Activated Src and Ras induce gefitinib resistance by activation of signaling pathways downstream of epidermal growth factor receptor in human gallbladder adenocarcinoma cells

Qin, Baoli; Ariyama, Hiroshi; Baba, Eishi; Tanaka, Risa; Kusaba, Hitoshi; Harada, Mine; Nakano, Susumu

doi:10.1007/s00280-006-0219-4

Cited by 37 publications

(26 citation statements)

References 30 publications

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“…An alternative possibility is that KRJ-I cells may be characterized by a constitutively active K-Ras and/or SRC phenotype. Both these products activate the same downstream targets as the EGFR, namely Akt and Erk, and resistance to gefitinib due to activation of these products has been demonstrated in human gallbladder adenocarcinoma cell lines (Qin et al 2006). We have sequenced codons 12 and 13 (exon 1) of the K-RAS gene which represent common mutational hotspots within this gene (Kahn et al 1987) but have detected no mutations (M Kidd, unpublished data).…”

Section: Discussionmentioning

confidence: 98%

Further delineation of the continuous human neoplastic enterochromaffin cell line, KRJ-I, and the inhibitory effects of lanreotide and rapamycin

Kidd

Eick²,

Modlin³

et al. 2007

Journal of Molecular Endocrinology

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Small intestinal carcinoids (SICs) are the most prevalent gastrointestinal carcinoid and characterized by local invasion metastasis and protean symptomatology. The proliferative and secretory regulation of the cell of origin, the enterochromaffin (EC) cell has not been characterized. The absence of either a pure preparation of normal EC cells or human EC carcinoid cell lines has hindered the development of therapeutic agents. We therefore further characterized the neoplastic SIC cell line, KRJ-I by assessing its secretory (serotonin (5-HT)) and proliferative responses and defining its log growth phase transcriptome. Electron microscopy demonstrated oval, lobulated nuclei and substance P, and 5-HT-positive cytoplasmic vesicles. RT-PCR detected transcripts for chromogranin A (CHGA), VMAT1 (SLC18A1), tryptophan hydroxylase (TPH1), substance P (TAC1), guanylin (GUCA2A), and SERT (SLC6A4). By immunohistochemistry, all cells were positive for CHGA, SERT, VMAT1, and TPH1. Transcriptome analysis (Affymetrix U133 Plus chips) identified somatostatin SSTR2/3, adrenergic a1C and b1, dopamine D2, nicotinic-type cholinergic A5, A6, B1, muscarinic acetylcholine M4, and 5-HT-2A receptors. The presence of transcripts for SSTR1, SSTR2, and SSTR3 receptors was confirmed by RT-PCR and sequencing. Isoproterenol (ISO) resulted in a dose-dependent increase in intracellular cAMP (EC 50 Z340 nM) and 5-HT (EC 50 Z81 nM) which was completely inhibited by the cAMP antagonist 2 0 ,5 0 -dideoxyadenosine (10 mM). Preincubation with a SSTR agonist, lanreotide, inhibited Ip-stimulated 5-HT secretion (IC 50 Z420 nM). Both lanreotide (10 nM) and rapamycin (50 nM) inhibited proliferation (20G12 and 35G5% respectively) in serum-free medium whereas gefitinib (1 nM-10 mM) inhibited proliferation at micromolar concentrations. KRJ-I is a neoplastic EC cell line that can be used as an in vitro model of SICs as it will allow elucidation and clarification of the secretory and proliferative mechanism(s) of neoplastic EC cells and the molecular signatures that characterize each of these responses.

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Section: Discussionmentioning

confidence: 98%

Further delineation of the continuous human neoplastic enterochromaffin cell line, KRJ-I, and the inhibitory effects of lanreotide and rapamycin

Kidd

Eick²,

Modlin³

et al. 2007

Journal of Molecular Endocrinology

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“…Gene amplification, such as cMET amplification, involved in 20% of acquired resistance cases in NSCLC patients treated with EGFRTKIs, and modulation of other pathways also contribute to resistance to EGFR-TKIs in NSCLC (19)(20)(21). Previous studies showed that SRC activation induced gefitinib resistance by modulation of both the extracellular signalregulated kinase and Akt signaling pathways in human gallbladder adenocarcinoma cells (49). Oncogenic SRC protein also induced the epithelial-mesenchymal transition, which is associated with the loss of E-cadherin complexes at cell-cell adhesions and, consequently, with an increased invasive/metastatic potential and worse NSCLC prognosis (50).…”

Section: Discussionmentioning

confidence: 99%

Association of Polymorphisms inAKT1andEGFRwith Clinical Outcome and Toxicity in Non–Small Cell Lung Cancer Patients Treated with Gefitinib

