UNC50 prompts G1/S transition and proliferation in HCC through regulation of epidermal growth factor receptor trafficking
ABSTRACT: Background: UNC50 has long been recognized as a Golgi apparatus protein in yeast, and is involved in nicotinic receptor trafficking in Caenorhabditis elegans, but little is known about UNC50 gene function in human biology despite it being conserved from yeast to high eukaryotes. Objectives: We investigated the relation between UNC50 and human hepatocellular carcinoma (HCC) and the potential mechanisms underlying HCC development. Methods: UNC50 mRNA expression patterns in 12 HCC and adjacent non-cancerous tissues determined using northern blotting were confirmed by real-time PCR in another 44 paired tissues. Microarray experiments were used to screen for global effects of UNC50 knockdown in the Hep3B cell line, and were confirmed by real-time PCR, western blotting, flow cytometry, and tetrazolium assay in both UNC50 overexpression and knockdown Hep3B cells. Results: UNC50 expression levels were upregulated in HCC tissues in comparison with the adjacent non-cancerous tissues. UNC50 knockdown reduced mRNA levels of the downstream targets of the epidermal growth factor receptor (EGFR) pathway: cyclin D1 (CCND1), EGF, matrix metalloproteinase-7 (MMP7), aldose reductase-like 1 (AKR1B10), cell surface–associated mucin 1 (MUC1), and gastrin (GAST). Moreover, UNC50 influenced EGF, inducing cell cycle entry by affecting cell surface EGFR amounts. Conclusions: UNC50 is a potential oncogene that promotes HCC progression by affecting the EGFR pathway. To gain insight into the role UNC50 plays in HCC progression, we used microarray analyses to identify indirect evidence of UNC50 gene function via the knockdown strategy in Hep3B cells. Hep3B cells transfected with the shRNA expression plasmids shR-467, shR-554, shR-749, and shR-MOCK were purified with 1ug/ml puromycin, and the total RNA from each cell was extracted and analyzed with oligo microarrays.
Project description:Bmi1 plays a pivotal role in hepatic carcinoma (HCC), but its targets in HCC is unknown. To screen the potential targets, we transfected HCC cell line Huh7 and Hep3B with Bmi1 shRNA lenti-virus. After confirming the Bmi1 was knocked down using western blotting, we extracted total RNA and then run the microarray detection. Gene expression profiles in Bmi1 KO cells were compared with those in Bmi1 WT cells to screen potential targets of Bmi1. Overall design: Bmi1 gene was knocked down using shRNA lenti-virus in HCC cell lines and the potential target genes were screened.
Project description:Emerging evidences indicate that microRNAs (miRNAs) are often deregulated and have fundamental roles in hepatocellular carcinoma (HCC). However, the mechanism underlying miRNA dysregulation in HCC is still elusive. In this report, we used an integrated analysis strategy combining methylated DNA immunoprecipitation chip (MeDIP-chip) and miRNA expression microarray data to study the correlation between aberrant methylation and dysregulation of miRNA in HCC. In all, we showed that global miRNA methylation profiles were significantly different between cancerous and normal hepatocytes, and abnormal methylation was an important mechanism governing miRNA expression in HCC. MeDIP-chip was processed in cancerous hepatocytes SK-HEP-1, HepG2, MHCC97-H and normal hepatocytes PHHC-4-1, PHHC-4-2, PHHC-4-3 (3 technical repeat of PHHC-4). MiRNA microarray were processed for cancerous hepatocytes SK-HEP-1, HepG2, Hep3B, Huh7, MHCC97-H, MHCC97-L, SMMC-7721 and normal hepatocytes PHHC-1, PHHC-2, PHHC-3. Then an integrated analysis strategy combining MeDIP-chip and miRNA expression microarray [GSE20077] were used to study the correlation of aberrant DNA methylation and dysregulation of miRNAs.
Project description:Orton2009 - Modelling cancerous mutations in
the EGFR/ERK pathway - EGF Model
This model studies the aberrations in
ERK signalling for different cancer mutations. The authors alter a
previously existing EGF model (Brown et al 2004) to include new
interactions that better fit empirical data.
Predictions show that the ERK signalling is a robust mechanism
taking different courses for different cancer mutations. Most
parameter values are used from the previous model and the new
parameters are estimated using experimental data performed by
the authors on PC12 cells (adrenal gland, rat). The authors
provide an SBML version of the model in the paper.
This model is described in the article:
modelling of cancerous mutations in the EGFR/ERK signalling
Orton RJ, Adriaens ME, Gormand A,
Sturm OE, Kolch W, Gilbert DR.
BMC Syst Biol 2009 Oct; 3: 100
The Epidermal Growth Factor Receptor (EGFR) activated
Extracellular-signal Regulated Kinase (ERK) pathway is a
critical cell signalling pathway that relays the signal for a
cell to proliferate from the plasma membrane to the nucleus.
