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A Risk Model Based on Sorafenib-Response Target Genes Predicts the Prognosis of Patients with HCC

Sorafenib is used to treat digestive system tumors in patients who do not respond to or cannot tolerate surgery. However, the roles and inhibitory mechanisms of sorafenib against hepatocellular carcinoma (HCC) are unclear. Differentially expressed genes in tissues from responders and nonresponders to sorafenib were investigated using the HCC GSE109211 data set. Biological functions and mechanisms were studied using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases. The expression levels of differential expressed target genes were identified in HCC tissues, using The Cancer Genome Atlas database, and their prognostic and diagnostic values were explored using survival and receiver operating characteristic curve analysis. A nomogram and risk model of sorafenib-response target genes enabled the evaluation of the prognosis of patients with HCC. The relationship between risk scores and levels of infiltrating immune cells was visualized via correlation analysis. We identified 1620 sorafenib-response target genes involved in the PPAR signaling pathway, antigen processing and presentation, and ferroptosis. SLC41A3, SEC61A1, LRP4, PPM1G, and HSP90AA1 were independent risk factors for a poor prognosis for patients with HCC and had diagnostic value. A risk model based on SLC41A3, SEC61A1, LRP4, PPM1G, and HSP90AA1 expression showed that patients with HCC in the high-risk group had a worse prognosis. Consensus-clustering analysis (performed with K set to 2) distinguished two clusters (the cluster 1 and cluster 2 groups). Patients in cluster 1 survived significantly longer than those in cluster 2. The risk score correlated with the levels of T cells, cytotoxic lymphocytes, CD8+ T cells, macrophages, memory B cells, follicular helper T cells, and other immune cells. The high risk based on the sorafenib-response targets SLC41A3, SEC61A1, LRP4, PPM1G, and HSP90AA1 represented the poor prognosis for patients with HCC and significantly correlated with the levels of immune infiltrating cells in HCC.


Introduction
Liver cancer has high morbidity and mortality rates and is one of the most common cancers in the world. Hepatocellular carcinoma (HCC) is the most common type of liver cancer [1,2]. Ultrasonography and alpha-fetoprotein (AFP) screening are often used for the early diagnosis of HCC. However, ultrasonography and AFP have certain limitations in HCC [1]. Previous data have shown that some genes, microRNAs, and long noncoding RNAs (lncRNAs) have important biological roles in the progression of HCC [2][3][4][5][6][7]. For example, Cheng et al. reported that kinesin family members 14 (KIF14) and 23 (KIF23) are upregulated in HCC tissues. KIF14 and KIF23 upregulation were associated with shorter overall survival (OS), recurrence-free survival (RFS), and disease-specific survival (DFS) in patients with HCC. Knocking down KIF14 and KIF23 inhibited cell proliferation, promoted cell invasion and HCC migration, and upregulated Bax expression. Decreased expression of KIF14 and KIF23 was shown to enhance the chemosensitivity of HCC cells to cisplatin and sorafenib [4]. He et al. reported that the expression level of lncRNA ZFPM2-AS1 was significantly higher in HCC tissues than in the adjacent normal tissues. High ZFPM2-AS1 expression levels were associated with a shorter OS. ZFPM2-AS1 silencing can inhibit the proliferation, migration, and invasion of HCC cells and promote apoptosis in vitro. ZFPM2-AS1 can regulate GDF10 expression by competitively binding to miR-139, and miR-139 overexpression and GDF10 downregulation can reverse the cellular phenotype induced by ZFPM2-AS1 [6]. Currently, biomarkers for diagnosing HCC and evaluating the prognosis of patients with HCC are lacking.
e results of previous studies have shown that sorafenib has anticancer efficacy, and it is often used in patients with advanced cancer that cannot be treated surgically [8][9][10][11][12][13][14]. For example, sorafenib can inhibit breast cancer (BC) cell proliferation, migration, and invasion and exerts cytotoxic effects. Sorafenib has been found to promote mitochondrial superoxide production and inhibit BC stem cell self-renewal, the epithelial-mesenchymal transition (EMT), and the ERK signaling pathway [12]. Sorafenib can also inhibit HCC cell viability, proliferation, and migration in dose-dependent manners. Sorafenib can destroy the mitochondrial morphology of HCC cells, reduce oxidative-phosphorylation activity, lower the mitochondrial membrane potential, reduce ATP synthesis, and lead to cell death [11]. Furthermore, the OS of sorafenib-treated patients with HCC was compared to that of patients with HCC who did not receive sorafenib. Among patients with grade 1/2, the median OS was 6.1 months in sorafenib-treated patients and 3.1 months in sorafenib-naïve patients [13,14]. However, the signaling mechanisms of sorafenib against HCC have not been fully elucidated. Using microarrays, Pinyol et al. analyzed gene expression levels in tissues from patients with HCC who were treated with sorafenib in order to explore potential biomarkers and improve patient survival [15]. In this study, the data from Pinyol et al. were used to explore the target genes in response to sorafenib treatment. e biological functions and signaling mechanisms of the response target genes were investigated. e response target genes with prognostic and diagnostic significance for patients with HCC were screened and identified, and sorafenib-response target-gene nomogram and risk model were constructed to assess their utilities in determining patient prognosis.

