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Homeobox genes encode sequence-specific transcription factors (SSTFs) that recognize specific DNA sequences and regulate organogenesis in all eukaryotes. They are essential in specifying spatial and temporal cell identity and as a result, their mutations often cause severe developmental defects. Pitx genes belong to the PRD class of the highly evolutionary conserved homeobox genes in all animals. Vertebrates possess three Pitx paralogs, Pitx1, Pitx2, and Pitx3 while non-vertebrates have only one Pitx gene. The ancient role of regulating left-right (LR) asymmetry is conserved while new functions emerge to afford more complex body plan and functionalities. In mouse, Pitx1 regulates hindlimb tissue patterning and pituitary development. Pitx2 is essential for the development of the oral cavity and abdominal wall while regulates the formation and symmetry of other organs including pituitary, heart, gut, lung among others by controlling growth control genes upon activation of the Wnt/s-catenin signaling pathway. Pitx3 is essential for lens development and migration and survival of the dopaminergic neurons of the substantia nigra. Pitx gene mutations are linked to various congenital defects and cancers in humans. Pitx gene family has the potential to offer a new approach in regenerative medicine and aid in identifying new drug targets.

Tumour cells adapt to nutrient deprivation in vivo, yet strategies targeting the nutrient poor microenvironment remain unexplored. In melanoma, tumour cells often experience low glutamine levels, which promote cell dedifferentiation. Here, we show that dietary glutamine supplementation significantly inhibits melanoma tumour growth, prolongs survival in a transgenic melanoma mouse model, and increases sensitivity to a BRAF inhibitor. Metabolomic analysis reveals that dietary uptake of glutamine effectively increases the concentration of glutamine in tumours and its downstream metabolite, αKG, without increasing biosynthetic intermediates necessary for cell proliferation. Mechanistically, we find that glutamine supplementation uniformly alters the transcriptome in tumours. Our data further demonstrate that increase in intra-tumoural αKG concentration drives hypomethylation of H3K4me3, thereby suppressing epigenetically-activated oncogenic pathways in melanoma. Therefore, our findings provide evidence that glutamine supplementation can serve as a potential dietary intervention to block melanoma tumour growth and sensitize tumours to targeted therapy via epigenetic reprogramming., The availability of nutrients within the tumour microenvironment can influence tumour growth. Here, the authors find that, in a melanoma mouse model, mice fed a high glutamine diet have reduced tumour growth, increased sensitivity to Braf inhibitors and elevated a-ketoglutarate levels.

Genetic-driven deregulation of the Wnt pathway is crucial but not sufficient for colorectal cancer (CRC) tumourigenesis. Here, we show that environmental glutamine restriction further augments Wnt signaling in APC mutant intestinal organoids to promote stemness and leads to adenocarcinoma formation in vivo via decreasing intracellular alpha-ketoglutarate (aKG) levels. aKG supplementation is sufficient to rescue low-glutamine induced stemness and Wnt hyperactivation. Mechanistically, we found that aKG promotes hypomethylation of DNA and histone H3K4me3, leading to an upregulation of differentiation-associated genes and downregulation of Wnt target genes, respectively. Using CRC patient-derived organoids and several in vivo CRC tumour models, we show that aKG supplementation suppresses Wnt signaling and promotes cellular differentiation, thereby significantly restricting tumour growth and extending survival. Together, our results reveal how metabolic microenvironment impacts Wnt signaling and identify aKG as a potent antineoplastic metabolite for potential differentiation therapy for CRC patients.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers. It thrives in a nutrient-poor environment; however, the mechanisms by which PDAC cells undergo metabolic reprogramming to adapt to metabolic stress are still poorly understood. Here, we show that microRNA-135 is significantly increased in PDAC patient samples compared to adjacent normal tissue. Mechanistically, miR-135 accumulates specifically in response to glutamine deprivation and requires ROS-dependent activation of mutant p53, which directly promotes miR-135 expression. Functionally, we found miR-135 targets phosphofructokinase-1 (PFK1) and inhibits aerobic glycolysis, thereby promoting the utilization of glucose to support the tricarboxylic acid (TCA) cycle. Consistently, miR-135 silencing sensitizes PDAC cells to glutamine deprivation and represses tumor growth in vivo. Together, these results identify a mechanism used by PDAC cells to survive the nutrient-poor tumor microenvironment, and also provide insight regarding the role of mutant p53 and miRNA in pancreatic cancer cell adaptation to metabolic stresses., Pancreatic ductal adenocarcinoma must adapt to a nutrient-poor microenvironment. Here, the authors show that miR-135 accumulates in response to glutamine deprivation and inhibits aerobic glycolysis by targeting phosphofructokinase-1, thereby redirecting glucose carbon to the TCA cycle and allowing pancreatic cancer cells survival.

