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.

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.

DCLK1 is detectable in plasma of patients with Barrett's esophagus and esophageal adenocarcinoma.

BACKGROUND: Doublecortin-like kinase 1 (DCLK1), a putative tumor stem cell marker has been shown to be highly expressed in the stromal and epithelial compartments in colon and pancreatic cancer as well as Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). AIM: To prospectively investigate whether the immunohistochemical expression of DCLK1 was associated with detectable DCLK1 plasma expression in patients with existing BE and EAC. METHODS: Immunohistochemistry was performed on paraffin-embedded sections using DCLK1 antibody and scored based on staining intensity and tissue involvement. Purified human plasma samples were subjected to Western blot and ELISA analysis. RESULTS: Forty (40) patients were enrolled: 10 controls (normal endoscopy) and 30 with BE/EAC (13 nondysplastic BE [NDBE], 9 dysplastic BE [DBE] and 8 EAC). Mean epithelial DCLK1 staining was as follows: controls = 0.11, NDBE = 3.83, DBE = 6.0, EAC = 7.17. Mean stromal DCLK1 staining was as follows: NDBE = 5.83, DBE = 5.375, EAC = 10.83. DCLK1 was detected by plasma Western blot in 1 control and in all patients with BE/EAC p < 0.0005. Plasma DCLK1 was elevated by ELISA in EAC compared to other groups, p < 0.05. CONCLUSIONS: Increased expression of DCLK1 was observed in the epithelium, stroma and plasma of patients with BE/EAC. Furthermore, the presence of detectable DCLK1 in plasma of BE/EAC patients may provide a less invasive, detection tool in those patients as well as represent a novel molecular marker distinguishing between normal esophageal mucosa and BE or EAC.

Small molecule kinase inhibitor LRRK2-IN-1 demonstrates potent activity against colorectal and pancreatic cancer through inhibition of doublecortin-like kinase 1.

BACKGROUND: Doublecortin-like kinase 1 (DCLK1) is emerging as a tumor specific stem cell marker in colorectal and pancreatic cancer. Previous in vitro and in vivo studies have demonstrated the therapeutic effects of inhibiting DCLK1 with small interfering RNA (siRNA) as well as genetically targeting the DCLK1+ cell for deletion. However, the effects of inhibiting DCLK1 kinase activity have not been studied directly. Therefore, we assessed the effects of inhibiting DCLK1 kinase activity using the novel small molecule kinase inhibitor, LRRK2-IN-1, which demonstrates significant affinity for DCLK1. RESULTS: Here we report that LRRK2-IN-1 demonstrates potent anti-cancer activity including inhibition of cancer cell proliferation, migration, and invasion as well as induction of apoptosis and cell cycle arrest. Additionally we found that it regulates stemness, epithelial-mesenchymal transition, and oncogenic targets on the molecular level. Moreover, we show that LRRK2-IN-1 suppresses DCLK1 kinase activity and downstream DCLK1 effector c-MYC, and demonstrate that DCLK1 kinase activity is a significant factor in resistance to LRRK2-IN-1. CONCLUSIONS: Given DCLK1's tumor stem cell marker status, a strong understanding of its biological role and interactions in gastrointestinal tumors may lead to discoveries that improve patient outcomes. The results of this study suggest that small molecule inhibitors of DCLK1 kinase should be further investigated as they may hold promise as anti-tumor stem cell drugs.

Inhibition of Notch signaling reduces the number of surviving Dclk1+ reserve crypt epithelial stem cells following radiation injury.

We have previously reported that doublecortin-like kinase 1 (Dclk1) is a putative intestinal stem cell (ISC) marker. In this report, we evaluated the use of Dclk1 as a marker of surviving ISCs in response to treatment with high-dose total body irradiation (TBI). Both apoptotic and mitotic Dclk1(+) cells were observed 24 h post-TBI associated with a corresponding loss of intestinal crypts observed at 84 h post-TBI. Although the Notch signaling pathway plays an important role in regulating proliferation and lineage commitment within the intestine, its role in ISC function in response to severe genotoxic injury is not yet fully understood. We employed the microcolony assay to functionally assess the effects of Notch inhibition with difluorophenacetyl-l-alanyl-S-phenylglycine t-butyl ester (DAPT) on intestinal crypt stem cell survival following severe (>8 Gy) radiation injury. Following treatment with DAPT, we observed a nearly 50% reduction in the number of surviving Dclk1(+) crypt epithelial cells at 24 h after TBI and similar reduction in the number of surviving small intestinal crypts at 84 h. These data indicate that inhibition of Notch signaling decreases ISC survival following radiation injury, suggesting that the Notch signaling pathway plays an important role in ISC-mediated crypt regeneration. These results also suggest that crypt epithelial cell Dclk1 expression can be used as one potential marker to evaluate the early survival of ISCs following severe radiation injury.

