PON2 mediates mitochondrial dysfunction in tracheal epithelial cells in response to a quorum sensing molecule N-(-3-oxododecanoyl)-l-homoserine lactone.

The opportunistic bacterium Pseudomonas aeruginosa secretes the quorum-sensing molecule N-(3-oxododecanoyl)-l-homoserine lactone (C12) to co-ordinate gene expression profiles favorable for infection. Recent studies have demonstrated that high concentrations of C12 impair many aspects of host cell physiology, including mitochondrial function and cell viability. The cytotoxic effects of C12 are mediated by the lactonase enzyme, Paraoxonase 2 (PON2), which hydrolyzes C12 to a reactive metabolite. However, the influence of C12 on host cell physiology at concentrations observed in patients infected with P. aeruginosa is largely unknown. Since the primary site of P. aeruginosa infections is the mammalian airway, we sought to investigate how PON2 modulates the effects of C12 at subtoxic concentrations using immortalized murine tracheal epithelial cells (TECs) isolated from wild-type (WT) or PON2-knockout (PON2-KO) mice. Our data reveal that C12 at subtoxic concentrations disrupts mitochondrial bioenergetics to hinder cellular proliferation in TECs expressing PON2. Subtoxic concentrations of C12 disrupt normal mitochondrial network morphology in a PON2-dependent manner without affecting mitochondrial membrane potential. In contrast, higher concentrations of C12 depolarize mitochondrial membrane potential and subsequently trigger caspase signaling and apoptotic cell death. These findings demonstrate that different concentrations of C12 impact distinct aspects of host airway epithelial cell physiology through PON2 activity in mitochondria.

Palbociclib treatment alters nucleotide biosynthesis and glutamine dependency in A549 cells.

BACKGROUND: Aberrant activity of cell cycle proteins is one of the key somatic events in non-small cell lung cancer (NSCLC) pathogenesis. In most NSCLC cases, the retinoblastoma protein tumor suppressor (RB) becomes inactivated via constitutive phosphorylation by cyclin dependent kinase (CDK) 4/6, leading to uncontrolled cell proliferation. Palbociclib, a small molecule inhibitor of CDK4/6, has shown anti-tumor activity in vitro and in vivo, with recent studies demonstrating a functional role for palbociclib in reprogramming cellular metabolism. While palbociclib has shown efficacy in preclinical models of NSCLC, the metabolic consequences of CDK4/6 inhibition in this context are largely unknown. METHODS: In our study, we used a combination of stable isotope resolved metabolomics using [U-13C]-glucose and multiple in vitro metabolic assays, to interrogate the metabolic perturbations induced by palbociclib in A549 lung adenocarcinoma cells. Specifically, we assessed changes in glycolytic activity, the pentose phosphate pathway (PPP), and glutamine utilization. We performed these studies following palbociclib treatment with simultaneous silencing of RB1 to define the pRB-dependent changes in metabolism. RESULTS: Our studies revealed palbociclib does not affect glycolytic activity in A549 cells but decreases glucose metabolism through the PPP. This is in part via reducing activity of glucose 6-phosphate dehydrogenase, the rate limiting enzyme in the PPP. Additionally, palbociclib enhances glutaminolysis to maintain mitochondrial respiration and sensitizes A549 cells to the glutaminase inhibitor, CB-839. Notably, the effects of palbociclib on both the PPP and glutamine utilization occur in an RB-dependent manner. CONCLUSIONS: Together, our data define the metabolic impact of palbociclib treatment in A549 cells and may support the targeting CDK4/6 inhibition in combination with glutaminase inhibitors in NSCLC patients with RB-proficient tumors.

Inhibition of 6-phosphofructo-2-kinase (PFKFB3) suppresses glucose metabolism and the growth of HER2+ breast cancer.

