Synergistic Simvastatin and Metformin Combination Chemotherapy for Osseous Metastatic Castration-Resistant Prostate Cancer

Abstract

Docetaxel (DTX) chemotherapy remains a standard-of-care for metastatic castration-resistant prostate cancer (CRPC). DTX modestly increases survival, yet results in frequent occurrence of side-effects and resistant disease. An alternate chemotherapy with greater efficacy and minimal side-effects is needed. Acquisition of metabolic aberrations promoting increased survival and metastasis in CRPC cells include constitutive activation of Akt, loss of adenosine monophosphate-activated protein kinase (AMPK) activity due to Ser-485/491 phosphorylation, and over-expression of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMG-CoAR). We report that combination of simvastatin (SIM) and metformin (MET), within pharmacological dose range (500nM to 4µM SIM and 250µM to 2mM MET), significantly and synergistically reduces C4-2B3/B4 CRPC cell viability and metastatic properties with minimal adverse effects on normal prostate epithelial cells. Combination of SIM and MET decreased Akt Ser-473 and Thr-308 phosphorylation and AMPKα Ser-485/491 phosphorylation, increased Thr-172 phosphorylation and AMPKα activity as assessed by increased Ser-79 and Ser-872 phosphorylation of acetyl-CoA carboxylase and HMG-CoAR, respectively; decreased HMG-CoAR activity, and reduced total cellular cholesterol and its synthesis in both cell lines. Studies of C4-2B4 orthotopic NCr-nu/nu mice further demonstrated that combination of SIM and MET (3.5–7.0µg/g body weight SIM and 175–350µg/g body weight MET) daily by oral gavage over 9-week period significantly inhibited primary ventral prostate tumor formation, cachexia, bone metastasis, and biochemical failure more effectively than 24µg/g body weight DTX intraperitoneally-injected every three weeks, 7.0µg/g/day SIM, or 350µg/g/day MET treatment alone, with significantly less toxicity and mortality than DTX, establishing combination SIM and MET as a promising chemotherapeutic alternative for metastatic CRPC.

INTRODUCTION

Recurrence and/or progression of castration-resistant prostate cancer (CRPC) following androgen deprivation therapy (ADT) is diagnosed by rising serum prostate specific antigen (PSA) and appearance of primarily osseous metastases (1). In the United States, approximately 30,000 men die each year from metastatic castration-resistant prostate cancer (mCRPC), a majority of them succumbing to metastasis- and treatment- related complications (2). Docetaxel (DTX) is the most commonly prescribed first-line chemotherapy for mCRPC. (2). Although DTX provides a modest (2.4-month) increase in median overall survival, many mCRPC patients cannot tolerate this cytotoxic chemotherapy due to advanced age, medical co-morbidities, or limited bone marrow reserves (3). Most patients receiving DTX eventually discontinue use due to development of DTX-resistant disease (3). Other recently FDA-approved treatments for mCRPC include sipuleucel-T, cabazitaxel, and abiraterone; however, these expensive treatment modalities only provide a median survival benefit of 2–5 months (1–4). Identification of an effective, low-cost alternate chemotherapy with fewer side effects may lead to increased survival and greatly benefit patient quality-of-life.

Metabolic aberrations promoting survival and metastasis have been identified in human CRPC bone metastasis specimens. These include increased protein expression of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMG-CoAR), the mevalonate pathway rate-limiting enzyme dominant in cholesterol, isoprenoid, and androgen synthesis; reduced activity of 5’-adenosine monophosphate-activated protein kinase (AMPK), the primary enzyme regulating cellular energy homeostasis; and significantly elevated levels of cholesterol and fatty acids (5). Additionally, high-intensity immunostaining of prostate cancer specimens for p-Akt and low-intensity staining for p-ERK are predictive of progression to castration-resistance (6); and phosphatase and tensin homolog (PTEN) deletion occurs in 70% of human specimens during advanced stage CRPC (7). PTEN dephosphorylates phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3), inhibiting Akt recruitment to the plasma membrane and activation; PTEN deletion leads to constitutively-active Akt.

A growing body of evidence suggests that hypercholesterolemia and Type 2 Diabetes (T2D) is correlated with increased cancer progression. Hypercholesterolemia and T2D are frequently present as co-morbidities associated with obesity (8), and both are associated with increased risk of developing advanced prostate cancer (9–12). Statins directly inhibit HMG-CoAR activity by binding its active site and are approved for treatment of hypercholesterolemia (13). Metformin (MET), an indirect activator of AMPKα, is FDA-approved for treatment of T2D (14). Use of lipophilic statins or MET has been associated with reduced risk of advanced prostate cancer and shown to reduce probability of biochemical failure, recurrence, and death from mCRPC, post surgery and radiotherapy (15–20). Lehman et al. demonstrated in a T2D population that, among non-statin users, the predictive prostate cancer hazard ratio (HR) for MET use versus sulfonylurea (anti-diabetic drug which stimulates insulin release from pancreatic beta cells) use was 2.15 [95% confidence interval 1.83–2.52], and among sulfonylurea users, the HR for statin use was 0.60 [95% confidence interval 0.49–0.70]; however, T2D patients taking both MET and statin had greatly reduced incidence of prostate cancer [HR 0.32, 95% confidence interval 0.25–0.42] versus T2D patients taking neither medication (21). Individually, lipophilic statins and MET have shown effectiveness in the inhibition of cell cycle progression, proliferation, and metastatic properties and the induction of cell death in androgen-refractory human prostate cancer PC-3 and DU145 cells in both in vitro and xenograft models (22–26). Moreover, treatment of prostate cancer cells with statins or MET has been shown to decrease Akt phosphorylation and activity (19, 24). Therefore, given the metabolic aberrations present in mCRPC, and that SIM inhibits HMG-CoAR and Akt activity while MET inhibits Akt and activates AMPKα, we hypothesize that combination SIM and MET could synergistically inhibit osseous mCRPC in both in vitro and in vivo models.

