Background
Colorectal cancer (CRC) is a malignant tumor with high morbidity and mortality, easy recurrence, and easy metastasis. CRC is mostly asymptomatic and difficult to detect in the early stage, and most patients are already in the advanced stage when they are diagnosed [
1]. Besides, advanced CRC infiltrates lymph nodes and is prone to metastasis of abdominal implantation or metastasis to other organs. The prognosis of the patient is very poorer and the survival rate is extremely lower [
2]. Reducing the probability of CRC metastasis and finding new targets is the key to improving the survival of CRC patients.
Epithelial-mesenchymal transition (EMT) is one of the main mechanisms of tumor metastasis and invasion [
3]. It also has the effect of promoting the malignant proliferation of tumor cells, reducing apoptosis and senescence, and promoting immune suppression. Loss of E-cadherin expression and loss of cell polarity are the key steps of EMT [
4]. The main E-cadherin inhibitors that have been discovered are Snail, Zeb, E47, and KLF8, which combine with the promoter of E-cadherin and inhibit its expression. Twist, Goosecoid, E2.2, and FoxC2 indirectly inhibit the activity of E-cadherin. The three protein complexes (Par, Crumbs, and Scribble) that maintain apical-basal polarity in epithelial cells are also regulated by EMT-induced genes, and cell polarity is lost after inhibition [
5]. TGF-β family, Wnts, Notch, EGF, HGF, FGF, HIF and other signaling pathways play an important role in regulating the above process. Therefore, the EMT process is also critical in the development, metastasis, and invasion of colorectal cancer.
EMT can affect the occurrence and development of CRC, the prognosis of metastasis, and the effect of chemotherapy and immunotherapy [
6]. However, the current research still lacks systematic research on the overall genes that regulate the EMT process and its prognosis and treatment effects with CRC. Therefore, we use TCGA and GEO data as training and validation sets to screen out differentially expressed EMT-related genes (EMT-RDGs) and lncRNAs. Construct a prognostic model to study their relationship with the prognosis, immune infiltration, drug sensitivity, and resistance of CRC patients, and provide a basis for clinical treatment of CRC.
Discussion
The prone to invasion and metastasis of CRC is one of the main factors leading to poor prognosis of patients. EMT is one of the core mechanisms of tumor invasion and metastasis, and it also promotes tumor cell proliferation [
11]. Therefore, we constructed a prognostic risk model for nine prognostic-related EMT-RDGs screened in the TCGA and GEO datasets and evaluated the reliability of the model and its relationship with survival and immunity. At the same time, we also analyzed the relationship between the expression, mutation, methylation of nine key EMT-RDGs and survival, immunity, and drug treatment response.
The transcription factor TCF15 has been found to affect the proliferation and differentiation of many types of cells, such as promoting hematopoietic stem cell quiescence and long-term self-renewal, [
12] inducing the proliferation and differentiation of embryonic stem cells [
13]. However, we found that TCF15 is low expressed in CRC, which may reduce the number of epithelial cells and promote the occurrence and metastasis of EMT of CRC.
miR-185 targeted SIX2 inhibiting the growth of HCC cells and the progression of EMT provides a new target for molecular therapy of liver malignancies [
14]. Therefore, the potential of SIX2 in other tumor EMT is obvious, but its role in the CRC is not yet clear. We found that SIX2 is highly expressed in CRC, and reducing the expression will prolong the survival time of CRC.
NOG is one of the key genes of mesenchymal-epithelial interaction [
15]. Studies have found that NOG disorders are related to the survival risk of nasopharyngeal carcinoma [
16]. Increased expression of NOG significantly promotes breast cancer bone metastasis [
17]. We found that low-expressed NOG has a good prognosis in CRC, indicating that NOG has the potential to be used in the treatment of CRC and even other tumors.
FGF8 is a mesodermal marker gene. When it is highly expressed, EMT is up-regulated and cell polarity is lost [
18]. In tumors, high expression of FGF8 affects EMT through the BRG1/Snai1/E-cadherin pathway and promotes tumor proliferation and invasion of gastric cancer [
19]. However, the high expression of FGF8 predicts a good prognosis in our results, which is contrary to the results for gastric cancer, and further research is needed to clarify its role.
The role of TBX5 in the EMT process is mainly to initiate the formation of mesenchymal limb progenitors [
20]. Up-regulation of TBX5 promotes the formation of mesoderm during EMT and affects the differentiation of cardiomyocytes [
21]. The increase of TBX5 drives the mesenchymal phenotype of breast cancer, promotes the EMT process, and inhibits the expression of the immune response network [
22]. The high expression of TBX5 in our results indicates a high risk of poor prognosis.
SNAIL1 is a well-known tumor EMT inducer, and its role in CRC is relatively clear. The high expression of SNAIL has been found to induce a poor prognosis of CRC due to the induction of EMT phenotype. And SNAIL affects the EMT process of CRC through various mechanisms such as β-Catenin-LEF1 complexes [
23]. Down-regulation of SNAIL1 mediated MYB and ISC markers (such as WiNTRLINC1) may help reduce EMT-related proliferation of CRC cells [
24]. These studies are consistent with our findings.
