From Inflammation to Oncogenesis: Tracing Serum DCLK1 and miRNA Signatures in Chronic Liver Diseases.

Chronic liver diseases, fibrosis, cirrhosis, and HCC are often a consequence of persistent inflammation. However, the transition mechanisms from a normal liver to fibrosis, then cirrhosis, and further to HCC are not well understood. This study focused on the role of the tumor stem cell protein doublecortin-like kinase 1 (DCLK1) in the modulation of molecular factors in fibrosis, cirrhosis, or HCC. Serum samples from patients with hepatic fibrosis, cirrhosis, and HCC were analyzed via ELISA or NextGen sequencing and were compared with control samples. Differentially expressed (DE) microRNAs (miRNA) identified from these patient sera were correlated with DCLK1 expression. We observed elevated serum DCLK1 levels in fibrosis, cirrhosis, and HCC patients; however, TGF-ß levels were only elevated in fibrosis and cirrhosis. While DE miRNAs were identified for all three disease states, miR-12136 was elevated in fibrosis but was significantly increased further in cirrhosis. Additionally, miR-1246 and miR-184 were upregulated when DCLK1 was high, while miR-206 was downregulated. This work distinguishes DCLK1 and miRNAs' potential role in different axes promoting inflammation to tumor progression and may serve to identify biomarkers for tracking the progression from pre-neoplastic states to HCC in chronic liver disease patients as well as provide targets for treatment.

The crosstalk between macrophages and cancer cells potentiates pancreatic cancer cachexia.

With limited treatment options, cachexia remains a major challenge for patients with cancer. Characterizing the interplay between tumor cells and the immune microenvironment may help identify potential therapeutic targets for cancer cachexia. Herein, we investigate the critical role of macrophages in potentiating pancreatic cancer induced muscle wasting via promoting TWEAK (TNF-like weak inducer of apoptosis) secretion from the tumor. Specifically, depletion of macrophages reverses muscle degradation induced by tumor cells. Macrophages induce non-autonomous secretion of TWEAK through CCL5/TRAF6/NF-κB pathway. TWEAK promotes muscle atrophy by activating MuRF1 initiated muscle remodeling. Notably, tumor cells recruit and reprogram macrophages via the CCL2/CCR2 axis and disrupting the interplay between macrophages and tumor cells attenuates muscle wasting. Collectively, this study identifies a feedforward loop between pancreatic cancer cells and macrophages, underlying the non-autonomous activation of TWEAK secretion from tumor cells thereby providing promising therapeutic targets for pancreatic cancer cachexia.

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.

Acetyl-Coenzyme A Synthetase 2 Potentiates Macropinocytosis and Muscle Wasting Through Metabolic Reprogramming in Pancreatic Cancer.

BACKGROUND & AIMS: Rapid deconditioning, also called cachexia, and metabolic reprogramming are two hallmarks of pancreatic cancer. Acetyl-coenzyme A synthetase short-chain family member 2 (ACSS2) is an acetyl-enzyme A synthetase that contributes to lipid synthesis and epigenetic reprogramming. However, the role of ACSS2 on the nonselective macropinocytosis and cancer cachexia in pancreatic cancer remains elusive. In this study, we demonstrate that ACSS2 potentiates macropinocytosis and muscle wasting through metabolic reprogramming in pancreatic cancer. METHODS: Clinical significance of ACSS2 was analyzed using samples from patients with pancreatic cancer. ACSS2-knockout cells were established using the clustered regularly interspaced short palindromic repeats-associated protein 9 system. Single-cell RNA sequencing data from genetically engineered mouse models was analyzed. The macropinocytotic index was evaluated by dextran uptake assay. Chromatin immunoprecipitation assay was performed to validate transcriptional activation. ACSS2-mediated tumor progression and muscle wasting were examined in orthotopic xenograft models. RESULTS: Metabolic stress induced ACSS2 expression, which is associated with worse prognosis in pancreatic cancer. ACSS2 knockout significantly suppressed cell proliferation in 2-dimensional and 3-dimensional models. Macropinocytosis-associated genes are upregulated in tumor tissues and are correlated with worse prognosis. ACSS2 knockout inhibited macropinocytosis. We identified Zrt- and Irt-like protein 4 (ZIP4) as a downstream target of ACSS2, and knockdown of ZIP4 reversed ACSS2-induced macropinocytosis. ACSS2 upregulated ZIP4 through ETV4-mediated transcriptional activation. ZIP4 induces macropinocytosis through cyclic adenosine monophosphate response element-binding protein-activated syndecan 1 (SDC1) and dynamin 2 (DNM2). Meanwhile, ZIP4 drives muscle wasting and cachexia via glycogen synthase kinase-ß (GSK3ß)-mediated secretion of tumor necrosis factor superfamily member 10 (TRAIL or TNFSF10). ACSS2 knockout attenuated muscle wasting and extended survival in orthotopic mouse models. CONCLUSIONS: ACSS2-mediated metabolic reprogramming activates the ZIP4 pathway, and promotes macropinocytosis via SDC1/DNM2 and drives muscle wasting through the GSK3ß/TRAIL axis, which potentially provides additional nutrients for macropinocytosis in pancreatic cancer.

