Background
APC is a tumor suppressor gene that is mutated in patients with familial adenomatous polyposis (FAP) and most sporadic colorectal cancers [
1,
2]. The
Apc mutation dysregulates the Wnt signaling pathway and triggers cellular transformation, resulting in the development of adenomatous polyps [
3]. It was suggested that the
APC gene mutation is required, but is not sufficient, for the development of cancer in the colon. Since tumorigenesis is considered the result of multiple genetic changes, several efforts have made to identify those tumorigenesis-promoting changes. Several genetic changes, including activation mutations in
Ki-RAS/N-RAS, mutations in the tumor suppressor
TP53, and deletion of a region of chromosome 18 containing
SMAD2,
SMAD4, and
DCC have been identified [
4]. Despite improvements in our understanding of this disease, the molecular events underlying the development and progression of intestinal tumors are still largely unknown and may be a key to the development of more effective and novel therapeutic strategies. Therefore, understanding the
APC gene mutation associated changes for intestinal tumorigenesis is important.
Similar to humans with germline mutations in
APC,
Apc
Min/+
mice have a heterozygous mutation in the
Apc gene, predisposing the mice to intestinal and colon tumor development. These mice start developing intestinal polyps by ~4 weeks of age, with progression to dysplasia at 18–21 weeks; adenocarcinoma is also evident at ~26-34 weeks [
5‐
8]. Eight-to-twelve-week-old
Apc
Min/+
mice are a good model with which to study the pathogenesis of FAP, while 26-to-34-week-old
Apc
Min/+ mice develop intestinal high-grade dysplasia and adenocarcinoma, and are a particularly relevant model for studying tumor progression and developing therapeutic strategies [
6,
7].
Apc
Min/+ mice develop high-grade dysplasia and adenocarcinoma and are a clinically relevant disease model, since a large number of patients diagnosed with advanced colon cancer are elderly and have unresectable or widespread disease [
9].
Doublecortin-like kinase 1 (Dclk1) is a microtubule-associated protein kinase and has been identified as a tuft cell marker in the small intestine [
10]. Dclk1 has been reported to mark tumor stem cells in the intestine and pancreas [
11‐
15]. Emerging evidence has confirmed that the majority of human malignancies are initiated and maintained by a distinct population of cells that display stem cell properties and self-renewal ability [
16]. More recently, it has been shown that the development and progression of colon and pancreatic cancer depend upon Dclk1
+ cancer stem cells [
11,
13‐
15]. We reported that Dclk1 is overexpressed in many cancers, including colon, pancreas, liver, and esophageal cancer [
12,
17‐
20]. Previous work from others and us supported the idea that DCLK1 expression is critical for cancer stem cells, cancer growth, EMT, and metastasis [
11,
12,
15,
18,
21‐
23]. These data provide a basis for Dclk1 as a regulatory factor for tumor growth and advancement.
Recent studies have indicated that neoplastic cells have active pro-survival signaling pathways for proliferation, resistance, self-renewal, and survival [
24‐
26]. Furthermore, progression of cancer, including metastasis and secondary tumor formation of cancer cells with self-renewal ability, is often linked to altered expression of pro-survival signaling pathways [
27]. Understanding the diversity of pro-survival signaling pathways that underlie cancer formation and progression is essential for developing a new generation of effective anticancer drugs for combinatorial therapeutic strategies. The interdependence between pro-survival signaling and tumor self-renewal ability with enhanced Dclk1 highlights the collective mechanism involved in tumor growth and survival. However, the precise mechanism by which Dclk1 supports intestinal tumor progression is poorly understood. With the aid of the
Apc
Min/+
mouse model, we assessed the contribution of Dclk1 to intestinal tumorigenesis using small interfering RNAs targeting Dclk1 incorporated into poly(lactic-co-glycolic acid) nanoparticles (siDclk1-NPs). We found that Dclk1 is involved in enhancing the pro-survival signaling pathways and tumor cells’ self-renewal ability to facilitate intestinal tumor growth and progression.
Methods
TCGA Colon Adenocarcinoma (COAD) Data
The RNA-seq datasets from February 2015 combining data from 329 patients with colon adenocarcinomas included in the Cancer Genome Atlas (TCGA) dataset were downloaded through the UCSC cancer genome browser (
https://www.xenabrowser.net), as previously described [
28].
Determination of DCLK1-correlated pro-survival signaling in APC mutant COAD
APC mutant/
APC non-mutant samples and samples with high/low DCLK1 expression levels were sorted by R v3.2. Patients whose DCLK1 expression levels was in the top 25% or bottom 25% were considered DCLK1-high or DCLK1-low, respectively. The corrplot function (R package corrplot) was used to confirm the correlation between the expression levels of DCLK1 and other genes. A heatmap was produced using the heatmap.2 function (R package gplots) [
28].
