Inhibition of Akt activity induces the mesenchymal-to-epithelial reverting transition with restoring E-cadherin expression in KB and KOSCC-25B oral squamous cell carcinoma cells

Kyoung-Ok Hong1, Ji-Hong Kim1, Ji-Soo Hong1, Hye-Jung Yoon1, Jae-Il Lee1, Sam-Pyo Hong1, Seong-Doo Hong1
1Department of Oral Pathology, School of Dentistry and Dental Research Institute, Seoul National University, Seoul, Korea

Tóm tắt

The Akt/PKB family of kinases is frequently activated in human cancers, including oral squamous cell carcinoma (OSCC). Akt-induced epithelial-to-mesenchymal transition (EMT) involves downregulation of E-cadherin, which appears to result from upregulation of the transcription repressor Snail. Recently, it was proposed that carcinoma cells, especially in metastatic sites, could acquire the mesenchymal-to-epithelial reverting transition (MErT) in order to adapt the microenvironments and re-expression of E-cadherin be a critical indicator of MErT. However, the precise mechanism and biologic or clinical importance of the MErT in cancers have been little known. This study aimed to investigate whether Akt inhibition would restore the expression of E-cadherin and β-catenin, reduce that of Vimentin, and induce the MErT in OSCC cells with low or negative expression of E-cadherin. We also investigate whether inhibition of Akt activity would affect the E-cadherin repressors and signaling molecules like NF-κB, ERK, and p38. We screened several OSCC cell lines in order to select suitable cell line models for inducing MErT, using immunoblotting and methylation specific-PCR. We examined whether Akt inhibitor phosphatidylinositol ether lipid analogues (PIA) treatment would restore the expression of E-cadherin and β-catenin, reduce that of Vimentin, and induce the MErT in KB and KOSCC-25B cells using RT-PCR, immunoblotting, immunofluorescence analysis, and in vitro migration assay. We also investigated whether inhibition of Akt activity would affect the E-cadherin repressors, including Snail, Twist, and SIP-1/ZEB-2 and signaling molecules like NF-κB, ERK, JNK, and p38 using RT-PCR, immunoblotting, and immunofluorescence analysis. Of the 7 OSCC cell lines, KB and KOSCC-25B showed constitutively activated phosphorylated Akt and low or negative expression of E-cadherin. Inhibition of Akt activity by PIA decreased NF-κB signaling, but did not affect phosphorylation of ERK, JNK, and p38 in KB and KOSCC-25B cells. Akt inhibition led to downregulation of Snail and Twist expression. In contrast, inhibition of Akt activity by PIA did not induce any changes in SIP-1/ZEB-2 expression. PIA treatment induced the expression of E-cadherin and β-catenin, reduce that of Vimentin, restored their epithelial morphology of a polygonal shape, and reduced tumor cell migration in KB and KOSCC-25B cells, which was the corresponding feature of MErT. All of these findings suggest that Akt inhibition could induce the MErT through decreased NF-κB signaling and downregulation of Snail and Twist in OSCC cells. A strategy involving Akt inhibition might be a useful therapeutic tool in controlling cancer dissemination and metastasis in oral cancer patients.

