PTPL1/PTPN13 regulates breast cancer cell aggressiveness through direct inactivation of Src kinase

Abstract

The protein tyrosine phosphatase PTPL1/PTPN13, the activity of which is decreased through allelic loss, promoter methylation, or somatic mutations in some tumors, has been proposed as a tumor suppressor gene. Moreover, our recent clinical study identified PTPL1 expression level as an independent prognostic indicator of a favorable outcome for patients with breast cancer. However, how PTPL1 can affect tumor aggressiveness has not been characterized. Here, we first show that PTPL1 expression, assessed by immunohistochemistry, is decreased in breast cancer and metastasis specimens compared with nonmalignant tissues. Second, to evaluate whether PTPL1 plays a critical role in breast cancer progression, RNA interference experiments were performed in poorly tumorigenic MCF-7 breast cancer cells. PTPL1 inhibition drastically increased tumor growth in athymic mice and also enhanced several parameters associated with tumor progression, including cell proliferation on extracellular matrix components and cell invasion. Furthermore, the inhibition of Src kinase expression drastically blocked the effects of PTPL1 silencing on cell growth. In PTPL1 knockdown cells, the phosphorylation of Src on tyrosine 419 is increased, leading to the activation of its downstream substrates Fak and p130cas. Finally, substrate-trapping experiments revealed that Src tyrosine 419 is a direct target of the phosphatase. Thus, by identification of PTPL1 as the first phosphatase able to inhibit Src through direct dephosphorylation in intact cells, we presently describe a new mechanism by which PTPL1 inhibits breast tumor aggressiveness.

Figure 1. Evaluation of PTPL1 expression in breast cancer specimens

A microarray of tissue sections from 5 benign tissues, 24 primary cancers and 10 paired primary cancers and lymph node metastases was immunostained with an antibody against PTPL1. A, Representative staining of two benign tissues and two paired primary cancers and lymph node metastases (magnification Gx200). B, Differences in the expression of PTPL1 between benign and cancer or metastasis groups were assessed using the Mann-Whitney test. (P=0.0004 and P=0.0013, respectively) C, Differences in the expression of PTPL1 between cancer and metastasis groups were assessed using the Wilcoxon signed rank test (P=0.014).

Figure 2. Implication of PTPL1 in breast cancer cells aggressiveness

A, Expression of PTPL1 in stable cell clones was monitored by western blot using anti-PTPL1 antibodies. The loading control was obtained by re-probing the membrane with anti-actin. B, Six-week-old mice were injected with clonal cell lines in the mammary glands. Tumor volume was measured at the indicated time, and the mean ± 95% confidence interval is shown (n=11 per group). *P<0.0012 for mean of PTPL1-shRNAs versus mean of control-shRNAs (Student’s t-test).

Figure 3. Cell growth on extra-cellular matrices of siRNA-PTPL1 transfected cells

A, Five days after siRNA transfection, MCF-7 cells were lysed and cell extracts were analyzed by immunoblotting with anti-PTPL1 and anti-actin antibodies. B–C, Mock-transfected (control-siRNA) and PTPL1-siRNA (PTPL1-siRNA) cell lines were coated on Matrigel. B, Phase contrast optical photomicrographs after five days of culture and photographs after p-iodonitrotetrazolium violet staining. C, Colonies on Matrigel with a diameter greater than 40 μm were counted on four different fields per well. The data given are the mean ± s.d. of quadruplet samples. *P=0.003 and **P<0.001 versus control-siRNA. One representative experiment out of four is shown. D, One day after siRNA transfection, cells were plated on fibronectin coated wells and the DNA was quantified at different days, as described in the Materials and Methods. Cell growth was expressed relative to day one (mean ± s.d. of six wells from two independent experiments out of four). **P=0.0001 versus control-siRNA.

