Giulia Guzzo

Aerobic glycolysis tunes YAP/TAZ transcriptional activity

Increased glucose metabolism and reprogramming toward aerobic glycolysis are a hallmark of cancer cells, meeting their metabolic needs for sustained cell proliferation. Metabolic reprogramming is usually considered as a downstream consequence of tumor development and oncogene activation; growing evidence indicates, however, that metabolism on its turn can support oncogenic signaling to foster tumor malignancy. Here, we explored how glucose metabolism regulates gene transcription and found an unexpected link with YAP/TAZ, key transcription factors regulating organ growth, tumor cell proliferation and aggressiveness. When cells actively incorporate glucose and route it through glycolysis, YAP/TAZ are fully active; when glucose metabolism is blocked, or glycolysis is reduced, YAP/TAZ transcriptional activity is decreased. Accordingly, glycolysis is required to sustain YAP/TAZ pro‐tumorigenic functions, and YAP/TAZ are required for the full deployment of glucose growth‐promoting activity. Mechanistically we found that phosphofructokinase (PFK1), the enzyme regulating the first committed step of glycolysis, binds the YAP/TAZ transcriptional cofactors TEADs and promotes their functional and biochemical cooperation with YAP/TAZ. Strikingly, this regulation is conserved in Drosophila, where phosphofructokinase is required for tissue overgrowth promoted by Yki, the fly homologue of YAP. Moreover, gene expression regulated by glucose metabolism in breast cancer cells is strongly associated in a large dataset of primary human mammary tumors with YAP/TAZ activation and with the progression toward more advanced and malignant stages. These findings suggest that aerobic glycolysis endows cancer cells with particular metabolic properties and at the same time sustains transcription factors with potent pro‐tumorigenic activities such as YAP/TAZ.

VEGF-Targeted Therapy Stably Modulates the Glycolytic Phenotype of Tumor Cells

Anti-VEGF therapy perturbs tumor metabolism, severely impairing oxygen, glucose, and ATP levels. In this study, we investigated the effects of anti-VEGF therapy in multiple experimental tumor models that differ in their glycolytic phenotypes to gain insights into optimal modulation of the metabolic features of this therapy. Prolonged treatments induced vascular regression and necrosis in tumor xenograft models, with highly glycolytic tumors becoming treatment resistant more rapidly than poorly glycolytic tumors. By PET imaging, prolonged treatments yielded an increase in both hypoxic and proliferative regions of tumors. A selection for highly glycolytic cells was noted and this metabolic shift was stable and associated with increased tumor aggressiveness and resistance to VEGF blockade in serially transplanted mice. Our results support the hypothesis that the highly glycolytic phenotype of tumor cells studied in xenograft models, either primary or secondary, is a cell-autonomous trait conferring resistance to VEGF blockade. The finding that metabolic traits of tumors can be selected by antiangiogenic therapy suggests insights into the evolutionary dynamics of tumor metabolism.

Metabolic reprogramming identifies the most aggressive lesions at early phases of hepatic carcinogenesis.

Metabolic changes are associated with cancer, but whether they are just bystander effects of deregulated oncogenic signaling pathways or characterize early phases of tumorigenesis remains unclear. Here we show in a rat model of hepatocarcinogenesis that early preneoplastic foci and nodules that progress towards hepatocellular carcinoma (HCC) are characterized both by inhibition of oxidative phosphorylation (OXPHOS) and by enhanced glucose utilization to fuel the pentose phosphate pathway (PPP). These changes respectively require increased expression of the mitochondrial chaperone TRAP1 and of the transcription factor NRF2 that induces the expression of the rate-limiting PPP enzyme glucose-6-phosphate dehydrogenase (G6PD), following miR-1 inhibition. Such metabolic rewiring exclusively identifies a subset of aggressive cytokeratin-19 positive preneoplastic hepatocytes and not slowly growing lesions. No such metabolic changes were observed during non-neoplastic liver regeneration occurring after two/third partial hepatectomy. TRAP1 silencing inhibited the colony forming ability of HCC cells while NRF2 silencing decreased G6PD expression and concomitantly increased miR-1; conversely, transfection with miR-1 mimic abolished G6PD expression. Finally, in human HCC patients increased G6PD expression levels correlates with grading, metastasis and poor prognosis. Our results demonstrate that the metabolic deregulation orchestrated by TRAP1 and NRF2 is an early event restricted to the more aggressive preneoplastic lesions.

Inhibition of glucose-6-phosphate dehydrogenase sensitizes cisplatin-resistant cells to death

The mechanisms of cisplatin resistance, one of the major limitations of current chemotherapy, has only partially been described. We previously demonstrated that cisplatin-resistant ovarian cancer cells (C13), are characterized by reduced mitochondrial activity and higher glucose-dependency when compared to the cisplatin-sensitive counterpart (2008). In this work we further characterized the role of metabolic transformation in cisplatin resistance. By using transmitochondrial hybrids we show that metabolic reprogramming of cisplatin-resistant cell is not caused by inherent mtDNA mutations. We also found that C13 cells not only present an increased glucose-uptake and consumption, but also exhibit increased expression and enzymatic activity of the Pentose Phosphate pathway (PPP) enzyme Glucose-6-Phosphate Dehydrogenase (G6PDH). Moreover, we show that cisplatin-resistant cells are more sensitive to G6PDH inhibition. Even if the metabolomic fingerprint of ovarian cancer cells remains to be further elucidated, these findings indicate that PPP offers innovative potential targets to overcome cisplatin resistance.

