A Gprc5a tumor suppressor loss of expression signature is conserved, prevalent, and associated with survival in human lung adenocarcinomas

Abstract

Increasing the understanding of the impact of changes in oncogenes and tumor suppressor genes is essential for improving the management of lung cancer. Recently, we identified a new mouse lung-specific tumor suppressor-the G protein-coupled receptor 5A (Gprc5a). Microarray analysis of the transcriptomes of lung epithelial cells cultured from normal tracheas of Gprc5a knockout and wild-type mice defined a loss-of-Gprc5a gene signature, which revealed many aberrations in cancer-associated pathways. To assess the relevance of this mouse tumor suppressor to human lung cancer, the loss-of-Gprc5a signature was cross species compared with and integrated with publicly available gene expression data of human normal lung tissue and non-small cell lung cancers. The loss-of-Gprc5a signature was prevalent in human lung adenocarcinomas compared with squamous cell carcinomas or normal lung. Furthermore, it identified subsets of lung adenocarcinomas with poor outcome. These results demonstrate that gene expression patterns of Gprc5a loss in nontumorigenic mouse lung epithelial cells are evolutionarily conserved and important in human lung adenocarcinomas.

Figure 1

Derivation of a mouse loss-of-Gprc5a signature. (A) Normal epithelial cells cultured fromtracheas of Gprc5a wild-type and knockout mice (WT-NLE and NULL-NLE, respectively) were used for global gene expression analysis to understand molecular consequences of Gprc5a loss. (B) A mouse loss-of-Gprc5a gene signature encompassing 1586 gene features was derived based on selection criteria described in the Materials and methods section. (C and D) Functional analyses of the mouse loss-of-Gprc5a signature using global functional categories from IPA. The value of -log(significance) represents the inverse log of the P values of the modulation of the depicted functional categories (C) and pathways (D) between the WT-NLE and NULL-NLE cells. The number of genes displaying more than a two-fold change is indicated above each bar. Red arrows indicate the predicted activation of the biological processes and pathways by IPA, whereas the blue arrows indicate the predicted inhibition of the processes and pathways (C and D). *Blue arrow signifies predicted inhibition of the G₂/M cell cycle checkpoint despite the predicted activation of the DNA damage pathway by IPA.

Figure 2

The mouse loss-of-Gprc5a signature differentiates human lung adenocarcinomas from normal lung or SCCs. Dendrograms of hierarchical cluster analysis of mixed mouse-human data sets including lung adenocarcinomas and adjacent normal lung samples from the studies by Stearman et al. (A) and Su et al. (B) as well as the WT-NLE and NULL-NLE mouse cells. Normalized GPRC5A expression in the integrated data is represented in the single row under the dendrograms as red (high) or green (low) and is quantified in the human normal lung and adenocarcinomas (C); P values were obtained by the Student's t test. Human orthologs of the mouse loss-of-Gprc5a signature were integrated with data of human NSCLC samples from the study by Bild et al. and analyzed by hierarchical cluster analysis after filtering and retaining of genes with a fold change of at least two from the median in at least 12 patient samples (D) and by principal component analysis (E). (F) Log-rank statistics and Kaplan-Meier plots were used to assess for the OS of lung adenocarcinoma patients and SCC patients separated independently according to clustering patterns (blue, Gprc5a-WT cluster; red, Gprc5a-NULL cluster). The number of censored patients of total number of cluster patients is indicated between the parentheses next to the survival plot arms.

Figure 3

The mouse loss-of-Gprc5a gene signature is associated with poor survival in human lung adenocarcinoma. (A) The mouse loss-of-Gprc5a gene signature was integrated with and analyzed in the NCI Director's Challenge data sets (n = 748) by hierarchical cluster analysis after filtering and retaining of genes with a fold change of at least two from the median in at least eight patient samples. (B) Kaplan-Meier plot for the OS of lung adenocarcinoma patients separated according to clustering patterns (blue, Gprc5a-WT cluster; red, Gprc5a-NULL cluster). (C)Human orthologs of the loss-of-Gprc5a signature and present in all array platforms of the studies by Shedden et al., Bild et al. (Duke cohort), and Bhattacharjee et al. (Harvard cohort) were identified (n = 547). Lung adenocarcinomas from the NCI Director's Challenge were used as a training set (n = 442), and those from the Duke and Harvard cohorts were pooled as a validation set (DH cohort). (D) Kaplan-Meier plots for the survival of lung adenocarcinoma patients clustered into Gprc5a-WT cluster (blue) or Gprc5a-NULL cluster (red) as predicted by the prediction algorithms. The number of censored patients of the total number of cluster patients is indicated between the parentheses next to the survival plot arms. CCP indicates compound covariate predictor; LDA, linear discriminator analysis; LOOCV, leave-one-out cross-validation; NC, nearest centroid; NN-1/-3, nearest neighbors 1 and 3; SVM, support vector machine.