Tài liệu tham khảo

Birchmeier C, Birchmeier W, Brand-Saberi B: Epithelial-mesenchymal transitions in cancer progression. Acta Anat. 1996, 156: 217-226. 10.1159/000147848. Mizunuma H, Miyazawa J, Sanada K, Imai K: The LIM-only protein, LMO4, and the LIM domain-binding protein, LDB1, expression in squamous cell carcinomas of the oral cavity. Br J Cancer. 2003, 88: 1543-1548. 10.1038/sj.bjc.6600952. Lee JM, Dedhar S, Kalluri R, Thompson EW: The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol. 2006, 172: 973-981. 10.1083/jcb.200601018. Christiansen JJ, Rajasekaran AK: Reassessing epithelial to mesenchymal transition as a prerequisite for carcinoma invasion and metastasis. Cancer Res. 2006, 66: 8319-8326. 10.1158/0008-5472.CAN-06-0410. Testa JR, Bellacosa A: AKT plays a central role in tumorigenesis. Proc Natl Acad Sci USA. 2001, 98: 10983-10985. 10.1073/pnas.211430998. Nakayama H, Ikebe T, Beppu M, Shirasuna K: High expression levels of NFκB, IκBα and Akt kinase in squamous cell carcinoma of the oral cavity. Cancer. 2001, 92: 3037-3044. 10.1002/1097-0142(20011215)92:12<3037::AID-CNCR10171>3.0.CO;2-#. Sun M, Wang G, Paciga JE, Feldman RI, Yuan ZQ, Ma XL, Shelley SA, Jove R, Tsichlis PN, Nicosia SV, et al: AKT1/PKBα kinase is frequently elevated in human cancers and its constitutive activation is required for oncogenic transformation in NIH3T3 cells. Am J Pathol. 2001, 159: 431-437. Brognard J, Clark AS, Ni Y, PDennis PA: Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation. Cancer Res. 2001, 61: 3986-3997. Hynes NE, Lane HA: ERBB receptors and cancer: the complexity of targeted inhibitors. Nat Rev Cancer. 2005, 5: 341-354. 10.1038/nrc1609. Yamamoto K, Altschuler D, Wood E, Horlick K, Jacobs S, Lapetina EG: Association of phosphorylated insulin-like growth factor-I receptor with the SH2 domains of phosphorylated 3-kinase p85. J Biol Chem. 1992, 267: 11337-11343. Woodgett JR: Recent advances in the protein kinase B signaling pathway. Curr Opin Cell Biol. 2005, 17: 150-157. 10.1016/j.ceb.2005.02.010. Castillo SS, Brognard J, Petukhov PA, Zhang C, Tsurutani J, Granville CA, Li M, Jung M, West KA, Gills JG, et al: Preferential inhibition of Akt and killing of Akt-dependent cancer cells by rationally designed phosphatidylinositol ether lipid analogues. Cancer Res. 2004, 8: 2782-2792. 10.1158/0008-5472.CAN-03-1530. Wells A, Yates C, Shepard CR: E-cadherin as an indicator of mesenchymal to epithelial reverting transitions during the metastatic seeding of disseminated carcinomas. Clin Exp Metastasis. 2008, 25: 621-8. 10.1007/s10585-008-9167-1. Baum B, Settleman J, Quinlan MP: Transitions between epithelial and mesenchymal states in development and disease. Semin Cell Dev Biol. 2008, 19: 294-308. 10.1016/j.semcdb.2008.02.001. Lee G, Kim YB, Kim JH, Kim MS, Shin KH, Won YS, Lee JI, Choung PH, Hyun BH, Min BM: Characterization of novel cell lines established from three human oral squamous cell carcinomas. Int J Oncol. 2002, 20: 1151-1159. Hong JS, Pai HK, Hong KO, Kim MA, Kim JH, Lee JI, Hong SP, Hong SD: CXCR-4 knockdown by small interfering RNA inhibits cell proliferation and invasion of oral squamous cell carcinoma cells. J Oral Pathol Med. 2009, 38: 214-219. Herman JG, Graff JR, Myohanen S, Kelkin BB, Baylin SB: Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci. 1996, 93: 9821-9826. 10.1073/pnas.93.18.9821. Lee JK, Kim MJ, Hong SP, Hong SD: Inactivation patterns of p16/INK4A in oral squamous cell carcinomas. Exp Mol Med. 2004, 36: 165-71. Graff JR, Herman JG, Lapidus RG, Chopra H, Xu R, Jarrard DF, Isaacs WB, Pitha PM, Davidson NE, Baylin SB: E-cadherin expression is silenced by DNA hypermethylation in human breast and prostate carcinomas. Cancer Res. 1995, 55: 5195-5199. Morali OG, Delmas V, Moore R, Jeanney C, Thiery JP, Larue L: IGF-II induces rapid β-catenin relocation to the nucleus during epithelium to mesenchyme transition. Oncogene. 