Chandrakesan et al. Molecular Cancer (2017) 16:30 DOI 10.1186/s12943-017-0594-y

RESEARCH Open Access Dclk1, a tumor stem cell marker, regulates pro-survival signaling and self-renewal of intestinal tumor cells Parthasarathy Chandrakesan1,2,3*† , Jiannan Yao1,7†, Dongfeng Qu1,2,3, Randal May1,3, Nathaniel Weygant1, Yang Ge1,7, Naushad Ali1,3, Sripathi M. Sureban1,3, Modhi Gude1,4, Kenneth Vega1,5, Eddie Bannerman-Menson8, Lijun Xia6, Michael Bronze1, Guangyu An7* and Courtney W. Houchen1,2,3,8*

Abstract 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 ApcMin/+ 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 ApcMin/+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 ApcMin/+mice than in wild-type controls. Intestinal epithelial cells of ApcMin/+mice showed increased expression of pro-survival signaling, pluripotency and self-renewal ability. Furthermore, the enteroids formed from the intestinal Dclk1+ cells of ApcMin/+mice display higher pluripotency and pro-survival signaling. Dclk1 knockdown in ApcMin/+ 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. (Continued on next page)

* Correspondence: [email protected]; [email protected]; [email protected] †Equal contributors 1Division of Digestive Diseases and Nutrition, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA 7Beijing Chao-Yang Hospital Department of Oncology, Capital Medical University, Beijing, China Full list of author information is available at the end of the article

© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chandrakesan et al. Molecular Cancer (2017) 16:30 Page 2 of 14

(Continued from previous page) 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. Keywords: Dclk1, APC mutation, Cancer stem cells, Intestinal epithelial cells, Pro-survival signaling, Self-renewal, Nanoparticles, Colorectal cancer

Background that the majority of human malignancies are initiated APC is a tumor suppressor gene that is mutated in pa- and maintained by a distinct population of cells that dis- tients with familial adenomatous polyposis (FAP) and play stem cell properties and self-renewal ability [16]. most sporadic colorectal cancers [1, 2]. The Apc muta- More recently, it has been shown that the development tion dysregulates the Wnt signaling pathway and triggers and progression of colon and pancreatic cancer depend cellular transformation, resulting in the development of upon Dclk1+ cancer stem cells [11, 13–15]. We reported adenomatous polyps [3]. It was suggested that the APC that Dclk1 is overexpressed in many cancers, including gene mutation is required, but is not sufficient, for the colon, pancreas, liver, and esophageal cancer [12, 17–20]. development of cancer in the colon. Since tumorigenesis Previous work from others and us supported the idea that is considered the result of multiple genetic changes, sev- DCLK1 expression is critical for cancer stem cells, cancer eral efforts have made to identify those tumorigenesis- growth, EMT, and metastasis [11, 12, 15, 18, 21–23]. promoting changes. Several genetic changes, including These data provide a basis for Dclk1 as a regulatory factor activation mutations in Ki-RAS/N-RAS, mutations in the for tumor growth and advancement. tumor suppressor TP53, and deletion of a region of Recent studies have indicated that neoplastic cells have chromosome 18 containing SMAD2, SMAD4, and DCC active pro-survival signaling pathways for proliferation, have been identified [4]. Despite improvements in our resistance, self-renewal, and survival [24–26]. Further- understanding of this disease, the molecular events more, progression of cancer, including metastasis and underlying the development and progression of intes- secondary tumor formation of cancer cells with self- tinal tumors are still largely unknown and may be a key renewal ability, is often linked to altered expression of to the development of more effective and novel thera- pro-survival signaling pathways [27]. Understanding the peutic strategies. Therefore, understanding the APC diversity of pro-survival signaling pathways that underlie gene mutation associated changes for intestinal tumori- cancer formation and progression is essential for devel- genesis is important. oping a new generation of effective anticancer drugs for Similar to humans with germline mutations in APC, combinatorial therapeutic strategies. The interdependence Min/+ Apc mice have a heterozygous mutation in the Apc between pro-survival signaling and tumor self-renewal gene, predisposing the mice to intestinal and colon ability with enhanced Dclk1 highlights the collective tumor development. These mice start developing intes- mechanism involved in tumor growth and survival. How- tinal polyps by ~4 weeks of age, with progression to dys- ever, the precise mechanism by which Dclk1 supports plasia at 18–21 weeks; adenocarcinoma is also evident at intestinal tumor progression is poorly understood. With Min/+ Min/+ ~26-34 weeks [5–8]. Eight-to-twelve-week-old Apc the aid of the Apc mouse model, we assessed the con- mice are a good model with which to study the patho- tribution of Dclk1 to intestinal tumorigenesis using small genesis of FAP, while 26-to-34-week-old ApcMin/+ mice interfering RNAs targeting Dclk1 incorporated into develop intestinal high-grade dysplasia and adenocarcin- poly(lactic-co-glycolic acid) nanoparticles (siDclk1-NPs). oma, and are a particularly relevant model for studying We found that Dclk1 is involved in enhancing the pro- tumor progression and developing therapeutic strategies survival signaling pathways and tumor cells’ self-renewal [6, 7]. ApcMin/+ mice develop high-grade dysplasia and ability to facilitate intestinal tumor growth and progression. adenocarcinoma and are a clinically relevant disease model, since a large number of patients diagnosed with Methods advanced colon cancer are elderly and have unresectable TCGA Colon Adenocarcinoma (COAD) Data or widespread disease [9]. The RNA-seq datasets from February 2015 combining Doublecortin-like kinase 1 (Dclk1) is a microtubule- data from 329 patients with colon adenocarcinomas in- associated protein kinase and has been identified as a cluded in the Cancer Genome Atlas (TCGA) dataset tuft cell marker in the small intestine [10]. Dclk1 has were downloaded through the UCSC cancer genome been reported to mark tumor stem cells in the intestine browser (https://www.xenabrowser.net), as previously and pancreas [11–15]. Emerging evidence has confirmed described [28]. Chandrakesan et al. Molecular Cancer (2017) 16:30 Page 3 of 14

