Esophageal cancer is the eighth most common cancer and the sixth most common cause of cancer-related death in the world. It is a severe malignancy originated from the epithelium of the esophagus. Approximately 80% of the new cases occur in less developed regions and 60% of these cases occur in China. In China, the incidence of esophageal cancer in men is about twice of that in women, ranging the fourth most common cancer in men following lung, stomach, and liver cancers. In women, it is the fifth most common cancer. Esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) are the 2 main subtypes of esophageal cancer and in China, ESCC accounts for 90% to 95% of esophageal cancer.[3,4] Most victims live in the “esophageal cancer belt,” which stretches from North Central China westwards through Central Asia to Iran. During the last 3 decades, as a result of socioeconomic development and lifestyle changes, mortality caused by ESCC has remarkably decreased by 41.6% in China. However, the 5-year survival rate for ESCC is still dismal, ranging from 15% to 20%.
The pathogenesis of esophageal cancer has not been fully elucidated, which impedes the curative treatment of this cancer. Some studies have suggested that environmental factors such as heavy alcohol drinking, cigarette smoking, and poor diet habits may be associated with the development of esophageal cancer.[7–10] However, only a small portion of individuals exposed to these risk factors is affected, suggesting that there is difference in susceptibility to esophageal cancer among individuals. It is now well recognized that genetic makeup is an important factor affecting the occurrence and development of complex diseases. Genetic variations may cause diseases directly or through the interactions with environmental risk factors resulting in complex diseases.[11–13] Therefore, it is interesting and important to identify genetic and genomic variations that play etiological roles in ESCC and to clarify their functions. Up to date, 2 main approaches have been used to identify disease-associated variations. One is genome-wide association studies (GWAS), which have been proved to be an effective way to search for genetic susceptibility genes and genetic variants. Another is next-generation sequencing (NGS) technologies, which are able to reveal the profiles of somatic mutations and other genomic changes in cancer cells. In recent years, by employing both GWAS and NGS, we and other groups have made great efforts in elucidating the genetic variations and somatic genomic changes that play important roles in the development and progression of ESCC, which may also provide basis for individualized prevention and clinic care of the cancer. This review describes the progress of these studies and discusses the potential use of the results in precise medicine of ESCC.
GWAS of esophageal cancer
GWAS offers a powerful approach in searching for genes confer susceptibility to complex disease. It is a cost-effective way to analyze the genetic differences among affected individuals with a specific disease and individuals without the disease. Several GWAS of esophageal cancer have been reported and the first was conducted by Cui et al in 2009 in Japanese. This study revealed that single nucleotide polymorphisms (SNPs) in ADH1B (rs1229984) and ALDH2 (rs671) are associated with ESCC in a Japanese population. These 2 genes are involved in the metabolism of alcohol and tobacco by-products. Individuals with both genetic and lifestyle-related risk factors had a nearly 190 times higher risk for ESCC than those had neither of the risk factors. In 2010, Wang et al performed a GWAS of ESCC in Chinese populations and identified 2 previously unknown susceptibility loci, PLCE1 at 10q23 and C20orf54 at 20p13. These 2 genes have important biological functions. PLCE1 is involved in regulation of cell growth, differentiation, apoptosis, and angiogenesis. C20orf54 is responsible for transporting riboflavin, and deficiency of riboflavin has been documented as a risk factor for ESCC. At the same time, Abnet et al reported a GWAS of gastric cancer and ESCC in Chinese subjects and found a notable non-synonymous SNP rs2274223 in PLCE1 associated with ESCC and gastric cancer. They showed that the association was stronger for tumors in the cardia but significant association was absent for tumor in the non-cardia stomach. In 2011, our group reported a GWAS in 2031 individuals with ESCC and 2044 controls of Chinese descent. In this study with larger sample size, 3 new susceptibility loci, 5q11, 6p21 and 21q22, were identified. Furthermore, we also found that 3 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. In 2012, another GWAS of ESCC was published by our group, in which we identified 6 new ESCC susceptibility loci. Of them, 2 loci at 2q22 and 13q33, respectively, had significant associations only in alcohol drinkers. Gene–environment interaction analysis revealed that drinkers with both of the ADH1B and ALDH2 risk alleles had a 4-fold increased risk for ESCC compared with drinkers without these risk alleles. In 2014, we in collaboration with other groups conducted a joint analysis of 3 GWAS in Chinese populations. This analysis consisted of 5337 ESCC cases and 5787 controls in GWAS stage and a total of 9654 ESCC cases and 10,058 controls in replication stages. In a logistic regression model adjusted for age, sex, study, and 2 eigenvectors, 2 new loci at 5q31 and 17p13 were identified associated with ESCC. Furthermore, this joint study also identified a new susceptibility locus in the human lymphocyte antigen class II region at 6p21 that is unique to the populations in the Taihang Mountain region where is at high risk of ESCC, suggesting a relevance to some infections.
