Phosphorylation of the androgen receptor by PIM1 in hormone refractory prostate cancer

Abstract

Integration of cellular signaling pathways with androgen receptor (AR) signaling can be achieved through phosphorylation of AR by cellular kinases. However, the kinases responsible for phosphorylating the AR at numerous sites and the functional consequences of AR phosphorylation are only partially understood. Bioinformatic analysis revealed AR serine 213 (S213) as a putative substrate for PIM1, a kinase overexpressed in prostate cancer. Therefore, phosphorylation of AR serine 213 by PIM1 was examined using a phosphorylation site-specific antibody. Wild-type PIM1, but not catalytically inactive PIM1, specifically phosphorylated AR but not an AR serine-to-alanine mutant (S213A). In vitro kinase assays confirmed that PIM1 can phosphorylate AR S213 in a ligand-independent manner and cell type-specific phosphorylation was observed in prostate cancer cell lines. Upon PIM1 overexpression, AR phosphorylation was observed in the absence of hormone and was further increased in the presence of hormone in LNCaP, LNCaP-abl and VCaP cells. Moreover, phosphorylation of AR was reduced in the presence of PIM kinase inhibitors. An examination of AR-mediated transcription showed that reporter gene activity was reduced in the presence of PIM1 and wild-type AR, but not S213A mutant AR. Androgen-mediated transcription of endogenous PSA, Nkx3.1 and IGFBP5 was also decreased in the presence of PIM1, whereas IL6, cyclin A1 and caveolin 2 were increased. Immunohistochemical analysis of prostate cancer tissue microarrays showed significant P-AR S213 expression that was associated with hormone refractory prostate cancers, likely identifying cells with catalytically active PIM1. In addition, prostate cancers expressing a high level of P-AR S213 were twice as likely to be from biochemically recurrent cancers. Thus, AR phosphorylation by PIM1 at S213 impacts gene transcription and is highly prevalent in aggressive prostate cancer.

Figure 1. Phosphorylation of AR WT by PIM1 Kinase

A) 293 cells were transiently transfected with either AR WT or AR mutant S213A and vector only, PIM1, or HA-myr-Akt. Cells were steroid starved in 10% cFBS overnight and then treated with vehicle or 10nM R1881 for 2 hours. Total protein lysates were subjected to Western blots against P-AR S213, AR (total), PIM1, HA (HA-epitope tagged myristoylated (Myr)-Akt), and tubulin (loading control). B 293 cells were transiently transfected with AR and PIM1 WT or PIM1 K67M and treated as in A. Protein lysates were subjected to Western blot analysis using antibodies against P-AR S213, AR, endogenous PIM1, exogenous PIM1, and tubulin.

Figure 2. Lambda Phosphatase Treatment Abolishes Phosphorylation of AR by PIM1

293 cells were transiently transfected with AR and vector only or PIM1. Cells were steroid starved overnight and then treated with vehicle or 10nM R1881 for 2 hours. Total protein lysates were mock treated or lambda phosphatase treated for 30 minutes. Lanes 9–12 (Inputs) are protein lysates prior to lambda phosphatase treatment. Protein lysates were subjected to Western blots against P-AR S213, AR, and ERK-1 (loading control).

Figure 3. PIM1 Phosphorylates AR in vitro

A 293 cells were transiently transfected with FLAG-AR. Cells were steroid starved overnight and then treated with vehicle or 10nM R1881 for 2 hours. FLAG-AR was immunopurified and then subjected to kinase reactions in the presence of recombinant GSK3, PIM1, or Akt. The proteins were immunoblotted for P-AR S213, AR, PIM1, and P-Akt S473. B and C Kinase reactions using recombinant His-Akt or GST-PIM1 with their known substrates, GSK3 and Bad, respectively. Kinase activity was detected using antibodies against P-GSK3α/β S21/9, P-Akt S473, and P-Bad S112. D Recombinant AR and PIM1 were combined in a kinase reaction in the presence of R1881. Phosphorylation was detected by P-AR S213 antibody. AR and PIM1 antibodies were also used as controls for the presence of AR and PIM1.

