STAP-2 protein promotes prostate cancer growth by enhancing epidermal growth factor receptor stabilization

Abstract

Signal-transducing adaptor family member-2 (STAP-2) is an adaptor protein that regulates various intracellular signaling pathways and promotes tumorigenesis in melanoma and breast cancer cells. However, the contribution of STAP-2 to the behavior of other types of cancer cells is unclear. Here, we show that STAP-2 promotes tumorigenesis of prostate cancer cells through up-regulation of EGF receptor (EGFR) signaling. Tumor growth of a prostate cancer cell line, DU145, was strongly decreased by STAP-2 knockdown. EGF-induced gene expression and phosphorylation of AKT, ERK, and STAT3 were significantly decreased in STAP-2-knockdown DU145 cells. Mechanistically, we found that STAP-2 interacted with EGFR and enhanced its stability by inhibiting c-CBL-mediated EGFR ubiquitination. Our results indicate that STAP-2 promotes prostate cancer progression via facilitating EGFR activation.

Keywords: STAT3; adaptor protein; c-CBL; prostate cancer; protein degradation; signal transduction.

Figure 1.

STAP-2 enhances proliferation of DU145 cells in vitro and in vivo. A, qPCR analysis for mRNA levels of STAP-2 in various cancer cell lines and human tissues. B and C, expression levels of STAP-2 in STAP-2–knockdown DU145 cells were determined by qPCR (B) and immunoblotting (IB) (C). TCL, total cell lysates. D, viable cells of control or STAP-2 shRNA–expressing DU145 cells were measured by a WST assay. E, control or STAP-2 shRNA–expressing DU145 cells were cultured in DMEM containing 1% FBS for the indicated times, and then the surviving cells were measured by a WST assay. F, STAP-2–knockdown DU145 cells were transfected with pcDNA3.1-Myc-STAP-2, and then their cell proliferation was measured by a WST assay at 72 h post-transfection, as in D. An aliquot of total cell lysates was immunoblotted by anti-Myc and anti-actin antibodies. G, expression levels of STAP-2 in STAP-2–knockdown LNCaP cells were determined by immunoblotting. H, viable cells of control or STAP-2 shRNA–expressing LNCaP cells were measured by a WST assay. I, DU145 cells (5.0 × 10⁶ cells) were subcutaneously injected into BALB/c nude mice (day 0), and tumor volume was measured. Tumors were established in 5 of 5 mice (shControl) and 3 of 5 mice (shSTAP-2). J, weight of tumors and spleens from the tumor-bearing mice at day 33. n = 3; mean values and S.E. (error bars) are depicted. *, p < 0.05; **, p < 0.01; ***, p < 0.005 (paired Student's t test).

Figure 2.

STAP-2 up-regulates EGFR signaling. A, control or STAP-2 shRNA–expressing DU145 cells were stimulated with 30 ng/ml EGF for the indicated periods. The lysate was immunoprecipitated (IP) with anti-EGFR antibody and blotted (IB) with the indicated antibodies. TCL, total cell lysates. B, band intensity of phosphorylated EGFR, STAT3, AKT, and ERK in the DU145 cell lysates was calculated and normalized by the β-actin band using ImageJ software. The data from three independent experiments and S.D. (error bars) are depicted. C, HEK293T cells were transfected with expression vectors of EGFR-HA and Myc-STAP-2 together with APRE or SRE reporter vectors. Luciferase activity was determined at 48 h post-transfection. An aliquot of total cell lysates was immunoblotted by anti-HA, anti-Myc, and anti-actin antibodies. D and E, gene expression was measured by qPCR in control or STAP-2 shRNA–expressing DU145 cells with or without 30 ng/ml EGF stimulation for 1 h. F, control or STAP-2 shRNA–expressing LNCaP cells were stimulated with EGF, and the lysates were blotted as in A. G, band intensity of phosphorylated proteins in the LNCaP cell lysates was calculated as in B. H, control or STAP-2 shRNA–expressing LNCaP cells were stimulated with EGF, and then gene expression was measured by qPCR as in E. I, control or STAP-2 shRNA–expressing DU145 cells were treated with 1 μm gefitinib for 3 days, and then viable cells were measured by a WST assay. n = 3; mean values and S.E. (error bars) are depicted. N.S., not significant; *, p < 0.05; **, p < 0.01 (paired Student's t test).

