that led to synthetic lethality are marked with a red circle or black rectangle (n≥2).

[0069] Figure 26 shows that mitogens lethally sensitize human colorectal cancer HCT15 cells to checkpoint or proteasome inhibition. Representative clonogenic assays with HEPG2 cells. 3.0 × 10 2 cells / cm 2 were plated in complete medium in the presence or absence of LB-100 (5µM - AC), PMA (2ng / ml – DI), FGF-2 (10-15ng / ml – JO), Okadaic Acid (10nM - PR) combined or not with (Bortezomib (10nM), SAHA (1.15µM), VE-822 (100nM) or Chloroquine (5µM) and cultured for 15 days and then fixed / stained. Culture media were renewed every 2 or 2.3 days. Data are presented as mean ± SD of values ​​from triplicates and statistical significance was determined using one-way ANOVA followed by Tukey's test for multiple comparisons. *p<0.05.

[0070] Figure 27 shows combined treatment with mitogenic stress inhibitors in human pancreatic cancer cells MiaPaCa2.4 × 104 MiaPaCa2 cells were plated in 1.93 cm² wells (upper panel). After 24 h, they were treated in the presence or absence of a combination of the mitogen FGF-2 (27.1), EGF (27.2), LB-100 (27.3) or GATPT (27.4) and specific inhibitors of: proteasome (Bortezomib - A), Histone deacetylase (SAHA - B), ATR (VE-822 - C) and autophagy (Chloroquine - D) at the concentrations described in each figure and maintained in a humidified atmosphere at 37°C and 5% CO2, for 72 h. Then, the cells were washed in PBSA (1x), fixed and stained in Crystal Violet solution (0.5g / L), 37% formaldehyde (2.7% v / v) and 100% methanol (1% v / v) at room temperature for 20 minutes. Afterwards, the plates were washed 3x in water distilled. The plates were photographed at UVITEC, and the demarcated area was quantified using ImageJ-FIJI (bottom panel). Combinations that led to synthetic lethality are marked with a red circle or black rectangle (n≥1).

[0071] Figure 28 shows combined treatment with mitogenic stress inhibitors in human pancreatic cancer PANC1 cells. 4 x 10 4PANC1 cells were plated in 1.93 cm² wells (upper panel). After 24 h, they were treated in the presence or absence of a combination of the mitogen FGF-2 (28.1), PMA (28.2), LB-100 (28.3) or GATPT (28.4) and specific inhibitors of: proteasome (Bortezomib - A), Histone deacetylase (SAHA - B), ATR (VE-822 - C) and autophagy (Chloroquine - D) at the concentrations described in each figure and maintained in a humidified atmosphere at 37°C and 5% CO2, for 72 h. The cells were then washed in PBSA (1x), fixed, and stained in Crystal Violet (0.5g / L), 37% formaldehyde (2.7% v / v), and 100% methanol (1% v / v) at room temperature for 20 minutes. The plates were then washed 3x in distilled water. The plates were photographed using the UVITEC, and the demarcated area was quantified using the ImageJ-FIJI program (bottom panel). Combinations that led to synthetic lethality are marked with a red circle or black rectangle (n≥2).

[0072] Figure 29 shows a Western blot comparing the pERK / ERK ratio in the Panc1 tumor line. Lysates were prepared from cells grown in complete medium in the presence or absence of FGF-2 (20ng / ml), PMA (10ng / ml), EGF (100nM) for 10 min, 4h and 24h. HPRT was used as a loading control (n = 1).

[0073] Figure 30 shows combined treatment with mitogenic stress inhibitors in human lung cancer A549 cells. 4 x 10 4 A549 cells were plated in 1.93 cm² wells (upper panel). After 24 h, they were treated with or without a combination of the mitogen FGF-2 (30.1), PMA (30.2) or GATPT (30.3) and specific inhibitors of: proteasome (Bortezomib - A), Histone deacetylase (SAHA - B), ATR (VE-822 - C) and autophagy (Chloroquine - D) at the concentrations described in each figure and maintained in a humidified atmosphere at 37°C and 5% CO2, for 72 hours. Then, the cells were washed in PBSA (1x), inserted and stained in Crystal Violet solution (0.5g / L), 37% formaldehyde (2.7% v / v) and 100% methanol (1% v / v) at room temperature for 20 minutes. Afterwards, the plates were washed 3x in distilled water. The plates were photographed in UVITEC and the quantification of the demarcated area was done in the ImageJ-FIJI program (lower panel). Matches that occurred to synthetic lethality are marked with a red circle or black rectangle (n≥2).

[0074] Figure 31 shows combined treatment with mitogenic stress inhibitors in murine melanoma B16 cells. 4 x 10 4B16 cells were plated in 1.93 cm² wells (upper panel). After 24 h, they were treated in the presence or absence of a combination of the mitogen EGF (31.1), PMA (31.2), LB-100 (31.3) or GATPT (31.4) and specific inhibitors of: proteasome (Bortezomib - A), Histone deacetylase (SAHA - B), ATR (VE-822 - C) and autophagy (Chloroquine - D) at the concentrations described in each figure and maintained in a humidified atmosphere at 37°C and 5% CO2, for 72 h. The cells were then washed in PBSA (1x), fixed, and stained in Crystal Violet (0.5g / L), 37% formaldehyde (2.7% v / v), and 100% methanol (1% v / v) at room temperature for 20 minutes. Afterwards, the plates were washed 3x in distilled water. The plates were photographed in UVITEC, and the demarcated area was quantified using ImageJ-FIJI (bottom panel). The combinations that led to synthetic lethality are marked with a red circle or black rectangle (n≥1).

[0075] Figure 32 shows a Western blot comparing the pERK / ERK ratio in the murine tumor cell line B16. Lysates were prepared from cells grown in complete medium in the presence or absence of PMA (10 ng / ml), GATPT (200 nM) and LB-100 (10 µM) for 10 min, 4 h and 24 h. HPRT was used as loading control (n = 1).

[0076] Figure 33 shows combined treatment with mitogenic stress inhibitors in murine triple-negative breast cancer 4T1 cells. 4 x 10 44T1 cells were plated in 1.93 cm² wells (upper panel). After 24 h, they were treated in the presence or absence of a combination of the mitogens FGF-2 (33.1), PMA (33.2), LB-100 (33.3), GATPT (33.4), EGF (33.5), BCI (33.6) and Okadaic Acid (33.7) and specific inhibitors of: proteasome (Bortezomib - A), Histone deacetylase (SAHA - B), ATR (VE-822 - C) and autophagy (Chloroquine - D) at the concentrations described in each figure and maintained in a humidified atmosphere at 37 °C and 5% CO2, for 72 h. The cells were then washed in PBSA (1x), fixed, and stained in Crystal Violet (0.5g / L), 37% formaldehyde (2.7% v / v), and 100% methanol (1% v / v) at room temperature for 20 minutes. Afterwards, the plates were washed 3x in distilled water. The plates were photographed in UVITEC, and the demarcated area was quantified using ImageJ-FIJI (bottom panel). The combinations that led to synthetic lethality are marked with a red circle or black rectangle (n≥1).

[0077] Figure 34 shows the combined treatment of mitogenic stress inhibitors in human TNBC MDA-MB-231 cells. 4 x 10 4 MDA-MB-231 cells were plated in 1.93 cm² wells (upper panel). After 24 h, they were treated in the presence or absence of a combination of the mitogen FGF-2 (Fig 34.1), PMA (Fig 34.2) or LB-100 (Fig 34.3) and specific inhibitors of: proteasome (Bortezomib - A), Histone deacetylase (SAHA - B), ATR (VE-822 - C) and autophagy (Chloroquine - D) at the concentrations described in each figure and maintained in a humidified atmosphere at 37°C and 5% CO2 for 72 h. Then, the cells were washed in PBSA (1x), fixed and stained in Crystal Violet solution (0.5 g / L), 37% formaldehyde (2.7% v / v) and 100% methanol (1% v / v) at room temperature. at room temperature for 20 minutes. Afterwards, the plates were washed 3 times in distilled water. The plates were photographed on the UVITEC, and the demarcated area was quantified using ImageJ-FIJI (bottom panel). Combinations that led to synthetic lethality are marked with a red circle or black rectangle (n≥2).

