nach oben

Journal of Cancer Research and Clinical Oncology

Erschienen in:

01.11.2011 | Original Paper

Down-regulation of JAK1 by RNA interference inhibits growth of the lung cancer cell line A549 and interferes with the PI3K/mTOR pathway

verfasst von: Dan Liu, Yi Huang, Jing Zeng, Bojiang Chen, Na Huang, Na Guo, Lunxu Liu, Hong Xu, Xianming Mo, Weimin Li

Erschienen in: Journal of Cancer Research and Clinical Oncology | Ausgabe 11/2011

Einloggen, um Zugang zu erhalten

Abstract

Purpose

The mammalian Janus kinase (JAK) family plays a critical role in cytokine/growth factor signalling pathways and is associated with human cancers. In this study, we explored the role of JAK1 in the non-small cell lung cancer (NSCLC) cell line A549 and its molecular crosstalk with the phosphatidyl inositol-3-kinase (PI3K)/mammalian target of the rapamycin (mTOR) pathway.

Methods

One hundred and two NSCLC and 50 normal lung specimens were collected after surgical resection. JAK1 expression and phosphorylation were determined via immunohistochemical staining (IHC) assay. A stable knockdown of JAK1 was performed in A549 cells by RNA interference. Stable cell proliferation, cell cycle, apoptosis, and invasion were characterised in vitro. Tumourigenicity was analysed in vivo. The NSCLC xenograft protein expression of PI3K/mTOR pathway molecules was determined by Western blot assay.

Results

JAK1 expression was higher in NSCLC tissues than in normal lung tissues (P < 0.01). JAK1 knockdown in A549 cells significantly inhibited cell proliferation and invasion while promoting cell arrest at G0/G1 phase (all P < 0.05). The xenograft model showed that JAK1 suppression inhibited tumour growth compared with normal control (P < 0.05). Moreover, JAK1 knockdown inhibited mTOR or P70 ribosomal protein S6 kinase (P70S6K) phosphorylation, but increased glycogen synthase kinase-3α (GSK-3α) and B-cell lymphoma-extra large (Bcl-xl) phosphorylation. Total protein expression and Akt1/2 phosphorylated status remained unchanged.

Conclusion

Our study suggests that JAK1 expression and phosphorylation is abnormal in NSCLC tissues. The knockdown of JAK1 significantly inhibits tumourigenicity of the A549 cell line and demonstrates that crosstalk between the JAK1 and PI3K/mTOR pathways is involved.

Akar U, Ozpolat B, Mehta K, Lopez-Berestein G, Zhang D, Ueno NT, Hortobagyi GN, Arun B (2010) Targeting p70S6K prevented lung metastasis in a breast cancer xenograft model. Mol Cancer Ther 9(5):1180–1187PubMedCrossRef

Cousens LP, Goulette FA, Darnowski JW (2005) JAK-mediated signaling inhibits Fas ligand-induced apoptosis independent of de novo protein synthesis. J Immunol 174(1):320–327PubMed

Dal Col J, Zancai P, Terrin L, Guidoboni M, Ponzoni M, Pavan A, Spina M, Bergamin S, Rizzo S, Tirelli U, De Rossi A, Doglioni C, Dolcetti R (2008) Distinct functional significance of Akt and mTOR constitutive activation in mantle cell lymphoma. Blood 111(10):5142–5151PubMedCrossRef

Diehl JA, Cheng M, Roussel MF, Sherr CJ (1998) Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes Dev 12(22):3499–3511PubMedCrossRef

Ferrajoli A, Faderl S, Ravandi F, Estrov Z (2006) The JAK-STAT pathway: a therapeutic target in hematological malignancie. Curr Cancer Drug Targets 6(8):671–679PubMedCrossRef

Freed DH, Borowiec AM, Angelovska T, Dixon IM (2003) Induction of protein synthesis in cardiac fibroblasts by cardiotrophin-1: integration of multiple signaling pathways. Cardiovasc Res 60(2):365–375PubMedCrossRef

Freitas S, Moore DH, Michael H, Kelley MR (2003) Studies of apurinic/apyrimidinic endonuclease/ref-1 expression in epithelial ovarian cancer: correlations with tumor progression and platinum resistance. Clin Cancer Res 9(13):4689–4694PubMed

Gamero AM, Larner AC (2001) Vanadate facilitates interferon alpha-mediated apoptosis that is dependent on the Jak/Stat pathway. J Biol Chem 276(17):13547–13553PubMed