MATERIALS AND METHODS

Cell culture and treatment

All reagents were purchased from Fisher Scientific unless otherwise noted. C4-2B3, B4 and B5 cells were obtained in January 2012 from Dr. R.A. Sikes (University of Delaware, Newark, DE), PC-3 D12 were gift from Dr. R.W. Watson (University College Dublin, Dublin, Ireland) obtained in February 2012, MDA-MB-231 were gift from Dr. H.-Y. Kao (Case Western Reserve University, Cleveland, OH) obtained in July 2012, and MDA-MB-231(SA) were gift from Dr. T. Guise (Indiana University, Indianapolis, IN) obtained in October 2012. These cell lines were established in the laboratory of the donors with published reference and were not authenticated by the recipient. These cell lines were obtained from donor laboratory at passage 2–3, propagated by our laboratory and frozen at −150°C until use and all experiments performed using passages 5–15. Normal human prostate epithelial PrEC cells were purchased from Clonetics^® in March 2009, which authenticates cell lines utilizing immunostaining, morphology, and flow cytometry; transformed prostate epithelial RWPE1 cells and human prostate cancer LNCaP cells were purchased from American Type Culture Collection (Manassas, VA) in June 2011, authenticated using short tandem repeat profiling. These cell lines were frozen at −150°C until propagation in January 2012 and experimentation using passages 2–4 for PrEC and passages 5–15 for RWPE-1 and LNCaP. The cells were grown in appropriate culture medium containing 1% penicillin-streptomycin in a humidified 5% CO₂ at 37°C. For demonstration of Akt constitutive activation, C4-2B3/B4 cells were switched to serum-free RPMI-1640 overnight, and treated with 100–300 ng/mL recombinant human (hrIGF-1, Gibco Life Technologies, Grand Island, NY), representative of low-end and high-end of normal physiological plasma range in men ≥50 years of age, and 2 µM Akt Inhibitor VIII (Santa Cruz Biotechnology, Santa Cruz, CA). Cells were treated with DTX concentrations ranging from 1 nM-10 µM for 24h to simulate pharmacological plasma levels in patients with metastatic prostate cancer treated with 75–100 mg/m² intravenously, with a plasma C_max of 5–10µM within 1h, diminishing to C_min = 1–10nM 24h post-treatment (27). Cells were treated with activated SIM and MET (AK Scientific Inc. Union City, CA), alone or in combination, for 96h to simulate chronic daily use, at concentrations corresponding to pharmacological plasma ranges for hypercholesterolemic and T2D patients.

Simvastatin activation

The prodrug SIM activated to SIM acid before in vitro and in vivo use per manufacturer protocol, neutralized (pH=7.3), and filter sterilized. >93% lactone-to-acid conversion confirmed by EI-GC/MS. Stock solution stored at 4°C, and prepared fresh every 90 days.

Methylene blue assay

Cells were cultured in 24-well plates; following treatment, cells washed with PBS, stained with 2g/L methylene blue solution 1h, and excess stain removed with ddH₂O. Plates were examined under light microscope and photographed. For semi-quantification, bound methylene blue eluted with 0.1N HCl with shaking, and absorbance measured spectrophotometrically at λ=650nm (FLUOstar Omega, BMG LabTech).

Total and free cholesterol estimation

Cholesterol levels were measured by GC/MS using a protocol modified from Bederman et al. (28). Detail provided in Supplementary Materials and Methods section.

Western blotting

Lysates of exponentially-growing cells and of mouse ventral prostate tissue was prepared by homogenization using stainless steel beads (Next Advance, Averill Park, NY) as described previously (29). 40µg protein denatured at 95°C, resolved over 4–20% SDS-PAGE (Bio-Rad, Hercules, CA), and transferred to a nitrocellulose membrane. Following Ponceau S visualization and blocking with 5% nonfat dry milk TBST, pH 7.4 (USB, Santa Clara, CA) for 1h, membrane probed with primary antibody overnight at 4°C (Table S1), incubated with corresponding HRP-conjugated secondary antibody (Santa Cruz Biotechnology), and detected using Pierce ECL reagent (Rockford, IL). Bands visualized upon autoradiography film (Denville Scientific, Metuchen, NJ) exposure and bands quantitated using Image J software (NIH).

Synergistic quantification of drug-combination

Synergistic action of drug in combination was performed per Chou-Talalay method using CompuSyn software (30).

Cell migration assay

Two-dimensional cell motility was examined by scratch assay (31). Photographic images were captured using digital camera Zeiss Telaval 31 light microscope (Carl Zeiss) at 25X magnification connected to SPOT Insight Color Digital Camera Model 3.2.0 and SPOT Basic imaging software (SPOT Imaging Solutions). Image J used to calculate scratch area.

Invasion assay

Invasion assay was performed using 24-well hanging insert (Millipore, Billerica, MA) pre-coated with Matrigel™ Matrix (BD Biosciences, San Jose, CA) as previously described (32).

Anchorage-independent cell growth assay

Anchorage-independent assay in C4-2B3/B4 cells were performed as previously described (33).

Cholesterol synthesis by D₂O incorporation

De novo cholesterol synthesis was estimated by published procedure with modification (28, 34). Detail provided in Supplementary Materials and Methods section.

Blood glucose estimation

Blood glucose (mg/dL) was measured using TRUEresult meter (CVS Pharmacy) and TRUEtest test strips (Chinook Diabetics, Louisville, CO).

Plasma alanine aminotransferase measurement

Plasma ALT was measured using enzymatic kinetic spectrophotometric kit (Sekisui Diagnostics LLC, Lexington, MA) according to manufacturer’s instructions.

Plasma PSA estimation

Plasma PSA was measured by ELISA (Abnova) as per manufacturer’s instructions using FLUOstar Omega spectrophotometer.

