Cancer stem cell (CSC) marker doublecortin-like kinase 1 (DCLK1) contributes greatly to the malignancy of gastrointestinal cancers, and DCLK1-targeted agents have potential therapeutic value. However, the molecular pathways regulated by DCLK1-S (DCLK1 isoform 4), a shortened splice variant of DCLK1, still remain obscure. Here we found that the expression of DCLK1-S is significantly increased in human esophageal squamous cell carcinoma (ESCC) tissues and associated with malignant progression and poor prognosis. Functional studies indicated that silencing total of DCLK1 mediated by CRISPR/Cas9 inhibited ESCC cell proliferation, migration, and invasion. Conversely, these changes were largely reversed after DCLK1-S rescue or overexpression. More importantly, DCLK1-S significantly enhanced primary tumor formation and metastatic lung colonization in vivo. The Cancer Genome Atlas database and molecular analysis showed that DCLK1-S was closely related to the epithelial–mesenchymal transition (EMT) process in patients with ESCC. Further RNA sequencing and Kyoto Encyclopedia of Genes and Genomes analysis demonstrated that MAPK signaling pathway was significantly enriched. Our in vitro study proclaimed that DCLK1-S induced MMP2 expression in ESCC cells via MAPK/ERK signaling, leading to the activation of EMT. In addition, administration of ERK1/2 blocker SCH772984 attenuated the proliferative and migratory phenotype induced by DCLK1-S. In conclusion, these findings suggest that DCLK1-S may be a key molecule in MAPK/ERK/MMP2 pathway–mediated progression of ESCC, and that it has potential as a biomarker or therapeutic target to improve outcomes in patients with ESCC.

Implications

: DCLK1-S induces ESCC progression by activating the MAPK/ERK/MMP2 axis and may serve as a prognostic biomarker or therapeutic target for patients with ESCC.

This article is featured in Highlights of This Issue, p. 1971

Esophageal squamous cell carcinoma (ESCC) has a high incidence rate and mortality worldwide and is the major histologic type of esophageal cancer in Asia (1–3). Because of a lack of early symptoms, most patients with ESCC are diagnosed with late-stage disease leading to poor prognosis and a high recurrence rate. Only 15% to 25% of patients with ESCC survive to 5 years after diagnosis (4, 5). Despite abundant research efforts, the molecular mechanism of ESCC progression has not been fully clarified, limiting opportunities for individualized diagnosis, prognosis evaluation, and treatment of patients with ESCC. There is an urgent need to seek new biomarkers and therapeutic targets that can predict efficacy and prognosis and improve treatment for patients with ESCC.

Doublecortin-like kinase 1 (DCLK1), a microtubule-associated protein kinase originally found in post mitotic neurons, plays an important role in maintaining neurogenesis and neuronal migration (6). In recent years, it has been identified as a specific gastrointestinal cancer stem cell (CSC) marker in colon and pancreatic cancer (7–9), and numerous studies now support a causative role for aberrant DCLK1 expression in various human cancer types such as colorectal cancer (10), pancreatic cancer (11), and renal cancer (12). In our previous study, we found that the expression of DCLK1 is increased on the protein level in colon cancer, and its expression is closely associated with poor prognosis which is consistent with other findings (13). In addition, we confirmed that DCLK1 can promote the progression of intestinal tumors by regulating pro-survival signaling pathways and enhancing self-renewal of tumor cells (14). Importantly, accumulating evidence indicates that DCLK1 is a promising target for tumor therapy, and small-molecule kinase inhibitors targeting DCLK1 have been developed (15, 16).

The human DCLK1 gene is characterized by two promoters, a 5′ (α) promoter and a 3′ (β) promoter, which are responsible for transcription of long (L)-isoform (DCLK1 isoform 1, DCLK1 isoform 2, ∼82 KDa) and short (S)-isoform (DCLK1 isoform 3, DCLK1 isoform 4, ∼47 KDa; Supplementary Fig. S1; refs. 17, 18). The L-isoform and S-isoform differ respectively by the presence or absence of the doublecortin domain. Recent studies shown that DCLK1-S expression is an independent diagnostic and prognostic marker for patients with colon cancer (19, 20). Despite these accumulated knowledge, the role of DCLK1 and its individual isoforms in ESCC remains unclear.

