Effects In Triple-negative Breast Cancer Research Articles

Abstract P6-05-02: Estrogen receptor beta repurposes EZH2 to inhibit oncogenic NFκB signaling in triple negative breast cancer

Abstract Background: Triple Negative Breast Cancer (TNBC) affects approximately 15-20% of BC patients, yet accounts for a disproportionately higher rate of BC morbidity and mortality, in part due to lack of targeted therapies. Using well-validated antibodies, we have found that estrogen receptor beta (ERβ) protein is expressed in approximately 20% of TN breast tumors. We have demonstrated that ligand-mediated activation of ERβ with estradiol (E2) or ERβ-selective agonists decreases tumor cell proliferation, invasion, and migration in vitro, as well as primary tumor growth and metastatic spread in vivo. As such, we aimed to elucidate the mechanisms by which ERβ elicits its anti-cancer effects in TNBC. Methods: RNAseq and ChIPseq were used to identify ERβ and NFκB regulomes and cistromes in TNBC cells. The global effects of ERβ on H3K27Ac and H3K27Me3 were also assessed via ChIPseq. The influence of ERβ on NFκB signaling was determined using co-immunoprecipitation, luciferase assays transcriptional readouts and ChIPseq. The involvement of EZH2 in mediating ERβ’s suppression of NFκB signaling and cancer cell phenotypes was determined using pharmacological approaches. Results: Pathway analysis of ERβ-regulated genes identified NFκB and inflammatory signaling as being highly suppressed in response to E2 treatment. Numerous NFκB target genes were among the most down-regulated genes by ERβ. ChIPseq revealed that ERβ primarily associated with estrogen response elements (EREs), but was also highly enriched at NFκB binding sites following E2 treatment. However, ERβ had no impact on TNFα-mediated NFκB phosphorylation or nuclear localization, but was shown to physically associate with NFκB protein. Using an NFκB reporter construct and qPCR, ERβ was shown to block TNFα-mediated induction of NFκB signaling and NFκB target gene expression. Globally, RNAseq identified 373 genes to be significantly regulated by TNFα in TNBC cells, of which 107 were blocked in the presence of E2+TNFα. Interestingly, E2+TNFα treatment induced novel NFκB binding sites compared to TNFα alone as identified by ChIPseq for NFκB. ChIPseq also demonstrated that ligand-mediated activation of ERβ significantly diminished an activating histone mark (H3K27Ac) at many of these NFκB target genes while enhancing a repressive mark (H3K27Me3). These modifications were shown to occur through the recruitment of the histone methyltransferase, EZH2, to enhancer elements of these NFκB target genes. Drug-mediated blockade of EZH2 activity reversed suppression of NFκB target gene expression by ERβ. Finally, knockdown of NFκB expression inhibited the anti-cancer effects of ERβ while expression of a constitutively active form of NFκB enhanced ERβ’s inhibitory effects. Conclusions: Our data suggest that ERβ elicits its anti-cancer effects, in part, via formation of a novel co-repressor complex consisting of ERβ, NFκB, and EZH2. These data are in keeping with prior observations of the importance of NFκB signaling as it relates to TNBC cell proliferation and invasion, and that decreased expression of NFκB target genes is associated with improved outcomes in TNBC patients. Currently, a Mayo Breast SPORE prospective study is underway to investigate the efficacy of estradiol for the treatment of metastatic ERβ+TNBC and to further evaluate the cross-talk between ERβ and NFκB signaling. Citation Format: Kirsten GM Aspros, Adam W Nelson, Zhenqing Ye, Zhifu Sun, Igor Chernukhin, Jason S Carroll, James N Ingle, Matthew P Goetz, John R Hawse. Estrogen receptor beta repurposes EZH2 to inhibit oncogenic NFκB signaling in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-05-02.

