p53 engages the cGAS/STING cytosolic DNA sensing pathway for tumor suppression.

Tumor suppression by TP53 involves cell-autonomous and non-cell-autonomous mechanisms. TP53 can suppress tumor growth by modulating immune system functions; however, the mechanistic basis for this activity is not well understood. We report that p53 promotes the degradation of the DNA exonuclease TREX1, resulting in cytosolic dsDNA accumulation. We demonstrate that p53 requires the ubiquitin ligase TRIM24 to induce TREX1 degradation. The cytosolic DNA accumulation resulting from TREX1 degradation activates the cytosolic DNA-sensing cGAS/STING pathway, resulting in induction of type I interferons. TREX1 overexpression sufficed to block p53 activation of the cGAS/STING pathway. p53-mediated induction of type I interferon (IFNB1) is suppressed by cGAS/STING knockout, and p53's tumor suppressor activities are compromised by the loss of signaling through the cGAS/STING pathway. Thus, our study reveals that p53 utilizes the cGAS/STING innate immune system pathway for both cell-intrinsic and cell-extrinsic tumor suppressor activities.

Serine Supports Epithelial and Immune Cell Function in Colitis.

Inflammatory bowel diseases (IBD) are chronic inflammatory disorders of the gastrointestinal tract that are largely driven by immune cell activity, and mucosal healing is critical for remission. Serine is a nonessential amino acid that supports epithelial and immune cell metabolism and proliferation; however, whether these roles affect IBD pathogenesis is not well understood. Herein, the study showed that serine synthesis increased selectively in the epithelial cells of colons from patients with IBD and murine models of colitis. Inhibiting serine synthesis impaired colonic mucosal healing and increased susceptibility to acute injury in mice, effects associated with diminished epithelial cell proliferation. Dietary removal of serine similarly sensitized mice to acute chemically induced colitis but ameliorated inflammation in chronic colitis models. The anti-inflammatory effect of exogenous serine depletion in chronic colitis was associated with mitochondrial dysfunction of macrophages, resulting in impaired nucleotide production and proliferation. Collectively, these results suggest that serine plays an important role in both epithelial and immune cell biology in the colon and that modulating its availability could impact IBD pathogenesis.

Metabolic regulation of the mitochondrial immune checkpoint.

Disrupting mitochondrial function in malignant cells is a promising strategy to enhance anticancer immunity. We have recently demonstrated that depriving colorectal cancer cells of serine results in mitochondrial dysfunction coupled with the cytosolic accumulation of mitochondrial DNA and consequent activation of CGAS- and STING-dependent tumor-targeting immune responses.

Serine Depletion Promotes Antitumor Immunity by Activating Mitochondrial DNA-Mediated cGAS-STING Signaling.

Serine is critical for supporting cancer metabolism, and depriving malignant cells of this nonessential amino acid exerts antineoplastic effects, in large part, through disrupting metabolic pathways. Given the intricate relationship between cancer metabolism and the immune system, the metabolic defects imposed by serine deprivation might impact tumor-targeting immunity. In this study, we demonstrated that restricting endogenous and exogenous sources of serine in colorectal cancer cells results in mitochondrial dysfunction, leading to mitochondrial DNA (mtDNA) accumulation in the cytosol and consequent cGAS-STING1-driven type I IFN secretion. Depleting mtDNA or blocking its release attenuated cGAS-STING1 activation during serine deprivation. In vivo studies revealed that serine deprivation limits tumor growth, accompanied by enhanced type I IFN signaling and intratumoral infiltration of immune effector cells. Notably, the tumor-suppressive and immune-enhancing effects of serine restriction were impaired by T-cell depletion and IFN receptor blockade. Moreover, disrupting cGAS-STING1 signaling in colorectal cancer cells limited the immunostimulatory and tumor-suppressive effects of serine deprivation. Lastly, serine depletion increased the sensitivity of tumors to an immune checkpoint inhibitor targeting PD-1. Taken together, these findings reveal a role for serine as a suppressor of antitumor immunity, suggesting that serine deprivation may be employed to enhance tumor immunogenicity and improve responsiveness to immune checkpoint inhibitors. Significance: Depriving cancer cells of serine provokes mitochondrial perturbations that induce cytosolic mitochondrial DNA accumulation and subsequent activation of cGAS-STING signaling, stimulating tumor-targeting immune responses that can be enhanced with PD-1 targeted therapy. See related commentary by Borges and Garg, p. 2569.