Quantitation of SIM acid and MET in plasma and ventral prostate

Concentration SIM acid and MET was determined by GC/MS using modified protocols (35–37). Detail provided in Supplementary Materials and Methods section.

Animal studies

Animal experiments using male NCr-nu/nu mice were performed in accordance with recommendations in Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and protocol approved by CWRU School of Medicine Institutional Animal Care and Use Committee. Detail provided in Supplementary Materials and Methods section.

Metastases examination

Spinal column and femur metastases were examined using x-ray imaging. Mice were anesthetized with 2% isoflurane, and x-rays obtained at 34 kV and 500 µA for 6s integration time. Images captured on Rad-Icon digital detector with Shadow Cam software. Cachexic mice and mice with palpatable primary tumors monitored with sequential x-ray images. All x-ray images were evaluated by radiologist and areas of suspected metastasis within spinal column and femurs were noted.

Histology analysis

Mouse ventral prostate and primary tumors, spinal columns, femurs, kidney metastases, rectus femoris muscle, and left lateral liver lobe tissues were fixed with phosphate-buffered 10% (w/v) formalin followed by bone decalcification, and paraffin embedding. 5µm sections were stained with H&E. Spinal column and femur slides were immunostained using human-specific anti-androgen receptor antibody (Cell Signaling [#5153], Danvers, MA) and evaluated by a pathologist.

Statistical analysis

Quantitative values represented as Mean or Median ± STD or SEM of at least three independent experiments. Significance was determined by two-tailed, unpaired Student’s t test, Kruskal-Wallis test, or ANOVA, followed by Tukey multiple comparison procedure (SAS 9.3). Comparisons resulting in P<0.05 considered statistically significant and identified with an asterisk (*), P<0.01 identified with double asterisk (**), and P<0.001 identified with triple asterisk (***).

RESULTS

C4-2B cell lines as model for osseous mCRPC

In our studies, C4-2B3, C4-2B4, and C4-2B5 cell lines were utilized as in vitro models of osseous mCRPC. The C4-2B3-5 strains readily form large, poorly-differentiated tumors with frequent (25–37%) metastasis to the spinal column and femurs when orthotopically-injected into castrated athymic mice (38). Metabolic aberrations in these cells include significantly increased protein expression of the 80–97 kDa full-length smooth endoplasmic reticulum membrane-bound glycoprotein and the ~65kDa cleavage product of HMG-CoAR, which retains enzymatic activity within the cytoplasm, compared to PrEC cells. Furthermore, complete lack of low molecular weight C-terminal cleavage products of HMG-CoAR, indicative of dysregulated negative-regulation and breakdown, was noted in these cells (Figure S1). This was associated with significantly elevated (2–fold) total cellular cholesterol (Figure S2). Consistent with previous report (39), the free-to-total cellular cholesterol ratio was not affected by progression to castration-resistance (attributed to unaltered cholesterol acyltransferase activity); approximately 80–95% of cellular cholesterol was in free form in all cell lines characterized, despite increase in total cellular cholesterol (Figure S3).

The C4-2B3-5 strains exhibit minimal ERK1/2 phosphorylation and constitutive activity of Akt (Figure S4A-C). In these cells, Akt phosphorylation is not affected by presence or absence of FBS or by stimulation with human recombinant insulin-like growth factor-1. Significantly increased fatty acid and cholesterol were noted in clinical mCRPC specimens (5), indicative of inhibited AMPK activity. AMPK is a heterotrimeric protein consisting of a catalytic α subunit and two regulatory β and γ subunits (40–41). Activation of AMPK involves AMP/ADP binding to γ-subunit regulatory sites, causing conformational changes to allosterically activate the α-subunit and allowing for α-subunit activation loop Thr-172 phosphorylation by upstream kinases (40–41). Despite detectable p-Thr-172 AMPKα in C4-2B cell lines, AMPKα kinase activity is limited, as seen by reduced p-Ser-79 of acetyl-CoA carboxylase and increased ACC protein expression (41), (Figure S4B). Akt-dependent inhibition of AMPKα activity, via Ser-485/491 phosphorylation of AMPKα₁/α₂, has been previously demonstrated in cardiac and skeletal muscle, brown adipose tissue, and granulosa cells (42–44). We hypothesized that constitutively-active Akt may lead to inhibition of AMPKα activity in C4-2B3-5 cells. Using Akt Inhibitor VIII we observed a marked decrease in p-Ser-485/491 AMPKα₁/α₂ and concomitant increase in p-Thr-172 AMPKα and p-Ser-79 ACC establishing the role of Akt in suppressing AMPKα activity in these cells (Figure S4D, lanes 3 & 7).

Combination SIM and MET synergistically inhibits C4-2B3/B4 cell viability

DTX plasma concentrations, following chemotherapy, initially peak at 5–10µM within an hour, declining to ≤1nM within 24h (27). Treatment of C4-2B3/B4 cells with DTX in the pharmacological range (1nM-10µM) for 24h led to decrease cell viability as an initial response followed by resistance to further viability inhibition by DTX after 100nM concentration. DTX treatment also causes a significant adverse effect on viability of PrEC cells (Figure 1A). In contrast, 1:500 SIM and MET combination treatment for 96h (time-point chosen from individual dose-response curves for SIM and MET (Figure S5) and synergy determination per Chou Talalay method) (Supplementary Methods and Figure 1B, Figure S6; Table 1, Table S2), significantly decreased viability of C4-2B3/B4 cells at 500nM-4µM SIM and 250µM-2mM MET concentrations, with minimal effect on PrEC cells only at highest combination (Figure 1C).

Figure 1. Combination SIM and MET synergistically inhibits mCRPC cell viability more effectively than DTX, with minimal adverse viability effect on PrEC normal prostate epithelial cells.