PURPOSE: Human epidermal growth factor receptor-2 (HER2) has been implicated in the progression of multiple tumor types, including breast cancer, and many downstream effectors of HER2 signaling are primary regulators of cellular metabolism, including Ras and Akt. A key downstream metabolic target of Ras and Akt is the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 isozyme (PFKFB3), whose product, fructose-2,6-bisphosphate (F26BP), is a potent allosteric activator of a rate-limiting enzyme in glycolysis, 6-phosphofructo-1-kinase (PFK-1). We postulate that PFKFB3 may be regulated by HER2 and contribute to HER2-driven tumorigenicity. METHODS: Immunohistochemistry and Kaplan-Meier analysis of HER2+ patient samples investigated the relevance of PFKFB3 in HER2+ breast cancer. In vitro genetic and pharmacological inhibition of PFKFB3 was utilized to determine effects on HER2+ breast cancer cells, while HER2 antagonist treatment assessed the mechanistic regulation on PFKFB3 expression and glucose metabolism. Administration of a PFKFB3 inhibitor in a HER2-driven transgenic breast cancer model evaluated this potential therapeutic approach in vivo. RESULTS: PFKFB3 is elevated in human HER2+ breast cancer and high PFKFB3 transcript correlated with poorer progression-free (PFS) and distant metastatic-free (DFMS) survival. Constitutive HER2 expression led to elevated PFKFB3 expression and increased glucose metabolism, while inhibition of PFKFB3 suppressed glucose uptake, F26BP, glycolysis, and selectively decreased the growth of HER2-expressing breast cancer cells. In addition, treatment with lapatinib, an FDA-approved HER2 inhibitor, decreased PFKFB3 expression and glucose metabolism in HER2+ cells. In vivo administration of a PFKFB3 antagonist significantly suppressed the growth of HER2-driven breast tumors and decreased 18F-2-deoxy-glucose uptake. CONCLUSIONS: Taken together, these data support the potential clinical utility of PFKFB3 inhibitors as chemotherapeutic agents against HER2+ breast cancer.

Estradiol stimulates glucose metabolism via 6-phosphofructo-2-kinase (PFKFB3).

Estradiol (E2) administered to estrogen receptor-positive (ER(+)) breast cancer patients stimulates glucose uptake by tumors. Importantly, this E2-induced metabolic flare is predictive of the clinical effectiveness of anti-estrogens and, as a result, downstream metabolic regulators of E2 are expected to have utility as targets for the development of anti-breast cancer agents. The family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) control glycolytic flux via their product, fructose-2,6-bisphosphate (F26BP), which activates 6-phosphofructo-1-kinase (PFK-1). We postulated that E2 might promote PFKFB3 expression, resulting in increased F26BP and glucose uptake. We demonstrate that PFKFB3 expression is highest in stage III lymph node metastases relative to normal breast tissues and that exposure of human MCF-7 breast cancer cells to E2 causes a rapid increase in [(14)C]glucose uptake and glycolysis that is coincident with an induction of PFKFB3 mRNA (via ER binding to its promoter), protein expression and the intracellular concentration of its product, F26BP. Importantly, selective inhibition of PFKFB3 expression and activity using siRNA or a PFKFB3 inhibitor markedly reduces the E2-mediated increase in F26BP, [(14)C]glucose uptake, and glycolysis. Furthermore, co-treatment of MCF-7 cells with the PFKFB3 inhibitor and the anti-estrogen ICI 182,780 synergistically induces apoptotic cell death. These findings demonstrate for the first time that the estrogen receptor directly promotes PFKFB3 mRNA transcription which, in turn, is required for the glucose metabolism and survival of breast cancer cells. Importantly, these results provide essential preclinical information that may allow for the ultimate design of combinatorial trials of PFKFB3 antagonists with anti-estrogen therapies in ER(+) stage IV breast cancer patients.

The ZEB1 transcription factor acts in a negative feedback loop with miR200 downstream of Ras and Rb1 to regulate Bmi1 expression.

Ras mutations are frequent in cancer cells where they drive proliferation and resistance to apoptosis. However in primary cells, mutant Ras instead can cause oncogene-induced senescence, a tumor suppressor function linked to repression of the polycomb factor Bmi1, which normally regulates cell cycle inhibitory cyclin-dependent kinase inhibitors (cdki). It is unclear how Ras causes repression of Bmi1 in primary cells to suppress tumor formation while inducing the gene in cancer cells to drive tumor progression. Ras also induces the EMT transcription factor ZEB1 to trigger tumor invasion and metastasis. Beyond its well-documented role in EMT, ZEB1 is important for maintaining repression of cdki. Indeed, heterozygous mutation of ZEB1 is sufficient for elevated cdki expression, leading to premature senescence of primary cells. A similar phenotype is evident with Bmi1 mutation. We show that activation of Rb1 in response to mutant Ras causes dominant repression of ZEB1 in primary cells, but loss of the Rb1 pathway is a hallmark of cancer cells and in the absence of such Rb1 repression Ras induces ZEB1 in cancer cells. ZEB1 represses miR-200 in the context of a mutual repression loop. Because miR-200 represses Bmi1, induction of ZEB1 leads to induction of Bmi1. Rb1 pathway status then dictates the opposing effects of mutant Ras on the ZEB1-miR-200 loop in primary versus cancer cells. This loop not only triggers EMT, surprisingly we show it acts downstream of Ras to regulate Bmi1 expression and thus the critical decision between oncogene-induced senescence and tumor initiation.