Chagas disease in Oklahoma.

Chagas disease, caused by infection with the protozoan Trypanosoma cruzi, is one of the leading public health problems in the Western Hemisphere. The parasite is mainly transmitted by contact with infected insect vectors but other forms of transmission are important in endemic areas. In the United States, while the disease is largely restricted to immigrants from endemic countries in Latin America, there is some risk of local acquisition. T. cruzi circulates in a sylvatic cycle between mammals and local triatomine insects in the southern half of the country, where human residents may be at risk for incidental infection. There are several reported cases of locally-acquired Chagas disease in the United States, but there is a paucity of information in Oklahoma. We present a brief summary of the available data of Chagas disease in Oklahoma to raise awareness and serve as a foundation for future research.

Circular RNA ANAPC7 Inhibits Tumor Growth and Muscle Wasting via PHLPP2-AKT-TGF-ß Signaling Axis in Pancreatic Cancer.

BACKGROUND & AIMS: Pancreatic cancer has the highest prevalence of cancer-associated cachexia among all cancers. ZIP4 promotes pancreatic cancer progression by regulating oncogenic miR-373, and perturbation of circular RNAs (circRNAs) is associated with cancer aggressiveness. This study aimed to identify circRNAs involved in ZIP4/miR-373-driven cancer growth and cachexia and decipher the underlying mechanism. METHODS: Differentially expressed circRNAs and potential targets of microRNA were identified through in silico analysis. The RNA interactions were determined by means of biotinylated microRNA pulldown, RNA immunoprecipitation, and luciferase reporter assays. The function of circRNA in ZIP4-miR-373 signaling axis were examined in human pancreatic cancer cells, 3-dimensional spheroids and organoids, mouse models, and clinical specimens. Mouse skeletal muscles were analyzed by means of histology. RESULTS: We identified circANAPC7 as a sponge for miR-373, which inhibited tumor growth and muscle wasting in vitro and in vivo. Mechanistic studies showed that PHLPP2 is a downstream target of ZIP4/miR-373. CircANAPC7 functions through PHLPP2-mediated dephosphorylation of AKT, thus suppressing cancer cell proliferation by down-regulating cyclin D1 and inhibiting muscle wasting via decreasing the secretion of transforming growth factor-ß through STAT5. We further demonstrated that PHLPP2 induced dephosphorylation of CREB, a zinc-dependent transcription factor activated by ZIP4, thereby forming a CREB-miR-373-PHLPP2 feed-forward loop to regulate tumor progression and cancer cachexia. CONCLUSION: This study identified circANAPC7 as a novel tumor suppressor, which functions through the CREB-miR-373-PHLPP2 axis, leading to AKT dephosphorylation, and cyclin D1 and transforming growth factor-ß down-regulation to suppress tumor growth and muscle wasting in pancreatic cancer.

Zinc-Dependent Regulation of ZEB1 and YAP1 Coactivation Promotes Epithelial-Mesenchymal Transition Plasticity and Metastasis in Pancreatic Cancer.

