TY - JOUR
AU - Danesi, Romano
AB - Abstract Among the potential mechanisms involved in resistance to tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer, the manifestation of stem-like properties in cancer cells seems to have a crucial role. Alterations involved in the development of TKI resistance may be acquired in a very early phase of tumorigenesis, supporting the hypothesis that these aberrations may be present in cancer stem cells (CSCs). In this regard, the characterization of tumor subclones in the initial phase and the identification of the CSCs may be helpful in planning a specific treatment to target selected biomarkers, suppress tumor growth, and prevent drug resistance. The aim of this review is to elucidate the role of CSCs in the development of resistance to TKIs and its implication for the management of patients. Among the potential mechanisms involved in resistance to tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC), the manifestation of stem-like properties in cancer cells seems to have a crucial role. Mechanisms of resistance may include appearance of stem-like properties by the over-expression of CSC antigens or increased expression of ALDH and ABC transporters (SP+ cells); de-regulation of signal transduction pathways (i.e., Notch, Wnt/β-catenin, and Hedgehog); acquisition of genetic (EGFR and KRAS mutations, ALK translocation) and epigenetic (i.e., miRNA) alterations. In this regard, the characterization of tumor subclones at initial phase and the identification of the CSCs may be more effective in planning a specific treatment targeting specific biomarkers, suppressing tumor growth, and preventing drug resistance. Open in new tabDownload slide Open in new tabDownload slide Cancer stem cells, Treatment resistance, Tyrosine kinase inhibitors, Biomarkers, Tumor heterogeneity Significance Statement Resistance to treatment in non-small cell lung cancer is one of the major obstacles for clinicians. Recently, mutations in different genes have emerged as potential biomarkers of acquired resistance to treatment, however, little is known about the role of cancer stem cells. The present review addresses how cancer stem cells can contribute to treatment resistance and how it can be overcome. Introduction The efficacy of targeted therapies in tumors harboring actionable mutations, such as those occurring in EGFR or ALK genes, is significantly reduced when resistance occurs, and despite the success of EGFR- and ALK-tyrosine kinase inhibitors (TKIs) [1], tumors become refractory to TKIs and the disease progresses after an initial response [2]. The resistance is strongly related to tumor heterogeneity and is often classified as “de novo” resistance, in which cancer cells initially respond to treatment and acquire resistance due to the appearance of new mutations as a result of clonal selection [3]. However, adaptive mechanism of resistance may also occur, in which cells are able to survive to treatment pressure, remaining in a quiescent state. Tumor heterogeneity may also be explained by the cancer stem cell (CSC) models, in which cells with stemness properties maintain the CSC pool, contributing to the generation of cancer stem-like cells (CSLC) [4]. The existence of highly malignant, chemo-resistant CSLC has a crucial role in tumorigenesis, growth, metastasis, resistance to treatments, and risk of relapse [5-7]. Despite the large improvements in terms of survival due to the EGFR- (i.e., gefitinib, erlotinib, and afatinib) and ALK-TKIs (i.e., crizotinib, ceritinib, alectinib, and brigatinib) in EGFR and ALK-positive NSCLC, the acquired resistance seems to be multifactorial, including new mutations and new mechanisms of activation. In this view, the role of CSCs is crucial to inspire the development of new “druggable” targets and new personalized treatments [8]. This review focuses on the role of CSCs in the development of spatial and temporal heterogeneity of NSCLC and how they can mediate resistance to TKIs [9, 10]. Role of Cancer Stem Cells in Tumor Heterogeneity The enhanced knowledge regarding the causes and consequences of tumor heterogeneity reveals a complex etiology of TKI resistance. In this regard, tumor heterogeneity may confer primary and secondary resistance to any targeted therapy [11]. Interestingly, several studies provide insights into the heterogeneity of clonal events, demonstrating that they can be detected in an early phase of tumor development [12]. Moreover, it has been shown that different levels of extracellular receptors, expression of stress-responsive genes, and the presence of both sensitizing and desensitizing mutations to TKIs may characterize different regions of the tumors [13], suggesting the co-existence of TKI-sensitive and TKI-resistant subclones [14], thereby posing a challenge to the current approach of personalized medicine. In addition, some researchers provided evidence of co-existence of both wild-type and mutant EGFR within the same tumor [15-17]. Currently, two hypotheses address the challenge of tumor heterogeneity: (a) the mechanism of mutation induction and (b) the presence of heterogeneous subclonal lineages derived from distinctive CSCs that dynamically maintain tumor plasticity [6, 18, 19]. By conducting deep sequencing analysis of 17 treatment-naïve lung adenocarcinomas, Govindan and colleagues suggested that EGFR mutations may be acquired at a very early phase of tumorigenesis and can be considered a founder mutation of the tumor due to their existence in all subclones. In this regard, they suggested that the characterization of tumor subclones in the initial phase and the identification of CSCs may be more effective in planning a specific treatment, targeting the developmental signaling pathways (i.e., the Notch, hedgehog, and transforming growth factor-beta [TGF-β] pathways), suppressing tumor growth and preventing drug resistance [7, 20]. Since there is co-existence of different CSC subpopulations generating a complex heterogeneous tumor, a specific treatment targeting CSCs has limited value and may be insufficient for cancer eradication because other CSC subpopulations could potentially propagate into the tumor. In recent years, several models of CSC biology have been proposed to explain how tumor heterogeneity develops and contributes to the early stage of tumor formation, disease progression, and drug resistance (Fig. 1) [21]. The most prevalent CSC theory proposes that tumor heterogeneity arises as a result of a hierarchical unidirectional path through which CSCs self-renew and generate more differentiated neoplastic progenitor cells, also called non-CSCs, explaining the existence of different subpopulations with distinct genetic and epigenetic characteristics [22]. In fact, in contrast to normal stem cells, these cells show genetic mutations and other molecular features, such as epigenetic modifications and phenotypic changes that, together with marked deregulation and persistent activation of highly conserved signal transduction pathways, could contribute to heterogeneity and progression of many tumors, including lung cancer [23]. Figure 1 Open in new tabDownload slide Models of CSCs biology explaining tumor heterogeneity. The classical CSCs model described that tumor heterogeneity reflects a unidirectional path through which CSCs self-renew and differentiate into neoplastic progenitor cells (non-CSCs). The stemness phenotype model (SPM) introduces the concept of interconversion between CSCs and non-CSCs depending on the signals of the microenvironment. Critical to classical CSC model is also the notion of plastic CSC model, describing that non-CSCs can undergo a de-differentiation process and re-enter the stem cell/CSC state. At last, in the complex system