Glutamine Supplementation as an Anticancer Strategy: A Potential Therapeutic Alternative to the Convention.

Glutamine, a multifaceted nonessential/conditionally essential amino acid integral to cellular metabolism and immune function, holds pivotal importance in the landscape of cancer therapy. This review delves into the intricate dynamics surrounding both glutamine antagonism strategies and glutamine supplementation within the context of cancer treatment, emphasizing the critical role of glutamine metabolism in cancer progression and therapy. Glutamine antagonism, aiming to disrupt tumor growth by targeting critical metabolic pathways, is challenged by the adaptive nature of cancer cells and the complex metabolic microenvironment, potentially compromising its therapeutic efficacy. In contrast, glutamine supplementation supports immune function, improves gut integrity, alleviates treatment-related toxicities, and improves patient well-being. Moreover, recent studies highlighted its contributions to epigenetic regulation within cancer cells and its potential to bolster anti-cancer immune functions. However, glutamine implementation necessitates careful consideration of potential interactions with ongoing treatment regimens and the delicate equilibrium between supporting normal cellular function and promoting tumorigenesis. By critically assessing the implications of both glutamine antagonism strategies and glutamine supplementation, this review aims to offer comprehensive insights into potential therapeutic strategies targeting glutamine metabolism for effective cancer management.

Supraphysiological glutamine as a means of depleting intracellular amino acids to enhance pancreatic cancer chemosensitivity.

Limited efficacy of systemic therapy for pancreatic ductal adenocarcinoma (PDAC) patients contributes to high mortality. Cancer cells develop strategies to secure nutrients in nutrient-deprived conditions and chemotherapy treatment. Despite the dependency of PDAC on glutamine (Gln) for growth and survival, strategies designed to suppress Gln metabolism have limited effects. Here, we demonstrated that supraphysiological concentrations of glutamine (SPG) could produce paradoxical responses leading to tumor growth inhibition alone and in combination with chemotherapy. Integrated metabolic and transcriptomic analysis revealed that the growth inhibitory effect of SPG was the result of a decrease in intracellular amino acid and nucleotide pools. Mechanistically, disruption of the sodium gradient, plasma membrane depolarization, and competitive inhibition of amino acid transport mediated amino acid deprivation. Among standard chemotherapies given to PDAC patients, gemcitabine treatment resulted in a significant enrichment of amino acid and nucleoside pools, exposing a metabolic vulnerability to SPG-induced metabolic alterations. Further analysis highlighted a superior anticancer effect of D-glutamine, a non-metabolizable enantiomer of the L-glutamine, by suppressing both amino acid uptake and glutaminolysis, in gemcitabine-treated preclinical models with no apparent toxicity. Our study suggests supraphysiological glutamine could be a means of inhibiting amino acid uptake and nucleotide biosynthesis, potentiating gemcitabine sensitivity in PDAC.

Plasma Metabolomics Predicts Chemotherapy Response in Advanced Pancreatic Cancer.

Pancreatic cancer (PC) is one of the deadliest cancers. Developing biomarkers for chemotherapeutic response prediction is crucial for improving the dismal prognosis of advanced-PC patients (pts). To evaluate the potential of plasma metabolites as predictors of the response to chemotherapy for PC patients, we analyzed plasma metabolites using high-performance liquid chromatography-mass spectrometry from 31 cachectic, advanced-PC subjects enrolled into the PANCAX-1 (NCT02400398) prospective trial to receive a jejunal tube peptide-based diet for 12 weeks and who were planned for palliative chemotherapy. Overall, there were statistically significant differences in the levels of intermediates of multiple metabolic pathways in pts with a partial response (PR)/stable disease (SD) vs. progressive disease (PD) to chemotherapy. When stratified by the chemotherapy regimen, PD after 5-fluorouracil-based chemotherapy (e.g., FOLFIRINOX) was associated with decreased levels of amino acids (AAs). For gemcitabine-based chemotherapy (e.g., gemcitabine/nab-paclitaxel), PD was associated with increased levels of intermediates of glycolysis, the TCA cycle, nucleoside synthesis, and bile acid metabolism. These results demonstrate the feasibility of plasma metabolomics in a prospective cohort of advanced-PC patients for assessing the effect of enteral feeding as their primary source of nutrition. Metabolic signatures unique to FOLFIRINOX or gemcitabine/nab-paclitaxel may be predictive of a patient's response and warrant further study.