293P - In vitro and in vivo rescue of resistance to BET inhibitors by targeting PLK1 in triple negative breast cancer

BackgroundNovel therapeutic strategies are needed for the treatment of triple negative breast cancer (TNBC). Inhibition of bromo and extraterminal domains (BET) has shown an anti-proliferative effect in TNBC as well as a synergistic interaction with polo-like kinase (PLK) inhibitors. As for many other therapeutic interventions, resistance to BET inhibitors is expected to occur at a given treatment point. MethodsWe generated two resistant models to the BET inhibitor JQ1, MDA-MB 231R and HS578TR. Western-blot, flow cytometry analysis, genomic and pharmacologic inhibition were executed to evaluate the anti-proliferative activity and biochemical effect. Nude mice were used to explore the in vivo pharmacological efficacy. ResultsWe report the generation of two resistant models to the BET inhibitor JQ1. In both models, resistant cells were particularly sensitive to PLK1 inhibition, and reduced cell proliferation in 2D and 3D cell cultures. Although PLK1 levels were similar in sensitive and resistant cell lines, pharmacological inhibition of BRD4 using JQ1 reduced PLK1 to a less extent in the resistant model, effect not observed with BRD4 gene downregulation. PLK1 inhibitor volasertib induced G2/M arrest in both cell lines, and this effect was more evident in resistant cells, in addition to an increase in pH3 and pCDK1. Combination of volasertib and JQ1 induced apoptosis that was partially caspase dependent. A slight activation of Erk1/2 and pS6 was observed in the resistant model, but the inhibition of these kinases did not have a different effect on proliferation compared with the sensitive one. Finally, JQ1-resistant cells xenografted in mice displayed resistance to JQ1 that was reversed after administration of the PLK1 inhibitor volasertib. ConclusionsPLK1 inhibition reverts resistance to BET inhibitors in our in vivo and in vitro models. These findings open avenues for further drug combinations in the clinical setting. Legal entity responsible for the studyUniversidad de Castilla-La Mancha. FundingInstituto de Salud Carlos III; ACEPAIN; CRIS CANCER; Diputación Albacete; UCLM. DisclosureAll authors have declared no conflicts of interest.

1234P - Anti-cancer and immunomodulatory effects of cobimetinib in triple negative breast cancer

BackgroundImmune check point inhibitor therapy has shown efficacy in Triple-negative breast cancer (TNBC), highlighting the immune-responsive microenvironment of TNBC. Recently, anti-cancer therapeutics have been shown to induce immunogenic cell death (ICD). MEK/ERK cascade mediates T cell receptor signaling and involves in immune response. MEK inhibitor cobimetinib is approved for advanced melanoma and is studied in several clinical trials including breast cancer. Here, we aimed to explore the anti-tumor and immunomodulatory effects of cobimetinib in TNBC. MethodsDamage-associated molecular patterns (DAMPs), cell-surface translocation of calreticulin (CRT), extracellular release of ATP, and increase in high-mobility group box protein B1 (HMGB1) release from dying tumor cells, were examined by immunoblotting and flow cytometric analysis. The pan-caspase inhibitor z-VAD-FMK was used to validate cobimetinib-induced signaling. The immunocompetent and immunodeficient mice were conducted to investigate the efficacy of cobimetinib. ResultsCobimetinib significantly induced cell apoptosis and DAMPs in TNBC cells (human MDA-MB-231, MDA-MB-468, and murine 4T1 cells). Cobimetinib downregulated p-ERK and activated caspase-8, in association with increased DAMPs such as HMGB1 and CRT. Cobimetinib-induced CRT was attenuated by ectopic ERK expression. Moreover, cobimetinib-induced cell apoptosis and increased CRT expressions were restored by the pan-caspase inhibitor. The anti-tumor activity of cobimetinib was superior in immunocompetent than in immunodeficient mice. DAMPs and caspase-8 cascade were upregulated in cobimetinib-treated 4T1 xenografts. Interestingly, the immunomodulation of cobimetinib in 4T1-bearing mice were different from that in non-tumor bearing immunocompetent mice. Importantly, cobimetinib-treated 4T1-bearing immunocompetent mice exhibited increased total number of CD8+ T cells, effector CD4+ T cells, regulatory T cells and reduced the number of myeloid-derived suppressor cells, compared to vehicle-treated mice. ConclusionsOur findings suggest that the MEK inhibitor cobimetinib acts as an ICD-inducer and exerts immunomodulatory effects in TNBC. Legal entity responsible for the studyChun-Yu Liu. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

18P - Anti-cancer effects of differentiation-inducing factor-1 in triple negative breast cancer