(A) Quantification (Mean ± S.D.) of cell viability by methylene blue staining after DTX treatment (24h) over pharmacological range (1nM-10µM) in PrEC, C4-2B3 and C4-2B4 cells. n ≥ 3 per group. (B) Classic isobolograms for (1:500) combination (96h) at IC₅₀, IC₇₅, and IC₉₀ for C4-2B3 and C4-2B4, respectively. Points below the “lines of additivity” indicate synergism. (C) Quantification (Mean ± S.D.) of cell viability by methylene blue staining following treatment with (1:500) combination of SIM and MET (96h) over the pharmacological range (500nM-4µM SIM and 250µM-2mM MET) in PrEC, C4-2B3 and C4-2B4 cells. n ≥ 3 per group. *, †, ‡ P < 0.05; **, ††, ‡‡ P <0.01; ***, †††, ‡‡‡ P < 0.001 compared to untreated control determined by two-tailed Student’s t test.

Table 1. 1:500 SIM and MET combination synergistically inhibits cell viability in C4-2B3 and C4-2B4 CRPC cell lines.

Slope (m), correlation coefficients (r), and median-effect dosages (D_m) from median-effect plots for SIM alone, MET alone, and (1:500) SIM + MET combination treatment for 96h, and combination indexes (CI) and dose-reduction indexes (DRI) at the fraction of cells affected by treatment (f_a) = 0.50, 0.75, 0.90, and 0.95 for (1:500) SIM + MET combination treatment for 96h in C4-2B3 and C4-2B4 cells.

		Parameters			CI Value				DRI Value
Cell Lines	Treatment	m	r	Dm	IC50	IC75	IC90	IC95	IC50	IC75	IC90	IC95
C4-2B3	SIM	0.81 ± 0.11	0.983	4.09µM					2.63	6.51	16.1	29.8
	MET	1.12 ± 0.09	0.994	1.61mM					2.07	3.54	6.05	8.71
	Combination (1:500)	3.24 ± 0.64	0.964	1.21µM SIM + 610mM MET	0.862	0.436	0.227	0.148
C4-2B4	SIM	0.73 ± 0.16	0.958	3.50µM					3.37	10.8	34.6	76.5
	MET	0.97 ± 0.11	0.986	1.93mM					2.48	5.52	12.3	21.1
	Combination (1:500)	2.44 ± 0.29	0.973	1.55µM SIM + 780mM MET	0.699	0.274	0.110	0.060

Combination SIM and MET inhibits metastatic potential of C4-2B3/B4 cells

Mortality and poor prognosis in patients with CRPC is related to metastasis of prostate cancer cells to bone and soft tissues (1–2). Metabolic aberrations identified in mCRPC are intricately involved in enhanced tumor cell invasion and migration (45–46). Accordingly, we assessed the potential of combining SIM and MET to inhibit C4-2B3/B4 invasion, migration, and adhesion-independent cell growth metastatic properties. The effect of 1:500 combination of SIM and MET on cell migration was assessed by scratch assay and allowing cells to migrate for 48h. As shown in Figure 2A, a gradient of 1:500 SIM and MET combinations along the pharmacological range significantly prevented wound closure over 48h in C4-2B3/B4 cells. In fact, SIM and MET displayed significant dose-reduction when used in combination; similar wound closure inhibition observed with 1µM SIM + 500µM MET combination as was seen with 4µM SIM or 2mM MET treatment individually. In contrast to individual treatment with 4µM SIM or 2mM MET for 48h, which inhibited wound closure by 43% and 30% in C4-2B3 and 58% and 49% in C4-2B4, respectively, 4µM SIM + 2mM MET combination completely prevented scratch closure after 48h treatment in both cell lines. We also determined adhesion-independent cell growth by soft agar colony formation. The number of colonies observed per 2-mm field after 10d was significantly fewer when pre-treated with 4µM SIM + 2mM MET combination compared to untreated control, 4µM SIM or 2mM MET alone (Figure 2B). In addition, invasion was assessed by transwell assay in which C4-2B3/B4 cells were pretreated for 96h, then given opportunity to invade through a Matrigel-coated transwell toward serum-free or 10% FBS-media stimulation for 48h. C4-2B3/B4 cells demonstrate highly-invasive character; 36–40% crosses the Matrigel-coated transwell even in the absence of FBS-invasive stimulation conditions (Figure 2C). 4µM SIM + 2mM MET combination was significantly more effective in the inhibition of C4-2B3/B4 invasion compared to untreated control, 4µM SIM or 2mM MET treatment individually.

Figure 2. Combination SIM and MET significantly inhibits metastatic properties of C4-2B3 and C4-2B4 mCRPC cells.

(A) Representative images and quantification (Mean ± S.D.) of cell migration by scratch assay (percent wound closure) following 24h and 48h treatment with untreated (UNT), 4µM SIM (IC₅₀), 2mM MET (IC₅₀), and gradient (1:500) SIM + MET combination in C4-2B3 and C4-2B4 cells. Scratch area at 0, 24, & 48h quantified from images using Image J software. n = 3 per group. * P < 0.05, ** P < 0.01, *** P < 0.001 compared to untreated control determined by two-tailed Student’s t test. (B) Representative photographs and quantification (Mean ± S.D.) of adhesion-independent cell growth by colony formation in soft agar in C4-2B3 and C4-2B4 cells. Cells were pre-treated for 96h; then 1 × 10⁴ cells grown in agar for 10d before colonies per 2-mm visual field were counted. n = 3 per group. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by ANOVA. (C) Quantification (Mean ± S.D.) of C4-2B3 and C4-2B4 cellular invasion through Matrigel™ transwell. Cells were pre-treated for 96h with untreated, 4µM SIM (IC₅₀), 2mM MET (IC₅₀), or (1:500) SIM + MET combination, then 1 × 10⁵ cells seeded into top of Matrigel™-coated transwell and incubated 48h; serum-free (SF) and 10% FBS RPMI-1640 media used for invasion stimulus. n = 3 per group. * P < 0.05, ** P < 0.01, *** P < 0.001 compared to respective SF- or 10% FBS-stimulated untreated control as determined by two-tailed Student’s t test.

