Bioinformatics Analysis and Insights for The Role of COMMD7 in Hepatocellular Carcinoma

Background: The tumorigenesis and development of hepatocellular carcinoma (HCC) is a process involving multiple factors. The COMMDs family proteins were reported to play important roles in various disease and cancers including HCC. We previously found COMMD7 acted as a HCC-promotion factor; however, further understanding on COMMD7 was needed. We conducted these bioinformatics analysis for the purpose of comprehensive understanding of the functional role of COMMD7 in HCC. Methods: The bioinformatics analysis of COMMD7 were launched by online platforms including KEGG, GEPIA, cBioportal, Gene Ontology and The Kaplan-Meier plotter. Data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) were downloaded, and the data analysis and processing were conducted by RStudio (version 1.3.959) software. Results: The expression pro�le results of COMMD7 in TCGA and GTEx database suggested that COMMD7 expressed highly in liver tumor tissues and positively related with poorer prognosis (p<0.01); COMMD7 also contributed to the early development of HCC as its higher expression resulted in progression from stage I to stage III (p<0.01). Based on our previous studies, COMMD7 may target NF-κB signaling and CXCL10 to enhance the proliferation of hepatoma cells so that promoting the development of HCC.


Background
Chronic liver disease and primary liver cancer are a massive global problem, as the most prevalent form of primary liver cancer, hepatocellular carcinoma is a global pandemic with a signi cant increase in morbidity and mortality worldwide [1].Recent epidemiology statistical data con rms that HCC represents the fourth leading cause of cancer-related death and ranks sixth in terms of new cases worldwide [2].With a poor 5-year survival, liver cancer is the second most lethal tumor after pancreatic cancer [3].The tumorigenesis and development of HCC is a complex process involving multiple genes and factors [4] (Fig. 1).It has been reported that the mutation of p53 [5], AKT/β-catenin[6], HFE [7], TSC1/2[8] and MBL [9] genes were frequently occurred in aggressive HCC.Meanwhile, the activation of these genes such as mTOR, PI3K, MAP kinase, STAT3, JAK were proved to be related with the progression of HCC [10][11][12][13].
The COpper Metabolism MURR1 Domain-containing (COMMD) family consists of ten members ranging from COMMD1 to COMMD10, which is characterized by a highly conservative COMM domain at the carboxyl terminal [17].The COMMDs family members have been frequently reported to involve in human disease and cancers (Table 1).For example, the most researched COMMD1 induces apoptosis in human lung cancer cells by inhibition of NF-κB signaling [18,19].COMMD1 disrupts HIF-1α/β dimerization and inhibits human tumor cell invasion [20], and downregulation of COMMD1 promotes tumor development by modulating a positive feedback loop that ampli es in ammatory and stemness-associated properties of cancer cells.COMMD3/8 complex selectively recruited GRK6 and induced GRK6-mediated phosphorylation of the receptor and activation of β-arrestin-mediated signaling thus regulates immune response [21].COMMD4 was de ned as an anti-cancer therapeutic target and prognostic factor in nonsmall cell lung cancer [22].COMMD5 acts as an adaptor protein to coordinate endosomal tra cking and plays an important role inEGFR transporting and activity in renal cell carcinoma [23].COMMD6 may modulate the ubiquitination and degradation of NF-κB subunits and regulate ribonucleoprotein and spliceosome complex biogenesis in tumors [24].COMMD9 participates in TFDP1/E2F1 activation and plays a critical role in non-small cell lung cancer [25].COMMD10 may play a tumor suppressive role in renal clear cell carcinoma through the miR-590-3p-COMMD10-Cul2-RBX1-NF-κB/HIF/NRF2 pathway and regulate the chemotherapy resistance of various tumor cells to cisplatin [26,27].In addition, the COMMDs family proteins also regulates plasma LDL Levels and attenuates atherosclerosis through forming COMMD/CCDC22/CCDC93 to stabilize the CCC complex in endosomal LDLR tra cking [28].
As a key member of COMMDs family, COMMD7 consists of 200 amino acids and located on the 20th chromosome.Yet the bio-function of COMMD7 was rarely reported.Goodall et al, found that the SNP of COMMD7 may associated with platelet function of FC and PC [29].While Bajuna Rashid Salehe con rmed the association of the SNP rs6141803 of COMMD7 with cardiovascular diseases (CVDs) by interfering platelet function [30].Our preliminary research revealed that COMMD7 was highly expressed in hepatocellular carcinoma and might play as an oncogenic role in HCC, and it may promote HCC through activating NF-κB signaling by regulating the expression of chemokine CXCL10.
In this study, we rstly launched the expression pro le data and bioinformatics analysis of COMMD7 in HCC, combining with recent studies, we summarized the functional role of COMMD7 comprehensively, providing an overview for the latest research advances of it, which may help to direct targeted therapy of HCC.