NFYA promotes malignant behavior of triple-negative breast cancer in mice through the regulation of lipid metabolism.

Two splicing variants exist in NFYA that exhibit high expression in many human tumour types. The balance in their expression correlates with prognosis in breast cancer, but functional differences remain unclear. Here, we demonstrate that NFYAv1, a long-form variant, upregulates the transcription of essential lipogenic enzymes ACACA and FASN to enhance the malignant behavior of triple-negative breast cancer (TNBC). Loss of the NFYAv1-lipogenesis axis strongly suppresses malignant behavior in vitro and in vivo, indicating that the NFYAv1-lipogenesis axis is essential for TNBC malignant behavior and that the axis might be a potential therapeutic target for TNBC. Furthermore, mice deficient in lipogenic enzymes, such as Acly, Acaca, and Fasn, exhibit embryonic lethality; however, Nfyav1-deficient mice exhibited no apparent developmental abnormalities. Our results indicate that the NFYAv1-lipogenesis axis has tumour-promoting effects and that NFYAv1 may be a safe therapeutic target for TNBC.

Combination L-Glutamine with Gemcitabine and Nab-Paclitaxel in Treatment-Naïve Advanced Pancreatic Cancer: The Phase I GlutaPanc Study Protocol.

Advanced pancreatic cancer is underscored by progressive therapeutic resistance and a dismal 5-year survival rate of 3%. Preclinical data demonstrated glutamine supplementation, not deprivation, elicited antitumor effects against pancreatic ductal adenocarcinoma (PDAC) alone and in combination with gemcitabine in a dose-dependent manner. The GlutaPanc phase I trial is a single-arm, open-label clinical trial investigating the safety of combination L-glutamine, gemcitabine, and nab-paclitaxel in subjects (n = 16) with untreated, locally advanced unresectable or metastatic pancreatic cancer. Following a 7-day lead-in phase with L-glutamine, the dose-finding phase via Bayesian design begins with treatment cycles lasting 28 days until disease progression, intolerance, or withdrawal. The primary objective is to establish the recommended phase II dose (RP2D) of combination L-glutamine, gemcitabine, and nab-paclitaxel. Secondary objectives include safety of the combination across all dose levels and preliminary evidence of antitumor activity. Exploratory objectives include evaluating changes in plasma metabolites across multiple time points and changes in the stool microbiome pre and post L-glutamine supplementation. If this phase I clinical trial demonstrates the feasibility of L-glutamine in combination with nab-paclitaxel and gemcitabine, we would advance the development of this combination as a first-line systemic option in subjects with metastatic pancreatic cancer, a high-risk subgroup desperately in need of additional therapies.

Gut microbiome and pancreatic cancer cachexia: An evolving relationship.

Nearly 80% of patients with pancreatic ductal adenocarcinoma (PDAC) develop cachexia along their disease course. Cachexia is characterized by progressive weight loss, muscle wasting, and systemic inflammation and has been linked to poorer outcomes and impairments in quality of life. Management of PDAC cachexia has historically involved a multidisciplinary effort comprised of nutritional support, pancreatic enzyme replacement therapy, and/or pharmacologic interventions. Despite current interventions to mitigate PDAC cachexia, a significant proportion of patients continue to die from complications associated with cachexia underscoring the need for novel insights and treatments for this syndrome. We highlight the feasibility and effectiveness of a recent enteral feeding prospective trial at our institution to improve cachexia outcomes in patients with advanced PDAC. Additionally, we were among the first to characterize the stool microbiome composition in patients with advanced PDAC receiving enteral feeding for the treatment of cachexia. Novel insights into the relationship between enteral nutritional support, cachexia, and the gut microbiome are presented. These promising results are discussed in the context of a potential ability to modulate the stool microbiome as a new interventional strategy to mitigate PDAC cachexia.

