Glutamine and amino acid metabolism as a prognostic signature and therapeutic target in endometrial cancer.

INTRODUCTION: Endometrial cancer (EC) is the most common female reproductive system cancer in developed countries with growing incidence and associated mortality, which may be due to the growing prevalence of obesity. Metabolism reprogramming including glucose, amino acid, and lipid remodeling is a hallmark of tumors. Glutamine metabolism has been reported to participate in tumor proliferation and development. This study aimed to develop a glutamine metabolism-related prognostic model for EC and explore potential targets for cancer treatment. METHOD: Transcriptomic data and survival outcome of EC were retrieved from The Cancer Genome Atlas (TCGA). Differentially expressed genes related to glutamine metabolism were recognized and utilized to build a prognostic model by univariate and multivariate Cox regressions. The model was confirmed in the training, testing, and the entire cohort. A nomogram combing prognostic model and clinicopathologic features was established and tested. Moreover, we explored the effect of a key metabolic enzyme, PHGDH, on the biological behavior of EC cell lines and xenograft model. RESULTS: Five glutamine metabolism-related genes, including PHGDH, OTC, ASRGL1, ASNS, and NR1H4, were involved in prognostic model construction. Kaplan-Meier curve suggested that patients recognized as high risk underwent inferior outcomes. The receiver operating characteristic (ROC) curve showed the model was sufficient to predict survival. Enrichment analysis recognized DNA replication and repair dysfunction in high-risk patients whereas immune relevance analysis revealed low immune scores in the high-risk group. Finally, a nomogram integrating the prognostic model and clinical factors was created and verified. Further, knockdown of PHGDH showed cell growth inhibition, increasing apoptosis, and reduced migration. Promisingly, NCT-503, a PHGDH inhibitor, significantly repressed tumor growth in vivo (p = 0.0002). CONCLUSION: Our work established and validated a glutamine metabolism-related prognostic model that favorably evaluates the prognosis of EC patients. DNA replication and repair may be the crucial point that linked glutamine metabolism, amino acid metabolism, and EC progression. High-risk patients stratified by the model may not be sufficient for immune therapy. PHGDH might be a crucial target that links serine metabolism, glutamine metabolism as well as EC progression.

PHGDH Inhibitor CBR-5884 Inhibits Epithelial Ovarian Cancer Progression via ROS/Wnt/ß-Catenin Pathway and Plays a Synergistic Role with PARP Inhibitor Olaparib.

PHGDH attaches importance to serine biosynthesis in cancer cells and maintaining mitochondrial redox homeostasis. However, the role of PHGDH inhibitor CBR-5884 in cell ROS level and its downstream pathways has not been explored in epithelial ovarian cancer. Thus, we investigated the function and possible mechanism of PHGDH inhibitor CBR-5884 on epithelial ovarian cancer in vitro and in vivo. A2780, OVCAR3, and ES-2 were treated with CBR-5884 at different concentrations or different time points. Results showed that CBR-5884 inhibited epithelial ovarian cancer cell proliferation, migration, and invasion and increases cell ROS level. Meanwhile, CBR-5884 exerts antitumor effect through activating ROS/Wnt/ß-catenin pathway. Besides, CBR-5884 exerts antitumor effect in vivo. What's more, we explored the effect of CBR-5884 with or without PARP inhibitor Olaparib, which showed that the two together had a larger effect. In conclusion, PHGDH inhibitor CBR-5884 inhibits epithelial ovarian cancer proliferation, migration, and invasion through activating ROS/Wnt/ß-catenin pathway and plays a synergistic role with PARP inhibitor olaparib, which provided a theoretical basis for PHGDH inhibitor CBR-5884 in clinical treatment.

Biophysical and biochemical properties of PHGDH revealed by studies on PHGDH inhibitors.

