Ubiquitin E3 ligase CRL4(CDT2/DCAF2) as a potential chemotherapeutic target for ovarian surface epithelial cancer.

Cullin-RING ubiquitin ligases (CRLs) are the largest family of E3 ligases and require cullin neddylation for their activation. The NEDD8-activating enzyme inhibitor MLN4924 reportedly blocked cullin neddylation and inactivated CRLs, which resulted in apoptosis induction and tumor suppression. However, CRL roles in ovarian cancer cell survival and the ovarian tumor repressing effects of MLN4924 are unknown. We show here that CRL4 components are highly expressed in human epithelial ovarian cancer tissues. MLN4924-induced DNA damage, cell cycle arrest, and apoptosis in ovarian cancer cells in a time- and dose-dependent manner. In addition, MLN4924 sensitized ovarian cancer cells to other chemotherapeutic drug treatments. Depletion of CRL4 components Roc1/2, Cul4a, and DDB1 had inhibitory effects on ovarian cancer cells similar to MLN4924 treatment, which suggested that CRL4 inhibition contributed to the chemotherapeutic effect of MLN4924 in ovarian cancers. We also investigated for key CRL4 substrate adaptors required for ovarian cancer cells. Depleting Vprbp/Dcaf1 did not significantly affect ovarian cancer cell growth, even though it was expressed by ovarian cancer tissues. However, depleting Cdt2/Dcaf2 mimicked the pharmacological effects of MLN4924 and caused the accumulation of its substrate, CDT1, both in vitro and in vivo. MLN4924-induced DNA damage and apoptosis were partially rescued by Cdt1 depletion, suggesting that CRL4(CDT2) repression and CDT1 accumulation were key biochemical events contributing to the genotoxic effects of MLN4924 in ovarian cancer cells. Taken together, these results indicate that CRL4(CDT2) is a potential drug target in ovarian cancers and that MLN4924 may be an effective anticancer agent for targeted ovarian cancer therapy.

Death domain-associated protein DAXX promotes ovarian cancer development and chemoresistance.

BACKGROUND: The role of DAXX in ovarian cancer development and metastasis has not been investigated before now. RESULTS: Overexpression of DAXX enhanced ovarian cancer cell proliferation, colony formation, and migration, whereas Daxx depletion had the opposite effects. CONCLUSION: DAXX promotes ovarian cancer cell proliferation and chemoresistance. SIGNIFICANCE: ModulatingDAXXmay be an effective strategy for preventing the recurrence and chemoresistance of ovarian cancers. Understanding the genes involved in apoptosis and DNA damage responses may improve therapeutic strategies for ovarian cancer. The death domain-associated protein DAXX can be either a pro-apoptotic or an anti-apoptotic factor, depending on the cell type and context. In this study, we found that DAXX was highly expressed in human ovarian surface epithelial tumors but not in granulosa cell tumors. In cultured ovarian cancer cells, DAXX interacted with promyelocytic leukemia protein (PML) and localized to subnuclear domains (so-called PML nuclear bodies). A role for DAXX in ovarian cancer cell proliferation, metastasis, and radio/chemoresistance was examined. Overexpression of DAXX enhanced multiple ovarian cancer cell lines' proliferation, colony formation, and migration, whereas Daxx depletion by RNA interference had the opposite effects. When transplanted into nude mice, ovarian cancer cells that overexpressed DAXX displayed enhanced tumorigenesis capability in vivo, whereas Daxx depletion inhibited tumor development. Importantly, Daxx induced tumorigenic transformation of normal ovarian surface epithelial cells. Daxx also protected ovarian cancer cells against x-irradiation- and chemotherapy-induced DNA damage by interacting with PML. Taken together, our results suggest that DAXX is a novel ovarian cancer oncogene that promotes ovarian cancer cell proliferation and chemoresistance in ovarian cancer cells. Thus, modulating DAXX-PML nuclear body activity may be an effective strategy for preventing the recurrence and chemoresistance of ovarian cancers.