BackgroundBreast cancer is the most common cancer in women and its metastasis markedly exacerbates patients’ prognosis. In particular, triple negative breast cancer (TNBC), which lacks estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor 2 (HER2), is an aggressive subtype of breast cancer frequently forming metastatic lesions. Differentiation-inducing factor-1 (DIF-1) identified in Dictyostelium discoideum inhibits cell proliferation and migration of various mammalian cancer cells. However, the effect of DIF-1 on TNBC has not been examined. Here, we investigated whether DIF-1 shows anti-proliferative and anti-metastatic effects on TNBC by in-vivo and in-vitro experiments. MethodsIn in-vivo experiments, we used cancer xenograft model mice in which murine TNBC 4T1/Luc cells (1.0 × 106 cells/mL) were injected into mammary fat pads to evaluate the effects of intragastric administration of DIF-1 (300mg/kg/day) on the primary tumor growth and lung metastasis. In in-vitro experiments, we carried out assays for cell proliferation, migration and invasion to evaluate the anti-proliferative and anti-metastatic effects of DIF-1. We also conducted Western blotting and real-time RT-PCR to identify the mechanisms for DIF-1’s anti-cancer effects. ResultsIn vivo, administration of DIF-1 significantly suppressed the primary tumor growth and lung metastasis without adverse effects such as weight loss and myelosuppression. In vitro, DIF-1 reduced cyclin D1 and c-Myc by inhibiting transcription and promoting degradation of the proteins, which resulted in the suppression of cell proliferation. DIF-1 also suppressed cell migration and invasion and reduced the protein expressions of snail, twist, vimentin and MMP-2, crucial factors in epithelial-mesenchymal transition (EMT). ConclusionsDIF-1 exerts anti-cancer effects in TNBC by reducing cell cycle accelerators and reversing EMT. Our findings suggest that using DIF-1 as a lead compound could develop a novel anti-cancer agent for TNBC. Legal entity responsible for the studyThe study protocol was approved by the Committee on Ethics of Animal Experiments of Kyushu University. Animal handling and procedures were carried out in compliance with the Guidelines for Animal Experiments, Kyushu University, and the Law (No. 105) and Notification (No. 6) of the Japanese Government. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Diffuse distribution of tumor-infiltrating lymphocytes is a marker for better prognosis and chemotherapeutic effect in triple-negative breast cancer.

High-density tumor-infiltrating lymphocytes (TILs) are a prognostic marker for triple-negative breast cancer (TNBC). However, lymphocytic infiltration is heterogeneous in its pattern. We aimed to explore the utility of TIL distribution patterns against TIL density for predicting TNBC prognosis and chemotherapeutic effects. Primary invasive TNBC cases were retrieved from a single institutional cohort, and archived samples were reviewed by two board-certificated pathologists. We used 154 consecutive surgical specimens from patients with standard adjuvant therapy, and 80 biopsies taken before primary systemic chemotherapy. The average density of stromal TILs was scored at 10% intervals, while the distribution pattern of TILs was evaluated as diffuse or non-diffuse. The association between TILs and prognosis or pathological complete response (pCR) was statistically analyzed. A diffuse pattern of TILs at primary surgery correlated with better prognosis (relapse-free survival [RFS], hazard ratio [HR] 3.71, 95% confidence interval [CI] 1.60-8.57; overall survival [OS], HR 3.87, 95% CI 1.46-10.27), as well as high TIL density (≥ 50%; RFS, HR 4.51, 95% CI 2.06-9.90; OS, HR 3.28, 95% CI 1.32-8.14). Diffuse TIL pattern and nodal status were independent prognostic factors in multivariate analysis. Diffuse TIL pattern upon biopsy was associated with higher pCR rate (diffuse, 46%; non-diffuse, 21%; P = 0.032). All high TIL cases had diffuse patterns and the best outcome. Interobserver concordance was moderate (k = 0.53-0.55; distribution pattern) to good (weighted k = 0.67-0.69; density), and it was faster to assess the distribution pattern than to assess the density of TIL. Showing similar clinical impacts to the TIL density, diffuse TILs could be a predictive marker for better prognosis and higher pCR. The assessment of TIL distribution pattern is simple, faster, and practical. Heterogeneous tumor immunity may contribute to further stratification of TNBC treatment.

IL-6 Impairs the Activity of Vitamin D3 in the Regulation of Epithelial-Mesenchymal Transition in Triple Negative Breast Cancer.

