The occurrence and progression of CRC is a complicated procedure comprising various steps and genes. A study report has revealed that CRC cells have a particular biological behavior, such as an increased proliferative capacity, the recurrence tendency and the ability to metastasize29. Even though surgery, radiotherapy, and chemotherapy have contributed to the progress in the treatment of CRC, an effective procedure for the prediction of tumor progression or the prognosis of patients is not available at present30,31. The tumor-lymph node-metastasis staging (TNM staging) is a commonly used staging system, but it does not function in the accurate prediction of the prognosis of patients with CRC, and the prognosis pattern can be distinct even for CRC patients having the same stage of tumor32. Detailed research on the mechanisms involved in tumors has led to increasing benefits of prognosis prediction at the molecular level. For instance, Li et al. assessed the prognostic value of methylation regions in CRC and detected four differentially methylated regions, namely MUC12, TBX20, CHN2, and B3GNT7, as potential prognostic indicators for CRC and created a prognostic prediction score on their basis33. Moreover, many other reports have focused on transcriptional-level analysis, including research on mRNAs, long non-coding RNAs (lncRNAs), or miRNAs34–36.
APA is a major post-transcriptional regulatory mechanism that regulates the nuclear export, stability, and translation efficiency of mature mRNA37,38. New data indicated that aberrant APA patterns happening in different cancer types are involved in multiple oncogenic procedures in cancer occurrence and progression9. Earlier reports have indicated that CFIm25 promotes the protein expression of oncogenes, including IGF1R, by regulating their APA, thereby enhancing the proliferation and inhibiting the apoptosis of lung cancer cells39. The 3′UTR in the mRNAs of the PRELID1 gene was greatly shortened in ER-positive breast cancer tissues, which has the potential to improve the mRNA stability and translation efficiency of the PRELID1 gene substantially. Increased levels of PRELID1 expression led to enhanced tumor cell growth and substantially lowered the survival of cancer patients40. Prior research on the function of APA events has aimed only at a single gene or a few genes and their prognostic value has not been explained comprehensively. Considering the value of APA events in tumorigenesis and progression, they were investigated to gain comprehensive knowledge related to the prognostic value of APA events in CRC using TCGA analysis.
The CRC samples were systematically analyzed and a total of 197 survival-related APA events in 194 genes were obtained. GO and KEGG analyses of the parental genes for these events indicated that the enrichment events occurred frequently in cell adhesion molecule binding, perinuclear region of cytoplasm, Golgi vesicle transport, Pentose phosphate pathway, and other functions and pathways. These biological activities are also closely correlated with the invasion and metastasis of CRC cells and are regarded as the major damaging factors affecting the survival of patients with CRC. Furthermore, a correlation network was constructed between prognostic-related APA events and SFs to further probe into the regulatory role of SFs in APA events in patients with CRC. To explore the prognostic value of APA events, a prediction signature was developed in this study according to the prognosis-related APA events in the training set. Afterward, patients with CRC were sorted into high and low-risk groups based on their median risk scores, and a major difference in OS was revealed between both groups. It was observed that the 1, 3, and 5-year AUC values for patients with CRC were all higher than 0.75, showing good predictive ability. The signature showed similar predictive potential in both the test set and the entire set, highlighting its good utility and reproducibility in predicting OS in patients with CRC.