Combination SIM and MET treatment ameliorates metabolic aberrations in C4-2B3/B4 cells

Next we sought to determine whether 1:500 SIM and MET combination mitigates metabolic aberrations observed in C4-2B3/B4 cells. Treatment of cells with 4µM SIM and 2mM MET in combination decreased Thr-308 and Ser-473 phosphorylation of Akt at an earlier time-point (by 24h) and more effectively than either drug alone; suppression of Akt phosphorylation continued throughout the duration (Figure 3A). Combination treatment also decreased inhibitory p-Ser-485/491 AMPKα₁/α₂, and concomitantly increased p-Thr-172 AMPKα and AMPKα kinase activity, with notable increase in Ser-79 ACC and Ser-872 HMG-CoAR phosphorylation. SIM and MET combination treatment modestly decreased ACC protein expression at 48h and 72h time-points in C4-2B3/B4 cells, compared to untreated controls. Protein expression of the 80–97 kDa full-length HMG-CoAR glycoprotein remained relatively unchanged, whereas expression of the ~65 kDa HMG-CoAR cleavage product clearly increased following SIM and MET combination treatment in both C4-2B3/B4 cells, compared to untreated, 4µM SIM or 2mM MET alone. Despite this, treatment of C4-2B3/B4 cells with 4µM SIM + 2mM MET combination for 48h significantly reduced total cellular cholesterol compared to untreated control (56% and 35% decrease in C4-2B3/B4, respectively, Figure 3B), restoring cholesterol concentrations within C4-2B3/B4 to approximately those of RWPE-1 cells (Figure S2C), and significantly reduced HMG-CoAR activity as determined by quantization of de novo cholesterol synthesis by deuterium incorporation (74% and 53% reduction in de novo cholesterol synthesis in C4-2B3/B4, respectively, (Figure 3C and S7) (47). Therefore, increase in HMG-CoAR expression is likely compensation for direct and indirect HMG-CoAR enzymatic inhibition by SIM and MET.

Figure 3. Combination SIM and MET ameliorates metabolic aberrations of C4-2B3 and C4-2B4 mCRPC cells.

(A) Western blot analysis of Akt, AMPKα, ACC, and HMGCR phosphorylation and expression in C4-2B3 and C4-2B4 cells following 24, 48, 72h treatment time course of untreated (U), 4µM SIM IC₅₀ (S), 2mM MET IC₅₀ (M), or 4µM SIM + 2mM MET combination (C). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as loading control. (B) Quantification (Mean ± S.D.) of total cholesterol (µg per 1 × 10⁶ cells) following untreated (UNT) or 4µM SIM + 2mM MET combination (Combo) treatment for 48h in C4-2B3 and C4-2B4 cells as determined by EI-GC/MS. n = 5 per group. (C) Quantification (Mean ± S.D.) of de novo cholesterol synthesis (ng per 1 × 10⁶ cells) over 48h in untreated (UNT) or 4µM SIM + 2mM MET combination (Combo) treated C4-2B3 and C4-2B4 cells as determined by deuterium incorporation and quantization by EI-GC/MS. n = 5 per group. ** P < 0.01, *** P < 0.001 compared to untreated control determined by two-tailed Student’s t test.

Combination SIM and MET significantly inhibits primary tumor growth and metastasis in mouse model of CRPC

To demonstrate in vivo efficacy of combination SIM and MET for treatment of CRPC, castrated male NCr-nu/nu mice were orthotopically inoculated with C4-2B4 cells within the ventral prostate. Tumors were allowed to seed for a week, followed by 9 weeks of SIM and MET treatments (Supplementary Methods and depicted in Figure S8). Orthotopic implantation of C4-2B4 cells resulted in poorly-differentiated primary tumor formation in 90% of animals (Figure 4A and S9), cachexia in 20% of animals, significantly increased genitourinary (GU) tract weight (Figure 4B), increased ventral prostate proliferative index (Figure 4C), biochemical failure determined by readily detectable prostate specific antigen (PSA) (Figure 4D), metastasis to spinal column and femurs in 20% of animals (Figure 5A), premature animal death (n = 2 mice sacrificed early). No soft tissue metastases were observed in any treatment group. SIM or MET individual treatments reduced the incidence of primary ventral prostate tumor (38% and 44% of mice, respectively); ventral prostate/primary tumor histopathology in the SIM and MET groups ranged from high-grade prostatic intraepithelial neoplasia (HGPIN) to poorly-differentiated high-grade tumors, similar to those observed in the control group (Figure 4A and S9). Individual treatment with SIM or MET did not significantly decrease GU tract weight or ventral prostate/primary tumor proliferative index compared to untreated controls (Figure 4B, C). However, the SIM group demonstrated a significant decrease in the plasma PSA versus untreated control, consistent with previous reports (24, 48); MET treatment did not affect plasma PSA (Figure 4D). SIM and MET individual treatment led to slightly reduced cachexia incidence (13% and 11% of mice, respectively). Two untreated mice, who also displayed cachexia, were determined to have femoral bone metastases (Figure 5A). Although only one mouse in the SIM group showed signs of cachexic wasting, two mice—the cachexic mouse and another, both with large primary tumors—presented with bone metastases; the cachexic mouse had metastases within the lumbar vertebrae, and the second mouse presented with femoral head metastases (Figure 5A). In our studies, SIM did not prevent progression to metastatic disease. Despite similar incidence of primary tumor and cachexia as the SIM group, no bone metastases were identified in any mouse within the MET group (Figure 5B). 24µg/g body weight/day DTX treatment every 3 wk for 3 cycles resulted in the inhibition of primary tumor growth (Figure 4A and S9), yet caused toxicity, intestinal blockage, and mortality of 75% of mice (Figure S10). Of the two mice surviving to the end of the 9 wk experiment, one displayed normal ventral prostate histology with no noted metastases and the other had HGPIN with small femur metastasis (Figure 4A, 5A). DTX did not significantly reduce GU tract weight, ventral prostate proliferative index, or plasma PSA concentration compared to the control group (Figure 4B-D). In sharp contrast, high-dose (HD) or low-dose (LD) SIM and MET combination completely inhibited primary ventral prostate tumor growth (no primary tumors detected in either treatment group); all HD and LD group mice had normal prostate glandular structure with reduced GU tract weight (Figure 4A-B, S9). HD and LD combination significantly reduced proliferation in the ventral prostate tissue assessed through PCNA proliferative index (Figure 4C), and prevented biochemical failure, evidenced by undetectable plasma PSA (Figure 4D). HD and LD combination treatment completely eliminated incidence of cachexic wasting and prevented bone metastasis (Figure 5B). Also of importance, no HD or LD group mice demonstrated any apparent signs of toxicity from treatment, as determined by monitoring of body weight (Figure S10), plasma alanine aminotransferase (Figure S11) liver and muscle histology or mortality (Figure S12 and S13). The in vivo results suggest that both HD and LD combination treatment are significantly more effective and less toxic than DTX in the inhibition of CRPC progression and metastasis.