Objective:The present study aimed to investigate the possible role of IL-6 and 1α,25-dihydroxyvitamin D3 (1,25D) signaling in epithelial-mesenchymal transition (EMT) and stemness in triple-negative breast cancer (TNBC) cell line.Methods:TNBC cell line, HCC 1806, was treated with IL-6 and 1,25D for three and six days. Also, the role of vitamin D receptor (VDR) was studied by transfection of TNBC cell line with VDR gene and transfection efficiency was assessed using Human VDR enzyme-linked immunosorbent assay (ELISA). Changes in E-cadherin gene expression were analyzed by quantitative real-time PCR (qRT-PCR). Also, changes in CD44+ cells were analyzed by flow cytometry. Finally, morphological changes were investigated by light microscopy after 6 days. Results:Treatment of HCC1806 cells with IL-6 has no significant effect either on E-cadherin gene expression or CD44+ cells, (p > 0.05). However, E-cadherin gene expression was significantly up-regulated after treatment with 1,25D for 6 days, (p < 0.05). Also, CD44+ cells were significantly reduced after treatment with 1,25D either for 3 or 6 days, (p < 0.05). Transfection of TNBC cell line with VDR gene significantly up-regulated VDR protein expression, (p < 0.05). In addition, overexpression of VDR in TNBC cells and treatment with 1,25D significantly up-regulated E-cadherin gene expression, (p < 0.05) and reduced CD44+ cells, (p < 0.05). Moreover, transfection with VDR and treatment with a combination of 1,25D and IL-6 significantly down-regulated E-cadherin gene expression and increased CD44+ cells compared with transfected cells with VDR treated with 1,25D alone, (p < 0.05). No significant morphological changes were observed in treated cells, 6 days post-treatment. Conclusion:The presence of IL-6 in the breast tumor microenvironment may impair the activity of vitamin D3 signaling, limiting its anti-tumor effects in TNBC.

Abstract 703: An anthelmintic drug, flubendazole, exerts antitumor effects in triple-negative breast cancer via targeting cancer stem-like properties

Abstract Triple-negative breast cancer (TNBC) remains challenging due to the absence of established molecular targets for the phenotype. These tumors tend to be more aggressive with a higher risk of recurrence and metastasis, leading to poorer clinical outcomes. Flubendazole (FLU) is a widely used anthelmintic agent that also exhibits anticancer activity in several cancer types. The objective of the present study was to investigate the effects of FLU on cell proliferation, apoptosis and breast cancer stem cell (BCSC)-like properties in TNBC. The effect of FLU on TNBC cell lines in vitro was evaluated in terms of apoptosis, cell cycle distribution, caspase-3/-7 activity and BCSC-like characteristics. An orthotopic allograft model with 4T1 mammospheres was used to examine the effect of FLU on tumor growth and angiogenesis in vivo. FLU efficiently induced apoptosis with accompanied by activation of caspase-3 and -7, and PARP cleavage in TNBC. FLU-induced apoptosis is associated with the accumulation of G2/M phase cells by tubulin degradation, leading to mitotic catastrophe. Interestingly, FLU significantly suppressed BCSC-like properties in TNBC, as evidenced by a marked inhibition of ALDH1 activity, the CD44high/CD24low and CD49fhigh/CD24high subpopulations, and the subsequent impairment of mammosphere formation in vitro. This appears to contribute toward the suppression of BCSC-enriched tumor burden and intra-tumoral angiogenesis in TNBC allografts, together with a significant downregulation of ALDH1A1 and CD49f expression in vivo. Our findings suggest that FLU kills not only rapid proliferating tumor cells but also effectively eradicates BCSC-like cells in vitro and in vivo. These findings warrant further investigation of flubendazole as a potential treatment for triple-negative breast cancer. Citation Format: Yoon-Jae Kim, Eunhye Oh, Tae-Min Cho, Seojin Jang, Jung Min Park, Soeun Park, Minsu Park, Ji Young Kim, Jae Hong Seo. An anthelmintic drug, flubendazole, exerts antitumor effects in triple-negative breast cancer via targeting cancer stem-like properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 703.

C-terminal HSP90 inhibitor L80 elicits anti-metastatic effects in triple-negative breast cancer via STAT3 inhibition

Triple-negative breast cancer (TNBC) is an aggressive heterogeneous disease with a divergent profile. It has an earlier tendency to form metastases and is associated with poor clinical outcomes due to the limited treatment options available. Heat-shock protein (HSP90) represents a potential treatment target as it promotes tumor progression and metastasis by modulating the maturation and stabilization of signal transduction proteins. We sought to investigate the efficacy of the C-terminal HSP90 inhibitor L80 on cell proliferation, breast cancer stem cell (BCSC)-like properties, tumor growth and metastasis. L80 suppressed cell viability and concomitantly inhibited AKT/MEK/ERK/JAK2/STAT3 signaling in TNBC cells but did not induce cytotoxicity in normal cells. L80 effectively targeted BCSC-like traits, together with significant reductions in the CD44high/CD24low-population, ALDH1 activity and mammosphere forming-ability. In support of the in vitro observations, L80 administration caused significant impairment in tumor growth, angiogenesis and distant metastases in an orthotopic allograft model with BCSC-enriched cells in vivo. These phenomena were associated with the suppression of BCSC-like characteristics and STAT3 dysfunction. Our findings highlight properties of the L80 compound that may be useful in suppressing metastatic TNBC.

