WWOX inhibits metastasis of triple-negative breast cancer cells through modulation of microRNAs

Introduction

Breast cancer is the most common cancer diagnosed in women worldwide and the second leading cause of cancer death in the USA. Ductal carcinoma in situ (DCIS) is the most common type of non-invasive breast cancer, yet, it is the precursor of invasive breast cancer (IBC), which causes over 90% of breast cancer-related deaths. Molecular characterization of genes involved in breast cancer metastasis (metastagenes) would greatly aid in future formulation of therapeutic interventions. The WW domain-containing oxidoreductase (WWOX) encompasses an active common fragile site, FRA16D at chromosome 16q23.2, a region that is implicated in breast carcinogenesis. WWOX is reduced or lost in about two thirds of breast cancer cases and its loss of expression associates with poor survival in triple-negative breast cancer (TNBC) patients. Both in vitro and in vivo evidence supports tumor suppressor function of WWOX in breast cancer. Nevertheless, the role of WWOX in breast cancer metastasis is largely unknown.

Material and method

Human breast cancer cell lines MDA-MB-231 and HCC70 along with another metastatic cell line MDA-MB-435 were used to characterize the effect of WWOX on cancer progression and metastasis. The work included in-vitro assays such as Matrigel invasion, wound healing assay, anchorage independent growth and proliferation. Moreover, cell lines were tested in in-vivo system including orthotopic injection in a mammary fat pad of female NOD/SCID mice in addition to tail-vein injection in NOD/SCID mice as well. Molecular analysis include: miRNA analysis, ChIP, IP and more.

Results and discussion

We found that stable expression of WWOX in metastatic breast cancer cells attenuated matrigel invasion in vitro and reduced tumorigenicity, metastatic seeding and colonization in NOD/SCID mice in vivo. Mechanistically, WWOX was found to regulate the cell phenotype by changing both epithelial and mesenchymal markers through regulation of microRNA expression. In particular, WWOX, likely through negative regulation of Myc expression, promotes accumulation of miR-146a which targets Fibronectin and supports an epithelial phenotype. Interestingly, reduced expression of miR-146a is associated with poor survival of TNBC patients.

Conclusion

These findings indicate that WWOX loss promotes TNBC development and enhances invasion and metastasis through modulation of microRNA gene expression.