Introduction
Rectal cancer is a common malignant tumor of the digestive tract. By the end of 2022, according to the latest epidemiological survey, rectal cancer was the third most common tumor worldwide. For patients with stage I rectal cancer, if the tumor is close to the anus, local tumor resection + postoperative radiotherapy can be given, and the same efficacy as radical surgery can be obtained while preserving the anus. For patients with stage II-III rectal cancer, preoperative concurrent chemoradiotherapy and postoperative concurrent chemoradiotherapy reduced locoregional recurrence rates and significantly improved long-term survival compared with surgery alone. Compared with postoperative concurrent chemoradiotherapy, preoperative concurrent chemoradiotherapy achieves similar long-term survival, and on this basis further reduces the locoregional recurrence rate and the incidence of adverse reactions, and improves the sphincter preservation rate, and preoperative concurrent chemoradiotherapy becomes the standard method for stage II–III surgically respectable rectal cancer [
1]. For patients with locally advanced inoperable rectal cancer, preoperative concurrent chemoradiotherapy can make some patients obtain the chance of surgery, while for patients who are still inoperable, palliative reduction can be performed. After preoperative concurrent chemoradiotherapy for locally advanced rectal cancer, radical surgical pathology confirmed a pCR rate between 12 and 20% [
2]. Patients who achieved pCR had improved overall and disease-free survival and reduced local recurrence rates compared with those who did not achieve pCR. At present, there is no elucidation on the related genes predicting the sensitivity of radiotherapy for rectal cancer and the pCR status after preoperative concurrent chemoradiotherapy for rectal cancer in clinical and basic research, so this paper intends to explore the related genes of radiotherapy sensitivity for rectal cancer from the multi-omics direction.
Tumor radiosensitivity is controlled by multiple factors such as DNA damage repair, regulation of cell cycle checkpoints, regulation of signal transduction pathways, and tumor microenvironment. The target of ionizing radiation is DNA. DNA double-strand breaks (DSBs) are lethal lesions, and their repair is mainly achieved by non-homologous end joining (NHEJ) and homologous recombination (HR). HR occurs mainly in the S and G
2 phases of the cell cycle, and NHEJ is mainly achieved by DNA-PKcs, KU70, and KU80, and inhibition of important targets in DSB repair such as ATM or DNA-PK significantly improves radiosensitivity [
3]. Cell cycle checkpoint inhibitors increase radiosensitivity in tumor cells. The main pathways associated with radiosensitivity are PI3k–AkT, NF-κB, MAPK and TGFβ, the first three of which are associated with cell survival, and TGFβ may affect radioresistance by controlling ATM activation. Hypoxic status, hypoxia-inducible factor (HIF), tumor angiogenesis are also associated with radiotherapy. Other studies such as cancer stem cells, microRNAs and radiosensitivity have also been reported, but most biomarkers lack sensitivity and specificity.
In recent years, significant progress has been made in cancer immunotherapy, but in clinical practice, immunotherapy alone has brought benefits to only a small proportion of cancer patients, while radiation therapy can release tumor-specific antigens from tumor cells, activate dendritic cells with antigen presentation around the antigen, drain activated dendritic cells to lymph nodes and T cells to produce systemic anti-tumor immune responses, and at the same time, the induced use of immunotherapy can also change the tumor microenvironment, promote tumor angiogenesis, improve hypoxia, and produce a synergistic effect with radiotherapy, and radiotherapy combined with immunotherapy can transform immune-cold tumors into immune-hot tumors and enhance anti-tumor immune effects [
4]. However, the specific role of radiotherapy combined with immunization in the treatment of rectal cancer has not been elucidated. Nanomaterials have been extensively studied in cancer therapy as vectors that may improve drug delivery. Such vectors not only bring numerous advantages such as stability, biocompatibility, and cellular uptake but have also been shown to overcome some cancer-related resistances [
5‐
7].
In summary, the mechanisms involved in chemoradiotherapy and immunotherapy sensitivity in rectal cancer remain elusive, therefore, we comprehensively explore the gene differences between radiotherapy sensitive group and radiotherapy insensitive group in rectal cancer, find five genes closely related to prognosis in differential genes, and then comprehensively explain the distribution of cell signaling pathways, immune infiltration, correlation of immune genes, sensitivity of chemotherapeutic drugs, and transcription factor regulation of these five genes in order to find appropriate biomarkers to guide clinical practice.
Discussion
In this study, we found that 600 up-regulated genes and 553 down-regulated genes were significantly different in the expression levels between the radiotherapy sensitive and radiotherapy insensitive groups of rectal cancer. Functional enrichment analysis showed that differential genes were significantly enriched in a variety of tumor biological processes. Through the random survival forest analysis of these 1153 differential genes, we finally screened five key genes, which were the radiosensitivity up-regulated genes of rectal cancer: TOP2A, MATR3, APOL6, JOSD1 and the radiosensitivity down regulated gene of rectal cancer: HOXC6.
