Prolyl Hydroxylase2Regulates Glycolysis In Colon Cancer Cells

Introduction Tumour cells reprogramme the metabolic pathways to meet their needsduring the process of malignant transformation. The best characterized metabolic phenotypeobserved in tumour cells is the Warburg effect, which is a shift from ATP generation throughoxidative phosphorylation to ATP generation through glycolysis, even under normal oxygenconcentrations. An increased glycolytic rate renders cancer cells more resistant to apoptosis,capable of producing biomacromolecule for anabolism, and more vulnerable to invasion.Additionally, metabolic reprogramming of cancer is also one of the important reasons thattumor is unsensitivity to chemo-or radi1otherapy. To date, the molecular basis and exactmechanism for tumor glycolysis remain unclear.Solid tumors frequently contain regions with low oxygen concentrations. To conquerhypoxia, tumor cell usually initiate glycolylsis for its energy supply and the expression of keycomponents of the angiogenesis, which eventually contributes to continued growth andsurvival of a tumor. Thus, sensing the variation of in vivo oxygen and passing the hypoxicmessage to downstream molecules to initiate an adaptable metabolism is a vital process. Asthe most important oxygen sensor, prolyl hydroxylases (PHDs) may be the one that can senseoxygen and regulate glycolysis in the tumor.PHDs hydroxylate HIF-1α at2specific prolyl residues in the oxygen-dependentdegradation domain, allowing capture by the von Hippel-Lindau tumor suppressor protein,ubiquitination, and subsequent proteasomal degradation. Of the3PHD isoformsinhumans,PHD2is the key limiting enzyme that targets HIF-1α for degradation under normoxicconditions. In addition, PHD2is low-expressed or absently expressed in many cancer cellsand tumor tissues, and the decreased expression levels of PHD2are significantly correlatedwith some hereditary lesions which ultimatally develop to manlignancies. As such, PHD2may serve as a potential tumour inhibitor. It is also reported that PHD2plays a critical role inSupported by the National Natural Science Foundation of China (30873015). tumor vasculature which is considered to occur after tumor glycolysis. Nevertheless, the roleof PHD2in tumor glycolysis is still unknown. In the present study, we will focus on theinvestigation of glycolysis effects including glucose uptake, lactate production and energeticmolecular quantitification, and confirm the regulatory effect of PHD2in glycolysis in coloncancer cells by RNA interference or gene overexpression of PHD2. Then, as an attempt tofind out the underlying mechanism, we plan to explore which signaling pathway is involvedin active glycolysis regulated by PHD2. This study may deepen our understanding of tumorglycolysis and provide new target for cancer therapy by reversing abnormal glycolysis.Objectives To investigate the regulatory effect of PHD2in glycolysis of colon cancercells and uncover the underlying mechanism.Methods Firstly, the expression levels of PHD2and glycolysis-related factors in humancolorectal cancer (CRC) tissues and cells were detected by immunohistochemistry,immunobloting and quantity-PCR. A Pearson chi-square analysis was conducted to analyzethe corelationship between PHD2and glycolysis-related factors. Kaplan-Meier method,log-rank test, and multivariate Cox regression analysis were performed based on tumor stageto evaluate the prognostic value of PHDs and clinicopathological features in CRC patients.Secondly, the PHD2levels as well as glycolysis-related factors including HK2, PDK1and Glut1were detected in pGC SIL-PHD2LS174T, pGC-FU-PHD2SW480, and LS174Tcells treated with DMOG. Liquid scintillation assay, enzyme-labeled assay, and HPLC wereused to test the glucose uptake, lactate production and ATP/ADP ratio in CRC cells,respectively. The Mitotracker Green was used to test the number of mitochondria.Thirdly, liquid scintillation assay, enzyme-labeled assay, and HPLC were used to explorethe glucose uptake, lactate production and ATP/ADP ratio of LS174T cells with PHD2andHIF-1α knock-down. Western blot and ELISA were used to detect the active protein levels ofLKB1/AMPK and IKK/NF-κB signaling pathway. Liquid scintillation assay, enzyme-labeledassay, and HPLC were used to analyze the glycolytic activity in PHD2-inhibited LS174T cellstreated with inhibitors of NF-κB pathway. The reaction of PHD2and NF-κB signal proteinswas examed by CoIP.Results Firstly, compared with normal tissures or adenoma, the levels of PHD2weresignificantly low-expressed in colorectal cancer. Low expression of PHD2was detected inCRC cell lines SW480, SW620, HT-29, LoVo, CaCo-2, RKO and HCT116by Western blot analysis, except for LS174T, PHD2of which was relatively-high expressed. The mRNAlevels of HK2, PDK1and Glut1in colorectal cancer tissues were negatively correlated withthat of paired para-cancer tissues. Kaplan-Meier survival analysis demonstrated that thesurvival of CRC patients with low PHD2expression was significantly shorter than that of thepatients with high PHD2expression (P=0.032), specifically in patients who had early-stagetumors (stage I and II; P=0.015). Multivariate Cox regression analysis indicated that lowPHD2expression was a statistically significant independent risk factor (HR=2.248,95%CI=1.125-4.139, P=0.011).Secondly, DMOG surppressed the protein levels of PHD2in LS174T cells inconcentration-dependent manner. The glycolysis levels of LS174T cells were significantlyupregulated after DMOG treatment. Similarly, the glycolysis levels of LS174T cellstransfected with pGC SIL-PHD2were also significantly increased. The expressions of Glut1,HK2and PDK1were inhibited in PHD2-overexpressed SW480; in addition, its glycolysislevels were significantly surppressed accordingly.Thirdly, the upregulation of glycolysis activity in pGC SIL-PHD2LS174T cells werenotreversed by introducing shHIF-1α. IKK-NF-κB signaling was confirmed to participate in theincreased glycolysis in colon cancer cells. Moreover, a concomitant increase ofphosphorylated AMPK was found in LS174T cells treated with an increased concentration ofDMOG.Conclusions Firstly, PHD2is abnormally weakly or not expressed in CRC, and highlyexpressed in colorectal normal tissues. Low PHD2expression in CRC could be a novelindependent factor for unfavorable prognosis, especially for those with early-stage tumors.Secondly, PHD2plays an important role in glycolysis activity of CRC cells.Thirdly, PHD2-IKK-NK-κB signaling is an important participant in glycolytic regulationin CRC cells, which is independent of HIF-1α.