1,25-Dihydroxyvitamin D₃ and PI3K/AKT Inhibitors Synergistically Inhibit Growth and Induce Senescence in Prostate Cancer Cells

Abstract

BACKGROUND

1-Alpha, 25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) inhibits proliferation of multiple cancer cell types including prostate cells and upregulates p21 and/or p27, while loss of Pten and PI3K/AKT activation stimulates survival and down regulates p21 and p27. We hypothesized that inhibition of the PI3K/AKT pathway synergizes with the antiproliferative signaling of 1,25(OH)₂D₃.

METHODS

Viability, cell cycle and senescence of cells were evaluated upon combinational treatment with 1,25(OH)₂D₃ and pharmacological PI3K/AKT inhibitors.

RESULTS

Pharmacological inhibitors of PI3K or Akt and 1,25(OH)₂D₃ synergistically inhibited growth of DU145, LNCaP, primary human prostate cancer cell strains and Pten null mouse prostatic epithelial cells (MPEC). The inhibitors used included API-2 (Triciribine) and GSK690693 which are currently in clinical trials for treatment of cancer. A novel mechanism for antiproliferative effects of 1,25(OH)₂D₃ in prostate cells, induction of senescence, was discovered. Combination of 1,25(OH)₂D₃ and AKT inhibitor cooperated to induce G₁ arrest, senescence, and p21 levels in prostate cancer cells. As AKT is commonly activated by PTEN loss, we evaluated the role of Pten in responsiveness to 1,25(OH)₂D₃ using shRNA knockdown and by in vitro knockout of Pten. MPEC that lost Pten expression remained sensitive to the antiproliferative action of 1,25(OH)₂D₃, and showed higher degree of synergism between AKT inhibitor and 1,25(OH)₂D₃ compared to Pten-expressing counterparts.

CONCLUSIONS

These findings provide the rationale for the development of therapies utilizing 1,25(OH)₂D₃ or its analogs combined with inhibition of PI3K/AKT for the treatment of prostate cancer.

INTRODUCTION

Prostate cancer is the most commonly diagnosed cancer and the second most common cause of cancer death in American men [1]. Vitamin D deficiency has been associated with the development of clinical prostate cancer [2,3].

Vitamin D is a hormone that can be obtained from the diet or produced endogenously by a series of reactions that culminate in the most active metabolite of vitamin D, 1α,25(OH)₂-vitamin D₃ (1,25(OH)₂D₃). 1,25(OH)₂D₃ elicits antiproliferative effects in a variety of cancer cell types including cell lines derived from prostate [4,5]. The ability of 1,25(OH)₂D₃ to inhibit prostate growth was also demonstrated in primary prostatic cells from histologically normal, benign prostatic hyperplasia, and prostate cancer specimens [6], in multiple prostate cancer cell lines [7–9], in xenograft models of prostate cancer [10,11] as well as in Dunning rat prostate model [12]. The anticancer mechanisms of 1,25(OH)₂D₃’s action include induction of cell cycle arrest, promotion of differentiation, inhibition of proliferation and angiogenesis, as well as inhibition of invasive and migratory potential of cancer cells (reviewed in Ref. [13]).

Classical actions of 1,25(OH)₂D₃ are mediated through the vitamin D receptor (VDR) which is a member of a superfamily of nuclear steroid hormone receptors. Upon 1,25(OH)₂D₃ binding VDR translocates to the nucleus, dimerizes with retinoid X receptor (RXR) and modulates the expression of target genes. Although a number of 1,25(OH)₂D₃ responsive genes are known, the exact mechanism of growth regulation by 1,25(OH)₂D₃ is not completely defined; however, an increase in p21 and/or p27 is an almost universal feature [4].

One of the central contributing factors which facilitates the survival of prostate cancer cells is attributed to the phosphoinositol-3 kinase PI3K-AKT pathway. Activated AKT phosphorylates a host of proteins that affect cell growth, cell cycle entry, and cell survival. The AKT pathway presents an attractive target for anticancer therapies and several AKT inhibitors have been developed that demonstrate anticancer activity in preclinical and clinical studies [14]. In prostate cancer, activation of AKT occurs most frequently due to the loss of tumor suppressor phosphatase and a tensin homologue deleted in chromosome ten (PTEN) [15–17]. Loss of PTEN protein occurs in 20% of primary prostate tumors and this loss is highly correlated with advanced tumor grade and stage with 50% of metastatic tumors exhibiting a loss of PTEN protein [18]. Moreover, loss of heterozygosity (LOH) is found in 20–60% of metastatic tumors [19]. Data suggest that advancing disease is associated with a progressive loss of PTEN or an accumulation of mutations in the PTEN gene. Loss of PTEN and activation of AKT has been shown to downregulate the expression of p21 and p27 by a number of mechanisms [20–24]. Since the antiproliferative effects of 1,25(OH)₂D₃ involve upregulation of p21 and/or p27 [4] while activation of PI3K/AKT downregulates their expression [20–24], we hypothesized that pharmacological inhibitors of AKT will cooperate with the antiproliferative actions of 1,25(OH)₂D₃ in prostate cancer cells.

Our results demonstrate that inhibition of PI3K or AKT synergized with 1,25(OH)₂D₃ to inhibit the growth of human prostate cancer cell lines and primary human prostate cancer strains, and led to the cooperative induction of G₁ arrest and senescence. Responsiveness to the antiproliferative effects of 1,25(OH)₂D₃ was not lost upon reduction of Pten expression or its deletion. We observed a higher susceptibility to synergism between 1,25(OH)₂D₃ and AKT inhibitor in MPECs with lost Pten expression compared to the cells expressing Pten. These findings provide the rationale for prostate cancer therapies involving use of AKT inhibitors and 1,25(OH)₂D₃ in adjunctive therapy.

