Aditya Bele

A Novel Interaction of Ecdysoneless (ECD) Protein with R2TP Complex Component RUVBL1 Is Required for the Functional Role of ECD in Cell Cycle Progression

ABSTRACT Ecdysoneless (ECD) is an evolutionarily conserved protein whose germ line deletion is embryonic lethal. Deletion of Ecd in cells causes cell cycle arrest, which is rescued by exogenous ECD, demonstrating a requirement of ECD for normal mammalian cell cycle progression. However, the exact mechanism by which ECD regulates cell cycle is unknown. Here, we demonstrate that ECD protein levels and subcellular localization are invariant during cell cycle progression, suggesting a potential role of posttranslational modifications or protein-protein interactions. Since phosphorylated ECD was recently shown to interact with the PIH1D1 adaptor component of the R2TP cochaperone complex, we examined the requirement of ECD phosphorylation in cell cycle progression. Notably, phosphorylation-deficient ECD mutants that failed to bind to PIH1D1 in vitro fully retained the ability to interact with the R2TP complex and yet exhibited a reduced ability to rescue Ecd-deficient cells from cell cycle arrest. Biochemical analyses demonstrated an additional phosphorylation-independent interaction of ECD with the RUVBL1 component of the R2TP complex, and this interaction is essential for ECDs cell cycle progression function. These studies demonstrate that interaction of ECD with RUVBL1, and its CK2-mediated phosphorylation, independent of its interaction with PIH1D1, are important for its cell cycle regulatory function.

Acetylation of Mammalian ADA3 Is Required for Its Functional Roles in Histone Acetylation and Cell Proliferation

ABSTRACT Alteration/deficiency in activation 3 (ADA3) is an essential component of specific histone acetyltransferase (HAT) complexes. We have previously shown that ADA3 is required for establishing global histone acetylation patterns and for normal cell cycle progression (S. Mohibi et al., J Biol Chem 287:29442–29456, 2012, http://dx.doi.org/10.1074/jbc.M112.378901). Here, we report that these functional roles of ADA3 require its acetylation. We show that ADA3 acetylation, which is dynamically regulated in a cell cycle-dependent manner, reflects a balance of coordinated actions of its associated HATs, GCN5, PCAF, and p300, and a new partner that we define, the deacetylase SIRT1. We use mass spectrometry and site-directed mutagenesis to identify major sites of ADA3 acetylated by GCN5 and p300. Acetylation-defective mutants are capable of interacting with HATs and other components of HAT complexes but are deficient in their ability to restore ADA3-dependent global or locus-specific histone acetylation marks and cell proliferation in Ada3-deleted murine embryonic fibroblasts (MEFs). Given the key importance of ADA3-containing HAT complexes in the regulation of various biological processes, including the cell cycle, our study presents a novel mechanism to regulate the function of these complexes through dynamic ADA3 acetylation.

Breast Cancer Subtypes: Two Decades of Journey from Cell Culture to Patients

Recent molecular profiling has identified six major subtypes of breast cancers that exhibit different survival outcomes for patients. To address the origin of different subtypes of breast cancers, we have now identified, isolated, and immortalized (using hTERT) mammary stem/progenitor cells which maintain their stem/progenitor properties even after immortalization. Our decade long research has shown that these stem/progenitor cells are highly susceptible to oncogenesis. Given the emerging evidence that stem/progenitor cells are precursors of cancers and that distinct subtypes of breast cancer have different survival outcome, these cellular models provide novel tools to understand the oncogenic process leading to various subtypes of breast cancers and for future development of novel therapeutic strategies to treat different subtypes of breast cancers.

Mammalian ECD protein is a novel negative regulator of the PERK arm of the unfolded protein response

ABSTRACT Mammalian Ecdysoneless (ECD) is a highly conserved ortholog of the DrosophilaEcd gene product whose mutations impair the synthesis of Ecdysone and produce cell-autonomous survival defects, but the mechanisms by which ECD functions are largely unknown. Here we present evidence that ECD regulates the endoplasmic reticulum (ER) stress response. ER stress induction led to a reduced ECD protein level, but this effect was not seen in PKR-like ER kinase knockout (PERK-KO) or phosphodeficient eukaryotic translation initiation factor 2α (eIF2α) mouse embryonic fibroblasts (MEFs); moreover, ECD mRNA levels were increased, suggesting impaired ECD translation as the mechanism for reduced protein levels. ECD colocalizes and coimmunoprecipitates with PERK and GRP78. ECD depletion increased the levels of both phospho-PERK (p-PERK) and p-eIF2α, and these effects were enhanced upon ER stress induction. Reciprocally, overexpression of ECD led to marked decreases in p-PERK, p-eIF2α, and ATF4 levels but robust increases in GRP78 protein levels. However, GRP78 mRNA levels were unchanged, suggesting a posttranscriptional event. Knockdown of GRP78 reversed the attenuating effect of ECD overexpression on PERK signaling. Significantly, overexpression of ECD provided a survival advantage to cells upon ER stress induction. Taken together, our data demonstrate that ECD promotes survival upon ER stress by increasing GRP78 protein levels to enhance the adaptive folding protein in the ER to attenuate PERK signaling.

