Hypertonicity-Affected Genes Are Differentially Expressed in Clear Cell Renal Cell Carcinoma and Correlate with Cancer-Specific Survival

Abstract

The heterogeneity of renal cell carcinoma (RCC) subtypes reflects the cell type of origin in the nephron, with consequences for therapy and prognosis. The transcriptional cues that determine segment-specific gene expression patterns are poorly understood. We recently showed that hypertonicity in the renal medulla regulates nephron-specific gene expression. Here, we analyzed a set of 223 genes, which were identified in the present study by RNA-Seq to be differentially expressed by hypertonicity, for the prediction of cancer-specific survival (CSS). Cluster analyses of these genes showed discrimination between tumor and non-tumor samples of clear cell RCC (ccRCC). Refinement of this gene signature to a four-gene score (OSM score) through statistical analyses enabled prediction of CSS in ccRCC patients of The Cancer Genome Atlas (TCGA) (n = 436) in univariate (HR = 4.1; 95% CI: 2.78−6.07; p = 4.39 × 10 −13 ), and multivariate analyses including primary tumor (T); regional lymph node (N); distant metastasis (M); grading (G)(p = 2.3 × 10 −5 ). The OSM score could be validated in an independent ccRCC study (n = 52) in univariate (HR = 1.29; 95% CI = 1.05-1.59; p = 0.011) and multivariate analyses (p = 0.016). Cell culture experiments using RCC cell lines demonstrated that the expression of the tumor suppressor ELF5 could be restored by hypertonicity. The innovation of our novel gene signature is that these genes are physiologically regulated only by hypertonicity, thereby providing the possibility to be targeted for therapy.

Deletion of Von Hippel–Lindau Interferes with Hyper Osmolality Induced Gene Expression and Induces an Unfavorable Gene Expression Pattern

“…In a recent study, we have shown that the hyperosmotic gene expression pattern is lost in ccRCC samples and that this has also consequences for patients' outcome [16]. In the mentioned study, the initial gene list was generated in rat primary collecting duct cells [15,16] and we were able to develop a translational comparison from healthy rat cell to human renal cancer and survival prediction, showing the translational potential of the data [16].…”

“…We have shown that the expression of hyperosmolality induced genes is reduced in RCC samples and that a gene signature of osmolality affected genes can be used for the prediction of patient's clinical outcome [16]. We have, therefore, analyzed if this is also the case in the present study.…”

“…We have shown that this environment is also important to regulate a specific gene expression pattern of several kidney-specific genes [15]. Further, we have recently shown that the expression of osmolality affected genes is inversely regulated in the ccRCC samples compared to normal tissue, and we were able to generate an Osm-score that allows the prediction of patients' survival [16]. We were also able to induce the expression of the E74-like factor 5 (ELF5), a tumor suppressor in RCC [17], in the 786-0, VHL deficient, RCC cell line under hyperosmotic cell culture conditions.…”

“…These data also show that Vhl has an influence on the expression of hyperosmolality affected genes. Vhl-KO reduces, for example, the expression of Fxyd2, Fxyd4, Rnf183, and Ranbp3l, which are prognostically favorable in patients with ccRCC [16]. Since the Human Pathology Atlas does not discriminate between the RCC entities, we have used selected genes and queried the KIRC TCGA database if they could serve as prognostic markers for patients with ccRCC.…”

“…We were also able to induce the expression of the E74-like factor 5 (ELF5), a tumor suppressor in RCC [17], in the 786-0, VHL deficient, RCC cell line under hyperosmotic cell culture conditions. Interestingly, the expression level was higher in 786-0 that ectopically expressed wild type VHL [16], suggesting that VHL somehow interferes with hyperosmolality associated gene expression.…”