Bile Acid Metabolism Mediates Cholesterol Homeostasis and Promotes Tumorigenesis in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) incidence has risen steadily over the last decade. Elevated lipid uptake and storage is required for ccRCC cell viability. As stored cholesterol is the most abundant component in ccRCC intracellular lipid droplets, it may also play an important role in ccRCC cellular homeostasis. In support of this hypothesis, ccRCC cells acquire exogenous cholesterol through the high-density lipoprotein receptor SCARB1, inhibition or suppression of which induces apoptosis. Here, we showed that elevated expression of 3 beta-hydroxy steroid dehydrogenase type 7 (HSD3B7), which metabolizes cholesterol-derived oxysterols in the bile acid biosynthetic pathway, is also essential for ccRCC cell survival. Development of an HSD3B7 enzymatic assay and screening for small-molecule inhibitors uncovered the compound celastrol as a potent HSD3B7 inhibitor with low micromolar activity. Repressing HSD3B7 expression genetically or treating ccRCC cells with celastrol resulted in toxic oxysterol accumulation, impaired proliferation, and increased apoptosis in vitro and in vivo. These data demonstrate that bile acid synthesis regulates cholesterol homeostasis in ccRCC and identifies HSD3B7 as a plausible therapeutic target. SIGNIFICANCE: The bile acid biosynthetic enzyme HSD3B7 is essential for ccRCC cell survival and can be targeted to induce accumulation of cholesterol-derived oxysterols and apoptotic cell death.

Bcl-xL Enforces a Slow-Cycling State Necessary for Survival in the Nutrient-Deprived Microenvironment of Pancreatic Cancer.

Solid tumors possess heterogeneous metabolic microenvironments where oxygen and nutrient availability are plentiful (fertile regions) or scarce (arid regions). While cancer cells residing in fertile regions proliferate rapidly, most cancer cells in vivo reside in arid regions and exhibit a slow-cycling state that renders them chemoresistant. Here, we developed an in vitro system enabling systematic comparison between these populations via transcriptome analysis, metabolomic profiling, and whole-genome CRISPR screening. Metabolic deprivation led to pronounced transcriptional and metabolic reprogramming, resulting in decreased anabolic activities and distinct vulnerabilities. Reductions in anabolic, energy-consuming activities, particularly cell proliferation, were not simply byproducts of the metabolic challenge, but rather essential adaptations. Mechanistically, Bcl-xL played a central role in the adaptation to nutrient and oxygen deprivation. In this setting, Bcl-xL protected quiescent cells from the lethal effects of cell-cycle entry in the absence of adequate nutrients. Moreover, inhibition of Bcl-xL combined with traditional chemotherapy had a synergistic antitumor effect that targeted cycling cells. Bcl-xL expression was strongly associated with poor patient survival despite being confined to the slow-cycling fraction of human pancreatic cancer cells. These findings provide a rationale for combining traditional cancer therapies that target rapidly cycling cells with those that target quiescent, chemoresistant cells associated with nutrient and oxygen deprivation. SIGNIFICANCE: The majority of pancreatic cancer cells inhabit nutrient- and oxygen-poor tumor regions and require Bcl-xL for their survival, providing a compelling antitumor metabolic strategy.

Quantitative subcellular acyl-CoA analysis reveals distinct nuclear metabolism and isoleucine-dependent histone propionylation.

Quantitative subcellular metabolomic measurements can explain the roles of metabolites in cellular processes but are subject to multiple confounding factors. We developed stable isotope labeling of essential nutrients in cell culture-subcellular fractionation (SILEC-SF), which uses isotope-labeled internal standard controls that are present throughout fractionation and processing to quantify acyl-coenzyme A (acyl-CoA) thioesters in subcellular compartments by liquid chromatography-mass spectrometry. We tested SILEC-SF in a range of sample types and examined the compartmentalized responses to oxygen tension, cellular differentiation, and nutrient availability. Application of SILEC-SF to the challenging analysis of the nuclear compartment revealed a nuclear acyl-CoA profile distinct from that of the cytosol, with notable nuclear enrichment of propionyl-CoA. Using isotope tracing, we identified the branched chain amino acid isoleucine as a major metabolic source of nuclear propionyl-CoA and histone propionylation, thus revealing a new mechanism of crosstalk between metabolism and the epigenome.