MATERIALS AND METHODS

Materials

1,25(OH)₂D₃ (Biomol, Plymouth Meeting, PA) was reconstituted in 100% ethanol and stored at −80°C. LY294002 (Sigma–Aldrich Co., St Louis, MO), GSK690693 [25] (a generous gift from GlaxoSmithKline, Collegeville, PA) and API-2 [26] (Calbiochem, La Jolla, CA) were reconstituted in DMSO and stored at −20°C.

shRNA Infection

WFU3 MPEC [27] were infected with lentivirus expressing shRNA targeting Pten (gaa cct gat cat tat aga tat t) or control shRNA (gggc cat ggc acg tac ggc aag). Lentivirus production and infection procedure were previously described [28]. MPEC were clonally selected using serial dilution as described [29] and Pten status was confirmed by Immunoblot.

MPECs With Acute Deletion of Pten

Prostate-specific Pten-knockout mice were generated by crossing Pten^loxP/loxP mice [30] with mice of the ARR2Probasin-cre transgenic line PB-cre4, wherein the Cre recombinase is under the control of a modified rat prostate-specific probasin promoter, as previously reported [31]. Pten^lox/lox anterior mouse prostatic epithelial cells (MPECs) were isolated from 8-weekold Pten^{lox/lox; pbCre-} animals as described [27] and infected with self-deleting Cre-recombinase lentivirus (Pten^−/−) [32]. Deletion was validated by PCR and Immunoblot.

Tissue Culture

LNCaP and DU145 cells (both from American Type Culture Collection, Manassas, VA) were grown in RPMI-1640 supplemented with 10% FBS and 1% penicillin–streptomycin. MPEC were grown as described previously [27]. Human prostate epithelial cancer cell strain WFU273Ca was isolated from fresh human prostate (prostate cancer, Gleason grade 6) validated for histological origin and maintained as previously described [33]. Acquisition of the human specimen from radical prostatectomies was performed at Wake Forest University School of Medicine in compliance with Institutional Research Board approval. Briefly, a small piece of tissue was removed and minced. The tissue was digested with collagenase overnight. To remove the collagenase and the majority of the stromal cells, the tissue was rinsed and centrifuged. The tissue was inoculated into a tissue culture dish coated with collagen type I (Collagen Corporation, Palo Alto, CA) and grown in medium PFMR-4A [34] supplemented with growth factors and hormones as described [33]. The histology of each specimen was verified by inking and fixing the prostate after dissection and serially sectioning the marked area as well as the sections immediately adjacent to the area of the dissection. The cells that grew out from the tissue were aliquoted and stored in liquid nitrogen. The frozen aliquots were thawed to produce secondary cultures, which were grown in medium MCDB 105 (Sigma) supplemented with growth factors and hormones as described [33].

Growth Assays for Synergism Determination

Cells were plated at 10⁴ cells per 35mm dish in triplicate. To determine synergism cells were treated with increasing doses of AKT inhibitor, increasing doses of 1,25(OH)₂D₃ or multiple combinations of AKT inhibitor and 1,25(OH)₂D₃. Briefly, 48 hr after plating the cell growth medium was replaced with 1 ml of experimental medium containing twice (2×) the indicated concentration of a PI3K/AKT inhibitor or vehicle (DMSO, 1× = 0.1%, v/v). One hour later 1 ml of medium containing twice the final concentration of 1,25(OH)₂D₃ or vehicle (ethanol, 1× = 0.1%, v/v) was added to each dish. An AKT inhibitor was applied 1 hr prior to the 1,25(OH)₂D₃ treatment as API-2 was shown to reduce phosphorylation of AKT and phosphorylation of AKT’s downstream targets (Bad, AFX, and GSK-3β) as early as at 1 hr post-treatment [26]. In addition, our preliminary tests demonstrated that application of AKT inhibitor 1 hr prior to 1,25(OH)₂D₃ treatment demonstrated moderately stronger synergism of growth inhibition compared to 0, 4, 8, and 24 hr between the treatments (data not shown).

DU145, LNCaP cells and MPEC remained in the experimental medium until the vehicle control cells reached 80–90% confluence, typically 5–7 days. For WFU273Ca and for all experiments using GSK690693 the experimental medium was replaced every 48 hr. Viable cells were counted with a hemacytometer after trypan blue exclusion. Results of representative experiments are shown.

Flow Cytometry

Flow cytometry was performed as described [5] using Becton Dickinson FACSCaliber and analyzed by the Cell Quest Pro v.6.0 program (Becton Dickinson, Mansfiled, MA). Data were processed with ModFit LT v.2.0 software (Verity Software House, Topsham, MN). Each treatment was performed in triplicate and the experiment was conducted three times.

Senescence-Associated (SA)-β-Galactosidase (gal) Activity

Cells were cultured and treated as described above. After 5–7 days of treatment (SA)-β-galactosidase activity was evaluated by the method of Dimri et al. [35]. For positive control SA-β-gal activity was evaluated in cells treated with 100 nM Doxorubicin; for negative control SA-β-gal activity was evaluated in cells treated with 100 nM Doxorubicin at pH 7 which inhibits SA-β-gal activity. Digital images were taken from 10 random areas at 20× magnification. Digital images were evaluated in Photoshop CS2 9.0.2 (Adobe Systems, San Jose, CA). The number of SA-β-gal positive cells was counted in each image and presented as percent of total cell number ± SE.

Immunoblot

Cells were collected from monolayer by light trypsinization and cell pellets were resuspended in lysis buffer (20 mM HEPES, 10 mM NaCl, 3 mM MgCl₂, 0.1% NP40, 10% glycerol, 0.2 mM EDTA) containing freshly added 1 mM DTT and 0.4 mM PMSF. Tubes with cells were left on ice for 15 min. Fifty micrograms of protein were subjected to electrophoresis on SDS–PAGE gels. Proteins were transferred onto prewetted Hybond-P PVDF (polyvinylidene diflouride) membrane according to manufactures’ protocol (Amersham Pharmacia Biotech, Buckinghamshire, UK). Membranes were incubated with appropriate dilutions of Pten (sc-7974) (Santa Cruz Biotechnology, Santa Cruz, CA), p21 (556430) and p27 (610241) antibodies (BD Pharmigen, San Diego, CA). Secondary antibodies conjugated to horse radish peroxidase (Santa Cruz Biotechnology) were used. ECL Plus Western Blotting Detection System kit (Amersham-Pharmacia) was used for detection of proteins. Images were analyzed by ImageQuant TL v. 7.0 (GE Healthcare, Piscataway, NJ).