Adipose tissue plays a major role in maintaining organismal metabolic equilibrium. Control over the fate decision from mesenchymal stem cells (MSCs) to adipocyte differentiation involves coordinated command of phosphorylation. Protein phosphatase 2A plays an important role in Wnt pathway and adipocyte development, yet how PP2A complexes actively respond to adipocyte differentiation signals and acquire specificity in the face of the promiscuous activity of its catalytic subunit remains unknown. Here, we report the PP2A phosphatase B subunit B56α is specifically induced during adipocyte differentiation and mediates PP2A to dephosphorylate GSK3β, thereby blocking Wnt activity and driving adipocyte differentiation. Using an inducible B56α knock-out mouse, we further demonstrate that B56α is essential for gonadal adipose tissue development invivo and required for the fate decision of adipocytes over osteoblasts. Moreover, we show B56α expression is driven by the adipocyte transcription factor PPARγ thereby establishing a novel link between PPARγ signaling and Wnt blockade. Overall, our results reveal B56α is a necessary part of the machinery dictating the transition from pre-adipocyte to mature adipocyte and provide fundamental insights into how PP2A complex specifically and actively regulates unique signaling pathway in biology.

Cancer cells depend on glutamine to sustain their increased proliferation and manage oxidative stress, yet glutamine is often depleted at tumor sites due to excessive cellular consumption and poor vascularization. We have previously reported that p53 protein, while a well-known tumor suppressor, can contribute to cancer cell survival and adaptation to low glutamine conditions. However, the TP53 gene is frequently mutated in tumors, and the role of mutant p53 (mutp53) in response to metabolic stress remains unclear. Here, we demonstrate that tumor-associated mutp53 promotes cancer cell survival upon glutamine deprivation both in vitro and in vivo. Interestingly, cancer cells expressing mutp53 proteins are more resistant to glutamine deprivation than cells with wild type p53 (wtp53). Depletion of endogenous mutp53 protein in human lymphoma cells leads to cell sensitivity to glutamine withdrawal, while expression of mutp53 in p53 null cells results in resistance to glutamine deprivation. Furthermore, we found that mutp53 proteins hyper-transactivate p53 target gene CDKN1A upon glutamine deprivation, thus triggering cell cycle arrest and promoting cell survival. Together, our results reveal an unidentified mechanism by which mutp53 confers oncogenic functions by promoting cancer cell adaptation to metabolic stresses.

