Cellular prostatic acid phosphatase (cPAcP) is a unique prostate-specific tumor suppressor and its loss of expression is associated with prostate carcinogenesis [8]. The data also revealed that cPAcP is involved in regulating androgen-stimulated PCa cell growth, and its expression is associated with androgen-sensitive cell proliferation [7, 16]. Previously, we identified that cPAcP is one of molecular targets by HDAC inhibitors in PCa growth suppression. In HDAC inhibitor-treated AR-positive PCa cells, cPAcP is elevated and cell growth is suppressed; conversely, knockdown cPAcP expression by shRNA reduces the degree of growth suppression by HDAC inhibitors [16]. Furthermore, cPAcP in addition to AR plays a critical role in regulating androgen sensitivity of PCa cell proliferation. While AR is essential to androgen sensitivity; the presence of AR alone is not sufficient for androgen responsiveness of growth stimulation. For example, CR PCa cells still express functional AR but are androgen unresponsive. Instead, the expression of cPAcP in those cells is associated with androgen sensitivity. Since cPAcP expression is responsive to but not regulated by androgens, cPAcP level is not consistent with AR level. Importantly, upon VPA pretreatment, the androgen responsiveness of cells was increased, higher than control cells. As a clinical correlation, we selected cPAcP as a surrogate marker and further explored the role of cPAcP expression in PCa cell growth suppression by various HDAC inhibitors and examined whether HDAC inhibitors treatment will indeed alter androgen responsiveness of different PCa cells.
First, we analyzed the basal level of AR, PAcP and PSA proteins in different PCa cell lines. As shown in Figure 1, in regular culture condition, MDA PCa2b AS and AI cells, and LNCaP C-33, C-81, C4-2 and C4-2B cells all express AR protein. Among them, LNCaP cells had an overall higher levels of AR protein than MDA PCa2b cells, and LNCaP C-33 cells expressed the highest AR protein level among these cells examined (Figure 1), correlating with the degree of androgen-stimulated cell growth (data not shown). On the contrary, MDA PCa2b cells express higher levels of cPAcP than LNCaP cells (Figure 1) and have slower cell proliferation [30]. Furthermore, in MDA PCa2b and LNCaP cell models, cPAcP level decreased in AI cells, lower than that in the corresponding AS cells, respectively. The low PAcP level in AI LNCaP C-81, C4-2 and C4-2B could be seen upon prolonged exposure (data not shown). The PSA protein level is also decreased in AI cells. In comparison, PC-3 and DU 145 cells do not express a detectable level of AR, PAcP and PSA proteins (Figure 1) despite prolonged exposure (data not shown).
We determined the effect of HDAC inhibitor on cPAcP protein level in AR-positive PCa cells and examined their relationship since cPAcP protein functions as a tumor suppressor in PCa cells. We first examined VPA effect on cPAcP protein level. As shown in Figure 2a–c, upon VPA treatment, cPAcP protein levels were greatly elevated in LNCaP C-33 and C4-2B cells, compared with control cells received the solvent alone (Figure 2a, b, Lane #3 vs. #1, right panel), which were decreased by subsequent DHT treatment (Figure 2a, b, Lane #4 vs. #3, right panel). cPAcP protein level had only a slight elevation in VPA-treated MDA PCa2b-AS cells (Figure 2c, Lane #3 vs. #1, right panel), which could be in part due to a very high basal level of cPAcP protein in those cells (Figure 1). Similar phenomenon was observed in LNCaP C-81, C4-2 and MDA PCa2B AI PCa cells (data not shown; [16]). Therefore, the observations on cPAcP protein levels that are increased in all VPA-pretreated PCa cells and then decreased by DHT treatment are inversely correlated with cell growth (Figure 2, Column #1 vs. #3, #3 vs. #4, left panel).
