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CD44 expression in intestinal epithelium and colorectal cancer is independent of p53 status.

Zeilstra J, Joosten SP, Vermeulen L, Koster J, Medema JP, Versteeg R, Spaargaren M, Pals ST - PLoS ONE (2013)

Bottom Line: CD44 marks stem cell-like cells in a number of tumour types, including colorectal cancer (CRC), while aberrant CD44 expression conveys increased tumourigenic, invasive, and metastatic potential.In the present study, we therefore explored the relation between p53 mutational status and CD44 expression in a cohort of 90 localized primary CRCs and studied the effect of radiation-induced p53 activation on CD44 expression.Moreover, DNA-damage induced p53 activation did not result in repression of CD44 expression, neither in colon cancer cells nor in normal intestinal epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

ABSTRACT
CD44 marks stem cell-like cells in a number of tumour types, including colorectal cancer (CRC), while aberrant CD44 expression conveys increased tumourigenic, invasive, and metastatic potential. Previous data indicate that CD44 is a direct target of p53-mediated transcriptional repression in breast cancer. Since inactivating p53 mutations are frequent genetic events in CRC these could unleash expression of CD44. In the present study, we therefore explored the relation between p53 mutational status and CD44 expression in a cohort of 90 localized primary CRCs and studied the effect of radiation-induced p53 activation on CD44 expression. Interestingly, we observed that, in contrast to breast cancer, loss of function p53 mutations were not associated with elevated CD44 expression in colon cancer. Moreover, DNA-damage induced p53 activation did not result in repression of CD44 expression, neither in colon cancer cells nor in normal intestinal epithelial cells. Our data demonstrate that CD44 expression in normal and malignant intestinal epithelial cells is not regulated by p53, implying that regulation of this potentially important therapeutic target is tissue and cancer-type specific.

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p53 protein does not repress CD44 expression in colon cancer cells.(A) Immunoblotting analysis of p53, p21 and CD44 protein levels in RKO colon cancer cells treated with ionizing radiation. Actin was used as loading control. (B) qRT-PCR results showing relative gene expression levels for CDKN1A (p21), CD44, and c-MYC. Data represent mean ± SEM of duplicate experiments;.(*, P<0.05 compared with t = 0).
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pone-0072849-g003: p53 protein does not repress CD44 expression in colon cancer cells.(A) Immunoblotting analysis of p53, p21 and CD44 protein levels in RKO colon cancer cells treated with ionizing radiation. Actin was used as loading control. (B) qRT-PCR results showing relative gene expression levels for CDKN1A (p21), CD44, and c-MYC. Data represent mean ± SEM of duplicate experiments;.(*, P<0.05 compared with t = 0).

Mentions: The above findings do not exclude the possibility that wild-type p53 may (partially) suppress CD44 expression in normal and neoplastic intestinal epithelium upon activation by genotoxic stress. To address this possibility, we determined the effects of DNA damage-induced p53 activation on CD44 levels in human RKO colon cancer cells. These cells express wild-type p53 and K-Ras, and are diploid [26]. Of particular interest, RKO cells also contain wild type APC and CTNNB1 genes and lack constitutive β-catenin/TCF-4-mediated transcription [27]. This is of importance since the transcriptional regulation of CD44 by p53 might be masked by constitutive Wnt pathway activation, leading to β-catenin/TCF-4-mediated CD44 expression. RKO cells were exposed to 10 Gy of γ-radiation after which expression of CDKN1A (p21) and CD44 and were analysed by real-time qRT-PCR. Expression of c-MYC, a direct Wnt target gene [28], [29] was also assayed to control for the maintenance of a steady state of β-catenin/TCF-4-driven transcriptional activity. In addition, p53, p21, and CD44 protein levels were analysed by immunoblotting. As expected, ionizing radiation-induced DNA damage resulted in p53 stabilization (Figure 3A) and the consequent transactivation of p21 was observed at all time points (Figure 3A and B). However, CD44 gene expression and CD44 protein levels did not decrease over time (Figure 3A and B), while c-MYC mRNA levels remained stable (Figure 3B). These data indicate that p53 is unable to repress CD44 expression in human colon cancer cells.


