. "10.1186%2Fs12943-015-0392-3" . _:b41359973 . _:b41360017 . _:b581303797 . . _:b41359980 . _:b41359990 . . _:b41359966 . _:b581303792 . _:b41359988 "Immunohistochemical analyses to detect PCNA, cleaved caspase-3, pIGF-1R, and pSrc expression in the xenograft and PDX tumors were performed as described previously [>>52<<]. In brief, formalin-fixed and paraffin-embedded tissue sections (4 \u03BCm) were deparaffinized, dehydrated, and treated with methanol containing 3\u00A0% hydrogen peroxide." . _:b41360011 . _:b41360021 "The canonical activation of IGF-1R results from the binding of ligands (IGF1 and IGF2) to IGF-IR, insulin receptor (IR) (with a lower affinity than insulin), and hybrid receptors of IGF-1R/IR [6, >>11<<], leading to the autophosphorylation of tyrosine residues 1131, 1135, and 1136 in the activation loop of the IGF-1R \u03B2-chain (the corresponding residues in the human IR are 1158, 1162, and 1163, respectively)." . _:b581303819 . _:b41360003 "De novo and acquired resistance to inhibitors of RTK have been suggested to occur due to the mutation, amplification, and increased/decreased expression of signaling molecules involved in multiple counter-regulatory pathways [>>39<<]. However, unlike EGFR, neither activating mutations nor gene amplifications of IGF-1R are common [40]." . _:b41359970 "EGF stimulation increased EGFR, Akt, Src, and IGF-1R phosphorylation in A549 and H460 cells but not in H522, a low EGFR-expressing cell line [>>23<<] (Fig.\u00A02b). This EGF-induced IGF-1R phosphorylation was suppressed by treatment with the clinically available small molecular Src inhibitor dasatinib [24] (Fig.\u00A02c), by transfection with an siRNA against Src (Fig.\u00A02d), and by treatment" . _:b581303810 . . _:b41360007 . . _:b41359973 "Previous reports suggested that Src can directly phosphorylate IGF-1R at the sites of ligand-induced autophosphorylation [12, >>13<<]. Consistent with this finding, in vitro kinase assays showed the ability of Src, derived from A549 cells or recombinant protein (rSrc), to phosphorylate recombinant IGF-1R protein (GST-IGF-1R) (Fig." . . _:b41359977 . _:b581303793 . _:b41360013 _:b41360016 . _:b41360013 _:b41360017 . _:b41360013 _:b41360018 . _:b41360013 _:b41360019 . _:b41359961 . . _:b41360013 _:b41360020 . _:b41360013 _:b41360021 . _:b41360013 _:b41360022 . _:b41360013 _:b41360023 . _:b41360013 _:b41360024 . _:b41360013 _:b41360025 . _:b41359971 "This EGF-induced IGF-1R phosphorylation was suppressed by treatment with the clinically available small molecular Src inhibitor dasatinib [>>24<<] (Fig.\u00A02c), by transfection with an siRNA against Src (Fig.\u00A02d), and by treatment with the EGFR TKI erlotinib, but the IGF-1R TKI linsitinib exhibited relatively minimal effects on the suppression of EGF-induced IGF-1R phosphorylation" . _:b41360013 _:b41360026 . _:b41360012 "In addition, several combination strategies, including the combination of EGFR TKIs with inhibitors of downstream EGFR targets, such as PI3K or STAT5, have been tested in preclinical models of EGFR-mutant NSCLCs [>>44<<]. Although these strategies are promising, their clinical efficacy remains to be evaluated. Hence, there is a need for alternative strategies to address the problem of resistance to EGFR TKIs. In this regard, our current findings suggest" . _:b41360026 . _:b41360018 "The insulin-like growth factor receptor (IGF-1R) signaling pathway plays a critical role in cancer cell survival, causing resistance to numerous anticancer drugs [>>5<<\u20137]. Therefore, effective regimens to inactivate the IGF-1R pathway may sensitize cancer cells to anticancer therapies and provide clinical benefits to cancer patients." . _:b41360006 . . _:b41360005 . _:b41359981 . . . _:b41360026 . . _:b41359989 "The detailed procedures for immunohistochemical analysis and scoring of tissue microarray were described in our previous report [>>15<<]." . . _:b41360002 . _:b41360013 _:b41360014 . _:b41360013 _:b41360015 . _:b41359983 . . _:b581303789 "3"^^ . _:b41359975 . _:b41360018 . . _:b581303788 "3"^^ . _:b41359985 . _:b581303791 "3"^^ . _:b41360020 "The canonical activation of IGF-1R results from the binding of ligands (IGF1 and IGF2) to IGF-IR, insulin receptor (IR) (with a lower affinity than insulin), and hybrid receptors of IGF-1R/IR [>>6<<, 11], leading to the autophosphorylation of tyrosine residues 1131, 1135, and 1136 in the activation loop of the IGF-1R \u03B2-chain (the corresponding residues in the human IR are 1158, 1162, and 1163, respectively)." . _:b581303790 "3"^^ . _:b41359991 . . _:b41360004 "However, unlike EGFR, neither activating mutations nor gene amplifications of IGF-1R are common [>>40<<]. We have demonstrated that the activation of the IGF-1R pathway through the increased expression of tissue-derived IGFs and the loss of IGFBP-3 is an early event in lung carcinogenesis [18, 41]. However, an analysis of previously studied" . . _:b41359979 . _:b581303785 "4"^^ . _:b41360011 "Recent studies suggest that second- and third-generation EGFR inhibitors are more potent than gefitinib or erlotinib (at least in pre-clinical models) against the drug-resistant mutation [>>43<<]. In addition, several combination strategies, including the combination of EGFR TKIs with inhibitors of downstream EGFR targets, such as PI3K or STAT5, have been tested in preclinical models of EGFR-mutant NSCLCs [44]. Although these" . _:b581303784 "6"^^ . _:b581303787 "3"^^ . _:b41359992 . _:b581303786 "4"^^ . _:b581303797 "2"^^ . "PMC0" . _:b581303796 "2"^^ . _:b41359997 . _:b581303791 . _:b581303796 . _:b41359998 "IR has also been suggested to mediate intrinsic and/or acquired resistance to IGF-1R mAbs [>>30<<, 35]. In this regard, dual IGF-1R/IR TKI regimens may be more effective in blocking the IGF-1R pathway in patients with NSCLC. However, the dual IGF-1R/IR TKI linsitinib has shown moderate efficacy and/or severe toxicity in multiple" . . _:b581303799 "2"^^ . _:b41360015 "Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related deaths worldwide [1], and the 5-year survival rate for patients with advanced NSCLC remains less than 20\u00A0% [>>2<<]. Although a small subset of patients with specific genetic and epigenetic abnormalities has shown clinical response to specific targeted therapies [3], most patients with NSCLCs are insensitive to chemotherapy and radiation1." . _:b41360014 . _:b581303798 "2"^^ . _:b41359999 . _:b41359993 . _:b41360019 . _:b581303793 "3"^^ . _:b41359978 "Notably, co-targeting Src and IGF-1R effectively suppressed the colony formation of EGFR TKI-resistant NSCLC cells (H1975, PC-9/GR and PC-9/ER) [>>28<<] (Additional file 18:" . _:b41359986 "The pBabe-Puro EGFR WT plasmid was purchased from Addgene (catalog # 11011, Cambridge, MA, USA) [>>51<<]. The mutant Src (Y527F) expression vector was kindly provided by Dr. Faye M. Johnson (MD Anderson Cancer Center, Houston, TX, USA)." . _:b41359968 "Transfection with the constitutively active Src phosphorylated IGF-1R, EGFR (Y1068 and Y845), Src, and FAK (Y576, a Src-specific phosphorylation site [>>21<<]), and Akt (S473) but not FAK (Y397, an integrin signaling-induced autophosphorylation site [22]) or ERK1/2 in H226Br and H226B cells (Fig." . _:b41359976 . _:b581303792 "3"^^ . _:b41359962 . . _:b581303795 "2"^^ . _:b41359996 . _:b581303805 . . _:b41359963 . _:b41360005 "We have demonstrated that the activation of the IGF-1R pathway through the increased expression of tissue-derived IGFs and the loss of IGFBP-3 is an early event in lung carcinogenesis [>>18<<, 41]. However, an analysis of previously studied tissue specimens from NSCLC patients and a panel of NSCLC cell lines revealed no correlation between the expression levels of pIGF-1R and IGFs. The previous reports, including; 1) the" . _:b41359964 . . _:b581303794 "2"^^ . _:b41359965 . _:b581303805 "2"^^ . _:b581303798 . _:b41359969 . _:b41359966 . _:b581303804 "2"^^ . _:b581303813 . _:b41359967 . _:b581303807 "2"^^ . _:b581303812 . _:b581303816 . _:b581303806 "2"^^ . _:b41359995 . _:b581303817 . _:b581303818 . _:b581303815 . _:b41360003 . . _:b581303819 . _:b581303820 . _:b41359974 . _:b41359961 . _:b581303801 "2"^^ . _:b581303814 . _:b41359962 . _:b581303808 . _:b581303800 "2"^^ . _:b581303809 . _:b581303810 . . _:b41359987 . . _:b581303811 . _:b581303809 . _:b41359963 . _:b581303812 . _:b581303803 "2"^^ . _:b581303813 . _:b581303814 . _:b41359988 . _:b581303808 . _:b581303815 . _:b41359972 . _:b581303802 "2"^^ . _:b581303811 . _:b41359973 . _:b41359992 . _:b581303810 . _:b41359974 . _:b41360021 . _:b41360013 "background" . _:b41359975 . _:b581303820 . . . _:b41359968 . _:b581303813 "2"^^ . _:b41359969 . _:b41360001 "However, the dual IGF-1R/IR TKI linsitinib has shown moderate efficacy and/or severe toxicity in multiple preclinical and clinical studies for various types of cancer3 [36, >>37<<]. Furthermore, the major components of the IGF-1R pathway, such as pIGF-1R, pAkt, pERK, pIRS, and pS6K, could not predict response to linsitinib [38]. Therefore, identifying and targeting the mechanism of resistance against the IGF-1R" . _:b581303812 "2"^^ . _:b41360023 "Previous reports have suggested that Src, a non-receptor protein tyrosine kinase, can directly phosphorylate IGF-1R [12, >>13<<]. Src also plays an important role in cancer cell survival and resistance to targeted anticancer therapies by acting as a common signaling facilitator that is activated by a myriad of redundant signaling pathways [12, 14]. The elevated" . _:b41359970 . _:b581303815 "2"^^ . _:b581303817 . _:b41359971 . _:b581303814 "2"^^ . _:b41360019 "However, patients who show a promising initial response to anti-IGF-1R monoclonal antibodies (mAbs) appear to rapidly acquire resistance to these mAbs [>>8<<\u201310]. Similarly, therapeutic efficacy of IGF-1R TKIs has been modest in a variety of human cancers, including NSCLC2 thus, there is an urgent need to understand the signaling pathways that confer inherent and/or acquired resistance to" . . _:b581303816 . _:b41359980 . _:b581303787 . _:b581303820 . _:b581303809 "2"^^ . _:b581303819 . _:b41359981 . . _:b581303808 "2"^^ . _:b581303818 . _:b41359982 . _:b581303811 "2"^^ . _:b581303790 . . _:b41359984 . _:b41359983 . _:b581303810 "2"^^ . _:b41359976 . _:b41360000 "However, the dual IGF-1R/IR TKI linsitinib has shown moderate efficacy and/or severe toxicity in multiple preclinical and clinical studies for various types of cancer3 [>>36<<, 37]. Furthermore, the major components of the IGF-1R pathway, such as pIGF-1R, pAkt, pERK, pIRS, and pS6K, could not predict response to linsitinib [38]. Therefore, identifying and targeting the mechanism of resistance against the IGF-1R" . . . _:b41359977 . _:b41359967 . _:b581303820 "2"^^ . . _:b41359978 . _:b41359979 . _:b41359997 "has shown that the inactivation of IGF-1R by the IGF-1R mAb resulted in enhanced signaling through IGF-dependent integrin/Src signaling and the Akt/mTOR-mediated protein synthesis of EGFR and Akt, leading to drug resistance [33, >>34<<]. IR has also been suggested to mediate intrinsic and/or acquired resistance to IGF-1R mAbs [30, 35]." . _:b581303800 . _:b41359988 . _:b581303817 "2"^^ . _:b41359989 . . _:b581303816 "2"^^ . _:b41359990 . _:b581303819 "2"^^ . _:b41360022 . _:b41359978 . _:b41360010 . _:b41359991 . _:b581303818 "2"^^ . _:b41359991 "discussion" . . _:b41359992 "These results, together with previous reports and our current findings showing the simultaneous over-expression/activation of multiple membrane-associated receptors in NSCLCs [>>29<<\u201331], suggest that combination regimens of molecularly targeted agents against IGF-1R and Src have a great potential for anticancer therapies." . _:b41359984 . . _:b41360024 . _:b41359993 . _:b41359985 . _:b41359986 . _:b41359966 "Based on previous findings suggesting that Src can activate IGF-1R in a ligand-independent manner [>>19<<], we examined the relationship between