A Multilevel Systemic Pan-cancer Analysis: Collagen Triple Helix Repeat Containing-1 is a Potential Target for Tumor Immunotherapy

Background: Cancer is one of the leading causes of pathological death in humans. Although CTHRC1 is a prooncogene highly expressed in a variety of tumor tissues, the specic biological mechanisms of CTHRC1 involvement in cancer development need to be elucidated. Methods: In the present study, nine online bioinformatics databases were employed to explore the potential prognostic and grading value of CTHRC1 in generalized cancer as well as its potential role in regulating tumor immunity. Results: Data from GEPIA2.0, Oncomine, TNMplot, Kaplan-Meier Plotter and TISIDB database had consistently demonstrated that CTHRC1 was associated with the expression, prognosis and typing in most cancer tissues. Cbioportal and SMART analysis revealed that genomic changes and methylation of CTHRC1 in most tumor tissues. Finally, Sangerbox and TIMER database analysis suggested that CTHRC1 was involved in the changes of immune cell components in tumor immune microenvironment, with certain heterogeneity. Meanwhile, CTHRC1 was correlated with TMB, MSI, neoantigen and tumor immune checkpoint, especially CD276. Conclusion: CTHRC1 had the potential as a prognostic and grading molecular marker for pan-cancer. And CTHRC1-related targeting agents may be a novel breakthrough in tumor immunotherapy.


Introduction
According to the World Health Organization (WHO), cancer will be the deadliest diseases in the 21st century [1]. Despite various tumor treatment methods are actively pursued, several issues remain. Such as poor tumor results, rapid disease progression, and high mortality. the mobilization of the immune system and enhancement of antitumor immunity are the main principles of cancer immunotherapy. As against traditional treatment, immunotherapy is characterized with high speci city and low side effects. The development of cancer immunotherapy represents a novel direction of cancer treatment Collagen triple helix repeat containing-1(CTHRC1) is rst discovered in injured arteries of rats, which encodes protein that belongs to a highly conserved glycosylated protein [2]. In the eld of tumor, CTHRC1 activates invasion and migration of gastric cancer (GC) cell lines SGC7901 and HGC27 via hiF-1/CXCR4 axis [3]. He et al. report that high expression of CTHRC1 protein in lung cancer tissues, which affects lung cancer cells' invasion and metastasis by acting on MMP7/MMP9 [4]. Zheng et al. found that CTHRC1 promotes HeLa cell growth via activating the Wnt/PCP signaling pathway [5]. Over-expression of CTHRC1 is found to be a marker of worse prognosis in several different malignancies, including colorectal cancer[6], lung cancer [7], gastric cancer[8] and liver cancer [9]. Those results implicate CTHRC1 is not only involved in regulating tumor cells growth, proliferation and apoptosis via multiple pathways, but also has the potential as a prognostic marker for pan-cancer.
The tumor microenvironment (TME) acts a pivotal part in the occurrence, development, metastasis and drug sensitivity of tumors. CTHRC1 can promote M2 macrophage polarization via STAT6 signaling pathway in ovarian cancer tissues [10]. CTHRC1 regulates the integrin-akt signaling pathway to promote M2 macrophage polarization and invasion and metastasis of endometrial carcinoma cells [11]. CTHRC1overexpressed patients with urological cancers have a poor prognosis in part because of alterations in TME [12]. Above studies suggest that CTHRC1 plays a certain role in regulating tumor immunity. However, there are no published reports focusing on the prognosis and immunomodulatory mechanism of CTHRC1 in pan-cancer.
Here, the expression differences, prognostic potential, immunotyping and molecular typing of CTHRC1 in human cancer were comprehensively investigated by multiple online analysis tools based The Cancer Genome Atlas (TCGA) resources. Next, Understanding CNV and methylation information of CTHRC1 in tumor tissues, and its role in tumor immunity through comprehensive and multi-dimensional analysis.
Overall, the main objective of this study was to evaluate potential value of CTHRC1 in immunotherapy of malignant tumors.

