CTHRC1 expression is a novel shared diagnostic and prognostic biomarker of survival in six different human cancer subtypes

CTHRC1 transcriptional expression in human cancers

By exploring the UALCAN databases, we analyzed the CTHRC1 expression across 24 different cancer tissues paired with normal samples. Our results revealed the notably elevated CTHRC1 transcriptional level in all these cancer tissues relative to normal control including head and neck squamous cell carcinoma (HNSC), kidney renal clear cell carcinoma (KIRC), liver hepatocellular carcinoma (LIHC), Lung adenocarcinoma (LUAD), stomach adenocarcinoma (STAD), and Uterine corpus endometrial carcinoma (UCEC) (Fig. 1).

CTHRC1 prognostic values in six different types of human cancers

Via KM plotter tool, next, we investigated whether CTHRC1 higher transcriptional level was associated with the OS duration of the cancer patients or not. We observed that higher CTHRC1 transcriptional level significantly (p > 0.05) reduced the OS duration of the patients HNSC (HR = 1.4, 95% CI 1.05–1.85, p = 0.018), KIRC (HR = 2.06, 95% CI 1.46–2.9, p = 2.4e − 05), LIHC (HR = 2.07, 95% CI 1.4–3.06, p = 0.00021), LUAD (HR = 1.55, 95% CI 1.14–2.12, p = 0.005), STAD (HR = 1.65, 95% CI 1.17–2.34, p = 0.004), and UCEC (HR = 1.67, 95% CI 1.08–2.57, p = 0.00021) patients (Fig. 2).

Validation of the CTHRC1 prognostic values in HNSC, KIRC, LIHC, LUAD, STAD, and UCEC using independent cohorts via OShnscc, OSkirc, OSlihc, OSluca, OSucec, and GEPIA databases

For validating the prognostic values of CTHRC1 in KIRC in HNSC, KIRC, LIHC, LUAD, STAD, and UCEC using new independent cohorts, we utilized a variety of publically accessible online tools including OShnscc (for validating the CTHRC1 prognostic value in HNSC), OSkirc (for validating the CTHRC1 prognostic value in KIRC), OSlihc (for validating the CTHRC1 prognostic value in LIHC), OSluca (for validating the CTHRC1 prognostic value in LUAD), OSucec (for validating the CTHRC1 prognostic value in UCEC), and GEPIA (for validating the CTHRC1 prognostic value in STAD). The analysis results by these tools have validated the findings of KM plotter and demonstrated that higher expression of CTHRC1 is associated with the reduced OS duration of the, HNSC (HR = 1.4926, 95% CI 0.9557–2.3311, p = 0.0483), KIRC (HR = 0.3888, 95% CI 0.1098–1.3764, p = 0.043), LIHC (HR = 1.411, 95% CI 1.0286–1.9356, p = 0.0328), LUAD (HR = 1.4006, 95% CI 0.4987–3.934, p = 0.5225), STAD (HR = 1.4, 95% CI p = 0.049), and UCEC (HR = 0.7251, 95% CI 0.3612–1.4555, p = 0.0365) (Fig. 3). Taken together the results of CTHRC1 prognostic value analysis, it was observed that the higher expression level of SCTHRC1 is vital in the tumorgenesis of the HNSC, KIRC, LIHC, LUAD, STAD, and UCEC. Therefore the next part of our study will mainly focus on the unique role of CTHRC1 in these six types of human cancers.

Correlation between CTHRC1 transcriptional expression level and different clinicopathological features of HNSC, KIRC, LIHC, LUAD, STAD, and UCEC

Using UALCAN and KM plotter tool, transcriptional expression analysis of CTHRC1 and its correlation analysis with the OS duration of the cancer patients showed that CTHRC1 level was significantly (p > 0.05) elevated and associated with the reduced OS duration of the HNSC, KIRC, LIHC, LUAD, STAD, and UCEC patients. Therefore, we next also explored the correlation between CTHRC1 expression level and different clinicopathological parameters of HNSC, KIRC, LIHC, LUAD, STAD, and UCEC patients. Results of the analysis revealed that CTHRC1 also significantly (p > 0.05) overexpressed in HNSC, KIRC, LIHC, LUAD, STAD, and UCEC patients of different clinicopathological characteristics stratified by cancer staging (stage 1, 2, 3, and 4), race grouping (Caucasian, African-American, and Asian), gender grouping (male and female), and age grouping (20–40 years, 41–60 years, 61–80 years, and 81–100 years) (Figs. 4, 5, 6, 7, 8, 9). Clinicopathological features based distribution of the HNSC, KIRC, LIHC, LUAD, STAD, and UCEC cohorts are given in Table 1.

Transcription expression level validation of CTHRC1 using independent HNSC, KIRC, LIHC, LUAD, STAD, and UCEC cohorts

To further validate the transcription expression level of CTHRC1, we re-analyze its expression using independent cohorts of HNSC, KIRC, LIHC, LUAD, STAD, and UCEC from Affymetrix U133A and U133Plus2 microarray platforms via GENT2 platform. The results of re-analysis also revealed its significant (p > 0.05) overexpression in HNSC, KIRC, LIHC, LUAD, STAD, and UCEC patients relative to normal controls (Fig. 10). Information of the HNSC, KIRC, LIHC, LUAD, STAD, and UCEC datasets used for CTHRC1 expression validation is given in Table 2.

