Repurposing diacerein to suppress colorectal cancer growth by inhibiting the DCLK1/STAT3 signaling pathway.

Double cortin-like kinase 1 (DCLK1) exhibits high expression levels across various cancers, notably in human colorectal cancer (CRC). Diacerein, a clinically approved interleukin (IL)-1ß inhibitor for osteoarthritis treatment, was evaluated for its impact on CRC proliferation and migration, alongside its underlying mechanisms, through both in vitro and in vivo analyses. The study employed MTT assay, colony formation, wound healing, transwell assays, flow cytometry, and Hoechst 33342 staining to assess cell proliferation, migration, and apoptosis. Additionally, proteome microarray assay and western blotting analyses were conducted to elucidate diacerein's specific mechanism of action. Our findings indicate that diacerein significantly inhibits DCLK1-dependent CRC growth in vitro and in vivo. Through high-throughput proteomics microarray and molecular docking studies, we identified that diacerein directly interacts with DCLK1. Mechanistically, the suppression of p-STAT3 expression following DCLK1 inhibition by diacerein or specific DCLK1 siRNA was observed. Furthermore, diacerein effectively disrupted the DCLK1/STAT3 signaling pathway and its downstream targets, including MCL-1, VEGF, and survivin, thereby inhibiting CRC progression in a mouse model, thereby inhibiting CRC progression in a mouse model.

A nasal cell atlas reveals heterogeneity of tuft cells and their role in directing olfactory stem cell proliferation.

The olfactory neuroepithelium serves as a sensory organ for odors and forms part of the nasal mucosal barrier. Olfactory sensory neurons are surrounded and supported by epithelial cells. Among them, microvillous cells (MVCs) are strategically positioned at the apical surface, but their specific functions are enigmatic, and their relationship to the other specialized epithelial cells is unclear. Here, we establish that the family of MVCs comprises tuft cells and ionocytes in both mice and humans. Integrating analysis of the respiratory and olfactory epithelia, we define the distinct receptor expression of TRPM5+ tuft-MVCs compared with Gɑ-gustducinhigh respiratory tuft cells and characterize a previously undescribed population of glandular DCLK1+ tuft cells. To establish how allergen sensing by tuft-MVCs might direct olfactory mucosal responses, we used an integrated single-cell transcriptional and protein analysis. Inhalation of Alternaria induced mucosal epithelial effector molecules including Chil4 and a distinct pathway leading to proliferation of the quiescent olfactory horizontal basal stem cell (HBC) pool, both triggered in the absence of olfactory apoptosis. Alternaria- and ATP-elicited HBC proliferation was dependent on TRPM5+ tuft-MVCs, identifying these specialized epithelial cells as regulators of olfactory stem cell responses. Together, our data provide high-resolution characterization of nasal tuft cell heterogeneity and identify a function of TRPM5+ tuft-MVCs in directing the olfactory mucosal response to allergens.

Discovery of a doublecortin-like kinase 1 inhibitor to prevent inflammatory responses in acute lung injury.

Doublecortin-like kinase 1 (DCLK1) is a microtubule-associated protein kinase involved in neurogenesis and human cancer. Recent studies have revealed a novel functional role for DCLK1 in inflammatory signaling, thus positioning it as a novel target kinase for respiratory inflammatory disease treatment. In this study, we designed and synthesized a series of NVP-TAE684-based derivatives as novel anti-inflammatory agents targeting DCLK1. Bio-layer interferometry binding screening and kinase assays of the NVP-TAE684 derivatives led to the discovery of an effective DCLK1 inhibitor (a24), with an IC50 of 179.7 nM. Compound a24 effectively inhibited lipopolysaccharide (LPS)-induced inflammation in macrophages with higher potency than the lead compound. Mechanistically, compound a24 inhibited LPS-induced inflammation by inhibiting DCLK1-mediated IKKß phosphorylation. Furthermore, compound a24 showed in vivo anti-inflammatory activity in an LPS-challenged acute lung injury model. These findings suggest that compound a24 may serve as a novel candidate for the development of DCLK1 inhibitors and a potential therapeutic agent for the treatment of inflammatory diseases.