Figure 4. Phosphorylation of Endogenous AR in Response to PIM1 Overexpression

A LNCaP, LNCaP-abl, and VCaP cells were transiently infected with either vector only or PIM1. Cells were steroid starved overnight and then treated with vehicle or 10nM R1881 for 2 hours. Protein lysates were subjected to Western blot analysis using antibodies against P-AR S213, AR, PIM1, and tubulin. B VCaP stable cell lines overexpressing vector only (vo) or PIM1 (right panel) were grown on ultra-low attachment plates. Spheres that grew in the anchorage independent condition were counted under a microscope. The graph represents three independent experiments performed in triplicate. Each point represents the number of spheres in a replicate and the horizontal bar denotes the mean (vo: 245.8 ± 21.4 and PIM1: 274.8 ± 22.0). The p-value was calculated using an unpaired t-test.

Figure 5. PIM1/2 Inhibition Decreases P-AR S213

A) VCaP cells were steroid starved overnight then treated with 10nM R1881 and PIM kinase inhibitor SGI-1776 as indicated for 4 hours. Total protein lysates were analyzed by Western blot with antibodies against P-AR S213, AR (total), P-4EBP1 Thr 37/46, and tubulin. B) VCaP and LNCaP cells were transiently infected with either vector only or PIM1. Cells were steroid starved overnight and then treated with vehicle or 10nM R1881 in the presence of absence of 20µM SGI-1776 for 4 hours. Protein lysates were subjected to Western blot analysis using antibodies against P-AR S213, AR, PIM1, and tubulin. C) 293 cells were transiently transfected with FLAG-AR. Cells were steroid starved overnight and then treated with vehicle or 10nM R1881 for 2 hours. FLAG-AR was immunopurified and incubated with recombinant PIM1 in the presence and absence of PIM1/2 inhibitor V. Kinase reactions were subjected to Western blot analysis using antibodies against P-AR S213, AR, and PIM1. D) VCaP cells were steroid starved and then treated with 10 nM R1881 and PIM1/2 inhibitor V for 2 hours.

Figure 6. Regulation of AR mediated Transcription by PIM1

A and B) 293 cells were transiently transfected with AR WT or AR mutant S213A, lacZ reporter, ARR3-Luciferase reporter, and increasing amounts of PIM1 in the presence and absence of 10nM R1881. Insets show AR and PIM1 protein levels for A and B. Data was normalized to 10 nM R1881 in the absence of PIM1 and set to 100% activity. C) VCaP cells were transfected with lacZ reporter, ARR3-Luciferase reporter, and increasing amounts of PIM1 in the presence and absence of 10nM R1881. D) LNCaP cells were transfected as above in the presence of 10nM R1881. E and F LNCaP cells stably expressing vector only (vo), or PIM1 (pools#1 and #2) were steroid starved overnight and then treated in the presence and absence of 10nM R1881 for 24 hours. qPCR was performed to quantify transcript levels of PSA and Nkx3.1 (E) and AR and PIM1 protein expression was determined (F). Graphs are representative of at least three independent experiments. Error was calculated using the standard deviation.

Figure 7. PIM1 Affects AR Target Gene Expression

A) LNCaP stable cell lines overexpressing vector only (vo pool #1) or PIM1 (pool #2) were treated as in Figure 6E and the gene expression of IGFBP5, CCNA1, IL6, and CAV2 was quantified by qPCR. B) VCaP cells were transfected with siRNA against PIM1 and treated as above. Gene expression of PSA and IGFBP5 was quantified by qPCR and protein expression of AR and PIM1 determined C. Graphs are representative of at least three independent experiments. Error was calculated using the standard deviation.

Figure 8. Expression of Phosphorylated AR S213 in Hormone Refractory Prostate Cancers

A) Immunohistochemistry on prostate tissues using antibodies against P-AR S213 (A–C) or PIM1 (D–F). For P-AR S213, a separate histoscore was given for nuclear and cytoplasmic staining positivity while PIM1 was present only in the nucleus. The total histoscore ranges from 0 for a completely negative sample to a maximum score of 300. Magnification is 10X with insets of the same tissue at a 40X magnification. B) Histoscores (H-Scores) of hormone refractory (HR) vs non- hormone refractory prostate cancers and Gleason score 5–7 vs Gleason 8–10 were compared. The distribution of H-Scores is shown with the mean (horizontal bar). The differences between the means were determined to be statistically significant by t-test.