Colorectal cancer (CRC) stands as a significant global health burden, ranking third among the leading causes of cancer-related mortality for both males and females. In 2020 alone, an estimated 515,637 deaths occurred among males, while females accounted for 419,536 deaths [52]. The overall survival (OS) rates for patients diagnosed with metastatic colorectal cancer indicate a challenging scenario, with approximately 70-75% surviving beyond one year, 30-35% surviving beyond three years, and less than 20% surviving beyond five years post-diagnosis [53]. The primary treatment approach for unresectable metastatic colorectal adenocarcinoma (COAD) involves systemic therapy, encompassing cytotoxic chemotherapy, biological therapies targeting cell growth factors, immunotherapy, and their combinations. Notably, recent clinical trials have demonstrated improved OS by tailoring treatments to the molecular and pathological features of individual tumors, underscoring the importance of analyzing genomic, transcriptomic, and proteomic profiles to identify effective treatments for specific somatic types [54-56].
Here, we employed an integrative analysis approach to identify COAD-specific differentially expressed genes by analyzing common DEGs from the GEO and GEPIA2 datasets within a pan-cancer model. Our investigation highlighted GUCA2A as a significantly differentiated gene specific to COAD. Through an examination of TCGA-COAD data, we observed downregulation of GUCA2A mRNA levels in COAD tissues compared to adjacent normal tissues and normal colon samples. Notably, our predictive analysis revealed a strong association between lower GUCA2A expression levels and shorter OS in COAD patients, indicating the potential prognostic value of GUCA2A in COAD. Additionally, the ROC curve analysis demonstrated the robust diagnostic performance of GUCA2A expression in differentiating COAD tissue from normal tissue, with 98% sensitivity, 95% specificity, and an impressive 99.6% AUC, further supporting its potential as a robust diagnostic marker for COAD. These findings align with the study by Zhang et al.[53], which reported a significantly shorter OS in COAD patients with lower GUCA2A expression levels compared to those with higher expression levels. Furthermore, Liu et al. [57] employed Cox regression analysis to identify genes associated with colorectal cancer prognosis and constructed a 3-gene signature, including CLCA1-CLCA4-GUCA2A, which exhibited predictive power for prognosis in colorectal cancer.
To gain insights into the genomic alterations associated with GUCA2A in COAD, we conducted further investigations. Our analysis revealed the occurrence of five GUCA2A missense mutations in COAD, with P75S, A98T, A108V, and G114R classified as deleterious mutations, while A98T, A108V, and G114R were classified as probably damaging mutations. Additionally, the analysis of GUCA2A methylation data revealed differential methylation patterns in COAD compared to healthy individuals, with one probe exhibiting significant differential methylation. In line with the expression profile, we observed a significant downregulation of GUCA2A promoter methylation in COAD, indicating the potential involvement of mutations and methylation in GUCA2A in altering the expression of GUCA2A protein and contributing to CRC pathogenesis.
The co-expression analysis conducted in our study revealed a strong positive correlation between GUCA2A and GUCA2B. These two genes encode peptide hormones that function as endogenous ligands for the guanylate cyclase-C (GUCY2C) receptor [58]. Specifically, GUCA2A and GUCA2B encode guanylin (GU) and uroguanylin (UG), respectively, while GUCY2C encodes guanylyl cyclase C (GC-C). The activation of GC-C occurs through GN and UG, which share structural and functional similarities. The GN and UG peptides play a crucial role in the transduction signaling that regulates homeostasis, as well as the transport and secretion of fluids and electrolytes in the gastrointestinal tract during the process of digestion [51, 59].
Previous studies have highlighted the significance of GUCY2C signaling in the mediation of mucosal wounding and inflammation by controlling the production of resistin-like molecule β [60]. Downregulation of GUCA2A, GUCA2B, and GUCY2C has been observed in inflammatory bowel disease, suggesting their potential involvement in the pathogenesis of this condition [61]. Furthermore, recent research has demonstrated that GUCY2C can exert inhibitory effects on tumor progression in the intestine, and the loss of GUCY2C signaling cascade increases susceptibility to colorectal cancer (CRC) [62, 63]. The disruption of intestinal homeostasis and the development of CRC are often associated with the loss of GUCA2A and GUCA2B [64-66]. Bashir et al. presented evidence suggesting that the loss of GUCA2A could lead to the silencing of GUCY2C, thereby contributing to the development of microsatellite instability tumors [67]. These findings collectively indicate the potential involvement of GUCA2A, GUCA2B, and GUCY2C in crucial biological processes, including gastrointestinal fluid regulation, inflammation mediation, and CRC development. Further investigation into the precise mechanisms underlying the interplay between these genes and their roles in intestinal homeostasis and tumorigenesis is warranted. Understanding these processes at a molecular level could potentially lead to the development of novel therapeutic strategies and interventions targeting GUCY2C signaling for the management and prevention of CRC.
