Identification of hub genes and pathways in lung metastatic colorectal cancer

Colorectal cancer (CRC) is one of the most common and prevalent malignant cancers with the third highest incidence frequency and the second highest mortality rate among all cancers worldwide [1, 2]. In 2022, 151,030 new CRC cases and 52,580 CRC-related deaths were estimated to have occurred in the United States [3]. Approximately 90% of patients with primary CRC cases at early stage can be cured by surgical resection. However, most patients with CRC are diagnosed at advanced stages with recurrence in distant organs, and thus do not have the opportunity to undergo radical surgery [4].

Metastasis is the predominant cause of CRC patient death. According to a recent study, 20% of CRC patients who are newly diagnosed have metastatic disease, and 25% of people with localized CRC will eventually develop metastases. Fewer than 20% of metastatic CRC patients survive for five years [5]. In fact, the lungs are the second most prevalent location of CRC metastasis, accounting for approximately 20–30% of cases [6]. However, limited therapeutic methods are available due to the lack of understanding in the biology of colorectal lung metastases. Therefore, a better understanding of the molecular mechanism of lung metastatic CRC is urgently needed to improve existing treatments and reduce CRC patients’ mortality.

Previous studies have demonstrated that a number of different molecules participate in the development of CRC metastases. For instance, CXCL12/CXCR4, the chemokine receptor pairs, are thought to be associated with liver metastasis and tumour recurrence in CRC [7]. CXCR7 activation is thought to promote the spread of CRC cells to the lung instead of the liver [8]. In addition, some genetic changes, such as WNT pathway activation and RAS mutation, may be linked to an increased proportion of lung metastases [9, 10]. However, these results are scarcely sufficient to provide a comprehensive picture of CRC lung metastases.

Recently, bioinformatics analyses emerged as an efficient and promising tool to screen significantly aberrantly expressed genes and genetic pathways involved in carcinogenesis, which could provide a rationale to identify potential therapeutic targets cancer and understand a cancer prognosis [11‐13]. In particular, many studies utilized integrated microarrays analysis and reported that certain vital genes or pathways potentially are involved in CRC liver metastasis or lymph node metastasis [12, 13]. However, studies were quite limited in CRC lung metastases. In this study, the GEO2R tool was utilized to identify differentially expressed genes (DEGs) between primary CRC and lung metastatic CRC tissues based on the GSE41258 and GSE68468 profiles. Subsequently, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome Gene Sets analyses were conducted to uncover enriched top biological processes and pathways regulated by the DEGs. The top 10 hub genes related to lung metastasis in CRC and the protein–protein interaction (PPI) network were identified using the search tool Retrieval of Interacting Genes (STRING) and Cytoscape. In addition, the expression and prognostic values of these the hub genes in CRC patients were validated by analyzing the database of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Furthermore, SFTPD, one of the hub genes specifically upregulated in lung metastatic CRC, was validated to promote cellular proliferation and lung metastasis in CRC in vitro and in vivo. In conclusion, the present study may contribute to identifying key genes and pathways for the diagnosis and prognosis of CRC patients with lung metastases, as well as yield novel and viable therapeutic targets.

Understanding the underlying molecular mechanisms of CRC lung metastasis would greatly benefit diagnosis, management and prognosis evaluation. In the present study, we identified 57 highly expressed DEGs and 18 poorly expressed DEGs between primary CRC samples and lung metastatic CRC samples by analyzing microarrays in the GEO database. The enrichment of these deregulated genes revealed that core pathways and hub genes could lead to new insights into CRC lung metastasis.

As suggested by GO analysis, the dysregulated genes were mainly enriched in inflammatory response, chemotaxis, chemokine activity, immune response, immunoglobulin receptor binding, antigen binding, cell adhesion and positive regulation of ERK1/2 cascade. This is plausible since inflammatory responses are important in the progression of cancer, including tumour initiation and metastasis. As main inflammatory mediators, chemokine activity, chemotaxis or aberrant immune reactions are critical tumourigenic signals of CRC [20, 21]. Cell adhesion molecules play a significant role in cell permeability, polarity and migration, which are the vital steps in CRC progression and metastasis [22]. In addition, it has been reported that the dysfunction of ERK/MAPK pathway is a crucial trigger for the progression of most cancers [23]. Moreover, the DEGs were also found to be enriched in the formation of ECM, extracellular exosomes, extracellular space and so on, indicating that the interaction with the extracellular environment could be triggered during the CRC lung metastatic process.

