SHP2 promotes proliferation of breast cancer cells through regulating Cyclin D1 stability via the PI3K/AKT/GSK3β signaling pathway

Abstract

Objective: The tyrosine phosphatase SHP2 has a dual role in cancer initiation and progression in a tissue type-dependent manner. Several studies have linked SHP2 to the aggressive behavior of breast cancer cells and poorer outcomes in people with cancer. Nevertheless, the mechanistic details of how SHP2 promotes breast cancer progression remain largely undefined. Methods: The relationship between SHP2 expression and the prognosis of patients with breast cancer was investigated by using the TCGA and GEO databases. The expression of SHP2 in breast cancer tissues was analyzed by immunohistochemistry. CRISPR/Cas9 technology was used to generate SHP2-knockout breast cancer cells. Cell-counting kit-8, colony formation, cell cycle, and EdU incorporation assays, as well as a tumor xenograft model were used to examine the function of SHP2 in breast cancer proliferation. Quantitative RT-PCR, western blotting, immunofluorescence staining, and ubiquitination assays were used to explore the molecular mechanism through which SHP2 regulates breast cancer proliferation. Results: High SHP2 expression is correlated with poor prognosis in patients with breast cancer. SHP2 is required for the proliferation of breast cancer cells in vitro and tumor growth in vivo through regulation of Cyclin D1 abundance, thereby accelerating cell cycle progression. Notably, SHP2 modulates the ubiquitin-proteasome-dependent degradation of Cyclin D1 via the PI3K/AKT/GSK3β signaling pathway. SHP2 knockout attenuates the activation of PI3K/AKT signaling and causes the dephosphorylation and resultant activation of GSK3β. GSK3β then mediates phosphorylation of Cyclin D1 at threonine 286, thereby promoting the translocation of Cyclin D1 from the nucleus to the cytoplasm and facilitating Cyclin D1 degradation through the ubiquitin-proteasome system. Conclusions: Our study uncovered the mechanism through which SHP2 regulates breast cancer proliferation. SHP2 may therefore potentially serve as a therapeutic target for breast cancer.

Keywords: Cyclin D1; GSK3β; PI3K/AKT; SHP2; breast cancer; proliferation.

Figure 1

High expression of SHP2 is associated with poor prognosis in patients with breast cancer. (A, B) The overall survival rate in patients with elevated SHP2 expression was significantly poorer than that in patients with low SHP2 expression, on the basis of the TCGA and GSE20685 databases (P < 0.001 and P = 0.043, respectively). (C, D) The relapse-free survival rate in patients with elevated SHP2 expression was significantly poorer than that in patients with low SHP2 expression, on the basis of the GSE21653 and the GSE2034 databases (P = 0.039 and P = 0.035, respectively). (E) The expression of SHP2 in breast cancer tissue was detected via IHC staining. The expression of SHP2 was scored according to the stained area and staining intensity, and divided into 2 groups with high and low SHP2 expression.

Figure 2

Knockout of SHP2 inhibits the proliferation ability of breast cancer cells. (A) Schematic representation of PTPN11-targeting gRNA sequences. Two different guide RNAs were used to target the PTPN11 gene. (B) Generation of stable SHP2-knockout breast cancer cell lines with a CRISPR/Cas9-mediated gene editing method. DNA sequencing analysis confirmed that the deleted mutations were introduced into the PTPN11 genomic region of the 2 breast cancer cell lines. (C) Western blot analysis of the expression of SHP2 in control and SHP2 knockout cells with β-actin as loading control. (D) CCK8-based assays showed that SHP2 knockout significantly slowed the proliferation rate of the 2 breast cancer cells. Data are presented as mean ± SD, and statistical analysis was carried out with two-way ANOVA (****P < 0.0001). (E) Fewer colonies were observed in the 2 breast cancer SHP2 knockout lines than in the control cells. All data are presented as mean ± SD. Experiments were repeated 3 times. Statistical analysis was carried out with one-way ANOVA (****P < 0.0001).

Figure 3

SHP2 knockout delays G1-to-S phase transition through downregulation of Cyclin D1 abundance in breast cancer cells. (A) SHP2 knockout increased the percentage of G1 phase cells and decreased the percentage of G2/S phase cells. The cell cycle was evaluated through flow cytometry assays. The percentages of cells in each cell cycle phase are shown as mean ± SD from 3 independent experiments (****P < 0.0001). (B) EdU incorporation assays showed that the proportion of cells in the S phase was lower in SHP2 knockout cells than control cells. Data are presented as mean ± SD (**P < 0.01, ****P < 0.0001). (C) SHP2 knockout markedly decreased the protein level of Cyclin D1, whereas the expression of Cyclin B1 and Cyclin E1 was not altered. (D) SHP2 knockout decreased the mRNA expression of Cyclin D1. Quantitative PCR analysis of the mRNA expression of Cyclin B1, Cyclin D1, and Cyclin E1 in control and SHP2 knockout cells. Data are presented as mean ± SD. Statistical analysis was carried out with one-way ANOVA (*P < 0.1, **P < 0.01).

