The PIK3CA E542K and E545K mutations promote glycolysis and proliferation via induction of the β-catenin/SIRT3 signaling pathway in cervical cancer

Wei Jiang, Tiancong He, Shuai Liu, Yingying Zheng, Libing Xiang, Xuan Pei, Ziliang Wang, Huijuan Yang, Wei Jiang, Tiancong He, Shuai Liu, Yingying Zheng, Libing Xiang, Xuan Pei, Ziliang Wang, Huijuan Yang

Abstract

Background: The study aims to present the effect of PIK3CA E542K and E545K mutations on glucose metabolism and proliferation and identify their underlying mechanisms in cervical cancer.

Methods: The maximum standard uptake value (SUVmax) of tumors was detected by18F-FDG PET/CT scan. In vitro, glycolysis analysis, extracellular acidification rate analysis, and ATP production were used to evaluate the impact of PIK3CA E542K and E545K mutations on glucose metabolism. The expression level of key glycolytic enzymes was evaluated by western blotting and immunohistochemical staining in cervical cancer cells and tumor tissues, respectively. Immunofluorescence analysis was used to observe the nuclear translocation of β-catenin. The target gene of β-catenin was analyzed by using luciferase reporter system. The glucose metabolic ability of the xenograft models was assessed by SUVmax from microPET/CT scanning.

Results: Cervical cancer patients with mutant PIK3CA (E542K and E545K) exhibited a higher SUVmax value than those with wild-type PIK3CA (P = 0.037), which was confirmed in xenograft models. In vitro, enhanced glucose metabolism and proliferation was observed in SiHa and MS751 cells with mutant PIK3CA. The mRNA and protein expression of key glycolytic enzymes was increased. AKT/GSK3β/β-catenin signaling was highly activated in SiHa and MS751 cells with mutant PIK3CA. Knocking down β-catenin expression decreased glucose uptake and lactate production. In addition, the nuclear accumulation of β-catenin was found in SiHa cells and tumors with mutant PIK3CA. Furthermore, β-catenin downregulated the expression of SIRT3 via suppressing the activity of the SIRT3 promotor, and the reduced glucose uptake and lactate production due to the downregulation of β-catenin can be reversed by the transfection of SIRT3 siRNA in SiHa cells with mutant PIK3CA. The negative correlation between β-catenin and SIRT3 was further confirmed in cervical cancer tissues.

Conclusions: These findings provide evidence that the PI3K E542K and E545K/β-catenin/SIRT3 signaling axis regulates glucose metabolism and proliferation in cervical cancers with PIK3CA mutations, suggesting therapeutic targets in the treatment of cervical cancers.

Trial registration: FUSCC 050432-4-1212B. Registered 24 December 2012 (retrospectively registered).

Keywords: Cervical cancer; Glycolysis; PIK3CA E542K and E545K mutations; SIRT3; β-Catenin.

Conflict of interest statement

Ethics approval and consent to participate

This study was approved by the Review Board and Ethical Committee of Fudan University (FUSCC 050432–4-1212B).

Consent for publication

Written informed consent was obtained from all of the patients.

