Doublecortin-like Kinase 1 Overexpression Research Articles

Targeting the PI3K/AKT/mTOR pathway offer a promising therapeutic strategy for cholangiocarcinoma patients with high doublecortin-like kinase 1 expression

BackgroundCholangiocarcinoma (CCA), characterized by high heterogeneity and extreme malignancy, has a poor prognosis. Doublecortin-like kinase 1 (DCLK1) promotes a variety of malignant cancers in their progression. Targeting DCLK1 or its associated regulatory pathways can prevent the generation and deterioration of several malignancies. However, the role of DCLK1 in CCA progression and its molecular mechanisms remain unknown. Therefore, we aimed to investigate whether and how DCLK1 contributes to CCA progression.MethodsThe expression of DCLK1 in CCA patients was detected using Immunohistochemistry (IHC). We established DCLK1 knockout and DCLK1 overexpression cell lines for Colony Formation Assay and Transwell experiments to explore the tumor-promoting role of DCLK1. RT-PCR, Western blot and multiple fluorescent staining were used to assess the association between DCLK1 and epithelial–mesenchymal transition (EMT) markers. RNA sequencing and bioinformatics analysis were performed to identify the underlying mechanisms by which DCLK1 regulates CCA progression and the EMT program.ResultsDCLK1 was overexpressed in CCA tissues and was associated with poor prognosis. DCLK1 overexpression facilitated CCA cell invasion, migration, and proliferation, whereas DCLK1 knockdown reversed the malignant tendencies of CCA cells, which had been confirmed both in vivo and in vitro. Furthermore, we demonstrated that DCLK1 was substantially linked to the advancement of the EMT program, which included the overexpression of mesenchymal markers and the downregulation of epithelial markers. For the underlying mechanism, we proposed that the PI3K/AKT/mTOR pathway is the key process for the role of DCLK1 in tumor progression and the occurrence of the EMT program. When administered with LY294002, an inhibitor of the PI3K/AKT/mTOR pathway, the tumor’s ability to proliferate, migrate, and invade was greatly suppressed, and the EMT process was generally reversed.ConclusionsDCLK1 facilitates the malignant biological behavior of CCA cells through the PI3K/AKT/mTOR pathway. In individuals with cholangiocarcinoma who express DCLK1 at high levels, inhibitors of the PI3K/AKT/mTOR signaling pathway may be an effective therapeutic approach.

Role of DCLK1/Hippo pathway in type II alveolar epithelial cells differentiation in acute respiratory distress syndrome

BackgroundDelay in type II alveolar epithelial cell (AECII) regeneration has been linked to higher mortality in patients with acute respiratory distress syndrome (ARDS). However, the interaction between Doublecortin-like kinase 1 (DCLK1) and the Hippo signaling pathway in ARDS-associated AECII differentiation remains unclear. Therefore, the objective of this study was to understand the role of the DCLK1/Hippo pathway in mediating AECII differentiation in ARDS.Materials and methodsAECII MLE-12 cells were exposed to 0, 0.1, or 1 μg/mL of lipopolysaccharide (LPS) for 6 and 12 h. In the mouse model, C57BL/6JNarl mice were intratracheally (i.t.) injected with 0 (control) or 5 mg/kg LPS and were euthanized for lung collection on days 3 and 7.ResultsWe found that LPS induced AECII markers of differentiation by reducing surfactant protein C (SPC) and p53 while increasing T1α (podoplanin) and E-cadherin at 12 h. Concurrently, nuclear YAP dynamic regulation and increased TAZ levels were observed in LPS-exposed AECII within 12 h. Inhibition of YAP consistently decreased cell levels of SPC, claudin 4 (CLDN-4), galectin 3 (LGALS-3), and p53 while increasing transepithelial electrical resistance (TEER) at 6 h. Furthermore, DCLK1 expression was reduced in isolated human AECII of ARDS, consistent with the results in LPS-exposed AECII at 6 h and mouse SPC-positive (SPC+) cells after 3-day LPS exposure. We observed that downregulated DCLK1 increased p-YAP/YAP, while DCLK1 overexpression slightly reduced p-YAP/YAP, indicating an association between DCLK1 and Hippo-YAP pathway.ConclusionsWe conclude that DCLK1-mediated Hippo signaling components of YAP/TAZ regulated markers of AECII-to-AECI differentiation in an LPS-induced ARDS model.Graphical

Abstract 12723: Smooth Muscle-Derived CD34 + Cells After Vascular Injury Contribute to Vascular Remodeling