Determination of DCLK1-correlated pro-survival signaling anti-CD45, anti-CD31, anti-EpCAM, and anti-Dclk1 in APC mutant COAD antibodies conjugated with the respective fluorochromes APC mutant/APC non-mutant samples and samples for 30 min. The cells were washed and sorted using an − − with high/low DCLK1 expression levels were sorted by Influx-V cell sorter (Cytopeia). CD45 CD31 EpCAM R v3.2. Patients whose DCLK1 expression levels was in +Dclk1+ cells were collected and then subjected to the top 25% or bottom 25% were considered DCLK1- enterosphere formation assays [18, 30]. high or DCLK1-low, respectively. The corrplot function (R package corrplot) was used to confirm the correlation Clonogenic assay between the expression levels of DCLK1 and other FACs isolated Dclk1+ IECs were plated in 48-well plates genes. A heatmap was produced using the heatmap.2 at a density of 100 cells per well in RPMI medium function (R package gplots) [28]. containing 0.3% soft agar. The cell suspensions were plated in a 48-well plate above a layer of solidified 1% DCLK1 network with pro-survival signaling utilizing the soft agar in plain RPMI medium. The plates were GeneMANIA database incubated at 37 °C under 5% CO2. The cells were Datasets, including physical interactions, pathway, and gen- followed for enterosphere/enteroid formation, as de- etic interactions, were collected from the public domain scribed previously [18, 30, 31]. GeneMANIA database. The dataset relevant to DCLK1 and the pro-survival signaling network was produced from RNA isolation and real-time RT-PCR analysis the GeneMANIA database (http://www.genemania.org). Total RNA isolated from small intestinal epithelial cells was subjected to reverse transcription. The complemen- Animals tary DNA (cDNA) was subsequently used to perform All animal experiments were performed with approval real-time PCR with SYBR™ chemistry (Molecular Probes, and authorization from the Institutional Review Board Eugene, OR) using gene-specific primers for specific and the Institutional Animal Care and Use Committee transcripts. The crossing threshold value assessed by at the University of Oklahoma Health Sciences Center real-time PCR was noted for the transcripts and normal- (Oklahoma City, Oklahoma). ApcMin/+ mice on the ized to β-actin. C57BL/6 J background were obtained from The Jackson Laboratory and were maintained by breeding ApcMin/+ Immunoblot analysis males to C57BL/6 J females. Mice were genotyped with Twenty-five micrograms of the total protein was size- a PCR assay to identify carriers of the Min allele of Apc. separated in a 4–12% SDS polyacrylamide gel and Min/+ Same sex (male) C57BL/6 J Apc and Apc+/+ litter- transferred electrophoretically onto a PVDF mem- mates at 30 week of age were used in the present study. brane with a wet-blot transfer apparatus (Bio-Rad, The average life span of ApcMin/+ mice on the C57BL/6 J Hercules, CA). The membrane was blocked and in- background is ∼ 20 weeks, although the mice in our facil- cubated overnight with a primary antibody and was ity have healthier survival, as observed in several previ- subsequently incubated with horseradish peroxidase- ous studies [5–8]. ApcMin/+ mice (i.e., >30 weeks of age) conjugated secondary antibody. The proteins were de- were carefully monitored and sacrificed before becoming tected using ECL Western blotting detection reagents moribund. (Amersham-Pharmacia, Piscataway, NJ). Actin (42-kD) wasusedasaloadingcontrol. Intestinal Epithelial Cell (IEC) Isolation Small intestines (ileum) were attached to a paddle, were Small interfering RNAs immersed in Ca2+-free standard Krebs-buffered saline The Dclk1 siRNA (siDclk1; Cat. # S234357) sequence (in mmol/l: 107 NaCl, 4.5 KCl, 0.2 NaH2PO4, 1.8 targeting the coding region of Dclk1 (accession No. Na2HPO4, 10 glucose, and 10 EDTA) at 37 °C for 15– NM_019978) and scrambled siRNAs (siScr; Cat. # 20 min, and were gassed with 5% CO2,95%O2. Individ- AM4636) not matching any of the mouse genes were ual crypt units were then separated by intermittent obtained (Ambion Inc., Austin, TX, USA). DCLK siRNA (30 s) vibration into ice-cold phosphate buffered saline (h) (# SC45618), RELA siRNA (h) (#SC29410) and and were collected by centrifugation [18, 29–31]. We NOTCH1 siRNA (h) (#SC36095) were obtained from utilized the whole intestinal epithelilal cells. Santa Cruz Biotechnology (SCBT, TX, USA).

FACS Synthesis and characterization of Dclk1 siRNA NPs and Freshly isolated IECs were washed and resuspended in treatment RPMI glutamax medium. To avoid endothelial and stro- Poly(lactide-co-glycolide acid nanoparticles (PLGA NPs) mal contamination, isolated cells were incubated with were synthesized using a double emulsion solvent Chandrakesan et al. Molecular Cancer (2017) 16:30 Page 4 of 14

