USP8 and TP53 Drivers are Associated with CNV in a Corticotroph Adenoma Cohort Enriched for Aggressive Tumors.

CONTEXT: Pituitary corticotroph adenomas are rare tumors that can be associated with excess adrenocorticotropin (ACTH) and adrenal cortisol production, resulting in the clinically debilitating endocrine condition Cushing disease. A subset of corticotroph tumors behave aggressively, and genomic drivers behind the development of these tumors are largely unknown. OBJECTIVE: To investigate genomic drivers of corticotroph tumors at risk for aggressive behavior. DESIGN: Whole-exome sequencing of patient-matched corticotroph tumor and normal deoxyribonucleic acid (DNA) from a patient cohort enriched for tumors at risk for aggressive behavior. SETTING: Tertiary care center. PATIENTS: Twenty-seven corticotroph tumors from 22 patients were analyzed. Twelve tumors were macroadenomas, of which 6 were silent ACTH tumors, 2 were Crooke's cell tumors, and 1 was a corticotroph carcinoma. INTERVENTION: Whole-exome sequencing. MAIN OUTCOME MEASURE: Somatic mutation genomic biomarkers. RESULTS: We found recurrent somatic mutations in USP8 and TP53 genes, both with higher allelic fractions than other somatic mutations. These mutations were mutually exclusive, with TP53 mutations occurring only in USP8 wildtype (WT) tumors, indicating they may be independent driver genes. USP8-WT tumors were characterized by extensive somatic copy number variation compared with USP8-mutated tumors. Independent of molecular driver status, we found an association between invasiveness, macroadenomas, and aneuploidy. CONCLUSIONS: Our data suggest that corticotroph tumors may be categorized into a USP8-mutated, genome-stable subtype versus a USP8-WT, genome-disrupted subtype, the latter of which has a TP53-mutated subtype with high level of chromosome instability. These findings could help identify high risk corticotroph tumors, namely those with widespread CNV, that may need closer monitoring and more aggressive treatment.

Regulation of 17 Beta-hydroxysteroid dehydrogenases in cancer: regulating steroid receptor at pre-receptor stage

Increased intracelullar hormone concentration levels have been shown to be the cause of several endocrine-related cancers including breast, prostate, endometrial, ovarian, cervix, testicular, thyroid, and osteosarcoma. Deregulated expression of steroidogenic enzymes in these tumors seems to be the source of a positive balance in active steroids that bind to the corresponding nuclear receptor, thus ultimately stimulating cell proliferation. Among these enzymes, 17 Beta-hydroxysteroid (..) (AU)

Regulation of 17ß-hydroxysteroid dehydrogenases in cancer: regulating steroid receptor at pre-receptor stage.

Increased intracelullar hormone concentration levels have been shown to be the cause of several endocrine-related cancers including breast, prostate, endometrial, ovarian, cervix, testicular, thyroid, and osteosarcoma. Deregulated expression of steroidogenic enzymes in these tumors seems to be the source of a positive balance in active steroids that bind to the corresponding nuclear receptor, thus ultimately stimulating cell proliferation. Among these enzymes, 17ß-hydroxysteroid dehydrogenases catalyze the interconversion between 17-ketosteroids and 17-hydroxysteroids on the last steps of sex hormones biosynthesis and metabolism. To date, 14 isoforms have been identified in vertebrates although only 13 are present in humans. Development and clinical evaluation of specific inhibitors to block their activity is currently under progress especially against the best characterized members 1 to 5. Selectivity and potency of these drugs constitute the main challenge in this new approach to cancer and steroid-dependent diseases treatment at the "pre-receptor level". Here we review the current state of knowledge regarding the deregulation of the expression of some of these enzymes in endocrine-related tumors.

Androgen-induced activation of gonadotropin-regulated testicular RNA helicase (GRTH/Ddx25) transcription: essential role of a nonclassical androgen response element half-site.

GRTH, a testis-specific member of the DEAD-box family of RNA helicases essential for spermatogenesis, is present in Leydig cells (LC) and germ cells. In LC, it exerts an autocrine negative regulation on androgen production induced by gonadotropin. GRTH is transcriptionally upregulated by gonadotropin via cyclic AMP/androgen through androgen receptors (AR). For studies of GRTH regulation by androgen in LC, we utilized in vitro/in vivo models. Androgen-induced GRTH expression was prevented by an AR antagonist. Two putative atypical ARE half-sites are present at bp -200 and -827 (ARE1 and ARE2). Point mutation of ARE2 prevented androgen-induced AR binding/function and upregulation of GRTH transcription. Chromatin immunoprecipitation (ChIP) assays showed recruitment of AR, SRC-1, Med-1, transcription factor IIB (TFIIB), and polymerase II (PolII) to GRTH ARE2 (bp -980/-702) and to the promoter region (bp -80/+63). ChIP3C assays revealed short-range chromosomal looping between AR/ARE2 and the core transcriptional machinery at the promoter. Knockdown of Med-1 and/or SRC-1 demonstrated the presence of a nonproductive complex which included AR, TFIIB, and PolII and the essential role of these coactivators in the transcriptional activation of GRTH. Our findings provide new insights into the molecular mechanism of androgen-regulated transcription in LC.