Moreover, we performed an intersectional analysis to identify genes and proteins that share a similar expression pattern with GUCA2A. Through this analysis, we constructed a GUCA2A protein-protein interaction (PPI) network consisting of 11 proteins. Among the identified common genes, TMIGD1, SLC26A3, NHERF4, and GUCA2B stood out for their potential significance in colorectal cancer (CRC). TMIGD1 has been implicated as a tumor suppressor gene that plays a crucial role in the intestinal epithelium. De La Cena et al. reported that the loss of TMIGD1 leads to adverse effects on the brush border membrane, junctional polarity, and maturation of the intestinal epithelium [68]. Their study demonstrated that TMIGD1 acts as a tumor suppressor by inhibiting tumor cell proliferation and migration, and by arresting the cell cycle at the G2/M phase. Moreover, TMIGD1 was found to induce the expression of key cell cycle inhibitor proteins, p21CIP1 and p27KIP1, which are responsible for regulating cell cycle progression. Importantly, TMIGD1 expression was progressively downregulated in sporadic human CRC, and its downregulation correlated with poor overall survival. These findings suggest that TMIGD1 could serve as a potential therapeutic target and a novel tumor suppressor gene, shedding light on the pathogenesis of CRC [68]. Similarly, another study by Mu et al. identified TMIGD1 as one of the highly downregulated genes in CRC, indicating its potential role in promoting CRC progression and invasion [69]. SLC26A3 is a transporter protein involved in the exchange of chloride and bicarbonate ions in intestinal cells, predominantly expressed in the apical domain of various intestinal segments. Studies have consistently reported a significant decrease in SLC26A3 expression levels in patients with CRC, suggesting its potential involvement in CRC progression. However, some studies propose that SLC26A3 is primarily expressed in differentiated colon cells rather than proliferating cells, potentially serving as a marker for differentiation [70, 71]. NHERF4 is a regulatory protein that interacts with GUCY2C and negatively modulates its activation induced by heat-stable enterotoxin [72]. Additionally, NHERF4 stimulates the activity of SLC9A3 in the presence of high calcium ions [73]. NHERF1, a closely related member of the NHERF family, has been identified as a key regulator of CRC progression through its interaction with the VEGFR2 pathway. High expression of NHERF1 has been associated with CRC progression, metastasis, and significantly worse overall survival, recurrence-free survival, and disease-specific survival. Knockdown of NHERF1 has been shown to increase apoptosis and reduce the expression of XIAP/survivin, underscoring the critical role of NHERF1 in CRC cell survival [74, 75]. The identification of TMIGD1, SLC26A3, and NHERF4 in our GUCA2A PPI network suggests their potential involvement in CRC pathogenesis.
Recent studies have emphasized the crucial role of different classes of non-coding RNAs, including mRNAs, miRNAs, and lncRNAs, in various biological processes and their associations with human diseases [76, 77]. Computational models have been developed to predict potential associations between miRNAs/lncRNAs and human diseases, providing valuable tools for disease-association prediction [78-81]. In 2011, the concept of competitive endogenous RNA (ceRNA) was introduced, which involves non-coding RNAs, such as lncRNAs or circRNAs, acting as competitive binding partners for miRNAs, thereby reducing the repression of target mRNAs by miRNAs [82]. In our study, we focused on investigating the involvement of miRNAs and circRNAs in the regulatory network of GUCA2A, aiming to identify more effective biomarkers and gain insights into the pathogenesis of colorectal adenocarcinoma (COAD) at different molecular levels. We constructed a comprehensive ceRNA network comprising 8 miRNAs that target GUCA2A and 183 circRNAs acting as miRNA sponges. Among the identified miRNAs, hsa-miR-1207-5p has been reported to promote the proliferation of breast cancer cells by directly regulating STAT6 [83]. Moreover, studies have demonstrated a significant decrease in circulating miR-1207-5p levels, which is associated with poor prognosis and serves as a highly diagnostic marker in colorectal cancer (CRC) [84]. Additionally, Ng et al. found a correlation between high tumor levels of miR-187-3p and poor prognosis in colorectal cancer [85].
We further aimed to explore the relationship between GUCA2A expression and the immune properties of the tumor microenvironment in colorectal adenocarcinoma. Our findings revealed significant correlations between GUCA2A expression and various immune cell populations within the tumor microenvironment. We observed significant correlations between GUCA2A expression and the abundance of several immune cell subsets, including CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, type 17 T helper cells, type 2 T helper cells, activated B cells, natural killer (NK) cells, natural killer T cells, eosinophils, mast cells, monocytes, and neutrophils. These correlations highlight the potential involvement of GUCA2A in modulating the immune response in COAD. Previous studies have indicated the significance of T helper 17 cells in the progression of colorectal cancer [86]. The presence of these cells within the tumor microenvironment has been associated with disease progression. Furthermore, infiltration of B cells, NK cells, and macrophages has been linked to a favorable prognosis in colorectal cancer [87-90]. The significant correlations observed between T and B cells, as well as activated NK cells, and GUCA2A expression suggest that these immune cell populations may contribute to the impact of GUCA2A on the survival of COAD patients. Our findings suggest that GUCA2A may interact with immune cells and influence the immune landscape within the COAD tumor microenvironment. Further investigations are warranted to elucidate the underlying mechanisms by which GUCA2A influences immune cell populations and its potential implications for the prognosis and treatment of COAD. Understanding the intricate interplay between GUCA2A expression and immune cell populations will provide valuable insights into the immune-mediated mechanisms driving COAD progression. This knowledge could potentially contribute to the development of immunotherapeutic strategies targeting GUCA2A and its associated immune pathways in the treatment of COAD patients.