Nuclear DNA topoisomerase II-alpha (TOP2A) is located at 17q21.2 and regulates chromosome condensation and chromatid segregation by altering the topological state of DNA during DNA replication and transcription [
16]. TOP2A was found to be highly expressed in non-small cell lung cancer, hepatocellular carcinoma, and breast cancer and is associated with tumor proliferation and poor prognosis [
17‐
19]. TOP2A is considered a major target of the chemotherapeutic agent etoposide [
20]. It has also been shown that TOP2A is associated with the efficacy of radiotherapy, and Terry et al. found that TOP2A is indirectly involved in the formation of chromatid breaks in radiation-induced DNA double-strand breaks and plays a role in individual differences in chromatid radiosensitivity [
21]. Upregulated expression of TOP2A is associated with recurrence after radiotherapy in prostate cancer patients [
22]. Wnt/b-Zhang et al. found that inhibition of TOP2A inhibited the activity of the catenin signaling pathway in medulloblastomas, thereby reducing tumorigenicity and radioresistance of medulloblastomas [
23] cells. In this study, TOP2A was found to be significantly positively associated with immune cells T cells resting helper, Dendritic follicular cells activated, Macrophages M1, and negatively associated with Dendritic follicular cells. TOP2A was significantly negatively correlated with immunomodulatory genes such as chemokines, immunosuppressive agents, MHC, MHC receptors. Correlation studies with chemosensitivity showed that TOP2A was significantly associated with chemosensitivity to bleomycin, camptothecin, cisplatin, adriamycin, gemcitabine, and mitomycin. TOP2A expression correlates with MSI. Related signal pathway enrichment analysis showed that the pathways enriched by GO of TOP2A gene included chromosome segregation and dna recombination, and the pathways enriched by KEGG included alpha linolenic acid metabolism and arachidonic acid metabolism.
Matrin3 (MATR3) is one of the nuclear matrix proteins and is a DNA and RNA-binding protein. Yang et al. found that MATR3 acts as a tumor suppressor in breast cancer cells, overexpression of MATR3 promotes apoptosis and inhibits epithelial-mesenchymal transition, migration and invasion of cells, and low expression of MATR3 is associated with poor prognosis in breast cancer patients [
24]. Kuriyama et al. found that knockdown of MATR3 expression inhibited the proliferation of malignant melanoma cells [
25]. Nho et al. found that decreased expression of MATR3 in oral squamous cell carcinoma cells induced apoptosis [
26]. Durılewicz et al. found that low expression of MATR3 was an independent poor prognostic factor in non-small cell lung cancer [
27]. In this study, MATR3 was found to be significantly positively associated with immune cells T cells follicular helper and Dendritic cells activated, and negatively associated with Dendritic cells resting. MATR3 was significantly negatively correlated with immunomodulatory genes such as immunosuppressive agents, MHC. Correlation studies with chemosensitivity identified MATR3 as significantly associated with chemosensitivity to bleomycin, camptothecin, cisplatin, doxorubicin, gemcitabine, mitomycin. MATR3 expression correlates with MSI. Related signaling pathway enrichment analysis revealed that the pathways enriched by GO of MATR3 gene were defense response funto gus, mrna 3 end processing and other pathways, and the pathways enriched by KEGG were basal transcription factors, cell cycle and other pathways.
HOXC6 is one of the members of the HOX family and maps on 12q13.3 of the human chromosome [
28]. HOXC6 is aberrantly expressed in head and neck squamous cell carcinoma, gastrointestinal malignancies, and breast cancer [
29,
30]. Ramachandran et al. found that low expression of HOXC6 induced apoptosis in prostate cancer cells and HOXC6 could serve as a therapeutic target for prostate cancer [
31]. Several previous studies have shown that high expression of HOXC6 is associated with poor prognosis in cancer patients. Du et al. found that increased HOXC6 expression in esophageal squamous cell carcinoma predicted poor prognosis [
32]. Zhang et al. found that gastric cancer patients with high HOXC6 expression had shorter survival time than patients with low HOXC6 expression [
33]. Zhou et al. found that patients with high HOXC6 expression faced a higher risk of death than those with low HOXC6 expression in prostate cancer patients [
34]. In this study, there was a significant positive correlation between HOXC6 and immune cells and Macrophages M1. HOXC6 was significantly positively associated with immunomodulatory genes such as immune activators. Correlation studies with chemosensitivity found no apparent correlation between HOXC6 and chemosensitivity to bleomycin, camptothecin, cisplatin, adriamycin, gemcitabine, mitomycin. HOXC6 expression did not correlate with MSI. Related signaling pathway enrichment analysis revealed that the pathways enriched by GO of HOXC6 gene were exploration behavior, muscle fiber development and ecm receptor interaction, dilated cardiomyopathy and other pathways.
