Michelle van Geldermalsen

Targeting ASCT2-mediated glutamine uptake blocks prostate cancer growth and tumour development

Glutamine is conditionally essential in cancer cells, being utilized as a carbon and nitrogen source for macromolecule production, as well as for anaplerotic reactions fuelling the tricarboxylic acid (TCA) cycle. In this study, we demonstrated that the glutamine transporter ASCT2 (SLC1A5) is highly expressed in prostate cancer patient samples. Using LNCaP and PC‐3 prostate cancer cell lines, we showed that chemical or shRNA‐mediated inhibition of ASCT2 function in vitro decreases glutamine uptake, cell cycle progression through E2F transcription factors, mTORC1 pathway activation and cell growth. Chemical inhibition also reduces basal oxygen consumption and fatty acid synthesis, showing that downstream metabolic function is reliant on ASCT2‐mediated glutamine uptake. Furthermore, shRNA knockdown of ASCT2 in PC‐3 cell xenografts significantly inhibits tumour growth and metastasis in vivo, associated with the down‐regulation of E2F cell cycle pathway proteins. In conclusion, ASCT2‐mediated glutamine uptake is essential for multiple pathways regulating the cell cycle and cell growth, and is therefore a putative therapeutic target in prostate cancer.

Targeting glutamine transport to suppress melanoma cell growth

Amino acids, especially leucine and glutamine, are important for tumor cell growth, survival and metabolism. A range of different transporters deliver each specific amino acid into cells, some of which are increased in cancer. These amino acids consequently activate the mTORC1 pathway and drive cell cycle progression. The leucine transporter LAT1/4F2hc heterodimer assembles as part of a large complex with the glutamine transporter ASCT2 to transport amino acids. In this study, we show that the expression of LAT1 and ASCT2 is significantly increased in human melanoma samples and is present in both BRAFWT (C8161 and WM852) and BRAFV600E mutant (1205Lu and 451Lu) melanoma cell lines. While inhibition of LAT1 by BCH did not suppress melanoma cell growth, the ASCT2 inhibitor BenSer significantly reduced both leucine and glutamine transport in melanoma cells, leading to inhibition of mTORC1 signaling. Cell proliferation and cell cycle progression were significantly reduced in the presence of BenSer in melanoma cells in 2D and 3D cell culture. This included reduced expression of the cell cycle regulators CDK1 and UBE2C. The importance of ASCT2 expression in melanoma was confirmed by shRNA knockdown, which inhibited glutamine uptake, mTORC1 signaling and cell proliferation. Taken together, our study demonstrates that ASCT2‐mediated glutamine transport is a potential therapeutic target for both BRAFWT and BRAFV600E melanoma.

Adipocyte lipolysis links obesity to breast cancer growth: adipocyte-derived fatty acids drive breast cancer cell proliferation and migration

BackgroundObesity is associated with increased recurrence and reduced survival of breast cancer. Adipocytes constitute a significant component of breast tissue, yet their role in provisioning metabolic substrates to support breast cancer progression is poorly understood.ResultsHere, we show that co-culture of breast cancer cells with adipocytes revealed cancer cell-stimulated depletion of adipocyte triacylglycerol. Adipocyte-derived free fatty acids were transferred to breast cancer cells, driving fatty acid metabolism via increased CPT1A and electron transport chain complex protein levels, resulting in increased proliferation and migration. Notably, fatty acid transfer to breast cancer cells was enhanced from “obese” adipocytes, concomitant with increased stimulation of cancer cell proliferation and migration. This adipocyte-stimulated breast cancer cell proliferation was dependent on lipolytic processes since HSL/ATGL knockdown attenuated cancer cell responses.ConclusionsThese findings highlight a novel and potentially important role for adipocyte lipolysis in the provision of metabolic substrates to breast cancer cells, thereby supporting cancer progression.

Intron retention is regulated by altered MeCP2-mediated splicing factor recruitment

While intron retention (IR) is considered a widely conserved and distinct mechanism of gene expression control, its regulation is poorly understood. Here we show that DNA methylation directly regulates IR. We also find reduced occupancy of MeCP2 near the splice junctions of retained introns, mirroring the reduced DNA methylation at these sites. Accordingly, MeCP2 depletion in tissues and cells enhances IR. By analysing the MeCP2 interactome using mass spectrometry and RNA co-precipitation, we demonstrate that decreased MeCP2 binding near splice junctions facilitates IR via reduced recruitment of splicing factors, including Tra2b, and increased RNA polymerase II stalling. These results suggest an association between IR and a slower rate of transcription elongation, which reflects inefficient splicing factor recruitment. In summary, our results reinforce the interdependency between alternative splicing involving IR and epigenetic controls of gene expression.

