MYC is sufficient to generate mid-life high-grade serous ovarian and uterine serous carcinomas in a p53-R270H mouse model.

Genetically engineered mouse models (GEMM) have fundamentally changed how ovarian cancer etiology, early detection, and treatment is understood. However, previous GEMMs of high-grade serous ovarian cancer (HGSOC) have had to utilize genetics rarely or never found in human HGSOC to yield ovarian cancer within the lifespan of a mouse. MYC, an oncogene, is amongst the most amplified genes in HGSOC, but it has not previously been utilized to drive HGSOC GEMMs. We coupled Myc and dominant negative mutant p53-R270H with a fallopian tube epithelium-specific promoter Ovgp1 to generate a new GEMM of HGSOC. Female mice developed lethal cancer at an average of 15.1 months. Histopathological examination of mice revealed HGSOC characteristics including nuclear p53 and nuclear MYC in clusters of cells within the fallopian tube epithelium and ovarian surface epithelium. Unexpectedly, nuclear p53 and MYC clustered cell expression was also identified in the uterine luminal epithelium, possibly from intraepithelial metastasis from the fallopian tube epithelium (FTE). Extracted tumor cells exhibited strong loss of heterozygosity at the p53 locus, leaving the mutant allele. Copy number alterations in these cancer cells were prevalent, disrupting a large fraction of genes. Transcriptome profiles most closely matched human HGSOC and serous endometrial cancer. Taken together, these results demonstrate the Myc and Trp53-R270H transgene was able to recapitulate many phenotypic hallmarks of HGSOC through the utilization of strictly human-mimetic genetic hallmarks of HGSOC. This new mouse model enables further exploration of ovarian cancer pathogenesis, particularly in the 50% of HGSOC which lack homology directed repair mutations. Histological and transcriptomic findings are consistent with the hypothesis that uterine serous cancer may originate from the fallopian tube epithelium.

BRD4 promotes resection and homology-directed repair of DNA double-strand breaks.

Double-strand breaks (DSBs) are one of the most toxic forms of DNA damage and represent a major source of genomic instability. Members of the bromodomain and extra-terminal (BET) protein family are characterized as epigenetic readers that regulate gene expression. However, evidence suggests that BET proteins also play a more direct role in DNA repair. Here, we establish a cell-free system using Xenopus egg extracts to elucidate the gene expression-independent functions of BET proteins in DSB repair. We identify the BET protein BRD4 as a critical regulator of homologous recombination and describe its role in stimulating DNA processing through interactions with the SWI/SNF chromatin remodeling complex and resection machinery. These results establish BRD4 as a multifunctional regulator of chromatin binding that links transcriptional activity and homology-directed repair.

BRCA1-BARD1 regulates transcription through BRD4 in Xenopus nucleoplasmic extract.

The tumor suppressor BRCA1 is considered a master regulator of genome integrity. Although widely recognized for its DNA repair functions, BRCA1 has also been implicated in various mechanisms of chromatin remodeling and transcription regulation. However, the precise role that BRCA1 plays in these processes has been difficult to establish due to the widespread consequences of its cellular dysfunction. Here, we use nucleoplasmic extract derived from the eggs of Xenopus laevis to investigate the role of BRCA1 in a cell-free transcription system. We report that BRCA1-BARD1 suppresses transcription initiation independent of DNA damage signaling and its established role in histone H2A ubiquitination. BRCA1-BARD1 acts through a histone intermediate, altering acetylation of histone H4K8 and recruitment of the chromatin reader and oncogene regulator BRD4. Together, these results establish a functional relationship between an established (BRCA1) and emerging (BRD4) regulator of genome integrity.

p97 Promotes a Conserved Mechanism of Helicase Unloading during DNA Cross-Link Repair.

Interstrand cross-links (ICLs) are extremely toxic DNA lesions that create an impassable roadblock to DNA replication. When a replication fork collides with an ICL, it triggers a damage response that promotes multiple DNA processing events required to excise the cross-link from chromatin and resolve the stalled replication fork. One of the first steps in this process involves displacement of the CMG replicative helicase (comprised of Cdc45, MCM2-7, and GINS), which obstructs the underlying cross-link. Here we report that the p97/Cdc48/VCP segregase plays a critical role in ICL repair by unloading the CMG complex from chromatin. Eviction of the stalled helicase involves K48-linked polyubiquitylation of MCM7, p97-mediated extraction of CMG, and a largely degradation-independent mechanism of MCM7 deubiquitylation. Our results show that ICL repair and replication termination both utilize a similar mechanism to displace the CMG complex from chromatin. However, unlike termination, repair-mediated helicase unloading involves the tumor suppressor protein BRCA1, which acts upstream of MCM7 ubiquitylation and p97 recruitment. Together, these findings indicate that p97 plays a conserved role in dismantling the CMG helicase complex during different cellular events, but that distinct regulatory signals ultimately control when and where unloading takes place.

