Poster Presentation 31st Lorne Cancer Conference 2019

The Function of DCLK1 isoforms in Gastric Cancer (#133)

Annalisa L.E. Carli 1 , Shoukat Sterle 1 , Janson Tse 1 , Ryan O'Keefe 1 , Isabelle Lucet 2 , Matthias Ernst 1 , Michael Buchert 1
  1. Olivia Newton-John Cancer Research Institute, Heidelberg, VIC, Australia
  2. The Walter and Eliza Hall Institute of Medical Research, Parkville , VIC, Australia

Doublecortin-like kinase 1 (DCLK1) is known for its involvement in neurogenesis and neuronal migration, but has also a proposed role as a cancer stem cell marker and potential driver of gastric tumourigenesis. A recent meta-analysis demonstrated a positive correlation of DCLK1 expression levels with advanced malignancy and reduced overall survival1. Analysis of the Cancer Genome Atlas (TCGA) RNAseq data for stomach cancer (STAD) shows that DCLK1 expression is most elevated within the Genomic Stable (GS) molecular subtype (TCGA classification) and the diffuse subtype (Lauren classification). Both subtypes correlate with poor prognosis and worse overall survival.

DCLK1 has four different isoforms, two full-length splice forms (740aa, 729aa) and two short –kinase only– splice forms (433aa, 422aa). The expression of the short splice forms correlates with poor prognosis in colon cancer. In addition, DCLK1 induces an epithelial to mesenchymal transition (EMT) in pancreatic cancer cells. However, the cellular functions and effectors of DCLK1 and its isoforms have yet to be determined. To address this question, we have generated expression constructs for the four different DCLK1 isoforms and stably transfected them individually in DCLK1-KO cell-lines. All constructs carry either an N- or C-terminal Flag/Strep2 tag for easy isolation of DCLK1 interaction partners. The DCLK1-KOs cells were created using the CRISPR-STOP method2, changing a single nucleotide to create a stop codon which is more efficient than CRISPR-Cas9 which produces double-strand DNA breaks relying on non-homologous end joining (NHEJ) repair to cause frameshift mutations. This approach will enable us to study the specific cellular functions, localizations and effectors of each DCLK1 isoform. In addition, 19% of all DCLK1 mutations (pan-cancer) are located in the 3’-UTR. A total of 2866 different miRNAs are predicted to bind to the 3’-UTRs of the 4 isoforms (miRWalk3), of which 616 can bind to all four isoforms and 27 are unique for individual isoforms. Linking mutation sites to miRNAs gives insights into the importance of miRNAs in regulating DCLK1.

  1. 1. Shi W, Li F, Li S, Wang J, Wang Q, Yan X, et al. Increased DCLK1 correlates with the malignant status and poor outcome in malignant tumors: a meta-analysis. Oncotarget. 2017 Nov 21;8(59):100545–57.
  2. 2. Kuscu C, Parlak M, Tufan T, Yang J, Szlachta K, Wei X, et al. CRISPR-STOP: Gene silencing through base-editing-induced nonsense mutations. Nat Methods. 2017;14(7):710–2.
  3. 3. Dweep H, Gretz N, Sticht C. MiRWalk database for miRNA-target interactions. Methods Mol Biol. 2014;1182:289-305.