Competing interests

The authors declare that they have no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
PIK3CA E542K, E545K mutation and its correlation with 18F-FDG PET/CT SUVmax. a The mutation occurrence of PIK3CA E542, E545, and H1047 in cervical cancer, breast cancer, and endometrial carcinoma. b cDNA sequence of wild-type PIK3CA and mutated at E542K and E545K. c The representative 18F-FDG PET/CT imaging in patients with wild-type and mutant PIK3CA. d Statistical analysis of SUVmax in groups with wild-type and mutant PIK3CA (n = 52; P = 0.037)
Fig. 2
Fig. 2
PIK3CA E542K and E545K mutations promote proliferation and glucose metabolism in cervical cancer cell. a Transfection of FLAG-tagged PI3K E542K and E545K in SiHa and MS751 cells. b Detection of proliferation by colony formation in SiHa and MS751 cells with wild-type and mutant PIK3CA (**P < 0.01). c The effect of PIK3CA E542K and E545K mutation on glucose uptake (**P < 0.01). d The effect of PIK3CA E542K and E545K mutation on lactate production (**P < 0.01). e ECAR analysis in SiHa and MS751 cells with wild-type and mutant PIK3CA.fPIK3CA E542K and E545K mutation affected ATP production (**P < 0.01). g Relative mRNA expression of key enzymes of glycolysis in SiHa and MS751 cells with wild-type and mutant PIK3CA (*P < 0.05; **P < 0.01). h The effect of PIK3CA E542K and E545K mutation on the expression level of key glycolytic enzyme
Fig. 3
Fig. 3
PIK3CA E542K and E545K mutations enhance the expression and nuclear accumulation of β-catenin. a The expression of AKT/GSK3 β/β-catenin in SiHa and MS751 cells with wild-type and mutant PIK3CA. b The effect of knocking down β-catenin on proliferation in SiHa and MS751 cells with wild-type and mutant PIK3CA. c The effect of knocking down β-catenin on glucose uptake and lactate production in SiHa and MS751 cells with wild-type and mutant PIK3CA (**P < 0.01). d The location of β-catenin was analyzed by immunofluorescence staining in SiHa cells. e The expression of β-catenin at nucleus in SiHa cells with wild-type and mutant PIK3CA by western blotting. f The representative imaging of β-catenin in membrane, cytoplasm, and nucleus by IHC in 60 patients with cervical cancer. g The corresponding proportion of β-catenin in membrane, cytoplasm, and nucleus in tissues of patients with wild-type and E542K, E545K mutant PIK3CA
Fig. 4
Fig. 4
SIRT3 is a negative effector of β-catenin in regulating glucose metabolism. a Relative mRNA expression of SIRT3, GLUT4, and LDHB in SiHa cells with PIK3CA E542K and E545K mutations. b The expression of SIRT3 in SiHa and MS751 cells with wild-type and mutant PIK3CA. c Effect of knocking down β-catenin on the expression of SIRT3 and key glycolytic enzymes. d Relative SIRT3 promotor activity in SiHa cells with downregulation of β-catenin (**P < 0.01). e Knocking down SIRT3 rescued the effect of shβ-catenin on glucose uptake, lactate production (**P < 0.01). f Knocking down SIRT3 rescued the effect of shβ-catenin on key glycolytic enzymes
Fig. 5
Fig. 5
PIK3CA E542K and E545K mutations positively regulate proliferation and glucose metabolism in xenograft mice. a The tumors were separated from the xenograft mice with SiHa and MS751 cells harbored wild type and E545K mutant PIK3CA. b The growth curve of xenograft tumors from SiHa and MS751 cells with wild type and E545K mutant PIK3CA. c Tumor weight of xenograft mice with SiHa and MS751 cells harbored wild type and E545K mutant PIK3CA. d The weight of xenograft mice with SiHa and MS751 cells harbored wild type and E545K mutant PIK3CA. e Immunohistochemical staining of xenograft tumor tissues. f Representative 18F-FDG microPET/CT imaging of tumor-bearing mice. The tumors are indicated with arrows. g The tumor SUVmax in xenograft mice with wild type and mutant PIK3CA
Fig. 6
Fig. 6
Immunohistochemical analyses of β-catenin, SIRT3, GLUT4, and LDHB expression in tissues of patients with cervical cancer. a Representative pictures of β-catenin/SIRT3 and GLUT4, LDHB in negative, weak staining, intermediate staining, and strong staining. b The statistical analysis of H score of β-catenin/SIRT3 and GLUT4, LDHB in cervical cancer tissues
Fig. 7
Fig. 7
Mechanism model of PIK3CA E542K and E545K mutation-mediate regulation of proliferation and glucose metabolism via the β-catenin/SIRT3 in cervical cancer

