DCLK1 Expression Research Articles

Succinate‐Induced Tuft Cell Hyperplasia Enhances FFA2/5‐HT Mediated Anion Secretion in Mouse Duodenum

Tuft cells are one of luminal chemosensing components in the small intestine, while the role of tuft cell in mucosal secretory responses to luminal chemicals such as short-chain fatty acids (SCFA) has not been elucidated. We thus hypothesized that tuft cell hyperplasia may increase the secretory responses to luminal SCFA due to the increased cholinergic signals since tuft cells express choline acetyltransferase (ChAT). Tuft cell hyperplasia was induced by oral sodium succinate (NaSuc, 100 mM) in the drinking water for 7 days. Sodium propionate (NaP, 10 mM, m), the selective free fatty acid receptor 2 (FFA2) agonist 4-CMTB (10 µM, s), or the TRPA1 agonist allyl isothiocyanate (AITC, 1 mM, m) were applied to the mucosal (m) or serosal (s) baths of an Ussing chamber mounted with muscle-stripped mucosa-submucosa preparations of mouse proximal duodenum or jejunum. The density of tuft cells (doublecortin-like kinase 1; DCLK1), enteroendocrine L cells (proglucagon, glucagon-like peptide-2), and enterochromaffin (EC) cells (5-HT, tryptophan hydroxylase 1; TPH1) were assessed by real-time PCR and immunostaining. NaSuc treatment significantly increased the number of DCLK1-positive tuft cells in the duodenal and jejunal villi. mRNA expression of DCLK1 and FFA2 were increased in the duodenal and jejunal mucosa, whereas TPH1 was increased only in the duodenal mucosa, and ChAT was increased in the jejunal mucosa, but proglucagon was unchanged in both. 5-HT-positive mast cell density rather than EC cell density was increased in the duodenum. Isc in response to luminal NaP was enhanced in the jejunum, which was atropine sensitive. In contrast, Isc responses to serosal 4-CMTB and luminal AITC were enhanced in the duodenum, which were mediated by 5-HT3 and 5-HT4 receptors. These results suggest that NaSuc differentially enhances secretory responses, with 5-HT signals in the duodenum and with cholinergic signals in the jejunum. These data may be implicated in the pathogenesis of functional dyspepsia (FD), since increased duodenal mast cell density and enhanced 5-HT signals are present in FD patients.

Celastrol and Triptolide Suppress Stemness in Triple Negative Breast Cancer: Notch as a Therapeutic Target for Stem Cells.

Triple negative breast cancer (TNBC) is observed in ~15% of breast cancers and results in poor survival and increased distant metastases. Within the tumor are present a small portion of cancer stem cells that drive tumorigenesis and metastasis. In this study, we aimed to elucidate whether the two natural compounds, celastrol and triptolide, inhibit stemness in TNBC. MDA-MB-231, BT20, and a patient-derived primary cells (PD-TNBC) were used in the study. Mammosphere assay was performed to assess the stemness. Both celastrol and triptolide treatment suppressed mammosphere formation. Furthermore, the compound suppressed expression of cancer stem cell marker proteins DCLK1, ALDH1, and CD133. Notch signaling plays a critical role in stem cells renewal. Both celastrol or triptolide reduced Notch -1 activation and expression of its downstream target proteins HES-1 and HEY-1. However, when NICD 1 was ectopically overexpressed in the cells, it partially rescued proliferation and mammosphere formation of the cells, supporting the role of notch signaling. Together, these data demonstrate that targeting stem cells and the notch signaling pathway may be an effective strategy for curtailing TNBC progression.

DCLK1, a promising colorectal cancer stem cell marker, regulates tumor progression and invasion through miR-137 and miR-15a dependent manner.

Cancer stem cells (CSCs) are thought to be a major player in tumor initiation, progression, and metastasis. Targeting CSCs for elimination presents a promising therapeutic strategy; however, this approach will require a stronger understanding of CSC biology and identification of CSC-specific markers. The present study was conducted to examine the correlation between DCLK1 and miR-137 and miR-15a levels in colorectal cancer. A total of 222 samples, including 181 colorectal cancer specimens, 24 adenomatosis, and 17 non-adenomatosis colonic polyps, were stained for DCLK1 expression using immunohistochemistry. Also, expression of miR-137 and miR-15a was assessed in colorectal cancer with high and low DCLK1 expression levels. Most colorectal cancer specimens (76%) showed strong expression of DCLK1, whereas only 21% of adenomatous and none of non-adenomatous colonic polyps showed strong DCLK1 expression. A significant difference in DCLK1 expression was found between colorectal cancer, adenomatous, and non-adenomatous colonic polyps (P < 0.001). Higher expression of DCLK1 was more frequently detected in colorectal cases with larger tumor size (P = 0.03), poor differentiation (P = 0.03), and lymph node involvement (P = 0.04). Comparison of miR-137 and miR-15a in colorectal cancer cases revealed a significant inverse correlation with DCLK1 expression (P = 0.03 and P = 0.04, respectively). DCLK1 may act as a candidate marker for colorectal cancer stem cells. The critical role of DCLK1 in colorectal cancer suggests that it may represent an early diagnostic marker and therapeutic target; however, further investigation is warranted.

