Elevated serum DAND5 is associated with metastasis and predicts poor prognosis in colorectal cancer

Abstract

Background

The biologic and clinical significance of DAND5 remains unknown in colorectal cancer (CRC).

Objective

Herein, we investigated the function of DAND5 and evaluated its clinical significance in both serum and matched primary tumors in patients with CRC.

Methods

The role of DAND5 was explored in CRC cells and clinical significance of DAND5 was investigated in CRC patients (n = 217) and healthy controls (n = 63).

Results

Knockdown of DAND5 significantly decreased CRC cell proliferation, migration and invasion partly associated with epithelial–mesenchymal transition phenotype. Serum DAND5 levels in CRC were significantly higher than in normal controls and accurately distinguished CRC from healthy subjects. High serum DAND5 levels were significantly correlated with tumor differentiation, large tumor size, advanced Tumor Node Metastasis (TNM) stage, lymph node and liver metastasis, high carcinoembryonic antigen level, recurrence, poor overall and disease-free survival. Serum DAND5 level, together with lymph node metastasis, were independent prognostic factors for CRC patients. High DAND5 protein expression in CRC tissues was increased according to TNM stage. A significant positive correlation existed between serum DAND5 levels and matched DAND5 expression in CRC tissues.

Conclusion

Our data provide novel evidence for the clinical significance of DAND5 as a potential biomarker for CRC prognosis.

Keywords

DAND5 colorectal cancer prognosis metastasis biomarker

Introduction

Colorectal cancer (CRC) is one of the most common cancers in developed countries and remains a serious public health problem.¹ Survival rates of patients with CRC have increased in the past few years, possibly as a result of earlier diagnosis and improved treatment regimens; nonetheless, approximately 30–50% of patients who undergo curative resection subsequently experience local tumor recurrence or metastasis.² The incidence of disease recurrence is 25% in the absence of regional node involvement, suggesting that conventional pathology may fail to detect occult nodal metastasis.^3,4 Therefore, identification of CRC patients at a higher risk of developing metastatic disease might enable us to stratify and select the candidate patients who need standard (5-FU) or intensive (FOLFOX) adjuvant chemotherapy, which is often given to prevent a possible cancer recurrence after primary treatments, such as surgery or radiation. Several CRC screening tests, including fecal occult blood test and colonoscopy, have been available for years.⁵ However, compliance with these tests has been far from adequate. Classical clinicopathologic parameters, such as Tumor Node Metastasis (TNM) staging and carcinoembryonic antigen (CEA) level, have been used for predicting outcome of cancer patients, but most of them are reported to be insensitive for accurate individual prognosis.⁶ Therefore, novel and reliable biomarkers, particularly noninvasive biomarkers in serum, for accurate identification of patients at higher risk of developing metastatic CRC disease are urgently needed.

Recently, several potential molecular markers have been reported to diagnose the existence of CRC. For instance, Toiyama et al. showed that serum ANGPTL2 was a novel diagnostic and recurrence-predictive biomarker in CRC patients.⁷ Fan et al. demonstrated that p-p38 was a negative independent prognostic factor for CRC. Complementing TNM staging with p-p38 might refine the risk definition more accurately for a subset of patients.⁸ One recent study provided evidence that RacGAP1 could serve as a potential biomarker for identifying patients with lymph node metastasis and poor prognosis in CRC.⁹ DAND5 (also known as Coco) belongs to the DAN (differential screening selected gene aberrative in neuroblastoma) family of secreted proteins.^10,11 Gao and colleagues,¹² using a gain-of-function cDNA screen, demonstrated that DAND5, a secreted antagonist of TGF-β ligands, induced dormant breast cancer cells to undergo reactivation in the lung. Mechanistic studies indicated that DAND5 was a particularly potent mediator of metastatic reactivation and that it exerted its effect by inhibiting lung-derived bone morphogenetic protein (BMP) ligands. In particular, DAND5 is the only BMP inhibitor whose expression correlates with lung metastatic capacity. Overexpression of DAND5 activates dormant metastatic 4T07 cells, whereas the knockdown of DAND5 induces the majority of 4T1 cells to enter a state of dormancy. Recently, Chi et al.¹³ showed that positive DAND5 staining cases displayed a higher occurrence of breast cancer events. DAND5-positive patients exhibited generally poor disease-free survival. Moreover, high serum DAND5 levels were associated with increased risk of breast cancer events and poor disease-free survival. These findings suggest that DAND5 is linked with poor prognosis and may act as a promising predictor for breast cancer. However, the functional role and clinical significance of DAND5 in CRC remains largely unknown.

