In vivo characterization of glutamine metabolism identifies therapeutic targets in clear cell renal cell carcinoma.

Targeting metabolic vulnerabilities has been proposed as a therapeutic strategy in renal cell carcinoma (RCC). Here, we analyzed the metabolism of patient-derived xenografts (tumorgrafts) from diverse subtypes of RCC. Tumorgrafts from VHL-mutant clear cell RCC (ccRCC) retained metabolic features of human ccRCC and engaged in oxidative and reductive glutamine metabolism. Genetic silencing of isocitrate dehydrogenase-1 or isocitrate dehydrogenase-2 impaired reductive labeling of tricarboxylic acid (TCA) cycle intermediates in vivo and suppressed growth of tumors generated from tumorgraft-derived cells. Glutaminase inhibition reduced the contribution of glutamine to the TCA cycle and resulted in modest suppression of tumorgraft growth. Infusions with [amide-15N]glutamine revealed persistent amidotransferase activity during glutaminase inhibition, and blocking these activities with the amidotransferase inhibitor JHU-083 also reduced tumor growth in both immunocompromised and immunocompetent mice. We conclude that ccRCC tumorgrafts catabolize glutamine via multiple pathways, perhaps explaining why it has been challenging to achieve therapeutic responses in patients by inhibiting glutaminase.

In vivo isotope tracing reveals a requirement for the electron transport chain in glucose and glutamine metabolism by tumors.

In mice and humans with cancer, intravenous 13C-glucose infusion results in 13C labeling of tumor tricarboxylic acid (TCA) cycle intermediates, indicating that pyruvate oxidation in the TCA cycle occurs in tumors. The TCA cycle is usually coupled to the electron transport chain (ETC) because NADH generated by the cycle is reoxidized to NAD+ by the ETC. However, 13C labeling does not directly report ETC activity, and other pathways can oxidize NADH, so the ETC's role in these labeling patterns is unverified. We examined the impact of the ETC complex I inhibitor IACS-010759 on tumor 13C labeling. IACS-010759 suppresses TCA cycle labeling from glucose or lactate and increases labeling from glutamine. Cancer cells expressing yeast NADH dehydrogenase-1, which recycles NADH to NAD+ independently of complex I, display normalized labeling when complex I is inhibited, indicating that cancer cell ETC activity regulates TCA cycle metabolism and 13C labeling from multiple nutrients.

Uridine Diphosphate Glucuronosyl Transferase 2B28 (UGT2B28) Promotes Tumor Progression and Is Elevated in African American Prostate Cancer Patients.

Prostate cancer (PCa) is the second most diagnosed cancer in the United States and is associated with metabolic reprogramming and significant disparities in clinical outcomes among African American (AA) men. While the cause is likely multi-factorial, the precise reasons for this are unknown. Here, we identified a higher expression of the metabolic enzyme UGT2B28 in localized PCa and metastatic disease compared to benign adjacent tissue, in AA PCa compared to benign adjacent tissue, and in AA PCa compared to European American (EA) PCa. UGT2B28 was found to be regulated by both full-length androgen receptor (AR) and its splice variant, AR-v7. Genetic knockdown of UGT2B28 across multiple PCa cell lines (LNCaP, LAPC-4, and VCaP), both in androgen-replete and androgen-depleted states resulted in impaired 3D organoid formation and a significant delay in tumor take and growth rate of xenograft tumors, all of which were rescued by re-expression of UGT2B28. Taken together, our findings demonstrate a key role for the UGT2B28 gene in promoting prostate tumor growth.

Optimized protocol for stable isotope tracing and steady-state metabolomics in mouse HER2+ breast cancer brain metastasis.

Analyzing the metabolic dependencies of tumor cells is vital for cancer diagnosis and treatment. Here, we describe a protocol for 13C-stable glucose and glutamine isotope tracing in mice HER2+ breast cancer brain metastatic lesions. We describe how to inject cancer cells intracardially to generate brain metastatic lesions in mice. We then detail how to perform 13C-stable isotope infusion in mice with established brain metastasis. Finally, we outline steps for sample collection, processing for metabolite extraction, and analyzing mass spectrometry data. For complete details on the use and execution of this protocol, please refer to Parida et al. (2022).

Metabolic diversity within breast cancer brain-tropic cells determines metastatic fitness.

