Search

PDF file - 95KB, Supplementary Table S3-A. Human tumor pY proteome. S3-B. Comparison analysis of unique pTyr sites iden_fy from di_erent samples.

PDF file - 104KB, Supplementary Figure 1A-B: PC9GR cells acquired drug resistance to both reversible and irreversible EGFR-TKI. A: PC9 and PC9GR cells were treated for 72 h with increasing concentrations of erlotinib or CL387,785. Data generated by cell viability assay (CellTiter-Glo) are expressed as a percentage of the value for untreated cells. Determinations were done in triplicate. B: PC9 and PC9GR cells were incubated for 1 hour in the absence or presence of erlotinib (1 �M) as indicated. Cell lysates were subjected to protein expression analysis with antibodies to pEGFR, pSrc, pAkt, and pErk along with antibodies to β-actin as a loading control. C-D: Gene status in PC9 and PC9GR cells. C: PC9GR cells acquired T790M while retaining exon 19 deletion as determined by PCR-invader assay. Values of red bars above zero represent positive results with the detection probe for indicated mutation sequences of EGFR, while values of light blue bars are quality controls showing good PCR quality, as previously described (1). The value of red bars = (fluorescence of the samples with detection probe for mutation sequences) - (fluorescence of the normal control with the same probe � 2). The value of light blue bars = (fluorescence of the samples with detection probe for wild type sequences) - (fluorescence of the normal control with the same probe � 0.8). DEL1 represents E746-A750del type1 (2235-2249del GGAATTAAGAGAAGC). DEL2 represents E746-A750del type2 (2236-2250del GAATTAAGAGAAGCA). DEL3 represents L747-P753del insS. D: FISH analysis (CEP7 (green)/D7S522 (red)) was done in PC9 and PC9GR cells as previously described (2). No additional copy of D7S522 compared to CEP 7 was found in these cells, explaining that neither PC9 nor PC9GR cells has MET amplification.

PDF file - 139KB, Effects of ligands exposure with or without EGFR-TKI in PC9 and PC9GR cells. A-F: PC9 (A, C, and E) or PC9GR cells (B, D, and F) were incubated for 1 hour in the absence or presence of 100 nM erlotinib (A, C, and E) or afatinib (B, D, and F) with indicated concentrations of HGF (A and B), IGF1 (C and D), or Gas6 (E and F). Cell lysates were subjected to protein expression analysis with antibodies to pMET (A and B), IGF1R (C and D), or pAXL (E and F) along with pEGFR, pAkt, pErk, and antibodies to β-actin as a loading control.

PDF file - 54KB, A: Example of peak area (EIC) for EGFR pY 1172. B: Example of peak area (EIC) for MET pY 1234.

Purpose: Irreversible EGFR-tyrosine kinase inhibitors (TKI) are thought to be one strategy to overcome EGFR-TKI resistance induced by T790M gatekeeper mutations in non–small cell lung cancer (NSCLC), yet they display limited clinical efficacy. We hypothesized that additional resistance mechanisms that cooperate with T790M could be identified by profiling tyrosine phosphorylation in NSCLC cells with acquired resistance to reversible EGFR-TKI and harboring T790M.Experimental Design: We profiled PC9 cells with TKI-sensitive EGFR mutation and paired EGFR-TKI–resistant PC9GR (gefitinib-resistant) cells with T790M using immunoaffinity purification of tyrosine-phosphorylated peptides and mass spectrometry–based identification/quantification. Profiles of erlotinib perturbations were examined.Results: We observed a large fraction of the tyrosine phosphoproteome was more abundant in PC9- and PC9GR-erlotinib–treated cells, including phosphopeptides corresponding to MET, IGF, and AXL signaling. Activation of these receptor tyrosine kinases by growth factors could protect PC9GR cells against the irreversible EGFR-TKI afatinib. We identified a Src family kinase (SFK) network as EGFR-independent and confirmed that neither erlotinib nor afatinib affected Src phosphorylation at the activation site. The SFK inhibitor dasatinib plus afatinib abolished Src phosphorylation and completely suppressed downstream phosphorylated Akt and Erk. Dasatinib further enhanced antitumor activity of afatinib or T790M-selective EGFR-TKI (WZ4006) in proliferation and apoptosis assays in multiple NSCLC cell lines with T790M-mediated resistance. This translated into tumor regression in PC9GR xenograft studies with combined afatinib and dasatinib.Conclusions: Our results identified both codrivers of resistance along with T790M and support further studies of irreversible or T790M-selective EGFR inhibitors combined with dasatinib in patients with NSCLC with acquired T790M. Clin Cancer Res; 20(15); 4059–74. ©2014 AACR.