Accordingly, the aim of this study was to investigate the function of DAND5 and evaluate the clinical significance of DAND5 in both serum and matched primary tumors in CRC.

Methods

Patients and specimens

This study is based on a retrospective design. For the DAND5 serum enzyme-linked immunosorbent assay (ELISA), serum samples were obtained from consecutively enrolled CRC patients (n = 217) and age-matched healthy volunteers (n = 63). The samples from healthy controls were obtained when they underwent routine body examination in Wuxi People’s Hospital. For the immunohistochemistry (IHC) assay, 217 paraffin-embedded primary CRC tissues and their matched normal tissues were collected from January 2010 to February 2015. All the specimens were collected from patients with CRC undergoing surgery in Wuxi People’s Hospital. None of the patients had received preoperative chemotherapy or radiotherapy. All patients were postoperatively classified based on histopathological analysis by using the Union for International Cancer Control TNM staging system. There were 57 patients with TNM stage T1, 61 patients with TNM stage T2, 56 patients with TNM stage T3 and 43 patients with TNM stage T4 cancer. For postoperative oncological follow-up, patients received laboratory tests and radiological examination to monitor local recurrence and/or distal metastasis. Exclusion criteria included inflammatory bowel disease, familial adenomatous polyposis, hereditary nonpolyposis colon cancer, or other rare and complex types of tumors. Patients who had incomplete follow-up records were also excluded in our study. The study was conducted with the approval of the ethics committee of Wuxi People’s Hospital. Written informed consent from patients for using their tissue specimens was also obtained.

Collection of serum

Peripheral venous blood samples were collected from all participants in fasting state and were gently subjected to centrifugation at 1500 × g for 15 min. Serum supernatants were collected, aliquoted, and stored at –80℃ until use. All of the samples were taken in accordance with the regulations and approval of the institutional review board of our Hospital. Serum CEA was measured with a commercial kit (ABBOTT Laboratories, CA, USA).

Measurement of serum DAND5

Serum DAND5 was measured by ELISA as described previously.¹³ Briefly, a Human DAND5 Duo Set kit for ELISA (R&D Systems, Inc., USA) was used to perform serum DAND5 ELISA assays according to the manufacturer’s protocol. The plates were coated with the DAND5 capture antibody in phosphate buffered saline (PBS). After incubation overnight at 4℃, the plates were then washed and blocked with washing buffer. Subsequently, 10 μl of standard preparations of different densities, as well as patient blood serum samples, were added to the wells, and the plate was incubated overnight at 4℃. After the plate was washed, a 100 μl solution containing the detection antibody (300 µg/ml) was added and incubated at room temperature for 2 h. The wells were washed again and then incubated with a 100 μl solution containing streptavidin-conjugated horseradish peroxidase at room temperature for 20 min. Following multiple washes, the wells were treated with 100 μl of substrate solution and incubated in the dark at room temperature for 20 min. After the addition of 50 μl of stop solution to each well, the absorbance was detected at 450 nm. The online tool “Four Parameter Logistic Fit” was used to calculate the absolute concentration of DAND5 in the blood serum samples. The intra- and inter-assay coefficients of variation were <5% and <10%, respectively. All samples were measured in triplicate.

Immunohistochemistry

Expression levels of DAND5 in postoperative paraffin-embedded tumor specimens from CRC patients were detected by IHC. The Envision and diaminobenzidine (DAB) Color Kit was purchased from Gene Tech Company Limited (Shanghai, China). The staining procedures strictly followed the supplier’s recommendation. The expression of DAND5 was evaluated according to the scoring system that we described preciously.¹⁴ Briefly, the expression of DAND5 was scored according to the immunoreactive score, which was calculated as a product of staining intensity and percentage of positive cells. Staining Intensity was divided into four grades: score 0 (negative), score 1 (weak), score 2 (moderate) and score 3 (strong). Percentage of positive cells was divided into five grades: score 0 (none), score 1 (≤10%), score 2 (11–50%), score 3 (51–80%) and score 4 (>80%). According to a previous report,¹³ for statistical analyses, the cases scoring at least 3 points in our study were designated as high expression of DAND5. Immunohistochemistry staining was scored independently by two pathologists blind to clinical data.