A multicenter study to standardize reporting and analyses of fluorescence-activated cell-sorted murine intestinal epithelial cells.

Fluorescence-activated cell sorting (FACS) is an essential tool for studies requiring isolation of distinct intestinal epithelial cell populations. Inconsistent or lack of reporting of the critical parameters associated with FACS methodologies has complicated interpretation, comparison, and reproduction of important findings. To address this problem a comprehensive multicenter study was designed to develop guidelines that limit experimental and data reporting variability and provide a foundation for accurate comparison of data between studies. Common methodologies and data reporting protocols for tissue dissociation, cell yield, cell viability, FACS, and postsort purity were established. Seven centers tested the standardized methods by FACS-isolating a specific crypt-based epithelial population (EpCAM+/CD44+) from murine small intestine. Genetic biomarkers for stem/progenitor (Lgr5 and Atoh 1) and differentiated cell lineages (lysozyme, mucin2, chromogranin A, and sucrase isomaltase) were interrogated in target and control populations to assess intra- and intercenter variability. Wilcoxon's rank sum test on gene expression levels showed limited intracenter variability between biological replicates. Principal component analysis demonstrated significant intercenter reproducibility among four centers. Analysis of data collected by standardized cell isolation methods and data reporting requirements readily identified methodological problems, indicating that standard reporting parameters facilitate post hoc error identification. These results indicate that the complexity of FACS isolation of target intestinal epithelial populations can be highly reproducible between biological replicates and different institutions by adherence to common cell isolation methods and FACS gating strategies. This study can be considered a foundation for continued method development and a starting point for investigators that are developing cell isolation expertise to study physiology and pathophysiology of the intestinal epithelium.

Hepatitis C virus-induced cancer stem cell-like signatures in cell culture and murine tumor xenografts.

Hepatitis C virus (HCV) infection is a prominent risk factor for the development of hepatocellular carcinoma (HCC). Similar to most solid tumors, HCCs are believed to contain poorly differentiated cancer stem cell-like cells (CSCs) that initiate tumorigenesis and confer resistance to chemotherapy. In these studies, we demonstrate that the expression of an HCV subgenomic replicon in cultured cells results in the acquisition of CSC traits. These traits include enhanced expression of doublecortin and CaM kinase-like-1 (DCAMKL-1), Lgr5, CD133, α-fetoprotein, cytokeratin-19 (CK19), Lin28, and c-Myc. Conversely, curing of the replicon from these cells results in diminished expression of these factors. The putative stem cell marker DCAMKL-1 is also elevated in response to the overexpression of a cassette of pluripotency factors. The DCAMKL-1-positive cells isolated from hepatoma cell lines by fluorescence-activated cell sorting (FACS) form spheroids in Matrigel. The HCV RNA abundance and NS5B levels are significantly reduced by the small interfering RNA (siRNA)-led depletion of DCAMKL-1. We further demonstrate that HCV replicon-expressing cells initiate distinct tumor phenotypes compared to the tumors initiated by parent cells lacking the replicon. This HCV-induced phenotype is characterized by high-level expression/coexpression of DCAMKL-1, CK19, α-fetoprotein, and active c-Src. The results obtained by the analysis of liver tissues from HCV-positive patients and liver tissue microarrays reiterate these observations. In conclusion, chronic HCV infection appears to predispose cells toward the path of acquiring cancer stem cell-like traits by inducing DCAMKL-1 and hepatic progenitor and stem cell-related factors. DCAMKL-1 also represents a novel cellular target for combating HCV-induced hepatocarcinogenesis.

Identification of the putative intestinal stem cell marker doublecortin and CaM kinase-like-1 in Barrett's esophagus and esophageal adenocarcinoma.

