Yoannis Imbert-Fernandez

Targeting 6-Phosphofructo-2-Kinase (PFKFB3) as a Therapeutic Strategy against Cancer

In human cancers, loss of PTEN, stabilization of hypoxia inducible factor-1α, and activation of Ras and AKT converge to increase the activity of a key regulator of glycolysis, 6-phosphofructo-2-kinase (PFKFB3). This enzyme synthesizes fructose 2,6-bisphosphate (F26BP), which is an activator of 6-phosphofructo-1-kinase, a key step of glycolysis. Previously, a weak competitive inhibitor of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), was found to reduce the glucose metabolism and proliferation of cancer cells. We have synthesized 73 derivatives of 3PO and screened each compound for activity against recombinant PFKFB3. One small molecule, 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15), was selected for further preclinical evaluation of its pharmacokinetic, antimetabolic, and antineoplastic properties in vitro and in vivo. We found that PFK15 causes a rapid induction of apoptosis in transformed cells, has adequate pharmacokinetic properties, suppresses the glucose uptake and growth of Lewis lung carcinomas in syngeneic mice, and yields antitumor effects in three human xenograft models of cancer in athymic mice that are comparable to U.S. Food and Drug Administration–approved chemotherapeutic agents. As a result of this study, a synthetic derivative and formulation of PFK15 has undergone investigational new drug (IND)-enabling toxicology and safety studies. A phase I clinical trial of its efficacy in advanced cancer patients will initiate in 2013 and we anticipate that this new class of antimetabolic agents will yield acceptable therapeutic indices and prove to be synergistic with agents that disrupt neoplastic signaling. Mol Cancer Ther; 12(8); 1461–70. ©2013 AACR.

6-Phosphofructo-2-kinase (PFKFB3) promotes cell cycle progression and suppresses apoptosis via Cdk1-mediated phosphorylation of p27

The control of glucose metabolism and the cell cycle must be coordinated in order to guarantee sufficient ATP and anabolic substrates at distinct phases of the cell cycle. The family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) are well established regulators of glucose metabolism via their synthesis of fructose-2,6-bisphosphate (F2,6BP), a potent allosteric activator of 6-phosphofructo-1-kinase (Pfk-1). PFKFB3 is overexpressed in human cancers, regulated by HIF-1α, Akt and PTEN, and required for the survival and growth of multiple cancer types. Although most functional studies of the role of PFKFB3 in cancer progression have invoked its well-recognized function in the regulation of glycolysis, recent observations have established that PFKFB3 also traffics to the nucleus and that its product, F2,6BP, activates cyclin-dependent kinases (Cdks). In particular, F2,6BP stimulates the Cdk-mediated phosphorylation of the Cip/Kip protein p27 (threonine 187), which in turn results in p27’s ubiquitination and proteasomal degradation. As p27 is a potent suppressor of the G1/S transition and activator of apoptosis, we hypothesized that the known requirement of PFKFB3 for cell cycle progression and prevention of apoptosis may be partly due to the ability of F2,6BP to activate Cdks. In this study, we demonstrate that siRNA silencing of endogenous PFKFB3 inhibits Cdk1 activity, which in turn stabilizes p27 protein levels causing cell cycle arrest at G1/S and increased apoptosis in HeLa cells. Importantly, we demonstrate that the increase in apoptosis and suppression of the G1/S transition caused by siRNA silencing of PFKFB3 expression is reversed by co-siRNA silencing of p27. Taken together with prior publications, these observations support a model whereby PFKFB3 and F2,6BP function not only as regulators of Pfk-1 but also of Cdk1 activity, and therefore serve to couple glucose metabolism with cell proliferation and survival in transformed cells.

