MINIREVIEW EGF receptor in relation to tumor development: molecular basis of responsiveness of cancer cells to EGFR-targeting tyrosine kinase inhibitors Kenji Takeuchi and Fumiaki Ito Department of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan Keywords cancer; epidermal growth factor receptor (EGFR); gefitinib; non-small cell lung cancer (NSCLC); tyrosine kinase inhibitor (TKI) Correspondence K Takeuchi, Department of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka 573-0101, Japan Fax: +81 72 866 3117 Tel: +81 72 866 3118 E-mail: takeuchi@pharm.setsunan.ac.jp (Received 17 July 2009, revised 17 September 2009, accepted 13 October 2009) doi:10.1111/j.1742-4658.2009.07450.x The function of the epidermal growth factor receptor (EGFR) is dysregulated in various types of malignancy as a result of gene amplification, mutations, or abnormally increased ligand production Therefore, the tyrosine kinase activity of the EGFR is a promising therapeutic target EGFR tyrosine kinase inhibitors, such as gefitinib (Iressa), show evident anticancer effects in patients with non-small cell lung cancer The induction of apoptosis has been considered to be the major mechanism for these gefitinibmediated anticancer effects Lung cancer cells harboring mutant EGFRs become dependent on them for their survival and, consequently, undergo apoptosis following the inhibition of EGFR tyrosine kinase by gefitinib Gefitinib has been shown to inhibit cell survival and growth signaling pathways such as the extracellular signal-regulated kinase ⁄ pathway and the Akt pathway, as a consequence of the inactivation of EGFR However, the precise downstream signaling molecules of extracellular signal-regulated kinase ⁄ and Akt have not yet been elucidated In this minireview we have highlighted the effect of tyrosine kinase inhibitors on members of the Bcl-2 family of proteins, which are downstream signaling molecules and serve as the determinants that control apoptosis We also discuss tyrosine kinase inhibitor-induced apoptosis via c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase Introduction The epidermal growth factor receptor (EGFR) is composed of an extracellular ligand-binding domain, a transmembrane domain and an intracellular tyrosine kinase domain The binding of a ligand to the extracellular domain of the EGFR induces receptor dimerization, activation of the intracellular kinase domain and autophosphorylation of tyrosine residues within the cytoplasmic domain of the receptor The tyrosinephosphorylated motifs of the EGFR recruit various adaptors or signaling molecules [1,2] The EGFR is able to activate a variety of signaling pathways through its association with these molecules Extra- Abbreviations BH, Bcl-2 homology domain; Bim, Bcl-2 interacting mediator of cell death; CDK, cyclin-dependent kinase; CRE, cAMP-response element; CREB, CRE-binding protein; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; HNSCC, head and neck squamous cell carcinomas; IAP, inhibitor of apoptosis protein; JNK, c-Jun NH2-terminal kinase; KIP, kinase inhibitor proteins; MEK, MAPK ⁄ ERK kinase; MKK, MAPK kinase; MKP-1, mitogen-activated protein kinase phosphatase-1; MOMP, mitochondrial outer membrane permeabilization; NSCLC, non-small cell lung cancer; Pak1, p21-activated kinase 1; PI3K, phosphatidylinositol 3-kinase; PUMA, p53 up-regulated modulator of apoptosis; RB, retinoblastoma; TKI, tyrosine kinase inhibitor 316 FEBS Journal 277 (2010) 316–326 ª 2009 The Authors Journal compilation ª 2009 FEBS K Takeuchi and F Ito Molecular mechanisms of sensitivity to EGFR-TKIs Fig Major signaling pathways downstream of the activated EGFR Activation of several signaling cascades triggered predominately by the ERK1 ⁄ and the PI3K ⁄ Akt pathways results, in turn, in the inactivation of pro-apoptotic Bcl-2 proteins (e.g PUMA, Bax, Bim and Bad), Fas, and CDK inhibitors (e.g p27KIP1, p21WAF1 and p15INK4b), and also in the activation of Pak1 cellular signal-regulated kinase (ERK)1 ⁄ 2, which is one of the three major groups of mitogen-activated protein kinases (MAPKs) in mammals, is activated by the EGFR tyrosine kinase and plays an essential role in cell proliferation In contrast, EGFR signaling inhibits the activation of the other two MAPKs, namely p38 MAPK and c-Jun NH2-terminal kinase (JNK) Furthermore, the phosphatidylinositol 3-kinase (PI3K) ⁄ Akt pathway, which is activated