Genetic Perturbation of Pyruvate Dehydrogenase Kinase 1 Modulates Growth, Angiogenesis and Metabolic Pathways in Ovarian Cancer Xenografts.

Pyruvate dehydrogenase kinase 1 (PDK1) blockade triggers are well characterized in vitro metabolic alterations in cancer cells, including reduced glycolysis and increased glucose oxidation. Here, by gene expression profiling and digital pathology-mediated quantification of in situ markers in tumors, we investigated effects of PDK1 silencing on growth, angiogenesis and metabolic features of tumor xenografts formed by highly glycolytic OC316 and OVCAR3 ovarian cancer cells. Notably, at variance with the moderate antiproliferative effects observed in vitro, we found a dramatic negative impact of PDK1 silencing on tumor growth. These findings were associated with reduced angiogenesis and increased necrosis in the OC316 and OVCAR3 tumor models, respectively. Analysis of viable tumor areas uncovered increased proliferation as well as increased apoptosis in PDK1-silenced OVCAR3 tumors. Moreover, RNA profiling disclosed increased glucose catabolic pathways-comprising both oxidative phosphorylation and glycolysis-in PDK1-silenced OVCAR3 tumors, in line with the high mitotic activity detected in the viable rim of these tumors. Altogether, our findings add new evidence in support of a link between tumor metabolism and angiogenesis and remark on the importance of investigating net effects of modulations of metabolic pathways in the context of the tumor microenvironment.

In situ Metabolic Profiling of Ovarian Cancer Tumor Xenografts: A Digital Pathology Approach.

Metabolic profiling of cancer is a rising interest in the field of biomarker development. One bottleneck of its clinical exploitation, however, is the lack of simple and quantitative techniques that enable to capture the key metabolic traits of tumor from archival samples. In fact, liquid chromatography associated with mass spectrometry is the gold-standard technique for the study of tumor metabolism because it has high levels of accuracy and precision. However, it requires freshly frozen samples, which are difficult to collect in large multi-centric clinical studies. For this reason, we propose here to investigate a set of established metabolism-associated protein markers by exploiting immunohistochemistry coupled with digital pathology. As case study, we quantified expression of MCT1, MCT4, GLS, PHGDH, FAS, and ACC in 17 patient-derived ovarian cancer xenografts and correlated it with survival. Among these markers, the glycolysis-associated marker MCT4 was negatively associated with survival of mice. The algorithm enabling a quantitative analysis of these metabolism-associated markers is an innovative research tool that can be exported to large sets of clinical samples and can remove the variability of individual interpretation of immunohistochemistry results.

Phosphorylated Acetyl-CoA Carboxylase Is Associated with Clinical Benefit with Regorafenib in Relapsed Glioblastoma: REGOMA Trial Biomarker Analysis.

PURPOSE: Preclinical studies show that antiangiogenic therapy exacerbates tumor glycolysis and activates liver kinase B1/AMP kinase (AMPK), a pathway involved in the regulation of tumor metabolism. We investigated whether certain metabolism-related in situ biomarkers could predict benefit to regorafenib in the phase II randomized REGOMA trial. PATIENTS AND METHODS: IHC and digital pathology analysis were used to investigate the expression in glioblastoma (GBM) sections of monocarboxylate transporter 1 and 4 (MCT1 and MCT4), associated with OXPHOS and glycolysis, respectively, phosphorylated AMPK (pAMPK), and phosphorylated acetyl-CoA carboxylase (pACC), a canonical target of AMPK activity. The status of each biomarker was associated with clinical endpoints, including overall survival (OS) and progression-free survival (PFS) in patients with relapsed GBM treated either with regorafenib or lomustine. RESULTS: Between November 2015 and February 2017, 119 patients were enrolled (n = 59 regorafenib and n = 60 lomustine) and stratified for surgery at recurrence, and baseline characteristics were balanced. Biomarker analysis was performed in 84 patients (71%), including 42 patients of the regorafenib arm and 42 patients of the lomustine arm. Among all markers analyzed, only pACC showed predictive value in terms of OS. In fact, median OS was 9.3 months [95% confidence interval (CI), 5.6-13.2] for regorafenib and 5.5 months (95% CI, 4.2-6.6) for lomustine for pACC-positive patients, HR, 0.37 (95% CI, 0.20-0.70); log rank P = 0.0013; test for interaction = 0.0453. No statistically significant difference was demonstrated for PFS according to pACC status. CONCLUSIONS: We found that AMPK pathway activation is associated with clinical benefit from treatment with regorafenib in relapsed GBM.

