Limits...
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.

Show MeSH

Related in: MedlinePlus

Over-expression of P4HA1 increases cell proliferation and invasionA, Microarray data of selected genes in stable P4HA1 knockdown in DU145 cells. B, Immunoblot analysis showing P4HA1 in RWPE cells (Inset). Stable RWPE-P4HA1 over-expressing cells showed increased cell proliferation than untreated or lacZ over-expressing cells. C, Wound healing assay in stable RWPE-P4HA1 over-expressing cells. An artificial wound was created using a 0.2 ml pipette tip on a confluent monolayer of cells. Images were taken at 0 and 72 h after scratching. The black lines show the margin of scratched area in which double headed arrow indicates scratch width (W) and black arrow indicates complete healing (H) of scratch wound. D, Immunoblot and qPCR analysis of MMP1 in RWPE cells over-expressing lacZ and P4HA1. E, Matrigel invasion assay was performed using lenti-lacZ or lenti-P4HA1 cells. Untreated cells, Non-T siRNA, three independent MMP1 specific siRNA treated cells or in the presence of MMP1 inhibitor were also used in the invasion assay and the invaded cells were counted. Inset, photomicrographs of invaded cells. F, Immunoblot analysis of PrEC cells expressing P4HA1 and qPCR analysis of MMP1 in PrEC cells that are uninfected or lacZ and adeno-P4HA1 infected. G, Matrigel invasion assay using uninfected PrEC cells or cells that are infected with lacZ, adeno-P4HA1 alone or in the presence of 10 μM MMP1 inhibitor. Inset, photomicrographs of invaded cells and H, Matrigel invasion assay using parental RWPE cells or lacZ, adeno-P4HA1 infected cells and adeno-P4HA1 infected cells in the presence of 10 μM MMP1 inhibitor. Inset, photomicrographs of invaded cells and immunoblot analysis of P4HA1. I, Immunoblot analysis of the secreted MMP1 from RWPE cells transiently over-expressing lacZ or P4HA1. The recombinant MMP1 (rhMMP1) served as positive control for MMP1 and CALR is used as a loading control. All bar graphs are shown with ± SEM. See also Supplementary Figures S11 -14.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4196154&req=5

Figure 6: Over-expression of P4HA1 increases cell proliferation and invasionA, Microarray data of selected genes in stable P4HA1 knockdown in DU145 cells. B, Immunoblot analysis showing P4HA1 in RWPE cells (Inset). Stable RWPE-P4HA1 over-expressing cells showed increased cell proliferation than untreated or lacZ over-expressing cells. C, Wound healing assay in stable RWPE-P4HA1 over-expressing cells. An artificial wound was created using a 0.2 ml pipette tip on a confluent monolayer of cells. Images were taken at 0 and 72 h after scratching. The black lines show the margin of scratched area in which double headed arrow indicates scratch width (W) and black arrow indicates complete healing (H) of scratch wound. D, Immunoblot and qPCR analysis of MMP1 in RWPE cells over-expressing lacZ and P4HA1. E, Matrigel invasion assay was performed using lenti-lacZ or lenti-P4HA1 cells. Untreated cells, Non-T siRNA, three independent MMP1 specific siRNA treated cells or in the presence of MMP1 inhibitor were also used in the invasion assay and the invaded cells were counted. Inset, photomicrographs of invaded cells. F, Immunoblot analysis of PrEC cells expressing P4HA1 and qPCR analysis of MMP1 in PrEC cells that are uninfected or lacZ and adeno-P4HA1 infected. G, Matrigel invasion assay using uninfected PrEC cells or cells that are infected with lacZ, adeno-P4HA1 alone or in the presence of 10 μM MMP1 inhibitor. Inset, photomicrographs of invaded cells and H, Matrigel invasion assay using parental RWPE cells or lacZ, adeno-P4HA1 infected cells and adeno-P4HA1 infected cells in the presence of 10 μM MMP1 inhibitor. Inset, photomicrographs of invaded cells and immunoblot analysis of P4HA1. I, Immunoblot analysis of the secreted MMP1 from RWPE cells transiently over-expressing lacZ or P4HA1. The recombinant MMP1 (rhMMP1) served as positive control for MMP1 and CALR is used as a loading control. All bar graphs are shown with ± SEM. See also Supplementary Figures S11 -14.

Mentions: In order to evaluate P4HA1-mediated effects in prostate cancer progression, we performed gene expression analysis using RNA from P4HA1 knockdown prostate cell lines. We identified multiple genes that were induced or repressed upon P4HA1 knockdown including those involved in tumor growth and invasion such as MMP1, MMP2 and FLRT3, among others (Figure 6A). We validated the activation of FLRT3 and the down-regulation of MMP1 both at mRNA and protein level upon P4HA1 stable (Supplementary Fig. S11A,) and transient (Supplementary Fig. S12A,) knockdown. MMP1 and FLRT3 are known to play a role in metastasis, cell de-adhesion and wound healing [38, 39]. To test the role of FLRT3 in cell migration, we performed a phenotype rescue experiment where we knocked down FLRT3 in DU145-P4HA1 stable knockdown cell lines using two independent duplexes targeting FLRT3 (Supplementary Fig. S11C). FLRT3 knockdown cells healed the gaps faster than in control and P4HA1 stable knockdowns in wound healing assay indicating a critical role for FLRT3 in P4HA1-mediated cell migration (Supplementary Fig. S11D).