Figure 4

SHP2 knockout promotes Cyclin D1 degradation through the ubiquitin–proteasome pathway. (A) The half-life of Cyclin D1 in SHP2 knockout cells was significantly shorter than that in control cells. The cells were treated with cycloheximide for the indicated times, and the expression of Cyclin D1 was analyzed by western blotting. (****P < 0.0001). (B) The expression level of Cyclin D1 in SHP2 knockout cells was restored by MG132 treatment. The cells were treated with 10 μM of MG132 for the indicated times, and the expression of Cyclin D1 was analyzed by western blotting (****P < 0.0001). (C) Immunofluorescence staining showed that MG132 treatment resulted in an elevation of Cyclin D1 in SHP2 deleted cells. Cyclin D1 was mainly localized in the nuclei in control cells, whereas the increased Cyclin D1 after MG132 treatment in SHP2 deleted cells was mainly localized in the cytoplasm. The quantification of Cyclin D1 nucleus/cytoplasm ratio is shown in the right panel (****P < 0.0001). (D) Significantly greater ubiquitinated Cyclin D1 was observed in SHP2 knockout cells than in control cells. The control and SHP2 knockout cells were treated with MG132 or left untreated, and were then lysed and immunoprecipitated with anti-Cyclin D1. The enriched proteins were analyzed by western blotting with anti-Cyclin D1 and anti-ubiquitin antibodies.

Figure 5

GSK3β-induced T286 phosphorylation of Cyclin D1 is responsible for Cyclin D1 proteasomal degradation. (A) SHP2 knockout increased the protein levels of phosphorylated Cyclin D1 at T286 in the presence of MG132 (10 μM). (B) Immunofluorescence staining showed that MG132 treatment considerably increased the level of phosphorylated Cyclin D1 (T286). Cells were treated with 10 μM of MG132 for 6 h, fixed, and stained with anti-p-Cyclin D1 (T286) antibodies. The quantification of the p-Cyclin D1 (T286) nucleus/cytoplasm ratio is shown in the right panel. Statistical analysis was carried out with one-way ANOVA (****P < 0.0001). (C) Western blot analysis of the expression of total and phosphorylated GSK3β (Ser9) in cell lysates from the control and SHP2 knockout cells. (D) The protein level of Cyclin D1 in SHP2 deleted cells recovered after treatment with the GSK3β inhibitor CHIR99021. Control and SHP2 deleted cells were treated with 20 μM of CHIR99021 for 6 h or left untreated, and the expression of Cyclin D1 was analyzed by western blotting. (E) Immunofluorescence staining showed that CHIR99021 treatment significantly increased the nuclear expression of Cyclin D1 in the control and SHP2 deleted cells. The quantification of the Cyclin D1 nucleus/cytoplasm ratio is shown in the right panel (****P < 0.0001).

Figure 6

SHP2-deficiency-mediated dephosphorylation and activation of GSK3β through inhibition of the PI3K/AKT signaling pathway. (A) SHP2 knockout decreased the protein levels of phosphorylated AKT (T308) and phosphorylated ERK (T202/Y204) in 2 breast cancer cell lines. (B) Western blot analysis of the levels of Cyclin D1, total and phosphorylated GSK3β (Ser9), and total and phosphorylated ERK (T202/Y204) in 2 breast cancer cell lines pretreated with different concentrations of PD98059 for 6 h. (C) Inhibition of the PI3K/AKT pathway with LY294002 decreased the expression of phosphorylated GSK3β (Ser9) in 2 breast cancer cell lines. The cells were pretreated with different concentrations of LY294002 for 6 h, lysed, and analyzed via western blot.

Figure 7

Rescued expression of SHP2 in SHP2 deleted cells restores the proliferation ability of breast cancer cells. (A) Rescued expression of SHP2 restores the expression of phosphorylated AKT (T308), phosphorylated ERK (T202/Y204), phosphorylated GSK3β (Ser9), and Cyclin D1 in SHP2 knockout cells. SHP2 knockout cells were infected with lentivirus for SHP2 expression. The cells were then lysed and analyzed via western blotting. (B) CCK-8 assays showed that the re-expression of SHP2 in SHP2 deleted cells restored the cell proliferation ability. Data are shown as mean ± SD. Statistical analysis was performed with two-way ANOVA (****P < 0.0001). (C) Rescued expression of SHP2 restored the colony formation ability of SHP2 knockout cells. Statistical analysis was carried out with one-way ANOVA (****P < 0.0001). (D) Rescued SHP2 expression resulted in a smaller proportion of G1 phase cells than that in SHP2 knockout cells. Data are shown as mean ± SD. Statistical analysis was carried out with one-way ANOVA (****P < 0.0001). (E) EdU incorporation assays showed that the re-expression of SHP2 increased the proportion of cells in S phase. Data are expressed as mean ± SD from 6 independent fields (****P < 0.0001). (F) Rescued SHP2 in SHP2 deleted cells restored the nuclear expression of Cyclin D1. Data are expressed as mean ± SD from 5 independent fields (****P < 0.0001). (G) SHP2 knockout inhibited tumor growth in vivo, whereas the re-expression of SHP2 rescued tumor growth defects caused by SHP2 deletion. Statistical analysis was performed with two-way ANOVA (****P < 0.0001). (H) SHP2 knockout in breast cancer cells resulted in a significant decrease in tumor weight, whereas the rescued expression of SHP2 in SHP2 deleted cells resulted in tumors heavier than those in the control group. All data are expressed as mean ± SD (****P < 0.0001). (I) SHP2 knockout decreased the expression of Ki67 and Cyclin D1 in tumor sections, whereas rescued expression of SHP2 restored the expression of these 2 proteins (**P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 8

A proposed schematic model: CRISPR/Cas9-mediated knockout of SHP2 inhibits breast cancer proliferation by regulating Cyclin D1 stability via the PI3K/AKT/GSK3β signaling pathway.