The current study assesses the biological role and underlying mechanisms of the short transcripts (DCLK1-S, ∼47 KDa), also termed isoform 4 in Uniprot (O15075-4), in human ESCC for the first time. We demonstrate that high expression of DCLK1-S in ESCC is associated with poor patient survival. Mechanistically, our findings show that DCLK1-S induces the epithelial–mesenchymal transition (EMT) process and ESCC cell proliferation by activating the MAPK/ERK/MMP2 axis. In short, we report that DCLK1-S is a new molecular determinant of invasion and metastasis of ESCC using in vitro and in vivo assays, which may serve as a prognostic biomarker or therapeutic target for patients with ESCC.

Tissue qPCR analysis

Human ESCC tissue cDNA microarray (product number: cDNA-HEsoS095Su01) containing 67 primary ESCC tumor and 28 paracancerous matching tissue cDNA samples was purchased from Shanghai Outdo Biotech. A summary of patient cohort characteristics is presented in Table 1. The expression of DCLK1-L/S was detected by real-time PCR as described below.

IHC

Primary tumor tissues and metastatic tissues (lymph node or lung) of 5 patients with ESCC were collected from the Beijing Chao-Yang Hospital (Beijing, P.R. China). The tissue microarray (product number: HEsoSqu060PG01) containing 30 pairs ESCC tissues and matched adjacent tissues were purchased from Shanghai Outdo Biotech. Ethical approval was granted by ethics committee of Shanghai Outdo Biotech Company. In brief, all the tissues were incubated with DCLK1 antibody (Ab31704, Abcam) at 4°C overnight. After incubating with a secondary antibody, diaminobenzidine (DAB) chromogen was added to detect the IHC signal. All images were photographed and analyzed according to the IHC scoring standard, which was calculated by multiplying the score of the staining intensity and the percentage of positive cells. The DCLK1 staining intensity was scored as 0 to 3 (0, negative; 1, weak; 2, moderate; 3, strong). The percentage of DCLK1-positive cells were scored as follows: 1 (0%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (76%–100%). Scores for intensity and proportion of positive cells were multiplied as final histoscores of the samples and the DCLK1 staining pattern was classified as low expression (0–3) and high expression (4–12).

The Cancer Genome Atlas ESCC data

Clinical and gene expression information for 204 patients with esophageal cancer (version 2017-10-13) included in The Cancer Genome Atlas (TCGA) dataset was downloaded from the UCSC cancer genome browser (http://xena.ucsc.edu). In addition, isoform-specific RNA sequencing (RNA-seq) data generated during TCGA PAN-CANCER project as described previously (12), was obtained and included transcripts for DCLK1 isoform 3 (ENST00000615680) and DCLK1 isoform 4 (ENST00000379893). Heatmaps were produced using the heatmap.2 function (R package gplots). The clinical characteristics of patients with ESCC in TCGA are presented in Supplementary Table S1.

Cell culture

Human ESCC cell lines KYSE410 (DSMZ, catalog no. ACC-381, RRID: CVCL_1352), KYSE450 (DSMZ catalog no. ACC-387, RRID: CVCL_1353), KYSE510 ((DSMZ catalog no. ACC-374, RRID: CVCL_1354), and KYSE70 (JCRB catalog no. JCRB0190, RRID: CVCL_1356) and HEK-293T (CCLV catalog no. CCLV-RIE 1018, RRID: CVCL_0063) cells were obtained from Cancer Hospital of the Chinese Academy of Medical Sciences (Beijing, P.R. China) in 2017. All ESCC cell lines were cultured in RPMI1640 medium (Gibco) with 10% FBS (Gibco). HEK-293T cells were cultured in DMEM medium (Gibco) with 10% FBS. All the cells were incubated in a 37°C humidified incubator with 5% CO2. All experiments were performed with Mycoplasma-free cells, which were regularly tested using Mycoplasma Detection Kit (Yeasen).

