Zhihua Liu

Identification of genomic alterations in oesophageal squamous cell cancer

Oesophageal cancer is one of the most aggressive cancers and is the sixth leading cause of cancer death worldwide. Approximately 70% of global oesophageal cancer cases occur in China, with oesophageal squamous cell carcinoma (ESCC) being the histopathological form in the vast majority of cases (>90%). Currently, there are limited clinical approaches for the early diagnosis and treatment of ESCC, resulting in a 10% five-year survival rate for patients. However, the full repertoire of genomic events leading to the pathogenesis of ESCC remains unclear. Here we describe a comprehensive genomic analysis of 158 ESCC cases, as part of the International Cancer Genome Consortium research project. We conducted whole-genome sequencing in 17 ESCC cases and whole-exome sequencing in 71 cases, of which 53 cases, plus an additional 70 ESCC cases not used in the whole-genome and whole-exome sequencing, were subjected to array comparative genomic hybridization analysis. We identified eight significantly mutated genes, of which six are well known tumour-associated genes (TP53, RB1, CDKN2A, PIK3CA, NOTCH1, NFE2L2), and two have not previously been described in ESCC (ADAM29 and FAM135B). Notably, FAM135B is identified as a novel cancer-implicated gene as assayed for its ability to promote malignancy of ESCC cells. Additionally, MIR548K, a microRNA encoded in the amplified 11q13.3-13.4 region, is characterized as a novel oncogene, and functional assays demonstrate that MIR548K enhances malignant phenotypes of ESCC cells. Moreover, we have found that several important histone regulator genes (MLL2 (also called KMT2D), ASH1L, MLL3 (KMT2C), SETD1B, CREBBP and EP300) are frequently altered in ESCC. Pathway assessment reveals that somatic aberrations are mainly involved in the Wnt, cell cycle and Notch pathways. Genomic analyses suggest that ESCC and head and neck squamous cell carcinoma share some common pathogenic mechanisms, and ESCC development is associated with alcohol drinking. This study has explored novel biological markers and tumorigenic pathways that would greatly improve therapeutic strategies for ESCC.

Genome-wide association study identifies three new susceptibility loci for esophageal squamous-cell carcinoma in Chinese populations

Esophageal squamous-cell carcinoma (ESCC) is one of the most prevalent cancers worldwide and occurs at a relatively high frequency in China. To identify genetic susceptibility loci for ESCC, we conducted a genome-wide association study on 2,031 individuals with ESCC (cases) and 2,044 controls of Chinese descent using 666,141 autosomal SNPs. We evaluated promising associations in an additional 6,276 cases and 6,165 controls of Chinese descent from different areas of China. We identified seven susceptibility loci on chromosomes 5q11, 6p21, 10q23, 12q24 and 21q22 (ranging from P = 7.48 × 10−12 to P = 2.44 × 10−31); among these loci, 5q11, 6p21 and 21q22 were newly identified. Three variants in high linkage disequilibrium on 12q24 confer their risks to ESCC in a gene-lifestyle interaction manner, with more pronounced risk enhancement seen in tobacco and alcohol users. Furthermore, the identified variants had a cumulative association with ESCC risk (Ptrend = 7.92 × 10−56). These findings highlight the involvement of multiple genetic loci and gene-environment interaction in the development of esophageal cancer.

The role of P53 and MDM2 polymorphisms in the risk of esophageal squamous cell carcinoma.

The tumor suppressor P53 pathway plays a crucial role in preventing carcinogenesis and genetic variations of this pathway may be associated with cancer susceptibility. We tested this hypothesis by examining the contribution of functional polymorphisms in P53 and MDM2 to risk of esophageal squamous cell carcinoma (ESCC). DNA from 758 ESCC patients and 1,420 controls were genotyped for P53 codon 72Arg>Pro and MDM2 309T>G polymorphisms. Odds ratios (OR) and 95% confidence intervals (CI) of ESCC were estimated by logistic regression. We observed an increased risk of ESCC associated with the P53 Pro/Pro (OR, 1.83; 95% CI, 1.43-2.35; P < 0.001) or MDM2 GG (OR, 1.49; 95% CI, 1.16-1.91; P = 0.002) genotype, compared with the P53 Arg/Arg or MDM2 TT genotype, respectively. Interaction between these P53 and MDM2 polymorphisms increased risk of ESCC in a multiplicative manner, with the OR being 3.10 (95% CI, 2.07-4.69) for subjects carrying both P53 Pro/Pro and MDM2 GG genotypes. Significant interactions were observed between these polymorphisms and smoking, with risk being the highest (OR, 5.29; 95% CI, 2.91-9.61) in smokers having both P53 Pro/Pro and MDM2 GG genotypes. The MDM2 GG genotype was also associated with risk of developing poorly differentiated and advanced ESCC compared with the GT or TT genotype (OR for high-grade and stages III-IV versus low-grade and stages I-II = 1.60; 95% CI, 1.00-2.64; P = 0.049). The P53 and MDM2 polymorphisms may be genetic determinants for the development of ESCC.

