TNBC Treatment Research Articles

Abstract 2025: A distinct role of RhoB in simvastatin-induced cytotoxicity in breast cancer cells

Abstract Purpose Statins, a class of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are initially developed as cholesterol-lowering drugs by inhibiting the mevalonate pathway. They have recently gained attention for their potential anticancer properties, but the mechanisms of their anticancer effects remain elusive. This study aims to investigate the antiproliferative effects of statins in breast cancer cell lines. Methods We screened a panel of breast cancer cell lines (Estrogen receptor positive (ER+): CAMA1, MCF7, T47D, ZR-75-1 and Hcc1428; triple negative (TNBC): MDA MB231, MDA MB468, BT549, Hs578T and Hcc1806) and assessed the sensitivity of these cells to simvastatin in relation to their hormone receptor status using cell proliferation assay. Next, to elucidate the mechanisms underlying simvastatin sensitivity, we evaluated the (i) expression of key enzymes in the mevalonate pathway using qPCR and western blot analysis and (ii) the role of sterol biosynthesis metabolites by exogenously adding these compounds and testing their effects on simvastatin-induced cytotoxicity. Finally, we performed functional assays following siRNA knockout to identify the key small GTPase involved. Results We found TNBC cell lines to be more susceptible to simvastatin compared to ER+ cell lines (mean IC50 of 7.98μM [95%CI 2.75-13.22] versus 41.74μM [95%CI 6.71-76.78]). Simvastatin treatment induced robust apoptosis in TNBC but not the ER+ cells. There was no significant difference in mRNA expression of HMGCR, HMGCS, MVK, MVD, IDI1 enzymes involved in the mevalonate pathway between TNBC and ER+ breast cancer cell lines. Supplementation with geranylgeranyl pyrophosphate but not farnesyl pyrophosphate, inhibited simvastatin-mediated cell death, suggesting the involvement of geranylgeranylated proteins such as Rho family of GTPases. Here, we identified RhoB to be a key player in simvastatin-mediated cytotoxicity. RhoB showed a significant increase in transcript and protein levels in response to simvastatin treatment in the TNBC cells. Interestingly, the increase in protein expression was not associated with an increase in its prenylation as the accumulation of RhoB was observed only in the cytosolic fraction. Finally, silencing RhoB was able to abrogate the cytotoxic effects mediated by simvastatin in TNBC cells. Conclusions Taken together, our data reveals a critical role of RhoB in the anticancer activity of simvastatin, which appears to be exclusive to TNBC cells suggesting that HMG-CoA reductase inhibitors may provide new therapeutic strategies for TNBC treatment. Citation Format: Serena Seah, Tingting Wang, Soo Chin Lee. A distinct role of RhoB in simvastatin-induced cytotoxicity in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2025. doi:10.1158/1538-7445.AM2017-2025

Abstract 1919: Twist1 modulates paclitaxel-induced cancer stem cell enrichment in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancers, is highly refractory to chemotherapy and has a high incidence of metastasis and tumor relapse. Targeted therapies for TNBC do not exist because this subtype lacks druggable hormone receptors and HER2 amplification or overexpression. Although Taxane-based chemotherapy is a standard of care for TNBC treatment, paclitaxel (Taxol)-induced cancer stem cell (CSC) enrichment presents a significant challenge to the success of breast cancer treatment. Thus, there is a critical need to understand the mechanisms by which chemotherapy induces CSC enrichment in residual tumors following anticancer therapies. Here we report that Taxol-induced enrichment for CSCs correlates well with a marked upregulation of Twist1 expression in human TNBC cells. Knockdown of Twist1 inhibits the clonogenic capacity of TNBC cells in vitro and TNBC tumorigenesis in vivo. Mammosphere formation assays indicate that silencing of Twist1 greatly diminishes the tumor sphere-forming potential of breast CSCs. Furthermore, limited dilution assays and stem cell xenotransplantation reveal that knockdown of Twist1 expression decreases the frequency of CSCs by 72-fold in human TNBC cells. More importantly, we show that silencing of Twist1 blocks Taxol-induced CSC enrichment in residual tumor cells that have survived drug treatment. Together, these results demonstrate a previously unrecognized role for Twist1 in mediating Taxol-induced CSC enrichment in residual tumors of human TNBC and suggest that targeted inhibition of the Twist1 signaling pathway may represent a novel therapeutic strategy to tackle therapy-induced CSC enrichment in breast cancer treatment. Citation Format: Aimin Yang, Xiaoyuan He, Bradley A. Schulte, Steven L. Carroll, Stephen P. Ethier, Gavin Y. Wang. Twist1 modulates paclitaxel-induced cancer stem cell enrichment in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1919. doi:10.1158/1538-7445.AM2017-1919

Targeted Degradation of BET Proteins in Triple-Negative Breast Cancer.