Real-time PCR

Total RNA was isolated from ESCC cells with TRIzol reagent (Invitrogen). The first strand cDNA was synthesized with 1 μg total RNA using reverse transcription Kit (Yeasen). Quantitative real-time PCR was performed using SYBR Green Premix (Yeasen) with GAPDH as a housekeeping gene on 7500 sequence detection system (Applied Biosystems). The DCLK1-L and DCLK1-S specific primers (19) were as follows: DCLK1-L, forward primer: 5′-GGAGTGGTGAAACGCCTGTAC-3′, reverse primer: 5′-GGTTCCATTAACTGAGCTGG-3′, DCLK1-S, forward primer: 5′-ACACTAAGACTGTGTCCATGTTAGAACT-3′, reverse primer: 5′-AAGCCTTCCTCCGACACTTCT-3′; GAPDH, forward primer: 5′-AATCCCATCACCATCTTCCA-3′, reverse primer: 5′-TGGACTCCACGACGTACTCA-3′. Relative quantification of gene expression was calculated by the 2−ΔΔCT method.

Western blotting

Cell protein was extracted using RIPA lysis (Beyotime Biotechnology) with 1 mmol/L phenylmethylsulfonyl fluoride (Beyotime) and 1 mmol/L protease inhibitor cocktail (Beyotime). Total protein concentration was determined by BCA protein assay kit (Thermo Fisher Scientific). A total of 30 μg of total proteins were loaded and separated on 10% SDS polyacrylamide gels (PAGE) and semi-dry transferred onto polyvinylidene difluoride membrane (Millipore Corporation). The membrane was blocked in 8% milk (BD, 232100) for 1 hour and incubated overnight with primary antibodies. After that, the membranes were washed by 1×TBST for three times and incubated with secondary antibody at room temperature for 1 hour. The proteins were detected on a Bio-Rad ChemiDoc MP using ECL reagents (Millipore). The antibodies used were anti-DCLK1 (Abcam catalog no. ab31704, RRID:AB_873537), anti-GAPDH (Cell Signaling Technology, catalog no. 8884, RRID: AB_11129865), anti-E-cadherin (Cell Signaling Technology, catalog no. 3195, RRID:AB_2291471), anti-ZEB1 (Cell Signaling Technology, catalog no. 3396, RRID:AB_1904164), anti-Snail (Cell Signaling Technology, catalog no. 3879, RRID:AB_2255011), anti-Slug (Cell Signaling Technology, catalog no. 9585, RRID:AB_2239535; all Cell Signaling Technology), and goat anti-rabbit IgG secondary antibodies (OriGene Technologies, Inc.).

Cloning of CRISPR/cas9 expression vector and rescue expression construct

For knocking out DCLK1-S in ESCC cells, CRISPR/Cas9 technology was used in the knockout (KO) experiment. The specifically targeting DCLK1-S single-guide RNA (sgRNA) sequence is as follows: Oligo1: 5′- CACCGGAGTAGAGAGCTGACTACCA-3′, Oligo2: 5′-AAACTGGTAGTCAGCTCTCTACTCC-3′. The lentiCRISPR v2 vector (RRID: Addgene_52961) was digested using the BsmBI restriction enzyme (New England Biolabs) and ligated with the sticky end harboring double-stranded sgRNA. For rescue expression construct, GenEZ DCLK1-S ORF clone was cloned into the linearized pCDH-MSCV-MCS-EF1α-puro vector used for stable transfection of human ESCC cells.

Generation of lentiviral particles and stable cell lines

All constructed lentiviral plasmids and lentiCRISPR v2 vector harboring no gene targeting sgRNA were respectively transfected into HEK-293T cells with packaging plasmids psPAX2 (RRID: Addgene_12260) and pMD2.G (RRID: Addgene_12259) using lipofectamine 3000 (Invitrogen). After 48 to 72 hours post-transfection, the virus was harvested and cleared through a 0.45 μmol/L filter. After 3 days of virus infection, ESCC cells were selected with 2 μg/mL puromycin (Gibco) to obtain stable cell lines. Protein was subsequently extracted from the screened cells for Western blotting. All validated DCLK1-S KO cells were then amplified and stored.