ESCC is more prevalent in Asian countries while EAC is more prevalent in Western countries. Levine et al reported a GWAS in 2390 EAC cases and 3175 individuals with Barrett esophagus, in which they identified 3 new susceptibility loci, CRTC1 at 19p13, BARX1 at 9q22, and near the transcription factor FOXP1 at 3p14. In 2016, Gharahkhani et al combined 6167 patients with Barrett esophagus, 4112 individuals with EAC and 17,159 representative controls in 4 GWAS in Europe, North America, and Australia and identified 8 new risk loci associated with either Barrett esophagus or EAC, of which the locus (rs9823696) near HTR3C and ABCC5 was specifically associated with EAC. These findings of GWAS suggest a great difference in genetic susceptibility factors for ESCC and EAC, which might reflect the difference in environmental etiological factors for these 2 subtypes of esophageal malignancies.
Gene–environment interaction in the development of esophageal cancer
Cancer is a complex disease resulting from environmental exposures, multiple genetic loci, and gene–environment interactions. As well known, not all exposed individuals develop cancer, indicating that genetic susceptibility to environmental risk factors might play an important role. However, most published GWAS only focus on analyzing a single genetic variation, ignoring the effects of gene–gene and gene–environment interactions. Therefore, it would be important to use effective statistical methods in the in-depth analysis of GWAS data, which may be helpful to comprehensively reveal the genetic basis of certain cancers. Many previous studies have pointed out that alcohol drinking is a risk factor for developing esophageal cancer.[14,17,18,22–25] In cells, alcohol is oxidized by alcohol dehydrogenases (ADHs) to carcinogenic aldehyde, which is further oxidized by aldehyde dehydrogenases (ALDHs) to non-toxic acetate.[26,27] We have identified ESCC-associated risk variants at 4q23, a locus the ADH cluster is located, and each had a significant interaction with alcohol drinking in its association with ESCC. We also confirmed the association between the ALDH2 variants at 12q24 and risk of ESCC. Furthermore, our analysis showed that drinkers having both ADH1B and ALDH2 risk alleles had a 4-fold increased risk for ESCC compared with drinkers without theses risk alleles. It is well known that alcohol drinking not only increases risk of esophageal cancer, but also increases risk for other gastrointestinal caners including gastric cancer and colorectal cancer. Based on these results and other epidemiological evidence, we suggest avoiding drinking as a effective measure for prevention of ESCC, especially among individuals who carry variant genotypes of alcohol-metabolizing genes. Further studies are also needed to identify risk gene–gene and gene–environment interactions in ESCC.
