Nivetha Ganesan

Annexin A1 Preferentially Predicts Poor Prognosis of Basal-Like Breast Cancer Patients by Activating mTOR-S6 Signaling

Introduction Annexin A1 (ANXA1) is an anti-inflammatory protein reported to play a role in cell proliferation and apoptosis, and to be deregulated in breast cancer. The exact role of annexin A1 in the biology of breast cancer remains unclear. We hypothesized that the annexin A1 plays an oncogenic role in basal subtype of breast cancer by modulating key growth pathway(s). Methods By mining the Cancer Genome Atlas (TCGA)-Breast Cancer dataset and manipulating annexin A1 levels in breast cancer cell lines, we studied the role of annexin A1 in breast cancer and underlying signaling pathways. Results Our in-silico analysis of TCGA-breast cancer dataset demonstrated that annexin A1 mRNA expression is higher in basal subtype compared to luminal and HER2 subtypes. Within the basal subtype, patients show significantly poorer overall survival associated with higher expression of annexin A1. In both TCGA patient samples and cell lines, annexin A1 levels were significantly higher in basal-like breast cancer than luminal and Her2/neu-positive breast cancer. Stable annexin A1 knockdown in TNBC cell lines suppressed the mTOR-S6 pathway likely through activation of AMPK but had no impact on the MAPK, c-Met, and EGFR pathways. In a cell migration assay, annexin A1-depleted TNBC cells showed delayed migration as compared to wild-type cells, which could be responsible for poor patient prognosis in basal like breast cancers that are known to express higher annexin A1. Conclusions Our data suggest that annexin A1 is prognostic only in patients with basal like breast cancer. This appears to be in part due to the role of annexin A1 in activating mTOR-pS6 pathway.

Regulation of miRNA-29c and its downstream pathways in preneoplastic progression of triple-negative breast cancer

Little is understood about the early molecular drivers of triple-negative breast cancer (TNBC), making the identification of women at risk and development of targeted therapy for prevention significant challenges. By sequencing a TNBC cell line-based breast cancer progression model we have found that miRNA-29c is progressively lost during TNBC tumorigenesis. In support of the tumor suppressive role of miRNA 29c, we found that low levels predict poor overall patient survival and, conversely, that ectopic expression of miRNA-29c in preneoplastic cell models inhibits growth. miRNA-29c exerts its growth inhibitory effects through direct binding and regulation of TGFB-induced factor homeobox 2 (TGIF2), CAMP-responsive element binding protein 5 (CREB5), and V-Akt murine thymoma viral oncogene homolog 3 (AKT3). miRNA-29c regulation of these gene targets seems to be functionally relevant, as TGIF2, CREB5, and AKT3 were able to rescue the inhibition of cell proliferation and colony formation caused by ectopic expression of miRNA-29c in preneoplastic cells. AKT3 is an oncogene of known relevance in breast cancer, and as a proof of principle we show that inhibition of phosphoinositide 3-kinase (PI3K) activity, a protein upstream of AKT3, suppressed proliferation in TNBC preneoplastic cells. We explored additional opportunities for prevention of TNBC by studying the regulation of miRNA-29c and identified DNA methylation to have a role in the inhibition of miRNA-29c during TNBC tumorigenesis. Consistent with these observations, we found 5 aza-cytadine to relieve the suppression of miRNA-29c. Together, these results demonstrate that miRNA-29c loss plays a key role in the early development of TNBC.

Suppression of Akt-mTOR Pathway-A Novel Component of Oncogene Induced DNA Damage Response Barrier in Breast Tumorigenesis

DNA damage has been thought to be directly associated with the neoplastic progression by enabling mutations in tumor suppressor genes and activating/and amplifying oncogenes ultimately resulting in genomic instability. DNA damage causes activation of the DNA damage response (DDR) that is an important cellular mechanism for maintaining genomic integrity in the face of genotoxic stress. While the cellular response to genotoxic stress has been extensively studied in cancer models, less is known about the cellular response to oncogenic stress in the premalignant context. In the present study, by using breast tissues samples from women at different risk levels for invasive breast cancer (normal, proliferative breast disease and ductal carcinoma in situ) we found that DNA damage is inversely correlated with risk of invasive breast cancer. Similarly, in MCF10A based in vitro model system where we recapitulated high DNA damage conditions as seen in patient samples by stably cloning in cyclin E, we found that high levels of oncogene induced DNA damage, by triggering inhibition of a major proliferative pathway (AKT), inhibits cell growth and causes cells to die through autophagy. These data suggest that AKT-mTOR pathway is a novel component of oncogene induced DNA damage response in immortalized ‘normal-like’ breast cells and its suppression may contribute to growth arrest and arrest of the breast tumorigenesis.

