Abstract
Purpose
Metastasis is a primary cause of colorectal cancer (CRC)-related death, and cancer stem cells (CSCs) are thought to be majorly responsible for initiating metastatic behaviors. Doublecortin-like kinase 1 (DCLK1) was recently discovered to be a marker for gastrointestinal CSCs. Here, we aimed to explore whether DCLK1 is associated with CRC metastasis through clinical and in vitro investigations.
Methods
The expression levels of DCLK1 mRNA and protein in human CRC tissues were analyzed through quantitative RT-PCR and immunohistochemistry staining, respectively. Human CRC cell line SW480 was selected to explore the effect of DCLK1 overexpression on cell migration and invasion. Besides, the associations between DCLK1 and epithelial–mesenchymal transition (EMT) were determined.
Results
Compared to normal colorectal tissues, DCLK1 expression was significantly up-regulated in human CRC tissues and correlated well with high lymphatic metastasis and poor prognosis in patients. DCLK1 expression was inversely associated with overall survival in CRC patients. Overexpression of DCLK1 in SW480 cells markedly promoted cell migration and invasion. Furthermore, we validated that DCLK1 could facilitate EMT in cancer cells by up-regulation of the mesenchymal markers Vimentin and ZEB1 and down-regulation of the epithelial marker E-cadherin in SW480 cells.
Conclusions
DCLK1 up-regulation may play a contributory role in CRC metastasis and poor prognosis via activation of EMT. DCLK1 may serve as an independent predictor for CRC prognosis.
Keywords: DCLK1, Colorectal cancer, Metastasis, Prognosis, Epithelial–mesenchymal transition
Introduction
Colorectal cancer (CRC) ranks the third most common cancer in the world (Schreuders et al. 2015). In the USA, the estimated number of invasive CRC cases has been about 140,000 and the mortality data have reached 50,000 per year (Siegel et al. 2016). Of note, around 90 % of CRC-related mortalities are attributed to metastases (Christofori 2006), whereas approximately half of CRC patients develop distant metastases, especially liver metastasis (Stein and Schlag 2007). CRC metastasis is a complex process that consists of multiple sequential steps, including the invasion of cancer cells into surrounding tissues, intravasation, survival in circulation, arrest at distant organ sites, extravasation, and growth in distant organs (Chaffer and Weinberg 2011). So far, the molecular mechanisms underlying cancer metastasis remain obscure.
Doublecortin-like kinase 1 (DCLK1) is one kind of microtuble-associated proteins (MAPs), which consists of an N terminus that is 65 % similar to doublecortin (DCX) and an additional 360 amino acid C-terminal domain encoding a putative Ca2+-/calmodulin-dependent protein kinase (Lin et al. 2000). DCLK1 is predominantly expressed in the lower two-thirds of intestinal crypt epithelium and in occasional crypt-based columnar cells (May et al. 2009). Recently, it has been noted that cancer stem cells (CSCs) are mainly responsible for relapse of tumor and initiation of metastatic behaviors (Kantara et al. 2015). Notably, many studies have disclosed that DCLK1 specifically marks colorectal CSCs (Gagliardi et al. 2012). It has been reported that siRNA-mediated knockdown of DCLK1 or application of small molecule kinase inhibitors for DCLK1 could result in tumor growth arrest (Sureban et al. 2011, 2014; Weygant et al. 2014). It therefore indicates that DCLK1 may be involved with cancer metastasis and may serve as a potentially therapeutic target for suppressing cancers.
In this study, we aimed to explore the clinical implication of DCLK1 in CRC and the underlying mechanisms through in vitro investigations. We found that DCLK1 expression was not only markedly up-regulated in human CRC tissues but also correlated well with cancer metastasis and poor prognosis. We further showed that overexpression of DCLK1 in CRC cell line SW480 could promote cell migration and invasion. Moreover, DCLK1 overexpression could activate EMT in the cells, which was likely one of the mechanisms underlying its involvement with metastasis.