Introduction: Vascular remodeling after injury participates in several atherosclerotic cardiovascular diseases. Vascular progenitors are thought to play vital roles in vascular remodeling, while the specific contribution of newly-generated progenitors from non-progenitors after injury is neglected. Methods: We generated a Cd34 lineage tracing system and marked newly-generated CD34 + cells in vascular injury models. Single-cell transcriptomes revealed cellular changes of CD34 + cells during vascular remodeling. To explore the cell origin of these CD34 + cells, we applied bone marrow (BM) transplantation, pseudo-time analysis of femoral artery cells, and further generated a smooth muscle (SM) lineage tracing system. To examine the role of Doublecortin Like Kinase 1 (DCLK1) in SM-derived CD34 + cell generation, overexpression of DCLK1 in primary VSMCs and genetic knockout of DCLK1 in SM lineage tracing system was performed. Results: A cluster of CD34 + cells was identified in femoral artery wire injury, which were CD34 negative before injury but CD34 positive after injury. These newly-generated CD34 + cells co-expressed VSMC or immune cell markers in neointima. BM transplantation clarified that vascular resident newly-generated CD34 + cells expressed VSMC makers or CD68, while BM CD34 + cells turned to immune cells but not VSMCs in neointima. These non-BM newly-generated CD34 + cells partly derived from VSMCs after injury. DCLK1 expression increased along the VSMCs-to-CD34 + cells transition. DCLK1 overexpression in VSMCs increased progenitor cell marker expression and decreased contractile VSMC marker expression. Genetic depletion of DCLK1 in VSMCs impaired SM-derived CD34 + cell generation during vascular remodeling. Conclusions: A novel cluster ‘SM-derived CD34 + cells’ was defined after vascular injury. These findings enriched our knowledge in pathological process of vascular remodeling and provided an avenue for future clinical interventions.

Doublecortin-like kinase 1 activates Hippo pathway to promote migration, invasion and proliferation of pancreatic cancer cells

Objective: To explore the mechanism of Doublecortin-like kinase 1 (DCLK1) in promoting cell migration, invasion and proliferation in pancreatic cancer. Methods: The correlation between DCLK1 and Hippo pathway was analyzed using TCGA and GTEx databases and confirmed by fluorescence staining of pancreatic cancer tissue microarrays. At the cellular level, immunofluorescence staining of cell crawls and western blot assays were performed to clarify whether DCLK1 regulates yes associated protein1 (YAP1), a downstream effector of the Hippo pathway. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to analyze the expressions of YAP1 binding transcription factor TEA-DNA binding proteins (TEAD) and downstream malignant behavior-promoting molecules CYR61, EDN1, AREG, and CTGF. Transwell test of the DCLK1-overexpressing cells treated with the Hippo pathway inhibitor Verteporfin was used to examine whether the malignant behavior-promoting ability was blocked. Analysis of changes in the proliferation index of experimental cells used real-time label-free cells. Results: TCGA combined with GTEx data analysis showed that the expressions of DCLK1 and YAP1 molecules in pancreatic cancer tissues were significantly higher than those in adjacent tissues (P<0.05). Moreover, DCLK1was positively correlated with the expressions of many effectors in the Hippo pathway, including LATS1 (r=0.53, P<0.001), LATS2 (r=0.34, P<0.001), MOB1B (r=0.40, P<0.001). In addition, the tissue microarray of pancreatic cancer patients was stained with multicolor fluorescence, indicated that the high expression of DCLK1 in pancreatic cancer patients was accompanied by the up-regulated expression of YAP1. The expression of DCLK1 in pancreatic cancer cell lines was analyzed by the CCLE database. The results showed that the expression of DCLK1 in AsPC-1 and PANC-1 cells was low. Thus, we overexpressed DCLK1 in AsPC-1 and PANC-1 cell lines and found that DCLK1 overexpression in pancreatic cancer cell lines promoted YAP1 expression and accessible to the nucleus. In addition, DCLK1 up-regulated the expression of YAP1 binding transcription factor TEAD and increased the mRNA expression levels of downstream malignant behavior-promoting molecules. Finally, Verteporfin, an inhibitor of the Hippo pathway, could antagonize the cell's malignant behavior-promoting ability mediated by high expression of DCLK1. We found that the number of migrated cells with DCLK1 overexpressing AsPC-1 group was 68.33±7.09, which was significantly higher than 22.00±4.58 of DCLK1 overexpressing cells treated with Verteporfin (P<0.05). Similarly, the migration number of PANC-1 cells overexpressing DCLK1 was 65.66±8.73, which was significantly higher than 37.00±6.00 of the control group and 32.33±9.61 of Hippo pathway inhibitor-treated group (P<0.05). Meanwhile, the number of invasive cells in the DCLK1-overexpressed group was significantly higher than that in the DCLK1 wild-type group cells, while the Verteporfin-treated DCLK1-overexpressed cells showed a significant decrease. In addition, we monitored the cell proliferation index using the real-time cellular analysis (RTCA) assay, and the proliferation index of DCLK1-overexpressed AsPC-1 cells was 0.66±0.04, which was significantly higher than 0.38±0.01 of DCLK1 wild-type AsPC-1 cells (P<0.05) as well as 0.05±0.03 of DCLK1-overexpressed AsPC1 cells treated with Verteporfin (P<0.05). PANC-1 cells showed the same pattern, with a proliferation index of 0.77±0.04 for DCLK1-overexpressed PANC-1 cells, significantly higher than DCLK1-overexpressed PANC1 cells after Verteporfin treatment (0.14±0.05, P<0.05). Conclusion: The expression of DCLK1 is remarkably associated with the Hippo pathway, it promotes the migration, invasion, and proliferation of pancreatic cancer cells by activating the Hippo pathway.

