Background: The extracellular signal-regulated kinase (ERK) pathway regulates cell growth, and is hyper-activated and associated with drug resistance in hepatocellular carcinoma (HCC). Metabolic pathways are profoundly dysregulated in HCC. Whether an altered metabolic state is linked to activated ERK pathway and drug response in HCC is unaddressed. Methods: We deprived HCC cells of glutamine to induce metabolic alterations and performed various assays, including metabolomics (with 13 C-glucose isotope tracing), microarray analysis, and cell proliferation assays. Glutamine-deprived cells were also treated with kinase inhibitors (e.g. Sorafenib, Erlotinib, U0126 amongst other MEK inhibitors). We performed bioinformatics analysis and stratification of HCC tumour microarrays to determine upregulated ERK gene signatures in patients. Findings: In a subset of HCC cells, the withdrawal of glutamine triggers a severe metabolic alteration and ERK phosphorylation (pERK). This is accompanied by resistance to the anti-proliferative effect of kinase inhibitors, despite pERK inhibition. High intracellular serine is a consistent feature of an altered metabolic state and contributes to pERK induction and the kinase inhibitor resistance. Blocking the ERK pathway facilitates cell proliferation by reprogramming metabolism, notably enhancing aerobic glycolysis. We have identified 24 highly expressed ERK gene signatures that their combined expression strongly indicates a dysregulated metabolic gene network in human HCC tissues. Interpretation: A severely compromised metabolism lead to ERK pathway induction, and primes some HCC cells to pro-survival phenotypes upon ERK pathway blockade. Our findings offer novel insights for understanding, predicting and overcoming drug resistance in liver cancer patients.

The p38 MAPK pathway is known to influence the anti-tumor effects of several chemotherapeutics, including that of organometallic drugs. Previous studies have demonstrated the important role of p38 both as a regulator and a sensor of cellular reactive oxygen species (ROS) levels. Investigating the anti-cancer properties of novel 1,8-naphthalimide derivatives containing Rh(I) and Ru(II) N-heterocyclic carbene (NHC) ligands, we observed a profound induction of ROS by the complexes, which is most likely generated from mitochondria (mtROS). Further analyses revealed a rapid and consistent activation of p38 signaling by the naphthalimide-NHC conjugates, with the Ru(II) analogue—termed MC6—showing the strongest effect. In view of this, genetic as well as pharmacological inhibition of p38α, attenuated the anti-proliferative and pro-apoptotic effects of MC6 in HCT116 colon cancer cells, highlighting the involvement of this signaling molecule in the compound’s toxicity. Furthermore, the influence of MC6 on p38 signaling appeared to be dependent on ROS levels as treatment with general- and mitochondria-targeted anti-oxidants abrogated p38 activation in response to MC6 as well as the molecule’s cytotoxic- and apoptogenic response in HCT116 cells. Altogether, our results provide new insight into the molecular mechanisms of naphthalimide-metal NHC analogues via the ROS-induced activation of p38 MAPK, which may have therapeutic interest for the treatment of various cancer types.

BackgroundWe have previously identified 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the bioactive form of vitamin D3, as a potent regulator of energy-utilization and nutrient-sensing pathways in prostate cancer cells. In the current study, we investigated the effects of 1,25(OH)2D3 on breast cancer (BCa) cell metabolism using cell lines representing distinct molecular subtypes, luminal (MCF-7 and T-47D), and triple-negative BCa (MDA-MB-231, MDA-MB-468, and HCC-1143).Methods1,25(OH)2D3’s effect on BCa cell metabolism was evaluated by employing a combination of real-time measurements of glycolysis/oxygen consumption rates using a biosensor chip system, GC/MS-based metabolomics, gene expression analysis, and assessment of overall energy levels. The influence of treatment on energy-related signaling molecules was investigated by immunoblotting.ResultsWe show that 1,25(OH)2D3 significantly induces the expression and activity of the pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase (G6PD) in all BCa cell lines, however differentially influences glycolytic and respiratory rates in the same cells. Although 1,25(OH)2D3 treatment was found to induce seemingly anti-oxidant responses in MCF-7 cells, such as increased intracellular serine levels, and reduce the expression of its putative target gene thioredoxin-interacting protein (TXNIP), intracellular reactive oxygen species levels were found to be elevated. Serine accumulation in 1,25(OH)2D3-treated cells was not found to hamper the efficacy of chemotherapeutics, including 5-fluorouracil. Detailed analyses of the nature of TXNIP’s regulation by 1,25(OH)2D3 included genetic and pharmacological inhibition of signaling molecules and metabolic enzymes including AMP-activated protein kinase and G6PD, as well as by studying the ITCH (E3 ubiquitin ligase)-TXNIP interaction. While these investigations demonstrated minimal involvement of such pathways in the observed non-canonical regulation of TXNIP, inhibition of estrogen receptor (ER) signaling by tamoxifen mirrored the reduction of TXNIP levels by 1,25(OH)2D3, demonstrating that the latter’s negative regulation of ER expression is a potential mechanism of TXNIP modulation.ConclusionsAltogether, we propose that regulation of energy metabolism contributes to 1,25(OH)2D3’s anti-cancer effects and that combining 1,25(OH)2D3 with drugs targeting metabolic networks in tumor cells may lead to synergistic effects.Electronic supplementary materialThe online version of this article (10.1186/s40170-018-0183-6) contains supplementary material, which is available to authorized users.

Background Aerobic glycolysis, discovered by Otto Warburg, is a hallmark of cancer metabolism even though not yet fully understood. The low activity of the cancerous pyruvate kinase isozyme (M2) is thought to play an important role by facilitating the conversion of glycolytic intermediates to other anabolic pathways to support tumors’ high proliferation rate. Methods Five breast cancer cell lines representing different molecular subtypes were used in this study where real time measurements of cellular bioenergetics and immunoblotting analysis of energy- and nutrient-sensing pathways were employed to investigate the potential effects of PKM2 allosteric activator (DASA-58) in glucose rewiring. Results In this study, we show that DASA-58 can induce pyruvate kinase activity in breast cancer cells without affecting the overall cell survival. The drug is also able to reduce TXNIP levels (an intracellular glucose sensor) probably through depletion of upstream glycolytic metabolites and independent of AMPK and ER signaling. AMPK shows an induction in phosphorylation (T172) in response to treatment an effect that can be potentiated by combining DASA-58 with other metabolic inhibitors. Conclusions Altogether, the multifaceted metabolic reprogramming induced by DASA-58 in breast cancer cells increases their susceptibility to other therapeutics suggesting the suitability of the intracellular glucose sensor TXNIP as a marker of PK activity.

Transforming growth factor β (TGF-β) signaling plays a fundamental role in metazoan development and tissue homeostasis. However, the molecular mechanisms concerning the ubiquitin-related dynamic regulation of TGF-β signaling are not thoroughly understood. Using a combination of proteomics and an siRNA screen, we identify pVHL as an E3 ligase for SMAD3 ubiquitination. We show that pVHL directly interacts with conserved lysine and proline residues in the MH2 domain of SMAD3, triggering degradation. As a result, the level of pVHL expression negatively correlates with the expression and activity of SMAD3 in cells, Drosophila wing, and patient tissues. In Drosophila, loss of pVHL leads to the up-regulation of TGF-β targets visible in a downward wing blade phenotype, which is rescued by inhibition of SMAD activity. Drosophila pVHL expression exhibited ectopic veinlets and reduced wing growth in a similar manner as upon loss of TGF-β/SMAD signaling. Thus, our study demonstrates a conserved role of pVHL in the regulation of TGF-β/SMAD3 signaling in human cells and Drosophila wing development.