dactolisib and Triple-Negative-Breast-Neoplasms

Taking NVP-BEZ235 (BEZ235) as an example to screen drug response-related genes (DRRGs) and explore their potential value in triple-negative breast cancer (TNBC). Through high-throughput technique, multidimensional transcriptome expression data (mRNA, miRNA and lncRNA) of BEZ235-treated and -untreated MDA-MB-468 cell lines were obtained. Combined with transcriptome data of the MDA-MB-468 cells and TCGA-TNBC tissues, differential gene expression analysis and WGCNA were performed to identify DRRGs associated with tumor trait by simulating the drug response microenvironment (DRM) of BEZ235-treated patients. Based on DRRGs, we constructed a ceRNA network and verified the expression levels of three key molecules by RT-qPCR, which not only demonstrated the successful construction of a BEZ235-treated cell line model but also explained the antitumor mechanism of BEZ235. Four molecular subtypes related to the DRM with survival difference were proposed using cluster analysis, namely glycolysis subtype, proliferation depression subtype, immune-suppressed subtype, and immune-activated subtype. A novel prognostic signature consisting of four DRRGs was established by Lasso-Cox analysis, which exhibited outstanding performance in predicting overall survival compared with several excellent reported signatures. The high- and low-risk groups were characterized by enrichment of metabolism-related pathways and immune-related pathways, respectively. Of note, the low-risk group had a better response to immune checkpoint blockade. Besides, pRRophetic analysis found that patients in the low-risk group were more sensitive to methotrexate and cisplation, whereas more resistant to BEZ235, docetaxel and paclitaxel. In conclusion, the DRRGs exemplified by BEZ235 are potential biomarkers for TNBC molecular typing, prognosis prediction and targeted therapy. The novel DRRGs-guided strategy for predicting the subtype, survival and therapy efficacy, might be also applied to more cancers and drugs other than TNBC and BEZ235.

Topics: Cell Line, Tumor; Docetaxel; Humans; Imidazoles; Triple Negative Breast Neoplasms; Tumor Microenvironment

Nearly half of human cancers harbor p53 mutations, and mutant p53 (mutp53) promotes carcinogenesis, metastasis, tumor recurrence and chemoresistance. mutp53 is observed in 30% of breast carcinomas, including triple-negative breast cancer (TNBC), and thus mutp53 is a promising target for treatment of TNBC. In this study, we investigated the effect of a phosphatidylinositide 3 kinase/mammalian target of rapamycin dual inhibitor, NVP-BEZ235 (BEZ235), on two TNBC cell lines with mutp53: MDA-MB-231 and MDA-MB-468. Cell growth, migration and colony-formation abilities were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, scratch assay, transwell and soft agar assay, revealing that BEZ235 can inhibit the growth, migration and colony-formation abilities of TNBC cells. In addition, BEZ235 caused degradation of mutp53 in these cells. We investigated the underlying mechanism by inhibiting proteasome function using MG132 and inhibiting autophagy using 3-methyladenine and shRNAs. We observed that BEZ235 may induce autophagy through repression of the Akt/mammalian target of rapamycin signaling pathway. The observed interplay between mutp53 and autophagy in TNBC cells was examined further by knockdown of ATG5 and ATG7, revealing that degradation of mutp53 induced by BEZ235 may be independent of the ubiquitin-proteasome pathway and autophagy mediated by ATG5 and ATG7. Moreover, we found evidence of positive feedback between mutp53 and autophagy in TNBC cells. In conclusion, BEZ235 may exert antitumor effects against TNBC cells by targeting mutp53, and this may have implications for the development of future therapies.

Topics: Antineoplastic Agents; Autophagy; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Female; Humans; Imidazoles; Mutant Proteins; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proteolysis; Proto-Oncogene Proteins c-akt; Quinolines; Signal Transduction; TOR Serine-Threonine Kinases; Transfection; Triple Negative Breast Neoplasms; Tumor Suppressor Protein p53