Cell culture

Human CRC cell lines RKO and HCT116 were purchased from American Type Culture Collection (Rockville, MD), and cultured as recommended as monolayers in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), penicillin (100 U/ml)/streptomycin (100 µg/ml)/amphotericin B (0.25 µg/ml) from Invitrogen (Carlsbad, CA) in a humidified incubator at 37℃ in a 5% CO₂ atmosphere.

EdU proliferation assay

The proliferation of RKO and HCT116 cells was examined using the Cell-Light EdU Apollo488 In Vitro Imaging Kit (RiboBio) according to the manufacturer’s protocol. Briefly, cells were incubated with 10 μM EdU for 2 h before fixation with 4% paraformaldehyde, permeabilization by 0.3% Triton X-100 and EdU staining. Cell nuclei were stained with 5 µg/ml DAPI (4’,6-diamidino-2-phenylindole) for 10 min. The number of Edu-positive cells was counted under a microscope in five random fields (×100). All assays were independently performed in triplicate.

Wound-healing assay

RKO and HCT116 cells (1 × 10⁶ cells per well) were seeded on 6-well plates. Twenty-four hours later, the cells were transfected with siRNA for DAND5. Twenty-four hours after transfection, RKO and HCT116 cells were wounded in serum-free medium, 1% bovine serum albumin (BSA) with a sterile 200 μl pipette tip to remove cells. The progress of migration was photographed (after identification of each wounded zone) in six regions, immediately and during 2 days after wounding (0 h–24 h–48 h), using an inverted microscope (Nikon TMS-F, 301655) equipped with a digital camera (Nikon Digital shot DS-L1). Cell migration was expressed as the migration rate: (original scratch width – new scratch width)/original scratch width ×100%.

Transient transfection with siRNA

siRNA for DAND5 and control siRNA were designed and synthesized by Guangzhou RiboBio (Guangzhou, China). The sequence of the negative control was also designed by RiboBio. Twelve hours prior to transfection, cells were plated onto a 6-well or a 96-well plate (Nest Biotech, Shanghai, China) at 30–50% confluence. TurboFect siRNA Transfection Reagent (Fermentas, Vilnius, Lithuania) was then used to transfect siRNA into cells according to the manufacturer’s protocol. These complexes were added to each well at a final concentration of 10 nM. The cells were incubated at 37℃ in a 5% humidified CO₂ atmosphere. Cells were collected after 48–72 h for further experiments.

Colony formation assay

Cells were plated in six-well culture plates at 200 cells/well. Each cell group had two wells. After incubation for 14 days at 37 ℃, cells were washed twice with PBS and stained with hematoxylin solution. The number of colonies containing >50 cells was counted under a microscope. The colony formation efficiency was calculated as (number of colonies/number of cells inoculated) × 100%. All assays were independently performed in triplicate.

Transwell invasion assay

The invasion assay was performed using Transwell inserts precoated with Matrigel (BD Biosciences). Briefly, cells were seeded into the upper chamber of the insert, which were suspended in serum-free culture medium. Then, medium with 10% fetal bovine serum was added into the lower chamber. After incubation for 24 h, the cells in upper chamber were carefully scraped with cotton buds. The invaded cells on the bottom surface of chambers were fixed with 4% paraformaldehyde for 20 min, and stained with Giemsa solution. The stained cells were imaged and counted in five fields with random choice.

Western blotting

Protein samples separated by SDS-PAGE were transferred to nitrocellulose membranes. After being blocked in 5% BSA (w/v) at room temperature for 1 h, the membranes were rinsed and incubated at 4℃ overnight with primary antibody. The membranes were then washed and incubated with secondary antibody at room temperature for 1 h. Signals were detected using enhanced chemiluminescence reagents (Pierce, Rockford, IL, USA). The monoclonal anti-DAND5 (dilution 1:2000) antibody was purchased from Origene (Rockville, MD, USA). The monoclonal anti-E-cadherin (dilution 1:2000), anti-Snail (dilution 1:1000) and anti-β-actin (dilution 1:2000) antibodies were purchased from Cell signaling Technology (Boston, MA, USA).

