Glutamine and cancer: cell biology, physiology, and clinical opportunities

A Variant of SLC1A5 Is a Mitochondrial Glutamine Transporter for Metabolic Reprogramming in Cancer Cells

How mitochondrial glutaminolysis contributes to redox homeostasis in cancer cells remains unclear. Here we report that the mitochondrial enzyme glutamate dehydrogenase 1 (GDH1), commonly upregulated in human cancers, is predominantly important for redox homeostasis in cancer cells by controlling the intracellular levels of its product alpha-ketoglutarate (a-KG) and subsequent metabolite fumarate. Mechanistically, fumarate binds to and activates a ROS scavenging enzyme glutathione peroxidase 1 (GPx1) to regulate redox homeostasis, which provides a proliferative advantage to cancer cells and tumor growth. Our findings not only provide novel insights into understanding of the role of glutaminolysis in redox homeostasis but also suggest a novel signaling function of fumarate that regulates GPx1, allowing additional crosstalk between glutaminolysis, TCA cycle and redox status. Targeting GDH1 by shRNA or a newly identified small molecule inhibitor R162 resulted in imbalanced redox homeostasis, leading to attenuated cancer cell proliferation and tumor growth. Thus, our findings provide proof-of-principle suggesting GDH1 as a promising therapeutic target in the treatment of human cancers associated with elevated glutamine metabolism. Citation Format: Lingtao Jin, Dan Li, Gina Alesi, Jun Fan, Hee-Bum Kang, Lu Zhou, Titus Boggon, Peng Jin, Robert Egnatchik, Ralph DeBerardinis, Kelly Magliocca, Chuan He, Martha Arellano, Hanna Khoury, Dong Shin, Fadlo Khuri, Sumin Kang. Glutamate dehydrogenase 1 signals through antioxidant glutathione peroxidase 1 to regulate redox homeostasis and tumor growth. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A17.

How mitochondrial glutaminolysis contributes to redox homeostasis in cancer cells remains unclear. Here we report that the mitochondrial enzyme glutamate dehydrogenase 1 (GDH1), commonly upregulated in human cancers, is predominantly important for redox homeostasis in cancer cells by controlling the intracellular levels of its product alpha-ketoglutarate (a-KG) and subsequent metabolite fumarate. Mechanistically, fumarate binds to and activates a ROS scavenging enzyme glutathione peroxidase 1 (GPx1) to regulate redox homeostasis, which provides a proliferative advantage to cancer cells and tumor growth. Our findings not only provide novel insights into understanding of the role of glutaminolysis in redox homeostasis but also suggest a novel signaling function of fumarate that regulates GPx1, allowing additional crosstalk between glutaminolysis, TCA cycle and redox status. Targeting GDH1 by shRNA or a newly identified small molecule inhibitor R162 resulted in imbalanced redox homeostasis, leading to attenuated cancer cell proliferation and tumor growth. Thus, our findings provide proof-of-principle suggesting GDH1 as a promising therapeutic target in the treatment of human cancers associated with elevated glutamine metabolism. Citation Format: Lingtao Jin, Dan Li, Gina Alesi, Jun Fan, Hee-Bum Kang, Lu Zhou, Titus Boggon, Kelly Magliocca, Chuan He, Martha Arellano, Hanna Khoury, Dong Shin, Fadlo Khuri, Sumin Kang. Glutamate dehydrogenase 1 signals through antioxidant glutathione peroxidase 1 to regulate redox homeostasis and tumor growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3046. doi:10.1158/1538-7445.AM2015-3046

Glutamate Dehydrogenase 1 Signals through Antioxidant Glutathione Peroxidase 1 to Regulate Redox Homeostasis and Tumor Growth

