Contributed equally
Collagen triple helix repeat containing 1 (CTHRC1) is a secreted glycoprotein that decreases the deposition of collagen matrix and accelerates tumor metastasis. However, the relationship between CTHRC1 and the outcomes of head and neck squamous cell carcinoma (HNSCC) and tumor-infiltrating lymphocytes remains unclear. In the present study, the transcriptional level of CTHRC1 and its association with overall survival (OS) and relapse-free survival (RFS) time in diverse cancer types were evaluated using The Cancer Genome Atlas, Tumor Immune Estimation Resource (TIMER), ONCOMINE and Kaplan-Meier plotter databases. The association of CTHRC1 expression level with the clinicopathological parameters of patients with HNSCC from The University of ALabama at Birmingham CANcer data analysis Portal (UALCAN) database were also evaluated. Enrichment analysis of CTHRC1 was carried out using gene set enrichment analysis software. CIBERSORT and TIMER databases were used to evaluate the relationship between the expression level of CTHRC1 and the proportion of tumor-infiltrating immune cells (TICs) in multiple cancer types. Moreover, immunohistochemistry was used to verify the expression of CTHRC1 in clinical samples of HNSCC. CTHRC1 was upregulated in HNSCC and high expression of CTHRC1 was associated with worsening clinicopathologic parameters and shorter OS and RFS times. There were eight HALLMARK gene sets, 1,231 immune signature gene sets and 14 KEGG gene sets significantly enriched in the high CTHRC1 expression group, while no gene set was enriched in the low CTHRC1 expression group. The expression of CTHRC1 was closely correlated with the proportion of TICs, where the expression of CTHRC1 was significantly positively correlated with the amount of infiltrated M0 and M2 macrophages, and significantly negatively associated with the levels of M1 macrophages. These findings suggest that CTHRC1 is an adverse prognostic marker and is associated with immune cell infiltration in HNSCC.
Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignant tumors, accounting for ~90% of the total number of head and neck tumors (
Collagen triple helix repeat containing 1 (CTHRC1) was first discovered in the injured arteries of rats (
The RNA-seq data (workflow type, HTSeq-FPKM) and relevant clinical data for the ‘TCGA-HNSC’ cohort, including 502 tumor samples and 44 normal samples, were downloaded from the TCGA database (
Expression levels of CTHRC1 were analyzed in multiple cancer types using the ONCOMINE database (
The TIMER database (
KM plotter contains data and clinical information from the Gene Expression Omnibus, TCGA and European Genome-Phenome Archive databases. The prognostic value of the mRNA expression levels of CTHRC1 in 21 cancer types was evaluated using the KM plotter (
UALCAN (
Based on the validated leukocyte gene signature matrix (LM22), the CIBERSORT (
R 64.4.0.0 software (
GSEA were downloaded from the Broad Institute Website (version 4.0.2,
From January 2020 to December 2020, 70 patients with primary HNSCC who underwent radical resection of tumors, did not receive preoperative radiotherapy or chemotherapy in the People's Hospital of Tongxu County (Kaifeng, China) and provided written informed consent were enrolled in the present study, and their tumor tissue samples were collected. A total of 11 patients with benign head and neck lesions were included, informed consent was signed and normal tissue samples adjacent to benign lesions were collected. Patients with HNSCC were between 32 and 79 years old, with a mean age of 59.9 years. Human tissue specimens were obtained from patients who had provided written informed consent and the present study and tissue collection were approved by the Ethics Supervision Committee of The People's Hospital of Tongxu County (Kaifeng, China; approval no. TX20NP003) and the National Human Genetic Resources Sharing Service Platform (2005DKA21300). The study protocol was approved by the Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China; approval no. 2020IEC-J050).