Testis-specific miRNA-469 up-regulated in gonadotropin-regulated testicular RNA helicase (GRTH/DDX25)-null mice silences transition protein 2 and protamine 2 messages at sites within coding region: implications of its role in germ cell development.

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), a testis-specific member of the DEAD-box family, is an essential post-transcriptional regulator of spermatogenesis. Failure of expression of Transition protein 2 (TP2) and Protamine 2 (Prm2) proteins (chromatin remodelers, essential for spermatid elongation and completion of spermatogenesis) with preservation of their mRNA expression was observed in GRTH-null mice (azoospermic due to failure of spermatids to elongate). These were identified as target genes for the testis-specific miR-469, which is increased in the GRTH-null mice. Further analysis demonstrated that miR-469 repressed TP2 and Prm2 protein expression at the translation level with minor effect on mRNA degradation, through binding to the coding regions of TP2 and Prm2 mRNAs. The corresponding primary-microRNAs and the expression levels of Drosha and DGCR8 (both mRNA and protein) were increased significantly in the GRTH-null mice. miR-469 silencing of TP2 and Prm2 mRNA in pachytene spermatocytes and round spermatids is essential for their timely translation at later times of spermiogenesis, which is critical to attain mature sperm. Collectively, these studies indicate that GRTH, a multifunctional RNA helicase, acts as a negative regulator of miRNA-469 biogenesis and consequently their function during spermatogenesis.

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), a negative regulator of luteinizing/chorionic gonadotropin hormone-induced steroidogenesis in Leydig cells: central role of steroidogenic acute regulatory protein (StAR).

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) is a testis-specific gonadotropin-regulated RNA helicase that is present in Leydig cells (LCs) and germ cells and is essential for spermatid development and completion of spermatogenesis. Normal basal levels of testosterone in serum and LCs were observed in GRTH null (GRTH(-/-)) mice. However, testosterone production was enhanced in LCs of GRTH(-/-) mice compared with WT mice by both in vivo and in vitro human chorionic gonadotropin stimulation. LCs of GRTH(-/-) mice had swollen mitochondria with a significantly increased cholesterol content in the inner mitochondrial membrane. Basal protein levels of SREBP2, HMG-CoA reductase, and steroidogenic acute regulatory protein (StAR; a protein that transports cholesterol to the inner mitochondrial membrane) were markedly increased in LCs of GRTH(-/-) mice compared with WT mice. Gonadotropin stimulation caused an increase in StAR mRNA levels and protein expression in GRTH(-/-) mice versus WT mice, with no further increase in SREBP2 and down-regulation of HMG-CoA reductase protein. The half-life of StAR mRNA was significantly increased in GRTH(-/-) mice. Moreover, association of StAR mRNA with GRTH protein was observed in WT mice. Human chorionic gonadotropin increased GRTH gene expression and its associated StAR protein at cytoplasmic sites. Taken together, these findings indicate that, through its negative role in StAR message stability, GRTH regulates cholesterol availability at the mitochondrial level. The finding of an inhibitory action of GRTH associated with gonadotropin-mediated steroidogenesis has provided insights into a novel negative autocrine molecular control mechanism of this helicase in the regulation of steroid production in the male.

Transcriptional regulation of type 11 17ß-hydroxysteroid dehydrogenase expression in prostate cancer cells.

Type 11 hydroxysteroid (17-beta) dehydrogenase (HSD17B11) catalyzes the conversion of 5α-androstan-3α,17ß-diol into androsterone suggesting that it may play an important role in androgen metabolism. We previously described that overexpression of C/EBPα or C/EBPß induced HSD17B11 expression in HepG2 cells but this process was not mediated by the CCAAT boxes located within its proximal promoter region. Here, we study HSD17B11 transcriptional regulation in prostate cancer (PC) cells. Transfection experiments showed that the region -107/+18 is sufficient for promoter activity in PC cells. Mutagenesis analysis indicated that Sp1 and C/EBP binding sites found in this region are essential for promoter activity. Additional experiments demonstrated that ectopic expression of Sp1 and C/EBPα upregulated HSD17B11 expression only in PC cell lines. Through DAPA and ChIP assays, specific recruitment of Sp1 and C/EBPα to the HSD17B11 promoter was detected. These results show that HSD17B11 transcription in PC cells is regulated by Sp1 and C/EBPα.