One of the hallmarks of cancer is the ability to reprogram cellular metabolism to increase the uptake of necessary nutrients such as glucose and glutamine. Driven by oncogenes, cancer cells have increased glutamine uptake to support their highly proliferative nature. However, as cancer cells continue to replicate and grow, they lose access to vascular tissues and deplete local supply of nutrients and oxygen. We previously showed that many tumor cells situate in a low glutamine microenvironment in vivo, yet the mechanisms of how they are able to adapt to this metabolic stress are still not fully understood. Here, we report that IκB-kinase β (IKKβ) is needed to promote survival and its activation is accompanied by phosphorylation of the metabolic sensor, p53, in response to glutamine deprivation. Knockdown of IKKβ decreases the level of wild-type and mutant p53 phosphorylation and its transcriptional activity, indicating a novel relationship between IKKβ and p53 in mediating cancer cell survival in response to glutamine withdrawal. Phosphopeptide mass spectrometry analysis further reveals that IKKβ phosphorylates p53 on Ser392 to facilitate its activation upon glutamine deprivation, independent of the NF-κB pathway. The results of this study offer an insight into the metabolic reprogramming in cancer cells that is dependent on a previously unidentified IKKβ–p53 signaling axis in response to glutamine depletion. More importantly, this study highlights a new therapeutic strategy for cancer treatment and advances our understanding of adaptive mechanisms that could lead to resistance to current glutamine targeting therapies.

Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH) enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON) or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer., Author summary Cancer cells residing within the intratumoral microenvironment are subject to severe glutamine shortages. Herein, we provide mechanistic insight by which glutamine deficiency leads to cellular sensitivity to alkylating agents. We find that glutamine deficiency inhibits the DNA repair activity of the ALKBH enzymes, leading to accumulation of DNA alkylation damage and thereby increasing cellular sensitivity to alkylating agents. This study provides a critical molecular basis to combine glutaminase inhibitors with alkylating agents for more effective treatment of cancers. These findings extend our understanding of the role of metabolic stress, in particular glutamine deficiency, in tumor development and therapeutic response.

Epigenetic alterations contribute to tumor development, progression, and therapeutic response. Many epigenetic enzymes use metabolic intermediates as cofactors to modify chromatin structure. Emerging evidence suggests that fluctuation in metabolite levels may regulate activities of these chromatin-modifying enzymes. Here, we summarize recent progress in understanding the cross-talk between metabolism and epigenetic control of gene expression in cancer. We focus on how metabolic changes, due to diet, genetic mutations, or tumor microenvironment, regulate histone methylation status and, consequently, affect gene expression profiles to promote tumorigenesis. Importantly, we also suggest some potential therapeutic approaches to target the oncogenic role of metabolic alterations and epigenetic modifications in cancer. Clin Cancer Res; 23(15); 4004–9. ©2017 AACR.

Summary: Cancer cells heavily depend on the amino acid glutamine to meet the demands associated with growth and proliferation. Due to the rapid consumption of glutamine, cancer cells frequently undergo glutamine starvation in vivo. We and others have shown that p53 is a critical regulator in metabolic stress resistance. To better understand the molecular mechanisms by which p53 activation promotes cancer cell adaptation to glutamine deprivation, we identified p53-dependent genes that are induced upon glutamine deprivation by using RNA-seq analysis. We show that Slc7a3, an arginine transporter, is significantly induced by p53. We also show that increased intracellular arginine levels following glutamine deprivation are dependent on p53. The influx of arginine has minimal effects on known metabolic pathways upon glutamine deprivation. Instead, we found arginine serves as an effector for mTORC1 activation to promote cell growth in response to glutamine starvation. Therefore, we identify a p53-inducible gene that contributes to the metabolic stress response. : Lowman et al. show that glutamine deprivation induces Slc7a3 to increase intracellular arginine levels in a p53-dependent manner. This induction transiently sustains mTORC1 activity and contributes to cellular adaptation to low glutamine conditions. Keywords: p53 activation, Slc7a3, glutamine deprivation, arginine

