APOC1, the smallest apolipoprotein (only 6.6 KDa), is a component of both triglyceride-rich lipoproteins and high-density lipoproteins.9 Previous studies have shown that APOC1 contributes to lipid transport and metabolism in serum and plasma.10,13 Interesting, emerging researches highlight the relationship between APOC1 and cancers. The upregulation of APOC1 was previously reported to associated with advanced tumor progression and dismal prognosis in patients with pancreatic cancer, colorectal cancer, lung cancer and papillary thyroid cancer.21–24 Nevertheless, the expression level and pattern of APOC1 in ESCC has still not been investigated. In this study, we conducted bioinformatic analysis using public data to explore the potential functions of APOC1 in ESCC.
We initially used TIMER2.0 to review the differential expression of APOC1 between tumor and normal tissues in various types of cancers and observed increased expression of APOC1 in esophageal cancer. Then, we further found that APOC1 overexpression occurred in multiple cohorts of ESCC and the mRNA expression level of APOC1 displayed favorable diagnostic value for ESCC accessed by ROC curves. In addition, we evaluated the clinical implication of APOC1 overexpression in ESCC and observed that APOC1 overexpression was correlated with gender and TNM stage. Meanwhile, Kaplan-Meier plotter analysis illustrated that ESCC patients with high APOC1 expression had unfavorable OS and RFS (all P < 0.05). We further confirmed the prognostic value of APOC1 by analyzing clinical information of 94 ESCC patients from TCGA. The KM analysis showed that increased expressions of APOC1 were associated significantly with poor OS (p < 0.05) and the multivariate analysis indicated that APOC1 overexpression was an independent prognostic factor of unfavorable OS in ESCC. These above results suggest that APOC1 may serve as a valuable diagnostic and prognostic markers of ESCC.
To probe the potential mechanisms of APOC1 upregulation in ESCC, we analyzed APOC1 co-expressed networks using GSEA. GO enrichment analysis revealed that APOC1 was mainly associated with adaptive immune response, cellular defense response and lymphocyte mediated immunity. These results suggest that APOC1 may participate in the immune response of ESCC. Moreover, the KEGG pathway analysis primarily included natural killer cell mediated cytotoxicity, phagosome, AMPK signaling pathway and hippo signaling pathway. AMPK is a crucial energy-sensing enzyme maintaining cellular energy homeostasis. It can be activated by cellular stress which increases the AMP/ATP ratio and leads to the production of metabolic poisons, the progression of hypoxia, glucose starvation, etc.25 A research reported that metformin exerted its anti-proliferative effects on ESCC cells through inducing the activation of AMPK pathway, suggesting that AMPK pathway may play a crucial role in the inhibition of the growth of ESCC.26 The primary functions of Hippo pathway are restricting tissue growth and modulating cell proliferation, differentiation and migration. Recent studies suggest that deregulation of hippo pathway plays an important role in cancer initiation and progression.27 Gao et al reported that miR-31 could promote ESCC tumorigenesis by inhibiting LATS2 expression via the hippo pathway.28 These above researches are consistent with our results, suggesting that APCO1 may participate in tumorigenesis and progression of ESCC through these signaling pathways.
To further gain insight into the regulators potentially responsible for APOC1 overexpression, we investigated networks of kinases, miRNAs and transcription factors. We observed that APOC1 in ESCC was related to the network of kinases including LCK, SYK, LYN, ITK and HCK. Additionally, LYN and HCK showed significantly overexpressed in ESCC tissues in the GSE23400 dataset (all p < 0.05). And HCK overexpression was associated with the poor prognosis and pathological stage in ESCC patients (all p < 0.05). In fact, HCK is the important paralog of LYN. HCK plays an important role in the regulation of innate immune responses, including neutrophil, monocyte, macrophage and mast cell functions, phagocytosis, cell survival and proliferation, cell adhesion and migration. Excessive HCK activation enhances cell proliferation and survival by associating with oncogenic fusion proteins and functionally interacting with receptor tyrosine kinases.29,30 Consistent with previous reports, we speculate that APOC1 may regulate immune system process via HCK kinase in ESCC and further studies are needed to verify this hypothesis.
IRF family, a broad class of cytokines elicited on challenge to the host defense, are important for mobilizing immune responses to pathogens.31 Moreover, statistical data indicated that IRF-2 expression was tightly correlated with progression of ESCC.32 Reduction of the ratio of IRF-1/IRF-2 might lead to the enhancement of tumorigenicity of ESCC cells.33 STAT3 plays important roles in the progression of various cancers by regulating the proliferation, invasion, angiogenesis and immune surveillance evasion.34,35 Previous researches have shown that the STAT3 pathway was activated in some ESCC cells and STAT3 overexpression indicated the poor prognosis of ESCC patients who had undergone curative resection.36 Our study suggests that IRF and STAT3 are potential regulators of APOC1 and that APOC1 may act through these factors to regulate the immune response of ESCC.