The tissue samples were fixed using 10% formalin solution at room temperature for 24 h, dehydrated, embedded in paraffin and sectioned into 4 µm sections. The expression of CTHRC1 protein was assessed using the immunohistochemistry ultrasensitive TMS-P method. After the sections were deparaffinized, hydrated and washed, they were placed in EDTA repair solution (pH 9.0), and boiled using a microwave oven for 2 min, and then cooled to room temperature naturally. Then sections were blocked using 3% H2O2 solution for 15 min at room temperature in the dark room. The sections were washed and incubated overnight at 4°C with CTHRC1 antibodies (1:200; cat no. 16534-1-AP; Wuhan Sanying Biotechnology). After washing three times with phosphate buffered saline, the sections were stained with secondary antibodies using the Ready-to-use Ultrasensitive™ SP kit (cat. no. Kit-9720; Fuzhou Maxin Biotechnology Development Co., Ltd.) and incubated for 30 min in a wet box at 37°C. DAB horseradish peroxidase color development kit (cat no. P0202; Beyotime Institute of Biotechnology) was used for color development. The sections were re-stained using hematoxylin staining solution (cat no. C0107; Beyotime Institute of Biotechnology) for 2 min at room temperature, dehydrated using ethanol, sealed and imaged using a Pannoramic MIDI scanner (3DHISTECH, Ltd.) and ImageJ software (version 1.8.0, National Institutes of Health) was used for image analysis. The cells on the microarrays were scored according to the staining intensity of the marker, as follows: 0, no coloring; 1, light yellow; 2, brown-yellow; and 3, tan. The proportion of cells under each score was then recorded, and the H-score value of each specimen was calculated. H-scores were defined as: (1× percentage of cells staining at 1) + (2× percentage of cells staining at 2) + (3× percentage of cells staining at 3).
SPSS 26.0 software (IBM Corp.) was used for statistical analyses. Fisher's exact test was used to analyze the relationship between clinical characteristics and CTHRC1 mRNA expression levels. The Mann-Whitney U test was used for calculating statistical differences in the H-scores in TMA and the ratio differentiation level of 21 types of immune cells in high and low CTHRC1 expression groups in HNSCC. Pearson correlation coefficient analysis was used to assess the correlation value between two kinds of cells. P<0.05 was considered to indicate a statistically significant difference.
To evaluate the distinct prognostic and potential therapeutic value of CTHRC1 in HNSCC, the mRNA expression level of CTHRC1 was studied using the TCGA, ONCOMINE and TIMER databases. Data from the TCGA database showed that CTHRC1 was highly expressed in 16 cancer types, including HNSCC (
Since clinical pathology can determine the progression and prognosis of diseases, the association of transcriptional levels of CTHRC1 with clinicopathological parameters in patients with HNSCC was investigated using UALCAN. As presented in
Since CTHRC1 was differentially expressed in diverse cancer types, the relationship between CTHRC1 expression levels and the survival time of patients with HNSCC was next evaluated using the KM plotter. Patients were grouped, into CTHRC1 high and CTHRC1 low expression groups, using the auto select best cutoff function on the KM plotter website (
In order to further investigate the potential function of CTHRC1, GSEA of the data from patients with HNSCC was conducted. A total of eight HALLMARK gene sets (
The immune system serves pivotal roles in the tumorigenesis of HNSCC (
The infiltration of 22 TICs in each HNSCC sample and the CIBERSORT algorithm was used to further evaluate the correlation between the transcriptional levels of CTHRC1 and the immune microenvironment (
HNSCC is among the six most common types of human tumor. Despite the development of medical technology the survival rate of patients with HNSCC is still low (
CTHRC1 is a secretory glycoprotein which negatively regulates the deposition of collagen matrix and is involved in vascular remodeling and cell migration (
In the present study, analysis indicated that upregulation of CTHRC1 was mainly involved in tumor and immune-related pathways, such as angiogenesis, apical junction, EMT and the KRAS and TGF-β signaling pathways. The EMT signaling pathway is associated with visibility, acquisition of mobility and self-renewal (
There are several limitations and challenges in the present study. Firstly, the sample size is small. Larger studies in HNSCC are needed to confirm the results. Secondly, CTHRC1 expression may not be a highly specific diagnostic and prognostic biomarker of HNSCC in humans, but be a shared diagnostic and prognostic biomarker of survival in different human cancers (
In conclusion, the upregulation of CTHRC1 in patients with HNSCC is related to the pathological grade, Tumor-Node-Metastasis stage, lymphatic metastasis, HPV status, TP53 mutation and TICs, which may lead to poor prognosis in patients with HNSCC. In addition, upregulation of CTHRC1 may activate tumor and immune related pathways, leading to the infiltration of immune cells and macrophage polarization in the tumor microenvironment in HNSCC. The present study identified a potential role of CTHRC1 in immunology and its adverse prognostic value in HNSCC. CTHRC1 should therefore be considered as a prognostic marker and therapeutic target for HNSCC.
Not applicable.
The data used in the present study may be accessed from the TCGA (
LW and JHZ proposed the idea for and designed the study. RLZ, MLY and RZ completed the data analysis work, RLZ and MLY drew the figures, MLY and LW drafted the manuscript and JHZ reviewed the manuscript. RLZ, MLY and LW confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript.