Materials and methods
Tissue specimens
A total of 101 paraffin-embedded human colorectal specimens were obtained from patients treated at Beijing Chao-Yang Hospital, Capital Medical University, Beijing, China, from 2007 to 2011. The specimens included 16 normal colorectal epithelium tissues, 71 primary colorectal cancer tissues, and 14 lymphatic metastases. In addition, a total of 38 human primary CRC tissues were freshly collected after surgical resection from March 2013 to November 2013. The patients were staged according to the tumor node metastasis (TNM) staging system of the American Joint Committee on Cancer (AJCC) (O’Connell et al. 2004). The patients included in this study had a negative medical history for any other malignant diseases. All patient tissues were taken before chemical and radiation therapies. This study was approved by the Clinical Research Ethics Committee of Beijing Chao-Yang Hospital, Capital Medical University, Beijing, China. Informed consent was obtained from each patient.
Detection of mRNA levels and DCLK1 protein in patients
For determination of DCLK1 mRNA levels, total RNA was isolated from 38 human fresh colorectal tissues using TRIzol extraction method. Concentration and purification of RNA were determined spectrophotometrically by measuring its optical density (A260/280 > 2.0, A260/230 > 1.8) using NanoDrop ND-2000 Spectrophotometer (Thermo Scientific Wilmington, DE, USA). cDNA was synthesized using 1 μg total RNA. Real-time PCR analyses were conducted to quantify mRNA expression using SYBR Premix (Applied Biosystems) with GAPDH as an internal control. The sequences of the primers were as follows: DCLK1 forward, 5′-TTTGACCCGAACTCTGTCGG-3′, DCLK1 reverse, 5′-GTGAAGACACTGCCCGAGAA-3′; GAPDH forward, 5′-AATCCCATCACCATCTTCCA-3′, GAPDH reverse 5′-TGGACTCCACGACGTACTCA-3′. Real-time PCR was performed using the Applied Biosystems 7500 Sequence Detection System.
Immunohistochemical staining was performed to assess DCLK1 protein levels in human tissues. In brief, formalin-fixed, paraffin-embedded colorectal tissues were cut into 5-μm sections by microtome. A rabbit polyclonal anti-DCLK1 antibody (Abgent) was used for staining all samples. Firstly, we performed heat-induced epitope retrieval using a pressure cooker. Then, the primary antibody was incubated overnight at 4 °C. DCLK1 staining intensity in tissue samples was evaluated by 2 independent observers to assign scores of both percentage of positive tissues 0 (<5 %), 1 (5–25 %), 2 (25–50 %), 3 (51–75 %), and 4 (>75 %) and staining intensity (0 = none, 1 = mild, 2 = moderate, 3 = strong). The two scores were combined to calculate the staining index (SI), which generated scores of 0, 1, 2, 3, 4, 6, 9, and 12. According to the log rank test results, immunostaining for DCLK1 was considered to be a high expression when SI was equal or greater than 4 and low expression with a SI score of 0–3.
Cell culture and assessment of DCLK1 mRNA and protein
For human colorectal cancer cell lines, HCT116, HT29, SW480, SW620, and RKO were obtained from the American Type Culture Collection (ATCC). HCT116 and HT29 were cultured in McCoy’s 5A basic medium (Gibco, USA). SW480 and SW620 were cultured in Leibovitz’s L-15 basic medium (Gibco, USA). RKO was cultured in Dulbecco’s modified Eagle’s medium (Gibco, USA). All of these media were supplemented with 10 % fetal bovine serum (FBS) (Gibco, USA). The cells were maintained under the conditions recommended by the ATCC.
Total RNA was individually isolated from these cell lines, and DCLK1 mRNA expression was quantified as described above. Moreover, equal amounts of cell lysate were subjected to western blot analysis. The concentration of total proteins was determined by BCA protein assay. The proteins were separated on a 10 % SDS-polyacrylamide gel and transferred onto a PVDF membrane. The membrane was blocked in 5 % nonfat dry milk for 1 h and incubated overnight with the primary antibodies. Subsequently, the membrane was incubated with secondary antibody for 1 h at room temperature. The proteins were detected using a LICOR Odyssey Infrared Imager. Protein density quantification was performed in Image Studio Lite (LICOR). The following primary antibodies were used: anti-DCLK1 (Abgent) and anti-β-actin (Cell Signaling Technology, USA).