Triple-negative breast cancers (TNBC) remain clinically challenging with a lack of options for targeted therapy. In this study, we report the development of a second-generation BET protein degrader, BETd-246, which exhibits superior selectivity, potency, and antitumor activity. In human TNBC cells, BETd-246 induced degradation of BET proteins at low nanomolar concentrations within 1 hour of exposure, resulting in robust growth inhibition and apoptosis. BETd-246 was more potent and effective in TNBC cells than its parental BET inhibitor compound BETi-211. RNA-seq analysis revealed predominant downregulation of a large number of genes involved in proliferation and apoptosis in cells treated with BETd-246, as compared with BETi-211 treatment that upregulated and downregulated a similar number of genes. Functional investigations identified the MCL1 gene as a critical downstream effector for BET degraders, which synergized with small-molecule inhibitors of BCL-xL in triggering apoptosis. In multiple murine xenograft models of human breast cancer, BETd-246 and a further optimized analogue BETd-260 effectively depleted BET proteins in tumors and exhibited strong antitumor activities at well-tolerated dosing schedules. Overall, our findings show that targeting BET proteins for degradation represents an effective therapeutic strategy for TNBC treatment. Cancer Res; 77(9); 2476-87. ©2017 AACR.

Abstract 2086: Noscapine chemosensitization enhances docetaxel anticancer activity and tumor stroma disruption against triple negative breast cancer

Abstract Purpose: The use of Noscapine (Nos) as a chemosensitizer followed by docetaxel (DTX) treatment therapy could be a novel approach for the treatment for breast cancer and possibly reduce the adverse side effects associated with DTX based chemotherapy. The goal of this study was to examine the chemo-sensitizing effect of Nos to DTX and also tumor stromal disruption effect of Nos in mice bearing xenograft TNBC tumors. Methods: Effect of Nos chemosensitization on DTX cytotoxicity was evaluated in MDA-MB-231 cells by trypan blue dye method. Apoptosis was measured by AnnexinV/FITC method using flow cytometer. Expression of different proteins like phospho-p38, pJNK, bcl-2, α-tubulin, Akt, pAkt, survivin was evaluated by immunoblot. Alpha-tubulin binding assay was done by fluorescent microscopy. In vivo antifibrotic efficacy of Nos and uptake of coumarin-6 loaded fluorescent liposomes was evaluated by picro-sirius red staining and fluorescent microscopy respectively in xenograft breast tumors. Results: MDA-MB-231 TNBC cells were exposed at sub-therapeutic dose of Nos (4 μM) which increased the cytotoxicity of DTX by 3.0-fold. Flow-cytometric analysis showed significant increase (30 percent) of late apoptotic cells in Nos chemosensitized, DTX-treated MDA-MB-231 cells compared with DTX alone treatment. Further, chemosensitization of TNBC cells with Nos at different time intervals (6 h, 12 h and 24 h), the effect on stress transducer p38 stress activator protein kinase was significantly activated (p<0.01). Also, Nos (4 μM) chemosensitized cells followed by DTX treatment showed downregulation of bcl2 (2.3-fold), survivin (1.3-fold) and pAKT (1.9-fold) expression further illustrating the role of Nos in inducing apoptosis. Interestingly, at this dose of Nos (4 μM), there was no impact on alpha-tubulin. Also, in vivo studies with 100 mg/kg Nos given orally to MDA-MB-231 tumor bearing athymic nude mice revealed significant reduction in tumor collagen-1 levels in Nos treated group compared to control as determined by picro-sirius staining. In Nos treated tumors, uptake of coumarin-6 loaded fluorescent liposomes was 7-fold higher compared to controls suggesting antifibrotic role of Nos. Conclusion: In conclusion, chemosensitization with sub-therapeutic dose of oral noscapine could be a promising approach to increase anticancer activity of DTX and can further enhance uptake of liposomes suggesting that this approach may have potential in TNBC treatment. Citation Format: Ravi Doddapaneni, Ketan Patel, Nusrat Chowdhury, Mandip Sachdeva. Noscapine chemosensitization enhances docetaxel anticancer activity and tumor stroma disruption against triple negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2086.

