pathways involved in activation and repression of EMT have also been reported [22]. Several tyrosine kinase receptors, including mesenchymal-epithelial transition (MET), fibroblast growth factor, insulin growth factor, epithelial growth factor family members, and, more recently, platelet derived growth factor, also play critical roles in regulation of EMT-like morphogenetic events that occur during development [23]. The three members of the Snail family, known as Snail (Snail1), Slug (Snail2), and Smuc (Snail3), encode zinc-finger type transcription factors [18]. The carboxyl-terminal domain, containing four to six C2H2-type zinc fingers, is the most conserved feature of the Snail family of proteins [18]. Evidence has indicated a fundamental role of Snail in EMT through its suppression of E-cadherin [24]. Up-regulation and nuclear accumulation of Snail have shown correlation with the EMT program in tumor tissues [25]. In addition, Kudo-Saito et al. [26] reported on acceleration of cancer metastasis by Snail-induced EMT through enhanced invasion. The transcriptional repressor zinc-finger E-box binding homeobox 1 (ZEB1) is a crucial inducer of EMT in various human tumours, and was recently shown to promote invasion and metastasis of tumour cells [27]. ZEB1 promotes EMT through repression of genes such as E-cadherin, which is involved in maintenance of the epithelial phenotype and activation of those required for transformation to the mesenchymal phenotype [28]. Repression of multiple key determinants of epithelial differentiation and cell-cell adhesion, including the cell polarity genes Crumbs3, HUGL2, and Pals1-associated tight junction protein, by ZEB1 has also been demonstrated [29]. Results of reporter assays showed an association of ZEB1 with endogenous promoters in vivo, as well as strong repression of promoter activities [29]. Early evidence from studies conducted using mouse models, as well as a wide range of primary human carcinomas, demonstrated induction of an EMT by ZEB proteins, linking their expression with increased aggressiveness and metastasis [30]. The increase in expression of Bmi-1 was accompanied by down- regulation of E-cadherin expression and up-regulation of vimentin expression in lung squamous cell carcinoma tissues [31]. Over-expression of Bmi-1 has been reported to increase the motility and invasive properties of immortalized human mammary epithelial cells (HMLEs), which is concurrent with increased expression of mesenchymal markers, decreased expression of epithelial markers, stabilization of Snail, and dysregulation of theAkt/GSK3b pathway [32]. Silencing of endogenous Bmi-1 expression has been shown to result in reversal of EMT and reduction of motility [33]. Some data have suggested that Bmi-1 expression may be regulated by Twist1 [34]. 2. Internal stimuli The prominent idea is that, although some factors, such as transforming growth factor- (TGF-), could possibly trigger EMT, accumulation of particular gene mutations would be required in order to unlock or maintain the EMT program. Indeed, gene alterations concerning extracellular receptors and/or consequent signal transduction are common in cancer [20]. Pirozzi et al. [35] reported that treatment with TGF-1 induced Fariz Nurwidya, EMT in Lung Cancer morphologic changes in NSCLC cell lines, characterized by loss of epithelial morphology assuming a fibroblast-like appearance. Treatment with TGF-1 has also been shown to promote a shift from epithelial to mesenchymal phenotype [35]. Induced autocrine expression of TGF- resulted in changes in markers and phenotype that were consistent with EMT, a modest effect on growth rate, and a shift to a more invasive phenotype [36]. In addition, several other mechanisms having the capacity to suppress E-cadherin expression, including somatic mutations, promoter hypermethylation, and histone deacetylation, have also been described [30]. R o l e o f E M T i n R e s i s t a n c e A c q u i r e d D r u g EMT may well play a role in determining sensitivity to EGFR-TKI. Thomson et al. [37] reported that human NSCLC lines containing wild-type EGFR, grown both in culture and as xenografts, display a range of sensi- tivities to EGFR inhibition, which is dependent on the degree to which they have undergone EMT. NSCLC lines expressing the epithelial cell junction protein E-cadherin showed greater sensitivity to EGFR inhibition in vitro and in xenografts. By contrast, NSCLC lines that had undergone EMT, expressing vimentin and/or fibronectin, were insensitive to the growth inhibitory effects of EGFR kinase inhibition in vitro and in xenografts. In another study by Xie et al. [38], highly up-regulated expression of Notch-1 was observed in gefitinib-resistant PC9/AB2 lung cancer cells. Promotion of the EMT phenotype by the Notch-1 receptor intracellular domain (N1IC), the activated form of the Notch-1 receptor, was observed in PC9 cells. Silencing of Notch-1 using siRNAresulted in reversal of the EMT phenotype and restored sensitivity to gefitinib in PC9/AB2 cells. In addition, involvement of Notch-1 reduction in inhibition of anoikis, as well as colony-formation activity of PC9/AB2 cells, was also observed. Taken together, these results provide strong molecular evidence that gefitinib-acquired resistance in lung cancer cells undergoing EMT occurs through activation of Notch-1 signaling. Using cancer specimens, Uramoto et al. [39] investigated the association between EMT status and acquired resistance. Immunohistochemical staining was performed for analysis of protein expression of epithelial and mesenchymal markers in tumour samples from patients with lung adenocarcinoma. All patients showed positive expression of epithelial markers in sensitive tumours. Findings from the study suggest that epithelial markers tended toward a slight decrease in resistance, compared with sensitive tumours. By contrast, mesenchymal markers tended toward a slight increase; however, the differences were not statistically significant [39]. However, according to findings from another study, continuous exposure of A549, a lung adenocarcinoma cell line, to gefitinib also resulted in induction of EMT [40]. Rho et al. [40] reported on use of a gefitinib- resistant subline (A549/GR) derived from the parental A549 cell line VOLUME 44 NUMBER 3 SEPTEMBER 2012 153