Keywords

1 Epithelial Mesenchymal Transition in Cancer: Overview

Epithelial–mesenchymal transition (EMT), an evolutionarily conserved process, is essential for embryonic development, gastrulation, neural crest formation, and organ development [1]. EMT has been established as an important step in tissue repair, organ fibrosis, and cancer progression [2–4]. EMT is a dynamic and reversible process, during which epithelial cells transition from polarized, cobblestone-like cells to migratory, spindle-shaped mesenchymal cells. In addition to morphological changes , cells undergoing EMT also exhibit changes at the molecular level by losing expression of epithelial markers such as E-cadherin, ZO-1 and occludin, and gaining expression of mesenchymal markers including N-cadherin, vimentin, and fibronectin . Several signaling pathways regulate EMT including TGFs, BMPs, FGF, EGF, HGF, Wnt/beta-catenin and Notch, in which both transcriptional and post-transcriptional processes are involved [5, 6].

The similarities between transcriptional and epigenetic regulatory pathways in developmental and pathological EMTs suggest that the developmental EMT program is hijacked during tumor invasion and metastasis [3, 7–10]. To identify key EMT molecular pathways that govern the metastatic process, many studies have focused on cell-based experimental models. These studies have shown that EMT confers tumor cells with invasive and metastatic abilities, resistance to therapies, as well as cancer stem cell (CSC) phenotypes that have a major impact on cancer progression [11]. Consistent with the demonstration that EMT activators such as Twist, can induce EMT and breast CSC phenotypes [12, 13], enrichment of CSC/EMT signatures in residual tumors remaining after neoadjuvant chemotherapy was demonstrated [14]. However, a recent study showed that the homeobox factor “paired-related homeobox transcription factor 1” (Prrx1) is an EMT inducer conferring migratory and invasive properties. However, in contrast to other EMT-activators, Prrx1 suppresses CSC phenotypes [15]. This study suggests that unlike the classical EMT transcription factors, Prrx1 contributes to metastasis by uncoupling stemness from EMT.

While cancer cell intrinsic EMT signaling pathways have been well elucidated, the contribution of the tumor microenvironment (TME) in providing EMT activating signals to the cancer cells have only recently been investigated [16, 17]. Several paracrine and autocrine signals trigger induction of EMT resulting in mesenchymal and CSC states in cancer [18–20]. Following EMT, the disseminated mesenchymal cells undergo mesenchymal to epithelial transition (MET) at the site of metastasis [1, 21, 22].

The clinical relevance of EMT has been an area of long standing controversy, mainly due to the lack of evidence of EMT in clinical carcinomas and metastasis [23–26]. More recent efforts have been directed towards demonstrating EMT directly in vivo in mice and humans, and until now the direct role of EMT in vivo has remained elusive. In this chapter, we will focus on EMT in cancer progression, with emphasis on lung cancer, and discuss opportunities for novel anti-EMT therapeutic approaches.

2 Epithelial Mesenchymal Transition in Physiological Processes and Cancer

Three types of EMT have been proposed [27]. Type 1 EMT describes the transition of cells into the mesenchyme during embryogenesis and organ development, and does not involve pathological events [1]. Type 2 EMT is important for wound healing, tissue repair and organ fibrosis, where inflammatory cells produce EMT-inducing factors including TGFβ, PDGF, FGF and Matrix metalloproteinases (MMPs), which induce EMT in normal epithelial cells leading to extensive organ fibrosis [27, 28]. Type 3 EMT is associated with cancer progression and metastases [3, 8, 9]. In addition, following primary EMT, mesenchymal cells are capable of reversing back to epithelial phenotypes through mesenchymal to epithelial transition (MET), which is critical for organ formation including kidney organogenesis and somitogenesis [3, 29]. In cancer, histological analysis has revealed morphological similarities between primary tumors and their metastatic lesions [29], and it has been reported that E-cadherin levels are elevated in lymph node metastases relative to matched primary tumor samples. These data suggest that EMT in primary tumors may be followed by MET at distant metastatic sites [30, 31]. Consistent with these correlative clinical findings, recent studies have demonstrated that re-differentiation of disseminated tumor cells in the metastatic site through MET is critical for colonization [21, 24, 32]. The involvement of EMT in cancer progression is widely recognized; however, the potential role of MET is unclear, and constitutes an area of intense investigation.