Kinome-wide siRNA screen identifies a DCLK2-TBK1 oncogenic signaling axis in clear cell renal cell carcinoma.

TANK-binding kinase 1 (TBK1) is a potential therapeutic target in multiple cancers, including clear cell renal cell carcinoma (ccRCC). However, targeting TBK1 in clinical practice is challenging. One approach to overcome this challenge would be to identify an upstream TBK1 regulator that could be targeted therapeutically in cancer specifically. In this study, we perform a kinome-wide small interfering RNA (siRNA) screen and identify doublecortin-like kinase 2 (DCLK2) as a TBK1 regulator in ccRCC. DCLK2 binds to and directly phosphorylates TBK1 on Ser172. Depletion of DCLK2 inhibits anchorage-independent colony growth and kidney tumorigenesis in orthotopic xenograft models. Conversely, overexpression of DCLK2203, a short isoform that predominates in ccRCC, promotes ccRCC cell growth and tumorigenesis in vivo. Mechanistically, DCLK2203 elicits its oncogenic signaling via TBK1 phosphorylation and activation. Taken together, these results suggest that DCLK2 is a TBK1 activator and potential therapeutic target for ccRCC.

Protein kinase D1 - A targetable mediator of pancreatic cancer development.

Members of the Protein kinase D (PKD) kinase family each play important cell-specific roles in the regulation of normal pancreas functions. In pancreatic diseases PKD1 is the most widely characterized isoform with roles in pancreatitis and in induction of pancreatic cancer and its progression. PKD1 expression and activation increases in pancreatic acinar cells through macrophage secreted factors, Kirsten rat sarcoma viral oncogene homolog (KRAS) signaling, and reactive oxygen species (ROS), driving the formation of precancerous lesions. In precancerous lesions PKD1 regulates cell survival, growth, senescence, and generation of doublecortin like kinase 1 (DCLK1)-positive cancer stem cells (CSCs). Within tumors, regulation by PKD1 includes chemoresistance, apoptosis, proliferation, CSC features, and the Warburg effect. Thus, PKD1 plays a critical role throughout pancreatic disease initiation and progression.

DCLK1 and its oncogenic functions: A promising therapeutic target for cancers.

Doublecortin-like kinase 1 (DCLK1), a significant constituent of the protein kinase superfamily and the doublecortin family, has been recognized as a prooncogenic factor that exhibits a strong association with the malignant progression and clinical prognosis of various cancers. DCLK1 serves as a stem cell marker that governs tumorigenesis, tumor cell reprogramming, and epithelial-mesenchymal transition. Multiple studies have indicated the capable of DCLK1 in regulating the DNA damage response and facilitating DNA damage repair. Additionally, DCLK1 is involved in the regulation of the immune microenvironment and the promotion of tumor immune evasion. Recently, DCLK1 has emerged as a promising therapeutic target for a multitude of cancers. Several small-molecule inhibitors of DCLK1 have been identified. Nevertheless, the biological roles of DCLK1 are mainly ambiguous, particularly with the disparities between its α- and ß-form transcripts in the malignant progression of cancers, which impedes the development of more precisely targeted drugs. This article focuses on tumor stem cells, tumor epithelial-mesenchymal transition, the DNA damage response, and the tumor microenvironment to provide a comprehensive overview of the association between DCLK1 and tumor malignant progression, address unsolved questions and current challenges, and project future directions for targeting DCLK1 for the diagnosis and treatment of cancers.

Design and synthesis of doublecortin-like kinase 1 inhibitors and their bioactivity evaluation.

Doublecortin-like kinase 1 (DCLK) is a microtubule-associated serine/threonine kinase that is upregulated in a wide range of cancers and is believed to be related to tumour growth and development. Upregulated DCLK1 has been used to identify patients at high risk of cancer progression and tumours with chemotherapy-resistance. Moreover, DCLK1 has been identified as a cancer stem cell (CSC) biomarker in various cancers, which has received considerable attention recently. Herein, a series of DCLK1 inhibitors were prepared based on the previously reported XMD8-92 structure. Among all the synthesised compounds, D1, D2, D6, D7, D8, D12, D14, and D15 showed higher DCLK1 inhibitory activities (IC50 40-74 nM) than XMD8-92 (IC50 161 nM). Compounds D1 and D2 were selective DCLK1 inhibitors as they showed a rather weak inhibitory effect on LRRK2. The antiproliferative activities of these compounds were also preliminarily evaluated. The structure-activity relationship revealed by our compounds provides useful guidance for the further development of DCLK1 inhibitors.

