High CTHRC1 expression may be closely associated with angiogenesis and indicates poor prognosis in lung adenocarcinoma patients
This study aimed to investigate the prognostic value of the potential biomarker collagen triple helix repeat containing 1 (CTHRC1) in lung adenocarcinoma (LUAD) patients.
A total of 210 LUAD patients diagnosed between 2003 and 2016 in the Department of Pathology of the First Affiliated Hospital of Sun Yat-sen University were included in this study. The expression of CTHRC1 and vascular endothelial growth factor (VEGF), and microvessel density (MVD, determined by CD34 immunostaining) were evaluated by immunohistochemistry in LUAD tissues. The association between the expression of these proteins and clinicopathological features or clinical outcomes was analyzed.
Here, we confirmed that CTHRC1 expression was associated with prognosis and can serve as a significant predictor for overall survival (OS) and progression-free survival (PFS) in LUAD. Additionally, we observed that CTHRC1 expression was positively associated with tumor angiogenesis markers, such as VEGF expression (P < 0.001) and MVD (P < 0.01). Then, we performed gene set enrichment analysis (GESA) and cell experiments to confirm that enhanced CTHRC1 expression can promote VEGF levels. Based on and cox regression analysis, a predictive model that included CTHRC1, VEGF and MVD was constructed and confirmed as a more accurate independent predictor for OS (P = 0.001) and PFS (P < 0.001) in LUAD than other parameters.
These results demonstrated that high CTHRC1 expression may be closely related to tumor angiogenesis and poor prognosis in LUAD. The predictive model based on the CTHRC1 level and tumor angiogenesis markers can be used to predict LUAD patient prognosis more accurately.
KeywordsLung adenocarcinoma (LUAD) Collagen triple helix repeat containing 1 (CTHRC1) Vascular endothelial growth factor (VEGF) Microvessel density (MVD) Tumor angiogenesis
collagen triple helix repeat containing 1
vascular endothelial growth factor
non-small cell lung cancer
formalin-fixed and paraffin-embedded
receiver operating characteristic
gene set enrichment analysis
molecular signatures database
The Cancer Genome Atlas
normalized enrichment score
- NOM P value
areas under the curve
pancreatic ductal adenocarcinoma
quantitative real-time PCR
green fluorescent protein
vascular endothelial growth factor A
anti-vascular endothelial growth factor
human umbilical vein endothelial cells
extracellular-signal-regulated protein kinase
c-Jun N-terminal kinase
hypoxia-inducible factor 1α
protein kinase B
mammalian target of rapamycin
hepatitis B virus
According to the most recently reported global cancer statistics, lung cancer remains the leading cause of cancer-related deaths [1, 2, 3] and the mortality rate is 18.4% . Lung adenocarcinoma (LUAD), the most commonly diagnosed histologic subtype of lung cancer , causes more than 500,000 deaths globally each year [5, 6]. Currently, the adenocarcinoma subtype of NSCLC had more effective responses to recently developed targeted therapies such as pemetrexed, gefitinib, bevaciuzumab, and crizotinib than the non-adenocarcinoma subtype [7, 8, 9]. Despite improvements in chemotherapeutic interventions and surgical resection, the prognosis for patients with LUAD remains poor, with a dismal 5-year survival rate of 16% , which is due to the high occurrence of tumor recurrence and distant metastases [10, 11]. Accordingly, the discovery of effective prognostic and diagnostic biomarkers in early stage will have a significant impact on further improving treatment outcomes in LUAD.
Collagen triple helix repeat containing 1 (CTHRC1) is a chondrocyte-specific, secreted glycoprotein that was originally discovered in a rat model of balloon-injured arteries [12, 13, 14, 15, 16, 17]. The overexpression of CTHRC1 is frequently detected in several solid tumors, such as melanoma, breast ductal carcinoma, gastric cancer, hepatocellular carcinoma and colorectal cancer [18, 19, 20, 21, 22]. According to our previous studies, CTHRC1 overexpression was significantly correlated with metastasis in patients with non-small cell lung cancer (NSCLC) . However, more evidence still needed to determine the prognostic value of CTHRC1 in LUAD, which is the most common subtype of lung cancer.