GEPIA2 (Gene Expression Pro ling Interactive Analysis) dataset
GEPIA2 is an updated and enhanced version of GEPIA web server which features 198619 isoforms and 84 cancer subtypes based on tumor and normal samples from the TCGA and the GTEx databases, exhibiting a gene signature quanti cation by single-cell sequencing studies, and provides tools such as correlation analysis and survival analysis [31,32].

The Kaplan-Meier plotter
The prognostic value of COMMD7 mRNA expression was evaluated by an online database, Kaplan-Meier Plotter (https://kmplot.com/),which contains gene expression data and survival information of 364 clinical liver cancer patients (https://kmplot.com/analysis/index.php?p=service&cancer=liver_rnaseq) [33].Toanalyze the overall survival (OS), progression-free survival (PFS), and recurrence-free survival (RFS) of patients with liver cancer.COMMD7 was loaded as the only gene symbol, patient samples were split into two groups (high expression vs. Low expression) and assessed by a Kaplan-Meier survival plot, with the hazard ratio (HR) with 95% con dence intervals (CI) and logrank p value.

TCGA data and cBioPortal
The Cancer Genome Atlas (TCGA) contains both sequencing and pathological data on 30 different cancers [34].The hepatocellular carcinoma (TCGA, rehose legacy) dataset including data from 373 cases with pathology reports was selectedfor further analysis of COMMD7 using cBioPortal (http://www.cbioportal.org).Thegenomic pro les included mutations, mRNA expression z-scores (RNA Seq V2 RSEM) and protein expression Z-scores.Co-expression and correlations among COMMD7 and other COMMDs family members were calculated according to the cBioPortal's online instruction.

Statistical analysis
RStudio is a powerful data processing tool and is frequently applied in bioinformatics analysis [35].The data from TCGA were processed and analyzed by RStudio, and heatmap drawing of COMMD7 with other COMMDs family members were conducted in R (version 4.0.0,R Development Core Team) within the RStudio platform (version 1.3.959).

Expression pro le of COMMD7 in patients with HCC
In order to understand the expression pro le of COMMD7 in liver cancer, we performed bioinformatics analysis of COMMD7.As shown in Fig. 2, according to the 369 liver cancer tissues and 160 normal tissues included in Gene Expression Pro ling Interactive Analysis (GEPIA) dataset (http://gepia2.cancerpku.cn/), the mRNA expression level of COMMD7 in liver cancer tissues is much higher, as the transcripts per million (TPM) in tumor tissues was nearly 1.5 times higher than in normal tissues (p < 0.01; Fig. 2a).The protein relative expression level of COMMD7 in tumors and non-tumors was consistent with that of mRNA (Fig. 2b).Further, we detected the expression dynamic changes of COMMD7 during the development of HCC.The results showed that the expression of COMMD7 gradually increased from stage I to stage III (p < 0.01), indicating that COMMD7 was closely related with the early progression of HCC (Fig. 2c).
Our team also clari ed that COMMD7 was highly expressed in liver cancer tissue specimens.We veri ed the correlation of COMMD7 with early stage of HCC by detecting the mRNA expression level of COMMD7 in 68 pairs of liver cancer and adjacent tissues [36].Subsequently, a self-prepared monoclonal antibody to COMMD7 was uesd to detect the protein level of COMMD7 in 7 liver cancer cell lines including HepG2, Huh7, Hep3B, HLE, HLF, SK-Hep-1 and PLC/PRF5.The results revealed that COMMD7 protein expressed totally higher in these hepatoma cells than in normal hepatocytes, especially in HepG2 and SK-Hep-1 [36,37].For further study, we constructed a pGenesil-COMMD7-shRNA plasmid both in HepG2 and SK-Hep-1cells by means of short-hairpin RNA targeted technology.As a result, silence of COMMD7 signi cantly reduced cell proliferation and colony formation, while increased apoptosis and led to cell cycle arrest at Sphase in both two cell lines [38].

Association of increased mRNA of COMMD7 with poor prognosis of patients with HCC
We further explored the critical e ciency of COMMD7 in the survival of patients with HCC.Kaplan-Meier Plotter tools were used to analyze the correlation between the mRNA level of COMMD7 and the survival of 364 HCC patients in the database (http://kmplot.com/analysis/index.php?p=service&cancer=liver).The Kaplan-Meier curve and log-rank test analysis revealed that the increased mRNA level of COMMD7 was signi cantly associated with lower overall survival (OS) and recurrence-free survival (RFS) (p < 0.01) (Fig. 3) in patients with HCC.