by the EGFR, has been implicated in both cell proliferation and survival Potential targets of these MAPK and PI3K ⁄ Akt signaling pathways include apoptosis-related molecules (Bcl-2 family members and Fas) and cell-cycle regulatory molecules (e.g p27KIP1; Fig 1) The EGFR therefore plays an important role in both cell proliferation and survival EGFR function is dysregulated in various types of malignancy [1,2] as a result of gene amplification, mutations (resulting in a constitutively active EGFR) or abnormally increased ligand production (reviewed in [3]) Moreover, enforced expression of mutant EGFRs in transgenic mice promotes the development of lung carcinomas [4,5] Therefore, EGFR-tyrosine kinase is a promising therapeutic target Small molecules that are active orally against the EGFR [e.g gefitinib (Iressa) and erlotinib (Tarceva)] show evident anticancer effects in patients with non-small cell lung cancer (NSCLC) [6–8] Beneficial responsiveness to these EGFR-targeting tyrosine kinase inhibitors (TKIs) in patients with NSCLC is closely associated with EGFR mutations such as del746-750 and L858R in the kinase domain [9–11] Lung cancer cells harboring mutant EGFRs become dependent on them for their survival and, consequently, undergo apopto- sis following inhibition of EGFR tyrosine kinase by gefitinib Gefitinib has been shown to inhibit cell growth and survival signaling pathways, such as the ERK1 ⁄ pathway and the Akt pathway, as a consequence of inactivation of the EGFR [12] With reference to Fig 1, which presents an overview of the intracellular signaling pathways activated by the EGFR tyrosine kinase, we will describe some of the diverse actions of TKIs on cell growth, cell survival and cell motility Akt and ERK1 ⁄ signaling pathways as target pathways for TKIs Akt induces the phosphorylation of pro-caspase-9, thereby inhibiting its protease activity [13] Furthermore, hepatocyte growth factor significantly inhibits adriamycin-induced apoptosis in the human gastric adenocarcinoma cell line MKN74 through phosphorylation of pro-caspase-9 via the Akt signaling pathway [14] Akt also phosphorylates Bad [15], a pro-apoptotic member of the Bcl-2 family, and the forkhead transcription factor FKHR [16], a pro-apoptotic transcription factor Therefore, the Akt signaling pathway has emerged as the major mechanism by which growth factors promote cell survival (reviewed in [17]) A link between the Akt pathway and gefitinibresponsiveness was reported by Engelman et al [18]: the Akt pathway is down-regulated in response to gefitinib only in NSCLC cell lines that are growth-inhibited by gefitinib Thus, activated Akt has been indicated as a molecular determinant of a response to EGFR-targeting drugs However, the NSCLC cell line H3255, harboring the L858R mutation in EGFR exon FEBS Journal 277 (2010) 316–326 ª 2009 The Authors Journal compilation ª 2009 FEBS 317 Molecular mechanisms of sensitivity to EGFR-TKIs K Takeuchi and F Ito 21, and PC-9, harboring a deletion (del746-750) in EGFR exon 19, are highly sensitive to gefitinib; and this sensitivity to gefitinib is associated with dependence on both Akt and ERK1 ⁄ pathways [10,19] Apoptosis The induction of apoptosis has been considered as the major mechanism for gefitinib-mediated anticancer effects Mammals have two distinct, but ultimately converging, apoptosis signaling pathways: the extrinsic (also called ‘death receptor’) pathway, which is activated by death receptors; and the intrinsic (also called ‘mitochondrial’ or ‘Bcl-2-regulated’) pathway [20] The intrinsic pathway is characterized by the permeabilization of the outer mitochondrial membrane and the release of several pro-apoptotic factors into the cytoplasm For mitochondrial outer membrane permeabilization (MOMP), a coordinated effort between numerous Bcl-2 proteins must be engaged (reviewed in [21]) The Bcl-2 proteins can be divided into three groups according to their function (Fig 2) Members of the Bcl-2 protein family are distinguished by the presence of up to four different Bcl-2 homology domains (designated BH1–4) The multidomain proapoptotic Bcl-2 proteins, Bax and Bak, contain BH1–3 domains and only induce MOMP following apoptotic stimuli, resulting in the release of cytochrome c, activation of the caspase cascade and cellular destruction [22] To prevent cell death, Bax and Bak are bound and inhibited by the anti-apoptotic