CCK8 and colony formation assay

ESCC cells were seeded in 96-well plate at a density of 5 × 103 cells/well. At different timepoints (24, 48, 72, and 96 hours) medium containing 10% Cell Counting Kit-8 (CCK8, Dojindo) was added to each well and cultured at 37°C for 1 hour. Optical density was measured at 450 nm by spectrophotometer. For colony formation assay, 200 to 300 cells were cultured in 6-well plates and cultivated at 37°C incubator for 2 weeks. Cells were fixed with 4% paraformaldehyde and then stained with 0.1% crystal violet. After that, the numbers of colonies were counted using Image J (RRID:SCR_003070).

Transwell migration and invasion assay

Transwell chamber technique (Corning) was used to detect the migration and invasion ability of ESCC cells. For migration assay, serum-free medium containing 10 × 104 cells were seeded into the upper chamber. For invasion assay, the reduced growth factor matrigel and PBS were inoculated into the transwell upper chamber at a ratio of 1:8 at 37°C for 2 hours and serum-free medium containing 15 × 104 cells were seeded into the upper chamber. Both the lower chamber contained complete medium with 10% FBS as a chemoattractant. After incubation at 37°C for 24 hours, cells migrating to the underside were fixed with 4% paraformaldehyde for 15 minutes and stained with 0.1% crystal violet. Then, 4 to 5 fields/well were selected for counting under the microscope.

In vivo experiments

All the animals were purchased from Charles River Laboratories and maintained under specific pathogen-free conditions. For tumor xenografts, 2 × 106 cells were diluted in 100 μL PBS and transplanted into the back of 6-week-old BALB/c female nude mice (n = 7 per group). Measurements were performed every 4 to 5 days after the tumor showed significant growth. The volume of tumor was calculated by formula: Volume = (Length × Width2)/2. After 24 days, the xenografted tumors were harvested and photographed. The metastatic tumor model was established in 6-week-old mice by tail vein injection of 2.0 × 106 cells/mouse. After 8 weeks, the mice were killed and the lungs were excised and fixed with formalin for hematoxylin and eosin (H&E) staining. All animal experiments were performed at the Medical Research Center of Beijing Chao-Yang Hospital (Beijing, P.R. China) according to the guidelines approved by the Institutional Animal Care and Use Committee of Capital Medical University (Beijing, P.R. China).

Statistical analysis

Data were analyzed using GraphPad Prism 6.0 (GraphPad Software, RRID:SCR_002798). For parametric analyses, statistical differences were determined with the Student t test. For nonparametric analyses, statistical differences were determined using the Mann–Whitney test. Categorical data were analyzed using the Fisher exact test. Kaplan–Meier curves and log-rank tests were used to evaluate differences in survival times between groups. The cut-off threshold between DCLK1-L/S high and low group was determined by the median value of mRNA expression in tumor tissue. Quantitative data were expressed as the mean ± SD. P < 0.05 were considered statistically significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

DCLK1-S expression is upregulated in ESCC tissues and high DCLK1-S expression is correlated with poor survival in patients

First, IHC was performed to assess DCLK1 total proteins using an anti-DCLK1 antibody in matched ESCC tissue microarray (n = 30). The staining intensity of DCLK1 was scored from 0 to 3 as shown in Fig. 1A. DCLK1 total proteins expression was higher in tumor tissues compared with paracancerous tissue (Fig. 1B and C), with a rate for high DCLK1 expression of 73% (22/30) and 30% (9/30), respectively. In addition, lymph node metastasis tissue also had higher DCLK1 total protein expression relative to primary esophageal carcinoma (Supplementary Fig. S2).

