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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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Collagen prolyl hydroxylase P4HA1 is overexpressed in prostate cancer and is associated with disease progressionA, P4HA1 gene expression from NGS data and B, quantitative real-time PCR of RNA from benign, prostate carcinoma (PCa) and metastatic prostate cancer (MET) tissues. C, P4HA1 protein expression by immunoblot analysis of prostate tissue extracts using P4HA1 antibody. β-actin was used as a loading control. D, Immunohistochemical analysis of P4HA1 in benign prostate epithelia (left), primary PCa (center) and metastatic PCa (right). E, P4HA1 staining intensity product scores for benign, primary prostate cancer and metastatic prostate cancer. A progression box plot representing the mean ± SEM values of P4HA1 protein expression in the tissue microarray for each of the indicated groups. P4HA1 expression progressively increases with the severity of the disease (p=0.001). F, Fluorescent in situ hybridization (FISH) using P4HA1 locus specific probe in PC3 cells showing 5-10 copies of P4HA1 on the isochromosome 10q. G, FISH analysis showing 4-5 copies of P4HA1 (green) in a metastatic prostate cancer sample (right) compared to a normal prostate sample with two copies of P4HA1 and control probe (red, left). See also Supplementary Figure S1.
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Figure 1: Collagen prolyl hydroxylase P4HA1 is overexpressed in prostate cancer and is associated with disease progressionA, P4HA1 gene expression from NGS data and B, quantitative real-time PCR of RNA from benign, prostate carcinoma (PCa) and metastatic prostate cancer (MET) tissues. C, P4HA1 protein expression by immunoblot analysis of prostate tissue extracts using P4HA1 antibody. β-actin was used as a loading control. D, Immunohistochemical analysis of P4HA1 in benign prostate epithelia (left), primary PCa (center) and metastatic PCa (right). E, P4HA1 staining intensity product scores for benign, primary prostate cancer and metastatic prostate cancer. A progression box plot representing the mean ± SEM values of P4HA1 protein expression in the tissue microarray for each of the indicated groups. P4HA1 expression progressively increases with the severity of the disease (p=0.001). F, Fluorescent in situ hybridization (FISH) using P4HA1 locus specific probe in PC3 cells showing 5-10 copies of P4HA1 on the isochromosome 10q. G, FISH analysis showing 4-5 copies of P4HA1 (green) in a metastatic prostate cancer sample (right) compared to a normal prostate sample with two copies of P4HA1 and control probe (red, left). See also Supplementary Figure S1.

Mentions: Gene expression profiling studies and transcriptome sequence analysis showed up-regulation of P4HA1 in metastatic prostate cancer (Figure 1A) [17-19]. In order to validate this observation, we performed real-time qPCR using RNA from multiple prostate cancer and benign tissue samples. Real-time qPCR analysis confirmed the overexpression of P4HA1 in metastatic prostate cancer tissues relative to benign prostate samples (Figure 1B) as did immunoblot analysis using P4HA1-specific antibody (Figure 1C). We conducted Oncomine Platform (Life Technologies, Ann Arbor, MI) database analyses on publicly available microarray datasets and found that P4HA1 is over-expressed in prostate adenocarcinoma (Supplementary Fig. S1A; p=8.57E-4) and metastatic samples (Supplementary Fig. S1B; p=2.22E-7) compared with normal tissues [20, 21]. Similarly, elevated levels of P4HA1 protein was observed in metastatic prostate cancer cell lines relative to benign cell lines (Supplementary Fig. S1C). However, P4HA2 mRNA expression levels were relatively lower than P4HA1 in malignant prostate cancer tissues and cell lines (Supplementary Fig. S1D, E). Moreover, no appreciable difference was observed in P4HA2 levels between benign and metastatic tissues and cell lines (Supplementary Fig. S1D, E), suggesting non-overlapping functions between the two isoforms. We investigated the expression of P4HA1 protein in large number of prostate cancer samples by immunohistochemical (IHC) analysis that showed weak or no reactivity in benign tissues but strong staining in the aggressive prostate cancer tissue and metastatic prostate tumors (Figure 1D). Statistical analysis of the tissue microarray IHC analysis suggested a significant progressive increase in P4HA1 expression with disease progression (p=0.001) (Figure 1E). Fluorescence in situ hybridization using P4HA1 locus specific FISH probe revealed copy number gain in aggressive prostate cancer cell line PC3 (Figure 1F). Similarly, a small subset of metastatic prostate cancer tissues were found to have copy number gains of P4HA1 (Figure 1G, right panel).