GSE109211 Data Set.
e data for HCC patients treated with sorafenib in the GSE109211 data set of the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo) database were analyzed using GEO2R. e patients were grouped based on the effectiveness of sorafenib treatment, with 46 patients in the nonresponder group and 21 patients in the responder group. In addition, the platform-annotation file (GPL13938 : Illumina HumanHT-12 WG-DASL V4.0 expression beadchip) in the GSE109211 data set was downloaded.

Biological Functions and Signaling Mechanisms.
e Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases are often used to investigate the biological functions and signaling mechanisms involving multiple genes. In this study, the R clusterProfiler package was used to investigate the biological processes, cellular components, and molecular functions associated with sorafenib-response targets based on GO annotation. e KEGG database was used to analyze possible signaling mechanisms involved in the response to sorafenib [16].

Protein-Protein Interaction (PPI) Network.
e sorafenib-response targets were entered into the Search Tool for the Retrieval of Interacting Genes (STRING; https://stringdb.org/) website, and human species were selected to explore the PPI relationships between the sorafenib-response targets [16]. Cytoscape software (version 3.8.2) was used to visualize the PPI network, and the CytoHubba plugin was used to explore key genes in the PPI network of sorafenib-response target genes based on the connectivity scores. e expression levels of key genes in the PPI network and their prognostic value in HCC were visualized using the Gene Expression Profiling Interactive Analysis (GEPIA) online database.

Identifying the Expression Levels of Sorafenib-Response Targets Using e Cancer Genome Atlas (TCGA) Database.
Published data for 374 HCC tissues and 50 normal liver tissues were downloaded from the official website of the TCGA (https://portal.gdc.cancer.gov/projects/) database. e sorafenib-responsive target genes in the GEO database were merged with the gene-expression data in the TCGA database, and then, the expression levels of sorafenib-responsive target genes in normal and HCC tissues were analyzed using the Limma package. Aberrantly expressed sorafenib-responsive genes were visualized using heat maps and dot plots.

Prognostic and Diagnostic Values of the Sorafenib-Response Target Genes.
e differentially expressed sorafenibresponse gene data in the TCGA database were merged with the prognostic data for the patients with HCC. Patients with incomplete prognostic information were excluded. e influence of the sorafenib-response target genes in the OS of patients with HCC was explored using Kaplan-Meier (K-M) survival analysis, where P < 0.001 was used as the screening criterion. A receiver operating characteristic (ROC) curve was used to evaluate the impact of the prognosis-related response genes on the disease diagnosis. e closer the area under the curve (AUC) was to 1, the greater the diagnostic value [17]. Target Genes.  e  relationships between the OS of patients and the expression  levels of BAMBI, BRD9, CCT3, CDC123, DEGS1, DENR,  DHX37, EIF3B, GAPDH, HM13, HSP90AA1, IQCA1,  LRP4, MCM8, PIGU, PPFIA4, PPM1G, RRP7A, SEC61A1,  SLC25A39, SLC41A3, SOX11, SPC25, TAGLN2, ZC3H3, and ZNF207 expression levels were explored using univariate Cox regression analysis. On this basis, multivariate Cox regression analysis and the Akaike information criterion (AIC) method were performed to screen independent prognostic factors and construct a risk model [18,19].

