Association of β-Catenin, APC, SMAD3/4, Tp53, and Cyclin D1 Genes in Colorectal Cancer: A Systematic Review and Meta-Analysis

Objectives Accumulating evidence indicates that the expression and/or variants of several genes play an essential role in the progress of colorectal cancer (CRC). The current study is a meta-analysis undertaken to estimate the prognosis and survival associated with CTNNB1/β-catenin, APC, Wnt, SMAD3/4, TP53, and Cyclin D1 genes among CRC patients. Methods The authors searched PubMed, EMBASE, and Science Direct for relevant reports published between 2000 and 2020 and analyzed them to determine any relationship between the (immunohistochemically/sequencing-detected) gene expression and variants of the selected genes and the survival of CRC patients. Results The analysis included 34,074 patients from 64 studies. To evaluate association, hazard ratios (HRs) were estimated for overall survival (OS) or disease-free survival (DFS), with a 95% confidence interval (CIs). Pooled results showed that β-catenin overexpression, APC mutation, SMAD-3 or 4 loss of expression, TP53 mutations, and Cyclin D1 expression were associated with shorter OS. β-Catenin overexpression (HR: 0.137 (95% CI: 0.131–0.406)), loss of expression of SMAD3 or 4 (HR: 0.449 (95% CI: 0.146–0.753)), the mutations of TP53 (HR: 0.179 (95% CI: 0.126–0.485)), and Cyclin D1 expression (HR: 0.485 (95% CI: 0.772–0.198)) also presented risk for shorter DFS. Conclusions The present meta-analysis indicates that overexpression or underexpression and variants of CTNNB1/β-catenin, APC, SMAD3/4, TP53, and Cyclin D1 genes potentially acted as unfavorable biomarkers for the prognosis of CRC. The Wnt gene was not associated with prognosis.


Introduction
Globally, cancer is the second leading cause of death after heart disease, and it is a prominent health issue. More specifically, colorectal cancer (CRC) is the third leading cause of death among men and women [1]. Unlike many other types of cancer, the survival rate for CRC has not changed a great deal. Recent studies showed that the prognostication of CRC depends upon the clinicopathological factors and the stages of tumor characteristics and reported the association with survival times and clinical outcomes [2][3][4]. Several susceptibility studies on the association of a genetic variant and CRC have been reported [5]. e solid tumors of CRC have served as genetic and biological paradigms and instigated to conduct studies on early detection [6], prevention [7], risk stratification [8], and treatments [9]. However, a greater understanding and identification of genetic biomarkers involving molecular and genetic pathways with improved sensitivity and specificity could improve screening for and expedite the diagnosis of CRC, yielding better outcomes. Currently, the prediction of outcomes in CRC relies heavily on traditional cancer characterization methods, including clinicopathological characteristics, such as staging, tumor size, invasion, tumor sidedness, and metastasis. It contributes to CRC's high mortality rate and tendency for poor prognosis with disappointing survival rates [10]. e uses of molecular prognostic biomarkers to forecast the progression of the condition and likely survival have interested scholars for some time [11]. However, CRC is a very diverse disease, and it is associated with complex interactions between genetic biomarkers and environmental risk factors. In addition, transduction pathways, namely transforming growth factor β-suppressor of mothers against decapentaplegic (TGFβ-SMADs), wingless/integrated (Wnt), and tumor suppressor protein (p53), play an essential role in the initiation and development of CRC [4]. e tumor protein p53 gene (Tp53) located at chromosome 17p13 consists of 90% of missense mutations. Furthermore, studies have reported that genetic variations, particularly at codon 72 Pro/Arg gene polymorphism of the Tp53 gene, could affect the prognosis and treatment of CRC [12]. e Wnt signaling pathway is of particular interest because of its vital function in embryogenesis and tissue homeostasis. Many studies have identified the excessive activation of Wnt signaling as playing a major role in CRC [13]. A genome-scale analysis has recognized that 90% of patients with CRC carried genetic variations in the Wnt signaling pathway, particularly the loss-of-functional variations of adenomatous polyposis coli (APC) and variations that activate the mutations of β-catenin [14]. e membranous expression of β-catenin applies a restrictive impact on the movements of tumor cells and their growth. e increases in cell motility, growth, and transformation promote tumorigenesis because of the loss of β-catenin expression on the cell surface [12]. Pre-existing intracellular β-catenin can cause abnormality in Wnt/ β-catenin-TCF signaling, leading to the progression of CRC. e hyperactivation of Wnt/β-catenin signaling enhances the invasive and metastatic possibility of CRC cells, while the knockdown of β-catenin in CRC cells reduces cell proliferation and further invasion [15]. Studies have reported the detection of nuclear β-catenin expression using immunohistochemical methods, and they have reported an association with a high burden of tumor and poor CRC survival [15].
Somatic mutations at the APC gene are found in approximately 75% of CRC cases. Several studies have suggested worse outcomes for CRC patients with wild-type APC (APC-WT) in comparison to mutant-type APC (APC-MT) [16]. However, the prognostic implication of this genomic alteration is not well-defined, especially in metastatic CRCs. SMAD4/DPC4 is a tumor suppressor gene that regulates cell growth and a common intracellular mediator that could alter the TGFβ signaling to promote tumor progression. Studies have reported an association of SMAD4 genetic variation with tumor invasion, metastasis, and prognosis in various cancers [17].
In light of inconsistent results in the literature, the authors perceived a need for a meta-analysis that would explore the prognostic value of selected genes in CRC. e objectives were to estimate the pooled risk (hazard ratio, HR) identified (between the years 2000 and 2020) for each of these genes for overall survival (OS) and disease-free survival (DFS) in CRC patients.
us, this meta-analysis comprehensively explores the prognostic role of selected genes in the β-catenin and related pathway implicated in the development and progression of CRC.