Genome-wide association analyses of esophageal squamous cell carcinoma in Chinese identify multiple susceptibility loci and gene-environment interactions

We conducted a genome-wide association study (GWAS) and a genome-wide gene-environment interaction analysis of esophageal squamous-cell carcinoma (ESCC) in 2,031 affected individuals (cases) and 2,044 controls with independent validation in 8,092 cases and 8,620 controls. We identified six new ESCC susceptibility loci, of which four, at chromosomes 4q23, 16q12.1, 22q12 and 3q27 had a significant marginal effect (P = 1.78 × 10−39 to P = 2.49 × 10−11) and two of which, at 2q22 and 13q33, had a significant association only in the gene–alcohol drinking interaction (gene-environment interaction P (PG × E) = 4.39 × 10−11 and PG × E = 4.80 × 10−8, respectively). Variants at the 4q23 locus, which includes the ADH cluster, each had a significant interaction with alcohol drinking in their association with ESCC risk (PG × E = 2.54 × 10−7 to PG × E = 3.23 × 10−2). We confirmed the known association of the ALDH2 locus on 12q24 to ESCC, and a joint analysis showed that drinkers with both of the ADH1B and ALDH2 risk alleles had a fourfold increased risk for ESCC compared to drinkers without these risk alleles. Our results underscore the direct genetic contribution to ESCC risk, as well as the genetic contribution to ESCC through interaction with alcohol consumption.

Overexpression of Aurora-A Contributes to Malignant Development of Human Esophageal Squamous Cell Carcinoma

Purpose: Aurora-A/STK15/BTAK, a centrosome-associated oncogenic protein, is implicated in the control of mitosis. Overexpression of Aurora-A has been shown to result in chromosomal aberration and genomic instability. Multiple lines of evidence indicate that Aurora-A induces cell malignant transformation. In the current study, we are interested in investigating the expression of Aurora-A in human esophageal squamous cell carcinoma (ESCC) and characterizing the association of Aurora-A with ESCCmalignant progression. Experimental Design: Aurora-A protein expression was examined in 84 ESCC tissues and 81 paired normal adjacent tissues by either immunohistochemistry or Western blot analysis. In addition, a gene-knockdown small interfering RNA technique was used in ESCC cells to investigate whether Aurora-A contributes to the ability of a tumor to grow invasively. Results: The amount of Aurora-A protein in ESCC was considerably higher than that in normal adjacent tissues. Overexpression of Aurora-A was observed in 57 of 84 (67.5%) ESCC samples. In contrast, <2% of normal adjacent tissue displayed high expression of Aurora-A. Interestingly, overexpression of Aurora-A seemed to correlate with the invasive malignancy of ESCC. Disruption of endogenous Aurora-A using small interfering RNA technique substantially suppressed cell migrating ability. Conclusion: The findings presented in this report show that Aurora-A expression is elevated in human esophageal squamous cell carcinoma and is possibly associated with tumor invasion, indicating that overexpression of Aurora-A may contribute to ESCC occurrence and progression.

Human papillomavirus infection in women in Shenzhen City, People's Republic of China, a population typical of recent Chinese urbanisation.

Select cancer registries report that cervical cancer is relatively rare in the Peoples Republic of China, but may not be representative of the entire country. We carried out a survey of human papillomavirus (HPV) prevalence in 3 samples of women, i.e., general population, factory workers, and tertiary sector workers, in Shenzhen City in 2004. All participants were interviewed and offered gynaecological examination. HPV detection in exfoliated cervical cells was performed using a GP5+/6+ PCR‐based assay. Overall HPV prevalence was 18.4% among the general population (n = 534), 11.2% among factory workers (n = 269) and 18.8% among tertiary sector workers (n = 224). Corresponding prevalence for high‐risk HPV types was 13.5%, 8.2% and 13.8%, respectively. The most commonly found HPV types were HPV16, 52, 58, 31 and 39. HPV prevalence significantly increased with age in the general population, whereas it was highest below age 25 years in tertiary sector workers. Associations of HPV prevalence with indicators of sexual behaviour were stronger among tertiary sector workers than in the other samples of women. High HPV prevalence in all age groups and the appearance of a ‘western‐type’ peak in HPV prevalence among young women employed in the tertiary sector raise important questions concerning the real cervical cancer burden, and its control, in urban China.