Human tissue specimens were obtained from patients who provided written informed consent and the present study and tissue collection was approved by the Ethics Supervision Committee of The People's Hospital of Tongxu County (Kaifeng, China; approval no. TX20NP003) and the National Human Genetic Resources Sharing Service Platform (2005DKA21300). The study protocol was approved by the Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China; approval no. 2020IEC-J050).
Not applicable.
The authors declare that they have no competing interests.
CTHRC1 expression levels in different types of human cancer. (A) The transcription levels of human CTHRC1 in different tumor types in the TCGA database were assessed using TIMER. (B) CTHRC1 transcriptional levels in adjacent and tumor tissues in patients with HNSCC from the TCGA database. (C) Expression level of CTHRC1 in different cancer datasets compared with normal tissues in the ONCOMINE database, the number indicated the number of data sets included by The National Center for Biotechnology Information that matched the conditions. (D) Scatter plot of CTHRC1 expression (using H-score) in HNSCC tissues compared with normal tissues. (E) Representative immunohistochemistry of CTHRC1 in clinical specimens. *P<0.05, **P<0.01, ***P<0.001. TCGA, The Cancer Genome Atlas; ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangio carcinoma; CNS, central nervous system; COAD, colon adenocarcinoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; HPV, human papillomavirus; neg, negative; pos, positive; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LGG, (brain) lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MESO, mesothelioma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumor; THCA, thyroid carcinoma; THYM, thymoma, UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma; UVM, uveal melanoma; CTHRC1, collagen triple helix repeat containing 1; TPM, transcripts per million.
Relationship between mRNA expression levels of CTHRC1 and the clinical characteristics of patients with HNSCC. Relationship between CTHRC1 mRNA expression level and (A) age, (B) TP53 mutation, (C) HPV status, (D) tumor grade, (E) nodal metastasis status and (F) cancer stages in HNSCC patients. *P<0.05, **P<0.01, ***P<0.001. CTHRC1, collagen triple helix repeat containing 1; HNSCC, head and neck squamous cell carcinoma; HPV, human papillomavirus; TCGA, The Cancer Genome Atlas; Yrs, years.
KM survival analysis of CTHRC1 in head and neck squamous cell carcinoma from the KM Plotter database. (A) Overall survival and (B) relapse-free survival. CTHRC1, collagen triple helix repeat containing 1; HR, hazard ratio; KM, Kaplan-Meier.
Gene sets for enrichment analysis for patients with head and neck squamous cell carcinoma with high or low CTHRC1 expression. Each row represents a specific set of genes with a unique color. Enriched gene sets with high expression of CTHRC1 in (A) HALLMARK, (B) KEGG and (C) IMMUNE SIGNITURE. Representative gene sets are shown. Nominal P<0.01, false discovery rate Q<0.06. CTHRC1, collagen triple helix repeat containing 1; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Correlation between tumor-infiltrating immune cell proportions and CTHRC1 expression in HNSCC in the Tumor Immune Estimation Resource database. (A) HNSCC, (B) HPVneg HNSCC and (C) HPVpos HNSCC. CTHRC1, collagen triple helix repeat containing 1; HNSCC, head and neck squamous cell carcinoma; HPV, human papillomavirus; neg, negative; pos, positive.
TIC profiles and correlation analysis of HNSCC samples. (A) The proportion of 22 types of TICs in HNSCC tumor samples. (B) Heatmap showing the correlation between 22 kinds of TICs, the number in each box indicates the corresponding correlation value between two kinds of cells. CTHRC1, collagen triple helix repeat containing 1; HNSCC, with head and neck squamous cell carcinoma; TICs, tumor-infiltrating immune cells.
Association between the percentage of each TIC and the CTHRC1 expression level. (A-J) The association of the percentage of 10 types of TICs with the transcriptional levels of CTHRC1. The linear model of immune cell proportional tendency and CTHRC1 expression was fitted by the gray line, and Pearson coefficient was used to test the correlation. (K) Violin plot indicating the ratio differentiation level of 21 types of immune cells in high and low CTHRC1 expression groups in head and neck squamous cell carcinoma tumor samples. (L) Venn plot displaying 10 kinds of TICs correlated with CTHRC1 expression codetermined by difference and correlation tests displayed in violin and scatter plots, respectively. CTHRC1, collagen triple helix repeat containing 1; NK, natural killer; TICs, tumor-infiltrating immune cells.
Relationship between clinical characteristics and CTHRC1 expression in patients with head and neck squamous cell carcinoma.