Angiogenesis, the process in which capillaries sprout from pre-existing vessels, has long been regarded as the principal mechanism of tumor vascularization, throughout cancer occurrence, growth, migration, invasion and distant metastasis in lung cancer [24, 25, 26]. Notably, Pyagay et al.  found that CTHRC1 expression cannot be detected in normal arteries but is only transiently expressed in injured arteries. Additionally, it was reported that the overexpression of CTHRC1 can significantly promote tumor angiogenesis in pancreatic tumors and gastrointestinal stromal tumors [27, 28]. However, the relationship between CTHRC1 expression and tumor angiogenesis in LUAD remains unclear.
Therefore, in the current study, we aimed to examine the association of CTHRC1 expression and tumor angiogenesis and to provide more significant evidence for its application in LUAD prognosis.
Materials and methods
Correlation of CTHRC1 and clinicopathologic characteristics in patients with LUAD
(n = 210)
(n = 137)
(n = 73)
Representative paraffin-embedded tissues were arrayed with a tissue-arraying instrument with 2.0-mm diameter core and were sectioned (4 um) for further analysis. CTHRC1 (Abcam, Cambridge, UK) was used in a 1:100 dilution [29, 30], VEGF (ZSGB-Bio, Beijing, China) and CD34 (ZSGB-Bio, Beijing, China) was used in ready to use dilution . Samples were incubated with antibodies against CTHRC1 (Abcam, Cambridge, UK), VEGF (ZSGB-Bio, Beijing, China) and CD34 (ZSGB-Bio, Beijing, China). The protocol for the IHC staining of tumor tissues from humans was described previously . Brown particles in the cytoplasm represent CTHRC1- or VEGF- positive staining. The expression intensities of CTHRC1 and VEGF were semiquantitatively evaluated according to the immunostaining intensity and positive cell distribution. The percentage of positive tumor cells was determined in at least three areas at 400× magnification and was averaged. The mean percentage was then assigned to one of five categories (Additional file 1: Fig. S1a–j): 0, no cancer cells stained; 1, 0–10% of cancer cells stained; 2, 11–50% of cancer cells stained; 3, 51–75% of cancer cells stained, 4, more than 75% of cancer cells stained. The intensity of immunostaining was scored as follows: 0, colorless; 1, tan; 2, brownish-yellow; and 3, dark brown. A weighted score was obtained by multiplying the positive cell percentage and staining intensity for each case. Microvessel density (MVD) was evaluated by the technique of Weidner et al.  and was based on the average CD34 positive cell count from IHC staining. Tumor slides were scanned first at low magnification (100×) to select three fields with the highest vascularization where the cell membrane of vascular endothelial cells was present and (or) there was brown staining, and then the microvessels were counted at high magnification (400×) (Additional file 1: Fig. S1k–o). Microvessels with a clearly defined lumen or a well-defined linear vessel shape were selected for counting and branching vessel structures were regarded as a single vessel. The mean value of three fields was considered as the microvessel density (MVD) for each case. Based on the receiver operative characteristic (ROC) analysis, the optimal cutoff value of CTHRC1, VEGF and MVD was confirmed: a staining index of 7.5 and 5 or greater was used to define tumors with high CTHRC1 and VEGF expression, respectively, and a staining index below 7.5 or 5 was defined as low expression; an evaluation of 28.5 or greater was used to define tumors with a high MVD, while an evaluation below 28.5 was used to define tumors with a low MVD [23, 34, 35].