evaporation technique, as described previously [18, 20]. bottom of each well as chemoattractant, and the cells The amount of encapsulated siRNA was quantified using were incubated for 24 h at 37 °C under 5% CO2. After- a spectrophotometer (DU-800, Beckman Coulter, Brea, wards, a cotton swab was used to scrape non-invasive/ CA). The size, polydispersity index, and zeta-potential migratory cells off the top of Transwells; the remaining measurements of synthesized siRNA NPs were deter- cells were fixed with 100% methanol, stained with mined using diffraction light scattering (DLS) utilizing 0.1% crystal violet, and allowed to dry. After drying, Zeta PALS (Brookhaven Instruments, Holtsville, NY). all invading cells were counted from each Transwell. Min/+ Sex- and age-matched littermates of C57BL/6 J Apc Results are reported as the number of cells invaded mice were injected i.p. with 0.25 nmol of siRNA prepar- and/or migrated. ation on every third day for a total of six doses. Colony formation assay Immunohistochemistry/immunofluorescence HT-29 and DLD1 were transiently transfected with si- Standard immunohistochemistry and immunofluores- DCLK1, siNFkB-p65, and si-NOTCH1 (from Santa Cruz cence protocols were used with specific antibodies, as Biotechnology), along with scramble siRNA. After 48 h, described previously [18, 30]. cells were seeded and passaged into new 6-well plates (100 cells/well). Cells were allowed to grow for one Antibodies week, then were fixed with glacial acetic acid/methanol We used the following antibodies: Dclk1, Lgr5, Bmi1, solution (1:3) and washed with PBS. Colonies were Hes1, Tcf4, Cox1, Cox2, EpCam, CD45, CD31 (all stained with 0.5% Crystal violet for 10 min and were from Abcam, Cambridge, MA), CXCL1, CyclinD1, washed with tap water to remove excess stain. Colonies cMYC, β − catenin (Santa Cruz Biotechnology, USA), were then counted under a stereomicroscope using a Notch1, NfkB-p65, CyclinD1, Ras, β-actin (Cell 1-cm2 grid. Four squares from four quadrants were Signaling, Danvers, MA, USA), anti-rabbit IgG, anti- counted for each well. mouse IgG, anti-goat IgG (Jackson ImmunoResearch, West Grove, PA, USA), Alexa Fluor® 488 donkey anti- Statistical analysis rabbit IgG, and Alexa Fluor® 568 donkey anti-goat Statistical analyses were conducted using GraphPad IgG (Invitrogen, USA). Prism 6.00 (GraphPad Software, La Jolla) and R system v3.2 for statistical computing. Pearson product–moment siRNA-mediated knockdown of DCLK1 correlation was used for analysis and correlation of gene HT-29 and DLD1 human colon cancer cells were pur- expressions between two groups. Colon cancer chased from the American Type Culture Collection recurrence-free survival analysis was performed using (ATCC) and were maintained in DMEM medium Kaplan Meier Survival analyses. P values of <0.05 = *, containing 10% fetal bovine serum (FBS). For siRNA- <0.01 = **, and 0.001 = *** were considered statistically mediated knockdown studies, cells were seeded into 6- significant. cm petri dishes and were allowed to attach overnight. After attachment, 25 nM of commercially validated Results siRNA targeting human DCLK1 or NOTCH1 or RELA DCLK1 is correlated with pro-survival signaling in colon (siRNA; Santa Cruz Biotechnology) or 25 nM human adenocarcinoma scrambled sequence (siSCR) not targeting any known Mutation and/or loss of function of APC is the cause of genes were complexed with Lipofectamine 3000 (Invitro- more than 80% of colon cancers. DCLK1+ tumor stem gen) and added to the dishes in fresh cell culture cells are vital to the development and progression of Min/+ medium. After 48 h of treatment, cells were collected colon cancer in Apc mice models. Indeed, the for migration, invasion, colony formation, and self- DCLK1 signature in APC mutant colon cancer predicts renewal (clonogenic) analysis. recurrence-free-survival (Additional file 1: Figure S1). Kaplan-Meier survival analysis demonstrated that the Migration and invasion assay APC-derived DCLK1 signature could be used to For the invasion assay, matrigel-coated Transwells (BD strongly predict recurrence-free survival in colon can- Biosciences) were prepared by retrieving in serum-free cer. The recurrence-free survival analysis of APC mu- media for 2 h at 37 °C. For the migration assay, Trans- tant patients with high DCLK1 expression (n = 31) wells (BD Biosciences) were also used. Subsequently, tended to be associated with a poor prognosis com- HT-29 and DLD1 cells (5000/well) pre-transfected with pared with APC mutant patients with low DCLK1 either 25 nM siRNA or siSCR for 48 h were seeded into expression (n = 35; P = 0.0171). each Transwell in triplicate in serum-free media. Cell Although the DCLK1 expression levels are important culture medium containing 10% FBS was added to the in APC mutant colon cancer to predict the cancer Chandrakesan et al. Molecular Cancer (2017) 16:30 Page 5 of 14

stemness, the role of DCLK1 in regulating the prosurvi- additional parameters (Fig. 1c). From the GeneMANIA val signaling pathways for intestinal cancer progression network, it is apparent that DCLK1 has networked with is largely unknown. Analysis of TCGA colon adenocar- pro-survival signaling, and DCLK1 shows its interaction cinoma (COAD) cancer datasets demonstrated a strong with pro-survival signaling via downstream factors/ correlation between DCLK1 mRNA expression and pro- adaptor factors, exception is PTGS1 directly networking survival signaling, including CTNNB1, NOTCH1, RELA, with DCLK1. These findings suggest that DCLK1 in colon PTGS1, and PTGS2, as determined by the Pearson cancers may have a relationship with pro-survival signal- product–moment correlation, as previously described ing and, by regulating pro-survival signaling, may assist in (Fig. 1a). DCLK1 was most strongly correlated with the development and progression of colon cancer. COX1 signaling in colon cancers (Additional file 2: Figure S2). Furthermore, heatmaps demonstrating dys- ApcMin/+ mice with intestinal adenocarcinoma showed regulated expression of the pro-survival signaling signa- increased expression of tumor stem cells ture are higher in DLCK1-high patients than in DCLK- Tumor stem cells (TSCs) are highly tumorigenic cells low patients from the TCGA COAD dataset (Fig. 1b). that have the ability to self-renew, giving rise to other The GeneMANIA webserver was utilized to predict malignant stem cells; TSCs are also phenotypically di- interactions between the DCLK1 and pro-survival sig- verse cancer cells that are considered to be the source of naling in the network using the parameters limited to tumor initiation and maintenance [32, 33]. To investi- physical interactions, genetic interactions, and pathways gate the role of Dclk1+ TSCs in intestinal tumorigenesis to score nodes and source organism Homo sapiens as under Apc mutation, we analyzed the level of Dclk1