IGF-1R and Src in NSCLC." . . _:b41359971 . _:b41359987 . _:b581303814 . _:b41359996 . _:b41359968 . _:b41360019 . _:b41359997 . . _:b41359998 . . _:b41359999 . _:b41359993 . _:b41359992 . . . _:b41359993 . . . _:b581303816 . . _:b41359994 . _:b41359981 "Because the clinical development of TKIs of IGF-1R and Src is ongoing [45, >>46<<], our findings directly impact the current clinical management of patients with NSCLC." . . . _:b41359995 . _:b41360018 . _:b41360004 . _:b581303788 . . _:b581303811 . . _:b41360005 . _:b581303794 . _:b41360006 . . _:b41360000 . _:b41360008 "The previous reports, including; 1) the mutation and/or overexpression of various molecules involved in Src activation, such as EGFR, KRas, and HER2 [16, >>29<<], has been implicated in the mechanism of resistance to IGF-1R TKIs [16]; and 2) the aberrant overexpression and activation of IGF-1R and Src in many NSCLCs [42], and our current results, indicating; 1) a strong positive correlation" . _:b41360007 . . . _:b41360000 . _:b41359972 "Previous reports suggested that Src can directly phosphorylate IGF-1R at the sites of ligand-induced autophosphorylation [>>12<<, 13]. Consistent with this finding, in vitro kinase assays showed the ability of Src, derived from A549 cells or recombinant protein (rSrc), to phosphorylate recombinant IGF-1R protein (GST-IGF-1R) (Fig." . _:b41360009 "The previous reports, including; 1) the mutation and/or overexpression of various molecules involved in Src activation, such as EGFR, KRas, and HER2 [16, 29], has been implicated in the mechanism of resistance to IGF-1R TKIs [>>16<<]; and 2) the aberrant overexpression and activation of IGF-1R and Src in many NSCLCs [42], and our current results, indicating; 1) a strong positive correlation between pSrc and pIGF-1R in a large TMA of NSCLC tissues and in the majority" . _:b41360001 . . . _:b41360006 "We have demonstrated that the activation of the IGF-1R pathway through the increased expression of tissue-derived IGFs and the loss of IGFBP-3 is an early event in lung carcinogenesis [18, >>41<<]. However, an analysis of previously studied tissue specimens from NSCLC patients and a panel of NSCLC cell lines revealed no correlation between the expression levels of pIGF-1R and IGFs. The previous reports, including; 1) the mutation" . _:b41360002 . _:b41359983 "Considering the extensive toxicities of Src inhibitors and IGF-1R TKIs [>>49<<, 50], further studies are warranted to evaluate the efficacy of co-targeting IGF-1R and Src in additional preclinical and clinical settings." . . _:b41359990 "The quantitative analysis of blots using densitometry was performed with Image J software (National Institute of Health, Bethesda, MD, USA) [>>53<<]. The statistical significance of the data obtained from the in vitro experiments was analyzed using two-sided Student\u2019s t-tests. The statistical significance of data obtained from the in vivo experiments was analyzed via one-way or" . _:b41360003 . . _:b41360026 "The elevated expression and/or activation of Src and multiple membrane-associated receptors, such as IGF-1R, IR integrins, HER2/neu, and EGFR (all of which trigger Src activation), have been reported in NSCLCs [>>14<<\u201317]. Hence, it is likely that Src acts as a downstream node that links signalings among several collateral membrane associated receptors." . _:b581303802 . _:b41360012 . . _:b581303784 . _:b41360010 "molecules involved in Src activation, such as EGFR, KRas, and HER2 [16, 29], has been implicated in the mechanism of resistance to IGF-1R TKIs [16]; and 2) the aberrant overexpression and activation of IGF-1R and Src in many NSCLCs [>>42<<], and our current results, indicating; 1) a strong positive correlation between pSrc and pIGF-1R in a large TMA of NSCLC tissues and in the majority of NSCLC cell lines; 2) the suppression of EGFR- or integrin\u03B23 -mediated activation of" . . _:b41360013 . . _:b41360022 "Previous reports have suggested that Src, a non-receptor