David O. Azorsa

A Nuclear Factor, ASC-2, as a Cancer-amplified Transcriptional Coactivator Essential for Ligand-dependent Transactivation by Nuclear Receptors in Vivo

Many transcription coactivators interact with nuclear receptors in a ligand- and C-terminal transactivation function (AF2)-dependent manner. We isolated a nuclear factor (designated ASC-2) with such properties by using the ligand-binding domain of retinoid X receptor as a bait in a yeast two-hybrid screening. ASC-2 also interacted with other nuclear receptors, including retinoic acid receptor, thyroid hormone receptor, estrogen receptor α, and glucocorticoid receptor, basal factors TFIIA and TBP, and transcription integrators CBP/p300 and SRC-1. In transient cotransfections, ASC-2, either alone or in conjunction with CBP/p300 and SRC-1, stimulated ligand-dependent transactivation by wild type nuclear receptors but not mutant receptors lacking the AF2 domain. Consistent with an idea that ASC-2 is essential for the nuclear receptor function in vivo, microinjection of anti-ASC-2 antibody abrogated the ligand-dependent transactivation of retinoic acid receptor, and this repression was fully relieved by coinjection of ASC-2-expression vector. Surprisingly, ASC-2 was identical to a gene previously identified during a search for genes amplified and overexpressed in breast and other human cancers. From these results, we concluded that ASC-2 is a bona fidetranscription coactivator molecule of nuclear receptors, and its altered expression may contribute to the development of cancers.

Activating Signal Cointegrator 2 Belongs to a Novel Steady-State Complex That Contains a Subset of Trithorax Group Proteins

ABSTRACT Many transcription coactivators interact with nuclear receptors in a ligand- and C-terminal transactivation function (AF2)-dependent manner. These include activating signal cointegrator 2 (ASC-2), a recently isolated transcriptional coactivator molecule, which is amplified in human cancers and stimulates transactivation by nuclear receptors and numerous other transcription factors. In this report, we show that ASC-2 belongs to a steady-state complex of approximately 2 MDa (ASC-2 complex [ASCOM]) in HeLa nuclei. ASCOM contains retinoblastoma-binding protein RBQ-3, α/β-tubulins, and trithorax group proteins ALR-1, ALR-2, HALR, and ASH2. In particular, ALR-1/2 and HALR contain a highly conserved 130- to 140-amino-acid motif termed the SET domain, which was recently implicated in histone H3 lysine-specific methylation activities. Indeed, recombinant ALR-1, HALR, and immunopurified ASCOM exhibit very weak but specific H3-lysine 4 methylation activities in vitro, and transactivation by retinoic acid receptor appears to involve ligand-dependent recruitment of ASCOM and subsequent transient H3-lysine 4 methylation of the promoter region in vivo. Thus, ASCOM may represent a distinct coactivator complex of nuclear receptors. Further characterization of ASCOM will lead to a better understanding of how nuclear receptors and other transcription factors mediate transcriptional activation.

Nonsense-mediated decay microarray analysis identifies mutations of EPHB2 in human prostate cancer

The identification of tumor-suppressor genes in solid tumors by classical cancer genetics methods is difficult and slow. We combined nonsense-mediated RNA decay microarrays and array-based comparative genomic hybridization for the genome-wide identification of genes with biallelic inactivation involving nonsense mutations and loss of the wild-type allele. This approach enabled us to identify previously unknown mutations in the receptor tyrosine kinase gene EPHB2. The DU 145 prostate cancer cell line, originating from a brain metastasis, carries a truncating mutation of EPHB2 and a deletion of the remaining allele. Additional frameshift, splice site, missense and nonsense mutations are present in clinical prostate cancer samples. Transfection of DU 145 cells, which lack functional EphB2, with wild-type EPHB2 suppresses clonogenic growth. Taken together with studies indicating that EphB2 may have an essential role in cell migration and maintenance of normal tissue architecture, our findings suggest that mutational inactivation of EPHB2 may be important in the progression and metastasis of prostate cancer.

EphB2 expression across 138 human tumor types in a tissue microarray : High levels of expression in gastrointestinal cancers

Purpose: To comprehensively evaluate ephrin receptor B2 (EphB2) expression in normal and neoplastic tissues. EphB2 is a tyrosine kinase recently implicated in the deregulation of cell-to-cell communication in many tumors. Experimental Design: EphB2 protein expression was analyzed by immunohistochemistry on tissue microarrays that included 76 different normal tissues, >4,000 samples from 138 different cancer types, and 1,476 samples of colon cancer with clinical follow-up data. Results: We found most prominent EphB2 expression in the intestinal epithelium (colonic crypts) with cancer of the colorectum displaying the highest EphB2 positivity of all tumors. Positivity was found in 100% of 118 colon adenomas but in 33.3% of 45 colon carcinomas. EphB2 expression was also observed in 75 tumor categories, including serous carcinoma of the endometrium (34.8%), adenocarcinoma of the esophagus (33.3%), intestinal adenocarcinoma of the stomach (30.2%), and adenocarcinoma of the small intestine (70%). The occasional finding of strong EphB2 positivity in tumors without EphB2 positivity in the corresponding normal cells [adenocarcinoma of the lung (4%) and pancreas (2.2%)] suggests that deregulation of EphB2 signaling may involve up-regulation of the protein expression. In colon carcinoma, loss of EphB2 expression was associated with advanced stage (P < 0.0001) and was an indicator of poor overall survival (P = 0.0098). Conclusions: Our results provide an overview on the EphB2 protein expression in normal and neoplastic tissues. Deregulated EphB2 expression may play a role in several cancer types with loss of EphB2 expression serving as an indicator of the possible pathogenetic role of EphB2 signaling in the maintenance of tissue architecture of colon epithelium.