Type 10 17ß-hydroxysteroid dehydrogenase expression is regulated by C/EBPß in HepG2 cells.

17ß-hydroxysteroid dehydrogenases (HSD17Bs) are enzymes that stereospecifically reduce or oxidize a keto- or hydroxy group at C17 of the steroid scaffold, respectively. Fourteen mammalian HSD17Bs have been identified so far. We previously described that the HSD17B8 gene is regulated by C/EBPß in the hepatocarcinoma cell line HepG2. Here, we analyze the 5'-flanking region of 14 promoters (HSD17B1-14) looking for CCAAT boxes and binding sites for CCAAT enhancer binding factors (C/EBPs). All promoters were found to have binding sites for these transcription factors except HSD17B1. Ectopic expression of C/EBPα or C/EBPß in HepG2 cells showed that HSD17B11 expression was induced by both transcription factors while HSD17B10 expression was only induced by C/EBPß. The first 500bp of the 5'-flanking region of both genes contain two putative binding sites for C/EBPs. Gene reporter assays showed that C/EBPß transactivated HSD17B10 but not HSD17B11. Additional experiments showed that several isoforms of C/EBPß are involved in HSD17B10 regulation. Mutation of the CCAAT box located at -30/-19 induced HSD17B10 promoter activity when only LIP was expressed, while impaired LAP-induced HSD17B10 transactivation in HepG2 cells when LAP isoforms are expressed. Taken together, our findings reveal that HSD17B10 is regulated by several isoforms of C/EBPß in HepG2 cells.

Caveolin-1 promotes resistance to chemotherapy-induced apoptosis in Ewing's sarcoma cells by modulating PKCalpha phosphorylation.

Caveolin-1 (CAV1) has been implicated in the regulation of several signaling pathways and in oncogenesis. Previously, we identified CAV1 as a key determinant of the oncogenic phenotype and tumorigenic activity of cells from tumors of the Ewing's Sarcoma Family (ESFT). However, the possible CAV1 involvement in the chemotherapy resistance commonly presented by an ESFT subset has not been established to date. This report shows that CAV1 expression determines the sensitivity of ESFT cells to clinically relevant chemotherapeutic agents. Analyses of endogenous CAV1 levels in several ESFT cells and ectopic CAV1 expression into ESFT cells expressing low endogenous CAV1 showed that the higher the CAV1 levels, the greater their resistance to drug treatment. Moreover, results from antisense- and shRNA-mediated gene expression knockdown and protein re-expression experiments demonstrated that CAV1 increases the resistance of ESFT cells to doxorubicin (Dox)- and cisplatin (Cp)-induced apoptosis by a mechanism involving the activating phosphorylation of PKCalpha. CAV1 knockdown in ESFT cells led to decreased phospho(Thr(638))-PKCalpha levels and a concomitant sensitization to apoptosis, which were reversed by CAV1 re-expression. These results were recapitulated by PKCalpha knockdown and re-expression in ESFT cells in which CAV1 was previously knocked down, thus demonstrating that phospho(Thr(638))-PKCalpha acts downstream of CAV1 to determine the sensitivity of ESFT cells to chemotherapeutic drugs. These data, along with the finding that CAV1 and phospho(Thr(638))-PKCalpha are co-expressed in approximately 45% of ESFT specimens tested, imply that targeting CAV1 and/or PKCalpha may allow the development of new molecular therapeutic strategies to improve the treatment outcome for patients with ESFT.

PCPH/ENTPD5 expression confers to prostate cancer cells resistance against cisplatin-induced apoptosis through protein kinase Calpha-mediated Bcl-2 stabilization.

