Disruption of Ovarian Cancer STAT3 and p38 Signaling with a Small-Molecule Inhibitor of PTP4A3 Phosphatase

Abstract

Protein tyrosine phosphatase type IVA member 3 (PTP4A3 or PRL-3) is a nonreceptor, oncogenic, dual-specificity phosphatase that is highly expressed in many human tumors, including ovarian cancer, and is associated with a poor patient prognosis. Recent studies suggest that PTP4A3 directly dephosphorylates SHP-2 phosphatase as part of a STAT3-PTP4A3 feedforward loop and directly dephosphorylates p38 kinase. The goal of the current study was to examine the effect of a PTP4A phosphatase inhibitor, 7-imino-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione (JMS-053), on ovarian cancer STAT3, SHP-2, and p38 kinase phosphorylation. JMS-053 caused a concentration- and time-dependent decrease in the activated form of STAT3, Y⁷⁰⁵ phospho-STAT3, in ovarian cancer cells treated in vitro. In contrast, the phosphorylation status of two previously described direct PTP4A3 substrates, SHP-2 phosphatase and p38 kinase, were rapidly increased with JMS-053 treatment. We generated A2780 and OVCAR4 ovarian cancer cells resistant to JMS-053, and the resulting cells were not crossresistant to paclitaxel, cisplatin, or teniposide. JMS-053-resistant A2780 and OVCAR4 cells exhibited a 95% and 50% decrease in basal Y⁷⁰⁵ phospho-STAT3, respectively. JMS-053-resistant OVCAR4 cells had an attenuated phosphorylation and migratory response to acute exposure to JMS-053. These results support a regulatory role for PTP4A phosphatase in ovarian cancer cell STAT3 and p38 signaling circuits. SIGNIFICANCE STATEMENT: This study demonstrates that chemical inhibition of PTP4A phosphatase activity with JMS-053 decreases STAT3 activation and increases SHP-2 phosphatase and p38 kinase phosphorylation activation in ovarian cancer cells. The newly developed JMS-053-resistant ovarian cancer cells should provide useful tools to further probe the role of PTP4A phosphatase in ovarian cancer cell survival and cell signaling.

Graphical abstract

Fig. 1.

Generation of durable JMS-053–resistant A2780 and OVCAR4 cells. (A) A2780 cells with 33-fold resistance to a 72-hour exposure to JMS-053 compared with wild-type A2780 cells. Viability determined by hemocytometer after incubation with Trypan blue. Black circles indicate A2780 WT, and red squares indicates JMS-053–resistant cells (A2780 RES). N = 3; bar = S.D. unless smaller than the symbol. (B) OVCAR4 cells with fourfold resistance to a 72-hour exposure to JMS-053 (OVCAR4 RES) compared with OVCAR4 WT cells. Viability determined by hemocytometer after incubation with Trypan blue. Black circles indicate OVCAR4 WT cells, and red squares indicates OVCAR4 RES cells. N = 3; bar = S.D. unless smaller than the symbol. (C) Retention of JMS-053 resistance in A2780 RES cells. Each symbol represents the mean value obtained from three technical replicates, and the bars indicate the S.D. unless smaller than the symbol. Black circles indicate A2780 WT cells, and red squares indicate A2780 RES cells. (D) Retention of JMS-053 resistance in OVCAR4 RES cells. Each symbol represents the mean value obtained from three technical replicates, and the bars indicate the S.D. unless smaller than the symbol. Black circles indicate OVCAR4 WT cells, and red squares indicates OVCAR4 RES cells.

Fig. 2.

Growth rates and colony-forming efficacy of JMS-053–resistant OvCa cells. A2780 RES (A) and OVCAR4 RES (B) cells have similar growth rates compared with parental wild-type cells. A2780 cells (6 × 10⁵) and OVCAR4 cells (6.5 × 10⁵) were plated in T-25 cm² tissue culture flasks, and cell growth rate was determined by hemocytometer. A2780 WT and OVCAR4 WT are indicated by black circles; A2780 RES and OVCAR4 RES cells are indicated by red rectangles. Bars = S.D. unless smaller than the symbols, N = 4. Representative growth curves of three biologic replicates are shown. (C) OVCAR4 RES cells have lower colony-forming efficiency compared with OVCAR4 WT cells. Each symbol represents the mean number of colonies with >50 cells counted 10 days after plating 100 cells from six biologic replicates. Bars = S.D. P = 0.011 with unpaired two tailed t test.

