Silencing of protein kinase D2 induces glioma cell senescence via p53-dependent and -independent pathways

Abstract

Background: Glioblastoma multiforme (GBM) is a highly aggressive tumor of the central nervous system with a dismal prognosis for affected patients. Aberrant protein kinase C (PKC) signaling has been implicated in gliomagenesis, and a member of the PKC-activated protein kinase D (PRKD) family, PRKD2, was identified as mediator of GBM growth in vitro and in vivo.

Methods: The outcome of PRKD2 silencing and pharmacological inhibition on glioma cell proliferation was established with different glioma cell lines. Western blotting, senescence assays, co-immunoprecipitation, fluorescence activated cell sorting, quantitative PCR, and immunofluorescence microscopy were utilized to analyze downstream signaling.

Results: RNA-interference (21-mer siRNA) and pharmacological inhibition (CRT0066101) of PRKD2 profoundly inhibited proliferation of p53(wt) (U87MG, A172, and primary GBM2), and p53(mut) (GM133, T98G, U251, and primary Gli25) glioma cells. In a xenograft experiment, PRKD2 silencing significantly delayed tumor growth of U87MG cells. PRKD2 silencing in p53(wt) and p53(mut) cells was associated with typical hallmarks of senescence and cell cycle arrest in G1. Attenuated AKT/PKB phosphorylation in response to PRKD2 silencing was a common observation made in p53(wt) and p53(mut) GBM cells. PRKD2 knockdown in p53(wt) cells induced upregulation of p53, p21, and p27 expression, decreased phosphorylation of CDK2 and/or CDK4, hypophosphorylation of retinoblastoma protein (pRb), and reduced transcription of E2F1. In p53(mut) GM133 and primary Gli25 cells, PRKD2 silencing increased p27 and p15 and reduced E2F1 transcription but did not affect pRb phosphorylation.

Conclusions: PRKD2 silencing induces glioma cell senescence via p53-dependent and -independent pathways.

Fig. 1.

Silencing and pharmacological inhibition of PRKD2 inhibits GBM cell growth. (A) Effect of PRKD2 RNAi on glioma cell proliferation. Two different siRNA constructs (siP5; siP6) were used to silence PRKD2 expression. Untreated cells and cells transfected with nontargeting siRNA (siScr) were used as controls. Cells were harvested and counted on day 6 post silencing. Results represent mean ± SD of relative cell numbers normalized to untreated cells from one representative experiment done in triplicate. The p53 status of the cells is indicated. Silencing efficacy is shown in the right panel. (B) Effects of pharmacological PRKD inhibition on GBM cell proliferation. The indicated p53^wt and p53^mut GBM cells were incubated in the presence of vehicle dimethyl sulfoxide (DMSO) or 0.5 and 1 µM CRT0066101 (added in DMSO). Cells were harvested and counted 3 days post CRT0066101/DMSO addition. Results represent mean ± SD of relative cell numbers normalized to untreated cells. Data from one representative experiment performed in triplicate are shown. (C) Analysis of cell cycle distribution and DNA synthesis of U87MG (upper panels) and GM133 (lower panels) cells. Two days post transfection control (untreated and siScr transfected) and PRKD2-silenced (siP5) cells were stained with propidium iodide (PI) (left panel) or bromodeoxyuridine (BrdU) (right panel) and analyzed by flow cytometry. Results are expressed as percentage of total cells and represent the mean values from 3 (PI) and 2 (BrdU) independent experiments. (**P < .01, *P < .05, one-way ANOVA). (D) Detection of Annexin V positive U87MG and GM133 cells. Four days post transfection cells (untreated, siScr, and siP5 transfected) were stained with APC Annexin V (‘A’) and PI and analyzed by fluorescence activated cell sorting. The percentages of A^-/PI⁻ (viable), A⁺/PI⁻ (early apoptotic), A⁺/PI⁺ (late apoptotic) and A⁻/PI⁺ are shown. Mean values from 2 experiments are presented. (E) PRKD2 silencing in glioma xenografts. Forty-eight hours after transfection, 1 × 10⁶ control or treated U87MG cells (siScr and siP5) were subcutaneously injected into the flank of SCID mice (n = 5 per group). Xenograft size was measured 3 times a week, and tumor volume was calculated. Results are expressed as mean ± SEM. Bar indicates delayed growth onset of siPRKD2 xenografts compared with siSCR xenografts (one-way ANOVA, **P < .01). Two animals (shown in grey circles) receiving silenced cells did not develop tumors until day 76 post inoculation.

