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The miR-124-prolyl hydroxylase P4HA1-MMP1 axis plays a critical role in prostate cancer progression.

Chakravarthi BV, Pathi SS, Goswami MT, Cieślik M, Zheng H, Nallasivam S, Arekapudi SR, Jing X, Siddiqui J, Athanikar J, Carskadon SL, Lonigro RJ, Kunju LP, Chinnaiyan AM, Palanisamy N, Varambally S - Oncotarget (2014)

Bottom Line: The invasive properties of P4HA1 overexpressing cells were reversed by blocking MMP1.Our studies indicate P4HA1 copy number gain in a subset of metastatic prostate tumors and its expression is also regulated by microRNA-124.Chick chorioallantoic membrane (CAM) assay and mice xenograft investigations show that P4HA1 is required for tumor growth and metastasis in vivo.

View Article: PubMed Central - PubMed

Affiliation: Michigan Center for Translational Pathology; Department of Pathology, University of Michigan.

ABSTRACT
Collagen prolyl hydroxylases (C-P4HAs) are a family of enzymes involved in collagen biogenesis. One of the isoforms of P4HA, Prolyl 4-hydroxylase, alpha polypeptide I (P4HA1), catalyzes the formation of 4-hydroxyproline that is essential for the proper three-dimensional folding of newly synthesized procollagen chains. Here, we show the overexpression of P4HA1 in aggressive prostate cancer. Immunohistochemical analysis using tissue microarray demonstrated that P4HA1 expression was correlated with prostate cancer progression. Using in vitro studies, we showed that P4HA1 plays a critical role in prostate cancer cell growth and tumor progression. Expression profiling studies using P4HA1 modulated prostate cells suggested regulation of Matrix metalloproteases 1. The invasive properties of P4HA1 overexpressing cells were reversed by blocking MMP1. Our studies indicate P4HA1 copy number gain in a subset of metastatic prostate tumors and its expression is also regulated by microRNA-124. MiR-124 in turn is negatively regulated by transcriptional repressors EZH2 and CtBP1, both of which are overexpressed in aggressive prostate cancer. Chick chorioallantoic membrane (CAM) assay and mice xenograft investigations show that P4HA1 is required for tumor growth and metastasis in vivo. Our observations suggest that P4HA1 plays a critical role in prostate cancer progression and could serve as a viable therapeutic target.

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CtBP1 and EZH2 maintain P4HA1 expression by down-regulating miR-124A and B, qPCR analysis of miR-124 and C, Immunoblot analysis of P4HA1 in CtBP1 and EZH2 stable knockdown DU145 and PC3 cells. D, qPCR analysis of miR-124 and E, Immunoblot analysis of P4HA1, CtBP1 and EZH2 in RWPE cells following infection with control, lacZ adenovirus or CtBP1, EZH2 or EZH2ΔSET mutant adenovirus for 48 h (Asterisk indicates truncated (EZH2SET)). F, Conventional Chromatin immunoprecipitation (ChIP)-PCR analysis for the CtBP1 and G, EZH2 occupancy on miR-124-1 promoter in PC3 cells. ChIP was performed in PC3 Non-T shRNA and respective knockdown cells. ChIP was performed using antibodies against CtBP1, EZH2 and a control IgG. Inset: Schematic representation of the miR-124-1 genomic region on chromosome 8 showing gene and amplicon positions. Error bars: n = 3. All bar graphs are shown with ± SEM. See also Supplementary Figures S8-S10.
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Figure 5: CtBP1 and EZH2 maintain P4HA1 expression by down-regulating miR-124A and B, qPCR analysis of miR-124 and C, Immunoblot analysis of P4HA1 in CtBP1 and EZH2 stable knockdown DU145 and PC3 cells. D, qPCR analysis of miR-124 and E, Immunoblot analysis of P4HA1, CtBP1 and EZH2 in RWPE cells following infection with control, lacZ adenovirus or CtBP1, EZH2 or EZH2ΔSET mutant adenovirus for 48 h (Asterisk indicates truncated (EZH2SET)). F, Conventional Chromatin immunoprecipitation (ChIP)-PCR analysis for the CtBP1 and G, EZH2 occupancy on miR-124-1 promoter in PC3 cells. ChIP was performed in PC3 Non-T shRNA and respective knockdown cells. ChIP was performed using antibodies against CtBP1, EZH2 and a control IgG. Inset: Schematic representation of the miR-124-1 genomic region on chromosome 8 showing gene and amplicon positions. Error bars: n = 3. All bar graphs are shown with ± SEM. See also Supplementary Figures S8-S10.