Verifying the Values of Risk Model and Constructing the
Nomogram of Prognostic Genes. After grouping patients based on the median expression of risk model factors, the relationships between risk-model genes and the clinicopathological characteristics of patients were explored. e relationships between the high-and low-risk models and the prognosis of patients with HCC were evaluated by performing K-M survival analysis, and the AUC of the risk model at 3 and 5 years was analyzed via ROC analysis. COX regression analysis was used to explore the effectiveness of the risk model in assessing the prognosis of patients with HCC [17]. e roles of risk-model factors in patient prognosis were incorporated into the nomogram.

Consensus
Clustering and Prognostic Assessment of Sorafenib-Response Target Genes. Principal component analysis (PCA) was performed by dividing the data from 374 patients with HCC (from the TCGA database) into two groups according to the gene-expression levels of SLC41A3, SEC61A1, LRP4, PPM1G, and HSP90AA1 using the R Consensus ClusterPlus package [18]. K-M survival analysis consensus clustering was used to separate the patients into two groups based on their survival rates.

Risk Model and Immune Infiltration.
e MCPcounter and CIBERSORT algorithms were separately applied to TCGA gene-expression data obtained from patients with HCC in order to calculate immune cell levels [20]. e TCGA HCC risk-model score was merged with patient immune cell data. e patients were divided into two risk groups, and the expression levels of immune cells in both groups were investigated.

Statistical Analysis.
Gene-expression levels in TCGA HCC tissues were analyzed using the Limma package. COX regression analysis, K-M survival analysis, and ROC analysis were used to explore the value of sorafenib-response targets in the prognosis and diagnosis of patients with HCC. Immune cell levels in the risk models were identified using the t-test. e threshold for statistical significance was set at P < 0.05.

Sorafenib-Response Targets in HCC.
Sixty-five samples from sorafenib-treated patients with HCC were included in the GSE109211 data set (Figure 1(a)). e gene-expression levels in the nonresponder and responder groups were analyzed using GEO2R, and the results showed that the tissue samples were consistent (Figure 1(a)). Compared with the tissues of the nonresponder group, those from the responder group had 1620 differentially expressed genes (DEGs) ( Table S1). Among them, 799 DEGs were upregulated and 821 genes were downregulated. Figures 1(b) and 1(c) show the top 20 DEGs according to the fold changes in expression.

Functions and Mechanisms of Sorafenib-Response Targets.
KEGG pathway indicated that the sorafenib-response targets were enriched for terms such as complement and coagulation cascades, PPAR signaling pathway, antigen processing and presentation, ribosomes, cholesterol metabolism, amino acid biosynthesis, ferroptosis, and other terms (Figure 2(a) and Table 1). GO annotation showed that the sorafenibresponse targets were enriched for terms such as translational initiation, protein targeting to ER, protein localization to the endoplasmic reticulum, response to toxic substance, mRNA catabolic process, protein activation cascade, protein targeting, drug catabolic process, RNA catabolic process, protein targeting to membrane, regulation of production of molecular mediator of the immune response, humoral immune response, epithelial tube formation, epidermal cell differentiation, positive regulation of cytokine production involved in immune response, and other terms (Figure 2(b)-2(d) and Table S2).

Determining the Expression Levels of Sorafenib-Response
Target Genes. In the TCGA database, 393 sorafenib-responsive target genes were abnormally expressed in HCC tissues versus normal liver tissues (Table S3). Among them, 352 response target genes were upregulated in HCC tissues and 41 were downregulated. Figure 4 shows a heat map with the top 20 sorafenib-responsive target genes based on fold changes in expression.

