Introduction
Esophageal cancer (ESCA) ranks seventh for incidence (604,000) and sixth for mortality (544,000), which means it is responsible for one in eighteen cancer deaths [
1]. Traditional prognostic methods, such as histopathology and tumor staging systems, are of limited use and early detection remains a difficult goal [
2]. Due to the lack of specific methods for early diagnosis and treatment, ESCA patients’ five-year survival rate is remains dismal [
3]. The poor outcome urges to identify robust biomarkers for predicting the prognosis of ESCA patients.
Zinc finger protein encoded by nearly 5% of the human genome is the largest family of transcription factor proteins, which has finger-like DNA binding do-mains and plays an important role in many biological processes [
4]. So far, zinc finger motifs have been classified into eight different classes based on their mainchain conformations and secondary structures around zinc-binding sites, including Cys2his2(C2H2)-like, ZN2/Cys6, Treble clef, zinc band, Gag joint, Taz2 domain-like, zinc-binding ring and metallothionein [
5]. Due to the diversity of zinc finger motifs and these domains, ZFPs play different gene regulatory roles in different cellular environments and stimuli. ZNF306 promotes the development of colorectal cancer by transcriptionally activating integrin β4 and vascular endothelial growth factor [
6]. ZNF384 promotes the proliferation of Hepatocellular carcinoma by directly up-regulating the expression of cyclin D1 [
7]. Upregulation of ZNF554 is a potential tumor suppressor and its decreased expression may lead to the loss of oncogene suppression, activation of tumor pathways, and shorter survival of patients with malignant glioma [
8]. The overexpression of ZNF655 promoted the progression of glioma by binding to the promoter of AURKA [
9]. ZNF410 represents a special class of gene regulators, a conserved TF, which has a unique regulatory role on chromatin subcomplexes [
10].Taken together, these studies indicate that ZNF genes may function as oncogenes involved in the occurrence and progression of cancer.
With the development of large-scale genome sequencing technologies, the integration of prognostic-related genetic markers has improved the level of early diagnosis of cancer compared with traditional clinical parameters. In the current study, we screened prognostic-associated ZNF family genes from TCGA dataset and validated the prognostic value of the six-gene signature using GEO dataset. We also constructed a nomogram based on the riskScore and clinical characteristics to predict individual overall survival (OS). In conclusion, our work may contribute to the early diagnosis of ESCA patients.
Discussion
Esophageal cancer is one of the most common cancers with high morbidity and mortality [
15]. Current therapeutic strategies for ESCA include surgery, chemotherapy, radiotherapy, molecular targeted therapy and their combination [
16,
17]. In addition, immunotherapy is also playing an increasingly significant role [
2,
18]. However, the prognosis remains poor and the overall Five-year survival rate is very low [
19]. Therefore, achieving early diagnosis and effective treatment remains challenging. Identification of novel biomarkers will help assess prognosis, screen out patients in need of Immune intervention and drug therapy. Zinc finger proteins (ZFPs) primarily function as transcription factors in tumorigenesis and tumor progression involved in various tumor, such as esophageal squamous cell carcinoma cells [
20],lung cancer [
21], hepatocellular carcinoma [
7], kidney renal clear cell carcinoma [
22], oral squamous cell carcinoma [
23]. Transcription factors (TFS) are proteins that play important roles in complex biological processes, such as metabolism, autophagy, apoptosis, immune response, stem cell maintenance and differentiation [
24].
The TCGA database of 163 cases of esophageal cancer has improved our ability to diagnose, treat, and prevent cancer [
25]. Based on the mRNA expression matrix and clinical data from the TCGA-ESCA cohort, we identified six prognostic-associated ZNF genes that may be clinically valuable biomarkers. Patients with ESCA were divided into two subgroups with different survival outcomes based on a prognostic model of six ZNF family genes. We also established a risk score model to predict the prognoses of patients with ESCA based on these prognostic genes. Importantly, the ability of the prognostic model to distinguish high-and low-risk patients, and to estimate OS, was similarly validated in the GSE53624 dataset. Moreover, we combined risk score with other clinical variables to conduct a nomogram to establish a quantitative prognostic evaluation method for patients with ESCA.