Gene set enrichment analysis (GSEA)
JavaGSEA-3.0 was downloaded from the official website. Before performing the analysis, we downloaded the related Molecular Signatures Database (MSigDB) and obtained microarray data from 40 LUAD patients and from the microarray data of 514 LUAD cases from The Cancer Genome Atlas (TCGA) database (https://www.cbioportal.org/, accessed September 18, 2019). These two cohorts were divided into a high-CTHRC1 group and a low-CTHRC1 group based on the level of CTHRC1 expression. GSEA was then carried out to determine the functions or pathways that showed statistically significant, concordant differences between the two groups. A positive enrichment score (ES) and a normalized enrichment score (NES) indicate that the majority of genes in this gene set were positively associated with our predefined group statuses. A normalized P-value (NOM P-value) of < 0.05 was considered statistically significant.
Primary normal lung epithelial cells (BEAS-2B) were purchased from American Type Culture Collection (ATCC) and cultured in a keratinocyte serum-free medium (Invitrogen, Carlsbad, CA) supplemented with epidermal growth factor (EGF) (Invitrogen), bovine pituitary extract, and antibiotics (100 μg/mL streptomycin and 100 U/mL penicillin). Lung adenocarcinoma cell lines (A549, GLC-82, SPC-A1, PC9, H1299 and H1975) and non-adenocarcinoma cell lines (L78 and H460) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, USA) supplemented with 10% fetal bovine serum (HyClone, San Angelo, TX, USA).
Stable clone establishment
CTHRC1 shRNA vectors (GV248, CTHRC1-shRNA, and Control shRNA) and CTHRC1-overexpression vectors (GV358, CTHRC1, and Control vector) were purchased from GeneChem (Shanghai, China), and were manipulated according to the protocol provide by the manufacturer. The transfection efficiency of the template was detected by fluorescence microscope (Axio Observer Z1, Zeiss), western blotting and quantitative real-time PCR.
Western blotting (WB)
Western blotting was performed as previously described . Targeted membranes were incubated with the antibodies for CTHRC1 (Abcam, USA), VEGFA (Abcam, USA) and Actin (Cell Signaling Technology, USA) in 5% milk/tris-buffered saline Tween-20 (TBST) at 4 °C overnight, and then were washed with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, USA) for 1 h with shaking. After enhanced chemiluminescence (ECL) (Merck Millipore, Germany) reaction, the immunoreactive bands were observed through a Gel Imaging System (Syngene, USA).
Total RNA extraction and Quantitative Real-time PCR (qRT-PCR)
Total RNA from tissue specimens and cells was extracted with TRIzol method Invitrogen, USA). RNA was reverse transcribed into cDNA using the two-step method with PrimeScript™ RT reagent kit with gDNA Eraser (Takara, China), according to the manufacturer’ s instructions. Then, qRT-PCR was performed with the SYBR® Premix Ex TaqTM kit (Takara, China), according to the manufacturer’ s protocol. The following primers were used: CTHRC1, forward 5′-TGGACACCCAACTACAAGCA-3′ and reverse 5′-GAACAAGTGCCAACCCAGAT-3′; GAPDH, forward 5′-ACCCACTCCTCCACCTTTG-3′ and reverse 5′-CTCTTGTGCTCTTGCTGGG-3′. GAPDH was used as an internal reference, with the 2−ΔΔCt method used for quantitation .
Statistical analysis was performed with SPSS 16.0 (SPSS Inc., Chicago, IL, USA). A two-tailed p-value of < 0.05 was considered statistically significant. Bonferroni correction was used to adjust the statistical significance level in multiple testing. Fisher’s exact tests, Chi-square tests, a Student’s t-test and one-way ANOVA were used for comparisons between groups. The MVD was presented as the mean ± the standard deviation (SD). The concordance rate of linear regression plots was assessed using the Pearson test. Receiver operative characteristic (ROC) analysis was performed to select the optimal cutoff value and to determine the predictive value of the factors. The Kaplan–Meier method was used to examine the overall survival (OS) and progression-free survival (PFS), and the log-rank test was used to compare curves for two or more groups. To investigate the independent prognostic factors for OS and PFS, a Cox proportional hazard regression model was applied.