Fig. 1 DCLK1 expression is positively correlated with genes of pro-survival signaling pathways and tumor stem cell markers. a Color indicates correlation of DCLK1 and other genes: 1) negative (green), and 2) positive (red). b Heatmap of pro-survival signaling pathways and tumor stem cell markers gene expression levels by dividing colon cancer patients into two groups based on DCLK1 expression levels from TCGA. Patients with the top 25% or bottom 25% DCLK1 expression levels were considered DCLK1-high or DCLK1-low, respectively. c A gene network from GeneMANIA shows the relationships for genes from the list (nodes) connected (with edges) according to the functional association networks from the databases. Based on the physical interactions, pathway, and genetic interactions, in the network representation, all the nodes are connected and related to DCLK1 Chandrakesan et al. Molecular Cancer (2017) 16:30 Page 6 of 14

Min/+ expression and Dclk1+ cells in the intestines of Apc enriched with the tumor stem cell markers (Additional and wild-type (WT) mice. Compared with WT intestinal file 3: Figure S3). Min/+ sections, the Dclk1 staining increased in Apc mouse intestines (Fig. 2a). The intensity of Dclk1 staining is 5–10 Amplified pro-survival signaling in the IECs of ApcMin/+ Min/+ fold higher in the intestinal sections from Apc mice mice are critical for tumorigenesis than those of WT mice (Fig. 2a and b). Furthermore, Pro-survival signaling pathways critical for cell survival Min/+ FACS analysis of the isolated IECs of Apc mice and proliferation in IECs are required for adenoma and showed 25-30% Dclk1+ cells compared with 1-3% adenocarcinoma formation [34–36]. In contrast to nor- Dclk1+ cells from WT mice (Fig. 2c). We analyzed mal intestinal epithelium, neoplastic cells have active the expression of tumor stem cell markers, and found pro-survival signaling pathways for proliferation, resist- a massive increase in mRNA and protein levels of the ance, self-renewal, and survival [37, 38]. We detected Dclk1, Lgr5, Bmi1, and Musashi1 (Fig. 2d) in the greater expression of β-catenin, Notch, and phospho- Min/+ Min/+ IECs of Apc mice compared with WT control and total NFκB-p65 in the IECs from Apc mice mice, corroborating the presence of highly tumori- than in IECs from WT mice. The downstream targets genic cells in the Apc mutant intestinal tumors. Fur- COX1, COX2, CyclinD1, Tcf4, Ras, Hes-1, and CXCL1 Min/+ Min/+ thermore, FACS isolated Dclk1+ cells from Apc were also upregulated in the IECs from Apc mice mice were utilized for Dclk1, Lgr5 and Bmi1 mRNA compared with IECs from WT mice (Fig. 2e and f). expression analysis and found that Dclk1+ cells are Thus, Apc loss may act as a primary window for the

Fig. 2 Increased expression of Dclk1 and Dclk1+ cells in the intestinal adenomas and adenocarcinomas of ApcMin/+ mice is associated with enhanced expression of tumor stem cell markers and pro-survival signaling. a IHC for Dclk1 in the small intestines of WT and ApcMin/+ mice. b Staining intensity was scored and is represented as a bar graph. c FACS data representing the % Dclk1+ cells isolated from the small intestines of WT and ApcMin/+ mice. d Differences in the number of Dclk1+ cells in staining and FACS corroborate with protein and mRNA levels of Dclk1 in the isolated IECs of WT and ApcMin/+ mice; protein and mRNA levels analyzed by western blot and RT-PCR of Bmi1, Lgr5, and Musashi1 in isolated IECs from WT and ApcMin/+ mice. f Protein expression levels of pro-survival signaling and their downstream targets in the isolated IECs of WT and ApcMin/+ mice, analyzed by western blot. e mRNA expression levels of pro-survival signaling and their downstream targets in the isolated IECs of WT and ApcMin/+ mice, analyzed by RT-PCR. All quantitative data are expressed as means ± SD of a minimum of three independent experiments. P values of <0.05 = *, <0.01 = **, and 0.001 = *** were considered statistically significant Chandrakesan et al. Molecular Cancer (2017) 16:30 Page 7 of 14

dysregulation of pro-survival signaling pathways to sup- higher in DLCK1-high patients than in DCLK-low pa- port the transformation of IECs towards neoplasia and tients from the TCGA COAD dataset (Fig. 1b). promote progression. Further, these pro-survival signal- From the GeneMANIA network, it is apparent that ing pathways are required for cell and stem cell homeo- DCLK1 has networked with BMI1, and lacks a direct stasis under non-neoplastic conditions; however, they network with LGR5 within the limited parameters of in- can act as oncogenic factors for tumor maintenance and teractions (Additional file 3: Figure S3). Therefore, we growth during neoplasia (24, 26). investigated whether Dclk1+ tumor stem cells with the Apc mutation have enriched co-expression of other Dclk1+ tumor stem cells from the intestines of ApcMin/+ tumor stem cell markers, and enhanced self-renewal, mice are highly clonogenic, and the enteroids formed and pro-survival signaling pathways. Dclk1+ cells iso- Min/+ display increased pro-survival signals and stemness lated from the small intestines of Apc mice formed FACS data revealed more Dclk1+ cells from the isolated a 5-fold increase in enteroids, which were also larger Min/+ IECs of Apc mice than from IECs of WT controls. than the enteroids observed in WT mice (Fig. 3a and b). Min/+ Further, analysis of TCGA colon adenocarcinoma Next, we analyzed whether Dclk1+ enteroids of Apc (COAD) cancer datasets demonstrated a correlation be- mice are enriched with tumor stem cell markers and tween DCLK1 mRNA expression and tumor stem cell pro-survival signaling pathways. We collected Dclk1+ markers LGR5 and BMI1, as determined by the Pearson enteroids and analyzed them for the expression of tumor product–moment correlation, as previously described stem cell markers and pro-survival signaling pathways. (Fig. 1a). Furthermore, heatmaps demonstrating dysreg- We found enhanced Dclk1 expression in the enteroids of Min/+ ulated expression of the tumor stem cell markers are Apc mice compared with WT mice (Fig. 3c and d).