Abstract 3829: Dclk1 expressing crypt epithelial cells become tumor initiating stem cells in an inflammation associated environment

Abstract Background: Chronic intestinal inflammation is a major risk factor for the development of CRC and consequently individuals suffering from inflammatory bowel diseases (IBD) are at increased risk. However, the molecular mechanism by which inflammation initiates colonic tumorigenesis in the cell type is unclear and the regulatory factors that mediate the complex interplay between inflammation and the colonic tumor initiation are unknown. Doublecortin-like kinase 1 (Dclk1) is a microtubule-associated kinase, which marks TSCs in colon cancer, and is predominantly expressed in a unique intestinal epithelial cell type termed the tuft cells. Tuft cells are chemosensory cells that regulate host immunity and inflammation and is highly increased in CRC and, ablation of Dclk1 expressing cells in the ApcMin/+ mice completely regressed the tumors without affecting the normal stem cell population. Our aim is to elucidate the molecular mechanism that is responsible for inflammation-associated colon cancer and to determine the role of Dclk1 expressing tuft cells in this process. Experimental Procedure: YMCA non-malignant colonic epithelial cells were used. Loss and gain of Dclk1 expression in the YMCA cells were carried with siRNA transfection and lentivirus overexpression vector infection respectively. Dclk1 expressing cells were selected for stable cell generation. DSS and LPS were used. Cell self-renewal, secondary and tertiary organoid generation, survival, DNA damage, DNA damage response (DDR) and apoptosis were assessed. Protein and mRNA expressions by western-blot and RT-PCR. Results: Here, we report that increased expression of Dclk1 is the key to the generation of tumor-initiating cells under the influence of an inflammatory environment. We observed increased self-renewal of Dclk1+ YMCA cells treated with DSS and further increase in self-renewal was observed with DSS+LPS treatment. Silencing Dclk1 failed to initiate self-renewal under the influence of DSS and LPS treatment. Dclk1+ YMCA cells displayed enhanced inflammatory mediators, survival with colony formation and increased survival pathways (AKT/mTOR/Beta-catenin/NFkB pathways) and DDR (ATM/ATR) with DSS or LPS treatment. However, the DNA damage and cell apoptosis were not increased in vector control cells and significantly increased in Dclk1 knockdown cells after DSS or LPS treatment. Conclusion: Our data demonstrate that the increased expression of Dclk1 is associated with tumor-initiating features, and survival ability in an inflammatory environment, which is important to understand the cell type and inflammation associated mechanisms in tumor initiation. Based on these findings we proposed that specific targeting of Dclk1-expressing colonic crypt epithelial cells has the potential to result in ablation of tumor initiation. Citation Format: Parthasarathy Chandrakesan, Janani Panneerselvam, Dongfeng Qu, Chinthalapally Rao, Michael Bronze, Courtney Houchen. Dclk1 expressing crypt epithelial cells become tumor initiating stem cells in an inflammation associated environment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3829.

LncRNA SNHG1 promotes EMT process in gastric cancer cells through regulation of the miR-15b/DCLK1/Notch1 axis

BackgroundGastric cancer (GC) is a malignant tumour originating from the gastric mucosa epithelium that seriously threatens human health. DCLK1, miR-15b and lncRNA SNHG1 play potential roles in the occurrence of GC, but the mechanism remains unclear.MethodsGene expression of DCLK1, miR-15b and lncRNA SNHG1 was investigated by qRT-PCR. Protein expression was detected by Western blotting. Migration and invasion of gastric cancer cells was tested by a Transwell assay and wound healing assay. Cell proliferation was measured by an MTT assay. Finally, the correctness of the prediction results was confirmed by a dual-luciferase reporter assay.ResultsThe expression of DCLK1, Notch1, and SNHG1 was increased in GC tissues, while the expression of miR-15b was decreased. Overexpression of lncRNA SNHG1 promoted the expression of DCLK1 and Nothc1 in GC cells. Moreover, miR-15b targeted DCLK1 to regulate Notch1 expression and inhibited the EMT process in GC cells. SNHG1 enhanced the effects of DCLK1/Notch1 on the EMT process through regulating miR-15b expression.ConclusionSNHG1 enhances the EMT process in GC cells through DCLK1-mediated Notch1 pathway, which can be a potential target for treating GC.