Clinicopathologic patient characteristics
The clinical and histopathologic characteristics of the 210 LUAD patients are summarized in Table 1. The patient cohort consisted of 114 (54.3%) males and 96 (45.7%) females, and the mean age at diagnosis was 58.5 years (range, 33–81 years). Of the 210 patients with a known smoking status, most (79.5%) were nonsmokers. The tumor size was 2.67 ± 1.8 cm (range, 0.4–15 cm). Tumors were classified as well, moderately and poorly differentiated for 10 (4.8%), 181 (86.2%) and 19 (9.0%) patients, respectively. The number of patients with pathologic TNM stage I, II, III, and IV was 130 (61.9%), 25 (11.9%), 36 (17.1%) and 19 (9.0%), respectively.
Association between CTHRC1 expression and clinicopathologic characteristics in LUAD
Association between CTHRC1 expression and patient survival in LUAD
Univariate analysis of factors associated with overall survival and progression-free survival in LUAD patients
Multivariate analysis of factors associated with overall survival and progression-free survival in LUAD patients
Association between CTHRC1 expression and tumor angiogenesis markers
Correlation of CTHRC1 expression with VEGF expression and MVD
Low (n = 137)
High (n = 73)
Enhanced CTHRC1 expression can promote VEGF expression in LUAD in vitro
Combining CTHRC1 expression with tumor angiogenesis markers to construct a predictive model
Univariate analysis of clinicopathologic characteristics demonstrated that tumor angiogenesis markers (VEGF and MVD) were significantly associated with OS (P < 0.05; Table 2) and PFS (P < 0.05; Table 2). Based on our results described above, it is reasonable to simultaneously consider CTHRC1 expression and tumor angiogenesis markers as a predicted panel. Therefore, we established a predictive model that included CTHRC1, VEGF and MVD, and all patients were divided into three groups: patients were assigned to the low-risk group if the three biomarkers were low; to the moderate-risk group if more than one of the three biomarkers was high; and to the high-risk group if the three biomarkers were high.
CTHRC1 expression concomitant with VEGF expression and MVD predicts prognosis in LUAD
Multivariate analysis of the predictive model with clinicopathologic characteristics in LUAD patients
The predictive model
Since CTHRC1 was first identified in a screen for differentially expressed genes in balloon-injured rats , an increasing number of scientists have investigated the prognostic value of CTHRC1 in tumors. Liu et al.  found that patients with higher CTHRC1 expression exhibited a remarkably shorter OS in four different pancreatic ductal adenocarcinoma (PDAC) cohorts. Additionally, some reports have demonstrated that patients with higher CTHRC1 levels tend to have poor prognosis in many tumors, such as Wilm’s tumor, esophageal squamous cell carcinoma, colorectal cancer and cervical squamous cell carcinoma [29, 38, 39, 40]. Similarly, we have previously analyzed the relationship between CTHRC1 expression and clinicopathologic features in NSCLC and found that higher CTHRC1 expression predicted poor prognosis . However, large sample studies are scarce that focus on CTHRC1 expression and LUAD, which is the most common diagnostic subtype of lung cancer. In this study, we used a large number of clinical samples to verify the strong relationship between the CTHRC1 expression status and LUAD prognosis. Moreover, our results showed that higher CTHRC1 expression can serve as an independent predictive biomarker for poor OS and PFS in LUAD.
During tumorigenesis and development, tumor tissue is often under hypoxic and hyponutrition conditions . There is also a volume of work demonstrating that under these conditions, a large number of new vessels will form due to the activation of angiogenic factors that are secreted by cancer cells, providing the oxygen and nutrients needed for tumor growth [41, 42, 43]. VEGF has been identified as the most important factor of many angiogenic factors related to tumor growth, indicating that VEGF could be a critical target for antiangiogenic therapy [44, 45, 46]. Wei et al.  indicated that neoadjuvant bevacizumab (a humanized anti-vascular endothelial growth factor (anti-VEGF) monoclonal antibody) in combination with chemotherapy appeared to be effective and safe in patients with unresectable stage III LUAD. However, few investigations have studied the clinical applications of anti-VEGF therapy in large LUAD samples, probably because of the difficulty in the early diagnosis of LUAD. Thus, an effective predictive panel is urgently needed to increase the accuracy of early diagnosis and to promote the development of anti-VEGF therapy in LUAD.
