1,25-dihydroxyvitamin D3 and PI3K/AKT inhibitors synergistically inhibit growth and induce senescence in prostate cancer cells

BACKGROUND: 1-Alpha, 25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) 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)(2)D(3). METHODS: Viability, cell cycle and senescence of cells were evaluated upon combinational treatment with 1,25(OH)(2)D(3) and pharmacological PI3K/AKT inhibitors. RESULTS: Pharmacological inhibitors of PI3K or Akt and 1,25(OH)(2)D(3) 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)(2)D(3) in prostate cells, induction of senescence, was discovered. Combination of 1,25(OH)(2)D(3) and AKT inhibitor cooperated to induce G(1) 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)(2)D(3) 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)(2)D(3), and showed higher degree of synergism between AKT inhibitor and 1,25(OH)(2)D(3) compared to Pten-expressing counterparts. CONCLUSIONS: These findings provide the rationale for the development of therapies utilizing 1,25(OH)(2)D(3) or its analogs combined with inhibition of PI3K/AKT for the treatment of prostate cancer

Supplementary Figures 1-9 from Interleukin-1α Mediates the Antiproliferative Effects of 1,25-Dihydroxyvitamin D<sub>3</sub> in Prostate Progenitor/Stem Cells

Supplementary Figures 1-9 from Interleukin-1α Mediates the Antiproliferative Effects of 1,25-Dihydroxyvitamin D&lt;sub&gt;3&lt;/sub&gt; in Prostate Progenitor/Stem Cells</jats:p

BAD Dephosphorylation and Decreased Expression of MCL-1 Induce Rapid Apoptosis in Prostate Cancer Cells

<div><p>PTEN loss and constitutive activation of the PI3K signaling pathway have been associated with advanced androgen-independent prostate cancer. PTEN-deficient prostate cancer C42Luc cells survive in serum-free media and show relative resistance to apoptosis even in the presence of the PI3K inhibitor ZSTK474. Yet, when ZSTK474 is combined with the translation inhibitor cycloheximide, C42Luc cells undergo apoptosis within 6 hours. We identified dephosphorylation of BAD (<u>B</u>cl2-<u>a</u>ssociated <u>d</u>eath promoter) as a main apoptosis-regulatory molecule downstream from PI3K, and loss of MCL-1 (<u>M</u>yeloid <u>c</u>ell <u>l</u>eukemia -1) as a major target of cycloheximide. The combination of MCL-1 knockdown and expression of phosphorylation-deficient mutant BAD2SA is sufficient to trigger rapid apoptosis in prostate cancer cells. These results establish the mechanism for the synergistic induction of apoptosis by the combination of a PI3K inhibitor and of a protein synthesis inhibitor in PTEN-deficient prostate cancer cells.</p></div

Combination of ZSTK474 and cycloheximide induces rapid apoptosis in C42Luc cells.

<p><b>A</b>) C42Luc cells were treated with either 5 µM ZSTK474, 100 µg/mL cycloheximide, or the combination, and apoptosis recorded for 24 hrs using time-lapse microscopy. The cumulative percentage of cells entering apoptosis (rounding and membrane blebbing), is shown at specific time points over 24 h. At least 100 cells were counted for each treatment. Error bars show standard deviations from the average of four randomly chosen fields. *, <i>p</i><0.05. <b>B</b>) Caspase-3 activation assay in C42Luc cells treated with 5 µM ZSTK474, 100 µg/mL cycloheximide, or the combination. After 6 h of treatment, cells were lysed, and caspase-3 activity in cell lysates was measured with a fluorogenic substrate (DEVD-AFC). Data are presented as fold-induction of fluorescence intensity normalized to the control (DMSO). *, <i>p</i><0.0001. <b>C</b>) Immunofluorescence staining of cleaved caspase 3 (red) and TUNEL (green) in C42Luc cells treated with DMSO, 5 µM ZSTK474, 100 µg/mL cycloheximide, or the combination. DAPI was used to visualize nuclei. Each row of panels represents the same field of view. <b>D</b>) Western blot of C42Luc cells treated with either 5 µM ZSTK474, 100 µg/mL cycloheximide, or the combination. Whole cell lysates were collected at the indicated times and probed for pBAD (Ser112) and total BAD.</p

Bim knockdown decreased sensitivity of C42Luc cells to apoptosis.