Targeting the HGF/c-MET pathway in advanced pancreatic cancer: a key element of treatment that limits primary tumour growth and eliminates metastasis

BackgroundStromal–tumour interactions facilitate pancreatic cancer (PC) progression. The hepatocyte growth factor (HGF)/c-MET pathway is upregulated in PC and mediates the interaction between cancer cells and stromal pancreatic stellate cells (PSCs). This study assessed the effect of HGF/c-MET inhibition plus gemcitabine (G) on the progression of advanced PC.MethodsOrthotopic PC was produced by implantation of luciferase-tagged human cancer cells + human PSCs into mouse pancreas. Tumours were allowed to develop without treatment for 4 weeks. Mice were then treated for 6 weeks with one of the following: IgG, G, HGF inhibitor (Hi), c-MET inhibitor (Ci), Hi + Ci, Hi + G, Ci + G, or Hi + Ci + G.ResultsBioluminescence imaging showed similar tumour sizes in all mice at the initiation of treatments. Triple therapy (Hi + Ci + G): (1) completely eliminated metastasis; (2) significantly reduced tumour size as assessed by bioluminescence and at necropsy; (3) significantly reduced proliferating cancer cell density and stem cell marker DCLK1 expression in tumours. In vitro 3D culture studies supported our in vivo findings.ConclusionEven at an advanced disease stage, a two-pronged approach, targeting (a) HGF/c-MET with relevant inhibitors and (b) cancer cells with chemotherapy, completely eliminated metastasis and significantly decreased tumour growth, suggesting that this is a promising treatment approach for PC.

Suppressing STAT5 signaling affects osteosarcoma growth and stemness

Osteosarcoma (OS) is the most common primary bone tumor that primarily affects children and adolescents. Studies suggested that dysregulation JAK/STAT signaling promotes the development of OS. Cells treated with pimozide, a STAT5 inhibitor suppressed proliferation and colony formation and induced sub G0/G1 cell cycle arrest and apoptosis. There was a reduction in cyclin D1 and CDK2 expression and Rb phosphorylation, and activation of Caspase-3 and PARP cleavage. In addition, pimozide suppressed the formation of 3-dimensional osteospheres and growth of the cells in the Tumor in a Dish lung organoid system. Furthermore, there was a reduction in expression of cancer stem cell marker proteins DCLK1, CD44, CD133, Oct-4, and ABCG2. More importantly, it was the short form of DCLK1 that was upregulated in osteospheres, which was suppressed in response to pimozide. We further confirmed by flow cytometry a reduction in DCLK1+ cells. Moreover, pimozide inhibits the phosphorylation of STAT5, STAT3, and ERK in OS cells. Molecular docking studies suggest that pimozide interacts with STAT5A and STAT5B with binding energies of −8.4 and −6.4 Kcal/mol, respectively. Binding was confirmed by cellular thermal shift assay. To further understand the role of STAT5, we knocked down the two isoforms using specific siRNAs. While knockdown of the proteins did not affect the cells, knockdown of STAT5B reduced pimozide-induced necrosis and further enhanced late apoptosis. To determine the effect of pimozide on tumor growth in vivo, we administered pimozide intraperitoneally at a dose of 10 mg/kg BW every day for 21 days in mice carrying KHOS/NP tumor xenografts. Pimozide treatment significantly suppressed xenograft growth. Western blot and immunohistochemistry analyses also demonstrated significant inhibition of stem cell marker proteins. Together, these data suggest that pimozide treatment suppresses OS growth by targeting both proliferating cells and stem cells at least in part by inhibiting the STAT5 signaling pathway.

Cucurbitacin B and I inhibits colon cancer growth by targeting the Notch signaling pathway

Cancer stem cells (CSCs) have the ability to self-renew and induce drug resistance and recurrence in colorectal cancer (CRC). As current chemotherapy doesn’t eliminate CSCs completely, there is a need to identify novel agents to target them. We investigated the effects of cucurbitacin B (C-B) or I (C-I), a natural compound that exists in edible plants (bitter melons, cucumbers, pumpkins and zucchini), against CRC. C-B or C-I inhibited proliferation, clonogenicity, induced G2/M cell-cycle arrest and caspase-mediated-apoptosis of CRC cells. C-B or C-I suppressed colonosphere formation and inhibited expression of CD44, DCLK1 and LGR5. These compounds inhibited notch signaling by reducing the expression of Notch 1–4 receptors, their ligands (Jagged 1-2, DLL1,3,4), γ-secretase complex proteins (Presenilin 1, Nicastrin), and downstream target Hes-1. Molecular docking showed that C-B or C-I binds to the ankyrin domain of Notch receptor, which was confirmed using the cellular thermal shift assay. Finally, C-B or C-I inhibited tumor xenograft growth in nude mice and decreased the expression of CSC-markers and notch signaling proteins in tumor tissues. Together, our study suggests that C-B and C-I inhibit colon cancer growth by inhibiting Notch signaling pathway.

176P - DCLK1 promotes tumour invasion and metastasis through epithelial-mesenchymal transition and the MEK/ERK pathway in esophageal squamous cell carcinoma