APOL6 is a member of the apolipoprotein L gene family and acts as a regulator of lipid metabolism, which promotes differentiation and lipogenesis in 3T3-L1 preadipocytes [
35]. Liu et al. found that the expression of APOL6 was associated with multiple programmed cell death through bioinformatics studies, suggesting that it may regulate multiple programmed cell death processes. In vitro experiments found that up-regulation of APOL6 could promote the necrosis and pyroptosis of pancreatic cancer cells, while necrosis and pyroptosis could enhance anti-tumor immune effects, which also indirectly explained the up-regulation of APOL6 in immunotherapy responders [
36]. Grace R. Raji et al. found that miR-643 is horizontally transferred from cisplatin-resistant cells and confers chemoresistance in receptor drug-sensitive cells by targeting APOL6 [
37]. In this study, significant positive correlations were found between APOL6 and immune cells T cells follicular helper, Dendritic cells activated, Macrophages M1, and significant negative correlations with Dendritic resting cells, Mast activated cells, etc. APOL6 was significantly positively associated with immunomodulatory genes such as chemokines, immunosuppressive agents, MHC, MHC receptors. Correlation studies with chemosensitivity identified APOL6 as significantly associated with chemosensitivity to bleomycin, camptothecin, cisplatin, adriamycin, gemcitabine, mitomycin. APOL6 expression correlates with MSI. Related signaling pathway enrichment analysis revealed that the pathways enriched by GO of APOL6 gene were defense response to virus, response to virus and other pathways, and the pathways enriched by KEGG were antigen processing and presentation, glycerophospholipid metabolism and other pathways.
JOSD1 is a member of the smallest family of DUBs MJDs, which contains only a highly conserved Josephine domain located 22 q13.1 on human chromosome [
38,
39]. Mutations in JOSD1 gene have been identified in melanoma, endometrial, bladder, and ovarian cancers [
38]. Previous studies have shown that JOSD1 stabilizes target proteins by cleaving the K48 ubiquitin chain [
40,
41]. JOSD1 promotes chemoresistance by stabilizing MCL1 in gynecologic tumors [
40]. Jing et al. found that under epigenetic regulation of BRD4, JOSD1 was overexpressed in HNSCC, and increased expression of JOSD1 was positively correlated with proliferation and chemoresistance of HNSCC cells, while highly expressed JOSD1 was also associated with poor prognosis of HNSCC patients [
42]. In this study, JOSD1 was found to be negatively correlated with Mast cells activated significantly. JOSD1 was significantly positively associated with immunomodulatory genes such as chemokines, immunosuppressive agents, MHC, MHC receptors. Correlation studies with chemosensitivity revealed that JOSD1 was significantly associated with chemosensitivity to gemcitabine, but not to bleomycin, camptothecin, cisplatin, adriamycin, or mitomycin. Expression of JOSD1 did not correlate with microsatellite instability (MSI). Related signaling pathway enrichment analysis revealed that: the pathways enriched in GO of JOSD1 gene were atp synthesis coupled electron transport, b cell homeostasis and pathways enriched in KEGG were apoptosis, b cell receptor signaling pathway.
We investigated the association of five core genes with clinical parameters and found that APOL6 was significantly associated with patient age, overall stage, M stage, N stage, and survival status, HOXC6 was significantly associated with patient overall stage, T stage, and N stage, JOSD1 was significantly associated with patient M stage and survival status, and MATR3 and TOP2A were significantly associated with patient age as well as survival status. At the same time, we constructed nomogram model to predict the prognosis of patients, and the age, gender, total stage of rectal cancer, T stage, N stage, M stage and the expression of five key genes of patients contributed to different extents, and predicted the 1- and 3-year survival, indicating that nomogram has a good predictive efficacy and can guide clinical practice. Patients with microsatellite instability-high (MSI-H) have a high tumor mutation burden and increased numbers of tumor-infiltrating lymphocytes and exhibit high sensitivity to immunotherapy [
43,
44]. The keynote-177 study has shown that pembrolizumab has emerged as a new standard of first-line treatment for patients with metastatic dMMR/MSI-H colorectal cancer [
45]. We found that TOP2A, APOL6, MATR3 were correlated with MSI and could be used as potential indicators of sensitivity to immunotherapy for rectal cancer. In parallel, we visualized cytoscape to construct a comprehensive transcriptional regulatory network of five key genes involved in radiotherapy in rectal cancer.
We found a new key gene related to the prognosis of rectal cancer, which may be a new biomarker for rectal cancer. We validated the expression of five core genes in human rectal cancer tissue samples in cancer and adjacent non-cancerous tissues and found that MATR3 expression was significant difference and there was no significant difference in the expression of the remaining four core genes. However, our study still has some limitations. First, the sample size retrieved from the database was limited. Second, the findings lack in vitro and in vivo experimental validation. In subsequent studies, we will perform cell function experiments, xenograft experiments in nude mice, and molecular mechanism experiments on irradiated human rectal cancer cell lines after overexpression or knockdown of TOP2A, MATR3, APOL6, JOSD1, and HOXC6. Despite these shortcomings, preliminary findings can still provide very meaningful and constructive information.
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