Characteristic | Low expression of CTHRC1, n=251 (%) | High expression of CTHRC1, n=250 (%) | P-value |
---|---|---|---|
Sex, n | 0.978 | ||
Female | 67 (26.7) | 67 (26.8) | |
Male | 184 (73.3) | 183 (73.2) | |
Age, n | 0.072 | ||
<60 years | 121 (48.4) | 100 (40.0) | |
≥60 years | 129 (51.6) | 150 (60.0) | |
HPV | 0.009 | ||
status, n | |||
Negative | 196 (81.3) | 216 (90.0) | |
Positive | 45 (18.7) | 24 (10.0) | |
Tumor | 0.118 | ||
stage, n | |||
1 | 20 (9.0) | 26 (11.7) | |
2 | 73 (32.7) | 59 (26.6) | |
3 | 54 (24.2) | 42 (18.9) | |
4 | 76 (34.1) | 95 (42.8) | |
Node | 0.412 | ||
stage, n | |||
0 | 93 (45.8) | 78 (38.0) | |
1 | 32 (15.8) | 33 (16.1) | |
2 | 75 (36.9) | 90 (43.9) | |
3 | 3 (1.5) | 4 (2.0) | |
Metastasis | >0.999 | ||
stage, n | |||
0 | 97 (99.0) | 89 (100.0) | |
1 | 1 (1.0) | 0 (0) | |
Pathological | 0.070 | ||
Stage, n | |||
I | 10 (4.6) | 15 (6.9) | |
II | 44 (20.4) | 26 (12.0) | |
III | 41 (19.0) | 37 (17.1) | |
IV | 121 (56.0) | 139 (64.1) | |
Grade, n | 0.016 | ||
1 | 39 (16.2) | 22 (9.1) | |
2 | 150 (62.2) | 150 (62.2) | |
3 | 50 (20.7) | 69 (28.6) | |
4 | 2 (0.8) | 0 (0) | |
Radiation | 0.985 | ||
therapy, n | |||
No | 77 (34.8) | 73 (34.9) | |
Yes | 144 (65.2) | 136 (65.1) |
A total of 501 patients were included, including 251 patients in the low expression group and 250 patients in the high expression group, certain patients had incomplete clinical information available. In each characteristic, patients lacking that information were excluded. CTHRC1, collagen triple helix repeat containing 1; HPV, human papillomavirus.
Correlation analysis between collagen triple helix repeat containing 1 and related gene markers of tumor-infiltrating immune cells in Tumor Immune Estimation Resource.
None |
Purity |
||||
---|---|---|---|---|---|
Cell type | Gene marker | Cor |
P-value | Cor | P-value |
CD8+ T cell | CD8A | −0.005 | 9.19×10−1 | −0.277 | 4.03×10−10 |
CD8B | 0.048 | 2.90×10−1 | −0.242 | 5.06×10−8 | |
T cell (general) | CD3D | 0.029 | 5.22×10−1 | −0.298 | 1.35×10−11 |
CD3E | 0.110 | 1.49×10−2 | −0.299 | 1.17×10−11 | |
CD2 | 0.116 | 9.77×10−3 | −0.285 | 1.15×10−10 | |
B cell | CD19 | 0.028 | 5.36×10−1 | −0.261 | 4.33×10−9 |
CD79A | 0.053 | 2.43×10−1 | −0.228 | 3.05×10−7 | |
Monocyte | CD86 | 0.358 | 2.55×10−16 | −0.295 | 2.26×10−11 |
CSF1R | 0.415 | 5.94×10−22 | −0.304 | 4.98×10−12 | |
TAM | CCL2 | 0.394 | 1.07×10−19 | −0.258 | 5.98×10−9 |
CD68 | 0.272 | 9.12×10−10 | −0.172 | 1.24×10−4 | |
IL10 | 0.332 | 4.03×10−14 | −0.313 | 1.21×10−12 | |
M1 Macrophage | NOS2 | 0.122 | 6.55×10−3 | 0.071 | 1.16×10−1 |
IRF5 | 0.103 | 2.26×10−2 | −0.001 | 9.75×10−1 | |