Fatty Acid Signaling Impacts Prostate Cancer Lineage Plasticity in an Autocrine and Paracrine Manner.

Prostate cancer (PCa) affects an estimated 250,000 men every year and causes 34,000 deaths annually. A high-fat diet and obesity are associated with PCa progression and mortality. This study's premise was the novel observation of crosstalk between PCa epithelia and cancer-associated fibroblasts (CAF) in response to palmitate-mediated lineage plasticity. We found that cholesterol activated canonical Hedgehog (Hh) signaling by increasing cilium Gli activity in PCa cells, while palmitate activated Hh independent of Gli. Exogenous palmitate activated SOX2, a known mediator of lineage plasticity, in PCa cells cocultured with CAF. Stroma-derived Wnt5a was upregulated in CAF while cocultured with PCa cells and treated with palmitate. Wnt5a knockdown in CAF inhibited Hh and SOX2 expression in PCa cells from cocultures. These findings supported our proposed mechanism of a high-fat diet promoting Hh signaling-mediated transformation within the tumor microenvironment. SOX2 and Wnt5a expression were limited by the CD36 neutralizing antibody. Mice xenografted with PCa epithelia and CAF tumors were fed a high-fat diet, leading to elevated SOX2 expression and lineage plasticity reprogramming compared to mice fed an isocaloric rodent diet. CD36 inhibition with enzalutamide elevated apoptosis by TUNEL, but limited proliferation and SOX2 expression compared to enzalutamide alone. This study revealed a mechanism for a high-fat diet to affect prostate cancer progression. We found that saturated fat induced lineage plasticity reprogramming of PCa by interaction with CAF through Wnt5a and Hh signaling.

Cardiomyocytes disrupt pyrimidine biosynthesis in nonmyocytes to regulate heart repair.

Various populations of cells are recruited to the heart after cardiac injury, but little is known about whether cardiomyocytes directly regulate heart repair. Using a murine model of ischemic cardiac injury, we demonstrate that cardiomyocytes play a pivotal role in heart repair by regulating nucleotide metabolism and fates of nonmyocytes. Cardiac injury induced the expression of the ectonucleotidase ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which hydrolyzes extracellular ATP to form AMP. In response to AMP, cardiomyocytes released adenine and specific ribonucleosides that disrupted pyrimidine biosynthesis at the orotidine monophosphate (OMP) synthesis step and induced genotoxic stress and p53-mediated cell death of cycling nonmyocytes. As nonmyocytes are critical for heart repair, we showed that rescue of pyrimidine biosynthesis by administration of uridine or by genetic targeting of the ENPP1/AMP pathway enhanced repair after cardiac injury. We identified ENPP1 inhibitors using small molecule screening and showed that systemic administration of an ENPP1 inhibitor after heart injury rescued pyrimidine biosynthesis in nonmyocyte cells and augmented cardiac repair and postinfarct heart function. These observations demonstrate that the cardiac muscle cell regulates pyrimidine metabolism in nonmuscle cells by releasing adenine and specific nucleosides after heart injury and provide insight into how intercellular regulation of pyrimidine biosynthesis can be targeted and monitored for augmenting tissue repair.

Peripubertal high-fat diet promotes c-Myc stabilization in mammary gland epithelium.

