ARTICLE https://doi.org/10.1038/s41467-019-09471-9 OPEN Mad1 destabilizes p53 by preventing PML from sequestering MDM2 Jun Wan1, Samuel Block2, Christina M. Scribano1, Rebecca Thiry1, Karla Esbona3, Anjon Audhya 2,4 & Beth A. Weaver1,4,5 Mitotic arrest deficient 1 (Mad1) plays a well-characterized role in the mitotic checkpoint. However, interphase roles of Mad1 that do not impact mitotic checkpoint function remain 1234567890():,; largely uncharacterized. Here we show that upregulation of Mad1, which is common in human breast cancer, prevents stress-induced stabilization of the tumor suppressor p53 in multiple cell types. Upregulated Mad1 localizes to ProMyelocytic Leukemia (PML) nuclear bodies in breast cancer and cultured cells. The C-terminus of Mad1 directly interacts with PML, and this interaction is enhanced by sumoylation. PML stabilizes p53 by sequestering MDM2, an E3 ubiquitin ligase that targets p53 for degradation, to the nucleolus. Upregulated Mad1 displaces MDM2 from PML, freeing it to ubiquitinate p53. Upregulation of Mad1 accelerates growth of orthotopic mammary tumors, which show decreased levels of p53 and its downstream effector p21. These results demonstrate an unexpected interphase role for Mad1 in tumor promotion via p53 destabilization. 1 Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, WI 53705, USA. 2 Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI 53705, USA. 3 Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, WI 53705, USA. 4 Carbone Cancer Center, University of Wisconsin-Madison, Madison, WI 53705, USA. 5 Department of Oncology/McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI 53705, USA. Correspondence and requests for materials should be addressed to B.A.W. (email: [email protected]) NATURE COMMUNICATIONS | (2019) 10:1540 | https://doi.org/10.1038/s41467-019-09471-9 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09471-9 ad1 was initially discovered in a landmark screen provide molecular insight into a novel interphase role of Mad1 in Mdemonstrating that mitosis is regulated by a cell cycle destabilizing p53 and promoting tumor initiation and growth. checkpoint, termed the mitotic (or spindle assembly) 1 checkpoint . The mitotic checkpoint ensures accurate chromo- Results some segregation by delaying separation of the replicated sister Mad1 accumulates into PML NBs. Upregulated Mad1 localizes chromatids until each sister chromatid pair is stably attached to – to kinetochores and the nuclear envelope, as expected, but also opposite spindle poles through its kinetochores2 6. Mad1 plays forms nuclear puncta2,16. This fraction of Mad1 does not colo- an evolutionarily conserved role in the mitotic checkpoint by calize with nucleoli (Supplementary Fig. 1a–b), but does show recruiting its binding partner Mad2 to the kinetochores of – substantial colocalization with Myc and HA tagged SUMO1 and unattached chromatids7 9. At unattached kinetochores, Mad2 is SUMO2 as well as endogenous SUMO1 in MDA-MB-231 breast converted into an active mitotic checkpoint inhibitor that delays – cancer cells and in HeLa cervical cancer cells (Fig. 1a–c, f, sister chromatid separation10 13. Once the kinetochores of all Supplementary Fig. 1b–e). SUMO1 and SUMO2 are highly sister chromatids are stably attached to spindle microtubules, – concentrated in PML NBs34 36, suggesting that Mad1 puncta the mitotic checkpoint is satisfied, and Mad1 and Mad2 are localize there. Indeed, Mad1 nuclear puncta substantially colo- no longer recruited. Loss of Mad1 is lethal, and cells with calize with endogenous as well as HA-tagged PML during reduced expression of Mad1 missegregate chromosomes to both interphase and mitosis (Fig. 1d–f, Supplementary Fig. 1f–g). become aneuploid1,14. Thus, Mad1 is essential and plays a highly Under stressful culture conditions, a small portion of endogenous conserved role in ensuring accurate chromosome segregation Mad1 also colocalizes with PML (Supplementary Fig. 1h–j). during mitosis. Arsenic trioxide is therapeutic in acute PML, since it induces Although Mad1 plays a well-characterized role during mitosis, proteasomal degradation of PML-RAR, as well as endogenous and expression of many mitotic proteins peaks during mitosis, – PML36 38. Arsenic treatment was used to confirm that upregu- Mad1 expression levels remain constant throughout the cell cycle2. lated Mad1 localizes to PML NBs. 12 and 24 h after arsenic During interphase, Mad1 recruits Mad2 to nuclear pores at the treatment, PML protein levels were decreased and PML NBs nuclear envelope, which permits the production of mitotic – substantially dispersed (Fig. 1g, h). Substantially fewer Mad1 checkpoint inhibitors during