Threonine 32 (Thr32) of FoxO3 is critical for TGF-β-induced apoptosis via Bim in hepatocarcinoma cells

Abstract

Transforming growth factor- β (TGF- β) exerts apoptotic effects on various types of malignant cells, including liver cancer cells. However, the precise mechanisms by which TGF- β induces apoptosis remain poorly known. In the present study, we have showed that threonine 32 (Thr32) residue of FoxO3 is critical for TGF- β to induce apoptosis via Bim in hepatocarcinoma Hep3B cells. Our data demonstrated that TGF- β induced FoxO3 activation through specific de-phosphorylation at Thr32. TGF- β-activated FoxO3 cooperated with Smad2/3 to mediate Bim up-regulation and apoptosis. FoxO3 (de)phosphorylation at Thr32 was regulated by casein kinase I- ϵ (CKI- ϵ). CKI inhibition by small molecule D4476 could abrogate TGF- β-induced FoxO/Smad activation, reverse Bim up-regulation, and block the sequential apoptosis. More importantly, the deregulated levels of CKI- ϵ and p32FoxO3 were found in human malignant liver tissues. Taken together, our findings suggest that there might be a CKI-FoxO/Smad-Bim engine in which Thr32 of FoxO3 is pivotal for TGF- β-induced apoptosis, making it a potential therapeutic target for liver cancer treatment.

Keywords

apoptosis / TGF-β / FoxO3 / casein kinase I-ϵ / hepatocarcinoma

References

[1] Akagi M, Yasui W, Akama Y, Yokozaki H, Tahara H, Haruma K, Kajiyama G, Tahara E (1996) Inhibition of cell growth by transforming growth factor β1 is associated with p53-independent induction of p21 in gastric carcinoma cells. Jpn J Cancer Res87: 377-384

[2] Biggs WH III, Cavenee WK, Arden KC (2001) Identification and characterization of members of the FKHR (FOX O) subclass of winged-helix transcription factors in the mouse. Mamm Genome12: 416-425

[3] <?Pub Caret1?>Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, Blenis J, Greenberg ME (1999) Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell96: 857-868

[4] Chen Q, Gong B, Mahmoud-Ahmed AS, Zhou A, Hsi ED, Hussein M, Almasan A (2001) Apo2L/TRAIL and Bcl-2-related proteins regulate type I interferon-induced apoptosis in multiple myeloma. Blood98: 2183-2192

[5] Conery AR, Cao Y, Thompson EA, Townsend CM Jr, Ko TC, Luo K (2004) Akt interacts directly with Smad3 to regulate the sensitivity to TGF-β induced apoptosis. Nat Cell Biol6: 366-372

[6] Derynck R, Zhang YE (2003) Smad-dependent and Smad-independent pathways in TGF-β family signalling. Nature425: 577-584

[7] Engel ME, Datta PK, Moses HL (1998) RhoB is stabilized by transforming growth factor β and antagonizes transcriptional activation. J Biol Chem273: 9921-9926

[8] Enroth S, Andersson R, Bysani M, Wallerman O, Termen S, Tuch BB, De La Vega FM, Heldin CH, Moustakas A, Komorowski J, Wadelius C (2014) Nucleosome regulatory dynamics in response to TGF-β. Nucleic Acids Res42: 6921-6934

[9] Fish KJ, Cegielska A, Getman ME, Landes GM, Virshup DM (1995) Isolation and characterization of human casein kinase I epsilon (CKI), a novel member of the CKI gene family. J Biol Chem270: 14875-14883

[10] Graves PR, Haas DW, Hagedorn CH, Paoli-Roach AA, Roach PJ (1993) Molecular cloning, expression, and characterization of a 49-kilodalton casein kinase I isoform from rat testis. J Biol Chem268: 6394-6401

[11] Green DR, Reed JC (1998) Mitochondria and apoptosis. Science281: 1309-1312

[12] Hagenbuchner J, Kuznetsov A, Hermann M, Hausott B, Obexer P, Ausserlechner MJ (2012) FOXO3-induced reactive oxygen species are regulated by BCL2L11 (Bim) and SESN3. J Cell Sci125: 1191-1203

