IRS-2 deubiquitination by USP9X maintains anchorage-independent cell growth via Erk1/2 activation in prostate carcinoma cell line

Abstract

Insulin-like growth factors (IGFs) have been shown to induce proliferation of many types of cells. Insulin receptor substrates (IRSs) are major targets of IGF-I receptor (IGF-IR) tyrosine kinase activated by IGFs, and are known to play important roles in the activation of downstream signaling pathways, such as the Erk1/2 pathway. Dysregulation of IGF signaling represents a central tumor promoting principle in human carcinogenesis. Prostate carcinoma is highly dependent on the IGF/IGF-IR/IRS axis. Here we identified the deubiquitinase, ubiquitin specific peptidase 9X (USP9X) as a novel binding partner of IRS-2. In a human prostate carcinoma cell line, small interfering RNA (siRNA)-mediated knockdown of USP9X reduced IGF-IR as well as IRS-2 protein levels and increased their ubiquitination. Knockdown of USP9X suppressed basal activation of the Erk1/2 pathway, which was significantly restored by exogenous expression of IRS-2 but not by IGF-IR, suggesting that the stabilization of IRS-2 by USP9X is critical for basal Erk1/2 activation. Finally, we measured anchorage-independent cell growth, a characteristic cancer feature, by soft-agar colony formation assay. Knockdown of USP9X significantly reduced anchorage-independent cell growth of prostate carcinoma cell line. Taken all together, our findings indicate that USP9X is required for the promotion of prostate cancer growth by maintaining the activation of the Erk1/2 pathway through IRS-2 stabilization.

Keywords: IGF-I; IRS-2; USP9X; prostate cancer; ubiquitin.

Figure 1. USP9X interacts with IRS-1/2

(A) HEK293 cells expressing FLAG-IRS-1, IRS-2 or FLAG alone were cultured under serum-free conditions for 24 hours. The cell lysates were immunoprecipitated with anti-FLAG antibody, followed by western blotting using indicated antibodies. (B) PC3 cells were serum-starved for 24 hours, and then the lysates were subjected to immunoprecipitation and western blotting using indicated antibodies. (C, D) After serum-starvation for 24 hours, PC3 cells were treated with 100ng/ml of IGF-I for 5 minutes. The cell lysates were analyzed by immunoprecipitation and western blotting using indicated antibodies. (E) The schematic diagrams show the domain structure of USP9X and deletion mutants. (F, G) The plasmids expressing the deletion mutants of USP9X together with Myc-IRS-1 (F) or IRS-2 (G) were transfected into HEK293T cells. After serum-starvation for 24 hours, the lysates were immunoprecipitated with anti-FLAG antibody, followed by western blotting using indicated antibodies.

Figure 2. USP9X deubiquitinates IRS-2 to prevent its proteasomal degradation

(A) PC3 cells were transfected with control siRNA or two different siRNAs targeting USP9X (#1 and #2), and cultured in the serum-free medium for 24 hours. The cell lysates were subjected to western blotting using indicated antibodies. HSP90 was used as loading control (left panel). This result is a representative of three independent experiments. The graphs represent the protein levels of IRS-1 and IRS-2 (right panel). (B) After serum-starvation for 22 hours, PC3 cells were treated with WP1130 for 2 hours. The cell lysates were analyzed by western blotting using indicated antibodies. HSP90 was used as internal control. (C) HEK293T cells were transfected with siRNA of USP9X or control. 24 hours after transfection, the cells were transfected with FLAG-IRS-2 and serum-starved for 20 hours followed by the treatment with MG132 (20 μM) for 2 hours. The cell lysates were denatured by boiled for 10 minutes in 1% SDS-containing buffer. FLAG-IRS-2 was immunoprecipitated by anti-FLAG antibody and the immunoprecipitates were analyzed by western blotting using indicated antibodies. (D) PC3 cells transfected with USP9X siRNA were serum-starved for 24 hours, and treated with cycloheximide (1 μg/ml) for the indicated time (top). The cell lysates were analyzed by western blotting using anti-IRS-2 antibody, and the protein levels of IRS-2 were normalized according to the levels at the time point of 0 hour (bottom). (E) PC3 cells transfected with control siRNA or USP9X siRNA were serum-starved for 24 hours, and treated with cycloheximide (1 μg/ml) and MG132 (20 μM) for the indicated time. The cell lysates were subjected to western blotting using indicated antibodies (top). The protein levels of IRS-2 were normalized according to the levels at the time point of 0 hours (bottom).

