Profiling of gene expression changes caused by p53 gain-of-function mutant alleles in prostate cancer cells

Clifford G Tepper, Jeffrey Gregg, Xu Bao Shi, Ruth Louise Vinall, Colin A. Baron, Philip E. Ryan, Pierre Yves Desprez, Hsing-Jien Kung, Ralph W deVere White

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

BACKGROUND. Tumor suppressor p53 mutations are associated with the transition of prostate cancer to metastatic, hormone-refractory disease and stable expression of p53 gain-of-function (p53GOF) alleles support growth of LNCaP in androgen-depleted medium. In this study, we performed gene expression profiling of four LNCaP-p53GOF sublines to test the hypothesis that different p53GOF mutants mediated androgen independence via modulation of a common set of genes. METHODS. Expression profiling was performed using Affymetrix HG-U95Av2 arrays followed by hierarchical clustering to identify expression patterns associated with particular molecular alterations. p53GOF-mediated regulation of Id-1 expression was validated by RT-PCR and dual-lUCiferase reporter assays. RNA interference was used to investigate the effects of Id-1 and Id-3 suppression. RESULTS. LNCaP-p53GOF sublines possessed a molecular signature consisting of 95 differentially regulated genes that could be segregated into two clusters of transcripts indUCed (n = 50) and repressed (n = 45) by p53 GOF expression. To begin validating these genes as effectors of the p53 mutants, we evaluated one of the overexpressed genes, Id-1. RT-PCR confirmed the microarray results and revealed elevated Id-1 levels in LNCaP-p53-P151S (loss-of-function only mutant), thereby implicating p53 mutational inactivation, but not gain-of-function, as a basis for Id-1 deregulation. Reporter assays demonstrated enhanced Id-1 promoter activity in an LNCaP-p53GOF subline. The contribution of Id-1 to p53GOF-mediated biology was demonstrated by the ability of RNAi-mediated gene silencing to decrease both basal and androgen-independent proliferation. CONCLUSIONS. While different p53GOF mutants result in overall distinct expression profiles, they share a common set of differentially-expressed genes that can be used to signify their presence and provide insight into mechanisms underlying androgen independence.

Original languageEnglish (US)
Pages (from-to)375-389
Number of pages15
JournalProstate
Volume65
Issue number4
DOIs
StatePublished - Dec 1 2005

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Gene Expression Profiling
Prostatic Neoplasms
Alleles
Androgens
Genes
RNA Interference
Genetic Suppression
Polymerase Chain Reaction
Gene Silencing
Luciferases
Cluster Analysis
Hormones
Growth
Neoplasms

Keywords

  • Androgen independence
  • Id-1
  • Microarray
  • RNA interference

ASJC Scopus subject areas

  • Urology

Cite this

Profiling of gene expression changes caused by p53 gain-of-function mutant alleles in prostate cancer cells. / Tepper, Clifford G; Gregg, Jeffrey; Shi, Xu Bao; Vinall, Ruth Louise; Baron, Colin A.; Ryan, Philip E.; Desprez, Pierre Yves; Kung, Hsing-Jien; deVere White, Ralph W.

In: Prostate, Vol. 65, No. 4, 01.12.2005, p. 375-389.

Research output: Contribution to journalArticle

Tepper, Clifford G ; Gregg, Jeffrey ; Shi, Xu Bao ; Vinall, Ruth Louise ; Baron, Colin A. ; Ryan, Philip E. ; Desprez, Pierre Yves ; Kung, Hsing-Jien ; deVere White, Ralph W. / Profiling of gene expression changes caused by p53 gain-of-function mutant alleles in prostate cancer cells. In: Prostate. 2005 ; Vol. 65, No. 4. pp. 375-389.
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T1 - Profiling of gene expression changes caused by p53 gain-of-function mutant alleles in prostate cancer cells

AU - Tepper, Clifford G

AU - Gregg, Jeffrey

AU - Shi, Xu Bao

AU - Vinall, Ruth Louise

AU - Baron, Colin A.

