Gadisetti V.R. Chandramouli

BRCA1, histone H2AX phosphorylation, and male meiotic sex chromosome inactivation.

In mammalian spermatogenesis, the X and Y chromosomes are transcriptionally silenced during the pachytene stage of meiotic prophase (meiotic sex chromosome inactivation, MSCI), forming a condensed chromatin domain termed the sex or XY body. The nucleosomal core histone H2AX is phosphorylated within the XY chromatin domain just prior to MSCI, and it has been hypothesized that this triggers the chromatin condensation and transcriptional repression. Here, we show that the kinase ATR localizes to XY chromatin at the onset of MSCI and that this localization is disrupted in mice with a mutant form of the tumor suppressor protein BRCA1. In the mutant pachytene cells, ATR is usually present at nonsex chromosomal sites, where it colocalizes with aberrant sites of H2AX phosphorylation; in these cells, there is MSCI failure. In rare pachytene cells, ATR does locate to XY chromatin, H2AX is then phosphorylated, a sex body forms, and MSCI ensues. These observations highlight an important role for BRCA1 in recruiting the kinase ATR to XY chromatin at the onset of MSCI and provide compelling evidence that it is ATR that phosphorylates H2AX and triggers MSCI.

Regulation of HIF prolyl hydroxylases by hypoxia‐inducible factors

Hypoxia and induction of hypoxia‐inducible factors (HIF‐1α and HIF‐2α) is a hallmark of many tumors. Under normal oxygen tension HIF‐alpha subunits are rapidly degraded through prolyl hydroxylase dependent interaction with the von Hippel‐Lindau (VHL) tumor suppressor protein, a component of E3 ubuiquitin ligase complex. Using microarray analysis of VHL mutated and re‐introduced cells, we found that one of the prolyl hydroxylases (PHD3) is coordinately expressed with known HIF target genes, while the other two family members (PHD1 and 2) did not respond to VHL. We further tested the regulation of these genes by HIF‐1 and HIF‐2 and found that siRNA targeted degradation of HIF‐1α and HIF‐2α results in decreased hypoxia‐induced PHD3 expression. Ectopic overexpression of HIF‐2α in two different cell lines provided a much better induction of PHD3 gene than HIF‐1α. In contrast, we demonstrate that PHD2 is not affected by overexpression or downregulation of HIF‐2α. However, induction of PHD2 by hypoxia has HIF‐1‐independent and ‐dependent components. Short‐term hypoxia (4 h) results in induction of PHD2 independent of HIF‐1, while PHD2 accumulation by prolonged hypoxia (16 h) was decreased by siRNA‐mediated degradation of HIF‐1α subunit. These data further advance our understanding of the differential role of HIF factors and putative feedback loop in HIF regulation. Published 2004 Wiley‐Liss, Inc.

Gene Expression Profiles Associated with Response to Chemotherapy in Epithelial Ovarian Cancers

Purpose: The goal of this study was to determine whether distinct gene expression profiles are associated with intrinsic and/or acquired chemoresistance in epithelial ovarian carcinoma. Experimental Design: Gene expression profiles were generated from 21 primary chemosensitive tumors and 24 primary chemoresistant tumors using cDNA-based microarrays. Gene expression profiles of both groups of primary tumors were then compared with those of 15 ovarian carcinomas obtained following platinum-based chemotherapy (“postchemotherapy” tumors). A theme discovery tool was used to identify functional categories of genes involved in drug resistance. Results: Comparison of primary chemosensitive and chemoresistant tumors revealed differential expression of 85 genes (P < 0.001). Comparison of gene expression profiles of primary chemosensitive tumors and postchemotherapy tumors revealed more robust differences with 760 genes differentiating the two groups (P < 0.001). In contrast, only 230 genes were differentially expressed between primary chemoresistant and postchemotherapy groups (P < 0.001). Common to both gene lists were 178 genes representing transcripts differentially expressed between postchemotherapy tumors and all primary tumors irrespective of intrinsic chemosensitivity. The gene expression profile of postchemotherapy tumors compared with that of primary tumors revealed statistically significant overrepresentation of genes encoding extracellular matrix–related proteins. Conclusions: These data show that gene expression profiling can discriminate primary chemoresistant from primary chemosensitive ovarian cancers. Gene expression profiles were also identified that correlate with states of intrinsic and acquired chemoresistance and that represent targets for future investigation and potential therapeutic interventions.