model both genetic (G) and epigenetic (E) changes occur within a single tumor and can produce different cell niche where several tumor-initiating cell types may co-exist. In addition, cell-cell communication and cell-niche interaction also contribute to produce different tumor cell subpopulations and so, tumor heterogeneity. Abbreviation: CSCs, cancer stem cells. Figure 1 Open in new tabDownload slide Models of CSCs biology explaining tumor heterogeneity. The classical CSCs model described that tumor heterogeneity reflects a unidirectional path through which CSCs self-renew and differentiate into neoplastic progenitor cells (non-CSCs). The stemness phenotype model (SPM) introduces the concept of interconversion between CSCs and non-CSCs depending on the signals of the microenvironment. Critical to classical CSC model is also the notion of plastic CSC model, describing that non-CSCs can undergo a de-differentiation process and re-enter the stem cell/CSC state. At last, in the complex system model both genetic (G) and epigenetic (E) changes occur within a single tumor and can produce different cell niche where several tumor-initiating cell types may co-exist. In addition, cell-cell communication and cell-niche interaction also contribute to produce different tumor cell subpopulations and so, tumor heterogeneity. Abbreviation: CSCs, cancer stem cells. In addition, emerging evidence supports the existence of a contextually regulated equilibrium between CSCs and neoplastic progenitors, including the ability of progenitor cells to undergo de-differentiation to form CSCs. In the stemness phenotype model (SPM) [24], as well as in the dynamic CSC model [25], it has been proposed that CSCs and differentiated cells can interconvert to each other, depending on signals from the tumor microenvironment that are able to drive phenotypic changes without additional mechanisms (i.e., genetic mutations). Furthermore, as described in a more complex system model, while genetic mutations may produce new tumor cell populations and epigenetic changes might enable cells to produce progeny with a more or less restricted fate, cell-cell interaction and cell-niche communication could significantly contribute to the production of different subpopulations of cells characterized by resistance to treatment and the expression of different cell markers [26, 27], considered to be important targets for successful therapy. Although lung cancer is the most common lethal form of cancer, less is known about the biology of lung CSCs than other solid tumor stem cells. The acquisition of genetic and epigenetic alterations by CSCs and the influence of the microenvironment determine the CSC-mediated drug resistance, enhancing tumorigenic and metastatic activities [28]. In fact, CSCs are responsible for resistance to most of the available anti-cancer treatments, including conventional chemotherapy and radiotherapy [29, 30]. Few studies have also examined the effects of the molecular targeted therapies, particularly of the EGFR-TKIs on NSCLC-CSCs [31]. CSCs Markers and Modulation of Signaling Pathways in NSCLC NSCLC-CSCs have specific biological characteristics involved in generation, development, metastasis, and recurrence of tumors. Recent evidence has highlighted that in NSCLC the presence of this small fraction of tumor cells is increased after treatment and is able to regenerate the primary tumor [32]. These cells are characterized by an overexpression of common CSC antigens, such as CD44, CD117, CD133, and CD166, and by an increased expression of aldehyde dehydrogenase (ALDH) and ATP-binding cassette (ABC) transporters (side population [SP] positive cells) (Table 1) [32, 33]. In detail, CD133, also known as prominin-1, is a plasma membrane glycoprotein able to maintain stemness by suppressing differentiation. Drug-resistant cells derived from NSCLC cell lines have been reported to be enriched in CD133pos cells and to display higher levels of cytokines, like angiogenic and growth factors, leading to an increased tumorigenic and metastatic potential of the CSCs [33]. ALDH is a family of intracellular detoxifying enzymes capable of modulating gene expression, cellular detoxification, and differentiation and with a significant role in the development of drug resistance in several tumors, thus representing a prominent marker in lung cancer [34, 35]. Indeed, ALDHhigh cells isolated from NCI-H358 and NCI-H125 lung cancer cell lines were enriched in tumorigenic CD133pos cancer cells [34], and the high expression of ALDH1 protein has been reported to correlate with poor prognosis in NSCLC patients and high resistance to chemotherapeutic agents [34, 47, 48]. Similarly, Huang and colleagues explored the association of ALDH1A1 expression with EGFR-TKI resistance in CSCs, demonstrating that the PC-9 cancer cell line ALDH1A1high was resistant to gefitinib [36]. In addition, results obtained from clinical studies showed that lung cancer cells resistant to gefitinib contained significantly increased proportions of ALDH1A1high CSCs [36]. Treatment with EGFR-TKIs has also been shown to induce overexpression of ALDH, increasing CSCs and epithelial to mesenchymal transition (EMT) markers, including Nanog, Oct-4, Sox-2, c-Met, and β-catenin, and conferring resistance to EGFR-TKIs treatment [49]. A similar induction of ALDH1high CSCs was observed during the development of resistance to the EGFR-TKI erlotinib [50] and in the HCC827-ACR afatinib-resistant cell line [51]. In addition, it is well known that among CSCs there exists a subpopulation of cells, called SP, able to pull out drugs and responsible in part for the resistance to therapy. Molecules involved in drug efflux are members of the ABC family of transporters [52], which are significantly upregulated, along with genes involved in NSCLC-CSCs stemness, and are able to induce resistance to different treatments [53]. Among all ABC transporters, BCRP/ABCG2 and P-gp/MDR1/ABCB1 are selectively expressed in SP cells. The BCRP/ABCG2 transporter is associated with tumor growth, progression, and metastasis, and its overexpression confers multidrug resistance to a number of anti-cancer drugs [54, 55]. Interestingly, EGFR-TKIs have been found to interact with this transporter [56]. Ho and colleagues have reported an increased expression of BCRP/ABCG2, P-gp/MDR1/ABCB1, MRP1/ABCC1 in SP cells of various NSCLC cell lines [46] and, similarly, in primary tumors obtained from lung cancer patients [57], suggesting that their overexpression was directly correlated with CSC-mediated resistance in NSCLC. Chen and colleagues showed that acquired resistance to gefitinib in wild-type EGFR-expressing cancer cells was associated with increased expression of BCRP/ABCG2, which in turn leads to gefitinib efflux from cells, and was correlated with poor response to gefitinib in both cancer cell lines and lung cancer patients with wild-type EGFR [58]. The afatinib-resistant cell line HCC827-ACR also exhibits an overexpression of P-gp/MDR1/ABCB1, suggesting a strong correlation between the presence of the ABC transporter and acquired resistance to afatinib [51]. More recently, Katayama and colleagues described P-gp/MDR1/ABCB1 overexpression as a mechanism of resistance to ceritinib in ALK-rearranged NSCLC [59]. Nevertheless, the use of these markers to identify CSC populations is more complex and is accompanied by limitations. Indeed, several articles have reported that, in addition to the correlation between CD133 positivity and CSLCs, there is also evidence that CD133-negative cells have stem-like properties, casting doubt on CD133 as a CSC marker [60-62]. In addition, Akunuru and colleagues demonstrated that in A549 and H441 NSCLC cell lines and primary patient samples, the