Alterations of the microenvironment of hepatocellular carcinoma in different unfolded protein response activity states

Liver cancer is one of the most common malignant tumors of the digestive system and has become a leading cause of cancer-associated death worldwide. As the most prevalent subtype, hepatocellular carcinoma (HCC) accounts for over 90% of liver cancer [1]. At present, the therapeutic strategies for HCC mainly include surgical resection, transarterial chemoembolization (TACE), radiotherapy, molecular targeted drug therapy and immunotherapy [2]. Among them, the application of immunotherapy, especially immune checkpoint inhibitors (ICI), has greatly changed the clinical treatment of HCC. However, due to the immunosuppressive effect of tumor microenvironment (TME), the prognosis of HCC patients is still not ideal [3, 4]. Recently, genome-wide association studies have identified a large number of long noncoding RNA (lncRNA) associated with a variety of tumor. Many researches have revealed that lncRNAs and circular RNAs could be translated into functional small proteins, which may be useful in the development of novel treatment strategies. Polenkowski et al. [5]. Have showed that Linc013026 encodes a 68 amino acid micropeptide Linc013026-68AA, which could enhance the cell proliferation in HCC. Niehus et al. [6]. Have reported that long antisense noncoding RNA EVA1A-AS could suppress the expression of anti-proliferating gene EVA1A in HCC, indicating this long antisense noncoding RNA may be a promising target for HCC therapy. Tran et al. [7]. Have revealed that long intergenic noncoding RNA Linc00176 promotes cell proliferation and cell survival in HCC, another study [8] have showed that C20orf204 is a splice variant of Linc00176, and its encoded protein C20orf204-189AA could enhance the hepatocellular carcinoma cell proliferation and ribosomal RNA transcription, suggesting that C20orf204-189AA maybe a potential target for cancer therapy. These evidence all indicate the great potential of long non-coding RNA-derived peptides as a target in the treatment of HCC.

The unfolded protein response (UPR) is a stress response mechanism and a conserved cell survival strategy, which is triggered by the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER), also known as ER stress [9]. The activation of UPR could reduce the synthesis of proteins, degrade unfolded proteins, and increase the expression of chaperone proteins, thereby reduce ER stress. In mammals, the functions of UPR are achieved by three ER-localized proteins: activating transcription factor 6 (ATF6), protein kinase R (PKR)-like ER kinase (PERK) and inositol requiring enzyme 1 (IRE1) [10]. In solid tumors, cancer cells are often challenged by various intrinsic and microenvironmental stresses during oncogenesis and development. An increasing number of evidences have indicated that UPR plays an important role in many biological processes of HCC, including promoting cancer cells survival, regulating angiogenesis and modulating the drug resistance to cancer therapy [11].

In recent years, there have been a lot of reports about the correlation between UPR and immunosuppressive TME. Increasing evidence indicated the involvement of UPR in the impairment of T‑cell function, T-cell exhaustion, and the activation of immunosuppressive signaling pathway in TME. A recent study by Liu et al. [12]. Revealed that ER stress could help liver cancer cells escape immunosurveillance through upregulating PD-L1 expression in macrophages and consequently suppressing T-cell function. Song et al. [13]. Have shown that the activation of IRE1α-XBP1 signaling pathway could inhibit T-cell infiltration and IFN-γ expression, thereby promote progression of tumor in ovarian cancer. Another study suggested that the palmitic acid-mediated ER stress activation could induce M1-type polarization through upregulation of PERK in macrophages [14]. Given the importance of understanding the molecular mechanism of UPR in the microenvironment modification of HCC, more studies are needed to provide deeper insight into the role of UPR in HCC microenvironment. The aim of this study is to analyze the alterations of infiltrating cells in microenvironment of HCC and key regulatory genes in high UPR activity state and to provide new ideas for personalized immunotherapy strategy of HCC.