Therapeutic activity of DCC-2036, a novel tyrosine kinase inhibitor, against triple-negative breast cancer patient-derived xenografts by targeting AXL/MET.

Triple-negative breast cancer (TNBC) is insensitive to endocrine therapies and targeted therapies to human epidermal growth factor receptor-2 (HER2), estrogen receptor (ER) and progesterone receptor (PR). New targets and new targeted therapeutic drugs for TNBC are desperately needed. Our study confirmed that DCC-2036 inhibited the proliferation, invasion, migration and epithelial-mesenchymal transition (EMT) of TNBC cells as well as induced apoptosis. Moreover, the antiproliferative activity of DCC-2036 was more efficient than that of most clinical drugs. In addition, the combination of DCC-2036 and cisplatin or lapatinib had synergistic effects on TNBC cells. Mechanistically, DCC-2036 targeted AXL/MET, especially AXL, and regulated the downstream PI3K/Akt-NFκB signaling to exert its antitumor effect in TNBC. DCC-2036 also inhibited the growth and metastasis of xenografted MDA-MB-231 cells (AXL/MET-high TNBC cells) but not MDA-MB-468 cells (AXL-low TNBC cells) in NSG mice in vivo. Furthermore, DCC-2036 significantly inhibited tumor growth and invasion of AXL/MET-high TNBC PDX tumors but not AXL/MET-low TNBC PDX tumors. These results highlighted the roles of AXL/MET in cancer growth and metastasis and further verified that the critical targets of DCC-2036 are AXL and MET, especially AXL. In addition, there was no significant toxicity of DCC-2036 even at a high dosage. Therefore, DCC-2036 may be a potential compound to treat TNBC, especially for tumors with AXL/MET overexpression.

ERβ-mediated induction of cystatins results in suppression of TGFβ signaling and inhibition of triple-negative breast cancer metastasis

Triple-negative breast cancer (TNBC) accounts for a disproportionately high number of deaths due to a lack of targeted therapies and an increased likelihood of distant recurrence. Estrogen receptor beta (ERβ), a well-characterized tumor suppressor, is expressed in 30% of TNBCs, and its expression is associated with improved patient outcomes. We demonstrate that therapeutic activation of ERβ elicits potent anticancer effects in TNBC through the induction of a family of secreted proteins known as the cystatins, which function to inhibit canonical TGFβ signaling and suppress metastatic phenotypes both in vitro and in vivo. These data reveal the involvement of cystatins in suppressing breast cancer progression and highlight the value of ERβ-targeted therapies for the treatment of TNBC patients.

Antitumor efficacy of the Runx2-dendritic cell vaccine in triple-negative breast cancer in vitro.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with a poor prognosis and limited effective treatment. The rise in immunotherapeutic strategies prompted the establishment of a genetic vaccine against TNBC in vitro using a possible biological marker of TNBC. In the present study, different detection methods were used to evaluate the distribution and expression of runt-associated transcription factor 2 (Runx2) in various breast cancer cell lines. Following the development of the Runx2-dendritic cell (DC) vaccine using a lentivirus, the transfection efficacy was recorded. The T lymphocytes co-cultured with the vaccine were collected to assess the antitumor potency. Increased levels of Runx2 were expressed in breast cancer cells; however, different breast cancer cell lines expressed various levels of Runx2. Runx2 demonstrated particularly high expression in TNBC cells, compared with non-TNBC cells. A Runx2 lentivirus transfection system was successfully engineered, and Runx2 was transduced into dendritic cells whilst maintaining stable expression. The sustained and stable cytotoxic T cells induced in the transfected group had higher and more specific antitumor efficacy against TNBC, compared with the other cell lines. Runx2 may be a novel target for TNBC treatment. The Runx2-DC vaccine may induce specific and efficient antitumor effects in TNBC in vitro.

Determination of the Relative Efficacy of Eicosapentaenoic Acid and Docosahexaenoic Acid for Anti-Cancer Effects in Human Breast Cancer Models.