[13] Higaki M, Shimokado K (1999) Phosphatidylinositol 3-kinase is required for growth factor-induced amino acid uptake by vascular smooth muscle cells. Arterioscler Thromb Vasc Biol19: 2127-2132

[14] Hua X, Liu X, Ansari DO, Lodish HF (1998) Synergistic cooperation of TFE3 and smad proteins in TGF-β -induced transcription of the plasminogen activator inhibitor-1 gene. Genes Dev12: 3084-3095

[15] Huynh H, Nguyen TT, Chan E, Tran E (2003) Inhibition of ErbB-2 and ErbB-3 expression by quercetin prevents transforming growth factor alpha (TGF-alpha)- and epidermal growth factor (EGF)-induced human PC-3 prostate cancer cell proliferation. Int J Oncol23: 821-829

[16] Karube K, Nakagawa M, Tsuzuki S, Takeuchi I, Honma K, Nakashima Y, Shimizu N, Ko YH, Morishima Y, Ohshima K, Nakamura S, Seto M (2011) Identification of FOXO3 and PRDM1 as tumorsuppressor gene candidates in NK-cell neoplasms by genomic and functional analyses. Blood118: 3195-3204

[17] Kato M, Yuan H, Xu ZG, Lanting L, Li SL, Wang M, Hu MC, Reddy MA, Natarajan R (2006) Role of the Akt/FoxO3a pathway in TGF- β 1-mediated mesangial cell dysfunction: a novel mechanism related to diabetic kidney disease. J Am Soc Nephrol17: 3325-3335

[18] Knippschild U, Gocht A, Wolff S, Huber N, Lohler J, Stoter M (2005) The casein kinase 1 family: participation in multiple cellular processes in eukaryotes. Cell Signal17: 675-689

[19] Kops GJ, Burgering BM (1999) Forkhead transcription factors: new insights into protein kinase B (c-akt) signaling. J Mol Med (Berl)77: 656-665

[20] Liao XD, Tang AH, Chen Q, Jin HJ, Wu CH, Chen LY, Wang SQ (2003) Role of Ca²⁺ signaling in initiation of stretch-induced apoptosis in neonatal heart cells. Biochem Biophys Res Commun310: 405-411

[21] Massague J, Weis-Garcia F(1996) Serine/threonine kinase receptors: mediators of transforming growth factor β family signals. Cancer Surv27: 41-64

[22] Massague J, Seoane J, Wotton D (2005) Smad transcription factors. Genes Dev19: 2783-2810

[23] Miyazono K (2000) TGF-beta/SMAD signaling and its involvement in tumor progression. Biol Pharm Bull23: 1125-1130

[24] Naka K, Hoshii T, Muraguchi T, Tadokoro Y, Ooshio T, Kondo Y, Nakao S, Motoyama N, Hirao A (2010) TGF-β-FOXO signalling maintains leukaemia-initiating cells in chronic myeloid leukaemia. Nature463: 676-680

[25] Nass SJ, Li M, Amundadottir LT, Furth PA, Dickson RB (1996) Role for Bcl-xL in the regulation of apoptosis by EGF and TGF-β in c-myc overexpressing mammary epithelial cells. Biochem Biophys Res Commun227: 248-256

[26] Ni YG, Wang N, Cao DJ, Sachan N, Morris DJ, Gerard RD, Kuro O, Rothermel BA, Hill JA (2007) FoxO transcription factors activate Akt and attenuate insulin signaling in heart by inhibiting protein phosphatases. Proc Natl Acad Sci U S A104: 20517-20522

[27] Park JT, Kato M, Yuan H, Castro N, Lanting L, Wang M, Natarajan R (2013) FOG2 protein down-regulation by transforming growth factor-β1-induced microRNA-200b/c leads to Akt kinase activation and glomerular mesangial hypertrophy related to diabetic nephropathy. J Biol Chem288: 22469-22480

[28] Philips N, McFadden K (2004) Inhibition of transforming growth factor-β and matrix metalloproteinases by estrogen and prolactin in breast cancer cells. Cancer Lett206: 63-68

