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The second BRCT domain of BRCA1 proteins interacts with p53 and stimulates transcription from the p21WAF1/CIP1 promoter.

Inherited mutations in the breast and ovarian cancer susceptibility gene BRCA1 are associated with high risk for developing breast and ovarian cancers. Several studies link BRCA1 to transcriptional regulation, DNA repair, apoptosis and growth/tumor suppression. BRCA1 associates with p53 and stimulates transcription in both p53 dependent and p53-independent manners. BRCA1 splice variants BRCA1a (p110) and BRCA1b (p100) associates with CBP/p300 co-activators. Here we show that BRCA1a and BRCA1b proteins stimulate p53-dependent transcription from the p21WAF1/CIP1 promoter. In addition, the C-terminal second BRCA1 (BRCT) domain is sufficient for p53 mediated transactivation of the p21 promoter. Previous studies emphasized the importance of the BRCT domain, which shows homology with p53 binding protein (53BP1), in transcriptional activation, growth inhibition and tumor suppression. Our findings demonstrate an additional function for this domain in protein – protein interaction and co-activation of p53. We also found that BRCA1a and BRCA1b proteins interact with p53 in vitro and in vivo. The p53 interaction domain of BRCA1a/1b maps, in vitro, to the second BRCT domain (aa 1760 – 1863). The BRCT domain binds to the central domain of p53 which is required for sequence specific DNA binding. These results demonstrate for the first time the presence of a second p53 interaction domain in BRCA1 proteins and suggests that BRCA1a and BRCA1b proteins, like BRCA1, function as p53 co-activators. This BRCT domain also binds in vitro to CBP. These results suggest that one of the mechanisms by which BRCA1 proteins function is through recruitment of CBP/p300 associated HAT/FAT activity for acetylation of p53 to specific promoters resulting in transcriptional activation.

c-Fos oncogene regulator Elk-1 interacts with BRCA1 splice variants BRCA1a/1b and enhances BRCA1a/1b-mediated growth suppression in breast cancer cells

Elk-1, a c-Fos protooncogene regulator, which belongs to the ETS-domain family of transcriptional factors, plays an important role in the induction of immediate early gene expression in response to a variety of extracellular signals. In this study, we demonstrate for the first time the in vitro and in vivo interaction of Elk-1 with BRCA1 splice variants BRCA1a and BRCA1b using GST-pull down assays, co-imunoprecipitations/Western blot analysis of cell extracts from breast cancer cells and mammalian two-hybrid assays. We have localized the BRCA1 interaction domain of Elk-1 protein to the conserved ETS domain, a motif involved in DNA binding and protein–protein interactions. We also observed binding of BRCA1 proteins to other ETS-domain transcription factors SAP1, ETS-1, ERG-2 and Fli-1 but not to Elk-1 splice variant ΔElk-1 and c-Fos protooncogene. Both BRCA1a and BRCA1b splice variants function as growth suppressors of human breast cancer cells. Interestingly, our studies reveal that although both Elk-1 and SAP-1 are highly homologous members of a subfamily of ETS domain proteins called ternary complex factors, it is only Elk-1 but not SAP-1 that can augment the growth suppressive function of BRCA1a/1b proteins in breast cancer cells. Thus Elk-1 could be a potential downstream target of BRCA1 in its growth control pathway. Furthermore, we have observed inhibition of c-Fos promoter activity in BRCA1a transfected stable breast cancer cells and over expression of BRCA1a/1b attenuates MEK-induced SRE activation in vivo. These results demonstrate for the first time a link between the growth suppressive function of BRCA1a/1b proteins and signal transduction pathway involving Elk-1 protein. All these results taken together suggest that one of the mechanisms by which BRCA1a/1b proteins function as growth/tumor suppressors is through inhibition of the expression of Elk-1 target genes like c-Fos.

Induction of apoptosis by Elk-1 and ΔElk-1 proteins

Elk-1, an ets related gene codes for at least two splice variants Elk-1, which regulates c-fos transcription and ΔElk-1, both of which function as transcriptional activators. To investigate the role of Elk-1 and ΔElk-1 proteins in apoptosis; we have developed rat fibroblast cell lines and human breast cancer cell lines expressing Elk-1 and ΔElk-1. The expression of Elk-1 and ΔElk-1 proteins in the Elk-1/ΔElk-1 transfectants were analysed by immunofluorescence, immunohistochemistry, and Western blot analysis. The Elk-1 unlike ΔElk-1 transfectants showed a shortened and flattened morphology compared to the parental cells. We have found that calcium ionophore treatment of Rat-1 Elk-1, MCF-7 Elk-1, Rat-1 ΔElk-1 and MCF-7 ΔElk-1 transfectants resulted in programmed cell death. These results indicate that constitutive expression of Elk-1 and ΔElk-1 proteins triggers apoptosis in Rat-1 fibroblasts and breast cancer cells when treated with calcium ionophore.