The rate-limiting serine biogenesis enzyme PHGDH is overexpressed in cancers. Both serine withdrawal and genetic/pharmacological inhibition of PHGDH have demonstrated promising tumor-suppressing activities. However, the enzyme properties of PHGDH are not well understood and the discovery of PHGDH inhibitors is still in its infancy. Here, oridonin was identified from a natural product library as a new PHGDH inhibitor. The crystal structure of PHGDH in complex with oridonin revealed a new allosteric site. The binding of oridonin to this site reduced the activity of the enzyme by relocating R54, a residue involved in substrate binding. Mutagenesis studies showed that PHGDH activity was very sensitive to cysteine mutations, especially those in the substrate binding domain. Conjugation of oridonin and other reported covalent PHGDH inhibitors to these sites will therefore inhibit PHGDH. In addition to being inhibited enzymatically, PHGDH can also be inhibited by protein aggregation and proteasome-mediated degradation. Several tested PHGDH cancer mutants showed altered enzymatic activity, which can be explained by protein structure and stability. Overall, the above studies present new biophysical and biochemical insights into PHGDH and may facilitate the future design of PHGDH inhibitors.

Exogenous and Endogenous Serine Deficiency Exacerbates Hepatic Lipid Accumulation.

Serine is involved in the regulation of hepatic lipid metabolism. However, whether exogenous or endogenous serine deficiency affects lipid accumulation in the liver and related mechanisms is unclear. Here, we investigated the effects of serine deficiency on hepatic fat accumulation in mice fed a serine-deficient diet or in mice supplemented with the D-3-phosphoglycerate dehydrogenase (PHGDH) inhibitor NCT-503. Both treatments produced an increase in body weight and liver weight and higher triglyceride content in the liver. Both treatments also exacerbated hepatic inflammatory responses and oxidative stress. Importantly, NCT-503 supplementation significantly inhibited PHGDH activity and decreased the serine content in the liver. Dietary serine deficiency significantly affected the colonic microbiota, characterized by a decreased ratio of Firmicutes/Bacteroidetes and decreased proportion of Bifidobacterium. Dietary serine deficiency additionally resulted in significantly decreased colonic and serum acetate and butyrate levels. The collective results indicate that NCT-503 supplementation may contribute to overaccumulation of hepatic lipid, by causing hepatic serine deficiency, while dietary serine deficiency may produce similar outcomes by affecting the gut-microbiota-liver axis.

Metabolic shift to serine biosynthesis through 3-PG accumulation and PHGDH induction promotes tumor growth in pancreatic cancer.

Cancer cells craftily adapt their energy metabolism to their microenvironment. Nutrient deprivation due to hypovascularity and fibrosis is a major characteristic of pancreatic ductal adenocarcinoma (PDAC); thus, PDAC cells must produce energy intrinsically. However, the enhancement of energy production via activating Kras mutations is insufficient to explain the metabolic rewiring of PDAC cells. Here, we investigated the molecular mechanism underlying the metabolic shift in PDAC cells under serine starvation. Amino acid analysis revealed that the concentrations of all essential amino acids and most nonessential amino acids were decreased in the blood of PDAC patients. In addition, the plasma serine concentration was significantly higher in PDAC patients with PHGDH-high tumors than in those with PHGDH-low tumors. Although the growth and tumorigenesis of PK-59 cells with PHGDH promoter hypermethylation were significantly decreased by serine starvation, these activities were maintained in PDAC cell lines with PHGDH promoter hypomethylation by serine biosynthesis through PHGDH induction. In fact, DNA methylation analysis by pyrosequencing revealed that the methylation status of the PHGDH promoter was inversely correlated with the PHGDH expression level in human PDAC tissues. In addition to PHGDH induction by serine starvation, PDAC cells showed enhanced serine biosynthesis under serine starvation through 3-PG accumulation via PGAM1 knockdown, resulting in enhanced PDAC cell growth and tumor growth. However, PHGDH knockdown efficiently suppressed PDAC cell growth and tumor growth under serine starvation. These findings provide evidence that targeting the serine biosynthesis pathway by inhibiting PHGDH is a potent therapeutic approach to eliminate PDAC cells in nutrient-deprived microenvironments.