Fig. 3 Dclk1+ cells isolated from the IECs of ApcMin/+ mice display enhanced self-renewal ability and enriched tumor stem cell markers and pro-survival signaling. a Enteroids formation of isolated Dclk1+ cells (100 cell per well) from the small intestines of WT and ApcMin/+ mice. b Stacked bar and line graph represent the quantification of the number of enteroids formed and spheroid volume from the Dclk1+ cells isolated from WT and ApcMin/+ mice. c & d mRNA and protein expression of Dclk1, Bmi1, Lgr5, and Msi1 in the isolated IECs of ApcMin/+ mice compared with WT mice. e & f mRNA and protein expression of pro-survival signaling and their downstream targets in the isolated IECs of ApcMin/+ mice compared with WT mice. All quantitative data are expressed as means ± SD of a minimum of three independent experiments. P values of <0.05 = *, <0.01 = **, and 0.001 = *** were considered statistically significant Chandrakesan et al. Molecular Cancer (2017) 16:30 Page 8 of 14

Strikingly, the expression levels of Lgr5, Bmi1, and regulate tumor formation and progression [34–36]. In Musashi1 were significantly higher in the enteroids of the present study, we found that Dclk1+ cells isolated Min/+ Min/+ Apc mice than in WT mice (Fig. 3c and d). from the intestinal epithelium of Apc mice display These data suggest that this enhancement in Dclk1 enhanced expression of pro-survival signaling pathways expression, along with other tumor stem cell markers, and self-renewal ability (Fig. 3). To test whether Dclk1 may provide Dclk1+ cells with greater tumor stem cell regulates pro-survival signaling pathways in the isolated Min/+ function and self-renewal ability. IECs from Apc mice, we silenced Dclk1 expression Min/+ We also collected the enteroids and examined the pro- using siDclk1-NPs. Depleting Dclk1 in Apc mice tein and gene expression of pro-survival signaling path- had a potent inhibitory effect on the pro-survival β- ways. We found a significant increase in the expression of catenin, Notch1, and NF-κB-p65 signaling pathways in β-catenin, Notch1, and the NF-κB-p65 active form (phos- the isolated IECs (Fig. 4e and f). To further clarify phorylation at Ser536, Ser276) and total form in the whether the inhibitory activity of β-catenin, Notch1, and Min/+ enteroids of Apc mice compared with WT mice NF-κB-p65 signaling caused by Dclk1 depletion is con- (Fig. 3e and f). Downstream targets cyclinD1, c-Myc, Tcf4, nected with an associated change in target gene expres- Ras, and Hes-1 were also increased in the enteroids of sion, downstream targets COX1, COX2, CyclinD1, Tcf4, Min/+ Apc mice compared with WT. Thus, the increased Ras, Hes-1, and Cxcl1 were quantified and were ob- Min/+ pro-survival signaling and tumor stem cell markers in the served to be reduced in the IECs from Apc mice Dclk1+ enteroids may be the source for increased self- treated with si-Dclk1-NP (Fig. 4e and f). renewal ability and survival upon loss of Apc function. These data suggest that the Dclk1 expression level is indispensable for the activation of pro-survival signaling Knocking down Dclk1 expression decreased the intestinal pathways upon loss of Apc function. However, the ex- tumorigenesis in ApcMin/+ mice pression of pro-survival signaling was not altered in the Dclk1 expression levels and Dclk1-expressing cells are en- IECs of WT mice treated with si-Dclk1-NP, except for a Min/+ hanced in the IECs of Apc mice. These findings sug- marginal increase in CyclinD1, suggesting that cell gest that Dclk1 may play a crucial role in development and cycling may be controlled by Dclk1 expression levels progression of intestinal tumors under the loss of Apc (Fig. 4e and f). Together, knocking down Dclk1 function. To test our hypothesis that Dclk1 is required for decreased the pro-survival signaling in the IECs of Min/+ intestinal tumorigenesis under the loss of Apc function, we Apc mice but not significantly in the WT mice. inhibited Dclk1 gene expression using siDclk1-NPs, along with si-Scramble-Nanoparticles (siScr-NPs) as a control, in Dclk1 is required for intestinal tumorigenesis in ApcMin/+ Min/+ Apc and WT mice. Histological observations of intes- mice tinal sections revealed fewer polyps and reduced dysplasia Stemness and self-renewal ability are the key features in Min/+ in the intestines of Apc mice treated with siDclk1- tumorigenesis, for tumor initiation and progression [33]. NPs compared with siScr-NPs (Additional file 4: Figure To determine whether Dclk1 is critical for intestinal S4). Compared with siScr-NPs treatment, siDclk1-NPs tumorigenesis, we inhibited Dclk1 gene expression using Min/+ treatment reduced the intensity of Dclk1 staining (~3fold) siDclk1-NPs, along with siScr-NPs as control, in Apc Min/+ in intestinal sections from Apc mice (Fig. 4a). Further- mice. The self-renewal ability of Dclk1+ cells and their more, FACS analysis of Dclk1+ cells from the isolated IECs clonal populations is reduced with siDclk1-NP treatment, Min/+ of Apc mice showed that siDclk1-NPs treatment sig- as evidenced by fewer and smaller enteroids formed from Min/+ nificantly reduced the number of Dclk1+ cells compared Dclk1+ cells of Apc mice (Fig. 5a-c). Next, we ana- Min/+ with siScr-NPs treatment (Fig. 4b). Significantly lower ex- lyzed whether Dclk1+ enteroids of Apc mice treated pression of Dclk1 and the other tumor stem cell markers, with siDclk1-NP displayed diminished tumor stem cell Lgr5, Bmi1, and Musashi1, were detected in the isolated markers and pro-survival signaling pathways. We found Min/+ Min/+ IECs of siDclk1-NP–treated Apc mice (Fig. 4c and d). reduced Dclk1 expression in the enteroids of Apc However, expression of Bmi1 and Msi1 was either un- mice treated with siDclk1-NP compared with siScr-NPs changed or increased in the siDclk1-NP–treated WT mice (Fig. 5d). Expression levels of Lgr5, Bmi1, and Musashi1 Min/ (Fig. 4c and d). No change in crypt architecture or abnor- were also significantly reduced in the enteroids of Apc + malities was observed in the WT mice treated with mice treated with siDclk1-NP (Fig. 5d). siDclk1-NPs or siScr-NPs as previously reported [18]. In addition, we examined the protein and gene expres- sion of pro-survival signaling pathways and found that Dclk1 regulates pro-survival signaling pathways to the expression of β-catenin, Notch1, and the NF-κB-p65 support intestinal tumorigenesis in ApcMin/+ mice active form (phosphorylation at Ser536, Ser276) and (Min/+) Conserved pro-survival signaling pathways, most notably total form in the enteroids of Apc mice treated the β-catenin, Notch, and NFkB pathways, coordinately with siDclk1-NP was less than the expression in mice Chandrakesan et al. Molecular Cancer (2017) 16:30 Page 9 of 14

