Galeterone and VNPT55 disrupt Mnk-eIF4E to inhibit prostate cancer cell migration and invasion

Abstract

Metastatic castration-resistant prostate cancer (mCRPC) accounts for a high percentage of prostate cancer mortality. The proprietary compound galeterone (gal) was designed to inhibit proliferation of androgen/androgen receptor (AR)-dependent prostate cancer cell in vitro and in vivo and is currently in phase III clinical development. Additionally, clinical studies with gal revealed its superb efficacy in four different cohorts of patients with mCRPC, including those expressing splice variant AR-V7. Preclinical studies with gal show that it also exhibits strong antiproliferative activities against AR-negative prostate cancer cells and tumors through a mechanism involving phosphorylation of eIF2α, which forms an integral component of the eukaryotic mRNA translation complex. Thus, we hypothesized that gal and its new analog, VNPT55, could modulate oncogenic mRNA translation and prostate cancer cell migration and invasion. We report that gal and VNPT55 profoundly inhibit migration and invasion of prostate cancer cells, possibly by down-regulating protein expression of several EMT markers (Snail, Slug, N-cadherin, vimentin, and MMP-2/-9) via antagonizing the Mnk-eIF4E axis. In addition, gal/VNPT55 inhibited both NF-κB and Twist1 transcriptional activities, down-regulating Snail and BMI-1 mRNA expression, respectively. Furthermore, profound up-regulation of E-cadherin mRNA and protein expression may explain the observed significant inhibition of prostate cancer cell migration and invasion. Moreover, expression of self-renewal proteins, β-catenin, CD44, and Nanog, was markedly depleted. Analysis of gal/VNPT55-treated CWR22Rv1 xenograft tissue sections also revealed that observations in vitro were recapitulated in vivo. Our results suggest that gal/VNPT55 could become promising agents for the prevention and/or treatment of all stages of prostate cancer.

Keywords: EMT; Gal/VNPT55; Mnk1/2; eIF4E.

Conflict of interest statement

Vincent C. O. Njar is the lead inventor of Galeterone (Gal or TOK-001) and VNPT55, patents and technologies thereof owned by the University of Maryland, Baltimore, and licensed to Tokai Pharmaceuticals, Inc. Puranik Purushottamachar and Andrew K. Kwegyir-Afful are co-inventors of VNPT55 and related compounds. A patent application to protect VNPT55 and related novel compounds has been filed. The other authors declare no potential conflict of interest.

© 2016 Federation of European Biochemical Societies.

Figures

Figure 1

Efficacy of Gal/VNPT55 on PC-3 xenografts. (a) PC-3 cells were inoculated into the flanks of male SCID mice and treated with either 0.15 mmol/kg gal (12 mice) or vehicle (24 mice) b.i.d. Mice were evaluated daily for the formation of palpable tumor. (b) Male SCID mice were inoculated with PC-3 cells and treated with either vehicle (0.3% hydroxypropyl cellulose, HPC) or 0.15 mmol/kg/b.i.d. d gal. Tumors were measured with calipers as described in materials and methods. (c) Excised PC-3 tumors were weighed following two weeks of treatment. (d) 1 × 106 LNCaP and CWR22Rv1 cells were seeded in 10 cm plates in 5% charcoal dextran supplemented RPMI and subsequently treated with gal (1–5 μM) for duration of 72 h. Immunoblot analysis was utilized to evaluate the expression of ERSR markers. (e) Tumors samples from 4 mice in each treatment group of LAPC4 xenografts were excised and analyzed by western blot for relative expression of ERSR markers, average expression were determined by densitometry (*p<0.05). (f) Cell viability assays were performed in DU145, PC-3 and CWR22Rv1 cells comparing efficacies of gal, VNPT55 and CGP-57380.