Progress in genomic research of esophageal cancer
The Cancer Genome Atlas (TCGA) project has recently conducted a comprehensive molecular analysis of 164 carcinomas of the esophagus derived from Western and Eastern populations. The results indicate that ESCC commonly has genomic amplifications in CCND1, SOX2, and TP63, while ERBB2, VEGFA, GATA4, and GATA6 are more frequently amplified in EAC. These results suggest that the 2 different histological subtypes of esophageal cancer are distinct in their molecular characteristics. ESCC emerges as a disease more reminiscent of other squamous cell carcinomas than EAC, which itself bears striking resemblance to gastric cancer. For this reason, treatment of ESCC and EAC should be different. Comprehensive analysis of genomic characteristics would provide a framework to facilitate more rational categorization of these tumors and a foundation for new therapies. With the advances in NGS technologies, the genomic changes of esophageal cancer have been revealed. It has been shown that NOTCH signaling pathway plays an important role in the development of esophageal cancer. Agrawal and colleagues have reported a difference between ESCC and EAC in genomic alterations, including general mutation spectra and loss of function mutations in NOTCH1. Inactivation mutations of NOTCH1 were identified in 21% of ESCC patients but not in EAC and there were more indels in ESCC than EAC. Furthermore, Cheng et al reported different patterns of NOTCH1 mutations between different stages of ESCC; the NOTCH1 mutation rate is higher among patients with stage I compare with those with stage III. NOTCH signaling is well known to play a role in developmental processes and in regulating the self-renewal of tissues.NOTCH1 and NOTCH3 have been shown to play important roles in the differentiation of esophageal cancer cells. These results indicate that mutations in NOTCH signaling pathway may be important drier mutations for the development and progression of ESCC and they commonly occur in early stage of the cancer. Epithelial growth factor receptor (EGFR) also plays a vital role in the development of esophageal cancer. EGFR is overexpressed in 50% of ESCC patients and high expression level is often correlated with lymph node metastasis and poor prognosis. However, EGFR is overexpressed only in 25% of EAC patients, indicating that EGFR mutation may be more important in ESCC and may serve as a therapeutic target. Another gene showing frequent mutation in ESCC is PI3KCA. One study reported that the PI3KCA mutation rate is 21% in ESCC and 6% in EAC. Other studies have also reported that alterations of genes involved in the PI3K/AKT signaling pathway, such as PTEN deletion and mTOR overexpression, are significantly associated with poor prognosis of ESCC.[38–40] In recent years, with the development of genomic analysis approaches, integrated analysis of somatic mutations, genetic variations, and lifestyle factors has open an avenue to elucidate the origins of specific mutations in cancer. Our group has identified 6 mutational signatures in ESCC via whole-genome sequencing of DNA and RNA of ESCC tissue samples from Chinese individuals. Interestingly, we found that signature 4 is unique in ESCC and is associated with alcohol intake and genetic variants in alcohol-metabolizing enzymes ALDH2 and ADH1B. The results provide strong evidence that alcohol intake is an important risk factor of ESCC. In addition, the study also reported 20 driver genes in ESCC, including TP53, NOTCH1 and MLL2. These findings provide a basis for precise prevention and treatment of ESCC.
Functional study in the post-GWAS era
Findings of cancer-associated genetic and genomic alterations by GWAS and NGS are very important in understanding the pathogenesis of human cancer. However, functional studies are needed to elucidate the acting mechanisms of these changes at the DNA level. Also, the specific functions of most genetic variants and somatic mutations remain to be fully elucidated. Integration of GWAS findings with biochemical and molecular and cell biological experiments is an important strategy in the post-GWAS era. In recent years, our laboratory as well as others has made great efforts to pursuit this post-GWAS task. A genetic variant in the promoter region of the carrier family 39 member 6 (SLC39A6) gene has been identified to be associated with the survival time in ESCC patients by our group in 2013. Recently, we have characterized the function of SLC39A6 in ESCC. We have shown that SLC39A6 is aberrantly overexpressed in ESCC and the high levels are correlated with shorter survival time in individuals with advanced ESCC, suggesting that SLC39A6 may play an important role in the development of ESCC. Further investigation showed that knockdown of SLC39A6 substantially reduces proliferation and metastasis of ESCC cells. SLC39A6 is a cellular zinc transporter and our study showed that overexpression of SLC39A6 significantly increases intracellular levels of zinc, which promotes the activation of PI3K signaling pathway. In addition, ESCC cells overexpressing SLC39A6 increase expression of genes involved in metastasis, such as matrix metalloproteinase (MMP) 1, MMP3, MYC and snail family transcriptional repressor 2 (SNAIL2). Another example of functional characterization of ESCC-associate genetic variant is about PLCE1, which has been linked to ESCC in previous GWAS.[15–17] We performed fine mapping of the PLCE1 locus and identified a 4-nucleotide insertion/deletion variation, which creates a silencer element that represses PLCE1 transcription via long-range interaction with PLCE1 promoter mediated by OCT2 binding. Decreased PLCE1 expression tends to facilitate ESCC cells to proliferate. Another recent study reported by our group has investigated the functions of 47 genes located in the 11 susceptible loci identified in our previous GWAS. By high content screening, we identified BRCA1-associated protein (BRAP) associated with ESCC cell migration and invasion. BRAP gene is frequently amplified in ESCC and over expression of BRAP is correlated with poor survival in ESCC patients. We found that BRAP overexpression results in increased activation of nuclear factor kappa B and high expression of matrix metallopeptidase 9 and vascular epithelial growth factor C, which leads to metastasis of ESCC. The above described example studies provide strong evidence that GWAS is effective in discovering cancer susceptible genes and most genetic variations identified by GWAS may really have functional significance. One of the most important challenges in the post-genome era is to characterize the biological functions of genetic variations and genomic alterations in diseases and to make them applicable in disease prevention and patient care.