The KEGG and Reactome Gene Sets analyses of DEGs and module analyses of the PPI network suggested that surfactant metabolism, phagosomes, cell–cell communication, and ECM organization may be involved in CRC lung metastasis, except for cell adhesion molecules and chemokine signaling pathways, which have already been found in GO enrichment. To date, no direct evidence of the role of surfactant metabolism in CRC lung metastases has been presented. Several studies reported the interaction between cancer metastasis and surfactant metabolism. It has been demonstrated that pulmonary and extra-pulmonary existence of surfactant proteins play important roles in film stabilization, viral defense and modulation of immune responses [24]. In the current study, the expression of SFTPB, SFTPC, SFTPD, and ABCA3, which are involved in the production, function, and metabolism of surfactant [25], was shown to be highly expressed in CRC lung metastases, suggesting that they may promote CRC lung metastasis. Phagosomes are dynamic organelles generated within cells by the uptake of particles larger than 0.5 μm, which are essential for pathogen eradication and antigen presentation in the process of innate and adaptive immunity [26]. Emerging evidence highlights the effect of immune microenvironment on colorectal metastasis [27]. This implies that DEGs associated with phagosomes formation and maturation might participate in CRC lung metastasis by influencing immunity.

Cell–cell communication is crucial for several biological events, including cell fate determination, proliferation, migration, and homeostasis. It has been well recognized that cell–cell communication between tumour microenvironments (e.g., stromal fibroblasts, epithelial cells, and multiple immune cell-types) and cancer cells drives CRC metastasis [28, 29]. ECM consists of various molecules, such as laminin, collagen, elastin and fibronectin, and plays a central role in tumour initiation, progression, and metastasis. Cross-talk between the ECM and CRC metastasis has been well clarified in the previous report [30]. Dysregulated ECM-related proteins induce both biochemical and biomechanical changes to promote cancer metastasis [18]. Herein, the upregulated expression of MGP, Biglycan, LTBP2 and PRELP may facilitate the interactions between CRC cells and ECM, and therefore promote cellular survival and colonization in CRC lung metastases. The enriched pathways modulated by DEGs in this study could provide some rationales for developing novel therapeutic targets in the treatment of CRC.

Of importance, the top 10 hub genes were identified in CRC lung metastases, including 8 upregulated genes and 2 downregulated genes. We validated the transcriptional expression of the hub genes in numerous primary and metastatic CRC cases in the GEO database. The expression of these hub genes was in accordance with data obtained through bioinformatics analysis in GSE41258 and GSE68468. The prognostic values of these hub genes were further analyzed in the TCGA and GEO database. High expression levels of CLU, SFTPD, CCL18, SPP1, APOE and BGN were positively associated with poor overall survival of CRC patients and low expression of MMP3 was associated with longer overall survival. Therefore, we hypothesized that CLU, SFTPD, CCL18, SPP1, APOE, BGN and MMP3 might be candidate biomarkers in CRC lung metastasis. To test this hypothesis, we examined protein levels of the seven genes in primary and highly lung metastatic MC38 cells, and paired CRC primary and lung metastatic tissues. Consistently, the protein expression levels of Clusterin, SFTPD, CCL18, Osteopontin, APOE, and Biglycan were significantly higher, and MMP3 was lower in lung metastatic CRC cells or tissues than in primary CRC cells or tissues.