The tumor microenvironment (TME) consists of various immune cells, stromal cells, blood vessels, lymphatic vessels and extracellular matrix, playing a crucial role in the initiation, progression and therapeutic resistance of cancer [15]. Hypoxia, chronic inflammation and immune suppression are the typical characteristics of TME, forming a very complex mechanism network. Endoplasmic reticulum (ER) is the central organelle where secreted proteins and membrane proteins are synthesized, folded, transported and modified. The persistent deregulation of ER homeostasis induced by hypoxia is the important feature of TME, which affects the survival of tumor cells and non-tumor cells [16]. The unfolded protein response (UPR), also known as the ER stress response, is an adaptive sensing-signaling network capable of reinstating ER homeostasis through three ER-resident transmembrane proteins that function as sensors of protein-folding stress: inositol-PRKR-like ER kinase (PERK), requiring protein 1α (IRE1α) and activating transcription factor 6 (ATF6) [10]. The overexpression of UPR sensors has been observed in a large number of solid cancers including that of brain, breast, liver and pancreas [17‐19]. Increasing evidence suggested that the activation of UPR signaling pathway in cancer cells can alleviate ER stress and promote tumor progression, and can also limit the effect of therapeutic approaches by affecting the function of the immune cells in the TME [20]. Studies have shown that UPR could help cancer cells to impair the function of CD8 + T cell [21]. Other studies indicated that cancer cells with high UPR activity could significantly change the function of macrophages, natural killer cells and myeloid cells in the TME [22].

In our study, we found high UPR activity had a significant effect on stromal cell components in the microenvironment of HCC including hepatocytes, HSC, ECs, adipocytes, etc. Analysis of the relative abundance of infiltrating cells showed these stromal cells decreased most significantly when UPR activity increased. Survival analysis indicated that patients with higher UPR activity and lower Stromal score have worse prognosis. Further analysis of the role of stromal cell components such as HSC, ECs and adipocytes in the occurrence and development of HCC is of great importance for understanding of the pathogenesis of HCC.

C-Type Lectin Domain Family 3 Member B (CLEC3B) is a member of the C-type lectin domain family and encodes a protein called tetranectin, primarily involved in the regulation of extracellular proteolysis by inducing plasminogen activation, which plays an important role in the tumor invasion and metastasis. At present, CLEC3B has been reported in a variety of tumor diseases, but in-depth studies about the molecular function of CLEC3B in cancer progression has been very limited. Lu et al. [23]. Reported that down-regulation of CLEC3B facilitates epithelial-mesenchymal transition, migration and invasion of lung adenocarcinoma cells. Sun et al. [24]. Suggested that CLEC3B might be associated with the immune infiltration and immune activation of lung cancer, especially in squamous cell carcinoma. Yang et al. [25]. Reported that CLEC3B expression was reduced in liver cancer tissues compared to normal liver tissues and was also identified as a tumor-associated endothelial cell marker gene. Another study showed that downregulation of exosomal CLEC3B could promote metastasis and angiogenesis via AMPK and VEGF signals in HCC [26]. So far, the relationship between CLEC3B and UPR activity and tumor-associated stromal cells in HCC has not been reported. Our study suggests that CLEC3B plays an important role in regulating the stromal cells of the TME during activation of the UPR pathway in HCC.

Receptor activity-modifying protein 3 (RAMP3) is an active modulator of various G protein-coupled receptors, including calcitonin-receptor-like receptor (CRLR). In the presence of RAMP3, CRLR could function as a receptor of adrenomedullin (ADM), which was reported in various cancers, including breast, pancreatic, liver and lung cancers and was identified as a potential therapeutic target against pathological angiogenesis. The regulatory effects of RAMP3 varied significantly in different cancers. Previous studies showed that the ADM-RAMP3 system was involved in the malignant transformation of cancer-associated fibroblasts (CAFs) in the cancer microenvironment and promoted cancer metastasis and the deficiency of the ADM-RAMP3 system suppressed metastasis through the modification of CAFs [27, 28]. While Fang et al. reported that RAMP3 expression was significantly downregulated in HCC tissues than in normal liver tissues, increased RAMP3 expression was an independent prognostic factor of favorable overall survival [29]. Our findings indicated lower expression of RAMP3 in HCC tissues with high UPR activity was related to worse prognosis and lower Stromal Score. Further investigation is warranted to explore the role of RAMP3 in the modification of tumor microenvironment of HCC in UPR activity state.

