Severe metabolic alterations in liver cancer lead to ERK pathway activation and drug resistance

in EBioMedicine (2020)

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 ... [more ▼]

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 13C-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. [less ▲]

ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells

in Molecular Cancer (2017), 8(june),

Background: Most melanoma patients with BRAFV600E positive tumors respond well to a combination of BRAF kinase and MEK inhibitors. However, some patients are intrinsically resistant while the majority of ... [more ▼]

Background: Most melanoma patients with BRAFV600E positive tumors respond well to a combination of BRAF kinase and MEK inhibitors. However, some patients are intrinsically resistant while the majority of patients eventually develop drug resistance to the treatment. For patients insufficiently responding to BRAF and MEK inhibitors, there is an ongoing need for new treatment targets. Cellular metabolism is such a promising new target line: mutant BRAFV600E has been shown to affect the metabolism. Methods: Time course experiments and a series of western blots were performed in a panel of BRAFV600E and BRAFWT/ NRASmut human melanoma cells, which were incubated with BRAF and MEK1 kinase inhibitors. siRNA approaches were used to investigate the metabolic players involved. Reactive oxygen species (ROS) were measured by confocal microscopy and AZD7545, an inhibitor targeting PDKs (pyruvate dehydrogenase kinase) was tested. Results: We show that inhibition of the RAS/RAF/MEK/ERK pathway induces phosphorylation of the pyruvate dehydrogenase PDH-E1α subunit in BRAFV600E and in BRAFWT/NRASmut harboring cells. Inhibition of BRAF, MEK1 and siRNA knock-down of ERK1/2 mediated phosphorylation of PDH. siRNA-mediated knock-down of all PDKs or the use of DCA (a pan-PDK inhibitor) abolished PDH-E1α phosphorylation. BRAF inhibitor treatment also induced the upregulation of ROS, concomitantly with the induction of PDH phosphorylation. Suppression of ROS by MitoQ suppressed PDH-E1α phosphorylation, strongly suggesting that ROS mediate the activation of PDKs. Interestingly, the inhibition of PDK1 with AZD7545 specifically suppressed growth of BRAF-mutant and BRAF inhibitor resistant melanoma cells. Conclusions: In BRAFV600E and BRAFWT/NRASmut melanoma cells, the increased production of ROS upon inhibition of the RAS/RAF/MEK/ERK pathway, is responsible for activating PDKs, which in turn phosphorylate and inactivate PDH. As part of a possible salvage pathway, the tricarboxylic acid cycle is inhibited leading to reduced oxidative metabolism and reduced ROS levels. We show that inhibition of PDKs by AZD7545 leads to growth suppression of BRAF-mutated and -inhibitor resistant melanoma cells. Thus small molecule PDK inhibitors such as AZD7545, might be promising drugs for combination treatment in melanoma patients with activating RAS/RAF/MEK/ERK pathway mutations (50% BRAF, 25% NRASmut, 11.9% NF1mut). [less ▲]

The role of HIF-1 in oncostatin M-dependent metabolic reprogramming of hepatic cells.

in Cancer and Metabolism (2016), 4

BACKGROUND: Hypoxia and inflammation have been identified as hallmarks of cancer. A majority of hepatocellular carcinomas are preceded by hepatitis B- or C-related chronic infections suggesting that liver ... [more ▼]