Figure 4. Combination SIM and MET significantly inhibits prostate tumor progression in an orthotopic mouse model of CRPC.

(A) Histopathology of representative mouse ventral prostates/tumors from six treatment groups. H&E staining, 100X magnification. Slides were analyzed by clinical pathologist (G.T. MacLennan). C4-2B4 xenografts produce poorly-differentiated, highly-vascularized tumors. Variation in treatment response with SIM and MET groups, HG-PIN to poorly-differentiated tumors. Normal glandular structure in LD and HD treatment groups. (B) Mouse terminal genitourinary (GU) tract weight (g) among six groups after 9 wk treatment. Statistical difference determined using Kruskal-Wallis ANOVA followed by Bonferroni multiple comparison procedure. Overall, there was a significant difference amongst six treatment groups (P = 0.0002). In pair-wise comparison, no significant differences were noted among the LD, HD, and DTX treatment groups. (C) Western blot analysis of proliferating cell nuclear antigen (PCNA) expression in representative ventral prostates from each group; α-tubulin loading control. Quantification of PCNA/α-tubulin (O.D.) ratio using Image J software. Statistical significance determined by Kruskal-Wallis ANOVA followed by Tukey multiple comparison procedure. Overall, there was a significant difference amongst the six groups (P = 0.022). (D) Terminal plasma prostate specific antigen (PSA, ng/mL) among the six groups. No PSA detected (ND) in any plasma sample from LD or HD combination group; therefore, limits of detection (<0.75 ng/mL) used for statistical calculations. Statistical significance determined by Kruskal-Wallis ANOVA followed by Tukey multiple comparison procedure. Overall, there was a significant difference in PSA concentration among the six treatment groups (P = <0.0001). Untreated (UNT), simvastatin (SIM), metformin (MET), low-dose combination (LD), high-dose combination (HD), docetaxel (DTX). Black bar: median, box: 25^th to 75^th percentiles, whiskers: range. Pair-wise comparisons depicted where UNT(a), SIM(b), MET(c), LD(d), HD(e), DTX(f); * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 5. MET treatment prevents bone metastasis in C4-2B4 orthotopically-inoculated mice.

(A) X-ray images and androgen receptor (AR)-stained immunohistochemistry (IHC) slides at 40X and 100X identifying C4-2B4 CRPC bone metastases in untreated (UNT) mouse #2 and mouse #9 femurs, SIM-treated mouse #7 femurs and mouse #5 spinal column, and DTX-treated mouse #4 femurs. Arrows indicate clinical radiologist-identified areas of potential bone metastasis; slides were cut and AR-IHC-stained based on radiologist recommendation. (B) Representative x-ray images and androgen receptor (AR) immunohistochemistry (IHC)-stained slides at 40X from femurs (left) and spinal columns (right) of MET-treated, low-dose combination (LD)-treated, and high-dose combination (HD)-treated mice. All MET, LD, and HD slides were AR-negative.

Combination SIM and MET lowers plasma cholesterol and blood glucose and are bioavailable within the ventral prostate

Next, we determined whether combination SIM and MET was efficacious in reducing plasma cholesterol and blood glucose, and SIM and MET bioavailability within the plasma and ventral prostate. Animals were orally-gavaged daily with β-hydroxyacid SIM, the same activated SIM used within the in vitro experiments, to prevent need for cytochrome P450 isoform 3A4 (CYP3A4)-humanized mice. Activated SIM is approximately 93.7% SIM acid (Figure S14). The concentration of SIM β-hydroxyacid and MET was quantified by electron ionization-gas chromatography-mass spectrometry (Supplementary Methods and Figure S15-S23). Both SIM acid and MET were readily detectable at appreciable concentrations in the plasma of respectively-treated groups collected terminally 1–8h post-gavage (Table S3). As expected, SIM acid and MET treatment lowered both plasma cholesterol and blood glucose to varying degrees. By 2 wk after treatment initiation, MET, LD and HD treatment lowered plasma cholesterol, and by 4 wk, both LD and HD combination treatment had significantly lowered plasma cholesterol concentration compared to untreated, SIM, MET and DTX treatment groups (Figure 6A). Plasma cholesterol remained lower within the SIM and HD groups than untreated controls throughout the remainder of the experiment, albeit not statistically significant. Plasma cholesterol in MET and LD groups significantly decreased initially at 4 wk, but rebounded to levels comparable to untreated mice by 7–9 wk. Lowering of blood glucose was noted by 2 wk after treatment initiation, particularly and significantly in the MET and HD groups, in which the blood glucose reduction was maintained throughout the 9 wk experiment (Figure 6B). Blood glucose was also lowered in SIM and LD groups 2–9 wk following treatment initiation, but was not statistically significant.

