Genomic signature of MTOR could be an immunogenicity marker in human colorectal cancer

Mammalian target of rapamycin (mTOR), the protein product of the MTOR gene, is a serine/threonine kinase and an important downstream effector of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway. In mammalian cells, the PI3K/AKT/mTOR pathway plays a crucial role in the regulation of cell survival, metabolism, growth, and protein synthesis in response to upstream signals during both normal physiological and abnormal pathological conditions [1]. This pathway represents one of the most deregulated signaling pathways in human cancer. mTOR is considered to serve as a master regulator of this signaling pathway, and recent findings have reported that mTOR activation plays a vital role in human cancer [2]. As reported, mTOR is aberrantly overactivated in more than 70% of cancers [3]. In lung cancer, the activation of the mTOR pathway is associated with a poor prognosis [4, 5]. In breast cancer, mTOR expression correlated with a worse prognosis, and p-mTOR was more commonly detected in triple-negative breast cancers [6, 7]. The abnormality of mTOR activation in cancer offers opportunities for targeted therapy. Several distinct classes of drugs have been developed to inhibit mTOR, including antibiotic allosteric mTOR inhibitors (rapamycin and its rapalogs), ATP-competitive mTOR inhibitors, and mTOR/PI3K dual inhibitors. Several mTOR inhibitors have reached various stages of clinical trials, but only temsirolimus and everolimus have been approved by the Food and Drug Administration for clinical use in the treatment of cancer patients. Preclinical studies have demonstrated the efficacy of most mTOR inhibitors in several types of cancers; however, early clinical trials have yielded mixed results because inhibitors (such as ATP-competitive mTOR inhibitors and mTOR/PI3K dual inhibitors) were often associated with dose-limiting toxicities, possibly due to low selectivity [8‐12]. The degree of dependence on mTOR activation varies, and even among patients with similar mutation profiles, the outcomes associated with mTOR inhibitor therapy have been reported to vary across different cancer types. Although temsirolimus has been approved for the treatment of patients with advanced-stage renal cell carcinoma (RCC), clinical experience has indicated that only 8.6% of patients experienced an objective response in phase III Global advanced-stage RCC trial [13]. Identifying predictive biomarkers capable of determining which patients might benefit from mTOR inhibitors remains necessary. Mutations in MTOR have been shown to alter the sensitivity to rapalog treatment in several cancer types [14‐16]. Therefore, the molecular alterations of MTOR warrant further investigation.

Colorectal cancer (CRC) is the second most common cause of cancer-related deaths worldwide [17] and represents one of the most common malignant tumors in China. The incidence and mortality of CRC in China ranked third and fifth, respectively, among all malignant tumors in 2015, associated with 388,000 new cases and 187,000 deaths [18]. Although developments in targeted therapy and immunotherapy have improved outcomes for some patients, the prognosis of CRC remains far from satisfactory for the majority of patients [19]. Therefore, the identification of potential prognostic biomarkers and novel treatment targets for CRC remains necessary. Several studies have examined the role of the PI3K/AKT/mTOR pathway in CRC, but most of these have focused on upstream regulators, such as phosphatase and tensin homolog (PTEN) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), with few studies focused on mTOR itself. Previous studies [20‐23] have demonstrated that mTOR is highly activated in CRC, and associated with the proliferation of CRC. However, the functional outcomes and the underlying activating mechanisms of MTOR in CRC remain to be investigated. Currently, the efficacy of mTOR inhibitors is limited in CRC, which may be related to the lack of study regarding MTOR function in this cancer type.

In this study, MTOR expression in CRC and the correlations between MTOR expression and prognosis were analyzed using datasets obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Differential gene expression analyses, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and gene set enrichment analysis (GSEA), were used to investigate the possible molecular functions of MTOR in CRC. We also analyzed the correlation between MTOR and tumor mutational burden (TMB), the tumor immune microenvironment, and microsatellite instability (MSI) status. Finally, for further exploration and verification, we examined the single-nucleotide variations in the MTOR sequence among an Asian population cohort from our database. Our findings indicate the prognostic value of MTOR in CRC and demonstrate the potential associations between MTOR and tumor mutation. We hope this study will contribute to new prognostic monitoring and treatment strategies for CRC patients.