Deregulation of the EGFR/ERK pathway due to alterations
affecting the expression or function of a number of pathway
components has long been associated with numerous forms of
cancer. Under normal conditions, Epidermal Growth Factor (EGF)
stimulates a rapid but transient activation of ERK as the
signal is rapidly shutdown. Whereas, under cancerous mutation
conditions the ERK signal cannot be shutdown and is sustained
resulting in the constitutive activation of ERK and continual
cell proliferation. In this study, we have used computational
modelling techniques to investigate what effects various
cancerous alterations have on the signalling flow through the
ERK pathway.We have generated a new model of the EGFR activated
ERK pathway, which was verified by our own experimental data.
We then altered our model to represent various cancerous
situations such as Ras, B-Raf and EGFR mutations, as well as
EGFR overexpression. Analysis of the models showed that
different cancerous situations resulted in different signalling
patterns through the ERK pathway, especially when compared to
the normal EGF signal pattern. Our model predicts that
cancerous EGFR mutation and overexpression signals almost
exclusively via the Rap1 pathway, predicting that this pathway
is the best target for drugs. Furthermore, our model also
highlights the importance of receptor degradation in normal and
cancerous EGFR signalling, and suggests that receptor
degradation is a key difference between the signalling from the
EGF and Nerve Growth Factor (NGF) receptors.Our results suggest
that different routes to ERK activation are being utilised in
different cancerous situations which therefore has interesting
implications for drug selection strategies. We also conducted a
comparison of the critical differences between signalling from
different growth factor receptors (namely EGFR, mutated EGFR,
NGF, and Insulin) with our results suggesting the difference
between the systems are large scale and can be attributed to
the presence/absence of entire pathways rather than subtle
difference in individual rate constants between the
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Project description:Lung adenocarcinoma cells harboring epidermal growth factor receptor (EGFR) mutations are sensitive to EGFR tyrosine kinase inhibitors (TKIs). Prolonged cancer treatment will induce the development of acquired resistance to EGFR TKI. To gain insight into the molecular mechanisms of EGFR-TKIs resistance, we generate EGFR-TKI-resistant HCC827-8-1 cells to be analyzed by microarray with their parental HCC827cells. gefitinib resistant HCC827-8-1 cells with three replications; gefitinib-sensitive HCC827 cells with three replications
Project description:Hepatocellular carcinoma (HCC) in young subjects is rare but more devastating. We hypothesize that genes and etiological pathways are unique to young HCC (yHCC; ≤40 years old at diagnosis) patients. We therefore compared the gene expression profiles between yHCCs and HCCs from elderly patients. Overall design: In this dataset, a total of 87 gene expression profiles of tissue samples were collected, which containing 48 primary HCC samples, as well as those of 39 non-cancerous tissues, from 61 patients. HCC tissues were collected from 16 young HCC and 32 elder HCC patients.
Project description:Sorafenib, an oral multikinase inhibitor, is the only approved agent for the treatment of advanced hepatocellular carcinoma (HCC). However, its benefits is modest, also because its mechanism of action remains elusive, therefore, a better understanding of its molecular action and molecular targets are needed. On the basis of our previous studies, here, we investigated the role of the nuclear protein 1 (NUPR1) in HCC and its role in the context of sorafenib treatment. NUPR1 is a stress-inducible protein over-expressed in different malignancies, however, its role in HCC is not yet fully understood. We found that NUPR1, is over-expressed in 53% of primary human HCC samples. Knockdown of NUPR1 significantly increased cell sensitivity to sorafenib and inhibits cell growth, migration and invasion of HCC cells in vitro and tumorigenicity in vivo. Moreover, NUPR1 silencing influenced expression of target genes RelB and IER3. Unsurprisingly, RelB and IER3 knockdown also inhibited HCC cells viability, growth and migration. By gene expression profiling of HCC cells following stable NUPR1 knockdown, we found that genes functionally involved in cell death and survival, cellular response to therapies, lipid metabolism, cell growth and proliferation, molecular transport and cellular movement were mostly suppressed. Network analysis of dynamic gene expression identified NF-κB and ERK as down-regulated gene nodes, and several genes known to be involved in hepatocarcinogenesis were also suppressed. In addition, we identified Runt-related transcription factor 2 (RUNX2) gene as a NUPR1 down-regulated gene. We also demonstrated that RUNX2 gene silencing inhibited HCC cells viability, growth, migration and increased cell sensitivity to sorafenib. Conclusion: We propose that NUPR1/RELB/IER3/RUNX2 pathway play pivotal role in hepatocarcinogenesis. The identification of NUPR1/RELB/IER3/RUNX2 pathway as a potential therapeutic target may contribute to the development of new treatment strategies for HCC management. To better understand the molecular mechanisms of NUPR1 gene action in ovarian HCC cells, we employed the Agilent Whole Human Genome microarrays, containing ~ 44,000 genes to identify global gene expression changes upon NUPR1 suppression in HCC cells. We compared the gene expression of the previously selected shRNA-mediated NURP1-knockdown Hep3B clone against the corresponding control (ctrl) clone. The microarray experiments were performed in duplicates, as two hybridizations were carried out for the NUPR1-suppressing cell clone against the corresponding control, using a fluorescent dye reversal (dye-swap) technique.