Epigenetic Landscape and Therapeutic Implication of Gene Isoforms of Doublecortin-Like Kinase 1 for Cancer Stem Cells.

While significant strides have been made in understanding cancer biology, the enhancement in patient survival is limited, underscoring the urgency for innovative strategies. Epigenetic modifications characterized by hereditary shifts in gene expression without changes to the DNA sequence play a critical role in producing alternative gene isoforms. When these processes go awry, they influence cancer onset, growth, spread, and cancer stemness. In this review, we delve into the epigenetic and isoform nuances of the protein kinase, doublecortin-like kinase 1 (DCLK1). Recognized as a hallmark of tumor stemness, DCLK1 plays a pivotal role in tumorigenesis, and DCLK1 isoforms, shaped by alternative promoter usage and splicing, can reveal potential therapeutic touchpoints. Our discussion centers on recent findings pertaining to the specific functions of DCLK1 isoforms and the prevailing understanding of its epigenetic regulation via its two distinct promoters. It is noteworthy that all DCLK1 isoforms retain their kinase domain, suggesting that their unique functionalities arise from non-kinase mechanisms. Consequently, our research has pivoted to drugs that specifically influence the epigenetic generation of these DCLK1 isoforms. We posit that a combined therapeutic approach, harnessing both the epigenetic regulators of specific DCLK1 isoforms and DCLK1-targeted drugs, may prove more effective than therapies that solely target DCLK1.

LGR5 Expression Predicting Poor Prognosis Is Negatively Correlated with WNT5A in Colon Cancer.

WNT/ß-catenin signaling is essential for colon cancer development and progression. WNT5A (ligand of non-canonical WNT signaling) and its mimicking peptide Foxy5 impair ß-catenin signaling in colon cancer cells via unknown mechanisms. Therefore, we investigated whether and how WNT5A signaling affects two promoters of ß-catenin signaling: the LGR5 receptor and its ligand RSPO3, as well as ß-catenin activity and its target gene VEGFA. Protein and gene expression in colon cancer cohorts were analyzed by immunohistochemistry and qRT-PCR, respectively. Three colon cancer cell lines were used for in vitro and one cell line for in vivo experiments and results were analyzed by Western blotting, RT-PCR, clonogenic and sphere formation assays, immunofluorescence, and immunohistochemistry. Expression of WNT5A (a tumor suppressor) negatively correlated with that of LGR5/RSPO3 (tumor promoters) in colon cancer cohorts. Experimentally, WNT5A signaling suppressed ß-catenin activity, LGR5, RSPO3, and VEGFA expression, and colony and spheroid formations. Since ß-catenin signaling promotes colon cancer stemness, we explored how WNT5A expression is related to that of the cancer stem cell marker DCLK1. DCLK1 expression was negatively correlated with WNT5A expression in colon cancer cohorts and was experimentally reduced by WNT5A signaling. Thus, WNT5A and Foxy5 decrease LGR5/RSPO3 expression and ß-catenin activity. This inhibits stemness and VEGFA expression, suggesting novel treatment strategies for the drug candidate Foxy5 in the handling of colon cancer patients.

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

BACKGROUND: Delay 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 METHODS: AECII 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. RESULTS: We 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. CONCLUSIONS: We conclude that DCLK1-mediated Hippo signaling components of YAP/TAZ regulated markers of AECII-to-AECI differentiation in an LPS-induced ARDS model.

Blocking of doublecortin-like kinase 1-regulated SARS-CoV-2 replication cycle restores cell signaling network.