Dietary fat consumption during accelerated stages of mammary gland development, such as peripubertal maturation or pregnancy, is known to increase the risk for breast cancer. However, the underlying molecular mechanisms are not fully understood. Here we examined the gene expression profile of mouse mammary epithelial cells (MMECs) on exposure to a high-fat diet (HFD) or control diet (CD). Trp53-/- female mice were fed with the experimental diets for 5 weeks during the peripubertal period (3-8 weeks of age). The treatment showed no significant difference in body weight between the HFD-fed mice and CD-fed mice. However, gene set enrichment analysis predicted a significant enrichment of c-Myc target genes in animals fed HFD. Furthermore, we detected enhanced activity and stabilization of c-Myc protein in MMECs exposed to a HFD. This was accompanied by augmented c-Myc phosphorylation at S62 with a concomitant increase in ERK phosphorylation. Moreover, MMECs derived from HFD-fed Trp53-/- mouse showed increased colony- and sphere-forming potential that was dependent on c-Myc. Further, oleic acid, a major fatty acid constituent of the HFD, and TAK-875, an agonist to G protein-coupled receptor 40 (a receptor for oleic acid), enhanced c-Myc stabilization and MMEC proliferation. Overall, our data indicate that HFD influences MMECs by stabilizing an oncoprotein, pointing to a novel mechanism underlying dietary fat-mediated mammary carcinogenesis.

Retinoblastoma Inactivation Induces a Protumoral Microenvironment via Enhanced CCL2 Secretion.

Cancer cell-intrinsic properties caused by oncogenic mutations have been well characterized; however, how specific oncogenes and tumor suppressors impact the tumor microenvironment (TME) is not well understood. Here, we present a novel non-cell-autonomous function of the retinoblastoma (RB) tumor suppressor in controlling the TME. RB inactivation stimulated tumor growth and neoangiogenesis in a syngeneic and orthotropic murine soft-tissue sarcoma model, which was associated with recruitment of tumor-associated macrophages (TAM) and immunosuppressive cells such as Gr1+CD11b+ myeloid-derived suppressor cells (MDSC) or Foxp3+ regulatory T cells (Treg). Gene expression profiling and analysis of genetically engineered mouse models revealed that RB inactivation increased secretion of the chemoattractant CCL2. Furthermore, activation of the CCL2-CCR2 axis in the TME promoted tumor angiogenesis and recruitment of TAMs and MDSCs into the TME in several tumor types including sarcoma and breast cancer. Loss of RB increased fatty acid oxidation (FAO) by activating AMP-activated protein kinase that led to inactivation of acetyl-CoA carboxylase, which suppresses FAO. This promoted mitochondrial superoxide production and JNK activation, which enhanced CCL2 expression. These findings indicate that the CCL2-CCR2 axis could be an effective therapeutic target in RB-deficient tumors. SIGNIFICANCE: These findings demonstrate the cell-nonautonomous role of the tumor suppressor retinoblastoma in the tumor microenvironment, linking retinoblastoma loss to immunosuppression.

Involvement of Prokineticin 2-expressing Neutrophil Infiltration in 5-Fluorouracil-induced Aggravation of Breast Cancer Metastasis to Lung.

Adjuvant chemotherapy is used for human breast cancer patients, even after curative surgery of primary tumor, to prevent tumor recurrence primarily as a form of metastasis. However, anticancer drugs can accelerate metastasis in several mouse metastasis models. Hence, we examined the effects of postsurgical administration with 5-fluorouracil (5-FU), doxorubicin, and cyclophosphamide, on lung metastasis process, which developed after the resection of the primary tumor arising from the orthotopic injection of a mouse triple-negative breast cancer cell line, 4T1. Only 5-FU markedly increased the numbers and sizes of lung metastasis foci, with enhanced tumor cell proliferation and angiogenesis as evidenced by increases in Ki67-positive cell numbers and CD31-positive areas, respectively. 5-FU-mediated augmented lung metastasis was associated with increases in intrapulmonary neutrophil numbers and expression of neutrophilic chemokines, Cxcl1 and Cxcl2 in tumor cells, with few effects on intrapulmonary T-cell or macrophage numbers. 5-FU enhanced Cxcl1 and Cxcl2 expression in 4T1 cells in a NFκB-dependent manner. Moreover, the administration of a neutrophil-depleting antibody or a Cxcr2 antagonist, SB225002, significantly attenuated 5-FU-mediated enhanced lung metastasis with depressed neutrophil infiltration. Furthermore, infiltrating neutrophils and 4T1 cells abundantly expressed prokineticin-2 (Prok2) and its receptor, Prokr1, respectively. Finally, the administration of 5-FU after the resection of the primary tumor failed to augment lung metastasis in the mice receiving Prokr1-deleted 4T1 cells. Collectively, 5-FU can enhance lung metastasis by inducing tumor cells to produce Cxcl1 and Cxcl2, which induced the migration of neutrophils expressing Prok2 with a capacity to enhance 4T1 cell proliferation. Mol Cancer Ther; 17(7); 1515-25. ©2018 AACR.

