Olga Aprelikova

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.

Impaired meiotic DNA-damage repair and lack of crossing-over during spermatogenesis in BRCA1 full-length isoform deficient mice

Breast tumor suppressor gene 1 (BRCA1) plays an essential role in maintaining genomic integrity. Here we show that mouse Brca1 is required for DNA-damage repair and crossing-over during spermatogenesis. Male Brca1Δ11/Δ11p53+/- mice that carried a homozygous deletion of Brca1 exon 11 and a p53 heterozygous mutation had significantly reduced testicular size and no spermatozoa in their seminiferous tubules. During spermatogenesis, homologous chromosomes from the mutant mice synapsed and advanced to the pachytene stage but failed to progress to the diplotene stage. Our analyses revealed that the Brca1 mutation affected cellular localization of several DNA damage-repair proteins. This included prolonged association of γH2AX with sites of DNA damage, reduced sex body formation, diminished Rad51 foci and absence of Mlh1 foci in the pachytene stage. Consequently, chromosomes from mutant mice did not form chiasmata, a point that connects exchanging homologous chromosomes. Brca1-mutant spermatocytes also exhibited decreased RNA expression levels of several genes that are involved in DNA-damage repair, including RuvB-like DNA helicase, XPB, p62 and TFIID. Of note, the premature termination of spermatogenesis at the pachytene stage was accompanied by increased apoptosis by both p53-dependent and p53-independent mechanisms. Thus, our study revealed an essential role of Brca1 in DNA-damage repair and crossing-over of homologous chromosomes during spermatogenesis.

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.

The role of miR-31 and its target gene SATB2 in cancer-associated fibroblasts

It is well established that there is a dynamic relationship between the expanding tumor and the host surrounding tissue. Cancer-associated fibroblasts (CAFs), the most common cellular population found in the tumor microenvironment, supporting tumor growth and dissemination. Here, we set out to determine the factors that may be involved in dramatic alteration of gene expression pattern in CAFs, focusing on microRNA and transcriptional regulators. We established matched pairs of human CAFs isolated from endometrial cancer and normal endometrial fibroblasts. MicroRNA and mRNA analyses identified differential expression of 11 microRNAs, with miR-31 being the most downregulated microRNA in CAFs (p=0.007). We examined several putative miR-31 target genes identified by microarray analysis and demonstrated that miR-31 directly targets the homeobox gene SATB2, which is responsible for chromatin remodeling and regulation of gene expression, and was significantly elevated in CAFs. The functional relevance of miR-31 and SATB2 were tested in in vitro models of endometrial cancer. Overexpression of miR-31 significantly impaired the ability of CAFs to stimulate tumor cell migration and invasion, without affecting tumor cell proliferation. Genetic manipulation of SATB2 levels in normal fibroblasts or CAFs showed that, reciprocally to miR-31, SATB2 increased tumor cell migration and invasion, while knock-down of endogenous SATB2 in CAFs reversed this phenotype. Introduction of SATB2 into normal fibroblasts stimulated expression of a number of genes involved in cell invasion, migration and scattering. These findings provide new insights into tumor-stroma interaction and document that miR

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.

miRNA signature associated with outcome of gastric cancer patients following chemotherapy

BackgroundIdentification of patients who likely will or will not benefit from cytotoxic chemotherapy through the use of biomarkers could greatly improve clinical management by better defining appropriate treatment options for patients. microRNAs may be potentially useful biomarkers that help guide individualized therapy for cancer because microRNA expression is dysregulated in cancer. In order to identify miRNA signatures for gastric cancer and for predicting clinical resistance to cisplatin/fluorouracil (CF) chemotherapy, a comprehensive miRNA microarray analysis was performed using endoscopic biopsy samples.MethodsBiopsy samples were collected prior to chemotherapy from 90 gastric cancer patients treated with CF and from 34 healthy volunteers. At the time of disease progression, post-treatment samples were additionally collected from 8 clinical responders. miRNA expression was determined using a custom-designed Agilent microarray. In order to identify a miRNA signature for chemotherapy resistance, we correlated miRNA expression levels with the time to progression (TTP) of disease after CF therapy.ResultsA miRNA signature distinguishing gastric cancer from normal stomach epithelium was identified. 30 miRNAs were significantly inversely correlated with TTP whereas 28 miRNAs were significantly positively correlated with TTP of 82 cancer patients (P<0.05). Prominent among the upregulated miRNAs associated with chemosensitivity were miRNAs known to regulate apoptosis, including let-7g, miR-342, miR-16, miR-181, miR-1, and miR-34. When this 58-miRNA predictor was applied to a separate set of pre- and post-treatment tumor samples from the 8 clinical responders, all of the 8 pre-treatment samples were correctly predicted as low-risk, whereas samples from the post-treatment tumors that developed chemoresistance were predicted to be in the high-risk category by the 58 miRNA signature, suggesting that selection for the expression of these miRNAs occurred as chemoresistance arose.ConclusionsWe have identified 1) a miRNA expression signature that distinguishes gastric cancer from normal stomach epithelium from healthy volunteers, and 2) a chemoreresistance miRNA expression signature that is correlated with TTP after CF therapy. The chemoresistance miRNA expression signature includes several miRNAs previously shown to regulate apoptosis in vitro, and warrants further validation.

A radiation hybrid map of 18 growth factor, growth factor receptor, hormone receptor, or neurotransmitter receptor genes on the distal region of the long arm of chromosome 5.

The distal portion of the long arm of human chromosome 5 contains an impressive number of genes encoding growth factors, growth factor receptors, and hormone/neurotransmitter receptors. The order of and relative distance between 18 of these genes was determined by radiation hybrid mapping. There is only a single gap in a contiguous radiation map from 5q22-5q35. For this set of radiation hybrids, one map unit (centiray) corresponds to 20-50 kb of DNA. Close physical proximity for several pairs of loci was predicted by the map. Two sets of these were found to be contained in single YAC clones. The physical map produced by radiation hybrid mapping should prove useful in efforts to identify four disease genes that have been assigned to distal 5q by linkage studies.

Silencing of miR-148a in cancer-associated fibroblasts results in WNT10B-mediated stimulation of tumor cell motility.

The tumor microenvironment has an important role in cancer progression. Here we show that miR-148a is downregulated in 15 out of 16 samples (94%) of cancer-associated fibroblasts (CAFs) compared with matched normal tissue fibroblasts (NFs) established from patients with endometrial cancer. Laser-capture microdissection of stromal cells from normal tissue and endometrial cancer confirmed this observation. Treatment of cells with 5-aza-deoxycytidine stimulated the expression of miR-148a in the majority of CAFs implicating DNA methylation in the regulation of miR-148a expression. Investigation of miR-148a function in fibroblasts demonstrated that conditioned media (CM) from CAFs overexpressing miR-148a significantly impaired the migration of five endometrial cancer cell lines without affecting their growth rates in co-culture experiments. Among predicted miR-148a target genes are two WNT family members, WNT1 and WNT10B. Activation of the WNT/β-catenin pathway in CAFs was confirmed by microarray analysis of gene expression and increased activity of the SuperTOPFlash luciferase reporter. We found elevated levels of WNT10B protein in CAFs and its level decreased when miR-148a was re-introduced by lentiviral infection. The 3′-UTR of WNT10B, cloned downstream of luciferase cDNA, suppressed luciferase activity when co-expressed with miR-148a indicating that WNT10B is a direct target of miR-148a. In contrast to the effect of miR-148a, WNT10B stimulated migration of endometrial cancer cell lines. Our findings have defined a molecular mechanism in the tumor microenvironment that is a novel target for cancer therapy.