G protein-coupled receptor 182 (GPR182) is identified as a new member of the atypical chemokine receptor (ACKR) family, and has the key characteristics of the known ACKRs. It has been reported that GPR182 was preferentially expressed in endothelial cells, including lymphatic endothelial cells, microvascular endothelial cells, liver sinusoidal endothelial cells and intestinal stem cells. The mRNA expression level of GPR182 is reported to be up-regulated in tumor-associated endothelial cells, but the function of GPR182 remains unclear. Torphy et al. [30]. Reported that up-regulated GPR182 could limit effector T cell infiltration into transplanted melanomas via scavenging chemokines and the ablation of GPR182 could improve antitumor immunity. Le Mercier et al. [31]. Demonstrated that GPR182 was a new atypical chemokine receptor for CXCL10, CXCL12, and CXCL13, which was involved in the regulation of hematopoietic stem cell homeostasis. Another study revealed that GPR182 was significantly downregulated in HCC tumorous versus peritumoral tissue, and the downregulation of endothelial GPR182 was also observed in tissues with liver fibrosis and cirrhosis [32]. In addition, a bioinformatics analysis showed that GPR182 was a prognostic tumor microenvironment-related gene in the TME of HCC and was correlated with 3-year survival rate of HCC patients [33].

Deoxyribonuclease1-like 3 (DNASE1L3) belongs to the deoxyribonuclease I family and is primarily secreted by macrophages and dendritic cells in the liver and spleen. Several studies suggested that DNASE1L3 was closely associated with autoimmune diseases, but little is known about the function of DNASE1L3 in cancers. Liu et al. [34]. Reported that the expression level of DNASE1L3 was gradually down-regulated with the progression of the tumor and was negatively correlated with the prognosis of colon cancer patients, and DNASE1L3 may change the TME of colon cancer by regulating the infiltration of multiple immune cells. Another study indicated that restoration of DNASE1L3 activity could enhance antitumor immunity and suppress colon cancer progression [35]. Sun et al. [36]. Suggested that DNASE1L3 was significantly downregulated in HCC tissue, and the high expression level of DNASE1L3 could inhibit the progression of HCC by delaying cell cycle progression through CDK2. In addition, Guo et al. [37]. Demonstrated that DNASE1L3 could enhance the apoptosis of DNA damaged HCC cell lines and the defects in DNASE1L3 enhances the accumulation of cytoplasmic DNA and triggers the activation of DNA sensors.

In the present study, differential expression analysis was performed to identify UPR activity-related genes, and survival analysis was used to evaluate the effect of 599 DEGs on the prognosis of HCC, totally 137 DEGs were found to be associated with the prognosis of HCC, among which, low expression of CLEC3B, RAMP3, GPR182 and DNASE1L3 were contributor to worse prognosis of HCC. Correlation analysis indicated that down-regulated gene CLEC3B, RAMP3, GPR182 and DNASE1L3 had strongest positive correlation with Stromal scores, in additions, the expression of CLEC3B, RAMP3, GPR182 and DNASE1L3 were also highly correlated with infiltrating cell HSC, LECs, ECs and microvascular endothelial cells, indicating the potential connection of these genes with infiltrating stromal cells in HCC microenvironment under different UPR activity.

HCC tissues with high UPR activity demonstrated lower Stromal scores and the decline of various infiltrating cells including HSC, LECs, microvascular endothelial cells, ECs and adipocyte, which possibly associated with poor prognosis of HCC patients. Down-regulated gene CLEC3B, RAMP3, GPR182 and DNASE1L3 may play an important regulatory role in the modification of microenvironment of HCC with high UPR activity.

Our study was based on transcriptome sequencing data from 371 HCC patients in the TCGA database, and the level of ER stress activity was also estimated based on the gene set in the UPR pathway provided by the MSigDB database. Due to limited funding, the establishment of a hepatocellular carcinoma ER stress model and the validation of the conclusions of this article have not yet been carried out.