SHISA3, an antagonist of the Wnt/β-catenin signaling, is epigenetically silenced and its ectopic expression suppresses growth in breast cancer.

Naveed Shahzad,Mariam Javed,Muhammad Akhtar Ali,Abdul Rauf Shakoori,Muhammad Umer,Zeeshan Mutahir,Tanveer Mustafa,Moazzam Ali,Munir Ahmad,Rana Salman Anjum,Kokab Farooq,Tehreem Munir,Fareeda Tasneem,Umair Hassan,Bilal Aslam,Qian Tao

doi:10.1371/journal.pone.0236192

Abstract

Breast cancer (BC) is the foremost cause of cancer related deaths in women globally. Currently there is a scarcity of reliable biomarkers for its early stage diagnosis and theranostics monitoring. Altered DNA methylation patterns leading to the silencing of tumor suppressor genes are considered as an important mechanism underlying tumor development and progression in various cancer types, including BC. Very recently, epigenetic silencing of SHISA3, an antagonist of β-catenin, has been reported in various types of tumor. However, the role of SHISA3 in BC has not been investigated yet. Therefore, we aimed at evaluating the contribution of SHISA3 in BC causation by analyzing its expression and methylation levels in BC cell lines (MDA-MB231, MCF-7 and BT-474) and in 103 paired BC tissue samples. The SHISA3 expression and methylation status was determined by qPCR and methylation specific PCR (MSP) respectively. The role of SHISA3 in BC tumorigenesis was evaluated by proliferation and migration assays after ectopic expression of SHISA3. The association between SHISA3 hypermethylation and clinicopathological parameters of BC patients was also studied. The downregulation of SHISA3 expression was found in three BC cell lines used and in all BC tissue samples. However, SHISA3 promoter region was hypermethylated in 61% (63/103) tumorous tissues in comparison to the 18% of their matched normal tissues. The 5-aza-2’-deoxycytidine treatment restored SHISA3 expression by reversing promoter hypermethylation in both MDA-MB231 and MCF-7 cells. Furthermore, ectopic expression of SHISA3 significantly reduced the proliferation and migration ability of these cells. Taken together, our findings for the first time reveal epigenetic silencing and tumor suppressing role of SHISA3 in BC. Henceforth, this study has identified SHISA3 as potentially powerful target for the development of new therapies against BC, as well as novel diagnostic and therapy response monitoring approaches.

Highlights

Breast cancer (BC) is one of the most widespread cancer with continuously increasing incidence rate and the foremost reason of deaths among women across the world [1]
We first determined the endogenous expression of SHISA3 in 3 BC cell lines and cultured normal human mammary epithelial cells (Normal) by Quantitative Real Time PCR (qPCR)
The SHISA3 was found to be downregulated in all three BC cell lines used as compared to the normal human mammary epithelial cells (Fig 1A)

Summary

Introduction

Breast cancer (BC) is one of the most widespread cancer with continuously increasing incidence rate and the foremost reason of deaths among women across the world [1]. One of the major reasons for high BC related deaths is late diagnosis especially in resource limited settings where majority of women are diagnosed during late stages due to weak and costly health care systems. While these data indicate the inefficiency of current strategies, they highlight the urgency to the development of more reliable approaches for early stage tumor diagnosis and therapy response monitoring for BC. In comparison with current gold standard BC diagnosis methods, like mammography, molecular biomarkers-based approaches are relatively easier to be integrated into portable and easy to use devices for POC applications [2, 3]

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Results

Discussion

Conclusion