Troglitazone suppresses glutamine metabolism through a PPAR-independent mechanism.

Enhanced glutamine metabolism is required for tumor cell growth and survival, which suggests that agents targeting glutaminolysis may have utility within anti-cancer therapies. Troglitazone, a PPARγ agonist, exhibits significant anti-tumor activity and can alter glutamine metabolism in multiple cell types. Therefore, we examined whether troglitazone would disrupt glutamine metabolism in tumor cells and whether its action was reliant on PPARγ activity. We found that troglitazone treatment suppressed glutamine uptake and the expression of the glutamine transporter, ASCT2, and glutaminase. In addition, troglitazone reduced 13C-glutamine incorporation into the TCA cycle, decreased [ATP], and resulted in an increase in reactive oxygen species (ROS). Further, troglitazone treatment decreased tumor cell growth, which was partially rescued with the addition of the TCA-intermediate, α-ketoglutarate, or the antioxidant N-acetylcysteine. Importantly, troglitazone's effects on glutamine uptake or viable cell number were found to be PPARγ-independent. In contrast, troglitazone caused a decrease in c-Myc levels, while the proteasomal inhibitor, MG132, rescued c-Myc, ASCT2 and GLS1 expression, as well as glutamine uptake and cell number. Lastly, combinatorial treatment of troglitazone and metformin resulted in a synergistic decrease in cell number. Therefore, characterizing new anti-tumor properties of previously approved FDA therapies supports the potential for repurposing of these agents.

Sequential inductions of the ZEB1 transcription factor caused by mutation of Rb and then Ras proteins are required for tumor initiation and progression.

Rb1 restricts cell cycle progression, and it imposes cell contact inhibition to suppress tumor outgrowth. It also triggers oncogene-induced senescence to block Ras mutation. Loss of the Rb1 pathway, which is a hallmark of cancer cells, then provides a permissive environment for Ras mutation, and Ras is sufficient for invasive tumor formation in Rb1 family mutant mouse embryo fibroblasts (MEFs). These results demonstrate that sequential mutation of the Rb1 and Ras pathways comprises a tumor initiation axis. Both Rb1 and Ras regulate expression of the transcription factor ZEB1, thereby linking tumor initiation to the subsequent invasion and metastasis, which is induced by ZEB1. ZEB1 acts in a negative feedback loop to block expression of miR-200, which is thought to facilitate tumor invasion and metastasis. However, ZEB1 also represses cyclin-dependent kinase (cdk) inhibitors to control the cell cycle; its mutation in MEFs leads to induction of these inhibitors and premature senescence. Here, we provide evidence for two sequential inductions of ZEB1 during Ras transformation of MEFs. Rb1 constitutively represses cdk inhibitors, and induction of ZEB1 when the Rb1 pathway is lost is required to maintain this repression, allowing for the classic immortalization and loss of cell contact inhibition seen when the Rb1 pathway is lost. In vivo, we show that this induction of ZEB1 is required for Ras-initiated tumor formation. ZEB1 is then further induced by Ras, beyond the level seen with Rb1 mutation, and this Ras superinduction is required to reach a threshold of ZEB1 sufficient for repression of miR-200 and tumor invasion.

MicroRNA regulation of the serine synthesis pathway in endocrine-resistant breast cancer cells.