CRC is a highly heterogeneous disease caused by the interaction between genetic and environmental factors. Multiple alternative genetic pathways exist in CRC development. CRC evolves from benign to malignant lesions, and many mutations in oncogenes and tumor suppressor genes are involved in CRC tumorigenesis and progression [30, 31]. CRC characterized by high mutation levels is thought to harbor an increased neoantigen burden, which is highly immunogenic and sensitive to immune checkpoint inhibitor (ICI) therapy [32]. The investigation of predictive and prognostic biomarkers has historically focused on alterations in the RAS/BRAF/MEK/MAPK and PI3K/AKT/mTOR pathways. For the RAS/BRAF/MEK/MAPK pathway, fruitful and solid evidence has emerged supporting the use of BRAF as a prognostic biomarker and RAS as a predictive biomarker in CRC [33]. However, biomarkers associated with the PI3K/AKT/mTOR pathway, such as PIK3CA and PTEN, are not recommended for use in clinical practice due to insufficient evidence, especially associated with CRC treatment [34, 35]. Therefore, identifying other components of the PI3K/AKT/mTOR pathway that may serve as prognostic biomarkers for targeted therapy and ICI remains necessary. In this study, we focused on mTOR, which is a master regulator of the PI3K/AKT signaling pathway. The role of MTOR in CRC has been under-characterized, and we aimed to dissect its biological functions and reveal its associations with responses to targeted therapies.

The pan-cancer analysis showed that MTOR mRNA is overexpressed in many cancers. However, analysis of RNA sequencing data from TCGA (COAD and READ) suggested no significant differences in mTOR expression between tumor tissues and normal tissues, which was inconsistent with previous findings [20‐22]. Considering the potential for ethnic differences to affect tumorigenesis, we further selected expression data associated with CRC among an Asian population from the GEO database for analysis and identified that the expression of MTOR in tumor tissues was upregulated compared with normal tissues in an ethnicity-dependent manner in an Asian cohort.

To analyze the prognostic value of MTOR among CRC patients, we divided CRC patients from the TCGA dataset into high and low MTOR expression groups based on the median and quartile levels. No significant difference was expected between groups based on the median level because the expression of MTOR in cancer tissues did not differ significantly from that in normal tissues. However, when analyzed according to the quartile level (top 25% and bottom 25%), the high MTOR expression group was significantly associated with poor survival, which suggested that differences in MTOR expression level were associated with prognosis. A similar trend was also found among CRC patients from the GEO database, although the differences in the GEO dataset did not reach significance, possibly due to the small sample size. These results indicated the potential for MTOR to serve as a prognostic marker in Asian CRC patients, although this possibility requires further clinical validation.

Pathway analysis and GSEA results showed that DEGs were enriched in the metabolism, cell adhesion, and translation pathways. MTOR was associated with a key fatty acid, glutathione, and oxidative phosphorylation metabolic pathways in CRC. Fatty acids are indispensable for the synthesis of membranes and signaling molecules, which are associated with cell proliferation [36]. As an antioxidant, glutathione has profound effects on cell survival while also conferring therapeutic resistance to cancer cells [37]. Oxidative phosphorylation is a primary source of energy. Studies have shown that mitochondrial DNA (mtDNA) content is higher in CRC than in normal tissue, which may indicate a higher contribution of oxidative phosphorylation in CRC [38]. MTOR serves as a key regulator of these metabolic pathways [39‐41]. In addition, MTOR regulates protein translation and synthesis to promote cell proliferation. The proliferation of CRC cells can be disrupted by reducing the phosphorylation level of eIF4E-binding protein 1 (4EBP1), which is a tumor suppressor protein activated by mTOR [42]. Cell adhesion is a key mediator of cancer progression and facilitates several behavioral hallmarks of cancer, including immune evasion and metastatic dissemination [43]. Further study found that mTOR complex (mTORC)1 and mTORC2 were both involved in the regulation of cell adhesion [44]. Therefore, MTOR might affect these signaling pathways, contributing to a poor prognosis in CRC patients.