Fig. 4 Dclk1 knockdown reduced the expression of Dclk1 and Dclk1+ cells and the associated expression of tumor stem cell markers and pro-survival signaling in the ApcMin/+ mice. a IHC for Dclk1 in the small intestines of ApcMin/+ mice treated with siDclk1-NPs and siScramble-NPs; staining intensity was scored and represented as a bar graph. b FACS data representing the % Dclk1+ cells isolated from the small intestines of ApcMin/+ mice treated with siDclk1-NPs compared with siScramble-NPs. c & d mRNA and protein expression levels of Dclk1, Bmi1, Lgr5, and Musashi1 in the isolated IECs of WT and ApcMin/+ mice treated with siDclk1-NPs and siScramble-NPs, analyzed by RT-PCR and western blot. f Protein expression levels of pro-survival signaling and their downstream targets in the isolated IECs of WT and ApcMin/+ mice, analyzed by western blot. e mRNA expression levels of pro-survival signaling and their downstream targets in the isolated IECs of WT and ApcMin/+ mice, analyzed by RT-PCR. All quantitative data are expressed as means ± SD of a minimum of three independent experiments. P values of <0.05 = *, <0.01 = **, and 0.001 = *** were considered statistically significant treated with siScr-NP (Fig. 5e). Downstream targets colon cancer cell lines and monitored for self-renewal cyclinD1, c-Myc, Tcf4, Ras, and Hes-1 were also reduced and colony formation ability and migration and invasion (Min/+) in the enteroids of Apc mice treated with siDclk1- in vitro. We utilized siRNAs against NOTCH1 and NF- NP (Fig. 5e). Together, these data suggest that Dclk1 is κB-p65 (RELA) to deplete their gene expression in important and required for i) intestinal tumorigenesis, ii) DLD1 and HT29 colon cancer cells. We also used siR- increased stemness during neoplasia, and iii) increased NAs against DCLK1 in the colon cancer cells as a pro- pro-survival signaling to support tumor progression. To- portional strategy. Protein and mRNA expression gether, the present data suggests that Dclk1 knockdown analysis confirmed a significant decrease in NOTCH1, downregulates pro-survival signaling, stemness, polyps, RELA, and DCLK1 protein and mRNA levels in cancer and, thus, tumorigenesis. cells treated with the respective siRNAs (Fig. 6a). The ef- fect of NOTCH1 and RELA knockdown on the self- Silencing the pro-survival signaling Notch1 and RELA in renewal ability of DLD1 and HT29 cells was analyzed human colon cancer cell lines reduced cancer cells’ self- with a clonogenic assay. We found that the number of renewal and progression colonospheres formed was significantly reduced with To further investigate whether DCLK1 regulates APC siRNA treatment against NOTCH1 and RELA5 com- mutant colon tumorigenesis via pro-survival signaling pared with the si-Scramble treatment (Fig. 6b). Indeed, pathways, we knocked down NOTCH1 and RELA in DCLK1 knockdown showed higher inhibitory action on Chandrakesan et al. Molecular Cancer (2017) 16:30 Page 10 of 14

