Genome-wide DNA methylation profiles in progression to in situ and invasive carcinoma of the breast with impact on gene transcription and prognosis

Background

Ductal carcinoma in situ (DCIS) of the breast is a precursor of invasive breast carcinoma. DNA methylation alterations are thought to be an early event in progression of cancer, and may prove valuable as a tool in clinical decision making and for understanding neoplastic development.

Results

We generate genome-wide DNA methylation profiles of 285 breast tissue samples representing progression of cancer, and validate methylation changes between normal and DCIS in an independent dataset of 15 normal and 40 DCIS samples. We also validate a prognostic signature on 583 breast cancer samples from The Cancer Genome Atlas. Our analysis reveals that DNA methylation profiles of DCIS are radically altered compared to normal breast tissue, involving more than 5,000 genes. Changes between DCIS and invasive breast carcinoma involve around 1,000 genes. In tumors, DNA methylation is associated with gene expression of almost 3,000 genes, including both negative and positive correlations. A prognostic signature based on methylation level of 18 CpGs is associated with survival of breast cancer patients with invasive tumors, as well as with survival of patients with DCIS and mixed lesions of DCIS and invasive breast carcinoma.

Conclusions

This work demonstrates that changes in the epigenome occur early in the neoplastic progression, provides evidence for the possible utilization of DNA methylation-based markers of progression in the clinic, and highlights the importance of epigenetic changes in carcinogenesis.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0435-x) contains supplementary material, which is available to authorized users.     

Molecular Signatures Associated with HCV-Induced Hepatocellular Carcinoma and Liver Metastasis

Hepatocellular carcinomas (HCCs) are a heterogeneous group of tumors that differ in risk factors and genetic alterations. In Italy, particularly Southern Italy, chronic hepatitis C virus (HCV) infection represents the main cause of HCC. Using high-density oligoarrays, we identified consistent differences in gene-expression between HCC and normal liver tissue. Expression patterns in HCC were also readily distinguishable from those associated with liver metastases. To characterize molecular events relevant to hepatocarcinogenesis and identify biomarkers for early HCC detection, gene expression profiling of 71 liver biopsies from HCV-related primary HCC and corresponding HCV-positive non-HCC hepatic tissue, as well as gastrointestinal liver metastases paired with the apparently normal peri-tumoral liver tissue, were compared to 6 liver biopsies from healthy individuals. Characteristic gene signatures were identified when normal tissue was compared with HCV-related primary HCC, corresponding HCV-positive non-HCC as well as gastrointestinal liver metastases. Pathway analysis classified the cellular and biological functions of the genes differentially expressed as related to regulation of gene expression and post-translational modification in HCV-related primary HCC; cellular Growth and Proliferation, and Cell-To-Cell Signaling and Interaction in HCV-related non HCC samples; Cellular Growth and Proliferation and Cell Cycle in metastasis. Also characteristic gene signatures were identified of HCV-HCC progression for early HCC diagnosis.

Conclusions

A diagnostic molecular signature complementing conventional pathologic assessment was identified.     

Elevated Pretherapy Serum IL17 in Primary Hepatocellular Carcinoma Patients Correlate to Increased Risk of Early Recurrence after Curative Hepatectomy

Background and Aims

Primary hepatocellular carcinoma (HCC) is usually presented in inflamed fibrotic/cirrhotic liver with extensive lymphocyte infiltration. We examined the associations between the HCC early recurrence and alterations in serum levels of inflammatory cytokines.

Methods

A cohort of 105 HCC patients with chronic hepatitis B virus infection were included. Pre-therapy, we quantified their serum concentrations of Th1-, Th2-, Th17-, Treg-related, and other cytokines that have been reported to be associated with poor prognosis in human cancers. IL17-producing T-cells were generated in vitro from HCC patients and co-cultured with HCC cell lines separated by a 0.4 µM transwell.

