Posts Tagged ‘Hepatocellular carcinoma’

Author and Reporter: Ritu Saxena, Ph.D.

On June 4, 2012, I authored a post on HBV and HCV-associated Liver Cancer: Important Insights from the Genome reporting about the major role of chromatin remodeling complexes and involvement of both interferon and oxidative stress pathways in hepatocellular malignant proliferation and transformation based on the genes showing recurrent mutations in the observed genes.

In this post, I have discussed the latest research on cyclin B1 and Sec62 expression in PBMCs of HCC patients and how their elevated expression correlates to significantly to negative prognostic value in terms of recurrence-free survival.

Researchers at the Changhai and Gongli Hospital in Shanghai, Military Medical University, People’s Republic of China recently identified the candidate biomarkers for HBV-related HCC recurrence after surgery. The research was published in the June 2012 issue of Molecular Cancer journal. According to the group findings, Cyclin B1 and Sec62 may serve as effective biomarkers and potential therapeutic targets for HBV-related HCC recurrence after surgery.

Research article: Identification of cyclin B1 and Sec62 as biomarkers for recurrence in patients with HBV-related hepatocellular carcinoma after surgical resection.

HCC background and Research Problem: Hepatocellular carcinoma is cancer of the liver. It is different from Metastaticc liver cancer, which starts in another organ (such as the breast or colon) and spreads to the liver. The most frequent factors causing HCC include chronic viral hepatitis (types B and C), alcohol intake and afla- toxin exposure.

In most cases, scarring of the liver referred to as cirrhosis is an important risk factor for HCC. Cirrhosis may be caused by:

However, patients with hepatitis B or C are at risk for liver cancer, even if they have not developed cirrhosis.

According to the data from International Agency for Research on Cancer, hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide, with over a half million deaths per annum.

In China, a very high infection rates with HBV have been reported. According to the recent statistics reported by Jemal et al in 2011, HCC cases occurring in China account for 55% of the total cases reported in the world.

Surgical resection, although provides an opportunity for cure, however, frequent recurrence post surgery has posed a major challenge to longterm survival. Pertinent to their research, authors state “Frequent tumor recurrence after surgery is related to its poor prognosis. Although gene expression signatures have been associated with outcome, the molecular basis of HCC recurrence is not fully understood..”.

Research: To determine the molecular basis of HCC, authors used the Peripheral blood mononuclear cells (PBMCs) to predict the recurrence of HCC after surgery. Use of PBMCs was in contrast to previous studies that used just the liver tissues. PBMCs have the advantage of being easily obtained in the clinical setting. Thus, identification of biomarkers using PBMCs would be a great way to predict the recurrence of HCC post surgery.

A microarray-based gene expression profiling was performed to indentify candidate genes related to HCC recurrence. In all, mRNA derived from 6 HCC cases (3 cases with recurrence and 3 without recurrence) were subjected to genome-wide analysis. Some critical genes were indentified including cyclin B1 (CCNB1), SEC62 homolog (S. cerevisiae) (SEC62), and baculoviral IAP repeat-containing 3 (BIRC3), suggesting that they probably play important roles in the pathogenesis of HCC recurrence. To confirm the results of microarray analysis, the mRNA and protein expressions of these 3 genes were measured in 80 HCC samples from HCC cases and 30 samples from healthy cases. The authors found that the transcriptional and protein expressions of cyclin B1, Sec62, and Birc3 in the PBMCs were significantly higher in HCC samples than those in the non-recurrent and normal samples.

Furthermore, to determine the clinicopathologic significance of cyclin B1, Sec62, and Birc3 in HCC, immunohistochemical analysis from 35 recurrent tissues and 45 nonrecurrent revealed that the protein levels of cyclin B1, Sec62, and Birc3 were substantially higher in the recurrent tissues than those in the non-recurrent samples. Thus, the immunohistochemical results from tissues were consistent with the previous transcriptional and protein results in PBMCs.

Conclusion of study:  The authors discussed that “In recent years, studies on malignant tumors has primarily focused on cell proliferation, migration, and apoptosis. Cyclin B1, Sec62, and Birc3, chosen in this study according to our microarray analysis, likely play important roles in cell proliferation and migration. They can exert a tumor-promoting effect on HCC by regulating cell cycle and protein translocation.” As derived from the statistical methods employed in the research, elevated cyclin B1 and Sec62 expression in PBMCs had a significantly negative prognostic value in terms of recurrence-free survival, which hints the potential use of these molecular markers to predict the risk of tumor recurrence after surgery and to act as therapeutic targets to reduce tumor recurrence and improve clinical therapies.

Thus, these results revealed that cyclin B1 and Sec62 may be candidate biomarkers and potential therapeutic targets for HBV-related HCC recurrence after surgery.


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Author: Ritu Saxena, PhD

Updated on July 5, 2013 (research article published in New England Journal of Medicine regarding the role of SALL4 gene in aggressive hepatocellular carcinoma)

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. The incidence of HCC varies considerably with the geographic area because of differences in the major causative factors. Chronic hepatitis B and C, mostly in the cirrhotic stage, are responsible for the great majority of cases of HCC worldwide.

Hepatitis B and C viruses (HBV/HCV) can be implicated in the development of HCC in an indirect way, through induction of chronic inflammation, or directly by means of viral proteins or, in the case of HBV, by creation of mutations by integration into the genome of the hepatocyte.

With the advent of genome sequencing methodologies, it was about time that the scientists look clues within the genome of HCC tumor cells that would provide clues for disease progression via virus integration into the liver cells.