Fig. 5 Dclk1 knockdown in the Apcmin/+ mice reduced the stemness and pro-survival signaling of Dclk1+ cells. a Enteroids formation of isolated Dclk1+ cells (100 cell per well) from the small intestines of ApcMin/+ mice treated with siDclk1-NPs and siScramble-NPs. b Stacked bar and c line graph represent the quantification of the number of enteroids formed and spheroid volume from the isolated Dclk1+ cells of ApcMin/+ mice. d mRNA and protein expression of Dclk1, Bmi1, Lgr5, and Msi1 in the isolated IECs of ApcMin/+ mice treated with siDclk1-NPs and siScramble-NPs. e & f mRNA and protein expression of pro-survival signaling and their downstream targets in the isolated IECs of ApcMin/+ mice treated with siDclk1-NPs and siScramble-NPs. All quantitative data are expressed as means ± SD of a minimum of three independent experiments. P values of <0.05 = *, <0.01 = **, and 0.001 = *** were considered statistically significant colon cancer cell self-renewal ability than NOTCH1 or decreased the expression of BMI1 (Additional file 6: RELA inhibition (Fig. 6b). Colon cancer cells’ colony- Figure S6). The present data suggest that pro-surivial forming ability, which represents the cells’ viability and signaling may support the colon cancer cell self-renewal survival, was reduced in cells treated with siRNAs and progression. Together, these results revealed that against NOTCH1 and RELA (Fig. 7a). Like colony for- DCLK1 controls the pro-survival signaling pathways in mation, colon cancer cells’ in vitro migration and inva- colon cancer cells to support the survival and stemness sion were significantly reduced with siRNA treatment of tumor cells vital for their progression. against Notch1 and NF-κB-p65, compared with si- Scramble treatment (Fig. 7b and c). However, the inhibi- Discussion tory effect of siNOTCH1 and siRELA on colon cancer Our previous studies demonstrated that Dclk1 overex- cell colony formation and invasion/migration was less pression is correlated with intestinal cancer progression than the effect of siDCLK1 treatments (Fig. 7b and c). and that silencing Dclk1 decreased the number and size DCLK1 knockdown in colon cancer cells also reduced of polyps, adenoma, and adenocarcinoma, suggesting the mRNA expression levels of NOTCH1, RELA and that Dclk1 plays an important active role in intestinal ERK1/2 (Additional file 5: Figure S5). Finally to test the tumorigenesis [18, 28, 39, 40]. Tumor cell self-renewal effect of MAPK inhibition in the regulation of BMI1 ex- and survival ability are the key features in tumorigenesis, pression in the colon cancer cells, ERK1/2 siRNA was for tumor progression [33]. Pro-survival signaling path- treated to the cells and found that their inhibition ways, most notably the ß-catenin, Notch, and NFkB Chandrakesan et al. Molecular Cancer (2017) 16:30 Page 11 of 14

Fig. 6 Silencing DCLK1 and pro-survival signaling NOTCH1 and RELA reduce the self-renewal ability of human colon cancer cells (DLD1 & HT29). a Protein and mRNA expression levels of DCLK1, NOTCH1, and RELA in the DLD1 and HT29 colon cancer cells transfected with si-DCLK1, si-NOTCH1, and siRELA compared with siScramble-transfected cells. b Self-renewal ability of DLD1 and HT29 cells after the knockdown of DCLK1, NOTCH1, and RELA; bar graph represents the average number of spheroids formed from DLD1 and HT29 cells after the knockdown of DCLK1, NOTCH1, and RELA. All quantitative data are expressed as means ± SD of a minimum of three independent experiments. P values of <0.05 were considered statistically significant pathways, coordinately regulate tumor cell survival and the intestinal epithelium [13]. Therefore, we suggest that self-renewal [34–36]. However, whether Dclk1 regulates the loss of Apc may induce the specific expansion of intestinal tumor cell survival and self-renewal for Lgr5+ cells derived Dclk1+ cells for intestinal tumori- tumorigenesis through pro-survival signaling pathways is genesis. However, the expansion of other stem cells Min/+ largely unknown. In the present study, we used Apc and the specific expansion of Dclk1+ cells need to be mice, an excellent model to evaluate human FAP and investigated in future. We also observed enhanced sporadic colorectal cancer [1, 2, 7]. Consistent with our self-renewal ability of Dclk1+ cells isolated from the Min/+ previous studies, we found increased expression of Dclk1 intestines of Apc mice. Min/+ in the IECs of Apc mice, which exhibit high-grade Cellular pro-survival signaling pathways are intercon- dysplasia and adenocarcinoma [12, 18, 20]. It has been nected, complex signaling networks, and their upregula- suggested that stem-like cells or stem cells are more tion is well illustrated in cancers [42]. The aberrant abundant in cancerous conditions, and that the loss of upregulation or constitutive activation of multiple Apc function increases the expansion of the tumor stem survival-signaling pathways in cancer cells promotes cell (TSC) compartment [3, 41]. Loss of Apc function proliferation and stemness, inhibits apoptosis, and in- significantly increased the expression of tumor stem cell creases survival and the ability to invade and migrate markers Dclk1, Lgr5, Bmi1, and Musashi in the IECs. into surrounding tissues and metastasize to distant sites However, how loss of Apc selects the cell type or stem [42, 43]. We found here that the ß-catenin, Notch, and cell type in the process of tumorigenesis is yet to be NFkB pro-survival signaling pathways are upregulated in Min/+ identified. Apc regulates the Wnt signaling, which is crit- the isolated IECs of Apc mice. We further deter- Min/+ ical for the maintenance of Lgr5+ stem cells and initi- mined that Dclk1+ cells of Apc mice display en- ation and progression of cancer [3]. It is recently hanced pro-survival signaling pathways, compared with − reported that Lgr5+ stem cells give rise to Dclk1+ cells in Dclk1 cells. These findings suggest that the enhanced Chandrakesan et al. Molecular Cancer (2017) 16:30 Page 12 of 14

