Vascular endothelial growth factor regulates myeloid cell leukemia-1 expression through neuropilin-1-dependent activation of c-MET signaling in human prostate cancer cells

Abstract

Background: Myeloid cell leukemia-1 (Mcl-1) is a member of the Bcl-2 family, which inhibits cell apoptosis by sequestering pro-apoptotic proteins Bim and Bid. Mcl-1 overexpression has been associated with progression in leukemia and some solid tumors including prostate cancer (PCa). However, the regulatory mechanism for Mcl-1 expression in PCa cells remains elusive.

Results: Immunohistochemical analyses revealed that Mcl-1 expression was elevated in PCa specimens with high Gleason grades and further significantly increased in bone metastasis, suggesting a pivotal role of Mcl-1 in PCa metastasis. We further found that vascular endothelial growth factor (VEGF) is a novel regulator of Mcl-1 expression in PCa cells. Inhibition of endogenous Mcl-1 induced apoptosis, indicating that Mcl-1 is an important survival factor in PCa cells. Neuropilin-1 (NRP1), the "co-receptor" for VEGF165 isoform, was found to be highly expressed in PCa cells, and indispensible in the regulation of Mcl-1. Intriguingly, VEGF165 promoted physical interaction between NRP1 and hepatocyte growth factor (HGF) receptor c-MET, and facilitated c-MET phosphorylation via a NRP1-dependent mechanism. VEGF165 induction of Mcl-1 may involve rapid activation of Src kinases and signal transducers and activators of transcription 3 (Stat3). Importantly, NRP1 overexpression and c-MET activation were positively associated with progression and bone metastasis in human PCa specimens and xenograft tissues.

Conclusions: This study demonstrated that Mcl-1 overexpression is associated with PCa bone metastasis. Activation of VEGF165-NRP1-c-MET signaling could confer PCa cells survival advantages by up-regulating Mcl-1, contributing to PCa progression.

Figure 1

Mcl-1 is a survival factor in human PCa cells. (a) IHC staining of Mcl-1 in human PCa tissue microarray consisting of normal adjacent tissues, primary PCa and bone metastases. (b) Intracardiac (i.c) injection of ARCaP_M cells in athymic mice resulted in metastases to bone and soft tissues. The ARCaP_M-C2 subclone was derived from metastatic bone tissues after two rounds of intracardiac inoculation of ARCaP_M cells. (c) Endogenous Mcl-1 expression in the ARCaP_M model was examined by RT-PCR and western blotting analyses. (d) Effect of Mcl-1 siRNA on ARCaP_M cell viability. Subconfluent ARCaP_M cells on a 6-well plate were transiently transfected with Mcl-1 siRNA (30 nM) for 72 h. Endogenous expression of Mcl-1 at the mRNA and protein levels, and cell viability of ARCaP_M cells as counted with trypan blue staining, were significantly inhibited by Mcl-1 siRNA treatment compared to the control. (e) Effects of Mcl-1 siRNA on ARCaP_M cell apoptosis. Subconfluent ARCaP_M cells were transfected with Mcl-1 siRNA or control siRNA for 72 h, expression of annexin V was measured by FACS.

Figure 2

VEGF₁₆₅ regulates Mcl-1 expression in PCa cells. (a) VEGF expression in ARCaP_M and ARCaP_M-C2 cells, as determined by RT-PCR and ELISA (conditioned medium). (b) Effects of recombinant VEGF₁₆₅ on cell proliferation as determined by MTS assay. Top, ARCaP_M cells were seeded in 96-well plates (1 × 10³cells/well) for 24 h, and serum-starved overnight. The cells were further incubated with varying concentrations of VEGF₁₆₅ in serum-free T-medium for 72 h. Bottom, ARCaP_M cells were incubated with PBS or VEGF₁₆₅ (50 ng/ml) in serum-free T-medium for varying times. (c) VEGF₁₆₅ effects on Mcl-1 expression. ARCaP_M cells were treated with VEGF₁₆₅ at indicated concentrations and times, and Mcl-1 mRNA expression was measured. For immunoblotting, ARCaP_M cells were incubated with VEGF₁₆₅ (10 ng/ml) for 72 h. (d) Effects of VEGF siRNA on Mcl-1 expression. ARCaP_M cells were transfected with VEGF siRNA or control siRNA (80 nM) for 72 h.