Summary and perspective
Precise prevention and treatment of ESCC has been an important research subject in China and some other parts of the world where ESCC is prevalent. With great efforts, important progresses have been achieved in the field of characterizing esophageal cancer genome. More than 10 susceptible genes or loci for ESCC have been identified by GWAS. These findings may provide important basis for precise prevention of ESCC. The interaction between alcohol consumption and genetic variations in alcohol-metabolizing genes in ESCC is a typical example. Excessive alcohol drinking and forced drinking are quite common in China and the frequencies of alcohol-metabolizing gene variations are relatively high in Chinese populations. In this context, limited drinking may be important for prevention of ESCC among individuals who are ADH and ALDH2 mutants and who are in low social and economic status. Moreover, whole-genome sequencing studies have resulted in a profile of ESCC genome; however, the genomic changes in ESCC cells are extremely complicated. Although some changes might be drugable or serve as markers for prognosis, the high complexity also points out big challenge in precision treatment of ESCC. Again, these findings also emphasize the importance of precision prevention of the cancer.
Despite significant progresses have been achieved in genetic and genomic studies on ESCC, further studies are warranted. More effective statistical approaches should be developed and utilized to analyze GWAS data, especially emphasizing on the gene–environment interaction. Furthermore, the function roles and the action mechanism of genetic variations identified by GWAS should be clarified through detailed functional studies. More importantly, it would be very interesting and important to integrate genetic susceptibility markers into prospective cohort studies to test their predictive effects on ESCC and early detection. For NGS of ESCC genome, studies with larger sample sizes are extremely needed to provide reliable results and to extend previously reported findings, especially to make the results useful in patient care.
Human genome consists of 2% protein coding sequences and 98% non-coding regions. It is now recognized that certain non-protein coding regions may generate non-coding RNAs (ncRNAs) such as microRNAs, long ncRNAs (lncRNAs) as well as PIWI-interacting RNAs (piRNAs), which have been shown to play important functions in normal tissues and in cancer cells including epigenetic regulation of gene expression and interaction with DNA, mRNA, and protein macromolecules. If aberrant expression and regulation, ncRNAs may demolish the homeostasis of normal cells and turn out to be oncogenic. Although some ncRNAs in ESCC have been investigated, their roles in the development and progression of ESCC are largely unknown. Therefore, in addition to examination of protein-coding gene changes, more research attentions should also be paid to ncRNAs in the future studies on ESCC. By integrating genetic and epigenetic results revealed by comprehensive studies with environmental and lifestyle factors, we may approach to precision medicine of ESCC.
QC contributed to the manuscript preparation based on the preliminary outline from DL, CW and DL were involved in manuscript review and revision, DL provided corrections and inputs to the progressing manuscript. All authors read and approved the final manuscript.
This work was supported by National Key Basic Research and Development Program (973 project, grants No. 2015CB553901 to DL and 2013CB910301 to CW), National Key R & D Program (grant No. 2016YFC1302100 to DL), and CAMS Innovation Fund for Medical Sciences (Grants No. 2016-12M-3-019 to DL and 2016-12M-4-002 to CW).
Conflicts of interest
The authors declared that they have no conflicts of interest.
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