Drug resistance in breast cancer cell populations has been shown to arise through phenotypic transition of cancer cells to a drug-tolerant state, for example through epithelial-to-mesenchymal transition or transition to a cancer stem cell state. However, many breast tumors are a heterogeneous mixture of cell types with numerous epigenetic states in addition to stem-like and mesenchymal phenotypes, and the dynamic behavior of this heterogeneous mixture in response to drug treatment is not well-understood. Recently, we showed that plasticity between differentiation states, as identified with intracellular markers such as cytokeratins, is linked to resistance to specific targeted therapeutics. Understanding the dynamics of differentiation-state transitions in this context could facilitate the development of more effective treatments for cancers that exhibit phenotypic heterogeneity and plasticity. In this work, we develop computational models of a drug-treated, phenotypically heterogeneous triple-negative breast cancer (TNBC) cell line to elucidate the feasibility of differentiation-state transition as a mechanism for therapeutic escape in this tumor subtype. Specifically, we use modeling to predict the changes in differentiation-state transitions that underlie specific therapy-induced changes in differentiation-state marker expression that we recently observed in the HCC1143 cell line. We report several statistically significant therapy-induced changes in transition rates between basal, luminal, mesenchymal, and non-basal/non-luminal/non-mesenchymal differentiation states in HCC1143 cell populations. Moreover, we validate model predictions on cell division and cell death empirically, and we test our models on an independent data set. Overall, we demonstrate that changes in differentiation-state transition rates induced by targeted therapy can provoke distinct differentiation-state aggregations of drug-resistant cells, which may be fundamental to the design of improved therapeutic regimens for cancers with phenotypic heterogeneity.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Cell Death; Cell Differentiation; Cell Division; Cell Line, Tumor; Dimethyl Sulfoxide; Epithelial-Mesenchymal Transition; Female; Humans; Imidazoles; Models, Biological; Pyridones; Pyrimidinones; Quinolines; Triple Negative Breast Neoplasms

Triple-negative breast cancer (TNBC) shows a higher malignant and poorer clinical outcome compared with other breast cancer subtypes. Albeit that chemotherapy is the first choice for TNBC treatment, rapid emergence of chemoresistance and variability of chemotherapeutic responses in TNBC patients call for novel therapeutic strategies. Here, we reported evidences highlighting that combination of BH3 mimetics and mTOR inhibitors could be a promising therapeutic strategy to improve TNBC treatment. Our results showed that combination of the BH3 mimetic ABT263 and typical mTOR inhibitors, BEZ235 or AZD8055, leads to efficient apoptosis in vitro. Tumor regression was significantly improved by combination therapy compared with either drug alone in the xenograft model. Further mechanistic investigations revealed that mTOR inhibitors induced the suppression of MCL-1; concomitantly, the expression level of PUMA was significantly upregulated in a FOXO3a-dependent manner. The specific changes of MCL-1 and PUMA facilitated the release of the apoptotic regulators, such as BIM, BAX, and BAK, to induce the activation of mitochondrial apoptotic pathway, thereby sensitizing the ABT263 activity in TNBC. Therefore, our findings provided evidences that mTOR inhibitors can enhance antitumor efficacy of BH3 mimetics via downregulating MCL-1 and upregulating PUMA in TNBC; it could be a promising therapeutic strategy to treat TNBC.

Topics: Aniline Compounds; Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Drug Synergism; Female; Forkhead Box Protein O3; Humans; Imidazoles; Mice, Nude; Models, Biological; Morpholines; Myeloid Cell Leukemia Sequence 1 Protein; Protein Biosynthesis; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Quinolines; Sulfonamides; TOR Serine-Threonine Kinases; Triple Negative Breast Neoplasms; Up-Regulation

Chemo-radio therapy (CRT) resistance is a main barrier in treating the triple negative breast cancer (TNBC). The success of conventional treatment may be ameliorated by elevating the responsiveness of the cancer cells to CRT. NVP-BEZ235 as a PI3K/AKT/mTOR dual inhibitor has been shown promising results in treating breast cancer cells. However, potential radiation-sensitizing effect of NVP-BEZ235 in TNBC remained unclear. In addition, SIRT-1 activation state and environmental cytokine were identified as being responsible for cancer cells responses to CRT. Herein, we investigate the role of interleukin 6 (IL-6) as a tumor environmental cytokine and SIRT1 in the effectiveness of NVP-BEZ235 plus radiotherapy.. TNBC cells were pre-treated with/without IL-6 and were exposed to single and combination of SRT1720 (SIRT1 activator)/EX-527 (SIRT1 inhibitor) and/or NVP-BEZ235 and/or gamma radiation. The effect of our treatments on cellular growth was determined by MTT and the cellular death and CSCs percentage were determined by Flow cytometry. Senescence detection kit was used to assay the effect of our treatments on cellular senescence induction.. Activation of SIRT1 via SRT1720 increased the efficacy of CRT in TNBC cells, especially when IL-6 exists in tumor microenvironment. Additionally, IL-6 pre-treatment followed by exposure to SRT1720 and NVP-BEZ235 significantly increased sensitivity of the cancer stem cells to radiation (p < 0.05).. Our result shows that combination of NVP-BEZ235 and SRT1720 may effectively improve late stage breast cancer cells therapeutics approach. Activation of SIRT1 and STAT3 in resistance breast cancer cells improves the in-vitro therapeutic efficacy of CRT.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Heterocyclic Compounds, 4 or More Rings; Humans; Imidazoles; Interleukin-6; Protein Kinase Inhibitors; Quinolines; Radiation-Sensitizing Agents; Signal Transduction; Triple Negative Breast Neoplasms