Among seven core genes, the expression levels of SPP1, APOE, and BGN were found to be upregulated, while MMP3 was downregulated in CRC lung metastases compared with primary CRC. Indeed, several studies have demonstrated that SPP1, APOE, and BGN could be involved in the CRC malignant phenotype [31‐33]. SPP1, encoding by Osteopontin, is an ECM protein which is reported to be overexpressed in a variety of malignancies such as ovarian cancer, breast cancer and CRC [31, 34, 35]. Osteopontin has been reported to boost the abilities of cell survival, migration, and angiogenesis to drive tumourgenesis and metastasis in CRC [31]. APOE, encoding Apolipoprotein E (APOE), is critical for lipoprotein metabolism [36]. Recent studies have demonstrated that APOE also contributes to DNA synthesis, cell proliferation, angiogenesis, and metastasis to facilitate tumorigenesis and progression [37]. Similar to previous reports that APOE was increased in CRC liver metastases [32], we found that APOE was elevated in CRC lung metastases and was positively associated with advanced stages and poor overall survival in CRC. BGN encodes Biglycan, which is a widely expressed ECM protein that provides stability and organization in tissues by interacting with other ECM proteins such as collagen and elastin [38]. Biglycan has been reported to trigger the activation of several pathways involved in tumorigenesis by orchestrating growth factors/cytokines and cell surface receptors [39]. In CRC, high level of Biglycan has been linked with metastatic progression, poor prognosis [33]. MMP3, also commonly known as matrix metallopeptidase 3, is encoded by MMP3 and belongs to a group of zinc-dependent proteolytic enzymes. Moran et al. reported that MMP3 expression was lower in CRC patients with high microsatellite instability (MSI) when compared with low or null MSI [40]. However, compelling evidence has shown that MMP3 promotes cancer invasion and metastasis by cleaving E-cadherin and disrupting its interaction with β-catenin [41, 42]. Some studies reporting that MMP3 exhibits anti-tumour activities depending in a substrate-depend manners [43, 44]. For instance, MMP3-mediated cleavage of IGF-BP3 and IGF-BP5 inhibits tumorigenesis in breast cancer [44]. Herein, MMP3 was shown to hamper CRC lung metastasis with unknown substrates, which needs further investigation.

Since retrospective clinical data reveal that 24.5% of metastatic CRC patients first develop lung metastases and lung metastases account for 32.9% of all metastatic CRCs [4], we focused on CRC lung metastasis in the present study. Here, we found that the expression levels of CLU, CCL18, and SFTPD were especially upregulated in CRC lung metastases instead of other metastases, and were positively associated with poor prognosis of CRC patients. Clusterin encoded by CLU, functions as a stress-activated molecular chaperone that is highly expressed in aggressive cancers by modulating different signaling networks [45]. It plays important roles in the regulation of protein homeostasis, pro-survival signaling and transcriptional networks [46]. Studies have demonstrated that high Clusterin expression is associated with a shorter survival time and that could be the biomarker for CRC patients [47, 48]. Therefore, targeting Clusterin might be a promising approach for the management of CRC. CCL18 encodes CC chemokine ligand 18 (CCL18), which is mainly expressed by macrophages and dendritic cells. CCL18 has been implicated in the stimulation of angiogenesis as well as cancer cell migration, invasion, and epithelial-to-mesenchymal transition. Recent studies have demonstrated that high expression of CCL18 in CRC patients is correlated with advanced tumour staging and liver metastasis [49, 50], which is similar to our findings in lung metastasis of CRC. Surfactant protein D (also known as SFTPD or SP-D), encoded by the SFTPD gene, is a collagenous glycoprotein that resides in the lungs and extra-pulmonary tissues [51]. To date, only one study has reported that SFTPD is negatively associated with pulmonary metastases in CRC [52]. However, our in vitro and in vivo results showed that SFTPD promotes cellular proliferation, migration, and invasion and further enhanced CRC cell lung metastasis. This inconsistent finding could be due to different cellular contexts and animal models.

In the current study, we highlighted that CLU, SFTPD, and CCL18 might serve as potential targets for the treatment of CRC lung metastasis. The effect of SFTPD on CRC lung metastasis was investigated through in vitro and in vivo experiments. Further investigation is warranted, especially to determine the precise mechanisms underlying the effect of these hub genes on CRC.

In summary, our bioinformatics analysis identified the DEGs and hub genes implicated in lung metastatic CRC, which may play critical roles in regulating CRC lung metastasis. A total of 75 DEGs and 10 hub genes were defined, and the enrichment analysis suggests that surfactant metabolism might play a dominant role in CRC lung metastasis. The 7 core hub genes, including CLU, SFTPD, CCL18, SPP1, APOE, BGN and MMP3, were significantly correlated with the advanced CRC stages and poor prognosis. Importantly, CLU, SFTPD and CCL18 might positively be correlated with specific lung tropism metastasis in CRC and represented as potential targets for the prevention and treatment of patients with CRC lung metastasis. Of note, our study demonstrated that SFTPD was critical to drive CRC lung metastasis. These findings may contribute to a profound understanding of CRC lung metastasis. Further studies are warranted to validate the results of these findings.