Statistical analysis

Data are presented as mean ± SD unless otherwise indicated. The data were analyzed by a two-tailed unpaired t-test. Serum DAND5 concentrations were used to draw receiver operating characteristic (ROC) curve, and the specificity, sensitivity, and area under the ROC curve (AUC) and cut-off value were determined. The AUCs were used to estimate model performance. The correlations between DAND5 expression and patient clinicopathologic parameters were assessed by Chi-square test. The survival and local recurrence curves were constructed using the Kaplan–Meier model and the log-rank test was applied to compare intergroup differences. Univariate and multivariate analyses based on the Cox proportional hazard model were performed to identify significant independent prognostic factors for CRC patients. A p-value < 0.05 was considered statistically significant. The statistical analysis was performed using SPSS statistical software.

Results

Knockdown of DAND5 expression decreased CRC cell proliferation, migration and invasion

Given that the expression of DAND5 is closely associated with breast cancer metastasis,¹⁵ we postulated that DAND5 could have an important role in CRC metastasis. It is well known that cell proliferation and migration are positively associated with metastasis; we investigated the effect of silencing DAND5 expression on CRC cell proliferation and migration. Inhibition of DAND5 expression in CRC cells was achieved by transfecting RKO and HCT116 cells with specific small-interfering RNA (siRNA)-siDAND5. The expression of DAND5 protein in siDAND5-treated RKO and HCT116 cells was significantly downregulated compared with cells transfected by control siRNA (Figure 1(a)). Further functional studies demonstrated that knockdown of DAND5 significantly decreased cell proliferation of RKO and HCT116 cells (Figure 1(b)). In addition, the cell migration ability of siDAND5-transfected RKO and HCT116 cells was remarkably decreased compared with control (Figure 1(c)).

Figure 1.

Knockdown of DAND5 expression in CRC cells decreased cell proliferation and migration. (a) The expression level of DAND5 was examined by Western blot in RKO (left panel) and HCT116 (right panel) cells treated with siDAND5 or control siRNA. Cell proliferation (b) was determined by measuring BrdU incorporation and cell migration (c) was measured by wound-healing assay in RKO and HCT116 cells transfected with siDAND5 or control siRNA. All values were expressed as mean ± SD from three independent experiments. **,p < 0.01 compared with the control group.

We further corroborated these effects by performing colony formation assays. As shown in Figure 2(a), the ability of colony formation was lower in DAND5 knockdown groups compared with controls. Moreover, we found that knockdown of DAND5 in RKO and HCT116 cells significantly decreased numbers of invaded cells through a Matrigel-coated membrane compared with controls (Figure 2(b)). Furthermore, to identify whether the role of DAND5 correlated with epithelial–mesenchymal transition (EMT) phenotype, we explored the expression of EMT markers in DAND5 knockdown cells. We found that epithelial molecule E-cadherin was significantly increased in DAND5 knockdown of RKO and HCT116 cells, whereas the expression of mesenchymal marker Snail was downregulated (Figure 2(c)). Taken together, these data indicate that knockdown of DAND5 expression suppresses cell proliferation, migration and invasion in CRC cells, and these effects are partly associated with EMT phenotype.

Figure 2.

The effect of knockdown of DAND5 expression on CRC cells colony formation, invasion and epithelial–mesenchymal transition. The colony formation assay (a) and invasion assay (b) were performed in RKO and HCT116 cells treated with siDAND5 or control siRNA. (c) Immunoblotting of E-cadherin and Snail in RKO and HCT116 cells treated with siDAND5 or control siRNA. All values were expressed as mean ± SD from three independent experiments. **, p < 0.01 compared with the control group.

Serum DAND5 levels were significantly elevated in CRC patients

Since serum DAND5 is easy to detect and could possibly serve as a serum biomarker suitable to predict the prognosis of patients with breast cancer,¹³ we evaluated the diagnostic potential of serum DAND5 levels in CRC patients. A total of 280 serum samples, including those from CRC patients (n = 217) and healthy controls (n = 63) were examined. We found that serum DAND5 levels in CRC patients were significantly elevated compared with healthy controls (Figure 3(a), p < 0.01). In addition, serum DAND5 levels increased in accordance with tumor TNM stage progression (p < 0.01) (Figure 3(b)). Significantly higher serum DAND5 levels were found in patients with lymph node metastasis as compared with patients without metastasis (Figure 3(c)). Moreover, to evaluate the potential significance of serum DAND5 as a noninvasive biomarker for the diagnosis of CRC, we performed ROC curve analysis. As shown in Figure 3(d), the AUC was 0.85 (95% confidence interval (CI), 0.79–0.89; p < 0.01). The cut-off value of DAND5 was 199.5 pg/ml with a 79.4% specificity and 74.2% sensitivity for CRC diagnosis. These data suggest that serum DAND5 level has a good capability in predicting CRC.