KDM3A promotes oral squamous cell carcinoma cell proliferation and invasion via H3K9me2 demethylation-activated DCLK1.

Oral squamous cell carcinoma (OSCC) is a frequently-diagnosed malignancy with high potential for proliferation and invasion. Histone methylation is known as a crucial mechanism that regulates pathological processes in various cancers, including OSCC. This study sought to delve into the molecular mechanism of lysine demethylase 3A (KDM3A) in OSCC cell proliferation and invasion. Expression levels of KDM3A, lysin-9 of di-methylated histone H3 (H3K9me2), and doublecortin-like kinase 1 (DCLK1) in cells were determined by reverse-transcription quantitative polymerase chain reaction or Western blot analysis. Cell proliferation and invasion were evaluated by cell counting kit-8, colony formation, and Transwell assays. The enrichment of KDM3A and H3K9me2 on the DCLK1 promoter was determined by chromatin immunoprecipitation assay. The functional rescue experiment was performed with DCLK1 overexpression vector and si-KDM3A in CAL-27 and SCC-9 cells. KDM3A was elevated in OSCC cells. KDM3A knockdown suppressed OSCC proliferation and invasion, along with increased H3K9me2 level in OSCC cells. KDM3A and H3K9me2 were enriched on the DCLK1 promoter and inhibiting H3K9me2 improved DCLK1 expression levels. DCLK1 overexpression neutralized the inhibition of KDM3A knockdown on OSCC proliferation and invasion. KDM3A facilitated OSCC proliferation and invasion by eliminating H3K9me2 to upregulate DCLK1 expression levels.

Cancer stem cell marker DCLK1 reprograms small extracellular vesicles toward migratory phenotype in gastric cancer cells.

Doublecortin-like kinase 1 (DCLK1) is a putative cancer stem cell marker, a promising diagnostic and prognostic maker for malignant tumors and a proposed driver gene for gastric cancer (GC). DCLK1 overexpression in a majority of solid cancers correlates with lymph node metastases, advanced disease and overall poor-prognosis. In cancer cells, DCLK1 expression has been shown to promote epithelial-to-mesenchymal transition (EMT), driving disruption of cell-cell adhesion, cell migration and invasion. Here, we report that DCLK1 influences small extracellular vesicle (sEV/exosome) biogenesis in a kinase-dependent manner. sEVs isolated from DCLK1 overexpressing human GC cell line MKN1 (MKN1OE -sEVs), promote the migration of parental (non-transfected) MKN1 cells (MKN1PAR ). Quantitative proteome analysis of MKN1OE -sEVs revealed enrichment in migratory and adhesion regulators (STRAP, CORO1B, BCAM, COL3A, CCN1) in comparison to MKN1PAR -sEVs. Moreover, using DCLK1-IN-1, a specific small molecule inhibitor of DCLK1, we reversed the increase in sEV size and concentration in contrast to other EV subtypes, as well as kinase-dependent cargo selection of proteins involved in EV biogenesis (KTN1, CHMP1A, MYO1G) and migration and adhesion processes (STRAP, CCN1). Our findings highlight a specific role of DCLK1-kinase dependent cargo selection for sEVs and shed new light on its role as a regulator of signaling in gastric tumorigenesis.

MicroRNA-223-5p suppresses the progression of nasopharyngeal carcinoma by targeting DCLK1.

The aim of the present study was to investigate the function of microRNA (miR)-223-5p in the malignant biological behavior of nasopharyngeal carcinoma (NPC) and elucidate the underlying molecular mechanism. The expression levels of miR-223-5p and doublecortin-like kinase 1 (DCLK1) were detected via reverse transcription-quantitative PCR analysis. Cell viability was evaluated using Cell Counting Kit-8 assay. Cell migration and invasion were measured via Transwell assays, while a luciferase reporter assay was conducted to identify the interaction between miR-223-5p and DCLK1. The results demonstrated that miR-223-5p expression was significantly downregulated, whereas DCLK1 expression was significantly upregulated in NPC tissues and cells. Moreover, both miR-223-5p overexpression and DCLK1 silencing markedly suppressed the progression of NPC. It was also observed that miR-223-5p directly targeted DCLK1 and decreased its expression. Furthermore, it was suggested that DCLK1 overexpression may partially reverse the suppressive effects of miR-223-5p on the progression of NPC. Collectively, the results of the present study indicated that miR-223-5p may suppress NPC progression by targeting DCLK1, thereby indicating a novel potential approach to the diagnosis and treatment of NPC.