PDF file - 91KB, A: Effects of dasatinib combined with afatinib on apoptosis in HCC4006-T790M and HCC827-T790M cells. Apoptosis assay was carried out using PE-conjugated caspase-3 antibody, following incubation of HCC4006-T790M or HCC827-T790M cells for 72 hours with DMSO, erlotinib (E), dasatinib (D), afatinib (A), and afatinib + dasatinib. Values are expressed as a percentage of caspase-3-positive cells. Determinations were done in triplicate. Bars, SD. *P < 0.001 versus DMSO or each single agent (Student's t-test). B: Validation of active pTyr sites from PC9 cell lines on lung cancer patient. Venn diagram was used to compare the pTyr sites identified from PC9 and human lung tumor tissue with positive EGFR mutation. Total 83 overlap active pTyr sites were obtained.

PDF file - 387KB, Effects of ligands exposure with or without EGFR-TKI in PC9 and PC9GR cells. A-F: PC9 (A, C, and E) or PC9GR cells (B, D, and F) were incubated for 1 hour in the absence or presence of 100 nM erlotinib (A, C, and E) or afatinib (B, D, and F) with indicated concentrations of HGF (A and B), IGF1 (C and D), or Gas6 (E and F). Cell lysates were subjected to protein expression analysis with antibodies to pMET (A and B), IGF1R (C and D), or pAXL (E and F) along with pEGFR, pAkt, pErk, and antibodies to β-actin as a loading control.

PDF file - 67KB, Supplementary Table S1: Quantitation of pY changes in between PC9 and PC9GR cell lines with and without erlotinib treatment. The cutoff was set as P value1.5; Supplementary Table S2: Cell viability IC50 in PC9GR cells and H1975 cells.

Disruption in circadian gene expression, whether due to genetic variation or environmental factors (e.g., light at night, shiftwork), is associated with increased incidence of breast, prostate, gastrointestinal and hematologic cancers and gliomas. Circadian genes are highly expressed in the ovaries where they regulate ovulation; circadian disruption is associated with several ovarian cancer risk factors (e.g., endometriosis). However, no studies have examined variation in germline circadian genes as predictors of ovarian cancer risk and invasiveness. The goal of the current study was to examine single nucleotide polymorphisms (SNPs) in circadian genes BMAL1, CRY2, CSNK1E, NPAS2, PER3, REV1 and TIMELESS and downstream transcription factors KLF10 and SENP3 as predictors of risk of epithelial ovarian cancer (EOC) and histopathologic subtypes. The study included a test set of 3,761 EOC cases and 2,722 controls and a validation set of 44,308 samples including 18,174 (10,316 serous) cases and 26,134 controls from 43 studies participating in the Ovarian Cancer Association Consortium (OCAC). Analysis of genotype data from 36 genotyped SNPs and 4600 imputed SNPs indicated that the most significant association was rs117104877 in BMAL1 (OR = 0.79, 95% CI = 0.68-0.90, p = 5.59 × 10(-4)]. Functional analysis revealed a significant down regulation of BMAL1 expression following cMYC overexpression and increasing transformation in ovarian surface epithelial (OSE) cells as well as alternative splicing of BMAL1 exons in ovarian and granulosa cells. These results suggest that variation in circadian genes, and specifically BMAL1, may be associated with risk of ovarian cancer, likely through disruption of hormonal pathways. ispartof: Journal of Genetics and Genome Research vol:2 issue:2 pages:017- ispartof: location:United States status: published