Figure 1. ccRCC tumors and cell lines activate bile acid metabolism.

A, Simplified schematic of the two branches of the “Bile Acid” pathway. Mammalian cells catabolize cholesterol through two independent pathways, the classical or the alternative, which are required for dietary lipid absorption.

B, Gene set enrichment analysis (GSEA) of RNAseq data provided by the TCGA KIRC project indicating that genes belonging to the “Bile Acid” pathway have higher expression in ccRCC tumors compared to normal kidney tissue. 538 ccRCC tumors and 72 normal kidney tissues were included. Generated metabolic gene sets were ranked based on normalized enrichment score changes in ccRCC compared to normal kidney tissue.

C, Metabolic gene set analysis of RNAseq data provided by the TCGA KIRC project. 538 ccRCC tumors and 72 normal kidney tissues were included. 2,752 genes encoding all human metabolic enzymes and transporters were classified according to KEGG. Generated metabolic gene sets were ranked based on their log2 median fold expression changes in ccRCC compared to normal tissue.

D, TCGA KIRC dataset analysis shows that expression of genes involved in the “Bile Acid” pathway are significantly up-regulated in ccRCC tumors vs. normal kidney tissue (left panel). BAAT, Bile Acid-CoA:Amino Acid N-Acyltransferase; AKR1D1, Aldo-Keto Reductase Family 1 Member D1; HSD3B7, Hydroxy-Delta-5-Steroid Dehydrogenase, 3 Beta- And Steroid Delta-Isomerase 7; CYP3A5, Cytochrome P450 Family 3 Subfamily A Member 5; CYP27A1, Cytochrome P450 Family 27 Subfamily A Member 1; CYP7A1, Cytochrome P450 Family 7 Subfamily A Member 1; CYP7B1, Cytochrome P450 Family 7 Subfamily B Member 1; CYP8B1, Cytochrome P450 Family 8 Subfamily B Member 1. Normalized RNASeq reads of HSD3B7 in 72 normal kidneys and 538 ccRCC tumors (TCGA KIRC) (right panel).

E, Normalized RNASeq reads of HSD3B7 in 72 normal kidneys and 538 ccRCC tumors grouped into stage I-IV (TCGA KIRC).

F, Kaplan-Meier analysis indicating that patients with tumors that expressed middle and high levels of HSD3B7 mRNA had a shorter overall survival (TCGA KIRC). 538 ccRCC tumors split into thirds based on HSD3B7 mRNA expression.

G, Real-time qPCR analysis of HSD3B7 mRNA performed on 17 tumor tissues and their normal counterparts. HSD3B7 gene expression is highly increased in tumors compared to normal tissues. Beta-2 microglobulin (B2M) was used as the housekeeping gene.

H, HSD3B7 protein expression in normal kidney tissue and ccRCC tumors assessed by immunoblots (top panel) with quantification by ImageJ (bottom panel). Tubulin (TUB) was used as the loading control.

I, HSD3B7 mRNA expression in ccRCC (KIRC), papillary RCC (KIRP) and chromophobe RCC (KICH) from TCGA. HSD3B7 gene expression is significantly increased in ccRCC compared to other subtypes.

(All experiments were performed in at least triplicates and statistical analysis was applied with *=P<0.05, **=P<0.01, ***=<0.001, n.s=non-significant).