XMD-17-51 Inhibits DCLK1 Kinase and Prevents Lung Cancer Progression.

Doublecortin-like kinase 1 (DCLK1) is a cancer stem cell marker that is highly expressed in various types of human cancer, and a protein kinase target for cancer therapy that is attracting increasing interest. However, no drug candidates targeting DCLK1 kinase have been developed in clinical trials to date. XMD-17-51 was found herein to possess DCLK1 kinase inhibitory activities by cell-free enzymatic assay. In non-small cell lung carcinoma (NSCLC) cells, XMD-17-51 inhibited DCLK1 and cell proliferation, while DCLK1 overexpression impaired the anti-proliferative activity of XMD-17-51 in A549 cell lines. Consequently, XMD-17-51 decreased Snail-1 and zinc-finger-enhancer binding protein 1 protein levels, but increased those of E-cadherin, indicating that XMD-17-51 reduces epithelial-mesenchymal transition (EMT). Furthermore, sphere formation efficiency was significantly decreased upon XMD-17-51 treatment, and XMD-17-51 reduced the expression of stemness markers such as β-catenin, and pluripotency factors such as SOX2, NANOG and OCT4. However, the percentage of ALDH+ cells was increased significantly following treatment with XMD-17-51 in A549 cells, possibly due to EMT inhibition. In combination, the present data indicated that XMD-17-51 inhibited DCLK1 kinase activity in a cell-free assay with an IC50 of 14.64 nM, and decreased DCLK1 protein levels, cell proliferation, EMT and stemness in NSCLC cell lines. XMD-17-51 has the potential to be a candidate drug for lung cancer therapy.

Doublecortin-like kinase 1 promotes hepatocyte clonogenicity and oncogenic programming via non-canonical \u03b2-catenin-dependent mechanism

Chronic liver injury is a risk factor for cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms that regulate the decision between normal injury repair and neoplastic initiation are unclear. Doublecortin-like kinase 1 (DCLK1), a tumor stem cell marker, is induced during cirrhosis and HCC. Here, we demonstrate that DCLK1-overexpressing primary human hepatocytes formed spheroids in suspension cultures. Spheroids derived from DCLK1-overexpressing hepatoma cells showed high level expression of active β-catenin, α-fetoprotein, and SOX9, suggesting that DCLK1 overexpression induces clonogenicity and dedifferentiated phenotypes in hepatoma cells. DCLK1 overexpression in hepatoma cells also increased phosphorylation of GSK-3β at Ser9. This was associated with an induction of a 48-kDa active β-catenin with a preserved hypophosphorylated N-terminus that interacted with nuclear TCF-4 resulting in luciferase reporter activity and cyclin D1 expression. DCLK1 downregulation inhibited 48-kDa β-catenin expression. The proteasome inhibitor bortezomib did not block the 48-kDa β-catenin, instead, caused a threefold accumulation, suggesting a proteasome-independent mechanism. Liver tissues from patients with cirrhosis and HCC revealed epithelial co-staining of DCLK1 and active β-catenin, and cleaved E-cadherin. Repopulated DCLK1-overexpressing primary human hepatocytes in humanized FRG mouse livers demonstrated active β-catenin. In conclusion, DCLK1 regulates oncogenic signaling and clonogenicity of hepatocytes by a novel non-canonical/atypical β-catenin-dependent mechanism.

Long non-coding RNA NEAT1 regulates Hodgkin's lymphoma cell proliferation and invasion via miR-448 mediated regulation of DCLK1.

To explore whether long non-coding RNA nuclear enriched abundant transcript 1 (lncRNA NEAT1) could regulate Hodgkin's lymphoma (HL) cell proliferation and invasion through miR-448, which could target doublecortin like kinase 1 (DCLK1) and mediate DCLK1 expression. Expressions of NEAT1, miR-448 and DCLK1 were evaluated by qRT-PCR or Western blot assay. Cell Counting Kit-8 (CCK-8) and transwell assay were utilized to detect cell proliferation and invasion capability in L428 cells respectively. The target relationship between NEAT1, miR-448 and DCLK1 was confirmed by Luciferase reporter assay. QRT-PCR results showed that NEAT1 expressed higher in HL tissues and cell lines than that in controls. In vitro experiments, NEAT1 downregulation could decrease cell proliferation and invasion capability in L428 cells. NEAT1 directly interacted with miR-448 and negatively regulated it. Moreover, DCLK1 was confirmed as a target of miR-448. DCLK1 expression was increased in L428 cells and positively regulated by NEAT1. NEAT1 overexpression upregulated the protein level of DCLK1 in L428 cells according to Western blot analysis. Additionally, DCLK1 overexpression could reverse the suppression on cell proliferation and invasion capability induced by NEAT1 knockdown or miR-448 overexpression. NEAT1 might be contributed to HL progression by promoting cell proliferation and invasion capability via miR-448 mediated DCLK1 expression.