BACKGROUND AND AIM: In Barrett's esophagus (BE), the normal esophageal squamous epithelium is replaced with a specialized metaplastic columnar epithelium. BE is a premalignant lesion that can progress to esophageal adenocarcinoma (EAC). Currently, there are no early molecular indicators that would predict progression from BE to EAC. As the only permanent residents of the epithelium, stem cells have been implicated in this metaplastic progression. The aim of the present study was to determine the expression of doublecortin and CaM kinase-like-1 (DCAMKL-1) and other putative gastrointestinal stem cell markers in normal esophageal mucosa (NEM), BE, and EAC. METHODS: Human NEM, BE, EAC, and multitissue microarrays were analyzed for DCAMKL-1, and immunohistochemically scored based on staining intensity and tissue involvement, with epithelia and stroma scored separately. Total RNA isolated from BE and paired NEM was subjected to real-time reverse-transcription-polymerase chain reaction analysis for DCAMKL-1, leucine-rich repeat-containing G-protein-coupled receptor (LGR5), and Musashi-1 (Msi-1) mRNA expression. RESULTS: DCAMKL-1 was minimally expressed in squamous NEM, but increased in BE (with and without dysplasia) and EAC tissues. In EAC, we found increased stromal DCAMKL-1 staining compared to adjacent epithelia. Within the submucosa of dysplastic BE tissues, an increase in the endothelial cell expression of DCAMKL-1 was observed. Finally, an upregulation of DCAMKL-1, LGR5, and Msi-1 mRNA was seen in BE compared to squamous NEM. CONCLUSIONS: In the present study, we report the progressive increase of DCAMKL-1 expression in BE from dysplasia to EAC. Furthermore, there was an increase in putative stem cell markers DCAMKL-1, LGR5, and Msi-1 mRNA. Taken together, these data suggest that the regulation of resident stem cells might play an important role in the progression of BE to EAC.

Reg IV regulates normal intestinal and colorectal cancer cell susceptibility to radiation-induced apoptosis.

BACKGROUND & AIMS: Regenerating (Reg) gene IV is predominantly expressed in gastrointestinal cells and highly up-regulated in many gastrointestinal malignancies, including colorectal cancer (CRC). Human CRC cells expressing higher levels of Reg IV gene and its protein product (Reg IV) are resistant to conventional therapies, including irradiation (IR). However, the underlying mechanism is not well defined. METHODS: A murine model of IR-induced intestinal injury and in vitro and in vivo models of human CRC were used to determine the role of Reg IV in regulation of normal intestinal and colorectal cancer cell susceptibility to IR-induced apoptosis. RESULTS: Treatments of recombinant human Reg IV (rhR4) protein protected normal intestinal crypt cells from IR-induced apoptosis by increasing the expression of antiapoptotic genes Bcl-2, Bcl-XL, and survivin. However, overexpression of Reg IV in human CRC cells was associated with increased resistance to IR-induced apoptosis. Therefore, we used antagonism of Reg IV as a tool to increase CRC cell susceptibility to IR-induced cell death. Two complementary approaches using specific monoclonal antibodies and small interfering RNAs were tested in both in vitro and in vivo models of human CRC. Both approaches resulted in increased apoptosis and decreased cell proliferation, leading to decreased tumor growth and increased animal survival. Furthermore, these approaches increased CRC cell susceptibility to IR-induced apoptosis. CONCLUSIONS: These results implicate Reg IV as an important modulator of gastrointestinal cell susceptibility to IR; hence, it is a potential target for adjunctive treatments for human CRC and other gastrointestinal malignancies.

Phosphoinositide 3-kinase signaling mediates beta-catenin activation in intestinal epithelial stem and progenitor cells in colitis.

BACKGROUND & AIMS: Mechanisms responsible for crypt architectural distortion in chronic ulcerative colitis (CUC) are not well understood. Data indicate that serine/threonine protein kinase Akt (Akt) signaling cooperates with Wingless (Wnt) to activate beta-catenin in intestinal stem and progenitor cells through phosphorylation at Ser552 (P-beta-catenin(552)). We investigated whether phosphoinositide 3-kinase (PI3K) is required for Akt-mediated activation of beta-catenin during intestinal inflammation. METHODS: The class IA subunit of PI3K was conditionally deleted from intestinal epithelial cells in mice named I-pik3r1KO. Acute inflammation was induced in mice and intestines were analyzed by biochemical and histologic methods. The effects of chemically blocking PI3K in colitic interleukin-10(-/-) mice were examined. Biopsy samples from patients were examined. RESULTS: Compared with wild-type, I-pik3r1KO mice had reduced T-cell-mediated Akt and beta-catenin signaling in intestinal stem and progenitor cells and limited crypt epithelial proliferation. Biochemical analyses indicated that PI3K-Akt signaling increased nuclear total beta-catenin and P-beta-catenin(552) levels and reduced N-terminal beta-catenin phosphorylation, which is associated with degradation. PI3K inhibition in interleukin-10(-/-) mice impaired colitis-induced epithelial Akt and beta-catenin activation, reduced progenitor cell expansion, and prevented dysplasia. Human samples had increased numbers of progenitor cells with P-beta-catenin(552) throughout expanded crypts and increased messenger RNA expression of beta-catenin target genes in CUC, colitis-associated cancer, tubular adenomas, and sporadic colorectal cancer, compared with control samples. CONCLUSIONS: PI3K-Akt signaling cooperates with Wnt to increase beta-catenin signaling during inflammation. PI3K-induced and Akt-mediated beta-catenin signaling are required for progenitor cell activation during the progression from CUC to CAC; these factors might be used as biomarkers of dysplastic transformation in the colon.