CD133pos and ALDH-high SP cells are a phenotypically distinct subpopulation enriched for CSCs activity. At the same time, non-SP CD133neg, and/or ALDH-low cells have been reported to be able to generate SP CD133pos and ALDH-high, suggesting that the elimination of one CSCs subpopulation may be insufficient for cancer eradication, because other CSCs subpopulations could potentially propagate the tumor [33]. Moreover, one of the most important mechanisms by which CSCs survive cancer treatment is represented by signals generated by the tumor microenvironment and is mainly due to a deregulation of signaling pathways [63, 64]. Notch, Wnt/β-catenin, and hedgehog signaling pathways, together with pathways involving NFκ-B, MAPK, PI3K, and EGF, are aberrantly active in CSCs and regulate self-renewal activity, EMT, and also play a significant role in tumor initiation and development [63], as well as in resistance to EGFR-TKIs, making them a key target for the eradication of CSC populations and impairing the metastatic behavior [65]. Table 1 Principal markers characterizing lung cancer stem cells (CSCs) Lung CSCs Markers . Role . References . Aldehyde Dehydrogenase (ALDH) Modulator of gene expression and cell differentiation [34-37] CD24 (CD24A) Role in cell-matrix (adhesion) and cell-cell (migration) [32] CD34 Role in adhesion or homing [32] CD44 (LHR) Regulatory protein for growth, proliferation, cell adhesion, motility, and migration. It is also involved in cell signaling cascades involved in tumor initiation and progression [38, 39] CD90 (Thy-1) Cell-cell and cell-matrix interactions, with implication in apoptosis, metastasis, inflammation, as well as cell adhesion, extravasation and migration [40] CD117 (c-KIT) Cytokine receptor expressed on the surface of hematopoietic stem cells able to bind the stem cell factor (SCF) playing a role in cell survival, proliferation, and differentiation [41, 42] CD133 (Prominin-1) Transmembrane glycoprotein with the function in maintaining stem cell properties by suppressing differentiation [32, 43] CD164 (Sialomucin core protein 24 or endolyn) Function as a cell adhesion molecule [44] CD166 (ALCAM) Important role in mediating adhesion interactions [45] Side population  (SP, Hoechst-negative) Role in the efflux of chemotherapy drugs, accounting for the resistance of cancer to chemotherapy [46] Lung CSCs Markers . Role . References . Aldehyde Dehydrogenase (ALDH) Modulator of gene expression and cell differentiation [34-37] CD24 (CD24A) Role in cell-matrix (adhesion) and cell-cell (migration) [32] CD34 Role in adhesion or homing [32] CD44 (LHR) Regulatory protein for growth, proliferation, cell adhesion, motility, and migration. It is also involved in cell signaling cascades involved in tumor initiation and progression [38, 39] CD90 (Thy-1) Cell-cell and cell-matrix interactions, with implication in apoptosis, metastasis, inflammation, as well as cell adhesion, extravasation and migration [40] CD117 (c-KIT) Cytokine receptor expressed on the surface of hematopoietic stem cells able to bind the stem cell factor (SCF) playing a role in cell survival, proliferation, and differentiation [41, 42] CD133 (Prominin-1) Transmembrane glycoprotein with the function in maintaining stem cell properties by suppressing differentiation [32, 43] CD164 (Sialomucin core protein 24 or endolyn) Function as a cell adhesion molecule [44] CD166 (ALCAM) Important role in mediating adhesion interactions [45] Side population  (SP, Hoechst-negative) Role in the efflux of chemotherapy drugs, accounting for the resistance of cancer to chemotherapy [46] Open in new tab Table 1 Principal markers characterizing lung cancer stem cells (CSCs) Lung CSCs Markers . Role . References . Aldehyde Dehydrogenase (ALDH) Modulator of gene expression and cell differentiation [34-37] CD24 (CD24A) Role in cell-matrix (adhesion) and cell-cell (migration) [32] CD34 Role in adhesion or homing [32] CD44 (LHR) Regulatory protein for growth, proliferation, cell adhesion, motility, and migration. It is also involved in cell signaling cascades involved in tumor initiation and progression [38, 39] CD90 (Thy-1) Cell-cell and cell-matrix interactions, with implication in apoptosis, metastasis, inflammation, as well as cell adhesion, extravasation and migration [40] CD117 (c-KIT) Cytokine receptor expressed on the surface of hematopoietic stem cells able to bind the stem cell factor (SCF) playing a role in cell survival, proliferation, and differentiation [41, 42] CD133 (Prominin-1) Transmembrane glycoprotein with the function in maintaining stem cell properties by suppressing differentiation [32, 43] CD164 (Sialomucin core protein 24 or endolyn) Function as a cell adhesion molecule [44] CD166 (ALCAM) Important role in mediating adhesion interactions [45] Side population  (SP, Hoechst-negative) Role in the efflux of chemotherapy drugs, accounting for the resistance of cancer to chemotherapy [46] Lung CSCs Markers . Role . References . Aldehyde Dehydrogenase (ALDH) Modulator of gene expression and cell differentiation [34-37] CD24 (CD24A) Role in cell-matrix (adhesion) and cell-cell (migration) [32] CD34 Role in adhesion or homing [32] CD44 (LHR) Regulatory protein for growth, proliferation, cell adhesion, motility, and migration. It is also involved in cell signaling cascades involved in tumor initiation and progression [38, 39] CD90 (Thy-1) Cell-cell and cell-matrix interactions, with implication in apoptosis, metastasis, inflammation, as well as cell adhesion, extravasation and migration [40] CD117 (c-KIT) Cytokine receptor expressed on the surface of hematopoietic stem cells able to bind the stem cell factor (SCF) playing a role in cell survival, proliferation, and differentiation [41, 42] CD133 (Prominin-1) Transmembrane glycoprotein with the function in maintaining stem cell properties by suppressing differentiation [32, 43] CD164 (Sialomucin core protein 24 or endolyn) Function as a cell adhesion molecule [44] CD166 (ALCAM) Important role in mediating adhesion interactions [45] Side population  (SP, Hoechst-negative) Role in the efflux of chemotherapy drugs, accounting for the resistance of cancer to chemotherapy [46] Open in new tab TKI Resistance Mediated by Genetic Alterations It is well known that the acquired resistance to EGFR/ALK-TKIs is associated in the majority of cases with the appearance of secondary mutations in the EGFR/ALK tyrosine kinase domain (i.e., the p.T790M mutation for EGFR-TKIs; the p.L1196M or other mutations for ALK-TKIs). Moreover, the activation of alternative pathways (amplification of c-MET), the alteration of the EGFR/ALK pathways (KRAS, BRAF, and PIK3CA mutations, loss of PTEN, aberrant expression of NF1), and the impairment of the EGFR/ALK-TKIs-mediated apoptosis pathway (BCL2-like 11/BIM deletion polymorphism) are the most important alterations responsible for TKI resistance [66]. Notably, two issues are still under debate: (a) if these resistance mutations truly appear after TKI exposure or, in contrast, they pre-exist before treatment and (b) if the mutations pre-exist in CSCs and become more prevalent in the tumor architecture under the selective pressure by TKIs. Currently there are several findings implying that CSCs contribute to TKI resistance in lung cancer by an “adaptive” resistance mechanism, which is assumed to occur rapidly after the initiation of therapy. However, this phenomenon is poorly described in the literature, and these mechanisms remain unclear. Recent data on ALK-mutant tumors obtained from an in vitro study [8] found that in selected cell lines (e.g., H3122, an ALK-translocated NSCLC cell line), there was an increased expression of CSC markers (ALDH1), as a response to targeted therapies (including ALK-TKI and