Project description:RNA-seq is a powerful tool for comprehensive characterization of whole transcriptome at both gene and exon levels and with a unique ability of identifying novel splicing variants. To date, RNA-seq analysis of HBV-related HCC has not been reported. In this study, we performed transcriptome analyses for 10 matched pairs of cancer and non-cancerous tissues from Chinese HBV-related hepatocelluar carcinoma patients using 36bp single-end sequencing approach on Solexa/Illumina GAII platform. On average, about 21.6 million sequencing reads and 10.6 million aligned reads were obtained for samples sequenced on each lane, which was able to identify > 50% of all the annotated genes for each sample. Furthermore, from by far the largest database of transcripts expressed in HCC tissues, we identified 1,378 significantly differently expressed genes (DEGs) and 24, 338 differentially expressed exons (DEEs). Comprehensive function analyses indicated that cell growth-related, metabolism-related and immune-related pathways were most significantly enriched by DEGs, pointing to a complex mechanism for HCC carcinogenesis. Positional gene enrichment analysis showed that DEGs were most significantly enriched at chromosome 8q21.3-24.3. The most interesting findings were from the analysis at exon levels where we characterized three major patterns of expression changes between gene levels and exon levels, implying a much complex landscape of transcript-specific differential expressions in HCC. Finally, we identified a novel highly up-regulated exon-exon junction in ATAD2 (ATPase family, AAA domain containing 2) gene in HCC tissues. Overall, to our best knowledge, our study represents the most comprehensive characterization of the HBV-related HCC transcriptome including exon level expression changes and novel splicing variants, which illustrated the power of RNA-seq and provided important clues for understanding the molecular mechanisms of HCC pathogenesis at system-wide levels. A comprehensive analysis of transcriptome for 10 match-paired HBV-related Chinese HCC and non-cancerous adjacent tissues. Processed data files: Exon-level results, gene-level results, differentially expressed exons, and differently expressed genes (DEGs).
Project description:Hep3B and Huh7 are two types of human hepatoma cell lines (HCC). In our laboratory, we cultured their stem-like cancer cells (HCSCs), Hep3B-C and Huh7-C. And we have demonstrated that these cells had enhanced stem cell properties, drug resistance, properties of EMT, and stronger tumor-initiating capabilities. To explore functionally crucial miRNAs in HCSCs, 2 samples of HCSCs and 2 samples of HCCs were sequenced by the Illumina Genome Analyzer II. Through differential expression analysis, we finally identified 9 up- and 9 down-regulated miRNAs which were consistently up- and down-regulated in two stem cells compared to the cancer cells. Expression analysis using total RNAs extracted from 2 HCSC cell lines (Hep3B-C and Huh7-C), and 2 HCC cell lines (Hep3B and Huh7).
Project description:Hep3B and Huh7 are two types of human hepatoma cell lines (HCC). In our laboratory, we cultured their stem-like cancer cells (HCSCs), Hep3B-C and Huh7-C. And we have demonstrated that these cells had enhanced stem cell properties, drug resistance, properties of EMT, and stronger tumor-initiating capabilities. To explore functionally crucial mRNAs in HCSCs, 2 samples of HCSCs and 2 samples of HCCs were sequenced by the Illumina Genome Analyzer II. Through differential expression analysis, we finally identified 115 up- and 402 down-regulated miRNAs which were consistently up- and down-regulated in two stem cells compared to the cancer cells. Expression analysis using total RNAs extracted from 2 HCSC cell lines (Hep3B-C and Huh7-C), and 2 HCC cell lines (Hep3B and Huh7).
Project description:It is known that exosomes (endosome derived vesicles) play important roles in the formation of the tumor microenvironment. Hepatocellular carcinoma (HCC) is a highly malignant cancer, whose malignancy is largely influenced by the tumor microenvironment. The possible role and the specific content of the HCC derived exosomes are however largely unknown. We performed super-SILAC-based mass spectrometry (MS) analyses to interrogate the differential proteins in the exosome of three human HCC cell lines, MHCC97H, MHCCLM3 and Hep3B cells. Exosomal proteins were systematically compared with multi-omics strategies, considering both proteomics and translatomics. With stringent data quality control (quantified unique peptides ≥ 2, FDR ≤ 0.01 at both protein and peptide level), 1907 exosomal proteins were confidently identified from the three HCC cell lines, out of which 469 and 443 exosomal proteins significantly altered in the highly malignant cell lines (MHCC97H/Hep3B and MHCCLM3/Hep3B), respectively. ClueGo and IPA analyses on the differentially expressed proteins (DEPs) revealed that translation and ubiquitination biological processes pathways were significantly more encapsulated in the exosome of higher malignant cell lines. We further observed significantly negative correlation of exosomal protein to cellular protein and translating mRNA in terms of relative abundances comparing the higher malignant cell lines with the low malignant cell line. The negatively correlated genes are also translation regulation-centric. In conclusion, we demonstrated that the exosomal enrichment of translation regulatory proteins is related to the malignant level of HCC cells.