Detection of Phosphorylated Proteins

Cells were grown to approximately 80% confluency, medium was replaced with fresh medium, and in 24 hr protein lysates were prepared. Cells were washed with cold PBS and lysis buffer (1%NP-40, 50 mM Tris–HCL, 150 mM NaCl, 5 mM EDTA with freshly added protease inhibitor cocktail, 100 mM DTT, 100 mM Na₃VO₄ and 500 mM NaF) was applied. Cells were left on ice for 20 min, collected, and centrifuged. Supernatants were stored at −80°C. Thirty to 50 µg of protein were subjected to electrophoresis on SDS–PAGE gels. Proteins were transferred onto Nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s protocols. Membranes were incubated with appropriate dilutions of pSer473 AKT (#4051), pThr308 AKT (#2965), AKT (#9272) antibodies (Cell Signaling Inc., Beverly, MA). Secondary antibodies conjugated to horse radish peroxidase (Santa Cruz Biotechnology) were used. Protein expression levels were detected using ECL Plus Western Blotting Detection System kit (Amersham-Pharmacia). Images were analyzed by ImageQuant TL v. 7.0 (GE Healthcare, Piscataway, NJ).

Statistical Analyses

Synergism was assessed with CalcuSyn software (Biosoft, Ferguson, MO) as previously described [5]. Briefly, the dose effect for each drug alone was determined based on the experimental observations using the median effect principle: the combination index (CI) for each combination was calculated according to the following equation:

$$\text{CI}=\left[\frac{{(\mathrm{D})}_1}{{({\mathrm{D}}_{\mathrm{x}})}_1}\right]+\left[\frac{{(\mathrm{D})}_2}{{{\mathrm{D}}_{\mathrm{x}})}_2}\right]+\left[\frac{{(\mathrm{D})}_1{(\mathrm{D})}_2}{{({\mathrm{D}}_{\mathrm{x}})}_1{({\mathrm{D}}_{\mathrm{x}})}_2}\right]$$

where (D)₁ and (D)₂ are the doses of drug that have × effect when used in combination and (D_x)₁ and (D_x)₂ are the doses of drug 1 and drug 2 that have the same x effect when used alone. CI = 1 represents the conservation isobologram and indicates additive effects. CI values < 1 indicate a more than expected additive effect (synergism). Dose Reduction Index (DRI) for each drug and dose was calculated using the equation:

$${(\text{DRI})}_1=\frac{{({\mathrm{D}}_{\mathrm{x}})}_1}{{\mathrm{D}}_1}\text{and}{(\text{DRI})}_2=\frac{{({\mathrm{D}}_{\mathrm{x}})}_2}{{\mathrm{D}}_2}$$

Statistical analyses for synergism experiments were performed using the statistical software package NCSS 2002 (Number Cruncher Statistical Systems, Kaysville, UT). Differences in growth data were determined by two-way ANOVA controlling for 1,25(OH)₂D₃ or PI3K/AKT inhibitor dose with post hoc analysis by Fisher’s test. Cell cycle distribution and senescence analyses were performed using two-way ANOVA with post hoc analysis by Fisher’s LSD test. In all cases, P ≤ 0.05 was considered significant. The effect of 1,25(OH)₂D₃-mediated growth inhibition in MPECs with Pten shRNA and scrambled control or Pten^−/− and Pten^lox/lox was evaluated using two-way ANOVA while adjusting for the within-clone correlation using a random effect in a corresponding mixed-effects model. Data were analyzed in PROC MIXED in SAS v9.2.

RESULTS

1,25(OH)₂D₃ and PI3K/Akt Inhibitors Synergistically Inhibit Prostate Cancer Cell Growth

To test whether inhibition of the PI3K/Akt pathway synergizes with 1,25(OH)₂D₃ treatment to inhibit the growth of prostatic cells we first utilized a pharmacological inhibitor of PI3K/AKT, LY294002 [36]. Cells were treated with increasing doses of 1,25(OH)₂D₃ or LY294002 or with multiple combinations of the two compounds and the number of viable cells was assessed. This was followed by assessment of synergism using the Chou–Talalay method [37] in which the combination index (CI) values < 1 indicates presence of synergism.

We found that LNCaP cells, which lack functional PTEN and are moderately sensitive to growth inhibition by 1,25(OH)₂D₃, demonstrated statistically significant synergism between 1,25(OH)₂D₃ and LY294002 at lower doses of 1,25(OH)₂D₃ (Fig. 1A and Table I). The combination of 1 nM 1,25(OH)₂D₃ treatment with 0.2, 1, or 5 µM LY294002 demonstrated statistically significant synergism as determined by CI values from isobologram analysis (Table I). The dose-reduction index (DRI) for each of the drugs is also depicted in Table I and presents a predictive measure of how much the dose of each drug in a synergistic combination may be reduced at a given effect level compared with the doses of each drug alone [37].

Fig. 1.

LY294002 and 1,25(OH)₂D₃ synergistically inhibits growth of LNCaP and DU145 cells. Growth inhibition of LNCaP (A) and DU145 (B) in response to LY294002 and 1,25(OH)₂D₃ alone or in combination. Cells were grown, treated and analyzed as described in Materials and Methods Section. Each point represents the mean and standard deviation of triplicate plates after normalization of cell number to controls (0.1% ethanol).

TABLE I.