Cholesterol Auxotrophy as a Targetable Vulnerability in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is characterized by large intracellular lipid droplets containing free and esterified cholesterol; however, the functional significance of cholesterol accumulation in ccRCC cells is unknown. We demonstrate that, surprisingly, genes encoding cholesterol biosynthetic enzymes are repressed in ccRCC, suggesting a dependency on exogenous cholesterol. Mendelian randomization analyses based on 31,000 individuals indicate a causal link between elevated circulating high-density lipoprotein (HDL) cholesterol and ccRCC risk. Depriving ccRCC cells of either cholesterol or HDL compromises proliferation and survival in vitro and tumor growth in vivo; in contrast, elevated dietary cholesterol promotes tumor growth. Scavenger Receptor B1 (SCARB1) is uniquely required for cholesterol import, and inhibiting SCARB1 is sufficient to cause ccRCC cell-cycle arrest, apoptosis, elevated intracellular reactive oxygen species levels, and decreased PI3K/AKT signaling. Collectively, we reveal a cholesterol dependency in ccRCC and implicate SCARB1 as a novel therapeutic target for treating kidney cancer. SIGNIFICANCE: We demonstrate that ccRCC cells are auxotrophic for exogenous cholesterol to maintain PI3K/AKT signaling pathway and ROS homeostasis. Blocking cholesterol import through the HDL transporter SCARB1 compromises ccRCC cell survival and tumor growth, suggesting a novel pharmacologic target for this disease. This article is highlighted in the In This Issue feature, p. 2945.

Preclinical antitumor activity of the oral platinum analog satraplatin.

PURPOSE: Satraplatin is an orally available platinum analog. The purpose of this study was to better characterize satraplatin's preclinical antitumor efficacy in a variety of sensitive and resistant human tumor cell lines and in a prostate cancer xenograft model and to evaluate the effect of satraplatin on PSA expression and/or secretion in a prostate cancer cell line. METHODS: Satraplatin and its primary metabolite JM-118 were preclinically tested for their cytotoxic activity in a range of cancer cells including: human prostate, those forming the NCI drug screening panel, and those resistant to anti-cancer drugs. Also, the antiproliferative efficacy of satraplatin was tested in vivo in a human prostate cancer model. The effect of satraplatin and JM-118 on PSA transcription was measured by quantitative real time PCR. RESULTS: Satraplatin and JM-118 inhibited in vitro and in vivo the growth of prostate cancer cells in a dose-dependent fashion. The IC50 cytotoxicity values for satraplatin ranged from 1 to 3 microM for androgen-insensitive cells and was 11 microM for the androgen-sensitive cell line. Interestingly, JM-118 was up to 16-fold more potent than satraplatin. Oral administration of satraplatin to nude mouse PC-3 xenograft models inhibited the growth of these human tumors. Satraplatin had no direct effect on PSA transcription and the observed decrease in secreted PSA correlated with a decrease in cell number. When evaluated in the NCI drug-screening panel, satraplatin was most active in leukemia and small cell lung cancer cell lines. Both satraplatin and JM-118 were tested on cells resistant to chemotherapeutic agents. Satraplatin and JM-118 were equally active in the cisplatin-resistant A129cp80 ovarian carcinoma cell line, with activity comparable to that observed in the parent line. Neither expression of MDR1, BCRP, MRP1, nor altered tubulin or topoisomerase I were found to mediate resistance to satraplatin or JM-118. Although these resistance mechanisms contribute to drug resistance for a number of chemotherapeutics, they do not appear to play a role in satraplatin resistance. CONCLUSIONS: These results demonstrate that satraplatin and JM-118 have preclinical antitumor activity in human prostate cancer and other tumor types as well, including several cell lines displaying drug resistance to cisplatin, docetaxel and mitoxantrone. In addition, the results suggest that PSA should be further evaluated as a relevant marker of clinical response in patients with prostate cancer treated with satraplatin.

MASPIT: three-hybrid trap for quantitative proteome fingerprinting of small molecule-protein interactions in mammalian cells.

Organic small molecules generally act by perturbing the function of one or more cellular target proteins, the identification of which is essential to an understanding of the molecular basis of drug action. Here we describe the application of methotrexate-linked small molecule ligands to a mammalian three-hybrid interaction trap for proteome-wide identification of small molecule targets, quantification of the targeting potency of unmodified small molecules for such targets in intact cells, and screening for inhibitors of small molecule-protein interactions. During the course of this study we also identified the pyrido[2,3-d]pyrimidine PD173955, a known SRC kinase inhibitor, as a potent inhibitor of several ephrin receptor tyrosine kinases. This finding could perhaps be exploited in the design of inhibitors for this kinase subfamily, members of which have been implicated in the pathogenesis of various diseases, including cancer.