Figure 4. Effect of PTPL1 downregulation on cell invasion and adhesion

A, Two days after transfection, cells were plated onto the upper well of a Matrigel coated Transwell Boyden Chamber and allowed to migrate toward the chemoattractant 10% FBS for 48 h. The percentage of cells that migrated through Matrigel-coated filters was quantified relative to the total number of seeded cells. The data represent the percentage of cells that migrated to the lower side of the filter relative to control-siRNA cells. The results are the means ± s.d of triplicate samples from two independent experiments (MCF-7; n=6) or quadruplet samples from one experiment (T47D). *P=0.036, **P=0.003. Photographs are representative brilliant blue stained filters. B, Five days after siRNA transfection, MCF-7 cells were plated in triplicate on Matrigel, collagen 4 or fibronectin for 30min. Data represent the percentage of adherent cells compared to the total seeded cells. The results are means ± s.d of triplicate. *P=0.011, **P=0.004, ***P=0.0001. One representative experiment out of four is shown. C, Immunofluorescence of cells plated on fibronectin was performed using a phosphotyrosine antibody. Representative cells from one experiment out of two. For control and PTPL1 siRNA transfected cells, left panels correspond to a 700-fold magnification and right panels correspond to an 1800-fold magnification.

Figure 5. Implication of Src kinase activity for PTPL1 biological activity

A, Five days after siRNA transfection, MCF-7 cells were lysed, and cell extracts were analyzed by immunoblot using anti-Src, anti-PTPL1 and anti-actin antibodies. B–C, One day after siRNA transfection, cells were plated on B) fibronectin and C) Matrigel. Outgrowth of transfected cells was monitored as described in Figure 2 C–D. The data given are the mean ± s.d. of B) triplicate C) quadruplet samples. One representative experiment out of three is shown. B) *P≤0.003. C)*P≤0.035. D, One day after siRNA transfection, MCF-7 cells were (second, third and fourth panel) or were not (first panel) transiently transfected with the Src expression vector. The phosphorylation state of specific tyrosine residues in Src (top blot) or FAK (middle blot) was monitored by western blot using anti-pSrcY419 and anti-pSrcY530 or anti-pFakY397 and anti-pFak576/577 antibodies. Equivalent amounts of Src or FAK expression were confirmed by re-probing the blots with anti-Src and anti-FAK antibodies. For Fak/P130 association (bottom blot), lysates were immunoprecipitated with anti-Fak antibody and immunoblotted with anti-P130cas. An equal immunoprecipitation level of FAK was confirmed by direct immunoblotting of the membrane after stripping with anti-Fak antibody.

Figure 6. Phosphorylation and localization of Src kinase in PTPL1 transfected cells

A, HEK293 cells were transiently cotransfected with Src and HA-tagged wt PTPL1 or PTPL1-YF/DA expression vectors. Total lysates were analyzed by direct immunoblotting with anti-pSrcY419 antibody (middle). Equivalent amounts of PTPL1 and Src were confirmed by re-probing the blots with anti-HA (top) and anti-Src (bottom) antibodies. B, HEK293 cells overexpressing the Src Y530F expression vector were serum-starved and stimulated with 10 mM pervanadate for 30 min. Cells were lysed and proteins were resolved by SDS-PAGE (7.5% gel; input) or incubated with the indicated GST fusion proteins immobilized on beads. Bound materials were immunoblotted with anti-pSrcY419 antibody. C, PTPL1 was immunoprecipitated from lysates prepared from HEK293 cells transfected as described in A) using an anti-HA antibody and immunoblotted for the presence of Src tyrosine-phosphorylated protein at residue 419 with anti-pSrcY419 antibody (bottom-left). Equivalent amounts of PTPL1 were confirmed by re-probing the blots with an anti-HA antibody (top-left). Src was immunoprecipitated from the same lysates using an anti-Src antibody and immunoblotted for the presence of PTPL1 using an anti-HA antibody (top-right). An equal expression level of Src was confirmed by direct immunoblotting of lysate with anti-Src antibody (bottom-right). Asterisks indicate IgG bands. D, Cells were treated for indirect FITC localization of PTPL1 constructs with anti-HA antibody (left) and indirect TRITC localization of Src (middle). Images represent merges of horizontal confocal sections (right). Bar, 250 nm.