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Simple Summary: The breakdown of glucose in the glycolytic pathway provides energy and essential building blocks to support cell division and growth in important body processes such as embryo development but also can promote disease pathologies including tumor formation. A family of enzymes called the PFKFBs are important regulators of this pathway, and the PFKFB3 family member plays a key role. Examining the functions of PFKFB3 in live animals is critical for fully understanding its role in glycolysis, and we have developed a new mouse model that can knock out PFKFB3 to evaluate these functions. In our studies, we have found that these mice have decreased PFKFB3 levels and activity and that knocking out PFKFB3 markedly impairs the growth of tumors that closely mimic human cancer. Our data establish a new and effective model for examining the regulation of glucose metabolism by PFKFB3 and highlight its importance in tumor growth. Rapidly proliferative processes in mammalian tissues including tumorigenesis and embryogenesis rely on the glycolytic pathway for energy and biosynthetic precursors. The enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) plays an important regulatory role in glycolysis by activating the key rate-limiting glycolytic enzyme, 6-phosphofructo-1-kinase (PFK-1). We have previously determined that decreased PFKFB3 expression reduced glycolysis and growth in transformed cells in vitro and suppressed xenograft growth in vivo. In earlier studies, we created a constitutive knockout mouse to interrogate the function of PFKFB3 in vivo but failed to generate homozygous offspring due to the requirement for PFKFB3 for embryogenesis. We have now developed a novel transgenic mouse model that exhibits inducible homozygous pan-tissue Pfkfb3 gene deletion (Pfkfb3fl/fl). We have induced Pfkfb3 genomic deletion in these mice and found that it effectively decreased PFKFB3 expression and activity. To evaluate the functional consequences of Pfkfb3 deletion in vivo, we crossed Cre-bearing Pfkfb3fl/fl mice with oncogene-driven tumor models and found that Pfkfb3 deletion markedly decreased their glucose uptake and growth. In summary, our studies reveal a critical regulatory function for PFKFB3 in glycolysis and tumorigenesis in vivo and characterize an effective and powerful model for further investigation of its role in multiple biological processes. [ABSTRACT FROM AUTHOR]

Despite the successful combination of therapies improving survival of estrogen receptor a (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 endocrineresistant, 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 endocrinetherapy 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. [ABSTRACT FROM AUTHOR]

Abstract 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-RasV12 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.

Abstract 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.

Abstract 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+CD25HIFoxp3+ 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

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 18 F-2-deoxy-glucose uptake., Conclusions: Taken together, these data support the potential clinical utility of PFKFB3 inhibitors as chemotherapeutic agents against HER2+ breast cancer.