BackgroundDoublecortin-like kinase1 (DCLK1), a recently discovered cancer stem cell (CSC) marker in the colon and pancreatic cancer, contributes greatly to the malignancy of gastrointestinal cancers. Also, higher DCLK1 expression is reported in esophageal adenocarcinoma (EAC) compared to Barrett esophagus and normal tissue. However, studies on the effect of DCLK1 in esophageal squamous cell carcinoma (ESCC), which accounts for about 90% of diagnosed EC in Asia areas, were not yet reported. MethodsCRISPR/Cas9 genome-editing technique was used to knockout DCLK1 in ESCC cells (Kyse450, Kyse70). Subsequently, the proliferative and invasive potential of these cells was evaluated using Real-Time Cell Analyzer for proliferation, wound healing assay for migration, and transwell assay for invasion. Epithelial-mesenchymal transformation (EMT) associated transcriptional factors and MEK/ERK pathway were tested by western blot. In addition, the correlation heatmap between DCLK1 expression and EMT associated transcriptional factors, and MEK/ERK pathway was analyzed in R among 95 ESCC patients from The Cancer Genome Atla (TCGA) database. ResultsCRISPR/Cas9 technology efficiently disrupted the DCLK1 gene and abrogated its expression in ESCC cell lines. DCLK1 deficiency significantly inhibited proliferation, migration and invasion of ESCC cells. DCLK1 deletion resulted in notable molecular changes including downregulation of mesenchymal markers such as Vimentin, ZEB1, Slug and Snail, and up-regulation of epithelial marker E-cadherin. Additionally, the phosphorylation of MEK/ERK pathway was inhibited in DCLK1-KO ESCC cells. Accordingly,evidence from TCGA ESCA demonstrated that human ESCCs expressing high levels of DCLK1 correlate with activated MEK/ERK pathway signaling suggesting greater EMT transition activity. ConclusionsCRISPR/Cas9-mediated knockout of DCLK1 results in a dramatic suppression of ESCC aggressiveness via inhibiting EMT and MEK/ERK pathway. Importantly, this study demonstrates DCLK1's functional significance in ESCC for the first time and provides a theoretical basis for the development of DCLK1-based biomarkers and targeted therapies. Legal entity responsible for the studyJiannan Yao. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

DCLK1 integrates induction of TRIB3, EMT, drug resistance and poor prognosis in colorectal cancer.

Doublecortin-like kinase 1 (DCLK1) promotes tumour proliferation in human colorectal cancer (CRC). To elucidate the mechanism and clinical relevance of this association, we performed expression analysis using commercially available colon carcinoma cell lines (SW480, HCT116, CaCO2, SW48 and SKCO1) and immunohistochemical analysis of 200 resected CRC samples for correlation with clinical features. DCLK1 showed a high level of expression, especially in SW480 and HCT116 cells. Silencing DCLK1 expression using short hairpin DCLK1 (shDCLK1) RNA inhibited the growth and invasion capacities of these cell lines, which showed signs of entering into the mesenchymal-epithelial transition (MET). We found evidence of a strong correlation of DCLK1 expression with that of Tribbles homolog 3 (TRIB3), and silencing TRIB3 also led to the MET phenotype in these cells. In the clinical samples, compared with samples showing low expression of DCLK1, high expression was associated with poor prognosis in terms of overall and recurrence-free survival (P < 0.0001). The results of univariate and multivariate analysis suggested that high expression of DCLK1 in clinical colon cancer samples was tied to poor prognosis, cancer invasion depth and lymph node metastasis. DCLK1 expression correlates with malignant grade of colon cancer and offers a potential treatment target.

Identification of WNT16 as a Predictable Biomarker for Accelerated Osteogenic Differentiation of Tonsil-Derived Mesenchymal Stem Cells In Vitro.

The application of mesenchymal stem cells (MSCs) for treating bone-related diseases shows promising outcomes in preclinical studies. However, cells that are isolated and defined as MSCs comprise a heterogeneous population of progenitors. This heterogeneity can produce variations in the performance of MSCs, especially in applications that require differentiation potential in vivo, such as the treatment of osteoporosis. Here, we aimed to identify genetic markers in tonsil-derived MSCs (T-MSCs) that can predict osteogenic potential. Using a single-cell cloning method, we isolated and established several lines of nondifferentiating (ND) or osteoblast-prone (OP) clones. Next, we performed transcriptome sequencing of three ND and three OP clones that maintained the characteristics of MSCs and determined the top six genes that were upregulated in OP clones. Upregulation of WNT16 and DCLK1 expression was confirmed by real-time quantitative PCR, but only WNT16 expression was correlated with the osteogenic differentiation of T-MSCs from 10 different donors. Collectively, our findings suggest that WNT16 is a putative genetic marker that predicts the osteogenic potential of T-MSCs. Thus, examination of WNT16 expression as a selection criterion prior to the clinical application of MSCs may enhance the therapeutic efficacy of stem cell therapy for bone-related complications, including osteoporosis.

Abstract 2997: 15-hydroxyprostaglandin dehydrogenase induced differentiation in colon cancer cells is regulated via Gli1