IMPORTANCE: Severe COVID-19 and post-acute sequelae often afflict patients with underlying co-morbidities. There is a pressing need for highly effective treatment, particularly in light of the emergence of SARS-CoV-2 variants. In a previous study, we demonstrated that DCLK1, a protein associated with cancer stem cells, is highly expressed in the lungs of COVID-19 patients and enhances viral production and hyperinflammatory responses. In this study, we report the pivotal role of DCLK1-regulated mechanisms in driving SARS-CoV-2 replication-transcription processes and pathogenic signaling. Notably, pharmacological inhibition of DCLK1 kinase during SARS-CoV-2 effectively impedes these processes and counteracts virus-induced alternations in global cell signaling. These findings hold significant potential for immediate application in treating COVID-19.

Targeting doublecortin-like kinase 1 reveals a novel strategy to circumvent chemoresistance and metastasis in ovarian cancer.

Ovarian cancer (OvCa) has a dismal prognosis because of its late-stage diagnosis and the emergence of chemoresistance. Doublecortin-like kinase 1 (DCLK1) is a serine/threonine kinase known to regulate cancer cell "stemness", epithelial-mesenchymal transition (EMT), and drug resistance. Here we show that DCLK1 is a druggable target that promotes chemoresistance and tumor progression of high-grade serous OvCa (HGSOC). Importantly, high DCLK1 expression significantly correlates with poor overall and progression-free survival in OvCa patients treated with platinum chemotherapy. DCLK1 expression was elevated in a subset of HGSOC cell lines in adherent (2D) and spheroid (3D) cultures, and the expression was further increased in cisplatin-resistant (CPR) spheroids relative to their sensitive controls. Using cisplatin-sensitive and resistant isogenic cell lines, pharmacologic inhibition (DCLK1-IN-1), and genetic manipulation, we demonstrate that DCLK1 inhibition was effective at re-sensitizing cells to cisplatin, reducing cell proliferation, migration, and invasion. Using kinase domain mutants, we demonstrate that DCLK1 kinase activity is critical for mediating CPR. The combination of cisplatin and DCLK1-IN-1 showed a synergistic cytotoxic effect against OvCa cells in 3D conditions. Targeted gene expression profiling revealed that DCLK1 inhibition in CPR OvCa spheroids significantly reduced TGFß signaling, and EMT. We show in vivo efficacy of combined DCLK1 inhibition and cisplatin in significantly reducing tumor metastases. Our study shows that DCLK1 is a relevant target in OvCa and combined targeting of DCLK1 in combination with existing chemotherapy could be a novel therapeutic approach to overcome resistance and prevent OvCa recurrence.

Mechanistic and evolutionary insights into isoform-specific 'supercharging' in DCLK family kinases.

Catalytic signaling outputs of protein kinases are dynamically regulated by an array of structural mechanisms, including allosteric interactions mediated by intrinsically disordered segments flanking the conserved catalytic domain. The doublecortin-like kinases (DCLKs) are a family of microtubule-associated proteins characterized by a flexible C-terminal autoregulatory 'tail' segment that varies in length across the various human DCLK isoforms. However, the mechanism whereby these isoform-specific variations contribute to unique modes of autoregulation is not well understood. Here, we employ a combination of statistical sequence analysis, molecular dynamics simulations, and in vitro mutational analysis to define hallmarks of DCLK family evolutionary divergence, including analysis of splice variants within the DCLK1 sub-family, which arise through alternative codon usage and serve to 'supercharge' the inhibitory potential of the DCLK1 C-tail. We identify co-conserved motifs that readily distinguish DCLKs from all other calcium calmodulin kinases (CAMKs), and a 'Swiss Army' assembly of distinct motifs that tether the C-terminal tail to conserved ATP and substrate-binding regions of the catalytic domain to generate a scaffold for autoregulation through C-tail dynamics. Consistently, deletions and mutations that alter C-terminal tail length or interfere with co-conserved interactions within the catalytic domain alter intrinsic protein stability, nucleotide/inhibitor binding, and catalytic activity, suggesting isoform-specific regulation of activity through alternative splicing. Our studies provide a detailed framework for investigating kinome-wide regulation of catalytic output through cis-regulatory events mediated by intrinsically disordered segments, opening new avenues for the design of mechanistically divergent DCLK1 modulators, stabilizers, or degraders.