Kinome-wide RNAi studies in human multiple myeloma identify vulnerable kinase targets, including a lymphoid-restricted kinase, GRK6

A paucity of validated kinase targets in human multiple myeloma has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in myeloma tumor lines bearing common t(4;14), t(14;16), and t(11;14) translocations to identify critically vulnerable kinases in myeloma tumor cells without regard to preconceived mechanistic notions. Fifteen kinases were repeatedly vulnerable in myeloma cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. Whereas several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly G protein-coupled receptor kinase, GRK6, appeared selectively vulnerable in myeloma. GRK6 inhibition was lethal to 6 of 7 myeloma tumor lines but was tolerated in 7 of 7 human cell lines. GRK6 exhibits lymphoid-restricted expression, and from coimmunoprecipitation studies we demonstrate that expression in myeloma cells is regulated via direct association with the heat shock protein 90 (HSP90) chaperone. GRK6 silencing causes suppression of signal transducer and activator of transcription 3 (STAT3) phosphorylation associated with reduction in MCL1 levels and phosphorylation, illustrating a potent mechanism for the cytotoxicity of GRK6 inhibition in multiple myeloma (MM) tumor cells. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma.

Intersex-like (IXL) is a cell survival regulator in pancreatic cancer with 19q13 amplification

Pancreatic cancer is a highly aggressive disease characterized by poor prognosis and vast genetic instability. Recent microarray-based, genome-wide surveys have identified multiple recurrent copy number aberrations in pancreatic cancer; however, the target genes are, for the most part, unknown. Here, we characterized the 19q13 amplicon in pancreatic cancer to identify putative new drug targets. Copy number increases at 19q13 were quantitated in 16 pancreatic cancer cell lines and 31 primary tumors by fluorescence in situ hybridization. Cell line copy number data delineated a 1.1 Mb amplicon, the presence of which was also validated in 10% of primary pancreatic tumors. Comprehensive expression analysis by quantitative real-time reverse transcription-PCR indicated that seven transcripts within this region had consistently elevated expression levels in the amplified versus nonamplified cell lines. High-throughput loss-of-function screen by RNA interference was applied across the amplicon to identify genes whose down-regulation affected cell viability. This screen revealed five genes whose down-regulation led to significantly decreased cell viability in the amplified PANC-1 cells but not in the nonamplified MiaPaca-2 cells, suggesting the presence of multiple biologically interesting genes in this region. Of these, the transcriptional regulator intersex-like (IXL) was consistently overexpressed in amplified cells and had the most dramatic effect on cell viability. IXL silencing also resulted in G(0)-G(1) cell cycle arrest and increased apoptosis in PANC-1 cells. These findings implicate IXL as a novel amplification target gene in pancreatic cancer and suggest that IXL is required for cancer cell survival in 19q13-amplified tumors.

CD9 Participates in Endothelial Cell Migration During In Vitro Wound Repair

CD9, a widely expressed membrane protein of the tetraspanin family, has been implicated in diverse functions, such as signal transduction, cell adhesion, and cell motility. We tested the effects of an anti-CD9 monoclonal antibody (ALMA.1) on the migration and proliferation of human vascular endothelial cells (ECs) during repair of an in vitro mechanical wound mimicking angiogenic processes. ALMA.1 induced dose-dependent inhibition of wound repair with a 35+/-1.5% decrease at 20 microg/mL. Only cell migration was affected, because the rate of proliferation of ECs at the lesion margin was not modified and because the inhibition of repair was also observed for nonproliferating irradiated ECs. Monoclonal antibodies against CD63 tetraspanin (H5C6) and control mouse IgG (MOPC-21) were inactive. CD9, one of the most abundant proteins at the surface of ECs, colocalized with beta(1) or beta(3) integrins on EC membranes in double-labeling immunofluorescence experiments with ALMA.1 and an anti-beta(1) (4B4) or anti-beta(3) (SDF.3) monoclonal antibody. Moreover, ALMA.1 and 4B4 had additive inhibitory effects on lesion repair, whereas 4B4 alone also inhibited EC proliferation. In transmembrane Boyden-type assays, ALMA.1 induced dose-dependent inhibition of EC migration toward fibronectin and vitronectin with 45+/-6% and 31+/-10% inhibition, respectively, at 100 microg/mL. 4B4 inhibited migration toward fibronectin at 10 microg/mL but had no effect in the case of vitronectin. Adhesion of ECs to immobilized anti-CD9 monoclonal antibodies induced tyrosine-phosphorylated protein levels similar to those observed during interactions with beta(1) or beta(3) integrins. These results point to the involvement of CD9 in EC adhesion and migration during lesion repair and angiogenesis, probably through cooperation with integrins. As such, CD9 is a potential target to inhibit angiogenesis in metastatic and atherosclerotic processes.