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)

Over-expression of P4HA1 increases cell proliferation and invasionA, Microarray data of selected genes in stable P4HA1 knockdown in DU145 cells. B, Immunoblot analysis showing P4HA1 in RWPE cells (Inset). Stable RWPE-P4HA1 over-expressing cells showed increased cell proliferation than untreated or lacZ over-expressing cells. C, Wound healing assay in stable RWPE-P4HA1 over-expressing cells. An artificial wound was created using a 0.2 ml pipette tip on a confluent monolayer of cells. Images were taken at 0 and 72 h after scratching. The black lines show the margin of scratched area in which double headed arrow indicates scratch width (W) and black arrow indicates complete healing (H) of scratch wound. D, Immunoblot and qPCR analysis of MMP1 in RWPE cells over-expressing lacZ and P4HA1. E, Matrigel invasion assay was performed using lenti-lacZ or lenti-P4HA1 cells. Untreated cells, Non-T siRNA, three independent MMP1 specific siRNA treated cells or in the presence of MMP1 inhibitor were also used in the invasion assay and the invaded cells were counted. Inset, photomicrographs of invaded cells. F, Immunoblot analysis of PrEC cells expressing P4HA1 and qPCR analysis of MMP1 in PrEC cells that are uninfected or lacZ and adeno-P4HA1 infected. G, Matrigel invasion assay using uninfected PrEC cells or cells that are infected with lacZ, adeno-P4HA1 alone or in the presence of 10 μM MMP1 inhibitor. Inset, photomicrographs of invaded cells and H, Matrigel invasion assay using parental RWPE cells or lacZ, adeno-P4HA1 infected cells and adeno-P4HA1 infected cells in the presence of 10 μM MMP1 inhibitor. Inset, photomicrographs of invaded cells and immunoblot analysis of P4HA1. I, Immunoblot analysis of the secreted MMP1 from RWPE cells transiently over-expressing lacZ or P4HA1. The recombinant MMP1 (rhMMP1) served as positive control for MMP1 and CALR is used as a loading control. All bar graphs are shown with ± SEM. See also Supplementary Figures S11 -14.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196154&req=5

Figure 6: Over-expression of P4HA1 increases cell proliferation and invasionA, Microarray data of selected genes in stable P4HA1 knockdown in DU145 cells. B, Immunoblot analysis showing P4HA1 in RWPE cells (Inset). Stable RWPE-P4HA1 over-expressing cells showed increased cell proliferation than untreated or lacZ over-expressing cells. C, Wound healing assay in stable RWPE-P4HA1 over-expressing cells. An artificial wound was created using a 0.2 ml pipette tip on a confluent monolayer of cells. Images were taken at 0 and 72 h after scratching. The black lines show the margin of scratched area in which double headed arrow indicates scratch width (W) and black arrow indicates complete healing (H) of scratch wound. D, Immunoblot and qPCR analysis of MMP1 in RWPE cells over-expressing lacZ and P4HA1. E, Matrigel invasion assay was performed using lenti-lacZ or lenti-P4HA1 cells. Untreated cells, Non-T siRNA, three independent MMP1 specific siRNA treated cells or in the presence of MMP1 inhibitor were also used in the invasion assay and the invaded cells were counted. Inset, photomicrographs of invaded cells. F, Immunoblot analysis of PrEC cells expressing P4HA1 and qPCR analysis of MMP1 in PrEC cells that are uninfected or lacZ and adeno-P4HA1 infected. G, Matrigel invasion assay using uninfected PrEC cells or cells that are infected with lacZ, adeno-P4HA1 alone or in the presence of 10 μM MMP1 inhibitor. Inset, photomicrographs of invaded cells and H, Matrigel invasion assay using parental RWPE cells or lacZ, adeno-P4HA1 infected cells and adeno-P4HA1 infected cells in the presence of 10 μM MMP1 inhibitor. Inset, photomicrographs of invaded cells and immunoblot analysis of P4HA1. I, Immunoblot analysis of the secreted MMP1 from RWPE cells transiently over-expressing lacZ or P4HA1. The recombinant MMP1 (rhMMP1) served as positive control for MMP1 and CALR is used as a loading control. All bar graphs are shown with ± SEM. See also Supplementary Figures S11 -14.
Mentions: In order to evaluate P4HA1-mediated effects in prostate cancer progression, we performed gene expression analysis using RNA from P4HA1 knockdown prostate cell lines. We identified multiple genes that were induced or repressed upon P4HA1 knockdown including those involved in tumor growth and invasion such as MMP1, MMP2 and FLRT3, among others (Figure 6A). We validated the activation of FLRT3 and the down-regulation of MMP1 both at mRNA and protein level upon P4HA1 stable (Supplementary Fig. S11A,) and transient (Supplementary Fig. S12A,) knockdown. MMP1 and FLRT3 are known to play a role in metastasis, cell de-adhesion and wound healing [38, 39]. To test the role of FLRT3 in cell migration, we performed a phenotype rescue experiment where we knocked down FLRT3 in DU145-P4HA1 stable knockdown cell lines using two independent duplexes targeting FLRT3 (Supplementary Fig. S11C). FLRT3 knockdown cells healed the gaps faster than in control and P4HA1 stable knockdowns in wound healing assay indicating a critical role for FLRT3 in P4HA1-mediated cell migration (Supplementary Fig. S11D).

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