Propionyl-CoA carboxylase subunit B regulates anti-tumor T cells in a pancreatic cancer mouse model.

Most human pancreatic ductal adenocarcinoma (PDAC) are not infiltrated with cytotoxic T cells and are highly resistant to immunotherapy. Over 90% of PDAC have oncogenic KRAS mutations, and phosphoinositide 3-kinases (PI3Ks) are direct effectors of KRAS. Our previous study demonstrated that ablation of Pik3ca in KPC (KrasG12D; Trp53R172H; Pdx1-Cre) pancreatic cancer cells induced host T cells to infiltrate and completely eliminate the tumors in a syngeneic orthotopic implantation mouse model. Now, we show that implantation of Pik3ca-/- KPC (named αKO) cancer cells induces clonal expansion of cytotoxic T cells infiltrating the pancreatic tumors. To identify potential molecules that can regulate the activity of these anti-tumor T cells, we conducted an in vivo genome-wide gene-deletion screen using αKO cells implanted in the mouse pancreas. The result shows that deletion of propionyl-CoA carboxylase subunit B gene (Pccb) in αKO cells (named p-αKO) leads to immune evasion, tumor progression and death of host mice. Surprisingly, p-αKO tumors are still infiltrated with clonally expanded CD8+ T cells but they are inactive against tumor cells. However, blockade of PD-L1/PD1 interaction reactivated these clonally expanded T cells infiltrating p-αKO tumors, leading to slower tumor progression and improve survival of host mice. These results indicate that Pccb can modulate the activity of cytotoxic T cells infiltrating some pancreatic cancers and this understanding may lead to improvement in immunotherapy for this difficult-to-treat cancer.

Glutamine synthetase limits ß-catenin-mutated liver cancer growth by maintaining nitrogen homeostasis and suppressing mTORC1.

Glutamine synthetase (GS) catalyzes de novo synthesis of glutamine that facilitates cancer cell growth. In the liver, GS functions next to the urea cycle to remove ammonia waste. As a dysregulated urea cycle is implicated in cancer development, the impact of GS's ammonia clearance function has not been explored in cancer. Here, we show that oncogenic activation of ß-catenin (encoded by CTNNB1) led to a decreased urea cycle and elevated ammonia waste burden. While ß-catenin induced the expression of GS, which is thought to be cancer promoting, surprisingly, genetic ablation of hepatic GS accelerated the onset of liver tumors in several mouse models that involved ß-catenin activation. Mechanistically, GS ablation exacerbated hyperammonemia and facilitated the production of glutamate-derived nonessential amino acids, which subsequently stimulated mechanistic target of rapamycin complex 1 (mTORC1). Pharmacological and genetic inhibition of mTORC1 and glutamic transaminases suppressed tumorigenesis facilitated by GS ablation. While patients with hepatocellular carcinoma, especially those with CTNNB1 mutations, have an overall defective urea cycle and increased expression of GS, there exists a subset of patients with low GS expression that is associated with mTORC1 hyperactivation. Therefore, GS-mediated ammonia clearance serves as a tumor-suppressing mechanism in livers that harbor ß-catenin activation mutations and a compromised urea cycle.

Correction: Tumor suppressor RARRES1 links tubulin deglutamylation to mitochondrial metabolism and cell survival.

[This corrects the article DOI: 10.18632/oncotarget.26600.].

The Ubiquitin E3 Ligase TRIM21 Promotes Hepatocarcinogenesis by Suppressing the p62-Keap1-Nrf2 Antioxidant Pathway.

BACKGROUND AND AIMS: TRIM21 is a ubiquitin E3 ligase that is implicated in numerous biological processes including immune response, cell metabolism, redox homeostasis, and cancer development. We recently reported that TRIM21 can negatively regulate the p62-Keap1-Nrf2 antioxidant pathway by ubiquitylating p62 and prevents its oligomerization and protein sequestration function. As redox homeostasis plays a pivotal role in many cancers including liver cancer, we sought to determine the role of TRIM21 in hepatocarcinogenesis. METHODS: We examined the correlation between TRIM21 expression and the disease using publicly available data sets and 49 cases of HCC clinical samples. We used TRIM21 genetic knockout mice to determine how TRIM21 ablation impact HCC induced by the carcinogen DEN plus phenobarbital (PB). We explored the mechanism that loss of TRIM21 protects cells from DEN-induced oxidative damage and cell death. RESULTS: There is a positive correlation between TRIM21 expression and HCC. Consistently, TRIM21-knockout mice are resistant to DEN-induced hepatocarcinogenesis. This is accompanied by decreased cell death and tissue damage upon DEN treatment, hence reduced hepatic tissue repair response and compensatory proliferation. Cells deficient in TRIM21 display enhanced p62 sequestration of Keap1 and are protected from DEN-induced ROS induction and cell death. Reconstitution of wild-type but not the E3 ligase-dead and the p62 binding-deficient mutant TRIM21 impedes the protection from DEN-induced oxidative damage and cell death in TRIM21-deficient cells. CONCLUSIONS: Increased TRIM21 expression is associated with human HCC. Genetic ablation of TRIM21 leads to protection against oxidative hepatic damage and decreased hepatocarcinogenesis, suggesting TRIM21 as a preventive and therapeutic target.

In-Source CID Ramping and Covariant Ion Analysis of Hydrophilic Interaction Chromatography Metabolomics.