Inhibiting PHGDH with NCT-503 reroutes glucose-derived carbons into the TCA cycle, independently of its on-target effect.

The small-molecule inhibitor of phosphoglycerate dehydrogenase, NCT-503, reduces incorporation of glucose-derived carbons into serine in vitro. Here we describe an off-target effect of NCT-503 in neuroblastoma cell lines expressing divergent phosphoglycerate dehydrogenase (PHGDH) levels and single-cell clones with CRISPR-Cas9-directed PHGDH knockout or their respective wildtype controls. NCT-503 treatment strongly reduced synthesis of glucose-derived citrate in all cell models investigated compared to the inactive drug control and independent of PHGDH expression level. Incorporation of glucose-derived carbons entering the TCA cycle via pyruvate carboxylase was enhanced by NCT-503 treatment. The activity of citrate synthase was not altered by NCT-503 treatment. We also detected no change in the thermal stabilisation of citrate synthase in cellular thermal shift assays from NCT-503-treated cells. Thus, the direct cause of the observed off-target effect remains enigmatic. Our findings highlight off-target potential within a metabolic assessment of carbon usage in cells treated with the small-molecule inhibitor, NCT-503.

Targeting Serine in Cancer: Is Two Better Than One?

Several recent preclinical studies have demonstrated that simultaneously blocking exogenous and endogenous sources of serine in malignant cells mediates superior anticancer effects as compared with limiting either source alone. Here, we critically summarize key developments in targeting serine to treat cancer and discuss persisting challenges for implementing such a therapeutic approach in patients.

Discovery of novel inhibitors of human phosphoglycerate dehydrogenase by activity-directed combinatorial chemical synthesis strategy.

Serine, the source of the one-carbon units essential for de novo purine and deoxythymidine synthesis plays a crucial role in the growth of cancer cells. Phosphoglycerate dehydrogenase (PHGDH) which catalyzes the first, rate-limiting step in de novo serine biosynthesis has become a promising target for the cancer treatment. Here we identified H-G6 as a potential PHGDH inhibitor from the screening of an in-house small molecule library based on the enzymatic assay. We adopted activity-directed combinatorial chemical synthesis strategy to optimize this hit compound. Compound b36 was found to be the noncompetitive and the most promising one with IC50 values of 5.96 ± 0.61 µM against PHGDH. Compound b36 inhibited the proliferation of human breast cancer and ovarian cancer cells, reduced intracellular serine synthesis, damaged DNA synthesis, and induced cell cycle arrest. Collectively, our results suggest that b36 is a novel PHGDH inhibitor, which could be a promising modulator to reprogram the serine synthesis pathway and might be a potential anticancer lead worth further exploration.

Epigenome-wide association meta-analysis of DNA methylation with coffee and tea consumption.

Coffee and tea are extensively consumed beverages worldwide which have received considerable attention regarding health. Intake of these beverages is consistently linked to, among others, reduced risk of diabetes and liver diseases; however, the mechanisms of action remain elusive. Epigenetics is suggested as a mechanism mediating the effects of dietary and lifestyle factors on disease onset. Here we report the results from epigenome-wide association studies (EWAS) on coffee and tea consumption in 15,789 participants of European and African-American ancestries from 15 cohorts. EWAS meta-analysis of coffee consumption reveals 11 CpGs surpassing the epigenome-wide significance threshold (P-value <1.1×10-7), which annotated to the AHRR, F2RL3, FLJ43663, HDAC4, GFI1 and PHGDH genes. Among them, cg14476101 is significantly associated with expression of the PHGDH and risk of fatty liver disease. Knockdown of PHGDH expression in liver cells shows a correlation with expression levels of genes associated with circulating lipids, suggesting a role of PHGDH in hepatic-lipid metabolism. EWAS meta-analysis on tea consumption reveals no significant association, only two CpGs annotated to CACNA1A and PRDM16 genes show suggestive association (P-value <5.0×10-6). These findings indicate that coffee-associated changes in DNA methylation levels may explain the mechanism of action of coffee consumption in conferring risk of diseases.