DCLK1 network with pro-survival signaling utilizing the GeneMANIA database
Datasets, including physical interactions, pathway, and genetic interactions, were collected from the public domain GeneMANIA database. The dataset relevant to DCLK1 and the pro-survival signaling network was produced from the GeneMANIA database (
http://www.genemania.org).
Animals
All animal experiments were performed with approval and authorization from the Institutional Review Board and the Institutional Animal Care and Use Committee at the University of Oklahoma Health Sciences Center (Oklahoma City, Oklahoma).
Apc
Min/+ mice on the C57BL/6 J background were obtained from The Jackson Laboratory and were maintained by breeding
Apc
Min/+ males to C57BL/6 J females. Mice were genotyped with a PCR assay to identify carriers of the
Min allele of
Apc. Same sex (male) C57BL/6 J
Apc
Min/+
and
Apc
+/+ littermates at 30 week of age were used in the present study. The average life span of
Apc
Min/+ mice on the C57BL/6 J background is ∼ 20 weeks, although the mice in our facility have healthier survival, as observed in several previous studies [
5‐
8].
Apc
Min/+ mice (i.e., >30 weeks of age) were carefully monitored and sacrificed before becoming moribund.
Intestinal Epithelial Cell (IEC) Isolation
Small intestines (ileum) were attached to a paddle, were immersed in Ca
2+-free standard Krebs-buffered saline (in mmol/l: 107 NaCl, 4.5 KCl, 0.2 NaH
2PO
4, 1.8 Na
2HPO
4, 10 glucose, and 10 EDTA) at 37 °C for 15–20 min, and were gassed with 5% CO
2, 95% O
2. Individual crypt units were then separated by intermittent (30 s) vibration into ice-cold phosphate buffered saline and were collected by centrifugation [
18,
29‐
31]. We utilized the whole intestinal epithelilal cells.
FACS
Freshly isolated IECs were washed and resuspended in RPMI glutamax medium. To avoid endothelial and stromal contamination, isolated cells were incubated with anti-CD45, anti-CD31, anti-EpCAM, and anti-Dclk1 antibodies conjugated with the respective fluorochromes for 30 min. The cells were washed and sorted using an Influx-V cell sorter (Cytopeia). CD45
−CD31
−EpCAM
+Dclk1
+ cells were collected and then subjected to enterosphere formation assays [
18,
30].
Clonogenic assay
FACs isolated Dclk1
+ IECs were plated in 48-well plates at a density of 100 cells per well in RPMI medium containing 0.3% soft agar. The cell suspensions were plated in a 48-well plate above a layer of solidified 1% soft agar in plain RPMI medium. The plates were incubated at 37 °C under 5% CO
2. The cells were followed for enterosphere/enteroid formation, as described previously [
18,
30,
31].
RNA isolation and real-time RT-PCR analysis
Total RNA isolated from small intestinal epithelial cells was subjected to reverse transcription. The complementary DNA (cDNA) was subsequently used to perform real-time PCR with SYBR™ chemistry (Molecular Probes, Eugene, OR) using gene-specific primers for specific transcripts. The crossing threshold value assessed by real-time PCR was noted for the transcripts and normalized to β-actin.
Immunoblot analysis
Twenty-five micrograms of the total protein was size-separated in a 4–12% SDS polyacrylamide gel and transferred electrophoretically onto a PVDF membrane with a wet-blot transfer apparatus (Bio-Rad, Hercules, CA). The membrane was blocked and incubated overnight with a primary antibody and was subsequently incubated with horseradish peroxidase-conjugated secondary antibody. The proteins were detected using ECL Western blotting detection reagents (Amersham-Pharmacia, Piscataway, NJ). Actin (42-kD) was used as a loading control.
Small interfering RNAs
The Dclk1 siRNA (siDclk1; Cat. # S234357) sequence targeting the coding region of Dclk1 (accession No. NM_019978) and scrambled siRNAs (siScr; Cat. # AM4636) not matching any of the mouse genes were obtained (Ambion Inc., Austin, TX, USA). DCLK siRNA (h) (# SC45618), RELA siRNA (h) (#SC29410) and NOTCH1 siRNA (h) (#SC36095) were obtained from Santa Cruz Biotechnology (SCBT, TX, USA).
Synthesis and characterization of Dclk1 siRNA NPs and treatment
Poly(lactide-
co-glycolide acid nanoparticles (PLGA NPs) were synthesized using a double emulsion solvent evaporation technique, as described previously [
18,
20]. The amount of encapsulated siRNA was quantified using a spectrophotometer (DU-800, Beckman Coulter, Brea, CA). The size, polydispersity index, and zeta-potential measurements of synthesized siRNA NPs were determined using diffraction light scattering (DLS) utilizing Zeta PALS (Brookhaven Instruments, Holtsville, NY). Sex- and age-matched littermates of C57BL/6 J
Apc
Min/+
mice were injected i.p. with 0.25 nmol of siRNA preparation on every third day for a total of six doses.