PHLDB2 has been proven to be a downstream effector of the EMT pathway, and it may be an important biomarker and target for a good prognosis of CRC when its expression is low [
25]. This is completely consistent with our results.
The overexpression of TIAM1 in lung adenocarcinoma is significantly related to advanced tumor grade and poor prognosis [
26]. Knockout of TIAM1 expression can reverse the proliferation, migration, and EMT transformation of HCC cells [
27]. The high expression of TIAM1 induced CRC proliferation and migration [
28]. We found that the low expression of TIAM1 in CRC showed a good prognosis in the TCGA data but a poor prognosis in the GEO data, but the difference was not significant and there was no statistical significance. Therefore, further verification is required.
TWIST is also a clear inducer of EMT and mesenchymal phenotypic marker. Reducing its expression in CRC promotes the increase of E-calonectin and reverses the EMT process [
29]. This is the same as our findings. It further illustrates the potential of TWIST as a CRC target and prognostic marker.
We found that nine key EMT-RDGs were closely associated with the metastasis of CRC, and first proposed that TBX5, FGF8, NOG, SIX2, and TCF15 are the role and potential of EMT-RDGs as prognostic markers and therapeutic targets in CRC. But it lacks in vivo experimental verification. Moreover, there were still few studies on the role of these genes' methylation and mutations in tumors. Therefore, we also studied the strong relationship and effects of the methylation and mutation status of these genes on the expression and prognosis. Targeting and monitoring the mutation status and methylation sites of these genes is also a potential tool to improve the prognosis of CRC. The EMT-RlncRNAs were selected to construct a prognostic model based on the TCGA data but fail to verify in GEO data due to the limited sample size.
EMT also promotes tumor immunosuppression. The current research on the relationship between immune cell infiltration and EMT mainly focuses on cancer-related fibroblasts, tumor-related macrophages, and EMT. TAM secretes a variety of cytokines and chemokines and promotes the paracrine transformation of adjacent epithelial tumor cells to EMT. In turn, the cytokines produced by tumor cells also promote the differentiation process of TAM, thereby forming a positive feedback loop between TAM and EMT in the process of tumor metastasis to promote tumor progression, invasion, and metastasis [
30]. TAM induces the EMT program by regulating the JAK2/STAT3/miR-506-3p/FoxQ1 axis to enhance CRC migration, invasion, and CTC-mediated metastasis [
31]. M2 macrophages promote the invasion and metastasis of lung cancer through EMT by up-regulating the expression of CRYAB and activating the ERK1/2/Fra-1/slug signaling pathway [
32]. Inhibition of M2 macrophages inhibits EMT and fibrosis of CRC [
33]. The high infiltration of M2 macrophages in the TCGA and GEO database was correlated to a high risk of prognosis and the high infiltration of M1 macrophages was correlated to a low risk of prognosis in CRC. However, research on other immune cells and EMT is still very few. When EMT decreases in CRC, CD14 + monocytes and CD19 + B cells also decrease, and the tumor increases infiltration of CD56 + NK cells [
34].
Highly infiltrating CD8 + tumor-infiltrating lymphocytes in CRC are accompanied by a decrease in SNAIl and an increase in E-cadherin expression, which are closely related to EMT, and are closely related to the good prognosis of CRC [
35]. However, in our results, the level of CD + 8 cell infiltration did not differ significantly between the high and low-risk groups. This may be due to the limited number of samples. EMT activation makes CRC cells more susceptible to NK cell-mediated NKG2D-mediated killing [
36]. We indeed found that the level of resting NK cells increased in the low-risk group. Therefore, increasing the proportion of NK cells could improve the prognosis of CRC patients.
In addition, the relationship between the non-response or response of targeted drugs and nine prognostic-related EMT-RDGs expression or mutations was analyzed. These mainly include anti-angiogenesis targeted drugs, PI3K-Akt-mTOR signaling pathway, and RAS/RAF/MAPK signaling pathway targeted drugs. We found that these prognostic-related EMT-RDGs are closely related to drug treatment response. 5-FU is the basic chemotherapeutic drug for CRC, and cetuximab is the standard targeted drug for the first-line treatment of RAS/RAF wild-type left semi-CRC. PHLDB2 is positively correlated with 5-FU sensitivity, and it is negatively correlated with cetuximab sensitivity. These findings may help enhance drug sensitivity, reverse CRC resistance to prolong PFS and OS.
Some of these drugs have been confirmed in clinical trials that the survival benefit of CRC patients. For example, Sorafenib did not show superior therapeutic effects in CRC (RESPECT trial) [
37]. Dabrafenib had shown a confirmed response rate in metastatic CRC with BRAF
V600E-mutation positive (NCT01750918) [
38]. Therefore, PLX4720, SB590885, 878739-06-1, and GDC-0879 are all potential drugs for BRAF
V600E-mutant CRC. Most PI3K-Akt signaling pathway drugs for the targeted therapy of CRC are still in the research stage of in vivo, in vitro and I phase of clinical trials. However, some of these drugs have not been studied or performed in a clinical trial about CRC, so they can be considered as a targeted therapy option for colorectal cancer patients in the future. Meanwhile, the nine hub EMT-RDGs might be the potential biomarkers of targeted therapy response, also could predict the effectiveness of targeted drugs and synergy of genes and drugs.
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