Nanoparticle-based delivery of siDCAMKL-1 increases microRNA-144 and inhibits colorectal cancer tumor growth via a Notch-1 dependent mechanism.

BACKGROUND: The development of effective drug delivery systems capable of transporting small interfering RNA (siRNA) has been elusive. We have previously reported that colorectal cancer tumor xenograft growth was arrested following treatment with liposomal preparation of siDCAMKL-1. In this report, we have utilized Nanoparticle (NP) technology to deliver DCAMKL-1 specific siRNA to knockdown potential key cancer regulators. In this study, mRNA/miRNA were analyzed using real-time RT-PCR and protein by western blot/immunohistochemistry. siDCAMKL-1 was encapsulated in Poly(lactide-co-glycolide)-based NPs (NP-siDCAMKL-1); Tumor xenografts were generated in nude mice, treated with NP-siDCAMKL-1 and DAPT (γ-secretase inhibitor) alone and in combination. To measure let-7a and miR-144 expression in vitro, HCT116 cells were transfected with plasmids encoding the firefly luciferase gene with let-7a and miR-144 miRNA binding sites in the 3'UTR. RESULTS: Administration of NP-siDCAMKL-1 into HCT116 xenografts resulted in tumor growth arrest, downregulation of proto-oncogene c-Myc and Notch-1 via let-7a and miR-144 miRNA-dependent mechanisms, respectively. A corresponding reduction in let-7a and miR-144 specific luciferase activity was observed in vitro. Moreover, an upregulation of EMT inhibitor miR-200a and downregulation of the EMT-associated transcription factors ZEB1, ZEB2, Snail and Slug were observed in vivo. Lastly, DAPT-mediated inhibition of Notch-1 resulted in HCT116 tumor growth arrest and down regulation of Notch-1 via a miR-144 dependent mechanism. CONCLUSIONS: These findings demonstrate that nanoparticle-based delivery of siRNAs directed at critical targets such as DCAMKL-1 may provide a novel approach to treat cancer through the regulation of endogenous miRNAs.

Identification of a novel putative pancreatic stem/progenitor cell marker DCAMKL-1 in normal mouse pancreas.

Stem cells are critical in maintaining adult homeostasis and have been proposed to be the origin of many solid tumors, including pancreatic cancer. Here we demonstrate the expression patterns of the putative intestinal stem cell marker DCAMKL-1 in the pancreas of uninjured C57BL/6 mice compared with other pancreatic stem/progenitor cell markers. We then determined the viability of isolated pancreatic stem/progenitor cells in isotransplantation assays following DCAMKL-1 antibody-based cell sorting. Sorted cells were grown in suspension culture and injected into the flanks of athymic nude mice. Here we report that DCAMKL-1 is expressed in the main pancreatic duct epithelia and islets, but not within acinar cells. Coexpression was observed with somatostatin, NGN3, and nestin, but not glucagon or insulin. Isolated DCAMKL-1+ cells formed spheroids in suspension culture and induced nodule formation in isotransplantation assays. Analysis of nodules demonstrated markers of early pancreatic development (PDX-1), glandular epithelium (cytokeratin-14 and Ep-CAM), and isletlike structures (somatostatin and secretin). These data taken together suggest that DCAMKL-1 is a novel putative stem/progenitor marker, can be used to isolate normal pancreatic stem/progenitors, and potentially regenerates pancreatic tissues. This may represent a novel tool for regenerative medicine and a target for anti-stem cell-based therapeutics in pancreatic cancer.

Selective blockade of DCAMKL-1 results in tumor growth arrest by a Let-7a MicroRNA-dependent mechanism.

