Molecular Characterization of Gleason Patterns 3 and 4 Prostate Cancer Using Reverse Warburg Effect-Associated Genes

Abstract

Background: Gleason scores (GS) 3+3 and 3+4 prostate cancers (PCa) differ greatly in their clinical courses, with Gleason pattern (GP) 4 representing a major independent risk factor for cancer progression. However, GP is not reliably ascertained by diagnostic biopsy, largely due to sampling inadequacies, subjectivity in the Gleason grading procedure, the high degree of heterogeneity of the disease, and a lack of more objective biomarker assays to stratify PCa aggressiveness. In most aggressive cancer types, the stromal microenvironment exhibits a reciprocal pro-tumourigenic metabolic phenotype consistent with the Reverse Warburg Effect (RWE). The RWE can be viewed as a physiologic response to the tumour phenotype that is both independent of tumour genotype and direct tumour sampling. This study aims to classify GP3 and GP4 based on differences in their stromal RWE-associated gene expression profiles. Methods: Gene expression profiling was conducted on RNA extracted from laser-capture microdissected (LCM) stromal tissue surrounding 20 GP3 and 21 GP4 cancer foci from PCa patients with GS 3+3 and GS ≥4+3 respectively. Genes were probed using a 102 gene NanoString probe-set targeted towards biological processes associated with the RWE. Differentially expressed genes were identified from normalized data by univariate analysis. A top-scoring pair (TSP) analysis was also completed on raw gene expression values. Results: Univariate analysis identified nine genes (FOXO1 (AUC: 0.884), GPD2, SPARC, HK2, COL1A2, ALDOA, MCT4, NRF2 and ATG5) that were differentially expressed between GP3 and GP4 stroma (p<0.05). However, following correction for false discovery only FOXO1 retained statistical significance at q<0.05. TSP analysis identified a significant gene pair, namely ATG5/GLUT1. Greater expression of ATG5 relative to GLUT1 correctly classified 77.4% of GP3/GP4 samples. Conclusions: The stroma associated with GP3 is distinct from that associated with GP4 tumour foci by the higher expression of FOXO1 and a greater expression of ATG5 relative to GLUT1. Together, FOXO1 and the relative expression of the ATG5/GLUT1 gene pair represent RWE-based classifiers for GP3 and GP4 PCa, that have the potential to distinguish between true low-risk (GS 3+3) and false low-risk (GS 3+3 harbouring unsampled ≥GP4) patient cases.