Fig. 1.

Silencing and pharmacological inhibition of PRKD2 inhibits GBM cell growth. (A) Effect of PRKD2 RNAi on glioma cell proliferation. Two different siRNA constructs (siP5; siP6) were used to silence PRKD2 expression. Untreated cells and cells transfected with nontargeting siRNA (siScr) were used as controls. Cells were harvested and counted on day 6 post silencing. Results represent mean ± SD of relative cell numbers normalized to untreated cells from one representative experiment done in triplicate. The p53 status of the cells is indicated. Silencing efficacy is shown in the right panel. (B) Effects of pharmacological PRKD inhibition on GBM cell proliferation. The indicated p53^wt and p53^mut GBM cells were incubated in the presence of vehicle dimethyl sulfoxide (DMSO) or 0.5 and 1 µM CRT0066101 (added in DMSO). Cells were harvested and counted 3 days post CRT0066101/DMSO addition. Results represent mean ± SD of relative cell numbers normalized to untreated cells. Data from one representative experiment performed in triplicate are shown. (C) Analysis of cell cycle distribution and DNA synthesis of U87MG (upper panels) and GM133 (lower panels) cells. Two days post transfection control (untreated and siScr transfected) and PRKD2-silenced (siP5) cells were stained with propidium iodide (PI) (left panel) or bromodeoxyuridine (BrdU) (right panel) and analyzed by flow cytometry. Results are expressed as percentage of total cells and represent the mean values from 3 (PI) and 2 (BrdU) independent experiments. (**P < .01, *P < .05, one-way ANOVA). (D) Detection of Annexin V positive U87MG and GM133 cells. Four days post transfection cells (untreated, siScr, and siP5 transfected) were stained with APC Annexin V (‘A’) and PI and analyzed by fluorescence activated cell sorting. The percentages of A^-/PI⁻ (viable), A⁺/PI⁻ (early apoptotic), A⁺/PI⁺ (late apoptotic) and A⁻/PI⁺ are shown. Mean values from 2 experiments are presented. (E) PRKD2 silencing in glioma xenografts. Forty-eight hours after transfection, 1 × 10⁶ control or treated U87MG cells (siScr and siP5) were subcutaneously injected into the flank of SCID mice (n = 5 per group). Xenograft size was measured 3 times a week, and tumor volume was calculated. Results are expressed as mean ± SEM. Bar indicates delayed growth onset of siPRKD2 xenografts compared with siSCR xenografts (one-way ANOVA, **P < .01). Two animals (shown in grey circles) receiving silenced cells did not develop tumors until day 76 post inoculation.

Fig. 2.

Interference with PRKD2 expression induces cellular senescence. (A) Untreated U87MG (left panels) and GM133 (right panels) cells and cells transfected with siScr or siP5 were stained for SA-β-Gal activity (blue) on day 3 (U87MG) and day 5 (GM133) post transfection. (B) Bar graphs represent cell surface area of control and transfected (siScr or siP5) cells. Three (U87MG) and 5 (GM133) days post transfection, 4 randomly chosen micrograph fields (in triplicates) were recorded. The total area occupied by the cells and the total cell number was calculated with ImageJ. Mean surface area/cell was calculated as total cell area/total cell number (mean ± SEM, one-way ANOVA, ***P < .001, **P < .01, *P < .05).

Fig. 3.