DCLK1 promotes malignant progression of breast cancer by regulating Wnt/β-Catenin signaling pathway.

The study was aimed to investigate the expression of doublecortin-like kinase-1 (DCLK1) in breast cancer (BCa) tissues and cells and further study its association with clinicopathology and prognosis of BCa patients. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to examine the expression of DCLK1 in 44 BCa tumor tissues, as well as adjacent normal tissues. Also, the interplay between DCLK1 level and clinical data or the prognosis of BCa patients was analyzed. QRT-PCR was further used to verify the level of DCLK1 in BCa cell lines. In addition, the DCLK1 knockdown model was constructed using lentivirus in BCa cell lines. Next, cell counting kit-8 (CCK-8) and cell clone formation and tranwell assays were used to analyze the effect of DCLK1 on the biological function of BCa cells. Finally, it was explored whether DCLK1 can act through the Wnt/β-Catenin signaling pathway. In this research, qRT-PCR results revealed that the level of DCLK1 in BCa tumor tissues was remarkably higher than in adjacent tissues. Compared to patients with a low-level of DCLK1, the pathology grading in patients with high-level was higher and the overall survival rate was lower. Similarly, proliferation, as well as the invasion and migration ability of cells in DCLK1 knockdown group was remarkably down-regulated when compared to negative control group. Moreover, the Western Blot results revealed that silencing DCLK1 remarkably decreased the expression of key proteins in Wnt/β-Catenin pathways such as β-Catenin, c-myc, and cyclinD1, thereby promoting the malignant progression of BCa. In addition, the Wnt/β-Catenin pathway inhibitor was found to be able to reverse the impact of DCLK1 overexpression on BCa cell proliferative and metastatic capacity. DCLK1 expression was found remarkably increased in BCa tissues and closely associated with the pathological stage, as well as poor prognosis of BCa patients. Furthermore, DCLK1 may promote the malignant progression of BCa by inhibiting the Wnt/β-Catenin pathway.

Application of RNA-sequencing to identify transcriptome modification by DCLK1 in colorectal cancer cells.

Doublecortin like kinase 1 (DCLK1) is a cancer stem cell marker for the colorectal cancer (CRC). It plays critical roles in the oncogenesis, progression and metastasis of CRC. DCLK1 can be an intriguing therapeutic target for CRC treatment. However, the molecular mechanism of how DCLK1 functions is unclear currently. In our research, we aim to apply RNA-Sequencing (RNA Seq) technology, a high throughput massively Next Generation Sequencing approach, to monitor transcriptome changes due to DCLK1 over-expression in the CRC cells. In order to achieve our goal, RNA from quadruplicate samples from two clones of isogenic DCLK1 stable over-expression cells and the parental wild type HCT116 cells was sent for RNA Seq on the Illumina NextSeq500 platform. Differentially expressed (DE) genes were evaluated by t-test (P <0.05 and fold-change ±1.5 or greater) using two methods: (1) FWER; and (2) Benjamani and Hochberg FDR (false discovery rate) which corrects for multiple comparisons. Gene networks and functional analysis were evaluated using Ingenuity Pathways Analysis (IPA). We identified 1463 DE genes common for both DCLK1 overexpression clone A and clone B cells. IPA results indicated that 72 canonical pathways were significantly modified by DCLK1 over-expression (P<0.05), among which 9 out of the top 10 pathways are involved in the cell cycle regulation, indicating that DCLK1 might play its tumorigenesis role via activation of pathways facilitating cell proliferation, repression of pathways inhibiting cells proliferation and function against pathways facilitating cell apoptosis. Cell cycle analysis results confirmed the IPA findings, which demonstrated that DCLK1 over-expression cells had much less G0/G1 cells but much more S and G2/M cells (P<0.05). In conclusion, DCLK1 over-expression significantly modified transcriptome profile of CRC cancer cells. Control of the cell cycle regulation might be one of the critical mechanism for DCLK1 function. Our findings provide more direct evidence for the development of DCLK1 as a therapeutic target for CRC treatment, and will be of great benefit for the discovery of novel therapeutic target within the DCLK1 molecular network for the treatment of colorectal cancer patients.