Abstract Prostaglandin E2 (PGE2) is a crucial regulator of colon cancer (CC), an inflammation associated cancer. Tumor suppressor 15-hydroxyprostglandin dehydrogenase (15-PGDH) is a PGE2 degrading enzyme but is often downregulated in CC. Interestingly, CC-patients with high levels of Cysteinyl leukotriene receptor 2 (CysLT2R) exhibit good prognoses. Previously, we have reported differentiation promoting role of 15-PGDH in CC cells when induced by LTC4, the CysLT2R ligand. However the mechanism is unclear. Hedgehog (Hh) signalling is involved in embryogenesis and development but hyper activated in colon carcinogenesis. LTC4-induced activation of 15-PGDH downregulated both mRNA and protein expression of GLI1 in HT-29 and Caco-2 colon cancer cells. Moreover, absence of Gli1 significantly reduced the differentiation promoting ability of 15-PGDH, with no change in the differentiation markers Sucrase isomaltase, Mucin-2 and Protocadherin-24. In addition, no noticeable change in the mRNA expression of WNT-specific target AXIN-2 coupled with restoration of the proliferation marker cMYC was observed. In HT-29-derived colonospheres depletion of mRNA expression of CC-CSC markers ALDH1A1 and DCLK1 remained regulated by Gli1. Elevation in Mucin-2 expression in zebrafish xenograft with HT-29 was also noted to be Gli1 dependent. GLI1 and MUCIN-2 mRNA expression in normal mucosa and matched tumor in colon cancer patient also exhibited higher co-relation along with concomitant elevation in DCLK1 expression in tumor in compared to normal mucosa. Tumor microarray (TMA) with low expression of CysLT2R and 15-PGDH exhibited higher Gli1 expression with no trace of Mucin-2. Therefore, restoring the expression of 15-PGDH in CC through activation of CysLT2R signalling and targeting Hh-Gli signalling would open new therapeutic possibilities with minimal collateral damage to the normal tissue. Citation Format: Shakti R. Satapathy, Lubna M. Mehdawi, Roger Olsson, Anita Sjolander. 15-hydroxyprostaglandin dehydrogenase induced differentiation in colon cancer cells is regulated via Gli1 [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 2997.

Abstract 1154: DCLK1 monoclonal antibody-based CAR-T cells as novel treatment strategy against multiple myeloma

Abstract Multiple Myeloma (MM) is characterized by the clonal expansion of malignant plasma cells in the bone marrow, and is the second most common hematological cancer. Cellular therapies including chimeric antigen T receptor cell (CAR-T) have emerged as a promising therapeutic strategy to combat this devastating disease. MM are derived from a rare population of cancer stem cells (CSC) that are responsible for initiation, progression, resistance to conventional therapy and recurrence. To date there have been no CAR-T therapies directed at CSC-specific antigens. In this report, we describe the effects of a novel CAR-T targeting the CSC marker DCLK1 on MM cells. Using mouse models, it has been demonstrated that DCLK1 marks the cell of origin of colon and pancreatic cancers. DCLK1 regulates epithelial to mesenchymal transition (EMT), and its upregulation is associated with poor overall survival in many cancer subtypes. Inhibition of DCLK1 activity following RNA silencing resulted in tumor xenograft growth arrest. These features lead us to explore the therapeutic potential of targeting DCLK1+ cells in MM. In order to create a novel DCLK1 CAR, we generated a new humanized DCLK1 monoclonal antibody, clone CBT-15 #8. This antibody recognizes a small extra cellular C-terminal epitope on human isoform 2 and 4 of DCLK1. Novel DCLK1 CAR-T cells containing the clone were generated and detected with anti-human FAB antibody by flow cytometry. Furthermore, we observed cell surface expression of DCLK1 on &amp;gt;93 percent of MM Cells (RPMI8226) by flow cytometry. The CAR-T cells had significantly higher cytotoxicity in MM (RPMI8226 and MM1S) cells than in leukemia K562 cells. The CAR-T cells secreted significantly higher levels of IFN-γ in vitro than T cells (p=0.0036). Further studies are underway to determine its efficacy in inhibiting tumor xenografts in vivo. This report is the first to link the TSC marker DCLK1 to MM and may represent a novel therapeutic strategy for MM and other hematologic malignancies. Citation Format: Sripathi M. Sureban, Robert Berahovich, Hua Zhou, Shirley Xu, Lijun Wu, Vita Golubovskaya, Dongfeng Qu, Randal May, Edwin Bannerman-Menson, Courtney W. Houchen. DCLK1 monoclonal antibody-based CAR-T cells as novel treatment strategy against multiple myeloma [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 1154.

Abstract 4398: KDM3A and DCLK1 interactions promote stemness and tumorigenesis in PDAC