Vector construction and cell transfection
The DCLK1 overexpression plasmid was generated by cloning the complete DCLK1 CDS into OriGene’s pCMV6-Entry Vector. The empty vector was used as a control. To establish a cell line that stably expressed DCLK1, the DCLK1 overexpressing and empty vectors were transfected into SW480 cells, respectively, and the cells were selected with G418 (Invitrogen, USA) for 20 days. Transfection was performed according to the manufacturer’s protocol.
Wound-healing assay
The DCLK1 and empty vector transfected SW480 cells (2 × 105) were, respectively, seeded into 6-well plates and allowed to grow to confluence. A sterile 200-μl pipette tip was used to scratch the confluent layer, and the detached cells were removed with washing. For each plate, 9 points were selected and marked, and the baseline images were taken at 10× magnification. Images were again taken at 24 and 48 h after wounding. The empty region between migrating monolayer edges was selected, and the width was measured.
Matrigel transwell invasion assay
Control or Matrigel-coated transwells (BD Biosciences) were prepared by soaking in serum-free media for 2 h at 37 °C in a 24-well plate. Subsequently, the DCLK1 and the control vectors transfected SW480 cells (5000/well) were seeded into each transwell in serum-free media. Leibovitz’s L-15 Cell culture medium containing 10 % FBS was added to the bottom of each well as chemoattractant, and the cells were incubated for 24 h. Afterward, a cotton swab was used to scrape noninvasive/migratory cells off the top of transwells, and the remaining cells were fixed with 100 % methanol, stained with 1 % toluidine blue/1 % borax, and allowed to dry. After drying, the film from the transwell inserts was removed with a scalpel blade and mounted on slides. For each sample, 5 fields were counted at 10× magnification. Percent invasion was calculated by dividing the number of invading cells (Matrigel-coated inserts) by the number of migrating cells (control inserts) and multiplying by 100.
Assay for mRNA and protein levels of EMT markers
First, total RNA was isolated from DCLK1 or the empty vector transfected SW480 cells, and the mRNA expression of EMT markers (E-cadherin, Vimentin, and ZEB1) was quantified using SYBR Premix (Applied Biosystems) with GAPDH as an internal control, as described above. The sequences of the primers for EMT markers were as follows: E-cadherin forward, 5′-CGAGAGCTACACGTTCACGG-3′, E-cadherin reverse, 5′-GGGTGTCGAGGGAAAAATAGG-3′; Vimentin forward, 5′-GGACCAGCTAACCAACGACA-3′, Vimentin reverse, 5′-AAGGTCAAGACGTGCCAGAG-3′; ZEB1 forward, 5′-AAGAATTCACAGTGGAGAGAGAAGCCA-3′, ZEB1 reverse, 5′-CGTTTCTTGCAGTTTGGGCATT-3′. Likewise, equal amounts of cell lysate were subjected to western blot analysis as describe above. The following primary antibodies were used: anti-E-cadherin, anti-Vimentin, anti-ZEB1, and anti-β-actin (Cell Signaling Technology, USA).
cBioPortal analysis of TCGA dataset
The ‘‘Colorectal Adenocarcinoma (TCGA, Provisional)’’ dataset that contained 631 total cases was queried. Genetic and transcriptional alterations in DCLK1 and the correlations between DCLK1 mRNA and Vimentin and ZEB1 mRNAs were analyzed using cBioPortal, a tool developed by the Computations Biology Center at Sloan Kettering, which was accessed at http://www.cbioportal.org/index.do (Cerami et al. 2012; Gao et al. 2013).
Statistical analysis
Statistical analysis was performed using a SPSS software package (SPSS Standard version 19.0, SPSS Inc). Differences in DCLK1 expression between human tissues were analyzed using one-way ANOVA. Associations between DCLK1 mRNA expression and clinicopathologic characteristics in 38 cases of fresh CRC tissues were evaluated using Mann–Whitney test. Associations between DCLK1 protein expression and clinicopathologic characteristics in 71 cases of CRC tissues were tested using Chi-square tests. For survival analysis, we analyzed all CRC patients by the Kaplan–Meier analysis. A log rank test was used to compare different survival curves. Multivariate survival analysis was performed on all parameters that were found to be significant in univariate analysis using the Cox regression model. Data derived from cell line experiments were presented as mean ± standard deviation (SD) and assessed by a two-tailed Student t test. P value <0.05 was considered statistically significant.