Prognostic
Value of the Risk Model. UHSP90AA1-expression levels correlated with the age, fibrosis Ishak score (FIS), and OS of patients with HCC (Table S4). LRP4-expression levels correlated with the T stage, pathologic stage, sex, adjacent hepatic tissue inflammation, vascular invasion, and OS in patients with HCC (Table S5). PPM1G-expression levels correlated with the T stage, pathologic stage, race, weight, body-mass index (BMI), histologic grade, AFP level, and OS of patients with HCC (Table S6). SEC61A1-expression levels correlated with the T stage, pathologic stage, sex, OS, and disease-specific survival (DSS) of patients with HCC (Table S7). SLC41A3expression levels correlated with the weight, BMI, histologic grade, AFP, and OS of patients with HCC (Table S8).   Overexpression   FABP6  LCN2  ZNF385D  IGDCC3  MYBPC1  KCNK12  GBX2  PSCA  AQP10  CLDN18  NPTX1  SOX11  DLL3  C1QL4  ADAMTS16  MIOX  CALY  MYO18B FAM178B ZNF695   showed that HCC patients in the high-risk group had a worse prognosis (Figure 7(d)). Time-dependent diagnostic analysis showed that the risk model had a significant diagnostic value (Figures 7(e) and 7(f )). Univariate and multivariate Cox regression analyses showed that the risk score was an independent risk factor for a poor prognosis for patients with HCC ( Figure S6). erefore, we constructed prognostic nomograms of the sorafenib-responsive targets,     Journal of Oncology SLC41A3, SEC61A1, LRP4, PPM1G, and HSP90AA1 ( Figure 8).

Consensus Clustering of Sorafenib-Response Targets Correlated with Distinct Clinical Outcomes in Patients with
HCC. Consensus-clustering analysis with the TCGA HCC data for the sorafenib-response targets, SLC41A3, SEC61A1, LRP4, PPM1G, and HSP90AA1, showed that K � 2 enabled the best grouping (Figures 9(a)-9(c)). Consensus-clustering analysis was performed with K � 2, and the patients were grouped into two clusters, namely, the cluster 1 and cluster 2 groups, with significant differences. PCA revealed a significant difference between the cluster 1 and cluster 2 groups based on data for 374 patients with HCC in the TCGA database (Figure 9(d)). K-M survival analysis showed that the survival times of patients with HCC in the cluster 1 group were significantly higher than those in the cluster 2 group (Figure 9(e)).

A Risk Model Based on the Sorafenib-Response Target
Genes Was Associated with HCC Immune Cell Infiltration. SLC41A3, SEC61A1, LRP4, PPM1G, and HSP90AA1 were differentially expressed between the high-and low-risk groups and correlated positively with risk scores ( Figure S7). e levels of immune cells in HCC tissues were calculated using the MCPcounter algorithm, and the results showed that the abundances of the B lineage, neutrophils, myeloid dendritic cells, T cells, cytotoxic lymphocytes, CD8 + T cells, monocytic lineage, and fibroblasts were abnormal in the high-and low-risk groups and that these differences were statistically significant ( Figure 10 and Table 3). e levels of immune cells in HCC tissues were calculated via CIBER-SORT analysis, and the results showed that the abundances of M0 macrophages, T cells, CD4 + memory T cells, activated CD4 + memory cells, resting mast cells, M1 macrophages, memory B cells, follicular helper T cells, naïve B cells, resting dendritic cells, eosinophils, neutrophils, resting NK cells, and monocytes differed significantly in the high-and lowrisk groups (Figure 11 and Table 4).

