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.

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.

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.

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.

Targeting Doublecortin-Like Kinase 1 (DCLK1)-Regulated SARS-CoV-2 Pathogenesis in COVID-19.

Host factors play critical roles in SARS-CoV-2 infection-associated pathology and the severity of COVID-19. In this study, we systematically analyzed the roles of SARS-CoV-2-induced host factors, doublecortin-like kinase 1 (DCLK1), and S100A9 in viral pathogenesis. In autopsied subjects with COVID-19 and pre-existing chronic liver disease, we observed high levels of DCLK1 and S100A9 expression and immunosuppressive (DCLK1+S100A9+CD206+) M2-like macrophages and N2-like neutrophils in lungs and livers. DCLK1 and S100A9 expression were rarely observed in normal controls, COVID-19-negative subjects with chronic lung disease, or COVID-19 subjects without chronic liver disease. In hospitalized patients with COVID-19, we detected 2 to 3-fold increased levels of circulating DCLK1+S100A9+ mononuclear cells that correlated with disease severity. We validated the SARS-CoV-2-dependent generation of these double-positive immune cells in coculture. SARS-CoV-2-induced DCLK1 expression correlated with the activation of ß-catenin, a known regulator of the DCLK1 promoter. Gain and loss of function studies showed that DCLK1 kinase amplified live virus production and promoted cytokine, chemokine, and growth factor secretion by peripheral blood mononuclear cells. Inhibition of DCLK1 kinase blocked pro-inflammatory caspase-1/interleukin-1ß signaling in infected cells. Treatment of SARS-CoV-2-infected cells with inhibitors of DCLK1 kinase and S100A9 normalized cytokine/chemokine profiles and attenuated DCLK1 expression and ß-catenin activation. In conclusion, we report previously unidentified roles of DCLK1 in augmenting SARS-CoV-2 viremia, inflammatory cytokine expression, and dysregulation of immune cells involved in innate immunity. DCLK1 could be a potential therapeutic target for COVID-19, especially in patients with underlying comorbid diseases associated with DCLK1 expression. IMPORTANCE High mortality in COVID-19 is associated with underlying comorbidities such as chronic liver diseases. Successful treatment of severe/critical COVID-19 remains challenging. Herein, we report a targetable host factor, DCLK1, that amplifies SARS-CoV-2 production, cytokine secretion, and inflammatory pathways via activation of ß-catenin(p65)/DCLK1/S100A9/NF-κB signaling. Furthermore, we observed in the lung, liver, and blood an increased prevalence of immune cells coexpressing DCLK1 and S100A9, a myeloid-derived proinflammatory protein. These cells were associated with increased disease severity in COVID-19 patients. Finally, we used a novel small-molecule inhibitor of DCLK1 kinase (DCLK1-IN-1) and S100A9 inhibitor (tasquinimod) to decrease virus production in vitro and normalize hyperinflammatory responses known to contribute to disease severity in COVID-19.

Replication stress identifies novel molecular classification associated with treatment outcomes in pancreatic cancer.

BACKGROUND: Replication stress is a prominent hallmark of tumor cells, which is crucial for maintaining genomic integrity. However, it remains poorly understood whether replication stress can serve as a surrogate biomarker to indicate prognosis and treatment response of pancreatic cancer. METHODS: Transcriptomic and clinical data were obtained from The Cancer Genome Atlas and literature. An integrated signature of 18 replication-stress associated genes (termed as REST18) was established using the cox proportional hazards regression analysis. Tumors were sorted into REST18-low and REST18-high groups. Survival analysis, gene set enrichment analysis and composition of immune cells were compared between these tumors. RESULTS: Patients with REST18-high tumors showed worse prognoses than those with REST18-low tumors in the TCGA database and the finding is validated in an independent cohort of pancreatic cancer. Comparison of REST18 model and other molecular classifications showed that REST18-high tumors are positively correlated to basal-like or squamous phenotypes, which have higher metastasis potential. DNA repair pathway is enriched in the REST18-high tumors. Analysis of tumor immune microenvironment found that REST18-high tumors are characterized with "immune-cold" features. Univariate and multivariate analysis show that REST18 is an independent risk factor for overall survival and predicts outcomes of chemotherapy in pancreatic cancer. CONCLUSION: REST18 is a novel biomarker to indicate prognosis and treatment response of chemotherapy in pancreatic cancer.

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.

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.

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.

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.

A subset of epithelial cells mimics regulatory T cells and contributes to immune evasion during development of pancreatic adenocarcinoma.

Pancreatic cancer is refractory to most current treatment options. Immunotherapy emerges as an effective and novel therapeutic strategy for several solid tumors. However, most of the clinical trials on immunotherapy have failed in pancreatic cancer. Understanding the underlying mechanism that drives immune evasion of pancreatic cancer is critical for overcoming resistance to therapy. Recently, Dr. He Ren and colleagues proposed a novel concept that a subset of epithelial cells in pancreatic cancer mimics the phenotype and function of regulatory T cells, named as "quasi-regulatory T cells." These cells contribute to enhanced immune evasion, angiogenesis, and metastasis of pancreatic cancer, thus providing potential therapeutic targets to improve the sensitivity of immunotherapy for this devastating disease. This ground-breaking concept will advance our understanding on the immune evasion of pancreatic cancer and chart novel paths towards the development of personalized treatment for pancreatic cancer.