BACKGROUND: Pancreatic cancer is characterized by extensive metastasis. Epithelial-mesenchymal transition (EMT) plasticity plays a critical role in tumor progression and metastasis by maintaining the transition between EMT and mesenchymal-epithelial transition states. Our aim is to understand the molecular events regulating metastasis and EMT plasticity in pancreatic cancer. METHODS: The interactions between a cancer-promoting zinc transporter ZIP4, a zinc-dependent EMT transcriptional factor ZEB1, a coactivator YAP1, and integrin α3 (ITGA3) were examined in human pancreatic cancer cells, clinical specimens, spontaneous mouse models (KPC and KPCZ) and orthotopic xenografts, and 3-dimensional spheroid and organoid models. Correlations between ZIP4, miR-373, and its downstream targets were assessed by RNA in situ hybridization and immunohistochemical staining. The transcriptional regulation of ZEB1, YAP1, and ITGA3 by ZIP4 was determined by chromatin immunoprecipitation, co-immunoprecipitation, and luciferase reporter assays. RESULTS: The Hippo pathway effector YAP1 is a potent transcriptional coactivator and forms a complex with ZEB1 to activate ITGA3 transcription through the YAP1/transcriptional enhanced associate domain (TEAD) binding sites in human pancreatic cancer cells and KPC-derived mouse cells. ZIP4 upregulated YAP1 expression via activation of miR-373 and inhibition of the YAP1 repressor large tumor suppressor 2 kinase (LATS2). Furthermore, upregulation of ZIP4 promoted EMT plasticity, cell adhesion, spheroid formation, and organogenesis both in human pancreatic cancer cells, 3-dimensional spheroid model, xenograft model, and spontaneous mouse models (KPC and KPCZ) through ZEB1/YAP1-ITGA3 signaling axis. CONCLUSION: We demonstrated that ZIP4 activates ZEB1 and YAP1 through distinct mechanisms. The ZIP4-miR-373-LATS2-ZEB1/YAP1-ITGA3 signaling axis has a significant impact on pancreatic cancer metastasis and EMT plasticity.

DCLK1 Regulates Tumor Stemness and Cisplatin Resistance in Non-small Cell Lung Cancer via ABCD-Member-4.

Chemoresistance cells have features similar to cancer stem cells. Elimination of these cells is an effective therapeutic strategy to clinically combat chemoresistance non-small cell lung cancer (NSCLC). Here, we demonstrate that Doublecortin-like kinase1 (DCLK1) is the key to developing chemoresistance and associated stemness in NSCLC. DCLK1 is highly expressed in human lung adenocarcinoma and strongly correlated with stemness. Silencing DCLK1 inhibits NSCLC cell primary and secondary spheroid formation, which is the prerequisite feature of tumor stem cells. DCLK1 inhibition reduced NSCLC cell migration/invasion in vitro and induced tumor growth inhibition in vivo. NSCLC cells responded differently to cisplatin treatment; indeed, the clonogenic ability of all NSCLC cells was reduced. We found that the cisplatin-resistant NSCLC cells gain the expression of DCLK1 compared with their parental control. However, DCLK1 inhibition in cisplatin-resistance NSCLC cells reverses the tumor cell resistance to cisplatin and reduced tumor self-renewal ability. Specifically, we found that DCLK1-mediated cisplatin resistance in NSCLC is via an ABC subfamily member 4 (ABCD4)-dependent mechanism. Our data demonstrate that increased expression of DCLK1 is associated with chemoresistance and enhanced cancer stem cell-like features in NSCLC. Targeting DCLK1 using gene knockdown/knockout strategies alone or in combination with cisplatin may represent a novel therapeutic strategy to treat NSCLC.

Doublecortin-like kinase 1 promotes hepatocyte clonogenicity and oncogenic programming via non-canonical ß-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 ß-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-3ß at Ser9. This was associated with an induction of a 48-kDa active ß-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 ß-catenin expression. The proteasome inhibitor bortezomib did not block the 48-kDa ß-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 ß-catenin, and cleaved E-cadherin. Repopulated DCLK1-overexpressing primary human hepatocytes in humanized FRG mouse livers demonstrated active ß-catenin. In conclusion, DCLK1 regulates oncogenic signaling and clonogenicity of hepatocytes by a novel non-canonical/atypical ß-catenin-dependent mechanism.

DCLK1-Isoform2 Alternative Splice Variant Promotes Pancreatic Tumor Immunosuppressive M2-Macrophage Polarization.