Results

All the 105 cases of HCC patients had liver cirrhosis. The patients who suffered from HCC early recurrence had higher pre-therapy serum levels of IL17 and lower levels of IL10 than those who did not suffer from recurrence after curative hepatectomy. After adjustment for general tumor clinicopathological factors, elevated serum levels of IL17 (≥0.9 pg/ml) was found to be an independent risk factor for HCC early recurrence with a hazard ratio of 2.46 (95%CI 1.34–4.51). Patients with bigger tumors (>5 cm in diameter) and elevated serum levels of IL17 had the highest risk of early recurrence as compared to those with only one of these factors (P = 0.009) or without any (P<0.001). These factors showed similar effects on the HCC patient overall survival. Intrahepatic infiltrated T-cells in HCC patients were identified as the major IL17-producing cells. Proliferation of HCC cells, QGY-7703, was augmented QGY-7703, was augmented in the presence of IL17-producing T-cells. This effect diminished after neutralizing antibody against human IL17A or TNFα was included.

Conclusion

Both tumors and IL17 from liver infiltrated T-cells contributed to HCC early recurrence and progression after curative resection. Pre-therapy serum IL17 levels may serve as an additional indicator for predicting high-risk patients.     

Clinical Value of Prognosis Gene Expression Signatures in Colorectal Cancer: A Systematic Review

Introduction

The traditional staging system is inadequate to identify those patients with stage II colorectal cancer (CRC) at high risk of recurrence or with stage III CRC at low risk. A number of gene expression signatures to predict CRC prognosis have been proposed, but none is routinely used in the clinic. The aim of this work was to assess the prediction ability and potential clinical usefulness of these signatures in a series of independent datasets.

Methods

A literature review identified 31 gene expression signatures that used gene expression data to predict prognosis in CRC tissue. The search was based on the PubMed database and was restricted to papers published from January 2004 to December 2011. Eleven CRC gene expression datasets with outcome information were identified and downloaded from public repositories. Random Forest classifier was used to build predictors from the gene lists. Matthews correlation coefficient was chosen as a measure of classification accuracy and its associated p-value was used to assess association with prognosis. For clinical usefulness evaluation, positive and negative post-tests probabilities were computed in stage II and III samples.

Results

Five gene signatures showed significant association with prognosis and provided reasonable prediction accuracy in their own training datasets. Nevertheless, all signatures showed low reproducibility in independent data. Stratified analyses by stage or microsatellite instability status showed significant association but limited discrimination ability, especially in stage II tumors. From a clinical perspective, the most predictive signatures showed a minor but significant improvement over the classical staging system.

Conclusions

The published signatures show low prediction accuracy but moderate clinical usefulness. Although gene expression data may inform prognosis, better strategies for signature validation are needed to encourage their widespread use in the clinic.     

Increased Expression of Cathepsin L: A Novel Independent Prognostic Marker of Worse Outcome in Hepatocellular Carcinoma Patients

Objectives

To investigate the expression and role of Cathepsin L (CTSL) in Hepatocellular carcinoma (HCC) tissue and cell line (MHCC-97H), and to evaluate the clinical and prognostic significance of CTSL protein in patients with HCC.

Methods

The expression of CTSL was examined in HCC tissue and MHCC-97H cells by Western-blotting, Real-time PCR and immunohistochemical staining. Cell growth curve assay and colony formation assay were used to verify the effect of CTSL on the proliferation and tumor progression ability of MHCC-97H cells. Tumor formation assay in nude mice was used to analyze the effect of CTSL on the tumorigenicity of MHCC-97H cells.

Results

The status of CTSL protein in carcinoma tissues is much higher than that in paracarcinoma tissues. The overall survival of the patients with high CTSL expression was significantly shorter than the low CTSL expression group. high CTSL expression was significantly correlated with advanced clinical staging, histological grade and tumor recurrence. In vitro experiments demonstrated that over-expression of CTSL in MHCC-97H cells promoted cell proliferation and tumor progression ability. Down-regulation of CTSL showed the opposite effects. Over-expression of CTSL increase the tumorigenicity of MHCC-97H cells by in vivo experiments. Moreover, multivariate analysis suggested that CTSL expression might be an independent prognostic indicator for the survival of HCC patients after curative surgery.

Conclusions

CTSL might involve in the development and progression of HCC as a oncogene, and thereby may be a valuable prognostic marker for HCC patients.     