A large proportion of the complexity and redundancy of LC-MS metabolomics data comes from adduct formation. To reduce such redundancy, many tools have been developed to recognize and annotate adduct ions. These tools rely on predefined adduct lists that are generated empirically from reversed-phase LC-MS studies. In addition, hydrophilic interaction chromatography (HILIC) is gaining popularity in metabolomics studies due to its enhanced performance over other methods for polar compounds. HILIC methods typically use high concentrations of buffer salts to improve chromatographic performance. Therefore, it is necessary to analyze adduct formation in HILIC metabolomics. To this end, we developed covariant ion analysis (COVINA) to investigate metabolite adduct formation. Using this tool, we completely annotated 201 adduct and fragment ions from 10 metabolites. Many of the metabolite adduct ions were found to contain cluster ions corresponding to mobile phase additives. We further utilized COVINA to find the major ionized forms of metabolites. Our results show that for some metabolites, the adduct ion signals can be >200-fold higher than the signals from the deprotonated form, offering better sensitivity for targeted metabolomics analysis. Finally, we developed an in-source CID ramping (InCIDR) method to analyze the intensity changes of the adduct and fragment ions from metabolites. Our analysis demonstrates a promising method to distinguish the protonated and deprotonated ions of metabolites from the adduct and fragment ions.

Glutamine Anabolism Plays a Critical Role in Pancreatic Cancer by Coupling Carbon and Nitrogen Metabolism.

Glutamine is thought to play an important role in cancer cells by being deaminated via glutaminolysis to α-ketoglutarate (aKG) to fuel the tricarboxylic acid (TCA) cycle. Supporting this notion, aKG supplementation can restore growth/survival of glutamine-deprived cells. However, pancreatic cancers are often poorly vascularized and limited in glutamine supply, in alignment with recent concerns on the significance of glutaminolysis in pancreatic cancer. Here, we show that aKG-mediated rescue of glutamine-deprived pancreatic ductal carcinoma (PDAC) cells requires glutamate ammonia ligase (GLUL), the enzyme responsible for de novo glutamine synthesis. GLUL-deficient PDAC cells are capable of the TCA cycle but defective in aKG-coupled glutamine biosynthesis and subsequent nitrogen anabolic processes. Importantly, GLUL expression is elevated in pancreatic cancer patient samples and in mouse PDAC models. GLUL ablation suppresses the development of KrasG12D-driven murine PDAC. Therefore, GLUL-mediated glutamine biosynthesis couples the TCA cycle with nitrogen anabolism and plays a critical role in PDAC.

The Pleiotropic Effects of Glutamine Metabolism in Cancer.

Metabolic programs are known to be altered in cancers arising from various tissues. Malignant transformation can alter signaling pathways related to metabolism and increase the demand for both energy and biomass for the proliferating cancerous cells. This scenario is further complexed by the crosstalk between transformed cells and the microenvironment. One of the most common metabolic alterations, which occurs in many tissues and in the context of multiple oncogenic drivers, is the increased demand for the amino acid glutamine. Many studies have attributed this increased demand for glutamine to the carbon backbone and its role in the tricarboxylic acid (TCA) cycle anaplerosis. However, an increasing number of studies are now emphasizing the importance of glutamine functioning as a proteogenic building block, a nitrogen donor and carrier, an exchanger for import of other amino acids, and a signaling molecule. Herein, we highlight the recent literature on glutamine's versatile role in cancer, with a focus on nitrogen metabolism, and therapeutic implications of glutamine metabolism in cancer.

Tumor suppressor RARRES1 links tubulin deglutamylation to mitochondrial metabolism and cell survival.

RARRES1, a retinoic acid regulated carboxypeptidase inhibitor associated with fatty acid metabolism, stem cell differentiation and tumorigenesis is among the most commonly methylated loci in multiple cancers but has no known mechanism of action. Here we show that RARRES1 interaction with cytoplasmic carboxypeptidase 2 (CCP2) inhibits tubulin deglutamylation, which in turn regulates the mitochondrial voltage dependent anion channel (VDAC1), mitochondrial membrane potential, AMPK activation, energy balance and metabolically reprograms cells and zebrafish to a more energetic and anabolic phenotype. Depletion of RARRES1 also increases expression of stem cell markers, promotes anoikis, anchorage independent growth and insensitivity to multiple apoptotic stimuli. As depletion of CCP2 or inhibition of VDAC1 reverses the effects of RARRES1 depletion on energy balance and cell survival we conclude that RARRES1 modulation of CCP2-modulated tubulin-mitochondrial VDAC1 interactions is a fundamental regulator of cancer and stem cell metabolism and survival.

Tumor suppressor RARRES1- A novel regulator of fatty acid metabolism in epithelial cells.

Retinoic acid receptor responder 1 (RARRES1) is silenced in many cancers and is differentially expressed in metabolism associated diseases, such as hepatic steatosis, hyperinsulinemia and obesity. Here we report a novel function of RARRES1 in metabolic reprogramming of epithelial cells. Using non-targeted LC-MS, we discovered that RARRES1 depletion in epithelial cells caused a global increase in lipid synthesis. RARRES1-depleted cells rewire glucose metabolism by switching from aerobic glycolysis to glucose-dependent de novo lipogenesis (DNL). Treatment with fatty acid synthase (FASN) inhibitor, C75, reversed the effects of RARRES1 depletion. The increased DNL in RARRES1-depleted normal breast and prostate epithelial cells proved advantageous to the cells during starvation, as the increase in fatty acid availability lead to more oxidized fatty acids (FAO), which were used for mitochondrial respiration. Expression of RARRES1 in several common solid tumors is also contextually correlated with expression of fatty acid metabolism genes and fatty acid-regulated transcription factors. Pathway enrichment analysis led us to determine that RARRES1 is regulated by peroxisome proliferating activated receptor (PPAR) signaling. These findings open up a new avenue for metabolic reprogramming and identify RARRES1 as a potential target for cancers and other diseases with impaired fatty acid metabolism.