As a widespread post-translational modification of proteins, the CAPN-mediated cleavage regulates numerous cellular processes, including proliferation, differentiation, cytoskeletal reorganisation, and apoptosis. The Human Genome Project has discovered 16 members of the CAPN family. Based on the molecular structure, CAPN1–3 and CAPN5–16 are the large catalytic subunits that contain domains DI, DII, DIII, and DVI, whereas CAPNS1 and CAPNS2 are the small regulatory subunits that contain domains DV and DVI. CAPNs are classified into classical (CAPN1–3, 8, 9, 11–14) and non-classical (CAPN5–7, 10, 15, 16), based on the presence or absence of domain DVI, respectively. CAPNs modulate key molecules in several signalling pathways that contribute to carcinogenesis or malignant metastasis, having a potential prognostic value in various malignancies; however, their role and prognostic value in PC remain unclear. This is the first report on the bioinformatic analysis of CAPNs to explore their prognostic value in PC.
Our results showed that the expression of CAPNs significantly increased or decreased in PC, indicating a potential prognostic value. CAPAN1, 2, 5, and 8 were prognostic biomarkers that were negatively correlated with OS and RFS, whereas CAPAN 10 was positively correlated with the same parameters. Moreover, the genetic alternation rate of CAPNs in patients with PC was estimated at 12.0%.
CAPN1 and CAPN2 were first discovered in 1964 and share a high sequence similarity of 62% in humans. Previous studies demonstrated that CAPN1 is overexpressed in various types of malignancies. For instance, CAPN1 and CAPN2 are overexpressed in colorectal and prostate cancers [36, 37]. Both CAPN1 and CAPN2 are expressed in small cell lung cancer and non-small cell lung cancer (NSCLC), and their upregulation increases the migration and invasion of lung cancer cells. Chen et al. demonstrated that CAPN1 promotes malignant behaviour in lung adenocarcinoma [22, 38]. A previous study described that CAPN2 upregulates the FAK-phosphatidylinositol 3-kinase /protein kinase B and extracellular signal-regulated kinase 1/2 signalling pathways to facilitate lung cancer metastasis [39]. Trillsch et al. demonstrated that CAPN1 cleaves the key epithelial-to-mesenchymal transition marker, E-cadherin and upregulates the canonical Wnt/β-catenin and non-canonical Wnt5A signalling pathways to facilitate tumour metastasis in advanced epithelial ovarian cancer [40]. Based on our findings, the transcriptional expression of CAPN1 and CAPN2 was significantly upregulated in PC tissues compared with that in normal tissues. CAPN1 but not CAPN2, was positively correlated with the advanced tumour stage, and both were negatively correlated with OS and RFS; thus, CAPN1 and CAPN2 have a potential prognostic value in PC.
CAPN3 is predominantly expressed in skeletal muscle, and the protein structure differs from that of conventional CAPNs in three sequences [1]. Previous studies showed that CAPN3 variants play a pro-apoptotic role in melanoma cells, and their downregulation contributes to cancer progression [41, 42]. Our results showed that the transcriptional expression of CAPN3 was not significantly upregulated in PC tissues compared with that in normal ones. Thus, CAPN3 might not be related to PC progression.
CAPNS1 mediates cell death through different cellular signalling pathways. It is known to prevent apoptosis via the PI3K/Akt pathway [43] and promote apoptosis via the calpain-protein phosphatase 2A (PP2A)-Akt-forkhead box O (FoxO) pathway [44]. Previous studies have demonstrated that CAPNS1 promotes tumour progression in NSCLC [45], gastric cancer [46], nasopharyngeal carcinoma [47], and intrahepatic cholangiocarcinoma [48]. Our results showed that the transcriptional expression of CPANS1 was significantly higher in PC tissues than in normal tissues, but no correlation was found with OS or RFS.
