Protein kinase D1 inhibits cell proliferation through matrix metalloproteinase-2 and matrix metalloproteinase-9 secretion in prostate cancer

Abstract

We and others previously showed that protein kinase D1 (PKD1) is downregulated in several cancers including prostate; interacts with E-cadherin, a major cell adhesion epithelial protein; and causes increased cell aggregation and decreased motility of prostate cancer cells. In this study, we show that PKD1 complexes with beta3-integrin, resulting in activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase-ERK pathway, which causes increased production of matrix metalloproteinase (MMP)-2 and MMP-9, that is associated with shedding of soluble 80 kDa E-cadherin extracellular domain. Interestingly, decreased cell proliferation following PKD1 transfection was rescued by MMP-2 and MMP-9 inhibitors and augmented by recombinant MMP-2 (rMMP-2) and rMMP-9 proteins, suggesting an antiproliferative role for MMPs in prostate cancer. Translational studies by in silico analysis of publicly available DNA microarray data sets show a significant direct correlation between PKD1 and MMP-2 expression in human prostate tissues. The study shows a novel mechanism for antiproliferative effects of PKD1, a protein of emerging translational interest in several human cancers, through increased production of MMP-2 and MMP-9 in cancer cells.

Figure 1

PKD1 increases production of MMP-2 and MMP-9 in prostate cancer cells. (A) Non-transfected (Cont.), GFP transfected (GFP) and PKD1-GFP transfected (PKD1) PC3 and DU145 cells were lysed and immunoblotted with GFP and β-actin antibodies, respectively. (B) Conditioned media were collected from same cells and subjected to gelatin zymography. (C) Same cell lysates as in (A) were immunoblotted with MMP-2, MMP-9 and β-actin, antibodies. (D) DU145 cells were transfected with shLuc and shPKD1 constructs. After 24h, cells were lysed and immunoblotted with PKD1 and β-actin antibodies, respectively (top). Conditioned media were collected from the same cell types and subjected to gelatin zymography (middle). Mean value of densitometric analysis of three independent experiments was shown in chart (bottom). Bar; standard deviation (SD) of mean, * P<0.05.

Figure 2

Mek-Erk phosphorylation is necessary for PKD1-induced MMP-2 and MMP-9 secretion in PC cells (A) Non-transfected (Cont.), GFP and PKD1-GFP transfected PC3 and DU145 cells lysates were immunoblotted with indicated antibodies. (B) DU145 cells were transfected with shLuc and shPKD1 constructs. After 24h, the cells were lysed and immunoblotted with PKD1, β-actin, p-Erk or Erk antibodies, respectively. The relative densitometric data of band intensity for p-Erk is shown in bottom of each panel. (C) PKD1 transfected stable PC3 cells were incubated with DMSO (Cont.) or U0126 (5µM) for 1h and cell lysates were immunoblotted with p-Erk, Erk, PKD1 or β-actin antibodies. Conditioned media were collected from each condition and subjected to gelatin zymography. Mean value of densitometric analysis of three independent experiments was shown in chart (bottom). Bar; standard deviation (SD) of mean, * P<0.05 (D) PC3-PKD1 cells were transfected with control siRNA or siErk. After 24 hours of transfection, cells were lysed and immunoblotted with Erk and β-actin antibodies. Conditioned media were collected from each condition and subjected to gelatin zymography (bottom of each panel). Relative densitometric data of band intensity were shown in bottom of each panel.

Figure 3

PKD1 and β3-integrin form a protein complex and regulate the secretion of MMP-2 and -9 in PC3 cells. (A) Equal amount of proteins were immunoprecipitated with anti-PKD1 or anti-β3-integrin and immunoblotted with β3-integrin and PKD1 antibodies. (B) PKD1-GFP transfected PC3 cells were incubated with RGE-peptide, a control peptide, and RGD-peptide, a specific inhibitor of β3-integrin, immunostained with β3-integrin (red), PKD1-GFP (green) and merged (Yellow). (C) RGE-peptide or RGD-peptide incubated PKD1 overexpressed PC3 cells were lysed and immunoblotted with p-Erk, Erk, PKD1 and β-actin antibodies. (D) Conditioned media were collected and subjected to gelatin zymography (top and bottom). Mean value of densitometric analysis of three independent experiments for top-figure was shown in chart (middle). Bar; standard deviation (SD) of mean, * P<0.05.

Figure 4

PKD1 enhances E-cadherin shedding through MMP-2 and MMP-9 and suppresses cell proliferation. (A) Conditioned media were collected from Control, GFP and PKD1-GFP over expressing DU145 cells and immunoblotted with E-cadherin antibody (first panel). Lysates from same cells were immunoblotted with E-cadherin and β-actin antibodies, respectively. (B) PKD1 over expressing DU145 cells were incubated with DMSO (Cont.), 5µM MMP-2 inhibitor or 200nM MMP-9 inhibitor for 24 hrs and conditioned media were immunoblotted with E-cadherin antibody. (C and D) MTS and [³H]-thymidine incorporation assay was performed with GFP and PKD1-GFP over expressing DU145 cells. The cells were incubated with DMSO (Cont.), inhibitor of MMP-2 or -9 for 48 h. Cell proliferation was measured by MTS and [³H]-thymidine incorporation assay and expressed as a percentage of control. Data are the means of three independent experiments with triplicate samples. Bar; standard deviation (SD) of mean, * P<0.05. Results from RT-PCR of PCNA and GAPDH from the cells treated with MMP-2 and -9 inhibitors are also shown (Fig.4C bottom).

Figure 5

Recombinant MMP-2 and MMP-9 suppressed proliferation and soft agar colony formation of PKD1-overexpressed PC cell. (A and B) GFP and PKD1-GFP over expressing DU145 cells were incubated with DMSO (Cont.), 300ng/ml rMMP-2, 300ng/ml rMMP-9 or both (rMMP-2/9) for 48 hrs. Cell proliferation was measured by MTS and [³H]-thymidine incorporation assay and percentage of cell proliferation was calculated compared to control. Bars; standard deviation (SD) of mean, * P<0.05. (B) Soft agar colony formation assay of same cells with control or rMMP-2 and rMMP-9 (rMMP-2/9) treatment. Picture was taken at 400X magnification. The numbers of colonies were counted and mean values of three independent experiments are shown. Bar; standard deviation (SD) of mean, * P<0.05.

Figure 6

The expression of MMP-2 positively correlates with PKD1 (PRKD1) in human prostate cancer tissue. (A) Box plots of PRKD1, MMP-2 and MMP-9 expression levels in Benign (Ben), Primary (Prim) and Metastatic (Meta) human prostate cancer tissue microarray data set (GSE3325). The box represents the interquartile range of data with various samples and line through that box represents the median of the distribution. The range is indicated by the whiskers on the plot. Benign tumor, n = 6; Primary tumor, n = 7; and Metastatic tumor samples, n = 6. (B) Scattered plots of PRKD1 versus MMP-2 and MMP-9 of all samples displayed in (A). (C) The heat map presentation of PRKD1, MMP-2 and MMP-9 expression in Normal (Norm), Primary tumor and Metastatic (Meta) prostate tissue from microarray data set GSE6919.