Giovannetti

Zucali

Peters

et al. 2010

Molecular Cancer Therapeutics

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EGFR mutations are strongly predictive of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor activity in non-small cell lung cancer (NSCLC), but resistance mechanisms are not completely understood. The interindividual variability in toxicity also points out to the need of novel pharmacogenetic markers to select patients before therapy. Therefore, we evaluated the associations between EGFR and AKT1 polymorphisms and outcome/toxicity in gefitinib-treated NSCLC patients. Polymorphic loci in EGFR, and AKT1, and EGFR and K-Ras mutations were assessed in DNA isolated from blood samples and/or paraffin-embedded tumor from 96 gefitinib-treated NSCLC patients. Univariate and multivariate analyses compared genetic variants with clinical efficacy and toxicity using Fisher's, log-rank test, and Cox's proportional hazards model. AKT1-SNP4 association with survival was also evaluated in 127 chemotherapy-treated/gefitinib-naive patients, whereas its relationship with AKT1 expression and gefitinib cytotoxicity was studied in 15 NSCLC cell lines. AKT1-SNP4 A/A genotype was associated with shorter time-to-progression (P = 0.04) and overall survival (P = 0.007). Multivariate analyses and comparison with the gefitinib-nontreated population underlined its predictive significance, whereas the in vitro studies showed the association of lower AKT1 mRNA levels with gefitinib resistance. In contrast, EGFR-activating mutations were significantly correlated with response, longer time-to-progression, and overall survival, whereas EGFR −191C/A (P < 0.001), −216 G/T (P < 0.01), and R497K (P = 0.02) polymorphisms were strongly associated with grade >1 diarrhea. AKT1-SNP4 A/A genotype seems to be a candidate biomarker of primary resistance, whereas EGFR −191C/A, −216G/T, and R497K polymorphisms are associated with diarrhea when using gefitinib in NSCLC patients, thus offering potential new tools for treatment optimization.

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“…Nested PCR to amplify EGFR (exons 18-21) and KRAS (exons 1, 2) were carried out as previously described [4,8]. cMET mutation analysis was carried out by direct sequencing, using the following primer sets for cMET exons 14 and 15 (juxtamembrane domain): hu-cMET-ex14F, 5#-CTTTAACAAGCTCTTTCTTTCT-3#; hu-cMET-ex14R 5#-TGTATAGGTATTTCTCAGAA-3#; hu-cMET-ex15F 5#-TTGTTCTTTAATAATTTTC-3#; hu-cMET-ex15F 5#-GAGTCGAAAAACAATTTATGCT-3#.…”

Section: Mutation Analysismentioning

confidence: 99%

“…Furthermore, SRC activation has been shown to induce gefitinib resistance by modulation of both AKT and ERK signaling pathways in human gallbladder adenocarcinoma cells [8], and oncogenic SRC and RAS proteins may induce the epithelial-mesenchymal transition (EMT) that is associated with loss of E-cadherin complexes at cell-cell adhesions and, consequently, with an increased invasive and metastatic potential [9]. A restored E-cadherin expression increased EGFR-TKIs sensitivity in lung cancer cells [10], while NSCLC patients with a strong E-cadherin staining had a significantly longer time to progression (TTP) and a trend toward longer survival with erlotinib/chemotherapy treatment versus chemotherapy alone [11].…”

Section: Introductionmentioning

confidence: 99%

Role of cMET expression in non-small-cell lung cancer patients treated with EGFR tyrosine kinase inhibitors

et al. 2008

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Background: Approximately 10% of unselected non-small-cell lung cancer (NSCLC) patients responded to the epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) treatment. However, resistance mechanisms are not well understood. We evaluated several potential biological markers of intrinsic EGFR-TKIs-resistance in NSCLC.Materials and methods: pAKT, pERK, cSRC, E-cadherin, cMET[pY1003], cMET[pY1230/1234/1235], and cMET[pY1349] immunohistochemistry, cMET FISH analysis, and EGFR-, KRAS-, and cMET mutation analysis were carried out on tumor samples from 51 gefitinib-treated NSCLC patients. Biological parameters and survival end points were compared by univariate and multivariate analyses. cMET expression was also investigated in two additional series of patients. The in vitro antiproliferative activity of gefitinib alone or in combination with hepatocyte growth factor and the cMET antibody DN-30 was assessed in NSCLC cells.Results: EGFR19 deletion and pAKT expression were significantly associated with response (P < 0.0001) and longer time to progression (TTP) (P = 0.007), respectively. Strong cMET[pY1003] membrane immunoreactivity was expressed in 6% of 149 tumors analyzed and was significantly associated with progressive disease (P = 0.019) and shorter TTP (P = 0.041). In vitro, the DN-30 combination synergistically (CI < 1) enhanced gefitinib-induced growth inhibition in all cMET[pY1003]-expressing cell lines studied.Conclusions: Activated cMET[pY1003] appears to be a marker of primary gefitinib resistance in NSCLC patients.cMET may be a target in treatment of NSCLC.

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Activated Src and Ras induce gefitinib resistance by activation of signaling pathways downstream of epidermal growth factor receptor in human gallbladder adenocarcinoma cells

Abstract: Our results suggest that activated Ras and Src could induce gefitinib resistance by activating either or both of Akt and Erk signaling pathways, thus providing a strategic rationale for assessment of these specific signaling molecules downstream of EGFR to customize treatment.

Cited by 37 publications

References 30 publications

Further delineation of the continuous human neoplastic enterochromaffin cell line, KRJ-I, and the inhibitory effects of lanreotide and rapamycin

Further delineation of the continuous human neoplastic enterochromaffin cell line, KRJ-I, and the inhibitory effects of lanreotide and rapamycin

Association of Polymorphisms inAKT1andEGFRwith Clinical Outcome and Toxicity in Non–Small Cell Lung Cancer Patients Treated with Gefitinib

Role of cMET expression in non-small-cell lung cancer patients treated with EGFR tyrosine kinase inhibitors

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