Serine metabolism antagonizes antiviral innate immunity by preventing ATP6V0d2-mediated YAP lysosomal degradation.

Serine metabolism promotes tumor oncogenesis and regulates immune cell functions, but whether it also contributes to antiviral innate immunity is unknown. Here, we demonstrate that virus-infected macrophages display decreased expression of serine synthesis pathway (SSP) enzymes. Suppressing the SSP key enzyme phosphoglycerate dehydrogenase (PHGDH) by genetic approaches or by treatment with the pharmaceutical inhibitor CBR-5884 and by exogenous serine restriction enhanced IFN-ß-mediated antiviral innate immunity in vitro and in vivo. Mechanistic experiments showed that virus infection or serine metabolism deficiency increased the expression of the V-ATPase subunit ATP6V0d2 by inhibiting S-adenosyl methionine-dependent H3K27me3 occupancy at the promoter. ATP6V0d2 promoted YAP lysosomal degradation to relieve YAP-mediated blockade of the TBK1-IRF3 axis and, thus, enhance IFN-ß production. These findings implicate critical functions of PHGDH and the key immunometabolite serine in blunting antiviral innate immunity and also suggest manipulation of serine metabolism as a therapeutic strategy against virus infection.

A retrospective overview of PHGDH and its inhibitors for regulating cancer metabolism.

Emerging evidence suggests that cancer metabolism is closely associated to the serine biosynthesis pathway (SSP), in which glycolytic intermediate 3-phosphoglycerate is converted to serine through a three-step enzymatic transformation. As the rate-limiting enzyme in the first step of SSP, phosphoglycerate dehydrogenase (PHGDH) is overexpressed in various diseases, especially in cancer. Genetic knockdown or silencing of PHGDH exhibits obvious anti-tumor response both in vitro and in vivo, demonstrating that PHGDH is a promising drug target for cancer therapy. So far, several types of PHGDH inhibitors have been identified as a significant and newly emerging option for anticancer treatment. Herein, this comprehensive review summarizes the recent achievements of PHGDH, especially its critical role in cancer and the development of PHGDH inhibitors in drug discovery.

Phosphoglycerate dehydrogenase (PHGDH) inhibitors: a comprehensive review 2015-2020.

Introduction:The phosphoglycerate dehydrogenase (PHGDH), a metabolic enzyme involved in the serine synthetic pathway (SSP), appears to play a central role in supporting cancer growth and proliferation. PHGDH is a dehydrogenase whose expression in cancers was first demonstrated in 2010. Because its silencing allows a significant reduction in tumor proliferation, it appears to be a promising target in the development of new anti-cancer agents.Areas covered: In this review, we will detail PHGDH inhibitors that were reported since 2015. These compounds will be ranked according to their chemical class and their site of action. Representative examples of each series will be presented as well as their inhibitory potency in vitro and/or in vivo. Finally, their most significant biological effects will be detailed.Expert opinion: Currently, and despite significant efforts, the search for PHGDH inhibitors has not yet led to the development of compounds that can be used therapeutically. The available inhibitors have either too weak inhibitory potency or limited selectivity. Therefore, it seems crucial, given the importance of this enzyme in the progression of cancer but also in other pathologies, to pursue the development of new chemical series.

Serine Biosynthesis Is a Metabolic Vulnerability in IDH2-Driven Breast Cancer Progression.