LAT1 is a putative therapeutic target in endometrioid endometrial carcinoma.

l‐type amino acid transporters (LAT1‐4) are expressed in various cancer types and are involved in the uptake of essential amino acids such as leucine. Here we investigated the expression of LAT1‐4 in endometrial adenocarcinoma and evaluated the contribution of LATs to endometrial cancer cell growth. Analysis of human gene expression data showed that all four LAT family members are expressed in endometrial adenocarcinomas. LAT1 was the most highly expressed, and showed a significant increase in both serous and endometrioid subtypes compared to normal endometrium. Endometrioid patients with the highest LAT1 levels exhibited the lowest disease‐free survival. The pan‐LAT inhibitor BCH led to a significant decrease in cell growth and spheroid area in four endometrial cancer cell lines tested in vitro. Knockdown of LAT1 by shRNA inhibited cell growth in HEC1A and Ishikawa cells, as well as inhibiting spheroid area in HEC1A cells. These data show that LAT1 plays an important role in regulating the uptake of essential amino acids such as leucine into endometrial cancer cells. Increased ability of BCH compared to LAT1 shRNA at inhibiting Ishikawa spheroid area suggests that other LAT family members may also contribute to cell growth. LAT1 inhibition may offer an effective therapeutic strategy in endometrial cancer patients whose tumours exhibit high LAT1 expression.

SAMHD1 enhances immunoglobulin hypermutation by promoting transversion mutation

Significance Antibody affinity rises during immune responses to viruses via antibody gene somatic hypermutation and Darwinian selection of mutated B cells—in a time frame of days. The enzyme activation-induced deaminase (AID) initiates hypermutation by deaminating genomic cytosines. Mutation is exacerbated by noncanonical G1-phase DNA repair pathways that deploy error-prone polymerases, including Pol η (gene Polh). In G1 phase, dNTP levels are restricted to inhibit viral replication. We derestricted G1-phase dNTP supply in hypermutating B cells, which increased virus susceptibility in vitro and caused changes in antibody hypermutation in vivo akin to Polh inactivation. We conclude that G1-phase dNTP paucity contributes to antibody hypermutation and that the evolution of antibody hypermutation included the repurposing of intracellular antivirus mechanisms based on dNTP starvation. Activation-induced deaminase (AID) initiates hypermutation of Ig genes in activated B cells by converting C:G into U:G base pairs. G1-phase variants of uracil base excision repair (BER) and mismatch repair (MMR) then deploy translesion polymerases including REV1 and Pol η, which exacerbates mutation. dNTP paucity may contribute to hypermutation, because dNTP levels are reduced in G1 phase to inhibit viral replication. To derestrict G1-phase dNTP supply, we CRISPR-inactivated SAMHD1 (which degrades dNTPs) in germinal center B cells. Samhd1 inactivation increased B cell virus susceptibility, increased transition mutations at C:G base pairs, and substantially decreased transversion mutations at A:T and C:G base pairs in both strands. We conclude that SAMHD1’s restriction of dNTP supply enhances AID’s mutagenicity and that the evolution of Ig hypermutation included the repurposing of antiviral mechanisms based on dNTP starvation.

Abstract B20: Glutamine metabolic vulnerabilities define triple-negative from luminal A breast cancer subsets

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.

Abstract A036: Blocking DNA and RNA synthesis by targeting glutamine metabolism in prostate cancer

Cancer cells greatly increase their uptake of nutrients (glucose, amino acids, and lipids), and metabolize them to provide the necessary building blocks for new cancer cells. It was recently shown that extracellular amino acids make up by far the majority of the carbon sources used by cancer cells for cell division, highlighting amino acid uptake as a viable therapeutic target. In addition, amino acids such as glutamine are critical in providing nitrogen for purine and pyrimidine metabolism. We have previously shown that glutamine uptake is mediated predominantly by ASCT2 in prostate cancer, with ASCT2 knockdown blocking cell growth in vitro and in vivo. Using the TCGA dataset, we have discovered increased expression of downstream glutamine metabolism enzymes in approximately 25% of prostate cancer patients. One of these enzymes, guanine monophosphate synthase (GMPS), is a glutamine amidotransferase involved in de novo purine biosynthesis and is responsible for the last step in the synthesis of the guanine nucleotide. Expression of GMPS correlates with increasing Gleason score in prostate cancer patient samples. Furthermore, patients with high GMPS expression had significantly reduced disease-free survival in the TCGA dataset. Immunofluorescent staining shows that GMPS is localized in both the cytoplasm and nucleus of LNCaP and PC-3 cells—consistent with a secondary role for GMPS in p53 stabilization. Inhibition of GMPS using decoyinine significantly decreased cell growth of both LNCaP and PC-3 cells. Knockdown of GMPS by shRNA significantly decreased cell growth, which could be rescued by addition of extracellular guanosine to the media, suggesting a direct effect on nucleotide synthesis. These results show the importance of downstream glutamine metabolism in prostate cancer, and suggest that GMPS is a potential therapeutic target in “glutamine-addicted” prostate cancers. Citation Format: Qian (Kevin) Wang, Michelle van Geldermalsen, Angel Pang, Blake Zhang, Jeff Holst. Blocking DNA and RNA synthesis by targeting glutamine metabolism in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A036.

