Esophageal squamous cell carcinoma (ESCC) is the most common type of human esophageal cancer with high mortality due to late stage diagnosis. Efforts have been made to figure out the genetic events underlying its carcinogenesis and progression, but the molecular mechanisms of these processes remain elusive. To identify the candidate genes involved in ESCC, literature about significantly mutated genes (SMGs) was extensively reviewed and gene expression profiles of GSE161533, GSE20347 and GSE77861 were downloaded from the Gene Expression Omnibus (GEO) database. Following the identification of 230 differentially expressed genes (DEGs), hub gene identification was performed by the plug-in MCODE in Cytoscape software. 14 hub genes were identified which were enriched in cell cycle, DNA replication and p53 signaling pathway. In summary, genes mentioned in this study may provide potential targets for treatment and diagnosis of ESCC and help us better understand the pathogenesis and progression of ESCC from genetic perspective.
Research Article
Review and screening of key genes in esophageal squamous cell carcinoma
https://doi.org/10.21203/rs.3.rs-498257/v1
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esophageal squamous cell carcinoma (ESCC)
cancer
genetics
mutated genes
Esophageal cancer is the eighth most common cancer and the sixth leading cause of cancer-related mortality in the world 1,2. ESCC is the major histological type accounting for about 90% of the 456,000 incident esophageal cancers each year 3. The 5-year survival rate for ESCC is about 18%, a number that reflects limited approaches of early diagnosis and treatment of ESCC 4. Thus, there is a great need to further figure out the molecular mechanisms and to develop better diagnostic and therapeutic methods for ESCC. The pathogenesis of ESCC is believed to be a multi-step process and the genetic determinants remain elusive. Increasing evidence shows that gene mutation plays a key role in ESCC tumorigenesis and tumor progression. These genes include upregulated genes ADAM29, AJUBA, CBX4/8, CCND1(BCL1/PRAD1), EGFR(ERBB1), ERBB2(HER-2), FAM135B, FGFR1, KMT2D(MLL2/MLL4/ALR), MMP14, MYC, NOTCH, NRF2(NFE2L2), PIK3CA, RB1, SOX2, TP53, XPO1, YAP1 and downregulated genes CDKN2A, CREBBP/EP300, CUL3, FAT1, FBXW7, KMT2C(MLL3), PTEN, TET2, TGFBR2, ZFP36L2, ZNF750 5-11(Table 1). Genes involved in cell cycle, the Notch signaling pathway, epigenetic processes and RTK/PI3K/AKT circuit are frequently altered 12. Cell cycle progression is changed mostly by TP53 mutation, CDKN2A deletion/mutation and CCND1 amplification 5. TP53 is the most significantly mutated genes (SMGs) in ESCC with mutation frequency reaching 93% 12. NOTCH plays a dual role as both a tumor suppressor pathway and an oncogenic pathway, for which further studies are warranted 13. Abudureheman et al. have shown that overexpression of KMT2D facilitates ESCC tumor progression, and that it may exert oncogenic role via activation of epithelial-to-mesenchymal transition (EMT) 14. In a large-sample study with ESCC in China 15, PIK3CA was significantly overexpressed in cancer tissue and its overexpression was independently associated with higher risk of local recurrence 15. EGFR and FGFR1 were the most often amplified RTK/RAS-related genes in ESCC 9, of which the inhibitors have been under therapeutic evaluation 16,17. It’s worth noting that conflicting results were found in studies about prognostic value of TP53 overexpression in ESCC 18-21, of which the biological functions were undoubted. In general, there are still significant gaps to fill to figure out the exact mechanism of carcinogenesis and to develop precision treatment means of ESCC.
Gene chip or gene profile is an advanced gene detection technique that can quickly detect all the genes within the same sample at one time 22. Last two decades have seen more and more studies of genetic alterations in cancers via microarray technology and bioinformatics analysis, which have helped us identify the differentially expressed genes (DEGs) and related pathways in ESCC. However, the results were always limited or inconsistent because of tissue or sample heterogeneity in independent studies, or the results were produced from a single cohort study. Thus, integrated bioinformatics analysis combined with gene profiling technique might be innovative and solve this disadvantage. In this work, we downloaded three microarray datasets GSE161533, GSE20347, GSE77861 from NCBI-Gene Expression Omnibus database (NCBI-GEO) (Available online: https://www.ncbi.nlm.nih.gov/geo) followed by DEGs identification via GEO2R analysis. Subsequently, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and protein-protein interaction (PPI) network analysis were conducted to help us understand the molecular mechanisms of carcinogenesis and progression of ESCC. In summary, 230 DEGs and 14 hub genes were found in this study, which may serve as potential biomarkers for individualized prevention, early diagnosis and precise treatment.