Despite the successful combination of therapies improving survival of estrogen receptor α (ER+) breast cancer patients with metastatic disease, mechanisms for acquired endocrine resistance remain to be fully elucidated. The RNA binding protein HNRNPA2B1 (A2B1), a reader of N(6)-methyladenosine (m6A) in transcribed RNA, is upregulated in endocrine-resistant, ER+ LCC9 and LY2 cells compared to parental MCF-7 endocrine-sensitive luminal A breast cancer cells. The miRNA-seq transcriptome of MCF-7 cells overexpressing A2B1 identified the serine metabolic processes pathway. Increased expression of two key enzymes in the serine synthesis pathway (SSP), phosphoserine aminotransferase 1 (PSAT1) and phosphoglycerate dehydrogenase (PHGDH), correlates with poor outcomes in ER+ breast patients who received tamoxifen (TAM). We reported that PSAT1 and PHGDH were higher in LCC9 and LY2 cells compared to MCF-7 cells and their knockdown enhanced TAM sensitivity in these-resistant cells. Here we demonstrate that stable, modest overexpression of A2B1 in MCF-7 cells increased PSAT1 and PHGDH and endocrine resistance. We identified four miRNAs downregulated in MCF-7-A2B1 cells that directly target the PSAT1 3'UTR (miR-145-5p and miR-424-5p), and the PHGDH 3'UTR (miR-34b-5p and miR-876-5p) in dual luciferase assays. Lower expression of miR-145-5p and miR-424-5p in LCC9 and ZR-75-1-4-OHT cells correlated with increased PSAT1 and lower expression of miR-34b-5p and miR-876-5p in LCC9 and ZR-75-1-4-OHT cells correlated with increased PHGDH. Transient transfection of these miRNAs restored endocrine-therapy sensitivity in LCC9 and ZR-75-1-4-OHT cells. Overall, our data suggest a role for decreased A2B1-regulated miRNAs in endocrine resistance and upregulation of the SSP to promote tumor progression in ER+ breast cancer.

Methionine-producing tumor micro(be) environment fuels growth of solid tumors.

BACKGROUND: Recent studies have uncovered the near-ubiquitous presence of microbes in solid tumors of diverse origins. Previous literature has shown the impact of specific bacterial species on the progression of cancer. We propose that local microbial dysbiosis enables certain cancer phenotypes through provisioning of essential metabolites directly to tumor cells. METHODS: 16S rDNA sequencing of 75 patient lung samples revealed the lung tumor microbiome specifically enriched for bacteria capable of producing methionine. Wild-type (WT) and methionine auxotrophic (metA mutant) E. coli cells were used to condition cell culture media and the proliferation of lung adenocarcinoma (LUAD) cells were measured using SYTO60 staining. Further, colony forming assay, Annexin V Staining, BrdU, AlamarBlue, western blot, qPCR, LINE microarray and subcutaneous injection with methionine modulated feed were used to analyze cellular proliferation, cell-cycle, cell death, methylation potential, and xenograft formation under methionine restriction. Moreover, C14-labeled glucose was used to illustrate the interplay between tumor cells and bacteria. RESULTS/DISCUSSION: Our results show bacteria found locally within the tumor microenvironment are enriched for methionine synthetic pathways, while having reduced S-adenosylmethionine metabolizing pathways. As methionine is one of nine essential amino acids that mammals are unable to synthesize de novo, we investigated a potentially novel function for the microbiome, supplying essential nutrients, such as methionine, to cancer cells. We demonstrate that LUAD cells can utilize methionine generated by bacteria to rescue phenotypes that would otherwise be inhibited due to nutrient restriction. In addition to this, with WT and metA mutant E. coli, we saw a selective advantage for bacteria with an intact methionine synthetic pathway to survive under the conditions induced by LUAD cells. These results would suggest that there is a potential bi-directional cross-talk between the local microbiome and adjacent tumor cells. In this study, we focused on methionine as one of the critical molecules, but we also hypothesize that additional bacterial metabolites may also be utilized by LUAD. Indeed, our radiolabeling data suggest that other biomolecules are shared between cancer cells and bacteria. Thus, modulating the local microbiome may have an indirect effect on tumor development, progression, and metastasis.

Cytochrome c oxidase is activated by the oncoprotein Ras and is required for A549 lung adenocarcinoma growth.

