Beta-catenin mediates alteration in cell proliferation, motility and invasion of prostate cancer cells by differential expression of E-cadherin and protein kinase D1

Abstract

We have previously demonstrated that Protein Kinase D1 (PKD1) interacts with E-cadherin and is associated with altered cell aggregation and motility in prostate cancer (PC). Because both PKD1 and E-cadherin are known to be dysregulated in PC, in this study we investigated the functional consequences of combined dysregulation of PKD1 and E-cadherin using a panel of human PC cell lines. Gain and loss of function studies were carried out by either transfecting PC cells with full-length E-cadherin and/or PKD1 cDNA or by protein silencing by siRNAs, respectively. We studied major malignant phenotypic characteristics including cell proliferation, motility, and invasion at the cellular level, which were corroborated with appropriate changes in representative molecular markers. Down regulation or ectopic expression of either E-cadherin or PKD1 significantly increased or decreased cell proliferation, motility, and invasion, respectively, and combined down regulation cumulatively influenced the effects. Loss of PKD1 or E-cadherin expression was associated with increased expression of the pro-survival molecular markers survivin, beta-catenin, cyclin-D, and c-myc, whereas overexpression of PKD1 and/or E-cadherin resulted in an increase of caspases. The inhibitory effect of PKD1 and E-cadherin on cell proliferation was rescued by coexpression with beta-catenin, suggesting that beta-catenin mediates the effect of proliferation by PKD1 and E-cadherin. This study establishes the functional significance of combined dysregulation of PKD1 and E-cadherin in PC and that their effect on cell growth is mediated by beta-catenin.

Fig. 1

Enhancement of prostate cancer (PC) cell proliferation by down-regulation of PKD1 and E-cadherin. A: Western blot analysis of E-cadherin/PKD1 following transfection with shRNA PKD1 or siRNA E-cadherin into PC cell lines (ALVA-41, LNCaP, C4-2, and DU-145). Nontransfected cells or empty vector transfected cells were used as controls. Successful protein down regulation is demonstrated by gene silencing. B: Effect of PKD1 and E-cadherin down regulation on the proliferation of PC cell lines. Four PC cell lines (ALVA-41, LNCaP, C4-2, and DU-145) were transfected with shRNA PKD1 and/or siRNA E-cadherin. Control cells were transfected with respective empty vector or control siRNA and additional control included untransfected cells. After 72 h of transfection, cell proliferation was measured by MTS assay, and proliferation of transfected cells was expressed as a percentage increase of proliferation of empty vector transfected control cells. Data are the means of three experiments with triplicate samples. Bars are mean ± SEM and * indicates statistically significant difference (P <0.05). Expression of cell proliferation marker PCNA is shown on the top on each panel, which is consistent with MTS assay results.

Fig. 2

Knockdown of PKD1 and E-cadherin increases cell motility and invasiveness. Effects of E-cadherin and PKD1 down regulation on (A) colony-forming ability and (B) invasion of ALVA-41, LNCaP, C4-2, and DU-145 cells were examined. Cells were transfected with shRNA PKD1, siRNA E-cadherin or respective controls, as indicated and additional control included untransfected cells. After 24 h, cells were cultured on soft agar to examine colony-forming ability or on Matrigel chambers to evaluate migration. Number of colonies were counted 21 days later and cells that migrated through the Matrigel were counted following 22 h of platting. Data are the means of three experiments with triplicate wells or plates. Bars are mean ± SEM and * indicates statistically significant difference (P <0.05).

Fig. 3

Inhibition of PC cell proliferation by over expression of PKD1 and E-cadherin. A: Western blot analysis of PKD1 and E-cadherin after ectopic expression of PKD1 and E-cadherin in PC cell lines (ALVA-41, LNCaP, C4-2, and DU-145). A significant increase in PKD1 and E-cadherin levels in transfected cell lines compared to controls is shown in the figure. B: Effect of PKD1 and E-cadherin over expression on the proliferation of PC cell lines. Four cell lines (ALVA-41, LNCaP, C4-2, and DU-145) were transfected with PKD1 and/or E-cadherin. Control cells were transfected with respective empty vector or left untransfected. Three days later, cell proliferation was measured by MTS assay, and proliferation of transfected cells was expressed as a percentage decrease of proliferation of empty vector transfected control cells. Data are the means of three experiments with triplicate samples. Bars are mean ± SEM and * indicates statistically significant difference (P <0.05). Expression of cell proliferation marker PCNA and cell apoptosis marker caspase C is shown on the top on each panel, which is consistent with MTS cell growth and viability assay.

Fig. 4

Inhibition of anchorage independent growth and invasiveness of PC cells by ectopic expression of PKD1 and E-cadherin. A: Cells transfected with PKD1 and/or E-cadherin or mock transfected were harvested 24 h after transfection and plated in media containing soft agar. Colonies were allowed to form for 21 days and then counted. B: PKD1, E-cadherin, or mock transfected cells were plated on Matrigel chambers to evaluate migration. Cells that migrated through the Matrigel were counted. Data are the means of three experiments with triplicate wells/plates. Bars are mean ± SEM and * indicates statistically significant difference (P <0.05).

Fig. 5

Loss of PKD1 and E-cadherin expression in LNCaP cells is associated with increased expression of survivin, cyclin-D and c-myc and gain of PKD1 and E-cadherin expression is associated with increased expression of caspases. A: LNCaP cells were transfected with shRNA of PKD1, siRNA E-cadherin or PKD1, E-cadherin expression vectors as indicated. Following 72 h of incubation, lysates were analyzed by Western blot with indicated antibodies. Loss or gain of PKD1 and E-cadherin is associated with increased expression of pro-survival proteins (survivin, β-catenin, and c-myc) or proapoptotic caspases, respectively. B: Enzyme activities of cell lysates toward tetrapeptide caspase substrates in LNCaP cells (LNCaP cells transfected with shRNA of PKD1, siRNA E-cadherin or PKD1, E-cadherin expression vectors as indicated) after 72 h of incubation. The chromogenic substrates were DEVD-pNA (caspase-3), IETD-pNA (caspase-8), and LEHDpNA (caspase-9). The caspase activity is expressed as fold relative to untreated controls and represented as the means of two experiments carried out in triplicate. Bars are mean ± SEM and * indicates statistically significant difference (P <0.05).

Fig. 6

A: Suppression of β-catenin rescues the increased cellular proliferative effect induced by knockdown of PKD1 and E-cadherin; four PC cell lines (ALVA-41, LNCaP, C4-2, and DU-145) were transfected with shRNA PKD1, siRNA E-cadherin or cotransfected with shRNAi of β-catenin as indicated. Control cells were transfected with respective control constructs. B: Co-transfection with β-catenin abrogates the proliferation inhibitory effect of ectopic expression of PKD1 and E-cadherin. PC cells were transfected with PKD1, E-cadherin or cotransfected with β-catenin as indicated. Control cells were transfected with respective control constructs. Three days later, cell proliferation was measured by MTS assay. Data are the means of two experiments with triplicate samples. Bars are mean ± SEM and * indicates statistically significant difference (P <0.05).