Abstract 4673: DCLK1 promotes hepatocellular carcinoma via atypical β-catenin-regulated signaling and immune suppression

Abstract Introduction: Genetic lineage tracing in mouse model has revealed that mature hepatocytes are the cell of origin of injury-induced hepatocellular carcinoma (HCC), the second most common cause of cancer-related deaths worldwide. Hepatocyte plasticity, defined as the ability to change cellular identity, plays a critical role in the repair process following liver injury. Inflammatory liver diseases often lead to cirrhosis and HCC. The molecular mechanisms that regulate the decision between hepatocyte injury repair and neoplastic initiation are unclear. Doublecortin-like kinase 1 (DCLK1), a microtubule-associated kinase, is expressed during liver injury, cirrhosis, and HCC, but not in normal liver. Here, we demonstrate a novel mechanism by which DCLK1 regulates injury-associated hepatocyte plasticity and immunosuppression in HCC. Methods: Cell culture, DEN/CCl4-induced injury in mouse livers, and humanized mouse model were used for analyzing hepatocyte plasticity and DCLK1+ cells-induced changes in hepatic cells and macrophages. Results: DCLK1-overexpressing primary human hepatocytes formed spheroids in 3D culture. DCLK1-overexpressing hepatoma cells exhibited a 48-kDa active β-catenin with preserved hypophosphorylated N-terminus, which interacted with TCF-4, resulting in enhanced luciferase reporter activity and cyclin D1 expression. Downregulation of DCLK1 using siRNAs inhibited expression of this β-catenin fragment. DCLK1 overexpression increased phosphorylation of GSK-3β at Ser9, thus preventing its complete proteasomal degradation. Liver tissues from patients with cirrhosis and HCC expressed the 48-kDa active β-catenin, DCLK1, cyclin D1, and 80-kDa E-cadherin ectodomain, suggesting a change in epithelial polarity. DCLK1-overexpressing primary human hepatocytes transplanted into FRG mice demonstrated β-catenin activation in the transplanted hepatocytes. Injury-induced DCLK1+ epithelial cells in mouse livers expressed PD-L1. DCLK1+ human hepatoma cells induced CD206 in macrophages in a dual co-culture assay. Conclusions: DCLK1 enhances hepatocyte plasticity, clonogenicity, and tumorigenic signaling via atypical β-catenin-dependent mechanism. DCLK1+ cells show ability to suppress the host anti-tumor immunity by expressing PD-L1 and inducing macrophage polarization. Citation Format: Naushad Ali, Parthasarathy Chandrakesan, Michael S. Bronze, Courtney W. Houchen. DCLK1 promotes hepatocellular carcinoma via atypical β-catenin-regulated signaling and immune suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4673.

The Essential Role of DCLK1 in Pathogenesis, Diagnostic Procedures and Prognostic Stratification of Colorectal Cancer.

Colorectal cancer (CRC) is the second most prevalent type of cancer among males and the third among females. CRC recurrence and poor prognosis may be related to the prevalence of chemotherapy-resistant cancer stem cells (CSCs). Recent studies have indicated the role of doublecortin-like kinase 1 (DCLK1) protein as a marker of CSC in CRC. This review focuses on the role of DCLK1 in CRC. Long-lived DCLK1-positive tuft cells can function as cancer-initiating cells. Numerous studies have shown DCLK1 overexpression to be significantly correlated with the stage of disease, the presence of metastasis and poor survival rate. DCLK1 may also be used to identify patients at high risk and those with chemotherapy-resistant tumors. DCLK1-specific drugs are examined as potential cancer treatments.

DCLK1 Plays a Metastatic-Promoting Role in Human Breast Cancer Cells.

Background Doublecortin-like kinase 1 (DCLK1) has been universally identified as a cancer stem cell (CSC) marker and is found to be overexpressed in many types of cancers including breast cancer. However, there is little data regarding the functional role of DCLK1 in breast cancer metastasis. In the present study, we sought to investigate whether and how DCLK1 plays a metastatic-promoting role in human breast cancer cells. Methods We used Crispr/Cas9 technology to knock out DCLK1 in breast cancer cell line BT474, which basically possesses DCLK1 at a higher level, and stably overexpressed DCLK1 in another breast cancer cell line, T47D, that basically expresses DCLK1 at a lower level. We further analyzed the alterations of metastatic characteristics and the underlying mechanisms in these cells. Results It was shown that, compared with the corresponding control cells, DCLK1 overexpression led to an increase in metastatic behaviors including enhanced migration and invasion of T47D cells. By contrast, forced depletion of DCLK1 drastically inhibited these metastatic characteristics in BT474 cells. Mechanistically, the epithelial-mesenchymal transition (EMT) program, which is critical for cancer metastasis, was prominently activated in DCLK1-overexpressing cancer cells, evidenced by a decrease in an epithelial marker ZO-1 and an enhancement in several mesenchymal markers including ZEB1 and Vimentin. In addition, DCLK1 overexpression induced the ERK MAPK pathway, which resultantly enhanced the expression of MT1-MMP that is also involved in cancer metastasis. Knockout of DCLK1 could reverse these events, further supporting a metastatic-promoting role for DCLK1. Conclusions Collectively, our data suggested that DCLK1 overexpression may be responsible for the increased metastatic features in breast cancer cells. Targeting DCLK1 may become a therapeutic option for breast cancer metastasis.

Doublecotin-Like Kinase 1 Increases Chemoresistance of Colorectal Cancer Cells through the Anti-Apoptosis Pathway.