Publication Search and Inclusion Criteria.
e authors searched the databases of PubMed, EMBASE and Science Direct for relevant published articles. Search terms included medical phrases related to SMAD 3, SMAD 4, β-catenin, Catenin beta 1(CTNNB1), APC, Wnt, Cyclin D1, Tp53, or p53 genes and their variants/polymorphisms, in combination with words related to CRC (tumor, neoplasms, carcinoma, CRC, colon cancer, or rectal cancer). In addition, terms related to prognosis (outcome or survival) were used to retrieve eligible studies from 2000 through to the end of 2020. Furthermore, the references in the selected published articles were searched to identify potentially relevant studies.
Eligible studies were selected based on the following criteria: (a) pathologically confirmed (i.e., via tissue samples) patients with CRC, (b) immunohistochemical/sequencing detection methods for the selected genes and OS, DFS, cancer-specific survival (CSS), or recurrence-free survival (RFS), (c) English language, and (d) full-text articles. Editorial letters, reviews, case reports, studies with duplicated/ repeated data, and studies lacking essential information and animal studies were excluded.

Data Extraction.
In accordance with the meta-analysis of observational studies in epidemiology (MOOSE) guidelines [18] and in compliance with PRISMA guidelines, the data were evaluated and extracted by two independent researchers, who entered them all onto the data extraction form. For data extraction, the details recorded were as follows: the first author, publication year, country, total number of cases, type of cancer, stages, reported genes, gene detection method, cut-off values used, hazard ratios (HRs) with their 95% confidence intervals (CIs), and P values. For inconsistencies, a consensus was reached on each item among the authors. e Newcastle-Ottawa scale (NOS) was used to evaluate the quality of the eligible studies.

Study Characteristics.
us, 64 studies involving 34,074 patients evaluating OS and DFS were analyzed in the current meta-analysis.

Quality of Eligible Studies.
e Newcastle-Ottawa Scale (NOS) was used to examine the methodological quality of the included studies. As previously described, a score of 9 implied the highest quality, while a score of ≥5 was considered to be high quality. Seventy-two studies included in our meta-analysis were of high quality, i.e., they had scores of 5 or more after quality assessment.

Prognostic Value of Gene Expression and Mutations in
Colorectal Cancer. Sixty-five studies, with 105 data points on genes where HR data was available, were included in the meta-analysis. ese are shown in Table 2. Twenty-eight enrolled studies provided the HRs, and 95% CI directly or indirectly reported the correlation between β-catenin overexpression and OS. e pooled HR of β-catenin overexpression in the nucleus, cytoplasm, or membranous with OS was 0.257 (95% CI: 0.003-0.511; Q = 53.978; P � 0.000) (Figure 2(a)), however, heterogeneity existed. e association of β-catenin overexpression with shorter DFS was analyzed. e pooled HR was 0.137 (95% CI: 0.131-0.406; Q = 48.832; P � 0.000) (Figure 2(b)). e above results suggested that β-catenin overexpression in the nucleus, membrane, or cytoplasm was associated with lower OS and DFS.

Discussion
Colorectal carcinogenesis is a complex multistage process that involves multiple genetic variations. e aberrant activation of the Wnt/β-catenin pathway has been identified as being involved in the progression of CRC [104] and early colorectal tumorigenesis [103]. In several studies, the β-catenin accumulation in the nucleus or cytoplasm was identified as a marker for poor prognosis. e variations of the APC or CTNNB1 genes are the main causes of the