dual PI3K/MEK therapy), together with an increased expression of alternative pathways, that is, PI3K-AKT-mTOR, previously associated not only with resistance to treatment, but also with the CSLC phenotype [67]. In vitro and in vivo results suggest that ALK activity may play an important role in maintaining the stemness of EML4-ALKpos NSCLC cells, characterized by increased expression of CSC-associated biomarkers such as ALDH, NANOG, and OCT4 [68]. It is still unclear whether CSLCs exist as a small subpopulation after initiation of treatment, or if there is just an upregulation of CSLC markers in response to drug treatment. Regarding the EGFR-mutant tumors, some studies report the pre-existence of the p.T790M before TKI treatments [69, 70]; similarly, MET amplification seems to exist before treatment [71]. Several preclinical studies highlighted the involvement of KRAS mutations in lung tumor initiation and maintenance [72]. The mechanisms by which KRAS mutations mediate TKI resistance in cells with stem cell-like properties remain elusive [73]. Lin and colleagues proposed that the activation of oncogenic KRAS, alone or in combination with the removal of tumor suppressor p53 in SPC+ cells, resulted in the development of alveolar tumors [74]. Previous results, published by Kim and colleagues, demonstrated that the expansion of bronchoalveolar stem cells stimulated by a constitutive oncogenic KRAS signaling may transform into adenocarcinomas. The authors’ hypothesis was that while lung tumors arise from expansion of stem cells, the advanced tumors retain characteristics of differentiated lineage(s) due to the influences from the microenvironment or continuous oncogenic signaling [75]. The mechanism of resistance may depend on KRAS alterations in lung CSCs. Recently, Ali and colleagues hypothesized that PKC and Notch pathways, by driving the CSC phenotype, may mediate the tumorigenic behavior of KRAS mutated lung adenocarcinoma cells and exhibit intrinsic drug resistance and therapeutic failure [72]. If acquired resistance is the result of the survival advantage of pre-existing resistant tumor subclones, it cannot be excluded that a limited group of cells with self-renewal properties, the CSCs, are carriers of the alterations commonly known to be the cause of TKI resistance. It has been widely reported that current anti-cancer therapies fail to destroy CSC clones and tend to favor the selection and expansion of resistant subclones, resulting in poor responses and outcomes, thus exerting a strong influence on intra-tumor heterogeneity. Moreover, it has been reported that pharmacological treatments could induce CSCs to drive the re-growth of tumors as a result of microenvironmental adaptation [76]. Shien and colleagues suggested that exposure to EGFR-TKIs may influence the mechanisms of acquired resistance. The exposure of EGFR-mutant lung cancer cell lines to high concentrations of gefitinib may cause the appearance of stem-like properties, including ALDH1 overexpression, increase of SP cells, and self-renewal capability [49]. Despite these interesting findings, it is still not entirely clear why cells with stemness properties appeared to be the major population only from the high-concentration exposure method. Probably, the EGFR-mutant lung cancer cells acquire novel genetic and epigenetic aberrations in response to chronic EGFR-TKI exposure, indicating that they are highly flexible in response to their microenvironment and tend to favor mechanisms in acquiring resistance to TKIs [76]. Published data show that lung adenocarcinoma cells resistant to gefitinib exhibit stemness characteristics [36], sometimes also associated with upregulation of interleukin-8 (IL-8), providing evidence for the role of chemokines in TKI-resistance through CSC regulation [77]. More recently Chiu and colleagues reported that suppression of FOXO-3a drives an EGFR mutation-independent mechanism of resistance to gefitinib, involving the enhancement of stemness of lung cancer in vitro and in vivo [78]. Understanding the mechanisms able to control CSCs, as well as their identification, isolation, and characterization, has major implications for lung cancer therapy. For this reason, targeting CSCs has become crucial in treating cancer and preventing tumor relapse [29, 79, 80]. However, while the development of acquired resistance is dependent on a clonal selection of drug-resistant cell clones already present at the initiation of tumor development, it has to be taken into account that a more complex development of acquired resistance may exist, based on the roles of CSCs and CSLCs. Phenotypic Alterations Other changes of tumor cells involved in acquired resistance to TKIs are EMT and the transformation to a small cell-like carcinoma [49, 81-83]. Della Corte et al. showed that in EGFR-mutated cells, the resistant phenotype is associated with the functional interaction between MET activation and amplification of the gene encoding the hedgehog receptor (SMO). In these cells, the combination of SMO and MET inhibitors exerted significant antitumor activity, while in EGFR wild-type NSCLC cells resistant to EGFR inhibitors, the effects of both SMO and EGFR influenced PI3K/AKT and MAPK signaling cascades with a strong anti-proliferative activity [84]. Bai et al. confirmed that the hedgehog signaling pathway was activated in EGFR-TKI—resistant NSCLC cells, while it was silenced in EGFR-TKI—sensitive NSCLC cells. Interestingly, these authors revealed that EGFR-TKI resistant phenotype is accompanied by EMT induction and ABCG2 overexpression [85]. Studies on H1299 NSCLC cell-lines harboring the EML4-ALK demonstrated that the induction of the EMT is consistent with CSC properties. In support of the hypothesis that H1299-EML4-ALK NSCLC cell lines display CSC characteristics, it was found that they display significantly higher levels of CD133pos cells, a lung CSC marker [32], suggesting that EML4-ALK confers subpopulations of NSCLC cells with CSC traits [86]. Moreover, cells within the tumor mass can regain stemness properties [19], making the involvement of CSCs in TKI resistance a complex topic for further investigation. Epigenetic Modifications Current evidence suggests that the involvement of epigenetic alterations, such as promoter methylation, histone modification, and the deregulation of specific microRNAs (miRNAs), may be implicated in the acquisition of resistance to several lung cancer treatments, including chemotherapy and targeted therapy [65]. In comparison to genomic mutations, epigenetic changes are reversible and subject to environmental pressure, generating a more complex tumor heterogeneity and contributing to the development of drug resistance. It has been reported that epigenetic modifications can regulate CSCs and influence cell-cycle exit and differentiation, pro-survival and EMT, migration and invasion, and can increase tumor initiation and its metastatic potential [87]. Few studies have been published in relation to epigenetics, CSCs, and EGFR-TKIs resistance. Promoter methylation involves DNA methylation of CpG islands in the promoter region of some genes, rendering them transcriptionally silent. Nevertheless, while the CpG island methylation in the promoter region of the EGFR gene is common in solid tumors, little is known about its role in affecting TKI resistance [88, 89]. Some in vitro studies report that the inhibition of DNA methylation using 5-aza-2′-deoxy cytidine (5-aza-CdR) in three NSCLC cell lines bearing different EGFR mutational status and levels of sensitivity to EGFR-TKIs enhanced the anti-tumor effects of gefitinib in NSCLC cells [90, 91], suggesting the EGFR gene promoter methylation as a potential mechanism for acquired resistance to gefitinib. Lin