[0078] Figure 35 shows the combined treatment with mitogenic stress inhibitors in human TNPC cells. 2 x 104 DU-145 cells were plated in 1.93 cm² wells (upper panel). After 24 h, they were treated in the presence or absence of a combination of the mitogen LB-100 (Fig 35.1), BCI (Fig 35.2), FGF-2 (Fig 35.3), EGF (35.4) or PMA (Fig 35.5), and specific inhibitors of: proteasome (Bortezomib - A), Histone deacetylase (SAHA - B), ATR (VE-822 - C) and autophagy (Chloroquine - D) at the concentrations described in each figure and maintained in a humidified atmosphere at 37°C and 5% CO2, for 72 h. The cells were then washed in PBSA (1x), fixed, and stained in a solution of Crystal Violet (0.5g / L), 37% formaldehyde (2.7% v / v), and 100% methanol (1% v / v) at room temperature for 20 minutes. Afterwards, the plates were washed 3x in distilled water. The plates were photographed using UVITEC, and the demarcated area was quantified using ImageJ-FIJI (bottom panel).Combinations that led to synthetic lethality are marked with a red circle or black rectangle (n≥2).

[0079] Figure 36 shows that mitogens promote sustained overactivation of MAPK-ERK1 / 2 and lethally sensitize human triple-negative prostate cancer DU-145 cells to proteasome inhibition and cell cycle control. (A). 2.0 x 10 4 (A. XTT assays, n=3), 2.0 x 10 2 (B. Representative clonogenic assay, n=3) or 0.2 x 10 3 cells / cm 2 (C. Growth curve, n=1) were plated in complete medium in the presence or absence of FGF-2 (20 ng / ml), LB-100 (10 µM), EGF (100 ng / ml), PMA (10 ng / ml), BCI (0.5uM) combined or not with Bortezomib (5 nM), SAHA (2µM) according to each figure. Cells were cultured and then counted or fixed / stained. Culture media were renewed every 2 or 3 days. Data are presented as mean ± SD of triplicate values, and statistical significance was determined using one-way ANOVA followed by Tukey's test for multiple comparisons (*p<0.05).

[0080] Figure 37 shows combined treatment with mitogenic stress inhibitors in human TNPC PC3 cells. 2 x 10 4PC-3 cells were plated in 1.93 cm² wells (upper panel). After 24 h, they were treated in the presence or absence of a combination of the mitogen FGF-2 (Fig. 37.1) or LB-100 (Fig. 37.2), and specific inhibitors of: proteasome (Bortezomib - A), Histone deacetylase (SAHA - B), ATR (VE-822 - C) and autophagy (Chloroquine - D) at the concentrations described in each figure and maintained in a humidified atmosphere at 37°C and 5% CO2, for 72 hours. Then, the cells were washed in PBSA (1x), fixed and stained in Crystal Violet solution (0.5 g / L), 37% formaldehyde (2.7% v / v) and 100% methanol (1% v / v) at room temperature for 20 minutes. The plates were then washed 3 times in distilled water. The plates were photographed using UVITEC, and the demarcated area was quantified using ImageJ-FIJI (bottom panel). Combinations that led to synthetic lethality are marked with a red circle or black rectangle (n≥1). DETAILED DESCRIPTION OF THE INVENTION

[0081] Although the present invention may be susceptible to different embodiments, preferred embodiments are shown in the following detailed description with the understanding that the present embodiment should be considered as exemplifying the principles of the invention and it is not intended to limit the present invention to what has been illustrated and described herein.

[0082] The present invention relates to a pharmaceutical composition comprising mitogens and inhibitors of stress control pathways for the cancer treatment. More specifically, the present invention relates to the classical growth factor FGF-2, which inhibits the proliferation of malignant adrenocortical cells of the Y1 lineage. The combination of mitogenic stimulation with inhibitors of proteotoxic cellular stress control pathways leads to the death of triple-negative breast cancer cells without compromising the survival of normal breast cells. EXAMPLES Cell lines and mice

[0083] The murine tumor cell lines Balb / 3T3-B61 and 4T1, the malignant human mammary cell lines MDA-MB-231 and MCF-7; the non-tumorigenic murine fibroblast cell line Balb / 3T3 clone A31; and the non-tumorigenic human mammary cell lines MCF-10A and MCF-12A were used. All these cell lines belong to the Butantan Foundation cell bank and are maintained in liquid nitrogen. When in culture, they were maintained in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, USA) or Roswell Park Memorial Institute (RPMI-1640, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Vitrocell, Brazil) in an incubator at 37°C and 5% CO2. The mouse strains used were: Balb / c from the Central Bioterium of the Butantan Institute and Balb / c-NUDE from the Central Bioterium of the Faculty of Medicine of the University of São Paulo. Viability assays

[0084] For this trial, 2 x 10 4cells per well in 24-well plates (2 cm²). The following day, treatment was initiated in the presence or absence of different concentrations of mitogen and stress control pathway inhibitor specific for each lineage. After 72 h, the cells were washed with phosphate-buffered saline (PBS), then fixed and stained for 20–30 minutes using a solution of PBS plus 1% formaldehyde, 1% methanol, and 0.05% Crystal Violet. The plates were then washed in water and after drying, they were scanned using UVITEC equipment (UVITEC Cambridge, UK) and analyzed using ImageJ software (NCBI). XTT survival assay

[0085] The XTT survival assay is a colorimetric test that quantifies cell proliferation, viability, and cytotoxicity. This test is based on the reduction of a yellow tetrazolium salt, known as XTT, to an orange formazan dye by metabolically active cells. This assay is analogous to the MTT colorimetric assay. A distinguishing feature of the assays is that in the XTT assay, the formazan dye formed is soluble in aqueous solutions, allowing it to be quantified directly using a multiwell scanning spectrophotometer (ELISA).

[0086] For the XTT survival assay, 24-well plates (2 cm²) were used and 2 x 10 4cells / well P24 for all cell lines in triplicate. After 24h of plating, the cell lines were treated in the absence or presence of FGF-2, LB-100, Bortezomib, and combined treatment (FGF-2 + Bortezomib and LB-100 + Bortezomib). After treatment, the cells were maintained for 72h in the incubator at 37°C and 5% CO2.

[0087] After 72 h of treatment, the XTT solution was prepared using medium without phenol red and FCS (kept on ice). The XTT solution comes in two vials (A and B), which were thawed in a 37°C water bath and shaken until diluted. When preparing the final solution, for a final volume of 1 ml used for each well, the final XTT solution will contain 880 µL of the culture medium without phenol red and 120 µL of the XTT solution A+B (in a 1:50 ratio, one part A to 50 parts B).

[0088] The medium was removed from the multi-well plates and washed once with PBS. Then, 300 µl of the XTT solution was added and placed in an incubator at 37°C with 5% CO2. After 30 min, the control was observed to be bright orange for up to 1 hour of incubation. Immediately after, 200 µL of the supernatant was removed and transferred to 96-well multiwell plates. As a blank control, triplicate samples were prepared with XTT solution. Absorbance readings were then taken at 450 nm and 660 nm. The specific absorbance for XTT is measured at 450 nm. The 660 nm absorbance reading is used to eliminate background signal contributed by cell debris or other nonspecific absorbance. The control absorbance should be close to 1.

[0089] Using the reading data, specific absorbance was calculated using the formula: specific absorbance = [Abs 450 nm – Abs 450 nm (Blank)] – Abs 660 nm. Finally, the control mean was calculated, and this value was established as 100% viability. Each value was divided by the control mean, and the data were transferred to Prisma for analysis and graphing. Growth curves

[0090] To analyze cell proliferation, 8 x 10³ cells per well were seeded in 12-well plates (4 cm²). After 24 h, point 0 was collected and treatments were initiated as indicated in each experiment. Every 2 days, cells were collected from the samples and washed with PBS. At each point, the cells were detached from the plate with 150 µL of trypsin, and then 350 µL of culture medium was added. Due to the low cell count, points 0, 2, and 4 had the entire volume (500 ml) added to the counting solution (10 ml), while the other points had 100 ml added to the counting solution (10 ml). The concentrations of the cell suspensions were measured with the Z Coulter particle counter (Beckman, USA) and for points 0-4 they were normalized with the following calculation: (particles counted / 5) / 2, for the other points they were normalized with the following calculation: (particles counted / 2). In vivo assays

[0091] Tumor induction: The procedure that was followed to wash, trypsinize, resuspend, count, and inject the cells was as follows:

[0092] The cells were washed twice with PBS, and then trypsin was added to loosen them. The cells were resuspended in RPMI or DMEM medium with 10% FBS and centrifuged for 4 minutes at 1200 rpm at room temperature in a benchtop centrifuge. The medium was discarded, and the cells were resuspended in culture medium (10 mL).