Histone deacetylase inhibitors, valproic acid and trichostatin-A induce apoptosis and affect acetylation status of p53 in ERG-positive prostate cancer cells

An ETS family member, ETS Related Gene (ERG) is involved in the Ewing family of tumors as well as leukemias. Rearrangement of the ERG gene with the TMPRSS2 gene has been identified in the majority of prostate cancer patients. Additionally, overexpression of ERG is associated with unfavorable prognosis in prostate cancer patients similar to leukemia patients. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) regulate transcription as well as epigenetic status of genes through acetylation of both histones and transcription factors. Deregulation of HATs and HDACs is frequently seen in various cancers, including prostate cancer. Many cellular oncogenes as well as tumor viral proteins are known to target either or both HATs and HDACs. Several studies have demonstrated that there are alterations of HDAC activity in prostate cancer cells. Recently, we found that ERG binds and inhibits HATs, which suggests that ERG is involved in deregulation of protein acetylation. Additionally, it has been shown that ERG is associated with a higher expression of HDACs. In this study, we tested the effect of the HDAC inhibitors valproic acid (VPA) and trichostatin-A (TSA) on ERG-positive prostate cancer cells (VCaP). We found that VPA and TSA induce apoptosis, upregulate p21/Waf1/CIP1, repress TMPRSS2-ERG expression and affect acetylation status of p53 in VCaP cells. These results suggest that HDAC inhibitors might restore HAT activity through two different ways: by inhibiting HDAC activity and by repressing HAT targeting oncoproteins such as ERG.

BRCA1a has antitumor activity in TN breast, ovarian and prostate cancers.

Breast cancer gene 1 (BRCA1) mutations predispose women to breast and ovarian cancers and men to increased risks for prostate cancer. We have previously showed BRCA1 splice variant BRCA1a/p110 to induce apoptosis of human breast cancer cells. In the current study, stable expression of BRCA1a/p110 resulted in inhibition of growth of estrogen receptor (ER)-positive and triple-negative (TN) human breast, ovarian, prostate and colon cancer cells and mouse fibroblast cells. Similar to wild-type BRCA1, only those cells with wild-type Rb were sensitive to BRCA1a-induced growth suppression and the status of p53 did not affect the ability of BRCA1a to suppress growth of tumor cells. BRCA1a also significantly inhibited tumor mass in nude mice bearing human CAL-51 TN breast cancer, ES-2 ovarian cancer and PC-3 prostate cancer xenografts. These results suggest that the majority of exon 11 sequences (residues 263–1365) are not required for the tumor suppressor function of BRCA1 proteins. This is the first report demonstrating antitumor activity of BRCA1a in human ER-positive and TN breast, hormone-independent ovarian and prostate cancer cells. Currently, there are no effective treatments against TN breast cancers and results from these studies will provide new treatments for one of the biggest needs in breast cancer research.

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. J. Cell. Physiol. 226: 3355–3367, 2011.

Role of protein-protein interactions in the antiapoptotic function of EWS-Fli-1.

In the majority of Ewing’s family tumors, chromosomal translocation t(11;22) leads to aberrant fusion of RNA-binding protein EWS with DNA-binding ETS transcriptional factor Fli-1. EWS-Fli-1 has altered the transcriptional activity and modulating its downstream target genes through this transcriptional activity is thought to be responsible for this tumor. We have previously shown that both EWS-Fli-1 and Fli-1 have antiapoptotic activity against several apoptotic inducers. Here, we show that the transcriptional activity of EWS-Fli-1 and Fli-1 is not essential for its antiapoptotic activity. We also demonstrate that EWS-Fli-1 and Fli-1 interact with CBP through its amino-terminal region and inhibit the CBP-dependent transcriptional activity of RXR. This activity appears to be independent of DNA-binding activity of EWS-Fli-1. Introduction of the dominant-negative form of CBP into Ewing’s sarcoma cells sensitizes these cells against genotoxic or retinoic-acid induced apoptosis. These results suggest that the ability of EWS-Fli-1/Fli-1 to target transcriptional cofactor(s) and modulate apoptotic pathways may be responsible for its antiapoptotic and tumorigenic activities.

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.

Abstract B21: Functional Role of ETS/ETV1-Fusion Proteins in Prostate Cancer and Other Cancers