Molecular Alterations in Primary Prostate Cancer after Androgen Ablation Therapy

Purpose: After an initial response to androgen ablation, most prostate tumors recur, ultimately progressing to highly aggressive androgen-independent cancer. The molecular mechanisms underlying progression are not well known in part due to the rarity of androgen-independent samples from primary and metastatic sites. Experimental Design: We compared the gene expression profiles of 10 androgen-independent primary prostate tumor biopsies with 10 primary, untreated androgen-dependent tumors. Samples were laser capture microdissected, the RNA was amplified, and gene expression was assessed using Affymetrix Human Genome U133A GeneChip. Differential expression was examined with principal component analysis, hierarchical clustering, and Students t testing. Analysis of gene ontology was done with Expression Analysis Systematic Explorer and gene expression data were integrated with genomic alterations with Differential Gene Locus Mapping. Results: Unsupervised principal component analysis showed that the androgen-dependent and androgen-independent tumors segregated from one another. After filtering the data, 239 differentially expressed genes were identified. Two main gene ontologies were found discordant between androgen-independent and androgen-dependent tumors: macromolecule biosynthesis was down-regulated and cell adhesion was up-regulated in androgen-independent tumors. Other differentially expressed genes were related to interleukin-6 signaling as well as angiogenesis, cell adhesion, apoptosis, oxidative stress, and hormone response. The Differential Gene Locus Mapping analysis identified nine regions of potential chromosomal deletion in the androgen-independent tumors, including 1p36, 3p21, 6p21, 8p21, 11p15, 11q12, 12q23, 16q12, and 16q21. Conclusions: Taken together, these data identify several unique characteristics of androgen-independent prostate cancer that may hold potential for the development of targeted therapeutic intervention.

Role of ETS transcription factors in the hypoxia-inducible factor-2 target gene selection

Tumor hypoxia often directly correlates with aggressive phenotype, metastasis progression, and resistance to chemotherapy. Two transcription factors [hypoxia-inducible factor-1alpha (HIF-1alpha) and HIF-2alpha] are dramatically induced in hypoxic areas and regulate the expression of genes necessary for tumor adaptation to the conditions of low oxygen; however, the relative contribution of these factors is controversial. We used RNA interference-mediated inactivation of HIF-1alpha or HIF-2alpha followed by microarray analysis to identify genes specifically regulated by either HIF-1 or HIF-2 in hypoxia. We found that, in the MCF7 cell line, the vast majority of hypoxia-responsive genes (>80%) were dependent on the presence of HIF-1alpha. However, a small group of genes were preferentially regulated by HIF-2alpha. Promoter analysis for this group of genes revealed that all of them have putative binding sites for ETS family transcription factors, and 10 of 11 HIF-2alpha-dependent genes had at least one potential hypoxia-responsive element (HRE) in proximity to an ETS transcription factor binding site. Knockdown of ELK-1, the most often represented member of ETS family, significantly reduced hypoxic induction of the HIF-2alpha-dependent genes. Physical and functional interaction between ELK-1 and HIF-2alpha were supported by coimmunoprecipitation of these two proteins, luciferase reporter assay using CITED2 promoter, and binding of ELK-1 protein to the promoters of CITED2 and WISP2 genes in proximity to a HRE. These data suggest that the choice of the target genes by HIF-1 or HIF-2 depends on availability and cooperation of HIFs with other factors recognizing their cognate elements in the promoters.