Induction and evaluation of murine colitis induced by T cell transfer.

Inflammatory bowel diseases (IBD) involve repetitive bouts of inflammation in the intestinal tract and can result in severe morbidity for patients. Moreover, long-standing IBD increases the risk for developing intestinal neoplasia. Although several factors including immune cell activity, microbiota and diet have been implicated in IBD pathogenesis, it is still considered a disease of idiopathic origin. Therefore, much work is needed to identify the critical mediators in IBD onset, severity and response to treatment. Mouse models are useful for identifying factors that contribute to IBD and the efficacy of therapy, which can then be tested in humans. There are currently multiple IBD models including the use of chemical induction, genetic manipulation and modulation of the immune response. The T cell transfer colitis model provides a quality mimic of human IBD that is T cell driven and results in inflammation in both the ileum and colon. Here, we have provided a detailed step-by-step protocol to induce inflammation and assess disease severity using this model. Such a detailed methodologic description will help to increase its utilization to advance IBD research.

Mutant p53 suppresses innate immune signaling to promote tumorigenesis.

Mutant p53 (mtp53) proteins can exert cancer-promoting gain-of-function activities. We report a mechanism by which mtp53 suppresses both cell-autonomous and non-cell-autonomous signaling to promote cancer cell survival and evasion of tumor immune surveillance. Mtp53 interferes with the function of the cytoplasmic DNA sensing machinery, cGAS-STING-TBK1-IRF3, that activates the innate immune response. Mtp53, but not wild-type p53, binds to TANK-binding protein kinase 1 (TBK1) and prevents the formation of a trimeric complex between TBK1, STING, and IRF3, which is required for activation, nuclear translocation, and transcriptional activity of IRF3. Inactivation of innate immune signaling by mtp53 alters cytokine production, resulting in immune evasion. Restoring TBK1 signaling is sufficient to bypass mtp53 and lead to restored immune cell function and cancer cell eradication. This work is of translational interest because therapeutic approaches that restore TBK1 function could potentially reactivate immune surveillance and eliminate mtp53 tumors.

Exogenous and Endogenous Sources of Serine Contribute to Colon Cancer Metabolism, Growth, and Resistance to 5-Fluorouracil.

Serine is a nonessential amino acid generated by the sequential actions of phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase (PSAT1), and phosphoserine phosphatase (PSPH). Increased serine biosynthesis occurs in several cancers and supports tumor growth. In addition, cancer cells can harness exogenous serine to enhance their metabolism and proliferation. Here we tested the relative contributions of exogenous and endogenous sources of serine on the biology of colorectal cancer. In murine tumors, Apc status was identified as a determinant of the expression of genes controlling serine synthesis. In patient samples, PSAT1 was overexpressed in both colorectal adenomas and adenocarcinomas. Combining genetic deletion of PSAT1 with exogenous serine deprivation maximally suppressed the proliferation of colorectal cancer cells and induced profound metabolic defects including diminished nucleotide production. Inhibition of serine synthesis enhanced the transcriptional changes following exogenous serine removal as well as alterations associated with DNA damage. Both loss of PSAT1 and removal of serine from the diet were necessary to suppress colorectal cancer xenograft growth and enhance the antitumor activity of 5-fluorouracil (5-FU). Restricting endogenous and exogenous serine in vitro augmented 5-FU-induced cell death, DNA damage, and metabolic perturbations, likely accounting for the observed antitumor effect. Collectively, our results suggest that both endogenous and exogenous sources of serine contribute to colorectal cancer growth and resistance to 5-FU. SIGNIFICANCE: These findings provide insights into the metabolic requirements of colorectal cancer and reveal a novel approach for its treatment. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/9/2275/F1.large.jpg.