Glutamine is an essential nutrient for cancer cell survival and proliferation. Enhanced utilization of glutamine often depletes its local supply, yet how cancer cells adapt to low glutamine conditions is largely unknown. Here, we report that IκB kinase β (IKKβ) is activated upon glutamine deprivation and is required for cell survival independently of NF-κB transcription. We demonstrate that IKKβ directly interacts with and phosphorylates 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase isoform 3 (PFKFB3), a major driver of aerobic glycolysis, at Ser269 upon glutamine deprivation to inhibit its activity, thereby down-regulating aerobic glycolysis when glutamine levels are low. Thus, due to lack of inhibition of PFKFB3, IKKβ-deficient cells exhibit elevated aerobic glycolysis and lactate production, leading to less glucose carbons contributing to tricarboxylic acid (TCA) cycle intermediates and the pentose phosphate pathway, which results in increased glutamine dependence for both TCA cycle intermediates and reactive oxygen species suppression. Therefore, coinhibition of IKKβ and glutamine metabolism results in dramatic synergistic killing of cancer cells both in vitro and in vivo. In all, our results uncover a previously unidentified role of IKKβ in regulating glycolysis, sensing low-glutamine-induced metabolic stress, and promoting cellular adaptation to nutrient availability.

© 2015 Hernandez-Davies et al. Background: V600 BRAF mutations drive approximately 50% of metastatic melanoma which can be therapeutically targeted by BRAF inhibitors (BRAFi) and, based on resistance mechanisms, the combination of BRAF and MEK inhibitors (BRAFi + MEKi). Although the combination therapy has been shown to provide superior clinical benefits, acquired resistance is still prevalent and limits the overall survival benefits. Recent work has shown that oncogenic changes can lead to alterations in tumor cell metabolism rendering cells addicted to nutrients, such as the amino acid glutamine. Here, we evaluated whether melanoma cells with acquired resistance display glutamine dependence and whether glutamine metabolism can be a potential molecular target to treat resistant cells. Methods: Isogenic BRAFi sensitive parental V600 BRAF mutant melanoma cell lines and resistant (derived by chronic treatment with vemurafenib) sub-lines were used to assess differences in the glutamine uptake and sensitivity to glutamine deprivation. To evaluate a broader range of resistance mechanisms, isogenic pairs where the sub-lines were resistant to BRAFi + MEKi were also studied. Since resistant cells demonstrated increased sensitivity to glutamine deficiency, we used glutaminase inhibitors BPTES [bis-2-(5 phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide] and L-L-DON (6-Diazo-5-oxo-l-norleucine) to treat MAPK pathway inhibitor (MAPKi) resistant cell populations both in vitro and in vivo. Results: We demonstrated that MAPKi-acquired resistant cells uptook greater amounts of glutamine and have increased sensitivity to glutamine deprivation than their MAPKi-sensitive counterparts. In addition, it was found that both BPTES and L-DON were more effective at decreasing cell survival of MAPKi-resistant sub-lines than parental cell populations in vitro. We also showed that mutant NRAS was critical for glutamine addiction in mutant NRAS driven resistance. When tested in vivo, we found that xenografts derived from resistant cells were more sensitive to BPTES or L-DON treatment than those derived from parental cells. Conclusion: Our study is a proof-of-concept for the potential of targeting glutamine metabolism as an alternative strategy to suppress acquired MAPKi-resistance in melanoma.

Despite advances in our understanding of protein kinase regulation in the DNA damage response, the mechanism that controls protein phosphatase activity in this pathway is unclear. Unlike kinases, the activity and specificity of serine/threonine phosphatases is governed largely by their associated proteins. Here we show that Tip41-like protein (TIPRL), an evolutionarily conserved binding protein for PP2A-family phosphatases, is a negative regulator of protein phosphatase 4 (PP4). Knockdown of TIPRL resulted in increased PP4 phosphatase activity and formation of the active PP4-C/PP4R2 complex known to dephosphorylate γ-H2AX. Thus, overexpression of TIPRL promotes phosphorylation of H2AX, and increases γ-H2AX positive foci in response to DNA damage, whereas knockdown of TIPRL inhibits γ-H2AX phosphorylation. In correlation with γ-H2AX levels, we found that TIPRL overexpression promotes cell death in response to genotoxic stress, and knockdown of TIPRL protects cells from genotoxic agents. Taken together, these data demonstrate that TIPRL inhibits PP4 activity to allow for H2AX phosphorylation and the subsequent DNA damage response.