CD44 expression in intestinal epithelium and colorectal cancer is independent of p53 status.

Zeilstra J, Joosten SP, Vermeulen L, Koster J, Medema JP, Versteeg R, Spaargaren M, Pals ST - PLoS ONE (2013)

p53 protein does not repress CD44 expression in colon cancer cells.(A) Immunoblotting analysis of p53, p21 and CD44 protein levels in RKO colon cancer cells treated with ionizing radiation. Actin was used as loading control. (B) qRT-PCR results showing relative gene expression levels for CDKN1A (p21), CD44, and c-MYC. Data represent mean ± SEM of duplicate experiments;.(*, P<0.05 compared with t = 0).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3756983&req=5

pone-0072849-g003: p53 protein does not repress CD44 expression in colon cancer cells.(A) Immunoblotting analysis of p53, p21 and CD44 protein levels in RKO colon cancer cells treated with ionizing radiation. Actin was used as loading control. (B) qRT-PCR results showing relative gene expression levels for CDKN1A (p21), CD44, and c-MYC. Data represent mean ± SEM of duplicate experiments;.(*, P<0.05 compared with t = 0).
Mentions: The above findings do not exclude the possibility that wild-type p53 may (partially) suppress CD44 expression in normal and neoplastic intestinal epithelium upon activation by genotoxic stress. To address this possibility, we determined the effects of DNA damage-induced p53 activation on CD44 levels in human RKO colon cancer cells. These cells express wild-type p53 and K-Ras, and are diploid [26]. Of particular interest, RKO cells also contain wild type APC and CTNNB1 genes and lack constitutive β-catenin/TCF-4-mediated transcription [27]. This is of importance since the transcriptional regulation of CD44 by p53 might be masked by constitutive Wnt pathway activation, leading to β-catenin/TCF-4-mediated CD44 expression. RKO cells were exposed to 10 Gy of γ-radiation after which expression of CDKN1A (p21) and CD44 and were analysed by real-time qRT-PCR. Expression of c-MYC, a direct Wnt target gene [28], [29] was also assayed to control for the maintenance of a steady state of β-catenin/TCF-4-driven transcriptional activity. In addition, p53, p21, and CD44 protein levels were analysed by immunoblotting. As expected, ionizing radiation-induced DNA damage resulted in p53 stabilization (Figure 3A) and the consequent transactivation of p21 was observed at all time points (Figure 3A and B). However, CD44 gene expression and CD44 protein levels did not decrease over time (Figure 3A and B), while c-MYC mRNA levels remained stable (Figure 3B). These data indicate that p53 is unable to repress CD44 expression in human colon cancer cells.

Bottom Line: CD44 marks stem cell-like cells in a number of tumour types, including colorectal cancer (CRC), while aberrant CD44 expression conveys increased tumourigenic, invasive, and metastatic potential.In the present study, we therefore explored the relation between p53 mutational status and CD44 expression in a cohort of 90 localized primary CRCs and studied the effect of radiation-induced p53 activation on CD44 expression.Moreover, DNA-damage induced p53 activation did not result in repression of CD44 expression, neither in colon cancer cells nor in normal intestinal epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

ABSTRACT
CD44 marks stem cell-like cells in a number of tumour types, including colorectal cancer (CRC), while aberrant CD44 expression conveys increased tumourigenic, invasive, and metastatic potential. Previous data indicate that CD44 is a direct target of p53-mediated transcriptional repression in breast cancer. Since inactivating p53 mutations are frequent genetic events in CRC these could unleash expression of CD44. In the present study, we therefore explored the relation between p53 mutational status and CD44 expression in a cohort of 90 localized primary CRCs and studied the effect of radiation-induced p53 activation on CD44 expression. Interestingly, we observed that, in contrast to breast cancer, loss of function p53 mutations were not associated with elevated CD44 expression in colon cancer. Moreover, DNA-damage induced p53 activation did not result in repression of CD44 expression, neither in colon cancer cells nor in normal intestinal epithelial cells. Our data demonstrate that CD44 expression in normal and malignant intestinal epithelial cells is not regulated by p53, implying that regulation of this potentially important therapeutic target is tissue and cancer-type specific.

Show MeSH
Related in: MedlinePlus