3 Epithelial Mesenchymal Transition in Primary Tumor and Metastatic Dissemination

Since the first description of EMT in cancer progression, EMT has been inherently related to metastasis [27, 33]. Accumulating evidence from in vitro experiments have shown that EMT represents a major mechanism for tumor cells to acquire critical metastatic features including enhanced mobility, invasion, and resistance to apoptotic stimuli. Furthermore, as a result of EMT, tumor cells acquire chemo-resistance and exhibit increased potential for initiating secondary tumors [34]. More importantly, EMT has also been implicated in conferring CSC properties [12, 35], a rare subpopulation of cancer cells with capacity of self-renewal, regeneration and differentiation into diverse types of cancer cells.

With the identification of a mesenchymal phenotype in the highly malignant breast CSCs, research focus has recently progressed towards understanding the role of EMT in metastasis in vivo. Using intravital imaging approaches, it was shown that single breast cancer cells gained mobility for hematogenous metastasis by activating EMT-promoting TGFβ-Smad2/3 signaling [36]. Indeed, EMT was also observed during metastasis in spontaneous tumor models in mice, where disseminated tumor cells in the lungs of MMTV-PyMT transgenic mice expressed a mesenchymal marker, FSP1, suggesting involvement of EMT in tumor dissemination [37]. Using a squamous cell carcinoma mouse model, activation of EMT-inducing transcription factor Twist was sufficient to promote carcinoma cells to undergo EMT and disseminate into blood circulation [38]. However, at the distant sites, turning off Twist1 to allow reversion of EMT was essential for disseminated tumor cells to proliferate and form overt metastases. Direct evidence of EMT has also been shown in a K-Ras mediated spontaneous pancreatic tumor model, which develops liver metastases [39]. Remarkably, EMT-positive cells were found in primary lesions, in the circulation, and as single cell deposits in the liver at a very early stage of primary tumor development, even before malignancy could be detected by rigorous histologic analysis. These post-EMT tumor cells gained expression of typical mesenchymal markers including fibronectin, Zeb1 and FSP1 and lost expression of E-cadherin. Importantly, the post-EMT tumor cells represent the majority of metastatic tumor cells that seeded the metastatic liver. However, more rigorous lineage tracing approaches are being developed to actually demonstrate the process of EMT in vivo. For example, using an EMT-lineage tracing strategy of mesenchymal specific (FSP1) Cre mediated β-galactosidase activity, Trimboli et al. compared the incidence of EMT events in three different oncogene-driven breast tumor models [40]. Significantly, post-EMT tumor cells were detected in the Myc-driven tumors, but not in the PyMT- or Neu-driven tumors. Notably, lung metastases were formed in almost all MMTV-PyMT and MMTV-neu mice, but not in MMTV-myc animals, suggesting that the contribution of EMT in metastasis may be tumor type specific. It is also possible that the β-galactosidase activity was not sensitive enough to monitor the relatively rare EMT events, and that better EMT-lineage tracing systems are required to clarify the biological contributions of post-EMT tumor cells in metastasis.

4 Epithelial Mesenchymal Transition in Lung Cancer

Lung cancer is a global public health problem with an estimated 1.3 million new cases each year [41]. In the United States, approximately 226,160 new cases of lung cancer are diagnosed per year with over 160,000 deaths. Despite advances in treatment options, including minimally invasive surgical resection, stereotactic radiation, and novel chemotherapeutic regimens, the 5-year survival rate in NSCLC remains only at approximately 15 %. Available targeted therapies such as EGFR tyrosine kinase inhibitors (TKIs, erlotinib and gefitinib) and EML4-ALK inhibitor (crizotinib) benefit only 15–20 % of NSCLC patients who carry specific drug-sensitive mutations. Even in these patients, acquired resistance is a major impediment to a durable therapeutic response [42–44]. Notably, EMT has been implicated in mediating resistance to therapy in lung cancer. A growing body of evidence supports the role of EMT in the progression of many cancers [2], and transcriptional factors and microRNAs involved in the EMT process have been identified in a number of signaling pathways. However, the role of EMT in lung cancer has not been extensively characterized.

