Elevated arginine levels in liver tumors promote metabolic reprogramming and tumor growth

Arginine auxotropy, due to reduced expression of urea cycle genes, is common in cancer. However, little is known about the levels of arginine in these cancers. Here, we report that arginine levels are elevated in hepatocellular carcinoma (HCC) despite reduced expression of urea cycle enzymes. Liver tumors accumulate high levels specifically of arginine via increased uptake and, more importantly, via suppression of arginine-to-polyamine conversion due to reduced arginase 1 (ARG1) and agmatinase (AGMAT) expression. Furthermore, the high levels of arginine are required for tumor growth. Mechanistically, high levels of arginine promote tumorigenesis via transcriptional regulation of metabolic genes, including upregulation of asparagine synthetase (ASNS). ASNS-derived asparagine further enhances arginine uptake, creating a positive feedback loop to sustain high arginine levels and oncogenic metabolism. Thus, arginine is a novel second messenger-like molecule that reprograms metabolism to promote tumor growth

USP29-mediated HIF1α stabilization is associated with Sorafenib resistance of hepatocellular carcinoma cells by upregulating glycolysis

Understanding the mechanisms underlying evasive resistance in cancer is an unmet medical need to improve the efficacy of current therapies. In hepatocellular carcinoma (HCC), aberrant expression of hypoxia-inducible factor 1 α (HIF1α) and increased aerobic glycolysis metabolism are drivers of resistance to therapy with the multi-kinase inhibitor Sorafenib. However, it has remained unknown how HIF1α is activated and how its activity and the subsequent induction of aerobic glycolysis promote Sorafenib resistance in HCC. Here, we report the ubiquitin-specific peptidase USP29 as a new regulator of HIF1α and of aerobic glycolysis during the development of Sorafenib resistance in HCC. In particular, we identified USP29 as a critical deubiquitylase (DUB) of HIF1α, which directly deubiquitylates and stabilizes HIF1α and, thus, promotes its transcriptional activity. Among the transcriptional targets of HIF1α is the gene encoding hexokinase 2 (HK2), a key enzyme of the glycolytic pathway. The absence of USP29, and thus of HIF1α transcriptional activity, reduces the levels of aerobic glycolysis and restores sensitivity to Sorafenib in Sorafenib-resistant HCC cells in vitro and in xenograft transplantation mouse models in vivo. Notably, the absence of USP29 and high HK2 expression levels correlate with the response of HCC patients to Sorafenib therapy. Together, the data demonstrate that, as a DUB of HIF1α, USP29 promotes Sorafenib resistance in HCC cells, in parts by upregulating glycolysis, thereby opening new avenues for therapeutically targeting Sorafenib-resistant HCC in patients

Live slow-frozen human tumor tissues viable for 2D, 3D, ex vivo cultures and single-cell RNAseq

Biobanking of surplus human healthy and disease-derived tissues is essential for diagnostics and translational research. An enormous amount of formalin-fixed and paraffin-embedded (FFPE), Tissue-Tek OCT embedded or snap-frozen tissues are preserved in many biobanks worldwide and have been the basis of translational studies. However, their usage is limited to assays that do not require viable cells. The access to intact and viable human material is a prerequisite for translational validation of basic research, for novel therapeutic target discovery, and functional testing. Here we show that surplus tissues from multiple solid human cancers directly slow-frozen after resection can subsequently be used for different types of methods including the establishment of 2D, 3D, and ex vivo cultures as well as single-cell RNA sequencing with similar results when compared to freshly analyzed material

Karşıyaka Prevalance and Awareness of Hypertension Study (KARHIP)

