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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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HIF1α modulates P4HA1 expression by down-regulating miR-124A, Immunoblot analysis of HIF1α -DU145 and -PC3 knockdown lysates under normoxia or in the presence of the hypoxia-mimetic agent CoCl2. HIF1α knockdown significantly reduced P4HA1, CtBP1 and EZH2. β-actin was used as a loading control and * denotes an additional band detected by the antibody. B and C, qPCR analysis of miR-124 in HIF1α-stable knockdown DU145 and PC3 cells. D, Benign prostate cancer cell line RWPE was treated with indicated concentrations of CoCl2 for 12 h and HIF1α, CtBP1, EZH2 and P4HA1 protein levels were measured by immunoblot analysis. E, Immunoblot analysis of HIF1α and P4HA1 in CoCl2-treated normal prostate cell line PrEC. F, miR-124 is down-regulated under hypoxia-mimicking conditions. qPCR analysis of miR-124 in samples from (E); ns, not significant. G, Conventional Chromatin immunoprecipitation (ChIP)-PCR analysis for the HIF1α occupancy on miR-124-1 promoter in PC3 cells following induction with 100 μM CoCl2 for 12 h. All bar graphs are shown with ± SEM. See also Supplementary Figures S5 – S7.
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Figure 4: HIF1α modulates P4HA1 expression by down-regulating miR-124A, Immunoblot analysis of HIF1α -DU145 and -PC3 knockdown lysates under normoxia or in the presence of the hypoxia-mimetic agent CoCl2. HIF1α knockdown significantly reduced P4HA1, CtBP1 and EZH2. β-actin was used as a loading control and * denotes an additional band detected by the antibody. B and C, qPCR analysis of miR-124 in HIF1α-stable knockdown DU145 and PC3 cells. D, Benign prostate cancer cell line RWPE was treated with indicated concentrations of CoCl2 for 12 h and HIF1α, CtBP1, EZH2 and P4HA1 protein levels were measured by immunoblot analysis. E, Immunoblot analysis of HIF1α and P4HA1 in CoCl2-treated normal prostate cell line PrEC. F, miR-124 is down-regulated under hypoxia-mimicking conditions. qPCR analysis of miR-124 in samples from (E); ns, not significant. G, Conventional Chromatin immunoprecipitation (ChIP)-PCR analysis for the HIF1α occupancy on miR-124-1 promoter in PC3 cells following induction with 100 μM CoCl2 for 12 h. All bar graphs are shown with ± SEM. See also Supplementary Figures S5 – S7.

Mentions: To identify upstream regulators of P4HA1, we analyzed the P4HA1 promoter sequences using Genomatix MatInspector and found several transcription factor binding sites including HIF1α that is known to transactivate P4HA1 [14, 25]. P4HA1 promoter sequence contains multiple HIF1α binding hypoxia-response elements (HRE) “C(G/A)(T/G)G” sites. In addition to inducing protein coding gene expression, hypoxia is known to modulate expression of several miRNAs [26-29]. Here we investigated the effect of HIF1α on P4HA1 and miR-124 expression. In addition, Genomatix MatInspector analysis showed that transcriptional repressor EZH2 and co-repressor CtBP1, both of which are overexpressed in prostate cancer, contain HIF1α binding sites at their promoters. Thus, we also investigated the role of HIF1α in regulating these transcriptional repressors. We performed knockdown of HIF1α in DU145 and PC3 cells and treated these cells with CoCl2 to induce hypoxic conditions and performed immunoblot analysis. Knockdown of HIF1α significantly reduced P4HA1 and CtBP1 and moderately EZH2 protein levels, indicating HIF1α role in transactivation of these genes (Figure 4A). Moreover, miR-124 levels were increased in these samples as assessed by qPCR (Figure 4B, C). To further investigate the HIF1α-P4HA1 axis, we incubated benign prostate RWPE cells under hypoxic conditions in the presence of CoCl2, a chemical inhibitor of HIF1α degradation that leads to HIF1α protein accumulation [30]. As expected, hypoxia increased the expression of HIF1α, P4HA1, CtBP1 and EZH2 as shown by immunoblot (Figure 4D) and qPCR analysis (Supplementary Fig. S5A), and also significantly repressed miR-124 levels (Supplementary Fig. S5B (p=0.0004)). Consistent with Cao et al.,[26], we also observed a down-regulation of miR-101 in these samples (Supplementary Fig. S5B). In normal prostate epithelial PrEC cells, we observed concomitant induction of HIF1α and P4HA1 protein levels in the presence CoCl2 (Figure 4E). Similar to RWPE, PrEC cells showed lower levels of miR-124 in hypoxic condition (Figure 4F).