Tumor cells are characterized by a delicate balance between elevated oxidative stress and enhanced antioxidant capacity. This intricate equilibrium, maintained within a threshold known as redox homeostasis, offers a unique perspective for cancer treatment by modulating reactive oxygen species (ROS) levels beyond cellular tolerability, thereby disrupting this balance. However, currently used chemotherapy drugs require larger doses to increase ROS levels beyond the redox homeostasis threshold, which may cause serious side effects. How to disrupt redox homeostasis in cancer cells more effectively remains a challenge. In this study, we found that sodium selenite and docosahexaenoic acid (DHA), a polyunsaturated fatty acid extracted from marine fish, synergistically induced cytotoxic effects in colorectal cancer (CRC) cells. Physiological doses of DHA simultaneously upregulated oxidation and antioxidant levels within the threshold range without affecting cell viability. However, it rendered the cells more susceptible to reaching the upper limit of the threshold of redox homeostasis, facilitating the elevation of ROS levels beyond the threshold by combining with low doses of sodium selenite, thereby disrupting redox homeostasis and inducing MAPK-mediated paraptosis. This study highlights the synergistic anticancer effects of sodium selenite and DHA, which induce paraptosis by disrupting redox homeostasis in tumor cells. These findings offer a novel strategy for more targeted and less toxic cancer therapies for colorectal cancer treatment.

To prevent cell death induced by elevated oxidative stress, cancer cells activate a series of antioxidant defense mechanisms to mitigate cytotoxicity, thereby enhancing the resistance to pro-oxidative therapy. However, the underlying antioxidant mechanisms in cancer cells remain inadequately understood. Through co-immunoprecipitation followed by quantitative mass spectrometry analysis, we for the first time identified that cytoplasmic ALDH1L1 translocates into mitochondria and co-localizes with mitochondrial transcription factor TFAM in cancer cells in a ROS-dependent feedback manner. Mitochondria-translocated ALDH1L1 maintains mitochondrial redox homeostasis by producing NADPH. Moreover, our findings revealed that the ROS-mediated oxidative modification of ALDH1L1 is necessary for its interaction with HSP90β and subsequent translocation into mitochondria via TOM70, where it binds to TFAM to prevent degradation by LONP1. Furthermore, we found that mitochondrial ALDH1L1 antagonized the double-edged role of ROS in cancer cell survival, indicating that disruption of ALDH1L1 expression promoted cancer cell proliferation and autophagy but concurrently diminished cellular capacity to counteract ROS-induced apoptosis. Consistently, ALDH1L1 knockout enhanced the anti-tumor effect of low-dose pro-oxidant Elesclomol, thereby achieving better efficacy and safety of pro-oxidant therapy. Furthermore, our results demonstrated that the combination of Elesclomol with HSP90 inhibitor Ganetespib exhibited synergistic anti-tumor effects. In conclusion, our findings that mitochondria-translocated ALDH1L1 functions as a feedback regulator of redox homeostasis in cancer cells to enhance the resistance to pro-oxidative therapy can provide critical insights into developing effective pro-oxidative therapies against tumors.

Background: Using small molecule smoothened (SMO) antagonists, Hedgehog (HH) signaling had been implicated to play an important role in a variety of cancers (e.g. Brain, Lung, Pancreas, digestive tract etc.). Recently, based on studies in a selected set of pancreatic and colorectal cancer cells it has been suggested: a) earlier observation of HH signaling dependence of various cancers by others and us is due to non-specific effect of SMO antagonists; b) HH producing cancer cells do not have a functional HH pathway; c) HH signaling is not required for the growth or survival of cancer cells rather it affects the growth of tumor by activating HH pathway in stroma cells. Given the reported prevalence of HH signaling in cancers and the suggested implication of this latter work on devising a successful cancer therapy, we decided to investigate the role of HH signaling in cancer in a manner independent of SMO antagonist. Lung cancer, being one of the deadliest cancers with the reported higher incidence of HH signaling, was used as a test system to evaluate the basic assumptions of this recently proposed model of HH role in cancer. Objective: Define the role of HH signaling in cancer cells. Methodology: The human non-small cell lung carcinoma (NSCLC) cell lines (HOP62, A549, U1752, H23, H157, H522) were used as a model system to study the relevance of HH signaling in cancer cells. The HH signaling was modulated by shRNA mediated knockdown of various HH signaling components (e.g. SMO, GLI1, SHH). The lentiviruses expressing shRNA were made and used to deliver the shRNA in NSCLC cells. HH responsiveness (quantitative real time RT-PCR and western blotting), cell proliferation (ATP quantitation), survival (Annexin-V labeling), anchorage independent growth (soft agar assay) and tumorigenesis (xenografts in Nu/Nu nude mice) were evaluated following shRNA mediated attenuation of HH signaling. Currently, HH pathway activation in cancer and stroma cells of primary lung cancer samples is being evaluated by immunohistochemistry (IHC) and RNA in situ hybridization (ISH). Preliminary Results and Conclusions: Exogenous expression of SHH in HOP62 and A549 induced the HH target gene GLI1 and PTCH1 expression while shRNA mediated knockdown of HH pathway components (e.g. SMO, SHH) inhibited the HH target gene expression. Further more, knockdown of HH pathway components, attenuated the proliferation, increased the apoptosis while decreased the anchorage independent growth of tested lung cancer cell lines. GLI1 shRNA mediated attenuation of HH signaling in A549 cells, greatly reduced their tumorigenicity in nude mice. We anticipate seeing activated HH signaling in cancer and stroma cells of primary lung tumor samples by IHC and ISH. Together, these results would indicate that cancer cells do elaborate a functional HH signaling pathway and require HH signaling for growth, survival and tumorigenicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-430. doi:10.1158/1538-7445.AM2011-LB-430