<p><b>A</b>) NOXA expression is decreased in C42Luc cells treated with the combination of ZSTK474 and cycloheximide. Western blot analysis of MCL-1, NOXA, BIM and β-actin (loading control) in cells treated for 4 and 6 hours with cycloheximide. <b>B</b>) Co-immunoprecipitation of MCL-1 and BIM from cells that expressed doxycycline-inducible FLAG-MCL-1. <b>C</b>) Apoptosis in C42Luc cells that express two BIM-specific shRNA and in parental cells was assessed by time lapse microscopy. Cells were treated with vehicle ((–) DMSO) or combination of cycloheximide and ZSTK474 (+). Amount of apoptosis (%) was determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074561#pone-0074561-g001" target="_blank">Figure 1A</a>. At least 100 cells were counted for each treatment. Error bars show standard deviations from the average of four randomly chosen fields. Data for 6 hours post-treatment are presented. Differences in% apoptosis between control cells (that express scrambled shRNA) and C42Luc BIM1 or C42Luc BIM2 cells were statistically significant (p<0.05 and p<0.0001, respectively). Insert shows decreased BIM expression in cells that stably expressed BIM-specific shRNA. <b>D</b>) Apoptosis in C42Luc cells that expressed one of two BIM-specific shRNA or control vector was assessed by measuring caspase activity. Cells were treated for 6 hours with vehicle ((–) DMSO) or the combination of cycloheximide and ZSTK474 (+). Differences in caspase activity between control cells and C42Luc BIM1 or C42Luc BIM2 cells were statistically significant (p = 0.002 and p = 0.05, respectively). <b>E</b>) C42Luc cells or C42LucBIM2 cells that stably express BIM-targeting shRNA were transfected with lentiviral vector that express GFP and either scrambled (scr) or MCL-1 –specific shRNA2. Percent of apoptosis in GFP-positive cells was determined by time-lapse microscopy. At least 100 cells were counted for each treatment. Error bars show average and standard error of four visual fields. Qualitatively similar results were obtained in C42LucBIM1 cells. Beta actin was used as a loading control in (A) and (C).</p

Loss of MCL-1 sensitizes prostate cancer cells to ZSTK474-induced apoptosis.

<p><b>A</b>) Western blot analysis of C42Luc cells treated with either 5 µM ZSTK474, 100 µg/mL cycloheximide, or the combination for 6 hours. Whole cell lysates were probed for MCL-1, BCL-2, and BCL-XL to compare the effects of the protein synthesis inhibitor and probed for pBAD (S112) and pAKT (S473 and T308) to verify the effects of the PI3K inhibitor. <b>B</b>) Analysis of apoptosis by time-lapse microscopy. C42Luc cells were transiently transfected with lentiviral vector that encodes GFP and MCL-1-specific shRNA or scrambled control shRNA, and treated with 5 µM ZSTK474 48 h after transfection. The percentage of apoptotic cells at indicated time points after treatments was determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074561#pone-0074561-g001" target="_blank">Figure 1A</a>. At least 100 cells were counted for each treatment. Error bars show standard deviations from the average of four randomly chosen fields. *, <i>p</i><0.005; #, <i>p</i><0.001 Inset shows Western blot of endogenous MCL-1 and β-actin (loading control) levels in C42Luc cells 48 hours after infection with lentiviral vector expressing MCL-1 specific or scrambled shRNA. <b>C</b>) Caspase-3 activation assay of C42Luc cells transfected with shRNA targeting MCL-1 or scrambled control shRNA. Caspase activation was measured 6 hours after cells were treated with either DMSO or 5 µM ZSTK474. Treatments were added 48 hours after transfection. *, <i>p</i><0.01; **, <i>p</i><0.05. <b>D</b>) Analysis of apoptosis by time-lapse microscopy in C42Luc cells transfected with MCL-1-specific shRNA (striped bars) or scrambled shRNA (checkered bars) and doxycycline-inducible Flag-MCL-1. The cumulative percentage of apoptotic cells at indicated time points was determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074561#pone-0074561-g001" target="_blank">Figure 1A</a> 24 hours after doxycycline treatment. At least 100 cells were counted for each treatment. Error bars show standard deviations from the average of four randomly chosen fields. *, <i>p</i><0.01 #, <i>p</i><0.05. Inset shows Western blot of Flag-tagged MCL-1 immunoprecipitated with anti-FLAG resin and probed for MCL-1 expression.</p

Knockdown of MCL-1 and ectopic expression of phosphorylation-deficient BAD are sufficient to induce apoptosis in prostate cancer cells.

<p>C42Luc cells were co-transfected with either MCL-1-specific shRNA or scrambled shRNA in combination with either the BAD2SA or WT-BAD construct. 48 hours post-transfection, apoptosis was analyzed by time-lapse microscopy. At least 100 cells were counted for each treatment. Error bars show standard deviations from the average of four randomly chosen fields. *, <i>p</i><0.05 **, <i>p</i><0.005 ***, <i>p</i><0.001.</p

DU145 and PC3 cells are less sensitive to apoptosis than C42Luc cells.

<p><b>A</b>) Treatment with combination of cycloheximide and ZSTK474 triggers MCL-1 loss in PC3, C42Luc, and DU145 cells, as well as BAD dephosphorylation in PTEN-deficient PC3 and C42Luc cells. Cell lysates prepared 6 hours after treatments were probed for pS112BAD, total BAD, and MCL-1, using β-actin as a loading control. Apoptosis in C42Luc, PC3, and DU145 cells was assessed by (<b>B</b>) measuring caspase 3 activity with fluorogenic substrate or (<b>C</b>) by time lapse microscopy. At least 100 cells were counted for each treatment. Error bars show standard deviations from the average of four randomly chosen fields. <b>D</b>) Knockdown of MCL-1 and ectopic expression of phosphorylation-deficient BAD were sufficient to induce apoptosis in prostate cancer cells. PC3 or DU145 cells were co-transfected with MCL-1-specific shRNA or scrambled control shRNA in combination with either a BAD2SA or WT-BAD expression construct. 48 hours post-transfection, apoptosis was analyzed by time-lapse microscopy. At least 100 cells were counted for each treatment. Error bars show standard deviations from the average of four randomly chosen fields.</p