Epidemiological studies have associated high fish oil consumption with decreased risk of breast cancer (BC). n-3 long chain polyunsaturated fatty acids (n-3 LCPUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) found in fish and fish oils exert anti-cancer effects. However, few studies have examined the relative efficacy of EPA and DHA alone and in mixtures on BC subtypes. This was the objective of the present review, as this research is a necessity for the translation of findings to human health and disease. The literature suggests that DHA has a greater anti-cancer effect in triple negative BC (TNBC). In estrogen positive (ER+) BC, DHA has a greater effect on cell viability, while both fatty acids have similar effects on apoptosis and proliferation. These effects are associated with preferential uptake of DHA into TNBC lipid rafts and EPA in ER+ BC. EPA:DHA mixtures have anti-cancer activity; however, the ratio of EPA:DHA does not predict the relative incorporation of these two fatty acids into membrane lipids as EPA appears to be preferentially incorporated. In summary, DHA and EPA should be considered separately in the context of BC prevention. The elucidation of optimal EPA:DHA ratios will be important for designing targeted n-3 LCPUFA treatments.

Value of FOXP3 expression in prediction of neoadjuvant chemotherapy effect in triple-negative breast cancer

Value of FOXP3 expression in prediction of neoadjuvant chemotherapy effect in triple-negative breast cancer

Overexpression of miR-509 Increases Apoptosis and Inhibits Invasion via Suppression of Tumor Necrosis Factor-α in Triple-Negative Breast Cancer Hs578T Cells.

Triple-negative breast cancer (TNBC) is associated with high recurrence rates of metastasis and death. miR-509 has been reported to be a tumor suppressor in many cancers, but its effect in TNBC has not yet been identified. In this article, we explored the effects of miR-509 on the malignant phenotype of TNBC cells, including proliferation, apoptosis, migration, and invasion. We transiently transfected TNBC cells, Hs578T, with miR-509 mimic. Upon transfection, the expression of miR-509 was upregulated about 50-fold compared with cells transfected with scramble mimic. Overexpression of miR-509 inhibited cell proliferation, induced cell apoptosis, and suppressed cell invasion of Hs578T cells. Moreover, tumor necrosis factor-α (TNF-α) was involved in miR-509-mediated suppressive effects of TNBC cells, as being treated with TNF-α could partially abolish the suppressive effects of miR-509. Collectively, these data suggest that miR-509 could reverse the malignant phenotype of TNBC cells, probably by suppressing TNF-α.

Antitumor Activity of KW-2450 against Triple-Negative Breast Cancer by Inhibiting Aurora A and B Kinases

Currently, no targeted drug is available for triple-negative breast cancer (TNBC), an aggressive breast cancer that does not express estrogen receptor, progesterone receptor, or HER2. TNBC has high mitotic activity, and, because Aurora A and B mitotic kinases drive cell division and are overexpressed in tumors with a high mitotic index, we hypothesized that inhibiting Aurora A and B produces a significant antitumor effect in TNBC. We tested this hypothesis by determining the antitumor effects of KW-2450, a multikinase inhibitor of both Aurora A and B kinases. We observed significant inhibitory activities of KW-2450 on cell viability, apoptosis, colony formation in agar, and mammosphere formation in TNBC cells. The growth of TNBC xenografts was significantly inhibited with KW-2450. In cell-cycle analysis, KW-2450 induced tetraploid accumulation followed by apoptosis or surviving octaploid (8N) cells, depending on dose. These phenotypes resembled those of Aurora B knockdown and complete pharmaceutical inhibition of Aurora A. We demonstrated that 8N cells resulting from KW-2450 treatment depended on the activation of mitogen-activated protein kinase kinase (MEK) for their survival. When treated with the MEK inhibitor selumetinib combined with KW-2450, compared with KW-2450 alone, the 8N cell population was significantly reduced and apoptosis was increased. Indeed, this combination showed synergistic antitumor effect in SUM149 TNBC xenografts. Collectively, Aurora A and B inhibition had a significant antitumor effect against TNBC, and this antitumor effect was maximized by the combination of selumetinib with Aurora A and B inhibition.

CXCR4 Protein Epitope Mimetic Antagonist POL5551 Disrupts Metastasis and Enhances Chemotherapy Effect in Triple-Negative Breast Cancer.

