Abstract 4906: TLK1-MK5 signaling axis contributes to prostate cancer cell motility and invasion
Authors: Khalil Md Imtiaz , Singh Vibha , De Benedetti Arrigo 
Published: September 2020

Background: Prostate cancer (PCa) is the 2nd leading death-causing malignancy in men where the majority of fatalities occur due to metastasis of cancer cells to adjacent and distal organs. We identified the novel interaction between two kinases that may initiate a signaling cascade promoting metastasis. One kinase is MAPK activated protein kinase 5 (MAPKAPK5 or MK5) which plays a debated role in cellular motility along with other cellular processes. Our observations are that MK5 interacts with another S/T kinase, called tousled like kinase 1 (TLK1), which may also have a potential role in cellular motility which is largely unstudied, apart from one report in breast cancer on TLK2. In PCa, TLK1-MK5 signaling axis might be crucial as androgen deprivation therapy leads to increased expression of both TLK1 and MK5 in metastatic castrate-resistant prostate cancer patients. Our working hypothesis is that TLK1-MK5 axis promotes PCa cell motility and invasion through actin rearrangement as well as focal adhesion proteins modification and disruption of this interaction may help to keep the tumor localized.

Method and Results: A previous proteomic screening from our lab identified MK5 as one of the novel interactors of TLK1 with high confidence (p&lt;0.00005). To validate this interaction, we overexpressed EGFP-MK5 in HEK 293 cells and performed a reciprocal Co-IP study targeting both TLK1 and EGFP-MK5 that confirmed their interaction in vivo. Additionally, a reciprocal His- and GST-pull down assays demonstrated the direct and physical interaction between TLK1 and MK5 in vitro. Another Co-IP experiment revealed that TLK1-MK5 interaction starts with the attachment of the cells to the ECM. We conducted several scratch wound repair and 3D chemotactic migration assays to find out if TLK1-MK5 axis enhances the migration rate of the cells. Inhibition of the kinase activity of TLK1 by using a specific inhibitor (thioridazine) decreases the migration rate of the HEK 293 cells, which supports the importance of TLK1 function in motility. Wound repair and chemotactic migration were also demonstrated to be faster in overexpressed EGFP-MK5 in LNCaP and MEF cells and compared also to MK5 knock out cells. An in-vitro kinase assay using [y-32P] ATP demonstrated that incubation of purified TLK1B and purified MK5 increases the phosphorylation of MK5 and its kinase activity. Mass spectrometric analysis identified three unique phosphorylation sites in MK5 (S160, S354, S386) by TLK1B which are mapped to the functional domains of MK5. We also measured the protein level of several focal adhesion proteins in TLK1 and MK5 co-expressed HEK 293 cells such as total FAK, phospho-FAK (Tyr397), total Paxillin, Phospho-Paxillin (Tyr118, Ser178), etc. and observed activating phosphorylation.

Conclusion: Our data support that TLK1-MK5 interaction is functionally involved in cell motility and migration and hence, disruption of this axis may inhibit the metastasis of PCa.

Citation Format: Md Imtiaz Khalil, Vibha Singh, Arrigo De Benedetti. TLK1-MK5 signaling axis contributes to prostate cancer cell motility and invasion [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4906.