Figure 6. Combination SIM and MET bioavailable and mitigates altered metabolism in an orthotopic mouse model of CRPC.

(A) Mouse plasma cholesterol (mg/dL, Mean ± S.D.) among the six treatment groups 2, 4, 7, and 9 wk following treatment initiation. Overall, there was a significant difference in plasma cholesterol concentration among the six treatment groups at wk 4 (P < 0.0001) and 9 (P = 0.008). Mouse plasma cholesterol concentration increases with age, accounting for acclivous trend. (B) Fasting blood glucose concentration (mg/dL, Mean ± S.D.) among the six mouse groups 1, 2, 3, 4, 5, 7, and 9 wk following treatment initiation. Overall, there was a statistically significant difference in fasted blood glucose between the treatment groups at wk 2 (P = 0.01), 5 (P = 0.003), and 9 (P = 0.009). (C) Quantification (Mean ± S.D.) of mouse ventral prostate/tumor cholesterol concentration (µg/mg of tissue) as determined using EI-GC/MS. (D) Western blot analysis of protein phosphorylation and expression of Akt, AMPKα, ACC, and HMGCR in ventral prostate/tumor tissue from representative mice of the six treatment groups; α-tubulin used as loading control. Untreated (UNT), simvastatin (SIM), metformin (MET), low-dose combination (LD), high-dose combination (HD), docetaxel (DTX). Statistical significance determined using ANOVA followed by Tukey multiple comparison procedure. Pair-wise comparisons depicted where UNT(a), SIM(b), MET(c), LD(d), HD(e), DTX(f); * P < 0.05, ** P < 0.01, *** P < 0.001.

SIM acid and MET were also bioavailable within the ventral prostate tissue (Table S3). Surprisingly, terminal ventral prostate cholesterol concentration was significantly higher in the SIM and LD groups than in untreated controls; and was significantly (p<0.001) elevated in the HD group versus the untreated group, and also significantly greater than in SIM, MET, and DTX treated groups (Figure 6C). MET or DTX treatment did not affect ventral prostate tissue cholesterol concentration. Representative ventral prostate tissue specimens from LD and HD groups demonstrated reduced Ser-473 and Thr-308 phosphorylation of Akt compared to untreated, SIM or MET groups, and showed concomitant decrease in p-Ser485/491 AMPKα₁/α₂ and increase of Ser-79 ACC and Ser-872 HMG-CoAR phosphorylation (Figure 6D). Despite significantly elevated ventral prostate cholesterol in the LD and HD groups, no change was noted in HMG-CoAR protein expression, with the exception of modestly increased low molecular weight cleavage products, indicative of increased negative feedback regulation (Figure 6D). SIM acid, present within ventral prostate tissue in pg/mg concentrations, significantly affects the ventral prostate cholesterol levels, perhaps through low-dose stimulation of HMG-CoAR and the hormetic bell-shape of its dose-response, and MET, despite inducing no change in ventral prostate cholesterol levels itself, does seem to amplify the SIM acid effect, perhaps through Ser-872 phosphorylation and indirect inhibition of HMG-CoAR activity. Changes in phosphorylation and activity of Akt, AMPKα, and HMG-CoAR observed within the ventral prostate tissue in LD and HD groups, indicates a comparable, but not identical, amelioration of metabolic aberration observed in cell culture studies.

Combination SIM and MET synergistically reduces cell viability of other models of DTX-resistant, hormone-refractory bone metastatic cancers

Like advanced prostate cancer, the primary site of metastasis in another hormonally-regulated cancer, breast cancer, is the bone (49). Therefore, we hypothesized that combination SIM and MET chemotherapy may also demonstrate a broader applicability to other models of osseous metastases, docetaxel-resistant, hormone-refractory prostate and breast cancer. PC-3 D12 is a DTX-resistant strain of the androgen-independent, bone metastasis-derived PC-3 PCa cell line (50). MDA-MB-231 was derived from triple-negative breast cancer; MDA-MB-231(SA) is a highly bone metastatic variant of the cell line MDA-MB-231 (49). PC-3 D12, MDA-MB-231, and MDA-MB-231(SA) cells demonstrate Akt, AMPKα, HMGCR, and ACC phosphorylation and protein expression patterns similar to those observed in C4-2B3/B4 omCRPC cells (Figure S24). In contrast to PC-3 which have an IC₅₀ ≤ 2nM DTX, the DTX IC₅₀ in PC-3 D12 is approximately 5.6µM (Figure S25A). Treatment of MDA-MB-231 and MDA-MB-231(SA) with DTX over the pharmacological range for 24h only led to maximal cell viability inhibition of 21% and 34%, respectively (Figure S25B,C). Therefore, all three of these cell lines display resistance to DTX treatment. In contrast to DTX, 1:500 SIM and MET combination along the pharmacological ranges of 500nM-4µM SIM and 250µM-2mM MET significantly decreased viability in all three cell lines compared to untreated control (Figure S25D-F); resulting in a viability reduction of 73–92% in PC-3 D12, 67–94% in MDA-MB-231, and 56–96% in MDA-MB-231(SA). Using the Chou-Talalay method, we determined that 1:500 combination SIM and MET is synergistic in all three tested cell lines (Figure S26 and Table S4, S5).

DISCUSSION

Here we report that combination SIM and MET acts synergistically, within the pharmacological range, to inhibit C4-2B3/B4 mCRPC cell viability, with minimal adverse effect on PrEC normal prostate epithelial cells, prevents invasion, migration, and colony formation, and inhibits primary tumor formation, cachexia, bone metastasis, and biochemical failure in a C4-2B4 orthotopic mouse model, through amelioration of CRPC metabolic aberrations.