Gene Expression Profiling of Breast, Prostate, and Glioma Cells following Single versus Fractionated Doses of Radiation

Studies were conducted to determine whether gene expression profiles following a single dose of radiation would yield equivalent profiles following fractionated radiation in different tumor cell lines. MCF7 (breast), DU145 (prostate), and SF539 (gliosarcoma) cells were exposed to a total radiation dose of 10 Gy administered as a single dose (SD) or by daily multifractions (MF) of 5 x 2 Gy. Following radiation treatment, mRNA was isolated at 1, 4, 10, and 24 h and processed for cDNA microarray analysis. To determine the influence of the tumor microenvironment on gene expression, one cell type (DU145) was evaluated growing as a solid tumor in athymic nude mice for both radiation protocols. Unsupervised hierarchical cluster map analysis showed significant differences in gene expression profiles between SD and MF treatments for cells treated in vitro, with MF yielding a more robust induction compared with SD. Several genes were uniquely up-regulated by MF treatment, including multiple IFN-related genes (STAT1, G1P2, OAS1, OAS3, G1P3, IFITM1) and TGF-beta-associated genes (EGR1, VEGF, THBS1, and TGFB2). DU145 cells grown in vivo exhibited a completely different set of genes induced by both SD and MF compared with the same cells exposed in vitro. The results of the study clearly show distinct differences in the molecular response of cells between SD and MF radiation exposures and show that the tumor microenvironment can significantly influence the pattern of gene expression after radiation exposures.

Global expression profiling identifies signatures of tumor virulence in MMTV-PyMT-transgenic mice: correlation to human disease.

FVB/N-Tg (MMTV-PyMT)634Mul-transgenic mice develop multifocal mammary tumors with a high incidence of pulmonary metastasis. We have demonstrated previously that mammary tumors derived from transgene-positive F1 progeny in particular inbred strains display altered latency, tumor growth rates, and metastatic rates when compared with the FVB/NJ homozygous parent. To identify genes with expression that might be critical in modifying the biological behavior of MMTV-PyMT tumors, we performed a detailed comparative analysis of expression profiles from mammary tumors arising in the parental FVB/NJ background and F1 progeny from crosses with I/LnJ, LP/J, MOLF/Ei, and NZB/B1NJ mice. Compared with normal mammary glands, gene expression profiles of tumors from all five strains exhibited up-regulation of genes involved in cell growth (e.g., Cks1 and CDC25C) and down-regulation of cell adhesion molecules, with many genes associated previously with human breast cancer such as STAT2, CD24 antigen, gelsolin, and lipocalin2. To identify genes with significant variation in expression between the five different genotypes, significance analysis of microarrays (SAM) and one-way ANOVA were used. Three definable groupings of tumors were identified: (a) tumors derived in the LP/J F1 and MOLF/Ei F1 strains in which tumor growth and dissemination are suppressed and latency prolonged; (b) the most aggressive tumors from the FVB/NJ parental strain and I/LnJ F1 genomic backgrounds; and (c) an intermediate virulence phenotype with tumors from NZB/B1NJ–F1 crosses. These array based assessments correlated well with a composite phenotype ranking using a “virulence” index. The gene expression signature that is associated with a high metastatic rate in the mouse contains the same 17 genes described recently as the signature gene set predictive of metastasis in human tumors (1) with 16 of the 17 genes exhibiting the same directional change in expression associated with human metastases. These results demonstrate that the genetic analysis of mouse models of tumorigenesis may be highly relevant to human cancer and that the metastatic phenotype of a tumor may be affected by the germline genetic configuration of the host.