[0093] The cells were counted and separated: 1 x 10 6 cells, 1 x 10 5 cells or 3 x 10 6cells for tumor lines B61, 4T1 and MDA-MB-231, respectively, in a 1.5 mL eppendorf tube. The cells were centrifuged again for 4 min at 1200 rpm and the medium was discarded. The cells were resuspended in 50 µL of RPMI or DMEM medium without FBS and transferred to a BD ultra-fine 31G (0.25 mm) insulin syringe. For the MDA-MB-231 cell line, a mixture of DMEM and matrigel medium (1:1) was used. Minimum injection volumes were used, and the cells were immediately injected into the animals, with the bevel tip facing down. For the B61 strain, the cells were inoculated into the back of male Balb / c mice; for the 4T1 strain, they were inoculated orthotopically into the right abdominal mammary gland of Balb / c females; and for the MDA-MB-231 strain, the inoculum was also in the abdominal mammary gland of Balb / c-NUDE females. The animals were randomly separated and treated as indicated in the protocols in Figures 5A, 8A, and 16A-B.For the drug diffusion assay, the B61 tumor line was inoculated on both flanks of the animals and treatment was performed only on the right side of the tumor (intratumoral). Quantification of metastases. .

[0094] 4T1 cells are resistant to 6-thioguanine and can be detected and quantified after organ explantation by plating their dissociated cells in 6-thioguanine-supplemented medium and counting the number of 6-TG-resistant clonogenic tumor cells. This procedure provides a sensitive assay for microdeposits of small metastatic cells, and allows quantitative monitoring of the development of spontaneous metastases at multiple distant sites.

[0095] The following protocol describes a procedure that was followed to harvest and prepare the organs of interest and detect 4T1 metastatic cells: (i) 1× Hank's balanced salt solution (HBSS); (ii) Culture medium: RPMI supplemented with 10% FBS, 1× antibiotic Ampicillin and Streptomycin, and 60 μM 6-thioguanine; (iii) For the lung, Collagenase D was used. (50 mg collagenase D (Sigma) was dissolved in 25 mL 1× HBSS (Vtotal 150 mg / 75 mL 1x HBSS).It was filtered, sterilized and 2.5 ml was aliquoted per 15 ml conical tube; (iv) Methanol; (v) 0.03% (w / v) methylene blue; (vi) 15 and 50 ml conical tubes; (vii) 6-well tissue culture plates; (viii) 1 ml tuberculin syringe and 27G needle; (ix) Dissection equipment: scissors, curved scissors and forceps; (x) Benchtop centrifuge; (xi) 6 and 10 cm tissue culture plates; (xii) Tissue culture incubator at 37 °C, 5% CO2; (xiii) Frosted-tipped glass slide or cell strainer; (ivx) 70 μm nylon strainers. .

[0096] Before animals were sacrificed, all tissue culture tubes and plates were labeled with the appropriate animal identification number. Example 1

[0097] As a proof of concept, derived from data from the Y1 cell line – a murine tumor cell line that has multiple copies of KRAS (COSTA; FORTI; MATOS; DERMARGOS et al., 2008; DIAS; FONSECA; ZEIDLER; ALBUQUERQUE et al., 2019) – the B61 cell line was used. This cell line was created from the Balb-3T3 cell line, derived from Balb / c mouse embryos. Through cotransfection, which inserted the HRASG12V gene and the geneticin resistance gene (G418), the B61 cell line acquired between 50 and 100 copies of HRASG12V (KOVARY; ARMELIN; ARMELIN, 1989). Therefore, Figure 2 presents the data obtained in vitro for the B61 and Balb / 3T3 clone A31 cell lines. Initially, a viability assay was used to define the lowest concentration of the mitogen combined with the proteasome inhibitor, which promotes death synergistically.

[0098] Figures 2A and 2B are representative 24-well plates demonstrating sensitivity as FGF-2 and bortezomib concentrations increase. However, as seen in the representative figure, the B61 cell line is more sensitive than the Balb / 3T3 cell line to the combinations used.

[0099] Figures 2C and 2D show a graphical representation, created using the SynergyFinder app, of the synergistic activity of the combinations for the B61 and Balb / 3T3 cell lines. Synergism and decreased cell viability were observed when the mitogen and the proteotoxic stress response pathway inhibitor were combined. This occurred at lower doses in the B61 tumor cell line compared to the normal Balb / 3T3 cell line. This comparison is more evident when we see the synergistic peak of the tumor cell line on the synergism map, shifted lower and to the left compared to the normal cell line. These data are corroborated by the ZIP score, which shows a higher value for the tumor cell line. Furthermore, it was observed that, at the concentrations used, FGF-2 did not present an IC for either cell line. 50 . However, for the Bortezomib, the B61 lineage showed an IC 50lower (6.78 nM) when compared with the Balb / 3T3 lineage (7.27 nM), therefore the normal Balb / 3T3 lineage only has synergism at concentrations that are toxic to the cell.

[0100] Finally, it was found that the tumor cell line (B61) has a pattern very similar to that of the non-tumor cell line (Balb / 3T3) for activation of the RAS-MEK-ERK pathway by FGF-2 (Figures 2E and 2F). However, only the tumor cell line was found to contain BIP, a marker of activation of the proteotoxic stress control pathway. BIP (Binding Immunoglobulin Protein), also known as GRP78 (Glucose-Regulated Protein 78), is a heat shock protein that plays a crucial role in the endoplasmic reticulum (ER) stress response. This response is activated under cellular stress conditions, such as the accumulation of misfolded proteins in the ER. The data indicate that only the tumor cell line is activated by FGF-2 when exposed to cellular stress.

[0101] After the crystal violet viability assay, a growth curve was generated for the two cell lines (B61 and Balb / 3T3), evaluating their sensitivity to different concentrations of FGF-2 alone or combined with bortezomib. Figure 3A shows that the B61 tumor cell line is sensitive to FGF-2 treatment alone, regardless of the dose used. In the normal Balb / 3T3 cell line, FGF-2 treatment does not affect cell growth rate when compared to the control (Figure 3B), even when higher concentrations of the mitogen were used. It is also noted that the dose used for bortezomib (5 nM) does not affect either cell line, but when used in conjunction with FGF-2, cell proliferation decreases. However, even with the combination, from day 4 onwards, the normal cell line begins a recovery process and has a growth rate above that of the tumor cell line.

[0102] These data are confirmed by clonogenic assays (Figures 3C-H). In these assays, it can be seen that bortezomib alone does not affect colony emergence, when compared to the control, in both strains. Treatment with FGF-2, however, showed smaller visible colonies and occupied a smaller area of ​​the plate in both treatments (Figures 4C, 4E, and 4G). However, in the normal strain, the decrease in relation to the control was smaller, demonstrating a lower sensitivity to the "anti-mitogenic effect" of FGF-2. The same pattern was observed for the combined treatment (Figures 3D, 3F, and 3H).

[0103] Given the in vitro results, the next step was to conduct in vivo trials to determine whether this new therapeutic approach could be effective in tumors with multiple copies of mutated Ras, such as the B61 tumor line. For this purpose, we used Balb / c mice to grow the tumors. The inoculation and treatment protocol are outlined in Figure 4A. After inoculation of the line into the right backs of the mice, it took an average of 7 days for solid, palpable tumors to appear. At this point, the animals were randomly divided into four groups: treated with saline (Control); treated with FGF-2; treated with bortezomib; and treated with the combination of FGF-2 and bortezomib. Treatments according to the regimens were administered at intervals of every 2 days.

[0104] As can be seen in Figure 4B, the animals in the combined group (FGF-2 + Bortezomib) showed a significant decrease in tumor volume when compared to the other groups and the control group. This is clearly visible in the figure of the removed and weighed tumors (Figures 4D and 4E, respectively). Another data extracted from this experiment is the percentage of weight gained or lost relative to the beginning of the treatments in the mice (Figure 4C). It can be noted that the concentration of Bortezomib used negatively affected the weight gain of the mice, even when treated in combination with FGF-2, but without statistical significance.

[0105] In experiments with the B61 tumor cell line, treatments were always administered directly into the tumors (intratumorally). This raised the question: would the treatment have the same effect if the drugs were administered at a distance from the tumor? To answer this question, the tumor was inoculated on both flanks of the mice, and the treatment was administered by injecting the drugs next to the right tumor. The experimental data, represented in Figure 6, show tumor volume, tumor weight, and animal weight under different treatment conditions.

[0106] The left tumor, which did not receive treatment, showed a progression in tumor volume over the days, similar to the right tumor under different treatments (Figure 5A). In Figure 5B, a scatterplot comparing tumor weights under different treatment conditions shows no difference between the left and right tumor weights. Furthermore, the weight loss due to the presence of both tumors and the treatments did not reach 20%, thus, euthanasia was not necessary before the end of the experiment (Figure 5C).