Fig. 7 Silencing DCLK1 and pro-survival signaling reduced the survival ability of human DLD1 & HT29 colon cancer cells. a Colony formation ability of DLD1 and HT29 cells after the knockdown of DCLK1, NOTCH1, and RELA; bar graph represents the average number of colonies formed from DLD1 and HT29 cells after the knockdown of DCLK1, NOTCH1, and RELA. b In vitro invasion and migration of DLD1 cells after the knockdown of DCLK1, NOTCH1, and RELA; bar graph represents the number of cells migrated and invaded after the knockdown of DCLK1, NOTCH1, and RELA. c In vitro invasion and migration of HT29 cells after the knockdown of DCLK1, NOTCH1, and RELA, bar graph represents the number of cells migrated and invaded after the knockdown of DCLK1, NOTCH1, and RELA. All quantitative data are expressed as means ± SD of a minimum of three independent experiments. P values of <0.05 were considered statistically significant pro-survival signaling pathways could be a vital factor [13, 20, 44, 47]. Indeed, the enterospheres formed from Min/+ for tumor progression by regulating tumor stem cells the isolated Dclk1+ cells of Apc mice treated with and/or tumor cell stemness. Several studies have indi- si-Dclk1-NPs display reduced pro-survival signaling cated that Dclk1 promotes the multistep process of can- pathways, which may be the reason for reduced self- cer formation and progression [13, 18, 31, 40, 44–46]. It renewal and tumor stem cells. has been suggested that Dclk1 can regulate pluripotency We further investigated the connection between pro- factors, miRNAs, and signaling pathways, including survival signaling pathways and Dclk1 expression in the Min/+ NOTCH and Runx2, in cancer and non-cancer cells Apc mice with high-grade dysplasia and intramuco- [46–48]. In the present study, we witnessed a similar sal adenocarcinoma. We observed that inhibition of phenomenon; the pro-survival signaling pathways were NOTCH and RELA reduced the colon cancer cell lines Min/+ upregulated in the IECs of Apc mice, in which DLD1 and HT29 self-renewal ability, survival/viability, Dclk1 expression is higher than in controls. We ob- and invasion/migration in vitro. However, DCLK1 served that silencing Dclk1 reduced the pro-survival sig- knockdown is highly effective at inhibiting the self- Min/+ naling pathways in the IECs of Apc mice. We have renewal, colony formation, and invasion/migration of previously demonstrated that downregulation of DCLK1 colon cancer cells than the NOTCH or RELA inhibition can up-regulate critical miRNAs in both in vitro and in in vitro. Furthermore, DCLK1 knockdown decreased the vivo cancer models and resulted in decreased pro- expression of NOTCH, RELA and MAPK in colon can- survival signaling and EMT-related transcription factors cer cells, suggesting that DCLK1 may act as a master Chandrakesan et al. Molecular Cancer (2017) 16:30 Page 13 of 14

regulator for multiple pro-survival signaling pathways, Authors’ contributions which could explain why any individual knockdown of P.C. was responsible for conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript writing; J.Y. pro-survival signaling is less effective than DCLK1 performed data interpretation, database analysis; D.Q. and N.W. generated knockdown in inhibiting cancer cells’ self-renewal and and maintained mouse models and analyzed the data; R.M. performed the progression. Our findings suggest that Dclk1 can regu- IHC; Y.G. conducted data analysis and interpretation; N.A. analyzed data; S.S. analyzed data; M.G., E.B., L.X., and M.B. analyzed and evaluated protein and late multiple signaling pathways for cancer formation gene expression data; G.A. G.A. supervised dataset generation and data and progression. However, the exact regulation mechan- analysis; C.H. supervised the whole project, interpreted results, and assisted ism of Dclk1 requires further clarification. in writing the manuscript. All authors discussed results, analyzed data, and edited the manuscript. All authors read and approved the final manuscript.

Conclusions Competing interests In conclusion, we found that Dclk1 was enhanced in Courtney Houchen and Eddie Bressenman are the founder and co-founder Apc of COARE Biotechnology, Inc. The other authors declare no competing mutant intestinal tumors, and elevated tumor stem- financial interests. ness and survival by regulating the pro-survival signaling pathways. We also determined that Dclk1 knockdown Consent for publication reduced tumor stemness, polyps, adenoma, and adeno- Not applicable. carcinoma by inhibiting pro-survival signaling and sup- Ethics approval and consent to participate pressing their downstream oncogenes. Together, these All animal experiments were performed with the approval and authorization results suggest that Dclk1, a tumor stem cell marker, of the Institutional Review Board and the Institutional Animal Care and Use may be a potential therapeutic target for colon cancer Committee, University of Oklahoma Health Sciences Center. therapy. Author details 1Division of Digestive Diseases and Nutrition, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, Additional files USA. 2OU Cancer Institute, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA. 3Department of Veterans Affairs Medical Center, 4 Additional file 1: Figure S1. DCLK1 gene expression predicts survival Oklahoma City, OK, USA. Access Endocrine, Diabetes and Thyroid center, 5 in APC mutant colon cancer. (TIF 9612 kb) Oklahoma City, OK, USA. Department of Medicine, National Jewish Health, Denver, CO, USA. 6Oklahoma Medical Research Foundation, Oklahoma City, Additional file 2: Figure S2. DCLK1 expression was positively OK, USA. 7Beijing Chao-Yang Hospital Department of Oncology, Capital correlated to COX1 signaling. (TIF 7698 kb) Medical University, Beijing, China. 8COARE Biotechnology, Oklahoma City, OK, Additional file 3: Figure S3. (a) Gene enrichment analysis in the FACs USA. isolated Dclk1+ and Dclk1- of ApcMin/+ mice small intestine. (b) Network of Dclk1 with BMI derived from GeneMANIA. (TIF 7440 kb) Received: 30 September 2016 Accepted: 18 January 2017 Additional file 4: Figure S4. DCLK1 knockdown decreased the intestinal tumorigenesis. (TIF 6996 kb) Additional file 5: Figure S5. DCLK1 knockdown decreased the mRNA References levels of NOTCH1, RELA and ERK1/2 in colon cancer cell lines. (TIF 6101 kb) 1. Fearnhead NS, Britton MP, Bodmer WF. The ABC of APC. Hum Mol Genet. 2001;10:721–33. Additional file 6: Figure S6. ERK1/2 knockdown decreased the mRNA 2. Rustgi AK. The genetics of hereditary colon cancer. Genes Dev. 2007;21: levels of BMI1 in DLD1 colon cancer cell line. (TIF 7467 kb) 2525–38. 3. Lewis A, Segditsas S, Deheragoda M, Pollard P, Jeffery R, Nye E, Lockstone H, Davis H, Clark S, Stamp G, et al. 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