Translational expression level of CTHRC1 in head and neck, kidney, liver, lung, stomach, and endometrial cancers

We also analyzed the CTHRC1 translational level in normal and head and neck, kidney, liver, lung, stomach, and endometrial cancers tissues using HPA. The obtained images from HPA revealed that CTHRC1 protein was not expressed or detected at a low level in head and neck (not expressed), kidney (not expressed), liver (low), lung (not expressed), and stomach (low), and endometrial normal tissues. However, its overexpression (medium) was detected in cancer tissues of the head and neck, kidney, liver, lung, stomach, and endometrial (Fig. 11).

CTHRC1 promoter methylation, genetic alterations, and CNVs analysis in HNSC, KIRC, LIHC, LUAD, STAD, and UCEC

Promoter methylation is a key epigenetic mechanism that regulates transcription gene expression and plays a vital role in tumorgenesis²⁷. Therefore, we further analyzed that whether CTHRC1 transcription expression level was influenced by its promoter methylation level or not using UALCAN. As highlighted in Fig. 12, the box plots indicated that CTHRC1 transcription expression level was significantly (p > 0.005) influenced by its promoter hypomethylation in HNSC and UCEC, therefore, we speculate that the up-regulation of CTHRC1 in HNSC and UCEC might be the outcome of its promoter hypomethylation. However, the observed significant hypermethylation of CTHRC1 promoter in KIRC, LIHC, LUAD, and STAD challenges the classical view of methylation where overexpression is always associated with hypomethylation. Therefore, further detailed work is required to be done to explore the connection between hypermethylation of CTHRC1 promoter and its expression in KIRC, LIHC, LUAD, and STAD.

In addition to promoter methylation, we also analyzed the contribution of genetic alterations and CNVs in the up-regulation of CTHRC1 via cBioPortal webserver using PanCancer Atlas datasets of HNSC, KIRC, LIHC, LUAD, STAD, and UCEC from TCGA. Our results revealed that CTHRC1 was genetically altered in only 4%, 0.6%, 9%, 4%, 5%, and 6% of the queued HNSC, KIRC, LIHC, LUAD, STAD, and UCEC samples, respectively, and deep amplification genetic abnormality was most frequent in these cancer subtypes (Fig. 13). Altogether, these results suggested that deep amplification may also participate in the overexpression of CTHRC1 in HNSC, KIRC, LIHC, LUAD, STAD, and UCEC.

Pathway enrichment analysis of CTHRC1

To explore the CTHRC1 enriched pathways, a protein–protein interaction (PPI) network of CTHRC1 associated genes was obtained using the STRING database and visualized through Cytoscape. In total 11 nodes and 138 edges were found in the obtained PPI network (Fig. 13A). Using David tool, we revealed that CTHRC1-related genes were involved in seven different pathways including Basal cell carcinoma, Melanogenesis, Wnt signaling pathway, Signaling pathways regulating pluripotency of stem cells, Hippo signaling pathway, Proteoglycans in cancer, and Notch signaling pathway (Fig. 14; Table 3).

CD8+ T immune cells infiltration and tumor purity of CTHRC1 in HNSC, KIRC, LIHC, LUAD, STAD, and UCEC patients

Tumor development is closely associated with immunity²⁸. As CD8+ T immune cells are the major components of the immune system, we evaluated the Spearman correlation between CTHRC1 expression and CD8+ T immune infiltration in HNSC, KIRC, LIHC, LUAD, STAD, and UCEC via TIMER database. Results of this analysis suggested a significant (p > 0.005) negative correlation between CD8+ T immune cells level and CTHRC1 higher expression in HNSC, KIRC, LUAD, and UCEC while a significant (p > 0.005) positive correlation between these two parameters in LIHC and STAD (Fig. 15). Collectivly these results highlighted a significant relationship between the CTHRC1 expression and CD8+ T immune cells infiltration. In addition, we have also explored the correlation between CTHRC1 expression and tumor purity in HNSC, KIRC, LIHC, LUAD, STAD, and UCEC using TIMER. Analysis results showed that CTHRC1 expression level was negatively related to the tumor purity in HNSC (Rho = − 0.05, p = 2.13e − 01), KIRC (Rho = − 0.192, p = 2.32e − 05), LIHC (Rho = − 0.367, p = 1.91e − 12), LUAD (Rho = − 0.21, p = 2.48e − 06), STAD (Rho = − 0.135, p = 8.25e − 03), and UCEC (Rho = − 0.07, p = 2.30e − 01) (Fig. 15).

Gene–drug interaction network analysis of the CTHRC1

In order to explore the relationship between CTHRC1 and available cancer therapeutic drugs, a gene–drug interaction network was developed using the CTD database. The expression of CTHRC1 could potentially influence by a variety of drugs. For example, cyciosperine and dicrotophos could elevate the expression level of CTHRC1 while valporic acid and doxorubicin could reduce CTHRC1 expression level (Fig. 16).