Our study revealed that MTOR expression was associated with a high somatic mutation burden. In theory, tumors with a higher number of genetic variations are statistically more likely to generate novel mutant proteins or neoantigens, which may be recognized as foreign invaders by the immune system and trigger a cytotoxic, tumor-killing response [45]. Relevant analyses revealed a correlation between TMB and the response rates and outcomes of ICI therapy [46]. ICI therapy has shown promising results in various types of cancers, particularly antibodies against the programmed cell death protein 1 (PD-1) T-cell coreceptor and its ligand B7-H1/programmed death-ligand 1 (PD-L1), which have induced durable tumor responses, even in late-stage patients who have failed to respond to multiple classical treatment strategies [47, 48]. Previous studies have indicated that immune infiltration levels are related to prognosis and the response to ICI therapy for several cancers, including esophageal squamous cell carcinoma, breast cancer, and CRC [49‐51]. In the TME, we found that the MTOR expression and CNAs significantly affected the immune cell infiltration of CD8⁺ T cells, B cells, neutrophils, and dendritic cells in CRC. Using CIBERSORT, our results showed that CD8⁺ T cells were negatively correlated with MTOR expression. In addition, we found that the expression levels of PDCD-1, CTLA4, and LAG3 were positively associated with MTOR expression. These results indicated that MTOR status could potentially be used to assess whether patients might benefit from ICI therapy.

Moreover, our results showed that the expression and function of MTOR were altered in MSI-H CRC patients. MSI is an intensively studied biomarker with prognostic and therapeutic values in CRC. MSI refers to changes in the lengths of short-tandem-repeat DNA sequences, the presence of which represents phenotypic evidence of deficient mismatch repair (dMMR). MMR is a highly conserved cellular process intended to correct erroneous insertions, deletions, and base–base mismatches that occur during DNA replication and recombination and have escaped the proofreading process. When the MMR system develops a malfunction, errors generated during DNA replication increase, including single-base substitutions, insertions, or deletions of short-tandem-repeat DNA sequences, resulting in MSI [52]. MSI is present in approximately 15% of CRC and 5% of metastatic CRC (mCRC) [53, 54]. Depending on the degree of instability, MSI tumors can be divided into MSI-H or MSI-L subsets. Several clinical studies have found that patients with MSI-H present with a durable and robust response to ICI therapy [55, 56]. Although the response rates to ICI among patients with MSI-H CRC have been variable in different trials, more somatic mutations and higher neoantigen burdens were identified in responsive tumors than in non-responsive tumors [57].

Few studies have reported the correlation between MTOR and MSI-H. Vilar et al. [58] reported that MSI-H cell lines responded better to therapies that preferentially target the PI3K/AKT/mTOR pathway but did not explore the expression of MTOR in MSI-H. Consistent with these results, we revealed that rapamycin preferentially targeted MSI-H cell lines via cytotoxicity experiments. Additionally, we found that the expression of MTOR increased in MSI-H CRC samples from a public database, which was then validated in both cell lines and CRC cohorts from our center. Lin et al. reported that MSI-H CRC tumors feature a significantly increased number of mTOR pathway mutations than MSS tumors [59]. In addition, evidence has shown that MTOR mutants can constitutively activate the mTOR signaling pathway and increase the sensitivity to rapalog treatment [60]. Therefore, whole-exome sequencing in CRC patients (MSI-H or MSS) was applied to detect variations among all SNP sites in MTOR. To the best of our knowledge, this is the first systematic study examining the relationship between MTOR mutations and CRC. Our analysis showed that the mutation frequency of MTOR in MSI-H patients was significantly increased compared with MSS patients, which can affect the protein activity of mTOR and influence the tumor response to rapamycin treatment. Furthermore, we observed a significant increase in the frequency of transition mutations in the MSI-H group compared with that in the MSS group, which may be attributed to dMMR. Studies on MMR function have shown that MMR has a higher repair efficiency for transition mutations than other mutations [61, 62]. These results suggested that MTOR mutations may present at a higher frequency in CRC patients with dMMR. Combined with the relationship between MTOR and high TMB, MTOR status could potentially be used to assess whether MSI-H patients might benefit from ICI therapy. Further research examining the efficacy of ICI therapy in MTOR-mutant MSI-H CRC remains necessary.