Abstract 4115: Inhibition of triple negative breast cancer cell invasion by the targeted interference of Sin3A function affecting Wnt and TGFβ signaling

Abstract Cancer cell invasion is an obligatory step for metastatic dissemination that contributes to rapid relapse and a poor survival in TNBC patients. Development of novel therapeutic strategies to block tumor invasion is an unmet need for TNBC treatment and for other tumor types. We reported that decoys with the SID sequence designed to bind and inhibit the function of PAH-2 domain of Sin3A protein markedly prolong survival in the adjuvant setting due to inhibition of metastatic dissemination to the lungs and bone marrow in TNBC mouse models. Here, we show that TNBC cell lines treated with SID decoys (peptides) display a strong in vitro inhibition of migration and invasion. This is accompanied by actin cytoskeleton reorganization with increased cortical actin, and inhibition of proteolytic enzymes (MMP9; MT-MMP1 and uPA) involved in extracellular matrix degradation. DNA microarray and Ingenuity pathway analysis (IPA) showed that the SID decoys inhibit Wnt and TGFβ signaling that is associated with epithelial to mesenchymal transition (EMT). Treatment with SID decoy peptide downregulated WNT/β-catenin-driven transactivation as measured by decreased promoter H3K4me3 and decreased expression of Wnt target genes like LEF1 and TCF7L2. We also show that SID decoys induce translocation of nuclear β-catenin to the cytoplasm in TNBC at 24 hours. Wnt/β-catenin is critical for EMT, cancer stem cell self-renewal, and early invasion in TNBC. TGIF1, a transcription factor that modulates TGFβ and Wnt signaling pathways and known to to interact with the PAH2 domain of Sin3A, can be dissociated from Sin3A complex by SID decoy treatment as measured by co-immunoprecipitation (Co-IP) and proximity linked assay. DNA microarray of SID peptide treated TNBC cells shows inhibition of TGFβ signaling evidenced by downregulation of MMP9, MT1-MMP and PLAU, known target genes of this pathway. This is in line with inhibition of the EMT program predicted by the IPA analysis in SID peptide treated TNBC. Taken together, the results indicate that SID decoys have potential value as therapeutic agents to revert the EMT program in TNBC that should translate into the inhibition of metastasis dissemination and eradication of residual disease in TNBC. To test this in clinic future investigations will involve the use of our previously identified small molecule mimetic of SID peptide, selamectin that is also a FDA approved drug. Use of a recently constructed cyclic stapled peptide that inhibits PAH-2 binding and invasion at <10nM is also anticipated. Citation Format: Yeon-Jin Kwon, Boris A. Leibovitch, Nidhi Bansal, Lutecia Pereira, Edgardo V. Ariztia, Kevin Petrie, Arthur Zelent, Ming-Ming Zhou, Eduardo F. Farias, Samuel Waxman. Inhibition of triple negative breast cancer cell invasion by the targeted interference of Sin3A function affecting Wnt and TGFβ signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4115.

Abstract C50: Sensitivity of triple-negative breast cancer cell lines to cytotoxic and targeted agents as a function of molecular subtype