Benzylserine inhibits breast cancer cell growth by disrupting intracellular amino acid homeostasis and triggering amino acid response pathways

BackgroundCancer cells require increased levels of nutrients such as amino acids to sustain their rapid growth. In particular, leucine and glutamine have been shown to be important for growth and proliferation of some breast cancers, and therefore targeting the primary cell-surface transporters that mediate their uptake, L-type amino acid transporter 1 (LAT1) and alanine, serine, cysteine-preferring transporter 2 (ASCT2), is a potential therapeutic strategy.MethodsThe ASCT2 inhibitor, benzylserine (BenSer), is also able to block LAT1 activity, thus inhibiting both leucine and glutamine uptake. We therefore aimed to investigate the effects of BenSer in breast cancer cell lines to determine whether combined LAT1 and ASCT2 inhibition could inhibit cell growth and proliferation.ResultsBenSer treatment significantly inhibited both leucine and glutamine uptake in MCF-7, HCC1806 and MDA-MB-231 breast cancer cells, causing decreased cell viability and cell cycle progression. These effects were not primarily leucine-mediated, as BenSer was more cytostatic than the LAT family inhibitor, BCH. Oocyte uptake assays with ectopically expressed amino acid transporters identified four additional targets of BenSer, and gas chromatography-mass spectrometry (GCMS) analysis of intracellular amino acid concentrations revealed that this BenSer-mediated inhibition of amino acid uptake was sufficient to disrupt multiple pathways of amino acid metabolism, causing reduced lactate production and activation of an amino acid response (AAR) through activating transcription factor 4 (ATF4).ConclusionsTogether these data showed that BenSer blockade inhibited breast cancer cell growth and viability through disruption of intracellular amino acid homeostasis and inhibition of downstream metabolic and growth pathways.

Abstract 1043: Targeting ASCT2-mediated glutamine uptake and metabolism in breast cancer

Although a nonessential amino acid in normal cell growth, the demand for glutamine is dramatically increased throughout malignant transformation to provide catabolic substrates for ATP production and anabolic substrates for macromolecule biosynthesis. To maintain glutamine availability for these metabolic processes, cancer cells overexpress cell surface transporters that function to exchange amino acids across the plasma membrane. One such transporter is ASCT2 (alanine, serine, cysteine-preferring transporter 2; SLC1A5), a sodium-dependent symporter that mediates uptake of small, neutral amino acids, including glutamine. Blocking ASCT2 to prevent glutamine uptake and glutaminolysis has been shown to successfully prevent tumor cell proliferation in melanoma, non-small cell lung cancer, prostate cancer and acute myeloid leukaemia. We recently showed that in breast cancer, although ASCT2 is highly expressed in most tumor subtypes, only aggressive triple-negative (TN) breast cancer cells require ASCT2-mediated uptake of glutamine to sustain cell growth in vitro and in vivo. Gene expression analysis of xenograft-derived tumor tissue and TN patient samples suggested coordinate regulation of ASCT2 and other glutamine metabolism-related genes, such as glutaminase (GLS) and glutamate-ammonia ligase (GLUL), with global activation of glutaminolytic energy production pathways in these tumors. The metabolism-regulating transcription factors, MYC and ATF4, were significantly correlated with these genes, suggesting a dynamic MYC and ATF4-driven transcriptional program in TN breast cancer. 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. We developed a targeted metabolomics approach that combined labelled 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 ASCT2 transporter function was blocked in vitro by pharmacological inhibitors or inducible shRNA knockdown, in combination with CB-839, a GLS inhibitor in Phase I clinical trials. To confirm the clinical utility of these findings, we also determined mRNA and protein expression of glutamine metabolism-related genes in tissue microarrays of TN patient samples. These data suggest a reliance on glutamine availability in TN breast cancers and reinforce the link between increased glutamine metabolism and clinically aggressive breast cancers, thus highlighting the therapeutic potential of targeting the ASCT2 glutamine uptake and metabolism pathway in these patients. Citation Format: Michelle van Geldermalsen, Qian Wang, Jeff Holst. Targeting ASCT2-mediated glutamine uptake and metabolism in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1043.