The SDF-1 receptor CXCR4 has been associated with early metastasis and poorer prognosis in breast cancers, especially the most aggressive triple-negative subtype. In line with previous reports, we found that tumoral CXCR4 expression in patients with locally advanced breast cancer was associated with increased metastases and rapid tumor progression. Moreover, high CXCR4 expression identified a group of bone marrow-disseminated tumor cells (DTC)-negative patients at high risk for metastasis and death. The protein epitope mimetic (PEM) POL5551, a novel CXCR4 antagonist, inhibited binding of SDF-1 to CXCR4, had no direct effects on tumor cell viability, but reduced migration of breast cancer cells in vitro. In two orthotopic models of triple-negative breast cancer, POL5551 had little inhibitory effect on primary tumor growth, but significantly reduced distant metastasis. When combined with eribulin, a chemotherapeutic microtubule inhibitor, POL5551 additively reduced metastasis and prolonged survival in mice after resection of the primary tumor compared with single-agent eribulin. Hypothesizing that POL5551 may mobilize tumor cells from their microenvironment and sensitize them to chemotherapy, we used a "chemotherapy framing" dosing strategy. When administered shortly before and after eribulin treatment, three doses of POL5551 with eribulin reduced bone and liver tumor burden more effectively than chemotherapy alone. These data suggest that sequenced administration of CXCR4 antagonists with cytotoxic chemotherapy synergize to reduce distant metastases.

Abstract 3526: OTX015 effects in triple-negative breast cancer (TNBC) models are independent of hypoxia conditions and synergistic with other anticancer agents

Abstract Background: TNBC is an aggressive and heterogeneous subtype group of breast cancers clinically defined by the lack of estrogen and progesterone receptors, as well as the human epidermal growth factor receptor 2 (HER2). Few therapeutic options have shown clinical benefit beyond cytotoxic chemotherapy. Clinical studies have demonstrated that more than 50% of human breast cancers present a much lower median O2 partial pressure than normal breast tissue, correlating with chemo- and radio-resistance. We previously reported the in vivo effects of the BET-bromodomain inhibitor OTX015 (OncoEthix SA, Switzerland) in TNBC models (Vázquez et al.; EORTC-ENA 2014; #580). The aim of this work was to evaluate the in vitro antitumor activity of OTX015 both in normoxic and hypoxic environments, as well as in combination with antitumor agents. Materials and Methods: OTX015 growth inhibition concentrations 50% (GI50) values were determined in HCC197, MDA-MB-231 and MDA-MB-468 human-derived TNBC cell lines after 48 and 72 h in normoxia and hypoxia (0.1% atmospheric O2), employing the MTT assay and cell counting. RT-PCR was performed with Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System at baseline, and after 24, 48 and 72 h of OTX015 treatments. OTX015 was combined with docetaxel or the mTOR inhibitor, everolimus, and the combination index (CI) determined by the Chou-Talalay method (CI &amp;lt; 0.9 synergy; CI = 0.9-1.1, additivity; CI &amp;gt; 1.1, antagonism). Results: OTX015 showed antiproliferative activity in the 3 cell lines after 48- and 72-h treatments in normoxia and hypoxia. MDA-MB-231 was the most sensitive in both conditions. Hypoxia significantly (p &amp;lt; 0.05) increased the antiproliferative activity of OTX015 in MDA-MB-468 cells. In addition, the antitumor effect of OTX015 was accompanied - in these cells only - with a marked (p &amp;lt; 0.05) reduction in the expression levels of c-MYC and n-MYC. The three cell lines presented a substantial increase in p21 expression following OTX015 exposure, correlating with G1 cell cycle arrest. Everolimus had an additive effect when simultaneously combined with OTX015 in HCC1937 and MDA-MB-231 cells (CI = 1.02 and 0.94, respectively), and was antagonistic in MDA-MB-468 cells (CI = 1.60). Likewise, docetaxel also had an additive effect when simultaneously combined with OTX015 in HCC1937 and MDA-MB-231 cells (CI = 1.03 and 0.95 respectively), and showed a slight synergism in MDA-MB-468 cells (CI = 0.87). Conclusion: These preclinical data support the further development OTX015 in the clinical setting for TNBC, alone or in combination with mTOR inhibitors. A single agent Phase Ib study in solid tumor patients including TNBC is underway. Citation Format: Ramiro Vázquez, Lucile Astorgues-Xerri, María E. Riveiro, Luca Beltrame, Sergio Marchini, Francesco Bertoni, Ivo Kwee, Mohamed Mohamed, Esteban Cvitkovic, Roberta Frapolli, Maurizio D'Incalci. OTX015 effects in triple-negative breast cancer (TNBC) models are independent of hypoxia conditions and synergistic with other anticancer agents. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3526. doi:10.1158/1538-7445.AM2015-3526

Differential cytotoxic and radiosensitizing effects of silver nanoparticles on triple-negative breast cancer and non-triple-negative breast cells.