Synergism Between 1,25(OH)₂D₃ and LY294002 in LNCaP and DU145 Cells^*

1,25(OH)2D3 (nM)	LY294002 (µM)	CIa	DRIb		Synergism
			1,25(OH)2D3	LY294002
LNCaP
1	0.2	0.171	8.2	20.7	Strong
1	1	0.298	10.9	4.8	Strong
1	0.5	0.675	26.3	1.6	Moderate
DU145
1	2.5	0.408	513.8	2.5	Intermediate
1	5	0.650	666.0	1.5	Intermediate
10	2.5	0.382	58.2	2.8	Intermediate
10	5	0.654	67.7	1.6	Intermediate
10	10	0.801	117.6	1.3	Moderate
100	2.5	0.489	6.4	3.0	Intermediate
100	5	0.585	9.5	2.1	Intermediate
100	10	0.701	14.9	1.6	Intermediate

^aCombination index (CI < 0.1 indicates very strong synergism; 0.1–0.3 strong synergism; 0.3–0.7 synergism; 0.70–0.85 moderate synergism; 0.85–0.90 slight synergism; 0.90–1.10 nearly additive synergism).

^bDose-reduction index. DRI is calculated using the equation: (DRI)₁ = (D_x)₁/D₁ and (DRI)₂ = (D_x)₂/D₂, where (D)₁ and (D)₂ are the doses of drug that have x effect when used in combination and (D_x)₁ and (D_x)₂ are the doses of drug 1 and drug 2 that have the same x effect when used alone.

^*Data are from assays depicted in Figure 1, only statistically significantly different means by ANOVA are depicted.

DU145 cells (wild-type PTEN) are known to be resistant to the antiproliferative effects of 1,25(OH)₂D₃ [38]. Consistent with this, we found that 1,25(OH)₂D₃ did not significantly affect growth of DU145 cells (Fig. 1B). While 100 nM 1,25(OH)₂D₃ did not inhibit growth of DU145 cells, pretreatment of DU145 cells with 5 µM LY294002 sensitized the cells to 1,25(OH)₂D₃ treatment leading to a reduction in the number of viable cells to 40% compared to treatment with LY294002 alone (Fig. 1B). Significant synergism was observed across multiple doses of both compounds (Table I).

As LY294002 has recently been reported to have many other off-target effects [39], we sought to validate synergism with AKT-inhibition specifically. To do so we used two different AKT inhibitors which currently are in clinical trials for cancer treatment: API-2 (Triciribine), which selectively inhibits Akt1/2/3 without inhibiting PI3K or PDK [26] and GSK690693, an ATP competitive AKT inhibitor [25].

The combination of 1,25(OH)₂D₃ with API-2 elicited synergistic growth inhibition of DU145 cells (Fig. 2A and Table II). DU145 cells treated with the combination of API-2 and 1,25(OH)₂D₃ demonstrated strong synergism at all doses tested as determined by CI values from isolobolgrams (Table II). Noteworthy, the treatment with API-2 and 1,25(OH)₂D₃ was applied only once after which the cells were allowed to grow until the cells in either of the treatment groups were 80–90% confluent.

Fig. 2.

AKT inhibitors API-2 and GSK690693 synergize with 1,25(OH)₂D₃ to inhibit growth of DU145 cells and human primary prostate cancer strain. Growth inhibition of DU145 cells (A) and human primary prostate cancer cell strain WFU273 Ca (B) in response to API-2 and 1,25(OH)₂D₃ alone or in combination. Growth inhibition of DU145 cells in response to GSK690693 and 1,25(OH)₂D₃ alone or in combination (C). Insert demonstrates PTEN and phospho-AKT levels in WFU273Ca cell strain as determined by Immunoblot. Growth inhibition of human primary prostate cancer cell strain WFU273Ca in response to GSK690693 and 1,25(OH)₂D₃ alone or in combination (D). Cells were grown, treated and analyzed as described in Materials and Methods Section.

TABLE II.

Synergism Between 1,25(OH)₂D₃ and AKT Inhibitors^*

1,25(OH)2D3 (nM)	AKT inhibitor (nM)	CIa	DRIb		Synergism
			1,25(OH)2D3	AKT inhibitor
DU145 (AKT inhibitor: API-2)
0.1	50	0.18 × 10−6	5.5 × 104	4.0 × 106	Very strong
0.1	100	0.12 × 10−6	8.7 × 104	4.4 × 106	Very strong
0.1	500	7.98 × 10−6	1.3 × 105	1.9 × 106	Very strong
0.1	1,000	9.82 × 10−6	1.2 × 105	7.4 × 105	Very strong
1	50	0.75 × 10−6	1.3 × 104	1.8 × 107	Very strong
1	100	0.55 × 10−6	1.8 × 104	1.6 × 107	Very strong
1	500	0.60 × 10−6	1.7 × 104	2.7 × 106	Very strong
1	1,000	0.54 × 10−6	1.9 × 104	1.6 × 106	Very strong
10	50	0.001	872,1	8.8 × 106	Very strong
10	100	0.001	1014.8	5.7 × 106	Very strong
10	500	0.001	1950.4	3.5 × 106	Very strong
10	1,000	0.001	1461.5	1.1 × 106	Very strong
100	50	0.007	135.6	1.9 × 107	Very strong
100	100	0.005	206.7	1.9 × 107	Very strong
100	500	0.006	180.3	3.1 × 106	Very strong
100	1,000	0.005	193.0	1.7 × 106	Very strong
WFU275CA (AKT inhibitor: API-2)
0.1	0.1	0.799	283.0	1.3	Moderate
1	0.05	0.720	12.2	1.6	Moderate
1	0.1	0.714	36.9	1.5	Moderate
10	0.025	0.216	9.0	9.6	Strong
10	0.05	0.169	22.5	8.0	Strong
10	0.1	0.176	52.0	6.4	Strong
DU145 (AKT inhibitor: GSK690693)
10	1	0.003	1.1 × 106	391.3	Very strong
10	10	0.070	6.5 × 104	14.3	Very strong
100	0.1	0.0001	2.3 × 104	2241.4	Very strong
100	1	0.004	3.9 × 104	268.7	Very strong
100	10	0.010	1.5 × 106	96.9	Very strong
100	100	0.061	6.6 × 106	16.3	Very strong
100	1,000	0.281	6.0 × 107	3.5	Strong
WFU273CA (AKT inhibitor: GSK690693)
0.1	1,000	0.773	5158.7	1.3	Moderate
1	10	0.062	37.1	28.5	Very strong
1	100	0.120	255.9	8.7	Strong
1	1,000	0.747	550.1	1.3	Moderate
10	10	0.115	10.5	51.7	Strong
10	100	0.234	14.1	6.1	Strong
10	1,000	0.344	215.4	3.0	Intermediate