Characterization of age signatures of DNA methylation in normal and cancer tissues from multiple studies

Background

DNA methylation (DNAm) levels can be used to predict the chronological age of tissues; however, the characteristics of DNAm age signatures in normal and cancer tissues are not well studied using multiple studies.

Results

We studied approximately 4000 normal and cancer samples with multiple tissue types from diverse studies, and using linear and nonlinear regression models identified reliable tissue type-invariant DNAm age signatures. A normal signature comprising 127 CpG loci was highly enriched on the X chromosome. Age-hypermethylated loci were enriched for guanine–and-cytosine-rich regions in CpG islands (CGIs), whereas age-hypomethylated loci were enriched for adenine–and-thymine-rich regions in non-CGIs. However, the cancer signature comprised only 26 age-hypomethylated loci, none on the X chromosome, and with no overlap with the normal signature. Genes related to the normal signature were enriched for aging-related gene ontology terms including metabolic processes, immune system processes, and cell proliferation. The related gene products of the normal signature had more than the average number of interacting partners in a protein interaction network and had a tendency not to interact directly with each other. The genomic sequences of the normal signature were well conserved and the age-associated DNAm levels could satisfactorily predict the chronological ages of tissues regardless of tissue type. Interestingly, the age-associated DNAm increases or decreases of the normal signature were aberrantly accelerated in cancer samples.

Conclusion

These tissue type-invariant DNAm age signatures in normal and cancer can be used to address important questions in developmental biology and cancer research.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-997) contains supplementary material, which is available to authorized users.     

Cytoplasmic and/or Nuclear Expression of β-Catenin Correlate with Poor Prognosis and Unfavorable Clinicopathological Factors in Hepatocellular Carcinoma: A Meta-Analysis

Background

The β-catenin is an important effector in WNT/β-catenin signaling pathway, which exerts a crucial role in the development and progression of hepatocellular carcinoma (HCC). Some researchers have suggested that the overexpression of β-catenin in cytoplasm and/or nucleus was closely correlated to metastasis, poor differentiation and malignant phenotype of HCC while some other researchers hold opposite point. So far, no consensus was obtained on the prognostic and clinicopathological significance of cytoplasmic/nuclear β-catenin overexpression for HCCs.

Methods

Systematic strategies were applied to search eligible studies in all available databases. Subgroup analyses, sensitivity analyses and multivariate analysis were performed. In this meta-analysis, we utilized either fixed- or random-effects model to calculate the pooled odds ratios (OR) and its 95% confidence intervals (CI).

Results

A total of 22 studies containing 2334 cases were enrolled in this meta-analysis. Pooled data suggested that accumulation of β-catenin in cytoplasm and/or nucleus significantly correlated with poor 1-, 3- and 5-year OS and RFS. Moreover, nuclear accumulation combined with cytoplasmic accumulation of β-catenin tended to be associated with dismal metastasis and vascular invasion while cytoplasmic or nuclear expression alone showed no significant effect. Besides, no significant association was observed between cytoplasmic and/or nuclear β-catenin expression and poor differentiation grade, advanced TNM stage, liver cirrhosis, tumor size, tumor encapsulation, AFP and etiologies. Additional subgroup analysis by origin suggested that the prognostic value and clinicopathological significance of cytoplasmic and/or nuclear β-catenin expression was more validated in Asian population. Multivariate analyses of factors showed that cytoplasmic and/or nuclear β-catenin expression, as well as TNM stage, metastasis and tumor size, was an independent risk factors for OS and RFS.

Conclusions

Cytoplasmic and/or nuclear accumulation of β-catenin, as an independent prognostic factor, significantly associated with poor prognosis and deeper invasion of HCC, and could serve as a valuable prognostic predictor for HCC.     

A signature inferred from Drosophila mitotic genes predicts survival of breast cancer patients

Introduction

The classification of breast cancer patients into risk groups provides a powerful tool for the identification of patients who will benefit from aggressive systemic therapy. The analysis of microarray data has generated several gene expression signatures that improve diagnosis and allow risk assessment. There is also evidence that cell proliferation-related genes have a high predictive power within these signatures.