Rottlerin induces Wnt co-receptor LRP6 degradation and suppresses both Wnt/β-catenin and mTORC1 signaling in prostate and breast cancer cells

Oxidative stress results from a disequilibrium between production and their elimination by cellular antioxidant systems. This leads to the accumulation of ROS that have a deleterious effect on vital biological macromolecules. It is now well established that cancer cells exhibit a pro-oxidant state due to metabolic and genetic abnormalities. The disequilibrium in redox homeostasis in cancer cells promotes genomic instability leading to the activation of oncogenes, mitochondrial dysfunction and an alteration in antioxidants activities. All these events can further escalate ROS levels, causing more DNA damage and genetic instability. This vicious cycle is “beneficial” for the process of carcinogenesis and numerous reports have indeed proven the pivotal role of ROS in cancer initiation, cell migration, invasion and metastasis. In order to cope with this sustained redox deregulation, cancer cells, very likely, utilize the full antioxidant capacity of their enzymatic and non-enzymatic systems. Cancer cells are thus highly dependent on their antioxidant systems and especially antioxidant enzymes. Keeping with this, targeting the enzymatic antioxidant system could be an efficient strategy to preferentially kill cancer cells by increasing intracellular ROS levels beyond a certain “threshold” of tolerance eventually leading to specific cancer cell death. In this review we present an overview on ROS generation and focus on the implication of ROS in cancer initiation, epithelial–mesenchymal transition, cell migration, invasion and metastasis, as well as the cancer stem-like phenotype. We finally present different therapeutic approaches that target the enzymatic antioxidant system in order to selectively kill cancer cells.

Sonodynamics has emerged as a new potential therapy for breast cancer in recent years. However, GSH-mediated redox systems in cancer cells make them tolerable to oxidative stress-related therapy. Herein, in this study, with G6PD, the gatekeeper enzyme of the pentose phosphate pathway, as the regulative target, a self-assembled thermosensitive chitosan-pluronic hydrogel coloaded with ICG (sono-sensitive agent) and RRx-001 (IR@CPGel) was successfully prepared to enhance SDT through interference with redox homeostasis. Both in vitro and in vivo antitumor investigations verified that when integrated with sonodynamic therapy applied in breast cancer treatment, local administration of IR@CPgel could enhance ROS generation under LIFU irradiation and trigger the intrinsic apoptotic pathway of cancer cells, thus effectively inhibiting tumor growth in a safe manner. Moreover, RRx-001 may interfere with redox homeostasis in cancer cells by downregulating G6PD expression. Due to this redox imbalance, proapoptotic signals, such as P21 and P53, were enhanced, and metastasis-related signals, including MMP-2, ZEB1 and HIF-1α, were effectively reduced. Taken together, this work aimed to enhance the efficacy of sonodynamic therapy through local administration of self-assembled IR@CPGel to interfere with redox homeostasis and thus amplify the oxidative stress microenvironment in tumor tissues. In a word, this work provides a new strategy for the SDT enhancement in breast cancer therapy.