Figure 3

NRP1 is required for basal expression and VEGF₁₆₅ induction of Mcl-1 in ARCaP_M cells. (a) Expression of VEGF-Rs in PCa cells and HUVEC. (b) Effect of ectopic expression of NRP1 on Mcl-1 basal level. Subconfluent ARCaP_M cells on 6-well plates were transfected with pCMV-NRP1 or pCMV-XL4 (16 μg) for 48 h (for RT-PCR) or 72 h (for immunoblotting). (c) Effects of NRP1 siRNA on Mcl-1 basal expression. ARCaP_M cells were transfected with NRP1 siRNA or control siRNA (60 nM) for 48 h (for RT-PCR) or 72 h (for immunoblotting). (d) Effects of NRP1 siRNA on VEGF₁₆₅ induction of Mcl-1. ARCaP_M cells were transfected with NRP1 siRNA or control siRNA for 48 h, respectively. Cells were then serum-starved overnight, and treated with VEGF₁₆₅ (10 ng/ml) or PBS for 2 h.

Figure 4

c-MET signaling is required for VEGF₁₆₅ induction of Mcl-1 in ARCaP_M cells. (a) Effects of c-MET inhibition on Mcl-1 expression. ARCaP_M cells were transfected with c-MET siRNA or control siRNA (30 nM) for 72 h. (b) Effects of recombinant HGF treatment (10 ng/ml) on Mcl-1 expression at RNA (0, 2, 4 and 6 h) and protein levels (72 h). (c) ARCaP_M cells were transfected with c-MET siRNA or control siRNA for 48 h, serum-starved overnight, and treated with VEGF₁₆₅ (10 ng/ml) for 2 h. (d) ARCaP_M cells were treated with PHA-665752 (0.5 μM) or dimethyl sulfoxide (DMSO) for 2 h, before treatment with VEGF₁₆₅ (10 ng/ml) or PBS for 2 h.

Figure 5

VEGF₁₆₅ induces c-MET activation through a NRP1-dependent mechanism. (a) Effects of NRP1 depletion on VEGF₁₆₅-mediated c-MET phosphorylation. ARCaP_M cells were transfected with NRP1 siRNA or control siRNA for 48 h, serum-starved overnight, then treated with VEGF₁₆₅ (10 ng/ml) for 60 min. (b) Immunoprecipitation assay of NRP1-c-MET interaction. Serum-starved ARCaP_M cells were treated with VEGF₁₆₅ (10 ng/ml) for the indicated times, and immunoprecipitated with anti-NRP1 (upper), or anti-c-MET (low) antibody. (c) Co-localization of NRP1 and c-MET or p-c-MET. Serum-starved ARCaP_M cells were treated with VEGF₁₆₅ (10 ng/ml) or PBS for the indicated times. Immunofluorescence staining of NRP1, c-MET or p-c-MET was performed and visualized by confocal microscopy. Arrows indicate co-localization of NRP1 and c-MET or p-c-MET.

Figure 6

A proposed model for VEGF₁₆₅ regulation of Mcl-1 in PCa cells. NRP1 may be constitutively associated with c-MET on plasma membrane. VEGF₁₆₅ engagement recruits c-MET into the protein complex and promotes its interaction with NRP1, thereby enhancing phosphorylation of c-MET. Src kinase-Stat3 signaling is subsequently activated, resulting in nuclear translocation of p-Stat3 and activation of Mcl-1 expression. Increased intracellular Mcl-1 protects PCa cells from apoptosis.

Figure 7

Src kinase-Stat3 signaling mediates VEGF₁₆₅ induction of Mcl-1 expression in ARCaP_M cells. (a) Effects of NRP1 inhibition on phosphorylation of Src kinases and Stat3. ARCaP_M cells were transfected with NRP1 siRNA or control siRNA for 72 h. (b) Effects of VEGF₁₆₅ on the phosphorylation of Src kinases and Stat3. ARCaP_M cells were treated with VEGF₁₆₅ (10 ng/ml) for the indicated time, and immunoblotting was performed on total lysates (left) and nuclear extracts and cytoplasmic proteins (right). (c) Effects of Src kinases on VEGF₁₆₅ regulation of Mcl-1. ARCaP_M cells were treated with PP2 (10 μM) or DMSO for 2 h before treatment with VEGF₁₆₅ or PBS. (d) Effects of Stat3 depletion on VEGF₁₆₅ induction of Mcl-1. ARCaP_M cells were transfected with Stat3 siRNA or control siRNA (80 nM) for 48 h, serum-starved overnight, then treated with VEGF₁₆₅ (10 ng/ml) or PBS for 2 h.

Figure 8

Expression of NRP1 and p-c-MET is associated with bone metastatic status of human PCa specimens and the ARCaP_M model. IHC analyses of NRP1 expression in human normal/benign, cancerous and metastatic prostatic tissue specimens (a) and primary and bone metastatic tissue specimens from ARCaP_M model (b), and of p-c-MET expression in human PCa progression (c) and ARCaP_M xenografts (d). Arrows indicate positively-stained cells.