Abstract P4-15-03: Regulation of miRNA-29c and its gene targets in preneoplastic progression of triple negative breast cancer

Introduction: Little is understood about the early molecular drivers of the triple negative breast cancer making identification of women at risk and development of targeted therapy for prevention a significant challenge. Methods: Here, by deep sequencing of TNBC- cell line based breast cancer progression system we have identified miRNA-29c and its functional gene targets to be potentially involved in the normal to preneoplastic transition during TNBC progression. We have used cell line based functional assays that are relevant in early tumorigenesis such cell proliferation (ki67), and colony formation assay to study the growth inhibitory potential of these miRNA and their gene targets. To identify direct gene targets of miRNA-29c, we cloned the 39untranslated region containing miRNA-29c binding sites from predicted gene targets in a luciferase reporter vector, pmiRGLO and studied the potential of miRNA-29c overexpression on the repression of luciferase reporter activity indicating their direct gene regulation. Results: Our deep sequencing results and their further validation by QPCR revealed miRNA-29c to be lost during the TNBC progression, and its forced expression to inhibit cell proliferation and colony formation of preneoplastic (MCF10AT1) and ductal carcinoma in situ (MCF10DCIS) cells. We found miRNA-29c to directly bind in 39UTR of TGIF2, CREB5, AKT3 and CDK6 and regulate their expression as shown by our luciferase assays. We also found miRNA-29c binding to 39UTR of these gene targets to be functionally relevant as TGIF2, CREB5 and AKT3 were able to rescue the inhibition in cell proliferation and colony formation assay caused by loss of miRNA-29c in preneoplastic cells. Further confirming the relevance of these miRNA-29c gene targets and pathways in TNBC tumorigenesis, inhibition of PI3K, which is upstream of AKT3, inhibits cell proliferation in MCF10AT1 and DCIS cells. We also examined the regulation of tumor suppressor miRNA-29c to study the mechanisms responsible for its loss during breast cancer development. We found c-myc and EZH2 driven epigenetic mechanism as well as DNA methylation in part to cause the loss of miRNA-29c during TNBC progression. Consistently, we found a pan HDAC inhibitor and a DNA methylation inhibitor to relieve the suppression of miRNA-29c. Conclusions: Together, these results indicate that loss of miRNA-29c plays a central role in preneoplastic development of breast cancer and efforts directed at inhibition of its target pathways or rescue of miRNA-29c itself may provide novel opportunities for prevention of TNBC.Introduction: Little is understood about the early molecular drivers of the triple negative breast cancer making identification of women at risk and development of targeted therapy for prevention a significant challenge. Methods: Here, by deep sequencing of TNBC- cell line based breast cancer progression system we have identified miRNA-29c and its functional gene targets to be potentially involved in the normal to preneoplastic transition during TNBC progression. We have used cell line based functional assays that are relevant in early tumorigenesis such cell proliferation (ki67), and colony formation assay to study the growth inhibitory potential of these miRNA and their gene targets. To identify direct gene targets of miRNA-29c, we cloned the 39untranslated region containing miRNA-29c binding sites from predicted gene targets in a luciferase reporter vector, pmiRGLO and studied the potential of miRNA-29c overexpression on the repression of luciferase reporter activity indicating their direct gene regulation. Results: Our deep sequencing results and their further validation by QPCR revealed miRNA-29c to be lost during the TNBC progression, and its forced expression to inhibit cell proliferation and colony formation of preneoplastic (MCF10AT1) and ductal carcinoma in situ (MCF10DCIS) cells. We found miRNA-29c to directly bind in 39UTR of TGIF2, CREB5, AKT3 and CDK6 and regulate their expression as shown by our luciferase assays. We also found miRNA-29c binding to 39UTR of these gene targets to be functionally relevant as TGIF2, CREB5 and AKT3 were able to rescue the inhibition in cell proliferation and colony formation assay caused by loss of miRNA-29c in preneoplastic cells. Further confirming the relevance of these miRNA-29c gene targets and pathways in TNBC tumorigenesis, inhibition of PI3K, which is upstream of AKT3, inhibits cell proliferation in MCF10AT1 and DCIS cells. We also examined the regulation of tumor suppressor miRNA-29c to study the mechanisms responsible for its loss during breast cancer development. We found c-myc and EZH2 driven epigenetic mechanism as well as DNA methylation in part to cause the loss of miRNA-29c during TNBC progression. Consistently, we found a pan HDAC inhibitor and a DNA methylation inhibitor to relieve the suppression of miRNA-29c. Conclusions: Together, these results indicate that loss of miRNA-29c plays a central role in preneoplastic development of breast cancer and efforts directed at inhibition of its target pathways or rescue of miRNA-29c itself may provide novel opportunities for prevention of TNBC. Citation Format: Bhardwaj A, Tachibana K, Ganesan N, Rajapakshe K, Singh H, Gunaratne P, Coarfa C, Bedrosian I. Regulation of miRNA-29c and its gene targets in preneoplastic progression of triple negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-15-03.