4.1 Epithelial Mesenchymal Transition and Prognosis in Lung Cancer

Several studies have suggested an association between EMT factors including E-cadherin, hypoxia inducible factor 1α (HIF-1α), twist, snail and poor prognosis in lung cancer [45]. Notably, expression of Twist, Slug, and Foxc2 was an independent predictor of recurrence-free and overall survival in stage I NSCLC [46]. Analysis of archived tissue from primary human lung tumors, brain metastases and adjacent bronchial epithelial specimens showed high expression of EMT associated markers in progressing primary lung cancer specimens, particularly in squamous cell carcinoma [47]. Compared to primary NSCLC, brain metastases showed decreased EMT phenotype expression, consistent with the notion that disseminated tumor cells undergo MET at the site of metastasis [1, 21]. It was suggested that overexpression of Forkhead box M1 (FOXM1) , a member of the Fox family of transcriptional factors, may have prognostic value for patients with NSCLC, and FOXM1 was shown to promote metastasis by inducing EMT through activation of the AKT/p70S6K pathway [48].

In NSCLC, invasive tumor growth is accompanied by desmoplastic stroma reaction and concomitant upregulation of EMT markers at the invasive front [49]. Previously, an analysis of surgically resected 533 NSCLC specimens by immunohistochemistry showed that EMT proteins periostin, versican and elastin confer prognostic value [50, 51]. Clinically relevant EMT biomarkers with significant prognostic value in lung adenocarcinoma were identified recently [52]. In this study, analysis of the secretome from a TGF-β induced model of EMT by mass spectrometry unraveled a 97-gene EMT signature with positive correlations to lymph node metastasis, advanced tumor stage and histological grade. Moreover, a refined 20-gene signature predicted survival of both adenocarcinoma and squamous carcinoma patients. Increased expression of BRF2, a RNA polymerase II transcription factor was significantly associated with the poor prognosis of NSCLC patients by virtue of promoting EMT [53]. In another study, downregulation of BRAF activated non-coding RNA promoted EMT, which was associated with poor prognosis in NSCLC [54]. Importantly, in some studies, survival data related to the EMT profile is lacking.

4.2 Epithelial Mesenchymal Transition and Lung Cancer Progression

The association of EMT and cancer progression has been shown in several types of cancer, including breast cancer, prostate cancer, pancreatic cancer and hepatocellular carcinoma. However, the role of EMT in lung cancer has not been extensively studied, and the role of EMT in the pathogenesis of several lung disorders is currently intensely debated. More recently, a number of signaling pathways and biomarkers have been implicated in EMT-induced lung cancer progression (Fig. 1).

Fig. 1
figure 1

Schema depicting potential EMT pathways in lung cancer

Full size image

EMT is orchestrated by several signaling pathways, including TGF-β/Smad and IL-6/JAK/STAT3 (signal transducer and activator of transcription 3) signaling. The JAK/STAT3 pathway was required for TGF-β-induced EMT and cancer cell migration and invasion via upregulation of p-Smad3 and Snail, and the IL-6/JAK/STAT3 and TGF-β/Smad signaling synergistically enhanced EMT in lung carcinomas [55]. In another study, activation of peroxisome proliferator-activated receptor-gamma (PPAR-γ) inhibited TGF-β-induced EMT in lung cancer cells and prevented metastasis by antagonizing Smad3 function [56]. TGF-β1-induced EMT in lung cancer cells resulted in the acquisition of a mesenchymal profile associated with elevated levels of stem cell markers [57–59]. In a related study, TGF-β1-induced EMT in lung cancer cells upregulated Neuropilin (NRP)-2, the high-affinity receptor for SEMA3F [60]. Notably, NRP2 blocked invasive potential of tumor xenografts and reversed TGF-β1-mediated growth inhibition. In NSCLC, Snail was shown to regulate Nanog during EMT via the Smad1/Akt/GSK3β signaling pathway [61].

Notch-1 signaling is critical in lung development and disease [62, 63], and has been shown to promote EMT [64]. It has been demonstrated that blocking Notch-1 signaling by Hey-1 or Jagged1 knockdown or a γ-secretase inhibitor (GSI) attenuates EMT [65]. Radiation-induced Notch-1 overexpression promoted survival and EMT in NSCLC via miR-34a [66]. In this context, induction of miR-34a decreased the expression of Notch-1 and its downstream targets including Hes-1, Cyclin D1, Survivin and Bcl-2 and blocked proliferation and invasion in NSCLC cells [67]. Analysis of the Kras (G12D)-driven NSCLC mouse model showed that conditional Notch1 and Notch2 receptor deletion revealed opposing roles in NSCLC progression [68]. In another study, transcriptional factors Notch2 and Six1 induced EMT and conferred malignant phenotypes to lung adenocarcinomas [69].