[0107] Therefore, it was concluded that the treatment of the present application diffuses normally throughout the body, allowing treatments to be administered by other routes, such as intraperitoneal. Response to the treatment of the present application in a triple-negative murine mammary cell line

[0108] Due to everything that triple-negative breast tumor represents when diagnosed (worse prognosis, low life expectancy, high recurrence, highly metastatic, without specific therapy), this type of aggressive tumor was chosen as a study model to serve as the basis for a preclinical protocol using the rationale proposed here.

[0109] To confirm that the use of a mitogen enhances and sensitizes a tumorigenic cell, making it more sensitive to inhibitors of stress control pathways, a triple-negative murine breast tumorigenic cell line, 4T1, was used. This cell line mimics triple-negative human breast tumors, with the advantage of using the Balb / c animal model, which is immunocompetent. Therefore, the inventors' initial work on this invention involved in vitro assays, as can be seen in Figure 6.

[0110] Data from 4T1 cells treated with the combination of FGF-2 plus bortezomib (Figure 7F) did not show synergism as observed with B61 cells. Therefore, a new approach was adopted: the use of a mitogen-like agent, LB-100, a specific inhibitor of the phosphatase PP2A. This phosphatase acts by dephosphorylating and consequently deactivating the MAP kinase pathway, particularly the RAS-MEK-ERK pathway. By keeping this pathway activated, the effect that occurs with FGF-2 was mimicked. Figure 6A shows how this cell line is resistant to the drugs used, requiring higher concentrations to reduce viability. This finding is confirmed by the XTT survival assay, which demonstrates the significance of the combined treatment (LB-100 + bortezomib) compared to individualized treatments based on the use of bortezomib at 2.5 nM.

[0111] Interestingly, although they do not produce a synergistic killing effect as expected, the growth factors FGF-2 and EGF alone increase the expression of pERK and BPI. This suggests that these mitogens may sensitize stress control pathways by maintaining the activated RAS-MEK-ERK pathway (Figure 6C), but this is not sufficient to lead to death of this cell line with bortezomib. This result is corroborated by the growth curve shown in Figure 6F. In Figure 6D, the clonogenic assay demonstrates the sensitivity of the 4T1 cell line to the combined treatment of LB-100 with bortezomib, which reinforces this. by the growth curve in Figure 6E.

[0112] The in vitro data motivated the performance of the in vivo tests, for which the protocol was followed as outlined in Figure 7A, where, 1 x 10 54T1 cells were inoculated per animal, which were subsequently randomly divided into 4 treatment groups: Control (50 μL PBS / animal), LB-100 (1 mg / kg / animal); Bortezomib (1 mg / kg / animal); and LB-100 + Bortezomib. The first treatment was administered 24 h after inoculation through subcutaneous (sc) injections in the inoculum region, and subsequent injections occurred every 72 h (ip). After 17 days, the animals were euthanized, and their tumors were removed for weighing and histological analysis.

[0113] The volumes of growing tumors were estimated periodically at the time of treatment for each animal (Figure 7B). At the end of the experiment, the tumors were dissected, and their volumes and weights were accurately measured (Figure 7E). Regarding animal weight, there is a similarity with the data from B61, where treatment alone with the proteasome inhibitor bortezomib led to weight loss in female mice, independent of the action of LB-100, which was not significant (Figure 7C), demonstrating that the concentrations used in the animals do not lead to weight loss. The results obtained show that the mean volumes and weights of tumors isolated from control animals and those treated with LB-100 and bortezomib, respectively, are similar. On the other hand, the mean tumors from animals treated with the LB-100 + bortezomib combination were significantly lower than the means for the other conditions, both in volume and weight (Figures 7B and 7E).

[0114] Figure 7D shows a panel of dissected tumors obtained after euthanasia. These tumors, viewed macroscopically, are solid and opaque white in color. In some, a reddish hue can be seen in the central portion of the tumor, suggesting the presence of dots. of more superficial necrosis, whereas in larger tumors the presence of a greater number of lobes can be seen, demonstrating significant growth.

[0115] Figure 8 shows a panel representing histological sections of the tumors, stained with hematoxylin and eosin (HE), for the 4T1 cell line. Note that, in all treatments, the tumors present a capsule (indicated by the red arrow) and vascularization, in addition to exhibiting an area of ​​necrosis in the central region (indicated by the black arrow). These observations led to a more detailed examination of the data obtained from the animals, specifically the histology of the tumors and harvested lungs.

[0116] Tumors from the different treatment groups were compared on histological slides to identify differences in tumors under different treatment conditions, as seen in Figure 8. Next, slides of the lungs, which are the preferred site of metastasis for triple-negative breast tumors, were analyzed. Visible metastasis was identified in only one of the control animals (Figure 9A), but the lungs showed signs of inflammation in the alveoli. It was then decided to quantify the presence and amount of pneumonitis in the lungs, which were classified as absent, low, moderate, or high. These data were obtained with the assistance of a pathologist, as can be seen in Figure 9B. Surprisingly, a change in the pneumonitis profile was observed in the treatments compared to the control, which showed pneumonitis present in most of the slides studied, mainly low or high grade.

[0117] With LB-100 treatment, there is still a predominance of slides positive for pneumonitis, with prevalence ranging from high to moderate, but an increase in the number of females with no pneumonitis was observed. With bortezomib treatment, all females presented pneumonitis, with a prevalence of moderate severity. In the combined treatment, it was found that half of the slides studied did not present pneumonitis, indicating that these animals have a better prognosis for this condition.

[0118] To further investigate whether the combined treatment could also inhibit metastasis formation, a quantification experiment was performed for distant micrometastasis sites, as described in the materials and methods. This experiment consisted of, after euthanasia and organ removal, the inguinal lymph node (the satellite lymph node closest to the breast where the 4T1 cell line was inoculated) and the lung. Mechanical and chemical digestion was performed to separate the solid tissues, and they were cultured in culture medium containing 6-thioguanine, a drug that inhibits cell proliferation. Since 4T1 is a drug-resistant cell line, we were able to maintain the plates under selective pressure with medium containing 6-thioguanine and count the colonies resulting from the proliferation of resistant cells, allowing us to estimate the possibility of micrometastasis.

[0119] As can be seen in Figure 10A, some plaques did not progress due to contamination. Those that remained uncontaminated were marked as positive or negative for 4T1 and digitized using UVITEC. The colonies formed, as can be seen in greater detail in Figure 10B, were counted manually. What can be seen (Figure 10C) is that, among the organs, there were more positive plaques with micrometastases in the satellite lymph nodes of the controls. In the individualized treatments, the lung presented more positive plaques. When we quantified the micrometastases in each plaque, in the lung, both the control group and the individualized treatments presented a higher number of colonies on average for each group than in the lymph node.

[0120] However, the combined treatment appeared to be an inhibitor of metastasis, as only 1 of the 6 lymph nodes was positive, as For the lung, of the 5 lung plaques, only 1 was positive. Table 1 contains a detailed and individualized summary for each female used in the experiment, showing that the females in the control group that presented larger tumors had more micrometastasis, and especially the female in the G21P control group was the one that presented the only metastasis visualized in the lung (Figure 9A). Further details can be seen in Table 1. Table 1

[0121] Therefore, the results obtained with the 4T1 cell line are particularly significant in the context of triple-negative breast cancer. (TNBC). The demonstration that combined treatment with LB-100, a phosphatase inhibitor, and bortezomib, a proteasome inhibitor, can reduce the risk of lung and lymph node metastasis is a promising advance. This suggests that this treatment combination may be effective in blocking the progression of TNBC, thus addressing a significant therapeutic challenge.

[0122] After verifying that the hypothesis of the present invention is true for a murine triple-negative breast tumor cell line, it was decided to transpose the hypothesis to human triple-negative breast cell lines, using the MDA-MB-231 cell line. The data obtained in the viability assays stained with crystal violet show that MDA-MB-231 is synergistically sensitive to both the mitogen FGF-2 and LB-100 when combined with bortezomib (Figures 11A to 11D). Figures 11E and 11F show the IC 50 of Bortezomib, which was 13.12 nM, and Figures 11F and 11G show the IC 50 estimated for FGF-2 and LB-100, respectively. Note that LB-100 behaves similarly to FGF-2 at the doses used.

[0123] Accordingly, to verify the ability of FGF-2 to stimulate ERK activity and BIP activation, a Western blot was performed using an estrogen-positive breast tumor cell line, MCF-7, as a control (Figure 13). As can be seen, when both cell lines are stimulated with FGF-2, ERK activation occurs. Furthermore, in both cases, there is an increase in the expression of IRE1α, a protein present in the endoplasmic reticulum and activated in the presence of malformed proteins, demonstrating an increase in proteotoxic stress. However, only MDA-MB-231 presents high BIP expression, indicating the greater sensitivity of the triple-negative breast tumor cell line to proteotoxic stress when compared to the tumor cell line sensitive to estrogen treatment. It can also be seen that stimulation with 17-β estradiol (E2) in a tumor cell line estrogen-sensitive breast (MCF-7), activates only IRE1α exactly like FGF-2, but does not show BIP expression (Figure 12).

