Pharmacological activation of FOXO3 suppresses triple-negative breast cancer in vitro and in vivo

Triple-negative breast cancer (TNBC) is the most lethal form of breast cancer. Lacking effective therapeutic options hinders treatment of TNBC. Here, we show that bepridil (BPD) and trifluoperazine (TFP), which are FDA-approved drugs for treatment of schizophrenia and angina respectively, inhibit Akt-pS473 phosphorylation and promote FOXO3 nuclear localization and activation in TNBC cells. BPD and TFP inhibit survival and proliferation in TNBC cells and suppress the growth of TNBC tumors, whereas silencing FOXO3 reduces the BPD- and TFP-mediated suppression of survival in TNBC cells. While BPD and TFP decrease the expression of oncogenic c-Myc, KLF5, and dopamine receptor DRD2 in TNBC cells, silencing FOXO3 diminishes BPD- and TFP-mediated repression of the expression of these proteins in TNBC cells. Since c-Myc, KLF5, and DRD2 have been suggested to increase cancer stem cell-like populations in various tumors, reducing these proteins in response to BPD and TFP suggests a novel FOXO3-dependent mechanism underlying BPD- and TFP-induced apoptosis in TNBC cells

Estrogen receptor-beta is a potential target for triple negative breast cancer treatment.

Triple Negative breast cancer (TNBC) is a subtype of breast cancer that lacks the expression of estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2. TNBC accounts for 15-20% of all breast cancer cases but accounts for over 50% of mortality. We propose that Estrogen receptor-beta (ERβ) and IGF2 play a significant role in the pathogenesis of TNBCs, and could be important targets for future therapy. Tissue microarrays (TMAs) from over 250 TNBC patients' were analyzed for ERβ and IGF2 expression by immunohistochemistry. Expression was correlated with clinical outcomes. In addition, TNBC cell lines Caucasians (CA): MB-231/BT549 and African Americans (AAs): MB-468/HCC70/HCC1806 were used to investigate the effect of hormonal and growth factor regulation on cell proliferation. TMAs from AAs had higher expression of ERβ and IGF2 expression when compared to CA. ERβ and IGF2 were found to be upregulated in our TNBC cell lines when compared to other cell types. TNBC cells treated with ERβ agonist displayed significant increase in cell proliferation and migration when compared to controls. AA tissue samples from TNBC patients had higher expression of ERβ. African-American breast cancer TNBC tissue samples from TNBC patients have higher expression of ERβ. In addition, TNBC cell lines were also found to express high levels of ERβ. IGF2 increased transcription of ERβ in TNBC cells. Understanding the mechanisms of IGF2/ERβ axis in TNBC tumors could provide an opportunity to target this aggressive subtype of breast cancer

Bcl2 is an independent prognostic marker of triple negative breast cancer (TNBC) and predicts response to anthracycline combination (ATC) chemotherapy (CT) in adjuvant and neoadjuvant settings

Background: TNBC represents a heterogeneous subgroup of BC with poor prognosis and frequently resistant to CT. Material and methods: The relationship between Bcl2 immunohistochemical protein expression and clinicopathological outcomes was assessed in 736 TNBC-patients: 635 patients had early primary-TNBC (EP-TNBC) and 101 had primary locally advanced (PLA)-TNBC treated with neo-adjuvant- ATC-CT. Results: Negative Bcl2 (Bcl2-) was observed in 70% of EP-TNBC and was significantly associated with high proliferation, high levels of P-Cadherin, E-Cadherin and HER3 (P’s<0.01), while Bcl2+ was significantly associated with high levels of p27, MDM4 and SPAG5 (P<0.01). After controlling for chemotherapy and other prognostic factors, Bcl2- was associated with 2-fold increased risk of death (P=0.006) and recurrence (P=0.0004). Furthermore, the prognosis of EP-TNBC/Bcl2- patients had improved both BC-specific survival (P=0.002) and disease-free survival (P = 0.003), if they received adjuvant-ATC-CT. Moreover, Bcl2- expression was an independent predictor of pathological complete response of primary locally advanced triple negative breast cancer (PLA-TNBC) treated with neoadjuvant-ATC-CT (P=0.008). Conclusion: Adding Bcl2 to the panel of markers used in current clinical practice could provide both prognostic and predictive information in TNBC. TNBC/Bcl2- patients appear to benefit from ATC-CT, whereas Bcl2+ TNBC seems to be resistant to ATC-CT and may benefit from a trial of different type of chemotherapy with/without novel-targeted agents. Key words: anthracyclin chemotherapy, Bcl2, predictive marker, prognostic marker, theraputic targets, triple negative breast cancer

Inhibition of fatty acid oxidation as a therapy for MYC-overexpressing triple-negative breast cancer.