Design, synthesis and biological evaluation of novel DCLK1 inhibitor containing purine skeleton for the treatment of pancreatic cancer.

Pancreatic cancer is a highly lethal form of malignancy that continues to pose a significant and unresolved health challenge. Doublecortin-like kinase 1 (DCLK1), a serine/threonine kinase, is found to be overexpressed in pancreatic cancer and holds promise as a potential therapeutic target for this disease. However, few potent inhibitors have been reported currently. Herein, a series of novel purine, pyrrolo [2,3-d]pyrimidine, and pyrazolo [3,4-d] pyrimidine derivatives were designed, synthesized, and evaluated their biological activities in vitro. Among them, compound I-5 stood out as the most potent compound with strong inhibitory activity against DCLK1 (IC50 = 171.3 nM) and remarkable antiproliferative effects on SW1990 cell lines (IC50 = 0.6 µM). Notably, I-5 exhibited higher in vivo antitumor potency (Tumor growth inhibition value (TGI): 68.6 %) than DCLK1-IN-1 (TGI: 24.82 %) in the SW1990 xenograft model. The preliminary mechanism study demonstrated that I-5 not only inhibited SW1990 cell invasion and migration, but also decreased the expression of prominin-1 (CD133) and cluster of differentiation 44 (CD44), which are considered as differentiation markers for SW1990 stem cells. All the results indicated that I-5, a novel DCLK1 inhibitor, shows promise for further investigation in the treatment of pancreatic cancer.

Molecular Mechanism of Mutational Disruption of DCLK1 Autoinhibition Provides a Rationale for Inhibitor Screening.

Doublecortin-like kinase 1 (DCLK1) is a prominent kinase involved in carcinogenesis, serving as a diagnostic marker for early cancer detection and prevention, as well as a target for cancer therapy. Extensive research efforts have been dedicated to understanding its role in cancer development and designing selective inhibitors. In our previous work, we successfully determined the crystal structure of DCLK1 while it was bound to its autoinhibitory domain (AID) at the active site. By analyzing this structure, we were able to uncover the intricate molecular mechanisms behind specific cancer-causing mutations in DCLK1. Utilizing molecular dynamics simulations, we discovered that these mutations disrupt the smooth assembly of the AID, particularly affecting the R2 helix, into the kinase domain (KD). This disruption leads to the exposure of the D533 residue of the DFG (Asp-Phe-Gly) motif in the KD, either through steric hindrance, the rearrangement of electrostatic interactions, or the disruption of local structures in the AID. With these molecular insights, we conducted a screening process to identify potential small-molecule inhibitors that could bind to DCLK1 through an alternative binding mode. To assess the binding affinity of these inhibitors to the KD of DCLK1, we performed calculations on their binding energy and conducted SPR experiments. We anticipate that our study will contribute novel perspectives to the field of drug screening and optimization, particularly in targeting DCLK1.

Proteomic investigation of neural stem cell to oligodendrocyte precursor cell differentiation reveals phosphorylation-dependent Dclk1 processing.

Oligodendrocytes are generated via a two-step mechanism from pluripotent neural stem cells (NSCs): after differentiation of NSCs to oligodendrocyte precursor/NG2 cells (OPCs), they further develop into mature oligodendrocytes. The first step of this differentiation process is only incompletely understood. In this study, we utilized the neurosphere assay to investigate NSC to OPC differentiation in a time course-dependent manner by mass spectrometry-based (phospho-) proteomics. We identify doublecortin-like kinase 1 (Dclk1) as one of the most prominently regulated proteins in both datasets, and show that it undergoes a gradual transition between its short/long isoform during NSC to OPC differentiation. This is regulated by phosphorylation of its SP-rich region, resulting in inhibition of proteolytic Dclk1 long cleavage, and therefore Dclk1 short generation. Through interactome analyses of different Dclk1 isoforms by proximity biotinylation, we characterize their individual putative interaction partners and substrates. All data are available via ProteomeXchange with identifier PXD040652.

Macrophage DCLK1 promotes obesity-induced cardiomyopathy via activating RIP2/TAK1 signaling pathway.