Cancer-specific metabolic phenotypes and their vulnerabilities represent a viable area of cancer research. In this study, we explored the association of breast cancer subtypes with different metabolic phenotypes and identified isocitrate dehydrogenase 2 (IDH2) as a key player in triple-negative breast cancer (TNBC) and HER2. Functional assays combined with mass spectrometry-based analyses revealed the oncogenic role of IDH2 in cell proliferation, anchorage-independent growth, glycolysis, mitochondrial respiration, and antioxidant defense. Genome-scale metabolic modeling identified phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) as the synthetic dosage lethal (SDL) partners of IDH2. In agreement, CRISPR-Cas9 knockout of PHGDH and PSAT1 showed the essentiality of serine biosynthesis proteins in IDH2-high cells. The clinical significance of the SDL interaction was supported by patients with IDH2-high/PHGDH-low tumors, who exhibited longer survival than patients with IDH2-high/PHGDH-high tumors. Furthermore, PHGDH inhibitors were effective in treating IDH2-high cells in vitro and in vivo. Altogether, our study creates a new link between two known cancer regulators and emphasizes PHGDH as a promising target for TNBC with IDH2 overexpression. SIGNIFICANCE: These findings highlight the metabolic dependence of IDH2 on the serine biosynthesis pathway, adding an important layer to the connection between TCA cycle and glycolysis, which can be translated into novel targeted therapies.

Unravelling the Allosteric Targeting of PHGDH at the ACT-Binding Domain with a Photoactivatable Diazirine Probe and Mass Spectrometry Experiments.

The serine biosynthetic pathway is a key element contributing to tumor proliferation. In recent years, targeting of phosphoglycerate dehydrogenase (PHGDH), the first enzyme of this pathway, intensified and revealed to be a promising strategy to develop new anticancer drugs. Among attractive PHGDH inhibitors are the α-ketothioamides. In previous work, we have demonstrated their efficacy in the inhibition of PHGDH in vitro and in cellulo. However, the precise site of action of this series, which would help the rational design of new inhibitors, remained undefined. In the present study, the detailed mechanism-of-action of a representative α-ketothioamide inhibitor is reported using several complementary experimental techniques. Strikingly, our work led to the identification of an allosteric site on PHGDH that can be targeted for drug development. Using mass spectrometry experiments and an original α-ketothioamide diazirine-based photoaffinity probe, we identified the 523Q-533F sequence on the ACT regulatory domain of PHGDH as the binding site of α-ketothioamides. Mutagenesis experiments further documented the specificity of our compound at this allosteric site. Our results thus pave the way for the development of new anticancer drugs using a completely novel mechanism-of-action.

In Vivo Evidence for Serine Biosynthesis-Defined Sensitivity of Lung Metastasis, but Not of Primary Breast Tumors, to mTORC1 Inhibition.

In tumors, nutrient availability and metabolism are known to be important modulators of growth signaling. However, it remains elusive whether cancer cells that are growing out in the metastatic niche rely on the same nutrients and metabolic pathways to activate growth signaling as cancer cells within the primary tumor. We discovered that breast-cancer-derived lung metastases, but not the corresponding primary breast tumors, use the serine biosynthesis pathway to support mTORC1 growth signaling. Mechanistically, pyruvate uptake through Mct2 supported mTORC1 signaling by fueling serine biosynthesis-derived α-ketoglutarate production in breast-cancer-derived lung metastases. Consequently, expression of the serine biosynthesis enzyme PHGDH was required for sensitivity to the mTORC1 inhibitor rapamycin in breast-cancer-derived lung tumors, but not in primary breast tumors. In summary, we provide in vivo evidence that the metabolic and nutrient requirements to activate growth signaling differ between the lung metastatic niche and the primary breast cancer site.

Inhibiting phosphoglycerate dehydrogenase counteracts chemotherapeutic efficacy against MYCN-amplified neuroblastoma.