A variety of genes among the APA parental genes in the signature were observed to play a significant role in the biological function of CRC progression. BCCIP, an interacting protein of BRCA2 and CDKN1A41, is strongly expressed in the progression of CRC42. Xu et al. Revealed that Celecoxib affected the function of p53 and inhibited recovery from the damage caused by irradiation by upregulating the expression of BCCIP. Moreover, Celecoxib elevated the radiosensitivity of CRC cells by regulating the expression of genes, including p21 and Cyclin B1, in a COX-2 independent manner43. PLK3 belongs to the serine-threonine kinase family and has a significant role in the cell cycle44. In case of DNA damage, PLK3 has been observed to phosphorylate and interact with WT in S20 and P53, which activates tumor suppressors and induces apoptosis45. PLK3 inhibits cancer cell growth and suppresses cellular glucose metabolism through the heat shock protein 90 (HSP90)/ signal transducer and activator of transcription 3 (STAT3)/ hexokinase 2 (HK2) pathways46. Consequently, low PLK3 expression in CRC tissues is correlated with a poor prognosis. Tissue inhibitor of metalloproteinases 3 (TIMP3), a member of the TIMP family, has been noted to inhibit tumor growth, angiogenesis, invasion, and metastasis47. TIMP3 was observed to enhance apoptosis susceptibility and facilitate apoptosis by stabilizing tumor necrosis factor-alpha (TNF- α) receptors on the surface of CRC cells48. Additionally, the overexpression of TIMP3 may reduce vascular density, promote apoptosis and inhibit malignant behaviors, including migration, invasion, and tumor growth of CRC cells49. Collectively, these APA events are closely correlated with CRC metastasis and the survival of patients and can act as potential prognostic and therapeutic targets for CRC.
Various clinical information has shown a correlation between genetic alterations and the responsiveness to immunotherapy50,51. In the current study, a substantially elevated mutation rate of adenomatous polyposis coli (APC) and tumor protein p53 (TP53) was observed in patients in the high-risk group. The APC mutations are early events in colorectal tumorigenesis and restoration of APC expression induces apoptosis in CRC cells with inadequate endogenous APC expression52. Recent reports have revealed that TP53 mutations not only affect CRC proliferation, apoptosis, and migration but also have a significant role in the immune response53,54. In the current study, TMB did not differentiate between patients' risk subtypes but did discriminate between the prognosis of patients. Subsequent stratified survival curves indicated the prognostic predictive ability of the risk scores independent of TMB, indicating that TMB and risk scores signify various aspects of immunobiology. Additionally, the risk scores and CNVs highlighted a different distribution between high and low-risk groups, but no substantial differences were found in the genome where CNVs occurred.
The tumor immune microenvironment (TIM) is a significant factor in the progression of tumors, in which the immune system protects tumor cells from the immune barrier. Tumor immune editing and resistance results in immune escape, promoting tumor cell proliferation, invasion, and metastasis55,56. Hence, we hypothesized and studied the correlation between APA events and TIM, which could provide potential prognostic and therapeutic targets for CRC patients. It was observed that the high-risk group showed decreased immune and stromal cell scores and presented low immune cell infiltration and immune checkpoint levels, while the low-risk group had low tumor purity and indicated considerable immune cell infiltration and increased immune checkpoint levels. These findings indicated that the signature can reveal the immune status of patients with CRC. Furthermore, the TIDE algorithm showed lower scores in the high-risk group, suggesting that patients in the high-risk group were more sensitive to immunotherapy. Moreover, 26 potential antitumor agents associated with the signature were identified in this study, which provided an additional reference for antitumor therapy for patients with distinct risk levels. The above discussion indicated that heterogeneity in TIM can produce various responses to immunotherapy or anti-cancer drugs. As our results were obtained from the bioinformatic analysis, additional clinical studies are needed for further confirmation.
At the end of this study, the nomogram was constructed for better prediction of the survival of patients with CRC and to visualize the predicted results, providing the additional direction for patient adherence and treatment outcomes. Moreover, the effectiveness and validity of the nomogram were compared with independent prognostic indicators, indicating that the nomogram can deliver better prognostic ability and more net gaining than other independent indicators in the clinical setting. The outcomes of this study indicated the reliable prognostic accuracy of the nomogram on the basis of seven APA events.
Certain limitations remained in the current study. First of all, the APA event set was generated using relatively lax criteria. These screening criteria allowed us to categorize a large number of potentially important APA events, but the reliability of this study could have been affected adversely. Moreover, due to the unavailability of other publicly present datasets, the signature used in this study was only verified internally. Evidently, independent datasets and prospective studies are required for further validation of the reliability of this signature. Lastly, the biological activity of these APA events in CRC needs to be examined in vivo and in vitro.