Abstract 1306: Synergistic function of a novel cell cycle regulator, Ecdysoneless with oncogenic Ras in the regulation of cell cycle and transformation

We have previously identified the mammalian ortholog of Drosophila ecdysoneless (Ecd) protein as a novel and essential regulator of Rb-E2F-dependent cell cycle progression. Consistent with role of Ecd in cell cycle, our recent studies show Ecd is overexpressed in breast cancer cell lines as well as in ductal carcinoma in situ and infiltrating ductal carcinomas of the breast. To explore the mechanism of Ecd overexpression and its correlation with oncogenesis, we overexpressed either Ecd alone, HRas-61L alone or both genes together in hTERT-immortalized human mammary epithelial cells (hMECs). These transfectants were then examined for their oncogenic properties. We observed while vector infected cells do not exhibit anchorage independence, all three transfectants showed increase in anchorage independent proliferation, particularly Ecd+Ras showed a significant increase in anchorage independence in comparison with either Ecd or Ras. Similarly, other oncogenic traits such as proliferation on matrigel, invasion and migration were dramatically increased when both genes were expressed in comparison with individual genes. Further analysis showed Ecd+Ras-expressing hMECs were unable to synchronize completely after growth factor deprivation and showed rapid progression into cell cycle, demonstrating synergy in altered cell cycle pathways in these cells. Most significantly, Ecd+Ras expressing hMECs were able to form tumors when implanted into mammary fat pads of NOD-SCID mice, whereas no tumors were observed with Ecd or Ras alone. These results demonstrate complete transformation of hMECs by overexpression of Ecd together with mutant Ras. Considering the known oncogenic role of Ras in human cancer and the known role of Ecd in cell cycle, these data are highly significant and present a novel synergistic pathway for mammary cell transformation. Current studies are directed towards defining the mechanism of synergy between Ecd and Ras in cellular transformation. Citation Format: Aditya Bele, Sameer Mirza, Riyaz Mir, Shakur Mohibi, Hamid Band, Vimla Band. Synergistic function of a novel cell cycle regulator, Ecdysoneless with oncogenic Ras in the regulation of cell cycle and transformation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1306. doi:10.1158/1538-7445.AM2014-1306

Abstract 3062: Ada3, a component of STAGA HAT complex, controls cell cycle by regulating c-myc

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The human homologue of the yeast Ada3 (alteration/deficiency in activation) protein is an essential component of various Histone acetyl transferase complexes (HATs). To gain insight into the physiological importance of Ada3, we established a conditional knockout mouse model and showed that loss of Ada3 led to embryonic lethality at an early stage of development. Using the derived Ada3lox/lox mouse embryonic fibroblast (MEF) cell lines we show that Ada3 deletion causes defects in histone acetylation. Also we show that Ada3 enhances catalytic activity of p300 HAT. We further showed that Ada3 plays an important role in cell proliferation; depletion of Ada3 from cells caused cell lethality. This proliferation defect was reversed by introduction of human Ada3 in Ada3 null MEFs. Ada3 null MEFs also showed delay in transition from G0-G1 phase of the cell cycle to the S phase. These cells also manifested decreased hyperphosphorylation of Retinoblastoma protein (Rb); an increased protein half life of CDK inhibitor p27Kip1 and decrease in Cdk2 kinase activity. The increase in p27 protein was a result of decreased Skp2 protein and mRNA levels which is a direct target of c-myc oncogene. We further show that mRNA levels of c-myc, a direct transcriptional target of Ada3 containing STAGA HAT complex, are reduced on deletion of Ada3. These data suggest an Ada3-myc-skp2-p27 pathway that controls mammalian cell cycle. Taken together, our data suggests an essential role of Ada3 in embryonic development and cell cycle progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3062. doi:1538-7445.AM2012-3062