Methods

We thus constructed a gene expression signature (the DM signature) using the human orthologues of 108 Drosophila melanogaster genes required for either the maintenance of chromosome integrity (36 genes) or mitotic division (72 genes).

Results

The DM signature has minimal overlap with the extant signatures and is highly predictive of survival in 5 large breast cancer datasets. In addition, we show that the DM signature outperforms many widely used breast cancer signatures in predictive power, and performs comparably to other proliferation-based signatures. For most genes of the DM signature, an increased expression is negatively correlated with patient survival. The genes that provide the highest contribution to the predictive power of the DM signature are those involved in cytokinesis.

Conclusion

This finding highlights cytokinesis as an important marker in breast cancer prognosis and as a possible target for antimitotic therapies.     

Tumor Endothelial Inflammation Predicts Clinical Outcome in Diverse Human Cancers

Background

Vascular endothelial cells contribute to the pathogenesis of numerous human diseases by actively regulating the stromal inflammatory response; however, little is known regarding the role of endothelial inflammation in the growth of human tumors and its influence on the prognosis of human cancers.

Methods

Using an experimental model of tumor necrosis factor-alpha (TNF-α)-mediated inflammation, we characterized inflammatory gene expression in immunopurified tumor-associated endothelial cells. These genes formed the basis of a multivariate molecular predictor of overall survival that was trained and validated in four types of human cancer.

Results

We report that expression of experimentally derived tumor endothelial genes distinguished pathologic tissue specimens from normal controls in several human diseases associated with chronic inflammation. We trained these genes in human cancer datasets and defined a six-gene inflammatory signature that predicted significantly reduced overall survival in breast cancer, colon cancer, lung cancer, and glioma. This endothelial-derived signature predicted outcome independently of, but cooperatively with, standard clinical and pathological prognostic factors. Consistent with these findings, conditioned culture media from human endothelial cells stimulated by pro-inflammatory cytokines accelerated the growth of human colon and breast tumors in immunodeficient mice as compared with conditioned media from untreated endothelial cells.

Conclusions

This study provides the first prognostic cancer gene signature derived from an experimental model of tumor-associated endothelial inflammation. These findings support the notion that activation of inflammatory pathways in non-malignant tumor-infiltrating endothelial cells contributes to tumor growth and progression in multiple human cancers. Importantly, these results identify endothelial-derived factors that could serve as potential targets for therapy in diverse human cancers.     

Gene Expression-Based Classification of Non-Small Cell Lung Carcinomas and Survival Prediction

Background

Current clinical therapy of non-small cell lung cancer depends on histo-pathological classification. This approach poorly predicts clinical outcome for individual patients. Gene expression profiling holds promise to improve clinical stratification, thus paving the way for individualized therapy.

Methodology and Principal Findings

A genome-wide gene expression analysis was performed on a cohort of 91 patients. We used 91 tumor- and 65 adjacent normal lung tissue samples. We defined sets of predictor genes (probe sets) with the expression profiles. The power of predictor genes was evaluated using an independent cohort of 96 non-small cell lung cancer- and 6 normal lung samples. We identified a tumor signature of 5 genes that aggregates the 156 tumor and normal samples into the expected groups. We also identified a histology signature of 75 genes, which classifies the samples in the major histological subtypes of non-small cell lung cancer. Correlation analysis identified 17 genes which showed the best association with post-surgery survival time. This signature was used for stratification of all patients in two risk groups. Kaplan-Meier survival curves show that the two groups display a significant difference in post-surgery survival time (p = 5.6E-6). The performance of the signatures was validated using a patient cohort of similar size (Duke University, n = 96). Compared to previously published prognostic signatures for NSCLC, the 17 gene signature performed well on these two cohorts.

Conclusions

The gene signatures identified are promising tools for histo-pathological classification of non-small cell lung cancer, and may improve the prediction of clinical outcome.     

Fibroblast Growth Factor Receptor 4 Polymorphism Is Associated with Liver Cirrhosis in Hepatocarcinoma

Background

Fibroblast growth factor receptor 4 (FGFR4) polymorphisms are positively correlated with tumor progression in numerous malignant tumors. However, the association between FGFR4 genetic variants and the risk of hepatocellular carcinoma (HCC) has not yet been determined. In this study, we investigated the potential associations of FGFR4 single nucleotide polymorphisms (SNPs) with HCC susceptibility and its clinicopathological characteristics.

