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Lung cancer signatures in plasma based on proteome profiling of mouse tumor models

ABSTRACT: Mouse models of cancer recapitulate many of the molecular and biological features of the human disease. We sought to exploit these experimental merits in a systematic comparative proteomics search for circulating proteins associated with lung tumor development. In-depth quantitative proteomics was applied to plasmas from three mouse models of lung adenocarcinoma driven by mutant EGFR or Kras or induced by urethane exposure and a mouse model of small cell lung cancer driven by loss of Trp53 and Rb. To further refine our lung cancer-specific and broad carcinoma signatures, we intersected these lung cancer proteome profiles with those from other well-established mouse models of pancreatic, ovarian, colon, prostate and breast cancer, as well as two mouse models of inflammation. A set of proteins regulated by Titf1/Nkx2-1, a master transcription factor in cells from the peripheral airways and a known lineage-survival oncogene in lung cancer was identified in plasmas of mouse models of lung adenocarcinoma. An EGFR network of proteins was discerned in the plasma of mice with lung tumors driven by a mutant human EGFR. Levels of these proteins returned toward baseline upon treatment with a tyrosine kinase inhibitor. Moreover, a distinct plasma signature was uncovered in the Trp53/Rb mutant small cell lung cancer model that included a set of proteins associated with neuroendocrine development. Our studies have identified novel plasma protein signatures among molecularly or histopathologically defined lung cancer subtypes. siRNA transfection experiments were performed in NCI-H3255 and HCC4019 lung adenocarcinoma cell lines using ON-TARGETplus SMARTpool small interfering RNAs (siRNAs) targeting TITF1 (L-019105-01-0005) along with a negative control (ON-TARGETplus siCONTROL nontargeting siRNA pool; D-001810-10-05) obtained from Dharmacon. 400000 cells were seeded in antibiotic-free RPMI-1640 media supplemented with 10% FBS, in 6-well culture plates. The next day, cells were transfected at a final concentration of 100nM siRNA using 6ul DharmaFECT 1 (Dharmacon) according to the manufacturer's instructions. 72-hours post-transfection, RNA was harvested using Trizol (Invitrogen) and protein using RIPA buffer for microarray expression and western blotting, respectively. RNA from TITF1 knockdown and control experiments was profiled by the MSKCC Genomics Core using the Illumina Human HT-12 v3.0 array platform according to manufacturer's instructions. Two biological replicates were profiled for each condition. Resulting data files were exported using GenomeStudio software, log2 transformed, quantile-normalized and analyzed using Partek Genomics Suite (v6.5). Average values of replicates for each gene were then compared between the TITF1 knockdown and non-targeting treatments for each cell line to identify candidate TITF1 regulated genes.

ORGANISM(S): Homo sapiens

SUBMITTER: Lockwood William

PROVIDER: S-ECPF-GEOD-28480 | biostudies-other |

REPOSITORIES: biostudies-other

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Project description:Mouse models of cancer recapitulate many of the molecular and biological features of the human disease. We sought to exploit these experimental merits in a systematic comparative proteomics search for circulating proteins associated with lung tumor development. In-depth quantitative proteomics was applied to plasmas from three mouse models of lung adenocarcinoma driven by mutant EGFR or Kras or induced by urethane exposure and a mouse model of small cell lung cancer driven by loss of Trp53 and Rb. To further refine our lung cancer-specific and broad carcinoma signatures, we intersected these lung cancer proteome profiles with those from other well-established mouse models of pancreatic, ovarian, colon, prostate and breast cancer, as well as two mouse models of inflammation. A set of proteins regulated by Titf1/Nkx2-1, a master transcription factor in cells from the peripheral airways and a known lineage-survival oncogene in lung cancer was identified in plasmas of mouse models of lung adenocarcinoma. An EGFR network of proteins was discerned in the plasma of mice with lung tumors driven by a mutant human EGFR. Levels of these proteins returned toward baseline upon treatment with a tyrosine kinase inhibitor. Moreover, a distinct plasma signature was uncovered in the Trp53/Rb mutant small cell lung cancer model that included a set of proteins associated with neuroendocrine development. Our studies have identified novel plasma protein signatures among molecularly or histopathologically defined lung cancer subtypes. siRNA transfection experiments were performed in NCI-H3255 and HCC4019 lung adenocarcinoma cell lines using ON-TARGETplus SMARTpool small interfering RNAs (siRNAs) targeting TITF1 (L-019105-01-0005) along with a negative control (ON-TARGETplus siCONTROL nontargeting siRNA pool; D-001810-10-05) obtained from Dharmacon. 400000 cells were seeded in antibiotic-free RPMI-1640 media supplemented with 10% FBS, in 6-well culture plates. The next day, cells were transfected at a final concentration of 100nM siRNA using 6ul DharmaFECT 1 (Dharmacon) according to the manufacturer's instructions. 72-hours post-transfection, RNA was harvested using Trizol (Invitrogen) and protein using RIPA buffer for microarray expression and western blotting, respectively. RNA from TITF1 knockdown and control experiments was profiled by the MSKCC Genomics Core using the Illumina Human HT-12 v3.0 array platform according to manufacturer's instructions. Two biological replicates were profiled for each condition. Resulting data files were exported using GenomeStudio software, log2 transformed, quantile-normalized and analyzed using Partek Genomics Suite (v6.5). Average values of replicates for each gene were then compared between the TITF1 knockdown and non-targeting treatments for each cell line to identify candidate TITF1 regulated genes.

Project description:Heart failure (HF) is a worldwide pandemic with an unacceptable high level of morbidity and mortality. Understanding the different pathophysiological mechanisms will contribute to prevention and individualized therapy of HF. We established mouse models for ischemic cardiomyopathy (ICM) and dilated cardiomyopathy (DCM) by inducing myocardial infarction (MI) and Coxsackievirus B3 infection, respectively. Isobaric tags for relative and absolute quantitation and liquid chromatography coupled with tandem mass spectrometry technology was used to identify the protein expression profiles in control and failing hearts. A total of 1,638 proteins were identified and compared in this proteomics analysis. Among them, 286 proteins were differently expressed. Gene ontology, KEGG pathway and ingenuity pathway analysis was performed to systematically assess the potential connections of the differentially expressed proteins to biological functions. Compared with control group, the differentially expressed proteins derived from the hearts of ICM and DCM mice were partially similar and mainly modulated in oxidative phosphorylation, metabolism and protein folding pathways. Moreover, difference still existed, the differentially expressed proteins between DCM and ICM hearts were significantly modulated in oxidative phosphorylation, metabolic and AMPK signaling pathways. Confirmatory western bolt analysis demonstrated that SDHB was down-regulated in both ICM and DCM hearts, while UQCRQ, GLUT4 and adiponectin were up-regulated in ICM hearts. Adenosine triphosphate (ATP) concentration significantly decreased in both DCM and ICM hearts. The protein expression of phospho-AMPK? decreased significantly in DCM hearts, but increased in ICM. In summary, oxidative phosphorylation, cardiac metabolism, and protein folding play critical roles in the pathogenesis of HF. The diverse changes in protein expression profiles between failing hearts induced by either MI or CVB3 infection demonstrated the heterogeneity of HF. Understanding the differences in proteome profiles could offer more precise therapeutic options for HF.

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Lung cancer signatures in plasma based on proteome profiling of mouse tumor models

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