We also conducted a gene-drug analysis to identify drugs that interact with GUCA2A, and we identified three drugs: lactose anhydrous, atropine, and volanesorsen sodium. The associations between these drugs and GUCA2A provide insights into potential therapeutic strategies and shed light on the underlying molecular mechanisms in colorectal cancer. Lactose intolerance has been found to have a significant relationship with sporadic CRC, suggesting that lactose intolerance may act as a risk factor for CRC development [91]. Interestingly, lactose consumption has been shown to lower the risk of CRC by activating the guanylate signaling pathway. The interaction between lactose anhydrous and GUCA2A highlights the potential role of this drug in modulating GUCA2A-mediated signaling pathways and its implications in CRC prevention. Another chemotherapy drug commonly used in colorectal cancer treatment is irinotecan. It has been observed that irinotecan can induce diarrhea as a side effect. Atropine, an anticholinergic agent, is used to prevent the development of irinotecan-induced diarrhea [92]. The interaction between atropine and GUCA2A suggests a potential mechanism through which atropine may modulate GUCA2A-associated pathways to alleviate diarrhea in patients receiving irinotecan-based chemotherapy. Further, Volanesorsen sodium is a drug used in the treatment of familial chylomicronemia syndrome or hypertriglyceridemia [93]. This drug targets apolipoprotein C3 (apoC3) to increase the clearance of chylomicrons and other triglyceride-rich lipoproteins, leading to a significant reduction in triglyceride (TG) levels by 70-80%. Elevated TG levels have been associated with an increased risk of pancreatitis, and there is also a significant relationship between hypertriglyceridemia and CRC [94]. The interaction between Volanesorsen sodium and GUCA2A suggests a potential link between GUCA2A and TG metabolism pathways, highlighting the importance of GUCA2A in modulating lipid-related pathways that may impact CRC risk. Additionally, our GUCA2A KEGG pathway analysis identified pancreatic secretion as a significant pathway associated with GUCA2A. The downregulation of GUCA2A expression may contribute to an elevation in TG levels, potentially increasing the risk of CRC. These findings provide valuable insights into the molecular pathways influenced by GUCA2A and its potential role in CRC development. The identification of drugs that interact with GUCA2A and the exploration of associated pathways provide a foundation for further research and potential therapeutic interventions. Understanding the mechanisms underlying the interactions between GUCA2A and these drugs may lead to the development of novel treatment approaches targeting GUCA2A-mediated pathways in CRC. Further investigations are warranted to validate these findings and explore the clinical implications of targeting GUCA2A and its associated pathways in the prevention and treatment of CRC. The identification of GUCA2A as a potential therapeutic target opens up new possibilities for precision medicine strategies in CRC management.
While this study contributes to the understanding of GUCA2A in COAD, it is important to acknowledge the limitations. Firstly, the microarray data obtained from the GEO database and the TCGA-RNASeq data were acquired from the GEPIA2 database. Utilizing data from different laboratories with diverse platforms may introduce systematic biases and variations in the results. Although efforts were made to minimize these biases through data processing and normalization, it is important to consider these potential limitations when interpreting the findings. Secondly, to further validate and enhance the credibility of our study's findings regarding the potential function of GUCA2A, in vivo and in vitro experiments are warranted. Experimental studies involving animal models or cell culture systems can provide more detailed mechanistic insights into the role of GUCA2A in colorectal cancer and its underlying molecular pathways. These experiments would help elucidate the functional significance of GUCA2A in tumor development, progression, and response to treatment. Thirdly, no anti-GUCA2A therapeutic monoclonal antibodies have been evaluated in clinical trials to date. Therefore, there is a lack of specific data available to assess the potential benefits of anti-GUCA2A targeting drugs in terms of the survival of COAD patients or inhibition of tumor growth. Future investigations should focus on exploring the feasibility and efficacy of anti-GUCA2A therapies, including the development and evaluation of novel anti-tumor immunotherapy drugs that specifically target GUCA2A. This would provide valuable insights into the clinical applicability of GUCA2A as a therapeutic target in COAD. In the future, a prospective study examining GUCA2A expression and its impact on immune infiltration in COAD patients is needed. By integrating comprehensive analyses of GUCA2A expression and immune cell infiltration, a deeper understanding of the interplay between GUCA2A and the tumor microenvironment can be obtained. Furthermore, testing newly developed anti-tumor immunotherapy drugs that target GUCA2A would provide valuable data on their efficacy, safety, and potential synergistic effects with existing treatment modalities. Addressing these limitations through further experimental studies, clinical trials, and prospective investigations will enhance our understanding of GUCA2A's functional role, clinical significance, and therapeutic potential in COAD management.