Gao B, Shen X, Kunos G, Meng Q, Goldberg ID, Rosen EM, Fan S (2001) Constitutive activation of JAK-STAT3 signaling by BRCA1 in human prostate cancer cells. FEBS Lett 488(3):179–184PubMedCrossRef

Gómez-Benito M, Balsas P, Carvajal-Vergara X, Pandiella A, Anel A, Marzo I, Naval J (2007) Mechanism of apoptosis induced by INF-α in human myeloma cells: role of Jak1 and Bim and potentiation by rapamycin. Cell Signal 19(4):844–854PubMedCrossRef

Harris TE, Lawrence JC Jr. (2003) TOR signaling. Sci STKE 212: re15

Hay N, Sonenberg N (2004) Upstream and downstream of mTOR. Genes Dev 18(16):1926–1945PubMedCrossRef

He B, You L, Xu Z, Mazieres J, Lee AY, Jablons DM (2004) Activity of the suppressor of cytokine signaling-3 promoter in human non-small-cell lung cancer. Clin Lung Cancer 5(6):366–370PubMedCrossRef

Huang HM, Lin YL, Chen CH, Chang TW (2005) Simultaneous activation of JAK1 and JAK2 confers IL-3 independent growth on Ba/F3 pro-B cells. J Cell Biochem 96(2):361–375PubMedCrossRef

Ihle JN (1995) The Janus protein tyrosine kinase family and its role in cytokine signaling. Adv Immunol 60:1–35PubMedCrossRef

Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ (2009) Cancer statistics, 2009. CA Cancer J Clin 59(4):225–249PubMedCrossRef

Jeong EG, Kim MS, Nam HK, Min CK, Lee S, Chung YJ, Yoo NJ, Lee SH (2008) Somatic mutations of JAK1 and JAK3 in acute leukemias and solid cancers. Clin Cancer Res 14(12):3716–3721PubMedCrossRef

Kaur N, Wohlhueter AL, Halvorsen SW (2002) Activation and inactivation of signal transducers and activators of transcription by ciliary neurotrophic factor in neuroblastoma cells. Cell Signal 14(5):419–429PubMedCrossRef

Kong D, Banerjee S, Huang W, Li Y, Wang Z, Kim HR, Sarkar FH (2008) Mammalian target of rapamycin repression by 3, 3′-diindolylmethane inhibits invasion and angiogenesis in platelet-derived growth factor-D-overexpressing PC3 cells. Cancer Res 68(6):1927–1934PubMedCrossRef

Kurdi M, Booz GW (2007) Jak inhibition, but not Stat1 knockdown, blocks the synergistic effect of IFN-gamma on Fas-induced apoptosis of A549 human non-small cell lung cancer cells. J Interferon Cytokine Res 27(1):23–31PubMedCrossRef

Lam P, Sian Lim K, Mei Wang S, Hui KM (2005) A microarray study to characterize the molecular mechanism of TIMP-3-mediated tumor rejection. Mol Ther 12(1):144–152PubMedCrossRef

Langlais P, Yi Z, Mandarino LJ (2011) The Identification of Raptor as a Substrate for p44/42 MAPK. Endocrinology 152(4):1264–1273PubMedCrossRef

Le A, Cooper CR, Gouw AM, Dinavahi R, Maitra A, Deck LM, Royer RE, Vander Jagt DL, Semenza GL, Dang CV (2009) Inhibition of lactate dehydrogenase A induces oxidative stress and inhibits tumor progression. Proc Natl Acad Sci USA 107(5):2037–2042CrossRef

Li J, Favata M, Kelley JA, Caulder E, Thomas B, Wen X, Sparks RB, Arvanitis A, Rogers JD, Combs AP, Vaddi K, Solomon KA, Scherle PA, Newton R, Fridman JS (2010) INCB16562, a JAK1/2 selective inhibitor, is efficacious against multiple myeloma cells and reverses the protective effects of cytokine and stromal cell support. Neoplasia 12(1):28–38PubMed

Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S (2002) The protein kinase complement of the human genome. Science 298(5600):1912–1934PubMedCrossRef

Memmott RM, Dennis PA (2009) The role of the Akt/mTOR pathway in tobacco carcinogen-induced lung tumorigenesis. Clin Cancer Res 16(1):4–10PubMedCrossRef

Noguchi M, Ropars V, Roumestand C, Suizu F (2007) Proto-oncogene TCL1: more than just a coactivator for Akt. FASEB J 21(10):2273–2284PubMedCrossRef