Abstract A02: OXPHOS inhibition and pentose phosphate pathway induction are early events priming preneoplastic lesions toward HCC development

Introduction: A shift towards a Warburg metabolism in which aerobic glycolysis is increased has long been associated to cancer cell transformation. However, whether the switch from oxidative phosphorylation to glycolysis can occur at early stages of cancer development, particularly in hepatocellular carcinoma (HCC), remains elusive. Materials and Methods: Preneoplastic hepatic lesions and Hepatocellular carcinomas were induced in rats subjected to the Resistant-Hepatocyte (RH) model, consisting of a single dose of dietthylnitrosamine (DENA) and a 2-week feeding a diet supplemented with 2-acetylaminoaminofluorene (2-AAF). In vitro experiments were performed in HCC cells obtained by perfusion of HCC-bearing rats or immortalized rat hepatocytes. Results and discussion: Using the Resistant-Hepatocyte (R-H) model, we show that the acquisition of the Warburg phenotype is a very early event in rat HCC development as demonstrated by concomitant MCT4 expression and oxidation/inhibition of pyruvate kinase M2 (PKM2). In keeping with this, we also observed inhibition of succinate dehydrogenase (SDH) by the chaperone tumor necrosis factor receptor-associated protein 1 (TRAP1) and an increase in the expression and activity of citrate synthase (CS). In these preneoplastic lesions, metabolic reprogramming towards the Pentose Phosphate Pathway (PPP) was indicated by a strong increase in the expression and activity of glucose-6-phosphate dehydrogenase (G6PD). G6PD increased expression was observed exclusively in the highly proliferating KRT-19 positive preneoplastic lesions, considered the HCC precursor lesions in the R-H model, and was associated with low levels of miR-1, a miRNA known to target G6PD. Accordingly, forced expression of miR-1 down-regulated G6PD expression in HCC cells. PPP induction has been suggested to be one of the mechanisms by which deregulated NRF2-KEAP1 signaling promotes cellular proliferation and tumorigenesis. Since in the R-H rat model a sustained activation of the NRF2/KEAP1 pathway occurs in KRT-19+ nodules, we investigated the effect of impairing NRF2 in cells derived from R-H rat HCC. Notably, NRF2 silencing decreases G6PD and increases miR-1 expression, consequently inhibiting PPP and PKM2 oxidation. Finally, an inverse correlation between miR-1 and its target gene G6PD was found in human HCC patients. Conclusion: Our results demonstrate that Warburg metabolic deregulation and PPP induction are early events in HCC development. Crucially, TRAP1 and NRF2 are key regulators of this metabolic reprogramming in preneoplastic hepatocytes Citation Format: Marta A. Kowalik, Giulia Guzzo, Andrea Morandi, Andrea Perra, Silvia Menegon, Maria M. Angioni, Silvia Giordano, Paola Chiarugi, Andrea Rasola, Amedeo Columbano. OXPHOS inhibition and pentose phosphate pathway induction are early events priming preneoplastic lesions toward HCC development. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A02.

Abstract 1009: Metabolic reprogramming discriminates aggressive vs. slowly growing preneoplastic lesions at early stages of HCC development

Introduction and aim: Among the several changes underlying metabolic reprogramming of cancer cells, increased glucose utilization and its uncoupling from oxygen availability is a well-established phenomenon and has been recognized as a hallmark of cancer. To what extent these metabolic changes are important for the progression of slow growing tumors and whether a metabolic rewiring occurs in the very early stages of neoplastic progression represent key questions on the significance of these metabolic alterations in cancer. Here, we compared the metabolic features of preneoplastic hepatic lesions with those of advanced hepatocellular carcinomas (HCCs) and of proliferating liver, following partial hepatectomy (PH). Materials and Methods: Expression levels, activity and modulation of several enzymes with key roles in glycolysis, pentose phosphate pathway (PPP) and oxidative phosphorylation (OXPHOS) were assessed in preneoplastic hepatic lesions and HCC, induced in rats exposed to the Resistant-Hepatocyte (R-H) model. In vitro experiments were performed on HCC cells obtained by perfusion of HCC-bearing rats. Expression of metabolic genes was also investigated in two different cohorts of human patients carrying HCC. Results and discussion: A switch from OXPHOS to PPP was observed in very early preneoplastic lesions generated 10 weeks after the treatment with DENA. Notably, this metabolic reprogramming was observed only in the most aggressive preneoplastic lesions, characterized by positivity for cytokeratin 19 (CK-19+). PPP induction, shown by a strong increase in the expression and activity of glucose 6-phosphate dehydrogenase (G6PD) was supported both by inhibition of pyruvate kinase activity and by TP53-inducible glycolysis and apoptosis regulator (TIGAR) induction. Importantly, such metabolic rewiring was not observed in normal hepatocytes, undergoing proliferation following 2/3 partial hepatectomy (PH). Activation of the NRF2/KEAP1 pathway and down-regulation of miR-1 accompanied the metabolic reprogramming in CK-19+ preneoplastic lesions. Accordingly, NRF2 silencing decreased G6PD and increased miR-1 expression, consequently inhibiting PPP, while forced expression of miR-1 downregulated G6PD expression in HCC cells. Finally, an inverse correlation between miR-1 and its target gene G6PD was found in human HCC patients. Conclusion: These results demonstrate that metabolic reprogramming takes place at early stages of hepatocarcinogenesis and is likely the consequence of the concomitant activation of the NRF2-KEAP1 pathway. Citation Format: Marta A. Kowalik, Giulia Guzzo, Andrea Morandi, Andrea Perra, Silvia Menegon, Ionica Masgras, Elena Trevisan, Maria M. Angioni, Francesca Fornari, Luca Quagliata, Giovanna M. Ledda-Columbano, Laura Gramantieri, Luigi Terracciano, Silvia Giordano, Paola Chiarugi, Andrea Rasola, Amedeo Columbano. Metabolic reprogramming discriminates aggressive vs. slowly growing preneoplastic lesions at early stages of HCC development. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1009.