Results
DCLK1 is up-regulated in human colorectal cancer
To assess the expression of DCLK1 in colorectal cancer, we first analyzed the genetic and transcriptional alterations of DCLK1 in a colorectal adenocarcinoma dataset (631 samples) from TCGA database using cBioPortal. It showed that there were 4 genomic and transcriptional changes (amplification, deep deletion, missense mutation, and mRNA up-regulation) detected in DCLK1 in 54 samples. Of these changes, amplification and mRNA up-regulation account for most of the alterations (77.8 %) (Fig. 1a), thus implying a probable up-regulation of DCLK1 in colorectal cancer.
Fig. 1.
DCLK1 is up-regulated in colorectal cancer. a Genetic and transcriptional alterations of DCLK1 in a TCGA colorectal cancer dataset analyzed using cBioPortal. b Immunohistochemistry staining of DCLK1 in normal epithelium (×400), primary CRC (×400) and lymphatic metastases (×400)
We further detected DCLK1 expression in human colorectal tissues (16 normal colorectal epitheliums, 71 primary CRC, and 14 lymphatic metastases) by immunohistochemistry staining. Compared to normal colorectal tissues, DCLK1 expression was significantly increased in patients with primary CRC (P = 0.039) or patients with lymphatic metastases (P = 0.000) (Table 1).
Table 1.
Immunohistochemistry staining results of DCLK1 in 101 cases
| Histologic classification | Level of DCLK1 expression | Total | P value | |||
|---|---|---|---|---|---|---|
| − | + | ++ | +++ | |||
| Normal epithelium | 4 | 7 | 3 | 2 | 16 | |
| Primary CRC | 11 | 17 | 32 | 11 | 71 | 0.039a |
| Lymphatic metastases | 0 | 0 | 6 | 8 | 14 | 0.000a ; 0.000b |
Level definition: − (0), + (1–3), ++ (4–8), +++ (9–12)
aCompared to normal epithelium
bCompared to primary CRC
DCLK1 up-regulation correlates with CRC metastasis and poor prognosis
Of note, DCLK1 expression was also significantly higher in patients with lymphatic metastases than in patients with primary CRC (P = 0.000) (Fig. 1b; Table 1), suggesting a possible contributing role of DCLK1 in metastasis.
To explore the role of DCLK1 in colorectal cancer, we first examined the mRNA expression levels of DCLK1 in 38 fresh colorectal cancer tissues by RT-PCR examination and analyzed the associations between DCLK1 mRNA level and clinicopathologic characteristics. We found that DCLK1 mRNA level was significantly correlated with lymphatic metastasis (P = 0.012) and TNM stage (P = 0.041), but not with age, gender, location, or differentiation. As is shown in Table 2, compared to the colorectal carcinoma tissues without any metastasis, DCLK1 mRNA expression was significantly higher in those with lymphatic metastasis (median 7.09 vs. 12.73).
Table 2.
