In this study, we found that both mRNA and protein expression of SPOCK2 were upregulated in ovarian serous cystadenocarcinoma along with a high expression of SPOCK2 mRNA in the most TCGA cancers. Further ROC curve analysis confirmed that SPOCK2 could be a promising diagnostic biomarker to distinguish HGSOC from benign tissues. To investigate factors that influence SPOCK expression, subgroup analyses based on clinical variables were done in all patients enrolled in the UALCAN dataset, suggesting that SPOCK2 expression is correlated with patient's race, tumor grade, and cancer stage. A critical element of this study is the evaluation of the effect of SPOCK2 on OS by using Kaplan-Meier and univariate logistic regression analyses, in which high SPOCK2 mRNA expression is associated with a shorter OS. Additionally, functional enrichment analysis of SPOCK2-correlated genes revealed that SPOCK2 may play a specific role in regulating functions of extracellular matrix. Exploratory analyses of TILs were conducted to examine the role of SPOCK2 in immune infiltration, showing that there were significant associations between SPOCK2 mRNA expression level and TILs abundance. Overall, this study has led to an improved understanding of SPOCK2 in HGSOC and provided potential therapeutic target.
SPOCK family comprised of SPOCK1, SPOCK2, and SPOCK3, and was originally identified by cDNA cloning, with amino acid homologies (Bradshaw et al. 2012; Vannahme et al. 1999). SPOCK proteins share a unique N-terminal region, followed by a follistatin-like (FS) domain, calcium-binding (EC) domain, thyroglobulin type-1 (TY) domain, and a C-terminal tail encompassing heparan/chondroitin sulfate glycosaminoglycan attachment sites at two serine residues (Alliel et al. 1993; Bonnet et al. 1996; Krajnc et al. 2020). To connect protein functions to the spatial organization of SPOCK2 protein domains, Krajnc et al. confirmed that the FS-EC-TY domain triplet formed a structure core and was responsible for executing functions (Krajnc et al. 2020). All three SPOCKs are extracellular proteoglycans highly expressed in brain, ovary, lung, testis, prostate, and kidney, and it is expected that SPOCKs might contribute to extracellular matrix and associate with tumor progression (Alliel et al. 1993; Bonnet et al. 1996; Vannahme et al. 1999). To gain insight into the biological functions of SPOCKs in human cancers, we focused on SPOCK2 as a presentative of the family because of its upregulated expression and prognostic potential in HGSOC patients (Ren et al. 2011). Nordgard et al. first described the correlation of SPOCK2 with human cancer and found that oligonucleotide polymorphism of SPOCK2 gene was associated with deletion of 16q chromosome in breast cancer (Nordgard et al. 2008). Encoded by the SPOCK2 gene, it is a 424-amino acid protein with a C-terminal tail that binds to glycosaminoglycans to form proteoglycans (Vannahme et al. 1999). In addition to being structural components of extracellular matrix, proteoglycans can serve as important biochemical signals that regulate cell proliferation, differentiation and migration, and modulate vascular networks and immune cell infiltration (Gordon-Weeks and Yuzhalin 2020). Thus, it is essential to determine whether abnormal changes of SPOCK2 exist in HGSOC and how its properties influence tumor progression.
By analyzing the expression level of SPOCK2 in HGSOC, we noted that both mRNA and protein level of SPOCK2 in HGSOC were significantly higher than those in normal ovary tissues, which were influenced by clinical features such as patient's race, tumor grade, and cancer stage. Few studies have been conducted to explore the underlying reasons of why these clinical characteristics could change genetic performance of SPOCK2. Nevertheless, some studies have focused on mechanisms of up- and down-regulated expression of SPOCK2 and concluded that promoter methylation of SPOCK2 gene may be one of reasons for abnormal expression of SPOCK2. Ren et al. carried out a retrospective study to compare the protein expression level and methylation status of SPOCK2 in malignant ovarian tissues and benign tissues (Ren et al. 2011). They found that the frequency of SPOCK2 hypermethylation in malignant ovarian tissues (45%) was significantly higher than 6.7% in the benign tissues; synchronously, the loss rate of SPOCK2 protein in malignant ovarian tissues was 44%, which was significantly higher than 13.3% in the benign tissues, indicating that SPOCK2 hypermethylation might lead to loss expression of protein. Alternatively, Chung et al. verified that treatment with 5-aza-2'-deoxycytidine, a demethylating agent, could restore expression of SPOCK2, suggesting that DNA methylation is related to transcriptional silencing of SPOCK2 in prostate, colon, and breast cancer (Chung et al. 2008). In addition to DNA methylation, future researches could focus on other epigenetic modifications to figure out how SPOCK2 expression in HGSOC is upregulated.
