Doublecortin-like kinase 1 (DCLK1) correlates with Notch pathway signaling, controls metastatic characteristics in Head and Neck cancer

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 and pancreas cancer. DCLK1 is also associated with poor prognosis in colon, renal and oropharyngeal cancers.

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 2 HNSCC cell lines (SCC25 and 93vu) with low DCLK1 expression 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:

DCLK1 is hyperphosphorylated but not over expressed in most of the HNSCC derived PDXs. In our comprehensive transcriptome-based computational analysis on hundreds of HNSCC patients from TCGA and GEO databases, we found that along with EMT pathways, high DCLK1 expression also correlates with NOTCH pathway signaling signatures in HNSCC. In 5 cell HNSCC cell lines, 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 signaling in all cell lines tested. Additionally, overexpression of DCLK1 in HNSCC cell lines leads to upregulation of activated Notch1. 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.