and colleagues demonstrated that the underlying biology of genes regulated by DNA methylation may have predictive value in NSCLC. They identified several candidate genes, including genes related to the EMT phenotype, such as AXL, that were differentially methylated in normal lung tissue versus NSCLC tumors and associated with erlotinib resistance [92]. Ogawa et al., performing a methylation-specific array, found that death-associated protein kinase (DAPK) was hypermethylated in an NSCLC cell line resistant to erlotinib. In vitro restoration of DAPK into the resistant NSCLC cells by transfection re-sensitized the cells to erlotinib, demonstrating that DAPK plays an important role in erlotinib resistance and that gene silencing through promoter methylation is one of the key mechanisms of resistance to anti-EGFR therapeutic agents [89]. The involvement of histone deacetylases (HDACs) in EGFR-TKI resistance has also been reported. Yu and colleagues demonstrated that inhibition of HDACs was able to induce a sensitization to erlotinib in a panel of EGFR-TKI-resistant NSCLC cell lines in vitro and in two erlotinib-resistant NSCLC xenograft models in vivo; a downregulation of the expression of HER2, c-Met, IGF1R, and AXL at both the transcriptional and protein levels was demonstrated, as well as a consensual inhibition of Akt and ERK activities [93]. In addition to this, HDAC inhibition can epigenetically restore the function of BIM, a BH3 pro-apoptotic member of the Bcl-2 protein family, and the death sensitivity to gefitinib and erlotinib in EGFR mutant forms of NSCLC where resistance to EGFR-TKI is associated with a common BIM polymorphism [94, 95]. Tanimoto and colleagues demonstrated that BIM polymorphism contributes also to resistance against osimertinib, both in vitro and in vivo [95]. There is evidence that miRNAs regulate certain genes associated with resistance to EGFR-TKIs [96]. In a recent study, Wang and colleagues identified miRNA profiles in plasma, involved in primary resistance to EGFR-TKIs in advanced NSCLC patients with EGFR activating mutations, finding high expression levels of miR-21, AmiR-27a, miR-34a, and miR-218 in 20 NSCLC patients with EGFR 19 deletion treated with first-line EGFR-TKIs [97]. In particular miR-21, together with miR-223 and miR-214, has been reported to generate gefitinib and erlotinib resistance via activation of the IGF1R/PI3K/AKT and ERK signaling pathways and suppression of PTEN [98-100]. Greater efficacy of EGFR-TKIs in NSCLC has been ascribed to miR-200c overexpression. These patients were able to regain gefitinib, erlotinib, and afatinib sensitivity through the regulation of EMT by PI3K/AKT and MEK/ERK pathways, and showed a correlation with higher disease control rate, longer progression-free survival, and overall survival [51, 101, 102]. Also, miR-30a-5p, as well as miR-138-5p, has been reported to overcome and/or revert EGFR-TKIs resistance in NSCLC through regulation of PI3K/AKT and G protein-coupled receptor 124, respectively, [103, 104]. In addition, the inhibition of hedgehog signaling sensitizes NSCLC-CSCs to erlotinib therapy through modulation of EMT-regulating miRNAs, upregulation of CSC markers (Sox2, Nanog, and EpCAM) and downregulation of the miR-200 and let-7 family of miRNAs [105]. The overexpression of miR-23, −103, −134, −147, −203, and −478b has been reported to be induced by TGF-β1, which is also able to promote the inhibition and/or activation of several targets and pathways, thereby influencing drug resistance to EGFR-TKIs [106]. As described above, approximately 4% of NSCLC patients have ALK-rearrangements. Unfortunately acquired resistance to crizotinib (ALK-TKIs) eventually developed in ALK-positive patients. Several miRNAs are involved in progression of ALK-positive tumor cells, but the relation between crizotinib resistance and miRNAs in NSCLC is still unknown. Future Therapies and CSCs Clinical Trials Even if targeting CSCs would be an interesting option to overcome treatment resistance, it still represents a big challenge. Unfortunately, there are no ongoing clinical trials targeting CSCs in TKI-pretreated NSCLC. However, a few clinical trials have been conducted in NSCLC patients receiving chemotherapy. The trial “Imetelstat as Maintenance Therapy After Initial Induction Chemotherapy in Non-Small Cell Lung Cancer (NSCLC)” (NCT01137968), evaluated the efficacy and safety of imetelstat (GRN163L), a telomerase inhibitor, as maintenance therapy for NSCLC patients who progressed to a platinum-based therapy. Another study, “Carboplatin and Pemetrexed Plus Demcizumab (OMP-21M18) in Subjects With Non-Squamous Non-Small Cell Lung Cancer” (NCT01189968) was focused on the safety and the optimal dose of demcizumab (OMP-21M18), a humanized monoclonal antibody developed to target CSCs, in combination with carboplatin and pemetrexed. The results of these trials are not yet available. Conclusion Despite the efforts made to improve the clinical outcomes of advanced NSCLC in the past decade, the death rate of patients is still very high, due to the development of resistance leading to tumor refractoriness and disease progression. The resistance is strongly related to tumor heterogeneity and is explained in part by the presence of CSCs. In this review, we have described the crucial role of CSCs in the development of spatial and temporal heterogeneity of NSCLC and how they can mediate TKIs resistance. Alterations involved in the development of TKI resistance may be acquired at a very early phase of tumorigenesis, supporting the hypothesis that these aberrations may be present in CSCs. Nowadays, approaches to specifically target the CSC population are often used as an adjunct to chemotherapy, radiotherapy, and targeted therapy. Therefore, due to the high tumor heterogeneity, the identification of CSCs at the initial phase could suggest a targeted approach to prevent drug resistance. Although the role of CSCs in drug resistance is an emerging topic, unfortunately, it lacks clinical application at the moment. The origin of CSCs, the role of the microenvironment, and phenotypic alterations together with genetic and epigenetic modifications are the hallmarks of tumor heterogeneity, which leads to resistance to targeted treatments in NSCLC. A deeper understanding of tumor and CSC biology will facilitate the understanding of the mechanisms underlying tumor evolution and resistance to targeted therapy. With the advances seen in this highly active field, improvements in the clinical outcome of patients with NSCLC can be expected in the foreseeable future. Author Contributions M.D.R., E.A., G.R., A.P., E.R., and S.C.: wrote the review; F.D.M., A.D.P., and R.D.: critically reviewed the manuscript. Disclosure of Potential Conflicts of Interest The authors indicated no potential conflicts of interest. References 1 Ge L , Shi R. Progress of EGFR-TKI and ALK/ROS1 inhibitors in advanced non-small cell lung cancer . Int J Clin Exp Med 2015 ; 8 : 10330 – 10339 . Google Scholar OpenURL Placeholder Text WorldCat 2 Russo A , Franchina T, Ricciardi GR et al. . A decade of EGFR inhibition in EGFR-mutated non small cell lung cancer (NSCLC): Old successes and future perspectives . Oncotarget 2015 ; 6 : 26814 – 26825 . Google Scholar Crossref Search ADS WorldCat 3 Cheng X , Chen H. Tumor heterogeneity and resistance to EGFR-targeted therapy in advanced nonsmall cell lung cancer: Challenges and perspectives . Onco Targets Ther 2014 ; 7 : 1689 – 1704 . Google Scholar OpenURL Placeholder Text WorldCat 4 Magee JA , Piskounova E, Morrison SJ. Cancer stem cells: Impact, heterogeneity, and uncertainty . Cancer Cell 2012 ; 21 : 283 – 296 . Google Scholar Crossref Search ADS WorldCat 5 Gainor JF , Shaw AT. Emerging