Abstract
Disclosure: T. Apaydin: None. S. Zonis: None. C. Zhou: None. R. Barrett: None. V. Chesnokova: None. S. Melmed: None. Deficient DNA repair and accumulated DNA damage underlie age-associated pathological changes, tissue fragility, and neoplastic development. ATM kinase, an activator of DNA damage responses (DDR), phosphorylates DDR effectors including CHK2, p53, and γH2AX to ensure DNA repair. We have shown that GH suppresses epithelial DDR activity by suppressing ATM phosphorylation, resulting in DNA damage accumulation due to repressed DNA repair. Wip1 dephosphorylates ATM thereby attenuating DNA damage repair. We found that GH significantly induced Wip1 expression (1.6-fold) in both human normal colon cells (hNCC) and in human 3-dimensional intestinal organoids, resulting in dephosphorylated ATM, CHK2, γH2AX, and p53. Mice bearing GH-secreting tumors with high circulating GH also exhibited induced colon Wip1 expression with lower phospho-ATM, γH2AX, and p53 as well as increased DNA damage. Twofold higher Wip1 expression was observed in blood buffy coats derived from patients with GH-secreting adenoma vs patients with non-functioning pituitary adenoma. Specificity of these observations was validated by showing that in GH-treated cells, blocking GH receptor (GHR) with pegvisomant or blocking STAT5 phosphorylation with the inhibitor CAS 285986-31-4 constrained Wip1 induction and restored ATM phosphorylation, indicating that GH effects on Wip1 are GHR/STAT5-mediated concordant with the observed decreased colon Wip1 expression in GHR-/- mice devoid of GH signaling. Furthermore, Wip1 inhibition with the small molecule inhibitor (GSK2830371) reversed GH-induced DNA damage by restoring phosphorylation of ATM and other key DDR proteins. To determine a mechanism for Wip1 induction, we tested homeodomain-interacting protein kinase 2 (HIPK2) which is regulated by AMPK and potentiates Wip1 proteasomal degradation and its activity. We observed that GH increased phospho-AMPK expression, while blocking AMPK kinase activity with Compound C abrogated GH induction of Wip1. Src upregulates AMPK and triggers HIPK2 cytoplasmic relocation thereby inactivating HIPK2. We found that GH, by activating Src/AMPK phosphorylation, elicits HIPK2 cytoplasmic relocation, thus suppressing Wip1 ubiquitination and increasing its stability. Consistent with these findings, blocking Src phosphorylation with the tyrosine kinase inhibitor dasatinib abolished Wip1 upregulation and induced phosphorylation of its downstream targets. These results indicate that GH suppresses DDR by inducing Wip1 expression, and suppression of Wip1 may prevent epithelial accumulation of unrepaired DNA damage. These findings identify Wip1 as a specific novel target for GH action mediating GH-attenuation of DNA repair. Presentation: Thursday, June 15, 2023