[0124] As can be seen in Figure 13, in the XTT survival assay, the combined treatment with LB-100 and Bortezomib was more efficient when compared with the data from the combined treatment with FGF-2 and Bortezomib (Figures 13A and 13B). These data are corroborated by the growth curves, which demonstrated greater sensitivity with LB-100 and the combined treatment of LB-100 and Bortezomib at the concentrations used for the MDA-MB-231 cell line (Figures 13C and 13D).

[0125] Based on the data with the tumor cell line (MDA-MB-231), which demonstrated the effect of the combined treatment of the present invention on decreasing cell viability and growth, in vitro confirmation was made that the treatments are not toxic to normal breast cells. For this, two cell lines were used: MCF-10A and MCF-12A (Figure 14). As can be seen, the dose of FGF-2 and the different concentrations of Bortezomib do not decrease the viability of these cells, allowing the use of these concentrations without harming normal breast cells.

[0126] Therefore, tumor growth was tested from 3.0 x 10 6Cells of the human tumor line MDA-MB-231 were inoculated into the right mammary glands of female Balb / c-nude mice. The protocol outlined in Figures 15A and 15B was followed. The main differences between the two protocols, besides the duration, were that prolonged use of bortezomib and LB-100 initially resulted in weight loss. This loss can be recovered by applying the treatment in cycles, with rest intervals (Figure 15B), which avoids compromising the experiment, as will be seen below.

[0127] Although the doses chosen for the XTT and growth assays of FGF-2 and Bortezomib did not show differences significant changes in the in vitro experiments, in vivo groups were performed to compare its effect with LB-100 (Figure 16), since, as seen in the viability assays and Western blot (Figures 11 and 12, respectively), the combination of FGF-2 + Bortezomib has an effect on generating proteotoxic stress. During the measurement of tumor volume, both the combined treatment with FGF-2 and Bortezomib and the combined treatment with LB-100 and Bortezomib inhibited tumor growth (Figures 16A and 16B, respectively). These data are congruent when observing the representative photos of the dissected breast tumors (Figures 16C and 16D).

[0128] It is important to note that, in the case of the combined treatment with LB-100, only 4 tumors were identified in 9 dissected breasts under the magnifying glass in one of the experimental groups. Another factor that differentiates the combined treatments concerns the weight of the dissected tumors. While a significant difference was observed between the combined treatment of LB-100 + Bortezomib in relation to the control and individual treatments (Figure 16F), the same was not observed for the combined treatment with FGF-2 + Bortezomib (Figure 16E). Regarding the weight of the animals, as occurred with the tumors formed by the B61 and 4T1 lines, there was no significant decrease in weight, nor did any animal present a 20% weight loss that would have led to euthanasia before the end of the experiment (Figures 16G and 16H).

[0129] As seen in 4T1, the tumors visualized macroscopically are solid and opaque whitish in color. However, unlike the murine lineage, during tumor extraction from the human MDA-MB-231 lineage, independent tumor masses of varying sizes were observed in some females, regardless of the treatment (Figure 16C-D). In Figure 17, it can be seen that the treatment with the combination of FGF-2 + Bortezomib (Figure 17D) differs from the others in that it presents mammary adipose tissue distributed throughout the tumor mass, being more present at the edges, but with irradiation to the tumor core. The regions of necrosis are apparently smaller than in 4T1 tumors and more diffuse, but still with more areas in the tumor core. This difference may be due to the greater volume and mass of 4T1 tumors compared to the volume and mass of MDA-MB-231.

[0130] Analysis of tumor slides allowed the collection of data on the total number of cells in mitosis and apoptotic cells in 5 random fields of each 2 non-serial sections per tumor. This count showed, using Pearson's correlation, that the combined treatment has a negative correlation between proliferation and death, while in the control and other treatments the correlation is close to zero. These results suggest that with FGF-2 + Bortezomib treatment, the more apoptotic cells were seen, the fewer cells in mitosis were seen (Figure 18). These experiments, performed with the triple-negative cell lines 4T1 and MDA-MB-231, suggest that mitogenic stimulation is effective in sensitizing highly aggressive cancer cells to inhibitors of stress control pathways in vivo. Demonstration of the absence of toxicity of the combined treatments.

[0131] In the toxicity assay, the enzyme pairs AST (Aspartate Aminotransferase) and ALT (Alanine Aminotransferase) and Urea and Creatinine were analyzed to evaluate hepatic and renal toxicity, respectively, in mice treated with saline (control), FGF-2, LB-100, Bortezomib and the combined treatments FGF-2 + Bortezomib and LB-100 + Bortezomib (Figure 19) at the concentrations used for treatment.

[0132] AST and ALT are enzymes found primarily in the liver. When liver cells are damaged, they release these enzymes into the bloodstream. Analyzing these enzymes is crucial for assessing liver function, as elevations in them indicate potential or existing liver damage. The graphs in Figures 19C and 19D show that there was a significant elevation of AST and ALT enzymes in the treated groups compared to the control group, suggesting that treatments alone or in combination do not induce significant liver toxicity.

[0133] Creatinine and urea are substances produced by the normal metabolism of muscle tissue and proteins, respectively. Both are removed from the blood through the kidneys, and their levels can indicate kidney function. Levels of these substances remained within the normal range in all treatment groups, indicating healthy kidney function (Figures 19E and 19F).

[0134] The maintenance of animal weight above 80% from the beginning of treatment corroborates the observation that the compounds are well tolerated, indicating that the treatments do not induce significant hepatic or renal toxicity (Figures 19A and 19B). In summary, the correlation between liver and renal enzymes and animal weight indicates a favorable safety profile for FGF-2, LB-100, and bortezomib when administered alone or in combination. These results suggest that these agents can be further explored for potential therapeutic applications without inducing significant hepatic or renal toxicities.

[0135] Previous and more recent results from the present invention have shown that FGF-2, a known mitogen, inhibits proliferation and increases replicative and proteotoxic stress in human tumor models of the Ewing's sarcoma family and in murine adrenal cancer cells (Y1 cells) dependent on amplified wtKras. Furthermore, FGF-2 can synergistically increase tumor cell death, sensitizing them to the action of genotoxic (VE-821 / 2) or proteotoxic (Bortezomib) inhibitors of the stress response pathway. This occurs depending on the RAS, RAF, and MEK signaling pathways.

[0136] To prove the hypothesis derived from the data from the Y1 cell line, a proof of concept was performed with the B61 tumor cell line, derived from non-tumorigenic Balb-3T3 cell line by transfection with Hras V12GThe in vitro results demonstrated that the B61 tumor cell line is much more sensitive to combined treatment than the normal Balb / c-3T3 cell line (Figures 2-4). This finding motivated us to test this proof of concept with in vivo experiments. The results demonstrated (Figure 4) that the combination of FGF-2 + Bortezomib virtually abolished tumor development, while the untreated control group and the groups that received the drugs separately showed robust tumor growth. The decreased tumor growth with the combined treatment corroborated the hypothesis that mitogenic activation, via the FGF-2 / FGFR / RAS / RAF / MEK / ERK pathway, leads to increased proteotoxic stress, making the cell more dependent on the ubiquitin-proteasome pathway, indicated by the induced expression of BIP in the B61, 4T1, and MDA-MB-231 cell lines when treated with FGF-2 (Figures 2E, 6C, and 12). This allows Bortezomib to work effectively.Furthermore, the results of this experiment are even more relevant when we show that the weight loss of the mice is not significant in the combined treatment of FGF-2 + Bortezomib (Figure 4C) and that the combined treatment spreads, not being necessary to apply the treatment next to the tumor (Figure 5).

[0137] Studies on the antitumor effects of FGF-2 indicate that a high concentration of activated oncoproteins, such as KRAS-GTP, is required, making cultures highly dependent on regulatory pathways of replicative stress (ATR), proteotoxic (proteasome pathway), and oxidative stress.