Oh H, Fujio Y, Kunisada K, Hirota H, Matsui H, Kishimoto T, Yamauchi-Takihara K (1998) Activation of phosphatidylinositol 3-kinase through glycoprotein 130 induces protein kinase B and p70 S6 kinase phosphorylation in cardiac myocytes. J Biol Chem 273(16):9703–9710PubMedCrossRef

O’Shea JJ, Gadina M, Schreiber RD (2002) Cytokine signaling in 2002: new surprises in the Jak/Stat pathway. Cell 109(Suppl):S121–S131PubMedCrossRef

Petritsch C, Beug H, Balmain A, Oft M (2000) TGF-beta inhibits p70 S6 kinase via protein phosphatase 2A to induce G(1) arrest. Genes Dev 14(24):3093–3101PubMedCrossRef

Ramanathan A, Schreiber SL (2009) Direct control of mitochondrial function by mTOR. Proc Natl Acad Sci USA 106(52):22229–22232PubMedCrossRef

Rane SG, Reddy EP (2000) Janus kinases: components of multiple signaling pathways. Oncogene 19(49):5662–5679PubMedCrossRef

Ravikumar B, Sarkar S, Davies JE, Futter M, Garcia-Arencibia M, Green-Thompson ZW, Jimenez-Sanchez M, Korolchuk VI, Lichtenberg M, Luo S, Massey DC, Menzies FM, Moreau K, Narayanan U, Renna M, Siddiqi FH, Underwood BR, Winslow AR, Rubinsztein DC (2011) Regulation of mammalian autophagy in physiology and pathophysiology. Physiol Rev 90(4):1383–1435CrossRef

Saxena NK, Sharma D, Ding X, Lin S, Marra F, Merlin D, Anania FA (2007) Concomitant activation of the JAK/STAT, PI3K/AKT, and ERK signaling is involved in leptin-mediated promotion of invasion and migration of hepatocellular carcinoma cells. Cancer Res 67(6):2497–2507PubMedCrossRef

Schindler CW (2002) Series introduction. JAK-STAT signaling in human disease. J Clin Invest 109(9):1133–1137PubMed

Shiojima I, Walsh K (2006) Regulation of cardiac growth and coronary angiogenesis by the Akt/PKB signaling pathway. Genes Dev 20(24):3347–3365PubMedCrossRef

Song L, Rawal B, Nemeth JA, Haura EB (2011) JAK1 activates STAT3 activity in non-small-cell lung cancer cells and IL-6 neutralizing antibodies can suppress JAK1-STAT3 signaling. Mol Cancer Ther 10(3):481–494PubMedCrossRef

Sussman MA, Volkers M, Fischer K, Bailey B, Cottage CT, Din S, Gude N, Avitabile D, Alvarez R, Sundararaman B, Quijada P, Mason M, Konstandin MH, Malhowski A, Cheng Z, Khan M, McGregor M (2011) Myocardial AKT: the omnipresent nexus. Physiol Rev 91(3):1023–1070PubMedCrossRef

Tang JM, He QY, Guo RX, Chang XJ (2006) Phosphorylated Akt overexpression and loss of PTEN expression in non-small cell lung cancer confers poor prognosis. Lung Cancer 51(2):181–191PubMedCrossRef

Tsikitis M, Zhang Z, Edelman W, Zagzag D, Kalpana GV (2005) Genetic ablation of Cyclin D1 abrogates genesis of rhabdoid tumors resulting from Ini1 loss. Proc Natl Acad Sci USA 102(34):12129–12134PubMedCrossRef

Upreti M, Galitovskaya EN, Chu R, Tackett AJ, Terrano DT, Granell S, Chambers TC (2008) Identification of the major phosphorylation site in Bcl-xL induced by microtubule inhibitors and analysis of its functional significance. J Biol Chem 283(51):35517–35525PubMedCrossRef

Verma A, Kambhampati S, Parmar S, Platanias LC (2003) Jak family of kinases in cancer. Cancer Metastasis Rev 22(4):423–434PubMedCrossRef

Vignot S, Faivre S, Aguirre D, Raymond E (2005) mTOR-targeted therapy of cancer with rapamycin derivatives. Ann Oncol 16(4):525–537PubMedCrossRef

Wilson W 3rd, Baldwin AS (2008) Maintenance of constitutive IkappaB kinase activity by glycogen synthase kinase-3alpha/beta in pancreatic cancer. Cancer Res 68(19):8156–8163PubMedCrossRef