BACKGROUND: Constitutive activation of Ras in immortalized bronchial epithelial cells increases electron transport chain activity, oxygen consumption and tricarboxylic acid cycling through unknown mechanisms. We hypothesized that members of the Ras family may stimulate respiration by enhancing the expression of the Vb regulatory subunit of cytochrome c oxidase (COX). RESULTS: We found that the introduction of activated H-Ras(V12) into immortalized human bronchial epithelial cells increased eIF4E-dependent COX Vb protein expression simultaneously with an increase in COX activity and oxygen consumption. In support of the regulation of COX Vb expression by the Ras family, we also found that selective siRNA-mediated inhibition of K-Ras expression in A549 lung adenocarcinoma cells reduced COX Vb protein expression, COX activity, oxygen consumption and the steady-state concentration of ATP. We postulated that COX Vb-mediated activation of COX activity may be required for the anchorage-independent growth of A549 cells as soft agar colonies or as lung xenografts. We transfected the A549 cells with COX Vb small interfering or shRNA and observed a significant reduction of their COX activity, oxygen consumption, ATP and ability to grow in soft agar and as poorly differentiated tumors in athymic mice. CONCLUSION: Taken together, our findings indicate that the activation of Ras increases COX activity and mitochondrial respiration in part via up-regulation of COX Vb and that this regulatory subunit of COX may have utility as a Ras effector target for the development of anti-neoplastic agents.

Small molecule inhibition of 6-phosphofructo-2-kinase suppresses t cell activation.

BACKGROUND: T cell activation is associated with a rapid increase in intracellular fructose-2,6-bisphosphate (F2,6BP), an allosteric activator of the glycolytic enzyme, 6-phosphofructo-1-kinase. The steady state concentration of F2,6BP in T cells is dependent on the expression of the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) and the fructose-2,6-bisphosphatase, TIGAR. Of the PFKFB family of enzymes, PFKFB3 has the highest kinase:bisphosphatase ratio and has been demonstrated to be required for T cell proliferation. A small molecule antagonist of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), recently has been shown to reduce F2,6BP synthesis, glucose uptake and proliferation in transformed cells. We hypothesized that the induction of PFKFB3 expression may be required for the stimulation of glycolysis in T cells and that exposure to the PFKFB3 antagonist, 3PO, would suppress T cell activation. METHODS: We examined PFKFB1-4 and TIGAR expression and F2,6BP concentration in purified CD3+ T cells stimulated with microbead-conjugated agonist antibodies specific for CD3 and the co-stimulatory receptor, CD28. We then determined the effect of 3PO on anti-CD3/anti-CD28-induced T cell activation, F2,6BP synthesis, 2-[1-14C]-deoxy-d-glucose uptake, lactate secretion, TNF-α secretion and proliferation. Finally, we examined the effect of 3PO administration on the development of delayed type hypersensitivity to methylated BSA and on imiquimod-induced psoriasis in mice. RESULTS: We found that purified human CD3+ T cells express PFKFB2, PFKFB3, PFKFB4 and TIGAR, and that anti-CD3/anti-CD28 conjugated microbeads stimulated a >20-fold increase in F2,6BP with a coincident increase in protein expression of the PFKFB3 family member and a decrease in TIGAR protein expression. We then found that exposure to the PFKFB3 small molecule antagonist, 3PO (1-10 µM), markedly attenuated the stimulation of F2,6BP synthesis, 2-[1-14C]-deoxy-D-glucose uptake, lactate secretion, TNF-α secretion and T cell aggregation and proliferation. We examined the in vivo effect of 3PO on the development of delayed type hypersensitivity to methylated BSA and on imiquimod-induced psoriasis in mice and found that 3PO suppressed the development of both T cell-dependent models of immunity in vivo. CONCLUSIONS: Our data demonstrate that inhibition of the PFKFB3 kinase activity attenuates the activation of T cells in vitro and suppresses T cell dependent immunity in vivo and indicate that small molecule antagonists of PFKFB3 may prove effective as T cell immunosuppressive agents.

Block copolymer micelles for controlled delivery of glycolytic enzyme inhibitors.

PURPOSE: To develop block copolymer micelles as an aqueous dosage form for a potent glycolytic enzyme inhibitor, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). METHODS: The micelles were prepared from poly(ethylene glycol)-poly(aspartate hydrazide) [PEG-p(HYD)] block copolymers to which 3PO was conjugated through an acid-labile hydrazone bond. The optimal micelle formulation was determined following the screening of block copolymer library modified with various aromatic and aliphatic pendant groups. Both physical drug entrapment and chemical drug conjugation methods were tested to maximize 3PO loading in the micelles during the screening. RESULTS: Particulate characterization showed that the PEG-p(HYD) block copolymers conjugated with 3PO (2.08∼2.21 wt.%) appeared the optimal polymer micelles. Block copolymer compositions greatly affected the micelle size, which was 38 nm and 259 nm when 5 kDa and 12 kDa PEG chains were used, respectively. 3PO release from the micelles was accelerated at pH 5.0, potentiating effective drug release in acidic tumor environments. The micelles retained biological activity of 3PO, inhibiting various cancer cells (Jurkat, He-La and LLC) in concentration ranges similar to free 3PO. CONCLUSION: A novel micelle formulation for controlled delivery of 3PO was successfully prepared.

