Abstract P4-15-03: Regulation of miRNA-29c and its gene targets in preneoplastic progression of triple negative breast cancer
Authors: Bhardwaj A , Tachibana K , Ganesan N , Rajapakshe K , Singh H , Gunaratne P , Coarfa C , Bedrosian I 
Published: March 2017

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 3'untranslated 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 3'UTR of TGIF2, CREB5, AKT3 and CDK6 and regulate their expression as shown by our luciferase assays. We also found miRNA-29c binding to 3'UTR 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 3'untranslated 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 3'UTR of TGIF2, CREB5, AKT3 and CDK6 and regulate their expression as shown by our luciferase assays. We also found miRNA-29c binding to 3'UTR 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.