Prostate cancer (PCa) frequently develops antiapoptotic mechanisms and acquires resistance to anticancer drugs. Therefore, identifying PCa drug resistance determinants should facilitate designing more effective chemotherapeutic regimens. Recently, we described that the PCPH protein becomes highly expressed in human prostatic intraepithelial neoplasia and in PCa, and that the functional interaction between PCPH and protein kinase Cdelta (PKCdelta) increases the invasiveness of human PCa. Here, we report that the functional interaction between PCPH and a different PKC isoform, PKCalpha, confers resistance against cisplatin-induced apoptosis to PCa cells. This interaction elicits a mechanism ultimately resulting in the posttranslational stabilization and subsequent elevated expression of Bcl-2. Stable knockdown of either PCPH, mt-PCPH, or PKCalpha in PCa cells decreased Ser70-phosphorylated Bcl-2 and total Bcl-2 protein, thereby increasing their cisplatin sensitivity. Conversely, forced expression of the PCPH protein or, in particular, of the mt-PCPH oncoprotein increased the levels of phosphorylated PKCalpha concurrently with those of Ser70-phosphorylated and total Bcl-2 protein, thus promoting cisplatin resistance. Consistently, Bcl-2 knockdown sensitized PCa cells to cisplatin treatment and, more importantly, reversed the cisplatin resistance of PCa cells expressing the mt-PCPH oncoprotein. Moreover, reexpression of Bcl-2 in PCPH/mt-PCPH knockdown PCa cells reversed the cisplatin sensitization caused by PCPH or mt-PCPH down-regulation. These findings identify PCPH and mt-PCPH as important participants in the chemotherapy response of PCa cells, establish a role for PCPH-PKCalpha-Bcl-2 functional interactions in the drug response process, and imply that targeting PCPH expression before, or simultaneously with, chemotherapy may improve the treatment outcome for PCa patients.

Estradiol induces type 8 17beta-hydroxysteroid dehydrogenase expression: crosstalk between estrogen receptor alpha and C/EBPbeta.

Hydroxysteroid (17-beta) dehydrogenase (HSD17B) are the enzymes responsible for the reversible interconversion of 17-hydroxy and 17-keto steroids. The human and mouse type 8 17beta-HSD (HSD17B8) selectively catalyze the conversion of estradiol (E2) to estrone (E1). We previously described thatHSD17B8 is transcriptionally regulated by C/EBPbeta, and that C/EBPbeta is bound to CCAAT boxes located at -5 and -46 of the transcription start site in basal conditions in HepG2 cells. Furthermore, ectopic expression of C/EBPbeta transactivated the HSD17B8 promoter activity. Here, we show that HSD17B8 expression is up-regulated in response to E2 in the estrogen receptor alpha (ERalpha) positive MCF-7 cells. Results showed that this induction is mediated by ERalpha because i) E2 did not induce HSD17B8 expression in ERalphanegative HepG2 cells, ii) ectopic expression of ERalpha restored E2-induced HSD17B8 expression, and iii) this induction was blocked by the anti-ER ICI 182,780. Additional experiments showed that no estrogen response element was necessary for this regulation. However, the CCAAT boxes located at the HSD17B8 proximal promoter were required for E2-induced transcription. Furthermore, co-immunoprecipitation studies revealed tethering of ERalphatoC/EBPbeta in response to E2 in cells expressing ERalpha. Additionally, chromatin immunoprecipitation assays demonstrated that, in response to E2, ERalpha is recruited to the CCAAT boxes in which C/EBPbeta is already bound. Taken together, our results reveal that ERalpha is involved in the transcriptional regulation of HSD17B8 gene in response to E2 through its interaction with C/EBPbeta.

PCPH/ENTPD5 expression enhances the invasiveness of human prostate cancer cells by a protein kinase C delta-dependent mechanism.

Previous reports showed that PCPH is mutated or deregulated in some human tumors, suggesting its participation in malignant progression. Immunohistochemical analyses showed that PCPH is not expressed in normal prostate, but its expression increases along cancer progression stages, being detectable in benign prostatic hyperplasia, highly expressed in prostatic intraepithelial neoplasia, and remaining at high levels in prostate carcinoma. Experiments designed to investigate the contribution of PCPH to the malignant phenotype of prostate cancer cells showed that PCPH overexpression in PC-3 cells, which express nearly undetectable PCPH levels, increased collagen I expression and enhanced invasiveness, whereas shRNA-mediated PCPH knockdown in LNCaP cells, which express high PCPH levels, down-regulated collagen I expression and decreased invasiveness. PCPH regulated invasiveness and collagen I expression by a mechanism involving protein kinase C delta (PKC delta): (a) PCPH knockdown in LNCaP cells decreased PKC delta levels relative to control cells; (b) PKC delta knockdown in LNCaP cells recapitulated all changes caused by PCPH knockdown; and (c) forced expression of PKC delta in cells with knocked down PCPH reverted all changes provoked by PCPH down-regulation and rescued the original phenotype of LNCaP cells. These results strongly suggested that the expression level and/or mutational status of PCPH contributes to determine the invasiveness of prostate cancer cells through a mechanism involving PKC delta. Data from immunohistochemical analyses in serial sections of normal, premalignant, and malignant prostate specimens underscored the clinical significance of our findings by showing remarkably similar patterns of expression for PCPH and PKC delta, thus strongly suggesting their likely coregulation in human tumors.