Previous research illustrated that miRNA which normally involve in post-transcriptional regulation of gene expression can contribute to human carcinogenesis.37 Thus, we tried to explore the potential regulatory miRNAs of APOC1. The miRNAs associated with APOC1 in our study participate in the process of carcinogenesis. In the current study, miR-26a and miR-26b inhibit ESCC cell proliferation through suppression of c-MYC pathway.38 Previous studies showed that the upregulation of miR-519 enhanced radiosensitivity of ESCC cells and facilitated ESCC cell apoptosis via targeting PI3K/AKT/mTOR signaling pathway. The low expression level of miR-519 indicated poor prognosis of ESCC patients.39 The expression of miR-202 was reported to aberrantly decreased in ESCC and the down-regulation of miR-202 was associated with the metastasis of tumor.40 Consistent with above literature evidence, deregulation of these miRNAs may partly contribute to APOC1 overexpression in ESCC.
Tumor-infiltrating immune cells (TIICs) are crucial parts of the tumor microenvironment, which are associated with patient outcome and tumor behavior.41 We first used the TIMER2.0 database to uncover the relationship between APOC1 expression and TIICs in esophageal cancer. The results indicated that APOC1 expression had a significant positive relationship with the infiltration levels of B cells, CD4 + T cells, neutrophils, macrophages and dendritic cells in esophageal cancer. Then we further studied the roles of immune infiltration in ESCC by using CIBERSORT to analyze the GSE23400 dataset. The CIBERSORT analysis indicated that the increased infiltration of activated NK cells, M0 macrophages cells, M1 macrophages cells, and the decreased infiltration of CD8 T cells, follicular helper T cells, regulatory T cells, monocytes and neutrophils were associated with the occurrence and development of ESCC. Analyses on the role of immune infiltration in human cancers typically focus on T cells, and the CD8 + T cells are one of the major anti-tumor immune cells in the tumor microenvironment. It has been reported that CD8 + T cells cooperate with CD4 + T cells to promote better prognosis of ESCC patients.42 Tregs are heterogeneous and have multiple context-dependent functions that are still not well-characterized. It may play a dual role in carcinogenesis, initially inhibiting inflammation that leads to tumorigenesis, but later suppressing anti-tumor immune responses.43 Besides T cells, the innate immune system including NK cells and macrophages also role importantly in the tumor microenvironment. Several studies have shown that the infiltration of NK cells significantly increases in ESCC. And IL-6 or IL-8 secreted by primary ESCC cells impairs the function of NK cells via the STAT3 signaling pathway.44 Tumor-associated macrophages (TAMs) have been reported to play a crucial role in inflammatory tumor microenvironment. TAM infiltration is related to poor responses to chemotherapy and unfavorable prognosis in ESCC.45 The above research findings combined with our study indicate that the infiltrating immune cells play crucial roles in ESCC and should be the highlight of future studies. Moreover, we analyzed the correlation between the immune cells in ESCC. Our results showed that M1 macrophages cells were positively correlated with activated NK cells, and M0 macrophages cells were negatively related to CD8 T cells and neutrophils. The potential mechanisms of these correlations require further studies.
To uncover the association between APOC1 overexpression and the immune infiltration in ESCC, we preformed the correlation analysis. Our results indicated that APOC1 was significantly positively correlated with M0 macrophages cells, M1 macrophages cells and activated NK cells, and negatively correlated with regulatory T cells, CD8 T cells, neutrophils and monocytes (|R|>0.3, P < 0.05). The correlation results are consistent with the previous studies of infiltration situation in ESCC and we speculate that APOC1 may stimulate M0 macrophages cells, M1 macrophages cells and activated NK cells or inhibit regulatory T cells, CD8 T cells, neutrophils and monocytes to participate in the tumorigenesis and progression of ESCC. Further experimental studies are needed to verify this assumption.
Furthermore, we performed the prognostic analysis of APOC1 expression levels in ESCC based on immune cells. The results showed that high APOC1 expression level had an unfavorable prognosis in the decreased B-cells, decreased CD4 + memory T-cells, decreased CD8 + T-cells, and enriched mesenchymal stem cells subgroups. These analyses suggest that the overexpression of APOC1 may partly affect the prognoses of ESCC patients due to immune infiltration, especially the decrease of CD8 + T cells.
In summary, this present study promotes our understanding of the association between APOC1 and ESCC and suggests that the upregulation of APOC1 may have an impact on the unfavorable prognosis of ESCC through the mechanism of immune infiltration. But this study also has some limitations. The study was preformed based on the data downloaded from public databases and there were few survival data of ESCC patients. It is essential to conduct additional experiments to verify our results and further clarify the role of APOC1 in ESCC.