BACKGROUND: Hypoxia and inflammation have been identified as hallmarks of cancer. A majority of hepatocellular carcinomas are preceded by hepatitis B- or C-related chronic infections suggesting that liver cancer development is promoted by an inflammatory microenvironment. The inflammatory cytokine oncostatin M (OSM) was shown to induce the expression of hypoxia-inducible factor-1 alpha (HIF-1 alpha) under normoxic conditions in hepatocytes and hepatoma cells. HIF-1 alpha is known to orchestrate the expression of numerous genes, many of which code for metabolic enzymes that play key roles in the adaptation of cellular metabolism to low oxygen tension. RESULTS: Here, we show that OSM-induced upregulation of HIF-1 alpha reprograms cellular metabolism in three clones of the human hepatocyte cell line PH5CH (PH5CH1, PH5CH7, and PH5CH8) towards a hypoxia-like metabolic phenotype but has no significant effect on cellular metabolism of HepG2 and JHH-4 hepatoma cells. Although we observed only minor changes in glucose uptake and lactate secretion in PH5CH8 upon OSM treatment, we identified more pronounced changes in intracellular fluxes based on stable isotope labeling experiments. In particular, glucose oxidation in the tricarboxylic acid (TCA) cycle is reduced through pyruvate dehydrogenase kinase 1 (PDK1)-mediated inhibition of the pyruvate dehydrogenase complex, thereby reducing the oxidative TCA cycle flux. As a result of the impaired mitochondrial glucose and glutamine oxidation, the reductive isocitrate dehydrogenase flux was increased. CONCLUSIONS: We provide evidence that connects the inflammatory mediator OSM to a hypoxia-like metabolic phenotype. In the human hepatocyte cell line PH5CH, OSM-mediated upregulation of HIF-1 alpha and PDK1 can induce hypoxia-like metabolic changes, although to a lesser extent than hypoxia itself. Since PDK1 is overexpressed in several cancers, it might provide a causal link between chronic inflammation and malignant cellular transformation. [less ▲]

DIFFERENTIAL EFFECTS OF IL-6-TYPE CYTOKINES, THEIR INTERPLAY WITH HYPOXIA AND RESULTING EFFECTS ON THE METABOLISM

Doctoral thesis (2015)

IL-6-type cytokines signal mainly via the Jak/STAT pathway using the common receptor chain gp130. They are implicated in various biological processes such as differentiation, apoptosis, tissue ... [more ▼]

IL-6-type cytokines signal mainly via the Jak/STAT pathway using the common receptor chain gp130. They are implicated in various biological processes such as differentiation, apoptosis, tissue regeneration and proliferation. However, one of their main functions is the regulation of inflammatory processes, especially the initial induction of an inflammatory response. They hereby exert key steps in the onset as well as in the termination of an inflammation and in the promotion of the shift from the innate to the adaptive immune response. In hepatocellular carcinoma (HCC) IL-6 has been identified as a key factor in the onset as well as in the progression of this cancer. In this study we first investigated the effects of the IL-6-type cytokine IL-27 on hepatic cells. By evaluating IL-27 signalling in several HCC cell lines as well as hepatic cells, we could demonstrate that IL-27 does, in contrary to other IL-6-type cytokines, activate STAT1 and STAT3. A transcriptional and protein read-out of STAT3 was not found in hepatic cells. Thus, IL-27 exerts interferon γ-like functions only in the liver. Interestingly, we could further show that the IL-27 signalling can still be repressed by the canonical IL-6-type cytokine feedback regulator SOCS3, following a pre-stimulation with other IL-6-type cytokines. The second part of this thesis focused on the effects of the IL-6-type cytokine Oncostatin M (OSM) on the cellular metabolism. We hereby evaluated to which extent OSM-mediated STAT3 activation can induce a Warburg-like, highly glycolytic, metabolic phenotype under normoxia. OSM induced the expression of HIF-1α, a key factor mediating the shift to a highly glycolytic phenotype, in several HCC cell lines and immortalized hepatocytes. In the non-neoplastic hepatocyte cell line, PH5CH8, this led to the induction of a more glycolytic metabolic phenotype. The observed changes in these immortalized hepatocytes did not fully resemble the metabolic state seen under hypoxia, since mainly the observed induction of PDK1 seems to explain the effect. Overall we could not observe strong effects on glycolytic enzymes or other canonical HIF-1α target genes involved in metabolism, on a transcriptional or protein expression level in the investigated cells. Interestingly we found that HIF-1α does not seem to be mediating all the early adaptions of the cellular metabolism to hypoxia. The early inhibition of the pyruvate dehydrogenase complex could be shown to be independent of the HIF-1α-mediated up-regulation of the pyruvate dehydrogenase kinase 1, but might rather be induced by reactive oxygen species, created during the switch from normoxia to hypoxia. In summary we could show that in the liver IL-27 exerts interferon γ-like effects. Additionally, cytokine-activated STAT3 does not per se induce a glycolytic metabolic phenotype and HIF-1α is not necessary for some of the early adaptions of the cellular metabolism to hypoxia. [less ▲]

Sequence interpretation. Functional annotation of mouse genome sequences.

in Science (2001), 291(5507), 1251-5