Figure 2

Mnk1-eIF4E overexpression enhances activity of gal/VNPT55 (a) Western blot analysis were conducted on CWR22Rv1 cells transfected with 50 nM of AR and Mnk1 siRNA alone and in combination for 3 days, complexes were washed off and cells lysed after an additional 72 h and analyzed for AR and Mnk1 protein expression. (b) CWR22Rv1 cells treated as in (a) were seeded at 2500 cells/well in a 96 well-plate, allowed to attach overnight and subsequently treated with gal and VNPT55 for an additional 72 h, cell viability was determined as in materials and methods, and GI50 values plotted in a barchart. Results of GI50 values are averages of 3 separate experiments. GI50 values show a significant increase in transfected cells (*p<0.05). (c, left and right panels) CWR22Rv1 cells were transfected with HA tagged eIF4E (HA-eIF4E) and Mnk plasmids for 96 h, western blot was used to analyze protein expression. (d) CWR22Rv1 cells were transfected with Mnk1 and eIF4E plasmids for 16 h, after which cells were counted and seeded at 2500 cells/well in 96-well plates. Cells were subsequently treated with increasing concentrations of gal/VNPT55 for 5 days. GI50 values were computed with graphpad prism and compared to vector-transfected controls (*p<0.05, **p<0.001) (e) CWR22Rv1 and DU145 cells were seeded in 96-well plates and treated with a combination of both gal/VNPT55 and CGP-57380 at a constant ratio of their respective GI50 values. After cell viability was determined with MTT reagent, the fractional effects of the compounds alone and in combination was determined and analyzed by the Calcusyn software to determine the combination indices (CI), to evaluate whether compounds act in synergy (CI < 1), additive (CI = 1) or antagonistic (CI > 1). (f) 1000 cells/well of CWR22Rv1 cells were seeded in 6-well plates and treated with gal/VNPT55 alone or in combination with CGP-57380 (left panel), colonies were stained and counted and presented as bar chart (right graph)

Figure 3

Gal and VNPT55 modulate eIF4E phosphorylation by depleting Mnk1/2 expression levels. (a–c) LNCaP, CWR22Rv1 and PC-3 cells were treated with gal and VNPT55 at 10 μM for 24 h. Cells were lysed with RIPA buffer and 50 and 100 μg of total protein used in analyzing total proteins and phosphorylated proteins respectively. (d) PC-3 cells were incubated with 5 and 10μM of CGP-57380 for 24 h and eIF4E phosphorylation analyzed by western blot analysis. (e) CWR22Rv1 cells were treated with increasing dose of gal/VNPT55 and Mnk1/2 protein expression analyzed by western blot with 50 μg total protein. (f) CWR22Rv1 cells were treated with cycloheximide and or gal and Mnk1 protein expression analyzed by western blot analysis. (g) LNCaP and CWR22Rv1 cells incubated with gal alone or in combination with MG132, as in materials and methods to determine the role of the proteasomal enzyme in gal/VNPT55 induced post-translational modulation of Mnk1. (h) LNCaP cells treated with 10 μM for 16 h were incubated with 5 μM MG132 for additional 8h. 500 μg total proteins was separated on a 10% Tris glycine gel, transferred to a Polyvinylidiene fluoride (PVDF) membrane and probed with ubiquitin antibodies. (i) LNCaP cells were treated with 10 μM of gal/VNPT55 and downstream targets of Mnk-eIF4E axis were evaluated. (j) RNA was collected from PC-3, DU145 and CWR22Rv1 cells incubated with 5 and 10 μM of gal/VNPT55 for 24 h. Mnk1 primers were used in quantitative real-time PCR to evaluate effects on mRNA expression levels. Results are represented as means ± S.E.M (*p<0.05).

Figure 4

Gal/VNPT55 decreases mesenchymal factors and enhances epithelial marker expression. (a) Lysates from PC-3 and DU145 cells treated with 5 and 10 μM were subjected to immunoblot analysis to evaluate N-Cadherin and ZO-1. (b) PC-3 and DU145 cells incubated with gal and VNPT55 at 10 μM for 24 h were stained with E-Cadherin polyclonal antibodies following protocol in materials and methods. Images were taken with Zeiss camera mounted immunofluorescence microscope. (c) PC-3, DU145 and CWR22Rv1 cells were analyzed for MMP-2/-9 protein expression after gal/VNPT55 treatment. (d) LNCaP cells were serum starved for 12 h and treated with 5, 10 and 20 μM gal/VNPT55 for 36 h in serum-free, pen-strep free RPMI media. Culture media was concentrated using Millipore 0.5 ml ultra-centrifugal columns. Conditioned media was separated on 0.1% gelatin Tris/Glycine gel to analyze proteolytic activity of MMPs (top), densitometric analyses (bottom bar chart) shows significant decrease in MMP-2 activity (*p<0.5, **p<0.001). (e) PC-3 cells were treated with gal/VNPT55 at 2.5 and 5 μM for 72 h and media concentrated as in (d) and a zymogram gel used to determine proteolytic activity of MMP-2/-9. (f) DU145 cells were treated as in (e), at 2.5 μM with gal/VNPT55 and analyzed with gelatin gels. Densitometric analysis shows a significant decrease in MMP-2/-9 activity. All zymogram assays were repeated at least three times and represented as means ± S.E.M (*p<0.05).