protein tyrosine kinase, can directly phosphorylate IGF-1R [>>12<<, 13]. Src also plays an important role in cancer cell survival and resistance to targeted anticancer therapies by acting as a common signaling facilitator that is activated by a myriad of redundant signaling pathways [12, 14]. The" . _:b41359967 "We also observed high levels of membrane-associated receptors including EGFR, integrins \u03B21 and \u03B23, and/or c-Met, all of which are involved in Src activation [>>14<<], in most of the NSCLC cell lines." . _:b41360014 . _:b581303789 . _:b41359994 "major role in mediating the activation of prosurvival pathways and contributes to resistance to various anticancer therapies, including chemotherapies, radiation therapy, and molecular targeted therapies, in multiple cancer types [5\u20137, >>11<<, 32]. These findings indicate that IGF-1R-targeted therapies represent a promising single-agent or combination regimen." . _:b41359989 . . _:b41360015 . _:b581303788 . _:b41359979 _:b41359980 . _:b41360008 . _:b41359979 _:b41359981 . _:b581303791 . _:b41359979 _:b41359982 . _:b41359979 _:b41359983 . _:b41360009 . _:b581303807 . _:b581303790 . . _:b41360010 . . . . _:b581303785 . . _:b41359999 "IR has also been suggested to mediate intrinsic and/or acquired resistance to IGF-1R mAbs [30, >>35<<]. In this regard, dual IGF-1R/IR TKI regimens may be more effective in blocking the IGF-1R pathway in patients with NSCLC. However, the dual IGF-1R/IR TKI linsitinib has shown moderate efficacy and/or severe toxicity in multiple" . _:b41360011 . _:b581303784 . _:b41360020 . . _:b581303787 . _:b41359976 "We then assessed the sensitivity of NSCLC cells to linsitinib in medium containing low (1\u00A0%) or high (10\u00A0%) fetal bovine serum (FBS), which contains various ligands for the membrane-associated receptors [>>27<<]. Linsitinib treatment decreased viability (Additional file 15: Figure S14A) and colony forming ability (Additional file 15:" . _:b581303806 . _:b41360021 . _:b41359979 _:b41359984 . _:b581303786 . _:b41360007 "The previous reports, including; 1) the mutation and/or overexpression of various molecules involved in Src activation, such as EGFR, KRas, and HER2 [>>16<<, 29], has been implicated in the mechanism of resistance to IGF-1R TKIs [16]; and 2) the aberrant overexpression and activation of IGF-1R and Src in many NSCLCs [42], and our current results, indicating; 1) a strong positive correlation" . _:b41360024 "Src also plays an important role in cancer cell survival and resistance to targeted anticancer therapies by acting as a common signaling facilitator that is activated by a myriad of redundant signaling pathways [>>12<<, 14]. The elevated expression and/or activation of Src and multiple membrane-associated receptors, such as IGF-1R, IR integrins, HER2/neu, and EGFR (all of which trigger Src activation), have been reported in NSCLCs [14\u201317]. Hence, it is" . _:b41360022 . _:b41360012 . _:b581303797 . . . _:b41359991 _:b41359992 . _:b41360023 . . _:b41359991 _:b41359993 . _:b581303796 . _:b41359977 "file 18: Figure S17B) and anchorage-independent colony formation (Additional file 18: Figure S17C) were also observed in both high-pSrc-expressing and low-pSrc-expressing NSCLC cells when another pharmacologic SFK inhibitor, PP2 [>>26<<], was combined with linsitinib. Notably, co-targeting Src and IGF-1R effectively suppressed the colony formation of EGFR TKI-resistant NSCLC cells (H1975, PC-9/GR and PC-9/ER) [28] (Additional file 18:" . _:b41359975 "Combined treatment with another SFK inhibitor PP2 [>>26<<] also decreased linsitinib-induced IGF-1R, Src, and Akt phoshsphorylation in H460 cells (Additional file 13:" . _:b41359991 _:b41359994 . _:b41359991 _:b41359995 . _:b41359991 _:b41359996 . _:b41360016 . _:b581303813 . _:b41359991 _:b41359997 . _:b581303799 . _:b41359991 _:b41359998 . _:b41359991 _:b41359999 . . _:b41360017 . _:b581303798 . _:b41360018 . _:b581303793 . . _:b581303803 . _:b41360019 . _:b581303792 . _:b41359961 "results" . _:b41359985 _:b41359986 . . _:b41359982 "It is noteworthy that a phase I trial of solid tumors treated with XL228 (a small molecule inhibitor targeting Src family kinases, IGF-1R, Aurora A, and Bcr-Abl in the low nanomolar range [>>47<<]) has shown promising preliminary clinical results in melanoma and solid tumors including NSLCLC [48]." . _:b41359985 _:b41359987 . _:b41359985 _:b41359988 . _:b41359985 _:b41359989 . . _:b41360023 . _:b41359985 _:b41359990 . _:b581303795 . _:b41359969 "with the constitutively active Src phosphorylated IGF-1R, EGFR (Y1068 and Y845), Src, and FAK (Y576, a Src-specific phosphorylation site [21]), and Akt (S473) but not FAK (Y397, an integrin signaling-induced autophosphorylation site [>>22<<]) or ERK1/2 in H226Br and H226B cells (Fig.\u00A02a). We next assessed whether Src activation via various signaling pathways would affect IGF-1R phosphorylation." . . _:b581303818 . _:b581303794 . _:b41359993 "a major role in mediating the activation of prosurvival pathways and contributes to resistance to various anticancer therapies, including chemotherapies, radiation therapy, and molecular targeted therapies, in multiple cancer types [>>5<<\u20137, 11, 32]. These findings indicate that IGF-1R-targeted therapies represent a promising single-agent or combination regimen." . _:b41360001 . _:b581303805 . _:b41360016 "Although a small subset of patients with specific genetic and epigenetic abnormalities has shown clinical response to specific targeted therapies [>>3<<], most patients with NSCLCs are insensitive to chemotherapy and radiation1." . _:b41359965 . _:b581303804 . _:b581303817 . _:b41359979 "conclusions" . _:b41360024 . _:b581303804 . . _:b581303807 . _:b41360025 . . _:b581303806 . . _:b41360026 . _:b41359965 "substantial levels of phosphorylated IGF-1R at tyrosine 1131 (tyrosine 1158 for IR) or tyrosines 1135 and 1136 (tyrosines 1162 and 1163 for IR) (pIGF-1R, hereafter) and IGF expression in tissue specimens from NSCLC patients [15, >>16<<]. In the current study, we investigated whether the ligand-dependent activation of the IGF-1R occurs in NSCLC by analyzing previously studied tissue specimens from NSCLC patients." . _:b581303801 . _:b41359982 . _:b581303800 . _:b41360008 . _:b581303803 . _:b581303802 . _:b41359984 "Considering the extensive toxicities of Src inhibitors and IGF-1R TKIs [49, >>50<<], further studies are warranted to evaluate the efficacy of co-targeting IGF-1R and Src in additional preclinical and clinical settings." . _:b581303808 . _:b41359970 . . . _:b581303812 . _:b41359974 "In contrast to the wild-type receptor, this mutant was unresponsive to IGF-stimulated IGF-1R tyrosine phosphorylation [>>25<<], confirming the importance of the site for receptor activity." . . _:b41359962 "previously demonstrated that pIGF-1R/IR levels are significantly higher in premalignant human lung epithelial tissue sample than in normal or reactive bronchial specimens and significantly correlated with the levels of IGF1 and IGF2 [>>18<<]. We have also shown that lung tumors can be formed by the lung-specific overexpression of IGF1 [18]." . . . _:b41360015 . _:b41359996 "laboratory has shown that the inactivation of IGF-1R by the IGF-1R mAb resulted in enhanced signaling through IGF-dependent integrin/Src signaling and the Akt/mTOR-mediated protein synthesis of EGFR and Akt, leading to drug resistance [>>33<<, 34]. IR has also been suggested to mediate intrinsic and/or acquired resistance to IGF-1R mAbs [30, 35]." . _:b581303799 . _:b41360016 . . _:b41359972 . . . . _:b41359964 "reported substantial levels of phosphorylated IGF-1R at tyrosine 1131 (tyrosine 1158 for IR) or tyrosines 1135 and 1136 (tyrosines 1162 and 1163 for IR) (pIGF-1R, hereafter) and IGF expression in tissue specimens from NSCLC patients [>>15<<, 16]. In the current study, we investigated whether the ligand-dependent activation of the IGF-1R occurs in NSCLC by analyzing