Figure 3.

STAP-2 interacts with EGFR. A and B, HEK293T cells were transfected with expression vectors of EGFR-HA, EGFR K721A-HA, and Myc-STAP-2, and then the lysates were immunoprecipitated (IP) with anti-HA antibody or anti-Myc antibody at 48 h post-transfection and blotted (IB). C, DU145 cells were transfected with Myc-STAP-2 expression vector, and at 48 h post-transfection, the cells were starved for 2 h following stimulation with 20 ng/ml EGF for 20 min. The lysates were immunoprecipitated with control IgG or anti-EGFR antibody and blotted. D, DU145 cells were stimulated with EGF as in C, and then the lysates were immunoprecipitated and blotted. E, schematic representation of deletion mutants of STAP-2. F and G, HEK293T cells were transfected with expression vectors of EGFR-HA and several mutant Myc-STAP-2s, and then the lysates were immunoprecipitated as in A. H, STAP-2–knockdown DU145 cells were transfected with expression vectors of mutant STAP-2, and their proliferation was measured as in Fig. 1F. I, HeLa cells were transfected with Myc-STAP-2 expression vector and then stimulated with 100 ng/ml EGF for 10 min at 48 h post-transfection. The cells were stained using DAPI (blue) and anti-Myc antibody (red). n = 3; mean values and S.E. (error bars) are depicted. N.S., not significant; *, p < 0.05 (paired Student's t test).

Figure 4.

STAP-2 increases EGFR stability via inhibiting its c-CBL-mediated ubiquitination. A, DU145 cells were transfected with STAP-2 and EGFR expression vectors and then treated with 10 μm cycloheximide in serum-free DMEM for 2 h at 48 h post-transfection. After stimulating the cells with 100 ng/ml EGF for the indicated periods, the cells were lysed and blotted (IB). TCL, total cell lysates. B, control or STAP-2 shRNA–expressing DU145 cells were starved for 2 h and then stimulated with 100 ng/ml EGF for 20 min. After biotinylation of the plasma membrane proteins, the lysates were pulled down with avidin-conjugated Sepharose beads and blotted. C, control or STAP-2 shRNA–expressing DU145 cells were starved for 2 h and then stimulated with 100 ng/ml EGF for 2 min. The lysates were immunoprecipitated (IP) with anti-EGFR antibody and blotted with the indicated antibodies. D, DU145 cells were transfected with expression vectors of FLAG-c-CBL and Myc-STAP-2. Samples were prepared at 48 h post-transfection as in C. E, control or STAP-2 shRNA–expressing DU145 cells were starved for 2 h with or without 100 μm chloroquine and then stimulated with 100 ng/ml EGF for the indicated times. The lysates were blotted as in B. F, control or STAP-2 shRNA–expressing DU145 cells were stimulated with EGF as in B and then stained with anti-EGFR (red) and anti-LAMP1 (green) antibodies. Their localization was observed by confocal microscopy, and the percentage of EGFR co-localized with LAMP1 is depicted in G. n = 10; mean values and S.D. (error bars) are depicted. *, p < 0.05 (paired Student's t test).

Figure 5.

Proposed model for the STAP-2–mediated up-regulation of EGFR signaling. EGF stimulation induces EGFR phosphorylation, leading to phosphorylation of STAP-2 and activation of its downstream signaling molecules, such as STAT3 and MAPK. Phosphorylated EGFR is ubiquitinated by c-CBL and then sorted to lysosomes, resulting in its degradation and down-regulation of EGFR signaling. In STAP-2 highly expressed cells, STAP-2 increases EGFR stability and activation of its downstream signaling by inhibiting c-CBL–mediated EGFR ubiquitination (left). EGFR ubiquitination and degradation were promoted in STAP-2 low expressing cells (right). Thus, STAP-2 up-regulates EGFR signaling by increasing its stability and enhances prostate cancer cell growth. P and Ub, phosphorylation and ubiquitination, respectively.