A novel Ubc9 -dependent pathway regulates SIRT1- ER-α Axis and BRCA1-associated TNBC lung metastasis.

Jingyao Xu,Vaishali Reddy,Yonte Burnam,E Shyam P Reddy,Collin Shumate,Veena N Rao,Victoria Lopez,Joel Okoli,Yulong Qin

doi:10.15761/imm.1000298

Abstract

Triple negative breast cancer (TNBC) is a heterogeneous disease and has a higher rate of recurrence and distant metastasis. African-American (AA) women have a higher frequency of BRCA1 mutations and TNBC compared to other populations. Basal-like tumors have a higher rate of brain, lung and distant nodal metastasis more than other TNBC subtypes, contributing to higher mortality rate. Our previous work suggested Ubc9, a SUMO E2-conjugating enzyme to induce proliferation and migration of BRCA1-incompetent TNBC cells and TNBC cell lines established from the pleural effusion metastasis of a woman with TNBC. To understand the downstream signaling axis involved in distant metastasis we have used clinically relevant BRCA1 mutant and lung metastatic TNBC cell lines and our results show deregulated expression of caveolin-1, VEGF and SIRT1 in these cells compared to normal mammary epithelial cells by immunofluorescence analysis. We observed SIRT1 to be induced by wild type BRCA1a and BRCA1a I26A mutant unlike the disease associated Ubc9 binding mutants in TNBC cells. Knock down of Ubc9 induced SIRT1 expression in TNBC and ER-α expression in breast cancer cells. This is the first report demonstrating a role for Ubc9 in repressing both SIRT1 and ER-α expression in BRCA1 associated TNBC cells. It also suggests that the BARD-dependent E3 Ubiquitin ligase and HR (homologous recombination) activity of BRCA1 may not be required for inducing SIRT1 expression. Our results suggest for the first time that in BRCA1 mutant TNBC Ubc9-mediated induction of VEGF, inhibition of caveolin-1, SIRT1 and ER-α expression as a novel molecular mechanism underlying TNBC EMT (epithelial mesenchymal transition) leading to lung metastasis with pleural effusion. Drugs that target Ubc9 to both induce SIRT1 and ER-α or using SIRT1 agonists in combination with chemotherapy can be used as a promising targeted therapeutic approach for treating basal-like metastatic BRCA1-linked TNBC thus reducing the mortality in patients with TNBC.

Highlights

Breast cancer is the most frequently diagnosed cancer in females in America, and it is the second leading cause of cancer deaths in America with 40,610 women expected to die from breast cancer in 2017 [1]
Wild Type BRCA1 protein stimulates caveolin-1 expression by binding to the caveolin-1 promoter and caveolin-1 induces BRCA1 protein expression [42,43]. To examine whether this occurs in a physiological relevant BRCA1 mutant cell line in vivo, we have studied the expression of caveolin-1 in normal human mammary epithelial cells MCF10A and a Basal-Like Triple Negative Breast Cancer (TNBC) cell line HCC1937 obtained from a patient with germ line BRCA1 mutation using immunofluorescence analysis
In this study we have found high levels of vascular endothelial growth factor (VEGF), reduced levels of caveolin-1, Sirtuin 1 (SIRT1) in highly migratory BRCA1 mutant TNBC cells and TNBC cells established from a pleural effusion metastasis of a women with TNBC compared to normal mammary epithelial cells

Summary

Introduction

Breast cancer is the most frequently diagnosed cancer in females in America, and it is the second leading cause of cancer deaths in America with 40,610 women expected to die from breast cancer in 2017 [1]. TNBC is notable for being an aggressive cancer with higher mean tumor size, higher grade tumors, higher rates of node positivity, high likelihood of recurrence, distinct metastasis patterns and poorer survival compared to other breast cancers [4]. TNBCs do not express estrogen receptor, (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) [4]. Because they do not express these receptors, TNBC’s lack targeted therapies that other breast cancers, such as ER+ breast cancer, have [4]. Most TNBC with BRCA1 mutations fall into the basal-like subtypes (BL1 and BL2), which have high rates of recurrence and distant metastasis, especially to the lungs and brain, contributing to higher mortality. BRCA1 proteins contain several functional domains, an N-terminal RING finger domain

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