In this study, we found that MTOR played an important role in tumorigenesis and was associated with the immunological status of CRC. The function of mTOR in immunity has been extensively studied. Several studies have reported that the mTOR signaling pathway affects the function of immune cells and cytokines in the TME [63‐65]. In our study, MTOR was shown to be associated with the infiltration of various immune cells. Moreover, our study found that the high expression of MTOR was related to high TMB. Tumors with higher numbers of genetic variations are more likely to generate novel mutant proteins or neoantigens. Therefore, MTOR is likely not only associated with immune cell function but also with tumor cell immunogenicity, suggesting that MTOR may play a central role in tumor immunity. Interestingly, we found that MTOR was associated with MSI status, which was characterized by high TMB and abundant TIICs. The consistent relationship identified between MTOR mutations and MSI suggests that MTOR may represent a marker for the prediction of MSI status and tumor immunogenicity. Yang et al. [66] reported that MSI CRC tumors had higher expression levels of thymocyte selection–associated high-mobility group box (TOX), an inhibitor of mTOR, compared with MSS tumors. Our research focused on MTOR mutations rather than MTOR expression levels, and the work by Yang et al. did not examine the relationship between TOX expression and MTOR mutation.

The significant contributions of MTOR to CRC tumorigenesis suggest that mTOR inhibitors may be effective for CRC therapy. Several clinical trials have studied the efficacy of mTOR inhibitors in CRC patients (A supplementary table shows this in more detail). For example, in a clinical phase I/II study, everolimus combined with mFOLFOX-6 and bevacizumab was found to be tolerable and demonstrated preliminary efficacy for mCRC therapy. The objective response rate was 53% in mCRC patients and was higher (86%) in those cases with PTEN deficiency [67]. Studies on other types of inhibitors, such as ATP-competitive mTOR inhibitors and mTOR/PI3K dual inhibitors, have also shown tumor growth inhibition effects against CRC cell lines and xenograft models [68, 69]. Moreover, studies have found that BEZ235, an mTOR/PI3K dual inhibitor, was capable of inducing a treatment response and overcoming resistance to everolimus in APC- and PIK3CA-mutant CRC cells [70, 71]. These findings suggested that different molecular subtypes might be associated with mTOR inhibition responses, which could be used to distinguish patients who will benefit from mTOR inhibition therapy. Further research examining the efficacy of mTOR inhibitors in MTOR-mutant remains necessary.

Moreover, the status of MTOR has been shown to regulate immunoreactions. Rapamycin is widely used as an immunosuppressant to prevent immune rejection in kidney transplant patients. Jung et al. found that rapamycin uniquely enhanced the number and function of CD8⁺ effector and central memory T cells [72]. In a mouse model of RCC, anti-PD-L1 combined with everolimus was more effective for tumor regression than individual treatment due to the upregulation of PD-L1 in tumor cells, which increased the tumor-infiltrating CD8⁺ T cells [73]. Similarly, our study demonstrated that the status of MTOR could be used to assess the immunological function of CRC patients and might serve as a potential indicator that can predict the optimum response to ICI therapy. mTOR inhibitors that promote cancer cell death and boost effector functions in T cells can be combined to improve ICI therapeutic outcomes. The relationship between MTOR mutations and dMMR suggests that CRC patients with dMMR are likely to benefit from combination therapy. It is worth noting that several studies have found the efficacy of rapamycin is likely to be based on dosage [74]. Like a double-edged sword, too low-dose rapamycin was insufficient to activate its therapeutic effects on the diseases, such as mitochondrial disease [75]. However, too high-dose rapamycin is clinically toxic. Low-dose rapamycin could enhance the quantity and quality of CD8⁺T cells, while the higher doses of rapamycin inhibited the T-cells response [76]. Therefore, it is meaningful and could be challenging, to further explore the most appropriate dose of rapamycin to strike a balance between stimulating immunity and anti-tumor activity.

In summary, our study found that MTOR plays an important role in CRC tumorigenesis and was associated with prognosis, metabolism, and immune. Furthermore, adenosine monophosphate-activated protein kinase (AMPK) has been found to be involved in regulating mTOR and mTOR regulated pathways in a feedback loop manner [77, 78]. 5-Aminoimidazole-4-carboxamide-1-β-4-ribofuranoside, an AMPK activator, could inhibit mTORC1 and promote the therapeutic effect of rapamycin in cancers [79]. The similarity of compounds to rapamycin in transcriptional signature have been previously shown to have rapamycin-like properties [80]. Therefore, elaborating this connection will be a future aspect of our study, providing new light on the role of AMPK and AMPK activators in CRC.

In conclusion, our study found that MTOR plays an important role in CRC tumorigenesis and was associated with prognosis, TMB, TIICs, and MSI status. Therefore, MTOR may represent a comprehensive indicator of prognosis and immunological status for CRC. Moreover, the mutation profile of MTOR may provide guidance for subsequent clinical research on mTOR inhibitors in CRC.