Abstract Triple-negative breast cancer is a heterogeneous disease with no approved targeted therapies. The disease course is very aggressive, characterized by relapse within 3 years of treatment and metastases to the viscera, including the brain. The prognosis in the setting of distant metastases is grim, as median survival is less than one year. Recently, insight into the molecular complexity of TNBC was provided by gene expression analysis, which identified six unique subtypes with distinct gene ontologies that differ with respect to drug sensitivity. These subtypes, each enriched in unique molecular components and pathways, suggest target-based strategies for treatment. A multiplicity of targeted drugs has entered clinical trials for the potential treatment of TNBC, but no uncontested victor has emerged that can appreciably extend overall survival. For instance, one of the most promising experimental drugs is iniparib, which significantly improved outcomes in a Phase II clinical trial of metastatic TNBC when combined with carboplatin/gemcitabine; however, overall survival was still unacceptably short at ~1 year. Therefore, there remains a dire need to identify novel therapeutic modalities for TNBC. A promising research strategy towards this goal is to determine the differential sensitivities of the TNBC molecular subtypes to combinations of cytotoxic and targeted drugs. We are determining the responses of a panel of TNBC cell lines representing different molecular subtypes, including the basal-like 1 (HCC1143 and MDA-MB-468), basal-like 2 (HCC1806 and HCC70), immunomodulatory (HCC1187), mesenchymal stem-like (MDA-MB-436, MDA-MB-231, and MDA-MB-157), and luminal androgen receptor (MFM-223) subtypes, to variable doses of carboplatin, capecitabine, cyclophosphamide, docetaxel, and everolimus, used alone and in combination. These drugs were chosen because they target molecular regulators of prominent signaling pathways specific to certain TNBC molecular subtypes, so we expect to find that sensitivity to these drugs is a function of subtype. The results of this study will enable characterization of TNBC subtypes in finer detail and provide a preclinical framework for the development of subtype-dependent regimens for TNBC treatment. Citation Format: Heather Colley, Roopali Saxena, Angela Ogden, Guilherme Cantuaria, Michelle D. Reid, Xiaoxian (Bill) Li, Uma Krishnamurthy, Padmashree CG Rida, Ritu Aneja. Sensitivity of triple-negative breast cancer cell lines to cytotoxic and targeted agents as a function of molecular subtype. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr C50.

Abstract A05: The tumor suppressor function of Plk2 in triple-negative breast cancer may be mediated through regulation of Plk1

Abstract Triple-negative breast cancers (TNBCs) are highly aggressive, associated with poor prognosis and lack targeted therapies. Current breast cancer therapies target the estrogen (ER), progesterone (PR) and human epidermal growth factor (HER2) receptors, which are absent in TNBCs. Developing new treatment strategies for TNBCs requires a better understanding of the signaling networks regulating TNBCs. Polo-like kinase 1 (Plk1) is a putative oncogene in TNBC. Plk1 is frequently overexpressed and promotes mitotic cell division, making it an attractive target for cancer therapy. Several inhibitors of Plk1 exist, one of which has accelerated to phase III clinical trials for acute myeloid leukemia. However, these drugs also inhibit Plk2, another polo-like kinase family member. The impact that the presence of Plk2 has on the effectiveness of Plk1 inhibitors as a cancer therapy is unknown. We reported recently that a loss of Plk2 in the developing mammary gland results in increased proliferation, hyperbranching, misoriented mitotic spindle assembly and defects in polarity (Villegas et al Development 2015). Loss of Plk2 was accompanied by increased expression of Plk1. Genetic rescue experiments, knocking down Plk1 in Plk2 null mouse mammary epithelium, and bimolecular fluorescence complementation assays, using wildtype Plk2 and a kinase dead mutant (KD) of Plk2 as bait, revealed that Plk2 regulates these processes through its direct interaction with Plk1. Our preliminary data suggest that loss of Plk2 results in increased Plk1 protein but not RNA expression. We propose that Plk2 functions as a tumor suppressor by decreasing Plk1 stability in TNBCs. Loss of Plk2, therefore, may sensitize tumors to treatment with Plk1 inhibitors if these tumors display Plk1 oncogene addiction. We hypothesize that Plk2, through targeted degradation of Plk1, inhibits tumorigenesis in TNBC. We observed that loss of Plk2 alone is not sufficient to generate mouse mammary tumors, however more lesions form after multiple pregnancies in Plk2 null glands than wildtype. To examine the tumor suppressor function of Plk2 through its regulation of Plk1 in TNBC, we are generating preclinical mouse mammary tumor models integrating the germline loss of Plk2 with p53 loss or c-Myc overexpression frequently observed in TNBC. Finally, to investigate the clinical relevance of Plk2 in TNBC, we will use available tissue microarrays of TNBC patient derived xenograft (PDX) mouse models to identify those that exhibit loss of Plk2. We will treat the identified PDX models with Plk1 inhibitors to confirm that Plk2 loss promotes Plk1 in human TNBC samples. With these studies, we expect to find that Plk2 is involved in the targeted degradation of Plk1 in TNBC, sensitizing this aggressive breast cancer subtype to treatment with Plk1 inhibitors. The results of these studies should help validate whether Plk2 is a new biomarker for determining which patients will benefit from Plk1 targeted TNBC treatment. Supported by Susan G. Komen Foundation grant SAC110031. Citation Format: Deanna Acosta, Elizabeth Villegas, Elena Kabotyanski, Celina Montemayor, Sarah J. Kurley, Rocio Dominguez-Vidana, Chad A. Shaw, Thomas F. Westbrook, Jeffrey M. Rosen. The tumor suppressor function of Plk2 in triple-negative breast cancer may be mediated through regulation of Plk1. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A05.

