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Collagen prolyl 4-hydroxylase 2 predicts worse prognosis and promotes glycolysis in cervical cancer.

ABSTRACT: P4HA2 is one of collagen prolyl 4-hydroxylase (P4H) isoform and increased in several types of human cancer. However, the role of P4HA2 during cervical tumorigenesis remains largely unknown. Here, we report that the protein level of P4HA2 is significantly increased in cervical cancer tissues. Silencing of P4HA2 inhibits cervical cancer cell proliferation, colony formation and migration. We also demonstrate decreased glucose uptake and lactate production in P4HA2 knockdown cells. Mechanistically, P4HA2 promotes cervical cancer cell glycolysis through upregulation of PGK1 and LDHA. We find a positive correlation between P4HA2 and PGK1/LDHA expression in cervical cancer tissues. Importantly, high expression of P4HA2, PGK1 or LDHA has a significantly shorter overall survival period and the survival prediction is enhanced by using combination of P4HA2 and PGK1/LDHA expression. Collectively, we identify P4HA2 as a regulator of glycolysis through PGK1 and LDHA, which may serve as a potential therapeutic target for cervical cancer.

SUBMITTER: Li Q

PROVIDER: S-EPMC6895525 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Collagen prolyl 4-hydroxylase 2 predicts worse prognosis and promotes glycolysis in cervical cancer.

Li Qingxian Q Wang Qingying Q Zhang Qinyi Q Zhang Jing J Zhang Jiawen J

American journal of translational research 20191115 11

P4HA2 is one of collagen prolyl 4-hydroxylase (P4H) isoform and increased in several types of human cancer. However, the role of P4HA2 during cervical tumorigenesis remains largely unknown. Here, we report that the protein level of P4HA2 is significantly increased in cervical cancer tissues. Silencing of P4HA2 inhibits cervical cancer cell proliferation, colony formation and migration. We also demonstrate decreased glucose uptake and lactate production in P4HA2 knockdown cells. Mechanistically, ...[more]

PMID: 31814898

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Project description:Increased collagen deposition provides physical and biochemical signals to support tumor growth and invasion during breast cancer development. Therefore, inhibition of collagen synthesis and deposition has been considered a strategy to suppress breast cancer progression. Collagen prolyl-4-hydroxylase ? subunit 2 (P4HA2), an enzyme hydroxylating proline residues in -X-Pro-Gly- sequences, is a potential therapeutic target for the disorders associated with increased collagen deposition. However, expression and function of P4HA2 in breast cancer progression are not well investigated.Gene co-expression analysis was performed in the published microarray datasets to identify potential regulators of collagen I, III, and IV in human breast cancer tissue. Expression of P4HA2 was silenced by shRNAs, and its activity was inhibited by 1, 4-DPCA, a prolyl-4-hydroxylase inhibitor. Three-dimensional culture assay was used to analyze roles of P4HA2 in regulating malignant phenotypes of breast cancer cells. Reduced deposition of collagen I and IV was detected by Western blotting and immunofluorescence. Control and P4HA2-silenced breast cancer cells were injected into fat pad and tail vein of SCID mice to examine effect of P4HA2 on tumor growth and lung metastasis.Using gene co-expression analysis, we showed that P4HA2 was associated with expression of Col1A1, Col3A1, and Col4A1 during breast cancer development and progression. P4HA2 mRNA levels were significantly upregulated in breast cancer compared to normal mammary tissue. Increased mRNA levels of P4HA2 correlated with poor clinical outcome in breast cancer patients, which is independent of estrogen receptor status. Silencing P4HA2 expression or treatment with the P4HA inhibitor significantly inhibited cell proliferation and suppressed aggressive phenotypes of breast cancer cells in 3D culture, accompanied by reduced deposition of collagen I and IV. We also found that knockdown of P4HA2 inhibited mammary tumor growth and metastasis to lungs in xenograft models.These results suggest the critical role of P4HA2 in breast cancer progression and identify P4HA2 as a potential therapeutic target and biomarker for breast cancer progression.

Project description:BackgroundWe describe a method for specific, quantitative and quick detection of human collagen prolyl 4-hydroxylase (C-P4H), the key enzyme for collagen prolyl-4 hydroxylation, in crude samples based on a sandwich ELISA principle. The method is relevant to active C-P4H level monitoring during recombinant C-P4H and collagen production in different expression systems. The assay proves to be specific for the active C-P4H alpha2beta2 tetramer due to the use of antibodies against its both subunits. Thus in keeping with the method C-P4H is captured by coupled to an anti-alpha subunit antibody magnetic beads and an anti-beta subunit antibody binds to the PDI/beta subunit of the protein. Then the following holoenzyme detection is accomplished by a goat anti-rabbit IgG labeled with alkaline phosphatase which AP catalyzes the reaction of a substrate transformation with fluorescent signal generation.ResultsWe applied an experimental design approach for the optimization of the antibody concentrations used in the sandwich ELISA. The assay sensitivity was 0.1 ng of C-P4H. The method was utilized for the analysis of C-P4H accumulation in crude cell extracts of E. coli overexpressing C-P4H. The sandwich ELISA signals obtained demonstrated a very good correlation with the detected protein activity levels measured with the standard radioactive assay. The developed assay was applied to optimize C-P4H production in E. coli Origami in a system where the C-P4H subunits expression acted under control by different promoters. The experiments performed in a shake flask fed-batch system (EnBase) verified earlier observations that cell density and oxygen supply are critical factors for the use of the inducer anhydrotetracycline and thus for the soluble C-P4H yield.ConclusionsHere we show an example of sandwich ELISA usage for quantifying multimeric proteins. The method was developed for monitoring the amount of recombinant C-P4H tetramer in crude E. coli extracts. Due to the specificity of the antibodies used in the assay against the different C-P4H subunits, the method detects the entire holoenzyme, and the signal is not disturbed by background expression of the separate subunits.

Dataset Information

Collagen prolyl 4-hydroxylase 2 predicts worse prognosis and promotes glycolysis in cervical cancer.

Publications

Collagen prolyl 4-hydroxylase 2 predicts worse prognosis and promotes glycolysis in cervical cancer.

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