Overexpression of Stefin A in Human Esophageal Squamous Cell Carcinoma Cells Inhibits Tumor Cell Growth, Angiogenesis, Invasion, and Metastasis

Purpose: Evidence is accumulating that an inverse correlation exists between stefin A level and malignant progression. The aim of this study is to investigate the role of stefin A in human esophageal squamous cell carcinoma cells and to evaluate the possibility of stefin A for cancer therapy. Experimental Design: We stably transfected stefin A cDNA into human EC9706 or KYSE150 esophageal squamous cell carcinoma cells. Subsequently, we evaluated the effect of stefin A overexpression on cell growth, cathepsin B activity, cell motility and invasion, tumor growth, and metastasis. Immunoanalysis was done to assess the expression of factor VIII and to support the localization of stefin A and cathepsin B. We also evaluated the effect of CA074Me, a selective membrane-permeant cathepsin B inhibitor. Results: Both transfection of stefin A and treatment with 10 μmol/L CA074Me significantly reduced cathepsin B activity and inhibited the Matrigel invasion. Combination of both further reduced cathepsin B activity and inhibited the Matrigel invasion. Overexpression of stefin A delayed the in vitro and in vivo growth of cells and significantly inhibited lung metastasis compared with 50% of lung metastasis in xenograft mice from EC9706 or empty vector cells. Transfection with stefin A showed a dramatic reduction of factor VIII staining in the tumors of xenograft mice. Conclusions: Our data strongly indicate that stefin A plays an important role in the growth, angiogenesis, invasion, and metastasis of human esophageal squamous cell carcinoma cells and suggest that stefin A may be useful in cancer therapy.

Role for Kruppel-like factor 4 in determining the outcome of p53 response to DNA damage.

Cells are incessantly exposed to many sources of genotoxic stress. A critical unresolved issue is how the resulting activation of the p53 tumor suppressor can lead to either cell cycle arrest or apoptosis depending on the extent of DNA damage. The present study shows that the level of Krüppel-like factor 4 (KLF4) expression is inversely correlated with the extent of DNA damage. KLF4 is activated by p53 following cytostatic, mild DNA damage, whereas it is strongly repressed via enhanced turnover of mRNA on severe DNA damage that irreversibly drives cells to apoptosis. Blocking the repression of KLF4 on severe DNA damage suppresses p53-mediated apoptosis, whereas ablation of the KLF4 induction on mild DNA damage shifts the p53 response from cell cycle arrest to cell death. Our results suggest that coordinate regulation of KLF4 expression depending on the extent of DNA damage may be an important mechanism that dictates the life and death decisions of p53.

Functional Role of S100A14 Genetic Variants and Their Association with Esophageal Squamous Cell Carcinoma

S100 proteins have been implicated in various human diseases, including certain types of cancer. Among them, S100A14 is down-regulated in esophageal squamous cell carcinoma (ESCC). In this study, we sought to identify functional genetic variants in the S100A14 locus and assessed their associations with susceptibility to ESCC. Thirty individual DNA samples were sequenced to search for genetic variations in S100A14, and the function of the variants was investigated by a set of biochemical assays. A case-control analysis was performed in 1,021 patients with ESCC and 1,253 control subjects. Odds ratios and 95% confidence intervals (95% CI) were computed by logistic regression model. Four single nucleotide polymorphisms, -43A>G, 461G>A, 1493A>G, and 1545A>T, were identified in the S100A14 locus and they are in absolute linkage disequilibrium. Among them, the 461G>A change was shown to diminish a P53-binding site and is therefore associated with decreased expression of S100A14 in vitro and in vivo in the target tissues. Case-control analysis showed that the 461A allele was associated with susceptibility to ESCC among smokers, with the ORs being 2.01 (95% CI, 1.50-2.69) or 2.10 (95% CI, 1.37-3.22) for the 461GA or 461AA genotype, respectively, compared with the 461GG genotype. These data constitute strong evidence in support of the notion that S100A14 might function as a cancer suppressor working in the P53 pathway and play a role in esophageal carcinogenesis.

Genomic analysis of oesophageal squamous-cell carcinoma identifies alcohol drinking-related mutation signature and genomic alterations

Approximately half of the worlds 500,000 new oesophageal squamous-cell carcinoma (ESCC) cases each year occur in China. Here, we show whole-genome sequencing of DNA and RNA in 94 Chinese individuals with ESCC. We identify six mutational signatures (E1–E6), and Signature E4 is unique in ESCC linked to alcohol intake and genetic variants in alcohol-metabolizing enzymes. We discover significantly recurrent mutations in 20 protein-coding genes, 4 long non-coding RNAs and 10 untranslational regions. Functional analyses show six genes that have recurrent copy-number variants in three squamous-cell carcinomas (oesophageal, head and neck and lung) significantly promote cancer cell proliferation, migration and invasion. The most frequently affected genes by structural variation are LRP1B and TTC28. The aberrant cell cycle and PI3K-AKT pathways seem critical in ESCC. These results establish a comprehensive genomic landscape of ESCC and provide potential targets for precision treatment and prevention of the cancer.