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TEAD1 and c-Cbl are novel prostate basal cell markers that correlate with poor clinical outcome in prostate cancer.

Knight JF, Shepherd CJ, Rizzo S, Brewer D, Jhavar S, Dodson AR, Cooper CS, Eeles R, Falconer A, Kovacs G, Garrett MD, Norman AR, Shipley J, Hudson DL - Br. J. Cancer (2008)

Bottom Line: RNA from basal and luminal cells isolated from benign tissue by immunoguided laser-capture microdissection was subjected to expression profiling.We identified 112 and 267 genes defining basal and luminal populations, respectively.Analyses of prostate cancer tissue microarray staining established that increased protein levels of either marker were associated with decreased patient survival independent of other clinicopathological metrics.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Carcinogenesis, The Bob Champion Prostate Stem Cell Team, The Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK.

ABSTRACT
Prostate cancer is the most frequently diagnosed male cancer, and its clinical outcome is difficult to predict. The disease may involve the inappropriate expression of genes that normally control the proliferation of epithelial cells in the basal layer and their differentiation into luminal cells. Our aim was to identify novel basal cell markers and assess their prognostic and functional significance in prostate cancer. RNA from basal and luminal cells isolated from benign tissue by immunoguided laser-capture microdissection was subjected to expression profiling. We identified 112 and 267 genes defining basal and luminal populations, respectively. The transcription factor TEAD1 and the ubiquitin ligase c-Cbl were identified as novel basal cell markers. Knockdown of either marker using siRNA in prostate cell lines led to decreased cell growth in PC3 and disrupted acinar formation in a 3D culture system of RWPE1. Analyses of prostate cancer tissue microarray staining established that increased protein levels of either marker were associated with decreased patient survival independent of other clinicopathological metrics. These data are consistent with basal features impacting on the development and clinical course of prostate cancers.

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The effects of siRNA-mediated knockdown of TEAD1 or c-Cbl in RWPE1 and PC3 cell lines. RWPE1 and PC3 cells were reverse transfected using two siRNA oligos per target gene. Mock transfection (M) and non-targeting siRNA (NT) were controls. (A) MTS proliferation assays were performed over 96 h post-transfection. Proliferation was normalised to that of the non-targeting control. Mean of three experiments, bars=s.e.m.; *P<0.05; ***P<0.01. Representative western blots at 72 h post-transfection are shown. (B) Transfected RWPE1 cells were grown in Matrigel for 4 days and the % failure for the formation of spherically polarised acini was calculated. Mean of three experiments, bars=s.e.m. Knockdown of TEAD1 and c-Cbl significantly increased the failure rate (P-values; T1_1=0.006, T1_3=0.009; cbl_8=0.08, cbl_9=0.003). Phase-contrast microscopy images of spheres: A=non-targeting control, B=T1_1 and C=cbl_9. Original magnification × 40, bar=50 μm. The level of knockdown remaining after 4 days was confirmed by western blotting. Representative blots for the three experiments are shown.
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fig4: The effects of siRNA-mediated knockdown of TEAD1 or c-Cbl in RWPE1 and PC3 cell lines. RWPE1 and PC3 cells were reverse transfected using two siRNA oligos per target gene. Mock transfection (M) and non-targeting siRNA (NT) were controls. (A) MTS proliferation assays were performed over 96 h post-transfection. Proliferation was normalised to that of the non-targeting control. Mean of three experiments, bars=s.e.m.; *P<0.05; ***P<0.01. Representative western blots at 72 h post-transfection are shown. (B) Transfected RWPE1 cells were grown in Matrigel for 4 days and the % failure for the formation of spherically polarised acini was calculated. Mean of three experiments, bars=s.e.m. Knockdown of TEAD1 and c-Cbl significantly increased the failure rate (P-values; T1_1=0.006, T1_3=0.009; cbl_8=0.08, cbl_9=0.003). Phase-contrast microscopy images of spheres: A=non-targeting control, B=T1_1 and C=cbl_9. Original magnification × 40, bar=50 μm. The level of knockdown remaining after 4 days was confirmed by western blotting. Representative blots for the three experiments are shown.

Mentions: To investigate whether TEAD1 or c-Cbl has a role in proliferation, two well-established prostate cell lines were transfected with siRNA oligonucleotides targeting each gene. Knockdown was confirmed by western blotting and the effect on proliferation quantitated by MTS assays carried out over a 96-h time course (Figure 4A). Although the proliferation of RWPE1 cells was not affected by knockdown of either TEAD1 or c-Cbl, the proliferation of PC3 cells was significantly reduced. Data were normalised to proliferation of cells transfected with non-targeting siRNA. Normalisation to proliferation of mock-transfected cells yielded similar results.