Methodology/Principal Findings

Four SNPs in FGFR4 (rs1966265, rs351855, rs2011077, and rs7708357) were analyzed among 884 participants, including 595 controls and 289 patients with HCC. The samples were further analyzed to clarify the associations between these gene polymorphisms and the risk of HCC, and the impact of these SNPs on the susceptibility and clinicopathological characteristics of HCC. After adjusting for other covariants, HCC patients who carrying at least one A genotype (GA and AA) at rs351855 were observed to have a higher risk of liver cirrhosis compared with those carrying the wild-type genotype (GG) (OR: 2.113, 95% CI: 1.188–3.831). Moreover, the patients with at least one A genotype were particularly showed a high level of alpha-fetoprotein (AFP).

Conclusions

Our findings suggest that genetic polymorphism in FGFR4 rs351855 may be associated with the risk of HCC coupled with liver cirrhosis and may markedly increase the AFP level in Taiwanese patients with HCC. In addition, this is the first study that evaluated the risk factors associated with FGFR4 polymorphism variants in Taiwanese patients with HCC.     

Chemically-induced cancers do not originate from bone marrow-derived cells

Background

The identification and characterization of cancer stem cells (CSCs) is imperative to understanding the mechanism of cancer pathogenesis. Growing evidence suggests that CSCs play critical roles in the development and progression of cancer. However, controversy exists as to whether CSCs arise from bone marrow-derived cells (BMDCs).

Methodology and Principal Findings

In the present study, n-nitrosodiethylamine (DEN) was used to induce tumor formation in female mice that received bone marrow from male mice. Tumor formation was induced in 20/26 mice, including 12 liver tumors, 6 lung tumors, 1 bladder tumor and 1 nasopharyngeal tumor. Through comparison of fluorescence in situ hybridization (FISH) results in corresponding areas from serial tumor sections stained with H&E, we determined that BMDCs were recruited to both tumor tissue and normal surrounding tissue at a very low frequency (0.2–1% in tumors and 0–0.3% in normal tissues). However, approximately 3–70% of cells in the tissues surrounding the tumor were BMDCs, and the percentage of BMDCs was highly associated with the inflammatory status of the tissue. In the present study, no evidence was found to support the existence of fusion cells formed form BMDCs and tissue-specific stem cells.

Conclusions

In summary, our data suggest that although BMDCs may contribute to tumor progression, they are unlike to contribute to tumor initiation.     

DNA Methylation Profiles and Their Relationship with Cytogenetic Status in Adult Acute Myeloid Leukemia

Background

Aberrant promoter DNA methylation has been shown to play a role in acute myeloid leukemia (AML) pathophysiology. However, further studies to discuss the prognostic value and the relationship of the epigenetic signatures with defined genomic rearrangements in acute myeloid leukemia are required.

Methodology/Principal Findings

We carried out high-throughput methylation profiling on 116 de novo AML cases and we validated the significant biomarkers in an independent cohort of 244 AML cases. Methylation signatures were associated with the presence of a specific cytogenetic status. In normal karyotype cases, aberrant methylation of the promoter of DBC1 was validated as a predictor of the disease-free and overall survival. Furthermore, DBC1 expression was significantly silenced in the aberrantly methylated samples. Patients with chromosome rearrangements showed distinct methylation signatures. To establish the role of fusion proteins in the epigenetic profiles, 20 additional samples of human hematopoietic stem/progenitor cells (HSPC) transduced with common fusion genes were studied and compared with patient samples carrying the same rearrangements. The presence of MLL rearrangements in HSPC induced the methylation profile observed in the MLL-positive primary samples. In contrast, fusion genes such as AML1/ETO or CBFB/MYH11 failed to reproduce the epigenetic signature observed in the patients.

Conclusions/Significance

Our study provides a comprehensive epigenetic profiling of AML, identifies new clinical markers for cases with a normal karyotype, and reveals relevant biological information related to the role of fusion proteins on the methylation signature.     