Discussion
Liver cancer is highly malignant and insensitive to cytotoxic chemotherapy, often resulting in a poor prognosis for cancer patients. Sorafenib was developed early as a small-molecule drug used in the first-line treatment of advanced liver cancer [8,21]. However, some patients with liver cancer do not respond to treatment with sorafenib and, thus, have a poor prognosis. erefore, in this study, we grouped patients with advanced HCC in terms of their response to sorafenib to explore the key response targets and provide new candidate molecules for improving the prognosis of such patients. e PPAR signaling pathway, antigen processing and presentation, and ferroptosis are associated with cancer growth and metastasis [22][23][24][25][26][27]. For example, the overexpression of the lncRNA TINCR inhibits colorectal cancer (CRC) cell proliferation and promotes apoptosis. Overexpression of miR-107 in CRC cells induces cancer cell proliferation and inhibits apoptosis. TINCR overexpression regulates the PPAR signaling pathway through the miR-107/ CD36 signaling axis to inhibit CRC progression [23]. Interferon gamma (IFNc) released by CD8 + T cells or natural killer cells plays a crucial role in antitumor host immunity. IFNc is involved in regulating tumor cell proliferation and apoptosis. IFNc enhances glutathione depletion, promotes cell cycle arrest in the G0/G1 phase, increases lipid peroxidation, and sensitizes HCC cells to ferroptosis activators. IFNc downregulates SLC3A2 and SLC7A1 mRNA and protein expression by activating the JAK/STAT signaling pathway in HCC cells [27]. In this study, we found that sorafenib-response target genes are involved in the PPAR signaling pathway, antigen processing and presentation, ferroptosis, and other mechanisms. Preliminary evidence suggests that the anticancer effects of sorafenib in HCC patients are related to these mechanisms. e results of previous studies have confirmed that SLC41A3, SEC61A1, LRP4, PPM1G, and HSP90AA1 play important roles in cancer progression [28][29][30][31][32][33][34][35][36][37][38][39]. SLC41A3 is a member of solute carrier family 41 and is involved in many cellular processes. e expression level of SLC41A3 in HCC tissues was higher than that in the normal tissues. SLC41A3 is associated with HCC metastasis, the disease grade, microvascular invasion, AFP, and prognosis and is an influencing factor for a short OS in patients with HCC [28]. Upregulated SEC61A1 expression promoted HCC cell proliferation, migration, and stem cell properties. miR-491-5p inhibits HCC cell proliferation and migration by regulating SEC61A1 expression. VPS9D1-AS1 can upregulate SEC61A1 expression by sponging miR-491-5p, which in turn is involved in HCC growth and metastasis [30]. PPM1G is overexpressed in HCC tissues and associated with HCC metastasis, the pathological grade, microvascular invasion, and hepatitis B virus (HBV) infection. High PPM1G expression is an independent prognostic factor for patients with HCC, and PPM1G regulates SRSF3 phosphorylation in HCC cells and contributes to the proliferation, invasion, and metastasis of HCC cells. PPM1G knockdown inhibits HCC cell growth, invasion, and tumor growth in vivo [34,35]. In this study, we found that SLC41A3, SEC61A1, LRP4, PPM1G, and HSP90AA1 were abnormally expressed in responder and nonresponder tissues from patients with HCC who were treated with sorafenib. In addition, compared with normal liver tissue, the expression levels of SLC41A3, SEC61A1, LRP4, PPM1G, and HSP90AA1 in HCC tissues were significantly higher and correlated significantly with a dismal prognosis for patients with HCC, which has significant diagnostic value. HSP90AA1 is associated with the age, FIS, and OS of patients with HCC. LRP4 is associated with the T stage, pathologic stage, sex, adjacent hepatic tissue inflammation, vascular invasion, and OS of patients with HCC. PPM1G is correlated with the T stage, pathologic stage, race, weight, BMI, histologic grade, AFP, and OS of patients with HCC. SEC61A1 is correlated with the T stage, pathologic stage, sex, OS, and DSS of patients with HCC. LC41A3 is correlated with the weight, BMI, histologic grade, AFP level, and OS of patients with HCC. e risk model constructed based on SLC41A3, SEC61A1, LRP4, PPM1G, and HSP90AA1 was associated with the OS of patients with HCC, where overexpression of these genes negatively impacted the prognosis. e prognostic nomogram constructed based on the sorafenib-response targets SLC41A3, SEC61A1, LRP4, PPM1G, and HSP90AA1 is expected to better assess the prognosis of patients with HCC and provide new candidate molecules.
Infiltrating immune cells in the immune microenvironment play crucial roles in the occurrence and development of HCC [39][40][41][42]. For example, the immune cell marker, CD39, is expressed in tumorinfiltrating lymphocytes and is a marker for identifying   Figure 11: e expression levels of immune cells were abnormal in the high-and low-risk groups.
tumor-reactive T cells. Simultaneous knockdown of PD-1, Tim-3, and Lag-3 enhances the antitumor activity of CD39 CAR-T cells. CD39 CAR-T cells showed increased interferon-c secretion and potent antitumor effects in a mouse model of patient-derived xenografts [39]. Amygdalin treatment rescued HBV-T cell viability and promoted IFNc and TNF-α production. Among HBV-T cells, the mean fluorescence intensity of CD8 + T cells decreased significantly. Amygdalin reduces the phosphorylation of STAT3 and JAK2 in HBV-T cells. Amygdalin suppresses HBV-related HCC cell proliferation, invasion, and migration through T cell-mediated tumor immunity [40]. e risk model constructed here, based on SLC41A3, SEC61A1, LRP4, PPM1G, and HSP90AA1 expression, was associated with the levels of immune infiltrating cells in HCC. e risk score was correlated with the B lineage, monocytic lineage, neutrophils, myeloid dendritic cells, T cells, cytotoxic lymphocytes, CD8 + T cells, fibroblasts, M0 macrophages, T cells, resting CD4 + memory T cells, activated CD4 + memory T cells, resting mast cells, M1 macrophages, memory B cells, follicular helper T cells, naïve B cells, resting dendritic cells, eosinophils, neutrophils, resting NK cells, and monocytes, which further indicates that the risk model constructed based on sorafenib-response target genes has an important value in predicting HCC progression.
In this study, we used information from the GEO and TCGA databases to identify key sorafenib-response targets for patients with advanced HCC and a prognostic geneexpression signature. e data obtained in this study offered the advantages of a large sample size and high reliability. However, a risk model based on sorafenib-response target genes needs to be established through basic experiments and clinical applications. Overall, the sorafenib-response targets SLC41A3, SEC61A1, LRP4, PPM1G, and HSP90AA1 were found to be independent risk factors for a poor prognosis for patients with HCC. Using the risk model constructed based on the expression of these five genes, we found that patients with HCC in the high-risk group had a worse prognosis and that the risk model had excellent diagnostic value. e survival times of patients with HCC in the cluster 1 and cluster 2 groups were significantly different. e risk model constructed based on SLC41A3, SEC61A1, LRP4, PPM1G, and HSP90AA1 is expected to enable the prognosis of patients with HCC and significantly correlated with the levels of infiltrating immune cells in HCC.