Xiang Z, Zhao Y, Mitaksov V, Fremont DH, Kasai Y, Molitoris A, Ries RE, Miner TL, McLellan MD, DiPersio JF, Link DC, Payton JE, Graubert TA, Watson M, Shannon W, Heath SE, Nagarajan R, Mardis ER, Wilson RK, Ley TJ, Tomasson MH (2008) Identification of somatic JAK1 mutations in patients with acute myeloid leukemia. Blood 111(9):4809–4812PubMedCrossRef

Xie HJ, Bae HJ, Noh JH, Eun JW, Kim JK, Jung KH, Ryu JC, Ahn YM, Kim SY, Lee SH, Yoo NJ, Lee JY, Park WS, Nam SW (2009) Mutational analysis of JAK1 gene in human hepatocellular carcinoma. Neoplasma 56(2):136–140PubMedCrossRef

Xiong H, Zhang ZG, Tian XQ, Sun DF, Liang QC, Zhang YJ, Lu R, Chen YX, Fang JY (2008) Inhibition of JAK1, 2/STAT3 signaling induces apoptosis, cell cycle arrest, and reduces tumor cell invasion in colorectal cancer cells. Neoplasia 10(3):287–297PubMed

Yeh YT, Ou-Yang F, Chen IF, Yang SF, Su JH, Hou MF, Yuan SS (2007) Altered p-JAK1 expression is associated with estrogen receptor status in breast infiltrating ductal carcinoma. Oncol Rep 17(1):35–39PubMed

Yoshizawa A, Fukuoka J, Shimizu S, Shilo K, Franks TJ, Hewitt SM, Fujii T, Cordon-Cardo C, Jen J, Travis WD (2010) Overexpression of phospho-eIF4E is associated with survival through AKT pathway in non-small cell lung cancer. Clin Cancer Res 16(1):240–248PubMedCrossRef

Zeng ZZ, Yellaturu CR, Neeli I, Rao GN (2002) 5(S)-hydroxyeicosatetraenoic acid stimulates DNA synthesis in human microvascular endothelial cells via activation of Jak/STAT and phosphatidylinositol 3-kinase/Akt signaling, leading to induction of expression of basic fibroblast growth factor 2. J Biol Chem 277(43):41213–41219PubMedCrossRef

Zhang F, Phiel CJ, Spece L, Gurvich N, Klein PS (2003) Inhibitory phosphorylation of glycogen synthase kinase-3 (GSK-3) in response to lithium. Evidence for autoregulation of GSK-3. J Biol Chem 278(35):33067–33077PubMedCrossRef

Zhang MX, Zhao X, Wang ZG, Zhao WM, Wang YS (2010) Constitutive activation of signal transducer and activator of transcription 3 regulates expression of vascular endothelial growth factor in human meningioma differentiation. J Cancer Res Clin Oncol 136(7):981–988PubMedCrossRef

Zhou H, Huang S (2010) The complexes of mammalian target of rapamycin. Curr Protein Pept Sci 11(6):409–424PubMedCrossRef

Zhou HY, Wong AS (2006) Activation of p70S6K induces expression of matrix metalloproteinase 9 associated with hepatocyte growth factor-mediated invasion in human ovarian cancer cells. Endocrinology 147(5):2557–2566PubMedCrossRef

Zhou X, Tan M, Stone Hawthorne V, Klos KS, Lan KH, Yang Y, Yang W, Smith TL, Shi D, Yu D (2004) Activation of the Akt/mammalian target of rapamycin/4E-BP1 pathway by ErbB2 overexpression predicts tumor progression in breast cancers. Clin Cancer Res 10(20):6779–6788PubMedCrossRef

Titel: Down-regulation of JAK1 by RNA interference inhibits growth of the lung cancer cell line A549 and interferes with the PI3K/mTOR pathway
verfasst von: Dan Liu
Yi Huang
Jing Zeng
Bojiang Chen
Na Huang
Na Guo
Lunxu Liu
Hong Xu
Xianming Mo
Weimin Li
Publikationsdatum: 01.11.2011
Verlag: Springer-Verlag
Erschienen in: Journal of Cancer Research and Clinical Oncology / Ausgabe 11/2011
Print ISSN: 0171-5216
Elektronische ISSN: 1432-1335
DOI: https://doi.org/10.1007/s00432-011-1037-6

Springer Medizin

Down-regulation of JAK1 by RNA interference inhibits growth of the lung cancer cell line A549 and interferes with the PI3K/mTOR pathway