Colorectal Cancer (CRC) is the third most common cancer diagnosed and the second leading cause of cancer-related deaths in the United States. Cancer Stem Cells (CSCs) are believed to be the primary reason for the recurrence of CRC. Specific stem cell marker, doublecortin-like kinase 1 (DCLK1) plays critical roles in the tumorigenesis and progression of CRC. Up-regulation of DCLK1 is correlated with poor prognosis. Whether DCLK1 is correlated with enhanced chemoresistance of CRC cells is unclear. We aim to reveal the association of DCLK1 with chemoresistance of CRC cells and the underlying molecular mechanisms. Stable DCLK1 over-expression cells (DCLK1+) were established using the HCT116 cells (WT). DCLK1+ and WT cells were treated with 5-Fluorouracil (5-Fu) at different doses for 24 or 48 hours. MTT assay was used to evaluate cell viability and IC50 of 5-Fu was determined. Quantitative real-time PCR was applied to determine the gene expression of caspase-3 (casp-3), casp-4, and casp-10. Cleaved casp-3 expression was investigated using Western blot and immunofluorescence. Our results demonstrated that IC50 of 5-Fu for the DCLK1+ cells was significantly higher than that of the WT cells for both 24 and 48-hour treatment (p=0.002 and 0.048 respectively), indicating increased chemoresistance of the DCLK1+ cells. Gene expression of casp-3, casp-4, and casp-10 were significantly inhibited in the DCLK1+ cells after 5-Fu treatment compared to the WT cells (p=7.616e-08, 1.575e-05 and 5.307e-08, respectively). Cleaved casp-3 amount and casp-3 positive cells were significantly decreased in the DCLK1+ cells after 5-Fu treatment compared to the WT cells (p=0.015). In conclusion, our results demonstrated that DCLK1 overexpression enhanced the chemoresistance of CRC cells to 5-Fu treatment by suppressing gene expression of key caspases in the apoptosis pathway and activation of the apoptosis pathway. DCLK1 can be an intriguing therapeutic target for the effective treatment of CRC patients.

MicroRNA-424 inhibits cell migration, invasion, and epithelial mesenchymal transition by downregulating doublecortin-like kinase 1 in ovarian clear cell carcinoma

Doublecortin-like kinase 1 (DCLK1) is overexpressed in many cancers and acts as a tumor stem cell marker. Here, we investigated the role of DCLK1 and microRNA-424 (miR-424) in ovarian clear cell carcinoma (OCCC), a histopathologically distinct subtype of epithelial ovarian cancer associated with poor prognosis and chemotherapy resistance. Analysis of samples from 30 OCCC patients showed that DCLK1 was upregulated and miR-424 was downregulated in tumors compared with adjacent non-tumor tissues. DCLK1 overexpression promoted OCCC cell proliferation, migration, and invasion, whereas DCLK1 knockdown reduced cell viability and invasion and induced growth arrest in vitro and in vivo. Dual-luciferase reporter assays revealed that miR-424 directly targets DCLK1 and downregulates its expression. Transfection of ES-2 cells with miR-424 mimics downregulated DCLK1 and suppressed the effects of DCLK1 overexpression on upregulating matrix metalloprotease-9 and promoting epithelial-mesenchymal transition (EMT). Taken together, these data demonstrate that miR-424 has the capacity to suppress cell invasion and EMT in OCCC by downregulating DCLK1, suggesting potential therapeutic targets and strategies for the treatment of this disease.

DCLK1 is up-regulated and associated with metastasis and prognosis in colorectal cancer.

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. 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. 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. 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.

Abstract 2493: Knocking down the expression of DCLK1 reduces mammary tumor formation, tumor cell self-renewal and metastasis: DCLK1 a novel therapeutic target in breast cancer