Tumor-associated M2-macrophages are one of the most abundant immunosuppressive cell types in the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME). However, the molecular mechanisms responsible for the generation of M2-macrophages are unclear. Here, we demonstrated that overexpression of DCLK1-isoform2 in AsPC1 and MIA PaCa2 cells resulted in the polarization of M1-macrophages toward an M2 phenotype via secreted chemokines/cytokines. These M2-macrophages enhanced parental PDAC cell migration, invasion, and self-renewal, and this was associated with increased expression of Snail and Slug. We observed distinct expression of Dclk-isoform2, marked infiltration of M2-macrophages, and a marginal increase of CD8+ T cells in 20-week-old KPCY mice pancreas compared with 5 weeks old. Utilizing an autochthonous mouse model of pancreatic adenocarcinoma, we observed distinct immunoreactive Dclk1 and arginase1 in tissues where CD8+ T-cell infiltration was low and observed a paucity of DCLK1 and arginase1 staining where CD8+ T-cell infiltration was high. Finally, we found that DCLK1-isoform2 tumor-educated M2-macrophages inhibit CD8+ T-cell proliferation and granzyme-B activation. Inhibition of DCLK1 in an organoid coculture system enhanced CD8+ T-cell activation and associated organoid death. We conclude that DCLK1-isoform2 is a novel initiator of alternate macrophage activation that contributes to the immunosuppression observed in the PDAC TME. These data suggest that tumor DCLK1-isoform2 may be an attractive target for PDAC therapy, either alone or in conjunction with immunotherapeutic strategies.

ZIP4 Increases Expression of Transcription Factor ZEB1 to Promote Integrin α3ß1 Signaling and Inhibit Expression of the Gemcitabine Transporter ENT1 in Pancreatic Cancer Cells.

BACKGROUND & AIMS: Pancreatic tumors undergo rapid growth and progression, become resistant to chemotherapy, and recur after surgery. We studied the functions of the solute carrier family 39 member 4 (SLC39A4, also called ZIP4), which regulates concentrations of intracellular zinc and is increased in pancreatic cancer cells, in cell lines and mice. METHODS: We obtained 93 pancreatic cancer specimens (tumor and adjacent nontumor tissues) from patients who underwent surgery and gemcitabine chemotherapy and analyzed them by immunohistochemistry. ZIP4 and/or ITGA3 or ITGB1 were overexpressed or knocked down with short hairpin RNAs in AsPC-1 and MIA PaCa-2 pancreatic cancer cells lines, and in pancreatic cells from KPC and KPC-ZEB1-knockout mice, and pancreatic spheroids were established; cells and spheroids were analyzed by immunoblots, reverse transcription polymerase chain reaction, and liquid chromatography tandem mass spectrometry. We studied transcriptional regulation of ZEB1, ITGA3, ITGB1, JNK, and ENT1 by ZIP4 using chromatin precipitation and luciferase reporter assays. Nude mice were given injections of genetically manipulated AsPC-1 and MIA PaCa-2 cells, and growth of xenograft tumors and metastases was measured. RESULTS: In pancreatic cancer specimens from patients, increased levels of ZIP4 were associated with shorter survival times. MIA PaCa-2 cells that overexpressed ZIP4 had increased resistance to gemcitabine, 5-fluorouracil, and cisplatin, whereas AsPC-1 cells with ZIP4 knockdown had increased sensitivity to these drugs. In mice, xenograft tumors grown from AsPC-1 cells with ZIP4 knockdown were smaller and more sensitive to gemcitabine. ZIP4 overexpression significantly reduced accumulation of gemcitabine in pancreatic cancer cells, increased growth of xenograft tumors in mice, and increased expression of the integrin subunits ITGA3 and ITGB1; expression levels of ITGA3 and ITGB1 were reduced in cells with ZIP4 knockdown. Pancreatic cancer cells with ITGA3 or ITGB1 knockdown had reduced proliferation and formed smaller tumors in mice, despite overexpression of ZIP4; spheroids established from these cells had increased sensitivity to gemcitabine. We found ZIP4 to activate STAT3 to induce expression of ZEB1, which induced expression of ITGA3 and ITGB1 in KPC cells. Increased ITGA3 and ITGB1 expression and subsequent integrin α3ß1 signaling, via c-Jun-N-terminal kinase (JNK), inhibited expression of the gemcitabine transporter ENT1, which reduced gemcitabine uptake by pancreatic cancer cells. ZEB1-knockdown cells had increased sensitivity to gemcitabine. CONCLUSIONS: In studies of pancreatic cancer cell lines and mice, we found that ZIP4 increases expression of the transcription factor ZEB1, which activates expression of ITGA3 and ITGB1. The subsequent increase in integrin α3ß1 signaling, via JNK, inhibits expression of the gemcitabine transporter ENT1, so that cells take up smaller amounts of the drug. Activation of this pathway might help mediate resistance of pancreatic tumors to chemotherapeutic agents.

