Inhibitory effects of a selective Jak2 inhibitor on adrenocorticotropic hormone production and proliferation of corticotroph tumor AtT20 cells.

PURPOSE

The primary cause of Cushing's disease is adrenocorticotropic hormone (ACTH)-producing pituitary adenomas. EGFR signaling induces mRNA-transcript levels and ACTH secretion from corticotroph tumors. The Jak-STAT pathway is located downstream of EGFR signaling; therefore, a Jak2 inhibitor could be an effective therapy for EGFR-related tumors. In this study, we determined the effect of a potent and selective Jak2 inhibitor, SD1029, on ACTH production and proliferation in mouse AtT20 corticotroph tumor cells.

MATERIALS AND METHODS

AtT20 pituitary corticotroph tumor cells were cultured after transfection with - or -specific siRNA. Expression levels of mouse , and mRNAs were evaluated using quantitative real-time polymerase chain reaction. ACTH levels were measured using ACTH ELISA. Western blot analysis was performed to examine protein expression of phosphorylated STAT3/STAT3. Viable cells and DNA fragmentation were measured using a cell-proliferation assay and cell-death detection ELISA, respectively. Cellular DNA content was analyzed using fluorescence-activated cell sorting.

RESULTS

SD1029 decreased and mRNA and ACTH levels, while increasing levels. The drug also decreased AtT20-cell proliferation and induced apoptosis, but did not alter cell-cycle progression. SD1029 also inhibited STAT3 phosphorylation. knockdown inhibited mRNA levels and cell proliferation. However, combined treatment with knockdown and SD1029 had no additive effect on mRNA levels or cell proliferation. knockdown inhibited the SD1029-induced decrease in mRNA levels and also partially inhibited the decrease in cell proliferation.

CONCLUSION

Both PTTG1 and GADD45β may be responsible, at least in part, for the Jak2-induced suppression of ACTH synthesis and cell proliferation. Accordingly, therapies that target EGFR-dependent Jak2/STAT3 may have clinical applications for treating Cushing's disease.