The Warburg effect describes an increase in aerobic glycolysis and enhanced lactate production in cancer cells. Lactate dehydrogenase A (LDH-A) regulates the last step of glycolysis that generates lactate and permits the regeneration of NAD(+). LDH-A gene expression is believed to be upregulated by both HIF and Myc in cancer cells to achieve increased lactate production. However, how oncogenic signals activate LDH-A to regulate cancer cell metabolism remains unclear. We found that the oncogenic receptor tyrosine kinase FGFR1 directly phosphorylates LDH-A. Phosphorylation at Y10 and Y83 enhances LDH-A activity by enhancing the formation of active, tetrameric LDH-A and the binding of LDH-A substrate NADH, respectively. Moreover, Y10 phosphorylation of LDH-A is common in diverse human cancer cells, which correlates with activation of multiple oncogenic tyrosine kinases. Interestingly, cancer cells with stable knockdown of endogenous LDH-A and rescue expression of a catalytic hypomorph LDH-A mutant, Y10F, demonstrate increased respiration through mitochondrial complex I to sustain glycolysis by providing NAD(+). However, such a compensatory increase in mitochondrial respiration in Y10F cells is insufficient to fully sustain glycolysis. Y10 rescue cells show decreased cell proliferation and ATP levels under hypoxia and reduced tumor growth in xenograft nude mice. Our findings suggest that tyrosine phosphorylation enhances LDH-A enzyme activity to promote the Warburg effect and tumor growth by regulating the NADH/NAD(+) redox homeostasis, representing an acute molecular mechanism underlying the enhanced lactate production in cancer cells.

Metabolic changes favoring aerobic glycolysis and glutaminolysis may influence the sensitivity of tumor cells to anticancer drugs. We previously showed that sulfasalazine, an inhibitor of xCT-dependent cystine transport, selectively induces oxidative damage in undifferentiated tumor cells that express CD44 variant (CD44v) at a high level, without affecting CD44v-negative differentiated tumor cells, in head and neck squamous cell carcinoma (HNSCC). However, whether metabolic changes contribute to the xCT dependency of CD44v-expressing HNSCC cells has remained unknown. Here we show that CD44v-expressing HNSCC cells manifest a highly glutaminolytic phenotype and constitutively generate reactive oxygen species through glutamine metabolism pathway, and that xCT plays a key role in maintaining redox homeostasis in these cells. Inhibition of xCT suppressed the consumption of glutamine without affecting that of glucose in these cells, indicating that CD44v-expressing HNSCC cells also rely on xCT for maintenance of glutaminolysis. Furthermore, administration of sulfasalazine, but not that of the anticancer drug cisplatin, selectively depleted CD44v-expressing undifferentiated tumor cells positive for the glutamine transporter ASCT2 in human HNSCC tumors formed in nude mice. Targeting of xCT is thus a potential therapeutic approach for ASCT2-expressing HNSCC tumors in which cellular metabolism is shifted toward enhanced glutaminolysis. Citation Format: Shogo Okazaki, Kenji Tsuchihashi, Oltea Sampetrean, Hideyuki Saya, Osamu Nagano. xCT inhibition disrupts redox homeostasis in CD44v-expressing tumor cells showing glutaminolytic metabolism in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 432. doi:10.1158/1538-7445.AM2017-432

Background: The pesticide atrazine does not bind to or activate the classical estrogen receptor (ER), but it up-regulates the aromatase activity in estrogen-sensitive tumor cells. The G protein estrogen receptor (GPR30/GPER) has been reported to be involved in certain biological responses to endogenous estrogens and environmental compounds exerting estrogen-like activity.Objectives: We aimed to evaluate the potential of atrazine to trigger GPER-mediated signaling in cancer cells and cancer-associated fibroblasts (CAFs).Methods and Results: Using gene reporter assays in diverse types of cancer cells, we found that atrazine did not transactivate endogenous ERα or chimeric proteins that encode the ERα and ERβ hormone binding domains. Conversely, atrazine was able to bind to GPER to induce ERK activation and the expression of estrogen target genes, which, interestingly, appeared to rely on both GPER and ERα expression. As a biological counterpart, atrazine stimulated the proliferation of ovarian cancer cells that depend on GPER and ERα, as evidenced by gene silencing experiments and the use of specific signaling inhibitors. Of note, through GPER, atrazine elicited ERK phosphorylation, gene expression, and migration in CAFs, thus extending its stimulatory role to these main players of the tumor microenvironment.Conclusions: Our results suggest a novel mechanism through which atrazine may exert relevant biological effects in cancer cells and CAFs. On the basis of our data, atrazine should be included among the environmental contaminants that may elicit estrogenic activity through GPER-mediated signaling.Citation: Albanito L, Lappano R, Madeo A, Chimento A, Prossnitz ER, Capello AR, Dolce V, Abonante S, Pezzi V, Maggiolini M. 2015. Effects of atrazine on estrogen receptor α– and G protein–coupled receptor 30–mediated signaling and proliferation in cancer cells and cancer-associated fibroblasts. Environ Health Perspect 123:493–499; http://dx.doi.org/10.1289/ehp.1408586

