Phosphoinositide‐dependent Kinase‐1 (PDPK1) regulates serum/glucocorticoid‐regulated Kinase 3 (SGK3) for prostate cancer cell survival

Abstract Prostate cancer (PCa) is the most common malignancy and is the second leading cause of cancer among men globally. Using a kinome‐wide lentiviral small‐hairpin RNA (shRNA) library screen, we identified phosphoinositide‐dependent kinase‐1 (PDPK1) as a potential mediator of cell survival in PCa cells. We showed that knock‐down of endogenous human PDPK1 induced significant tumour‐specific cell death in PCa cells (DU145 and PC3) but not in the normal prostate epithelial cells (RWPE‐1). Further analyses revealed that PDPK1 mediates cancer cell survival predominantly via activation of serum/glucocorticoid‐regulated kinase 3 (SGK3). Knock‐down of endogenous PDPK1 in DU145 and PC3 cells significantly reduced SGK3 phosphorylation while ectopic expression of a constitutively active SGK3 completely abrogated the apoptosis induced by PDPK1. In contrast, no such effect was observed in SGK1 and AKT phosphorylation following PDPK1 knock‐down. Importantly, PDPK1 inhibitors (GSK2334470 and BX‐795) significantly reduced tumour‐specific cell growth and synergized docetaxel sensitivity in PCa cells. In summary, our results demonstrated that PDPK1 mediates PCa cells’ survival through SGK3 signalling and suggest that inactivation of this PDPK1‐SGK3 axis may potentially serve as a novel therapeutic intervention for future treatment of PCa.


| INTRODUC TI ON
As the most prevalent form of non-cutaneous cancer, prostate cancer (PCa) is the second-highest incidence of cancer in male globally. 1 In 2018, approximately 1.3 million new patients were diagnosed with PCa, and nearly 360 000 deaths occurred globally. 1 In the early stages, PCa is mainly regulated by androgen, thus, androgen deprivation therapy (ADT) has become routine in clinical practice.
However, about 10%-20% of patients inevitably fail this therapy and progress to castration-resistant PCa (CRPC) with a median survival ranges between 15 and 36 months. 2 Despite the fact that our understanding of the clinical, molecular and pathologic characteristics of PCa is incomplete, the androgen receptor (AR), which is regarded as the primary oncoprotein in PCa and CRPC, is regularly expressed in a heterogeneous way, even in the context of AR gene amplification. 3 In AR-positive PCa, hormonal treatment resistance can arise via clonal selection, intracrine mechanisms or adaptation to decreased androgen (eg mutation, AR phosphorylation and bypass of the AR pathway). 4 While some of the early studies indicated that CRPC depends on AR activity, numerous new evidence suggests that other mechanisms have the capability to promote CRPC progression in a manner that is independent of AR activation. 5 For example, several studies have shown that phosphoinositide 3-kinase (PI3K) signalling is adequate for CRPC survival when AR activity is reduced or not present. [5][6][7] In contrast to primary PCa, AR gene expression signatures were found to be inversely correlated with cell proliferation signatures in a subset of CRPC patients. 8 Furthermore, AR activities have also been shown to possess a tumour and metastasis suppressor function, suggesting PCa disease progression can be driven by AR-independent mechanisms. 9,10 In order to identify genes and pathways that modify PCa growth in the context of suppressed AR signalling, we conducted in vitro high-throughput RNA interference (RNAi) screening using a lentiviral-shRNA library designed to target the whole kinome against the AR-negative PCa DU145 cell line. 11 Here, we describe the results of our screen that identified the 3-phosphoinositide-dependent protein kinase 1 (PDPK1) as an essential kinase critical for the proliferation and viability of a subset of PCa cells.

| Lentiviral human kinase shrna library screen
The MISSION LentiExpress™ Human Kinases shRNA library (Sigma-Aldrich, St Louis, MO, USA) was used to screen for candidate protein kinases mediating the growth of PCa cells.
Briefly, the AR-negative DU145 cells were seeded in a 384-well plate overnight, followed by transduction of lentiviral particles at multiplicities of infection (MOI) of 1 in the presence of 7.5 μg/mL polybrene (Sigma-Aldrich, St Louis, MO, USA). After 18h incubation, the medium containing the lentivirus particles was replaced with complete medium, and the cell viability was evaluated using the CellTiter-Glo® assay (Promega, Madison, WI, USA) at 72 hours post-transduction. Lentiviral particles carrying an empty vector (pLKO.1-puro), a non-target shRNA (NS) or a GFP expressing lentiviral construct were included as controls to examine transduction efficiency and well-to-well variation. All data were normalized against NS controls and hits were considered when shRNA targeting a specific gene achieved a Z-score of less than −2. 12,13