[0138] Once the hypothesis was validated in a model constructed by the present invention, the study was directed to triple-negative human breast tumor, for which there is no established effective therapy, using the MDA-MB-231 cell line as a model. The results demonstrated that the combination therapy of FGF-2 and Bortezomib causes a drastic blockade of the tumor growth compared to the control groups, whose tumors grew (Figures 16A and 16C). Even more relevant were the results obtained with the combination of LB-100 and bortezomib (Figures 16B and 16D). In addition to drastically inhibiting tumor growth compared to controls, it was observed that 5 of the 13 animals, in two groups with independent treatments, did not even develop tumors. It was also observed that there is a correlation between the decrease in proliferative cells and the increase in apoptotic cells in tumors treated with the FGF-2 + bortezomib combination compared to controls (Figure 18).

[0139] These data correlate with in vivo results with the LB-100 + Bortezomib combination in the murine TNBC cell line (4T1). This combination also decreased orthotopic tumor growth in the breast (Figures 6-8). Furthermore, this combination inhibited the development of murine triple-negative breast tumor metastases to distant organs in Balb / c mice (Figures 9 and 10 and Table 1), a highly relevant effect given the high mortality rate associated with metastasis in this tumor type.

[0140] Murine 4T1 breast tumor cells are similar to human MDA-MB-231 breast tumor cells and are widely used models for the study of TNBC and metastasis of these tumors (ARROYO-CRESPO; ARMIÑÁN; CHARBONNIER; DELADRIERE et al., 2019). The results suggest that increased mitogenic stimulation and the use of therapeutic inhibitors targeting stress control pathways are not only effective in sensitizing highly malignant cancer cells but also inhibiting the formation of lung and satellite lymph node metastases.

[0141] A potential concern with the proposed approach is that activation of mitogenic signaling could cause proliferation of normal or precancerous cells in patients. As can be seen from the lineage Balb / 3T3 clone A31 (Figures 2 and 3) and the normal breast cell lines MCF-10A and MCF-12A (Figure 14) did not decrease viability and survival in in vitro assays when exposed to the combined treatment. This is because normal cells have effective feedback mechanisms to limit mitogenic activity and uncontrolled proliferation (THOMA; NEURATH; WALDNER, 2021). Higher levels of oncogenic signaling and less effective feedbacks to control this signaling are hallmarks of cancer (HANAHAN, 2022; HANAHAN; MONJE, 2023; HANAHAN; WEINBERG, 2000; 2011). This notion suggests a therapeutic window to exploit this hyperactivation.

[0142] The good tolerance of LB-100 in mice and patients provides additional assurance that a therapeutic window will be found in clinical studies, as can be said for the use of FGF-2. Arguably, the most attractive aspect of the therapeutic approach described here is that the stress cancer cells must avoid to become resistant is hyperactivated oncogenic signaling itself. In preclinical models, LB-100 improved the efficacy of chemotherapeutics without worsening toxicity in experimental animals (HONG; HO; ZHANG; YANG et al., 2015).

[0143] It is also important to highlight that the use of mitogen-like drugs, such as LB-100, or bortezomib alone, are being studied in current clinical trials with the aim of developing new therapeutic approaches for tumors. Downregulation of DNA repair signaling has been proposed as the basis for LB-100-induced replication stress. Furthermore, protein phosphatase PP2A regulates several mitotic proteins, and LB-100 has been shown to induce mitotic catastrophe by dysregulating the activity of these proteins. LB-100 has shown promise in several studies by preventing MEK inactivation, resulting in the maintenance of mitogenic stimulation. This has been observed in studies involving pancreatic adenocarcinoma, fibrosarcoma, chondrosarcoma, thymus, atypical lung carcinoids, ovarian, testicular, breast, and prostate cancers. It is also worth noting that LB-100 reduced cell viability in in vitro and in vivo models of acute myeloid leukemia, inhibiting the G2 / M transition of the cell cycle and activating the caspase cascade.

[0144] On the other hand, the proteasome inhibitor bortezomib has been approved by the Food and Drug Administration (FDA) as a primary treatment for multiple myeloma, but is not yet widely recommended for solid tumors (TRAN; WU; SUNG; CHANGOU et al., 2020). However, upregulation of mitotic signaling alone is sufficient to trigger both replication stress and a myriad of mitotic defects such as proteotoxic stress.

[0145] The series of experiments reported here concluded that overstimulation of tumor proliferation, followed by adequate inhibition of stress control pathways, using lower drug concentrations than those used clinically, potentiates the death of triple-negative cancer cells without causing liver and kidney toxicity (Figure 19). These results are consistent with a therapeutic model in which further reinforcement of the mitogenic signaling underlying the oncogenic phenotype can force cancer cells to die without compromising the survival of normal cells. It is worth noting that the effective use of a natural or synthetic mitogen as a sensitizer for drugs that control intracellular stress response pathways, as discussed here, was proposed in a novel way by the present invention, based on results in different tumor models.Example 2 - New combinations of mitogens and different classes of inhibitors of stress control pathways in different aggressive tumor cell lines.

[0146] Another important approach used in cancer therapy is the disruption of homeostatic balance. To this end, inhibitors of cellular stress response pathways are being used, which play an important role in the survival of malignant cells. For example, it has been shown that replication, essential for tumorigenesis driven by RAS and EWS-FLI-13, promotes increased stress, and monotreatment with therapeutic inhibitors targeting specific stress causes tumor cytotoxicity. However, it leads to many undesirable side effects, even with advances in drug specificity and delivery mechanisms.

[0147] In this sense, even with the advancement of drugs and clinical protocols, the therapies currently available are insufficient, given the increase in the number of deaths from some types of aggressive tumors and the occurrence of metastases in distant organs (https: / / www.hopkinsmedicine.org / health / conditions-and-diseases / prostate-cancer / prostate-cancer-prognosis).

[0148] In this sense, studies that aim to better understand the biology of the tumor and the environment in which it is found, in line with the development of effective therapeutic procedures that provide fewer side effects, are of fundamental importance.

[0149] The results of the present invention demonstrated that FGF-2, a recognized oncogenic growth factor, paradoxically inhibited the proliferation of malignant murine Y1 adrenocortical cells by blocking the cell cycle at the S-G2 transition. Further studies demonstrated that FGF-2-stimulated Y1 cells were under severe proteotoxic stress and were highly dependent on ATR, suggesting a high incidence of single-stranded DNA damage and increased replication stress. These experimental observations lead to the conclusion that FGF-2-treated Y1 cells underwent increased mitogenic stimulation, which mobilized stress response pathways to ensure cell survival. This conclusion led to a far-reaching hypothesis: increased mitogenic stimulation could sensitize cancer cells to specific therapeutic inhibitors targeting stress to promote death in multiple malignant cell lines, independent of the genetic mutations that lead to malignant transformation.

[0150] This hypothesis was corroborated in cultures of mouse Y1 tumor cells and four human Ewing sarcoma cell lines treated with FGF-2 and bortezomib or VE-822, inhibitors of the proteasome (proteotoxic stress control pathway) and ATR (genotoxic stress control pathway), respectively. In these models, the combined treatment led to synergistic cell death.

[0151] To validate this hypothesis, efficient concentrations of mitogens or mitogen-like agents (Table 2) and inhibitors of stress control pathways (Table 3) were determined, some of which are widely used in clinical anticancer therapies and others are participating in ongoing clinical trials. Table 2 - Mitogens, signaling pathways in which they operate, and concentration ranges Mitogens Description Final concentration Okadaic Ac. PP1 and PP2A inhibitor 0-15 nM Table 3 - Inhibitors, stress response signaling pathways in which they operate, and concentration ranges Inhibitor Description Final concentration Bortezomib Proteasome inhibitor 0-20 nM (BORTEZOMIB) SAHA Histone deacetylase inhibitor 0-10 µM VE-822 ATR Inhibitor 0-1000 nM Chloroquine Autophagy Inhibitor 0-100 µM

[0152] The inventors of the present application were able to validate the rationale in a panel of cell lines that exhibit different mechanisms involved in the development of highly aggressive tumors that are commonly resistant to conventional cancer therapies (Table 4), aiming to generate translational developments. Table 4 - Cell lines with oncogenic drivers. Results

[0153] Below are the results of cell viability assays and / or clonogenic assays with the combinations of a mitogen and a stress control pathway inhibitor described above. Cultures left untreated or treated with each compound alone were used as controls. For some models, mitogenic pathway activation was analyzed through the pERK / ERK protein expression ratio. The data showed that the hypothesis was largely corroborated with different pairs of combinations leading to synergistic killing in in vitro assays. A. Glioblastoma Multiforme