BACKGROUND & AIMS: MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression. The tumor suppressor miRNA let-7a has been reported to be inhibited posttranscriptionally in embryonic stem cells and in human cancers. Microtubule-associated kinase DCAMKL-1 is a putative intestinal stem cell marker that is expressed in Apc(Min/+) adenomas. We investigated the role of DCAMKL-1 on expression of let-7a miRNA and the oncogene c-Myc and in tumorigenesis. METHODS: Human tissue microassay slides were immunostained for DCAMKL-1. HCT116 and SW480 cells were transfected with DCAMKL-1 small interfering RNA (siRNA) (si-DCAMKL-1) and analyzed for DCAMKL-1, c-Myc (using immunoblot and real-time reverse-transcription polymerase chain reaction [RT-PCR]), and pri-let-7a miRNA (using real-time RT-PCR) levels. A liposomal preparation of si-DCAMKL-1 was administered into HCT116 xenografts in nude mice, and tumor volumes were measured. A luciferase reporter assay, with a plasmid containing a let-7a-binding site at the 3' untranslated region, was utilized to measure let-7a in cell lines. Cells were isolated from normal mouse intestine using DCAMKL-1 and fluorescence-activated cell sorting (FACS) and subjected to pri-let-7a miRNA analysis. RESULTS: Expression of DCAMKL-1 was increased in human colorectal cancers. siRNA-mediated blockade of DCAMKL-1 resulted in H tumor xenograft growth arrest, increased pri-let-7a miRNA, a corresponding decrease in luciferase activity, and decreased expression of the oncogene c-Myc. DCAMKL-1(+) cells isolated by FACS demonstrated a significant decrease in pri-let-7a miRNA, compared with more differentiated cells. CONCLUSIONS: DCAMKL-1 is a negative regulator of let-7a miRNA biogenesis in intestinal stem and colorectal cancer cells; it could represent a novel target for anti-cancer stem cell-based strategies.

Doublecortin and CaM kinase-like-1 and leucine-rich-repeat-containing G-protein-coupled receptor mark quiescent and cycling intestinal stem cells, respectively.

It is thought that small intestinal epithelia (IE) undergo continuous self-renewal primarily due to their population of undifferentiated stem cells. These stem cells give rise to transit amplifying (daughter/progenitor) cells, which can differentiate into all mature cell types required for normal gut function. Identification of stem cells in IE is paramount to fully understanding this renewal process. One major obstacle in gastrointestinal stem cell biology has been the lack of definitive markers that identify small intestinal stem cells (ISCs). Here we demonstrate that the novel putative ISC marker doublecortin and CaM kinase-like-1 (DCAMKL-1) is predominantly expressed in quiescent cells in the lower two-thirds of intestinal crypt epithelium and in occasional crypt-based columnar cells (CBCs). In contrast, the novel putative stem cell marker leucine-rich-repeat-containing G-protein-coupled receptor (LGR5) is observed in rapidly cycling CBCs and in occasional crypt epithelial cells. Furthermore, functionally quiescent DCAMKL-1+ crypt epithelial cells retain bromo-deoxyuridine in a modified label retention assay. Moreover, we demonstrate that DCAMKL-1 is a cell surface expressing protein; DCAMKL-1+ cells, isolated from the adult mouse small intestine by fluorescence activated cell sorting, self-renew and ultimately form spheroids in suspension culture. These spheroids formed glandular epithelial structures in the flanks of athymic nude mice, which expressed multiple markers of gut epithelial lineage. Thus, DCAMKL-1 is a marker of quiescent ISCs and can be distinguished from the cycling stem/progenitors (LGR5+). Moreover, DCAMKL-1 can be used to isolate normal small intestinal stem cells and represents a novel research tool for regenerative medicine and cancer therapy.

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.

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.

Gastrin-mediated interleukin-8 and cyclooxygenase-2 gene expression: differential transcriptional and posttranscriptional mechanisms.