Gene mutation and co-expression analysis of COMMD7 in HCC
We analyzed COMMD7 gene alterations, expression correlation of COMMDs family members, and coexpression genes of COMMD7 by using the cBioPortal online tool for hepatocellular carcinoma (TCGA, Firehose Legacy, http://www.cbioportal.org/).AS a result, COMMD7 was altered in 41 samples out of 373 patients with HCC (11%) (Fig. 4a), and the altered group obtained better overall survival (median month overall survival 81.67 Vs 53.35, Fig. 4b and 4c).We also calculated the mRNA expression correlations of COMMD7 and other COMMDs family members with each other by Pearson's correction.
The results indicated that most of the COMMDs family members were expressed positively with COMMD7, however the expression of COMMD2 and COMMD10 were negatively correlated with COMMD7 (Fig. 4d).

Functional analysis of COMMD7 in liver cancer stem cells
We have previously knocked down COMMD7 gene in liver cancer stem cells (LCSCs) to observe the effect of COMMD7 in LCSCs.After gene-sequencing, we analyzed the function of COMMD7 by Gene Ontology (GO) in the Database for Annotation, Visualization, and Integrated Discovery (DAVID, https://david.ncifcrf.gov/ summary.jsp), the go enrichment including biological processes (BP), cellular components (CC), and molecular functions (MF) were launched.We found that GO:0055114 (oxidation-reduction process), GO:0098590 (plasma membrane region), GO:0070742 (C2H2 zinc nger domain binding) were most enriched for up-regulated genes after COMMD7 knock-down, while GO:0007399 (nervous system development), GO:0031012 (extracellular matrix), GO:0005509 (calcium ion binding) were signi cantly enriched for down-regulated genes(Fig.5), indicating that COMMD7 may mainly combined with targeted domain or ions to exert its functional effect.

Discussion
The bioinformatics analysis results suggested that COMMD7 expressed highly in liver tumor tissues, which donating poor prognosis and early progression of HCC.Since current studies on COMMD7 and HCC was primarily carried out in our center, herein we discussed the potential molecular mechanism by reviewing our previous work and research advances about COMMD7.

COMMD7 enhanced hepatoma cell proliferation through activating NF-κB signaling
The nuclear factor of κ-light-chain-enhancer of activated B cells (NF-κB) transcription factor is well-known for its pivotal role in the maintenance of cellular homeostasis including cell death and apoptosis, in ammation and immunity [39].Dysregulation of NF-κB pathway is frequently found in common human disease, and it is usually in a state of continuous activation in most cancers including Leukemia [40], lung cancer, HCC [41,42].The classical activation of NF-κB needs the 26 s proteasome induced ubiquitination and degradation of IKBa, NF-κB is then released from the cytoplasmic NF-κB/IkBa complex, and exposing the nuclear localization domain, forming a p50/RelA dimer, activating the transcription of tumor-related factors downstream.Additionally, NF-κB also can be activated in other ways such as nucleolar stress, tumor necrosis factor receptor (TNFR) family ligands CD40L [43], ROS [44], and angiotensin [45].Thus the inhibition of NF-κB activity is considered to be one promising strategy to manipulate the tumorigenesis and development of cancers.
The COMMDs family member COMMD1 has been widely reported to facilitate the ubiquitination of NF-κB subunit RelA and its subsequent proteasomal degradation, therefore down-regulates NF-κB activity as well as the transcription of its targeted cancer-related genes downstream [19], hence COMMD1 negatively regulates the in ammatory responses and acts as an anti-in ammatory effector to repress colitis-associated cancers [46] and even as a tumor suppressor [47] in certain cancers such as prostate cancer[48] and lung cancer [18].While in our previous studies, we found that COMMD7 is positively related to the expression and activation of NF-κB in HCC.An analysis of the expression relationship between COMMD1 and COMMD7 also showed their negative correlation (R=-0.6237,p < 0.001).
Furthermore, COMMD7 may be correlated with NF-κB through a positive feedback loop.Scilicet on one hand NF-κB directly binds to the promoter 1, 4, 5 and 6 sites of COMMD7, suggesting that NF-κB could target to COMMD7 and directly regulate its transcription in hepatocellular carcinoma cells [49]; on the other hand, COMMD7 silence in HepG2 cells resulted in 75% inhibition rate of NF-κB, indicating that COMMD7 conversely regulates the nuclear translocation of NF-κB as well as the transcriptions of consequent genes involved in HCC [38].While how COMMD7 regulates the activity of NF-κB need further study.You et al, demonstrated that oxidative stress was required for COMMD7-mediated NF-κB activation since they found that overexpression of COMMD7 augmented intracellular ROS level and p65 phosphorylation in Huh7 cells [50].Meanwhile, recent study manifested that COMMD7 activates NF-κB by upregulating protein inhibitor for activated stat4 (PIAS4) in Nanog + hepatocellular carcinoma stem cells to maintain its proliferation and metastasis [51].