Four databases de ne CTHRC1 transcript levels in various tumor tissues
GEPIA2.0 database (http://gepia2.cancer-pku.cn/#index) is developed based on RNA-seq data from thousands of tumor and normal samples of TCGA and GTEx [13]. The TIMER (https://cistrome.shinyapps.io/timer/) is a web-based public tool based on TCGA [14]. The Oncomine database ( https://software.oncomine.com ) is a public open database, which incorporates thousands of microarray datasets from human tumors [15]. The screening condition was that P<0.001, Change= 2. The TNMplot database (https://tnmplot.com/analysis/)belongs to a freely available bioinformatics website, which contains mainly RNA-seq data from TCGA, GTEX and TARGET [16]. Above databases was used to investigate the differential expression of CTHRC1 mRNA between tumor and normal tissue, as shown by box plots.
3.2 Two databases de ne the prognostic value of CTHRC1 in human cancers GEPIA2.0 can be used not only for the analysis of single gene expression differences, but also for the prognostic value of gene in cancers. Input CTHRC1, the selection condition: P < 0.05, Group Cutoff: Media. K-M plotter (http://kmplot.com/analysis/index.php?p=background) is a classic website focused on cancer prognosis and contains RNA-seq and gene chip data for 21 cancers [17]. Using microarray data from this website. we analyzed the prognostic value of CTHRC1 in some cancers. 3.4 Two databases were used to analyze CTHRC1 CNV and methylation in human cancer tissues Cbioportal (http://www.cbioportal.org/) is a bioinformatics analysis tool developed based on TCGA, ICGC and GEO data resources, which can study the mutation and copy number variation of CTHRC1 in different cancer types [19]. At the same time, it can also be combined with clinical data to investigate the relevance between CTHRC1 CNV and the prognosis of cancer patients. Input CTHRC1, and select the consistent sample TCGA Pan-Cancer Atlas. The Oncoprint module presents details of genomic alterations in CTHRC1 in cancer. Cancer summar Types survival module is used to understand the association of the CTHRC1 gene alteration with the prognosis. SMART (http://www.bioinfo-zs.com/smartapp/) is an online website built based on TCGA tumor methylation data, which can the relationship between different methylation sites of genes and expression differences, prognosis and grade [20]. On the home page, CTHRC1 was entered and the Click to check CpG-aggregated methylation module was selected to visually analyze CTHRC1 methylation in pantothenic cancer. Then, the prognostic module was selected to view the prognosis of different methylation sites in a variety of cancers.
3.5 Two databases jointly analyze the link between CTHRC1 and the tumor immune microenvironment Sangerbox (http://sangerbox.com/Tool), which is a medical bioinformatics platform built using TCGA resources [21]. Enter CTHRC1 to view links to TMB, MSI, neoantigens, and immune checkpoints in different cancer types. Gene module of The TIMER database was additionally used to understand the link between CTHRC1 and a variety of immune cell numbers in cancer tissues.

Statistical Analysis
All statistical methods were provided by the corresponding website. Statistical signi cance was considered at P < 0.05.

Differential expression of CTHRC1 in non-tumor and tumor tissues
In a rst step, signi cant difference was observed for expression of CTHRC1 mRNA in non-tumor and tumor tissues. By analyzing the gene chip data of Oncomine, expression levels of CTHRC1 are high in most tumor tissues, consisting of brain and CNS cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer and prostate cancer. In TIMER database, Compared with the normal counterparts, the transcript level of CTHRC1 was over-expressed in most cancer types, including BLCA (bladder urothelial carcinoma), BRCA (breast invasive carcinoma), CHOL (acute myeloid leukemia) and UCEC. The above results tentatively proposed that CTHRC1 was an oncogene and had potential as a diagnostic marker for pan-cancer.

CTHRC1 and prognosis of cancer patients
Our study next found that over-expression of CTHRC1 affects a poor prognosis of patients based on GEPIA2.0 (TCGA RNA-seq data) and KM-plotter (gene chip data). Detailed results are presented in the following sets of data. In LGG. CTHRC1 was not always highly expressed in a single immune subtype. In more detail, CTHRC1 showed the highest expression in C1 subtype of three distinct tumors, which include KIRC, LIHC and SARC. Highest expression in C2 subtype, which include BLCA, KIRP and PRAD. Highest expression in C3 subtype, which include KICH, OV and LGG. Highest expression in C4 subtype, which include BRCA and COAD. Furthermore, CTHRC1 expression is also differentially expressed in molecular subtypes of multiple tumors, which include BLCA, BRCA, COAD, KIRP, KICH, KIRC, LIHC, SARC, PRAD, OV and LGG. The above analysis indicated that CTHRC1 has a diagnostic index as a classi cation of pan-cancer.

