Gastric cancer is the leading cause of cancer-related mortality worldwide, and its rapid progression and invasive metastasis often contributes to its poor prognosis. Both Lauren’s classification and the World Health Organizations (WHO) classification [19, 20] recommend surgical resection, neoadjuvant, and adjuvant therapies as the primary treatment options for GC, but these have unfavorable prognosis. In recent years, cancer immunotherapies have received considerable attention for the treatment of various tumor types. Consequently, studies into the immunological regulation of GC have been increasing [21, 22, 23]. Among these immunotherapies, the blocking of immune checkpoints to target PD-L1 and CTLA4, has shown promising inhibitory effects. Whereas, in GC patients, the anti-CTLA4 drug tremelimumab has had insufficient therapeutic efficacy in clinical treatments [24], and PD-1 and PD-L1 inhibitors have had incomplete responses in most advanced GC patients [25, 26, 27]. Despite the substantial improvement in the survival of GC patients, identifying the population who can benefit from immunotherapy is still a challenge. Thus, there is an urgent need to identify more immune-related prognostic biomarkers for GC treatments.
The HTRA4 proteins are important members a of complicated protein network designed to promote cellular processes in stressful environments, and they are well conserved in the evolution of the HTRA (High-temperature requirement A) family of serine proteases [28, 29, 30]. There are four human HtrA proteins (HTRA1, HTRA2, HTRA3, and HTRA4), and of these, HTRA1, HTRA3, and HTRA4 have similar domain organizations. The HTRA 1, 2, and 3 proteases have been widely reported to function in many processes, such as extracellular matrix proteins, the protection of mitochondrial homeostasis, and cell apoptosis to promote cell death [31, 32, 33]. Moreover, HTRA1, HTRA2, and HTRA3 proteins are involved in neurodegenerative disorders and cancers. HTRA1 was the first sequenced protein of the human AB family [34], and its expression is regulated during the proliferation of the endometrium during pregnancy [35]. HTRA2 is expressed ubiquitously in human tissue, and its expression is extremely variable according to the type of tumor [36, 37, 38]. Like HTRA2, the expression of HTRA3 is also different according to the type of tumor [39], and previous research has described the functional mechanisms and role of HTRA1–3 proteins. However, the cellular functions of the HTRA4 protease are still poorly understood, especially in cancers.
HTRA4 can be secreted into the extracellular matrix to act as a similar signal secretory peptide as HTRA1 and HTRA3 [40.41]. It was previously suggested that HTRA4 plays an important role in the implantation of the embryo and could a biomarker with which to predict pre-eclampsia [6, 42, 43, 44]. In some malignancies, HTRA4 has been found to promote tumor progression. Analysis of microarray studies shows that HTRA4 is upregulated in glioblastoma multiforme, when compared to brain tissues in epilepsy patients [45]. Similarly, it has been indicated that HTRA4 is overexpressed in breast cancer tissues, when compared to normal breast tissues [46]. Conversely, HTRA4 has been found to have anti-tumor effects in other malignancies. HTRA4 is downregulated in hormone refractory metastatic prostate cancer when compared to primary prostate cancer [47]. Maser et al. indicated that the allelic ratio of HTRA4 is lower in glioblastoma and pancreatic cancer, when compared to normal DNA [48]. Recently, Wenta et al. suggested that HTRA4 stimulates the drug-induced death of cancer cells, and that this protease is a promising anti-cancer therapeutic target [18]. Nevertheless, knowledge regarding the HTRA4 biological features and its roles in cancers is currently limited. At present, there have been no previous studies on the role of HTRA4 in GC, and consequently this was the focus of this investigation.
Bioinformatics analysis using high throughput RNA-sequence data from the GEO and TCGA database shows obvious individual heterogeneity in the RNA data, and HTRA4 may be a potential identification biomarker for GC tissues. Increased expression of HTRA4 in GC tissues was accompanied by advanced clinical pathological characteristics, shorter survival time, poor prognosis, and more powerful T cell immune levels. To further investigate the functions of HTRA4 in GC prognosis, we conducted GO, KEGG, and GSEA analyses using the TCGA data. The results revealed that most immune-related pathways, including antigen processing and presentation, toll-like receptor signaling pathway, intestinal immune network for IgA production, natural killer cell mediated cytotoxicity, T-cell receptor signaling pathway, and primary immunodeficiency were differentially enriched in the HTRA4 high expression phenotype. CIBERSORT analysis shows that high HTRA4 expression levels are associated with higher infiltration levels of various T cells and macrophages. Therefore, these data suggested that HTRA4 might act as potential prognostic marker and immune-related therapeutic target in GC.
Tumor-infiltration lymphocytes (TILs) among stromal cells in the tumor microenvironment play an important role in the occurrence and development of tumors, which can influence the carcinogenic characteristics of adjacent tumor cells [49, 50]. TILs establish a complex interaction network, helping to improve and maintain the immune microenvironment, to promote immune effects, and thus promote tumor progression [51]. Immune infiltrates in tumors like tumor associated macrophages, tumor-infiltrating neutrophils, and various kinds of T cells are significantly relevant to patient prognosis and the efficacy of therapeutics [52, 53]. Our results demonstrated that there were four kinds of T cells in the HTRA4-high GC group than in the HTRA4-low GC group, suggesting that high expression of HTRA4 is associated with high immune cell levels. The CD8 + and CD4 + T cells in the tumor microenvironment have strong immunoenhancement activity. Therefore, our data elucidated that the immune effects induced by the T cells in the primary tumor microenvironment might result in a lower overall survival rate in HTRA4-high GC patients.
High HTRA4 expression levels were shown to correlate to the poor prognosis of GC in T2, T3, N1, N1&2&3, M1, stage 3 and HER2 + subgroups when HTRA4 was highly expressed in the GC tissues. The expression of HTRA4 remains a powerful forecaster of the prognosis within these subgroups, showing that HTRA4 was independent of these important clinicopathological parameters.
These results have improved our understanding of the relationship between HTRA4 and GC, but the study had some limitations. First, to discover the specific role of HTRA4 in the development of GC comprehensively and completely, all clinical information should be included, such as the details on the treatment methods received by the patients and successive survival follow-up times, but the information for the treatments and follow-up times was lacking or inconsistent as the public database experiments were performed in different cancer centers. Second, the number of normal gastric tissue samples in the current study was too small, and this should be improved in the future to ensure balanced sample sizes. Third, retrospective studies overall have their own limits, and thus a prospective study should be conducted in the future to avoid analysis bias. Fourth, the data from the current study were analyzed using the sequencing data from the GEO and TCGA databases only, it is necessary to conduct further research to prove the direct mechanisms and role of HTRA4 in GC.
In the current study, we have reported that high HTRA4 expression levels were significantly related to the progression, poor survival, and immune infiltration of GC. HTRA4 may promote tumorigenesis through abnormal inflammation and immune responses and may have the potential to become a new immune biomarker of GC. Thus, HTRA4 was identified as a reliable predictor of GC prognosis. The mechanisms by which HTRA4 accelerates the progression and metastasis of GC will be investigated further. This study provides a new and promising immunotherapeutic target, HTRA4, that has clinicopathological significance and will improve molecular pathogenesis assessments.