[0154] Glioblastoma multiforme is the most malignant and aggressive tumor cerebral and is characterized by high invasiveness, resistance to therapy, and recurrence. Treatment consists of a combination of resection, when possible, radiotherapy, and chemotherapy. Even so, the average life expectancy of patients with this type of tumor is only 10 to 15 months. In this study, we used the classic glioblastoma U251-MG cell model. A summary of the positive results is shown in Table 5. Table 5 - Different combinations of mitogens and stress response pathway inhibitors killed malignant cells derived from a glioblastoma. Lineage Reduction* Tumor Mitogen Cellular Stress Inhibitor (%) BORTEZOMIB 77 U251-MG GATPT VE-822 60 CHLOROQUINE 18 BORTEZOMIB 24 U251-MG PMA CHLOROQUINE 66 VE-822 24 BORTEZOMIB 17 U251-MG FGF-2 CHLOROQUINE 18 * lower % reduction in viability of the combined treatment compared to controls. B. Hepatocellular carcinoma (HCC)

[0155] Hepatocellular carcinoma is the fifth most common type of cancer worldwide and accounts for 80% of primary liver neoplasms. HCC is associated with mutations, chronic infections caused by the hepatitis B or C virus, and cirrhosis. Treatment (resection, transplantation, targeted therapy, immunotherapy, chemotherapy, radiation therapy, ablation, and embolization) and prognosis vary according to the stage of the disease: between 2009 and 2015, the 5-year survival rate ranged from 33% for patients with operable tumors to 2% for patients with distant metastases (American Cancer Society, available at https: / / www.cancer.org / cancer / liver-cancer / detection-diagnosis-staging / survival-rates.html - accessed on 03 / 03 / 21).

[0156] HepG2 is considered a pure human liver carcinoma cell line because it exhibits no traces of viral infection and is frequently used as a model for HCC. Interestingly, and yet not fully understood, this cell line exhibits networks of canonical pathways regulated up and down, compared to normal hepatocytes, which govern tumor progression. However, it does not present genetic mutations or complications with viral infection. We highlight the transcription factors EIF1 and EIF4 (cell cycle regulators and tumor suppressors), TP53 (involved in the cellular response to different types of stress), and MYC (cell cycle progression, apoptosis, and cellular transformation) with upregulated expression. Among the downregulated genes, we can highlight those involved in cholestasis and hepatitis, glutathione depletion, and response to inflammation and immune attack. The complete viability results for the HEPG-2 cell line are shown in Figures 23 and 24.Table 6 summarizes the main synergistic killing results found in this cell line. Table 6. Different combinations of mitogens and stress response pathway inhibitors killed malignant cells derived from a hepatocellular carcinoma. Cell Line Mitogen Reduction Inhibitor* Tumor Cell Stress (%). 53 HEPG2 GATPT VE-822 29 HEPG2 LB-100 CHLOROQUINE 47 * lower % reduction in viability of the combined treatment compared to controls C. Colorectal carcinoma

[0157] Colorectal cancer (CRC) is the second leading cause of death worldwide for both genders combined, even with advances in early diagnosis, polyp screening, and treatment developments. Although screening tests are recommended from the age of 50, this silent disease affects people below this age range.

[0158] CRC is a heterogeneous disease with three partially overlapping phenotypes due to different forms of DNA instability: chromosomal instability pathway (CIN – 85%), with aneuploid cells and large-scale rearrangements; microsatellite instability pathway (MSI – 15%), with deficiency in the DNA repair system; and CpG island phenotype (CIMP), which presents aberrant DNA methylation. The HCT15 cell line, derived from colorectal adenocarcinoma, is widely used in CRC studies and has been classified with the following molecular status: MSI (+), CIMP (+), CIN (-).

[0159] It is noteworthy that virtually all mitogens used sensitized the colorectal cancer cell line to specific inhibitors of proteotoxic and replicative stress pathways. Table 7 summarizes the main cell viability results that demonstrated synergistic killing. The complete results for the HCT15 tumor cell line are shown in Figures 25 and 26. Table 7. Different combinations of mitogens and stress response pathway inhibitors killed malignant cells derived from a colorectal carcinoma. Lineage Reduction* Tumor Mitogen Cellular Stress Inhibitor (%) BORTEZOMIB 15 Carcinoma SAHA 16 HCT15 FGF-2 Colorectal VE-822 84 CHLOROQUINE 34 BORTEZOMIB 39 EGF SAHA 20 BORTEZOMIB 53 PMA CHLOROQUINE 35 BORTEZOMIB 31 SAHA 38 LB-100 VE-822 45 CHLOROQUINE 40 BORTEZOMIB 44 GATPT SAHA 50 VE-822 39 CHLOROQUINE 36 * lower % reduction in viability of the combined treatment compared to controls; & result that needs to be confirmed D. Pancreatic Cancer

[0160] The pancreas is an organ with dual functions: endocrine and exocrine. The most common type of pancreatic cancer is adenocarcinoma (90%), which most often begins in the pancreatic ducts of the exocrine gland. Because it is generally diagnosed late, the average survival rate is 5 years after diagnosis, depending on the stage of detection and the possibility of resection. Classical treatment involves surgery, radiotherapy, and chemotherapy. To study the effects of the proposed combined treatment, we chose two cell lines derived from pancreatic adenocarcinomas: Panc-1 and MiaPaCa-2.

[0161] Table 8 summarizes the main cell viability results that demonstrated synergistic killing when treated with the described combination. The complete results for the MiaPaCa2 and Panc1 pancreatic tumor cell lines are shown in Figures 27 and 28, respectively. Using several mitogens, we analyzed the MAPK pathway activation profile, verifying the kinetics of pERK activation in Panc-1 cells, which are shown in Figure 29. Table 8. Different combinations of mitogens and stress response pathway inhibitors killed malignant cells derived from pancreatic adenocarcinoma. Lineage Reduction* Tumor Mitogen Cellular Stress Inhibitor (%) 25 Pancreas MiaPaCa2 FGF-2 BORTEZOMIB 33 BORTEZOMIB 46 EGF BORTEZOMIB 46 VE-822 20 BORTEZOMIB 16 LB-100 VE-822 48 BORTEZOMIB 27 GATPT CHLOROQUINE 25 BORTEZOMIB 36 PANC-1 FGF-2 CHLOROQUINE 17 17AAG 26 35 BORTEZOMIB 47 GATPT SAHA 36 VE-822 19 BORTEZOMIB 45 SAHA 34 LB-100 VE-822 50 CHLOROQUINE 21 * lower % reduction in viability of the combined treatment compared to the controls. E. Lung Cancer

[0162] Even with advances in clinical lung cancer treatment over the past decade, including screening strategies, the use of immunotherapy-based approaches to treat locally advanced and metastatic disease, and targeted approaches to molecularly defined subgroups, lung cancer remains the most prevalent type of cancer worldwide and caused more deaths in 2017 than breast, prostate, colorectal, and brain cancers combined. For this part of the study, a classical A549 human lung carcinoma cell line model was used.

[0163] Table 9 presents a summary of the main cell viability results that showed synergistic killing when treated with the described combination. The complete viability results with the A549 lung tumor cell line are in Figure 30. Table 9 - Different combinations of mitogens and stress response pathway inhibitors killed malignant cells derived from human lung carcinoma. Reduction* Tumor Cell Line Mitogen Stress Inhibitor (%) BORTEZOMIB 26 Lung A549 FGF-2 SAHA 27 BORTEZOMIB 22 PMA SAHA 23 CHLOROQUINE 8 BORTEZOMIB 40 SAHA 38 GATPT VE-822 24 CHLOROQUINE 25 * lower % viability reduction of the combined treatment compared to the controls F. Melanoma Skin Cancer

[0164] Skin cancer is the most common type of cancer, with 3% being melanoma, the most aggressive. This skin cancer can appear anywhere on the body in the form of spots, moles, or warts resulting from a disorder in melanocytes and primarily affects white-skinned adults. In Brazil, the estimated number of new melanoma cases is 8,500 / year (2020), with 23.4% of deaths (2019) (available at https: / / www.inca.gov.br / tipos-de-cancer / cancer-de-pele-melanoma - accessed in April 2021). The main treatment is surgery; however, when metastases are present, palliative treatment is used to delay disease progression and increase survival.

[0165] Cellular models derived from murine melanomas, of which B16 is the best known, have been used since the mid-20th century to study the central biological characteristics of this tumor type. The B16 cell line has a primary metastatic profile and high antioxidant capacity.28 and presents variant sublineages with greater or lesser metastatic capacity.