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the major leading cause of cancer related human death in USA. There is no possible treatment or target available in PDAC. It was proposed that the cancer stem cells (CSCs) can regulates malignancy in PDAC. DCLK1 is one of the quiescent cancer stem cell marker in PDAC that can regulate tumor progression. Identification of the key factors that influence stemness will help to target PDAC. Histone lysine demethylase KDM3A is an enzyme/protein which can regulate stem cell renewability and tumor progression, thereby KDM3A can interact with DCLK1 for tumor progression. KDM3A influences tumor growth and regulates stemness. Therefore, our goal is to find out the the role of KDM3A in tumor progression in PDAC through interactions with DCLK1. We observed expression of KDM3A in both PDAC patients’ samples and cells. Knockdown and overexpression of KDM3A were executed by using lentiviral vector. Tumor progression were also observed in orthotopic mice model. ChIP and RNA seq were performed to validate the data. KDM3A was overexpressed in human PDAC patient tissues and human pancreatic cancer cells with concomitant increase of CSC marker, DCLK1. Moreover, DCLK1 and KDM3A was found to be co-localized in patient tissue samples and identified binding sites of KDM3A with DCLK1 using ChIP-seq. Knockdown of KDM3A abrogates oncogenic potential whereas, overexpressed KDM3A in transformed HPNE cells showed malignant properties with enhanced invasive property, pancosphere formation, foci formation and tumor formation in mouse. Moreover, ChIP-seq and RNA seq suggested that KDM3A regulated DCLK1 expression in tumor progression. In conclusion, co-expression of DCLK1 with KDM3A influenced stemness and enhance tumor progression in PDAC. Citation Format: Chandrayee Ghosh, Kankaraj Palaniyandi, Santanu Paul, Prasad Dandawate, Sonia Rawal, Dharmalingam Subramaniam, Subhash Padhye, Sumedha Gunewardena, Sufi Thomas, Maura O'Neil, Roy Jensen, Danny Welch, Sally Milisky, Scott Weir, Tomoo Iwakuma, Shrikant Anant, Animesh Dhar. KDM3A and DCLK1 interactions promote stemness and tumorigenesis in PDAC [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 4398.

Sprouty2 restricts colonic tuft and goblet cell numbers by repressing epithelial IL‐33 expression

OBJECTIVETuft and goblet cells are secretory cell types of the intestinal epithelium that serve key roles in sensing and responding to inflammation. However, the intracellular mechanisms controlling their numbers and activity are incompletely defined. Here, we investigated the role of Sprouty2 (Spry2), an intracellular modulator of PI3K and MAPK signaling, in tuft and goblet cell development. Both PI3K and MAPK are implicated in control of differentiation in the intestine. Furthermore, both pathways have been linked to expression of IL‐33, a cytokine that indirectly influences secretory differentiation by stimulating IL‐13 release from immune cell populations. We tested the hypothesis that Spry2 controls IL‐33 production in the colon, thus altering intestinal secretory cell development.METHODSMucosal scrapings from wild type mice given dextran sodium sulphate (DSS) colitis, and colonoids treated with TNF, were subjected to qPCR analysis for Spry2 expression. Colons from mice with intestinal epithelial‐specific Spry2 deletion (Spryflox/flox;Villin‐Cre, here called Spry2KOIE) and littermate controls were collected at 8 weeks of age. Cytokine levels and epithelial marker expression were determined by immunostaining and qPCR. Signaling pathway alterations were assessed by dot blot array and confirmed by western blot. In vitro, murine colonoids were treated with IL‐33 or IL‐13 +/− signaling pathway inhibitors (LY294002, PI3K; CHIR99021, GSK3β) to determine the effects on cytokine and epithelial marker expression.RESULTSSpry2 levels were decreased in inflamed colons (59% decrease, p&lt;0.05) and TNF‐treated colonoids (45% decrease, p&lt;0.05), demonstrating regulation by inflammation. Spry2KOIE mice displayed increased colonic IL‐33 expression (80% increase, p&lt;0.05), more mesenchymal IL‐13+ cells, more Dclk1+ tuft cells (2.2‐fold, p&lt;0.01), and more Muc2+ goblet cells (1.57‐fold, p&lt;0.01). These mice also had elevated GSK3β phosphorylation (88% increase, p&lt;0.01) on Ser9 (an inhibitory site phosphorylated by Akt), and increased phospho‐Akt (p&lt;0.01). In vitro, inactivation of GSK3β in murine colonoids with CHIR99021 mimicked the increased IL‐33 observed in vivo, but interestingly did not alter Dclk1 or Muc2 expression. This effect was attenuated by PI3K inhibition. IL‐13, but not IL‐33, induced Dclk1 and Muc2 in colonoids, suggesting an epithelial‐mesenchymal crosstalk circuit drives alterations in tuft and goblet cells following Spry2 loss.CONCLUSIONInflammation‐driven loss of colonic epithelial Spry2 de‐represses PI3K/Akt signals that lead to elevated IL‐33 expression. IL‐33 may act, in turn, to promote mesenchymal IL‐13 release that drives a protective expansion of tuft and goblet cells in the epithelium.Support or Funding InformationNIH R01DK095004 (Frey); Crohn's and Colitis Foundation Career Development Award (Schumacher)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dclk1 in tuft cells promotes inflammation-driven epithelial restitution and mitigates chronic colitis.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by defective intestinal barrier integrity toward the microbiota and epithelial damage. Double cortin-like kinase 1 (Dclk1), a marker ofintestinal tuft cells, can regulate tissue regenerative responses, but its role in epithelial repair during bacterial-dependent chronic colitis is unclear. We addressed this question using our recently developed mouse model of spontaneous microbiota-dependent colitis induced by mucin-type O-glycan deficiency (DKO), which recapitulates most features of human UC. We generated DKO mice lacking intestinal epithelial Dclk1 (DKO;Dclk1ΔIEC) and analyzed colitis onset and severity using clinical and histologic indices, immune responses by qPCR and immunostaining, and epithelial responses using proliferation markers and organoid culture. We found 3-4-week-old DKO;Dclk1ΔIEC mice developed worsened spontaneous colitis characterized by reduced body weight, loose stool, severe colon thickening, epithelial lesions, and inflammatory cell infiltrates compared with DKO mice. The primary defect was an impaired epithelial proliferative response during inflammation. Dclk1 deficiency also reduced inflammation-induced proliferation and growth of colon organoids ex vivo. Mechanistically, Dclk1 expression was important for inflammation-induced Cox2 expression and prostaglandin E2 (PGE2) production in vivo, and PGE2 rescued proliferative defects in Dclk1-deficient colonic organoids. Although tuft cells were expanded in both DKO and DKO;Dclk1ΔIEC relative to WT mice, loss of Dclk1 was associated with reduced tuft cell activation (i.e., proliferation) during inflammation. Similar results were found in DKO vs. DKO;Dclk1ΔIEC mice at 3-6 months of age. Our results support that tuft cells, via Dclk1, are important responders to bacterial-induced colitis by enhancing epithelial repair responses, which in turn limits bacterial infiltration into the mucosa.