2001, 20: 4942-4950. 10.1038/sj.onc.1204660. Irie HY, Pearline RV, Grueneberg D, Hsia M, Ravichandron P, Kothari N, Natesan S, Brugge JS: Distinct roles of Akt1 and Akt2 in regulating cell migration and epithelial-mesenchymal transition. J Cell Biol. 2005, 171: 1023-1034. 10.1083/jcb.200505087. Boyer B, Valles AM, Edme N: Induction and regulation of epithelial-mesenchymal transitions. Biochem Phamacol. 2000, 60: 1091-1099. 10.1016/S0006-2952(00)00427-5. Lim J, Kim JH, Paeng JY, Kim MJ, Hong SD, Lee JI, Hong SP: Prognostic value of activated Akt expression in oral squamous cell carcinoma. J Clin Pathol. 2005, 58: 1199-1205. 10.1136/jcp.2004.024786. Grille SJ, Bellacosa A, Upson J, Klein-Szanto AJ, van Roy F, Lee-Kwon W, Donowitz M, Tsichlis PN, Larue L: The protein kinase Akt induces epithelial-mesenchymal transition and promotes enhanced motility and invasiveness of squamous cell carcinoma lines. Cancer Res. 2003, 63: 2172-2178. Peinado H, Portillo F, Cano A: Transcriptional regulation of cadherins during development and carcinogenesis. Int J Dev Biol. 2004, 48: 365-375. 10.1387/ijdb.041794hp. Nieto MA: The snail superfamily of zinc-finger transcription factors. Nat Rev Mol Cell Biol. 2002, 3: 155-166. 10.1038/nrm757. Karreth F, Tuveson DA: Twist induces an epithelial-mesenchymal transition to facilitate tumor metastasis. Cancer Biol Ther. 2004, 3: 1058-1059. Eger A, Aigner K, Sonderegger S, Dampier B, Oehler S, Schreiber M, Berx G, Cano A, Beug H, Foisner R: EF1 is a transcriptional repressor of E-cadherin and regulates epithelial plasticity in breast cancer cells. Oncogene. 2005, 24: 2375-2385. 10.1038/sj.onc.1208429. Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C, Savagner P, Gitelman I, Richardson A, Weinberg RA: Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell. 2004, 117: 927-939. 10.1016/j.cell.2004.06.006. Rosivatz E, Becker I, Specht K, Fricke E, Luber B, Busch R, Höfler H, Becker KF: Differential expression of the epithelial-mesenchymal transition regulators snail, SIP1, and twist in gastric cancer. Am J Pathol. 2002, 161: 1881-1891. Cano A, Perez-Moreno MA, Rodrigo I, Locascio A, Blanco MJ, del Barrio MG, Portillo F, Nieto MA: The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol. 2000, 2: 76-83. 10.1038/35000025. Batlle E, Sancho E, Franci C, Domínguez D, Monfar M, Baulida J, García De Herreros A: The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol. 2000, 2: 84-89. 10.1038/35000034. Takkunen M, Grenman R, Hukkanen M, Korhonen M, Garcia de Herreros A, Virtanen I: Snail-dependent and -independent epithelial-mesenchymal transition in oral squamous carcinoma cells. J Histochem Cytochem. 2006, 54: 1263-1275. 10.1369/jhc.6A6958.2006. Kang Y, Massague J: Epithelial-mesenchymal transitions: twist in development and metastasis. Cell. 2004, 118: 277-279. 10.1016/j.cell.2004.07.011. Larue L, Bellacosa A: Epithelial-mesenchymal transition in development and cancer: role of phosphatidylinositol 3' kinase/AKT pathways. Oncogene. 2005, 24: 7443-7454. 10.1038/sj.onc.1209091. Chua HL, Bhat-Nakshatri P, Clare SE, Morimiya A, Badve S, Nakshatri H: NF-kappaB represses E-cadherin expression and enhances epithelial to mesenchymal transition of mammary epithelial cells: potential involvement of ZEB-1 and ZEB-2. Oncogene. 2007, 26: 711-724. 10.1038/sj.onc.1209808. Julien S, Puig I, Caretti E, Bonaventure J, Nelles L, van Roy F, Dargemont C, de Herreros AG, Bellacosa A, Larue L: Activation of NF-kappaB by Akt upregulates Snail expression and induces epithelium mesenchyme transition. Oncogene. 2007, 26: 7445-7456. 10.1038/sj.onc.1210546. Huber MA, Azoitei N, Baumann B, Grünert S, Sommer A, Pehamberger H, Kraut N, Beug H, Wirth T: NF-κB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression. J Clin Invest. 2004, 114: 569-581.