Abstract 3982: Targeting of miRNA networks for prevention of basal-like breast cancers

Introduction: Approximately 75% of women who develop sporadic breast cancer have no known epidemiological risk factors. This creates a significant challenge for identification of at risk patients for prevention. We hypothesized that detailing the molecular signature of the precancerous state by subtype will provide opportunities to identify biomarkers of subtype specific breast cancer risk and identify pathways that can be targeted for subtype specific prevention. Experimental procedures: Using cell lines that model breast cancer progression and The Cancer Genome Atlas (TCGA) genome-characterization datasets, we have identified common microRNA (miRNA)-gene signatures as potential biomarkers for early detection and targets for prevention of basal/ triple negative breast cancer (TNBC). We have performed next generation RNA and small RNA seq of MCF10A cell line based TNBC progression model (10A P; parental, 10AT1; atypia, DCIS; ductal carcinoma in situ and Ca1d; invasive carcinoma) and used cell proliferation assay, western blotting, and miRNA analysis by QPCR to study the effects of target inhibition. Results: We have identified miRNA-29c as a lead candidate because it satisfied 3 key criteria necessary for subtype-tailored intervention. First, it found it to be expressed at high levels in MCF10A (immortalized normal line), and showed a steady decrease in the TNBC progression model. Second, it strongly inhibited migration of the metastatic TNBC cell line, MDA MB 231. Third, its downstream targets include druggable pathways such as src kinase, GSK-3β, Pan-ErbB TK, EGFR, MEK1/2, PI3K, PI3Kγ and c-met. One particularly interesting candidate is DNMT3A, the DNA methyl transferase, which we show to be inversely correlated to miRNA-29c and up-regulated in the progression from normal to cancer in the TNBC model. The miRNA-29c-DNMT3A connection that we uncovered strongly suggests an epigenetic component to tumorigenesis in basal like breast cancers and thus, we have tested natural occurring HDAC inhibitor and a small molecule drug, sulforaphane and sodium butyrate, for their ability to revert cellular phenotype of MCF 10A.AT1 and DCIS cells and found these compounds inhibit cell proliferation. We have also used small molecule synthetic inhibitors to target miRNA-29c regulated pathways and studied their effect on cell proliferation and target inhibition. In these analyses a small molecule inhibitor of PI3K/AKT, LY294002 effectively inhibited the AKT signaling and cell proliferation in MCF10A.AT1, and MCF10A.DCIS cells. Similarly, MEK inhibitors PD032590 and GSK1120212 inhibited MEK signaling and cell proliferation in both hyperplastic and DCIS cells. Conclusions: Our study suggests that miRNA-29c-network, may play a key role in the development of triple negative of breast cancer and this networks may provide novel opportunities at several levels for prevention of TNBC/ basal like breast cancer. Citation Format: Anjana Bhardwaj, Kazunoshin Tachibana, Nivetha Ganesan, Yinghong Pan, Kimal Rajapakshi, Cristian Coarfa, Preethi H. Gunaratne, Isabelle Bedrosian. Targeting of miRNA networks for prevention of basal-like breast cancers. [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 3982. doi:10.1158/1538-7445.AM2015-3982

Abstract 3906: Annexin A1 overexpression preferentially predicts poor prognosis of basal-like breast cancer patients by activating m TOR-S6 signaling