Triple negative breast cancer (TNBC) is the most lethal and aggressive kind of breast cancer. Studies with TNBC cells suggest that tumor environmental cytokines such as Transforming Growth Factor β1 (TGF-β1) have important roles in tumors fate. In the present study, we aimed to investigate, the effect of phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway dual inhibitor, NVP-BEZ235 and Caffeic acid phenyl ester (CAPE) on TNBC cell line (MDA-MB-231), stimulated with TGF-β1 for 14days in vitro. We found that TGF-β1 as a local tumor environmental cytokine plays important role in the progression and invasiveness of TNBC cells. NVP-BEZ235 inhibited the enhanced cell viability and CXCR4 expression induced by TGF-β1. In addition, the combined treatment of TNBC cell lines with CAPE and NVP-BEZ235 synergistically inhibited cell growth and reduced CXCR4 expression. Also, treatment of MDA-MB-231 cells with CAPE and NVP-BEZ235 led to decreasing the expression levels of p-FOXO3a in a time-dependent manner. Overall, these results suggest that tumor metastasis and progression in TNBC cells can be effectively reduced through the concurrent use of NVP-BEZ235 and CAPE. This could be of particular interest in assessing the effects of this therapy in the reduction of tumor metastasis and progression in other tumor types.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biomarkers, Tumor; Caffeic Acids; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Resistance, Neoplasm; Drug Synergism; Female; Forkhead Box Protein O3; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Neoplasm Proteins; Phenylethyl Alcohol; Phosphorylation; Protein Processing, Post-Translational; Quinolines; Receptors, CXCR4; Triple Negative Breast Neoplasms

Widely established targeted therapies directed at triple negative breast cancer (TNBC) are missing. Classical chemotherapy remains the systemic treatment option. Cisplatin has been tested in TNBC but bears the disadvantage of resistance development. The purpose of this study was to identify resistance mechanisms in cisplatin-resistant TNBC cell lines and select targeted therapies based on these findings.. The TNBC cell lines HCC38 and MDA-MB231 were subjected to intermittent cisplatin treatment resulting in the 3.5-fold cisplatin-resistant subclone HCC38CisR and the 2.1-fold more resistant MDA-MB231CisR. Activation of pro-survival pathways was explored by immunostaining of phospho-receptor tyrosine kinases. Targeted therapies (NVP-AEW541, lapatinib and NVP-BEZ235) against activated pathways were investigated regarding cancer cell growth and cisplatin sensitivity.. In HCC38CisR and MDA-MB231CisR, phosphorylation of epidermal growth factor receptor (EGFR) and insulin-like growth factor 1 receptor (IGF1R) was observed. In HCC38CisR, treatment with NVP-AEW541 increased potency of lapatinib almost seven-fold, but both compounds could not restore cisplatin sensitivity. However, the dual phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitor NVP-BEZ235 acted synergistically with cisplatin in HCC38CisR and fully restored cisplatin sensitivity. Similarly, NVP-BEZ235 increased cisplatin potency in MDA-MB231CisR. Furthermore, NVP-AEW541 in combination with lapatinib restored cisplatin sensitivity in MDA-MB231CisR.. Simultaneous inhibition of EGFR and IGF1R in cisplatin-resistant TNBC cell lines was synergistic regarding inhibition of proliferation and induction of apoptosis. Co-treatment with NVP-BEZ235 or with a combination of NVP-AEW541 and lapatinib restored cisplatin sensitivity and may constitute a targeted treatment option for cisplatin-resistant TNBC.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Drug Resistance, Neoplasm; Drug Synergism; Humans; Imidazoles; Lapatinib; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Pyrimidines; Pyrroles; Quinazolines; Quinolines; Signal Transduction; TOR Serine-Threonine Kinases; Triple Negative Breast Neoplasms