References

    1. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015;136(5):E359–E386.
    1. Vivanco I, Sawyers CL. The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nat Rev Cancer. 2002;2(7):489–501.
    1. Samuels Y, Diaz LA, Jr, Schmidt-Kittler O, Cummins JM, Delong L, Cheong I, et al. Mutant PIK3CA promotes cell growth and invasion of human cancer cells. Cancer Cell. 2005;7(6):561–573.
    1. Engelman JA, Luo J, Cantley LC. The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat Rev Genet. 2006;7(8):606–619.
    1. Miled N, Yan Y, Hon WC, Perisic O, Zvelebil M, Inbar Y, et al. Mechanism of two classes of cancer mutations in the phosphoinositide 3-kinase catalytic subunit. Science (New York, NY) 2007;317(5835):239–242.
    1. Huw LY, O'Brien C, Pandita A, Mohan S, Spoerke JM, Lu S, et al. Acquired PIK3CA amplification causes resistance to selective phosphoinositide 3-kinase inhibitors in breast cancer. Oncogenesis. 2013;2:e83.
    1. Hollander MC, Blumenthal GM, Dennis PA. PTEN loss in the continuum of common cancers, rare syndromes and mouse models. Nat Rev Cancer. 2011;11(4):289–301.
    1. Bader AG, Kang S, Vogt PK. Cancer-specific mutations in PIK3CA are oncogenic in vivo. Proc Natl Acad Sci U S A. 2006;103(5):1475–1479.
    1. Vasudevan KM, Barbie DA, Davies MA, Rabinovsky R, McNear CJ, Kim JJ, et al. AKT-independent signaling downstream of oncogenic PIK3CA mutations in human cancer. Cancer Cell. 2009;16(1):21–32.
    1. Ojesina AI, Lichtenstein L, Freeman SS, Pedamallu CS, Imaz-Rosshandler I, Pugh TJ, et al. Landscape of genomic alterations in cervical carcinomas. Nature. 2014;506(7488):371–375.
    1. Xiang L, Li J, Jiang W, Shen X, Yang W, Wu X, et al. Comprehensive analysis of targetable oncogenic mutations in Chinese cervical cancers. Oncotarget. 2015;6(7):4968–4975.
    1. Millis SZ, Ikeda S, Reddy S, Gatalica Z, Kurzrock R. Landscape of phosphatidylinositol-3-kinase pathway alterations across 19784 diverse solid tumors. JAMA oncology. 2016;2(12):1565–1573.
    1. Xiang L, Jiang W, Li J, Shen X, Yang W, Yang G, et al. PIK3CA mutation analysis in Chinese patients with surgically resected cervical cancer. Sci Rep. 2015;5:14035.
    1. Hao Y, Wang C, Cao B, Hirsch BM, Song J, Markowitz SD, et al. Gain of interaction with IRS1 by p110alpha-helical domain mutants is crucial for their oncogenic functions. Cancer Cell. 2013;23(5):583–593.
    1. Warburg O, Wind F, Negelein E. The metabolism of tumors in the body. J Gen Physiol. 1927;8(6):519–530.
    1. Boroughs LK, DeBerardinis RJ. Metabolic pathways promoting cancer cell survival and growth. Nat Cell Biol. 2015;17(4):351–359.
    1. Elstrom RL, Bauer DE, Buzzai M, Karnauskas R, Harris MH, Plas DR, et al. Akt stimulates aerobic glycolysis in cancer cells. Cancer Res. 2004;64(11):3892–3899.
    1. Kohn AD, Summers SA, Birnbaum MJ, Roth RA. Expression of a constitutively active Akt Ser/Thr kinase in 3T3-L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation. J Biol Chem. 1996;271(49):31372–31378.
    1. Yecies JL, Manning BD. Transcriptional control of cellular metabolism by mTOR signaling. Cancer Res. 2011;71(8):2815–2820.
    1. Hao Y, Samuels Y, Li Q, Krokowski D, Guan BJ, Wang C, et al. Oncogenic PIK3CA mutations reprogram glutamine metabolism in colorectal cancer. Nat Commun. 2016;7:11971.
    1. Wang Z, Liu Y, Lu L, Yang L, Yin S, Wang Y, et al. Fibrillin-1, induced by Aurora-a but inhibited by BRCA2, promotes ovarian cancer metastasis. Oncotarget. 2015;6(9):6670–6683.
    1. Ciriello G, Gatza ML, Beck AH, Wilkerson MD, Rhie SK, Pastore A, et al. Comprehensive molecular portraits of invasive lobular breast Cancer. Cell. 2015;163(2):506–519.
    1. Kandoth C, Schultz N, Cherniack AD, Akbani R, Liu Y, Shen H, et al. Integrated genomic characterization of endometrial carcinoma. Nature. 2013;497(7447):67–73.