After lung cancer prostate cancer is the most common age related cancer among men in the United State. African American men have the highest incidence of, and mortality rate from, this disease in the United States (American Cancer Society). According to the American Cancer Society, 29% of all cancer cases and 9% of all cancer deaths are a result of prostate cancer. By the age of 75, one out of nine men will develop prostate cancer. Individuals who are at higher risk include African-American men, men over 60 years of age and those with a family history of the disease. African-Americans also have twice the risk of developing prostate cancer as Caucasians. E26 transforming sequence (ETS) factors play an important role in cancers. ETV1, an ETS factor, is notable for its association in prostate cancers, where truncated ETV1 (dETV1) or its full length counterpart is overexpressed in approximately 10% of the patients. Formation of cancers may be brought on by deregulation of Wnt/β-catenin pathway. Mutations that stabilize β-catenin may contribute to loss of cell growth control in tumorigenesis. ETV19s interaction with components of the Wnt/β-catenin pathway may alter β-catenin interaction with downstream tumor suppressor genes, which are critical in regulating apoptosis and cell growth properties. Our results show that ETV1 regulates Wnt/β-catenin activity. We provide evidence that ETV1 activates kinases that regulate Wnt/β-catenin activity by post translational modification. Also, we demonstrate that therapeutic agents that reverse ETV1-regulated Wnt/β-catenin activity can be used to target ETV1-positive prostate cancer cells. These therapeutic agents will have a profound impact on prevention and treatment of prostate cancer and this may help to reduce health disparity seen in the minority patients. Citation Format: Sharif Morsalin, Chunshu Yang, Jinbo Fang, Yasuo Fujimura, Shubhalaxmi Kayarthodi, Huali Xu, Ujwala Gunnal, Clarence Clark, Julian Menter, Veena Rao, Shyam Reddy. Functional Role of ETS/ETV1-Fusion Proteins in Prostate Cancer and Other Cancers. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B21.

Abstract B33: BRCA1 RING domain mutations and function-based tools to predict risk for the development of TNBC

Breast cancer is the second leading cause of cancer-related deaths amongst women with 231,840 new cases projected for 2015. Majority of young African American women with BRCA1 mutations have a so-called Triple negative breast cancer with an aggressive phenotype. Currently there is no targeted therapy for TNBC. BRCA1 encodes a large protein and the two highly conserved regions are located at both ends, an N-terminal RING domain and two BRCT repeats at the C-terminus. The RING domain through interaction with BARD1 contains E3 ubiquitin ligase activity. Various BRCA1 mutations have been found throughout BRCA1 coding region, but the majority of cancer-associated mutations are nonsense or frame shift mutations which lead to chain termination. The C61G mutation has been linked to both breast and ovarian cancer development. In people from Ashkenazi Jewish descent, the most common mutations are 185delAG and 5382 ins C in BRCA1 and is 1:40. Our lab has cloned two naturally occurring splice variants of BRCA1 (BRCA1a and BRCA1b). These isoforms are the most evolutionary conserved of all the splice variants and code for multifunctional proteins. Our group has previously reported that BRCA1 RING domain, unlike K109R and cancer-predisposing mutant C61G BRCA1 proteins interact with the sole SUMO E2-conjugating enzyme Ubc9 and this facilitates both the entry of BRCA1 proteins to the nucleus and mediates ubiquitination of ER-alpha. The disease associated mutants do not bind Ubc9, remain stalled in the cytoplasm and have lost their growth suppressor function. We have identified a new nuclear trafficking pathway and malfunction of this by BRCA1 dysfunction can result in TNBC. The BARD1-dependent -E3 ubiquitin ligase activity of BRCA1 has been predicted to be required for its tumor suppressor function, as certain cancer –associated mutations in BRCA1 RING domain ablate this activity. Several mutations in BRCA1 RING domain have been identified however; their role in TNBC has yet to be elucidated. This work is based on the hypothesis that BRCA1 is a tumor suppressor gene and its RING domain can harbor several mutations some of which can result in loss of BRCA1 function resulting in TNBC and others can be gain of function mutants similar to WT BRCA1. We tested this hypothesis by introducing C61G, K109R and I26A mutations into TNBC cells and studied their growth inhibitory activity using colony suppression assays. Our results demonstrate for the first time that BRCA1 I26A to be a gain of function mutant similar to WT BRCA1. I26A mutant associates with Ubc9, lacks E3 ubiquitin ligase activity and suppresses growth of BRCA1 mutant TNBC and sporadic TNBC cells unlike K109R and C61G mutants. Clinically, the ability to predict which of these mutations can result in TNBC offers unprecedented prospects for early detection and cancer prevention. This is the first study demonstrating the physiological link between Ubc9 binding, loss of BARD1- dependent E3 ubiquitin ligase activity and growth suppression of I26A mutant BRCA1 protein in TNBC cells. BRCA1, by turning off or on Ubc9 binding, regulates growth of TNBC. This study will accelerate precision medicine and reduce cancer health disparities in health outcomes. Work supported in part by Georgia Cancer Coalition Distinguished Cancer Scholar award, NIH-NCRR-RCMI grant G-12-RR003034, U54 RR02613, 5P20RR11104 and NIHMD research endowment grant 2S21MD000101, MSM/TU/UAB CCC Partnership/U54 CA118638 and ING foundation to V.N.R. Patent issued No:8372,580; 2013. Citation Format: Jingyao Xu, Shanekkia Black, Kartik Aysola, Yunlong Qin, Vaishali Reddy, Karan Singh, Joel Okoli, Derrick Beech, Uma Krishnamurthy, Gabriela Oprea, Valerie M. Rice, E Shyam Reddy, Douglas Moellering, Yuchang Fu, Veena N. Rao. BRCA1 RING domain mutations and function-based tools to predict risk for the development of TNBC. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B33.