Obesity increases the risk for cardiovascular diseases and induces cardiomyopathy. Chronic inflammation plays a significant role in obesity-induced cardiomyopathy and may provide new therapeutic targets for this disease. Doublecortin-like kinase 1 (DCLK1) is an important target for cancer therapy and the role of DCLK1 in obesity and cardiovascular diseases is unclear. Herein, we showed that DCLK1 was overexpressed in the cardiac tissue of obese mice and investigated the role of DCLK1 in obesity-induced cardiomyopathy. We generated DCLK1-deleted mice and showed that macrophage-specific DCLK1 knockout, rather than cardiomyocyte-specific DCLK1 knockout, prevented high-fat diet (HFD)-induced heart dysfunction, cardiac hypertrophy, and fibrosis. RNA sequencing analysis showed that DCLK1 deficiency exerted cardioprotective effects by suppressing RIP2/TAK1 activation and inflammatory responses in macrophages. Upon HFD/palmitate (PA) challenge, macrophage DCLK1 mediates RIP2/TAK1 phosphorylation and subsequent inflammatory cytokine release, which further promotes hypertrophy in cardiomyocytes and fibrogenesis in fibroblasts. Finally, a pharmacological inhibitor of DCLK1 significantly protects hearts in HFD-fed mice. Our study demonstrates a novel role and a pro-inflammatory mechanism of macrophage DCLK1 in obesity-induced cardiomyopathy and identifies DCLK1 as a new therapeutic target for the treatment of this disease. Upon HFD/PA challenge, DCLK1 induces RIP2/TAK1-mediated inflammatory response in macrophages, which subsequently promotes cardiac hypertrophy and fibrosis. Macrophage-specific DCLK1 deletion or pharmacological inhibition of DCLK1 protects hearts in HFD-fed mice.

Exosomes from metastatic colon cancer cells drive the proliferation and migration of primary colon cancer through increased expression of cancer stem cell markers CD133 and DCLK1.

The exchange of biological material between the neighbouring cells is essential for homeostasis. In pathological conditions, such as cancer, the major challenge in cancer treatment is the abnormal expression of crucial proteins and miRNA exchanged between the cancer cells through extracellular vesicles called exosomes. Clinically, it has been noticed that the primary tumour and the distal metastases are interconnected and co-dependent. The exosomes are key factors responsible for preparing the pre-metastatic niche and communicating between the tumour and the distal metastatic site. Cancer stem cells (CSCs) are a subpopulation of cancer cells with self-renewal characteristics and are shown to be responsible for metastasis. This study aims to understand the effect of metastatic cell line-derived exosomes and their regulation of CSC marker expressions on primary colon cancer cell lines. We have identified that treatment of primary colon cancer cell lines with metastatic colon cancer cell-derived exosomes has significantly increased the proliferation, colony formation, cell migration, and invasion. In addition, there was a significant increase in the number and size of spheroids following the exosomes treatment. We found that this metastatic phenotype is due to the increased expression of CD133 and DCLK1 in primary colon cancer cells.

[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.

Appearance of tuft cells during prostate cancer progression.

Tuft cells are chemosensory epithelial cells that increase in number following infection or injury to robustly activate the innate immune response to alleviate or promote disease. Recent studies of castration resistant prostate cancer and its subtype, neuroendocrine prostate cancer, revealed Pou2f3+ populations in mouse models. The transcription factor Pou2f3 is a master regulator of the tuft cell lineage. We show that tuft cells are upregulated early during prostate cancer development, and their numbers increase with progression. Cancer-associated tuft cells in the mouse prostate express DCLK1, COX1, COX2, while human tuft cells express COX1. Mouse and human tuft cells exhibit strong activation of signaling pathways including EGFR and SRC-family kinases. While DCLK1 is a mouse tuft cell marker, it is not present in human prostate tuft cells. Tuft cells that appear in mouse models of prostate cancer display genotype-specific tuft cell gene expression signatures. Using bioinformatic analysis tools and publicly available datasets, we characterized prostate tuft cells in aggressive disease and highlighted differences between tuft cell populations. Our findings indicate that tuft cells contribute to the prostate cancer microenvironment and may promote development of more advanced disease. Further research is needed to understand contributions of tuft cells to prostate cancer progression.