Inhibition of USP2 eliminates cancer stem cells and enhances TNBC responsiveness to chemotherapy.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer that harbors enriched cancer stem cell (CSC) populations in tumors. Conventional chemotherapy is a standard treatment for TNBC, but it spares the CSC populations, which cause tumor recurrence and progression. Therefore, identification of the core molecular pathway that controls CSC activity and expansion is essential for developing effective therapeutics for TNBC. In this study, we identify that USP2 deubiquitinating enzyme is upregulated in CSCs and is a novel regulator of CSCs. Genetic and pharmacological targeting of USP2 substantially inhibits the self-renewal, expansion and chemoresistance of CSCs. We show that USP2 maintains the CSC population by activating self-renewing factor Bmi1 and epithelial-mesenchymal transition through Twist upregulation. Mechanistically, USP2 promotes Twist stabilization by removing ß-TrCP-mediated ubiquitination of Twist. Animal studies indicate that pharmacological inhibition of USP2 suppresses tumor progression and sensitizes tumor responses to chemotherapy in TNBC. Furthermore, the histological analyses reveal a positive correlation between USP2 upregulation and lymph node metastasis. Our findings together demonstrate a previously unrecognized role of USP2 in mediating Twist activation and CSC enrichment, suggesting that targeting USP2 is a novel therapeutic strategy to tackle TNBC.

'Dual hit' metabolic modulator LDCA selectively kills cancer cells by efficient competitive inhibition of LDH-A.

Herein, we synthesize and elucidate the potential of a novel 'dual hit' molecule, LDCA, to constitutively block lactate dehydrogenase isoform-A (LDH-A) to selectively subvert apoptosis and rigorously attenuate breast tumor progression in a mouse model, comprehensively delineating the therapeutic prospectus of LDCA in the field of cancer metabolics.

Role of metabolic modulator Bet-CA in altering mitochondrial hyperpolarization to suppress cancer associated angiogenesis and metastasis.

Solid tumors characteristically reflect a metabolic switching from glucose oxidation to glycolysis that plays a fundamental role in angiogenesis and metastasis to facilitate aggressive tumor outcomes. Hyperpolarized mitochondrial membrane potential is a manifestation of malignant cells that compromise the intrinsic pathways of apoptosis and confer a suitable niche to promote the cancer associated hallmark traits. We have previously reported that co-drug Bet-CA selectively targets cancer cells by inducing metabolic catastrophe without a manifest in toxicity. Here we report that the same molecule at a relatively lower concentration deregulates the cardinal phenotypes associated with angiogenesis and metastasis. In mice syngeneic 4T1 breast cancer model, Bet-CA exhibited effective abrogation of angiogenesis and concomitantly obliterated lung metastasis consistent with altered mitochondrial bioenergetics. Furthermore, Bet-CA significantly lowered vascular endothelial growth factor (VEGF) levels and obviated matrix metalloproteases (MMP-2/9) production directly to the criterion where abrogation of autocrine VEGF/VEGFR2 signalling loop was documented. In vitro studies anticipatedly documented the role of Bet-CA in inhibiting actin remodeling, lamellipodia formation and cell membrane ruffling to constitutively suppress cell motility and invasion. Results comprehensively postulate that Bet-CA, a mitochondria targeting metabolic modulator may serve as an excellent candidate for combating angiogenesis and metastasis.

A potent tumoricidal co-drug 'Bet-CA'--an ester derivative of betulinic acid and dichloroacetate selectively and synergistically kills cancer cells.

Selective targeting of cancer cells employing multiple combinations as co-drug holds promise for new generation therapeutics. Betulinic acid (BA), a plant secondary metabolite kills cancer cells and Dichloroacetate (DCA) is capable of reversing the Warburg phenotype by inhibiting pyruvate dehydrogenase kinase (PDK). Here, we report synthesis, characterization and tumoricidal potential of a co-drug Bet-CA, where a DCA molecule has been appended on C-3 hydroxyl group of BA to generate an ester derivative for increased solubility and subsequent cleavage by internal esterase(s) to release one unit each of BA and DCA. In vitro studies revealed pronounced synergistic cytotoxicity of Bet-CA against a broad spectrum of cancer cells and it selectively killed them when co-cultured with human fibroblasts. Bet-CA treatment increased reactive oxygen species (ROS) production, significantly altered mitochondrial membrane potential gradient (ΔΨm); followed by the release of cytochrome c (Cyt c) which prompted cells to undergo mitochondria mediated apoptosis. In vivo experimentation expectedly exhibited tumor inhibitory potential of Bet-CA and clinically achievable doses did not produce any apparent toxicity. Taken together, results suggestively raise an important corollary hypothesis stating that Bet-CA selectively and synergistically combats cancer without producing toxic manifestations and emerges to be the prospect for the new generation therapeutics.