BACKGROUND & AIMS: Gastrin induces the expression of cyclooxygenase (COX)-2 and interleukin (IL)-8; however, the mechanism(s), especially in gastric epithelial cells, is not well understood. Here, we have determined the intracellular mechanisms mediating gastrin-dependent gene expression. METHODS: AGS-E human gastric cancer cell line stably expressing cholecystokinin-2 receptor was treated with amidated gastrin-17. Real-time polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay were performed to determine COX-2 and IL-8 expression and Akt, Erk, and p38 phosphorylation. Gene promoter activity was determined by luciferase assay. Electrophoretic mobility shift assay analysis was performed for nuclear factor kappaB (NF-kappaB) and activator protein-1 activity. RNA stability was determined after actinomycin D treatment. HuR localization was determined by immunocytochemistry. RESULTS: Gastrin induced COX-2 and IL-8 expression in AGS-E cells, which was inhibited by phosphatidylinositol 3' kinase (PI3K) and p38 inhibitors. Gastrin-mediated Akt activation was observed to be downstream of p38. IL-8 expression was dependent on COX-2-mediated prostaglandin E(2) synthesis. In the presence of an NF-kappaB inhibitor MG132, IL-8 transcription was inhibited, but not that of COX-2. This was confirmed after knockdown of the p65 RelA subunit of NF-kappaB. Further studies showed that COX-2 gene transcription is regulated by activator protein-1. Gastrin increased the stability of both COX-2 and IL-8 messenger RNA (mRNA) in a p38-dependent manner, the half-life increasing from 31 minutes to 8 hours and approximately 4 hours, respectively. Gastrin, through p38 activity, also enhanced HuR expression, nucleocytoplasmic translocation, and enhanced COX-2 mRNA binding. CONCLUSIONS: Gastrin differentially induces COX-2 and IL-8 expression at the transcriptional and posttranscriptional levels by PI3K and p38 mitogen-activated protein kinase pathways, respectively.

Knockdown of RNA binding protein musashi-1 leads to tumor regression in vivo.

BACKGROUND & AIMS: In the gut, tumorigenesis is thought to arise from the stem cell population located near the base of intestinal and colonic crypts. The RNA binding protein musashi-1 (Msi-1) is a putative intestinal and progenitor/stem cell marker. Msi-1 expression is increased during rat brain development and in APC(min/+) mice tumors. This study examined a potential role of Msi-1 in tumorigenesis. METHODS: Msi-1 small interfering RNA (siRNA) was administered as a liposomal preparation to HCT116 colon adenocarcinoma xenografts in athymic nude mice and tumor volume was measured. Cell proliferation was assessed by hexosaminidase and 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyltetrazolium bromide MTT assays. siRNA-transfected cells were subjected to 12 Gy gamma-irradiation. Apoptosis was assessed by immunoreactive activated caspase-3 and mitosis was assessed by phosphorylated histone H3 staining. The tumor xenografts were stained similarly for phosphorylated histone H3, activated caspase-3, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, Notch-1, and p21(WAF1). Furthermore, siRNA-transfected cells were subjected to cell-cycle analysis and Western blot analyses for Notch-1 and p21(WAF1). RESULTS: Knockdown of Msi-1 resulted in tumor growth arrest in xenografts, reduced cancer cell proliferation, and increased apoptosis alone and in combination with radiation injury. siRNA-mediated reduction of Msi-1 lead to mitotic catastrophe in tumor cells. Moreover, there was inhibition of Notch-1 and up-regulation of p21(WAF1) after knockdown of Msi-1. CONCLUSIONS: Our results show the involvement of Msi-1 in cancer cell proliferation, inhibition of apoptosis, and mitotic catastrophe, suggesting an important potential mechanism for its role in tumorigenesis.

Identification of a novel putative gastrointestinal stem cell and adenoma stem cell marker, doublecortin and CaM kinase-like-1, following radiation injury and in adenomatous polyposis coli/multiple intestinal neoplasia mice.

In the gut, tumorigenesis arises from intestinal or colonic crypt stem cells. Currently, no definitive markers exist that reliably identify gut stem cells. Here, we used the putative stem cell marker doublecortin and CaM kinase-like-1 (DCAMKL-1) to examine radiation-induced stem cell apoptosis and adenomatous polyposis coli (APC)/multiple intestinal neoplasia (min) mice to determine the effects of APC mutation on DCAMKL-1 expression. Immunoreactive DCAMKL-1 staining was demonstrated in the intestinal stem cell zone. Furthermore, we observed apoptosis of the cells negative for DCAMKL-1 at 6 hours. We found DNA damage in all the cells in the crypt region, including the DCAMKL-1-positive cells. We also observed stem cell apoptosis and mitotic DCAMKL-1-expressing cells 24 hours after irradiation. Moreover, in APC/min mice, DCAMKL-1-expressing cells were negative for proliferating cell nuclear antigen and nuclear beta-catenin in normal-appearing intestine. However, beta-catenin was nuclear in DCAMKL-1-positive cells in adenomas. Thus, nuclear translocation of beta-catenin distinguishes normal and adenoma stem cells. Targeting DCAMKL-1 may represent a strategy for developing novel chemotherapeutic agents.