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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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Laser-capture microdissection pressure catapulting (LMPC) and expression profiling of basal and luminal prostate epithelial cells. (A) Snap-frozen BPH tissue was rapid immunostained for basal cell marker K14, with nuclei counter stained with haematoxylin (a). Basal and luminal epithelial cells were laser captured sequentially; luminal cells were selected (b), then captured (c), followed by selection (d) and capture (e) of K14-positive basal cells. Extracted RNA from five patients was used for expression profiling by cDNA microarray. Differentially expressed genes were identified through ANOVA (P=0.05). Genes were entered into hierarchical cluster analysis represented here by a dendrogram. (B) Semiquantitative RT–PCR of amplified RNA from three patient samples (1, 2 and 4) confirming differential basal (B) and luminal (L) expression of SNAP25, TEAD1 and integrin αV. Luminal expression of SNAP25 was confirmed in three out of three patients; basal expression of TEAD1 was confirmed in two out of three patients and basal expression of integrin αV was confirmed in one out of three patients.
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fig1: Laser-capture microdissection pressure catapulting (LMPC) and expression profiling of basal and luminal prostate epithelial cells. (A) Snap-frozen BPH tissue was rapid immunostained for basal cell marker K14, with nuclei counter stained with haematoxylin (a). Basal and luminal epithelial cells were laser captured sequentially; luminal cells were selected (b), then captured (c), followed by selection (d) and capture (e) of K14-positive basal cells. Extracted RNA from five patients was used for expression profiling by cDNA microarray. Differentially expressed genes were identified through ANOVA (P=0.05). Genes were entered into hierarchical cluster analysis represented here by a dendrogram. (B) Semiquantitative RT–PCR of amplified RNA from three patient samples (1, 2 and 4) confirming differential basal (B) and luminal (L) expression of SNAP25, TEAD1 and integrin αV. Luminal expression of SNAP25 was confirmed in three out of three patients; basal expression of TEAD1 was confirmed in two out of three patients and basal expression of integrin αV was confirmed in one out of three patients.

Mentions: Basal and luminal cells were laser captured as illustrated in Figure 1A. Two rounds of RNA amplification yielded 24–38 μg of RNA with fragment sizes ⩽1500 bp. Microarray hybridisations were performed for each of the five basal and luminal samples, hybridising each against universal reference cDNA. Hierarchical clustering of the gene set (Figure 1A) showed a clear segregation of basal and luminal samples. A 1.4-fold cutoff relative to reference cDNA was applied for all differentially expressed genes, yielding basal and luminal lists of 112 and 267 genes, respectively. Basal/luminal or luminal/basal fold-change ratios were calculated to directly compare the two populations (Supplementary Tables S2a and S2b). K14 was overexpressed in the basal population, confirming positive cell separation. A number of differentially expressed genes were selected for verification of the microarray data by semiquantitative PCR (Figure 1B). Luminal expression of SNAP25 was confirmed in three out of three patients and basal expression of TEAD1 was confirmed in two out of three patients. Basal expression of integrin αV was confirmed in just one out of three patients and c-Cbl failed to generate any product. Owing to the varying success of the PCR, due, most likely, to the poor quality of the starting RNA, all further localisation confirmation was carried out using antibody staining.


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)

Laser-capture microdissection pressure catapulting (LMPC) and expression profiling of basal and luminal prostate epithelial cells. (A) Snap-frozen BPH tissue was rapid immunostained for basal cell marker K14, with nuclei counter stained with haematoxylin (a). Basal and luminal epithelial cells were laser captured sequentially; luminal cells were selected (b), then captured (c), followed by selection (d) and capture (e) of K14-positive basal cells. Extracted RNA from five patients was used for expression profiling by cDNA microarray. Differentially expressed genes were identified through ANOVA (P=0.05). Genes were entered into hierarchical cluster analysis represented here by a dendrogram. (B) Semiquantitative RT–PCR of amplified RNA from three patient samples (1, 2 and 4) confirming differential basal (B) and luminal (L) expression of SNAP25, TEAD1 and integrin αV. Luminal expression of SNAP25 was confirmed in three out of three patients; basal expression of TEAD1 was confirmed in two out of three patients and basal expression of integrin αV was confirmed in one out of three patients.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2600693&req=5

fig1: Laser-capture microdissection pressure catapulting (LMPC) and expression profiling of basal and luminal prostate epithelial cells. (A) Snap-frozen BPH tissue was rapid immunostained for basal cell marker K14, with nuclei counter stained with haematoxylin (a). Basal and luminal epithelial cells were laser captured sequentially; luminal cells were selected (b), then captured (c), followed by selection (d) and capture (e) of K14-positive basal cells. Extracted RNA from five patients was used for expression profiling by cDNA microarray. Differentially expressed genes were identified through ANOVA (P=0.05). Genes were entered into hierarchical cluster analysis represented here by a dendrogram. (B) Semiquantitative RT–PCR of amplified RNA from three patient samples (1, 2 and 4) confirming differential basal (B) and luminal (L) expression of SNAP25, TEAD1 and integrin αV. Luminal expression of SNAP25 was confirmed in three out of three patients; basal expression of TEAD1 was confirmed in two out of three patients and basal expression of integrin αV was confirmed in one out of three patients.
Mentions: Basal and luminal cells were laser captured as illustrated in Figure 1A. Two rounds of RNA amplification yielded 24–38 μg of RNA with fragment sizes ⩽1500 bp. Microarray hybridisations were performed for each of the five basal and luminal samples, hybridising each against universal reference cDNA. Hierarchical clustering of the gene set (Figure 1A) showed a clear segregation of basal and luminal samples. A 1.4-fold cutoff relative to reference cDNA was applied for all differentially expressed genes, yielding basal and luminal lists of 112 and 267 genes, respectively. Basal/luminal or luminal/basal fold-change ratios were calculated to directly compare the two populations (Supplementary Tables S2a and S2b). K14 was overexpressed in the basal population, confirming positive cell separation. A number of differentially expressed genes were selected for verification of the microarray data by semiquantitative PCR (Figure 1B). Luminal expression of SNAP25 was confirmed in three out of three patients and basal expression of TEAD1 was confirmed in two out of three patients. Basal expression of integrin αV was confirmed in just one out of three patients and c-Cbl failed to generate any product. Owing to the varying success of the PCR, due, most likely, to the poor quality of the starting RNA, all further localisation confirmation was carried out using antibody staining.

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