Given the existence of DCLK1-long (L) and DCLK1-short (S) isoforms, qPCR was conducted to detect the mRNA expression of DCLK1-L/S isoforms in the ESCC cDNA microarray (n = 67) using specific primers. The results showed that DCLK1-S mRNA expression in tumor tissues was significantly higher than that in paracancerous tissue (Fig. 1D and E). Furthermore, we used the isoform-specific RNA-seq data generated during the TCGA PAN-CANCER project to identify the two short isoforms. We found that DCLK1 isoform 3 (UniProt: O15075-3; ENST00000615680) was rarely expressed in patients with ESCC compared with DCLK1 isoform 4 (UniProt: O15075-4; ENST00000379893; Supplementary Fig. S1B), which is similar to findings in renal cancer (12). Therefore, DCLK1 isoform 4 is the most prominent short isoform in patients with ESCC. Moreover, high DCLK1-S mRNA expression was correlated with higher histologic grade and lymph node metastasis (Table 1). As indicated in Fig. 1F, Kaplan–Meier analysis showed that higher expression of DCLK1-S mRNA led to a significantly poorer prognosis (25.5 vs. 12 months, P = 0.0306). However, similar results were not observed with the detection of DCLK1-L (Supplementary Fig. S3). The univariate analysis indicated that high DCLK1-S expression and lymph node metastasis correlated with a short overall survival (OS). Furthermore, multivariate analysis revealed that DCLK1-S expression was an independent risk factor for reduced OS (HR = 2.242; 95% confidence interval, 1.077–4.666; P = 0.031), as was lymph node metastasis (Table 2). These data suggest that DCLK1-S is an independent prognostic factor in patients with ESCC.

Inhibition of DCLK1 decreases progression, migration, and invasion of ESCC cells

DCLK1-S is > 98% homologous with the 3′-end of the DCLK1-L, and only six unique amino acids at the 5′-end (18, 19). To investigate the specific biological function of DCLK1-S in ESCC, qPCR and Western blot analysis were first used to determine the DCLK1 total expression in four ESCC cell lines (KYSE410, KYSE510, KYSE450, KYSE70). As shown in Fig. 2A and B, higher DCLK1 total expression levels were present in KYSE450 and KYSE70 compared with KYSE510 and KYSE410 cell lines. Subsequently, we designed sgRNA targeting all subtypes of DCLK1, which eventually led to complete silencing of total DCLK1 (DCLK1-KO) in KYSE450 and KYSE70 cells, which was confirmed by Western blot analysis (Fig. 2C). We found that DCLK1-KO significantly reduced ESCC cell proliferation (Fig. 2D). Meanwhile, DCLK1 deletion led to a markedly lower number of colonies compared with control cells (Fig. 2E). In addition, transwell assays with permeable filter and basement membrane were respectively used to measure the migration and invasion capacity of DCLK1-KO ESCC cells and the results demonstrated that KO of DCLK1 significantly reduced migration and invasion (Fig. 2F). Furthermore, we found that LRRK2-IN-1, an inhibitor of DCLK1, could also suppress cell growth and migration in ESCC (Supplementary Fig. S4). However, whether these inhibitory effects are attributed to DCLK1-S deserves further exploration.

Reexpression of DCLK1-S restores cell proliferation, migration, and invasion in vitro

To further verify the role of DCLK1-S in the proliferation and metastasis of ESCC cells, we generated and confirmed a DCLK-S clone for stable transfection of DCLK1-KO cells to restore the expression of DCLK1-S without affecting the expression of DCLK1-L (Fig. 3A). CCK8 and colony formation experiments showed that DCLK1-S reexpression could restore ESCC cell proliferation (Fig. 3B and C). In transwell assays, we observed DCLK1-S reexpression could fully restore migration and invasion potential compared in KO cells (Fig. 3D). Next, functional experiments of DCLK1-S overexpression were performed in KYSE410 and KYSE510 cells with low endogenous expression of DCLK1-S. Similarly, Western blot analysis validated the efficiency of DCLK1-S overexpression without altering the expression of DCLK1-L (Fig. 3E), as did qPCR (Supplementary Fig. S5). As expected, overexpression of DCLK1-S in KYSE410 and KYSE510 can accelerate cell proliferation in both CCK8 and colony formation experiments (Fig. 3F and G). In addition, pro-migratory and pro-invasive characteristics were observed in transwell experiments (Fig. 3H). These results confirm that the biological effects of the above observations are attributed to DCLK1-S.