Early assessment of KRAS mutation in cfDNA correlates with risk of progression and death in advanced non-small-cell lung cancer.

BACKGROUND: Liquid biopsy has the potential to monitor biological effects of treatment. KRAS represents the most commonly mutated oncogene in Caucasian non-small-cell lung cancer (NSCLC). The aim of this study was to explore association of dynamic plasma KRAS genotyping with outcome in advanced NSCLC patients. METHODS: Advanced NSCLC patients were prospectively enrolled. Plasma samples were collected at baseline (T1), after 3 or 4 weeks, according to treatment schedule (T2) and at first radiological restaging (T3). Patients carrying KRAS mutation in tissue were analysed in plasma with droplet digital PCR. Semi-quantitative index of fractional abundance of mutated allele (MAFA) was used. RESULTS: KRAS-mutated cohort included 58 patients, and overall 73 treatments (N = 39 chemotherapy and N = 34 immune checkpoint inhibitors) were followed with longitudinal liquid biopsy. Sensitivity of KRAS detection in plasma at baseline was 48.3% (95% confidence interval (CI): 35.0-61.8). KRAS mutation at T2 was associated with increased probability of experiencing progressive disease as best radiological response (adjusted odds ratio: 7.3; 95% CI: 2.1-25.0, p = 0.0016). Increased MAFA (T1-T2) predicted shorter progression-free survival (adjusted hazard ratio (HR): 2.1; 95% CI: 1.2-3.8, p = 0.0142) and overall survival (adjusted HR: 3.2; 95% CI: 1.2-8.4, p = 0.0168). CONCLUSIONS: Longitudinal analysis of plasma KRAS mutations correlated with outcome: its early assessment during treatment has great potentialities for monitoring treatment outcome in NSCLC patients.

Rewiring of Lipid Metabolism and Storage in Ovarian Cancer Cells after Anti-VEGF Therapy.

Anti-angiogenic therapy triggers metabolic alterations in experimental and human tumors, the best characterized being exacerbated glycolysis and lactate production. By using both Liquid Chromatography-Mass Spectrometry (LC-MS) and Nuclear Magnetic Resonance (NMR) analysis, we found that treatment of ovarian cancer xenografts with the anti-Vascular Endothelial Growth Factor (VEGF) neutralizing antibody bevacizumab caused marked alterations of the tumor lipidomic profile, including increased levels of triacylglycerols and reduced saturation of lipid chains. Moreover, transcriptome analysis uncovered up-regulation of pathways involved in lipid metabolism. These alterations were accompanied by increased accumulation of lipid droplets in tumors. This phenomenon was reproduced under hypoxic conditions in vitro, where it mainly depended from uptake of exogenous lipids and was counteracted by treatment with the Liver X Receptor (LXR)-agonist GW3965, which inhibited cancer cell viability selectively under reduced serum conditions. This multi-level analysis indicates alterations of lipid metabolism following anti-VEGF therapy in ovarian cancer xenografts and suggests that LXR-agonists might empower anti-tumor effects of bevacizumab.

Overcoming platinum-acquired resistance in ovarian cancer patient-derived xenografts.

BACKGROUND: Epithelial ovarian cancer is the most lethal gynecological cancer and the high mortality is due to the frequent presentation at advanced stage, and to primary or acquired resistance to platinum-based therapy. METHODS: We developed three new models of ovarian cancer patient-derived xenografts (ovarian PDXs) resistant to cisplatin (cDDP) after multiple in vivo drug treatments. By different and complementary approaches based on integrated metabolomics (both targeted and untargeted mass spectrometry-based techniques), gene expression, and functional assays (Seahorse technology) we analyzed and compared the tumor metabolic profile in each sensitive and their corresponding cDDP-resistant PDXs. RESULTS: We found that cDDP-sensitive and -resistant PDXs have a different metabolic asset. In particular, we found, through metabolomic and gene expression approaches, that glycolysis, tricarboxylic acid cycle and urea cycle pathways were deregulated in resistant versus sensitive PDXs. In addition, we observed that oxygen consumption rate and mitochondrial respiration were higher in resistant PDXs than in sensitive PDXs under acute stress conditions. An increased oxidative phosphorylation in cDDP-resistant sublines led us to hypothesize that its interference could be of therapeutic value. Indeed, in vivo treatment of metformin and cDDP was able to partially reverse platinum resistance. CONCLUSIONS: Our data strongly reinforce the idea that the development of acquired cDDP resistance in ovarian cancer can bring about a rewiring of tumor metabolism, and that this might be exploited therapeutically.