^aCombination index (CI < 0.1 indicates very strong synergism; 0.1–0.3 strong synergism; 0.3–0.7 synergism; 0.70–0.85 moderate synergism; 0.85–0.90 slight synergism; 0.90–1.10 nearly additive synergism).

^bDose-reduction index. DRI is calculated using the equation: (DRI)₁ = (D_x)₁/D₁ and (DRI)₂ = (D_x)₂/D₂, where (D)₁ and (D)₂ are the doses of drug that have x effect when used in combination and (D_x)₁ and (D_x)₂ are the doses of drug 1 and drug 2 that have the same x effect when used alone. DRI values for 1,25(OH)₂D₃ treatment in DU145 are high due to inability of the program to correctly calculate DRI based on the formula as the x effect of the 1,25(OH)₂D₃ alone is near zero.

^*Data are from assays depicted in Figure 2, only statistically significantly different means by ANOVA are depicted.

Next we tested for the presence of synergism in primary human prostate cancer epithelial cells. WFU273Ca human primary epithelial cell strain was isolated from fresh human prostate with histologically confirmed prostate cancer of Gleason grade 6. The strain was shown to be PTEN-positive by Immunoblot, while still demonstrating high levels of activated phosphorylated AKT (Fig. 2B, insert). The combination of API-2 and 1,25(OH)₂D₃ applied to WFU273Ca strain also demonstrated statistically significant synergism (Fig. 2B and Table II), with DRI values as high as 15.3 for 1,25(OH)₂D₃ and 14.6 for API-2.

Even though both cell types, the WFU273Ca cell strain and DU145 cell line, are PTEN-positive, WFU273Ca shows activation of AKT pathway as demonstrated by phospho-AKT levels (Fig. 2B, insert) while DU145 consistently demonstrated undetectable levels of pAKT (data not shown). The mechanistic basis for activated AKT in WFU273Ca is not known. The higher sensitivity of the WFU273Ca cell line to growth inhibition by AKT inhibitor API-2 might be explained by possible dependency of WFU273Ca on AKT activation for proliferation of the cells.

The combination of GSK690693 with 1,25(OH)₂D₃ also elicited strong to very strong synergistic growth inhibition in DU145 cells (Fig. 2C and Table II), as well as moderate to very strong synergism in human primary prostatic epithelial cells (Fig. 2D and Table II).

1,25(OH)₂D₃ and PI3K/Akt Inhibitors Cooperate to Inhibit Cell Cycle Progression and Induce Senescence

Having demonstrated synergism between 1,25(OH)₂D₃ and AKT inhibitors we sought to explore the mechanism. First, we performed video time-lapse microscopy analysis of DU145 cells treated with API-2 (0.25 µM) or 1,25(OH)₂D₃ (100 nM) alone as well as in combination of the two. We did not observe any considerable apoptosis in either of the treatments (data not shown). Next we evaluated cell cycle distribution of DU145 cells treated with API-2 or 1,25(OH)₂D₃. Cell cycle analysis demonstrated a small but statistically significant increase in G₁-arrested cells treated with the combination of API-2 at 0.25 µM with 1,25(OH)₂D₃ at 10 nM compared to either agent alone (Fig. 3).

Fig. 3.

AKT inhibitor API-2 and 1,25(OH)₂D₃ cooperate to induce G1-arrest in DU145 cells. DU145 cells were treated with the indicated doses for 24 hr and evaluated for cell cycle distribution as described in the Materials and Methods Section. Insert demonstrates the G1/S ratios. Values are means for the triplicates ± SD. Means without a common letter are significantly different by ANOVA (P < 0.05).

When we assessed the effects of drug combination on senescence in the human primary prostate cancer cell strain WFU273Ca, we found that 1,25(OH)₂D₃ was able to induce SA-β-Gal activity which was associated with morphological features consistent with senescence (Fig. 4A, B). Interestingly, inhibition of the AKT pathway with API-2 alone induced senescence in WFU273Ca cells with 0.5 µM API-2 causing 30% of the cells to undergo senescence, suggesting a role for activated AKT in the prevention of senescence in these cells (Fig. 4C). The combination of API-2 and 1,25(OH)₂D₃ cooperated to induce greater SA-β-Gal activity which was statistically significant by ANOVA (Fig. 4D). Treatment with 0.1 µM API-2 induced senescence in 6.5% of WFU273Ca cells and 10 nM 1,25(OH)₂D₃ induced senescence in 7.3% of the cells; while the combination of the two led to 24.0% of the cells to undergo senescence (Fig. 4D). DU145 cells also showed cooperativity for induction of SA-β-gal (Fig. 4E).

Fig. 4.