Transcriptional regulation of the human type 8 17beta-hydroxysteroid dehydrogenase gene by C/EBPbeta.

17beta-Hydroxysteroid dehydrogenases (17beta-HSD) regulate the intracellular concentration of active sex steroid hormones in target tissues. To date, at least 14 different isozymes have been identified. The type 8 17beta-hydroxysteroid dehydrogenase (17beta-HSD8) selectively catalyzes the conversion of estradiol (E2) to estrone (E1). To map the promoter region and to investigate its regulation, we cloned and fused a 1600 bp DNA fragment upstream of the 17beta-HSD8 transcriptional start site to a luciferase reporter gene. After transient transfection in HepG2 cells, this fragment was shown to possess promoter activity. Deletion constructs of the 5' flanking region of the 17beta-HSD8 gene led to the identification of the minimal promoter region within the first 75 bp upstream of the transcriptional start site. This region included two CCAAT boxes and sequences closely resembling the consensus Sp1 and NF-kappaB motifs. Site directed mutagenesis revealed that the CCAAT boxes were essential for transcription in HepG2. EMSA, supershift and chromatin immunoprecipitation reflected that these sequences were binding sites for C/EBPbeta. Furthermore, promoter activity was increased by the co-transfection of a C/EBPbeta expression vector, and this transactivation was through both CCAAT boxes. Our studies indicate that C/EBPbeta is essential for the transcription of the 17beta-HSD8 gene in the liver.

Caveolin-1 (CAV1) is a target of EWS/FLI-1 and a key determinant of the oncogenic phenotype and tumorigenicity of Ewing's sarcoma cells.

Tumors of the Ewing's sarcoma family (ESFT), such as Ewing's sarcoma (EWS) and primitive neuroectodermal tumors (PNET), are highly aggressive malignancies predominantly affecting children and young adults. ESFT express chimeric transcription factors encoded by hybrid genes fusing the EWS gene with several ETS genes, most commonly FLI-1. EWS/FLI-1 proteins are responsible for the malignant phenotype of ESFT, but only few of their transcriptional targets are known. Using antisense and short hairpin RNA-mediated gene expression knockdown, array analyses, chromatin immunoprecipitation methods, and reexpression studies, we show that caveolin-1 (CAV1) is a new direct target of EWS/FLI-1 that is overexpressed in ESFT cell lines and tumor specimens and is necessary for ESFT tumorigenesis. CAV1 knockdown led to up-regulation of Snail and the concomitant loss of E-cadherin expression. Consistently, loss of CAV1 expression inhibited the anchorage-independent growth of EWS cells and markedly reduced the growth of EWS cell-derived tumors in nude mice xenografts, indicating that CAV1 promotes the malignant phenotype in EWS carcinogenesis. Reexpression of CAV1 or E-cadherin in CAV1 knockdown EWS cells rescued the oncogenic phenotype of the original EWS cells, showing that the CAV1/Snail/E-cadherin pathway plays a central role in the expression of the oncogenic transformation functions of EWS/FLI-1. Overall, these data identify CAV1 as a key determinant of the tumorigenicity of ESFT and imply that targeting CAV1 may allow the development of new molecular therapeutic strategies for ESFT patients.

Acute viral gastroenteritis: proportion and clinical relevance of multiple infections in Spanish children.

Dual infections associated with acute infectious diarrhoea and its microbiological, epidemiological and clinical findings have been evaluated in patients selected from a comprehensive survey of children under 4 years old, admitted to hospital emergency rooms from October 1996 to November 1997. A total of 820 children (433 males and 387 females) were enrolled. Stools were tested for rotavirus, adenovirus, astrovirus and bacterial enteropathogens. Patients were grouped according to age, and the seasonality of mixed infections was evaluated. Clinical trends and severity of gastrointestinal disease by Ruuska's score were also analysed. Mixed infections were identified in 39 cases (5 %), of which 23 were males and 16 were females. The majority of cases were in the 7-18-month age group (26 cases) and occurred in autumn (67 %). Virus-virus co-infections were more frequent (26/39) than virus-bacteria co-infections (13/39). More than two infectious agents were detected in only four cases. The most common viral co-infections were rotavirus-astrovirus (13/26) and rotavirus-adenovirus (10/26). The present report is the first prospective analysis of clinical-epidemiological trends of dual infections in young Spanish children with acute viral gastroenteritis. Our results emphasize the clinical importance of mixed infections as a cause of severe diarrhoea in children.

Neurorretinitis por Bartonella spp. nohenselae / Non henselae Bartonella neuroretinitis

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