COMMD7 promoted HCC progress and metastasis by modulating the production of CXCL10
The role of immune system in tumor has been in the spotlight, cancer immunotherapy turns into hot issue.Chemokines are a class of small cytokines or signaling proteins secreted by cells which induce directional chemotaxis of vicinity response cells, these low-molecular-weight cytokines range from 8 kDa to 10 kDa are of critical importance in tumor immune response.C-X-C motif chemokine ligand 10 (CXCL10) belongs to interferon-γ (INF-γ) inducible chemokine family which orchestrates leukocyte tra cking and modulate innate and adaptive immune responses and exerts its biological function by binding with CXCR3 receptor [52].The role of CXCL10 in cancers and disease remains controversial, although it has been reported to suppress tumor by interferon-γ-mediated anti-tumor effect [53], more and more researches revealed it was bene cial for tumor growth, metastasis and recurrence.High serum level of CXCL10 could be a potential biomarker for many kinds of disease such as rheumatoid arthritis [54], psoriasis [55], cGVHD[56] and the CXCL10/CXCR3 axis mediates lung cancer cell migration [57].Besides, expression of CXCL10 positively correlates with obesity and type 2 diabetes and linked to poor outcomes after islet transplantation [58].In hepatocytes and liver cancer, CXCL10 was proved to enhance HCV and male HBV infection [59], the CXCL10/CXCR3 signaling promotes liver tumor growth via recruiting more endothelial progenitor cell (EPC) and inducing their mobilization, differentiation and neovessel formation [60].Likewise, CXCL10 upregulated at liver graft injury directly induced the mobilization and intragraft recruitment of Tregs, which further promoted HCC recurrence after transplantation [61].
Moreover, immune checkpoint inhibitor (ICI) therapy targeting anti-programmed cell death-1 (anti-PD-1) or its ligand (anti-PD-L1) is the backbone of numerous combination regimens aimed at improving the objective response and survival of patients with HCC[62], and Chow's recent study illustrated that CXCL10 is required for PD-1 blocked therapy responders [63].Taken together, targeting CXCL10 may attenuate early HCC progression and recurrence.
Interestingly, our present study discovered that CXCL10 dramatically upregulated in COMMD7 overexpressed Huh7 cell line, and the expression levels of CXCL10 in other HCC cell lines including HepG2, Hep3B and HLE have been detected to be consistent with the expression level of COMMD7.In addition, inhibition of CXCL10 impaired COMMD7 mediated HCC cell proliferation and metastasis, indicating that COMMD7 positively regulates CXCL10 production in HCC [64].Further, it has been reported that NF-κB directly binds to the promoter site of CXCL10 and regulates its expression [65].For example, the activation NF-κB is required for the induction of CXCL10 during the progress and metastasis of Melanoma[66] and breast cancer [67].Given that our previous evidence demonstrated that COMMD7 participated in NF-κB activation, therefore it probably promoted CXCL10 production by activating NF-κB, and the subsequent research also proved it [50].Overall, these researches expounded that COMMD7 could strengthen HCC progress and metastasis by modulating the production of CXCL10 via NF-κB activation.

Conclusions
In summary, we illuminated the expression pro le of COMMD7 in hepatoma cell lines and human liver tumor tissues, shedding light on the promoting effect of COMMD7 on HCC.The results further highlight that high expression of COMMD7 is related with the early progression of HCC and poor prognosis.More importantly, it plays a pivotal role in hepatoma cell proliferation and metastasis through activating NF-κB signaling and CXCL10 via PIAS4 and ROS.In conclusion, since these bioinformatics analysis results and researches direct the positive correlation of COMMD7 high expression with HCC, it is possible to be regarded as a potential biomarker for the prevention and treatment of HCC.However how COMMD7 interacts with these effectors and more detailed mechanism warrants further investigation.could be con rmed that all written informed consent had already been obtained.

Consent for publication
Not applicable. The expression pro le of COMMD7 in HCC (GEPIA).(a) Transcription level of COMMD7 in tumor(T) and non-tumor(N) samples.(b) Relative protein level of COMMD7 in tumor(T) and non-tumor(N) samples; (c) Correlation between COMMD7 expression and tumor stage in HCC patients.

Figure 3 The
Figure 3

Figure 5 The
Figure 5

Table 1
Studies about COMMDs family proteins in different cancers and diseases