Background: The study was planned to assess potential differences in hypertension prevalance and hypertension related demographic properties in an urban area with relatively higher income and cultural population compared to the national average. Methods: Fieldwork was done by educated and dedicated personnel at Karsiyaka Municipality Building by one by one interviewing poll, blood pressure measurement, rhythm and body composition analysis in February 2014. Hypertension was defined as an average systolic blood pressure ≥140 mmHg or an average diastolic blood pressure ≥90 mmHg. Results: Out of a total of 1417 (627 males and 790 females) people enrolled, 780 people were hypertensive (prevalence 55%). Hypertension prevalence in the middle age group (age 35-65) was 46% and in the geriatric age group (age>65) it was %79. 216 out of 780 hypertensive (27.7%) people were not aware of their disease. The proportion of people taking antihypertensive treatment was 69.4% and the proportion under control was 34.7%, whereas the control rate was 50.1% in 541 patients who were aware of their diseases. Conclusions: Hypertension prevalances in our study were similar to the PatenT 2 trial prevalances, which were 46 % for the middle age group and 78% for the geriatric age group. Compared to PatenT 2 data, the rate of hypertension awareness (54.7% vs 72.3%) and the rate of being under treatment (47.5% vs 69.4%) were higher. The rate of controlled hypertension was a little bit higher (28.7 % vs 34.7% ) in our group, whereas control rates in aware and treated groups were similar (53.9 % and 50.1 %) in both studies

Proteogenomic characterization of hepatocellular carcinoma

We performed a proteogenomic analysis of hepatocellular carcinomas (HCCs) across clinical stages and etiologies. We identified pathways differentially regulated on the genomic, transcriptomic, proteomic and phosphoproteomic levels. These pathways are involved in the organization of cellular components, cell cycle control, signaling pathways, transcriptional and translational control and metabolism. Analyses of CNA-mRNA and mRNA-protein correlations identified candidate driver genes involved in epithelial-to-mesenchymal transition, the Wnt-β- catenin pathway, transcriptional control, cholesterol biosynthesis and sphingolipid metabolism. The activity of targetable kinases aurora kinase A and CDKs was upregulated. We found that CTNNB1 mutations are associated with altered phosphorylation of proteins involved in actin filament organization, whereas TP53 mutations are associated with elevated CDK1/2/5 activity and altered phosphorylation of proteins involved in lipid and mRNA metabolism. Integrative clustering identified HCC subgroups with distinct regulation of biological processes, metabolic reprogramming and kinase activation. Our analysis provides insights into the molecular processes underlying HCCs

Standardizing Patient-Derived Organoid Generation Workflow to Avoid Microbial Contamination From Colorectal Cancer Tissues.

The use of patient-derived organoids (PDO) as a valuable alternative to in vivo models significantly increased over the last years in cancer research. The ability of PDOs to genetically resemble tumor heterogeneity makes them a powerful tool for personalized drug screening. Despite the extensive optimization of protocols for the generation of PDOs from colorectal tissue, there is still a lack of standardization of tissue handling prior to processing, leading to microbial contamination of the organoid culture. Here, using a cohort of 16 patients diagnosed with colorectal carcinoma (CRC), we aimed to test the efficacy of phosphate-buffered saline (PBS), penicillin/streptomycin (P/S), and Primocin, alone or in combination, in preventing organoid cultures contamination when used in washing steps prior to tissue processing. Each CRC tissue was divided into 5 tissue pieces, and treated with each different washing solution, or none. After the washing steps, all samples were processed for organoid generation following the same standard protocol. We detected contamination in 62.5% of the non-washed samples, while the use of PBS or P/S-containing PBS reduced the contamination rate to 50% and 25%, respectively. Notably, none of the organoid cultures washed with PBS/Primocin-containing solution were contaminated. Interestingly, addition of P/S to the washing solution reduced the percentage of living cells compared to Primocin. Taken together, our results demonstrate that, prior to tissue processing, adding Primocin to the tissue washing solution is able to eliminate the risk of microbial contamination in PDO cultures, and that the use of P/S negatively impacts organoids growth. We believe that our easy-to-apply protocol might help increase the success rate of organoid generation from CRC patients

Integrative proteogenomic characterization of hepatocellular carcinoma across etiologies and stages.