Phase II trial of the regulatory T cell-depleting agent, denileukin diftitox, in patients with unresectable stage IV melanoma.

BACKGROUND: We previously found that administration of an interleukin 2/diphtheria toxin conjugate (DAB/IL2; Denileukin Diftitox; ONTAK) to stage IV melanoma patients depleted CD4(+)CD25(HI)Foxp3(+) regulatory T cells and expanded melanoma-specific CD8(+) T cells. The goal of this study was to assess the clinical efficacy of DAB/IL2 in an expanded cohort of stage IV melanoma patients. METHODS: In a single-center, phase II trial, DAB/IL2 (12 µg/kg; 4 daily doses; 21 day cycles) was administered to 60 unresectable stage IV melanoma patients and response rates were assessed using a combination of 2-[(18)F]-fluoro-2-deoxy-glucose (FDG)-positron emission tomography (PET) and computed tomography (CT) imaging. RESULTS: After DAB/IL2 administration, 16.7% of the 60 patients had partial responses, 5% stable disease and 15% mixed responses. Importantly, 45.5% of the chemo/immuno-naïve sub-population (11/60 patients) experienced partial responses. One year survival was markedly higher in partial responders (80 ± 11.9%) relative to patients with progressive disease (23.7 ± 6.5%; p value < 0.001) and 40 ± 6.2% of the total DAB/IL2-treated population were alive at 1 year. CONCLUSIONS: These data support the development of multi-center, randomized trials of DAB/IL2 as a monotherapy and in combination with other immunotherapeutic agents for the treatment of stage IV melanoma. TRIAL REGISTRATION: NCT00299689.

Nuclear Pyruvate Kinase M2 (PKM2) Contributes to Phosphoserine Aminotransferase 1 (PSAT1)-Mediated Cell Migration in EGFR-Activated Lung Cancer Cells.

An elevated expression of phosphoserine aminotransferase 1 (PSAT1) has been observed in multiple tumor types and is associated with poorer clinical outcomes. Although PSAT1 is postulated to promote tumor growth through its enzymatic function within the serine synthesis pathway (SSP), its role in cancer progression has not been fully characterized. Here, we explore a putative non-canonical function of PSAT1 that contributes to lung tumor progression. Biochemical studies found that PSAT1 selectively interacts with pyruvate kinase M2 (PKM2). Amino acid mutations within a PKM2-unique region significantly reduced this interaction. While PSAT1 loss had no effect on cellular pyruvate kinase activity and PKM2 expression in non-small-cell lung cancer (NSCLC) cells, fractionation studies demonstrated that the silencing of PSAT1 in epidermal growth factor receptor (EGFR)-mutant PC9 or EGF-stimulated A549 cells decreased PKM2 nuclear translocation. Further, PSAT1 suppression abrogated cell migration in these two cell types whereas PSAT1 restoration or overexpression induced cell migration along with an elevated nuclear PKM2 expression. Lastly, the nuclear re-expression of the acetyl-mimetic mutant of PKM2 (K433Q), but not the wild-type, partially restored cell migration in PSAT1-silenced cells. Therefore, we conclude that, in response to EGFR activation, PSAT1 contributes to lung cancer cell migration, in part, by promoting nuclear PKM2 translocation.

Serine synthesis influences tamoxifen response in ER+ human breast carcinoma.