IGF1R inhibition in mammary epithelia promotes canonical Wnt signaling and Wnt1-driven tumors.

Triple-negative breast cancer (TNBC) is an aggressive disease subtype that, unlike other subtypes, lacks an effective targeted therapy. Inhibitors of the insulin-like growth factor receptor (IGF1R) have been considered for use in treating TNBC. Here, we provide genetic evidence that IGF1R inhibition promotes development of Wnt1-mediated murine mammary tumors that offer a model of TNBC. We found that in a double transgenic mouse model carrying activated Wnt1 and mutant Igf1r, a reduction in IGF1R signaling reduced tumor latency and promoted more aggressive phenotypes. These tumors displayed a squamous phenotype with increased expression of keratins 5/6 and β-catenin. Notably, cell lineage analyses revealed an increase in basal (CD29(hi)/CD24(+)) and luminal (CD24(+)/CD61+/CD29(lo)) progenitor cell populations, along with increased Nanog expression and decreased Elf5 expression. In these doubly transgenic mice, lung metastases developed with characteristics of the primary tumors, unlike MMTV-Wnt1 mice. Mechanistic investigations showed that pharmacologic inhibition of the IGF1R in vitro was sufficient to increase the tumorsphere-forming efficiency ofMMTV-Wnt1 tumor cells. Tumors from doubly transgenic mice also exhibited an increase in the expression ratio of the IGF-II-sensitive, A isoform of the insulin receptor versus the IR-B isoform, which when stimulated in vitro resulted in enhanced expression of β-catenin. Overall, our results revealed that in Wnt-driven tumors, an attenuation of IGF1R signaling accelerates tumorigenesis and promotes more aggressive phenotypes with potential implications for understanding TNBC pathobiology and treatment.

Abstract B166: MicroRNAs downstream of ADAM8 as therapeutic targets and non-invasive biomarkers for triple-negative breast cancer.

Abstract Triple-negative breast cancer (TNBC) represents ∼20% of breast cancers, is highly aggressive and lacks targeted therapies due to the absence of hormone and HER2 receptors. TNBCs respond only poorly to chemotherapy and recur more frequently than other breast cancers. Their recurrence often involves distant metastasis, especially to the brain and lungs with a poor prognosis, which lead to approximately 25% of the half million yearly deaths worldwide from breast cancer. Thus, there is a critical unmet need for early detection and new therapeutic targets for TNBC treatment, which was the focus of our current study. Recently, our lab has shown that the transmembrane protein ADAM8 (A Disintegrin And Metalloproteinase 8) plays a key role in invasion and metastasis of TNBCs and high ADAM8 levels correlate with a poor prognosis in breast cancer patients. MicroRNAs (miRNAs) play important functions in both normal and disease development. It is estimated that more than 60% of the protein-coding genes in the human genome are regulated by miRNAs. Furthermore, circulating tumor cell-associated miRNAs and free circulating miRNAs have been identified in whole blood, plasma and serum in various cancer subtypes. Here we tested two related hypotheses, that: (1) miRNAs regulated by the ADAM8 pathway are important mediators promoting invasion and migration of TNBC cells; and (2) these miRNAs represent potential therapeutic targets and/or noninvasive biomarkers for detection of TNBC recurrence. A microarray analysis was performed using TaqMan microRNA cards on RNA isolated from MDA-MB-231 cells after 72 h of transient siRNA-mediated knockdown of ADAM8. We identified 68 miRNAs that were differentially regulated upon ADAM8 knockdown. Thus far we have confirmed 13 miRNAs using real-time PCR. Of the miRNAs confirmed, miR-720 was selected as the initial candidate of study, since it has been previously identified as an oncomiR and also detected in blood from breast cancer patients. Transient ectopic ADAM8 overexpression in MDA-MB-231 and SUM149 TNBC cell lines induced levels of miR-720 whereas ADAM8 siRNA reduced them. Furthermore, knockdown of miR-720 using an antagomiR led to a significant decrease of the migratory and invasive phenotype of TNBC cells as judged by Boyden chamber and 3D Matrigel outgrowth assays, respectively. Consistently, we used a miRNA mimic to overexpress miR-720 activity and observed increased migration and invasion of TNBC cells. Importantly, a profound increase in levels of miR-720 was detected in the plasma of mice bearing orthotopic mammary tumors derived from control shRNA-MDA-MB-231 cells in contrast to shADAM8-MDA-MB-231 cells and non-tumor bearing mice, which suggests that miR-720 levels in blood correlate with ADAM8 expression in tumors. Taken together our data indicates that the miR-720 is induced by ADAM8 and miRNAs regulated downstream of ADAM8 have a strong potential as both biomarkers and therapeutic targets for the treatment of TNBC. Research was funded by grants from the DOD (W81XWH-11-1-0814) and the NIH (R01 CA129129) Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B166. Citation Format: Sonia G. Das, Mathilde Romagnoli, Nora D. Mineva, Gail E. Sonenshein. MicroRNAs downstream of ADAM8 as therapeutic targets and non-invasive biomarkers for triple-negative breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B166.