Cancer cells take up more glucose than normal tissue and favor aerobic glycolysis, generating lactate through a NADH-dependent enzyme, lactate dehydrogenase A (LDH-A). This is the last step of glycolysis that permits the regeneration of NAD+, which is needed as an electron acceptor to maintain cytosolic glucose catabolism. Therefore, most tumor cells are reliant on lactate production for their survival. LDH-A gene expression is believed to be upregulated by both HIF and Myc in cancer cells to achieve increased lactate production, and expression of LDH-A was previously implicated to be involved in tumour initiation and growth. However, how oncogenic signals activate LDH-A to regulate cancer cell metabolism remains unclear. Our phospho-proteomics studies revealed that oncogenic fibroblast growth factor (FGF) receptor type 1 (FGFR1) tyrosine kinase directly phosphorylates LDH-A. Structural and biochemical studies revealed that phosphorylation at Y10 and Y83 activates LDH-A by promoting the formation of active, tetrameric LDH-A and binding of LDH-A substrate NADH, respectively. Moreover, we found that LDH-A is commonly phosphorylated at Y10 in diverse human cancer cells by multiple oncogenic tyrosine kinases including BCR-ABL, FLT3 and JAK2, which represents an acute molecular mechanism underlying increased lactate production in cancer cells. Furthermore, cancer cells with stable knockdown of endogenous LDH-A and rescue expression of a catalytic hypomorph LDH-A mutant, Y10F, show decreased cell proliferation and ATP levels under hypoxia, increased mitochondrial respiration to sustain glycolysis by providing NAD+, and reduced tumor growth in xenograft nude mice. Our findings suggest that tyrosine phosphorylation activates LDH-A to promote the Warburg effect and tumor growth by regulating NADH/NAD+ redox homeostasis in cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2798. doi:10.1158/1538-7445.AM2011-2798

Cancer is classified into metabolic and/or genetic disorders; notably, the tryptophan catabolism pathway is vital in different cancer types. Here, we focused on the interaction and molecular connection between the cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) receptor and indoleamine-2,3-dioxygenase (IDO) enzyme.To test the impact of the selected immunotherapies on breast cancer cell migration and cell survival, we used in vitro assays. Also, we test the impact of anti-CTLA-4 antibody on the IDO-positive cells.The results of cell migration and clonogenic assays showed that anti-CTLA-4 antibody reduces cancer cell migration and clonogenic abilities of murine breast cancer cells. In addition, the result of flow cytometry showed that the anti-CTLA-4 antibody did not change the percentage of IDO-positive cancer cells. Notably, administrating an IDO blocker, 1-Methyl-DL-tryptophan (1MT), reduces the efficiency of the antiCTLA-4 antibody.The enzymatic blocking of the IDO reduces the efficiency of the anti-CTLA-4 antibody on cell migration and clonogenic abilities suggesting that there is an inhibitory interaction at the molecular level between functions of CTLA-4 and IDO. It is unclear via which mechanism(s) IDO interacts with CTLA-4 signaling and also why blocking IDO makes disruption in CTLA-4 signaling in cancer cells. Indeed, evaluating the role of IDO in CTLA-4 signaling in cancer cells may assist in clarifying a poor response to CTLA-4 immunotherapies by some patients. Hence, further investigation of the molecular interaction between CTLA-4 and IDO might help to improve the efficiency of CTLA-4 immunotherapy.