members of the Bcl-2 protein family (Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and A1), which contain four BH domains [22] The third subgroup, the BH3-only proteins, are structurally diverse and contain only one conserved domain (BH3) Often, the BH3-only proteins are subdivided into direct activators [Bid and Bcl-2 interacting mediator of cell death (Bim)] and de-repressors [Bad, Bik, Bmf, NOXA, and p53 up-regulated modulator of apoptosis (PUMA)] These de-repressors initiate apoptosis signaling by binding and antagonizing the anti-apoptotic Bcl-2 family members, thereby causing activation of Bax and Bak [23] Regulation of Bcl-2 family members can occur by a number of mechanisms, including up-regulation of synthesis, enhancement of degradation and phosphorylation In the event that cancer cells undergo apoptosis in response to gefitinib, inhibition of Akt- and ERK1 ⁄ 2-dependent pathways eventually change the expression level of one or more of these Bcl-2 family members Bad Bad is one of the ‘death-promoting’ members of the Bcl-2 family, and its pro-apoptotic activity is regulated primarily by phosphorylation at several sites [24] Activated Akt [13,25] and ERK1 ⁄ 2-p90 ribosomal S6 kinase-1 (p90Rsk-1) [26,27] pathways have been shown to promote survival signaling by phosphorylating Bad at Ser136 and Ser112, respectively These phosphorylated residues provide binding sites for 14-3-3 proteins, which subsequently sequester Bad Phosphorylation of Bad-Ser112 via ERK1 ⁄ pathway ( in Fig 2) is inhibited by either gefitinib or the MAPK ⁄ ERK kinase (MEK) inhibitor PD98059 in mammary epithelial cells and primary cultures of malignant breast carcinoma [28] Gefitinib has no effect on EGF-mediated Bad-Ser112 phosphorylation in the cells transfected with vectors encoding constitutively active p90Rsk-1 Thus, the EGF induces Bad phosphorylation through an ERK1 ⁄ pathway involving p90Rsk-1 It has also been reported that primary cultures of Bad) ⁄ ) mammary cancer cells are no longer sensitive to gefitinib-induced apoptosis, suggesting that Bad might be an important pro-apoptotic effector molecule downstream of the EGFR Fig Bcl-2 family proteins as targets of TKIs 318 FEBS Journal 277 (2010) 316–326 ª 2009 The Authors Journal compilation ª 2009 FEBS K Takeuchi and F Ito Molecular mechanisms of sensitivity to EGFR-TKIs PUMA Bax PUMA was initially identified as a critical mediator of apoptosis induced by the tumor suppressor p53 [29,30] and it can be directly activated by p53 through p53responsive elements in its promoter region PUMA can also induce p53-independent apoptosis in response to a wide variety of stimuli [31] Therefore, PUMA is a critical mediator of both p53-dependent and p53-independent apoptosis and mediates apoptosis through the Bcl-2 family proteins Bax ⁄ Bak [32] PUMA is induced by gefitinib, independently of p53, in head and neck squamous cell carcinomas (HNSCC) [33] This BH3only protein functions as a critical mediator of gefitinib-induced apoptosis, and in the Akt pathway and p73, p53 family proteins serve as key regulators of PUMA induction after EGFR inhibition (pathway in Fig 2) Overexpression of EGFR is found in more than 80% of HNSCC Thus, TKIs have emerged as promising treatments, not only for NSCLC but also for HNSCC Bax is a 21-kDa multi-BH domain pro-apoptotic protein and acts downstream of BH3-only proteins The induction of Bax expression can be sufficient to induce apoptosis and requires no additional death stimulus [41] Furthermore, Bax expression is associated with tumor development [42] The protein is normally found in the cytoplasm, where it is heterodimerized to antiapoptotic Bcl-2 family members such as Mcl-1 and Bcl-xL; however, once the cell is exposed to an apoptotic stimulus, Bax is translocated to the mitochondria [43] and induces mitochondrial dysfunction, characterized by the formation of large pores in the mitochondrial membrane [44] Stimulation of the Akt pathway inhibits Bax translocation from the cytoplasm to the mitochondria and promotes survival [45] Anti-apoptotic stimuli lead to the activation of Akt and to Ser184 phosphorylation of Bax [46] This phosphorylation promotes the sequestration of Bax in the cytoplasm and increases the ability of Bax to heterodimerize with the anti-apoptotic Bcl-2 family members Mcl-1 and Bcl-xL, thereby inhibiting activation of apoptosis signals Gefitinib is known