Abstract P6-01-19: Decoding the genetic basis of mammary mineralization and their putative role in promotion of distant metastases
Authors: Zheng C , Rizwan A , Paidi SK , Yu Z , Barman I , Glunde K 
Published: March 2017

Abstract
Background: Breast microcalcifications are the sole early stage diagnostic markers of breast cancer. The association of mineralization (especially type II microcalcifications) with both benign and malignant lesions often leads to unnecessary biopsies. The processes by which these ectopic microcalcifications form are unknown. In the current work, we attempted to explore the possibility of obtaining genes responsible for the formation of microcalcifications in breast cancer cell lines at cellular level and understand their potential involvement in disease progression and distant metastases.
Methods:The GEO dataset GSE16795 used in this study contains gene expression profiles of 28 human breast cancer cell lines that were divided into two groups - metastatic and non-metastatic. Gene expression levels of OPN were found to be significantly (p=0.0002) elevated for the metastatic group compared to the non-metastatic group. Hence, the human breast cancer cell lines - metastatic (MDA-MB-231 and SUM 149) and non-metastatic (BT-474 and T47D) from the American Type Culture Collection were cultured and their OPN expression at mRNA and protein levels determined by qRT-PCR and Western blotting were compared. Additionally the same cell lines were cultured in media enriched with an osteogenic cocktail containing 10mM β-glycerophosphate (Sigma, USA) and 50 mg/ml-1 ascorbic acid (Sigma, USA) for induction of microcalcifications.Next, several clones were generated using shRNA knockdown of OPN gene in MDA-MB-231 cells for further study. In vitro studies were conducted to assess the effects of OPN knockdown on the migration and invasion potential of MDA-MB-231 cells using transwell migration assays.
Results: The expression of OPN at both mRNA and protein levels are significantly higher for the metastatic cell lines when compared to non-metastatic cell lines. It can also be observed that OPN expression in the cells increases substantially with addition of exogenous phosphates in the form of osteogenic cocktail and thereby indicating that OPN possibly plays a crucial role in mediating formation of microcalcifications in these cells(P&lt;0.001). Metastatic cell line MDA-MB-231 was employed as a model system for further investigation in this study. There is a consistent inhibition of formation of cellular microcalcifications due to the knockdown of gene responsible for OPN, suggesting that OPN gene is directly associated with the formation and regulation of hydroxyapatite formation in MDA-MB-231 breast cancer. Further, the above observation is strengthened by the similarity of the trend of variation in level of mRNA expression of OPN and cellular calcification content across the knockdown clones. OPN knockdown cell lines show reduced cell migration and invasion in the assays compared to the control MDA-MB-231 cells.
Conclusion: The knockdown of OPN gene not only reduced the formation of microcalcifications in the cells in response to osteogenic cocktail but also affected their migration and invasion characteristics. The observed dual roles of the OPN gene encourage us to probe further into the possible existence of a direct relationship between microcalcifications and ability to metastasize to distant organs mediated by common genetic factors in the future.
Citation Format: Zheng C, Rizwan A, Paidi SK, Yu Z, Barman I, Glunde K. Decoding the genetic basis of mammary mineralization and their putative role in promotion of distant metastases [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 P6-01-19.