Montelukast, a CysLT1 receptor antagonist, reduces colon cancer stemness and tumor burden in a mouse xenograft model of human colon cancer

Inflammation is implicated in the etiology of sporadic colon cancer (CC), which is one of the leading causes of cancer-related deaths worldwide. Here, we report that inhibition of the inflammatory receptor CysLT1 through its antagonist, montelukast, is beneficial in minimizing stemness in CC and thereby minimizing tumor growth in a mouse xenograft model of human colon cancer. Upon treatment with montelukast, colonospheres derived from HT-29 and SW-480 human colon cancer cells exhibited a significant phenotypic change coupled with the downregulation of mRNA and protein expression of cancer stem cell (CSC) markers ALDH1 and DCLK1. Moreover, montelukast reduced the size of HT-29 cell-derived tumors in mice. The reduction in tumor size was associated with decreased levels of ALDH1A1, DCLK1, BCL2 mRNA and macrophage infiltration into the tumor tissue. Interestingly, this treatment elevated levels of the tumor suppressor 15-PGDH while reducing COX-2 expression. Our data highlight the association of CysLT1R with CSCs and demonstrate that inhibition of CysLT1R could prove beneficial in minimizing CSC-induced tumor growth. This work advances the notion that targeting CSCs is a promising approach to improve outcomes in those afflicted with colon cancer.

Analysis of the clinical significance of DCLK1+ colorectal cancer using novel monoclonal antibodies against DCLK1.

IntroductionDoublecortin-like kinase 1 (DCLK1) is considered a putative tumor stem cell (TSC) marker and a promising therapeutic target, as DCLK1+ progeny cells exhibit high expression in tumors. However, the biological function of DCLK1+ cells in tumorigenesis and tumor progression remains unclear.Materials and methodsWe generated rabbit monoclonal antibodies (mAbs) against DCLK1, DCLK1-42, and DCLK1-87 mAbs, using a novel chip-based immunospot array assay on a chip system. First, the specificity of two mAbs to DCLK1 was confirmed by Western blot, which were bound to DCLK1-long in normal colon cells and to DCLK1-short in a cancer cell line as well as colorectal cancer (CRC) cells.ResultsPrecise localization analysis using immunofluorescence revealed that both mAbs had cytoplasmic signal and exhibited a high degree of overlap with microtubules. Furthermore, bacterial display technology indicated that the antigenic epitope region of DCLK1-87 mAb was consistent with that of a commercial anti-DCLK1 polyclonal antibody. In addition, DCLK1-42 mAb has the common polyclonal antibody characteristic of binding to more than one site on DCLK1. By immunohistochemistry, it was found that DCLK1-87 mAb was more specific for DCLK1+ cell labeling than a commercial anti-DCLK1 polyclonal antibody. DCLK1 labeled with DCLK1-87 mAb might be a potential TSC marker because the tissue expression site covers the ALDH1 area in CRC tissues. Finally, we analyzed 100 pairs of cancer tissues and matching paracancerous tissue samples from patients with CRC who received 100 months of follow-up with the DCLK1-87 mAb. The results showed that patients with high DCLK1 expression exhibited a longer survival time than that of patients with low DCLK1 expression (P=0.0029).DiscussionOur results indicated that we successfully generated an efficient tool for the precise detection of DCLK1+ cells in cancer tissues. Moreover, we found that high DCLK1 expression in CRC patients appears to play a protective role against tumor progression.