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)

Collagen prolyl hydroxylase P4HA1 is overexpressed in prostate cancer and is associated with disease progressionA, P4HA1 gene expression from NGS data and B, quantitative real-time PCR of RNA from benign, prostate carcinoma (PCa) and metastatic prostate cancer (MET) tissues. C, P4HA1 protein expression by immunoblot analysis of prostate tissue extracts using P4HA1 antibody. β-actin was used as a loading control. D, Immunohistochemical analysis of P4HA1 in benign prostate epithelia (left), primary PCa (center) and metastatic PCa (right). E, P4HA1 staining intensity product scores for benign, primary prostate cancer and metastatic prostate cancer. A progression box plot representing the mean ± SEM values of P4HA1 protein expression in the tissue microarray for each of the indicated groups. P4HA1 expression progressively increases with the severity of the disease (p=0.001). F, Fluorescent in situ hybridization (FISH) using P4HA1 locus specific probe in PC3 cells showing 5-10 copies of P4HA1 on the isochromosome 10q. G, FISH analysis showing 4-5 copies of P4HA1 (green) in a metastatic prostate cancer sample (right) compared to a normal prostate sample with two copies of P4HA1 and control probe (red, left). See also Supplementary Figure S1.
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Figure 1: Collagen prolyl hydroxylase P4HA1 is overexpressed in prostate cancer and is associated with disease progressionA, P4HA1 gene expression from NGS data and B, quantitative real-time PCR of RNA from benign, prostate carcinoma (PCa) and metastatic prostate cancer (MET) tissues. C, P4HA1 protein expression by immunoblot analysis of prostate tissue extracts using P4HA1 antibody. β-actin was used as a loading control. D, Immunohistochemical analysis of P4HA1 in benign prostate epithelia (left), primary PCa (center) and metastatic PCa (right). E, P4HA1 staining intensity product scores for benign, primary prostate cancer and metastatic prostate cancer. A progression box plot representing the mean ± SEM values of P4HA1 protein expression in the tissue microarray for each of the indicated groups. P4HA1 expression progressively increases with the severity of the disease (p=0.001). F, Fluorescent in situ hybridization (FISH) using P4HA1 locus specific probe in PC3 cells showing 5-10 copies of P4HA1 on the isochromosome 10q. G, FISH analysis showing 4-5 copies of P4HA1 (green) in a metastatic prostate cancer sample (right) compared to a normal prostate sample with two copies of P4HA1 and control probe (red, left). See also Supplementary Figure S1.
Mentions: Gene expression profiling studies and transcriptome sequence analysis showed up-regulation of P4HA1 in metastatic prostate cancer (Figure 1A) [17-19]. In order to validate this observation, we performed real-time qPCR using RNA from multiple prostate cancer and benign tissue samples. Real-time qPCR analysis confirmed the overexpression of P4HA1 in metastatic prostate cancer tissues relative to benign prostate samples (Figure 1B) as did immunoblot analysis using P4HA1-specific antibody (Figure 1C). We conducted Oncomine Platform (Life Technologies, Ann Arbor, MI) database analyses on publicly available microarray datasets and found that P4HA1 is over-expressed in prostate adenocarcinoma (Supplementary Fig. S1A; p=8.57E-4) and metastatic samples (Supplementary Fig. S1B; p=2.22E-7) compared with normal tissues [20, 21]. Similarly, elevated levels of P4HA1 protein was observed in metastatic prostate cancer cell lines relative to benign cell lines (Supplementary Fig. S1C). However, P4HA2 mRNA expression levels were relatively lower than P4HA1 in malignant prostate cancer tissues and cell lines (Supplementary Fig. S1D, E). Moreover, no appreciable difference was observed in P4HA2 levels between benign and metastatic tissues and cell lines (Supplementary Fig. S1D, E), suggesting non-overlapping functions between the two isoforms. We investigated the expression of P4HA1 protein in large number of prostate cancer samples by immunohistochemical (IHC) analysis that showed weak or no reactivity in benign tissues but strong staining in the aggressive prostate cancer tissue and metastatic prostate tumors (Figure 1D). Statistical analysis of the tissue microarray IHC analysis suggested a significant progressive increase in P4HA1 expression with disease progression (p=0.001) (Figure 1E). Fluorescence in situ hybridization using P4HA1 locus specific FISH probe revealed copy number gain in aggressive prostate cancer cell line PC3 (Figure 1F). Similarly, a small subset of metastatic prostate cancer tissues were found to have copy number gains of P4HA1 (Figure 1G, right panel).

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