[29] Qi W, Weber CR, Wasland K, Roy H, Wali R, Joshi S, Savkovic SD (2011a) Tumor suppressor FOXO3 mediates signals from the EGF receptor to regulate proliferation of colonic cells. Am J Physiol Gastrointest Liver Physiol300: G264-G272

[30] Qi W, Weber CR, Wasland K, Savkovic SD (2011b) Genistein inhibits proliferation of colon cancer cells by attenuating a negative effect of epidermal growth factor on tumor suppressor FOXO3 activity. BMC Cancer11: 219

[31] Ramjaun AR, Tomlinson S, Eddaoudi A, Downward J (2007) Upregulation of two BH3-only proteins, Bmf and Bim, during TGF beta-induced apoptosis. Oncogene26: 970-981

[32] Remy I, Montmarquette A, Michnick SW (2004) PKB/Akt modulates TGF-β signalling through a direct interaction with Smad3. Nat Cell Biol6: 358-365

[33] Renard E, Chadjichristos C, Kypriotou M, Beauchef G, Bordat P, Dompmartin A, Widom RL, Boumediene K, Pujol JP, Galera P (2008) Chondroitin sulphate decreases collagen synthesis in normal and scleroderma fibroblasts through a Smad-independent TGF-β pathway—implication of C-Krox and Sp1. J Cell Mol Med12: 2836-2847

[34] Renault VM, Thekkat PU, Hoang KL, White JL, Brady CA, Kenzelmann BD, Venturelli OS, Johnson TM, Oskoui PR, Xuan Z, Santo EE, Zhang MQ, Vogel H, Attardi LD, Brunet A (2011) The pro-longevity gene FoxO3 is a direct target of the p53 tumor suppressor. Oncogene30: 3207-3221

[35] Runyan CE, Liu Z, Schnaper HW (2012) Phosphatidylinositol 3-kinase and Rab5 GTPase inversely regulate the Smad anchor for receptor activation (SARA) protein independently of transforming growth factor-β 1. J Biol Chem287: 35815-35824

[36] Seoane J, Le HV, Massague J (2002) Myc suppression of the p21 (Cip1) Cdk inhibitor influences the outcome of the p53 response to DNA damage. Nature419: 729-734

[37] Torregroza I, Evans T (2006) Tid1 is a Smad-binding protein that can modulate Smad7 activity in developing embryos. Biochem J393: 311-320

[38] Tremblay ML, Giguere V (2008) Phosphatases at the heart of FoxO metabolic control. Cell Metab7: 101 -103

[39] Tuazon PT, Traugh JA (1991) Casein kinase I and II–multipotential serine protein kinases: structure, function, and regulation. Adv Second Messenger Phosphoprotein Res23: 123-164

[40] Valderrama-Carvajal H, Cocolakis E, Lacerte A, Lee EH, Krystal G, Ali S, Lebrun JJ (2002) Activin/TGF-β induce apoptosis through Smad-dependent expression of the lipid phosphatase SHIP. Nat Cell Biol4: 963-969

[41] Vignais ML (2000) Ski and SnoN: antagonistic proteins of TGF-β signaling. Bull Cancer87: 135-137

[42] Vogt PK, Jiang H, Aoki M (2005) Triple layer control: phosphorylation, acetylation and ubiquitination of FOXO proteins. Cell Cycle4: 908-913

[43] Waddell DS, Liberati NT, Guo X, Frederick JP, Wang XF (2004) Casein kinase Iepsilon plays a functional role in the transforming growth factor-β signaling pathway. J Biol Chem279: 29236-29246

[44] Wildey GM, Howe PH (2009) Runx1 is a co-activator with FOXO3 to mediate transforming growth factor β (TGF-β )-induced Bim transcription in hepatic cells. J Biol Chem284: 20227-20239

[45] Yamamura Y, Hua X, Bergelson S, Lodish HF (2000) Critical role of Smads and AP-1 complex in transforming growth factor-β -dependent apoptosis. J Biol Chem275: 36295-36302

[46] Yan L, Lavin VA, Moser LR, Cui Q, Kanies C, Yang E (2008) PP2A regulates the pro-apoptotic activity of FOXO1. J Biol Chem283: 7411-7420

RIGHTS & PERMISSIONS

2014 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.