| Quantitative real-time PCR (QPCR) analysis
The total RNA extraction and first-strand cDNA synthesis were conducted using RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and

| Detection of apoptosis by annexin V flow cytometry
All floating and attached cells were stained for cell apoptosis assay

| Transfection
Plasmids for constitutively active myristoylated AKT and SGK3-S486D mutant were obtained from Addgene (Addgene plasmid # 9008) and Gene Universal (Newark, DE, USA), respectively. Plasmids were transfected into target cells using X-tremeGENE HP DNA transfection reagent (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's protocol.

| Drug combination analysis
Drug combinatory effects were analysed using the Chou-Talalay method and Highest Single Agent (HSA) models as described previously. 21,22 Briefly, cells were plated at 2.5 × 10 3 cells/well in 96-well format and treated with docetaxel and/or PDPK1 inhibitors (GSK2334470 and BX795) alone or in combination. The plates were terminated by MTT cell proliferation assay at 72 hours after treatment. 23

| Statistical analysis
All results were presented as mean ± standard deviation (SD) from at least three independent experiments. SPSS (version 19.0 INC, Chicago, IL) was used to evaluate the statistical significance based on Student's independent t test. A P-value <0.01 was considered statistically significant.

| Kinome-wide shRNA library screen identifies PDPK1 as putative target mediating PCa cell survival
To identify genes and pathways that modify PCa growth, we con- (also known as ERK4) 29 and ROCK2 [38][39][40] have been shown to inhibit PCa cell growth, independently validated our results.  Figure 1D). Interestingly, PDPK1 proteins were found to be phosphorylated in cells which express them, suggesting that PDPK1 proteins are constitutively active in these cells.

| Depletion of PDPK1 inhibits SGK3 phosphorylation
PDPK1 is known to phosphorylate AKT that regulates several signalling pathways altered in cancer. 42 However, recent studies have also shown that PDPK1 can activate many other members of AGC kinase family such as p70S6K, SGK, p90RSK and the members of PKC family, independent of AKT. [42][43][44] To evaluate whether the pro-

| PDPK1 mediates the survival of DU145 and PC3 cells via SGK3 signalling pathway
To test whether the pro-survival effects of PDPK1 is mediated through SGK3, we transfected a constitutively active SGK3 S486D mutant in DU145 and PC3 cells followed by PDPK1 depletion.
As shown in Figure 4, ectopic expression of SGK3 S486D significantly rescued the apoptosis induced by PDPK1 depletion (P < .01, Student's t test). In contrast, no such effects were observed in cells transfected with a constitutively active myristoylated AKT (Myr-AKT), suggesting that PDPK1 regulates cell survival in PCa cells through activation of SGK3 signalling ( Figure S3).

| Inhibition of PDPK1 enhances docetaxel sensitivity in PCa cells
Since PDPK1 up-regulation and activation have been recently shown to confer chemoresistance in breast, 45  and PDPK1 inhibitors (GSK2334470 and BX795) on a panel of PCa cells (DU145, PC3 and LNCaP). As shown in Figure 5A and Next, we investigated whether the inhibition of PDPK1 could synergize docetaxel sensitivity in PCa cells. As shown in Figure 5B, inhibition of PDPK1 enhances docetaxel sensitivity in DU145 and PC3 but not LNCaP cells, suggesting that PDPK1 inhibitors might potentiate sensitivity of refractory PCa cells to chemotherapy (Tables 1 and 2).   Vec S486D In an effort to define the mechanisms of PDPK1-mediated regulation of PCa cell survival, we observed that depletion of PDPK1

| CON CLUS IONS
In conclusion, we identified PDPK1 as a novel potential therapeutic target in PCa and demonstrated PDPK1 is mediating PCa cells' survival through activation of SGK3 in an AKT-independent manner. Our data further suggest that combination of PDPK1 inhibitors with docetaxel enhances their anti-cancer activity, possibly by targeting SGK3-dependent resistance mechanisms. Together, our results provide a strong rationale to investigate further the use of PDPK1 inhibitors in as novel therapeutic strategies for refractory PCa patients.

DATA S TAT E M E N T
The data that supports the findings of this study are available in the supplementary material of this article.