Abstract 193P
Background
Doublecortin-like kinase 1 (DCLK1) plays an important role as a cancer stem cell (CSC) marker in gastrointestinal cancer. Esophageal squamous cell carcinoma (ESCC) accounts for 95% of global esophageal cancer cases and is highly prevalent in Asia. High expression of DCLK1-BL (isoform 4) in ESCC patients is indicative of malignant progression and poor prognosis. Herein, we explore the role of DCLK1-BL in ESCC stemness and evaluate the therapeutic potential of targeting DCLK1 in this disease.
Methods
Spheroid and colony forming ability of three ESCC cell lines (KYSE-70, KYSE-410 and KYSE-510) were assessed after overexpression of DCLK1-iso4 and DCLK1-iso4-KD (kinase-dead, D226N). Western blot was used for molecular level detection of stemness markers. The DCLK1 kinase inhibitor DCLK1-IN-1 was used for ESCC treatment assays including MTT, spheroid formation, et al. Additionally, phospho-kinase array and RNA sequencing were used to explore the mechanism of DCLK1-targeted therapy.
Results
DCLK1-iso4 overexpression stimulated the number of spheroids in KYSE-70, KYSE-410 and KYSE-510 cell lines compared to controls (p<0.05), an effect which was attenuated in DCLK1-iso4-KD cells (p<0.01). Notably, LGR5 expression was doubled in DCLK1-iso4 cells compared to controls and downregulated in DCLK1-iso4-KD cells, suggesting a potential mechanism for DCLK1-BL kinase in stemness. Additionally, functional assays demonstrated that DCLK1-IN-1 induced cytotoxicity and inhibited spheroid formation in a concentration-dependent manner across ESCC cell lines. At the molecular level, DCLK1-IN-1 downregulated CSC markers (CD133, CD44) and pluripotency factors (c-Myc, KLF4, SOX2) in all cell lines. Phospho-kinase array revealed concurrent downregulation of AKT1/2/3 expression in KYSE-70 and KYSE-510 cells after 48-hour treatment of DCLK1-IN-1, which was supported by RNA-seq results.
Conclusions
These findings elucidate the pro-stemness effect of DCLK1-BL (DCLK1-isoform 4) activity in ESCC and demonstrate the potential of DCLK1 targeted therapy. DCLK1 kinase inhibitors are putative candidates to enhance therapeutic efficacy of ESCC, and further studies should be conducted in combination with currently approved ESCC therapies.
Clinical trial identification
Editorial acknowledgement
Legal entity responsible for the study
The authors.
Funding
Fujian University of Traditional Chinese Medicine.
Disclosure
All authors have declared no conflicts of interest.