CTHRC1 CNV and Methylation in tumors
The use of cBioPortal database was used to analyse the uniform sample (TCGA, PanCancer Atlas). CTHRC1 ampli cation (AMP) was the most prevalent actionable alteration in multiple tumors, consisting of OV, BLCA, BRCA, LIHC, PRAD, STAD, SARC, COAD, KICH, LGG and KIRC. And CTHRC1 CNV alteration was a high risk factor in cancer patients, consisting of OV, LGG, SARC, PRAD and KIRP. For instance, the group of CTHRC1 CNV alteration had a signi cantly poorer OS, PFS and DSS. More detailed data are shown in (Figure 4). DNA methylation was considered an essential factor impacting on the occurrence and progression of tumor. Currently, there is a paucity of research on the relationship between abnormal methylation of CTHRC1 gene and tumors. Thus, through the analysis results of the SMART database, we demonstrated that abnormal methylation of CTHRC1 gene in a variety of tumor tissues, including BLCA, BRCA, CESC, COAD, ESCA, HNSC, KIRC, LIHC, LUAD, LUSC, READ, THCA and UCEC. Also, we found that CTHRC1 hypermethylation was generally linked to good prognosis in BLCA, BRCA, HNSC, KIRC and UCEC. More detailed results are shown in (Figure 5).

CTHRC1 and human cancers immune microenvironment
The TIMER database found a link between CTHRC1 transcript levels and immune cell numbers in a variety of cancer types. For example, a signi cant positive association was observed between CTHRC1 and multiple immune cell types such as B cells, CD4 +T cells CD8 +T cells, neutrophils, macrophages and dendritic cells in BRCA. An additional thing we noticed was that CTHRC1 was not always related to speci c immune cell species. Like, CTHRC1 has the largest correlation coe cient with B cells in ten cancer types. Having the closest association with CD4+T cells in eight cancer types. Having the largest correlation coe cient with CD8+T cells in ten cancer types. Having the closest association with macrophages in ten cancer types. Having the closest association with neutrophils in ten cancer types. And having the closest association with dendritic cells in twelve cancer types. This suggests that CTHRC1 exerts different immune effects in different types of tumor tissues. The sangerbox platform analyzed 47 immune checkpoint genes and found a positive correlation between CTHRC1 and immune checkpoint genes in most tumor tissues. In addition, there was a signi cant positive correlation between CTHRC1 and CD276 in most tumor tissues. TMB, MSI and neoantigens were important evaluation metric of immunotherapy e cacy. There was a positive correlation between CTHRC1 with TMB in COAD, LAML, LGG, LUAD, PRAD, SARC and THYM, and a negative correlation between CTHRC1 with TMB in GBM, LIHC and UCEC. MSI and neoantigens exhibited the closest linkage with CTHRC1. In particular, UCEC patients with high CTHRC1 expression tend to show negative scores of TMB, MSI and neoantigens. This suggests that UCEC patients with high CTHRC1 expression have poor immune e cacy.

