Somatic mutations in the gene encoding the catalytic subunit of protein phosphatase 6 (Ppp6c) have been identified in malignant melanoma and are thought to function as a driver in B-raf- or N-ras-driven tumorigenesis. To assess the role of Ppp6c in carcinogenesis, we generated skin keratinocyte-specific Ppp6c conditional knockout mice and performed two-stage skin carcinogenesis analysis. Ppp6c deficiency induced papilloma formation with 7,12-dimethylbenz (a) anthracene (DMBA) only, and development of those papillomas was significantly accelerated compared with that seen following DMBA/TPA (12-O-tetradecanoylphorbol 13-acetate) treatment of wild-type mice. NF-κB activation either by tumor necrosis factor (TNF)-α or interleukin (IL)-1β was enhanced in Ppp6c-deficient keratinocytes. Overall, we conclude that Ppp6c deficiency predisposes mice to skin carcinogenesis initiated by DMBA. This is the first report showing that such deficiency promotes tumor formation in mice.

BRCA1 functions as a tumor suppressor in DNA repair and centrosome regulation. Previously, Obg-like ATPase 1 (OLA1) was shown to interact with BARD1, a heterodimer partner of BRCA1. OLA1 binds to BRCA1, BARD1, and γ-tubulin and functions in centrosome regulation. This study determined that overexpression of wild-type OLA1 (OLA1-WT) caused centrosome amplification due to centriole overduplication in mammary tissue-derived cells. Centrosome amplification induced by overexpression of the cancer-derived OLA1 mutant, which is deficient at regulating centrosome number, occurred in significantly fewer cells than in that induced by overexpression of OLA1-WT. Thus, it was hypothesized that overexpression of OLA1 with normal function efficiently induces centrosome amplification, but not that of OLA1 mutants, which are deficient at regulating centrosome number. We analyzed whether overexpression of OLA1 missense mutants of nine candidate phosphorylation residues, three residues modified with acetylation, and two ATP-binding residues caused centrosome amplification and identified five missense mutants that are deficient in the regulation of centrosome number. Three of them did not bind to BARD1. Two phosphomimetic mutations restored the binding to BARD1 and the efficient centrosome amplification by their overexpression. Knockdown and overexpression of BARD1 also caused centrosome amplification. BARD1 mutant reported in cancer failed to bind to OLA1 and rescue the BARD1 knockdown-induced centrosome amplification and reduced its centrosomal localization. Combined, these data reveal that the OLA1-BARD1 interaction is important for the regulation of centrosome number. Regulation of centrosome number by BRCA1/BARD1 together with OLA1 is important for the genome integrity to prevent tumor development. .

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Ppp6c, which encodes the catalytic subunit of phosphoprotein phosphatase 6 (PP6), is conserved among eukaryotes from yeast to humans. In mammalian cells, PP6 targets IκBε for degradation, activates DNA-dependent protein kinase to trigger DNA repair, and is reportedly required for normal mitosis. Recently, Ppp6c mutations were identified as candidate drivers of melanoma and skin cancer. Nonetheless, little is known about the physiological role of Ppp6c. To investigate this function in vivo, we established mice lacking the Ppp6c phosphatase domain by crossing heterozygous mutants. No viable homozygous pups were born, indicative of a lethal mutation. Ppp6c homozygous mutant embryos were identified among blastocysts, which exhibited a normal appearance, but embryos degenerated by E7.5 and showed clear developmental defects at E8.5, suggesting that mutant embryos die after implantation. Accordingly, homozygous blastocysts showed significant growth failure of the inner cell mass (ICM) in in vitro blastocyst culture, and primary Ppp6c exon4-deficient MEFs showed greatly reduced proliferation. These results establish for the first time that the Ppp6c phosphatase domain is indispensable for mouse embryogenesis after implantation.

Here, we address the function of protein phosphatase 6 (PP6) loss on K‐ras‐initiated tumorigenesis in keratinocytes. To do so, we developed tamoxifen‐inducible double mutant (K‐ras G12D‐expressing and Ppp6c‐deficient) mice in which K‐ras G12D expression is driven by the cytokeratin 14 (K14) promoter. Doubly‐mutant mice showed early onset tumor formation in lips, nipples, external genitalia, anus and palms, and had to be killed by 3 weeks after induction by tamoxifen, while comparably‐treated K‐ras G12D‐expressing mice did not. H&E‐staining of lip tumors before euthanasia revealed that all were papillomas, some containing focal squamous cell carcinomas. Immunohistochemical analysis of lips of doubly‐mutant vs K‐ras G12D mice revealed that cell proliferation and cell size increased approximately 2‐fold relative to K‐ras G12D‐expressing mutants, and epidermal thickness of lip tissue greatly increased relative to that seen in K‐ras G12D‐only mice. Moreover, AKT phosphorylation increased in K‐ras G12D‐expressing/Ppp6c‐deficient cells, as did phosphorylation of the downstream effectors 4EBP1, S6 and GSK3, suggesting that protein synthesis and survival signals are enhanced in lip tissues of doubly‐mutant mice. Finally, increased numbers of K14‐positive cells were present in the suprabasal layer of doubly‐mutant mice, indicating abnormal keratinocyte differentiation, and γH2AX‐positive cells accumulated, indicating perturbed DNA repair. Taken together, Ppp6c deficiency enhances K‐ras G12D‐dependent tumor promotion.