PRKD2 modulates AKT activity in glioma cells. (A) Three days post silencing, U87MG and GM133 were stimulated with 10 ng/ml PDGF for 5 and 30 minutes. Total cell lysates were immunoblotted, and activation of indicated proteins was monitored using phospho-specific antibodies. To ensure comparable loading of lysates, the corresponding pan-proteins or actin were used as controls. One representative immunoblot (of at least 3 independent experiments) is shown. AKT antibodies used recognize AKT isoforms 1, 2, and 3. (B) The effect of PRKD2 silencing on AKT phosphorylation was studied in cytosolic and nuclear fractions by Western blotting. C = untreated cells, S = siScr, P5 = siP5. (C) Co-immunoprecipitation experiments indicating physical interaction of PRKD2 and AKT. Protein extracts of U87MG cells (3 independent dishes, ‘1, 2, 3′) were immunoprecipitated with the indicated antibodies, followed by immunoblotting. The association of proteins was confirmed by reverse immunoprecipitation. Input levels were detected to ensure comparable loading. Sham precipitation (“-”) was performed to exclude unspecific immunoreactions. (D) The indicated cell lines were incubated in the presence of 5 and 10 µM triciribine. Ethanol was used as vehicle control. Five days after addition of triciribine/ethanol, cells were harvested and counted. Results represent mean ± SD (n = 4) of relative cell numbers normalized to untreated cells. (E) Two different siRNA constructs (si1; si2) were used to silence AKT1 and AKT3 expression. Untreated cells and cells transfected with nontargeting siRNA (siScr) were used as controls. Cells were harvested and counted on day 6 post silencing. Results represent mean ± SD of relative cell numbers normalized to untreated cells from one representative experiment done in triplicates.

Fig. 4.

PRKD2 silencing upregulates expression of tumor suppressor proteins. U87MG and GM133 cells were transfected with nontargeting (S) and PRKD2 targeting siRNA (P5), and expression analysis of tumor suppressor proteins was performed by immunoblotting in cytosolic and nuclear fractions 3 days post transfection. Untreated cells (“C”) were used as controls. Nuclear and cytoplasmic lysates were analyzed for expression of PRKD2, p53, p21, p27, and p15. Lamin A/C, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin was used as loading controls.

Fig. 5.

PRKD2 silencing interferes with the G1/S transition complex. U87MG (left panels) and GM133 (right panels) cells were transfected with nontargeting (S) and PRKD2 targeting (P5) siRNA. C = control, untreated cells. One representative immunoblot out of 3 independent experiments is shown. (A) Analysis of phosphorylated and total CDK2 and CDK4 proteins was performed by immunoblotting in cytosolic and nuclear fractions 3 days post transfection. Lamin A/C and β-actin were used as loading controls. (B) Phosphorylation of pRb on Ser⁷⁸⁰, Ser⁷⁹⁵, and Ser^807/811 and expression of total pRb was determined by immunoblotting in nuclear and cytoplasmic fractions. Lamin A/C and β-actin were used as loading controls. (C) CREB was analyzed for phosphorylation on Ser¹³³ by immunoblotting, and total CREB was used as loading control. The phospho-antibody used also detects the phosphorylated form of CREB-related protein ATF-1. The lower line shows c-Myc expression in control and silenced cells. (D) On day 4, (U87MG) and 6 (GM133) post transfection target gene expression was analyzed by qPCR using validated primer pairs. HPRT1 was used as housekeeping gene. Relative gene expression of target genes is presented in relation to scrambled RNAi. Results represent mean ± SD from 3 biological replicates (***P < .001). Gene expression ratios were calculated by REST as described in Materials and Methods.

Fig. 6.

Summary of signaling events leading to PRKD2-dependent induction of senescence in p53^wt and p53^mut GBM cells. RTK and/or GPCR signaling induces PKCs and downstream PRKD2 activation. Silencing of PRKD2 attenuates AKT activation (and MAPK activation in U87MG cells). In p53^wt U87MG and primary GBM2 cells, this leads to increased p53, p21, and p27 expression. As a consequence, diminished cyclin-CDK activation, hypophosphorylation of pRb, and reduced transcription of E2F target genes induce senescence. In p53^mut GM133 and Gli25 primary cells, PRKD2 silencing results in decreased AKT activation, upregulation of p15 and p27, G1/S arrest, and downregulated E2F1 transcription. Also this pathway induces senescence; however, the signaling modules downstream of p15 and p27 that are ultimately responsible for the induction of senescence remain to be elucidated.