Mentions: Previous studies demonstrated that miR-124 is down-regulated by epigenetic mechanisms, including DNA methylation and histone modification in various cancers [32-35]. Our previous work suggested EZH2-mediated down-regulation of multiple tumor suppressor miRs such as miR-26, 31, 181a, 181b, 200b, 200c and 203 [36, 37] in prostate and breast cancer. We had also shown that transcriptional co-repressor CtBP1 plays a role in prostate cancer progression by down regulating multiple tumor suppressor genes [16]. We hypothesized that EZH2 and CtBP1 may regulate P4HA1 expression by repressing miR-124. We observed an up-regulation of miR-124 in DU145 and PC3 cells upon stable knockdown of CtBP1 and EZH2 (Figures 5A, B) and a decrease in P4HA1 mRNA (Supplementary Fig. S8A, B) and protein (Figure 5C). Overexpression of CtBP1 and EZH2 resulted in repression of miR-124 (Figure 5D) and a concomitant increase in P4HA1 transcript (Supplementary Fig. S8C) and protein (Figure 5E). SET domain mutant EZH2 (EZH2ΔSET) or control adenoviruses did not repress the miR-124 expression. These data support the roles of CtBP1 and EZH2 in maintaining P4HA1 expression by down-regulating miR-124. Our study is corroborated through Oncomine co-expression analysis that shows CtBP1, EZH2 and P4HA1 are co- and over-expressed in prostate carcinoma samples (Supplementary Fig. S8D).


The miR-124-prolyl hydroxylase P4HA1-MMP1 axis plays a critical role in prostate cancer progression.

Chakravarthi BV, Pathi SS, Goswami MT, Cieślik M, Zheng H, Nallasivam S, Arekapudi SR, Jing X, Siddiqui J, Athanikar J, Carskadon SL, Lonigro RJ, Kunju LP, Chinnaiyan AM, Palanisamy N, Varambally S - Oncotarget (2014)