Figure 5

Gal and VNPT55 inhibit migration and invasion of PC cells in vitro. (a) To evaluate whether PC-3 and DU145 cells used in migration and invasion assays at indicated compound concentrations did not significantly compromise cell numbers and viability, we performed a 24 h MTT cell viability assay with gal/VNPT55 (1–10 μM. (b) PC-3 (top panel) and DU145 (bottom panel) cultured in 24-well plates to a confluent monolayer were scratched with a 200 μl pipette tip and subsequently treated with indicated compounds for 12 h. (c) Wounds were measured before and after the 12 h time point. Distance migrated were quantified by measuring the difference at time 0 and 12 h and normalized to control. (Distance migrated = Distance at time 0 h - distance at 12 h/Distance migrated by control). Experiments were repeated at least 3 times and represented as mean ± S.E.M, shows significant inhibition of cell migration (**p<0.001) (d) PC-3, DU145 and CWR22Rv1 were seeded in BME pre-coated inserts. Cells treated with gal, VNPT55 and CGP at 5 μM in the upper chamber in serum free RPMI media. The bottom chamber was filled with 1ml RPMI media supplemented with 10% FBS. Set-up was placed in 37 °C incubator for 36 h. Cells were fixed in 3.7% paraformaldehyde for 10 minutes and stained with 0.05% crystal violet; cells in upper chamber were wiped off with cotton swabs and invaded cells at the bottom of inserts analyzed by counting. (e) Schematic illustration of invasion assay set-up, with upper and lower chambers. (f) Quantified invaded cells shows a significant inhibition of PC cell invasion (*p < 0.05, **p<0.001). (g and h) PC-3 cells grown to a monolayer and scratched with 200 μl were treated with EGF (10ng/ml) alone and in combination with gal (5 μM), CGP (5 μM) and U0126 (5 μM). Wound healing was analyzed as in (b and c), gal and CGP significantly inhibited migration in the presence of EGF ligand (*p<0.05). All experiments were repeated at least three times and represented as mean ± S.E.M. (i) To evaluate whether activated MMP-9 could be inhibited by galeterone as in Figure 4g, we treated cells with gal, VNPT55 with or without EGF at (gal-5 μM + EGF-10ng/ml) and incubated for 72 conditioned media was concentrated and separated on zymogram gels. It was observed, just as in Figure 5g, that galeterone even in the presence of EGF, was able to inhibit collagenase activity. Experiments were repeated three times (*p<0.05, **p<0.001)

Figure 6

Gal/VNPT55 disrupts NF-κB and Twist1 transcriptional activity. (a) PC-3 and DU145 cells were treated with gal and VNPT55 for 24 h and 50 or 100 μg of total cell lysates separated on a 10% Tris/glycine gel to analyze p65, p52 and phosphorylated p65 (p-p65). (b) RNA collected from treated PC-3 cells were subjected to quantitative real-time PCR to analyze E-Cadherin, MMP-9, Twist1 and Snail mRNA expression (**p<0.001). (c) Protein expression of Snail, Slug, Twist1, RhoA, vimentin and Vascular endothelial growth factor (VEGF) were evaluated by immunoblot analysis after a 24 h treatment in PC-3 and DU145 cells. (d and e) PC-3 and DU145 cells pre-treated with 10 ng TNF-α for 2 h were subsequently treated with gal/VNPT55 at 10 μM for an additional 24 h. Total cell lysates were subjected to cell fractionation. [PC-3-Nu and DU145-Nu are nuclear fractions; PC-3-Cyt and DU145-Cyt are cytosolic fractions]. LSD1 was used as loading controls for nuclear fractions and lactate dehydrogenase (LDH) for cytosolic fraction controls. Twist1, p65 and p-p65 expression levels were analyzed in the different compartments. (f)PC-3 cells were pre-treated with 10 ng TNF-α for 2 h and then treated with gal/VNPT55 for 24 h. Cells were fixed in 3.7% paraformaldehyde and stained with Twist1 mouse monoclonal antibody and images taken

Figure 7

Gal/VNPT55 inhibits p65 and Twist1 chromatin binding (a) Schematic of NF-κB binding site in Snail promoter region (a, top panel). Gal/VNPT55 decreases p65 binding activity to Snail promoter region in PC-3 cells (a, bottom graph). (b) RNA collected from PC-3, DU145 and CWR22Rv1 cells were analyzed for BMI-1 expression levels after 24 h treatment (*p<0.05, **p<0.001). (c) Schematic representation of Twist1 binding site in BMI-1 promoter region. (d) DU145 and (e) CWR22Rv1 cells were treated with gal/VNPT55 for 36 h at 10 μM and interaction between Twist1 and BMI-1 promoter measured using chromatin immunoprecipitation (ChIP) assay. BMI-1 promoter fold enrichment was normalized to input controls and presented as mean ± SEM (**p<0.001).