Cancers as Wounds that Do Not Heal: Differences and Similarities between Renal Regeneration/Repair and Renal Cell Carcinoma

Cancers have been described as wounds that do not heal, suggesting that the two share common features. By comparing microarray data from a model of renal regeneration and repair (RRR) with reported gene expression in renal cell carcinoma (RCC), we asked whether those two processes do, in fact, share molecular features and regulatory mechanisms. The majority (77%) of the genes expressed in RRR and RCC were concordantly regulated, whereas only 23% were discordant (i.e., changed in opposite directions). The orchestrated processes of regeneration, involving cell proliferation and immune response, were reflected in the concordant genes. The discordant gene signature revealed processes (e.g., morphogenesis and glycolysis) and pathways (e.g., hypoxia-inducible factor and insulin-like growth factor-I) that reflect the intrinsic pathologic nature of RCC. This is the first study that compares gene expression patterns in RCC and RRR. It does so, in particular, with relation to the hypothesis that RCC resembles the wound healing processes seen in RRR. However, careful attention to the genes that are regulated in the discordant direction provides new insights into the critical differences between renal carcinogenesis and wound healing. The observations reported here provide a conceptual framework for further efforts to understand the biology and to develop more effective diagnostic biomarkers and therapeutic strategies for renal tumors and renal ischemia.

Microarray Analysis of Endometrial Carcinomas and Mixed Mullerian Tumors Reveals Distinct Gene Expression Profiles Associated with Different Histologic Types of Uterine Cancer

Previous studies using cDNA microarray have indicated that distinct gene expression profiles characterize endometrioid and papillary serous carcinomas of the endometrium. Molecular studies have observed that mixed mullerian tumors, characterized by both carcinomatous and sarcomatous components, share features that are characteristic of endometrial carcinomas. The objective of this analysis was to more precisely define gene expression patterns that distinguish endometrioid and papillary serous histologies of endometrial carcinoma and mixed mullerian tumors of the uterus. One hundred nineteen pathologically confirmed uterine cancer samples were studied (66 endometrioid, 24 papillary serous, and 29 mixed mullerian tumors). Gene expressions were analyzed using the Affymetrix Human Genome Arrays U133A and U133B Genechip set. Unsupervised analysis revealed distinct global gene expression patterns of endometrioid, papillary serous, mixed mullerian tumors, and normal tissues as grossly separated clusters. Two-sample t tests comparing endometrioid and papillary serous, endometrioid and mixed mullerian tumor, and papillary serous and mixed mullerian tumor pairs identified 1,055, 5,212, and 1,208 differentially expressed genes at P < 0.001, respectively. These data revealed that distinct patterns of gene expression characterize various histologic types of uterine cancer. Gene expression profiles for select genes were confirmed using quantitative PCR. An understanding of the molecular heterogeneity of various histologic types of endometrial cancer has the potential to lead to better individualization of treatment in the future.

Molecular determinants of tumor differentiation in papillary serous ovarian carcinoma

In epithelial ovarian cancer, tumor grade is an independent prognosticator whose molecular determinants remain unknown. We investigated patterns of gene expression in well‐ and poorly differentiated serous papillary ovarian and peritoneal carcinomas with cDNA microarrays. A 6500‐feature cDNA microarray was used for comparison of the molecular profiles of eight grade III and four grade I stage III serous papillary adenocarcinomas. With a modified F‐test in conjunction with random permutations, 99 genes whose expression was significantly different between grade I and grade III tumors were identified (P < 0.01). A disproportionate number of these differentially expressed genes were located on the chromosomal regions 20q13 and all exhibited higher expression in grade III tumors. Interphase fluorescent in situ hybridization demonstrated 20q13 amplification in two of the four grade III and none of the three grade I tumors available for evaluation. Several centrosome‐related genes also showed higher expression in grade III tumors. We propose a model in which tumor differentiation is inversely correlated with the overexpression of several oncogenes located on 20q13, a common amplicon in ovarian and numerous other cancers. Dysregulation of centrosome function is one potential mechanistic link between genetic/epigenetic changes and the poorly differentiated phenotype in ovarian cancer. Published 2003 Wiley‐Liss, Inc.