to induce apoptosis through shutdown of Akt signaling However, it has not been demonstrated whether this shutdown transmits the apoptotic signal via inhibition of Bax phosphorylation Regulation of Bax also occurs by mechanisms other than phosphorylation Gefitinib inhibits growth of human gallbladder adenocarcinoma cells (HAG-1) by arresting the cells in the G0 ⁄ G1 phase [47] This arrest is accompanied by depression of cyclin D1 mRNA as well as by the accumulation of p27 protein However, when HAG-1 cells are treated with gefitinib for more than 72 h, the apoptotic population increases Correspondingly, gefitinib up-regulates expression of total Bax, with a subsequent increase in p18 Bax that has been shown to be generated through the cleavage of full-length Bax during apoptosis (pathway in Fig 2) Cleavage of Bax into p18 Bax occurs in response to various stimuli, such as interferon-a [48] and chemotherapeutic agents [49] p18 Bax fragment is as efficient as full-length Bax in promoting cytochrome c release [49,50] and more potent than full-length Bax in inducing apoptotic cell death [51] It is also suggested that an increase in gefitinib-induced expression of total Bax is caused by the decreased degradation of Bax As ERK1 ⁄ and Akt are significantly inhibited in gefitinib-treated HAG-1 cells, simultaneous inhibition of these pathways by gefitinib may lead to the accumulation of Bax and subsequent apoptosis As described below, gefitinib initiates the intrinsic pathway of apop- Bim Bim is a member of the BH3-only proteins [34] Under conditions that promote cell growth, Bim is bound to dynein light chain (LC8) of the microtubular motor complex and is sequestered away from other Bcl-2 family members [35] Following a proapoptotic stimulus, however, Bim is localized to the mitochondria, where it initiates the mitochondrial cell death pathway by directly activating Bax ⁄ Bak [36] Bim expression is regulated by both transcriptional in Fig 2) and post-transcriptional levels (pathway Phosphorylation of Bim by ERK1 ⁄ targets Bim for degradation by the ubiquitin-proteasome system [37] Bim has recently been reported to mediate gefitinibinduced apoptosis [38–40] Bim knockdown by RNA interference protects the NSCLC cell line, H3255, potently against gefitinib, and the level of protection correlates with the extent of Bim reduction, indicating that Bim is essential for gefitinib-induced apoptosis of NSCLC cells The induction of Bim after treatment with gefitinib is a consequence of both transcriptional induction and dephosphorylation Thus, shutdown of the EGFR-MEK-ERK signaling cascade by gefitinib elicits Bim accumulation and causes apoptosis The T790M mutation of the EGFR, which renders gefitinib and erlotinib ineffective inhibitors of EGFR kinase activity, blocks gefitinb-induced up-regulation of Bim and apoptosis [38] These experiments point to an important role for the induction of Bim in gefitinib-triggered apoptosis of NSCLC cells FEBS Journal 277 (2010) 316–326 ª 2009 The Authors Journal compilation ª 2009 FEBS 319 Molecular mechanisms of sensitivity to EGFR-TKIs K Takeuchi and F Ito tosis through p38a-dependent Bax activation in intestinal epithelial cells (pathway in Fig 2) Accordingly, inhibition of Akt-dependent Bax phosphorylation at Ser184, generation of the p18 Bax fragment and p38a-dependent Bax activation are proposed for Bax activation Further studies are needed to understand the precise mechanism by which gefitinib induces apoptosis in a Bax-dependent manner Inhibitor of apoptosis protein family Induction of MOMP following apoptotic stimuli results in the release of cytochrome c and subsequent activation of caspase-9 and caspase-3, which are cystein proteases that cleave vital cellular targets and cause apoptosis Caspases can be inhibited by members of the inhibitor of apoptosis protein (IAP) family such as cIAP-1, cIAP-2, X-linked IAP and survivin Recent studies have suggested that activation of the PI3K ⁄ Akt pathway by EGFR signaling causes up-regulation of survivin expression [52] The levels of cIAP-2 are down-regulated by gefitinib or erlotinib in intestinal epithelial cells [53] Furthermore, the expression of cIAP-1 and of X-linked IAP is reduced by AG1478 in squamous cell carcinoma cell lines NA and Ca9-22 [54] As small interfering RNA (siRNA)-based depletion of IAP increases apoptosis in response to gefitinib, IAPs might