Figure 3.

Serum DAND5 concentrations in CRC patients and ROC curve analysis. (a) Serum DAND5 concentrations were measured by ELISA in CRC patients (n = 217) and healthy controls (n = 63). Serum DAND5 levels in 217 CRC patients subdivided by TNM staging (b) and lymph node metastasis (c). (d) Serum DAND5 concentrations in CRC patients and healthy controls were used to draw ROC curve, and the specificity, sensitivity, and the area under the ROC curve were determined. Data are presented as mean ± SD. **, p < 0.01.

Correlations between serum DAND5 levels and CRC clinicopathologic characteristics

A biomarker should have a significant relationship to clinical symptoms. To further investigate the clinical significance of DAND5 in CRC, the relationships between serum DAND5 levels and CRC clinicopathologic characteristics were statistically analyzed. As 199.5 pg/ml was the cut-off value of DAND5 for CRC diagnosis, serum DAND5 concentration higher than 199.5 pg/ml was defined as high serum level of DAND5 (n = 161, 74.2%), the others were low serum level of DAND5 (n = 56, 25.8%). As shown in Table 1, high serum DAND5 levels were significantly correlated with tumor differentiation (p < 0.01), large tumor size (p = 0.024), TNM stage (T3/T4) (p < 0.01), lymph node metastasis (p < 0.01), liver metastases (p < 0.01) and high CEA level ( > 5 ng/ml) (p < 0.01). In contrast, no statistically significant correlations were found between serum DAND5 levels and gender, age and tumor location.

Table 1.

Correlations between serum DAND5 levels and clinicopathological characteristics in CRC patients.

	Serum DAND5 levels (n = 217)
Characteristics	High (n = 161)	Low (n = 56)	p-value
Gender
male	83	30	0.85
female	78	26
Age
≤60	75	25	0.54
>60	86	31
Tumor location
colon	72	27	0.49
rectal	89	29
Tumor differentiation
Well and moderate	45	33	<0.01
poor	116	23
Tumor size
≤5 cm	51	31	0.024
>5 cm	110	25
TNM stage
T1–T2	43	34	<0.01
T3–T4	118	22
Lymph node metastasis
Absent	35	37	<0.01
Present	126	19
Liver metastases
Without	39	35	<0.01
With	122	21
CEA level (ng/ml)
≤5	23	45	<0.01
>5	138	11

Serum DAND5 levels significantly correlated with tumor progression and recurrence in CRC patients

Next, we examined whether serum DAND5 levels in CRC patients could serve as a predictor of patient outcome. The prognostic significance of serum DAND5 levels were evaluated using Kaplan–Meier survival analysis. As anticipated, patients with higher levels of serum DAND5 had significantly worse overall survival (Figure 4(a); p = 0.02). In addition, increased serum DAND5 concentrations were associated with decreased disease-free survival (Figure 4(b); p = 0.03).

Figure 4.

Kaplan–Meier curves for survival and local recurrence of CRC patients. (a and b) Patients with higher serum DAND5 levels had a worse overall survival (a) and disease-free survival (b) than those with lower serum DAND5 levels.

To identify whether high levels of serum DAND5 could serve as an independent prognostic marker for overall survival in CRC patients, we performed univariate and multivariate analysis. As shown in Table 2, the univariate analysis showed that poor overall survival in CRC patients was associated with high serum DAND5 levels (p < 0.01), large tumor size (p = 0.013), tumor differentiation (p = 0.024) and lymph node metastasis (p < 0.01). The multivariate analysis of these factors revealed that serum DAND5 levels and lymph node metastasis were significantly independent prognostic indicators for overall survival of CRC patients (p = 0.026 and p < 0.01). Moreover, univariate analysis also showed that lymph node metastasis (p = 0.027) and high serum DAND5 levels (p = 0.029) were significantly associated with disease-free survival. Multivariate analysis revealed that a high DAND5 serum level was an independent predictor for tumor recurrence for CRC patients (HR = 2.17; 95% CI = 1.06–5.84; p = 0.039; Table 2).

