Abstract 4902: A putative stem cell marker, doublecortin-like kinase (DCLK1), is involved in the development of hepatocellular carcinoma.

Introduction: Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide. We recently demonstrated that expression of doublecortin-like kinase DCLK1, also called DCAMKL1, tend to increase as the liver condition advances from normal to cirrhosis and HCC. A significant increase in the number of DCLK1+ cells is observed in patients who develop low-grade cirrhosis to dysplastic regenerative nodules. DCLK1 is a putative intestinal stem cell marker that is overexpressed in cells with tumor-initiating properties and regulates tumor growth and tumor-suppressor miRNAs. Therefore, we investigated the role of DCLK1 in HCC. Methods: Normal human hepatocytes (NHHs) were cultured in Matrigel containing Hepato-STIM media supplemented with epidermal growth factor. The NHH-derived 3D spheroids were analyzed for expression of phenotype markers and their ability to form tumor in immunodeficient mouse model. Cultured hepatoma cells (Huh7.5) were transplanted into the flanks of athymic nude to generate HCC-like tumor. The tumors were treated with PLGA nanoparticles (NPs) containing siRNA against DCLK1 (siDCLK1-NP) or scrambled siRNA (siSCR-NP) for two weeks. The tumor growth was monitored and cancer-related molecular markers in these tumors were analyzed after euthanizing the animals. Results: The NHH-derived 3D spheroids exhibited eccentric perinuclear staining of DCLK1 in most cells at early stages (2-3 weeks). However, asymmetric ductules associated with the spheroids lacked DCLK1 expression. Upon further growth, heterogeneous hepatic cell lineages appeared in the spheroids. The presence of hepatoblasts was characterized by co-staining of α-fetoprotein (AFP) with CK19. Other cell populations showed DCLK1 expression or the presence of cytoplasmic/membranous CK19. A number of cells in the spheroids were negative for CK19, AFP or DCLK1 whereas a few cells were positive for human albumin. Transplantation of these spheroids in the dorsal flanks of immunodeficient mice exhibited metastatic tumor development at multiple sites. The small cuboidal morphology, Alb- phenotypes, and presence of sporadic human Alb+ cells in the tumors suggest that most cells possessed undifferentiated characteristics. These tumors were highly enriched in DCLK1+ cells located near vascular areas. The treatment of Hu7.5-derived tumors with siDCLK1-NP resulted in a significant reduction in DCLK1 expression and concomitant decrease (∼60%) in tumor volume, whereas tumor growth was unaffected in the control animals treated with siSCR-NP. Conclusions: These observations indicate that hepatic DCLK1+ present in the early stages of spheroids may possess stem-like properties. DCLK1 appears to regulate tumorigenic behavior of hepatocytes and its down regulation leads to inhibition of cancer growth. Thus, DCLK1 is likely to be a novel target for HCC treatment. Citation Format: Naushad Ali. A putative stem cell marker, doublecortin-like kinase (DCLK1), is involved in the development of hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4902. doi:10.1158/1538-7445.AM2013-4902