AKT inhibitor API-2 and 1,25(OH)₂D₃ cooperate to induce G1 senescence and p21 levels. A: Representative photographs of 1,25(OH)₂D₃ and API-2 inducing (SA)-β-galactosidase activity in a cooperative manner in WFU273Ca cells. B–D:Quantitative data from multiple images of WFU273Ca cells treated as indicated. Analysis was performed as described in the Materials and Methods Section. Means ± SE are shown. Means without a common letter are significantly different by ANOVA (P < 0.05). E: Induction of senescence by 1,25(OH)₂D₃ alone or in presence of 0.25 µM API-2 in DU145 cells. Quantitative data from ten images of DU145 cells treated as indicated. Cells were grown, treated and (SA)-β-galactosidase activity was evaluated as described in Materials and Methods Section. Means ± SE are shown. Means without a common letter are significantly different by ANOVA (P < 0.05). F: API-2 and 1,25(OH)₂D₃ cooperatively induce p21 and p27 protein levels in DU145 cells. DU145 cells were treated with vehicle, 0.25µM API-2 or100 nM 1,25(OH)₂D₃ or the combination of the two and in 24 hr protein lysates were collected and Immunoblot was carried out as described in Materials and Methods Section.

As the antiproliferative effects of 1,25(OH)₂D₃ commonly involve upregulation of p21^Cip1 and/or p27^Kip1 [4] we sought to test the effect of AKT inhibition and 1,25(OH)₂D₃ treatment on protein levels of p21^Cip1 and p27^Kip1 in DU145. The result demonstrated that 24 hr of treatment with API-2 and 1,25(OH)₂D₃ cooperated to increase p21^Cip1 protein (Fig. 4F). While 0.25 µM API-2 treatment increased p21 by 3.7 fold and 100 nM 1,25(OH)₂D₃ did not have a significant effect, the combination of the two led to an 8.3-fold increase in the p21^Cip1 protein level compared to the base level. Although API-2 treatment modestly increased p27 levels, 1,25(OH)₂D₃, alone or in combination with API-2 did not demonstrate a significant effect on p27 protein levels.

Together these data suggest that synergism between API-2 and 1,25(OH)₂D₃ in prostate cancer cells might be a result of cooperative induction of cell cycle arrest and senescence possibly through induction of p21^Cip1 levels.

The Role of Pten Status in Sensitivity to Anti proliferative Action of 1,25(OH)₂D₃

Activation of AKT in prostate cancer occurs most frequently due to the loss of the tumor suppressor phosphatase and a tensin homologue deleted in chromosome ten (PTEN), which is found in 20% of primary tumors and more than 50% of metastatic tumors [18]. Thus, we wanted to evaluate the role of Pten in the antiproliferative signaling of 1,25(OH)₂D₃. To test this we generated MPEC in which Pten expression was suppressed using an shRNA approach. MPEC WFU3 [27] were infected with either lentivirus expressing scrambled shRNA or with lentivirus expressing shRNA targeting Pten. Single cell clones of each of the cell types were established and Pten status was verified by Immunoblot (Fig. 5B). All clones with Pten expression knocked down to undetectable levels were selected and activation of Akt pathway was confirmed by assessment of Akt phosphorylation. Clones infected with Pten shRNA activated Akt pathways as phospho-Ser473 and phospho-Thr308 Akt levels were increased compared to the Control clones (Fig. 5B).

Fig. 5.

In vitro suppression or loss of Pten is not sufficient to antagonize 1,25(OH)₂D₃-mediated growth suppression. A: Growth inhibition of WFU3MPECs infected with Pten shRNA or scrambled shRNA in response to 1,25(OH)₂D₃. Values are means for thetriplicates ± SD. B: Pten and phospho-Akt levels of WFU3MPEC clones infected with lentivirus expressing scrambled shRNA (control clones) or shRNA targeting Pten (Pten shRNA clones) as determined by Immunoblot. C: Growth inhibition of Pten^lox/lox and Pten^−/− MPECs in response to 1,25(OH)₂D₃. Pten^lox/lox MPECs isolated from prostates of Pten^lox/lox Cre-recombinase negative animals and infected with lentivirus expressing self-deleting Cre-recombinase to generate Pten^−/− MPEC. Values are means for the triplicates ±SD. D: Pten and phospho-Akt levels of Pten^lox/lox and Pten^−/− MPECs determined by Immunoblot. Protein lysate isolated from LNCaP cells was used as controls.

WFU3 control MPEC clones and WFU3 MPEC clones with Pten knocked down were treated with increasing doses of 1,25(OH)₂D₃ (Fig. 5A). Both cell types exhibited robust growth suppression by 1,25(OH)₂D₃ in a dose-dependant manner. There was a trend towards increased sensitivity of Pten shRNA clones to 1,25(OH)₂D₃-mediated growth inhibition at 10 nM dose (P = 0.041) but not at 100 nM (P = 0.655) as shown by two-way ANOVA. The overall test for any differences (across ethanol, 10, 100 nM) in Control and Pten shRNA clones was not significant (P = 0.089). These findings demonstrate that loss of Pten expression and activation of Akt pathway does not reduce responsiveness of the cells to the antiproliferative effects of 1,25(OH)₂D₃ and suggests a possible increase in the sensitivity of the cells with lost Pten to the antiproliferative actions of 1,25(OH)₂D₃.

Since gene suppression using an shRNA approach has the potential disadvantages of incomplete suppression of target gene expression and also possible off-target effects, we sought to establish a cell line with acute in vitro deletion of Pten. Thus, we isolated Pten^lox/lox MPEC from prostates of Pten^lox/lox Cre-recombinase negative animals and infected them with lentivirus expressing self-deleting Cre-recombinase [32]. PCR analysis (data not shown) and Immunoblot analysis (Fig. 5D) demonstrated the absence of Pten expression in the cells infected with Cre-recombinase. Deletion of Pten led to 5.6-fold increase in phosphor-Ser473 Akt level confirming activation of the Akt pathway (Fig. 5D). The control Pten^lox/lox MPEC and Pten^−/− (Pten^lox/lox infected with Cre-recombinase lentivirus) cells were treated with increasing concentrations of 1,25(OH)₂D₃. We observed that in vitro loss of Pten did not significantly affect the ability of 1,25(OH)₂D₃ to inhibit the growth of the cells (Fig. 5C).