Introduction: Annexin A1 is an anti-inflammatory protein reported to play a role in cell proliferation, apoptosis and to be deregulated in breast cancer. The role and regulation of annexin A1 in the biology and prognosis of breast cancer remain unclear because most of the studies did not consider specific differences between the different subtypes of breast cancer. Thus, in this study, we hypothesize that annexin A1 plays subtype-specific role in basal like breast cancer progression and metastasis. Methods: By mining The Cancer Genome Atlas-Breast Cancer Dataset (TCGA-BRCA) and manipulating annexin A1 levels in breast cancer cell lines, we studied the role of annexin A1 in breast cancer and underlying signaling pathways. Results: Our in-silico analysis of TCGA-BRCA dataset demonstrated that annexin A1 mRNA expression is higher in basal subtype compared to luminal and HER2 subtypes. Within the basal subtype, patients show significantly poorer survival associated with higher expression of annexin A1. In both TCGA patient samples and cell lines, annexin A1 levels were significantly higher in basal-like breast cancer tumors than luminal and Her2/neu-positive breast cancer tumors. Stable annexin A1 knockdown in TNBC cell lines suppressed the mTOR-S6 pathway. Specifically, annexin A1 knockdown inhibited phosphorylation of mTOR and S6 (18-73% inhibition in pmTOR and 50-60% inhibition in pS6). But annexin A1 knockdown had no impact on the MAPK, c-Met, and EGFR pathways. In cell migration assays, annexin A1-depleted TNBC cells showed delayed migration as compared to wild-type cells which we found to be through suppression of MMP9. Conclusions: Our data suggest that annexin A1 is prognostic only in patients with basal like breast cancer. This appears to be due in part to the role of annexin A1 in activating mTOR-pS6 pathway. Citation Format: Kazunoshin Tachibana, Anjana Bhardwaj, Nivetha Ganesan, Kimal Rajapakshe, Constance T. Albarracin, Preethi H. Gunaratn, Cristian Coarfa, Isabelle Bedrosian. Annexin A1 overexpression preferentially predicts poor prognosis of basal-like breast cancer patients by activating m TOR-S6 signaling. [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 3906. doi:10.1158/1538-7445.AM2015-3906

Abstract A115: Annexin A1: A novel gene target of miRNA-21

Introduction: Numerous studies have reported miRNA-21 (miR-21) to be deregulated in a wide variety of cancers, including breast. miR-21 regulates a number of processes and pathways relevant to tumorigenesis including PDCD4, a regulator of apoptosis; PTEN, a suppressor of PI3K/AKT signaling; Smad7 and MSH2, regulators of TGFβ signaling; and Sprouty 1, an inhibitor of ERK-MAPK signaling. Here we describe an additional gene target of miR-21, annexin A1 that may be particularly relevant in the biology of triple negative breast cancer. Methods: To identify post-transcriptionally regulated genes that may play a role in breast cancer progression, we screened proteome and transcriptome of MCF10A breast cancer progression panel, reported by Choong et al (2010) and Rhee et al (2008) respectively. This resulted in the identification of Annexin A1. To identify miRNAs that have the potential to regulate annexin A1, we performed an in silico screen for miRNAs targeting annexin A1 using the miRNA algorithm TargetScan, and found miR-21 as a potential regulator. To validate if miR-21 indeed regulates annexin A1 we performed transient transfections to modulate miRNA levels in cells and subsequently performed western blot analysis and luciferase assay to measure the annexin A1 protein and reporter levels respectively. To measure endogenous and modulated levels of miRNAs and their effect on mRNA targets we performed QPCR on RNA samples obtained cell lines. Results: Our in silico analysis, suggested that annexin A1 was a potential downstream target of miR-21 with probability of conserved site targeting (PCT ) Citation Format: Anjana Bhardwaj, Nivetha Ganesan, Constance T. Albarracin, Isabelle Bedrosian. Annexin A1: A novel gene target of miRNA-21. [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 A115.