Table1. Expression of significantly mutated genes (SMGs) and their functions in ESCC
|
Gene Symbol |
Expression in ESCC |
Function of the Product |
Reference |
|
ADAM29 |
upregulated |
a member of ADAM family which plays an important role in regulating cell-to-cell or cell-matrix interactions |
6,23 |
|
AJUBA |
upregulated |
functions as a regulator of the Hippo pathway |
7,24 |
|
BAP1 |
lack study |
a deubiquitinating enzyme involved in the regulation of cell growth |
25 |
|
CBX4/8 |
upregulated |
promotes cell proliferation, colony formation,and cell invasion |
8 |
|
CCND1 (BCL1/PRAD1) |
upregulated |
regulation of cell cycle |
7,26 |
|
CDKN2A |
downregulated |
regulation of cell cycle |
9 |
|
CREBBP |
lack study |
Serves as global transcriptional coactivators and integrators of numerous signaling pathways |
7 |
|
CUL3 |
downregulated |
core component of multiple cullin-RING-based BCR (BTB-CUL3-RBX1) E3 ubiquitin-protein ligase complexes |
10,11 |
|
DCDC1 |
lack study |
microtubule-binding protein which plays an important role in mediating dynein-dependent transport |
11 |
|
EGFR(ERBB1) |
upregulated |
stimulates proliferation of different cell types |
27,28 |
|
EP300 |
upregulated |
functions as histone acetyltransferase and regulates transcription via chromatin remodeling |
29 |
|
ERBB2 (HER-2/NEU/NGL/MLN19) |
upregulated |
regulates cell growth and differentiation |
30 |
|
FAM135B |
upregulated |
promotes cell proliferation likely through its direct interaction with growth factor GRN |
6,23,31 |
|
FAT1 |
downregulated |
inhibits cell proliferation and migration |
32 |
|
FBXW7 |
downregulated |
one of the F-box proteins inducing the degradation of positive cell-cycle regulators |
33-35 |
|
FGFR1 |
upregulated |
regulation of cell proliferation, differentiation, migration, and angiogenesis |
5,36 |
|
KDM6A(UTX) |
lack study |
a histone demethylase essential for cellular reprogramming |
37 |
|
KMT2C(MLL3) |
downregulated |
regulation of expression of genes involved in cell growth and migration |
38 |
|
KMT2D |
upregulated |
histone methyltransferase |
7,14 |
|
MMP14 |
upregulated |
endopeptidase that degrades various components of the extracellular matrix |
24 |
|
MYC |
upregulated |
transcription factor that binds DNA in a non-specific manner |
39 |
|
NOTCH |
upregulated |
regulation of squamous differentiation |
13 |
|
NRF2(NFE2L2) |
upregulated |
transcription factor that plays a key role in the response to oxidative stress |
40,41 |
|
PIK3CA |
upregulated |
the p110α catalytic subunit of PI3K |
15,42,43 |
|
PTCH |
lack study |
a key modulator of signaling in the Hh pathway |
44 |
|
PTEN |
downregulated |
a phosphatase which inhibits cell migration, spreading and focal adhesions |
45 |
|
RB1 |
upregulated |
inhibits cell migration and invasion |
46 |
|
SOX2 |
upregulated |
transcriptional regulator having crucial roles in maintenance of progenitor and neural stem cells and neuroendocrine differentiation |
8,47 |
|
TET2 |
downregulated |
dioxygenase that catalyzes the conversion of the modified genomic base 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) |
48,49 |
|
TGFBR2 |
downregulated |
a key mediator of TGF-β signaling |
50,51 |
|
TP53 |
upregulated |
regulation of cell cycle, apoptosis and DNA damage repairing |
18-21 |
|
XPO1 |
upregulated |
mediates the nuclear export of cellular proteins (cargos) bearing a leucine-rich nuclear export signal (NES) and of RNAs |
5 |
|
YAP1 |
upregulated |
the critical downstream regulatory target in the Hippo signaling pathway that plays a pivotal role in organ size control and tumor suppression |
24,52 |
|
ZFP36L2 |
downregulated |
zinc-finger RNA-binding protein that destabilizes several cytoplasmic AU-rich element (ARE)-containing mRNA transcripts |
10 |
|
ZNF750 |
downregulated |
transcription factor involved in epidermis differentiation |
53 |
Identification of DEGs in ESCC
After analyzing with GEO2R with adj. P value < 0.01, |log FC| > 1, DEGs (1504 in GSE161533, 1680 in GSE20347 and 972 in GSE77861) were identified. The intersection of three gene sets of DEGs contains 230 genes as shown in venn diagram (Fig.1), which consists of 144 upregulated genes and 86 downregulated genes between normal and ESCC tissues.