DCLK1-S is involved in the activation of EMT in ESCC

Given that EMT plays an important role in promoting the invasion and metastasis of ESCC (21, 22), and because the above results suggest that DCLK1-S may be a key factor in promoting the malignant phenotype of ESCC. We queried TCGA ESCC database including 204 patients diagnosed with esophageal carcinoma from 2013 to 2015 (Supplementary Fig. S6). First, DCLK1 total expression was observed to be closely correlated with EMT-related makers in TCGA database (Supplementary Fig. S7A). Consistently, Western blot analysis was used to assessed the effect of DCLK1-KO on the EMT program of ESCC cells, and the results showed that the expression of E-cadherin was significantly upregulated, but the EMT transcription factors Slug, Snail, and ZEB1 were significantly decreased after DCLK1 entire silencing in KYSE450 and KYSE70 cell lines (Supplementary Fig. S7B). To better verify the correlation between DCLK1-S and the EMT program, we reexamined the expression of DCLK1-S in TCGA database, and similar results for DCLK1-S were observed in the heatmap (n = 92). As shown in Fig. 4A, DCLK1-S expression positively correlated with mesenchymal markers ZEB1, ZEB2, Vimentin, FN1, CDH2, SNAL1, Twist1, and Twist2, but inversely correlated with the epithelial markers, such as CDH1, CLDN4, and CLDN7, suggesting that DCLK1-S correlates with EMT in patients with ESCC. Strikingly, Western blot detection showed that both DCLK1-S reexpression and DCLK1-S overexpression reversed the changes in above EMT-related molecules mediated by low DCLK1 levels (Fig. 4B and C). Together these results indicate that DCLK1-S activates the EMT program in ESCC cells, which contributes to ESCC malignancy.

DCLK1-S promotes ESCC progression via the MAPK/ERK/MMP2 pathway

Activation of MAPK/ERK pathway in ESCC has been proved to be closely related to various of malignant biological phenotypes in numerous studies (23–25). To further elucidate the potential mechanism of DCLK1-S in promoting tumorigenesis and progression, we evaluated the correlation between DCLK1 expression and MAPK/ERK pathway–related genes from TCGA database of patients with ESCC (n = 95). Results of the heatmap showed that gene expression changes during carcinogenesis, of representative MAPK/ERK signaling pathway genes, are associated with the expression of DCLK1, including MMP2, ELK1, FOXD3, AKT3, and MAP2K1 (Supplementary Fig. S7C). RNA-seq was performed to identify differential expression genes in the DCLK1-KO and DCLK1-S-Res cells. Not surprisingly, KEGG analysis showed that the most significantly enriched pathway was MAPK signaling (Fig. 5A). Consistent results were also observed using Western blot analysis. Knocking out total of DCLK1 significantly inhibited the protein levels of p-ERK1/2 and MMP2 compared with the control cells (Supplementary Fig. S7D), while their expression was significantly upregulated after DCLK1-S reexpression (Fig. 5B). To further confirm whether DCLK1-S regulates the malignant progression of ESCC by activating ERK1/2 pathway, ERK1/2 antagonist SCH772984 was used to evaluate the specificity of DCLK1-S–mediated proliferation and migration of ESCC cells. Blocking ERK1/2 pathway sharply attenuated the cell proliferation induced by DCLK1-S as detected by CCK8 and colony formation assays (Fig. 5C and D). In addition, the enhancement of migration and invasion mediated by DCLK1-S was also counteracted by ERK inhibitors (Fig. 5E). Moreover, the activation of EMT-related transcription factors and MMP2 stimulated by DCLK1-S also returned to normal after ERK inhibition (Fig. 5F). Taken together, these results strongly suggest that DCLK1-S can upregulate ERK1/2 phosphorylation, driving the expression of downstream MMP2, thus promoting the occurrence and progression of ESCC.