Abstract B030: The HBP1 transcriptional repressor: An unexpected window on breast cancer metabolism in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) represents 20-25% of sporadic breast cancers, lacks ER, PR, and overexpressed Her2 – and thus has no targeted treatment options. TNBC is the most clinically challenging subtype with exceptionally poor prognosis, high recurrence and metastases and currently represents an unmet medical need. While classified pathologically by negative criteria, TNBC is a heterogeneous group of breast cancers in need of improved molecular classification. Numerous studies have linked EGFR signaling to TNBC, but paradoxically, TNBC is refractory to the well-used EGFR inhibitors that have been efficacious in other cancers. In addition, Wnt signaling has been linked to TNBC, but its relationship to EGFR signaling and inhibitor resistance has not been explored. In the current work, the HBP1 transcriptional repressor may be a new and unappreciated bridge to both TNBC pathways. Previously, we have shown that decreases in HBP1: 1) triggered an increase in both Wnt and EGFR signaling to regulate proliferation and senescence; 2) were coincident with exceptional increased tumor growth and invasiveness in preclinical models of breast cancer; and 3) were associated with a poor breast cancer prognosis. While altered metabolism has been investigated in many cancers, the mechanisms by which EGFR and Wnt signaling might trigger a metabolic reprogramming have not been investigated in TNBC. Our data suggests that HBP1 may be a new integrating factor. Our new data show that HBP1 levels are significantly reduced in TNBC relative to other breast cancer subtypes in clinical specimens. We derived a new combined EGFR and HBP1 gene signature that predicted 90+% of TNBC patients in 4 large patient databases and additionally predicted a poor patient prognosis in non-TNBC patients. To understand how HBP1 decreases trigger a poor prognosis, a whole genome analysis revealed a surprising change in 300+ genes to enact a Warburg-like metabolic reprogramming. The Warburg effect is characterized by increased glycolytic flux with increased biosynthesis of amino acids, lipids, and nucleic acids'all to provide for the increased growth and proliferation demands of a tumor cell. The specific pattern was an increase in certain isoforms involved in glycolysis and TCA cycle and a decrease in the expression of genes involved in oxidative phosphorylation. We used gene-, biochemistry- and NMR-based approaches to investigate the metabolic alterations upon changes in HBP1 expression in TNBC cells and tumors. Our results show that hexokinase 2 (HK2), Pyruvate Kinase-M2 (PKM2), Lactate Dehydrogenase A (LDHA), malate dehydrogenase 2 (MDH2) are amongst the metabolic enzymes that are altered with HBP1 expression and with increased Wnt signaling. HK2, PKM2, LDHA and MDH2 have all been previously associated with the Warburg effect. The NMR and biochemical analysis showed that lactate and fumarate are amongst the metabolites that are altered. The results here provide important metabolomics information to impact MRI/MRS studies. While breast MRIs are used for assessing tumor morphology, MRS (magnetic resonance spectroscopy) methods can non-invasively visualize metabolic spectra in tumors or metastases. The metabolic changes from our studies potentially focus MRS/MRI applications for the future discovery of non-invasive MRS biomarkers to improve TNBC classification and treatment efficacy. (Supported by a grant to ASY and JB from the Dept. of Defense). Citation Format: Amy S. Yee, Kurtz Eric Paulson, Kai Wang, Wesley Field, Brian Pedro, Davis Vigneault, James B. Baleja. The HBP1 transcriptional repressor: An unexpected window on breast cancer metabolism in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B030.