Abstract 2576: Dclk1 can be a predictor for poor survival in patients with resected pancreatic cancer

Abstract Background: Doublecortin-like kinase-1 (Dclk1) shows higher expression levels in human pancreatic cancer compared with human normal pancreatic tissue. Dclk1 positively regulates tumor growth, invasion, metastasis, pluripotency factors, angiogenic factors, and epithelial-mesenchymal transition (EMT)-related genes in pancreatic cancer cells. We previously reported that Dclk1 inhibition in combination with gemcitabine (GEM) treatment offers a novel approach to treat pancreatic cancer. However, few studies focusing on the role of Dclk1 in pancreatic cancer using clinical specimens have been reported. Objective: To investigate the expression of Dclk1 in resected specimens of pancreatic cancer and its prognostic significance. Methods: Tumor specimens were obtained from 26 patients with pancreatic cancer (stage 3 and 4) who underwent resection between April 2005 and March 2015 in our department. Dclk1, Ki67, and phosphorylated MEK expression levels were examined using immunohistochemical staining. The expression levels of these markers were evaluated using Histoscore (Intensity [0-3] x Extent [0-4]). In order to identify proteins phosphorylated by Dclk1, we analyzed a protein microarray using a pancreatic cancer cell line. Results: In total, 42.3% (11/26) of samples expressed high levels of Dclk1 (Histoscore≧3.3) and were classified into the high Dclk1 group. There were no significant differences between patients in the high Dclk1 group and the low Dclk1 group in terms of clinical characteristics and surgical findings. Patients in the high Dclk1 group exhibited significantly shorter survival times than those in the low Dclk1 group (P &amp;lt; 0.05). The expression of phosphorylated MEK, which is observed on a mitogen-activated protein kinase (MAPK) pathway, decreased by &amp;gt;50% in Dclk1-inhibited pancreatic cancer cells by protein microarray. Interestingly, the expression of pMEK and Ki67 in resected specimens tended to increase in samples in the high Dclk1 group. Therefore, Dclk1 might contribute to the progression of pancreatic cancer through the phosphorylation of MEK. Conclusion: Dclk1 may be a marker for poor prognosis in patients with resected pancreatic cancer. Citation Format: Yoshihiro Takemoto, Arata Nishimoto, Yukari Hironaka, Yuki Suehiro, Naruji Kugimiya, Atsushi Suga, Eijiro Harada, Kimikazu Hamano. Dclk1 can be a predictor for poor survival in patients with resected pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2576.

Immune Cell Production of Interleukin 17 Induces Stem Cell Features of Pancreatic Intraepithelial Neoplasia Cells

Little is known about how the immune system affects stem cell features of pancreatic cancer cells. Immune cells that produce interleukin 17A (IL17A) in the chronically inflamed pancreas (chronic pancreatitis) contribute to pancreatic interepithelial neoplasia (PanIN) initiation and progression. We investigated the effects that IL17A signaling exerts on pancreatic cancer progenitor cells and the clinical relevance of this phenomena. We performed studies with Mist1Cre;LSLKras;Rosa26mTmG (KCiMist;G) and Kras(G12D);Trp53(R172H);Pdx1-Cre (KPC) mice (which upon tamoxifen induction spontaneously develop PanINs) and control littermates. Some mice were injected with neutralizing antibodies against IL17A or control antibody. Pancreata were collected, PanIN epithelial cells were isolated by flow cytometry based on lineage tracing, and gene expression profiles were compared. We collected cells from pancreatic tumors of KPC mice, incubated them with IL17 or control media, measured expression of genes regulated by IL17 signaling, injected the cancer cells into immune competent mice, and measured tumor growth. IL17A was overexpressed in pancreata of KCiMist mice from an adenoviral vector. Pancreata were collected from all mice and analyzed by histology and immunohistochemistry. Levels of DCLK1 and other proteins were knocked down in KPC pancreatic cancer cells using small interfering or short hairpin RNAs; cells were analyzed by immunoblotting. We obtained 65 pancreatic tumor specimens from patients, analyzed protein levels by immunohistochemistry, and compared results with patient survival times. We also analyzed gene expression levels and patient outcome using The Cancer Genome Atlas database. PanIN cells from KCiMist;G mice had a gene expression pattern associated with embryonic stem cells. Mice given injections of IL17-neutralizing antibodies, or with immune cells that did not secrete IL17, lost this expression pattern and had significantly decreased expression of DCLK1 and POU2F3, which regulate tuft cell development. KCiMist mice that overexpressed IL17 formed more PanINs, with more DCLK1-positive cells, than control mice. Pancreatic tumor cells from KPC mice and human Capan-2 cells exposed to IL17A had increased activation of NF-κB and mitogen-activated protein kinase signaling and increased expression of DCLK1 and ALDH1A1 (a marker of embryonic stem cells) compared with cells in control media. These cells also formed tumors faster that cells not exposed to IL17 when they were injected into immunocompetent mice. KPC cells with knockdown of DCLK1 expressed lower levels of ALDH1A1 after incubation with IL17 than cells without knockdown. Expression of the IL17 receptor C was higher in DCLK1-positive PanIN cells from mice compared with DCLK1-negative PanIN cells. In human pancreatic tumor tissues, high levels of DCLK1 associated with a shorter median survival time of patients (17.7 months, compared with 26.6 months of patients whose tumors had low levels of DCLK1). Tumor levels of POU2F3 and LAMC2 were also associated with patient survival time. In studies of mouse and human pancreatic tumors and precursors, we found that immune cell-derived IL17 regulated development of tuft cells and stem cell features of pancreatic cancer cells via increased expression of DCLK1, POU2F3, ALDH1A1, and IL17RC. Strategies to disrupt this pathway might be developed to prevent pancreatic tumor growth and progression.