AU - Ryan, Philip E.

AU - Desprez, Pierre Yves

AU - Kung, Hsing-Jien

AU - deVere White, Ralph W

PY - 2005/12/1

Y1 - 2005/12/1

N2 - BACKGROUND. Tumor suppressor p53 mutations are associated with the transition of prostate cancer to metastatic, hormone-refractory disease and stable expression of p53 gain-of-function (p53GOF) alleles support growth of LNCaP in androgen-depleted medium. In this study, we performed gene expression profiling of four LNCaP-p53GOF sublines to test the hypothesis that different p53GOF mutants mediated androgen independence via modulation of a common set of genes. METHODS. Expression profiling was performed using Affymetrix HG-U95Av2 arrays followed by hierarchical clustering to identify expression patterns associated with particular molecular alterations. p53GOF-mediated regulation of Id-1 expression was validated by RT-PCR and dual-lUCiferase reporter assays. RNA interference was used to investigate the effects of Id-1 and Id-3 suppression. RESULTS. LNCaP-p53GOF sublines possessed a molecular signature consisting of 95 differentially regulated genes that could be segregated into two clusters of transcripts indUCed (n = 50) and repressed (n = 45) by p53 GOF expression. To begin validating these genes as effectors of the p53 mutants, we evaluated one of the overexpressed genes, Id-1. RT-PCR confirmed the microarray results and revealed elevated Id-1 levels in LNCaP-p53-P151S (loss-of-function only mutant), thereby implicating p53 mutational inactivation, but not gain-of-function, as a basis for Id-1 deregulation. Reporter assays demonstrated enhanced Id-1 promoter activity in an LNCaP-p53GOF subline. The contribution of Id-1 to p53GOF-mediated biology was demonstrated by the ability of RNAi-mediated gene silencing to decrease both basal and androgen-independent proliferation. CONCLUSIONS. While different p53GOF mutants result in overall distinct expression profiles, they share a common set of differentially-expressed genes that can be used to signify their presence and provide insight into mechanisms underlying androgen independence.

AB - BACKGROUND. Tumor suppressor p53 mutations are associated with the transition of prostate cancer to metastatic, hormone-refractory disease and stable expression of p53 gain-of-function (p53GOF) alleles support growth of LNCaP in androgen-depleted medium. In this study, we performed gene expression profiling of four LNCaP-p53GOF sublines to test the hypothesis that different p53GOF mutants mediated androgen independence via modulation of a common set of genes. METHODS. Expression profiling was performed using Affymetrix HG-U95Av2 arrays followed by hierarchical clustering to identify expression patterns associated with particular molecular alterations. p53GOF-mediated regulation of Id-1 expression was validated by RT-PCR and dual-lUCiferase reporter assays. RNA interference was used to investigate the effects of Id-1 and Id-3 suppression. RESULTS. LNCaP-p53GOF sublines possessed a molecular signature consisting of 95 differentially regulated genes that could be segregated into two clusters of transcripts indUCed (n = 50) and repressed (n = 45) by p53 GOF expression. To begin validating these genes as effectors of the p53 mutants, we evaluated one of the overexpressed genes, Id-1. RT-PCR confirmed the microarray results and revealed elevated Id-1 levels in LNCaP-p53-P151S (loss-of-function only mutant), thereby implicating p53 mutational inactivation, but not gain-of-function, as a basis for Id-1 deregulation. Reporter assays demonstrated enhanced Id-1 promoter activity in an LNCaP-p53GOF subline. The contribution of Id-1 to p53GOF-mediated biology was demonstrated by the ability of RNAi-mediated gene silencing to decrease both basal and androgen-independent proliferation. CONCLUSIONS. While different p53GOF mutants result in overall distinct expression profiles, they share a common set of differentially-expressed genes that can be used to signify their presence and provide insight into mechanisms underlying androgen independence.

KW - Androgen independence

KW - Id-1

KW - Microarray

KW - RNA interference

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