Associations between DCLK1 mRNA expression and clinicopathologic characteristics in 38 cases of fresh primary CRC tissues
| Clinical information | Total | Expression of DCLK1 mRNA | P value | |
|---|---|---|---|---|
| Median | Interquartile range | |||
| Age | ||||
| ≤60 | 14 | 11.68 | 5.68–57.38 | 0.109 |
| >60 | 24 | 7.30 | 3.12–26.24 | |
| Gender | ||||
| Male | 24 | 9.46 | 4.07–31.63 | 0.486 |
| Female | 14 | 9.23 | 2.94–17.90 | |
| Location | ||||
| Colon | 26 | 8.14 | 3.20–8.74 | 0.330 |
| Rectal | 12 | 9.76 | 6.37–3.08 | |
| Differentiation | ||||
| High/medium | 15 | 6.05 | 2.85–14.54 | 0.194 |
| Low | 23 | 10.11 | 3.88–3.89 | |
| TNM stage | ||||
| I | 4 | 6.62 | 3.04–26.82 | 0.041* |
| III | 15 | 7.09 | 2.51–9.42 | |
| III | 19 | 12.73 | 6.05–42.71 | |
| Lymphatic metastasis | ||||
| Yes | 19 | 12.73 | 6.05–42.72 | 0.012* |
| No | 19 | 7.09 | 2.92–9.82 | |
* P < 0.05
P values in bold are statistically significant at the level of 0.05
We further investigated the associations between DCLK1 expression and clinicopathologic characteristics at the protein level in the above 71 cancerous tissues. In accordance with the mRNA analysis results, staining level of DCLK1 protein was also significantly correlated with TNM stage (P = 0.003) and lymphatic metastasis (P = 0.002), but not with age, gender, tumor location, or differentiation (Table 3). As cancer metastasis is the main cause of poor prognosis and death for CRC patients, we further evaluate the correlation between DCLK1 expression and overall survival (OS) in these patients using Kaplan–Meier analysis. As is shown in Fig. 2, the 5-year disease-specific survival (DSS) in patients with high DCLK1 staining (≥4) is significantly shorter than those with low DCLK1 staining (0–3) (85.7 vs. 52.5 %, P = 0.016). Moreover, the univariate Cox regression analysis results revealed that TNM stage, metastasis, and DCLK1 level are three prognostic factors for overall survival, while multivariate Cox regression indicated that lymphatic metastasis (P = 0.015) and DCLK1 level (P = 0.036) are independent predictors for poor survival of CRC patients (Table 4).
Table 3.
Associations between clinicopathologic characteristics and DCLK1 protein expression in 71 cases of primary CRC tissues
| Clinical information | Total | Level of DCLK1 expression | P value | |
|---|---|---|---|---|
| Low | High | |||
| Age | ||||
| ≤60 | 29 | 15 (51.7 %) | 14 (48.3 %) | 0.078 |
| >60 | 42 | 13 (30.9 %) | 29 (69.1 %) | |
| Gender | ||||
| Male | 44 | 19 (43.2 %) | 25 (56.8 %) | 0.410 |
| Female | 27 | 9 (33.3 %) | 18 (66.7 %) | |
| Location | ||||
| Colon | 34 | 11 (32.3 %) | 23 (67.7 %) | 0.242 |
| Rectal | 37 | 17 (45.9 %) | 20 (54.1 %) | |
| Differentiation | ||||
| High | 8 | 4 (50 %) | 4 (50 %) | 0.247 |
| Medium | 53 | 21 (40.4 %) | 32 (59.6 %) | |
| Low | 10 | 3 (30 %) | 7 (70 %) | |
| TNM stage | ||||
| I | 11 | 6 (54.5 %) | 5 (45.5 %) | 0.003** |
| II | 17 | 12 (70.6 %) | 5 (29.4 %) | |
| III | 32 | 9 (28.1 %) | 23 (71.9 %) | |
| IV | 11 | 1 (9 %) | 10 (91 %) | |
| Lymphatic metastasis | ||||
| Yes | 41 | 10 (24.4 %) | 31 (75.6 %) | 0.002** |
| No | 30 | 18 (60 %) | 12 (40 %) | |
** P < 0.01
P values in bold are statistically significant at the level of 0.01
Fig. 2.
High expression of DCLK1 correlates with poor prognosis in human CRC patients. Survival analysis in 71 primary CRC patients was performed. Patients with high (≥4) DCLK1 staining score owned much shorter survival time than those with low (0–3) DCLK1 staining score in Kaplan–Meier cancer-specific survival curve (P = 0.016)
Table 4.