2'-hydroxy C16-ceramide induces apoptosis-associated proteomic changes in C6 glioma cells.

Ceramide is a bioactive sphingolipid involved in regulation of numerous cell signaling pathways. Evidence is accumulating that differences in ceramide structure, such as N-acyl chain length and desaturation of sphingoid base, determine the biological activities of ceramide. Using synthetic (R)-2'-hydroxy-C16-ceramide, which is the naturally occurring stereoisomer, we demonstrate that this ceramide has more potent pro-apoptotic activity compared to its (S) isomer or non-hydroxylated C16-ceramide. Upon exposure to (R)-2'-hydroxy-ceramide, C6 glioma cells rapidly underwent apoptosis as indicated by caspase-3 activation, PARP cleavage, chromatin condensation, and annexin V stain. A 2D gel proteomics analysis identified 28 proteins whose levels were altered during the initial 3 h of exposure. Using the list of 28 proteins, we performed a software-assisted pathway analysis to identify possible signaling events that would result in the observed changes. The result indicated that Akt and MAP kinase pathways are among the possible pathways regulated by (R)-2'-hydroxy-ceramide. Experimental validation confirmed that 2'-hydroxy-ceramide significantly altered phosphorylation status of Akt and its downstream effector GSK3ß, as well as p38, ERK1/2, and JNK1/2 MAP kinases. Unexpectedly, robust phosphorylation of Akt was observed within 1 h of exposure to 2'-hydroxy-ceramide, followed by dephosphorylation. Phosphorylation status of MAPKs showed a complex pattern, in which rapid phosphorylation of ERK1/2 was followed by dephosphorylation of p38 and ERK1/2 and phosphorylation of the 46 kDa isoform of JNK1/2. These data indicate that (R)-2'-hydroxy-ceramide regulates multiple signaling pathways by affecting protein kinases and phosphatases with kinetics distinct from that of the extensively studied non-hydroxy-ceramide or its unnatural stereoisomer.

Central nervous system dysfunction in a mouse model of FA2H deficiency.

Fatty acid 2-hydroxylase (FA2H) is responsible for the synthesis of myelin galactolipids containing hydroxy fatty acid (hFA) as the N-acyl chain. Mutations in the FA2H gene cause leukodystrophy, spastic paraplegia, and neurodegeneration with brain iron accumulation. Using the Cre-lox system, we developed two types of mouse mutants, Fa2h(-/-) mice (Fa2h deleted in all cells by germline deletion) and Fa2h(flox/flox) Cnp1-Cre mice (Fa2h deleted only in oligodendrocytes and Schwann cells). We found significant demyelination, profound axonal loss, and abnormally enlarged axons in the CNS of Fa2h(-/-) mice at 12 months of age, while structure and function of peripheral nerves were largely unaffected. Fa2h(-/-) mice also exhibited histological and functional disruption in the cerebellum at 12 months of age. In a time course study, significant deterioration of cerebellar function was first detected at 7 months of age. Further behavioral assessments in water T-maze and Morris water maze tasks revealed significant deficits in spatial learning and memory at 4 months of age. These data suggest that various regions of the CNS are functionally compromised in young adult Fa2h(-/-) mice. The cerebellar deficits in 12-month-old Fa2h(flox/flox) Cnp1-Cre mice were indistinguishable from Fa2h(-/-) mice, indicating that these phenotypes likely stem from the lack of myelin hFA-galactolipids. In contrast, Fa2h(flox/flox) Cnp1-Cre mice did not show reduced performance in water maze tasks, indicating that oligodendrocytes are not involved in the learning and memory deficits found in Fa2h(-/-) mice. These findings provide the first evidence that FA2H has an important function outside of oligodendrocytes in the CNS.

Cryptorchidism induced in normal rats by the relaxin-like factor inhibitor.

Cryptorchidism is a serious problem, which affects 2-5% of the male population. Failure of the testes to descend into the scrotal region impairs germ cell development and is associated with a greater incidence of testicular cancer. The relaxin-like factor (RLF or insulin-like-3) has been shown to be critically important for the timely descent of the testicles in mice. We have discovered that the signal initiation site of the RLF can be eliminated without measurable effects on hormone binding to its receptor and that the resulting RLF derivative is a competitive inhibitor of RLF called RLFi. RLFi administered to pregnant rats causes dose-dependent gonadal retention in the offspring. The ability to control the severity of the syndrome by altering the concentration of RLFi and the timing of administration enables us to study in detail the structural changes that are associated with the action of RLF during critical stages of development. Targeted inhibition of the physiological migration pattern of testicles by RLFi lets one dissect the physiological process such as to find a window for clinical application of RLF and to search for ancillary factors that might play a role during normal development.