Liver cancer cell lines distinctly mimic the metabolic gene expression pattern of the corresponding human tumours

Background: Although metabolism is profoundly altered in human liver cancer, the extent to which experimental models, e.g. cell lines, mimic those alterations is unresolved. Here, we aimed to determine the resemblance of hepatocellular carcinoma (HCC) cell lines to human liver tumours, specifically in the expression of deregulated metabolic targets in clinical tissue samples. Methods: We compared the overall gene expression profile of poorly-differentiated (HLE, HLF, SNU-449) to well-differentiated (HUH7, HEPG2, HEP3B) HCC cell lines in three publicly available microarray datasets. Three thousand and eighty-five differentially expressed genes in ≥2 datasets (P < 0.05) were used for pathway enrichment and gene ontology (GO) analyses. Further, we compared the topmost gene expression, pathways, and GO from poorly differentiated cell lines to the pattern from four human HCC datasets (623 tumour tissues). In well- versus poorly differentiated cell lines, and in representative models HLE and HUH7 cells, we specifically assessed the expression pattern of 634 consistently deregulated metabolic genes in human HCC. These data were complemented by quantitative PCR, proteomics, metabolomics and assessment of response to thirteen metabolism-targeting compounds in HLE versus HUH7 cells. Results: We found that poorly-differentiated HCC cells display upregulated MAPK/RAS/NFkB signaling, focal adhesion, and downregulated complement/coagulation cascade, PPAR-signaling, among pathway alterations seen in clinical tumour datasets. In HLE cells, 148 downregulated metabolic genes in liver tumours also showed low gene/protein expression – notably in fatty acid β-oxidation (e.g. ACAA1/2, ACADSB, HADH), urea cycle (e.g. CPS1, ARG1, ASL), molecule transport (e.g. SLC2A2, SLC7A1, SLC25A15/20), and amino acid metabolism (e.g. PHGDH, PSAT1, GOT1, GLUD1). In contrast, HUH7 cells showed a higher expression of 98 metabolic targets upregulated in tumours (e.g. HK2, PKM, PSPH, GLUL, ASNS, and fatty acid synthesis enzymes ACLY, FASN). Metabolomics revealed that the genomic portrait of HLE cells co-exist with profound reliance on glutamine to fuel tricarboxylic acid cycle, whereas HUH7 cells use both glucose and glutamine. Targeting glutamine pathway selectively suppressed the proliferation of HLE cells. Conclusions: We report a yet unappreciated distinct expression pattern of clinically-relevant metabolic genes in HCC cell lines, which could enable the identification and therapeutic targeting of metabolic vulnerabilities at various liver cancer stages. - - - - - - - - - CORRECTION Published online 2.11.2018 in Journal of Experimental & Clinical Cancer Research, 37 (2018), Nr. 267; DOI: https://doi.org/10.1186/s13046-018-0939-4. In the publication of this article, there was an error in Fig. 5b. This has been updated in the original article on BioMed Central's website. The authors declare that the correction does not change the results or conclusions of this paper

Correction to: Liver cancer cell lines distinctly mimic the metabolic gene expression pattern of the corresponding human tumours

Table S5. Complete list of downregulated HMGs concordantly low in the p.d. cells (part of Fig. 2c). (DOCX 14 kb

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

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

PRRX1 expression and functions in hepatocellular carcinoma

Background & Aims: Hepatocellular carcinoma (HCC) is the most frequently diagnosed liver cancer and a leading cause of cancer-related death. Paired related homeobox 1 (PRRX1) is a transcriptional co-activator, which regulates cell growth, differentiation and, as I could recently show, is also linked to epithelial to mesenchymal transition (EMT). EMT is a hallmark of cancer progression, paving the way to tumor cell spreading into surrounding tissue, vessels and other organs. The transforming growth factor-β (TGF-β) is a known EMT inducer that also plays important roles in HCC. Whether PRRX1 has a functional relevance in HCC is still unknown. Thus, the aim of my work was to perform an in-depth analysis of PRRX1 expression and its co-expressed genes in HCC samples, as well as to clarify its function in liver carcinogenesis. Methods: The expression of PRRX1 in human HCC was assessed in online databases, including Oncomine, cBioPortal, and in microarray datasets encompassing > 1,400 liver tumor profiles. Bioinformatics analyses were performed for functional annotation of genes correlated with PRRX1 in HCC, and followed by Kaplan-Meier overall survival analyses. In vitro, PRRX1 expression was analyzed in HCC cell lines, treated or not with TGF-β, its receptor inhibitor Galunisertib. Further, PRRX1 expression was inhibited with small interfering RNA and the functional impact on cell migration, proliferation, clonogenicity, apoptosis and metabolism were measured. Result: PRRX1 is frequently upregulated in human HCC. Patients with high PRRX1 show elevated expression of TGFBR1. The genes positively correlated with PRRX1 display enrichment in crucial features of cancer, including stroma remodeling, signal transduction, downregulated metabolic pathways, focal adhesion and EMT. I could identify two EMT-related transcription factors, ZEB1 and ZEB2, as novel PRRX1-related genes in HCC. PRRX1 in combination with ZEBs significantly predicted survival outcome in HCC patient cohorts investigated. In HCC cell lines, TGF-β1 treatment increases expression of PRRX1. PRRX1 influences cell migration and modulates expression of EMT markers in a cell type dependent-manner. PRRX1 knockdown increases cell proliferation and clonogenicity. Further, knock down of PRRX1 promotes glucose consumption (Warburg effect), regulates core metabolic genes and levels of TCA cycle metabolites and amino acids. Moreover, depleting PRRX1 causes upregulation of genes regulating fatty acid biosynthesis and oxidation with influence on fatty acid levels. Conclusion: The findings of my study provide evidence for a functional role of PRRX1 in HCC, including a master control on pathways that facilitate tumor progression, including modulation of cell metabolism. The new findings on PRRX1 functions suggest further in-depth mechanistic studies on the relevance of PRRX1 in human liver cancer

Dysregulated paired related homeobox 1 impacts on hepatocellular carcinoma phenotypes

Abstract Background Hepatocellular carcinoma (HCC) is a major cause of cancer-related death. Paired related homeobox 1 (PRRX1) is a transcription factor that regulates cell growth and differentiation, but its importance in HCC is unclear. Methods We examined the expression pattern of PRRX1 in nine microarray datasets of human HCC tumour samples (n > 1100) and analyzed its function in HCC cell lines. In addition, we performed gene set enrichment, Kaplan-Meier overall survival analysis, metabolomics and functional assays. Results PRRX1 is frequently upregulated in human HCC. Pathway enrichment analysis predicted a direct correlation between PRRX1 and focal adhesion and epithelial-mesenchymal transition. High expression of PRRX1 and low ZEB1 or high ZEB2 significantly predicted better overall survival in HCC patients. In contrast, metabolic processes correlated inversely and transcriptional analyses revealed that glycolysis, TCA cycle and amino acid metabolism were affected. These findings were confirmed by metabolomics analysis. At the phenotypic level, PRRX1 knockdown accelerated proliferation and clonogenicity in HCC cell lines. Conclusions Our results suggest that PRRX1 controls metabolism, has a tumour suppressive role, and may function in cooperation with ZEB1/2. These findings have functional relevance in HCC, including in understanding transcriptional control of distinct cancer hallmarks.http://deepblue.lib.umich.edu/bitstream/2027.42/173541/1/12885_2021_Article_8637.pd

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

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