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

Mol Cancer. 2017 Feb 1;16(1):30. doi: 10.1186/s12943-017-0594-y.

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 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.

Keywords: APC mutation; Cancer stem cells; Colorectal cancer; Dclk1; Intestinal epithelial cells; Nanoparticles; Pro-survival signaling; Self-renewal.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenocarcinoma / genetics
  • Adenocarcinoma / metabolism
  • Adenocarcinoma / pathology
  • Animals
  • Cell Self Renewal / genetics*
  • Cell Survival / genetics*
  • Cluster Analysis
  • Colonic Neoplasms / genetics*
  • Colonic Neoplasms / metabolism*
  • Colonic Neoplasms / pathology
  • Disease Models, Animal
  • Epithelial-Mesenchymal Transition / genetics
  • Female
  • Gene Expression Profiling
  • Gene Knockdown Techniques
  • Genes, APC
  • Humans
  • Intracellular Signaling Peptides and Proteins / genetics*
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Male
  • Mice
  • Mice, Transgenic
  • Mutation
  • Neoplastic Stem Cells / metabolism*
  • Protein-Serine-Threonine Kinases / genetics*
  • Protein-Serine-Threonine Kinases / metabolism*
  • Receptor, Notch1 / metabolism
  • Signal Transduction*

Substances

  • Intracellular Signaling Peptides and Proteins
  • Receptor, Notch1
  • DCLK1 protein, human
  • Protein-Serine-Threonine Kinases