MicroRNA-140 mediates RB tumor suppressor function to control stem cell-like activity through interleukin-6.

We established an in vitro cell culture system to determine novel activities of the retinoblastoma (Rb) protein during tumor progression. Rb depletion in p53-null mouse-derived soft tissue sarcoma cells induced a spherogenic phenotype. Cells retrieved from Rb-depleted spheres exhibited slower proliferation and less efficient BrdU incorporation, however, much higher spherogenic activity and aggressive behavior. We discovered six miRNAs, including mmu-miR-18a, -25, -29b, -140, -337, and -1839, whose expression levels correlated tightly with the Rb status and spherogenic activity. Among these, mmu-miR-140 appeared to be positively controlled by Rb and to antagonize the effect of Rb depletion on spherogenesis and tumorigenesis. Furthermore, among genes potentially targeted by mmu-miR-140, Il-6 was upregulated by Rb depletion and downregulated by mmu-mir-140 overexpression. Altogether, we demonstrate the possibility that mmu-mir-140 mediates the Rb function to downregulate Il-6 by targeting its 3'-untranslated region. Finally, we detected the same relationship among RB, hsa-miR-140 and IL-6 in a human breast cancer cell line MCF-7. Because IL-6 is a critical modulator of malignant features of cancer cells and the RB pathway is impaired in the majority of cancers, hsa-miR-140 might be a promising therapeutic tool that disrupts linkage between tumor suppressor inactivation and pro-inflammatory cytokine response.

An in vitro system to characterize prostate cancer progression identified signaling required for self-renewal.

Mutations in RB and PTEN are linked to castration resistance and poor prognosis in prostate cancer. Identification of genes that are regulated by these tumor suppressors in a context that recapitulates cancer progression may be beneficial for discovering novel therapeutic targets. Although various genetically engineered mice thus far provided tumor models with various pathological stages, they are not ideal for detecting dynamic changes in gene transcription. Additionally, it is difficult to achieve an effect specific to tumor progression via gain of functions of these genes. In this study, we developed an in vitro model to help identify RB- and PTEN-loss signatures during the malignant progression of prostate cancers. Trp53-/- ; Rbf/f , Trp53-/- ; Ptenf/f , and Trp53-/- ; Rbf/f ; Ptenf/f prostate epithelial cells were infected with AD-LacZ or AD-Cre. We found that deletion of Rb, Pten or both stimulated prostasphere formation and tumor development in immune-compromised mice. The GO analysis of genes affected by the deletion of Rb or Pten in Trp53-/- prostate epithelial cells identified a number of genes encoding cytokines, chemokines and extracellular matrix remodeling factors, but only few genes related to cell cycle progression. Two genes (Il-6 and Lox) were further analyzed. Blockade of Il-6 signaling and depletion of Lox significantly attenuated prostasphere formation in 3D culture, and in the case of IL-6, strongly suppressed tumor growth in vivo. These findings suggest that our in vitro model may be instrumental in identifying novel therapeutic targets of prostate cancer progression, and further underscore IL-6 and LOX as promising therapeutic targets. © 2015 Wiley Periodicals, Inc.

Undifferentiated State Induced by Rb-p53 Double Inactivation in Mouse Thyroid Neuroendocrine Cells and Embryonic Fibroblasts.

