Synergistic anti-tumor effect of combined inhibition of EGFR and JAK/STAT3 pathways in human ovarian cancer

Abstract

Background: The EGFR signaling pathway is frequently activated in human ovarian cancer and associated with poor prognosis. However, inhibition of EGFR signaling in patients with recurrent ovarian cancer has been disappointing. It remains to be addressed whether ovarian cancer patients could benefit from targeting EGFR signaling. Here we investigated the mechanisms underlying the resistance to EGFR inhibition in ovarian cancer and developed a strategy to overcome it.

Results: We found that treatment of human ovarian cancer cells with an EGFR inhibitor, gefitinib, resulted in increased STAT3 phosphorylation in a dose- and time-dependent manner. Inhibiting STAT3 activation with a small molecule inhibitor of JAK, an upstream kinase that phosphorylates and activates STAT3, synergistically increased the anti-tumor activity of gefitinib in vitro. Similar results were obtained when STAT3 or JAK1 expression was knocked down. In contrast, inhibiting other signaling pathways, such as AKT/mTOR, MEK or SRC, was relatively less effective. The combined treatment resulted in simultaneous attenuation of multiple survival pathways and increased inhibition of ERK pathway. In addition, the dual inhibition showed a stronger suppression of xenograft tumor growth than either single inhibition.

Conclusions: Our findings demonstrate that feedback activation of STAT3 pathway might contribute to the resistance to EGFR inhibition. Combined blockade of both pathways appears to be more effective against human ovarian cancer than inhibition of each pathway alone both in vitro and in vivo. This study may provide a strategy to improve clinical benefit of targeting EGFR pathway in ovarian cancer patients.

Figure 1

Anti-tumor activity of gefitinib in ovarian cancer cells. (A) Human ovarian cancer cell lines were treated with the indicated concentrations of gefitinib. Cell viability was determined 72 h later. The IC50 was determined by the Chou-Talalay method. (B) Western blots showing the dose-dependent effect of gefitinib on downstream signaling in human ovarian cancer cells. SKOV3 and MDAH2774 cells were incubated with increasing amounts of gefitinib for 24 h. (C) Western blots showing the time-dependent effect of gefitinib on downstream signaling in SKOV3 cells. SKOV3 cells were incubated with 1 μM gefitinib and collected at the indicated time points. GAPDH and actin were used as a loading control for SKOV3 and MDAH2774 cells, respectively. Results are representative of 2–5 preparations.

Figure 2

Suppressing the JAK/STAT3 pathway with a small molecule inhibitor of JAK (JAKi) enhanced the anti-tumor activity of gefitinib in human ovarian cancer cells. (A) SKOV3 cells were treated with JAKi (AZD1480) or gefitinib alone or in combination at various concentrations in a fixed molar ratio 1:1. Cell viability was determined 72 h later. (B) SKOV3 cells were treated with JAKi (5 μM) or gefitinb (5 μM) either as single agent or in combination. Cell viability was determined 24 h, 48 h and 72 h later. (C) SKOV3 cells were incubated with increasing amounts of gefitinib in the presence of a fixed concentration of JAKi. Cell viability was determined 72 h later. OVCAR8 (D), MDAH2774 (E) and OVCAR 3 cells (F) were treated with gefitinib or JAKi either alone or in combination. Cell viability was determined 72 h later. Results are representative of 2–5 preparations.

Figure 3

Suppressing JAK/STAT3 signaling enhanced gefitinib-induced apoptosis in human ovarian cancer cells. SKOV3 (A&B) and MDAH2774 (C&D) cells were treated with gefitinib, JAKi, either alone or together, for 48 h. Apoptosis was determined by flow cytometry using Annexin V and PI staining (A&C) or for cleaved poly-ADP ribose polymerase (PARP) and cleaved caspase-3 by Western blot (B&D). **, P < 0.005; ***, P < 0.005, combination vs. JAKi alone or gefitinib alone. (E) SKOV3 cells were transfected with siRNA against STAT3, JAK1, or control siRNA and treated with gefitinib or (F) transfected with siRNA against EGFR and treated with JAKi. After 48 h, cells were harvested and evaluated for apoptosis using Annexin V staining. *, P < 0.05; **, P < 0.005, vs. control siRNA.

Figure 4

Western blots show that combination treatment with JAKi and gefitinib caused attenuation of multiple signaling pathways. SKOV3 (A) and MDAH2774 (B) cells were treated with JAKi, gefitinib or the combination for 2 h and 24 h. Results are representative of 2–4 preparations.

Figure 5

JAKi increased the anti-tumor activity of gefitinib in mice. (A) SKOV3 cells were implanted subcutaneously into the right flank of nude mice. Tumors were treated with vehicle, JAKi (30 mg/kg), gefitinib (150 mg/kg) or their combination by oral gavage once daily. Tumor growth was measured twice a week. Data represents means ± SD (n = 8-10). *, P < 0.05; ***, P < 0.005, combination vs. vehicle, or gefitinib alone or JAKi alone. (B) Western blots show the effects of treatment on the indicated intracellular signaling pathways in tumor tissue. (C) Relative levels of p-EGFR, p-STAT3, p-ERK were determined by measuring the density of each band and normalized to that of GAPDH. Densitometry data were relative changes in protein expression and were mean ± SD of 2–3 preparations. *, P < 0.05; ***, P < 0.0005.