[0166] Table 10 presents a summary of the main cell viability results that showed synergistic killing when treated with the proposed combination. The viability results with the murine tumor line B16 are shown in Figures 31(1-4). An activation profile of the MAPK pathway is also presented (Figure 32). Table 10 - Different combinations of mitogens and stress response pathway inhibitors killed malignant cells derived from rat melanoma. Line Reduction* Tumor Mitogen Cell Stress Inhibitor (%) Skin cancer BORTEZOMIB 27 B16 EGF murine melanoma VE-822 35 PMA BORTEZOMIB 10 BORTEZOMIB 26 LB-100 SAHA 21 BORTEZOMIB 36 GATPT VE-822 52 * lower % reduction in viability of the combined treatment compared to controls Triple Negative Breast Tumor (TNBC)

[0167] Figures 34 and 35 (4T1 and MDA-MB-231 cell lines, respectively) show viability results with other combinations of mitogens with stress response pathway inhibitors. Table 11 presents a summary of the main synergistic killing results for both cell lines. Table 11 - Different combinations of mitogens and stress response pathway inhibitors killed malignant cells derived from murine or human TNBC. Reduction* Tumor Cell line Mitogen Stress Inhibitor (%) BORTEZOMIB 22 4T1 LB-100 SAHA 27 BORTEZOMIB 50 SAHA 34 GATPT VE-822 30 CHLOROQUINE 41 BORTEZOMIB 21 TNBC AC. OCADAICO CHLOROQUINE 74 34 MDA-MB-231 FGF-2 BORTEZOMIB BORTEZOMIB 25 SAHA 18 LB-100 VE-822 33 CHLOROQUINE 21 BORTEZOMIB 34 SAHA 54 PMA VE-822 21 CHLOROQUINE 34 * lower % reduction in viability of the combined treatment compared to the controls. H. Triple Negative Prostate Tumor (TNPC)

[0168] In prostate cancer, a complex and heterogeneous disease, it is known that genetic events and many environmental factors are involved in the development or progression of cancer, but they still remain poorly defined.

[0169] Even with the development of new generations of drugs Despite antiandrogens, the problem of acquired resistance to hormonal therapy remains for the treatment of advanced prostate tumors. Two of the three main triple-negative prostate tumor cell models (DU-145 and PC-3), that is, unresponsive to hormonal therapy, were used in this viability trial. Table 12 summarizes the main synergistic killing results for both cell lines. Table 12. Different combinations of mitogens and stress response pathway inhibitors killed human TNPC-derived malignant cells. Reduction* Tumor Cell Line Mitogen Stress Inhibitor (%) DU-145 LB-100 BORTEZOMIB 69 BCI BORTEZOMIB 22 FGF-2 BORTEZOMIB 32 TNBC EGF VE-822 86 BORTEZOMIB 10 PC-3 LB-100 SAHA 30 * Lower % reduction in viability of the combined treatment compared to the controls.

[0170] The invention therefore provides the following aspects / embodiments:

[0171] 1. Pharmaceutical composition comprising a mitogenic agent and a stress control pathway inhibitor.

[0172] 2. Pharmaceutical composition according to aspect 1, in which the mitogenic agent is selected from the group comprising a growth factor that binds to tyrosine kinase receptors (FGFR), preferably Fibroblast Growth Factor type 2 (FGF-2), and a compound that inhibits the phosphatase PP2A (LB-100), keeping the MAPKinase pathway active. Other mitogenic agents in accordance with aspect 1 are: those that bind to tyrosine kinase receptors, such as EGF and HGF; PMA (activator of the PKC-ERK pathway); BCI, GATP, Okadaic Acid, which inhibit phosphatases, DUSP1 and DUSP6, DUSP3 and PP1A and PP2A, respectively, keeping the MAPKinase pathway active.

[0173] 3. Pharmaceutical composition, according to aspect 1, in that the inhibitor of the proteotoxic stress control pathway is preferably Bortezomib, that the genotoxic stress control pathway is VE-822, that the autophagy control pathway is Chloroquine and that the genotoxic stress control pathway by histone deacetylase is SAHA.

[0174] 4. Pharmaceutical composition, according to aspect 1, comprising FGF-2 and Bortezomib.

[0175] 5. Pharmaceutical composition, according to aspect 4, comprising 200ng of FGF-2 and 0.3 to 1.0 mg / kg of body weight of Bortezomib.

[0176] 6. Pharmaceutical composition, according to aspect 1, comprising LB-100 and Bortezomib.

[0177] 7. Pharmaceutical composition, according to aspect 6, comprising 1.0 to 1.25 mg / kg of body weight of LB-100 and 0.3 to 1.0 mg / kg of body weight of Bortezomib.

[0178] 8. Pharmaceutical composition according to any one of aspects 1 to 7, in which the combination of the mitogen agent with the stress control pathway inhibitor has a synergistic effect to cause death of tumor cell lines without causing significant damage to normal cell lines.

[0179] 9. Pharmaceutical composition, according to aspect 4, in which FGF-2 and Bortezomib cause a drastic blockage of tumor growth compared to the control groups.

[0180] 10. Pharmaceutical composition according to aspect 1, in which it increases mitogenic stimulation and the therapeutic inhibitor targeting proteotoxic stress control pathways sensitizes highly malignant cancer cells and inhibits the formation of lung and satellite lymph node metastasis.

[0181] 11. Use of the pharmaceutical composition as defined by any one of aspects 1 to 10, wherein it is for the preparation of a medicine for the treatment of cancer.

[0182] 12. Use according to aspect 11, wherein the cancer is selected from the group comprising triple-negative breast tumors (TNBC), glioblastoma multiforme, hepatocellular carcinoma, colorectal carcinoma, pancreatic cancer, lung cancer, melanoma skin cancer, triple-negative prostate tumor (TNPC).

[0183] Thus, although only some embodiments, aspects and exemplifications of the present invention have been shown, it will be understood that various omissions, substitutions and alterations of the present invention can be made by a person skilled in the art, without departing from the spirit and scope of the present invention.

[0184] It is expressly intended that all combinations of the elements that perform the same function in substantially the same manner to achieve the same results are within the scope of the present invention. Substitutions of elements from one described embodiment to another are also fully intended and contemplated.

Claims

CLAIMS 1. Pharmaceutical composition, characterized by the fact that it comprises a mitogenic agent and an inhibitor of the stress control pathway.

2. Pharmaceutical composition according to claim 1, characterized in that the mitogenic agent is selected from the group comprising a growth factor that binds to tyrosine kinase receptors (FGFR), preferably fibroblast type 2 (FGF-2), a compound that binds to tyrosine kinase receptors (preferably EGF and HGF), an activator of the PKC-ERK pathway (preferably PMA), and an agent that maintains the MAP Kinase pathway active, preferably selected from the group comprising an inhibitor of the DUSP1 or DUSP6 phosphatases (preferably BCI), an inhibitor of the DUSP3 phosphatase (preferably GATPT), an inhibitor of the PP1A or PP2A phosphatases (preferably Okadaic Acid), and an agent that maintains the MAPKinase pathway active. 3.Pharmaceutical composition according to claim 1, characterized by the fact that the inhibitor of the proteotoxic stress control pathway is preferably Bortezomib, that the genotoxic stress control pathway is VE-822, that the autophagy control pathway is Chloroquine and that the genotoxic stress control pathway by histone deacetylase is SAHA.

4. Pharmaceutical composition according to claim 1, characterized by the fact that it comprises FGF-2 and Bortezomib.

5. Pharmaceutical composition according to claim 4, characterized by the fact that it comprises 200ng of FGF-2 and 0.3 to 1.0 mg / kg of body weight of Bortezomib.

6. Pharmaceutical composition according to claim 1, characterized by the fact that it comprises LB-100 and Bortezomib, 7. Pharmaceutical composition according to claim 6. characterized by the fact that it comprises 1.0 to 1.25 mg / kg of body weight of LB-100 and from 0.3 to 1.0 mg / kg of body weight of Bortezomib.

8. Pharmaceutical composition according to any one of claims 1 to 5, characterized by the fact that the combination of the mitogenic agent with the stress control pathway inhibitor presents a synergistic effect to cause death of tumor cell lines without causing significant damage to normal cell lines.

9. Pharmaceutical composition according to claim 4, characterized by the fact that FGF-2 and Bortezomib cause a drastic blockade of tumor growth compared to the control groups.

10. Pharmaceutical composition according to claim 1, characterized by the fact that it increases mitogenic stimulation and the therapeutic inhibitor targeting the proteotoxic stress control pathways sensitizes highly malignant cancer cells and inhibits the formation of lung and satellite lymph node metastasis.

11. Use of the pharmaceutical composition as defined by any one of claims 1 to 8, characterized in that it is for the preparation of a medicament for the treatment of cancer.

12. Use according to claim 9, characterized in that the cancer is selected from the group comprising triple-negative breast tumors (TNBC), glioblastoma multiforme, hepatocellular carcinoma, colorectal carcinoma, pancreatic cancer, lung cancer, melanoma skin cancer, triple-negative prostate tumor (TNPC).

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