MicroRNAs have been shown to contribute to EMT in NSCLC. miR-132 suppressed the migration and invasion of NSCLC cells through targeting ZEB2 [70]. Expression of miR-149, downregulated in lung cancer, was inversely correlated with invasive and EMT phenotypes in NSCLC cells [71]. miR-149 targeted Forkhead box M1 (FOXM1) , and FOXM1 was involved in the EMT induced by TGF-β1. miR-200s have recently been shown to inhibit EMT and promote MET by direct targeting of E-cadherin transcriptional repressors ZEB1 and ZEB2 [72–75]. The observation that the miR-200 family enforces the epithelial phenotype and inhibits EMT and invasion in vitro suggests that these miRNAs are likely to suppress metastasis. Recently, it was shown that, while re-differentiation induced by expression of miR-200 is required for metastatic colonization in a lung tumor xenograft model, miR-200 also directly targets SEC23A, which stimulates the secretion of metastasis-suppressive proteins [32]. Interestingly, cancer cells established from a mouse model of lung adenocarcinoma, driven by oncogenic K-Ras and loss of function p53 mutations, display epithelial plasticity [76], and undergo EMT following TGF-β exposure, which is dependent on downregulation of mir-200 with concomitant stabilization of ZEB1 expression . Ceppi and colleagues have shown that miR-200c expression induces an aggressive, invasive, and chemoresistant phenotype, and that lower mir-200c levels were associated with poor grade of differentiation and higher metastatic potential in NSCLC patients [77]. In another study, immortalized human bronchial epithelial cells (HBECs) exposed to tobacco carcinogens exhibited EMT and stem-like features associated with miR-200 and miR-205. Notably, EMT was driven both by chromatin remodeling and promoter DNA methylation [78]. Some studies have reported conflicting roles of miR-200s in metastatic progression [76, 79, 80], possibly invalidating the therapeutic utility of miR-200s. Furthermore, it remains unclear whether metastasis-related functions of the miR-200s are mediated entirely or only partially through the ZEB–E-cadherin axis.

Osteopontin (OPN) , a prognostic marker in NSCLC [81, 82], through integrin αVβ3, activated the FAK, PI3K, Akt, ERK and NF-kB pathways, contributing to the migration of lung cancer cells [83]. Similarly, a role of pituitary tumor transforming gene (PTTG) , in regulating EMT by inducing expression of integrin αVβ3 and adhesion-complex proteins (FAK) in lung cancer cells was shown [84]. Zyxin was identified as a novel functional target and effector of TGF-β/Smad3 signaling that regulates lung cancer cell motility and EMT via Integrin α5β1 [85].

Inflammation is an important contributor of lung carcinogenesis. The inflammatory component of the TME has been shown to stimulate EMT in lung cancer by contributing to hypoxia, angiogenesis and differential regulation of miRNAs [86, 87]. Paracrine and autocrine contribution of signaling molecules in inducing EMT in lung cancer has been documented. For example, a role for IL-27 in regulating EMT and angiogenesis through modulation of the STAT pathways in human NSCLC was demonstrated [88]. Similarly, COX-2-dependent pathways via modulation of transcriptional repressors of E-cadherin, ZEB1 and Snail regulated EMT in NSCLC [89].

Although association between cigarette smoking and lung cancer is well documented, the molecular mechanisms underlying cigarette smoke-induced EMT processes that are critical for the progression and metastasis of lung cancer are not well understood. Cigarette smoking was shown to induce the repression of E-cadherin via transcription factors LEF-1 and Slug-mediated recruitment of histone deacetylase, HDAC [90]. In another study, cigarette smoke induced EMT through Rac1/Smad2 and Rac1/PI3K/Akt signaling pathways in pulmonary epithelial cells [91].

MMPs that degrade components of the extracellular matrix have been shown to induce EMT. MMP-3, MMP-7, and MMP-28 induce EMT in human A549 lung adenocarcinoma cells [92–94]. Recently MMP-induced upregulation of Rac1b contributed to EMT in a transgenic mice model of lung cancer [95].