Univariate and multivariate Cox regression analyses of overall survival in 71 colorectal cancer patients
| Variable | Classification | Univariate analysis | Multivariate analysis | ||
|---|---|---|---|---|---|
| HR (95 % CI) | P | HR (95 % CI) | P | ||
| Age | >60/≤60 | 1.585 (0.678–3.707) | 0.288 | ||
| Gender | Male/female | 1.511 (0.670–3.408) | 0.320 | ||
| Differentiation | Low/high–medium | 0.764 (0.344–1.695) | 0.508 | ||
| TNM stage | T3–T4/T1–T2 | 4.630 (1.578–13.585) | 0.005** | 3.706 (1.312–10.919) | 0.476 |
| Metastasis | Yes/no | 4.190 (1.559–11.261) | 0.005** | 3.448 (1.266–9.386) | 0.015* |
| DCLK1 | Low/high | 3.699 (1.261–10.848) | 0.017* | 2.864 (1.063–8.520) | 0.036* |
HR hazard ratio, CI confidence interval
* P < 0.05; ** P < 0.01
P values in bold are statistically significant at the level of 0.05
DCLK1 promotes CRC cell migration and invasion
To determine the potential contribution of DCLK1 to cancer metastasis, we investigated the effect of DCLK1 up-regulation with in vitro studies. We first measured the baseline levels of DCLK1 mRNA and protein in five CRC cell lines using quantitative RT-PCR and western blotting and found that SW480 cells have the lowest levels of DCLK1 in contrast to other CRC cells (Fig. 3a, b). Therefore, we selected SW480 cells to stably overexpress DCLK1 by plasmid transfection. The successful overexpression of DCLK1 in SW480 cells was confirmed by western blot detection (Fig. 3c, d). Both the wound-healing and transwell assays showed that up-regulation of DCLK1 expression markedly promoted the migration and invasion properties of SW480 cells (Fig. 3e–h). Collectively, these results suggest that DCLK1 may play a contributory role in CRC metastasis by enhancing cell migration and invasion.
Fig. 3.
DCLK1 promotes CRC cell migration and invasion in vitro. a Western blotting analysis of the relative protein levels of DCLK1 in 5 CRC cell lines (HCT116, HT29, SW480, SW620, and RKO). b Real-time PCR analysis of the relative mRNA levels of DCLK1 in 5 CRC cell lines. c, d Western blotting examination and quantitative analysis of DCLK1 in the control and DCLK1 overexpressing SW480 cell lines. e, f Wound-healing assays showing that DCLK1 promoted the migration speed of SW480. g, h Transwell analyses of the effect of DCLK1 on the migration and invasion abilities of SW480 cell line. The experiments were repeated at least 3 times, and error bars represent mean ± SD. Original magnification, (e, g), ×100
DCLK1 promotes EMT in CRC cells
To further explore the underlying mechanisms by which DCLK1 promotes cell migration and invasion, we analyzed the associations between DCLK1 and the reported metastasis-associated genes in a TCGA CRC database using cBioPortal. The results showed that DCLK1 mRNA had a highly positive correlation with ZEB1 mRNA (r = 0.718, P = 0.000) and Vimentin mRNA (r = 0.791, P = 0.000), respectively (Fig. 4a, b). Since ZEB1 and Vimentin are the canonical mesenchymal markers, it suggests that DCLK1 may exert biological effects through regulation of EMT. We therefore examined the effect of DCLK1 overexpression on the EMT markers in SW480 cells by both RT-PCR and western blotting assays. It showed that up-regulation of DCLK1 in SW480 cells led to an obvious decrease in E-cadherin expression, which is an epithelial marker, but significantly promoted the expression of mesenchymal markers such as Vimentin and ZEB1 at both mRNA and protein levels (Fig. 4c–e). Taken together, these in vitro findings suggest that DCLK1 can promote the EMT event in colorectal cancer cells.
Fig. 4.
DCLK1 promotes EMT in CRC. a, b TCGA database analysis using cBioPortal showed that DCLK1 mRNA expression correlated highly with ZEB1 (a) and Vimentin (b) in CRC patients. c Quantitative real-time PCR analysis of the effect of DCLK1 overexpression on EMT markers (E-cadherin, Vimentin, and ZEB1) at mRNA level. d, e Western blotting analysis of the effect of DCLK1 on EMT markers at protein level. The experiments were repeated at least three times, and error bars represent mean ± SD
Discussion
Cancer invasion and distant metastasis, which are likely regulated by various genetic and/or epigenetic changes, are the leading causes of more than 90 % of cancer-related deaths, including CRC-related deaths (Christofori 2006). Although CRC patients have undertaken successful resection or chemotherapy, their survival rates would significantly drop once localized or distant metastasis occurs. So far, the key factors that are responsible for initiation of metastatic behaviors have not been defined. Recent evidence suggests that cancer stem cells (CSCs) may play a central role in initiating cancer metastasis. CSCs are a small subpopulation in cancer and have been shown to be responsible for chemotherapy resistance, metastasis, and cancer recurrence (Mirzaei et al. 2015b; Mitra et al. 2015). Previous reports have suggested that DCLK1 may mark cancer stem cells in gastrointestinal and other cancers (Li and Bellows 2013; Mirzaei et al. 2015a; Nakanishi et al. 2013). DCLK1-positive tumor cell lines can reflect stem-like characteristics and possess much stronger self-renewal capability than low expression of DCLK1 ones (Li and Bellows 2013). Indeed, some reports demonstrate DCLK1 up-regulation in some cancers. For example, DCLK1 levels in pancreatic ductal adenocarcinoma (PDAC) patients were serologically higher than in normal controls (Qu et al. 2015). An elevated expression of DCLK1 protein has been identified in the plasma of patients with hepatocellular carcinoma (Sureban et al. 2015). In addition, DCLK1 was found to be increased in Barrett’s esophagus, a premalignant lesion that can progress to esophageal adenocarcinoma, and could be regarded as an early molecular indicator for predicting tumor progression (Vega et al. 2012).