Pten^lox/lox MPEC demonstrated higher sensitivity to 1,25(OH)₂D₃ compared to WFU3 MPEC used for the shRNA knock down of Pten. This could be attributed to the difference in the genetic background of the mouse strains from which the cell lines were established. The WFU3 MPECs used for the infection with shRNA-expressing virus were isolated from prostates of B1/6; 129/SVEV mice [27], while Pten^lox/lox MPEC were isolated from mice of mixed C57BL/6 and BALB/c background [31,40].

We also evaluated the effect of Pten status on the presence of synergism between 1,25(OH)₂D₃ and AKT inhibitor API-2. WFU3 Control clone 3MPEC (the clone with the lowest levels of phosphor-Ser473 Akt) and WFU3 Pten shRNA clone 4 MPEC (the clone with the highest levels of phospho- Ser473 Akt and phosphor- Thr308 Akt) were treated with 1,25(OH)₂D₃ (0.1, 1, 10, and 100 nM) or API-2 (10, 50, 100, and 500 nM) or with multiple combinations of the two compounds. Out of all dose combinations tested the control clone MPEC showed “intermediate” synergism at 10 nM 1,25(OH)₂D₃ combined with 50 or 100 nM API-2 (Fig. 6A and Table III). On the other hand, MPEC with Pten knock down showed “very strong” to “strong” synergism throughout all tested dose combinations of 1,25(OH)₂D₃ and API-2 as determined by the CI values (Fig. 6B and Table III). A similar trend was observed in the MPEC with Pten deleted using Cre-recombinase. Briefly, Pten^lox/lox MPEC demonstrated only “intermediate” synergism between 1,25(OH)₂D₃ and API-2 at 10 nM 1,25(OH)₂D₃ combined with 5 and 50 nM API-2, while the Pten^−/− MPEC demonstrated “strong” to “very strong” synergism at the highest dose of API-2 tested (500 nM) combined with any dose of 1,25(OH)₂D₃ (data not shown).

Fig. 6.

AKT inhibitor API-2 synergizes with 1,25(OH)₂D₃ to inhibit growth of MPEC with reduced Pten expression. Growth inhibition of Control shRNA clone 3 MPEC (A), Pten shRNA clone 4 (B) in response to API-2 and 1,25(OH)₂D₃ alone or in combination. MPEC were established, grown, treated and analyzed as described in Materials and Methods Section.

TABLE III.

Synergism Between 1,25(OH)₂D₃ and API-2 in MPEC^*

1,25(OH)2D3 (nM)	LY294002 (µM)	CIa	DRIb		Synergism
			1,25(OH)2D3	API-2
Control shRNA cl. 3 MPEC
10	50	0.370	4.7	6.3	Intermediate
10	100	0.334	13.8	5.5	Intermediate
Pten shRNA cl. 4 MPEC
0.1	500	0.018	57.2	9354.0	Very strong
1	50	0.112	8.9	4121.8	Strong
1	100	0.099	10.1	2257.8	Very strong
1	500	0.013	81.9	2122.8	Very strong
10	10	0.217	4.6	6.9 × 104	Strong
10	50	0.134	7.5	2.0 × 104	Strong
10	100	0.095	10.5	1.3 × 104	Very strong
10	500	0.013	78.8	1.1 × 104	Very strong
100	10	0.246	4.1	3.5 × 105	Strong
100	50	0.205	4.9	8.0 × 104	Strong
100	100	0.177	5.6	4.4 × 104	Strong
100	500	0.048	20.7	2.3 × 104	Very strong

^aCombination index (CI < 0.1 indicates very strong synergism; 0.1–0.3 strong synergism; 0.3–0.7 synergism; 0.70–0.85 moderate synergism; 0.85–0.90 slight synergism; 0.90–1.10 nearly additive synergism).

^bDose-response index. DRI is calculated using the equation: (DRI)₁ = (D_x)₁/D₁ and (DRI)₂ = (D_x)₂/D₂, where (D)₁ and (D)₂ are the doses of drug that have x effect when used in combination and (D_x)₁ and (D_x)₂ are the doses of drug 1 and drug 2 that have the same x effect when used alone.

^*Data are from assays depicted in Figure 6, only statistically significantly different means by ANOVA are depicted.

Together, these data demonstrate that suppression or loss of Pten in MPECs was not associated with increased resistance to growth-inhibitory qualities of 1,25(OH)₂D₃ in MPEC. In addition, our data suggests that loss of Pten might strengthen the synergistic effect between 1,25(OH)₂D₃ and AKT inhibition on cellular growth inhibition.

DISCUSSION

In this study we showed that AKT inhibitors in combination with 1,25(OH)₂D₃ synergistically inhibit the growth of prostate cancer cells. The effect was observed with multiple inhibitors of PI3K and/or AKT in MPEC with Pten knock down, prostate cancer cell lines, as well as primary human prostate cancer sample.

These findings might be important as AKT inhibitors (including the ones tested in this study) are in clinical trials for various cancers [14,25,41], and therefore the results could have fast clinical translation. However, a factor complicating the use of AKT inhibitors is that the AKT pathway presents one of the most important pathways for normal cell survival. It is not clear yet whether treatment with AKT inhibitors will demonstrate acceptable levels of toxicity at doses that are therapeutically effective.

1,25(OH)₂D₃ used as a single agent has been shown to possess anticancer qualities in a number of cancer models but has toxicities associated with calcium mobilization at doses that are therapeutically effective [4]. One strategy to overcome this problem is the creation of less calcemic 1,25(OH)₂D₃ analogs or organization of the treatment regimen in a way that allows reduction of side-effects. Another strategy is to combine 1,25(OH)₂D₃ with other agents to develop therapeutic interventions that allow dose reduction, and thus alleviation of toxicities, while maintaining the growth inhibitory potential. Clinical trials utilizing 1,25(OH)₂D₃ or its analogs in combination with chemotherapy in advanced prostate cancer have demonstrated the feasibility of the use of 1,25(OH)₂D₃ for treatment of advanced prostate cancer [42–44]. For instance, Beer et al. [42] reported an 81% response rate for the combination of 1,25(OH)₂D₃ and docetaxel in metastatic prostate cancer versus an expected response of 40% to 50% for docetaxel alone.