DCLK1-S promotes tumor growth and metastasis in vivo

To further investigate whether DCLK1-S expression affects tumor growth in vivo, we injected DCLK1-S-KO cells and DCLK1-S-Res KYSE450 cells into BALB/C nude mice subcutaneously. After 24 days of inoculation, the growth curve obtained by regular measurement of tumor size showed that DCLK1-S gene rescue significantly accelerated tumor growth compared with DCLK1-KO (Fig. 6A; P < 0.05). Moreover, the average weight of tumor tissue obtained from DCLK1-S-Res group (n = 7) was also significantly higher than that of DCLK1-S-KO group (n = 5), as shown in Fig. 6B (P < 0.05). Similarly, to further confirm the role of DCLK1-S in promoting ESCC metastasis, KYSE450-DCLK1-S-KO and DCLK1-S-Res cell were injected into the nude mice tail vein to establish a metastatic tumor model. Compared with the KO group, the physical condition of DCLK1-S-Res group was worsened. Although no metastatic nodules were observed in the liver, obvious lung metastases were present in mice injected with DCLK1-S-Res (Fig. 6C), which was confirmed by H&E staining (Fig. 6D). These findings provide convincing evidence that DCLK1-S promotes the colonization ability of ESCC cells in distant organs.

As the main histologic type of esophageal cancer, ESCC has become one of the most fatal gastrointestinal malignancies due to local invasion and distant metastasis, which necessitates further study of its etiology and new strategies to improve systemic therapy. Abnormal expression of DCLK1 in various human tumors has been reported frequently, including in colon, pancreatic, renal, and breast cancers (10–12, 26). To date, numerous studies have confirmed that DCLK1 plays a key role in regulating cell proliferation, migration, and invasion (10, 27). There are two promoters of the human DCLK1 gene, a 5′ (α) promoter and a 3′ (β) promoter, responsible for transcription of long/short transcripts (DCLK1-L/S isoform), respectively (19, 28). The precise role of DCLK1 isoforms have been continuously highlighted in diverse tumors, and some researchers suggest that DCLK1-S is the main oncogenic isoform in human colon adenocarcinomas (ref. 19). Our own work in renal cell carcinoma (RCC) found that DCLK1 alternative splice variants with a lengthened C-terminus (DCLK1-AL, isoform 2) are overexpressed in patients with RCC and predict overall and recurrence-free survival (12). Consistent with this phenomenon, the high expression of DCLK1-AL is also reported to increase the invasiveness, drug resistance and KRAS activation in pancreatic ductal adenocarcinoma (29). Together these findings highlight the importance of investigating DCLK1 isoforms individually in different cancer types where their function may vary.

Recent reports suggest that siRNA-mediated DCLK1 knockdown can reduce proliferation, migratory/invasive potential, and chemoresistance in ESCC cells (30), but the function and underlying mechanism of this phenomenon remains unclear. In the current study, higher levels of DCLK1-S were observed in ESCC tissue and associated with poorer OS in patients with ESCC. However, similar results for DCLK1-L (full-length DCLK1) were not observed in the current patients with ESCC, and the DCLK1-L expression was not associated with OS in patients with ESCC, which is consistent with the published studies of human colorectal cancer (19, 20). Considering our further findings that DCLK1 isoform 3 was rarely expressed in patients with ESCC, similar to findings in renal cancer (12), we believe that DCLK1 isoform 4 is the representative short isoform in patients with ESCC and may be a predictor of survival.

In our previous studies, we proposed that DCLK1 can enhance the proliferation and invasion of colon (13), renal (12), and breast cancers (26). Here, to determine the role of DCLK1-S in ESCC, we first constructed a total DCLK1 KO cell line (DCLK1-KO) and demonstrated that CRISPR/Cas9-mediated DCLK1 total ablation significantly restricted the proliferation, migration, and invasion of ESCC cells. Similar results were obtained with the use of LRRK2-IN-1, a DCLK1 inhibitor. DCLK1-S rescue experiments in the DCLK1-KO cell line reversed this phenotype and overexpression experiments significantly increased the proliferation and invasion of ESCC cells. Importantly, we found that both rescue and overexpression of DCLK1-S did not affect the protein and mRNA levels of DCLK1-L, which confirms that the biological effects of the previous in vitro observation can be attributed to DCLK1-S. Furthermore, in vivo experiments also confirmed the DCLK1-S–mediated enhancement of tumorigenesis and metastasis. In conclusion, this is the first study to demonstrate that DCLK1-S regulates the proliferation and invasion of ESCC cells in vitro and in vivo and our findings provide a basis for DCLK1-S as a therapeutic target for patients with ESCC.