interphase3,15 17. Interphase func- puncta formed 12 and 24 h after arsenic treatment, although tions of Mad1 that do not affect mitotic checkpoint signaling Mad1 protein levels remained constant (Fig. 1g, h, Supplementary have remained largely uncharacterized, although it is known that Fig. 1k). These data demonstrate that the formation of Mad1 Mad1 functions independently of Mad2 at the Golgi apparatus nuclear puncta is dependent upon PML NBs. to promote secretion of α5 integrin18,19. Mad1 is frequently Mad1 recruits Mad2 to unattached kinetochores and nuclear upregulated at both the mRNA and protein level in human breast pores3,16,39, and Mad1 interacts with Mad2 throughout the cell cancers, where Mad1 upregulation serves as a marker of poor cycle3,40. Mad2 is expressed in molar excess of Mad1, and the prognosis2,20,21. Mad1 upregulation causes a low rate of chro- – only known pool of Mad1 that does not recruit Mad2 is at the mosome missegregation, which is weakly tumor promoting2,22 24. Golgi apparatus41. To determine whether Mad1 recruits Mad2 to However, whether Mad1 upregulation has additional tumor- PML NBs, Mad1-3xFLAG and Mad2-GFP were expressed singly promoting activities during interphase has remained unclear. or in combination. Mad1-3xFLAG recruited Mad2-GFP to Upregulated Mad1 localizes to nuclear pores and kinetochores, nuclear puncta, which Mad2-GFP did not form when expressed as expected, but also forms punctate structures2,16. A fraction alone (Supplementary Fig. 1l). Thus, Mad1 recruits Mad2 to PML of these colocalize with markers of annulate lamellae, storage NBs, as it does to unattached kinetochores and nuclear pores. compartments for excess nuclear pore components, which are We have previously shown that Mad1 protein levels are predominantly cytoplasmic2,16,25. Nuclear Mad1 puncta have commonly increased in breast cancer patients, and that remained uncharacterized. Promyelocytic leukemia (PML) upregulated Mad1 forms punctate structures in breast cancer2. nuclear bodies (NBs) represent one prominent source of nuclear To determine if Mad1 puncta represent PML NBs, sections from puncta. The PML protein, which is fused to retinoic acid receptor nine primary breast cancers were costained with antibodies to alpha (RARα) due to a reciprocal translocation between chro- Mad1 and SUMO1. Indeed Mad1 puncta frequently colocalized mosomes 15 and 17 in >98% of acute PML patients, forms the with SUMO1 (Fig. 1i, j). Together, these results demonstrate that core of PML NBs26. >100 proteins localize to PML NBs, including upregulated Mad1 localizes to PML NBs in cultured cells and in proteins involved in cell cycle arrest, apoptosis, transcription, human cancer. and metabolism27. Though the proteins that localize to PML NBs are functionally diverse, most of these proteins, including PML itself, are sumoylated26,27. Here, we show that upregulated Mad1 Mad1 interacts with PML through its C terminal domain localizes to PML NBs. (CTD). As an initial test of whether Mad1 localization to PML Protein levels of the p53 tumor suppressor remain low in the NBs was due to an interaction with PML or with another com- absence of cellular stresses due to continuous ubiquitination by ponent of PML NBs, Mad1-3xFLAG was immunoprecipitated MDM2 followed by degradation28–30. In response to a variety from HEK293T cells. HA-PML was co-immunoprecipitated with of cellular stresses including DNA damage, PML sequesters Mad1-3xFLAG, supporting an interaction between these proteins MDM2 in the nucleolus, which physically separates MDM2 from (Supplementary Fig. 1m). To determine the region of Mad1 p53 and results in p53 stabilization31–34. Here, we demonstrate responsible for the interaction with PML, 10 deletion mutants a previously unexpected interphase role for Mad1 in preventing of Mad1 were generated (Fig. 2a, Supplementary Fig. 2a). p53 stabilization. The C-terminal domain (CTD) of Mad1 binds Immunoprecipitation experiments revealed that all constructs PML directly in a manner facilitated by sumoylation of PML. containing the CTD of Mad1 (aa 597–718) co-precipitated Upregulated Mad1 localizes to PML NBs, and localization is PML, while fragments lacking the Mad1 CTD did not (Fig. 2b). dependent on the SUMO interacting motif (SIM) within the A reciprocal immunoprecipitation experiment showed that Mad1 CTD. After DNA damage, upregulated Mad1 displaces immunoprecipitation of HA-PML co-precipitated all Mad1 MDM2 from PML, replaces MDM2 at nucleoli, and increases the fragments containing the CTD, but no fragments lacking the interaction of MDM2 with p53. Mad1-YFP promotes orthotopic Mad1 CTD (Fig. 2c). Thus, Mad1 interacts with PML in cell mammary tumors