Distinct High-Profile Methylated Genes in Colorectal Cancer

Background

Mutations and promoters'' methylation of a set of candidate cancer genes (CAN genes) are associated with progression of colorectal cancer (CRC). We hypothesized that these genes'' promoters are inactivated through epigenetic silencing and may show a different profile in high-risk populations. We investigated the status of CAN gene methylation and CHD5 protein expression in African American CRC tissue microarrays (TMA) using immunohistochemical staining.

Methodology/Principal Findings

The promoter methylation status of the CAN genes was studied by methylation-specific PCR (MSP) in 51 Iranians (a white population) and 51 African Americans (AA). Microsatellite instability (MSI) was analyzed as well. The differential frequency of methylation for each gene was tested by chi-square analysis between the two groups based on matched age and sex. CHD5 protein expression was evaluated in moderate to well differentiated and poorly differentiated carcinomas compared to matched normal tissue using TMA. In addition, the correlation between these epigenetic biomarkers and various clinicopathological factors, including, age, location, and stage of the disease were analyzed.Seventy-seven and 34% of tumors were distal in Iranian and African American patients, respectively. In both populations, the percentage of methylation was >65% for SYNE1, MMP2, APC2, GPNMB, EVL, PTPRD, and STARD8, whereas methylation was <50% for LGR6, RET, CD109, and RNF. The difference in methylation between the two populations was statistically significant for CHD5, ICAM5 and GPNMB. Thirty-one percent AA tumors showed MSI-H, compared to 28% in Iranians.

Conclusions/Significance

A significantly higher methylation rate was found for GPNMB, ICAM5, and CHD5 genes in AA patients compared to Iranians. These genes might play a role in the high incidence and aggressiveness of CRC in the AA population. The hypermethylation of the CAN genes can be considered as a marker of colon carcinogenesis.     

Bimodal signatures of germline methylation are linked with gene expression plasticity in the coral Acropora millepora

Background

In invertebrates, genes belonging to dynamically regulated functional categories appear to be less methylated than “housekeeping” genes, suggesting that DNA methylation may modulate gene expression plasticity. To date, however, experimental evidence to support this hypothesis across different natural habitats has been lacking.

Results

Gene expression profiles were generated from 30 pairs of genetically identical fragments of coral Acropora millepora reciprocally transplanted between distinct natural habitats for 3 months. Gene expression was analyzed in the context of normalized CpG content, a well-established signature of historical germline DNA methylation. Genes with weak methylation signatures were more likely to demonstrate differential expression based on both transplant environment and population of origin than genes with strong methylation signatures. Moreover, the magnitude of expression differences due to environment and population were greater for genes with weak methylation signatures.

Conclusions

Our results support a connection between differential germline methylation and gene expression flexibility across environments and populations. Studies of phylogenetically basal invertebrates such as corals will further elucidate the fundamental functional aspects of gene body methylation in Metazoa.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1109) contains supplementary material, which is available to authorized users.     

The prognostic significance of whole blood global and specific DNA methylation levels in gastric adenocarcinoma

Background

Epigenetics, particularly DNA methylation, has recently been elucidated as important in gastric cancer (GC) initiation and progression. We investigated the clinical and prognostic importance of whole blood global and site-specific DNA methylation in GC.

Methods

Genomic DNA was extracted from the peripheral blood of 105 Omani GC patients at diagnosis. DNA methylation was quantified by pyrosequencing of global DNA and specific gene promoter regions at 5 CpG sites for CDH1, 7 CpG sites for p16, 4 CpG sites for p53, and 3 CpG sites for RUNX3. DNA methylation levels in patients were categorized into low, medium, and high tertiles. Associations between methylation level category and clinicopathological features were evaluated using χ² tests. Survival analyses were carried out using the Kaplan-Meier method and log rank test. A backward conditional Cox proportional hazards regression model was used to identify independent predictors of survival.