Abstract 634: Suppression of Akt-mTOR pathway: a novel component of oncogene induced DNA damage response (DDR) barrier in breast.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: Activation of the DNA damage response (DDR) is an important cellular mechanism for maintaining genomic integrity in the face of genotoxic stress. While the cellular response to genotoxic stress has been extensively studied in cancer models, less is known about the cellular response to oncogenic stress in the premalignant context. Methods: DNA damage (γ-H2AX), cell proliferation (ki-67) and apoptosis (cleaved caspase-3) were assessed by immunohistochemistry in breast tissue samples from 3 cohorts of women with: i) histologically normal breast tissue (n=50), ii) histological changes of increased risk (LCIS, ADH, ALH) (n=54) but without a personal history of breast cancer and iii) ductal carcinoma in situ (n=46). DNA damage parameters were also assessed in a panel of cell lines derived from the MCF-10A line that represents the multi-step process of breast tumorigenesis (AT1, DCIS, CA1d). Lastly, we generated stable cell clones over expressing cyclin E in the normal mammary epithelial cell line, MCF10A. DNA damage was measured by immuno fluorescence of γ-H2AX in the cell lines models. Apoptosis and autophagy were measured by staining cells with acridine orange and annexin V respectively followed by cell sorting with flow cytometry. Protein expression of AKt-mTOR pathway was assessed by western blotting. Results: Breast tissues samples from women at different risk levels for invasive breast cancer revealed that DNA damage is inversely correlated with breast cancer risk with the highest levels of damage seen in histologically normal tissues and lowest levels in DCIS (p 10 foci= 19%) and lowest levels (> 10 foci= 0%) in the DCIS line and in the invasive cancer cell line CA1d (p<0.001). Overexpression of cyclin E in MCF10A cells resulted in about 4-fold increase in DNA damage and triggered the suppression of AKT-mTOR pathway with resultant cell death through autophagy. Conclusion: DNA damage is inversely associated with progression from normal mammary epithelium to carcinoma. DNA damage induced suppression of the pAKT-mTOR pathway in normal mammary epithelial cells appears to play a role in the tumorigenic barrier. Citation Format: Anjana Bhardwaj, Mei Liu, Yan Liu, Nivetha Ganesan, Daniel Rosen, Carol Etzel, Constance T. Albaracin, Qiang Hao, Ashley Gullett, Isabelle Bedrosian. Suppression of Akt-mTOR pathway: a novel component of oncogene induced DNA damage response (DDR) barrier in breast. [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 634. doi:10.1158/1538-7445.AM2013-634

Abstract 4160: miR-34a as a radio sensitizer of triple negative breast cancer.

Introduction: Triple negative breast cancer (TNBC) is known to be relatively resistant to radiation therapy resulting in a comparatively higher rates of locoregional recurrence compared with non-TNBC patients. The mechanisms behind this relative radio resistance remain unknown. The goal of this study is to investigate the role of three miRNAs namely, miR-34a, miR-21, and miR-210, which are implicated in DNA damage-repair process, in the response of TNBC cell lines to radiation therapy. Methods: Seven TNBC breast cancer cell lines were used for the study. The levels of miR-34a, miR-21 and miR-210 were evaluated by taqman- based quantitative PCR assay. These cells were exposed to 2 Gy radiation dose and colonogenic survival was assessed after about 2 weeks. Survival fraction after 2 Gy (SF2), which is the ratio of number of cell colonies formed after 2 Gy radiation vs. the number of cell colonies with out radiation, was determined. The SF2 factor is a reliable index to measure radio sensitivity, greater the SF2 factor more radio resistant the cells are. Results: The SF2 value of the TNBC cells varied from 0.31 to 0.74 with MDA-MB-468, HCC-70 being the most radio sensitive cell lines and MDA-MB-436, BT-20 being the most radio resistant cell lines. MDA-MB- 231, BT-549 and Hs 578T were found to be moderately radio resistant breast cancer lines. The oncomiR -21 expression levels in these 7 TNBC cell lines varied from 0.85 to 4.18 fold with a very poor correlation with SF2 (r2=0.08). The oncomiR 210 levels varied from 0.49 to 9.19 fold and also did not exhibit any correlation with radio resistance in TNBC cells (r2= 0.141). Whereas the tumor suppressor miR, miR-34a expression showed an inverse correlation with radio resistance (r2=0.8) in TNBC cells. The radio resistant cell line (e.g., MDA-MB-436, SF2 = 0.74) expressed significantly low levels of miR-34a (0.016 fold) relative to miR -34a levels (set as 1) in HCC 70, which was found to be a radiosensitive cell line with SF2 of 0.309. Conclusions: Our preliminary data indicates that miR 34a may play a role in the response of TNBC cells to radiotherapy. Citation Format: Anjana Bhardwaj, Nivetha Ganesan, David Molkentine, Uma Raju, Isabelle Bedrosian. miR-34a as a radio sensitizer of triple negative breast cancer. [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 4160. doi:10.1158/1538-7445.AM2013-4160