GO and KEGG enrichment analysis of DEGs
To annotate the DEGs, GO and KEGG enrichment analysis were performed using DAVID, with P value<0.05 considered significant. GO analysis results (Fig.2) showed that changes in biological processes (BP) were mainly enriched in oxidation-reduction process, positive regulation of cell proliferation, cell-cell adhesion and inflammatory response. Changes in cellular components (CC) of DEGs were significantly enriched in cytoplasm, extracellular exosome, cytosol and extracellular space. Changes in molecular function (MF) of DEGs were enriched in calcium ion binding, protein homodimerization activity, cadherin binding involved in cell-cell adhesion and actin binding. And KEGG analysis showed that DEGs mainly enriched in transcriptional misregulation in cancer and p53 signaling pathway.
PPI network construction and hub genes identification
Prediction of the functional interaction was conducted by STRING online and the PPI network of DEGs was constructed by Cytoscape (Fig.3). Subsequently, 14 hub genes were identified with MCODE score > 10 (Fig.4).
Hub gene analysis
Among all hub genes, only ESPL1 is down-regulated. GO and KEGG analysis network of hub genes was performed using ClueGO (Fig.5). Result showed that hub genes were mainly enriched in positive regulation of mitotic cell cycle phase transition, regulation of cytokinesis and DNA replication origin binding. Subsequently, we conducted an extensive literature search on the hub genes.
AURKA, which is significantly overexpressed in various cancers including ESCC 54 has been reported to contribute to the malignant development of ESCC 55 and Jin et al. have revealed part of the mechanism underlying this process 56. Ke et al. showed that downregulated CDC6 functioned as downstream molecule of RYBP in the inhibition of cell proliferation in ESCC 57. An association between overexpression of DTL and detrimental outcome in basal-like and luminal breast cancer and non-small cell lung adenocarcinomas but not esophagus-stomach cancer was found in another bioinformatic analysis 58. The gene ECT2 encoding guanine nucleotide exchange factor (GEF) functions as an oncogene in a wide spectrum of cancers 59. Sun et al. 60 showed that ECT2 promoted proliferation and metastasis of ESCC via the RhoA-ERK signaling pathway. And the overexpression of ECT2 often suggests a poor outcome in cancers, especially in breast cancer 61,62 , gastric cancer 63, non-small-cell lung cancer 64,65, and ESCC 65. ESPL1, the dysregulation of which plays an important role in the development of aneuploidy 66, is a candidate oncogene in luminal B breast cancers 67. Studies have revealed the significant association between high expression of FOXM1 and poor outcome of ESCC 68,69. GTSE1 was found to promote malignant behavior in hepatocellular carcinoma (HCC) 70, confer to cisplatin resistance in gastric cancer cells 71 and to be involved in breast cancer progression 72. Overexpression of KIF14 in ESCC was validated in a study, in which KIF14 was a downstream gene regulated by the miR-375/MMP13 axis 73. The overexpression of MCM10 in ESCC has been confirmed in an experiment using semiquantitative reverse transcription-PCR 74. MCM2, of which the diagnostic value has been confirmed in different types of cancers 75-77 was found to be a more reliable and useful marker than Ki67 in assessing tumor growth and tumor aggressiveness in patients with ESCC 78 and in screening patients at high risk of ESCC in mass surveys 79. RFC4 has been found to be overexpressed in several cancers 80, while study about its role in ESCC remains blank. RRM1 has been found to be an oncogene in lung cancer 81, the overexpression of which is involved in tumor progression 82 and is transforming to the therapeutic target 83,84. A large-scale, long-term follow-up retrospective analysis 85 showed that TOP2A expression was not only associated with perineural invasion and poorer differentiation, but it could be also an independent prognostic factor. Additionally, as TOP2A is a specific marker for the use of chemotherapeutic drugs such as anthracycline, therapy targeting TOP2A protein may be an appropriate way of individualized treatment and improving the prognosis of ESCC patients. Studies 86,87 using immunohistochemical analysis confirmed that UBE2C protein expression was upregulated in all ESCC cases, but absent in the histologically normal tumor surrounding tissues, pointing out its role as a diagnostic biomarker for ESCC. Besides, high expression of UBE2C is a marker of poor prognosis in ESCC 87.
Table2. Fuction and clinical significance of 14 hub genes with MCODE score ≥10. “→” indicates “was significantly associated with”
No competing interests reported.