Ubc9 mediates nuclear localization and growth suppression of BRCA1 and BRCA1a proteins.

BRCA1 gene mutations are responsible for hereditary breast and ovarian cancers. In sporadic breast tumors, BRCA1 dysfunction or aberrant subcellular localization is thought to be common. BRCA1 is a nuclear-cytoplasm shuttling protein and the reason for cytoplasmic localization of BRCA1 in young breast cancer patients is not yet known. We have previously reported BRCA1 proteins unlike K109R and cancer-predisposing mutant C61G to bind Ubc9 and modulate ER-α turnover. In the present study, we have examined the consequences of altered Ubc9 binding and knockdown on the subcellular localization and growth inhibitory function of BRCA1 proteins. Our results using live imaging of YFP, GFP, RFP-tagged BRCA1, BRCA1a and BRCA1b proteins show enhanced cytoplasmic localization of K109 R and C61G mutant BRCA1 proteins in normal and cancer cells. Furthermore, down-regulation of Ubc9 in MCF-7 cells using Ubc9 siRNA resulted in enhanced cytoplasmic localization of BRCA1 protein and exclusive cytoplasmic retention of BRCA1a and BRCA1b proteins. These mutant BRCA1 proteins were transforming and impaired in their capacity to inhibit growth of MCF-7 and CAL51 breast cancer cells. Interestingly, cytoplasmic BRCA1a mutants showed more clonogenicity in soft agar and higher levels of expression of Ubc9 than parental MCF7 cells. This is the first report demonstrating the physiological link between cytoplasmic mislocalization of mutant BRCA1 proteins, loss of ER-α repression, loss of ubiquitin ligase activity and loss of growth suppression of BRCA1 proteins. Thus, binding of BRCA1 proteins to nuclear chaperone Ubc9 provides a novel mechanism for nuclear import and control of tumor growth.

Mitochondrial localization, ELK-1 transcriptional regulation and growth inhibitory functions of BRCA1, BRCA1a, and BRCA1b proteins.

BRCA1 is a tumor suppressor gene that is mutated in families with breast and ovarian cancer. Several BRCA1 splice variants are found in different tissues, but their subcellular localization and functions are poorly understood at the moment. We previously described BRCA1 splice variant BRCA1a to induce apoptosis and function as a tumor suppressor of triple negative breast, ovarian and prostate cancers. In this study we have analyzed the function of BRCA1 isoforms (BRCA1a and BRCA1b) and compared them to the wild-type BRCA1 protein using several criteria like studying expression in normal and tumor cells by RNase protection assays, subcellular localization/fractionation by immunofluorescence microscopy and Western blot analysis, transcription regulation of biological relevant proteins and growth suppression in breast cancer cells. We are demonstrating for the first time that ectopically expressed GFP-tagged BRCA1, BRCA1a, and BRCA1b proteins are localized to the mitochondria, repress ELK-1 transcriptional activity and possess antiproliferative activity on breast cancer cells. These results suggest that the exon 9, 10, and 11 sequences (aa 263-1365) which contain two nuclear localization signals, p53, Rb, c-Myc, gamma-tubulin, Stat, Rad51, Rad50 binding domains, angiopoietin-1 repression domain are not absolutely required for mitochondrial localization and growth suppressor function of these proteins. Since mitochondrial dysfunction is a hallmark of cancer, we can speculate that the mitochondrial localization of BRCA1 proteins may be functionally significant in regulating both the mitochondrial DNA damage as well as apoptotic activity of BRCA1 proteins and mislocalization causes cancer. J. Cell. Physiol. 219: 634-641, 2009. (c) 2009 Wiley-Liss, Inc.

A Provocative Molecular Link between Mammographic Density and BRCA1-loss associated TNBC.

Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer that has a high mortality rate and disproportionately affects young African American (AA) women who carry mutations in the BRCA1 gene. Approximately 80% of breast cancers which develop in BRCA1-mutant carriers will have TNBC and the molecular mechanism facilitating tumor development is unclear. Our earlier work suggested Ubc9 to play a critical role in BRCA1 loss mediated TNBC cell migration and metastasis. Collagen is one of the major components of the stromal extracellular matrix (ECM) network that influences tissue density. Its re-organization act as a scaffold aiding cancer cells to migrate causing metastasis. Ubc9 is known to increase the production of collagen, a key component of fibroglandular breast tissue, as well as tumorigenesis. Our work is based on the hypothesis that loss of BRCA1 in women with high breast density causes abnormal Ubc9 levels which upregulates collagen, fibronectin and inhibits SIRT1, ß-catenin expression facilitating TNBC. We tested this hypothesis by studying the expression of total collagen, fibronectin, Ubc9, SIRT1, ß-catenin in BRCA1 mutant TNBC cells and tumor sample derived from patient with dense breasts using immunofluorescence, immunohistochemistry, and collagen assay. Our results suggest for the first time that mutation or loss of BRCA1 function in women with fibrocystic breasts can lead to over expression of Ubc9, induction of collagen and; fibronectin, inhibition of SIRT1 and nuclear accumulation of ß-catenin which could contribute to TNBC development. This network will aid not only in the identification of potential mechanism-based biomarkers that could detect disease early, but also enforce preventive measures that could reduce the risk for TNBC in women with high MD thus reducing the mortality associated with these cancers to achieve health equity.

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

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.

Triple Negative Breast Cancer - An Overview.

Triple Negative Breast Cancer (TNBC) is a heterogeneous disease that based on immunohistochemistry (IHC) is estrogen receptor (ER) negative, progesterone receptor (PR) negative and human epidermal growth factor receptor 2 (HER2) negative. TNBC is typically observed in young AA women and Hispanic women who carry a mutation in the BRCA1 gene. TNBC is characterized by a distinct molecular profile, aggressive nature and lack of targeted therapies. The purpose of this article is to review the current and future novel signalling pathways as therapeutic approaches to TNBC. Recent Identification of a new BRCA1 trafficking pathway holds promise in the future for the development of targeted therapies for TNBC.