Discussion
An extracellular matrix protein encoded by CTHRC1 was integral components of the immune microenvironment. Extracellular matrix (ECM) had been shown to be involved in tumor development via biological pathways, including activating intracellular signaling of cell cycle progression, inhibiting the function of tumor suppressor factors, participating in the invasion and metastasis of cancer cells, tumor suppressor gene mutation and evasion of immune surveillance, etc [22,23]. Actually, studies have found that CTHRC1 is involved in the progression of human cancer [24]. However, the speci c mechanism of CTHRC1 regulates tumor immune microenvironment remains unclear.
First step in this investigation, bioinformatics analysis based on four databases suggests that CTRHC1 is highly expressed in various tumor tissues. It was preliminarily suggested that CTHRC1 is a protooncogene involved in the progression of tumors. Next, GEPIA2.0 and KM-plotter databases found that high expression of CTHRC1 was a negative factor in 10 cancers, implicating CTHRC1 can be served as a pan-cancer diagnostic and prognostic biomarker. The correct classi cation of cancer patients is clinically conducive to selecting appropriate therapeutic regimens. For most cancers except STAD and ACC, CTHRC1 transcription levels were different in immune and molecular subtypes, which suggested that CTHRC1 may be a novel marker for the diagnosis of pan-cancer and participates in the regulation of tumor immunity. Oncogenic transformation always occurs along with genomic alterations. Liu et al. reported ampli cation of the levels of CTHRC1 drives proliferation and invasion of oral squamous cell carcinoma [25]. We demonstrated that CTHRC1 was shown to undergo some ampli cation in most cancers, with the most signi cant in OV. CTHRC1 gene ampli cation is a prevalent phenomenon in the vast majority of tumor types. And altered group often implies a worse prognosis in OV, SARC, LGG, KIRP and PRAD. Aberrant DNA methylation is one of the important characteristics of cancer epigenetics. Early research found that CTHRC1 gene demethylation increases expression and is associated with cell line growth and invasive abilities in gastric cancer [26]. As shown in the present study, the methylation level of distinct sites of CTHRC1 gene was signi cantly lower in most tumor tissues than in neighboring nontumor tissue samples. And CTHRC1 methylation levels are closely related to patient prognosis in BLCA, BRCA, HNSC, KIRC and UCEC. The results provide a deeper theoretical basis for relevant research of cancers epigenetics.
More recently, it has been reported that tumor in ltrating lymphocytes (TILs) could be used as an indicator of evaluating the prognosis and treatment e cacy for cancer patients [27,28]. Our study suggested that CTHRC1 mRNA levels were closely linked to the number of immune cells and played a critical role in regulating TME. Speci cally, a signi cant positive association was observed between CTHRC1 and number of macrophages in BLCA, BRCA, COAD and STAD. Interestingly, The most strongly positive relationship between CTHRC1 and number of dendritic cells in KICH, KIRP, PRAD and SARC. Thus, this suggests that CTHRC1 can participate in the regulation of tumor microenvironment immune status.
Noteworthy, the following phenomenon could have contributed to the poor prognosis of cancer patients. First, CTHRC1 has a correlation with the number of macrophages in most tumor tissues. There is a report that macrophages are involved in the process of tumor immune escape [29]. Second, CTHRC1 has a correlation with multiple types of immune checkpoints in most tumor tissues. For example, CTHRC1 has a positive correlation with PDCD1 in PRAD, which is similar to the results of a previous work [30].
Clinically, PD-1 and CTLA4 targeting have already been applied extensively in treatment of cancer [31]. TMB is a promising cancer predictive biomarker, and has signi cant signi cance in guiding immune checkpoint inhibitor (ICI) treatments [32,33]. MSI also be an independent prognostic biomarker of cancers [34][35][36]. In this current study, the association of CTHRC1 with tumor mutation burden (TMB) and microsatellite instability (MSI) also provided strong evidence that CTHRC1 was involved in the regulation of TME in human cancers. Particularly, CTHRC1 showed a negative correlation with TMB, MSI and neoantigens in UCEC. This may be the reason why some patients with UCEC do not respond well to ICI treatment. Future targeting of CTHRC1 in combination with PDCD1 inhibitors will be a novel direction for UCEC patients.

Conclusion
Altogether, our study is the rst to systematically investigated the link between CTHRC1 and pan-cancer at the mRNA level, CNV level and methylation level as well as prognostic effects by bioinformatics methods. And the study demonstrated that CTHRC1 is an unfavorable prognostic factor in a variety of cancers with a link to alterations in the immune microenvironment. However, there exists some limitations in the present study. First of all, the quantity and quality of public data resources, screening conditions and statistical methods can affect the authenticity and rigor of research. Validation by in vitro and in vivo experiments and pathological specimens is required. In addition, we lack immunological experiments to prove that CTHRC1 has immunomodulatory ability in cancers. Prospective studies of CTHRC1 targeting and the tumor immune microenvironment may help provide a clear anticancer strategy in the future.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
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Declaration of Competing Interest
The authors declare that there is no con icts of interest.     The correlation between CTHRC1 transcription levels and 50 immune checkpoints in pan-cancer.

Figure 7
The Correlation between CTHRC1 transcription levels and MSI, neoantigen, TMB, and ESTIMATE Score in pan-cancer. Note: MSI: microsatellite instability, TMB: tumor mutational burden.