Here we sought metabolic alterations specifically associated with MYCN amplification as nodes to indirectly target the MYCN oncogene. Liquid chromatography-mass spectrometry-based proteomics identified seven proteins consistently correlated with MYCN in proteomes from 49 neuroblastoma biopsies and 13 cell lines. Among these was phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in de novo serine synthesis. MYCN associated with two regions in the PHGDH promoter, supporting transcriptional PHGDH regulation by MYCN. Pulsed stable isotope-resolved metabolomics utilizing 13 C-glucose labeling demonstrated higher de novo serine synthesis in MYCN-amplified cells compared to cells with diploid MYCN. An independence of MYCN-amplified cells from exogenous serine and glycine was demonstrated by serine and glycine starvation, which attenuated nucleotide pools and proliferation only in cells with diploid MYCN but did not diminish these endpoints in MYCN-amplified cells. Proliferation was attenuated in MYCN-amplified cells by CRISPR/Cas9-mediated PHGDH knockout or treatment with PHGDH small molecule inhibitors without affecting cell viability. PHGDH inhibitors administered as single-agent therapy to NOG mice harboring patient-derived MYCN-amplified neuroblastoma xenografts slowed tumor growth. However, combining a PHGDH inhibitor with the standard-of-care chemotherapy drug, cisplatin, revealed antagonism of chemotherapy efficacy in vivo. Emergence of chemotherapy resistance was confirmed in the genetic PHGDH knockout model in vitro. Altogether, PHGDH knockout or inhibition by small molecules consistently slows proliferation, but stops short of killing the cells, which then establish resistance to classical chemotherapy. Although PHGDH inhibition with small molecules has produced encouraging results in other preclinical cancer models, this approach has limited attractiveness for patients with neuroblastoma.

Serine restriction alters sphingolipid diversity to constrain tumour growth.

Serine, glycine and other nonessential amino acids are critical for tumour progression, and strategies to limit their availability are emerging as potential therapies for cancer1-3. However, the molecular mechanisms driving this response remain unclear and the effects on lipid metabolism are relatively unexplored. Serine palmitoyltransferase (SPT) catalyses the de novo biosynthesis of sphingolipids but also produces noncanonical 1-deoxysphingolipids when using alanine as a substrate4,5. Deoxysphingolipids accumulate in the context of mutations in SPTLC1 or SPTLC26,7-or in conditions of low serine availability8,9-to drive neuropathy, and deoxysphinganine has previously been investigated as an anti-cancer agent10. Here we exploit amino acid metabolism and the promiscuity of SPT to modulate the endogenous synthesis of toxic deoxysphingolipids and slow tumour progression. Anchorage-independent growth reprogrammes a metabolic network involving serine, alanine and pyruvate that drives the endogenous synthesis and accumulation of deoxysphingolipids. Targeting the mitochondrial pyruvate carrier promotes alanine oxidation to mitigate deoxysphingolipid synthesis and improve spheroid growth, similar to phenotypes observed with the direct inhibition of SPT or ceramide synthesis. Restriction of dietary serine and glycine potently induces the accumulation of deoxysphingolipids while decreasing tumour growth in xenograft models in mice. Pharmacological inhibition of SPT rescues xenograft growth in mice fed diets restricted in serine and glycine, and the reduction of circulating serine by inhibition of phosphoglycerate dehydrogenase (PHGDH) leads to the accumulation of deoxysphingolipids and mitigates tumour growth. The promiscuity of SPT therefore links serine and mitochondrial alanine metabolism to membrane lipid diversity, which further sensitizes tumours to metabolic stress.

Down regulating PHGDH affects the lactate production of sertoli cells in varicocele.

BACKGROUND: Although varicocele is considered to be one of the leading causes of male infertility, the precise mechanism underlying how varicocele leads to male infertility is not completely understood. We found the lactate concentration on the varicocele side of the patients was decreased compare with peripheral venous blood. In the testicles, the lactate produced by the sertoli cells through the glycolysis pathway provides most of the energy needed for spermatogenesis, the reduction of lactate will affect spermatogenesis. The objective of this study was to investigate the mechanism of this abnormal energy metabolism phenomenon in varicocele. METHODS: In this study, we collected the testicular tissue from patients with varicocele, the glycolysis related proteins PHGDH was identified by iTRAQ proteomics technology. Experimental rat varicocele model was constructed according to our new clip technique, the mRNA and protein expression levels of PHGDH were examined with qRT-PCR and Western blotting. We constructed a sertoli cell of PHGDH down-regulation model, and then detected the glucose consumption, LDH activities and lactate production in the sertoli cells. Western blot was conducted to investigate the effects of PHGDH on the expression of phosphoserine phosphatase (PSPH) and Pyruvate kinase M2 (PKM2). Flow cytometry was used to detect the cell apoptosis and cell cycle in sertoli cells. RESULTS: The results showed that testicular protein PHGDH was down-regulated in patients with varicocele and in experimental rat varicocele model. Down-regulation of PHGDH in sertoli cells significantly decreased the glucose consumption, LDH activities and lactate production in the sertoli cells, indicating that the low expression of PHGDH ultimately led to a decrease in lactate production by affecting the glycolysis. The Western blot results showed that the down-regulation of PHGDH significantly reduced the expression of pathway protein PSPH and PKM2, leading to the reduction of lactate production. Moreover, PHGDH knockdown can promote apoptosis and inhibit cell cycle to affect cell growth. CONCLUSIONS: Overall, we conformed that varicocele lead to the decreasing of testis lactate production. Down-regulation of PHGDH in sertoli cells may mediate the process of abnormal glucose metabolism. Our study provide new insight into the mechanisms underlying metabolism-associated male infertility and suggests a novel therapeutic target for male infertility.

