Several studies have investigated the role of SMAD4 mutation of the prognosis and clinicopathological parameters in CRC, but the results are not consistent. Moreover, there is no meta-analysis to evaluate the impact of SMAD4 gene on the prognosis of CRC. Therefore, we conducted a meta-analysis and suggest that SMAD4 mutation is associated with poor prognosis in CRC. Compared with the SMAD4 wild-type controls, SMAD4 mutation is associated with worse OS (pooled HR = 1.46, 95% CI = 1.28–1.67, P < 0.001) and worse PFS/RFS (HR = 1.59, 95% CI = 1.14–2.22, P = 0.006). In order to further investigate the role of SMAD4 gene in colorectal cancer, we also analyzed the relationship between SMAD4 status with clinical pathological parameters of colorectal cancer, the results show that patients with SMAD4 mutation in colorectal cancer have higher TNM stages (I- III/IV; pooled OR = 0.78; 95%CI = 0.63–0.97; p = 0.025), that is, patients with SMAD4 mutation are more likely to occur distant metastasis. And SMAD4 mutant patients were more likely to feature mucinous differentiation (pooled OR = 2.23; 95%CI = 1.85–2.70, P = 0.000), tumors are more likely to occur in the colon (pooled OR = 1.15; 95%CI = 1.01–1.31; P = 0.042), more prone to lymph node metastasis (N0/N+; pooled OR = 1.42; 95%CI = 1.20–1.67; p = 0.000), and to harbor concurrent RAS mutations (pooled OR = 0.47; 95%CI = 0.30–0.73; P = 0.001). The important thing is all of these parameters often indicated a poor prognosis. Combined OR suggested that SMAD4 gene mutation has nothing to do with MSI and BRAF status. The role of SMAD4 gene affecting MSI or BRAF status remains to be elucidated. There is a meta-analysis showed that SMAD4 mutation (combined OR 2.04, 95% CI 1.41–2.95) were at a higher risk of distant metastasis[14], which is consistent with our results.
Over the past two decades, many studies have shown that SMAD4 mutation does not cause tumorigenesis by itself, but promote tumor progression caused by other genes[8].Ohtaki et al. reported that the frequency of SMAD4 mutations was significantly higher in tumors with liver metastasis than in those without such metastasis[22]. Inamoto et al. reported that SMAD4-deficient colorectal tumor cells secreted more CCL9 and CCL15, two chemokines that recruit CCR1 + myeloid cells through CCL9-CCR1 and CCL15-CCR1 axis, resulting in metastasis[23]. Vauthey et al. concluded that patients with SMAD4 mutations are less likely to undergo repeated hepatectomy for recurrent disease following initial tumor resection[24]. Alhopuro et al. showed that SMAD4 is a predictive biomarker for 5-fluorouracil (5-Fu) based chemotherapy in CRC patients[25]. Zhang et al. discovered a novel mechanism mediated by SMAD4 to trigger 5-Fu chemosensitivity through cell cycle arrest by inhibiting the PI3K/Akt/CDC2/survivin cascade[26]. Mei et al. suggested that SMAD4 mutations could be potential biomarkers for poor prognosis of cetuximab-based therapy [27], which needs to be further validated in a larger patient cohort. Lin et al. found that silencing SMAD4 reduces the sensitivity of colorectal cancer cells to cetuximab by promoting epithelial-mesenchymal transition(EMT), while the high expression of Smad4 may be clinically beneficial to cetuximab-based therapy[28].Mizuno et al. found that SMAD4 gene mutations were significantly associated with worse OS following hepatic resection, which was independent of RAS mutation status[11]. These findings indicate that SMAD4 genetic alteration has a key role in tumor progression and efficacy of target therapy for CRC patients.
In the current analysis, we found that SMAD4 gene alteration was significantly associated with loss of SMAD4 expression in CRC, and loss of SMAD4 disrupts canonical TGF-β signaling[29], because it is a transcription factor for signaling. In addition, it is reported that the loss of SMAD4 function is independently associated with the reduction of RFS and OS in CRC patients, especially patients with advanced disease[30]. In contrast, the median overall survival in CRC patients with high Smad4 expression is much longer than that with low Smad4 expression[31]. Germline mutations in genes in the TGF-β family signaling pathway strongly increase the risk of colonic neoplasia[32]. The canonical TGF-β/Smad4 signaling pathway acts as a tumor suppressor in the early stages, which is characterized by its anti-proliferative activity, ability to induce apoptosis and promote genome stability, while in the late stage of tumor, TGF-β acts as a tumor and metastasis promoter to stimulate tumor development[33].
Studies have suggested that EMT is a is a key step in tumor progression and metastasis, and theTGF-β1 signaling plays a key role in EMT[34]. EMT is a well-coordinated process in which epithelial cells lose cell connectivity and polarity and transform into mesenchymal cells with migration and invasion capabilities. Functional study results indicate that TGF-β-induced Smad4-dependent EMT followed by apoptosis in colorectal cancer cells[35, 36]. Siraj et al. identified TGF-β-induced EMT insufficient to obtain invasive potential, the activated Ras TGF-β will change the reaction, imparting tumorigenic and invasive potential[37]. Therefore, the synergistic effect between Ras-Raf-MAPK and TGF-β/Smad cascades are needed to obtain the aggressive phenotype in cancer.
At present, RAS has been recognized as tumor driver genes, predictive biomarkers and therapeutic targets in CRC. The expression of RAS up-regulates the expression of phosphotyrosine kinase receptors ERBB1 (EGFR) and ERBB2 (HER2) and induces an aggressive phenotype. Smad4-dependent signal transduction negatively regulates the expression of these receptors and inhibits the up-regulation of EGFR and ERBB2 induced by RAS, thereby play an anti-proliferative effect. The loss of oncogenic RAS and SMAD4 signals synergistically up-regulate the abnormal expression of EGFR and ERBB2, leading to the development of cancer and the metastasis and spread of the primary tumor[38, 39]. TGF-β can quickly activated RAS and ERK pathway[40], In contrast, the ERK pathway inhibits the TGF-β/Smad4 pathway by phosphorylating Smad2 and Smad3 at serine or threonine residues in the linker region, so epithelial cells with oncogenic RAS mutations usually exhibit loss of TGF-β antiproliferative response[8].Patients with RAS wild-type tumors and retained SMAD4 wild type had an overall survival longer than cases with mutations in both genes[41]. However, regardless of the RAS mutation status or other clinicopathological factors, SMAD4 mutations are significantly associated with poor OS. The precise cooperative mechanisms of SMAD4 with other genes of influence also requires further examination.
Given the relative frequency of SMAD4 mutations in CRC patients, routine SMAD4 testing may be appropriate. For individualized treatment of CRC, further research should be done for guiding clinical decision-making, which SMAD4 is a driver mutation and will be a novel target for precision medical treatment of CRC.
No heterogeneity or publication bias was found in this meta-analysis, and sensitivity analysis shows that our results are reliable. However, this analysis has several limitations. First, our meta-analysis included studies of qualified articles published in English and Chinese, did not include some relevant articles written in other languages or unpublished papers, which is likely to result in selection bias. Second, the use of specific therapies and tumor stage differed among the included articles. Third, the HR calculated from the data or extracted from the survival curve may not be as reliable as the HR calculated directly using the analysis of variance. Therefore, the results should be carefully interpreted. However, as far as we know, this is the first meta-analysis to demonstrate SMAD4 mutation by evaluating the pathological features and prognostication in CRC.