Table 2.

Univariate and multivariate analyses of OS and DFS in CRC patients (Cox proportional hazards regression model).

OS	Univariate analysis			Multivariate analysis
Factors	HR	95% CI	p-value	HR	95% CI	p-value
Gender (male/female)	0.96	0.28–1.45	0.16	—	—	—
Age (>60/≤60)	1.36	0.67–3.42	0.32	—	—	—
Tumor location (colon/rectal)	1.27	0.58–3.27	0.56	—	—	—
Tumor differentiation (well/poor)	2.63	1.24–4.87	0.024	1.27	0.75–3.26	0.61
Tumor size (>5/≤5 cm)	3.22	1.85–6.14	0.013	1.69	0.86–3.67	0.46
Lymph node metastasis (present/absent)	4.95	1.46–10.27	<0.01	3.65	1.62–9.92	<0.01
Serum DAND5 (high/low)	4.51	1.79–11.52	<0.01	3.06	1.58–5.64	0.026
DFS	Univariate analysis			Multivariate analysis
Factors	HR	95% CI	p-value	HR	95% CI	p-value
Lymph node metastasis (present/absent)	2.74	1.38–7.46	0.027	2.29	1.28–6.99	0.033
Serum DAND5 (high/low)	2.48	1.12–6.74	0.029	2.17	1.06–5.84	0.039

High DAND5 protein expression in CRC tissues was associated with high serum DAND5 levels

To validate the DAND5 protein expression levels in CRC tissues, IHC analysis was performed. As shown in Figure 5(a), adjacent normal tissues showed low or deficient staining of DAND5, whereas 37.3% (81/217) of CRC tumor tissues showed high IHC staining (Figure 5(b)). In addition, DAND5 staining scores were significantly increased according to TNM stage (Figure 5(c)). To further validate that circulating DAND5 levels accurately reflected concentrations found in CRC tissues, we determined the relationship between serum DAND5 levels from individual CRC patients and matched DAND5 intensity scores in CRC tissues. Notably, we found a significant positive correlation between serum DAND5 levels and matched DAND5 expression in CRC tissues (r = 0.50, p < 0.01; Figure 5(d)).

Figure 5.

DAND5 expression status in CRC tumor tissue and adjacent normal tissue. (a and b) Representative photomicrographs showing IHC analysis of DAND5 expression in adjacent normal tissue (a) and CRC tumor tissue (b), Bar = 50 µm. (c) DAND5 immunostaining scores in 217 CRC patients subdivided by TNM staging. Bar represents SD; line across the box indicates median value. (d) The correlations between serum DAND5 concentration and matched DAND5 intensity scores in CRC tissues were examined using Spearman’s correlation coefficient.

Discussion

To the best of our knowledge, this study presents the first analysis for the function and clinical significance of DAND5 in CRC. We found that knockdown of DAND5 expression by siRNA in CRC cell lines significantly decreased CRC cell proliferation and migration and invasion partly associated with EMT phenotype. We also observed that serum DAND5 levels in CRC were significantly higher than normal controls and had a high accuracy for distinguishing CRC. Previous study showed that 30.8 pg/ml was used as the positive/negative threshold of serum DAND5 in breast cancer patients.¹³ DAND5 serum concentration lower than 30.8 pg/ml was defined as DAND5 negative. In our study, 199.5 pg/ml was the cut-off value of DAND5 for CRC diagnosis, and serum DAND5 concentration higher than 199.5 pg/ml was defined as a high serum level of DAND5. High serum DAND5 levels were significantly correlated with advanced TNM stage, large tumor size, tumor differentiation, lymph node metastasis, recurrence, poor overall and disease-free survival. Moreover, we identified that serum DAND5 level together with lymph node metastasis were independent prognostic factors for CRC patients. Finally, using IHC analysis of clinical specimens, we confirmed that DAND5 protein expression in CRC tissues was higher than those in adjacent normal mucosa, and high DAND5 protein expression in CRC tissues was increased according to TNM stage. A significant positive correlation was found between serum DAND5 levels and matched DAND5 expression in CRC tissues. Collectively, these results indicate that evaluation of DAND5 in CRC patients presents a clinically promising biomarker that can facilitate disease risk assessment and severity in patients with CRC.