Since androgen sensitivity is an important clinical phenomenon; we examined VPA effect on the androgen responsiveness. Importantly, the PSA level was greatly elevated by 10 nM DHT in VPA-pretreated LNCaP cell lines by over 18-fold, higher than that of control cells without VPA-pretreatment with about fourfold increase (Figure 2a, b, Lane #4 vs. #2, right panel). In VPA-pretreated MDA PCa2b AS cells, PSA protein level was greatly elevated, which is further increased upon DHT treatment (Figure 2c, Lane #4 vs. #3, right panel); despite the fact that DHT alone only had a marginal effect on PSA protein level in the absence of VPA pretreatment, the similar trends were observed in LNCaP C-81, C4-2 and MDA PCa2B AI PCa cell lines (data not shown; [16]). In summary, our data show that VPA pretreatment can increase the degree of androgen sensitivity in cell proliferation by cell number counting and PSA protein level, an androgen-regulated marker, despite that in MDA PCa2b AS cells, VPA pretreatment only greatly enhanced DHT-increased PSA level (Figure 2c, Lane #3 vs. #4, right panel) but not cell growth (Figure 2c, Column #3 vs. #4, left panel). Furthermore, cPAcP protein level was indeed elevated by VPA treatment and then diminished by subsequent DHT treatment (Figure 2, Lane #3 vs. #4, right panels). Since VPA pretreatment greatly enhances DHT-upregulated PSA level, the data indicate that VPA can increase androgen responsiveness of AR-positive PCa cells.
We examined whether other HDAC inhibitors could similarly enhance the androgen responsiveness of PCa cells. LNCaP C-81 cells were used as the model for HDAC inhibitors pretreatment since C-81 cells exhibit many biochemical properties of CR PCa cells (16, 31, 32). C-81 cells were treated with HDAC inhibitors including NaB, SAHA, PxD101, MS-275 and AR42. Results of western blot analyses showed that cPAcP protein including the 38 kDa intermediate form and 50 kDa mature form were greatly elevated by all HDAC inhibitor treatments (Figure 3a–e, Lane #3 vs. #1, right panel) and were diminished by subsequently 1 and 10 nM DHT treatments, following the dose-dependent manner (Figure 3a, Lane #4 vs. #3, right panel; Figure 3b–e, Lane #5, 6 vs. Lane #4, right panel). Further analyses on Figures 2 and 3 reveal that the efficacy of HDAC inhibitor’s growth suppression is at least in part correlated with the degree of induced expression of cPAcP in addition to cPAcP basal level.
Furthermore, we validated DHT responsiveness by analyzing PSA protein level followed by semi-quantification in HDAC inhibitors-pretreated cells. In absence of DHT, PSA basal levels were greatly elevated by SAHA, PxD 101 and MS-275 treatments, and slightly increased in AR42-treated cells, but not significantly elevated in NaB-treated cells, respectively (Figure 3b–e, Lane #4 vs. #1; Figure 3a, Lane #3 vs. #1, right panel). Importantly, PSA expression levels were greatly elevated by 10 nM DHT in all HDAC inhibitor-pretreated cells by up to 12-fold of that in control cells without HDAC inhibitor pretreatment (Figure 3a, Lane #4 vs. #2; Figure 3b–e, Lane #6 vs. #3 right panel). However, the growth stimulation by DHT was only marginally increased in those HDAC inhibitors-pretreated cells comparing with control cells and only significantly increased in MS-275 and AR42 pretreated cells (Figure 3a, Column #4 vs. #3; Figure 3b–e, Column #5, 6 vs. #4, left panel). The data collectively indicate that HDAC inhibitors exhibit the significant efficacy of growth suppression and can enhance the androgen responsiveness of PSA levels. Nevertheless, the effect of HDAC inhibitors pretreatment on DHT-stimulated cell growth requires further investigation.
Since the expression of cPAcP correlates with growth suppression by HDAC inhibitors, we investigated whether cPAcP protein level can serve as a useful biomarker of identifying the PCa patient sub-population who is potentially responsive to HDAC inhibitors treatment. To mimic the clinical situation of chemotherapy under androgen ablation conditions, cell growth suppression was determined in steroid-reduced (SR) conditions. Six AR/cPAcP-positive PCa cell lines and four AR/cPAcP-negative PCa cell lines including NE 1–3 and NE 1–8 cells, two neuroendocrine prostate cancer cell lines, were examined. After 2 days of VPA treatment, all PCa cells were maintained in SR medium for 72 h. Interestingly, the growth of those AR/cPAcP-positive PCa cells were in general significantly decreased by VPA treatment with more than 50% suppression; while the growth of AR/cPAcP-negative PCa cells was suppressed by less than 50% (Figure 4). The data indicate that cPAcP can potentially serve as a biomarker for HDAC inhibitor treatment with clinical benefits. Those patients can be more sensitive to HDAC inhibitor treatment, i.e., higher growth suppression and enhanced androgen responsiveness.