Expression of the oncogenic transcription factor MYC is disproportionately elevated in triple-negative breast cancer (TNBC), as compared to estrogen receptor-, progesterone receptor- or human epidermal growth factor 2 receptor-positive (RP) breast cancer. We and others have shown that MYC alters metabolism during tumorigenesis. However, the role of MYC in TNBC metabolism remains mostly unexplored. We hypothesized that MYC-dependent metabolic dysregulation is essential for the growth of MYC-overexpressing TNBC cells and may identify new therapeutic targets for this clinically challenging subset of breast cancer. Using a targeted metabolomics approach, we identified fatty acid oxidation (FAO) intermediates as being dramatically upregulated in a MYC-driven model of TNBC. We also identified a lipid metabolism gene signature in patients with TNBC that were identified from The Cancer Genome Atlas database and from multiple other clinical data sets, implicating FAO as a dysregulated pathway that is critical for TNBC cell metabolism. We found that pharmacologic inhibition of FAO catastrophically decreased energy metabolism in MYC-overexpressing TNBC cells and blocked tumor growth in a MYC-driven transgenic TNBC model and in a MYC-overexpressing TNBC patient-derived xenograft. These findings demonstrate that MYC-overexpressing TNBC shows an increased bioenergetic reliance on FAO and identify the inhibition of FAO as a potential therapeutic strategy for this subset of breast cancer

Receptors for Insulin-Like Growth Factor-2 and Androgens as Therapeutic Targets in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) occurs in 10-15% of all breast cancer patients, yet it accounts for about half of all breast cancer deaths. There is an urgent need to identify new antitumor targets to provide additional treatment options for patients afflicted with this aggressive disease. Preclinical evidence suggests a critical role for insulin-like growth factor-2 (IGF2) and androgen receptor (AR) in regulating TNBC progression. To advance this work, a panel of TNBC cell lines was investigated with all cell lines showing significant expression of IGF2. Treatment with IGF2 stimulated cell proliferation in vitro (p &lt; 0.05). Importantly, combination treatments with IGF1R inhibitors BMS-754807 and NVP-AEW541 elicited significant inhibition of TNBC cell proliferation (p &lt; 0.001). Based on Annexin-V binding assays, BMS-754807, NVP-AEW541 and enzalutamide induced TNBC cell death (p &lt; 0.005). Additionally, combination of enzalutamide with BMS-754807 or NVP-AEW541 exerted significant reductions in TNBC proliferation even in cells with low AR expression (p &lt; 0.001). Notably, NVP-AEW541 and BMS-754807 reduced AR levels in BT549 TNBC cells. These results provide evidence that IGF2 promotes TNBC cell viability and proliferation, while inhibition of IGF1R/IR and AR pathways contribute to blockade of TNBC proliferation and promotion of apoptosis in vitro

Family history of breast and ovarian cancer and triple negative subtype in hispanic/latina women.

Familial breast and ovarian cancer prevalence was assessed among 1150 women of Mexican descent enrolled in a case-only, binational breast cancer study. Logistic regression was conducted to compare odds of triple negative breast cancer (TNBC) to non-TNBC according to family history of breast and breast or ovarian cancer among 914 of these women. Prevalence of breast cancer family history in a first- and first- or second-degree relative was 13.1% and 24.1%, respectively; that for breast or ovarian cancer in a first-degree relative was 14.9%. After adjustment for age and country of residence, women with a first-degree relative with breast cancer were more likely to be diagnosed with TNBC than non-TNBC (OR=1.98; 95% CI, 1.26-3.11). The odds of TNBC compared to non-TNBC were 1.93 (95% CI, 1.26-2.97) for women with a first-degree relative with breast or ovarian cancer. There were non-significant stronger associations between family history and TNBC among women diagnosed at age &lt;50 compared to ≥50 years for breast cancer in a first-degree relative (P-interaction = 0.14) and a first- or second-degree relative (P-interaction = 0.07). Findings suggest that familial breast cancers are associated with triple negative subtype, possibly related to BRCA mutations in Hispanic/Latina women, which are strongly associated with TNBC. Family history is an important tool to identify Hispanic/Latina women who may be at increased risk of TNBC, and could benefit from prevention and early detection strategies

Robust Identification of Target Genes and Outliers in Triple-negative Breast Cancer Data

Correct classification of breast cancer sub-types is of high importance as it directly affects the therapeutic options. We focus on triple-negative breast cancer (TNBC) which has the worst prognosis among breast cancer types. Using cutting edge methods from the field of robust statistics, we analyze Breast Invasive Carcinoma (BRCA) transcriptomic data publicly available from The Cancer Genome Atlas (TCGA) data portal. Our analysis identifies statistical outliers that may correspond to misdiagnosed patients. Furthermore, it is illustrated that classical statistical methods may fail in the presence of these outliers, prompting the need for robust statistics. Using robust sparse logistic regression we obtain 36 relevant genes, of which ca. 60\% have been previously reported as biologically relevant to TNBC, reinforcing the validity of the method. The remaining 14 genes identified are new potential biomarkers for TNBC. Out of these, JAM3, SFT2D2 and PAPSS1 were previously associated to breast tumors or other types of cancer. The relevance of these genes is confirmed by the new DetectDeviatingCells (DDC) outlier detection technique. A comparison of gene networks on the selected genes showed significant differences between TNBC and non-TNBC data. The individual role of FOXA1 in TNBC and non-TNBC, and the strong FOXA1-AGR2 connection in TNBC stand out. Not only will our results contribute to the breast cancer/TNBC understanding and ultimately its management, they also show that robust regression and outlier detection constitute key strategies to cope with high-dimensional clinical data such as omics data