previously studied tissue specimens from NSCLC patients." . _:b41359992 . _:b41359991 _:b41360008 . _:b581303795 . _:b41359991 _:b41360009 . _:b41359991 _:b41360010 . _:b41359991 _:b41360011 . _:b41359991 _:b41360012 . . _:b41360002 "Furthermore, the major components of the IGF-1R pathway, such as pIGF-1R, pAkt, pERK, pIRS, and pS6K, could not predict response to linsitinib [>>38<<]. Therefore, identifying and targeting the mechanism of resistance against the IGF-1R TKIs would be effective and clinically practical in controlling prosurvival pathways." . _:b41359998 . _:b41359991 _:b41360000 . _:b41359991 _:b41360001 . _:b41359991 _:b41360002 . _:b41359964 . _:b41359991 _:b41360003 . _:b41359991 _:b41360004 . _:b41360014 "Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related deaths worldwide [>>1<<], and the 5-year survival rate for patients with advanced NSCLC remains less than 20\u00A0% [2]." . _:b41359991 _:b41360005 . _:b41359991 _:b41360006 . _:b41359991 _:b41360007 . . _:b581303789 . _:b41360004 . _:b41359980 "Because the clinical development of TKIs of IGF-1R and Src is ongoing [>>45<<, 46], our findings directly impact the current clinical management of patients with NSCLC." . . . _:b41359961 _:b41359962 . . _:b41359961 _:b41359963 . _:b41360025 "Src also plays an important role in cancer cell survival and resistance to targeted anticancer therapies by acting as a common signaling facilitator that is activated by a myriad of redundant signaling pathways [12, >>14<<]. The elevated expression and/or activation of Src and multiple membrane-associated receptors, such as IGF-1R, IR integrins, HER2/neu, and EGFR (all of which trigger Src activation), have been reported in NSCLCs [14\u201317]. Hence, it is" . _:b41359961 _:b41359964 . _:b41359961 _:b41359965 . _:b41359961 _:b41359966 . _:b41359961 _:b41359967 . . _:b41359961 _:b41359968 . _:b41359961 _:b41359969 . _:b41359963 "We have also shown that lung tumors can be formed by the lung-specific overexpression of IGF1 [>>18<<]. These results indicate the importance of tissue-derived IGF expression in the activation of the IGF-1R pathway at an early stage of lung carcinogenesis. We recently reported substantial levels of phosphorylated IGF-1R at tyrosine 1131" . _:b581303809 . _:b581303786 . _:b41359961 _:b41359970 . _:b41359961 _:b41359971 . _:b41359961 _:b41359972 . _:b41360020 . _:b41359961 _:b41359973 . _:b41359961 _:b41359974 . _:b41359961 _:b41359975 . _:b41360019 . _:b41359961 _:b41359976 . _:b41360026 . _:b41359961 _:b41359977 . _:b41359961 _:b41359978 . _:b41359986 . _:b41359987 "Total RNA isolation and RT-PCR were performed as described previously [>>5<<] using the following primer sequences:" . _:b41360025 . _:b41360018 . _:b41359985 "methods" . _:b41359994 . _:b581303801 . . . . _:b581303784 . _:b581303785 . _:b581303786 . _:b581303787 . _:b41359995 "role in mediating the activation of prosurvival pathways and contributes to resistance to various anticancer therapies, including chemotherapies, radiation therapy, and molecular targeted therapies, in multiple cancer types [5\u20137, 11, >>32<<]. These findings indicate that IGF-1R-targeted therapies represent a promising single-agent or combination regimen." . _:b581303788 . _:b581303789 . _:b581303790 . _:b581303791 . _:b41360026 . . _:b41360017 "Moreover, acquired resistance to the therapies eventually emerges in the initially sensitive patients after continuous treatment [>>4<<]. As activation of the survival potential seem to provide cancer cells resistance to anticancer therapies, it is likely that effective anticancer therapies must occur in parallel with blockade of key survival pathways." . _:b581303785 . _:b581303800 . _:b581303801 . _:b581303802 . . _:b581303803 . _:b581303815 . _:b581303804 . _:b581303805 . _:b581303806 . _:b581303807 . _:b41360009 . _:b581303792 . _:b581303793 . _:b581303794 . _:b581303795 . . _:b581303796 . _:b581303797 . _:b41360013 . _:b581303798 . _:b581303799 .