E. Shyam P. Reddy

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.

Inhibition of apoptosis by normal and aberrant Fli-1 and erg proteins involved in human solid tumors and leukemias

Two ets family members, namely erg and Fli-1 are fused with two EWS familiy members namely EWS and TLS/FUS as a result of chromosome translocation in human solid tumors and leukemias. EWS-erg and EWS-Fli-1, which are involved in greater than 95% of Ewing family of tumors, were shown to function as transcriptional activators. TLS/FUS-erg, which is involved in human myeloid leukemias also functions as a transcriptional activator. Expression of these fusion proteins (EWS-erg and EWS-Fli-1) are shown to be essential for maintaining the oncogenic and tumorigenic properties of tumor cells. Cancer is thought to be caused not only by uncontrolled cell proliferation but also by deregulation of programmed cell death. Therefore, we have studied the role of normal (Fli-1 and erg) and aberrant fusion proteins (EWS-erg, EWS-Fli-1 and TLS/FUS-erg) in apoptosis. We have found that expression of normal (Fli-1 and erg) and aberrant fusion proteins inhibit the apoptosis of NIH3T3 cells induced by either serum deprivation or by treatment with calcium ionophore. We have also observed similar suppression of apoptosis in Ewings sarcoma cells expressing EWS-Fli-1 and EWS-erg proteins suggesting that these fusionproteins may be responsible for the decreased ability of these tumor cells to undergo apoptosis. Inhibition of the expression of these aberrant fusion proteins by antisense RNA technique resulted in increased susceptibility to apoptosis leading to the death of tumor cells. Therefore, our results suggest that one can use therapeutic agents which can down regulate the expression of fusion proteins in combination with chemotherapeutic agents as an effective treatment for these human solid tumors and leukemias.

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.

EWS-ATF-1 chimeric protein in soft tissue clear cell sarcoma associates with CREB-binding protein and interferes with p53-mediated trans-activation function

The recurrent t(12;22) (q13;q12) chromosomal translocation associated with soft tissue clear cell sarcoma results in a chimeric protein EWS-ATF-1 that acts as a constitutive transcriptional activator. The CBP/p300 transcriptional coactivator, which links various transcriptional factors to basal transcription apparatus, participates in transcriptional activation, growth and cell cycle control and differentiation. In this study, we show that EWS-ATF-1 associates constitutively with CBP both in vitro and in vivo. Both EWS and ATF-1 fusion domains are needed for this interaction. Here, we demonstrate that EWS-ATF-1 represses p53/CBP-mediated trans-activation function. Overexpression of CBP can counteract this repressive effect of EWS-ATF-1. Taken together, these findings suggest that one of the mechanisms by which EWS-ATF-1 may cause tumors is through targeting CBP/p300 resulting in the loss of function of p53. This novel mechanism may be responsible for the development of these and other related solid tumors.

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.

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.

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, γ‐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.

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.

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.

Abstract B37: EWS-ERG targets transcriptional co-factors in ERG-positive Ewing sarcoma.

Ewing sarcoma is a highly aggressive and malignant tumor that occurs in the bone or soft tissue. It affects children and adolescents with majority of incidence between the age of 10 and 20 and about 250 children and adolescents are diagnosed each year in US. There is a clear health disparity seen in Ewing sarcoma population. The overall survival rates for black and hispanic patients have shown to be significantly worse compared to white patients. Previously, Drs. Reddy and Rao have discovered ERG (ETS Related Gene) and several important members of ETS family genes and have shown that ERG gene encodes for sequence specific transcriptional activators. Ewing sarcoma is characterized by chromosomal translocations involving EWS gene and one of the ETS (Erythroblastosis virus E26 transformation–specific) families of transcription factors (ERG, FLI1, ETV1, ETV4, E1AF and FEV). Dr. Reddys laboratory has demonstrated that EWS gene codes for RNA binding protein and the fusion protein (EWS-ERG) functions as transcriptional activator. Our results have shown that EWS-ERG also inhibits transcriptional activation properties of RXR. This suggests that the aberrant fusion protein (EWS-ERG) may target transcriptional co-factors and thereby regulate RXR transcriptional activity. To understand the molecular mechanism of action of how the fusion protein targets nuclear receptor function and to provide a clue for the cancer health disparity seen in Ewing Sarcoma, we hypothesized that the aberrant fusion protein EWS-ERG targets CBP and other members of the transcriptional machinery causing transcriptional repression of RXR activity. Methods: We propose to study the interaction of EWS-ERG and CBP by co-immunoprecipitation approach and also investigate whether this interaction has a role in the inhibition of RXR transcriptional activity by luciferase assays. Results: 1. EWS-ERG inhibits RXR transcriptional activity. 2. EWS-ERG interacts with CBP in Ewing Sarcoma cells. 3. Overexpression of dominant negative mutant of CBP relieves EWS-ERG inhibition of RXR transcriptional activity. Conclusion: The transcriptional co-factor (CBP) is sequestered by the aberrant fusion protein EWS-ERG which inhibits the binding of RXR with CBP and other members of the transcriptional machinery causing transcriptional repression of RXR activity. This study may provide a clue for the cancer health disparity seen in Ewing Sarcoma and may have a profound impact on prevention, management and treatment of many types of cancers. Citation Format: Shubhalaxmi P. Kayarthodi, Yasuo Fujimura, Kunchala Rungasrisuriyachai, Huali Xu, Jinbo Fang, Chunshu Yang, Veena N. Rao, E.Shyam P. Reddy. EWS-ERG targets transcriptional co-factors in ERG-positive Ewing sarcoma. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr B37.