Our ZNF family gene-based signature included six genes, i.e., ZNF91, ZNF586, ZNF502, ZNF865, ZNF106 and ZNF225. ZNF91 is likely to play an important role in cell proliferation and/or anti-apoptosis, and may serve as a molecular marker for AML [
26]. Upregulation of ZNF91 could promote irradiation resistance by regulating the stem cell-like properties of NSCLC cells. Abnormal expression of ZNF91 is related to the occurrence and development of bladder cancer [
26,
27], colorectal cancer (CRC) [
28]and ovarian cancer [
29,
30]. Our results also showed that ZNF91 is upregulated and plays an oncogenic role in ESCA. Genome-wide differential gene/microRNA signatures show that ZNF502 might be a prognostic biomarker in cytogenetically normal acute myeloid leukemia [
31]. In our study, the expression of ZNF502 is low in esophageal carcinoma, which is associated with poor prognosis. ZNF106 is a RNA-binding protein that binds to the core splicing factor RNA-binding motif protein 39 and localizes to nuclear spots near the spliceosome [
32]. We found that mRNA level of 106 was significantly reduced in ESCA tissues, this was similar to the reported results that ZNF106 expression was downregulated and associated with a good predictive value in Bladder Cancer [
33,
34]. Little research has been done on the role of ZNF225 in ESCA, and a few evidences suggest ZNF225 inhibits autophagy and promotes apoptosis of hepatocellular carcinoma cells. Our findings on ZNF225 are supported by evidence that this ZNF protein serves as prognostic genes. In contrast, the roles of ZNF586, and ZNF865 in ESCA onset and development had not, to our knowledge, been as yet explored. Based on current knowledge, our findings suggest that the six prognosis-related ZNF family genes may exert important roles in the tumorigenesis and progression of ESCA.
By univariate Cox analysis, lasso regression and multivariate Cox analysis, we screened six prognosis-related ZNF family genes to construct the prognostic model. Survival and ROC curve analyses showed that these six genes had good diagnostic ability and could be used to screen out ESCA patients who had poor prognoses.when compared to previously reported models [
35,
36], our model(At 1, 3, and 5 years, the risk score’s AUC values were 0.848, 0.872, and 0.952, respectively) has greater predictive power. However, the specific molecular mechanisms of these six prognosis-related ZNF family genes in ESCA remain unclear, and the underlying molecular mechanisms should be explored. Subsequently, we assessed the relationship between risk score model and clinical variables and found that the risk score model had significantly distinct risk stratification ability in ESCA. Nomogram has long been used in oncology to calculate the prognosis of patients with esophageal cancer based on the relevant clinical parameters [
37‐
39]. We then established a nomogram to more intuitively predict 1-year, 3-year, and 5-year survival estimates in patients with ESCA and found that the risk score was more accurate than the pathological stage and age in predicting OS from TCGA and GEO dataset.
Next, we analyzed the differences in immune cell infiltration and response rates to chemotherapy sensitivity among different groups of patients with ESCA based on the model. ESCA was enriched in immune-suppressive cell populations, including Tregs, exhausted CD8 T, CD4 T and NK cells, M2 macrophages, and tDCs [
40]. Accumulating evidence regards the tumor immune microenvironment can potentially influence the patient’s response to immune checkpoint inhibitors, tumor immunity, such as PD-L1 expression on tumors, tumor-infiltrating lymphocytes and tumor-associated macrophages [
41]. Thus, our model can be used as an indicator to predict immune cell infiltration and immune response in patients with ESCA. At present, surgical resection, radiotherapy and chemotherapy are the main clinical treatment methods for ESCA, However, due to the limited efficacy and serious adverse effects of conventional treatment, the result is still unsatisfactory. As a new treatment method, target therapy has a good application prospect [
41,
42]. In our study, patients in the high-risk group were more sensitive to AP-24,534, BMS-509,744, CGP-082996, HG-6-64-1, MG-132, Midostaurin, Ruxolitinib, Sunitinib, TAE684and Thapsigargin. From what has been discussed above, our results revealed differences in immune cell infiltration and immune response between the groups. ZNF-gene signature for ESCA was able to predict chemotherapy sensitivity and may thus help guide treatment selection.
We further conducted GO and KEGG analyses to evaluate biological functions. Enrichment analysis of biological functions and pathways of the ZNF family gene indicated that these prognosis gene were significantly enriched in Cell cycle, DNA replication and Fanconi anemia pathway.
Summarily, this study found that ZNF genes were differentially expressed in ESCA tissues and the reason may be different from the mechanism in the process of tumor formation. As the largest transcription regulator family in mammals, zinc finger (ZNF) protein expression regulation mechanism is very complex, including Genetic variation [
43], Epigenetic modifications [
44] and Posttranslational regulation [
45]. We used the Kaplan-Meier analysis to study the prognostic significance of the six prognosis-related ZNF family genes and found that ZNF502, ZNF865, ZNF106 and ZNF225 gene expressions were related to good prognoses in patients with ESCA, while high ZNF586 and ZNF91 gene expressions were related to poor prognoses. We further confirmed the expressions of these genes at the tissue level. The results suggested that the signatures of these six genes may assess treatment outcomes and predict patient survival.
However, the current study has multiple limitations. Firstly, there are few normal tissues in TCGA database, which need to be verified by expanded samples. Second, the functional relationship between the ZNF gene signature members and non-tumor cells in the tumor microenvironment, especially infiltrating immune cells, could not be elucidated and requires future in vitro and in vivo studies. The effect on proliferation, invasion and migration of ZNF family genes in ESCA requires further be verified in vitro and in vivo.
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