Inhibition of 6-phosphofructo-2-kinase (PFKFB3) induces autophagy as a survival mechanism

BackgroundUnlike glycolytic enzymes that directly catabolize glucose to pyruvate, the family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFBs) control the conversion of fructose-6-phosphate to and from fructose-2,6-bisphosphate, a key regulator of the glycolytic enzyme phosphofructokinase-1 (PFK-1). One family member, PFKFB3, has been shown to be highly expressed and activated in human cancer cells, and derivatives of a PFKFB3 inhibitor, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), are currently being developed in clinical trials. However, the effectiveness of drugs such as 3PO that target energetic pathways is limited by survival pathways that can be activated by reduced ATP and nutrient uptake. One such pathway is the process of cellular self-catabolism termed autophagy. We hypothesized that the functional glucose starvation induced by inhibition of PFKFB3 in tumor cells would induce autophagy as a pro-survival mechanism and that inhibitors of autophagy could increase the anti-tumor effects of PFKFB3 inhibitors.ResultsWe found that selective inhibition of PFKFB3 with either siRNA transfection or 3PO in HCT-116 colon adenocarcinoma cells caused a marked decrease in glucose uptake simultaneously with an increase in autophagy based on LC3-II and p62 protein expression, acridine orange fluorescence of acidic vacuoles and electron microscopic detection of autophagosomes. The induction of autophagy caused by PFKFB3 inhibition required an increase in reactive oxygen species since N-acetyl-cysteine blocked both the conversion of LC3-I to LC3-II and the increase in acridine orange fluorescence in acidic vesicles after exposure of HCT-116 cells to 3PO. We speculated that the induction of autophagy might protect cells from the pro-apoptotic effects of 3PO and found that agents that disrupt autophagy, including chloroquine, increased 3PO-induced apoptosis as measured by double staining with Annexin V and propidium iodide in both HCT-116 cells and Lewis lung carcinoma (LLC) cells. Chloroquine also increased the anti-growth effect of 3PO against LLCs in vivo and resulted in an increase in apoptotic cells within the tumors.ConclusionsWe conclude that PFKFB3 inhibitors suppress glucose uptake, which in turn causes an increase in autophagy. The addition of selective inhibitors of autophagy to 3PO and its more potent derivatives may prove useful as rational combinations for the treatment of cancer.

Anacardic acid inhibits estrogen receptor alpha-DNA binding and reduces target gene transcription and breast cancer cell proliferation

Anacardic acid (AnAc; 2-hydroxy-6-alkylbenzoic acid) is a dietary and medicinal phytochemical with established anticancer activity in cell and animal models. The mechanisms by which AnAc inhibits cancer cell proliferation remain undefined. AnAc 24:1ω5 was purified from geranium (Pelargonium × hortorum) and shown to inhibit the proliferation of estrogen receptor α (ERα)–positive MCF-7 and endocrine-resistant LCC9 and LY2 breast cancer cells with greater efficacy than ERα-negative primary human breast epithelial cells, MCF-10A normal breast epithelial cells, and MDA-MB-231 basal-like breast cancer cells. AnAc 24:1ω5 inhibited cell cycle progression and induced apoptosis in a cell-specific manner. AnAc 24:1ω5 inhibited estradiol (E2)–induced estrogen response element (ERE) reporter activity and transcription of the endogenous E2 target genes pS2, cyclin D1, and cathepsin D in MCF-7 cells. AnAc 24:1ω5 did not compete with E2 for ERα or ERβ binding, nor did AnAc 24:1ω5 reduce ERα or ERβ steady-state protein levels in MCF-7 cells; rather, AnAc 24:1ω5 inhibited ER-ERE binding in vitro. Virtual screening with the molecular docking software Surflex evaluated AnAc 24:1ω5 interaction with ERα ligand binding (LBD) and DNA binding (DBD) domains in conjunction with experimental validation. Molecular modeling revealed AnAc 24:1ω5 interaction with the ERα DBD but not the LBD. Chromatin immunoprecipitation experiments revealed that AnAc 24:1ω5 inhibited E2-ERα interaction with the endogenous pS2 gene promoter region containing an ERE. These data indicate that AnAc 24:1ω5 inhibits cell proliferation, cell cycle progression, and apoptosis in an ER-dependent manner by reducing ER-DNA interaction and inhibiting ER-mediated transcriptional responses. Mol Cancer Ther; 9(3); 594–605

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

Background: The regulation of glucose metabolism by estradiol is poorly defined. Results: We find that estradiol stimulates glucose metabolism in part by stimulating the production of fructose 2,6-bisphosphate by PFKFB3. Conclusion: PFKFB3 is a downstream target of estradiol required to stimulate glucose metabolism. Significance: Combined targeting of PFKFB3 and the estrogen receptor may prove beneficial to ER+ stage IV breast cancer patients. 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 [14C]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, [14C]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.