Estrogen receptor-positive breast cancer (ER+ BC) is the most common form of breast carcinoma accounting for approximately 70% of all diagnoses. Although ER-targeted therapies have improved survival outcomes for this BC subtype, a significant proportion of patients will ultimately develop resistance to these clinical interventions, resulting in disease recurrence. Phosphoserine aminotransferase 1 (PSAT1), an enzyme within the serine synthetic pathway (SSP), has been previously implicated in endocrine resistance. Therefore, we determined whether expression of SSP enzymes, PSAT1 or phosphoglycerate dehydrogenase (PHGDH), affects the response of ER+ BC to 4-hydroxytamoxifen (4-OHT) treatment. To investigate a clinical correlation between PSAT1, PHGDH, and endocrine resistance, we examined microarray data from ER+ patients who received tamoxifen as the sole endocrine therapy. We confirmed that higher PSAT1 and PHGDH expression correlates negatively with poorer outcomes in tamoxifen-treated ER+ BC patients. Next, we found that SSP enzyme expression and serine synthesis were elevated in tamoxifen-resistant compared to tamoxifen-sensitive ER+ BC cells in vitro. To determine relevance to endocrine sensitivity, we modified the expression of either PSAT1 or PHGDH in each cell type. Overexpression of PSAT1 in tamoxifen-sensitive MCF-7 cells diminished 4-OHT inhibition on cell proliferation. Conversely, silencing of either PSAT1 or PHGDH resulted in greater sensitivity to 4-OHT treatment in LCC9 tamoxifen-resistant cells. Likewise, the combination of a PHGDH inhibitor with 4-OHT decreased LCC9 cell proliferation. Collectively, these results suggest that overexpression of serine synthetic pathway enzymes contribute to tamoxifen resistance in ER+ BC, which can be targeted as a novel combinatorial treatment option.

HNRNPA2B1 regulates tamoxifen- and fulvestrant-sensitivity and hallmarks of endocrine resistance in breast cancer cells.

Despite new combination therapies improving survival of breast cancer patients with estrogen receptor α (ER+) tumors, the molecular mechanisms for endocrine-resistant disease remain unresolved. Previously we demonstrated that expression of the RNA binding protein and N6-methyladenosine (m6A) reader HNRNPA2B1 (A2B1) is higher in LCC9 and LY2 tamoxifen (TAM)-resistant ERα breast cancer cells relative to parental TAM-sensitive MCF-7 cells. Here we report that A2B1 protein expression is higher in breast tumors than paired normal breast tissue. Modest stable overexpression of A2B1 in MCF-7 cells (MCF-7-A2B1 cells) resulted in TAM- and fulvestrant- resistance whereas knockdown of A2B1 in LCC9 and LY2 cells restored TAM and fulvestrant, endocrine-sensitivity. MCF-7-A2B1 cells gained hallmarks of TAM-resistant metastatic behavior: increased migration and invasion, clonogenicity, and soft agar colony size, which were attenuated by A2B1 knockdown in MCF-7-A2B1 and the TAM-resistant LCC9 and LY2 cells. MCF-7-A2B1, LCC9, and LY2 cells have a higher proportion of CD44+/CD24-/low cancer stem cells (CSC) compared to MCF-7 cells. MCF-7-A2B1 cells have increased ERα and reduced miR-222-3p that targets ERα. Like LCC9 cells, MCF-7-A2B1 have activated AKT and MAPK that depend on A2B1 expression and are growth inhibited by inhibitors of these pathways. These data support that targeting A2B1 could provide a complimentary therapeutic approach to reduce acquired endocrine resistance.

Lactate supports a metabolic-epigenetic link in macrophage polarization.

Lactate accumulation is a hallmark of solid cancers and is linked to the immune suppressive phenotypes of tumor-infiltrating immune cells. We report herein that interleukin-4 (IL-4)­induced M0 → M2 macrophage polarization is accompanied by interchangeable glucose- or lactate-dependent tricarboxylic acid (TCA) cycle metabolism that directly drives histone acetylation, M2 gene transcription, and functional immune suppression. Lactate-dependent M0 → M2 polarization requires both mitochondrial pyruvate uptake and adenosine triphosphate­citrate lyase (ACLY) enzymatic activity. Notably, exogenous acetate rescues defective M2 polarization and histone acetylation following mitochondrial pyruvate carrier 1 (MPC1) inhibition or ACLY deficiency. Lastly, M2 macrophage­dependent tumor progression is impaired by conditional macrophage ACLY deficiency, further supporting a dominant role for glucose/lactate mitochondrial metabolism and histone acetylation in driving immune evasion. This work adds to our understanding of how mitochondrial metabolism affects macrophage functional phenotypes and identifies a unique tumor microenvironment (TME)­driven metabolic-epigenetic link in M2 macrophages.

Selective loss of phosphoserine aminotransferase 1 (PSAT1) suppresses migration, invasion, and experimental metastasis in triple negative breast cancer.