4.3 Epithelial Mesenchymal Transition and Drug Resistance in Lung Cancer

Drug resistance constitutes a major challenge for the successful treatment of cancer patients. Cancer therapy is often associated with two major forms of drug resistance—de novo or acquired. Patients who are initially refractory to therapy display intrinsic or “de novo” drug resistance. Patients that initially respond to therapy typically relapse as a consequence of “acquired” drug resistance. EMT has been associated with resistance to chemotherapy, EGFR inhibitors, and other targeted drugs in cancers of the lung [96–98], bladder [99], head and neck [100], pancreas [101], and breast [102]. Intriguingly, EMT can trigger reversion to a CSC-like phenotype [12, 35], providing an association between EMT, CSCs and drug resistance.

In NSCLC , despite the initial response, patients with EGFR-mutant NSCLC eventually develop acquired resistance to EGFR TKIs. The EGFR-T790M secondary mutation is responsible for approximately half of acquired resistance cases, while MET amplification has been associated with acquired resistance in about 5–15 % of NSCLCs [43, 103]. Accumulating evidence suggests that reversible epigenetic changes that emerge during acquired drug resistance reflect changes in the differentiation state of the tumor, which is likely to reflect EMT and the emergence of chemoresistant cells with stem cell-like features [104, 105]. Notably, gefitinib inhibited invasive phenotype and EMT in drug-resistant NSCLC cells with MET amplification [106].

Overcoming de novo and acquired resistance to drug therapy remains a challenge in the clinical management of NSCLC, and approaches to reverse or inhibit EMT as a strategy for drug sensitization are being considered. For example, Buonato and colleagues showed that ERK 1/2 signaling maintained a mesenchymal phenotype in NSCLC cells, and prolonged exposure to MEK or ERK inhibitors restored epithelial phenotypes and overcome resistance to EGFR-targeted therapy [107]. Consistent with these observations, simultaneous EGFR and MEK inhibition are being considered in gastric cancer [108] and pancreatic cancer cells [109], and current clinical trials are evaluating erlotinib combined with MEK inhibitors in NSCLC.

In an attempt to explain resistance to EGFR TKIs, Sordella and colleagues have uncovered the existence of a subpopulation of lung cancer cells that are intrinsically resistant to erlotinib and display EMT phenotypes. These cells by virtue of secreting elevated amounts of TGF-β and IL-6 resisted Tarceva treatment independently of the EGFR pathway [110]. In a previous study, lung adenocarcinomas harboring EGFR mutations were shown to exhibit upregulated IL-6 which activated the gp130/JAK/STAT3 pathway [111]. In this context, Varmus and colleagues showed that inducible expression of EGFR kinase domain–activating mutations targeted to the lung epithelium gave rise to adenocarcinomas containing pSTAT3 and pAKT, demonstrating an association between this oncogene and activated STAT3 [112]. Interestingly, metformin that suppress the IL-6/STAT3 pathway mediated EMT, and sensitized EGFR-TKI-resistant human lung cancer cells to erlotinib or gefitinib [113]. In another study, the expression of Ras-related nuclear protein (Ran) GTPase was elevated in invasive NSCLC. Ran induced EMT and enhanced invasion in NSCLC cells through the activation of PI3K-AKT signaling [114].

In EGFR-TKI resistant lung cancer, activated Notch-1 was found to promote EMT associated with increased Snail and Vimentin expression, suggesting that gefitinib resistance was secondary to Notch-activated EMT [115]. Consistent with this observation, cisplatin was shown to induce the enrichment of multidrug resistant CD133+ CSCs by the activation of Notch signaling [116]. Consistent with this observation, Notch pathway activity identified cells with CSC-like properties and correlated with worse survival in human lung adenocarcinoma [117]. High Notch activity has also been shown to induce radiation resistance in NSCLC [118]. The Hedgehog (Hh) pathway is implicated in lung squamous cell carcinomas (SCC). Notably, activated Hh signaling was shown to regulate metastasis through EMT, and the Shh/Gli pathway was implicated in SCC recurrence, metastasis and resistance to chemotherapy [119]. In NSCLC, TGF-β1-mediated upregulation of shh induced EMT in NSCLC cells [120], and conferred resistance to EGFR-TKIs [121]. Importantly, both genetic and pharmacological inhibition of the Hh pathway reversed the EMT phenotype and improved the therapeutic efficacy of EGFR-TKIs [121].

The miR-134/487b/655 cluster was shown to regulate TGF-β1-induced EMT and induced resistance to gefitinib by targeting MAGI2 (membrane-associated guanylate kinase, WW, and PDZ domain-containing protein 2) in which suppression subsequently caused loss of PTEN stability in lung cancer cells [122].

