Abstract LB-302: PTP1B regulates the Moyamoya disease-associated E3 ligase, RNF213 and cellular dioxygenase activity to allow breast tumor survival in hypoxia

Abstract

Abstract Deletion of Ptpn1, which encodes Protein-Tyrosine Phosphatase-1B (PTP1B), delays the onset of Her2/Neu-driven breast cancers in mice, but the underlying mechanism(s) remains controversial. Moreover, the role of PTP1B in HER2+ human breast cancer is unresolved. We found that, unexpectedly, PTP1B protects HER2+ breast cancer (BC) cell lines and tumors from hypoxia-induced death. Although there was no consistent effect of PTPN1 depletion or PTP1B inhibition on growth factor signaling or proliferation of HER2+ BC cells in vitro, PTP1B-deficient HER2+ xenografts showed increased hypoxia, necrosis and impaired growth. PTPN1-knockdown (1B-KD) also sensitized HER2+ BC lines to hypoxia-induced death in vitro. Studies using catalytically inactive mutants or an allosteric PTP1B inhibitor demonstrated that the ability of PTP1B to promote survival in hypoxia requires catalytic activity. Metabolic analysis revealed increased non-mitochondrial oxygen consumption, accompanied by decreased α-ketoglutarate (α-KG) levels, in 1B-KD cells, suggestive of enhanced activity of one or more α-KG-dependent dioxygenases. Consistent with this notion, addition of the pan-oxygenase inhibitors IOXI or DMOG protected 1B-KD HER2+ BC cells from hypoxia-induced death. Studies with “substrate-trapping” mutants identified the product of the Moyamoya disease-associated gene RNF213, as a PTP1B substrate in HER2+ BC cells. Remarkably, RNF213-knockdown (RNF213-KD) rescued the effects of PTP1B-deficiency on non-mitochondrial oxygen consumption and hypoxia-induced death of HER2+ BC cells. RNF213-KD also partially restored growth of tumors evoked by 1B-KD HCC1954 cells. RNF213 is a 591kDa E3-ligase with RING finger and AAA+ ATPase domains, not previously implicated in PTP1B action. Preliminary proteomic characterization revealed that BT474 1B-KD cells have RNF213-dependent alterations in the ubiquitylome. Future work will determine how these changes affect α-KG-dependent dioxygenase(s) activity. Our results reveal a new function for PTP1B, acting via RNF213, to control one or more α-KG-dependent dioxygenases in HER2+ BC cells. This novel PTP1B/RNF213 hypoxia-regulatory pathway is critical for the survival of breast cancer and possibly other malignant cells in the tumor microenvironment. Note: This abstract was not presented at the meeting. Citation Format: Robert S. Banh, Caterina Iorio, Richard Marcotte, Yang Xu, Dan Cojocari, Anas Abdel Rahman, Judy Pawling, Ankit Sinha, Toshiaki Hitomi, Toshiyuki Habu, Akio Koizumi, Sarah Wilkins, Thomas Kislinger, Christopher J. Schofield, James W. Dennis, Bradly G. Wouters, Benjamin G. Neel. PTP1B regulates the Moyamoya disease-associated E3 ligase, RNF213 and cellular dioxygenase activity to allow breast tumor survival in hypoxia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-302. doi:10.1158/1538-7445.AM2015-LB-302