Abstract 3443: Doublecortin-like kinase 1 (DCLK1) is novel Notch pathway signaling regulator in HNSCC

Abstract

Abstract Introduction: Despite advancements in the field, the 5 year survival rate of Head and Neck Squamous Cell Carcinoma (HNSCC) still hovers at 60%. DCLK1 has been shown to regulate epithelial-to-mesenchymal transition (EMT) as well as serving as a cancer stem cell marker in colon, pancreatic and renal cancer. Although it was reported that DCLK1 is associated with poor prognosis in oropharyngeal cancers, very little is known about the molecular characterization of DCLK1 in HNSCC. Methods and Materials: In a quest for novel therapy targets for this disease, we performed an extensive and comprehensive quantitative phosphoproteomics scan on 10 HNSCC patient derived xenografts (PDXs) in order to find dysregulated phosphoproteins. We also performed a comprehensive transcriptome-based computational analysis on hundreds of HNSCC patients from TCGA and GEO databases. We used immunohistochemistry (IHC) to stain a tumor microarray (TMA) of 45 HNSCC samples. We have selected 6 cell HNSCC cell lines (JHU-011, JHU-022, JHU-029, FaDu, SCC22B and Cal27) that express DCLK1, and inhibited DCLK1 with LRRK2-in1 inhibitor and siRNA. We selected 1 HNSCC cell line with low DCLK1 expression, SCC25 and 2 normal keratinocyte cell lines, OKF6 and NOKSI, in which we overexpressed DCLK1. We assessed DCLK1 and active Notch1 expression in these cell lines by Western Blot and Immunohistochemistry (IHC). We evaluated metastatic characteristics with scratch assay, invasion assay, alamar blue proliferation assay, and colony formation assay. Results: We found that DCLK1 is hyperphosphorylated in most of the HNSCC derived PDXs. In our comprehensive transcriptome-based computational analysis on, we found that along with EMT and metastasis pathways, high DCLK1 expression also correlates with NOTCH pathway signaling signatures in HNSCC. Since Notch signaling pathway has a recognized role in tumorigenesis in HNSCC tumors, we have focused on investigating the role of DCLK1 in Notch pathway regulation. Towards this end, we performed a series of in vitro experiments in a collection of HNSCC cell lines. Our analyses revealed DCLK1 inhibition both with LRRK2in1 and siRNA resulted in substantially decreased proliferation, invasion, migration, and colony formation. Furthermore, these effects paralleled downregulation of active NOTCH1, and its downstream effectors, Hey1, Hes1 and Hes5 in all cell lines tested. Additionally, overexpression of DCLK1 in HNSCC cell lines and normal keratinocytes leads to upregulation of Notch signaling as well as increased proliferation. IHC staining on 45 HNSCC tumors indicates that 45% of the samples are positive for DCLK1 compared to 90% DCLK1 negative staining in normal oral tissue. Conclusion: Overall, our results demonstrate the novel role of DCLK1 in HNSCC as a regulator of the NOTCH signaling network and suggests its potential as a therapeutic target in HNSCC. Citation Format: Esther Channah Broner, Tejaswini Subbannayya, Alex Zhavoronkov, Mike Korzinkin, Artem Artemov, Ivan Ozerov, Ido Sloma, David Sidransky, Aditi Chatterjee, Evgeny Izumchenko. Doublecortin-like kinase 1 (DCLK1) is novel Notch pathway signaling regulator in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3443.