Fig. 3.

JMS-053–resistant cells do not have markedly altered PTP4A3 expression levels. (A) Differences in the PTP4A1-3 mRNA levels in A2780 WT and A2780 RES cells determined by reverse-transcription polymerase chain reaction (RT-PCR) from three technical replicates. Bars = S.D. (B) Differences in the PTP4A1-3 mRNA levels in OVCAR4 WT and OVCAR4 RES cells determined by RT-PCR from three technical replicates. Bars = S.D. (C) Western blots for PTP4A3 protein with lysates from A2780 and OVCAR4 cells. (D) Ratio of the PTP4A3 protein levels in wild-type and JMS-053–resistant A2780 and OVCAR4 cells as determined by western blotting and normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) loading control. Each symbol indicates the mean value from a technical replicate, with the bars representing the S.D. or the range from biologic replicates.

Fig. 4.

Cells with acquired resistance to JMS-053 have reduced basal STAT3 phosphorylation and activation levels. (A) Representative western blots of A2780 WT and A2780 RES Y⁷⁰⁵ p-STAT3, S⁷²⁷ p-STAT3, STAT3, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (B) Quantification of A2780 WT (gray bars) and A2780 RES (red bars) western blot signal normalized to GAPDH. N = 3 biologic replicates. Bars = S.D. One-sample two tailed t test with Wilcoxon test. NS = P > 0.05. (C) Western blots of A2780 WT and A2780 RES Y⁷⁰⁵ p-STAT3, S⁷²⁷ p-STAT3, STAT3 and GAPDH. (D) Quantification of OVCAR4 WT (gray bars) and OVCAR4 RES (red bars) western blot signal normalized to GAPDH. N = 4. Bars = S.D. Two-tailed t test with Wilcoxon test. NS, not significant.

Fig. 5.

Acute exposure of OVCAR4 cells to JMS-053 decreases STAT3 activation and phosphorylation. (A) Representative western blots of OVCAR4 WT cell protein lysates after exposure to JMS-053 measuring Y⁷⁰⁵ p-STAT3, S⁷²⁷ p-STAT3, STAT3, and glyceraldehyde-3-phosphate dehydrogenase. (B) Kinetics of Y⁷⁰⁵ p-STAT3 changes in OVCAR4 WT cells exposed to different concentrations of JMS-053. N = 3 or 4. Bars = S.D. Data analyzed with ANOVA and t test with Wilcoxon test. *P < 0.05 for the values found below the asterisk compared with vehicle control. **P < 0.05 for the two values found below the asterisks compared with vehicle control. ***P < 0.05 for the three values found below the compared with vehicle control. Black dotted line indicates value at 0 hours. (C) Kinetics of S⁷²⁷ p-STAT3 changes in OVCAR4 WT cells exposed to different concentrations of JMS-053. Black squares indicate lysates from cells treated with 1.5 μM JMS-053, blue triangles indicate lysates from cells treated with 4.5 μM JMS-053, and red inverted triangles indicate lysates from cells treated with 40 μM JMS-053. N = 3 or 4. Bars = S.D. Data analyzed with ANOVA and t test with Wilcoxon test. *P < 0.05 for the value found below the asterisk compared with vehicle control. Black dotted line indicates value at 0 hours.

Fig. 6.