Identification of differential sensitivity of cancer cells as compared to normal cells has the potential to reveal a therapeutic window for the use of silver nanoparticles (AgNPs) as a therapeutic agent for cancer therapy. Exposure to AgNPs is known to cause dose-dependent toxicities, including induction of oxidative stress and DNA damage, which can lead to cell death. Triple-negative breast cancer (TNBC) subtypes are more vulnerable to agents that cause oxidative stress and DNA damage than are other breast cancer subtypes. We hypothesized that TNBC may be susceptible to AgNP cytotoxicity, a potential vulnerability that could be exploited for the development of new therapeutic agents. We show that AgNPs are highly cytotoxic toward TNBC cells at doses that have little effect on nontumorigenic breast cells or cells derived from liver, kidney, and monocyte lineages. AgNPs induced more DNA and oxidative damage in TNBC cells than in other breast cells. In vitro and in vivo studies showed that AgNPs reduce TNBC growth and improve radiation therapy. These studies show that unmodified AgNPs act as a self-therapeutic agent with a combination of selective cytotoxicity and radiation dose-enhancement effects in TNBC at doses that are nontoxic to noncancerous breast and other cells.

Disruption of STAT3-DNMT1 interaction by SH-I-14 induces re-expression of tumor suppressor genes and inhibits growth of triple-negative breast tumor.

Epigenetic regulation of gene expression is an emerging target to treat several human diseases including cancers. In cancers, expressions of many tumor suppressor genes are suppressed by hyper-methylation in their regulatory regions. Herein, we describe a novel carbazole SH-I-14 that decreased the level of the acetyl-STAT3 at the K685 residue. Mutation analysis revealed that SH-I-14 disrupted STAT3-DNMT1 interaction by removing acetyl group from K685 of STAT3. Finally, the inhibition of STAT3-DNMT1 interaction by SH-I-14 resulted in re-expression of tumor suppressor genes such as VHL and PDLIM4 through de-methylation of their promoter regions. In addition, SH-I-14 showed anti-proliferative effect in triple-negative breast cancer (TNBC) cell lines in vitro and anti-tumor effect in a mouse xenograft model of MDA-MB-231 tumor. Taken together, our results suggest that targeting acetyl-STAT3 (K685) provides potential therapeutic opportunity to treat a subset of human cancers.

Teriflunomide, an immunomodulatory drug, exerts anticancer activity in triple negative breast cancer cells.

Triple-negative breast cancer (TNBC) is defined as a group of primary breast cancers lacking expression of estrogen, progesterone, and human epidermal growth factor receptor-2 (HER-2) receptors, characterized by higher relapse rate and lower survival compared with other subtypes. Due to lack of identified targets and molecular heterogeneity, conventional chemotherapy is the only available option for treatment of TNBC, but non-discordant positive therapeutic efficacy could not be achieved. Here, we demonstrated that these TNBC cells were sensitive to teriflunomide, which was a well-known immunomodulatory drug for treatment of relapsing multiple sclerosis (MS). Potent anti-cancer effects in TNBC invitro, including proliferation inhibition, cell cycle delay, cell apoptosis, and suppression of cell motility and invasiveness, could be achieved with this agent. Of note, we showed that multiple signals involved in TNBC proliferation, survival, migratory, and invasive potential were under regulation by teriflunomide. Among them, we identified down-regulation of growth factor receptors to abolish growth maintenance, suppression of c-Myc, and cyclin D1 to contribute to its anti-proliferative effect, modulation of components of cell cycle to induce S-phase arrest, degradation of Bcl-xL, and up-regulation of BAX via activation of MAPK pathway to induce apoptosis, and inhibition of epithelial-mesenchymal transition (EMT) process, matrix metalloproteinase-9 (MMP9) expression, and inactivation of Src/FAK to reduce TNBC migration and invasion. The results identified teriflunomide may be of therapeutic benefit for the more aggressive and difficult-to-treat breast cancer subtype, indicating the use of teriflunomide for clinical trials for treatment of TNBC patients.