CTHRC1 can be upregulated to promote tumor growth (in vitro and in vivo) by several mechanisms, such as the demethylation of the CTHRC1 promoter and canonical WNT signaling, and can be inhibited by a group of microRNAs to reduce cancer growth (in vitro and in vivo) in many tumors, such as gastric cancer, colorectal cancer and oral cancer [14, 18, 48, 49, 50]. However, little is known about the regulation and function of CTHRC1 in the cancer microenvironment. Recently, CTHRC1 overexpression was reported to be associated with MVD and to induce the migration and tube formation of human umbilical vein endothelial cells (HUVECs) by increasing the phosphorylation of extracellular-signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) in gastrointestinal stromal tumors . The administration of a CTHRC1-neutralizing inhibitor to a xenograft mouse model reduced the tumor burden and infiltration of Tie2-expressing monocytes (TEMs) in pancreatic tumor specimens, indicating that blocking the CTHRC1/angiopoietin-2 (Ang-2)/TEM axis during angiogenesis suppresses tumorigenesis . Specifically, Zhang et al.  discovered that CTHRC1 activated hypoxia-inducible factor 1α (HIF-1α) and VEGF by regulating the phosphoinositide-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway, and the knockdown of CTHRC1 resulted in the repression of hepatitis B virus (HBV)-associated carcinogenesis in nude mice. However, the relationship between CTHRC1 expression and tumor angiogenesis and tumor microenvironment in LUAD is still unclear. This encourages our future work to further explore the underlying mechanisms of how high CTHRC1 expression promotes angiogenesis in LUAD.
In this study, we observed that CTHRC1 expression, an independent predictor of prognosis in LUAD, was positively associated with tumor angiogenesis markers, and the GSEA data provided more evidence that gene expression related to the promotion of angiogenesis was more activated in the high-CTHRC1 group with LUAD than in the low-CTHRC1 group. Furthermore, we validated that enhanced CTHRC1 expression can promote VEGF expression in LUAD in vitro. Based on the aforementioned results, it was reasonable to combine CTHRC1 and tumor angiogenesis markers to construct a predictive model in LUAD. Additionally, we found that the constructed predictive model can be used as a more accurate prognostic panel, as it exhibited the best predictive power for OS and PFS in LUAD patients. However, the specific functional mechanism in this study remains to be studied, and we will further explore the potential mechanism between CTHRC1 expression and tumor angiogenesis in LUAD in our next study.
In summary, high CTHRC1 expression may be closely associated with angiogenesis, and the combination of CTHRC1 with angiogenesis markers in a predictive model can be used as a functional biomarker panel to predict prognosis in LUAD.
I am truly grateful for the endless support and care provided by my boyfriend Red Zhu, without which this study could not have been conducted.
Conception and design: ZK, YC, CZ; funding support: ZK; collection and assembly of data: YS, YC, YL, NJ, WJ, HW, LC, JL, YC, and KT; manuscript writing: YC. All authors read and approved the final manuscript.
This work was supported by grants from YFC (2017YFC1308800), National Natural Science Foundation of China to Zunfu Ke (30900650, 81372501, 81572260, 81773299, 81701834, 81502327, 81172232 and 31430030), and Guangdong Natural Science Foundation (2011B031800025, S2012010008378, S2012010008270, S2013010015327, 2013B021800126, 20090171120070, 9451008901002146, 2014A030313052, 2014J4100132, 2015A020214010, 2016A020215055, 201704020094, 2013B021800259, 2017B070705002, 16ykjc08 and 2015ykzd07).
Ethics approval and consent to participate
This study was approved by the institutional ethics committee of the First Affiliated Hospital of Sun Yat-sen University, and written informed consent was obtained from all patients.
Consent for publication
The authors declare that they have no competing interests.
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