Targeting the Intracellular MUC1 C-terminal Domain Inhibits Proliferation and Estrogen Receptor Transcriptional Activity in Lung Adenocarcinoma Cells

Mucin 1 (MUC1) is a diagnostic factor and therapy target in lung adenocarcinoma. MUC1 C-terminal intracellular domain (CD) interacts with estrogen receptor (ER) α and increases gene transcription in breast cancer cells. Because lung adenocarcinoma cells express functional ERα and ERβ, we examined MUC1 expression and MUC1–ER interaction. Because blocking MUC1 CD with an inhibitory peptide (PMIP) inhibited breast tumor growth, we tested whether PMIP would inhibit lung adenocarcinoma cell proliferation. We report that MUC1 interacts with ERα and ERβ within the nucleus of H1793 lung adenocarcinoma cells in accordance with MUC1 expression. PMIP was taken up by H23 and H1793 cells and inhibited the proliferation of H1793, but not H23 cells, concordant with higher MUC1 protein expression in H1793 cells. Lower MUC1 protein expression in H23 does not correspond to microRNAs miR-125b and miR-145 that have been reported to reduce MUC1 expression. PMIP had no effect on the viability of normal human bronchial epithelial cells, which lack MUC1 expression. PMIP inhibited estradiol-activated reporter gene transcription and endogenous cyclin D1 and nuclear respiratory factor-1 gene transcription in H1793 cells. These results indicate MUC1–ER functional interaction in lung adenocarcinoma cells and that inhibiting MUC1 inhibits lung adenocarcinoma cell viability. Mol Cancer Ther; 10(11); 2062–71. ©2011 AACR.

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

PurposeHuman 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.MethodsImmunohistochemistry 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.ResultsPFKFB3 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.ConclusionsTaken together, these data support the potential clinical utility of PFKFB3 inhibitors as chemotherapeutic agents against HER2+ breast cancer.

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

BackgroundConstitutive 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).ResultsWe 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.ConclusionTaken 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.

Simultaneous inhibition of the estrogen receptor and 6-phosphofructo-2-kinase (PFKFB3) for the treatment of ER+ breast cancer

Background The family of 6-phospofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) control glycolytic flux via their product, fructose-2,6-bisphosphate (F26BP), which activates the key control step, 6-phosphofructo-1-kinase (PFK-1). We recently demonstrated that exposure of estrogen receptor (ER)+ MCF-7 human breast cancer cells to estradiol (E2) causes a rapid increase in 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. Selective inhibition of PFKFB3 expression and activity using siRNA or a PFKFB3 inhibitor, PFK158, markedly reduced the E2-mediated increase in F26BP, C-glucose uptake and glycolysis. Importantly, co-treatment of MCF-7 cells with PFK158 and an ER antagonist synergistically induced apoptotic cell death in vitro. In the current study, we sought to determine if co-administration of PFK158, with ICI 182,780 (fulvestrant), would have an additive anti-tumor effect on ER+ human breast cancer xenografts in vivo.

Fructose-2,6-bisphosphate links glucose to survival and growth

Background Glucose starvation, as occurs in neoplastic tumors, causes G1 arrest and/or apoptosis. Although the mechanisms for these events are poorly understood, reductions in nucleotide synthesis and ATP are widely believed to restrict progression into the S phase and cause apoptosis, respectively. Recently, a side product of glycolysis and stimulator of PFK-1, fructose 2,6-bisphosphate (F2,6BP), was found to be synthesized in the nucleus of cells by the enzyme PFKFB3 and to stimulate proliferation in part by activating cyclin dependent kinases (Cdk). We hypothesized that glucose deprivation would reduce F2,6BP which in turn would suppress the activity of Cdks and cause G1 arrest and apoptosis.