Proteogenomic analyses of hepatocellular carcinomas (HCC) have focused on early-stage, HBV-associated HCCs. Here we present an integrated proteogenomic analysis of HCCs across clinical stages and etiologies. Pathways related to cell cycle, transcriptional and translational control, signaling transduction, and metabolism are dysregulated and differentially regulated on the genomic, transcriptomic, proteomic and phosphoproteomic levels. We describe candidate copy number-driven driver genes involved in epithelial-to-mesenchymal transition, the Wnt-β-catenin, AKT/mTOR and Notch pathways, cell cycle and DNA damage regulation. The targetable aurora kinase A and CDKs are upregulated. CTNNB1 and TP53 mutations are associated with altered protein phosphorylation related to actin filament organization and lipid metabolism, respectively. Integrative proteogenomic clusters show that HCC constitutes heterogeneous subgroups with distinct regulation of biological processes, metabolic reprogramming and kinase activation. Our study provides a comprehensive overview of the proteomic and phophoproteomic landscapes of HCCs, revealing the major pathways altered in the (phospho)proteome

Transcriptional Enhancer Factor Domain Family member 4 Exerts an Oncogenic Role in Hepatocellular Carcinoma by Hippo-Independent Regulation of Heat Shock Protein 70 Family Members.

Transcriptional enhancer factor domain family member 4 (TEAD4) is a downstream effector of the conserved Hippo signaling pathway, regulating the expression of genes involved in cell proliferation and differentiation. It is up-regulated in several cancer types and is associated with metastasis and poor prognosis. However, its role in hepatocellular carcinoma (HCC) remains largely unexplored. Using data from The Cancer Genome Atlas, we found that TEAD4 was overexpressed in HCC and was associated with aggressive HCC features and worse outcome. Overexpression of TEAD4 significantly increased proliferation and migration rates in HCC cells in vitro as well as tumor growth in vivo. Additionally, RNA sequencing analysis of TEAD4-overexpressing HCC cells demonstrated that TEAD4 overexpression was associated with the up-regulation of genes involved in epithelial-to-mesenchymal transition, proliferation, and protein-folding pathways. Among the most up-regulated genes following TEAD4 overexpression were the 70-kDa heat shock protein (HSP70) family members HSPA6 and HSPA1A. Chromatin immunoprecipitation-quantitative real-time polymerase chain reaction experiments demonstrated that TEAD4 regulates HSPA6 and HSPA1A expression by directly binding to their promoter and enhancer regions. The pharmacologic inhibition of HSP70 expression in TEAD4-overexpressing cells reduced the effect of TEAD4 on cell proliferation. Finally, by overexpressing TEAD4 in yes-associated protein (YAP)/transcriptional coactivator with PDZ binding motif (TAZ)-knockdown HCC cells, we showed that the effect of TEAD4 on cell proliferation and its regulation of HSP70 expression does not require YAP and TAZ, the main effectors of the Hippo signaling pathway. Conclusion: A novel Hippo-independent mechanism for TEAD4 promotes cell proliferation and tumor growth in HCC by directly regulating HSP70 family members

Genomic analysis of focal nodular hyperplasia with associated hepatocellular carcinoma unveils its malignant potential: a case report.

Background Focal nodular hyperplasia (FNH) is typically considered a benign tumor of the liver without malignant potential. The co-occurrence of FNH and hepatocellular carcinoma (HCC) has been reported in rare cases. In this study we sought to investigate the clonal relationship between these lesions in a patient with FNH-HCC co-occurrence. Methods A 74-year-old female patient underwent liver tumor resection. The resected nodule was subjected to histologic analyses using hematoxylin and eosin stain and immunohistochemistry. DNA extracted from microdissected FNH and HCC regions was subjected to whole exome sequencing. Clonality analysis were performed using PyClone. Results Histologic analysis reveals that the nodule consists of an FNH and two adjoining HCC components with distinct histopathological features. Immunophenotypic characterization and genomic analyses suggest that the FNH is clonally related to the HCC components, and is composed of multiple clones at diagnosis, that are likely to have progressed to HCC through clonal selection and/or the acquisition of additional genetic events. Conclusion To the best of our knowledge, our work is the first study showing a clonal relationship between FNH and HCC. We show that FNH may possess the capability to undergo malignant transformation and to progress to HCC in very rare cases