be a molecular target for the induction of apoptosis by TKIs Inhibition of cell proliferation EGFR signaling activates a variety of pathways such as those for cell survival, cell proliferation, cell motility, angiogenesis and expression of extracellular matrix proteins [55] Accordingly, TKIs against EGFR exert not only apoptosis-inducing action but also other divergent actions For instance, EGFR inhibition leads to the induction of cell-cycle arrest at the G1-S boundary [56] Cell-cycle regulation is important in growth control, and therefore deregulation of the cell-cycle machinery has been implicated in carcinogenesis [57] Cyclins and cyclin-dependent kinases (CDKs), in association with each other, play key roles in promoting the G1-to-S phase transition of the cell-cycle by phosphorylating the retinoblastoma (RB) protein Activation of cyclin–CDK complexes is counterbalanced by CDK inhibitors, including those of the kinase inhibitor proteins (KIP) family and the INK4 family (Fig 3) The KIP family consists of p27KIP1, p21WAF1 ⁄ CIP1 and p57KIP2; and the INK4 family consists of p15INK4b, p16INK4a, p18INK4c and p19INK4d AG1478, which, like gefitinib and erlotinib, acts as a specific inhibitor of the EGFR tyrosine kinase, has been shown to result in a dose-dependent up-regulation of p27KIP1 and in hypophosphorylation of the RB protein in human epidermoid carcinoma cell line A431 cells [56] These changes are temporally associated with recruitment of tumor cells in the G1 phase and a marked reduction in the proportion of cells in the S phase The G1 arrest and up-regulation of p27KIP1 resulting from EGFR blockade are caused by the interruption of PI3K signals In addition to p27KIP1, p21WAF1 ⁄ CIP1 is involved in gefitinib-induced growth inhibition in HNSCC [58] Another group of cell-cycle regulatory molecules – those of the INK4 family – has also been implicated in gefitinib-induced inhibition of cell growth Gefitinib up-regulates p15INK4b in human immortalized keratinocyte HaCaT cells and results in RB hypophosphorylation and G1 arrest [59] Moreover, mouse embryo fibroblasts lacking p15INK4b are resistant to the growth-inhibitory effects of gefitinib As the level of p15INK4b is increased by MEK inhibitors, but not by Akt inhibitors, the induction of Fig The cell-cycle is arrested at the G1 phase by TKI-induced CDK inhibitors EGFRTKIs result in the up-regulation of CDK inhibitors, including KIP family members and INK4 family members Members of the KIP family can inhibit the catalytic activity of CDK2, and Members of the INK4 family are specific inhibitors of the cyclin D–CDK4 ⁄ complex It is not yet known if EGFR-TKIs can stop G2 transition and G2 ⁄ M cell-cycle progression by up-regulation of KIP family members 320 FEBS Journal 277 (2010) 316–326 ª 2009 The Authors Journal compilation ª 2009 FEBS K Takeuchi and F Ito p15INK4b by inhibition of the ERK1 ⁄ pathway is associated with the antiproliferative effects of gefitinib Inhibition of cell motility The EGFR can transmit signals for re-organization of the cytoskeleton, the formation of lamellipodia, membrane ruffling and changes in cell morphology Accordingly, dysregulation of the EGFR contributes to the progression, invasion and maintenance of the malignant phenotype In keratinocyte and cutaneous squamous cancer cells, gefitinib blocks EGF-induced cytoskeleton remodeling and in vitro invasiveness, as well as cell growth [60] Gefitinib also effectively inhibits ERK1 ⁄ activation and p21-activated kinase (Pak1) activity (see Fig 1) Pak1 is a serine ⁄ threonine kinase and is a critical component of many growth factor receptor-mediated signal transduction pathways, leading to directional cell motility and cell invasiveness [61] Because deregulation of EGFR signaling is commonly associated with stimulation of ERK1 ⁄ and Pak1 pathways, gefitinib might lead to inhibition of invasiveness of human cancer cells through the inhibition of ERK1 ⁄ and Pak1 The use of gefitinib in cells with activated ERK1 ⁄ or Pak1 pathways might potentially lead to beneficial anti-cancer activity through the inhibition of not only cell survival but also cell invasiveness p38, JNK and Fas as target molecules of gefitinib p38 As described above, the treatment of intestinal epithelial cells with gefitinib results in a dramatic increase in apoptosis and activation of the intrinsic apoptotic pathway via trafficking of