Treatment of mCRPC is challenging, with limited success; DTX remains first-line chemotherapy, yet results in frequent occurrence of DTX-resistant disease (3). We found pharmacological dosages of DTX indiscriminately toxic to normal prostate epithelial cells, at concentrations as low as 10nM; yet, all mCRPC sub-lines utilized within this study exhibited resistance to DTX-induced inhibition of cell viability. DTX at 24 µg/g body weight dose was highly toxic to mice as 75% of animals experience systemic toxicity and bowel obstruction, due to DTX effect on dividing cells within intestinal tissue, leading to mortality. This dosage was chosen to simulate the human treatment regimen and for maximal efficacy on C4-2B4 cells; a lower dose may have resulted in less systemic toxicity to the mice, but may have also reduced inhibition of CRPC tumor growth and metastasis. Patients often discontinue DTX use due to side effects, such as secondary infections and fever due to neutropenia and immuno-depression, anemia, edema, peripheral neuropathy, allergic reactions, weakness, and development of DTX-resistant disease (3–4).

Our pre-clinical results suggest that combination SIM and MET may be an effective, convenient, inexpensive, and less toxic alternate chemotherapeutic option for treatment of mCRPC. Using CompuSyn software, we determined 1:500 SIM and MET combination is synergistic and was found to significantly inhibit viability and metastatic properties of C4-2B3/B4 cells more effectively than DTX, SIM or MET treatment alone, with minimal adverse effect on PrEC cells. In orthotopic model of mCRPC, combination SIM and MET, at dosages equivalent to low-to-mid range for treatment of hypercholesterolemia and T2D, completely inhibited C4-2B4 primary tumor growth, significantly reduced GU weight and ventral prostate tissue PCNA protein expression, completely inhibited cachexic wasting, metastasis to bone, and prevented biochemical failure, significantly better than SIM or MET alone or DTX chemotherapy, without adversely affecting animal health. Combination SIM and MET treatment abated metabolic aberrations noted in the C4-2B3/B4 mCRPC cells in a time-dependent fashion, decreasing Akt Ser-473 and Thr-308 phosphorylation, leading to a reduction of inhibitory p-Ser-485/491 of AMPKα₁/α₂, and simultaneously increasing Thr-172 phosphorylation and AMPKα kinase activity. SIM and MET combination treatment also inhibited HMG-CoAR activity and significantly diminished cellular cholesterol concentration in mCRPC cells to levels comparable with those found in RWPE-1 cells. Amelioration of metabolic aberrations was also noted in vivo studies, at both systemic level and directly in the ventral prostate tissue, where combination treatment reduced Akt phosphorylation and increased AMPKα catalytic activity. Whether amelioration of these metabolic aberrations (and potential inhibition of aerobic glycolysis and biomass production) is the mechanism by which SIM and MET treatment selectively kills CRPC cells and not normal epithelial cells warrants further investigation.

Presently, an ongoing clinical trial (http://clinicaltrials.gov/ct2/show/NCT01561482) is investigating use of combination SIM and MET for prevention of rising PSA following radical prostatectomy or radiation therapy for localized prostate cancer. This trial does not directly address the effect of combination SIM and MET in CRPC (as patients on ADT within 6 months prior to study enrollment are not eligible) or in metastatic disease (as subjects can only participate until metastatic progression). Provided the investigators account for patients with and without previous ADT use during data analysis, it will be interesting to know whether combination SIM and MET chemotherapy demonstrates different efficacies with respect to hormone-dependent and hormone-refractory cancers, since the same metabolic aberrations noted in CRPC and advanced bone metastatic prostate cancer were not identified in early androgen-dependent prostate cancer in our study (AMPKα still active, HMG-CoAR still demonstrate normal negative feedback regulation, and less cholesterol accumulation). Without accounting for previous ADT use, beneficial effects of combination SIM and MET chemotherapy may remain unseen within this clinical trial, as combination SIM and MET treatment may be more effective in CRPC patients.

Our in vitro experimentation in PC-3 D12, MDA-MB-231, and MDA-MB-231(SA) cell lines suggests that SIM and MET combination may have broader applicability as therapeutic option for metastatic DTX-resistant prostate cancer and triple-negative breast cancer. These cell lines demonstrate altered metabolism, similar to that observed in the C4-2B strains, and could explain why SIM and MET treatment is effective in these cells. Furthermore of interest would be investigation as to whether combination SIM and MET treatment could translate to other bone metastatic cancers, such as carcinoma of the lungs or kidneys.

With respectable safety profiles, SIM and MET combination treatment could be readily used by elderly patients and those who cannot tolerate or fail DTX. SIM and MET are oral drugs consumed daily, facilitating ease of use, in contrast to other FDA-approved chemotherapeutic drugs which are expensive. In conclusion, our studies have identified an effective, inexpensive alternate chemotherapy with an excellent safety record that would greatly benefit quality of life in patients suffering from mCRPC and perhaps other cancers metastasizing to bone.

List of Abbreviations

ACC

acetyl-Coenzyme A carboxylase

ADT

androgen deprivation therapy

AMPK

adenosine monophosphate-activated protein kinase

AR

androgen receptor

CI

combination index

CRPC

castration-resistant prostate cancer

DRI

dose-reduction index

DTX

docetaxel

EI-GC/MS

electron ionization-gas chromatography-mass spectrometry

F_a

fraction of cells affected by treatment

GU

genitourinary

HD

high-dose SIM and MET combination group

HMG-CoAR

3-hydroxy-3-methylglutaryl Coenzyme A reductase

HR

hazard ratio

IS

internal standard

LD

low-dose SIM and MET combination group

MC

methyl cellulose

mCRPC

metastatic castration-resistant prostate cancer

MET

metformin

PCNA

proliferating cell nuclear antigen

PSA

prostate-specific antigen

RT

retention time

SIM

simvastatin

T2D

type 2 diabetes.