Studying the Functions of TGF-ß Signaling in the Ovary.

In mammals, ovulation is a multistep physiological process that includes preovulatory follicle growth, oocyte meiotic maturation, cumulus-oocyte complex (COC) expansion, follicle rupture, and luteinization. TGF-ß signaling pathway has multiple functions in mammalian ovary, as its complexity in ovarian function has been demonstrated by mouse models with knockouts of TGF-ß receptors and SMADs. We describe the protocol that we use to study functions of TGF-ß signaling pathway in follicle development and ovulation. Because total knockout of TGF-ß pathway components often causes embryonic lethality, which prevents further investigation of these genes in ovarian functions, people have generated ovarian cell type-specific knockout mouse strains for TGF-ß signaling pathway genes. These mouse models are also described.

Oocyte-expressed yes-associated protein is a key activator of the early zygotic genome in mouse.

In early mammalian embryos, the genome is transcriptionally quiescent until the zygotic genome activation (ZGA) which occurs 2-3 days after fertilization. Despite a long-standing effort, maternal transcription factors regulating this crucial developmental event remain largely elusive. Here, using maternal and paternal mouse models of Yap1 deletion, we show that maternally accumulated yes-associated protein (YAP) in oocyte is essential for ZGA. Maternal Yap1-knockout embryos exhibit a prolonged two-cell stage and develop into the four-cell stage at a much slower pace than the wild-type controls. Transcriptome analyses identify YAP target genes in early blastomeres; two of which, Rpl13 and Rrm2, are required to mediate maternal YAP's effect in conferring developmental competence on preimplantation embryos. Furthermore, the physiological YAP activator, lysophosphatidic acid, can substantially improve early development of wild-type, but not maternal Yap1-knockout embryos in both oviduct and culture. These observations provide insights into the mechanisms of ZGA, and suggest potentials of YAP activators in improving the developmental competence of cultured embryos in assisted human reproduction and animal biotechnology.

BTG4 is a meiotic cell cycle-coupled maternal-zygotic-transition licensing factor in oocytes.

The mRNAs stored in oocytes undergo general decay during the maternal-zygotic transition (MZT), and their stability is tightly interconnected with meiotic cell-cycle progression. However, the factors that trigger decay of maternal mRNA and couple this event to oocyte meiotic maturation remain elusive. Here, we identified B-cell translocation gene-4 (BTG4) as an MZT licensing factor in mice. BTG4 bridged CNOT7, a catalytic subunit of the CCR4-NOT deadenylase, to eIF4E, a key translation initiation factor, and facilitated decay of maternal mRNA. Btg4-null females produced morphologically normal oocytes but were infertile, owing to early developmental arrest. The intrinsic MAP kinase cascade in oocytes triggered translation of Btg4 mRNA stored in fully grown oocytes by targeting the 3' untranslated region, thereby coupling CCR4-NOT deadenylase-mediated decay of maternal mRNA with oocyte maturation and fertilization. This is a key step in oocyte cytoplasmic maturation that determines the developmental potential of mammalian embryos.

CRL4 complex regulates mammalian oocyte survival and reprogramming by activation of TET proteins.

The duration of a woman's reproductive period is determined by the size and persistence of a dormant oocyte pool. Specific oocyte genes are essential for follicle maintenance and female fertility. The mechanisms that regulate the expression of these genes are poorly understood. We found that a cullin-ring finger ligase-4 (CRL4) complex was crucial in this process. Oocyte-specific deletion of the CRL4 linker protein DDB1 or its substrate adaptor VPRBP (also known as DCAF1) caused rapid oocyte loss, premature ovarian insufficiency, and silencing of fertility maintaining genes. CRL4(VPRBP) activates the TET methylcytosine dioxygenases, which are involved in female germ cell development and zygote genome reprogramming. Hence, CRL4(VPRBP) ubiquitin ligase is a guardian of female reproductive life in germ cells and a maternal reprogramming factor after fertilization.