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)

HIF1α modulates P4HA1 expression by down-regulating miR-124A, Immunoblot analysis of HIF1α -DU145 and -PC3 knockdown lysates under normoxia or in the presence of the hypoxia-mimetic agent CoCl2. HIF1α knockdown significantly reduced P4HA1, CtBP1 and EZH2. β-actin was used as a loading control and * denotes an additional band detected by the antibody. B and C, qPCR analysis of miR-124 in HIF1α-stable knockdown DU145 and PC3 cells. D, Benign prostate cancer cell line RWPE was treated with indicated concentrations of CoCl2 for 12 h and HIF1α, CtBP1, EZH2 and P4HA1 protein levels were measured by immunoblot analysis. E, Immunoblot analysis of HIF1α and P4HA1 in CoCl2-treated normal prostate cell line PrEC. F, miR-124 is down-regulated under hypoxia-mimicking conditions. qPCR analysis of miR-124 in samples from (E); ns, not significant. G, Conventional Chromatin immunoprecipitation (ChIP)-PCR analysis for the HIF1α occupancy on miR-124-1 promoter in PC3 cells following induction with 100 μM CoCl2 for 12 h. All bar graphs are shown with ± SEM. See also Supplementary Figures S5 – S7.
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Figure 4: HIF1α modulates P4HA1 expression by down-regulating miR-124A, Immunoblot analysis of HIF1α -DU145 and -PC3 knockdown lysates under normoxia or in the presence of the hypoxia-mimetic agent CoCl2. HIF1α knockdown significantly reduced P4HA1, CtBP1 and EZH2. β-actin was used as a loading control and * denotes an additional band detected by the antibody. B and C, qPCR analysis of miR-124 in HIF1α-stable knockdown DU145 and PC3 cells. D, Benign prostate cancer cell line RWPE was treated with indicated concentrations of CoCl2 for 12 h and HIF1α, CtBP1, EZH2 and P4HA1 protein levels were measured by immunoblot analysis. E, Immunoblot analysis of HIF1α and P4HA1 in CoCl2-treated normal prostate cell line PrEC. F, miR-124 is down-regulated under hypoxia-mimicking conditions. qPCR analysis of miR-124 in samples from (E); ns, not significant. G, Conventional Chromatin immunoprecipitation (ChIP)-PCR analysis for the HIF1α occupancy on miR-124-1 promoter in PC3 cells following induction with 100 μM CoCl2 for 12 h. All bar graphs are shown with ± SEM. See also Supplementary Figures S5 – S7.
Mentions: To identify upstream regulators of P4HA1, we analyzed the P4HA1 promoter sequences using Genomatix MatInspector and found several transcription factor binding sites including HIF1α that is known to transactivate P4HA1 [14, 25]. P4HA1 promoter sequence contains multiple HIF1α binding hypoxia-response elements (HRE) “C(G/A)(T/G)G” sites. In addition to inducing protein coding gene expression, hypoxia is known to modulate expression of several miRNAs [26-29]. Here we investigated the effect of HIF1α on P4HA1 and miR-124 expression. In addition, Genomatix MatInspector analysis showed that transcriptional repressor EZH2 and co-repressor CtBP1, both of which are overexpressed in prostate cancer, contain HIF1α binding sites at their promoters. Thus, we also investigated the role of HIF1α in regulating these transcriptional repressors. We performed knockdown of HIF1α in DU145 and PC3 cells and treated these cells with CoCl2 to induce hypoxic conditions and performed immunoblot analysis. Knockdown of HIF1α significantly reduced P4HA1 and CtBP1 and moderately EZH2 protein levels, indicating HIF1α role in transactivation of these genes (Figure 4A). Moreover, miR-124 levels were increased in these samples as assessed by qPCR (Figure 4B, C). To further investigate the HIF1α-P4HA1 axis, we incubated benign prostate RWPE cells under hypoxic conditions in the presence of CoCl2, a chemical inhibitor of HIF1α degradation that leads to HIF1α protein accumulation [30]. As expected, hypoxia increased the expression of HIF1α, P4HA1, CtBP1 and EZH2 as shown by immunoblot (Figure 4D) and qPCR analysis (Supplementary Fig. S5A), and also significantly repressed miR-124 levels (Supplementary Fig. S5B (p=0.0004)). Consistent with Cao et al.,[26], we also observed a down-regulation of miR-101 in these samples (Supplementary Fig. S5B). In normal prostate epithelial PrEC cells, we observed concomitant induction of HIF1α and P4HA1 protein levels in the presence CoCl2 (Figure 4E). Similar to RWPE, PrEC cells showed lower levels of miR-124 in hypoxic condition (Figure 4F).

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