activated Bax to the mitochondria [62] Akt is known to phosphorylate Bax and to prohibit its mitochondrial translocation However, the Akt pathway plays a minor role in the induction of apoptosis in intestinal epithelial cells Instead, p38 MAPK phosphorylation is associated with mitochondrial translocation of Bax and subsequent induction of in apoptosis following EGFR inhibition (pathway Fig 2) Furthermore, p38a, one of the four p38 isoforms, is required for Bax activation and apoptosis Because activation of p38 by UVB irradiation in human keratinocytes results in induction of a conformational change in Bax and its translocation to mitochondria [63], p38 may be an important upstream molecule of Bax activation in response to a variety of apoptosis-inducing stimuli Molecular mechanisms of sensitivity to EGFR-TKIs JNK and mitogen-activated protein kinase phosphatase-1 The activity of JNK, one of the MAPKs, is tightly controlled by both protein kinases, such as MAPK kinase (MKK4) or MAPK kinase (MKK7) and protein phosphatases such as MAPK phosphatase (MKP) Mitogen-activated protein kinase phosphatase1 (MKP-1) is a dual-specificity protein phosphatase, which can dephosphorylate both phosphothreonine and phosphotyrosine residues and subsequently block the activities of MAPKs [64] Although MKP-1 was initially characterized as an ERK-specific phosphatase [65], subsequent studies have determined that MKP-1 preferentially acts on JNK and p38 MAPK in response to various stresses [66] MKP-1 has been correlated with tumorigenesis Several observations have indicated that MKP-1 is overexpressed in human tumors Constitutive expression levels of MKP-1 in NSCLC cell lines are higher than those found in normal cells under basal growth conditions [67] Overexpression of MKP-1 has been reported to protect cells against apoptosis induced by UV irradiation, Fas ligand, cisplatin, paclitaxel, proteasome inhibitors or radiation therapy [68] These observations have established that MKP-1 plays an important role in resistance against many types of stresses, including anticancer drugs, in various cell lines MKP-1 may be a rational target to enhance anticancer drug activity Our recent results have shown that the activation of JNK induced by EGFR-TKI AG1478 is critical for the apoptotic action of AG1478 against the NSCLC cell line PC-9 [69] Various types of stimuli activate JNK through phosphorylation by the dual-specificity JNK kinases; but JNK kinases MKK4 and MKK7 are not activated by AG1478 treatment In contrast, JNK phosphatase (i.e MKP-1) is constitutively expressed in PC-9 cells and its expression level is reduced by AG1478 Furthermore, the inhibition of JNK activation by ectopic expression of MKP-1 or a dominant-negative form of JNK strongly suppresses AG1478-induced apoptosis Thus, JNK, which is activated through the decrease in the MKP-1 level, is critical for the apoptotic action of AG1478 against PC-9 cells Interestingly, AG1478 has no inhibitory activity towards MKP-1 expression in some resistant cell lines isolated from gefitinib-sensitive PC-9 cells (unpublished data of T Shin-ya, K Takeuchi and F Ito) Although MKP-1 expression has been implicated in cancer and in TKI sensitivity, the mechanism by which EGFR activation controls the MKP-1 expression level is unclear MKP-1 is encoded by an early response gene, which is transiently induced by mitogens and FEBS Journal 277 (2010) 316–326 ª 2009 The Authors Journal compilation ª 2009 FEBS 321 Molecular mechanisms of sensitivity to EGFR-TKIs K Takeuchi and F Ito stress signals such as serum, growth factors, cytokines, UV irradiation, heat shock, hypoxia and anticancer drugs [68] through both transcriptional [70,71] and post-translational [72,73] mechanisms As depicted in Fig 3, the human mkp-1 gene contains four exons and three introns coding for an inducible mRNA that is approximately 2.4 kb long [74] The promoter ⁄ enhancer region of the mkp-1 gene contains multiple activator protein (AP2), trans-acting transcription factor (SP1) and cAMP-response element (CRE) sites, but only one site for each of activator protein (AP1), neurofibromin (NF1) and TATA box [74] ERK1 ⁄ can phosphorylate Ser133 of the CRE-binding protein (CREB) through p90Rsk-2, and Ser133 phosphorylation is required for CREB-mediated transcription [75] These results suggest that EGFR signaling can induce the transcription of mkp-1 via