Figure 8

Gal/VNPT55 inhibits PC colony formation and downregulate protein expression of stem cell factors. Mnk1 knockdown exhibited similar effects. (a) PC-3 and DU145 were treated with gal/VNPT55 as indicated and stem cell factors analyzed by immunoblot. (b) 1000 cells/well (PC-3 and DU145), seeded in 6-well plates were treated with indicated concentrations of compounds for a period of 14 days. Media containing compounds were replaced every 3 days. Colonies were stained with 0.05% crystal violet. (c) Colonies from B were counted and presented as bar chart. Results are represented as averages from 3 separate experiments with S.E.M. (*p<0.5, **0.001).

Figure 9

Mnk1 knockdown mirrors activity of gal/VNPT55 (a and b) LNCaP (top panel) and CWR22Rv1 (bottom panel) cells were transfected with AR siRNA (25 and 50 nM) for 72 h and Mnk1/2 protein expression analyzed by western blot. LNCaP (top panel) and CWR22Rv1 (bottom panel) cells were transfected with Mnk1 siRNA (25 and 50 nM) for 72 h and full length AR analyzed. (c and d) PC-3 and DU145 cells were transfected with 25 and 50 nM Mnk1 siRNA for 72 h. EMT and stem cell factors were analyzed by western blot.

Figure 10

Gal and VNPT55 modulate components of the translational machinery in vivo. (a) Representative tumors-1 and 2 (T1 and T2) from gal and VNPT55 treated groups were excised, minced and lysed. Protein was quantified and western blotting performed to analyze effects on protein expression. Mice (n = 5) were administered with gal (0.15 mmol/kg/twice daily) and VNPT55 (0.15 mmol/kg/twice daily), by intraperitoneal injection, 5 days per week for 34 days. 50 μg of total cell lysate separated on a Tris/glycine gel shows that, in Gal treated xenograft tumors, Protein expression of β-Catenin, Mnk1/2, BMI-1 and Oct-4 are down-regulated. (b) Immunoblot analysis in VNPT55 treated groups also show that effects seen on expression of indicated protein in vitro were also observed in vivo. (c) Densitometry analysis of protein expression from western blot analysis was plotted to quantify the effects seen in vivo. (d) Representative images of Mnk1/2, BMI-1, Slug and peIF4E immunostaining in vehicle and gal/VNPT55 treated groups. Parafinised tumor sections were stained with Mnk1/2 BMI-1, Slug and peIF4E antibodies following protocol in materials and methods. Both gal and VNPT55 show strong depletion effects on Mnk1/2. (e) IHC staining in (e) was quantified using the ImageJ software and represented as a bar chart. (f) Schematic representation of multiple effects of Gal and VNPT55 in inhibiting EMT (1) NF-κB (p65) and p50 heterodimerizes and binds to its cognate sequences in promoter regions of target genes to activate them, gal/VNPT55 in addition to decreasing p65 phosphorylation levels also decrease binding to the chromatin. (2) Twist1 binds to BMI-1 gene to activate its transcription; ChIP analyses shows gal/VNPT55 decreases this interaction. (3) Twist1 also binds to E-Cadherin promoter to repress its transcription, with gal/VNPT55 inhibiting nuclear translocation of Twist1, this significantly inhibits the process and enhances E-Cadherin expression. (4) Mnk1/2 phosphorylation leads to phosphorylation of eIF4E and subsequent formation of the translation complex to translate oncogenic mRNAs. (5) Gal and VNPT55 deplete protein expression of Mnk1/2 and (6) ultimately inhibit the metastatic potential of Mnk-eIF4E axis. Gal and its new analog, VNPT55, decrease transcription and translation of oncogenic mRNA via inhibiting chromatin binding of transcription factors (NF-κB and Twist1) and depleting protein expression of Mnk1/2, respectively. Mnk1/2 depletion culminates in decrease in eIF4E phosphorylation and disruption of cap-dependent mRNA translation, thus inhibiting prostate cancer cell proliferation and epithelial mesenchymal transition (EMT).