Of the non-classical CAPNs that lack domain DVI, CAPN5 is highly expressed in the central nervous system, retina, and gastrointestinal tract, but its role in various cancers remains unclear [49–51]; CAPN6 is highly expressed in the placenta and embryos, correlated with malignant diseases, such as leiomyosarcoma, endometrial stromal sarcoma, and cervical cancer, and promotes angiogenesis by increasing the secretion of vascular endothelial growth factor [10, 49, 52–55]; CAPN7 promotes the migration and invasion of human endometrial stromal cell, but its role in cancer has not been fully elucidated [56]; CAPN10 plays a role in diabetes mellitus and recognised as a potential prognostic biomarker for oesophageal squamous cell carcinoma [57–59], and CAPN15 plays a critical role in the development of eye disorders [60]. Our study revealed that the transcriptional expression of CAPN5, 6, and 10 were significantly higher in PC tissues than in normal tissues. Besides, the expression of CAPN5 was negatively correlated with OS and RFS, whereas that of CAPN10 was positively correlated with the same parameters. Therefore, CAPN5 and CAPN10 could be used as prognostic biomarkers for PC, and the protective mechanism of the latter requires further investigation.
CAPN8 and 9 that show highly conserved gene organisation of other typical CAPN large subunits are predominantly expressed in the stomach and the gastrointestinal tract [61, 62]. It is known that CAPN8 is correlated with the progression of lung squamous cell carcinoma, whereas CAPN9 is considered a prognostic biomarker for clinical outcomes of gastric cancer [63]. Our study revealed that CAPN8 and CAPN9 were negatively correlated with OS and RFS and thus, could be used as prognostic biomarkers for survival in PC.
CAPN11 has a high sequence similarity to CAPN1 and CAPN2 and is overexpressed in the testis [64, 65]; however, information on its role in cancers is limited. CAPN12 is predominantly expressed in skin tissues [66, 67], and little is known about its function in various malignant diseases. We found that CAPN12 had significantly higher expression in PC tissues than in normal ones. Thus, further studies are needed to investigate the underlying molecular mechanism of CAPN12 in PC.
CAPN13 is considered the parent of all classical CAPNs and selectively, lowly expressed in the testis and lungs [68, 69]. CAPN14 has a sequence similarity of 36% with CAPN13 and is not detected in any of the previously studied tissues; however, it has been reported to be correlated with eosinophilic esophagitis [68, 70, 71]. In our study, CAPN13 was not differentially expressed in PC tissues, whereas CAPN14 was undetectable.
PC is characterised by extensive desmoplasia, hypoperfusion, and an immunosuppressive microenvironment. The tumour microenvironment includes cells associated with innate and adaptive immunity [72]. Host immune responses to tumour cells are represented by tumour-infiltrating immune cells [73, 74]. A previous study reported that a large number of tumour-infiltrating Tregs are associated with poor prognosis in patients with PC [75]. Besides, tumour-infiltrating CD4+T and CD8+T cells are positively correlated with survival [76]. De Monte et al. [77] found that Th2 rather than Th1 cells predominantly infiltrated in PC, and that their ratio is an independent prognostic biomarker. Therefore, the systematic analysis of tumour-infiltrating immune cells is important for indicating the effects of specific cell combinations in the immune microenvironment. Here, we found that CAPN1, 2, 5, and 8 were highly correlated with tumour-infiltrating Tfh cells that are a specialised subset of CD4+ T cells and play a crucial role in the regulation of antigen-specific B cell responses. In breast cancer, the differentiation of tumour-infiltrating CXCL13-producing Tfh cells plays a role in converting Treg-mediated immune suppression to de novo activation of adaptive anti-tumour humoral responses, and also Tfh cell activation in B cells induces anti-tumour response [78, 79]. We found that CAPN10 is correlated with the tumour-infiltrating Th2 cells and thus, could be used as a biomarker for response to immune checkpoint inhibitors.
Enrichment analysis indicated that the functions of CAPNs and the related differential proteins are mainly correlated with axonogenesis, cell-substrate adhesion, immune response-activating cell surface receptor signalling pathway, and cell junction organisation in PC, results that were consistent with those reported previously [4]. Furthermore, KEGG enrichment analysis that prognostic CAPNs in PC were associated with focal adhesion, regulation of actin cytoskeleton, and the rap1 signalling pathway, suggest their role in modulating tumour progression.
Our study had some limitations: 1) the number of enrolled patients in the used databases was relatively small and 2) all the data were analysed based on online databases. Therefore, extensive basic and clinical research is required to validate our findings.