EMT, an important process in embryonic development and wound healing, is commonly activated in tumorigenesis and metastasis of primary tumors (31, 32). In recent years, the effect of DCLK1 on EMT has been observed in many cancers (9, 10, 20, 33). Our findings show that DCLK1-S is closely associated with EMT-related genes in patients with ESCC and that the expression of E-cadherin, ZEB1, Slug, and Snail are regulated by DCLK1-S. Notably, DCLK1-S overexpression or rescue of DCLK1-S in KO cell lines lead to the activation/reactivation of the EMT phenotype in ESCC cells. These findings suggest that DCLK1-S may be an important inducer of EMT in ESCC.

Because of the complexity of tumorigenesis and metastasis, the potential mechanism of DCLK1-S in regulating ESCC proliferation and invasion and EMT, a downstream process induced by various transcription factors, remains to be further elucidated. It has been reported that Wnt/β-catenin signaling (34), TGFβ (35), and MAPK/ERK (36, 37) are key pathways involved in tumor progression. In our study, our bioinformatics analysis from TCGA ESCC database showed that DCLK1 is closely associated with the MAPK/ERK pathway in patients with ESCC. Further RNA-seq and KEGG analysis of DCLK1-KO and DCLK1-S rescue cells showed the most significant enrichment in the MAPK signaling pathway. Consistent with this result, we found that silencing DCLK1 in ESCC can block the ERK1/2 pathway, while DCLK1-S rescue increased ERK phosphorylation levels and activated ERK. In addition, MMP2 is one of the most important members of matrix metalloproteinases (38) and a key enzyme involved in proteolysis of the main components of basement membrane which has been reported to be involved in ESCC invasion and metastasis (39, 40). Findings show that ERK pathway can stimulate the expression of MMP2, which further affects the EMT process in tumor cells (24, 41). In this study, we found that changes in DCLK1-S levels altered the expression of MMP2 through the ERK1/2 pathway. This may in part explain the effect of DCLK1-S on the proliferation, migration, and invasion of ESCC cells. To further verify this phenomenon, we treated DCLK1-S overexpression cells with SCH772984, an ERK1/2 pathway inhibitor, and found that proliferation, migration, and the EMT phenotype of ESCC cells induced by DCLK1-S overexpression were significantly reversed. Taken together, these results suggest that the DCLK1-S higher expression in ESCC may trigger the activation of ERK1/2 pathway, leading to the expression of downstream gene MMP2, thus promoting tumor progression and metastasis, as shown in Fig. 6E.

In conclusion, we found that the expression of DCLK1-S was increased in patients with ESCC and associated with poor patient survival. Our results suggest that abnormal DCLK1-S expression may be involved in the carcinogenesis of ESCC by promoting malignant proliferation and regulating migratory and invasive capabilities. In addition, DCLK1-S expression activates MAPK/ERK/MMP2 pathway, revealing a potential driver pathway for EMT in ESCC. Taken together, we believe that DCLK1-S will be an important prognostic marker and a potential target for ESCC clinical therapy.

No disclosures were reported.

Y. Ge: Supervision. X. Fan: Formal analysis, writing–original draft. X. Huang: Methodology. N. Weygant: Writing–review and editing. Z. Xiao: Software. R. Yan: Methodology. H. Liu: Validation. J. Liu: Validation. G. An: Resources, supervision. J. Yao: Writing–review and editing.

This work was supported by National Natural Science Foundation of China (82003057, to J. Yao), and National Natural Science Foundation of China (81802738, to Y. Ge).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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