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

P4HA1 is essential for prostate cancer cell proliferation and invasionA and B, Transient knockdown of P4HA1 in prostate cancer cell lines reduces prostate cancer cell proliferation. Immunoblot analysis of protein using lysates from aggressive prostate cancer cell lines DU145 and PC3 treated with two specific and independent P4HA1 siRNA duplexes. β-actin was used as a loading control. Cell proliferation assay of DU145 and PC3 cells transfected with either P4HA1 siRNA duplex or non-targeting siRNA (Non-T siRNA) control. C and D, Knockdown of P4HA1 reduces DU145 and PC3 cell invasion. Boyden chamber matrigel invasion assay of DU145 or PC3 cells where P4HA1 was transiently knocked down using two independent P4HA1 siRNA duplexes. Non-T siRNA treated cells served as control. Invaded cells were stained with crystal violet and the absorbance was measured at 560 nm. All bar graphs are shown with ± SEM. See also Supplementary Figures S2 and S3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: P4HA1 is essential for prostate cancer cell proliferation and invasionA and B, Transient knockdown of P4HA1 in prostate cancer cell lines reduces prostate cancer cell proliferation. Immunoblot analysis of protein using lysates from aggressive prostate cancer cell lines DU145 and PC3 treated with two specific and independent P4HA1 siRNA duplexes. β-actin was used as a loading control. Cell proliferation assay of DU145 and PC3 cells transfected with either P4HA1 siRNA duplex or non-targeting siRNA (Non-T siRNA) control. C and D, Knockdown of P4HA1 reduces DU145 and PC3 cell invasion. Boyden chamber matrigel invasion assay of DU145 or PC3 cells where P4HA1 was transiently knocked down using two independent P4HA1 siRNA duplexes. Non-T siRNA treated cells served as control. Invaded cells were stained with crystal violet and the absorbance was measured at 560 nm. All bar graphs are shown with ± SEM. See also Supplementary Figures S2 and S3.

Mentions: To determine the functional significance of P4HA1 overexpression in prostate cancer we perturbed P4HA1 levels in prostate cells and tested them in cell proliferation, migration and invasion assays. We utilized both transient RNA interference and stable knockdown strategies targeting P4HA1 in aggressive prostate cancer cell lines, DU145 and PC3. The efficiency of P4HA1 knockdowns were confirmed by immunoblot (Figure 2A, B; Supplementary Fig. S2A) and qPCR (Supplementary Fig. S2B; Supplementary Fig. S3) analyses. We observed significant decrease in cell proliferation upon transient or stable knockdown of P4HA1 compared to control cells transfected with non-targeting si/sh RNAs (Figure 2A, B; Supplementary Fig. S2C, D, respectively). Next, we tested cell motility after stable P4HA1 knockdown in prostate cancer cells using wound healing assay. P4HA1 knockdown showed a wider wound area 24 hours post-wound generation relative to control cells, the delayed time to heal indicating an inability of P4HA1 knockdown cells to migrate (Supplementary Fig. S2E, F). Additionally, P4HA1 knockdown in DU145 and PC3 reduced the invasive potential of these cells as assessed by Boyden chamber matrigel invasion assay (Figure 2C, D). Together, these observations demonstrate the involvement of P4HA1 in the proliferation, migration and invasion of prostate cancer cells in vitro.


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)

P4HA1 is essential for prostate cancer cell proliferation and invasionA and B, Transient knockdown of P4HA1 in prostate cancer cell lines reduces prostate cancer cell proliferation. Immunoblot analysis of protein using lysates from aggressive prostate cancer cell lines DU145 and PC3 treated with two specific and independent P4HA1 siRNA duplexes. β-actin was used as a loading control. Cell proliferation assay of DU145 and PC3 cells transfected with either P4HA1 siRNA duplex or non-targeting siRNA (Non-T siRNA) control. C and D, Knockdown of P4HA1 reduces DU145 and PC3 cell invasion. Boyden chamber matrigel invasion assay of DU145 or PC3 cells where P4HA1 was transiently knocked down using two independent P4HA1 siRNA duplexes. Non-T siRNA treated cells served as control. Invaded cells were stained with crystal violet and the absorbance was measured at 560 nm. All bar graphs are shown with ± SEM. See also Supplementary Figures S2 and S3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: P4HA1 is essential for prostate cancer cell proliferation and invasionA and B, Transient knockdown of P4HA1 in prostate cancer cell lines reduces prostate cancer cell proliferation. Immunoblot analysis of protein using lysates from aggressive prostate cancer cell lines DU145 and PC3 treated with two specific and independent P4HA1 siRNA duplexes. β-actin was used as a loading control. Cell proliferation assay of DU145 and PC3 cells transfected with either P4HA1 siRNA duplex or non-targeting siRNA (Non-T siRNA) control. C and D, Knockdown of P4HA1 reduces DU145 and PC3 cell invasion. Boyden chamber matrigel invasion assay of DU145 or PC3 cells where P4HA1 was transiently knocked down using two independent P4HA1 siRNA duplexes. Non-T siRNA treated cells served as control. Invaded cells were stained with crystal violet and the absorbance was measured at 560 nm. All bar graphs are shown with ± SEM. See also Supplementary Figures S2 and S3.
Mentions: To determine the functional significance of P4HA1 overexpression in prostate cancer we perturbed P4HA1 levels in prostate cells and tested them in cell proliferation, migration and invasion assays. We utilized both transient RNA interference and stable knockdown strategies targeting P4HA1 in aggressive prostate cancer cell lines, DU145 and PC3. The efficiency of P4HA1 knockdowns were confirmed by immunoblot (Figure 2A, B; Supplementary Fig. S2A) and qPCR (Supplementary Fig. S2B; Supplementary Fig. S3) analyses. We observed significant decrease in cell proliferation upon transient or stable knockdown of P4HA1 compared to control cells transfected with non-targeting si/sh RNAs (Figure 2A, B; Supplementary Fig. S2C, D, respectively). Next, we tested cell motility after stable P4HA1 knockdown in prostate cancer cells using wound healing assay. P4HA1 knockdown showed a wider wound area 24 hours post-wound generation relative to control cells, the delayed time to heal indicating an inability of P4HA1 knockdown cells to migrate (Supplementary Fig. S2E, F). Additionally, P4HA1 knockdown in DU145 and PC3 reduced the invasive potential of these cells as assessed by Boyden chamber matrigel invasion assay (Figure 2C, D). Together, these observations demonstrate the involvement of P4HA1 in the proliferation, migration and invasion of prostate cancer cells in vitro.

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