In this light, synergism between 1,25(OH)₂D₃ with AKT inhibitors and DRI values demonstrated in this study suggests that more therapeutic efficacy can be achieved by combining AKT inhibitors and 1,25(OH)₂D₃ (or its analogs) potentially reducing systemic toxicities.

Mechanistically, combinational treatment with AKT inhibitor and 1,25(OH)₂D₃ showed no evidence of apoptosis, but moderate effect on cell cycle progression and larger effect on the induction of senescence was observed.

Treatment with 1,25(OH)₂D₃ alone did not have an effect on senescence in DU145 cells which is in agreement with the observation that these cells are not growth-inhibited by 1,25(OH)₂D₃. However, combined with AKT inhibitor API-2, 1,25(OH)₂D₃, increased the percentage of cells undergoing senescence.

In human primary prostate cancer cell strain treatment with 1,25(OH)₂D₃ alone was able to inhibit proliferation of the cells as well as to induce senescence as demonstrated by induction of β-galactosidase straining and senescence-associated cell morphology alterations. The ability of 1,25(OH)₂D₃ to induce senescence in prostate cells has not been demonstrated before and contributes to the understanding of the antiproliferative effects of 1,25(OH)₂D₃ in prostate cells which have not been clearly defined.

Senescent cells are described as a cell permanently arrested in the cell cycle. These cells are refractory to proliferation stimuli, exhibit altered cell morphology and gene expression while remaining viable and preserving metabolic activity (reviewed in Ref. [45]). There are multiple data demonstrating that senescence is a mechanism that limits cellular lifespan and presents a barrier for cellular immortalization and progression of tumorigenesis [46,47]. DNA damage or oncogene expression can induce cellular senescence and in order to become immortal cells have to overcome senescence by acquiring additional genetic alterations.

A process of senescence was demonstrated to have a significant role in human cancers as well. It was shown that human benign tumors contain senescent cells and that these cells disappear in their malignant counterparts [48,49]. In human prostate cancer Majumder et al. [50] demonstrated that markers of cellular senescence are elevated in PIN when compared to nondysplastic elithelial cells in the same tissue section. Chen et al. [51] showed that in specimens from early-stage human prostate cancer markers of senescence were present in areas of prostate hyperplasia/PIN and rarely in areas of carcinoma. These findings suggest a role for senescence as a barrier for progression of tumorigenesis in prostatic cells. The ability of 1,25(OH)₂D₃ to induce senescence in human primary prostate cancer cell strain demonstrated in the study supports utilization of 1,25(OH)₂D₃ for the treatment of earlier stages of human prostate cancer with the goal of prevention of disease progression. Ultimately, combination of 1,25(OH)₂D₃ with pharmacological AKT inhibitors might provide further benefits by stimulation of senescence, reduction of growth of cancer cell and blocking or slowing tumorigenesis.

It was previously demonstrated that p21 or p27 expression plays a critical role in the induction of senescence in a number of cell types [52–54] including prostate cells [50]. In addition, 1,25(OH)₂D₃ has been shown to increase the steady-state levels of p27 protein in prostate cancer cells [55]. Thus, we sought to explore the effect of AKT inhibition and 1,25(OH)₂D₃ treatment on the p21 and p27 levels. In agreement with the lack of response to antiproliferative action of 1,25(OH)₂D₃ DU145 that did not induce p21, p27 levels upon 1,25(OH)₂D₃ treatment. However, when AKT inhibitor API-2 induced p21 protein levels, the cooperative induction of p21 by the combinational treatment with API-2 and 1,25(OH)₂D₃ was observed, which correlated with sensitization of the cells to the antiproliferative effects of 1,25(OH)₂D₃ and induction of senescence. Thus, while 1,25(OH)₂D₃-sensitive cell strains undergo senescence upon 1,25(OH)₂D₃ treatment, the 1,25(OH)₂D₃-insensitive DU145 cell line required an AKT inhibitor treatment in order for 1,25(OH)₂D₃ to inhibit proliferation and induce senescence.

As activation of AKT in prostate cancer commonly occurs due to the loss of expression of functional PTEN [15–17], we evaluated the role of Pten in responsiveness to 1,25(OH)₂D₃, as well as the role of Pten in synergistic cell growth inhibition upon treatment with 1,25(OH)₂D₃ in combination with AKT inhibitors.

We demonstrated that loss of Pten in MPEC was not associated with decreased responsiveness of the cells to antiproliferative effects of 1,25(OH)₂D₃. There was a suggestion for increased sensitivity of cells with deleted Pten to lower dose of 1,25(OH)₂D₃ tested (10 nM) as shown by enhanced growth inhibition of Pten shRNA MPEC clones compared to the Control shRNA MPEC clones at that dose (P = 0.041).

More profound synergism between 1,25(OH)₂D₃ and AKT inhibitor API-2 was observed in MPEC that lost Pten expression as compared to the Pten-expressing counterparts. This finding, if confirmed by further studies, could be relevant to clinical application as cancer cells with lost Pten would be more sensitive to the growth-inhibitory effects of the combinational treatment leading to more selective growth inhibition of prostate cancer cells. However, in human prostate cancer cell lines synergism was observed in cells with functional Pten (DU145), as well as in cells lacking Pten activity (LNCaP). Thus, role of Pten status for the induction of synergism might be cell-type specific.

CONCLUSIONS

In summary, our results show that 1,25(OH)₂D₃ and AKT inhibitors synergistically inhibit prostate cancer growth through induction of cell cycle and senescence. We also have demonstrated that reduced Pten expression was not associated with decreased response to antiproliferative actions of 1,25(OH)₂D₃ suggesting that 1,25(OH)₂D₃ can be used for treatment independent of PTEN status of the tumor. These data may have implications for the clinical use of these agents in prostate cancer patients especially in patient with high risk.