HER2+ breast cancer patients are presented with either synchronous (S-BM), latent (Lat), or metachronous (M-BM) brain metastases. However, the basis for disparate metastatic fitness among disseminated tumor cells of similar oncotype within a distal organ remains unknown. Here, employing brain metastatic models, we show that metabolic diversity and plasticity within brain-tropic cells determine metastatic fitness. Lactate secreted by aggressive metastatic cells or lactate supplementation to mice bearing Lat cells limits innate immunosurveillance and triggers overt metastasis. Attenuating lactate metabolism in S-BM impedes metastasis, while M-BM adapt and survive as residual disease. In contrast to S-BM, Lat and M-BM survive in equilibrium with innate immunosurveillance, oxidize glutamine, and maintain cellular redox homeostasis through the anionic amino acid transporter xCT. Moreover, xCT expression is significantly higher in matched M-BM brain metastatic samples compared to primary tumors from HER2+ breast cancer patients. Inhibiting xCT function attenuates residual disease and recurrence in these preclinical models.

Integrated Metabolic Profiling and Transcriptional Analysis Reveals Therapeutic Modalities for Targeting Rapidly Proliferating Breast Cancers.

Metabolic dysregulation is a prominent feature in breast cancer, but it remains poorly characterized in patient tumors. In this study, untargeted metabolomics analysis of triple-negative breast cancer (TNBC) and patient with estrogen receptor (ER)-positive breast cancer samples, as well as TNBC patient-derived xenografts (PDX), revealed two major metabolic groups independent of breast cancer histologic subtypes: a "Nucleotide/Carbohydrate-Enriched" group and a "Lipid/Fatty Acid-Enriched" group. Cell lines grown in vivo more faithfully recapitulated the metabolic profiles of patient tumors compared with those grown in vitro. Integrated metabolic and gene expression analyses identified genes that strongly correlate with metabolic dysregulation and predict patient prognosis. As a proof of principle, targeting Nucleotide/Carbohydrate-Enriched TNBC cell lines or PDX xenografts with a pyrimidine biosynthesis inhibitor or a glutaminase inhibitor led to therapeutic efficacy. In multiple in vivo models of TNBC, treatment with the pyrimidine biosynthesis inhibitor conferred better therapeutic outcomes than chemotherapeutic agents. This study provides a metabolic stratification of breast tumor samples that can guide the selection of effective therapeutic strategies targeting breast cancer subsets. In addition, we have developed a public, interactive data visualization portal (http://brcametab.org) based on the data generated from this study to facilitate future research. SIGNIFICANCE: A multiomics strategy that integrates metabolic and gene expression profiling in patient tumor samples and animal models identifies effective pharmacologic approaches to target rapidly proliferating breast tumor subtypes.

A renal cell carcinoma tumorgraft platform to advance precision medicine.

Renal cell carcinoma (RCC) encompasses a heterogenous group of tumors, but representative preclinical models are lacking. We previously showed that patient-derived tumorgraft (TG) models recapitulate the biology and treatment responsiveness. Through systematic orthotopic implantation of tumor samples from 926 ethnically diverse individuals into non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice, we generate a resource comprising 172 independently derived, stably engrafted TG lines from 148 individuals. TG lines are characterized histologically and genomically (whole-exome [n = 97] and RNA [n = 102] sequencing). The platform features a variety of histological and oncogenotypes, including TCGA clades further corroborated through orthogonal metabolomic analyses. We illustrate how it enables a deeper understanding of RCC biology; enables the development of tissue- and imaging-based molecular probes; and supports advances in drug development.

The hexosamine biosynthesis pathway is a targetable liability in KRAS/LKB1 mutant lung cancer.

In non-small-cell lung cancer (NSCLC), concurrent mutations in the oncogene KRAS and the tumour suppressor STK11 (also known as LKB1) encoding the kinase LKB1 result in aggressive tumours prone to metastasis but with liabilities arising from reprogrammed metabolism. We previously demonstrated perturbed nitrogen metabolism and addiction to an unconventional pathway of pyrimidine synthesis in KRAS/LKB1 co-mutant cancer cells. To gain broader insight into metabolic reprogramming in NSCLC, we analysed tumour metabolomes in a series of genetically engineered mouse models with oncogenic KRAS combined with mutations in LKB1 or p53. Metabolomics and gene expression profiling pointed towards activation of the hexosamine biosynthesis pathway (HBP), another nitrogen-related metabolic pathway, in both mouse and human KRAS/LKB1 co-mutant tumours. KRAS/LKB1 co-mutant cells contain high levels of HBP metabolites, higher flux through the HBP pathway and elevated dependence on the HBP enzyme glutamine-fructose-6-phosphate transaminase [isomerizing] 2 (GFPT2). GFPT2 inhibition selectively reduced KRAS/LKB1 co-mutant tumour cell growth in culture, xenografts and genetically modified mice. Our results define a new metabolic vulnerability in KRAS/LKB1 co-mutant tumours and provide a rationale for targeting GFPT2 in this aggressive NSCLC subtype.