paradigms in the development of resistance to tyrosine kinase inhibitors in lung cancer . J Clin Oncol 2013 ; 31 : 3987 – 3996 . Google Scholar Crossref Search ADS WorldCat 6 Marte B. Tumour heterogeneity . Nature 2013 ; 501 : 327 . Google Scholar Crossref Search ADS WorldCat 7 Peacock CD , Watkins DN. Cancer stem cells and the ontogeny of lung cancer . J Clin Oncol 2008 ; 26 : 2883 – 2889 . Google Scholar Crossref Search ADS WorldCat 8 Jokinen E , Laurila N, Koivunen P et al. . Combining targeted drugs to overcome and prevent resistance of solid cancers with some stem-like cell features . Oncotarget 2014 ; 5 : 9295 – 9307 . Google Scholar Crossref Search ADS WorldCat 9 Passaro A , Lazzari C, Karachaliou N et al. . Personalized treatment in advanced ALK-positive non-small cell lung cancer: From bench to clinical practice . Onco Targets Ther 2016 ; 9 : 6361 – 6376 . Google Scholar Crossref Search ADS WorldCat 10 Passaro A , Guerini-Rocco E, Pochesci A et al. . Targeting EGFR T790M mutation in NSCLC: From biology to evaluation and treatment . Pharmacol Res 2017 ; 117 : 406 – 415 . Google Scholar Crossref Search ADS WorldCat 11 Burrell RA , McGranahan N, Bartek J et al. . The causes and consequences of genetic heterogeneity in cancer evolution . Nature 2013 ; 501 : 338 – 345 . Google Scholar Crossref Search ADS WorldCat 12 Izumchenko E , Chang X, Brait M et al. . Targeted sequencing reveals clonal genetic changes in the progression of early lung neoplasms and paired circulating DNA . Nat Commun 2015 ; 6 : 8258 . Google Scholar Crossref Search ADS WorldCat 13 Blackhall FH , Pintilie M, Wigle DA et al. . Stability and heterogeneity of expression profiles in lung cancer specimens harvested following surgical resection . Neoplasia 2004 ; 6 : 761 – 767 . Google Scholar Crossref Search ADS WorldCat 14 Gerlinger M , Rowan AJ, Horswell S et al. . Intratumor heterogeneity and branched evolution revealed by multiregion sequencing . N Engl J Med 2012 ; 366 : 883 – 892 . Google Scholar Crossref Search ADS WorldCat 15 Yatabe Y , Matsuo K, Mitsudomi T. Heterogeneous distribution of EGFR mutations is extremely rare in lung adenocarcinoma . J Clin Oncol 2011 ; 29 : 2972 – 2977 . Google Scholar Crossref Search ADS WorldCat 16 Nagai Y , Miyazawa H, Huqun et al. . Genetic heterogeneity of the epidermal growth factor receptor in non-small cell lung cancer cell lines revealed by a rapid and sensitive detection system, the peptide nucleic acid-locked nucleic acid PCR clamp . Cancer Res 2005 ; 65 : 7276 – 7282 . Google Scholar Crossref Search ADS WorldCat 17 Kamila W-K , Michał S, Paweł K et al. . EGFR activating mutations detected by different PCR techniques in Caucasian NSCLC patients with CNS metastases: Short report . Clin Exp Metastasis 2013 ; 30 : 1063 – 1071 . Google Scholar Crossref Search ADS WorldCat 18 Shackleton M , Quintana E, Fearon ER et al. . Heterogeneity in cancer: Cancer stem cells versus clonal evolution . Cell 2009 ; 138 : 822 – 829 . Google Scholar Crossref Search ADS WorldCat 19 Meacham CE , Morrison SJ. Tumour heterogeneity and cancer cell plasticity . Nature 2013 ; 501 : 328 – 337 . Google Scholar Crossref Search ADS WorldCat 20 Govindan R , Ding L, Griffith M et al. . Genomic landscape of non-small cell lung cancer in smokers and never-smokers . Cell 2012 ; 150 : 1121 – 1134 . Google Scholar Crossref Search ADS WorldCat 21 Kreso A , Dick JE. Evolution of the cancer stem cell model . Cell Stem Cell 2014 ; 14 : 275 – 291 . Google Scholar Crossref Search ADS WorldCat 22 Li Y , Laterra J. Cancer stem cells: Distinct entities or dynamically regulated phenotypes? . Cancer Res 2012 ; 72 : 576 – 580 . Google Scholar Crossref Search ADS WorldCat 23 Magdaleno SM , Barrish J, Finegold MJ et al. . Investigating stem cells in the lung . Adv Pediatr 1998 ; 45 : 363 – 396 . Google Scholar OpenURL Placeholder Text WorldCat 24 Cruz MH , Siden A, Calaf GM et al. . The stemness phenotype model . ISRN Oncol 2012 ; 2012 : 392647 . Google Scholar OpenURL Placeholder Text WorldCat 25 Vermeulen L , de Sousa e Melo F, Richel DJ et al. . The developing cancer stem-cell model: Clinical challenges and opportunities . Lancet Oncol 2012 ; 13 : e83 – e89 . Google Scholar Crossref Search ADS WorldCat 26 Laks DR , Visnyei K, Kornblum HI. Brain tumor stem cells as therapeutic targets in models of glioma . Yonsei Med J 2010 ; 51 : 633 – 640 . Google Scholar Crossref Search ADS WorldCat 27 Liu L , Li WY, Chen Q et al. . The biological characteristics of glioma stem cells in human glioma cell line SHG44 . Mol Med Rep 2012 ; 5 : 552 – 558 . Google Scholar Crossref Search ADS WorldCat 28 Baccelli I , Trumpp A. The evolving concept of cancer and metastasis stem cells . J Cell Biol 2012 ; 198 : 281 – 293 . Google Scholar Crossref Search ADS WorldCat 29 Morrison R , Schleicher SM, Sun Y et al. . Targeting the mechanisms of resistance to chemotherapy and radiotherapy with the cancer stem cell hypothesis . J Oncol 2011 ; 2011 : 941876 . Google Scholar Crossref Search ADS WorldCat 30 Perona R , Lopez-Ayllon BD, de Castro Carpeno J et al. . A role for cancer stem cells in drug resistance and metastasis in non-small-cell lung cancer . Clin Transl Oncol 2011 ; 13 : 289 – 293 . Google Scholar Crossref Search ADS WorldCat 31 Gottschling S , Herpel E, Eberhardt WE et al. . The gefitinib long-term responder (LTR)–a cancer stem-like cell story? Insights from molecular analyses of German long-term responders treated in the IRESSA expanded access program (EAP) . Lung Cancer 2012 ; 77 : 183 – 191 . Google Scholar Crossref Search ADS WorldCat 32 Eramo A , Lotti F, Sette G et al. . Identification and expansion of the tumorigenic lung cancer stem cell population . Cell Death Differ 2008 ; 15 : 504 – 514 . Google Scholar Crossref Search ADS WorldCat 33 Akunuru S , James Zhai Q, Zheng Y. Non-small cell lung cancer stem/progenitor cells are enriched in multiple distinct phenotypic subpopulations and exhibit plasticity . Cell Death Dis 2012 ; 3 : e352 . Google Scholar Crossref Search ADS WorldCat 34 Jiang F , Qiu Q, Khanna A et al. . Aldehyde dehydrogenase 1 is a tumor stem cell-associated marker in lung cancer . Mol Cancer Res 2009 ; 7 : 330 – 338 . Google Scholar Crossref Search ADS WorldCat 35 Patel M , Lu L, Zander DS et al. . ALDH1A1 and ALDH3A1 expression in lung cancers: Correlation with histologic type and potential precursors . Lung Cancer 2008 ; 59 : 340 – 349 . Google Scholar Crossref Search ADS WorldCat 36 Huang CP , Tsai MF, Chang TH et al. . ALDH-positive lung cancer stem cells confer resistance to epidermal growth factor receptor tyrosine kinase inhibitors . Cancer Lett 2013 ; 328 : 144 – 151 . Google Scholar Crossref Search ADS WorldCat 37 Ucar D , Cogle CR, Zucali JR et al. . Aldehyde dehydrogenase activity as a functional marker for lung cancer . Chem Biol Interact 2009 ; 178 : 48 – 55 . Google Scholar Crossref Search ADS WorldCat 38 Li G , Gao Y, Cui Y et al. . Overexpression of CD44 is associated with the occurrence and migration of non-small cell lung cancer . Mol Med Rep 2016 ; 14 : 3159 – 3167 . Google Scholar Crossref Search ADS WorldCat 39 Leung EL , Fiscus RR, Tung JW et al. . Non-small cell lung cancer cells expressing CD44 are enriched for stem cell-like properties . PLoS One 2010 ; 5 : e14062 . Google Scholar Crossref Search ADS WorldCat 40 Yan X , Luo H, Zhou X et al. . Identification of CD90 as a marker for lung cancer stem cells in A549 and H446 cell lines . Oncol Rep 2013 ; 30 : 2733 – 2740 . Google Scholar Crossref Search ADS WorldCat 41 Donnenberg AD , Zimmerlin L, Landreneau RJ et al. . KIT (CD117) expression in a subset of non-small cell lung carcinoma (NSCLC) patients . PLoS One 2012 ; 7 : e52885 . Google Scholar Crossref Search ADS WorldCat 42 Pelosi G , Barisella M, Pasini F et al. . CD117 immunoreactivity in stage I adenocarcinoma and squamous cell carcinoma of the lung: Relevance to prognosis in a subset of adenocarcinoma patients . Mod Pathol 2004 ; 17 : 711 – 721 . Google Scholar Crossref Search ADS WorldCat 43 Meng X , Li M, Wang X et al. . Both CD133+ and CD133- subpopulations of A549 and H446 cells contain cancer-initiating cells . Cancer Sci 2009 ; 100 : 1040 – 1046 . Google Scholar Crossref Search ADS WorldCat 44 Chen WL , Huang AF, Huang SM et al. . CD164 promotes lung tumor-initiating cells with stem cell activity and determines tumor growth and drug resistance via Akt/mTOR signaling . Oncotarget 2016 ; 8 : 54115 – 54135 . Google Scholar Crossref Search ADS WorldCat 45 Tachezy M , Zander H, Wolters-Eisfeld G et al. . Activated leukocyte cell adhesion molecule (CD166): An “inert” cancer stem cell marker for non-small cell lung cancer? . Stem Cells 2014 ; 32 : 1429 – 1436 . Google Scholar Crossref Search ADS WorldCat 46 Ho MM , Ng AV, Lam S et al. . Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells . Cancer Res 2007 ; 67 : 4827 – 4833 . Google Scholar Crossref Search ADS WorldCat 47 Okudela K , Woo T, Mitsui H et al. . Expression of the potential cancer stem cell markers, CD133, CD44, ALDH1, and beta-catenin, in primary lung adenocarcinoma–their prognostic significance . Pathol Int 2012 ; 62 : 792 – 801 . Google Scholar Crossref Search ADS WorldCat 48 Shien K , Toyooka S, Ichimura K et al. . Prognostic impact of cancer stem cell-related markers in non-small cell lung cancer patients treated with induction chemoradiotherapy . Lung Cancer 2012 ; 77 : 162 – 167 . Google Scholar Crossref Search ADS WorldCat 49 Shien K , Toyooka S, Yamamoto H et al. . Acquired resistance to EGFR inhibitors is associated with a manifestation of stem cell-like properties in cancer cells . Cancer Res 2013 ; 73 : 3051 – 3061 . Google Scholar Crossref Search ADS WorldCat 50 Corominas-Faja B , Oliveras-Ferraros C, Cuyas E et al. . Stem cell-like ALDH(bright) cellular states in EGFR-mutant non-small cell lung cancer: A novel mechanism of acquired resistance to erlotinib targetable with the natural polyphenol silibinin . Cell Cycle 2013 ; 12 : 3390 – 3404 . Google Scholar Crossref Search ADS WorldCat 51 Hashida S , Yamamoto H, Shien K et al. . Acquisition of cancer stem cell-like properties in non-small cell lung cancer with acquired resistance to afatinib . Cancer Sci 2015 ; 106 : 1377 – 1384 . Google Scholar Crossref Search ADS WorldCat 52 Arrigoni E , Galimberti S, Petrini M et al. . ATP-binding cassette transmembrane transporters and their epigenetic control in cancer: An overview . Expert Opin Drug Metab Toxicol 2016 ; 12 : 1419 – 1432 . Google Scholar Crossref Search ADS WorldCat 53 Dean M. ABC transporters, drug resistance, and cancer stem cells . J Mammary Gland Biol Neoplasia 2009 ; 14 : 3 – 9 . Google Scholar Crossref Search ADS WorldCat 54 Sarkadi B , Ozvegy-Laczka C, Nemet K et al. . ABCG2 – a transporter for all seasons . FEBS Lett 2004 ; 567 : 116 – 120 . Google Scholar Crossref Search ADS WorldCat 55 An Y , Ongkeko WM. ABCG2: The key to chemoresistance in cancer stem cells? . Expert Opin Drug Metab Toxicol 2009 ; 5 : 1529 – 1542 . Google Scholar Crossref Search ADS WorldCat 56 Shukla S , Chen ZS, Ambudkar SV. Tyrosine kinase inhibitors as modulators of ABC transporter-mediated drug resistance . Drug Resist Updat 2012 ; 15 : 70 – 80 . Google Scholar Crossref Search ADS WorldCat 57 Singh S , Trevino J, Bora-Singhal N et al. . EGFR/Src/Akt signaling modulates Sox2 expression and self-renewal of stem-like side-population cells in non-small cell lung cancer . Mol Cancer 2012 ; 11 : 73 . Google Scholar Crossref Search ADS WorldCat 58 Chen YJ , Huang WC, Wei YL et al. . Elevated BCRP/ABCG2 expression confers acquired resistance to gefitinib in wild-type EGFR-expressing cells . PLoS One 2011 ; 6 : e21428 . Google Scholar Crossref Search ADS WorldCat 59 Katayama R , Sakashita T, Yanagitani N et al. . P-glycoprotein mediates ceritinib resistance in anaplastic lymphoma kinase-rearranged non-small cell lung cancer . EBioMedicine 2016 ; 3 : 54 – 66 . Google Scholar Crossref Search ADS WorldCat 60 Levina V , Marrangoni AM, DeMarco R et al. . Drug-selected human lung cancer stem cells: Cytokine network, tumorigenic and metastatic properties . PLoS One 2008 ; 3 : e3077 . Google Scholar Crossref Search ADS WorldCat 61 LaBarge MA , Bissell MJ. Is CD133 a marker of metastatic colon cancer stem cells? . J Clin Invest 2008 ; 118 : 2021 – 2024 . Google Scholar OpenURL Placeholder Text WorldCat 62 Joo KM , Kim SY, Jin X et al. . Clinical and biological implications of CD133-positive and CD133-negative cells in glioblastomas . Lab Invest 2008 ; 88 : 808 – 815 . Google Scholar Crossref Search ADS WorldCat 63 Takebe N , Miele L, Harris PJ et al. . Targeting Notch, Hedgehog, and Wnt pathways in cancer stem cells: Clinical update . Nat Rev Clin Oncol 2015 ; 12 : 445 – 464 . Google Scholar Crossref Search ADS WorldCat 64 Salem ML , El-Badawy AS, Li Z. Immunobiology and signaling pathways of cancer stem cells: Implication for cancer therapy . Cytotechnology 2015 ; 67 : 749 – 759 . Google Scholar Crossref Search ADS WorldCat 65 MacDonagh L , Gray SG, Breen E et al. . Lung cancer stem cells: The root of resistance . Cancer Lett 2016 ; 372 : 147 – 156 . Google Scholar Crossref Search ADS WorldCat 66 Huang L , Fu L. Mechanisms of resistance to EGFR tyrosine kinase inhibitors . Acta Pharm Sin B 2015 ; 5 : 390 – 401 . Google Scholar Crossref Search ADS WorldCat 67 Dubrovska A , Kim S, Salamone RJ et al. . The role of PTEN/Akt/PI3K signaling in the maintenance and viability of prostate cancer stem-like cell populations . Proc Natl Acad Sci USA 2009 ; 106 : 268 – 273 . Google Scholar Crossref Search ADS WorldCat 68 Oh SJ , Noh KH, Lee YH et al. . Targeting stemness is an effective strategy to control EML4-ALK+ non-small cell lung cancer cells . Oncotarget 2015 ; 6 : 40255 – 40267 . Google Scholar Crossref Search ADS WorldCat 69 Inukai M , Toyooka S, Ito S et al. . Presence of epidermal growth factor receptor gene T790M mutation as a minor clone in non-small cell lung cancer . Cancer Res 2006 ; 66 : 7854 – 7858 . Google Scholar Crossref Search ADS WorldCat 70 Tokumo M , Toyooka S, Ichihara S et al. . Double mutation and gene copy number of EGFR in gefitinib refractory non-small-cell lung cancer . Lung Cancer 2006 ; 53 : 117 – 121 . Google Scholar Crossref Search ADS WorldCat 71 Turke AB , Zejnullahu K, Wu YL et al. . Preexistence and clonal selection of MET amplification in EGFR mutant NSCLC . Cancer Cell 2010 ; 17 : 77 – 88 . Google Scholar Crossref Search ADS WorldCat 72 Ali SA , Justilien V, Jamieson L et al. . Protein kinase Ciota drives a NOTCH3-dependent stem-like phenotype in mutant KRAS lung adenocarcinoma . Cancer Cell 2016 ; 29 : 367 – 378 . Google Scholar Crossref Search ADS WorldCat 73 Sutherland KD , Song JY, Kwon MC et al. . Multiple cells-of-origin of mutant K-Ras-induced mouse lung adenocarcinoma . Proc Natl Acad Sci USA 2014 ; 111 : 4952 – 4957 . Google Scholar Crossref Search ADS WorldCat 74 Lin C , Song H, Huang C et al. . Alveolar type II cells possess the capability of initiating lung tumor development . PLoS One 2012 ; 7 : e53817 . Google Scholar Crossref Search ADS WorldCat 75 Kim CF , Jackson EL, Woolfenden AE et al. . Identification of bronchioalveolar stem cells in normal lung and lung cancer . Cell 2005 ; 121 : 823 – 835 . Google Scholar Crossref Search ADS WorldCat 76 Suda K , Mizuuchi H, Maehara Y et al. . Acquired resistance mechanisms to tyrosine kinase inhibitors in lung cancer with activating epidermal growth factor receptor mutation–diversity, ductility, and destiny . Cancer Metastasis Rev 2012 ; 31 : 