A Pilot Proteogenomic Study with Data Integration Identifies MCT1 and GLUT1 as Prognostic Markers in Lung Adenocarcinoma

<div><p>We performed a pilot proteogenomic study to compare lung adenocarcinoma to lung squamous cell carcinoma using quantitative proteomics (6-plex TMT) combined with a customized Affymetrix GeneChip. Using MaxQuant software, we identified 51,001 unique peptides that mapped to 7,241 unique proteins and from these identified 6,373 genes with matching protein expression for further analysis. We found a minor correlation between gene expression and protein expression; both datasets were able to independently recapitulate known differences between the adenocarcinoma and squamous cell carcinoma subtypes. We found 565 proteins and 629 genes to be differentially expressed between adenocarcinoma and squamous cell carcinoma, with 113 of these consistently differentially expressed at both the gene and protein levels. We then compared our results to published adenocarcinoma versus squamous cell carcinoma proteomic data that we also processed with MaxQuant. We selected two proteins consistently overexpressed in squamous cell carcinoma in all studies, MCT1 (SLC16A1) and GLUT1 (SLC2A1), for further investigation. We found differential expression of these same proteins at the gene level in our study as well as in other public gene expression datasets. These findings combined with survival analysis of public datasets suggest that MCT1 and GLUT1 may be potential prognostic markers in adenocarcinoma and druggable targets in squamous cell carcinoma. Data are available via ProteomeXchange with identifier PXD002622.</p></div

Comparison with Existing NSCLC Proteomic Datasets.

<p>Mean intensities are given in log₂ scale. (A) The correlations between reporter ion intensities and peptide intensities from Kikuchi et al. were low (R = 0.3, <i>P</i> < 2.2E-16; ρ = 0.26, <i>P</i> < 2.2E-16). (B) As with the Kikuchi et al. data, correlations between reporter ion intensities and peptide intensities from Li et al. were also low (R = 0.23, <i>P</i> < 2.2E-16; ρ = 0.21, <i>P</i> < 2.2E-16).</p

Gene and Protein Expression Agreement.

<p>Gene and Protein Expression Agreement.</p

Proteogenomic Data Recapitulates NSCLC Histology.

<p>(A) Clustering of all identified proteins (7,241) from quantitative TMT analysis group tissues by ADC/SCC histology. (B) Clustering of Affymetrix array probes with standard deviation > 1 (11,008 or 18% of total probes) also groups tissues by ADC/SCC histology.</p

Differentially Expressed Proteins from Quantitative TMT Shared Between Proteomic Datasets.

<p>Italics denote entries that were also differentially expressed at the gene level. Log₂ fold-change was calculated as log₂(SCC/ADC).</p><p>Differentially Expressed Proteins from Quantitative TMT Shared Between Proteomic Datasets.</p

Overlap Between Proteomic Datasets.

<p>(A) Peptide overlap between proteomic datasets. (B) Protein overlap between proteomic datasets. (C) Differentially expressed protein overlap between proteomic datasets. D) Differentially expressed protein overlap between proteomic datasets, excluding proteins not sharing the same direction of change.</p

NSCLC Proteomics Summary.

<p>*Kikuchi et al. used 4 pools of the same 20 samples in their analyses.</p><p>NSCLC Proteomics Summary.</p

Gene and Protein Expression Show Minor Correlations Across Samples.

<p>Mean intensities are given in log₂ scale. (A) Pairwise protein expression and corresponding gene expression across all samples. (B) Pearson correlations grouped by protein and corresponding gene. Values with Pearson R > 0 are colored yellow. The mean Pearson correlation, including all positive and negative correlations, was 0.34.</p

Expression and Survival Analysis of MCT1 and GLUT1 in NSCLC Datasets.

<p>Gene expression is given in log₂ scale. (A) Expression of MCT1 and GLUT1 in TCGA data (ADC = 490, SCC = 491). (B) Expression of MCT1 and GLUT1 ADC, SCC, and matched normal samples (N-ADC and N-SCC, respectively) extracted from Sanchez-Palencia et al.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142162#pone.0142162.ref055" target="_blank">55</a>] (C) Survival analysis of ADC patients expressing low and high levels of MCT1 (median cutoff). (D) Survival analysis of ADC patients expressing low and high levels of GLUT1 (median cutoff).</p

Top Enriched Pathways from Combined Results.

<p>Top Enriched Pathways from Combined Results.</p