Kikuchi-Fujimoto Disease: An Atypical Presentation of a Rare Disease.

Kikuchi-Fujimoto disease (KFD), or necrotizing histiocytic lymphadenitis, is a rare cause of lymphadenopathy and fever. Although the clinical course is usually benign, KFD is often mistaken for malignancy or infection. Recognition of typical and atypical cases of KFD is necessary to avoid unnecessary interventions. Here we report an atypical presentation of KFD with diffuse lymphadenopathy and leukocytosis associated with high levels of circulating Epstein-Barr viral DNA.

ZIP4 Promotes Muscle Wasting and Cachexia in Mice With Orthotopic Pancreatic Tumors by Stimulating RAB27B-Regulated Release of Extracellular Vesicles From Cancer Cells.

BACKGROUND & AIMS: Cachexia, which includes muscle wasting, is a frequent complication of pancreatic cancer. There are no therapies that reduce cachexia and increase patient survival, so it is important to learn more about its mechanisms. The zinc transporter ZIP4 promotes growth and metastasis of pancreatic tumors. We investigated its effects on muscle catabolism via extracellular vesicle (EV)-mediated stimulation of mitogen-activated protein kinase 14 (p38 MAPK). METHODS: We studied nude mice with orthotopic tumors grown from human pancreatic cancer cell lines (AsPC-1 and BxPC-3); tumors were removed 8 days after cell injection and analyzed by histology. Mouse survival was analyzed by Kaplan-Meier curves. ZIP4 was knocked down in AsPC-1 and BxPC-3 cells with small hairpin RNAs; cells with empty vectors were used as controls. Muscle tissues were collected from mice and analyzed by histology and immunohistochemistry. Conditioned media from cell lines and 3-dimensional spheroid/organoid cultures of cancer cells were applied to C2C12 myotubes. The myotubes and the media were analyzed by immunoblots, enzyme-linked immunosorbent assays, and immunofluorescence microscopy. EVs were isolated from conditioned media and analyzed by immunoblots. RESULTS: Mice with orthotopic tumors grown from pancreatic cancer cells with knockdown of ZIP4 survived longer and lost less body weight and muscle mass than mice with control tumors. Conditioned media from cancer cells activated p38 MAPK, induced expression of F-box protein 32 and UBR2 in C2C12 myotubes, and also led to loss of myofibrillar protein myosin heavy chain and myotube thinning. Knockdown of ZIP4 in cancer cells reduced these effects. ZIP4 knockdown also reduced pancreatic cancer cell release of heat shock protein (HSP) 70 and HSP90, which are associated with EVs, by decreasing CREB-regulated expression of RAB27B. CONCLUSIONS: ZIP4 promotes growth of orthotopic pancreatic tumors in mice and loss of muscle mass by activating CREB-regulated expression of RAB27B, required for release of EVs from pancreatic cancer cells. These EVs activate p38 MAPK and induce expression of F-box protein 32 and UBR2 in myotubes, leading to loss of myofibrillar myosin heavy chain and myotube thinning. Strategies to disrupt these pathways might be developed to reduce pancreatic cancer progression and accompanying cachexia.

ZIP4 Promotes Pancreatic Cancer Progression by Repressing ZO-1 and Claudin-1 through a ZEB1-Dependent Transcriptional Mechanism.