To explore the molecular mechanisms of resistance to phosphatidyl inositol 3-kinase (PI3K) inhibitors in triple-negative breast cancer (TNBC) cells.. HCC70 cells (TNBC) were transfected with siFZD7, siWANT5B or siGSK3 using lipofectamine 2000 transfection reagent. The expression levels of key proteins of WNT/β-catenin and PI3K/AKT/mTOR pathways were determined by Western blot analysis. After HCC70, MCF-7 (ER-positive) and SK-BR3 (HER2-positive) cells were treated with PI3K/AKT/mTOR inhibitors, the inhibition rates of cell proliferation were measured by MTT assay, and half maximal inhibitory concentrations (IC50) were calculated. The altered activities of WNT/β-catenin and PI3K/AKT/mTOR proteins were detected by Western blot and luciferase report gene assay, respectively. The nuclear translocation of β-catenin protein was examined by immunofluorescence assay. Xenograft nude mouse model was used to evaluate the tumorigenicity of breast cancer cells treated with BKM120 in vivo. The expression levels of p-LRP6, p-4EBP1 and β-catenin proteins in the tumor tissues were determined by immunohistochemical staining.. The expression levels of FZD7, WANT5B and GSK3 proteins were significantly reduced in the HCC70 cells transfected with the target siRNAs. Meanwhile, the activity of WNT/β-catenin was enhanced and PI3K/AKT/mTOR pathway was inhibited. PI3K/AKT/mTOR inhibitors suppressed MCF-7 and SK-BR3 cell proliferation. The IC50 of GDC-094, BKM120, XL147, perifosine, everolimus, and BEZ235 in MCF-7 cells were 0.46 mmol/L, 1.44 mmol/L, 4.34 mmol/L, 11.35 μmol/L, 53.71 μmol/L and 12.87 μmol/L respectively, and 0.63 mmol/L, 0.58 mmol/L, 3.74 mmol/L, 13.22 μmol/L, 60.00 μmol/L and 11.38 μmol/L in the SK-BR3 cells, respectively. The results of luciferase report gene assay showed that the luciferase activities in HCC70, MCF-7 and SK-BR3 cells treated with BKM120 were 1.75±0.05, 1.13±0.02 and 0.43±0.01, respectively. The luciferase activities in HCC70 and SK-BR3 cells were significantly different from that of the control cells (1.00±0.02, P<0.05). The immunohistochemical analysis showed that BKM120 inhibited mTOR activity, and the enhanced WNT/β-catenin activity reversed the phenotype of inhibitory mTOR induced by BKM120. BKM120 suppressed the tumorigenic ability of MCF-7 and SK-BR3 cells in vivo, but had no effect on cultured HCC70 cells. The immunohistochemical analysis showed nuclear translocation of β-catenin protein and increased expression level of p-LRP-6 protein in transplanted tumor tissues from HCC70 cells treated with BKM120, increased the level of p-LRP-6 protein, and no changes of p-4EBP1 protein expression. However, no nuclear translocation of β-catenin protein and no decrease of p-LRP6 and p-4EBP1 protein levels in the transplanted tumor tissue of MCF-7 cells after treatment with BKM120.. The triple-negative breast cancer HCC70 cells have drugs-resistance to PI3K inhibitors. The WNT/β-catenin signaling pathway may regulate the PI3K/AKT/mTOR pathway, therefore, inducing the drug-resistance of TNBC cells to PI3K inhibitors.

Topics: Adaptor Proteins, Signal Transducing; Aminopyridines; Animals; beta Catenin; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Humans; Imidazoles; Mice; Morpholines; Phosphatidylinositol 3-Kinase; Phosphatidylinositol 3-Kinases; Phosphoproteins; Proto-Oncogene Proteins c-akt; Quinolines; Signal Transduction; TOR Serine-Threonine Kinases; Triple Negative Breast Neoplasms

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer. Despite response to chemotherapy, relapses are frequent and resistance to available treatments is often seen in the metastatic setting. Therefore, identification of new therapeutic targets is required. With this aim, we have profiled the activation status of 44 receptor tyrosine kinases (RTKs) and their major signaling pathways in patient-derived TNBC tumors. Frequent co-activation of several RTKs as well as the extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) and mammalian target of rapamycin (mTOR) routes was found. Pharmacologic targeting of the activated kinases indicated that agents that attack the mTOR route are more potent and efficient antitumoral treatments than agents targeting RTKs. mTOR signals through two multiprotein complexes, mTORC1 and mTORC2. We used a genetic approach to explore the contribution of each of the two mTOR branches to the regulation of cell number of TNBC cells. RNA interference experiments indicated that mTORC1 predominated over mTORC2 in the control of TNBC cell proliferation. Moreover, RNA interference of mTOR had a superior antiproliferative action than separately acting on mTORC1 or mTORC2. To analyze the relevance of mTOR targeting in vivo, we used mice with TNBC. Treatment of these mice with BEZ235, a drug that targets mTOR, slowed tumor growth. Mechanistically, BEZ235 delayed cell cycle progression without affecting cell viability. Our results show that TNBCs are particularly sensitive to inhibition of the mTOR pathway, and indicate that mTOR targeting may be a more efficient anti-TNBC therapy than exclusively acting on the mTORC1 branch of the pathway. This is relevant as most mTOR inhibitors used in the clinic act on mTORC1. Collectively with the fact that BEZ235 synergized with drugs commonly used in the treatment of TNBC, our data support the clinical development of agents that act on mTOR as a therapy for this disease.

Topics: Animals; Cell Cycle Checkpoints; Enzyme Activation; Female; Humans; Imidazoles; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Mice, Inbred BALB C; Multiprotein Complexes; Protein Kinase Inhibitors; Quinolines; Receptor Protein-Tyrosine Kinases; TOR Serine-Threonine Kinases; Triple Negative Breast Neoplasms