Two studies published in the recent issue of Nature Genetics (May 2012) explored the genome of HCC cells for genetic mutations that might be related to HBV and HCV highlighting the types of genetic mutations that underlie the liver cancer hepatocellular carcinoma, including forms of the disease related to hepatitis B and hepatitis C virus infection.

In the first study, Sung et al performed an extensive whole genome analysis using a large sample size of 88 Chinese individuals with HCC This was in the fact first unbiased, genome-wide, HBV-integration map in HCC leading to new recurrent integration sites and molecular mechanisms.

Although integration of viral DNA sequence within HCC genome has been reported in several studies, however, fewer cases of recurring mutations within genes during these integrations have been studied. The reason might be limited sample size in these studies. Tumor and non-tumor adjacent liver cells were surveyed in 81 HBV positive and 7 HBV negative HCC tumor samples. After the survey of whole genome of the 88 patients, several viral integration sites were discovered referred to as breakpoints. The breakpoints were found to be much more common in tumor than normal samples. Although the observed breakpoints were randomly distributed across the genome, a handful or frequently occurring sites referred to as ‘hotspots’ were discovered. The frequency of integration revealed that there were five genes with recurring integrations in HBV tumors- TERT, MLL4, CCNE5, SENP1, and ROCK1.

Apart from genome analysis, expression levels of the 5 genes implicated in the study were determined. In other words, the levels of proteins formed from the genes were compared and it was observed that samples with HBV integration had significantly higher level of protein expression of TERT, MLL4 and CCNE5 than the samples harboring no HBV integration sites. Although not statistically significant, overexpression of SENP1 and ROCK1 genes was also observed in HBV integration samples. This lead to an important conclusion from the study that the five genes that harbor recurrent HBV integrations might be implicated in HCC tumor development and that overexpression of these proteins is a probable molecular mechanism of tumorigenesis.

Interestingly, analysis of the HBV analysis revealed that almost 40% of the HBV genomes were cleaved at approximately 1,800 bp and then integrated into the human genome. The cleaved HBV sites had the necessary machinery (enhancers and ORF replication sites) for protein formation.

The study also confirmed the popular belief that HBV integrations might worsen the prognosis of HCC patients revealing a significant correlation between the number of HBV integrations and the survival of patients.An interesting observation from the study that had not been reported before was that HBV integration was associated with the occurrence of HCC at a younger age.

The study presented convincing evidence that chromosomal instability of HCC genome may originate from HBV integration.

A parallel study published in the same issue of Nature Genetics explored the genome of HCC tumors to gain insights into HBV and HCV-related genomic alterations. The research team sequenced whole-exon (protein forming genomic regions) of 27 liver tumors from 25 patients and compared with the corresponding genome sequences from matched white blood cell samples.

The study involved both HBV-related and HCV-related tumors along with two samples of tumors from individuals without HBV or HCV infection. The genome wide sequencing of HCC tumor cells revealed several mutations that included deletions and mutations of genes with predicted functional consequences. “Considering the high complexity and heterogeneity of [hepatocellular carcinomas] of both etiological and genetic aspects,” they concluded, “further molecular classification is required for appropriate diagnosis and therapy in personalized medicine.” Additionally, recurrent alterations were observed in the four genes – ARID1ARPS6KA3NFE2L2 and IRF2 that had not been previously described in HCC. The comprehensive mutation pattern observed in the study might be indicative of specific mutagenesis mechanisms occurring in tumor cells.

Authors said “Although no common somatic mutations were identified in the multicentric tumor pairs,” further stating “their whole-genome substitution patterns were similar, suggesting that these tumors developed from independent mutations, although their shared etiological backgrounds may have strongly influenced their somatic mutation patterns.”The researchers suggested a major role of chromatin remodeling complexes and involvement of both interferon and oxidative stress pathways in hepatocellular malignant proliferation and transformation based on the genes showing recurrent mutations in the observed genes.

Thus, in both the studies new genes recurrently altered in HCC were identified along with uncovering some important clues relating to the molecular mechanism of virus-associated HCC.

Role of SALL4 in HCC

The oncofetal gene SALL4 is a marker of a subtype of HCC with progenitor-like features and is associated with a poor prognosis. Investigators at Cancer Science Institute of Singapore, National University of Singapore studied the role of oncofetal gene, SALL4 in HCC and the results were published were in a recent issue of New England Journal of Medicine ((Yong KJ, et al, Oncofetal Gene SALL4 in Aggressive Hepatocellular Carcinoma. Yong and colleagues (2013) screened specimens from patients with primary HCC for the expression of SALL4 and carried out a clinicopathological analysis. Loss-of-function studies were then performed to evaluate the role of SALL4 in hepatocarcinogenesis and its potential as a molecular target for therapy. Furthermore, in vitro functional and in vivo xenograft assays were performed to assess the therapeutic effects of a peptide that targets SALL4.

According to the results, SALL4 is an oncofetal protein that is expressed in the human fetal liver and silenced in the adult liver, but it is reexpressed in a subgroup of patients who have HCC and an unfavorable prognosis. Gene-expression analysis showed the enrichment of progenitor-like gene signatures with overexpression of proliferative and metastatic genes in SALL4-positive HCC. Loss-of-function studies confirmed the critical role of SALL4 in cell survival and tumorigenicity. The peptide targeting SALL4 blocked ­SALL4-corepressor interactions that released suppression of PTEN and inhibited tumor formation in xenograft assays in vivo. In conclusion, the results from the study indicate that SALL4 is a marker for a progenitor subclass of HCC with an aggressive phenotype. The absence of SALL4 expression in the healthy adult liver enhances the potential of SALL4 as a treatment target in HCC.

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