Purpose: ZIP4 is overexpressed in human pancreatic cancer and promotes tumor growth. However, little is known about the role of ZIP4 in advanced stages of this dismal neoplasm. Our goal is to study the underlying mechanism and define a novel signaling pathway controlled by ZIP4-modulating pancreatic tumor metastasis.Experimental Design: The expression of ZIP4, ZO-1, claudin-1, and ZEB1 in human pancreatic cancer tissues, genetically engineered mouse model, xenograft tumor model, and pancreatic cancer cell lines were examined, and the correlations between ZIP4 and those markers were also analyzed. Functional analysis of ZO-1, claudin-1, and ZEB1 was investigated in pancreatic cancer cell lines and orthotopic xenografts.Results: Genetic inactivation of ZIP4 inhibited migration and invasion in pancreatic cancer and increased the expression of ZO-1 and claudin-1. Conversely, overexpression of ZIP4 promoted migration and invasion and increased the expression of ZEB1 and downregulation of the aforementioned epithelial genes. ZIP4 downregulation of ZO-1 and claudin-1 requires the transcriptional repressor ZEB1. Further analysis demonstrated that ZIP4-mediated repression of ZO-1 and claudin-1 leads to upregulation of their targets FAK and Paxillin. Silencing of ZIP4 caused reduced phosphorylation of FAK and Paxillin, which was rescued by simultaneous blocking of ZO-1 or claudin-1. Clinically, we demonstrated that ZIP4 positively correlates with the levels of ZEB1 and inversely associates with the expression of ZO-1 and claudin-1.Conclusions: These findings suggest a novel pathway activated by ZIP4-controlling pancreatic cancer invasiveness and metastasis, which could serve as a new therapeutic target for this devastating disease. Clin Cancer Res; 24(13); 3186-96. ©2018 AACR.

Alternative splice variants of DCLK1 mark cancer stem cells, promote self-renewal and drug-resistance, and can be targeted to inhibit tumorigenesis in kidney cancer.

Renal cell carcinoma (RCC) is a common and devastating disease characterized by a hypoxic microenvironment, epithelial-mesenchymal transition and potent resistance to therapy evidencing the presence of cancer stem cells (CSCs). Various CSC markers have been studied in RCC, but overall there is limited data on their role and most markers studied have been relatively nonspecific. Doublecortin-like kinase 1 (DCLK1) is a validated CSC marker in the gastrointestinal tract and evidence for an equivalent role in other cancers is accumulating. We used bioinformatics, immunohistochemistry, flow cytometry, spheroid self-renewal and chemoresistance assays in combination with overexpression and siRNA-knockdown to study the stem cell-supportive role of DCLK1 alternative splice variants (DCLK1 ASVs) in RCC. To target tumor cells expressing DCLK1 ASVs directly, we developed a novel monoclonal antibody (CBT-15) and delivered it systemically to RCC tumor xenografts. DCLK1 ASVs were overexpressed, enriched together with CSC markers and predictive of overall and recurrence-free survival in RCC patients. In vitro, DCLK1 ASVs were able to directly stimulate essential molecular and functional characteristics of renal CSCs including expression of aldehyde dehydrogenase, self-renewal and resistance to FDA-approved receptor tyrosine kinase and mTOR inhibitors, while targeted downregulation of DCLK1 reversed these characteristics. Finally, targeting DCLK1 ASV-positive cells with the novel CBT-15 monoclonal antibody blocked RCC tumorigenesis in vivo. These findings establish DCLK1 as a CSC marker with implications for therapy, disease progression and survival in RCC and demonstrate the therapeutic value of DCLK1-targeted monoclonal antibodies against renal CSCs.

Dclk1, a tumor stem cell marker, regulates pro-survival signaling and self-renewal of intestinal tumor cells.