Results

Older GC patients had increased methylation levels at specific CpG sites within the CDH1, p53, and RUNX-3 promoters. Male gender was significantly associated with reduced global and increased site-specific DNA methylation levels in CDH1, p16, and p53 promoters. Global DNA low methylation level was associated with better survival on univariate analysis. Patients with high and medium methylation vs. low methylation levels across p16 promoter CpG sites, site 2 in particular, had better survival. Multivariate analysis showed that global DNA hypermethylation was a significant independent predictor of worse survival (hazard ratio (HR) = 2.0, 95% CI: 1.1–3.8; p = 0.02) and high methylation mean values across p16 promoter sites 1–7 were associated with better survival with HR of 0.3 (95% CI, 0.1–0.8; p = 0.02) respectively.

Conclusions

Analysis of global and site-specific DNA methylation in peripheral blood by pyrosequencing provides quantitative DNA methylation values that may serve as important prognostic indicators.     

Dynamical Observation on Biological Progression of VX2 Liver Tumors to Identify the Optimal Time for Intervention in Animal Models

Purpose

Based on practice guideline of “management of hepatocellular carcinoma (HCC): update” published by American Association for the Study of Liver Diseases (AASLD) and “Barcelona Clinic Liver Cancer staging system (BCLC),” this study investigated how to enroll the optimal VX2 liver tumor model for HCC researches by dynamically observing the biological progression of the tumor.

Materials

Thirty-two healthy New Zealand white rabbits were implanted VX2 liver tumor by cell suspension method (n=24) and tissue fragment method (n=8). All the rabbits underwent CT scans on day 7, 14, 21 and 28 after implantation to observe the size of the tumors, the time when metastases and ascites occurred and the survival time. Appropriate intervention times were estimated corresponding to different clinical HCC stages by using tumor diameter-time curve.

Results

The VX2 liver tumors grew rapidly within 28 days after implantation. And the tumors in the cell suspension group grew faster than those of the tissue fragment group. The appropriate intervention time corresponding to very early stage, early stage and intermediate stage were <11 days, 11–16.9 days and >16.9 days, respectively in the cell suspension group, and <19.9 days, 19.9–25.5 days and >25.5 days, respectively in the tissue fragment group.

Conclusion

Preclinical animal research needs to improve on different levels to yield best predictions for human patients. Researchers should seek for an individualized proposal to select optimal VX2 liver tumor models for their experiments. This approach may lead to a more accurate determination of therapeutic outcomes.     

CCAAT/Enhancer-Binding Protein Homologous (CHOP) Protein Promotes Carcinogenesis in the DEN-Induced Hepatocellular Carcinoma Model

Background and Aims

C/EBP homologous protein (CHOP) plays pro-apoptotic roles in the integrated stress response. Recently, a tumor suppressive role for CHOP was demonstrated in lung cancer via regulation of tumor metabolism. To explore the role of CHOP in hepatocarcinogenesis, we induced hepatocellular carcinoma (HCC) in wild type (wt) and CHOP knockout (KO) mice using the carcinogen N-diethylnitrosamine (DEN).

Results

Analysis of tumor development showed reduced tumor load, with markedly smaller tumor nodules in the CHOP KO animals, suggesting oncogenic roles of CHOP in carcinogen-induced HCC. In wt tumors, CHOP was exclusively expressed in tumor tissue, with minimal expression in normal parenchyma. Analysis of human adenocarcinomas of various origins demonstrated scattered expression of CHOP in the tumors, pointing to relevance in human pathology. Characterization of pathways that may contribute to preferential expression of CHOP in the tumor identified ATF6 as a potential candidate. ATF6, a key member of the endoplasmic reticulum stress signaling machinery, exhibited a similar pattern of expression as CHOP and strong activation in wt but not CHOP KO tumors. Because HCC is induced by chronic inflammation, we assessed whether CHOP deficiency affects tumor-immune system crosstalk. We found that the number of macrophages and levels of IFNγ and CCL4 mRNA were markedly reduced in tumors from CHOP KO relative to wt mice, suggesting a role for CHOP in modulating tumor microenvironment and macrophage recruitment to the tumor.

Conclusion

Our data highlights a role for CHOP as a positive regulator of carcinogen-induced HCC progression through a complex mechanism that involves the immune system and modulation of stress signaling pathways.