SPOCK2 was highly expressed in HGSOC patients and was related to poorer OS. This study has, therefore, characterized SPOCK2 mRNA expression as a prognostic biomarker for prognosis. On the contrary, Lu et al. found that overexpressed SPOCK2 was significantly associated with better OS (HR = 0.302, P = 0.016) in patients with pancreatic ductal adenocarcinoma (Lu et al. 2021). Similarly, an in vitro experiment revealed that overexpression of SPOCK2 contribute to inhibit cell proliferation and invasion in endometrium-derived cancer cell lines. The researchers also found that inhibitory functions of SPOCK2 on tumor cell progression were achieved partly through suppressing expression or activation of MT1-MMP and MMP2 (Ren et al. 2020). However, another in vitro study confirmed that abundant distribution of SPOCK2 may contribute to glioma invasion by countering the inhibitory action of SPOCK1 and SPOCK3 and supporting MT-MMP-mediated cell migration (Nakada et al. 2003). In summary, these findings suggested that functions of SPOCKs may differ among human cancers. Although SPOCK2 protein was upregulated in HGSOC, high expression of SPOCK2 protein failed to be a prognostic indicator in this study. Considering that SPOCK2 is a potential biomarker for OS, a nomogram was constructed to predict 1-, 3-, and 5-year survival possibility. Our findings indicated that this nomogram model could help to differentiate more advanced HGSOC patients and determine more aggressive therapeutic regimens.
Functional enrichment analyses in this study demonstrated that biological processes of SPOCK2 in HGSOC often require gene network interactions. To further understand biological capabilities of SPOCK2 in HGSOC, correlated genes were used to perform functional enrichment analysis, finding that SPOCK2 may participate in extracellular matrix structure constituent, focal adhesion, cell junction organization, and growth factor binding. According to the results of previous studies, degradation of extracellular matrix surrounding focal adhesion sites played an important role in regulating metastatic process, which required the proteolytic activity of MT1-MMP. Researcher further verified that focal adhesion kinase (FAK), a focal adhesion component, was essential to recruit MT1-MMP molecules (Wang and McNiven 2012). Moreover, Nakada et al. found that SPOCK2 could abolish inactivation of MT1-MMP and MT3-MMP by other SPOCK family members and permit migration of glioma cells in the presence of SPOCK1 and SPOCK3 proteins (Nakada et al. 2003). Accordingly, SPOCK2 could promote remodeling of extracellular matrix and, therefore, was considered as an important molecule in cell migration. Overall, SPOCK2 played critical role in tumor development, suggesting that more future researches would be needed to explore properties of SPOCK2 in HGSOC.
During cancer progression, tumor cells can establish an overtly immunosuppressive tumor microenvironment that is composed of infiltrating immune cells, endothelial cells, and fibroblasts, often embedded within extracellular matrix (Anderson et al. 2017). Numerous studies indicated that abundance of tumor infiltrating immune cells is closely correlated with clinical survival (Hao et al. 2020a; Hao et al. 2020b). However, extracellular matrix surrounding the tumor islets can integrate and deliver multiple complex signals to leukocytes that affect their biological functions, thereby indicating that the complex relationships between extracellular matrix and TILs should be addressed (Pickup et al. 2014). Extracellular matrix can exert antineoplastic activity through providing migratory highways on which leukocytes can directly invade into inflammation-inflamed tumor areas in response to chemo-attractant matrix molecules released via MMP-mediated degradation (Pickup et al. 2014; Sorokin et al. 2010). In this regard, SPOCK2 could promote leukocyte migration and activate anti-tumor inflammation because its facilitative role of MT-MMP-mediated proteolysis that releases chemo-attractant fragments. Similarly, this study found that SPOCK2 positively correlated with infiltration of NK cells, Tem cells, Tcm cells, and Mast cells, indicating a pro-immunogenic role of SPOCK2. Besides inducing anti-tumor immunity, extracellular matrix can directly inhibit T cell activity through impairing the process of antigen presentation (Meyaard et al. 2008; Vesely et al. 2011). Coincidentally, we found that SPOCK2 negatively correlated with infiltration of aDC which is the most capable antigen-presenting cells, suggesting a pro-tumoral role of SPOCK2. On the basis of our findings and previous studies, we concluded that effects of abnormal SPOCK2 expression on TILs still remain elusive, appealing to more fundamental researches.
In conclusion, this study provided evidence for upregulated expression of SPOCK2 and found that SPOCK2 had diagnostic and prognostic potential for HGSOC. In addition to explorations of SPOCK2-related pathways that indicated a critical role in remodeling extracellular matrix, immune cell infiltration analyses provided new understandings for associations between SPOCK2 and TILs. Although a lack of in vivo/vitro experiments and use of public databases make it difficult to detail the mechanisms of SPOCK2 that contribute HGSOC progression, this article is expected to provide a new direction for predicting prognosis and exploring therapeutic targets. In the future, more fundamental experiments with clinical samples instead of data mining should be well-designed.