Abstract 3078: Mifepristone suppresses triple-negative breast cancer cell growth via miR-153 mediated down-regulation of KLF5 expression.

Abstract Triple-negative breast cancer (TNBC) is one of the most malignant breast cancers without effective therapeutic approaches currently. It is reported that KLF5 is over-expressed in TNBC; and KLF5 depletion suppresses TNBC cell proliferation, cell survival and tumor growth in vivo significantly, which implies that KLF5 may be a therapeutic target for TNBC treatment. As a progesterone receptor antagonist, mifepristone (MIF) has been found to have anti-tumor activity in multiple hormone-dependent cancers, including luminal type breast cancers. Interestingly, we found that MIF is also able to suppress KLF5 protein expression in TNBC cell and inhibit TNBC cell growth in vitro and in vivo. We further demonstrated that MIF does not suppress KLF5 mRNA expression at all, but down-regulates KLF5 protein level via up-regulating miR-153 expression in TNBC cells. We found miR-153 mimics can down-regulates KLF5 protein level in TNBC cells similar to MIF does; and consistently, miR-153 inhibitor rescues MIF-induced down-regulation of KLF5 protein expression. Taken together, our findings suggest that MIF is able to inhibit TNBC growth possibly via suppressing KLF5 expression; and our research may provide a new possible chemotherapeutic for the treatment of TNBC. Citation Format: Rong Liu, Ceshi Chen. Mifepristone suppresses triple-negative breast cancer cell growth via miR-153 mediated down-regulation of KLF5 expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3078. doi:10.1158/1538-7445.AM2013-3078

Inhibiting Aurora Kinases Reduces Tumor Growth and Suppresses Tumor Recurrence after Chemotherapy in Patient-Derived Triple-Negative Breast Cancer Xenografts

Triple-negative breast cancers (TNBC) have an aggressive phenotype with a relatively high rate of recurrence and poor overall survival. To date, there is no approved targeted therapy for TNBCs. Aurora kinases act as regulators of mammalian cell division. They are important for cell-cycle progression and are frequently overexpressed or mutated in human tumors, including breast cancer. In this study, we investigated the therapeutic potential of targeting Aurora kinases in preclinical models of human breast cancers using a pan-inhibitor of Aurora kinases, AS703569. In vitro, AS703569 was tested in 15 human breast cancer cell lines. TNBC cell lines were more sensitive to AS703569 than were other types of breast cancer cells. Inhibition of proliferation was associated with cell-cycle arrest, aneuploidy, and apoptosis. In vivo, AS703569 administered alone significantly inhibited tumor growth in seven of 11 patient-derived breast cancer xenografts. Treatment with AS703569 was associated with a decrease of phospho-histone H3 expression. Finally, AS703569 combined to doxorubicin-cyclophosphamide significantly inhibited in vivo tumor recurrence, suggesting that Aurora kinase inhibitors could be used both in monotherapy and in combination settings. In conclusion, these data indicate that targeting Aurora kinases could represent a new effective approach for TNBC treatment.

Identification of AGR3 as a potential biomarker though public genomic data analysis of triple-negative (TN) versus triple-positive (TP) breast cancer (BC).

