TLK1-MK5 axis drives prostate cancer cell motility and pathologic features of aggressiveness

Abstract

Background: Majority of prostate cancer (PCa) related fatalities occur due to metastasis of cancer cells to adjacent and distal organs. We identified the novel interaction between two kinases (TLK1-MK5) that in part may initiate a signaling cascade promoting PCa metastasis. In PCa, TLK1-MK5 signaling might be crucial as androgen deprivation therapy leads to increased expression of TLK1 and compensatory activation of MK5 in metastatic castration-resistant prostate cancer patients. Methods: We performed scratch wound repair and 3D chemotactic migration assays to determine the motility rates of different TLK1 and MK5 perturbed cells. Co-IP, His, and GST pull down, in vitro kinase (IVK) assays and mass spectrometry (MS) were conducted to determine TLK1-MK5 interaction and phosphorylation. Western blotting (WB), immunohistochemistry (IHC) and bioinformatic analysis were used to examine TLK1 and pMK5 levels in PCa cell lines, mice prostate tumors and PCa tissue microarray (TMA). Results: Both genetic depletion and pharmacologic inhibition of TLK1 and MK5 can significantly reduce wound healing rate in MEF and LNCaP cells. However, TLK1 overexpression alone in the MK5 −/− MEF cells did not increase the wound healing which suggested that TLK1 cannot enhance cellular migration in absence of MK5. Our reciprocal co-IPs, His- and GST pull down assays confirmed TLK1-MK5 interaction in cultured cells. Incubation of purified recombinant TLK1B and MK5 increases the phosphorylation of MK5 and its kinase activity. MS analysis identified three unique phosphorylation sites in MK5 (S160, S354, S386) by TLK1B. While our WB detected substantial amount of pMK5 S354 and TLK1 in all major PCa cell lines, anti-androgen treatment increased pMK5 S354 level in a dose-dependent manner and pharmacologic inhibition of TLK1 reduces pMK5 S354 level in LNCaP cells. IHC staining of TRAMP mice prostate tissues also exhibited increased pMK5 S354 level in aggressive tumor compared to benign regions. Finally, IHC analysis of PCa TMA indicated a correlation between elevated pMK5 S354 level and generally higher Gleason scores as well as nodal metastatic status of the tumors. Conclusion: Our data support that TLK1-MK5 signaling is functionally involved in driving PCa cell motility and clinical aggressiveness, hence, disruption of this axis may inhibit the metastasis of PCa.

Interaction of TLK1 and AKTIP as a Potential Regulator of AKT Activation in Castration-Resistant Prostate Cancer Progression

“…The cell proliferation assay was conducted, as previously described [ 30 ]. Briefly, 2000 cells/well were seeded in a 96-well plate, with 10% FCS containing media with or without different concentrations of J54.…”

Tousled-like kinase 1: a novel factor with multifaceted role in mCRPC progression and development of therapy resistance

“…However, studies aimed at identifying TLKs' role in prostate cancer progression and drug resistance are very limited. Notably, the TLK1 gene was identified by co-expression analysis using WGCNA as a key driver of PCa, highly enriched among candidate genes collected from expression Quantitative Trait Loci, somatic copy number alterations and prognostic analyses [28] . We also recently observed from data mining that TLK1 expression at mRNA level is increased in metastatic and high grade prostatic adenocarcinoma compared to the low risk tumors [29] .…”

“…Furthermore, ADT increases pMK5 S354 level in LNCaP cells, and this pMK5 protein is progressively increased in tumors of higher grades and nodal metastatic scores analyzed from TRAMP mice prostate tissue and human PCa TMA, suggesting that the TLK1>MK5 signaling may be associated with increased aggressiveness of PCa. Finally, disruption of TLK1>MK5 axis by TLK1 or MK5 inhibition drastically reduces PCa cells motility analyzed in a panel of PCa cell lines [ 29 ] . Figure 1 graphically depicts the currently known multifaceted roles of TLK1 in mCRPC progression and acquisition of drug resistance.…”

“…Notably, the TLK1 gene was identified by co-expression analysis using WGCNA as a key driver of PCa, highly enriched among candidate genes collected from expression Quantitative Trait Loci, somatic copy number alterations and prognostic analyses [ 28 ] . We also recently observed from data mining that TLK1 expression at mRNA level is increased in metastatic and high grade prostatic adenocarcinoma compared to the low risk tumors [ 29 ] . Moreover, androgen deprivation translationally increases TLK1B level in LNCaP cells and a PDX mice model through an mTORC1 dependent mechanism without increasing TLK1 mRNA expression; and mTOR inhibition by rapamycin reverses this phenomenon [ 30 , 31 ] .…”

The TLK1-MK5 axis regulates motility, invasion, and metastasis of prostate cancer cells

“…We previously demonstrated that a serine/threonine (S/T) protein kinase, Tousled-like kinase 1 (TLK1) could promote PCa cell motility and clinical aggressiveness by phosphorylating another S/T kinase, MAPK-activated protein kinase 5 (MAPKAPK5/MK5) (7,8). TLK1 is known as a genome maintenance regulator, which functions in regulating replication, transcription, cell cycle checkpoint control, DNA damage response and repair through its direct and indirect substrates (reviewed in (9)(10)(11)(12)).…”