Immunohistochemistry/immunofluorescence
Standard immunohistochemistry and immunofluorescence protocols were used with specific antibodies, as described previously [
18,
30].
Antibodies
We used the following antibodies: Dclk1, Lgr5, Bmi1, Hes1, Tcf4, Cox1, Cox2, EpCam, CD45, CD31 (all from Abcam, Cambridge, MA), CXCL1, CyclinD1, cMYC, β − catenin (Santa Cruz Biotechnology, USA), Notch1, NfkB-p65, CyclinD1, Ras, β-actin (Cell Signaling, Danvers, MA, USA), anti-rabbit IgG, anti-mouse IgG, anti-goat IgG (Jackson ImmunoResearch, West Grove, PA, USA), Alexa Fluor® 488 donkey anti-rabbit IgG, and Alexa Fluor® 568 donkey anti-goat IgG (Invitrogen, USA).
HT-29 and DLD1 human colon cancer cells were purchased from the American Type Culture Collection (ATCC) and were maintained in DMEM medium containing 10% fetal bovine serum (FBS). For siRNA-mediated knockdown studies, cells were seeded into 6-cm petri dishes and were allowed to attach overnight. After attachment, 25 nM of commercially validated siRNA targeting human DCLK1 or NOTCH1 or RELA (siRNA; Santa Cruz Biotechnology) or 25 nM human scrambled sequence (siSCR) not targeting any known genes were complexed with Lipofectamine 3000 (Invitrogen) and added to the dishes in fresh cell culture medium. After 48 h of treatment, cells were collected for migration, invasion, colony formation, and self-renewal (clonogenic) analysis.
Migration and invasion assay
For the invasion assay, matrigel-coated Transwells (BD Biosciences) were prepared by retrieving in serum-free media for 2 h at 37 °C. For the migration assay, Transwells (BD Biosciences) were also used. Subsequently, HT-29 and DLD1 cells (5000/well) pre-transfected with either 25 nM siRNA or siSCR for 48 h were seeded into each Transwell in triplicate in serum-free media. Cell culture medium containing 10% FBS was added to the bottom of each well as chemoattractant, and the cells were incubated for 24 h at 37 °C under 5% CO2. Afterwards, a cotton swab was used to scrape non-invasive/migratory cells off the top of Transwells; the remaining cells were fixed with 100% methanol, stained with 0.1% crystal violet, and allowed to dry. After drying, all invading cells were counted from each Transwell. Results are reported as the number of cells invaded and/or migrated.
HT-29 and DLD1 were transiently transfected with si-DCLK1, siNFkB-p65, and si-NOTCH1 (from Santa Cruz Biotechnology), along with scramble siRNA. After 48 h, cells were seeded and passaged into new 6-well plates (100 cells/well). Cells were allowed to grow for one week, then were fixed with glacial acetic acid/methanol solution (1:3) and washed with PBS. Colonies were stained with 0.5% Crystal violet for 10 min and were washed with tap water to remove excess stain. Colonies were then counted under a stereomicroscope using a 1-cm2 grid. Four squares from four quadrants were counted for each well.
Statistical analysis
Statistical analyses were conducted using GraphPad Prism 6.00 (GraphPad Software, La Jolla) and R system v3.2 for statistical computing. Pearson product–moment correlation was used for analysis and correlation of gene expressions between two groups. Colon cancer recurrence-free survival analysis was performed using Kaplan Meier Survival analyses. P values of <0.05 = *, <0.01 = **, and 0.001 = *** were considered statistically significant.
Discussion
Our previous studies demonstrated that Dclk1 overexpression is correlated with intestinal cancer progression and that silencing Dclk1 decreased the number and size of polyps, adenoma, and adenocarcinoma, suggesting that Dclk1 plays an important active role in intestinal tumorigenesis [
18,
28,
39,
40]. Tumor cell self-renewal and survival ability are the key features in tumorigenesis, for tumor progression [
33]. Pro-survival signaling pathways, most notably the ß-catenin, Notch, and NFkB pathways, coordinately regulate tumor cell survival and self-renewal [
34‐
36]. However, whether Dclk1 regulates intestinal tumor cell survival and self-renewal for tumorigenesis through pro-survival signaling pathways is largely unknown. In the present study, we used
Apc
Min/+
mice, an excellent model to evaluate human FAP and sporadic colorectal cancer [
1,
2,
7]. Consistent with our previous studies, we found increased expression of Dclk1 in the IECs of
Apc
Min/+
mice, which exhibit high-grade dysplasia and adenocarcinoma [
12,
18,
20]. It has been suggested that stem-like cells or stem cells are more abundant in cancerous conditions, and that the loss of
Apc function increases the expansion of the tumor stem cell (TSC) compartment [
3,
41]. Loss of
Apc function significantly increased the expression of tumor stem cell markers Dclk1, Lgr5, Bmi1, and Musashi in the IECs. However, how loss of
Apc selects the cell type or stem cell type in the process of tumorigenesis is yet to be identified.