TEAD1 and c-Cbl are novel prostate basal cell markers that correlate with poor clinical outcome in prostate cancer.

Knight JF, Shepherd CJ, Rizzo S, Brewer D, Jhavar S, Dodson AR, Cooper CS, Eeles R, Falconer A, Kovacs G, Garrett MD, Norman AR, Shipley J, Hudson DL - Br. J. Cancer (2008)

The effects of siRNA-mediated knockdown of TEAD1 or c-Cbl in RWPE1 and PC3 cell lines. RWPE1 and PC3 cells were reverse transfected using two siRNA oligos per target gene. Mock transfection (M) and non-targeting siRNA (NT) were controls. (A) MTS proliferation assays were performed over 96 h post-transfection. Proliferation was normalised to that of the non-targeting control. Mean of three experiments, bars=s.e.m.; *P<0.05; ***P<0.01. Representative western blots at 72 h post-transfection are shown. (B) Transfected RWPE1 cells were grown in Matrigel for 4 days and the % failure for the formation of spherically polarised acini was calculated. Mean of three experiments, bars=s.e.m. Knockdown of TEAD1 and c-Cbl significantly increased the failure rate (P-values; T1_1=0.006, T1_3=0.009; cbl_8=0.08, cbl_9=0.003). Phase-contrast microscopy images of spheres: A=non-targeting control, B=T1_1 and C=cbl_9. Original magnification × 40, bar=50 μm. The level of knockdown remaining after 4 days was confirmed by western blotting. Representative blots for the three experiments are shown.
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Related In: Results  -  Collection

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fig4: The effects of siRNA-mediated knockdown of TEAD1 or c-Cbl in RWPE1 and PC3 cell lines. RWPE1 and PC3 cells were reverse transfected using two siRNA oligos per target gene. Mock transfection (M) and non-targeting siRNA (NT) were controls. (A) MTS proliferation assays were performed over 96 h post-transfection. Proliferation was normalised to that of the non-targeting control. Mean of three experiments, bars=s.e.m.; *P<0.05; ***P<0.01. Representative western blots at 72 h post-transfection are shown. (B) Transfected RWPE1 cells were grown in Matrigel for 4 days and the % failure for the formation of spherically polarised acini was calculated. Mean of three experiments, bars=s.e.m. Knockdown of TEAD1 and c-Cbl significantly increased the failure rate (P-values; T1_1=0.006, T1_3=0.009; cbl_8=0.08, cbl_9=0.003). Phase-contrast microscopy images of spheres: A=non-targeting control, B=T1_1 and C=cbl_9. Original magnification × 40, bar=50 μm. The level of knockdown remaining after 4 days was confirmed by western blotting. Representative blots for the three experiments are shown.
Mentions: To investigate whether TEAD1 or c-Cbl has a role in proliferation, two well-established prostate cell lines were transfected with siRNA oligonucleotides targeting each gene. Knockdown was confirmed by western blotting and the effect on proliferation quantitated by MTS assays carried out over a 96-h time course (Figure 4A). Although the proliferation of RWPE1 cells was not affected by knockdown of either TEAD1 or c-Cbl, the proliferation of PC3 cells was significantly reduced. Data were normalised to proliferation of cells transfected with non-targeting siRNA. Normalisation to proliferation of mock-transfected cells yielded similar results.

Bottom Line: RNA from basal and luminal cells isolated from benign tissue by immunoguided laser-capture microdissection was subjected to expression profiling.We identified 112 and 267 genes defining basal and luminal populations, respectively.Analyses of prostate cancer tissue microarray staining established that increased protein levels of either marker were associated with decreased patient survival independent of other clinicopathological metrics.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Carcinogenesis, The Bob Champion Prostate Stem Cell Team, The Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK.

ABSTRACT
Prostate cancer is the most frequently diagnosed male cancer, and its clinical outcome is difficult to predict. The disease may involve the inappropriate expression of genes that normally control the proliferation of epithelial cells in the basal layer and their differentiation into luminal cells. Our aim was to identify novel basal cell markers and assess their prognostic and functional significance in prostate cancer. RNA from basal and luminal cells isolated from benign tissue by immunoguided laser-capture microdissection was subjected to expression profiling. We identified 112 and 267 genes defining basal and luminal populations, respectively. The transcription factor TEAD1 and the ubiquitin ligase c-Cbl were identified as novel basal cell markers. Knockdown of either marker using siRNA in prostate cell lines led to decreased cell growth in PC3 and disrupted acinar formation in a 3D culture system of RWPE1. Analyses of prostate cancer tissue microarray staining established that increased protein levels of either marker were associated with decreased patient survival independent of other clinicopathological metrics. These data are consistent with basal features impacting on the development and clinical course of prostate cancers.

Show MeSH
Related in: MedlinePlus