Platinum-based chemotherapy is the standard first-line approach for the treatment of NSCLC, but recurrence occurs in most patients [123]. Novel combination of chemotherapeutic agents have enhanced the overall median survival of NSCLC patients [124]. However, chemoresistance of tumor cells continues to be a challenge in the management of NSCLCs. Tumor cells often show initial sensitivity to chemotherapeutic drugs, but acquired resistance develops during the treatment, leading to tumor recurrence and further tumor progression. Analysis of cisplatin resistant lung cancer cells showed acquisition of the EMT phenotype, decreased connexin43 (Cx43) expression, and increased capability of invasion and migration [125]. In a related study, resistance of lung cancer cells to docetaxel was associated with EMT, and inhibition of ZEB1 reversed EMT and chemoresistance [126]. Integrinβ1 induced EGFR TKI resistance in NSCLC tumors was associated with an EMT phenotype. [127].

5 Therapeutic Potential of Targeting Epithelial Mesenchymal Transition in Lung Cancer

In lung cancer, EMT has been associated with key tumorigenic properties including increased invasion, angiogenesis and metastasis. Mechanistic insights on how EMT affects signaling pathways contributing to carcinogenesis is necessary to develop effective therapeutics. A number of signaling pathways including notch, wnt, hedgehog and PI3K-AKT, have been implicated in EMT. Furthermore, a growing body of evidence suggests that epithelial cells are more likely to initially respond to therapy, and that EMT confers acquisition of therapeutic resistance. As such, EMT, CSCs, and drug resistance have been described as an emerging axis of evil in cancer [128]. Targeting EMT has been considered a promising strategy against lung cancer, as it would provide novel translational and clinical studies for the benefit of advanced stage cancer patients with metastatic disease [129]. NSCLCs resistant to EGFR TKIs have been shown to downregulate EGFR and increase expression of platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), and AXL [130].

EMT is currently being investigated as a therapeutic target for overcoming drug resistance in lung cancer. For example, HGF-mediated activation of Met receptor induced EMT conferred an aggressive phenotype and induced chemoresistance in preclinical models. Notably, treatment with Met inhibitor resensitized cells to chemotherapy [131]. These findings have clinical relevance, as human NSCLC specimens expressing mesenchymal markers were associated with Met activation, predicted worse survival, and were upregulated in chemorefractory disease . These results support the rationale for Met inhibitor and chemotherapy-centered clinical trials, and suggest that the selection of SCLC patients based on mesenchymal biomarkers in combination with Met expression may be a superior alternative for clinical trials of Met inhibitors plus chemotherapy. Similarly, in drug-resistant NSCLC cells with MET amplification, gefitinib was shown to inhibit invasive phenotype and EMT [106].

ERK1/2 signaling was shown to maintain a mesenchymal phenotype in NSCLC cells associated with resistance to EGFR-TKIs. Prolonged exposure to MEK or ERK inhibitors restored epithelial phenotypes and overcame resistance of NSCLC to EGFR-targeted therapy [107]. For example, combination treatment with gefitinib and MEK inhibitors was effective in the treatment of gefitinib-resistant lung adenocarcinoma cells harboring EGFR mutations [132]. Indeed, current clinical trials have begun to evaluate erlotinib in combination with MEK inhibitors in NSCLC (NCT01229150). Similarly, the IL-6/STAT3 pathway-mediated EMT is also being exploited in EGFR-TKI-resistant NSCLC. Suppression of this pathway with metformin sensitized resistant lung cancer cells to erlotinib or gefitinib [113]. Metformin also inhibited IL-6-induced EMT and lung adenocarcinoma growth and metastasis [133].

A 76-gene EMT signature was found to predict resistance to EGFR and PI3K/Akt inhibitors, and AXL (a member of the RTK family), was identified as a potential therapeutic target for overcoming EGFR inhibitor resistance associated with the mesenchymal phenotype [134]. In this context, activated phospho-AXL was detected in 59.8 % of adenocarcinoma cases examined and correlated significantly with larger tumor size and with overall survival of the patients [135]. A recent study has shown that EMT rewires the mechanism of PI3K pathway activation -dependent proliferation in NSCLC cells [136, 137]. In epithelial cells, autocrine ERBB3 activation maintained PI3K signaling; however EMT altered the proliferative potential of cells by modulating ERBB3 expression.