Apc regulates the
Wnt signaling, which is critical for the maintenance of Lgr5+ stem cells and initiation and progression of cancer [
3]. It is recently reported that Lgr5
+ stem cells give rise to Dclk1
+ cells in the intestinal epithelium [
13]. Therefore, we suggest that the loss of
Apc may induce the specific expansion of Lgr5
+ cells derived Dclk1
+ cells for intestinal tumorigenesis. However, the expansion of other stem cells and the specific expansion of Dclk1
+ cells need to be investigated in future. We also observed enhanced self-renewal ability of Dclk1
+ cells isolated from the intestines of
Apc
Min/+
mice.
Cellular pro-survival signaling pathways are interconnected, complex signaling networks, and their upregulation is well illustrated in cancers [
42]. The aberrant upregulation or constitutive activation of multiple survival-signaling pathways in cancer cells promotes proliferation and stemness, inhibits apoptosis, and increases survival and the ability to invade and migrate into surrounding tissues and metastasize to distant sites [
42,
43]. We found here that the ß-catenin, Notch, and NFkB pro-survival signaling pathways are upregulated in the isolated IECs of
Apc
Min/+
mice. We further determined that Dclk1
+ cells of
Apc
Min/+
mice display enhanced pro-survival signaling pathways, compared with Dclk1
− cells. These findings suggest that the enhanced pro-survival signaling pathways could be a vital factor for tumor progression by regulating tumor stem cells and/or tumor cell stemness. Several studies have indicated that Dclk1 promotes the multistep process of cancer formation and progression [
13,
18,
31,
40,
44‐
46]. It has been suggested that Dclk1 can regulate pluripotency factors, miRNAs, and signaling pathways, including NOTCH and Runx2, in cancer and non-cancer cells [
46‐
48]. In the present study, we witnessed a similar phenomenon; the pro-survival signaling pathways were upregulated in the IECs of
Apc
Min/+
mice, in which Dclk1 expression is higher than in controls. We observed that silencing Dclk1 reduced the pro-survival signaling pathways in the IECs of
Apc
Min/+
mice. We have previously demonstrated that downregulation of DCLK1 can up-regulate critical miRNAs in both in vitro and in vivo cancer models and resulted in decreased pro-survival signaling and EMT-related transcription factors [
13,
20,
44,
47]. Indeed, the enterospheres formed from the isolated Dclk1
+ cells of
Apc
Min/+
mice treated with si-Dclk1-NPs display reduced pro-survival signaling pathways, which may be the reason for reduced self-renewal and tumor stem cells.
We further investigated the connection between pro-survival signaling pathways and Dclk1 expression in the Apc
Min/+
mice with high-grade dysplasia and intramucosal adenocarcinoma. We observed that inhibition of NOTCH and RELA reduced the colon cancer cell lines DLD1 and HT29 self-renewal ability, survival/viability, and invasion/migration in vitro. However, DCLK1 knockdown is highly effective at inhibiting the self-renewal, colony formation, and invasion/migration of colon cancer cells than the NOTCH or RELA inhibition in vitro. Furthermore, DCLK1 knockdown decreased the expression of NOTCH, RELA and MAPK in colon cancer cells, suggesting that DCLK1 may act as a master regulator for multiple pro-survival signaling pathways, which could explain why any individual knockdown of pro-survival signaling is less effective than DCLK1 knockdown in inhibiting cancer cells’ self-renewal and progression. Our findings suggest that Dclk1 can regulate multiple signaling pathways for cancer formation and progression. However, the exact regulation mechanism of Dclk1 requires further clarification.
Conclusions
In conclusion, we found that Dclk1 was enhanced in Apc mutant intestinal tumors, and elevated tumor stemness and survival by regulating the pro-survival signaling pathways. We also determined that Dclk1 knockdown reduced tumor stemness, polyps, adenoma, and adenocarcinoma by inhibiting pro-survival signaling and suppressing their downstream oncogenes. Together, these results suggest that Dclk1, a tumor stem cell marker, may be a potential therapeutic target for colon cancer therapy.
Acknowledgements
Authors thank Ms. Kathy Kyler, Staff Editor, OUHSC, for editing our manuscript.