Inosine Monophosphate Dehydrogenase Dependence in a Subset of Small Cell Lung Cancers.

Small cell lung cancer (SCLC) is a rapidly lethal disease with few therapeutic options. We studied metabolic heterogeneity in SCLC to identify subtype-selective vulnerabilities. Metabolomics in SCLC cell lines identified two groups correlating with high or low expression of the Achaete-scute homolog-1 (ASCL1) transcription factor (ASCL1High and ASCL1Low), a lineage oncogene. Guanosine nucleotides were elevated in ASCL1Low cells and tumors from genetically engineered mice. ASCL1Low tumors abundantly express the guanosine biosynthetic enzymes inosine monophosphate dehydrogenase-1 and -2 (IMPDH1 and IMPDH2). These enzymes are transcriptional targets of MYC, which is selectively overexpressed in ASCL1Low SCLC. IMPDH inhibition reduced RNA polymerase I-dependent expression of pre-ribosomal RNA and potently suppressed ASCL1Low cell growth in culture, selectively reduced growth of ASCL1Low xenografts, and combined with chemotherapy to improve survival in genetic mouse models of ASCL1Low/MYCHigh SCLC. The data define an SCLC subtype-selective vulnerability related to dependence on de novo guanosine nucleotide synthesis.

Applications of metabolomics to study cancer metabolism.

Reprogrammed metabolism supports tumor growth and provides a potential source of therapeutic targets and disease biomarkers. Mass spectrometry-based metabolomics has emerged as a broadly informative technique for profiling metabolic features associated with specific oncogenotypes, disease progression, therapeutic liabilities and other clinically relevant aspects of tumor biology. In this review, we introduce the applications of metabolomics to study deregulated metabolism and metabolic vulnerabilities in cancer. We provide examples of studies that used metabolomics to discover novel metabolic regulatory mechanisms, including processes that link metabolic alterations with gene expression, protein function, and other aspects of systems biology. Finally, we discuss emerging applications of metabolomics for in vivo isotope tracing and metabolite imaging, both of which hold promise to advance our understanding of the role of metabolic reprogramming in cancer.

Inhibition of the hexosamine biosynthetic pathway promotes castration-resistant prostate cancer.

The precise molecular alterations driving castration-resistant prostate cancer (CRPC) are not clearly understood. Using a novel network-based integrative approach, here, we show distinct alterations in the hexosamine biosynthetic pathway (HBP) to be critical for CRPC. Expression of HBP enzyme glucosamine-phosphate N-acetyltransferase 1 (GNPNAT1) is found to be significantly decreased in CRPC compared with localized prostate cancer (PCa). Genetic loss-of-function of GNPNAT1 in CRPC-like cells increases proliferation and aggressiveness, in vitro and in vivo. This is mediated by either activation of the PI3K-AKT pathway in cells expressing full-length androgen receptor (AR) or by specific protein 1 (SP1)-regulated expression of carbohydrate response element-binding protein (ChREBP) in cells containing AR-V7 variant. Strikingly, addition of the HBP metabolite UDP-N-acetylglucosamine (UDP-GlcNAc) to CRPC-like cells significantly decreases cell proliferation, both in-vitro and in animal studies, while also demonstrates additive efficacy when combined with enzalutamide in-vitro. These observations demonstrate the therapeutic value of targeting HBP in CRPC.

Genomic Aberrations Drive Clonal Evolution of Neuroendocrine Tumors.

Molecular features of castration-resistant neuroendocrine prostate cancer (CRPC-NE) are not well characterized. A recent study that investigated genomic aberrations of CRPC-NE tumors suggests their clonal evolution from CRPC adenocarcinoma. Furthermore, the existence of a distinct DNA methylation profile in CRPC-NE implicates a critical role for epigenetic modification in the development of CRPC-NE.

