PTPD1 supports receptor stability and mitogenic signaling in bladder cancer cells

Abstract

PTPD1, a cytosolic non-receptor protein-tyrosine phosphatase, stimulates the Src-EGF transduction pathway. Localization of PTPD1 at actin cytoskeleton and adhesion sites is required for cell scattering and migration. Here, we show that during EGF stimulation, PTPD1 is rapidly recruited to endocytic vesicles containing the EGF receptor. Endosomal localization of PTPD1 is mediated by interaction with KIF16B, an endosomal kinesin that modulates receptor recycling at the plasma membrane. Silencing of PTPD1 promotes degradation of EGF receptor and inhibits downstream ERK signaling. We also found that PTPD1 is markedly increased in bladder cancer tissue samples. PTPD1 levels positively correlated with the grading and invasiveness potential of these tumors. Transgenic expression of an inactive PTPD1 mutant or genetic knockdown of the endogenous PTPD1 severely inhibited both growth and motility of human bladder cancer cells. These findings identify PTPD1 as a novel component of the endocytic machinery that impacts on EGF receptor stability and on growth and motility of bladder cancer cells.

FIGURE 1.

PTPD1 is a component of endosomes. HEK293 cells were subjected to double immunostaining for PTPD1 and EEA1, Rab7, Rab11, or LAMP-1. Before fixation, cells were serum-deprived overnight and stimulated with EGF for 5 min (EEA1 and Rab7) and 30 min (Rab11 and LAMP-1). Fluorescence images were collected and analyzed by a confocal microscope. A merged composite for each immunostaining is shown. Magnification of selected areas is shown (insets). Bar, 10 μm.

FIGURE 2.

PTPD1 interacts and colocalizes with KIF16B through its FERM domain. A, lysates from HEK293 cells transiently transfected with FLAG-PTPD1 were immunoprecipitated (IP) with anti-KIF16B antibody or control IgG and immunoblotted (IB) with anti-FLAG and anti-KIF16B antibodies. B, lysates from HEK293 cells transiently co-transfected with FLAG-PTPD1 or FLAG-PTPD1_Δ1–325 and HA-KIF16B or HA-L1248A/F1249A mutant were immunoprecipitated with anti-HA and immunoblotted with anti-FLAG antibody. As a control, we used a lysate from untransfected cells. C and D, cells were transiently transfected with vector encoding Myc-tagged KIF16B, either wild type (C) or L1248A/F1249A mutant (D). Where indicated, a GFP vector was included in the transfection mixture (D). Twenty-four h after transfection, cells were serum-deprived overnight and then stimulated with EGF (100 ng/ml) for 30 min. Cells were subjected to double immunostaining with anti-Myc and anti-PTPD1 antibodies. Images were collected by confocal microscopy. A merged composite and magnified images are shown.

FIGURE 3.

KIF16B anchors PTPD1 to endosomes. A, lysates (L), supernatant (S), and endosomal (E) fractions were isolated from HEK293 lysates and immunoblotted (IB) for PTPD1, EEA1, KIF16B, and tubulin. B, total membranes (M) and endosomal (E) fractions were purified from HEK293 cells transiently transfected with Myc-KIF16B (either wild type or mutant) and FLAG-PTPD1 vectors. Protein fractions were immunoblotted with the indicated antibodies. C, quantitative analysis of the experiments shown in B. A mean value ± S.E. (error bars) of five independent experiments is shown.

FIGURE 4.

PTPD1 co-localizes with EGF receptor. J82 cells were serum-deprived overnight (top) and stimulated with EGF (100 ng/ml) for 15 min (middle) and 30 min (bottom). Before fixation, cells were pulsed with mouse anti-EGFR antibody (see “Experimental Procedures”), washed with phosphate-buffered saline, and formalin-fixed. Immunostaining was performed using anti-PTPD1 antibody.

FIGURE 5.

