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HSP90 is a molecular chaperone required for stabilization and activation of hundreds of client proteins, including many known oncoproteins. AUY922 (luminespib), a new-generation HSP90 inhibitor, exhibits potent preclinical efficacy against several cancer types including prostate cancer. However, clinical use of HSP90 inhibitors for prostate cancer has been limited by toxicity and treatment resistance. Here, we aimed to design an effective combinatorial therapeutic regimen that utilizes subtoxic doses of AUY922, by identifying potential survival pathways induced by AUY922 in clinical prostate tumors. We conducted a proteomic analysis of 30 patient-derived explants (PDE) cultured in the absence and presence of AUY922, using quantitative mass spectrometry. AUY922 significantly increased the abundance of proteins involved in oxidative phosphorylation and fatty acid metabolism in the PDEs. Consistent with these findings, AUY922-treated prostate cancer cell lines exhibited increased mitochondrial mass and activated fatty acid metabolism processes. We hypothesized that activation of fatty acid oxidation is a potential adaptive response to AUY922 treatment and that cotargeting this process will sensitize prostate cancer cells to HSP90 inhibition. Combination treatment of AUY922 with a clinical inhibitor of fatty acid oxidation, perhexiline, synergistically decreased viability of several prostate cancer cell lines, and had significant efficacy in PDEs. The novel drug combination treatment induced cell-cycle arrest and apoptosis, and attenuated the heat shock response, a known mediator of HSP90 treatment resistance. This combination warrants further preclinical and clinical investigation as a novel strategy to overcome resistance to HSP90 inhibition. Implications: Metabolic pathways induced in tumor cells by therapeutic agents may be critical, but targetable, mediators of treatment resistance.

Prostate cancer cells exhibit altered cellular metabolism but, notably, not the hallmarks of Warburg metabolism. Prostate cancer cells exhibit increased de novo synthesis of fatty acids (FA); however, little is known about how extracellular FAs, such as those in the circulation, may support prostate cancer progression. Here, we show that increasing FA availability increased intracellular triacylglycerol content in cultured patient-derived tumor explants, LNCaP and C4-2B spheroids, a range of prostate cancer cells (LNCaP, C4-2B, 22Rv1, PC-3), and prostate epithelial cells (PNT1). Extracellular FAs are the major source (∼83%) of carbons to the total lipid pool in all cell lines, compared with glucose (∼13%) and glutamine (∼4%), and FA oxidation rates are greater in prostate cancer cells compared with PNT1 cells, which preferentially partitioned extracellular FAs into triacylglycerols. Because of the higher rates of FA oxidation in C4-2B cells, cells remained viable when challenged by the addition of palmitate to culture media and inhibition of mitochondrial FA oxidation sensitized C4-2B cells to palmitate-induced apoptosis. Whereas in PC-3 cells, palmitate induced apoptosis, which was prevented by pretreatment of PC-3 cells with FAs, and this protective effect required DGAT-1–mediated triacylglycerol synthesis. These outcomes highlight for the first-time heterogeneity of lipid metabolism in prostate cancer cells and the potential influence that obesity-associated dyslipidemia or host circulating has on prostate cancer progression. Implications: Extracellular-derived FAs are primary building blocks for complex lipids and heterogeneity in FA metabolism exists in prostate cancer that can influence tumor cell behavior.

Although a nonessential amino acid in normal cells, the demand for glutamine is dramatically increased throughout malignant transformation to support increased metabolic demands; namely, provision of catabolic substrates for ATP production and anabolic substrates for the citric acid cycle and subsequent macromolecule biosynthesis, as well as potentiating the uptake of other critical amino acids by acting as an obligate exchange substrate. Elevated expression of glutamine metabolism-related genes, MYC-driven transcriptional events, and increased consumption and reliance on glutamine are all associated with aggressive breast cancers, including the high-risk triple-negative (TN) subtype. We recently showed that in breast cancer cells, glutamine uptake by the small neutral amino acid transporter, ASCT2, is required to sustain TN cell growth in vitro and in vivo. We therefore hypothesized that highly proliferative TN breast cancers that are sensitive to ASCT2 inhibition may have unique metabolic signatures that could be additionally exploited for therapeutic purposes. Using a targeted metabolomics approach, we combined labeled substrate tracing, liquid chromatography coupled tandem-mass spectrometry (LC-MS/MS), and gas chromatography mass spectrometry (GC-MS) to analyze intracellular levels of key tricarboxylic acid (TCA) cycle intermediates, glycolytic metabolites, fatty acid precursors, and amino acids in human breast cancer cell lines. These analyses revealed distinct metabolic effects when glutamine uptake was blocked in vitro by L-γ-glutamyl-p-nitroanilide (GPNA), a pharmacologic inhibitor of ASCT2. These data confirm a broad reliance on glutamine availability in TN breast cancers, reinforced by TCGA gene expression data showing a specific upregulation of multiple glutamine metabolism enzymes that is completely absent in the luminal A subtype. These data emphasize the link between increased glutamine metabolism and clinically aggressive breast cancers, thus highlighting the therapeutic potential of targeting glutamine metabolism pathways in these patients. Citation Format: Michelle van Geldermalsen, Lake-Ee Quek, Nigel Turner, Seher Balaban, Andrew Hoy, Qian Wang, Jeff Holst. Glutamine metabolic vulnerabilities define triple-negative from luminal A breast cancer subsets [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B20.