Conclusions
In our study, the high risk based on the sorafenib-response targets SLC41A3, SEC61A1, LRP4, PPM1G, and HSP90AA1 represented the poor prognosis for patients with HCC and significantly correlated with the levels of immune infiltrating cells in HCC.

Data Availability
e genomic data used to support the findings of this study have been deposited in the TCGA and GEO database. ese data can also be obtained from the corresponding author.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.  Figure S1. Hub sorafenib-response target genes in the PPI network. Abbreviation: PPI, protein-protein interaction. Figure S2. e expression levels and prognostic values of hub sorafenib-response genes in HCC based on patient information deposited in the GEPIA database. Abbreviations: GEPIA, Gene Expression Profiling Interactive Analysis; HCC, hepatocellular carcinoma. Figure S3. e prognostic values of sorafenib-response target genes in the TCGA database were determined by performing K-M survival analysis. Abbreviations: K-M, Kaplan-Meier; TCGA, e Cancer Genome Atlas. Figure S4. e diagnostic values of sorafenib-response target genes in the TCGA database were determined by performing ROC analysis. Abbreviations: ROC, receiver operating characteristic; TCGA, e Cancer Genome Atlas. Figure S5. Univariate Cox regression analysis showed that the overexpression of sorafenib-response target genes affected the dismal prognosis of patients with HCC. Abbreviation: HCC, hepatocellular carcinoma. Figure S6. Cox regression analysis showed that risk score was an independent risk factor for a poor prognosis for patients with HCC. Abbreviation: HCC, hepatocellular carcinoma. Figure S7. Sorafenib-response target genes associated with the highand low-risk groups. Table S1. Differentially expressed genes in the tissues of patients with HCC who responded to treatment with sorafenib. Abbreviation: HCC, hepatocellular carcinoma. Table S2. Functions of sorafenibresponse targets as determined by performing GO analysis. Abbreviation: GO, Gene Ontology.