phosphorylation of CREB However, induction of MKP-1 in mouse embryo fibroblasts following treatment with arsenite and irradiation with UVC is predominantly mediated by the p38 MAPK pathway Both p38 MAPK and ERK have been implicated in the transcriptional induction of MKP-1, and each may use a different set of transcription factors to enhance MKP-1 expression Several lines of evidence suggest that phosphorylation of MKP-1 protein plays an important role in the stabilization of MKP-1 (Fig 4) ERK1 ⁄ reduces MKP-1 degradation by phosphorylating the Ser359 and Ser364 residues of MKP-1 [72] ERK1 ⁄ is also responsible for the degradation of MKP-1 via the phosphorylation of Ser296 and Ser323 residues [76] Once phosphorylated, Skp2 (also called SCFSkp2 of Skp1 ⁄ Cul1 ⁄ F-box protein Skp2; ubiquitin-protein isopeptide ligase E3) targets MKP-1 for degradation via the ubiquitin proteasomal pathway [73] In addition to the transcriptional and post-translational control described here, it is suggested that transcription of the mkp-1 gene is also controlled at the level of transcriptional elongation [71] The mechanism responsible for the regulation of MKP-1 expression is complex, and both transcriptional down-regulation and degradation of MKP-1 may be effects observed in cells having an apoptotic response to EGFR-TKI AG1478 Fas Exposure of the human NSCLC cell line, A549, to gefitinib causes a marked increase in the expression of Fas protein and in the activation of caspases 2, and [77] Co-treatment of cells with Fas antagonist antibody significantly blocks gefitinib-induced apoptosis Furthermore, caspase-8 and caspase-3 inhibitors, but not a caspase-9 inhibitor, are capable of restoring cell viability Thus, Fas appears to play a major role in the initiation of gefitinib-induced apoptosis through activation of the caspase-8 ⁄ caspase-3 cascade Treatment of A549 cells with gefitinib results in the translocation of p53 from the cytosol to the nucleus Moreover, inhibition of p53 using antisense oligonucleotide causes down-regulation of Fas and a significant decrease in gefitinib-induced apoptosis p53 may thus play a role in determining gefitinib sensitivity by regulating Fas expression in NSCLC Conclusions Important regulators of cell survival and apoptosis are the Bcl-2 family of proteins Members of this family, such as Bcl-2 and Bcl-xL, can inhibit apoptosis, Fig Regulation of MKP-1 expression by EGFR signaling MKP-1 expression is controlled through both transcription and post-translation steps ERK1 ⁄ 2, JNK and p38 MAPK can activate transcription of the mkp-1 gene The promoter ⁄ enhancer region of the mkp-1 gene has the potential to bind many transcription factors AP1, activator protein 1; CAD, phosphatase catalytic domain; CRE, cAMP-responsive element; NF1, neurofibromin 1; SP1, trans-acting transcription factor 322 FEBS Journal 277 (2010) 316–326 ª 2009 The Authors Journal compilation ª 2009 FEBS K Takeuchi and F Ito whereas others promote apoptosis The balance between these pro-apoptotic and anti-apoptotic Bcl-2 family members determines the cellular fate (i.e survival or apoptosis) In cancer cells that undergo apoptosis in response to TKIs, shutdown of ERK1 ⁄ and PI3K ⁄ Akt signaling pathways following the inhibition of EGFR activation ultimately results in the disruption of the balance between pro-apoptotic and anti-apoptotic Bcl-2 proteins and subsequent apoptosis Bcl-2 family proteins are key molecules for regulating the permeabilization of the outer mitochondrial membrane and thus represent pivotal components in TKI-dependent apoptosis signaling TKIs change the transcription level of Bcl-2 family genes and the phosphorylation state of their proteins, thereby changing the amount and localization of Bcl-2 family members However, each type of cancer has its own way of disrupting the balance of the networks of signaling cascades following TKI treatment Therefore, understanding how Bcl-2 family members are regulated in each type of cancer is critical for understanding how TKIs cause apoptosis in each of them It is now clear that TKIs are unlikely to provide cures for the majority of patients with NSCLC Despite the initial dramatic efficacy of gefitinib and erlotinib in NSCLC patients with EGFR mutations, all patients ultimately develop resistance to TKIs A secondary mutation in the EGFR (T790M) 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