Limited Environmental Serine and Glycine Confer Brain Metastasis Sensitivity to PHGDH Inhibition.

A hallmark of metastasis is the adaptation of tumor cells to new environments. Metabolic constraints imposed by the serine and glycine-limited brain environment restrict metastatic tumor growth. How brain metastases overcome these growth-prohibitive conditions is poorly understood. Here, we demonstrate that 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the rate-limiting step of glucose-derived serine synthesis, is a major determinant of brain metastasis in multiple human cancer types and preclinical models. Enhanced serine synthesis proved important for nucleotide production and cell proliferation in highly aggressive brain metastatic cells. In vivo, genetic suppression and pharmacologic inhibition of PHGDH attenuated brain metastasis, but not extracranial tumor growth, and improved overall survival in mice. These results reveal that extracellular amino acid availability determines serine synthesis pathway dependence, and suggest that PHGDH inhibitors may be useful in the treatment of brain metastasis. SIGNIFICANCE: Using proteomics, metabolomics, and multiple brain metastasis models, we demonstrate that the nutrient-limited environment of the brain potentiates brain metastasis susceptibility to serine synthesis inhibition. These findings underscore the importance of studying cancer metabolism in physiologically relevant contexts, and provide a rationale for using PHGDH inhibitors to treat brain metastasis.This article is highlighted in the In This Issue feature, p. 1241.

Characterization of PHGDH expression in bladder cancer: potential targeting therapy with gemcitabine/cisplatin and the contribution of promoter DNA hypomethylation.

d-3-Phosphoglycerate dehydrogenase (PHGDH) conducts an important step in the synthesis of serine. Importantly, the PHGDH gene is often amplified in certain cancers. Our previous studies revealed that PHGDH gene amplification was associated with poor overall survival in clear cell renal cell carcinoma (ccRCC) and that metabolic reprogramming of serine synthesis through PHGDH recruitment allowed ccRCC cells to survive in unfavorable environments. There have been no investigations of the role of PHGDH expression in bladder cancer (BC). In this investigation, we examined the clinical importance of PHDGH in BC. Furthermore, we asked whether PHGDH expression could be exploited for BC therapy. Finally, we investigated the regulatory mechanisms that modulated the expression of PHGDH. Using data from The Cancer Genome Atlas, we found that patients with high-grade BC had significantly higher PHGDH expression levels than did those with low-grade BC. In addition, patients with high PHGDH expression did not survive as long as those with low expression. PHGDH downregulation by si-RNAs or an inhibitor in BC cell lines significantly inhibited proliferative ability and induced apoptosis. Furthermore, combined treatment using a PHGDH inhibitor and gemcitabine/cisplatin achieved synergistic tumor suppression compared to use of a single agent both in vitro as well as in vivo. Mechanistic analyses of PHGDH regulation showed that PHGDH expression might be associated with DNA copy number and hypomethylation in BC. These findings suggest novel therapeutic strategies could be used in BC. Finally, our data enhance our understanding of the role of PHGDH in BC.