807 – 814 . Google Scholar Crossref Search ADS WorldCat 77 Liu YN , Chang TH, Tsai MF et al. . IL-8 confers resistance to EGFR inhibitors by inducing stem cell properties in lung cancer . Oncotarget 2015 ; 6 : 10415 – 10431 . Google Scholar Crossref Search ADS WorldCat 78 Chiu CF , Chang YW, Kuo KT et al. . NF-kappaB-driven suppression of FOXO3a contributes to EGFR mutation-independent gefitinib resistance . Proc Natl Acad Sci USA 2016 ; 113 : E2526 – E2535 . Google Scholar Crossref Search ADS WorldCat 79 Hill RP , Marie-Egyptienne DT, Hedley DW. Cancer stem cells, hypoxia and metastasis . Semin Radiat Oncol 2009 ; 19 : 106 – 111 . Google Scholar Crossref Search ADS WorldCat 80 Signore M , Ricci-Vitiani L, De Maria R. Targeting apoptosis pathways in cancer stem cells . Cancer Lett 2013 ; 332 : 374 – 382 . Google Scholar Crossref Search ADS WorldCat 81 Barr S , Thomson S, Buck E et al. . Bypassing cellular EGF receptor dependence through epithelial-to-mesenchymal-like transitions . Clin Exp Metastasis 2008 ; 25 : 685 – 693 . Google Scholar Crossref Search ADS WorldCat 82 Uramoto H , Iwata T, Onitsuka T et al. . Epithelial-mesenchymal transition in EGFR-TKI acquired resistant lung adenocarcinoma . Anticancer Res 2010 ; 30 : 2513 – 2517 . Google Scholar OpenURL Placeholder Text WorldCat 83 Chung JH , Rho JK, Xu X et al. . Clinical and molecular evidences of epithelial to mesenchymal transition in acquired resistance to EGFR-TKIs . Lung Cancer 2011 ; 73 : 176 – 182 . Google Scholar Crossref Search ADS WorldCat 84 Della Corte CM , Bellevicine C, Vicidomini G et al. . SMO gene amplification and activation of the hedgehog pathway as novel mechanisms of resistance to anti-epidermal growth factor receptor drugs in human lung cancer . Clin Cancer Res 2015 ; 21 : 4686 – 4697 . Google Scholar Crossref Search ADS WorldCat 85 Bai XY , Zhang XC, Yang SQ et al. . Blockade of hedgehog signaling synergistically increases sensitivity to epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer cell lines . PLoS One 2016 ; 11 : e0149370 . Google Scholar Crossref Search ADS WorldCat 86 Guo F , Liu X, Qing Q et al. . EML4-ALK induces epithelial-mesenchymal transition consistent with cancer stem cell properties in H1299 non-small cell lung cancer cells . Biochem Biophys Res Commun 2015 ; 459 : 398 – 404 . Google Scholar Crossref Search ADS WorldCat 87 Garg M. MicroRNAs, stem cells and cancer stem cells . World J Stem Cells 2012 ; 4 : 62 – 70 . Google Scholar Crossref Search ADS WorldCat 88 Zhu J , Wang Y, Duan J et al. . DNA Methylation status of Wnt antagonist SFRP5 can predict the response to the EGFR-tyrosine kinase inhibitor therapy in non-small cell lung cancer . J Exp Clin Cancer Res 2012 ; 31 : 80 . Google Scholar Crossref Search ADS WorldCat 89 Ogawa T , Liggett TE, Melnikov AA et al. . Methylation of death-associated protein kinase is associated with cetuximab and erlotinib resistance . Cell Cycle 2012 ; 11 : 1656 – 1663 . Google Scholar Crossref Search ADS WorldCat 90 Li XY , Wu JZ, Cao HX et al. . Blockade of DNA methylation enhances the therapeutic effect of gefitinib in non-small cell lung cancer cells . Oncol Rep 2013 ; 29 : 1975 – 1982 . Google Scholar Crossref Search ADS WorldCat 91 Terai H , Soejima K, Yasuda H et al. . Longterm exposure to gefitinib induces acquired resistance through DNA methylation changes in the EGFRmutant PC9 lung cancer cell line . Int J Oncol 2015 ; 46 : 430 – 436 . Google Scholar Crossref Search ADS WorldCat 92 Lin SH , Wang J, Saintigny P et al. . Genes suppressed by DNA methylation in non-small cell lung cancer reveal the epigenetics of epithelial-mesenchymal transition . BMC Genomics 2014 ; 15 : 1079 . Google Scholar Crossref Search ADS WorldCat 93 Yu W , Lu W, Chen G et al. . Inhibition of histone deacetylases sensitizes EGF receptor-TK inhibitor-resistant non-small-cell lung cancer cells to erlotinib in vitro and in vivo . Br J Pharmacol 2017 ; 174 : 3608 – 3622 . Google Scholar Crossref Search ADS WorldCat 94 Nakagawa T , Takeuchi S, Yamada T et al. . EGFR-TKI resistance due to BIM polymorphism can be circumvented in combination with HDAC inhibition . Cancer Res 2013 ; 73 : 2428 – 2434 . Google Scholar Crossref Search ADS WorldCat 95 Tanimoto A , Takeuchi S, Arai S et al. . Histone deacetylase 3 inhibition overcomes BIM deletion polymorphism-mediated osimertinib resistance in EGFR-mutant lung cancer . Clin Cancer Res 2017 ; 23 : 3139 – 3149 . Google Scholar Crossref Search ADS WorldCat 96 Geng Q , Fan T, Zhang B et al. . Five microRNAs in plasma as novel biomarkers for screening of early-stage non-small cell lung cancer . Respir Res 2014 ; 15 : 149 . Google Scholar Crossref Search ADS WorldCat 97 Wang S , Su X, Bai H et al. . Identification of plasma microRNA profiles for primary resistance to EGFR-TKIs in advanced non-small cell lung cancer (NSCLC) patients with EGFR activating mutation . J Hematol Oncol 2015 ; 8 : 127 . Google Scholar Crossref Search ADS WorldCat 98 Li B , Ren S, Li X et al. . MiR-21 overexpression is associated with acquired resistance of EGFR-TKI in non-small cell lung cancer . Lung Cancer 2014 ; 83 : 146 – 153 . Google Scholar Crossref Search ADS WorldCat 99 Han J , Zhao F, Zhang J et al. . miR-223 reverses the resistance of EGFR-TKIs through IGF1R/PI3K/Akt signaling pathway . Int J Oncol 2016 ; 48 : 1855 – 1867 . Google Scholar Crossref Search ADS WorldCat 100 Wang YS , Wang YH, Xia HP et al. . MicroRNA-214 regulates the acquired resistance to gefitinib via the PTEN/AKT pathway in EGFR-mutant cell lines . Asian Pac J Cancer Prev 2012 ; 13 : 255 – 260 . Google Scholar Crossref Search ADS WorldCat 101 Li J , Li X, Ren S et al. . miR-200c overexpression is associated with better efficacy of EGFR-TKIs in non-small cell lung cancer patients with EGFR wild-type . Oncotarget 2014 ; 5 : 7902 – 7916 . Google Scholar Crossref Search ADS WorldCat 102 Zhou G , Zhang F, Guo Y et al. . miR-200c enhances sensitivity of drug-resistant non-small cell lung cancer to gefitinib by suppression of PI3K/Akt signaling pathway and inhibites cell migration via targeting ZEB1 . Biomed Pharmacother 2017 ; 85 : 113 – 119 . Google Scholar Crossref Search ADS WorldCat 103 Meng F , Wang F, Wang L et al. . MiR-30a-5p overexpression may overcome EGFR-inhibitor resistance through regulating PI3K/AKT signaling pathway in non-small cell lung cancer cell lines . Front Genet 2016 ; 7 : 197 . Google Scholar Crossref Search ADS WorldCat 104 Gao Y , Fan X, Li W et al. . miR-138-5p reverses gefitinib resistance in non-small cell lung cancer cells via negatively regulating G protein-coupled receptor 124 . Biochem Biophys Res Commun 2014 ; 446 : 179 – 186 . Google Scholar Crossref Search ADS WorldCat 105 Ahmad A , Maitah MY, Ginnebaugh KR et al. . Inhibition of Hedgehog signaling sensitizes NSCLC cells to standard therapies through modulation of EMT-regulating miRNAs . J Hematol Oncol 2013 ; 6 : 77 . Google Scholar Crossref Search ADS WorldCat 106 Kitamura K , Seike M, Okano T et al. . MiR-134/487b/655 cluster regulates TGF-beta-induced epithelial-mesenchymal transition and drug resistance to gefitinib by targeting MAGI2 in lung adenocarcinoma cells . Mol Cancer Ther 2014 ; 13 : 444 – 453 . Google Scholar Crossref Search ADS WorldCat Author notes Available online without subscription through the open access option © AlphaMed Press 2018 This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Concise Review: Resistance to Tyrosine Kinase Inhibitors in Non-Small Cell Lung Cancer: The Role of Cancer Stem Cells
JF - Stem Cells
DO - 10.1002/stem.2787
DA - 2018-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/concise-review-resistance-to-tyrosine-kinase-inhibitors-in-non-small-46ICx8Tamh
SP - 633
EP - 640
VL - 36
IS - 5
DP - DeepDyve
ER -