Abstract 2899: On the cutting edge of the leading edge: investigating the role of DCLK1 in invadopodia

Head and neck squamous cell carcinoma (HNSCC) patients suffer from high mortality despite advancing therapeutics. Five-year survival of HNSCC patients decreases dramatically as disease stage progresses. Locoregional invasion, an early step in the metastatic process, significantly contributes to HNSCC mortality. Initial steps of invasion involve the formation of protrusions from the cancer cell known as invadopodia. Mature invadopodia secrete matrix metalloproteases (MMPs) that degrade the extracellular matrix (ECM), providing a way forward for the tumor cell. Thus, understanding the molecular mechanisms that regulate invadopodia formation could reveal molecular targets to prevent locoregional invasion and metastasis. Others have shown that during the maturation phase of invadopodia, adaptor protein tyrosine kinase substrate with four Src homology 3 domains (TKS4) is expressed, MMP 2 and 9 are secreted, and microtubules polymerize elongating the invadopodia into the ECM. In neuronal cells, doublecortin like kinase 1 (DCLK1), a microtubule binding protein, stabilizes and bundles microtubules, contributing to neural elongation, migration, and axon formation. Although, DCLK1 has been implicated in the progression of several cancers including colorectal and pancreas, little is known about its role in invadopodia formation. In preliminary studies, we observed an increase in DCLK1 levels at the invasive front of patient HNSCC, specifically where stromal fibroblasts meet the tumor. Initial the cancer genome atlas (TCGA) analysis reveal DCLK1 is elevated as lymph node stage and histological grade progress. Further, TCGA analysis shows that both TKS4 and DCLK1 overexpression correlates with reduced 5-year survival in HNSCC patients and, DCLK1 and TKS4 are positively correlated. We demonstrate expression of TKS4 and DCLK1 in a panel of HNSCC cell lines. Further, TKS4 and DCLK1 coimmunoprecipitate, indicating they are either directly interactive with each other or are a part of the same molecular complex. Inhibition of the upstream activator of TKS4, c-Src, with dasatinib reduced the levels of TKS4 interacting with DCLK1. Further, shRNA knockdown of DCLK1 in HNSCC cells decreased migration and invasion through a transwell assay, inhibiting functional tumor cell invasion. Localization of DCLK1 to invadopodia was confirmed by confocal microscopy. To better understand the molecular partners of DCLK1 that are likely involved in invadopodia, we performed TCGA analysis and saw a positive correlation between kinesins KIF1A, B, C and select Rab GTPases to both DCLK1 and TKS4. Additionally, DCLK1 coimmunoprecipitated with Rab 40b and heavy chain kinesin. In summary, we have uncovered a fascinating biological role of DCLK1 in the function of invadopodia that promote tumor cell invasion and can be uniquely targeted for therapy. Citation Format: Levi K. Arnold, Michael Barry, Prabhu Ramamoorthy, Brendan Otteman, Shrikant Ananat, Sufi Thomas. On the cutting edge of the leading edge: investigating the role of DCLK1 in invadopodia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2899.