PCa is diagnosed in ~200,000 US men annually, resulting in approximately 30,000 deaths. ADT with anti-androgens is the standard of care for patients with locally advanced prostate cancer, metastatic prostate cancer, and biochemically recurrent disease after failure of localized treatments. ADT is known to provide remission of the disease, best evidenced by a decline of prostate-specific antigen (PSA) in about 90% of patients. However, after a mean time of 2–3 years, the disease progresses despite continuous hormonal manipulation [
3,
4]. This type of cancer is known as mCRPC, which has a poor prognosis with a mean survival of only 16–18 months [
5] with slight improvement with chemotherapy [
88]. The best window of opportunity is before the development of mCRPC [
6], and this requires a clear understanding of the process of PCa cells’ mechanisms of adaptation to ADT. The best-characterized model so far for studying this is the LNCaP. Androgen deprivation of LNCaP cells results in loss of AR function with a compensatory pro-survival activation of mTOR [
89] mediated by loss of the mTOR inhibitor, FK506-binding protein 5 (FKBP5), which is an AR-regulated gene [
90], and concomitant implementation of a cell division arrest by activation of the DDR mediated by ATR-Chk1 [
91] or ATM-Chk2 [
92]. However, what signals the DDR and ATR activation is poorly understood [
93]. Then, after a quiescent period of ADT adaptation of 2-3 weeks, androgen-independent colonies begin to form [
94]. We have reproduced these effects in TRAMP-C2 cells, another line that recapitulates the conversion from AS to AI growth typically observed in most PCa patients following ADT. An attractive strategy to prevent this process would be to bypass the cell cycle arrest via inhibition of ATM or ATR, causing the cells to undertake replication with damaged DNA that would cause mitotic catastrophe, a strategy that was implemented in LNCaP treated concomitantly with bicalutamide (BIC) and ATM inhibition [
92]. Nevertheless, a limitation of this approach is how to make the inhibition of ATM or ATR specific to PCa cells.
Figure 2 represents our working model of how TLK1/1B are key DDR regulators that allow PCa cells to survive ADT and reprogram into AI growth. Elucidation of optimal pharmacological targeting of these pathways will lead to more effective and potentially curative therapy for hormone naïve PCa and pre-empt CRPC.
Extensive studies also suggest the critical importance of the Hippo/YAP pathway in various solid tumors and in mCRPC [
99]. However, the fundamental mechanism of YAP hyperactivation in PCa onset and development remains elusive. Importantly, our preliminary studies discovered that TLK1 has a role in the Hippo pathway (via NEK1-mediated upregulation of YAP) and can directly hasten the conversion toward CRPC [
100]. Experimentally, we found that 2-fold overexpression of wt-NEK1 (but not the T141A mutant) hastens the progression of LNCaP cells to AI growth [
78]. The protective cell cycle arrest mediated by the TLK1-NEK1 DDR pathway seems insufficient to explain the rapid growth recovery observed in BIC-treated cells when NEK1 is overexpressed and suggests that NEK1 may have additional functions. We suspected it might regulate the Hippo pathway. It was reported that ectopic expression of YAP was sufficient to convert LNCaP cells from AS to AI in vitro [
101]. NEK1 was found to phosphorylate TAZ specifically at S309 [
102], resulting in increased CTGF expression (one of the TAZ/YAP transcriptional targets). TLK1 may regulate the Hippo pathway through its activity on NEK1. We showed that overexpression of the NEK1-T141A mutant results in reduced levels of YAP, along with evidence of an elevated cleaved product (Cl-YAP) [
100,
103]. Decreased YAP levels and Cl-YAP are also seen in control LNCaP cells treated with 1μM THD for 1h (+/- BIC), with similar effects obtained with J54 (our in-house, test, 2
nd generation TLK1 inhibitor, described later). In contrast, LNCaP cells that overexpress NEK1-wt show elevated expression of YAP and no evidence of Cl-YAP. We also showed that NEK1 interacts with YAP as coIP enriched it, and THD does not alter their affinity. As previously demonstrated [
20], the coIP also brought down TLK1. Inhibition of TLK1 with THD or J54 resulted in a dose and time-dependent degradation of YAP in mouse PCa-NT1 cells, showing conservation of function [
24]. All this suggests that TLK1 and NEK1 may be novel regulators of the Hippo pathway, leading to higher YAP levels or phospho-driven nuclear translocation. In contrast, reduced TLK1 activity could impair YAP-driven gene expression via inhibition of the TLK1-NEK1 axis, which seems vital for stabilizing YAP and its accumulation. A recent literature search showed that a known PTH TLK1 inhibitor (Cl-Promazine [
79]) increases pYAP-S127 and degradation in aggressive (YAP-driven) BCA cells [
104]. In further support of our arguments that NEK1 activity is critical for YAP stabilization, we found that CRISPR-mediated KO of NEK1 in the PCa line Neo-TAg1 (NT1) revealed that YAP expression was concomitantly reduced in all the KO-positive clones. Consistently, the expression of several YAP-target genes (e.g., CTGF, Zeb1, Twist1) that drive EMT and invasiveness of these cells was suppressed in the NEK1-KO clones [
103], and there was no apparent compensation from other NEKs, which are not reported to act on YAP [
105]. We have also tested by qRT-PCR the expression of several of these genes in LNCaP and C2 cells treated with BIC+J54, but in that case, the main effect was a dramatic 70-fold increase in BAX [
100], confirming an effect previously reported in response to PTHs treatment of cancer cells [
106]. This suggests that any residual YAP switches to the YAP/P73 transcriptional complex that implements the apoptotic program in response to DNA damage [
107]. This is in contrast to the reported effects of ADT alone, where in some models, AR inhibition results in YAP transcriptional activation; in turn, transcription of stemness and EMT-driver genes in a TEAD-driven manner induces sphere formation in vitro [
99]
. Conversely, THD alone can inhibit cell migration via suppression of EMT-related genes such as Claudin1, E-cadherin, N-cadherin, Twist1, Snail3, Slug, FOXC2, MMP3, MMP9 in HCC cells, though this was hastily attributed to its activity on dopamine receptors [
108].