Tetraspanin CD9 Is Associated With Very Late–Acting Integrins in Human Vascular Smooth Muscle Cells and Modulates Collagen Matrix Reorganization

CD9, a member of the tetraspanin family, and very late-acting (VLA) integrins are known to associate and form functional units on the surface of several cell types. We studied the changes in expression of CD9 and beta1-integrins (CD29, VLA) in human vascular smooth muscle cells (VSMCs) under in vitro culture conditions mimicking proliferative vascular diseases. We also investigated possible interactions between CD9 and VLA integrins in VSMCs. We found that CD9 is highly expressed in VSMCs and is subject to modulation, depending on the proliferative/contractile state of the cells. In the contractile phenotype, the levels of CD9, CD81, another tetraspanin, and CD29 are approximately 50% of those found in the proliferative phenotype. Coimmunoprecipitation experiments showed physical association between CD9 and CD29. CD9 was mainly associated with alpha2 and alpha3-integrins (CD49b and c) and also with alpha5-integrin to a weaker extent. Functionally, the addition of anti-CD9 monoclonal antibodies (MoAbs) doubled the extent of collagen gel contraction mediated by VSMCs, a model for the reorganization of the extracellular collagen matrix occurring in the vessel wall. Anti-CD29 MoAbs inhibited gel contraction, but anti-CD9 MoAbs counteracted this inhibitory effect of anti-CD29 MoAbs. Transfection of human CD9 into Chinese hamster ovary cells more than doubled the extent of Chinese hamster ovary cell-mediated collagen gel contraction (130% stimulation), confirming a role for CD9 in extracellular matrix reorganization. Thus, CD9 seems to be involved in the modulation of VLA integrin-mediated collagen matrix reorganization by VSMCs. These findings suggest that high CD9 expression is associated with a proliferative state of VSMCs. The role of CD9 could be to modulate the function of VLA integrins on the surface of VSMCs.

Association of steroid receptor coactivator AIB1 with estrogen receptor-alpha in breast cancer cells.

The steroid receptor coactivator AIB1 (amplified in breast cancer-1) is a transcriptional coactivator which has been found to be amplified in breast cancer. We have now investigated the role of the AIB1 protein in breast cancer cell lines. Although detectable levels of AIB1 were present in most cell lines, high levels of AIB1 expression were observed only in the ER-positive cell lines MCF-7 and BT-474 by western blot analysis. Newly developed monoclonal antibodies (mAbs) were used in several assays to show an association between AIB1 and estrogen receptor-α (ER). AIB1 and ER co-localized to the nucleus of ER positive cell lines as shown by immunofluorescence microscopy, and a functional association of native AIB1 and ER in MCF-7 nuclear extracts was shown by EMSA. Recombinant ER also recruited AIB1 protein from nuclear extracts, shown by EMSA and by precipitation of ER-complex proteins bound to a biotinylated-ERE DNA target. Additionally, anti-AIB1 mAbs were able to immunoprecipitate ER from nuclear extracts of chemically cross-linked cells but not from uncross-linked cells. Both immunoprecipitation and oligonucleotide precipitation studies demonstrated the presence of p300 and CBP as part of the ER transcriptional complex. These results suggest that AIB1 and ER do associate physically in ER-positive breast cancer cell lines. We propose that in AIB1 amplified breast cancers, a heightened AIB1/ER association may play a crucial role in the progression of these tumors.

Aurora B kinase is a potent and selective target in MYCN-driven neuroblastoma

Despite advances in multimodal treatment, neuroblastoma (NB) is often fatal for children with high-risk disease and many survivors need to cope with long-term side effects from high-dose chemotherapy and radiation. To identify new therapeutic targets, we performed an siRNA screen of the druggable genome combined with a small molecule screen of 465 compounds targeting 39 different mechanisms of actions in four NB cell lines. We identified 58 genes as targets, including AURKB, in at least one cell line. In the drug screen, aurora kinase inhibitors (nine molecules) and in particular the AURKB-selective compound, barasertib, were the most discriminatory with regard to sensitivity for MYCN-amplified cell lines. In an expanded panel of ten NB cell lines, those with MYCN-amplification and wild-type TP53 were the most sensitive to low nanomolar concentrations of barasertib. Inhibition of the AURKB kinase activity resulted in decreased phosphorylation of the known target, histone H3, and upregulation of TP53 in MYCN-amplified, TP53 wild-type cells. However, both wild-type and TP53 mutant MYCN-amplified cell lines arrested in G2/M phase upon AURKB inhibition. Additionally, barasertib induced endoreduplication and apoptosis. Treatment of MYCN-amplified/TP53 wild-type neuroblastoma xenografts resulted in profound growth inhibition and tumor regression. Therefore, aurora B kinase inhibition is highly effective in aggressive neuroblastoma and warrants further investigation in clinical trials.