Genetics Research 13
accumulation of nuclear β-catenin [105]. In contrast, β-catenin expression in the nucleus was associated with noninvasive tumors and more favorable outcomes [106] but remains controversial. e current meta-analysis has explored the cumulative prognostic significance of the different subcellular localizations of β-catenin expression among CRC subjects. e results indicated that the nuclear expression or decreased expression of β-catenin in the membrane was associated with lower OS, which is consistent with the published articles. Pooled data from a study [107] found that the reduced expression of β-catenin in the membrane to be significantly associated with poor survival among CRC patients, thus the majority of the selected studies are from nuclear β-catenin overexpression.
Wnt2 is an oncogene with the potential to activate canonical Wnt signaling during CRC tumorigenesis [21,22]. e role of Wnt5 in the progression of CRC is quite complex and appears to be inconsistent in findings. Several studies [21][22][23][24][25] proved that Wnt5a was silenced in most CRC celllines because of recurrent methylation in the promoter region. Wnt5a acts as a tumor suppressor by interfering with the canonical β-catenin signaling. However, it activates the noncanonical signaling pathways [100]. In this study, there   was no significant association of Wnt (2 and 5) to OS or DFS found among CRC patients, and it is well in accordance with the contradictory studies reported [23][24][25].
In our meta-analysis pertaining to SMAD genes, we found that the loss of SMAD 3 or SMAD4 staining was strongly associated with a worse prognosis for OS and DFS (including CSS/RFS). Several other individual reports are in alignment with our findings [87,92,93]. ese studies reported SMAD-4 to have a stronger association compared with SMAD-3 or other SMAD genes.
Most studies have shown the predictive value of Tp53 for overall survival in CRC to be poor. Dong et al. [108] reported 53% of Tp53 gene variation as the susceptibility for the development of CRC. Another study reported that, in mouse models, a high rate of spontaneous tumors was noted because of p53-deficiency [109]. Moreover, the deletion of p53 and the Tp53 gene variation led to tumor progression and tumor cell death.
A meta-analysis of Asian patients indicates that an association between Tp53 Arg72Pro polymorphism CC genotype might contribute to an increased risk of CRC [110]. e current meta-analysis included diverse populations, and the results pertaining to the association of Tp53 with shorter overall and DFS in CRCs may, therefore, be considered more generalizable.
In an independent study of 331 patients, the prognostic value of APC was evaluated, and the findings were validated on a public database of stage IV colon cancer from Memorial Sloan Kettering Cancer Center (MSKCC) [75]. e study found that APC-WT was present in 26% of metastatic CRC patients, and it was more prevalent in patients of younger age and those with right-sided tumors. APC-WT tumors      [75]. Overall, in most studies, APC-WT is associated with poor OS. Additionally, APC-WT tumors were associated with other activating alterations of the Wnt pathway, including RNF43 and CTNNB1. Cyclin D1 overexpression has been reported to occur in 40-70% of colorectal tumors [111]. Despite the wellestablished role of Cyclin D1 in cell cycle progression, previous data on Cyclin D1 and clinical outcomes in CRC have been conflicting. Cyclin D1 overexpression has also been significantly related to poor OS in Asian and non-Asian CRC patients [112]. Two mechanisms have been implicated, namely nuclear expression and cytoplasmic expression, wherein most studies found an association of the nuclear expression of Cyclin D1 with OS and DFS. Moreover, Cyclin D1 also has been shown as a poor prognosis marker when co-expressed with other genes, notably p53 [113]. ese results are consistent with the present meta-analysis's findings that shortened overall survival and DFS are associated with Cyclin D1 among CRC patients.
We acknowledge that this study has several limitations. Firstly, the element of bias cannot be ruled out because of the inclusion of retrospective studies. Secondly, all of the selected studies measured gene expression by immunohistochemistry and sequencing methods. Moreover, the cut-offs used in various studies differed between and across the genes studied. However, there was no subgroup analysis performed to investigate the potential effect of the technique on the combined results. irdly, some heterogeneity has been found because of location and the types of cancer. To eliminate variations across studies, a random-effects model was performed accordingly. Limited databases were used for article search, and only freely available full-text articles in the English language were used, which might affect the persuasive power of the pooled estimate, although to a limited extent. In addition, publication bias existed because only studies generating positive results or significant outcomes were suitable for publication. Future research might helpfully contribute further relevant analyses and well-designed extensive prospective studies, since they will address the limitations of the current meta-analysis.

Conclusion
e present meta-analysis has found that the genes associated with worst OS in CRC were β-catenin (cytoplasmic, membranous, and nuclear overexpression), APC (mutant type), Tp53 (mutated), SMAD-3 and SMAD-4 (loss of expression), and Cyclin D1 (high). e gene associated with shorter DFS in CRC patients was APC (mutant type). In  16 Genetics Research contrast, Wnt (2 and 5) genes were not associated with prognosis in CRC in this meta-analysis.

Data Availability
e data extraction sheets used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare no conflicts of interest.

Authors' Contributions
Hongfeng Yan took part in conceptualization, methodology, resources, writing-original draft, writing-review, and editing. Jianwu Yang took part in conceptualization, methodology, data curation, resources, writing-original draft, writing-review, and editing. Fuquan Jiang took part in conceptualization, resources, writing-review, editing, and supervision. All authors have read and approved the manuscript. Fuquan Jiang and Jianwu Yang shared equal correspondence.