31 Background: TNBCs have an aggressive clinical phenotype with worse prognosis than TPBC. Here we sought to identify other genomic changes in patients with TN vs. TPBC, by mining TCGA breast cancer genomic data using the NextBio Clinical platform. Methods: Breast cancer patients with decreased (n=51) and increased (n=46) ER/PR/HER2 mRNA levels were selected with the molecular filter in NB Clinical. Top-ranked differentially expressed genes were identified then analyzed via NB Research, which comprises a library of individually curated publically available research and clinical grade genomic data. Results: Whole transcriptome analysis of TNBC and TPBC patients identified anterior gradient-3 (AGR3), a protein disulfide isomerase, as most downregulated (TNBC vs. TPBC, fold change = -178.2, p=2.7E-32). AGR3 mRNA was reduced in 94% of TNBC and increased in 96% of TPBC patients. AGR3 was hypomethylated in 50% of TPBC vs. 4% of TNBC patients, suggesting regulation by methylation. When the entire breast cancer cohort was stratified by AGR3 expression, underexpression was prevalent in younger patients (<50 yr, 39%) compared to those with overexpression (<50 yr, 23%). Further, 63% of AGR3 underexpressing patients had severe TP53 mutations vs. 19% with AGR3 overexpression. Breast cancer was most correlated to AGR3 in the NextBio Disease Atlas, with supporting data from 110 studies. AGR3 expression was decreased in ER- vs. ER+, PR- vs. PR+, HER2- vs. HER2+, and G3 vs. G1 tumors, and AGR3 promoter was hypermethylated in ER- vs. ER+, consistent with the TCGA results. Body Atlas showed strongest AGR3 expression in mucosa, fallopian tube, breast, and epithelial cells, in line with its proposed role in epithelial barrier function. Knockdown Atlas results showed TP53 mutation associated with reduced AGR3 mRNA, and the PharmacoAtlas showed positive regulation of AGR3 by estrogen analogues. Conclusions: Using the NextBio Clinical and Research platforms, we identified AGR3 as a marker of TNBC vs. TPBC that correlates with TP53 mutation status. AGR3 may provide an alternate route of TNBC treatment. Its potential in regulating epithelial barrier function may provide insights into the mechanism of disease progression.

Abstract 15: Targeting cancer stem cells and vasculature by a novel anti-delta-like 4 ligand (DLL4) antibody inhibits triple-negative breast cancer tumor growth and delays tumor recurrence

Abstract Triple negative breast cancer (TNBC, ER−, PR− Her2−) accounts for about 10% of all types of breast cancer and is known to particularly aggressive and refractory to current therapies. Chemotherapeutic agents, such as taxanes, are currently the only treatment option for this type of breast cancer, however, these treatments often result in high rates of local and systemic relapse. It has been suggested that cancer stem cells drive tumor growth and progression and are preferentially resistant to many current therapies. Delta-like 4 ligand (DLL4) is an important component of the Notch signaling pathway which is known to mediate stem cell self-renewal and vascular development. We hypothesized that targeting cancer stem cells and the tumor vasculature by interfering with the DLL4/Notch pathway will improve treatment outcome. We developed anti-DLL4 antibodies that recognize either the human or murine protein and have potent binding and antagonist activities. We used these antibodies to investigate the role of DLL4 in triple negative breast cancer and to probe the mechanism in tumor and stromal cells in xenograft models derived from primary breast tumors. These studies showed that anti-DLL4 was efficacious as a single agent and in combination with paclitaxel (Taxol) against TNBC xenograft tumors. Gene expression analysis showed that anti-DLL4 affected vascular-related genes in the stroma and Notch target genes in the tumor and stroma. Inclusion of anti-DLL4 delayed breast tumor recurrence and decreased the growth rate of recurrent tumors following termination of paclitaxel treatment. Flow cytometric analysis showed that ESA+/CD44+/CD24low expressing breast cancer stem cells, BrCSC, were enriched followed by paclitaxel treatment, whereas the combination of anti-DLL4 and paclitaxel reversed paclitaxel-induced increase in this cell population. Further analysis indicated that blocking DLL4 signaling in tumor cells by anti-human DLL4 delayed tumor recurrence and decreased ESA+/CD44+/CD24low expressing BrCSC fraction and tumorigenic potential, whereas blocking DLL4 in host stroma and endothelial cells by anti-mouse DLL4 interfered with angiogenesis. The combination of both antibodies produced an additive effect. Our findings provide a rationale for targeting cancer stem cells and tumor vasculature through inhibition of the DLL4/Notch signaling for TNBC treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 15.