BACKGROUND: More than 80% of intestinal neoplasia is associated with the adenomatous polyposis coli (APC) mutation. Doublecortin-like kinase 1 (Dclk1), a kinase protein, is overexpressed in colorectal cancer and specifically marks tumor stem cells (TSCs) that self-renew and increased the tumor progeny in Apc Min/+ mice. However, the role of Dclk1 expression and its contribution to regulating pro-survival signaling for tumor progression in Apc mutant cancer is poorly understood. METHODS: We analyzed DCLK1 and pro-survival signaling gene expression datasets of 329 specimens from TCGA Colon Adenocarcinoma Cancer Data. The network of DCLK1 and pro-survival signaling was analyzed utilizing the GeneMANIA database. We examined the expression levels of Dclk1 and other stem cell-associated markers, pro-survival signaling pathways, cell self-renewal in the isolated intestinal epithelial cells of Apc Min/+ mice with high-grade dysplasia and adenocarcinoma. To determine the functional role of Dclk1 for tumor progression, we knocked down Dclk1 and determined the pro-survival signaling pathways and stemness. We used siRNA technology to gene silence pro-survival signaling in colon cancer cells in vitro. We utilized FACS, IHC, western blot, RT-PCR, and clonogenic (self-renewal) assays. RESULTS: We found a correlation between DCLK1 and pro-survival signaling expression. The expression of Dclk1 and stem cell-associated markers Lgr5, Bmi1, and Musashi1 were significantly higher in the intestinal epithelial cells of Apc Min/+ mice than in wild-type controls. Intestinal epithelial cells of Apc Min/+ mice showed increased expression of pro-survival signaling, pluripotency and self-renewal ability. Furthermore, the enteroids formed from the intestinal Dclk1+ cells of Apc Min/+ mice display higher pluripotency and pro-survival signaling. Dclk1 knockdown in Apc Min/+ mice attenuates intestinal adenomas and adenocarcinoma, and decreases pro-survival signaling and self-renewal. Knocking down RELA and NOTCH1 pro-survival signaling and DCLK1 in HT29 and DLD1 colon cancer cells in vitro reduced the tumor cells' ability to self-renew and survive. CONCLUSION: Our results indicate that Dclk1 is essential in advancing intestinal tumorigenesis. Knocking down Dclk1 decreases tumor stemness and progression and is thus predicted to regulate pro-survival signaling and tumor cell pluripotency. This study provides a strong rationale to target Dclk1 as a treatment strategy for colorectal cancer.

Intestinal tuft cells regulate the ATM mediated DNA Damage response via Dclk1 dependent mechanism for crypt restitution following radiation injury.

Crypt epithelial survival and regeneration after injury require highly coordinated complex interplay between resident stem cells and diverse cell types. The function of Dclk1 expressing tuft cells regulating intestinal epithelial DNA damage response for cell survival/self-renewal after radiation-induced injury is unclear. Intestinal epithelial cells (IECs) were isolated and purified and utilized for experimental analysis. We found that small intestinal crypts of VillinCre;Dclk1f/f mice were hypoplastic and more apoptotic 24 h post-total body irradiation, a time when stem cell survival is p53-independent. Injury-induced ATM mediated DNA damage response, pro-survival genes, stem cell markers, and self-renewal ability for survival and restitution were reduced in the isolated intestinal epithelial cells. An even greater reduction in these signaling pathways was observed 3.5 days post-TBI, when peak crypt regeneration occurs. We found that interaction with Dclk1 is critical for ATM and COX2 activation in response to injury. We determined that Dclk1 expressing tuft cells regulate the whole intestinal epithelial cells following injury through paracrine mechanism. These findings suggest that intestinal tuft cells play an important role in regulating the ATM mediated DNA damage response, for epithelial cell survival/self-renewal via a Dclk1 dependent mechanism, and these processes are indispensable for restitution and function after severe radiation-induced injury.

Survival of Patients with Gastrointestinal Cancers Can Be Predicted by a Surrogate microRNA Signature for Cancer Stem-like Cells Marked by DCLK1 Kinase.

Doublecortin-like kinase 1 (DCLK1) is a gastrointestinal (GI) tuft cell kinase that has been investigated as a biomarker of cancer stem-like cells in colon and pancreatic cancers. However, its utility as a biomarker may be limited in principle by signal instability and dilution in heterogeneous tumors, where the proliferation of diverse tumor cell lineages obscures the direct measurement of DCLK1 activity. To address this issue, we explored the definition of a miRNA signature as a surrogate biomarker for DCLK1 in cancer stem-like cells. Utilizing RNA/miRNA-sequencing datasets from the Cancer Genome Atlas, we identified a surrogate 15-miRNA expression signature for DCLK1 activity across several GI cancers, including colon, pancreatic, and stomach cancers. Notably, Cox regression and Kaplan-Meier analysis demonstrated that this signature could predict the survival of patients with these cancers. Moreover, we identified patient subgroups that predicted the clinical utility of this DCLK1 surrogate biomarker. Our findings greatly strengthen the clinical significance for DCLK1 expression across GI cancers. Further, they provide an initial guidepost toward the development of improved prognostic biomarkers or companion biomarkers for DCLK1-targeted therapies to eradicate cancer stem-like cells in these malignancies. Cancer Res; 76(14); 4090-9. ©2016 AACR.