In this study, we investigated whether DCLK1 expression is altered in human colorectal cancer and its clinical significance. We observed that DCLK1 expression was not only significantly up-regulated in human CRC tissues, but also correlated well with CRC metastasis. More importantly, DCLK1 expression was inversely associated with overall survival in patients and might serve as an independent predictor for poor prognosis. Our in vitro studies showed that overexpression of DCLK1 in SW480 cells could remarkably promote colorectal cancer cell migration and invasion abilities, which was partly in line with our clinical observations that DCLK1 might contribute to CRC metastasis. However, this in vitro study is limited by investigation on only one CRC cell line SW480. The results may require further validation with more CRC cells included.
We further intended to explore the underlying mechanisms by which DCLK1 participates in cancer metastasis. EMT has long been recognized to play a crucial role in promoting tumor metastasis (Tsai and Yang 2013). During EMT process, the polarized epithelial cells switch to highly mobile, non-polarized mesenchymal cells, and this provides tumor cells with the ability to migrate to distant regions and to invade normal tissues (Liang 2011). Loss of E-cadherin and gain of mesenchymal markers such as Vimentin or ZEB1 are regarded as characteristics of EMT. Indeed, EMT has also been reported to be associated with cancer stem cells in promoting tumor growth and metastasis in advanced malignancies (Liang 2011; Mitra et al. 2015; Tsai and Yang 2013). Through in vitro investigations, we demonstrated that up-regulation of DCLK1 could decrease the expression of E-cadherin (an epithelial cell marker) and increased Vimentin and ZEB1 expression. Both Vimentin and ZEB1 are classical mesenchymal markers. It is known that ZEB1 is a transcriptional factor that induces EMT process and has been shown to down-regulate E-cadherin in epithelial cells (Soini et al. 2011).
In summary, this study illustrates that DCLK1 expression is significantly up-regulated in human CRC and may serve as a biomarker for cancer metastasis. DCLK1 can participate in EMT process, which may be one of its molecular mechanisms for facilitating cancer metastasis.
Acknowledgments
This work was supported by grants from National Natural Science Foundation of China (No. 81572825, to Guangyu An). Beijing Municipal Health System High-level Talent Training Project (to Tao Wen), Beijing, P. R. China. National Natural Science Foundation of China (Youth fund project, No. 81502384, to Jian Liu).
Abbreviations
- DCLK1
Doublecortin-like kinase 1
- CRC
Colorectal cancer
- TCGA
The Cancer Genome Atlas
- AJCC
American Joint Committee on Cancer
- MAPs
Microtuble-associated proteins
- DCX
Doublecortin
- CSCs
Cancer stem cells
- EMT
Epithelial–mesenchymal transition
- TNM
Tumor node metastasis
- ATCC
American Type Culture Collection
- OS
Overall survival
- HR
Hazard ratio
- CI
Confidence interval
- SD
Standard deviation
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
Ethical approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
Informed consent
Informed consent was obtained from all individual participants included in the study.
Footnotes
Tianbo Gao and Min Wang contributed equally to this work.
Contributor Information
Jian Liu, Phone: +86 1085231375, Email: liujian2004811@126.com.
Guangyu An, Phone: +86 1085231569, Email: anguangyu@hotmail.com.
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