PTGS2 | −0.006 | 8.96×10−1 | 0.1 | 2.67×10−2 | |
M2 Macrophage | CD163 | 0.399 | 3.28×10−20 | −0.286 | 1.02×10−10 |
VSIG4 | 0.425 | 5.06×10−23 | −0.257 | 7.42×10−9 | |
MS4A4A | 0.438 | 1.69×10−24 | −0.287 | 8.35×10−11 | |
Neutrophils | CEACAM8 | 0.001 | 9.90×10−1 | 0.034 | 4.48×10−1 |
ITGAM | 0.416 | 5.06×10−23 | −0.137 | 2.27×10−3 | |
CCR7 | 0.194 | 1.53×10−5 | −0.322 | 2.23×10−13 | |
Natural killer cell | KIR2DL1 | −0.006 | 8.93×10−1 | −0.093 | 3.81×10−2 |
KIR2DL3 | −0.01 | 8.20×10−1 | −0.136 | 2.56×10−3 | |
KIR2DL4 | −0.134 | 2.89×10−3 | −0.183 | 4.30×10−5 | |
KIR3DL1 | −0.042 | 3.48×10−1 | −0.144 | 1.35×10−3 | |
KIR3DL2 | 0.047 | 2.97×10−1 | −0.147 | 1.05×10−3 | |
KIR3DL3 | −0.045 | 3.23×10−1 | −0.086 | 5.68×10−2 | |
KIR2DS4 | 0.017 | 7.12×10−1 | −0.148 | 9.93×10−4 | |
Dendritic cell | HLA-DPB1 | 0.223 | 6.05×10−7 | −0.302 | 7.78×10−12 |
HLA-DQB1 | 0.178 | 7.19×10−5 | −0.228 | 3.28×10−7 | |
HLA-DRA | 0.194 | 1.41×10−5 | −0.299 | 1.16×10−11 | |
HLA-DPA1 | −0.209 | 7.58×10−2 | −0.52 | 2.00×10−6 | |
BDCA-1 (CD1C) | −0.01 | 9.34×10−1 | −0.58 | 5.92×10−8 | |
BDCA-4 (NRP1) | 0.455 | 5.15×10−5 | 0.065 | 5.80×10−1 | |
CD11c (ITGAX) | −0.064 | 5.88×10−1 | −0.387 | 6.55×10−4 | |
Th1 | T-bet (TBX21) | 0.166 | 1.59×10−1 | −0.617 | 4.68×10−9 |
STAT4 | 0.133 | 2.64×10−1 | −0.496 | 7.06×10−6 | |
STAT1 | 0.291 | 1.26×10−2 | 0.05 | 6.69×10−1 | |
IFN-γ (IFNG) | 0.092 | 4.37×10−1 | −0.421 | 1.85×10−4 | |
TNF-α (TNF) | 0.112 | 1.03×10−1 | −0.467 | 2.7×10−5 | |
Th2 | GATA3 | 0.248 | 2.63×10−8 | −0.227 | 3.63×10−7 |
STAT6 | 0.056 | 2.12×10−1 | 0.069 | 1.26×10−1 | |
STAT5A | 0.188 | 2.81×10−5 | −0.132 | 3.28×10−3 | |
IL13 | 0.071 | 1.15×10−1 | −0.154 | 5.78×10−4 | |
Tfh | BCL6 | 0.192 | 1.03×10−1 | 0.25 | 3.19×10−2 |
IL21 | NA | NA | NA | NA | |
Th17 | STAT3 | 0.124 | 2.96×10−1 | 0.062 | 5.98×10−1 |
IL17A | 0.236 | 4.42×10−2 | −0.238 | 4.09×10−2 | |
Treg | FOXP3 | 0.312 | 7.25×10−3 | 0.009 | 9.37×10−1 |
CCR8 | 0.129 | 2.77×10−1 | −0.33 | 4.11×10−3 | |
STAT5B | 0.008 | 9.44×10−1 | −0.036 | 7.59×10−1 | |
TGFβ (TGFB1) | 0.49 | 1.08×10−5 | −0.263 | 2.35×10−2 | |
T cell exhaustion | PD-1 (PDCD1) | 0.109 | 3.58×10−1 | −0.581 | 5.89×10−8 |
CTLA4 | 0.206 | 8.04×10−2 | −0.504 | 4.78×10−6 | |
LAG3 | 0.195 | 9.89×10−2 | −0.259 | 2.61×10−2 | |
TIM-3 (HAVCR2) | −0.001 | 9.94×10−1 | −0.552 | 3.34×10−7 | |
GZMB | 0.062 | 6.05×10−1 | −0.406 | 3.33×10−4 |
None, correlation without adjustment.
Purity, correlation adjusted by purity.
Cor, R value of Spearman's correlation. TAM, tumor-associated macrophage; Th, T helper cell; Tfh, follicular helper T cell; Treg, regulatory T cell; NA, not available in database.