Abstract 4177: Synergistic role of ecdysoneless with oncogenic Ras in transformation of human mammary epithelial cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Precisely regulated cell proliferation is essential for embryonic development as well as homeostasis in adult organs and tissues, whereas uncontrolled cell proliferation is a hallmark of cancer. Thus, elucidating how the cell cycle machinery is controlled is an important area of research in cancer cell biology. A large body of evidence has established a basic paradigm of the control of cell cycle progression involving the Retinoblastoma (Rb) protein family in conjunction with the E2F family of transcription factors. During G0/G1, interaction of hypo-phosphorylated Rb proteins with E2Fs prevents the transcription of E2F target genes. Cyclin-CDK complexes generated during cell cycle progression hyper-phosphorylate Rb, leading to release of Rb from E2Fs; this allows E2F target gene transcription and cell cycle progression. We previously identified the mammalian ortholog of Drosophila ecdysoneless (Ecd) protein as a novel and essential regulator of Rb-E2F-dependent cell cycle progression. Loss of Ecd retards the separation of Rb from E2F, arrests cells at G1/S boundary and prevents cell cycle progression. Consistent with role of Ecd in cell cycle, our recent studies show Ecd is overexpressed in breast cancer cell lines as well as in ductal carcinoma in situ and infiltrating ductal carcinomas of the breast. To understand the mechanism of Ecd overexpression and its correlation with oncogenesis, we overexpressed either Ecd alone, 61L-HRas alone or both genes together in hTERT-immortalized hMECs. These transfectants were then examined for their oncogenic properties. We observed while vector infected cells do not exhibit anchorage independence, all three transfectants showed anchorage independent proliferation, particularly Ecd+Ras showed a dramatic increase in anchorage independence in comparison with either Ecd or H-Ras. Similarly, other oncogenic traits such as proliferation on matrigel, invasion and migration were dramatically increased when both genes were expressed in comparison with individual genes. Considering the known oncogenic role of Ras in human cancer and the known role of Ecd in cell cycle, these data are highly significant and present a novel synergistic pathway for mammary cell transformation. We are currently defining the molecular mechanism of this synergistic oncogenic effect. In addition, we are generating Ecd overexpressed transgenic mice to examine the role of Ecd in oncogenesis in in vivo models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4177. doi:1538-7445.AM2012-4177

Abstract 4593: Expression of Ecdysoneless in breast cancer progression predicts poor outcome in breast cancer patients

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Breast cancer is the most common type of cancer and second leading cause of cancer-related deaths in women in the United States. Our laboratory has identified the protein hEcd (human ortholog of Drosophila Ecdysoneless) as a novel regulator of cell cycle. Previous studies showed that Ecd regulates cell cycle by regulating the Rb-E2F pathway. Given the dysregulation of the cell cycle machinery in cancer, we examined expression of Ecd in normal, benign, ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDCs) of breast. In first cohort, we showed while normal and hyperplasia of breast barely showed Ecd expression, high Ecd expression was observed in DCIS and IDC patient tissue specimens. In this cohort of 104 IDC patents, Ecd expression was positively associated with higher grade (p=0.04).We then analyzed Ecd expression in a second larger cohort (954) and observed similar results, where increased Ecd expression was associated with tumors of higher histological grade (p=0.013), mitotic count (p=0.032), and Nottingham Prognostic Index score (p=0.014). Notably, Ecd expression was positively associated with HER2/neu (p=0.002) overexpression. Significantly, a positive association between Ecd expression and shorter breast cancer specific survival (BCSS) (p=0.008) and disease-free survival (DFS) (p=0.003) was observed in HER2/neu overexpressing patients. Taken together, our results demonstrate Ecd expression as a novel marker for breast cancer progression that predicts tumor progression and the clinical outcome in breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4593. doi:1538-7445.AM2012-4593

Abstract 3929: ADA3, a component of ATAC complex regulates DNA damage response

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Our long term research goal is to understand the biochemical mechanism of immortalization of human epithelial cells that represents early step in cancer progression. Over the years, we have generated several epithelial cell immortalization models, using viral oncogenes, mutated cancer genes, as well as radiation treatment of cells. In one of such immortalization model, we identified alteration/deficiency in activation (ADA3) protein as a novel human papilloma virus E6 oncoprotein binding protein, and as a regulator of p53 function. We and others have shown that human ADA3 protein is an essential component of various Histone acetyl transferase complexes (HATs) and thus controls transactivation of various transcriptional factors. To gain insight into the physiological importance of ADA3, we established a conditional knockout mouse model and showed that loss of ADA3 led to embryonic lethality at an early stage of development. Using ADA3lox/lox mouse embryonic fibroblast (MEF) cell lines, we further showed that ADA3 plays an important role in DNA damage and repair pathway. Adenovirus cre-mediated deletion of ADA3 led to a global reduction of histone acetylation, severe G2/M cell cycle arrest, various aberration in nuclear structures, and massive chromosome aberration. Significantly, cells lacking ADA3 showed defects in recruitment of DNA repair associated proteins, BRCA1 and 53BP1 to the DNA damage foci. These results implicate ADA3 as a major cell cycle and DNA repair response regulating protein. These findings in the context of oncogenesis and chemotherapy will be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3929. doi:10.1158/1538-7445.AM2011-3929