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.

Defective Intestinal Mucin-Type O-Glycosylation Causes Spontaneous Colitis-Associated Cancer in Mice.

BACKGROUND & AIMS: Core 1- and core 3-derived mucin-type O-linked oligosaccharides (O-glycans) are major components of the colonic mucus layer. Defective forms of colonic O-glycans, such as the Thomsen-nouveau (Tn) antigen, frequently are observed in patients with ulcerative colitis and colorectal cancer, but it is not clear if they contribute to their pathogenesis. We investigated whether and how impaired O-glycosylation contributes to the development of colitis-associated colorectal cancer using mice lacking intestinal core 1- and core 3-derived O-glycans. METHODS: We generated mice that lack core 1- and core 3-derived intestinal O-glycans (DKO mice) and analyzed them, along with mice that singly lack intestinal epithelial core 1 O-glycans (IEC C1galt1(-/-) mice) or core 3 O-glycans (C3Gnt(-/-) mice). Intestinal tissues were collected at different time points and analyzed for levels of mucin and Tn antigen, development of colitis, and tumor formation using imaging, quantitative polymerase chain reaction, immunoblot, and enzyme-linked immunosorbent assay techniques. We also used cellular and genetic approaches, as well as intestinal microbiota depletion, to identify inflammatory mediators and pathways that contribute to disease in DKO and wild-type littermates (controls). RESULTS: Intestinal tissues from DKO mice contained higher levels of Tn antigen and had more severe spontaneous chronic colitis than tissues from IEC C1galt1(-/-) mice, whereas spontaneous colitis was absent in C3GnT(-/-) and control mice. IEC C1galt1(-/-) mice and DKO mice developed spontaneous colorectal tumors, although the onset of tumors in the DKO mice occurred earlier (age, 8-9 months) than that in IEC C1galt1(-/-) mice (15 months old). Antibiotic depletion of the microbiota did not cause loss of Tn antigen but did reduce the development of colitis and cancer formation in DKO mice. Colon tissues from DKO mice, but not control mice, contained active forms of caspase 1 and increased caspase 11, which were reduced after antibiotic administration. Supernatants from colon tissues of DKO mice contained increased levels of interleukin-1ß and interleukin-18, compared with those from control mice. Disruption of the caspase 1 and caspase 11 genes in DKO mice (DKO/Casp1/11(-/-) mice) decreased the development of colitis and cancer, characterized by reduced colonic thickening, hyperplasia, inflammatory infiltrate, and tumors compared with DKO mice. CONCLUSIONS: Impaired expression of O-glycans causes colonic mucus barrier breach and subsequent microbiota-mediated activation of caspase 1-dependent inflammasomes in colonic epithelial cells of mice. These processes could contribute to colitis-associated colon cancer in humans.

Novel regenerative peptide TP508 mitigates radiation-induced gastrointestinal damage by activating stem cells and preserving crypt integrity.

In recent years, increasing threats of radiation exposure and nuclear disasters have become a significant concern for the United States and countries worldwide. Exposure to high doses of radiation triggers a number of potentially lethal effects. Among the most severe is the gastrointestinal (GI) toxicity syndrome caused by the destruction of the intestinal barrier, resulting in bacterial translocation, systemic bacteremia, sepsis, and death. The lack of effective radioprotective agents capable of mitigating radiation-induced damage has prompted a search for novel countermeasures that can mitigate the effects of radiation post exposure, accelerate tissue repair in radiation-exposed individuals, and prevent mortality. We report that a single injection of regenerative peptide TP508 (rusalatide acetate, Chrysalin) 24 h after lethal radiation exposure (9 Gy, LD100/15) appears to significantly increase survival and delay mortality by mitigating radiation-induced intestinal and colonic toxicity. TP508 treatment post exposure prevents the disintegration of GI crypts, stimulates the expression of adherens junction protein E-cadherin, activates crypt cell proliferation, and decreases apoptosis. TP508 post-exposure treatment also upregulates the expression of DCLK1 and LGR5 markers of stem cells that have been shown to be responsible for maintaining and regenerating intestinal crypts. Thus, TP508 appears to mitigate the effects of GI toxicity by activating radioresistant stem cells and increasing the stemness potential of crypts to maintain and restore intestinal integrity. These results suggest that TP508 may be an effective emergency nuclear countermeasure that could be delivered within 24 h post exposure to increase survival and delay mortality, giving victims time to reach clinical sites for advanced medical treatment.

Brief report: Dclk1 deletion in tuft cells results in impaired epithelial repair after radiation injury.

The role of Dclk1(+) tuft cells in the replacement of intestinal epithelia and reestablishing the epithelial barrier after severe genotoxic insult is completely unknown. Successful restoration requires precise coordination between the cells within each crypt subunit. While the mechanisms that control this response remain largely uncertain, the radiation model remains an exceptional surrogate for stem cell-associated crypt loss. Following the creation of Dclk1-intestinal-epithelial-deficient Villin-Cre;Dclk1(flox/flox) mice, widespread gene expression changes were detected in isolated intestinal epithelia during homeostasis. While the number of surviving crypts was unaffected, Villin-Cre;Dclk1(flox/flox) mice failed to maintain tight junctions and died at approximately 5 days, where Dclk1(flox/flox) mice lived until day 10 following radiation injury. These findings suggest that Dclk1 plays a functional role critical in the epithelial restorative response.