High throughput microscopy identifies bisphenol AP, a bisphenol A analog, as a novel AR down-regulator.

Prostate cancer remains a deadly disease especially when patients become resistant to drugs that target the Androgen Receptor (AR) ligand binding domain. At this stage, patients develop recurring castrate-resistant prostate cancers (CRPCs). Interestingly, CRPC tumors maintain dependency on AR for growth; moreover, in CRPCs, constitutively active AR splice variants (e.g., AR-V7) begin to be expressed at higher levels. These splice variants lack the ligand binding domain and are rendered insensitive to current endocrine therapies. Thus, it is of paramount importance to understand what regulates the expression of AR and its splice variants to identify new therapeutic strategies in CRPCs. Here, we used high throughput microscopy and quantitative image analysis to evaluate effects of selected endocrine disruptors on AR levels in multiple breast and prostate cancer cell lines. Bisphenol AP (BPAP), which is used in chemical and medical industries, was identified as a down-regulator of both full length AR and the AR-V7 splice variant. We validated its activity by performing time-course, dose-response, Western blot and qPCR analyses. BPAP also reduced the percent of cells in S phase, which was accompanied by a ~60% loss in cell numbers and colony formation in anchorage-independent growth assays. Moreover, it affected mitochondria size and cell metabolism. In conclusion, our high content analysis-based screening platform was used to classify the effect of compounds on endogenous ARs, and identified BPAP as being capable of causing AR (both full-length and variants) down-regulation, cell cycle arrest and metabolic alterations in CRPC cell lines.

Coactivator SRC-2-dependent metabolic reprogramming mediates prostate cancer survival and metastasis.

Metabolic pathway reprogramming is a hallmark of cancer cell growth and survival and supports the anabolic and energetic demands of these rapidly dividing cells. The underlying regulators of the tumor metabolic program are not completely understood; however, these factors have potential as cancer therapy targets. Here, we determined that upregulation of the oncogenic transcriptional coregulator steroid receptor coactivator 2 (SRC-2), also known as NCOA2, drives glutamine-dependent de novo lipogenesis, which supports tumor cell survival and eventual metastasis. SRC-2 was highly elevated in a variety of tumors, especially in prostate cancer, in which SRC-2 was amplified and overexpressed in 37% of the metastatic tumors evaluated. In prostate cancer cells, SRC-2 stimulated reductive carboxylation of α-ketoglutarate to generate citrate via retrograde TCA cycling, promoting lipogenesis and reprogramming of glutamine metabolism. Glutamine-mediated nutrient signaling activated SRC-2 via mTORC1-dependent phosphorylation, which then triggered downstream transcriptional responses by coactivating SREBP-1, which subsequently enhanced lipogenic enzyme expression. Metabolic profiling of human prostate tumors identified a massive increase in the SRC-2-driven metabolic signature in metastatic tumors compared with that seen in localized tumors, further implicating SRC-2 as a prominent metabolic coordinator of cancer metastasis. Moreover, SRC-2 inhibition in murine models severely attenuated the survival, growth, and metastasis of prostate cancer. Together, these results suggest that the SRC-2 pathway has potential as a therapeutic target for prostate cancer.

Metabolomic profiling identifies biochemical pathways associated with castration-resistant prostate cancer.

Despite recent developments in treatment strategies, castration-resistant prostate cancer (CRPC) is still the second leading cause of cancer-associated mortality among American men, the biological underpinnings of which are not well understood. To this end, we measured levels of 150 metabolites and examined the rate of utilization of 184 metabolites in metastatic androgen-dependent prostate cancer (AD) and CRPC cell lines using a combination of targeted mass spectrometry and metabolic phenotyping. Metabolic data were used to derive biochemical pathways that were enriched in CRPC, using Oncomine concept maps (OCM). The enriched pathways were then examined in-silico for their association with treatment failure (i.e., prostate specific antigen (PSA) recurrence or biochemical recurrence) using published clinically annotated gene expression data sets. Our results indicate that a total of 19 metabolites were altered in CRPC compared to AD cell lines. These altered metabolites mapped to a highly interconnected network of biochemical pathways that describe UDP glucuronosyltransferase (UGT) activity. We observed an association with time to treatment failure in an analysis employing genes restricted to this pathway in three independent gene expression data sets. In summary, our studies highlight the value of employing metabolomic strategies in cell lines to derive potentially clinically useful predictive tools.