PTPD1 regulates EGFR stability. A, J82 cells were transiently transfected with siRNA_PTPD1 or siRNAc. Twenty-four h after transfection, cells were serum-deprived overnight and then stimulated with EGF (100 ng/ml) for 30 min. Before fixation, cells were pulsed with mouse anti-EGFR, washed with phosphate-buffered saline, and formalin-fixed. Cells were subjected to immunostaining for PTPD1. Images were collected by confocal microscopy. A merged composite is shown on the right. B, quantification of fluorescence signals in cells subjected to pulse-chase experiments as described in A. Fluorescence values ± S.E. (error bars) were normalized to the value of control, serum-deprived cells. C, total lysates from J82 transfected cells (as in A) were subjected to immunoblot analysis with the indicated antibodies. A representative set of autoradiograms is shown. D, quantitative analysis of the experiments shown in C. A mean value ± S.E. of four independent experiments is shown. *, p < 0.01 versus control (siRNAc). P-ERK, phospho-ERK.

FIGURE 6.

PTPD1 is required for growth and motility of bladder cancer cells. A, immunoblot analysis for PTPD1 on total lysates from human bladder cells (RT4, J82, 5637, and HT), human neuroblastoma (SK), and human embryonic kidney (HEK293) cell lines. B, motility assays from J82 cells transiently transfected with vectors expressing the following transgenes: CMV (control), HA-PTPD1_C1108S, dominant negative Src (Src⁻), HA-PTPD1, siRNA_PTPD1, and siRNAc. Twenty-four h after transfection, cells were plated on a Transwell apparatus and incubated for an additional 24 h. Migrated cells were fixed, stained, and counted. Cumulative data are presented as mean ± S.E. (error bars) of 3–5 independent experiments. Values from control (CMV) cells were set as 100. C, fluorescence-activated cell sorter analysis on J82 transiently transfected with siRNA_PTPD1 or siRNAc. Cells were harvested and analyzed by FACS at 72 h (upper panels) and 96 h (lower panels) after transfection. The experiment shown is representative of four independent experiments that gave similar results. D, J82 cells (15,000 cells/well) were transiently transfected with siRNA_PTPD1 or siRNAc. DNA synthesis was monitored by thymidine (0.5 μCi/well) incorporation at 72 and 96 h after transfection. Data are expressed as mean ± S.E.

FIGURE 7.

PTPD1 is highly expressed in bladder carcinomas. A and B, tumor samples (T) were isolated from patients affected by high grade (lanes 2, 3, 4, 6, 7, and 8) or low grade (lanes 1, 5, and 9) urothelial carcinoma. Normal tissue (N) surrounding each neoplastic lesion was also isolated. Tissue samples were lysed, resolved on 8% SDS-polyacrylamide gels, and immunoblotted (IB) with the following antibody: anti-peptide PTPD1 (ab1) (A) or anti-polypeptide PTPD1 (ab2) (B), anti-ERK2, and anti-cytokeratins. C, tissue sections from normal bladder (a), hyperplastic bladder (b and c), and high grade (d) of urothelial carcinoma were immunostained with anti-PTPD1 antibody and analyzed by light microscopy. Higher resolution panels (a′, b′, c′, and d′) of each set of images are shown on the right. D, bladder lesions were grouped into three subcategories: normal/hyperplastic, low grade urothelial carcinoma, and high grade urothelial carcinoma. Cumulative data and relative abundance of PTPD1 in each category are shown. E, a tissue microarray of 505 bladder samples ranging from normal tissue to benign lesions and urothelial carcinomas was immunostained with anti-PTPD1 polyclonal antibody. Shown is an enlarged section of representative biopsies of normal and cancer lesions immunostained with anti-PTPD1 antibody. F, cumulative data are expressed as the percentage of PTPD1-positive samples within the two main categories (normal/hyperplastic lesions and urothelial carcinomas). p value is indicated on the right. G, PTPD1-positive urothelial carcinomas were scored for Ki-67 positivity. The cut-off value represents the percentage of Ki-67-positive cells versus total cells scored. H, inverse correlation between bladder stage disease (pTa, pT1, and pT3) and PTPD1 signal. The analysis was carried out on a total of 349 patients with urothelial carcinoma.