CtBP1 and EZH2 maintain P4HA1 expression by down-regulating miR-124A and B, qPCR analysis of miR-124 and C, Immunoblot analysis of P4HA1 in CtBP1 and EZH2 stable knockdown DU145 and PC3 cells. D, qPCR analysis of miR-124 and E, Immunoblot analysis of P4HA1, CtBP1 and EZH2 in RWPE cells following infection with control, lacZ adenovirus or CtBP1, EZH2 or EZH2ΔSET mutant adenovirus for 48 h (Asterisk indicates truncated (EZH2SET)). F, Conventional Chromatin immunoprecipitation (ChIP)-PCR analysis for the CtBP1 and G, EZH2 occupancy on miR-124-1 promoter in PC3 cells. ChIP was performed in PC3 Non-T shRNA and respective knockdown cells. ChIP was performed using antibodies against CtBP1, EZH2 and a control IgG. Inset: Schematic representation of the miR-124-1 genomic region on chromosome 8 showing gene and amplicon positions. Error bars: n = 3. All bar graphs are shown with ± SEM. See also Supplementary Figures S8-S10.
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Figure 5: CtBP1 and EZH2 maintain P4HA1 expression by down-regulating miR-124A and B, qPCR analysis of miR-124 and C, Immunoblot analysis of P4HA1 in CtBP1 and EZH2 stable knockdown DU145 and PC3 cells. D, qPCR analysis of miR-124 and E, Immunoblot analysis of P4HA1, CtBP1 and EZH2 in RWPE cells following infection with control, lacZ adenovirus or CtBP1, EZH2 or EZH2ΔSET mutant adenovirus for 48 h (Asterisk indicates truncated (EZH2SET)). F, Conventional Chromatin immunoprecipitation (ChIP)-PCR analysis for the CtBP1 and G, EZH2 occupancy on miR-124-1 promoter in PC3 cells. ChIP was performed in PC3 Non-T shRNA and respective knockdown cells. ChIP was performed using antibodies against CtBP1, EZH2 and a control IgG. Inset: Schematic representation of the miR-124-1 genomic region on chromosome 8 showing gene and amplicon positions. Error bars: n = 3. All bar graphs are shown with ± SEM. See also Supplementary Figures S8-S10.
Mentions: Previous studies demonstrated that miR-124 is down-regulated by epigenetic mechanisms, including DNA methylation and histone modification in various cancers [32-35]. Our previous work suggested EZH2-mediated down-regulation of multiple tumor suppressor miRs such as miR-26, 31, 181a, 181b, 200b, 200c and 203 [36, 37] in prostate and breast cancer. We had also shown that transcriptional co-repressor CtBP1 plays a role in prostate cancer progression by down regulating multiple tumor suppressor genes [16]. We hypothesized that EZH2 and CtBP1 may regulate P4HA1 expression by repressing miR-124. We observed an up-regulation of miR-124 in DU145 and PC3 cells upon stable knockdown of CtBP1 and EZH2 (Figures 5A, B) and a decrease in P4HA1 mRNA (Supplementary Fig. S8A, B) and protein (Figure 5C). Overexpression of CtBP1 and EZH2 resulted in repression of miR-124 (Figure 5D) and a concomitant increase in P4HA1 transcript (Supplementary Fig. S8C) and protein (Figure 5E). SET domain mutant EZH2 (EZH2ΔSET) or control adenoviruses did not repress the miR-124 expression. These data support the roles of CtBP1 and EZH2 in maintaining P4HA1 expression by down-regulating miR-124. Our study is corroborated through Oncomine co-expression analysis that shows CtBP1, EZH2 and P4HA1 are co- and over-expressed in prostate carcinoma samples (Supplementary Fig. S8D).

Bottom Line: The invasive properties of P4HA1 overexpressing cells were reversed by blocking MMP1.Our studies indicate P4HA1 copy number gain in a subset of metastatic prostate tumors and its expression is also regulated by microRNA-124.Chick chorioallantoic membrane (CAM) assay and mice xenograft investigations show that P4HA1 is required for tumor growth and metastasis in vivo.

View Article: PubMed Central - PubMed

Affiliation: Michigan Center for Translational Pathology; Department of Pathology, University of Michigan.

ABSTRACT
Collagen prolyl hydroxylases (C-P4HAs) are a family of enzymes involved in collagen biogenesis. One of the isoforms of P4HA, Prolyl 4-hydroxylase, alpha polypeptide I (P4HA1), catalyzes the formation of 4-hydroxyproline that is essential for the proper three-dimensional folding of newly synthesized procollagen chains. Here, we show the overexpression of P4HA1 in aggressive prostate cancer. Immunohistochemical analysis using tissue microarray demonstrated that P4HA1 expression was correlated with prostate cancer progression. Using in vitro studies, we showed that P4HA1 plays a critical role in prostate cancer cell growth and tumor progression. Expression profiling studies using P4HA1 modulated prostate cells suggested regulation of Matrix metalloproteases 1. The invasive properties of P4HA1 overexpressing cells were reversed by blocking MMP1. Our studies indicate P4HA1 copy number gain in a subset of metastatic prostate tumors and its expression is also regulated by microRNA-124. MiR-124 in turn is negatively regulated by transcriptional repressors EZH2 and CtBP1, both of which are overexpressed in aggressive prostate cancer. Chick chorioallantoic membrane (CAM) assay and mice xenograft investigations show that P4HA1 is required for tumor growth and metastasis in vivo. Our observations suggest that P4HA1 plays a critical role in prostate cancer progression and could serve as a viable therapeutic target.

Show MeSH
Related in: MedlinePlus