BackgroundGleason 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, Gleason grade is not reliably ascertained by diagnostic biopsy, largely due to sampling inadequacies, subjectivity in the Gleason grading procedure, and a lack of more objective biomarker assays to stratify prostate cancer aggressiveness. In most aggressive cancer types, the tumor microenvironment exhibits a reciprocal pro-tumorigenic metabolic phenotype consistent with the reverse Warburg effect (RWE). The RWE can be viewed as a physiologic response to the epithelial phenotype that is independent of both the epithelial genotype and of direct tumor sampling. We hypothesize that differential expression of RWE-associated genes can be used to classify Gleason pattern, distinguishing GP3 from GP4 PCa foci.MethodsGene expression profiling was conducted on RNA extracted from laser-capture microdissected 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 completed on raw gene expression values. Genes were analyzed for enriched Gene Ontology (GO) biological processes and protein-protein interactions using STRING and GeneMANIA.ResultsUnivariate 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. The 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. Enrichment for GO-biological processes revealed that catabolic glucose processes and oxidative stress response pathways were strongly associated with GP3 foci but not GP4. FOXO1 was identified as being a primary nodal protein.ConclusionsWe report that RWE-associated genes can be used to distinguish between GP3 and GP4 prostate cancers. Moreover, we find that the RWE response is downregulated in the stroma surrounding GP4, possibly via modulation of FOXO1.

中文翻译：

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使用反向 Warburg 效应相关基因对格里森模式 3 和 4 前列腺癌进行分子表征


背景格里森评分 (GS) 3+3 和 3+4 前列腺癌 (PCa) 在其临床过程中差异很大，其中格里森模式 (GP) 4 代表癌症进展的主要独立危险因素。然而，格里森分级并不能通过诊断活检可靠地确定，这主要是由于采样不足、格里森分级程序的主观性以及缺乏更客观的生物标志物测定来对前列腺癌的侵袭性进行分层。在大多数侵袭性癌症类型中，肿瘤微环境表现出与反向 Warburg 效应 (RWE) 一致的相互促肿瘤代谢表型。 RWE 可以被视为对上皮表型的生理反应，独立于上皮基因型和直接肿瘤取样。我们假设 RWE 相关基因的差异表达可用于对 Gleason 模式进行分类，从而区分 GP3 和 GP4 PCa 病灶。方法对从 PCa 的 20 个 GP3 和 21 个 GP4 癌灶周围激光捕获显微切割基质组织中提取的 RNA 进行基因表达谱分析分别为 GS 3+3 和 GS ≥4+3 的患者。使用针对与 RWE 相关的生物过程的 102 基因 NanoString 探针组探测基因。通过单变量分析从标准化数据中鉴定差异表达基因。对原始基因表达值完成了最高得分对 (TSP) 分析。使用 STRING 和 GeneMANIA 对基因进行了丰富的基因本体 (GO) 生物过程和蛋白质-蛋白质相互作用的分析。结果单变量分析确定了 9 个基因（FOXO1 (AUC: 0.884)、GPD2、SPARC、HK2、COL1A2、ALDOA、MCT4、NRF2 和ATG5) 在 GP3 和 GP4 基质之间差异表达 (p<0.05)。 然而，在纠正错误发现后，只有 FOXO1 在 q<0.05 处保留了统计显着性。 TSP 分析确定了一个重要的基因对，即 ATG5/GLUT1。相对于 GLUT1，ATG5 的表达量更高，正确分类了 77.4% 的 GP3/GP4 样本。 GO 生物过程的富集表明，葡萄糖分解代谢过程和氧化应激反应途径与 GP3 病灶密切相关，但与 GP4 无关。 FOXO1 被鉴定为主要淋巴结蛋白。结论我们报告 RWE 相关基因可用于区分 GP3 和 GP4 前列腺癌。此外，我们发现 RWE 反应在 GP4 周围的基质中下调，可能是通过 FOXO1 的调节。