Abstract Introduction: Breast cancer is a heterogeneous disease, and is comprised of a rare population of cells that display stem cell-like properties with high metastatic potential and relative resistance to conventional therapies. Doublecortin-like Kinase 1 (DCLK1) has recently been identified as a tumor stem cell marker in the intestine and pancreas and is overexpressed in many cancers including breast cancer. In this study, we targeted DCLK1 as a novel therapeutic strategy in breast cancer cells to reduce cancer cell self-renewal, tumorigenesis and metastasis. Methods: Anti-DCLK1-siRNA was transfected into breast cancer cell lines (MCF7 [luminal] and MDAMB231 [basal-triple negative breast cancer cell line]). For analysis of circulating tumor cells, MDAMB231/MCF7 cells were stably transfected with RFP (red fluorescence protein). Tumor cell colony formation, mammosphere formation (self-renewal), cell migration and invasion were assessed to evaluate the effect of DCLK1 knockdown and or DCLK1 overexpression on breast cancer cells self-renewal and metastatic potential. Xenograft model was used to identify the role of DCLK1 on breast cancer cell growth and circulating tumor cells in vivo. The data were generated utilizing experimental protocols for clonogenic culture, circulating tumor cells, immunohistochemistry, RT-qPCR and Western blotting. Results: Expression of DCLK1 was ∼36% higher in human breast cancer tissues than in normal breast tissue. Silencing DCLK1 via RNA interference decreased the colony formation (∼70%), and self-renewal ability (∼85%), of the breast cancer cell lines in vitro. In vitro migration and invasion assays demonstrated that knocking down DCLK1 led to less (40-80%) breast cancer cells migration and invasion. Furthermore, expression of EMT associated factors (Slug, Snail, Twist, Zeb-1, Zeb-2 and Vimentin) and pluripotency factors (c-Myc, Sox2, Nanog and Oct-4) were markedly reduced between 30 and 50%, after silencing DCLK1. Inhibition of DCLK1 in xenograft tumors resulted in reduced tumor growth (∼81%) and CTCs (∼95%) in vivo. However, overexpression of DCLK1 in the non-metastatic MCF7 breast cancer cell line increased its in vitro migration and invasion potency and pluripotency along with in vivo tumor formation and progression. Conclusions: DCLK1 expression is associated with breast cancer cell self-renewal, metastasis and tumorigenesis. Targeting DCLK1 reduced breast cancer cell self-renewal, CTCs, metastasis and tumor growth. DCLK1 inhibition may represent a novel targeted therapeutic approach to reduce the morbidity and mortality associated with breast cancer. Citation Format: PARTHASARATHY CHANDRAKESAN, Nathaniel Weygant, Dongfeng Qu, William Berry, Randal May, Naushad Ali, Sripathi Sureban, Eddie Bannerman-Menson, Michael Schlosser, Courtney Houchen. Knocking down the expression of DCLK1 reduces mammary tumor formation, tumor cell self-renewal and metastasis: DCLK1 a novel therapeutic target in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2493.

Abstract 1728: DCLK1 promotes tumor growth and invasion through Slug-mediated epithelial-mesenchymal transition in pancreatic neuroendocrine tumors

Abstract Introduction: Accumulating evidence suggests that Doublecortin-like kinase 1 (DCLK1) is a putative marker for intestinal and pancreatic cancer stem cells. (Nakanishi et al., Nat Genet 2013, Bailey et al., Gastroenterology 2013). Recently, we have reported that DCLK1 was highly and diffusely expressed in human rectal neuroendocrine tumors (NETs) (Ikezono et al., Oncol Lett, 2015). However, the function of DCLK1 has not yet been investigated in detail. Aims: The aims of the present study were to assess expression levels of DCLK1 in pancreatic NET (PNET) tissues and to identify critical functions of this molecule in PNET cells. Methods: Fifteen patients (8 male, 7 female; mean age, 56) with PNETs were enrolled in this study. The tumors were surgically resected between 1997 and 2012 in Kurume University Hospital. Immunohistochemistry (IHC) was employed to assess expression levels of DCLK1. QGP1, a human PNET cell line, was used in this study, and the cells were transfected with dclk1 cDNA to establish the DCLK1-overexpressing (QGP1-DOE) cells. The QGP1-DOE cells were subjected to dclk1 silencing to confirm acquired cellular characteristics by DCLK1 overexpression. Protein and mRNA expression levels were analyzed by Western blot and real-time PCR (ABI PRISM 7700), respectively. Results: In IHC, all of the 15 PNETs clearly and diffusely expressed DCLK1 in the tumor areas. QGP1-DOE cells robustly expressed full length of DCLK1, showing epithelial-mesenchymal transition (EMT)-like appearance. Of note, extremely high expression of the EMT regulator Slug was found in QGP1-DOE cells compared with control cells at both protein and mRNA levels. Expressions of N-cadherin and Vimentin were also upregulated, in concert with those of phospholylated (p-) FAK, Paxillin, and cyclin D1, in the QGP1-DOE cells. These cells increased cellular motility and proliferation. DCLK1 knockdown restored the above-mentioned expression profile of the EMT-associated molecules. Conclusion: We demonstrated robust expression of DCLK1 in human PNET tissues and PNET cells for the first time. Enforced expression of DCLK1 induced EMT via upregulating Slug expression and promoted proliferation and invasion probably through increasing expression levels of cyclin D1, pFAK, and Paxillin. Therefore, it is speculated that inhibition of DCLK1 expression is a novel therapeutic strategy for PNETs. Citation Format: Yu Ikezono, Hironori Koga, Jun Akiba, Mitsuhiko Abe, Fumitaka Wada, Toru Nakamura, Hideki Iwamoto, Atsutaka Masuda, Takahiko Sakaue, Hirohisa Yano, Osamu Tsuruta, takuji Torimura. DCLK1 promotes tumor growth and invasion through Slug-mediated epithelial-mesenchymal transition in pancreatic neuroendocrine tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1728.