The CXCR4/CXCL12 axis contributes to the pathology of NSCLC, and targeting this axis has been considered as a potential therapeutic approach for the treatment of NSCLC [138]. Importantly, elevated CXCR4 levels were observed in NSCLC cells high in self-renewal capacity and increased chemotherapeutic resistance [139]. Inhibition of CXCR4 suppressed the self renewal capacity of NSCLC cells [140], and a previous study had shown that the transcription factor 5T4 via CXCR4 may induce EMT and increase migration of NSCLC [141]. The therapeutic potential of CXCR4/CXCL12 axis is being considered for cancer treatment [142–144]. EMT-induced CSC phenotypes have been implicated in resistance to cisplatin, as cisplatin-treated patients with lung cancer showed enrichment of CD133+ stem cells due to activated Notch signaling, suggesting that blocking Notch signaling may reduce the recurrence of NSCLCs [116]. Similarly, the AKT/β-catenin/Snail signaling pathway has been associated with CSC-like properties and EMT features in NSCLC cells, implying the therapeutic potential of this pathway for the treatment of NSCLC [145].

6 Future Perspectives

Current EMT research efforts are directed towards understanding the interplay of multiple regulatory networks that contribute to the conversion of an epithelial tumor cell to a mesenchymal state resulting in acquisition of various acquired capabilities such as resistance to anoikis, oncogene-induced senescence, and resistance to apoptosis/chemotherapy and CSC properties. Various transcriptional and post-transcriptional processes have been identified; however, the mechanisms by which these pathways are interconnected during cancer progression are not completely understood. A variety of contextual paracrine and autocrine signaling factors that maintain mesenchymal and CSC phenotypes have begun to emerge [20], and recent studies have implicated the contribution of chromatin modification as a mechanism to attain widespread changes in gene expression that accompany the EMT process [9]. In lung cancer, EMT is associated with metastatic progression, resistance to EGFR inhibitors, chemotherapy, and other targeted drugs [96–98]. Acquired resistance to the EGFR inhibitor erlotinib resulted from the selection and expansion of a mesenchymal subpopulation [110], and restoring E-cadherin expression in mesenchymal-like NSCLC cells potentiated sensitivity to EGFR inhibitors [146] suggesting that a treatment approach eliciting a mesenchymal to epithelial transition (MET) may be useful for expanding the efficacy of EGFR inhibitors. In addition, growing evidence for AXL-mediated EGFR inhibitor resistance has been linked to EMT [147]. EMT regulators are being considered as potential molecular biomarkers and therapeutic targets for developing multi-targeted strategies for improving current cancer therapies and preventing disease relapse. For example, TGF-β has been shown to induce EMT in NSCLC [57], and clinical benefits of TGF-β signaling inhibitors is being considered [148]. Consistent with this notion, IN-1130, a novel inhibitor of TGF-β type I receptor, was shown to impair breast cancer lung metastasis through inhibition of EMT [149]. Similarly, Wnt signaling has emerged as a critical pathway in lung carcinogenesis, and Wnt pathway antagonists are being explored in NSCLC [150, 151]. Given that EMT contributes to resistance of EGFR-TKIs, inhibition of EMT constitutes a critical therapeutic strategy for overcoming to EGFR-TKis resistance in lung cancer.

Despite the significant and rapid progress in the EMT field, several issues have still remained unresolved. For example, circulating tumor cell (CTC) number in metastatic cancer patients is being considered as prognostic markers consistent with enhanced cell migration and invasion via loss of adhesion, a feature of EMT. Evidence of prognostic significance of CTC number emerged from a study of resectable NSCLC, demonstrating an association between increased CTC number and shorter disease free survival [152]. A hybrid EMT phenotype of CTCs was also demonstrated in patients with metastatic NSCLC [153]. In light of these studies, multiplex analysis and further detailed exploration of metastatic potential and EMT in CTCs is now warranted in a larger patient cohort.

Finally, the role of EMT in cancer progression has been a topic of debate in the scientific community mainly due to paucity of robust in vivo data demonstrating the importance of EMT in tumorigenesis. Furthermore, the clinical relevance of EMT is often questionable due to the lack of evidence of EMT in clinical carcinomas and metastasis [23, 25] [24, 26]. More recent efforts are directed towards EMT demonstration directly in vivo using lineage tracing approaches and live intravital microscopy imaging. These analyses may establish a more direct role of EMT in vivo during tumorigenesis.