Acute exposure of OVCAR4 WT cells to JMS-053 rapidly increases Y⁵⁸⁰ and Y⁵⁴² SHP-2 phosphorylation. (A) Representative western blot of OVCAR4 WT cell protein lysates after exposure to JMS-053 for 7 minutes measuring Y⁵⁸⁰ SHP-2 phosphorylation. (B) Representative western blot of OVCAR4 WT cell protein lysates after exposure to JMS-053 for 7 minutes measuring Y⁵⁴² SHP-2 phosphorylation. (C) Representative western blot of OVCAR4 WT cell protein lysates after exposure to JMS-053 for 7 minutes measuring SHP-2 protein levels. (D) Representative western blot of OVCAR4 WT cell protein lysates after exposure to JMS-053 for 7 minutes measuring glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (E) Y⁵⁸⁰ SHP-2 phosphorylation normalized to GAPDH in OVCAR4 WT cell protein lysates after exposure to JMS-053 for 7 minutes. N = 4. biologic replicates. Bars = S.D. ANOVA and Mann-Whitney test. (F) Kinetics of Y⁵⁸⁰ SHP-2 phosphorylation normalized to GAPDH in OVCAR4 WT cell protein lysates after exposure to JMS-053. Data analyzed with ANOVA and t test with Wilcoxon test. *P < 0.05 compared with vehicle treated samples. (G) Y⁵⁴² SHP-2 phosphorylation normalized to GAPDH in OVCAR4 WT cell protein lysates after exposure to JMS-053 for 7 minutes. N = 4 biologic replicates. Bars = S.D. NS = P > 0.05. ANOVA and Mann-Whitney test. (H) Kinetics of Y⁵⁴² SHP-2 phosphorylation normalized to GAPDH in OVCAR4 WT cell protein lysates after exposure to JMS-053. Data analyzed with ANOVA and t test with Wilcoxon test. *P < 0.05 compared with vehicle treated samples.

Fig. 7.

Acute exposure of OVCAR4 WT cells to JMS-053 increases p38 phosphorylation. (A) Representative western blots of OVCAR4 WT cell protein lysates after exposure to JMS-053 for various times measuring p-p38, p38 kinase, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (B) Kinetics of altered p38 phosphorylation by JMS-053 exposure of OVCAR4 WT cells. Fold increase of p-p38, which was normalized to GAPDH, and the corresponding value for lysates from OVCAR4 WT cells exposed to vehicle. Black squares indicate lysates from cells treated with 1.5 μM JMS-053, blue triangles indicate lysates from cells treated with 4.5 μM JMS-053, and red inverted triangles indicate lysates from cells treated with 40 μM JMS-053. N = 3-4. Bars = S.D. Data analyzed with ANOVA and t test with Wilcoxon test. *P < 0.05 for the values found below the asterisk compared with vehicle control. **P < 0.05 for the individual value found next to the asterisk compared with vehicle control.

Fig. 8.

OVCAR4 RES cells have attenuated phosphorylation responses to JMS-053. (A) OVCAR4 WT cells were treated with various concentrations of JMS-053 for 30 minutes (black circles) or 4 hours (blue squares), and the phosphorylation status of p38 kinase normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) loading control was determined by western blotting. Bar = S.D. Data analyzed with ANOVA and t test with Wilcoxon test. (B) OVCAR4 WT cells were treated with various concentrations of JMS-053 for 30 minutes (black circles) or 4 hours (blue squares), and the phosphorylation status of Y⁷⁰⁵ STAT3 normalized to the GAPDH loading control was determined by western blotting. Data analyzed with ANOVA and t test with Wilcoxon test. (C) OVCAR4 RES cells were treated with various concentrations of JMS-053 for 30 minutes (black circles) or 4 hours (blue squares), and the phosphorylation status of p38 kinase normalized to the GAPDH loading control was determined by western blotting. Bars = S.D. Data analyzed with ANOVA and t test with Wilcoxon test. (D) OVCAR4 RES cells were treated with various concentrations of JMS-053 for 30 minutes (black circles) or 4 hours (blue squares), and the phosphorylation status of Y⁷⁰⁵ STAT3 normalized to the GAPDH loading control was determined by western blotting. Bars = S.D. Data analyzed with ANOVA and t test with Wilcoxon test. (E) Confluent serum-deprived OVCAR4 WT and OVCAR4 RES were uniformly scratch wounded with a pipette and then exposed to 15 ng/mL IL-6 and either vehicle control or JMS-053 (4.5 μM) for 24 hours. Cell migration was calculated by determining the cell-free area at 0 hours and subtracting that from the remaining cell-free area at 24 hours. The data are from three independent biologic replicates and analyzed by t test. Bars = S.D.