Retinoblastoma tumor suppressor protein (RB) is inactivated more frequently during tumor progression than during tumor initiation. However, its exact role in controlling the malignant features associated with tumor progression is poorly understood. We established in vivo and in vitro models to investigate the undifferentiated state induced by Rb inactivation. Rb heterozygous mice develop well-differentiated thyroid medullary carcinoma. We found that additional deletion of Trp53, without change in lineage, converted these Rb-deficient tumors to a poorly differentiated type associated with higher self-renewal activity. Freshly prepared mouse embryonic fibroblasts (MEFs) of Rb(-/-) ; Trp53(-/-) background formed stem cell-like spheres that expressed significant levels of embryonic genes despite of lacking the ability to form colonies on soft agar or tumors in immune-deficient mice. This suggested that Rb-p53 double inactivation resulted in an undifferentiated status but without carcinogenic conversion. We next established Rb(-/-) ; N-ras(-/-) MEFs that harbored a spontaneous carcinogenic mutation in Trp53. These cells (RN6), in an Rb-dependent manner, efficiently generated spheres that expressed very high levels of embryonic genes, and appeared to be carcinogenic. We then screened an FDA-approved drug library to search for agents that suppressed the spherogenic activity of RN6 cells. Data revealed that RN6 cells were sensitive to specific agents including ones those are effective against cancer stem cells. Taken together, all these findings suggest that the genetic interaction between Rb and p53 is a critical determinant of the undifferentiated state in normal and tumor cells.

ATM mediates pRB function to control DNMT1 protein stability and DNA methylation.

The retinoblastoma tumor suppressor gene (RB) product has been implicated in epigenetic control of gene expression owing to its ability to physically bind to many chromatin modifiers. However, the biological and clinical significance of this activity was not well elucidated. To address this, we performed genetic and epigenetic analyses in an Rb-deficient mouse thyroid C cell tumor model. Here we report that the genetic interaction of Rb and ATM regulates DNMT1 protein stability and hence controls the DNA methylation status in the promoters of at least the Ink4a, Shc2, FoxO6, and Noggin genes. Furthermore, we demonstrate that inactivation of pRB promotes Tip60 (acetyltransferase)-dependent ATM activation; allows activated ATM to physically bind to DNMT1, forming a complex with Tip60 and UHRF1 (E3 ligase); and consequently accelerates DNMT1 ubiquitination driven by Tip60-dependent acetylation. Our results indicate that inactivation of the pRB pathway in coordination with aberration in the DNA damage response deregulates DNMT1 stability, leading to an abnormal DNA methylation pattern and malignant progression.

The inositol phosphatase SHIP2 negatively regulates insulin/IGF-I actions implicated in neuroprotection and memory function in mouse brain.

Impairment of insulin and IGF-I signaling in the brain is one of the causes of dementia associated with diabetes mellitus and Alzheimer's disease. However, the precise pathological processes are largely unknown. In the present study, we found that SH2-containing inositol 5'-phosphatase 2 (SHIP2), a negative regulator of phosphatidylinositol 3,4,5-trisphosphate-mediated signals, is widely expressed in adult mouse brain. When a dominant-negative mutant of SHIP2 was expressed in cultured neurons, insulin signaling was augmented, indicating physiological significance of endogenous SHIP2 in neurons. Interestingly, SHIP2 mRNA and protein expression levels were significantly increased in the brain of type 2 diabetic db/db mice. To investigate the impact of increased expression of SHIP2 in the brain, we further employed transgenic mice overexpressing SHIP2 and found that increased amounts of SHIP2 induced the disruption of insulin/IGF-I signaling through Akt. Neuroprotective effects of insulin and IGF-I were significantly attenuated in cultured cerebellar granule neurons from SHIP2 transgenic mice. Consistently, terminal deoxynucleotide transferase-mediated dUTP nick end labeling assay demonstrated that the number of apoptosis-positive cells was increased in cerebral cortex of the transgenic mice at an elderly age. Furthermore, SHIP2 transgenic mice exhibited impaired memory performance in the Morris water maze, step-through passive avoidance, and novel-object-recognition tests. Importantly, inhibition of SHIP2 ameliorated the impairment of hippocampal synaptic plasticity and memory formation in db/db mice. These results suggest that SHIP2 is a potent negative regulator of insulin/IGF-I actions in the brain, and excess amounts of SHIP2 may be related, at least in part, to brain dysfunction in insulin resistance with type 2 diabetes.