    1. Monga SP. Beta-catenin signaling and roles in liver homeostasis, injury, and tumorigenesis. Gastroenterology. 2015;148(7):1294–1310.
    1. Chocarro-Calvo A, Garcia-Martinez JM, Ardila-Gonzalez S, De la Vieja A, Garcia-Jimenez C. Glucose-induced beta-catenin acetylation enhances Wnt signaling in cancer. Mol Cell. 2013;49(3):474–486.
    1. Wu D, Pan W. GSK3: a multifaceted kinase in Wnt signaling. Trends Biochem Sci. 2010;35(3):161–168.
    1. Clevers H. Wnt/beta-catenin signaling in development and disease. Cell. 2006;127(3):469–480.
    1. Finkel T, Deng CX, Mostoslavsky R. Recent progress in the biology and physiology of sirtuins. Nature. 2009;460(7255):587–591.
    1. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646–674.
    1. Cairns RA, Harris IS, Mak TW. Regulation of cancer cell metabolism. Nat Rev Cancer. 2011;11(2):85–95.
    1. Nomura DK, Dix MM, Cravatt BF. Activity-based protein profiling for biochemical pathway discovery in cancer. Nat Rev Cancer. 2010;10(9):630–638.
    1. Dang L, White DW, Gross S, Bennett BD, Bittinger MA, Driggers EM, et al. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature. 2009;462(7274):739–744.
    1. Carvalho KC, Cunha IW, Rocha RM, Ayala FR, Cajaiba MM, Begnami MD, et al. GLUT1 expression in malignant tumors and its use as an immunodiagnostic marker. Clinics (Sao Paulo, Brazil) 2011;66(6):965–972.
    1. Jiang ZY, Zhou QL, Coleman KA, Chouinard M, Boese Q, Czech MP. Insulin signaling through Akt/protein kinase B analyzed by small interfering RNA-mediated gene silencing. Proc Natl Acad Sci U S A. 2003;100(13):7569–7574.
    1. Kidd EA, Siegel BA, Dehdashti F, Grigsby PW. The standardized uptake value for F-18 fluorodeoxyglucose is a sensitive predictive biomarker for cervical cancer treatment response and survival. Cancer. 2007;110(8):1738–1744.
    1. Schwarz JK, Siegel BA, Dehdashti F, Grigsby PW. Association of posttherapy positron emission tomography with tumor response and survival in cervical carcinoma. Jama. 2007;298(19):2289–2295.
    1. Brooks RA, Rader JS, Dehdashti F, Mutch DG, Powell MA, Thaker PH, et al. Surveillance FDG-PET detection of asymptomatic recurrences in patients with cervical cancer. Gynecol Oncol. 2009;112(1):104–109.
    1. Eguez L, Lee A, Chavez JA, Miinea CP, Kane S, Lienhard GE, et al. Full intracellular retention of GLUT4 requires AS160 Rab GTPase activating protein. Cell Metab. 2005;2(4):263–272.
    1. Larance M, Ramm G, Stockli J, van Dam EM, Winata S, Wasinger V, et al. Characterization of the role of the Rab GTPase-activating protein AS160 in insulin-regulated GLUT4 trafficking. J Biol Chem. 2005;280(45):37803–37813.
    1. Khatri S, Yepiskoposyan H, Gallo CA, Tandon P, Plas DR. FOXO3a regulates glycolysis via transcriptional control of tumor suppressor TSC1. J Biol Chem. 2010;285(21):15960–15965.
    1. Duvel K, Yecies JL, Menon S, Raman P, Lipovsky AI, Souza AL, et al. Activation of a metabolic gene regulatory network downstream of mTOR complex 1. Mol Cell. 2010;39(2):171–183.
    1. Kotliarova S, Pastorino S, Kovell LC, Kotliarov Y, Song H, Zhang W, et al. Glycogen synthase kinase-3 inhibition induces glioma cell death through c-MYC, nuclear factor-kappaB, and glucose regulation. Cancer Res. 2008;68(16):6643–6651.
    1. Kaidi A, Williams AC, Paraskeva C. Interaction between beta-catenin and HIF-1 promotes cellular adaptation to hypoxia. Nat Cell Biol. 2007;9(2):210–217.
    1. Verdin E, Hirschey MD, Finley LW, Haigis MC. Sirtuin regulation of mitochondria: energy production, apoptosis, and signaling. Trends Biochem Sci. 2010;35(12):669–675.
    1. Finley LW, Carracedo A, Lee J, Souza A, Egia A, Zhang J, et al. SIRT3 opposes reprogramming of cancer cell metabolism through HIF1alpha destabilization. Cancer Cell. 2011;19(3):416–428.

Source: PubMed

Sponzoři a spolupracovníci

Zdravotní podmínky

Drogové intervence

3
Předplatit