Breast cancer is the second leading cause of cancer-related deaths among women and 90% of these mortalities can be attributed to progression to metastatic disease. In particular, triple negative breast cancer (TNBC) is extremely aggressive and frequently metastasizes to multiple organs. As TNBCs are categorized by their lack of hormone receptors, these tumors are very heterogeneous and are immune to most targeted therapies. Metabolic changes are observed in the majority of TNBC and a large proportion upregulate enzymes within the serine synthesis pathway, including phosphoserine aminotransferase 1 (PSAT1). In this report, we investigate the role of PSAT1 in migration and invasion potential in a subset of TNBC cell types. We found that the expression of PSAT1 increases with TNBC clinical grade. We also demonstrate that suppression of PSAT1 or phosphoglycerate dehydrogenase (PHGDH) does not negatively impact cell proliferation in TNBC cells that are not dependent on de novo serine synthesis. However, we observed that suppression of PSAT1 specifically alters the F-actin cytoskeletal arrangement and morphology in these TNBC cell lines. In addition, suppression of PSAT1 inhibits motility and migration in these TNBC cell lines, which is not recapitulated upon loss of PHGDH. PSAT1 silencing also reduced the number of lung tumor nodules in a model of experimental metastasis; yet did not decrease anchorage-independent growth. Together, these results suggest that PSAT1 functions to drive migratory potential in promoting metastasis in select TNBC cells independent of its role in serine synthesis.

Loss of Rb1 Enhances Glycolytic Metabolism in Kras-Driven Lung Tumors In Vivo.

Dysregulated metabolism is a hallmark of cancer cells and is driven in part by specific genetic alterations in various oncogenes or tumor suppressors. The retinoblastoma protein (pRb) is a tumor suppressor that canonically regulates cell cycle progression; however, recent studies have highlighted a functional role for pRb in controlling cellular metabolism. Here, we report that loss of the gene encoding pRb (Rb1) in a transgenic mutant Kras-driven model of lung cancer results in metabolic reprogramming. Our tracer studies using bolus dosing of [U-13C]-glucose revealed an increase in glucose carbon incorporation into select glycolytic intermediates. Consistent with this result, Rb1-depleted tumors exhibited increased expression of key glycolytic enzymes. Interestingly, loss of Rb1 did not alter mitochondrial pyruvate oxidation compared to lung tumors with intact Rb1. Additional tracer studies using [U-13C,15N]-glutamine and [U-13C]-lactate demonstrated that loss of Rb1 did not alter glutaminolysis or utilization of circulating lactate within the tricarboxylic acid cycle (TCA) in vivo. Taken together, these data suggest that the loss of Rb1 promotes a glycolytic phenotype, while not altering pyruvate oxidative metabolism or glutamine anaplerosis in Kras-driven lung tumors.

Regulation of glucose metabolism by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases in cancer.

A high rate of glycolytic flux, even in the presence of oxygen, is a central metabolic hallmark of neoplastic tumors. Cancer cells preferentially utilize glycolysis in order to satisfy their increased energetic and biosynthetic requirements. This metabolic phenotype has been confirmed in human studies using positron emission tomography (PET) with (18)F-2-fluoro-deoxy-glucose which have demonstrated that tumors take up 10-fold more glucose than adjacent normal tissues in vivo. The high glucose metabolism of cancer cells is caused by a combination of hypoxia-responsive transcription factors, activation of oncogenic proteins and the loss of tumor suppressor function. Over-expression of HIF-1alpha and myc, activation of ras and loss of p53 function each have been found to stimulate glycolysis in part by activating a family of regulatory bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB). The PFKFB enzymes synthesize fructose-2,6-bisphosphate (F2,6BP) which allosterically activates 6-phosphofructo-1-kinase (PFK-1), a rate-limiting enzyme and essential control point in the glycolytic pathway. PFK-1 is inhibited by ATP when energy stores are abundant and F2,6BP can override this inhibition and enhance glucose uptake and glycolytic flux. It is therefore not surprising that F2,6BP synthesis is stimulated by several oncogenic alterations which simultaneously cause both enhanced consumption of glucose and growth. Importantly, these studies suggest that selective depletion of intracellular F2,6BP in cancer cells may suppress glycolytic flux and decrease their survival, growth and invasiveness. This review will summarize the requirement of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases for the regulation of glycolysis in tumor cells and their potential utility as targets for the development of antineoplastic agents.