Triple negative breast cancer: new perspectives for targeted therapies

Breast cancer is a heterogeneous disease, encompassing a large number of entities showing different morphological features and having clinical behaviors. It has became apparent that this diversity may be justified by distinct patterns of genetic, epigenetic, and transcriptomic aberrations. The identification of gene-expression microarray-based characteristics has led to the identification of at least five breast cancer subgroups: luminal A, luminal B, normal breast-like, human epidermal growth factor receptor 2, and basal-like. Triple-negative breast cancer is a complex disease diagnosed by immunohistochemistry, and it is characterized by malignant cells not expressing estrogen receptors or progesterone receptors at all, and human epidermal growth factor receptor 2. Along with this knowledge, recent data show that triple-negative breast cancer has specific molecular features that could be possible targets for new biological targeted drugs. The aim of this article is to explore the use of new drugs in this particular setting, which is still associated with poor prognosis and high risk of distant recurrence and death

Androgen receptor as a mediator and biomarker of radioresistance in triple-negative breast cancer.

Increased rates of locoregional recurrence have been observed in triple-negative breast cancer despite chemotherapy and radiation therapy. Thus, approaches that combine therapies for radiosensitization in triple-negative breast cancer are critically needed. We characterized the radiation therapy response of 21 breast cancer cell lines and paired this radiation response data with high-throughput drug screen data to identify androgen receptor as a top target for radiosensitization. Our radiosensitizer screen nominated bicalutamide as the drug most effective in treating radiation therapy-resistant breast cancer cell lines. We subsequently evaluated the expression of androgen receptor in &gt;2100 human breast tumor samples and 51 breast cancer cell lines and found significant heterogeneity in androgen receptor expression with enrichment at the protein and RNA level in triple-negative breast cancer. There was a strong correlation between androgen receptor RNA and protein expression across all breast cancer subtypes (R2 = 0.72, p &lt; 0.01). In patients with triple-negative breast cancer, expression of androgen receptor above the median was associated with increased risk of locoregional recurrence after radiation therapy (hazard ratio for locoregional recurrence 2.9-3.2)) in two independent data sets, but there was no difference in locoregional recurrence in triple-negative breast cancer patients not treated with radiation therapy when stratified by androgen receptor expression. In multivariable analysis, androgen receptor expression was most significantly associated with worse local recurrence-free survival after radiation therapy (hazard ratio of 3.58) suggesting that androgen receptor expression may be a biomarker of radiation response in triple-negative breast cancer. Inhibition of androgen receptor with MDV3100 (enzalutamide) induced radiation sensitivity (enhancement ratios of 1.22-1.60) in androgen receptor-positive triple-negative breast cancer lines, but did not affect androgen receptor-negative triple-negative breast cancer or estrogen-receptor-positive, androgen receptor-negative breast cancer cell lines. androgen receptor inhibition with MDV3100 significantly radiosensitized triple-negative breast cancer xenografts in mouse models and markedly delayed tumor doubling/tripling time and tumor weight. Radiosensitization was at least partially dependent on impaired dsDNA break repair mediated by DNA protein kinase catalytic subunit. Our results implicate androgen receptor as a mediator of radioresistance in breast cancer and identify androgen receptor inhibition as a potentially effective strategy for the treatment of androgen receptor-positive radioresistant tumors

Integrative analysis of the inter-tumoral heterogeneity of triple-negative breast cancer.

Triple-negative breast cancers (TNBC) lack estrogen and progesterone receptors and HER2 amplification, and are resistant to therapies that target these receptors. Tumors from TNBC patients are heterogeneous based on genetic variations, tumor histology, and clinical outcomes. We used high throughput genomic data for TNBC patients (n = 137) from TCGA to characterize inter-tumor heterogeneity. Similarity network fusion (SNF)-based integrative clustering combining gene expression, miRNA expression, and copy number variation, revealed three distinct patient clusters. Integrating multiple types of data resulted in more distinct clusters than analyses with a single datatype. Whereas most TNBCs are classified by PAM50 as basal subtype, one of the clusters was enriched in the non-basal PAM50 subtypes, exhibited more aggressive clinical features and had a distinctive signature of oncogenic mutations, miRNAs and expressed genes. Our analyses provide a new classification scheme for TNBC based on multiple omics datasets and provide insight into molecular features that underlie TNBC heterogeneity