It has been reported that DAND5 is involved in regulating organogenesis, body patterning, and tissue differentiation.^16–18 Gao et al. demonstrated that DAND5 was involved in metastasis in breast cancer.¹² One recent report revealed that secreted DAND5 promoted tumor growth and angiogenesis in vitro and in vivo.¹³ In this study, our in vitro experiments clearly demonstrated that knockdown of DAND5 expression significantly reduced cell proliferation and migration, suggesting that aberrant expression of DAND5 could induce a more metastatic phenotype in CRC cells. Recently, molecular biomarker-guided target therapy and prognosis prediction have been proven to be critical in the individual treatment of tumor patients. Complemented with a specific biomarker, tumor patient outcome might be defined more accurately.^19,20 In addition, local recurrence and distant metastasis remain the most important causes of CRC mortality; identifying robust biomarkers to complement the current cancer surveillance approaches would provide a warning sign for early clinical intervention.

Our results are the first to demonstrate that high levels of DAND5 in both serum samples and matched primary CRC tissues are associated with advanced TNM stage and lymph node metastasis. Chi and colleagues recently demonstrated that positive DAND5 in breast cancer tissues and serum samples were associated with increased risk of disease events occurrence and poor disease-free survival.¹³ This report supports our hypothesis that serum DAND5 in patients with CRC is likely secreted by primary CRC tissues. We also showed the potential role of serum DAND5 in the diagnosis of CRC. This was supported by the relatively high sensitivity and specificity in distinguishing CRC patients from healthy subjects. Moreover, we demonstrated that high DAND5 protein expression in CRC tissues was increased according to TNM stage. A significant positive correlation was found between serum DAND5 levels and matched DAND5 expression in CRC tissues.

Prognostic biomarkers for CRC patients are critical because they can provide useful information for clinical management. For example, prognostic biomarkers may be helpful for identifying patients who are likely to undergo recurrence, leading to a more precise therapeutic strategy.¹⁴ Currently, adjuvant chemotherapy provides significant survival benefit in Stage III CRC patients, it is controversial whether this treatment has any effect on patients with Stage II disease. Adjuvant chemotherapy was shown to increase the survival of certain populations of Stage II patients.²¹ Therefore, identification of high-risk patients among those with Stage II and III CRC would be of great benefit in selecting appropriate candidates for standard (5-FU) or intense (FOLFOX) adjuvant therapy. In the present study, our results showed that CRC patients with high serum DAND5 levels had a poorer overall and disease-free survival than patients with low serum DAND5 levels. Another important finding of our study was that lymph node metastasis and high serum DAND5 levels were significantly associated with disease-free survival, and high serum DAND5 levels were independent predictors for tumor recurrence for CRC patients. These results suggest that DAND5 detection combined with classic clinicopathological parameters might be beneficial to prognosis evaluation and personalized therapy of CRC.

In conclusion, this is the first study to demonstrate the clinical significance of DAND5 expression in CRC. Our results demonstrated that DAND5 levels were elevated in CRC patients. Serum DAND5 levels were significantly associated with pathological development of CRC. In addition, we found high serum DAND5 levels were correlated with recurrence and could serve as an independent prognostic factor for CRC patients. CRC patients with high serum DAND5 levels had worse overall and disease-free survival, suggesting that identification of serum DAND5 level is potentially helpful in identifying patients who need adjuvant chemotherapy and strict surveillance. Collectively, our results provide novel evidence for clinical significance of DAND5 as a potential biomarker for CRC prognosis. However, future larger prospective cohort studies are needed to further evaluate the clinical utility and prognostic use of DAND5 in CRC patients. It would also be interesting to see the influence of chemotherapeutic drugs on DAND5 expression in future studies.

Footnotes

Acknowledgements

Contributors: Xiaofei Miao, Ye Zhang, Jialin Sun, Songkui Cui, Qingyang Meng, Kuiyu Zhu, Xingqian Hu: acquisition of data or analysis and interpretation of data, Approved the version to be published; concept and design, drafted the article or revised it critically for important intellectual content, approved the version to be published: Xiaofei Miao, Ye Zhang, Tong Wang.

Funding

This work was supported by grants from the National Natural Science Foundation of China (No.81371683/H1819, 11104317), Medical Technology Major Projects of Wuxi Hospital Center (YGZX1105, YGZXM14044) and Clinical Medicine Project of Jiangsu Province (BL2014023).

Conflict of interests

The authors declare that there are no conflicts of interest.

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