Figure 3. USP9X interacts with IGF-IR and stabilizes it

(A) PC3 cells were transfected with control siRNA or USP9X siRNA, and serum-starved for 24 hours. The cells lysates were subjected to western blotting using indicated antibodies. HSP90 was used as internal control (top). This result is a representative of three independent experiments. The graph represents the protein level of IGF-IR (bottom). (B) HEK293T cells expressing IGF-IR-FLAG or FLAG alone were subjected to immunoprecipitation and western blotting using indicated antibodies. (C) HEK293T cells were transfected with control siRNA or USP9X siRNA. 24 hours after transfection, the cells were transfected with the plasmid expressing IGF-IR-FLAG and serum-starved for 20 hours. The cell lysate was denatured by boiling for 10 minutes in 1% SDS-containing buffer and analyzed by immunoprecipitation and western blotting using indicated antibodies.

Figure 4. USP9X maintains the activation of Erk pathway by stabilizing IRS-2

(A, B) PC3 cells were transfected with control, USP9X siRNA, serum-starved for 24 hours, and stimulated with 100 ng/ml of IGF-I for 5 minutes. The cell lysates were subjected to immunoprecipitation and western blotting using indicated antibodies. (C) PC3 cells transfected with control, USP9X or IRS-2 siRNA were serum-starved for 24 hours, and stimulated with 100 ng/ml of IGF-I for 5 minutes. The cell lysates were subjected to western blotting analysis using indicated antibodies. (D) PC3 cells transfected with siRNA of IGF-IR or control were cultured in the serum-free medium for 24 hours. The lysates were analyzed by western blotting using indicated antibodies. (E) PC3 cells were cultured in the serum-free medium containing BMS754807 at indicated concentrations for 24 hours. The cell lysates were subjected to western blotting using indicated antibodies. (F) PC3 cells stably expressing empty vector, FLAG-IRS-2 or IGF-IR-mRFP were transfected with control or USP9X siRNA and serum-starved for 24 hours. The cell lysates were analyzed by western blotting using indicated antibodies.

Figure 5. USP9X contributes to anchorage-dependent/independent cell growth induced by the activation of IGF-IR-IRS-2-Erk1/2 pathway

(A) PC3 cells were cultured in the serum-free medium containing solvent control, 300 nM of BMS754807 or 100 μM of PD98059 for 48 hours. The cells were incubated with or without 100 ng/ml of IGF-I for 20 hours. For the last 4 hours, we added [³H]-thymidine to the medium, and measured the thymidine incorporation into DNA. (B) PC3 cells were transfected with control, USP9X or IRS-2 siRNA. The cells were serum-starved for 48 hours and incubated with or without 100 ng/ml of IGF-I for 20 hours. For the last 4 hours, we added [³H]-thymidine to the medium, and measured the thymidine incorporation into DNA. (C) PC3 cells stably expressing empty vector or FLAG-IRS-2 were transfected with siRNA of USP9X or control and serum-starved for 44 hours. For the last 4 hours, we added [³H]-thymidine to the medium, and measured the thymidine incorporation into DNA. (D) PC3 cells were resuspended in 0.32 % agarose containing medium with solvent control, 100 nM of BMS754807 or 50 μM of PD98059, and cultured for 13 days. The formed colonies were visualized by crystal violet staining (top), and the number of colonies was normalized according to that of control dish (bottom). (E) PC3 cells were transfected with siRNA of control, USP9X or IRS-2, and resuspended in 0.32% agarose containing medium. After culture for 13 days, the formed colonies were visualized by crystal violet staining (top). The number of colonies was normalized according to that of control dish (bottom).