Novel FOXF1-Stabilizing Compound TanFe Stimulates Lung Angiogenesis in Alveolar Capillary Dysplasia.

Rationale: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is linked to heterozygous mutations in the FOXF1 (Forkhead Box F1) gene, a key transcriptional regulator of pulmonary vascular development. There are no effective treatments for ACDMPV other than lung transplant, and new pharmacological agents activating FOXF1 signaling are urgently needed. Objectives: Identify-small molecule compounds that stimulate FOXF1 signaling. Methods: We used mass spectrometry, immunoprecipitation, and the in vitro ubiquitination assay to identify TanFe (transcellular activator of nuclear FOXF1 expression), a small-molecule compound from the nitrile group, which stabilizes the FOXF1 protein in the cell. The efficacy of TanFe was tested in mouse models of ACDMPV and acute lung injury and in human vascular organoids derived from induced pluripotent stem cells of a patient with ACDMPV. Measurements and Main Results: We identified HECTD1 as an E3 ubiquitin ligase involved in ubiquitination and degradation of the FOXF1 protein. The TanFe compound disrupted FOXF1-HECTD1 protein-protein interactions and decreased ubiquitination of the FOXF1 protein in pulmonary endothelial cells in vitro. TanFe increased protein concentrations of FOXF1 and its target genes Flk1, Flt1, and Cdh5 in LPS-injured mouse lungs, decreasing endothelial permeability and inhibiting lung inflammation. Treatment of pregnant mice with TanFe increased FOXF1 protein concentrations in lungs of Foxf1+/- embryos, stimulated neonatal lung angiogenesis, and completely prevented the mortality of Foxf1+/- mice after birth. TanFe increased angiogenesis in human vascular organoids derived from induced pluripotent stem cells of a patient with ACDMPV with FOXF1 deletion. Conclusions: TanFe is a novel activator of FOXF1, providing a new therapeutic candidate for treatment of ACDMPV and other neonatal pulmonary vascular diseases.

BIR repeat-containing ubiquitin conjugating enzyme (BRUCE) regulation of ß-catenin signaling in the progression of drug-induced hepatic fibrosis and carcinogenesis.

BACKGROUND: BIR repeat-containing ubiquitin conjugating enzyme (BRUCE) is a liver tumor suppressor, which is downregulated in a large number of patients with liver diseases. BRUCE facilitates DNA damage repair to protect the mouse liver against the hepatocarcinogen diethylnitrosamine (DEN)-dependent acute liver injury and carcinogenesis. While there exists an established pathologic connection between fibrosis and hepatocellular carcinoma (HCC), DEN exposure alone does not induce robust hepatic fibrosis. Further studies are warranted to identify new suppressive mechanisms contributing to DEN-induced fibrosis and HCC. AIM: To investigate the suppressive mechanisms of BRUCE in hepatic fibrosis and HCC development. METHODS: Male C57/BL6/J control mice [loxp/Loxp; albumin-cre (Alb-cre)-] and BRUCE Alb-Cre KO mice (loxp/Loxp; Alb-Cre+) were injected with a single dose of DEN at postnatal day 15 and sacrificed at different time points to examine liver disease progression. RESULTS: By using a liver-specific BRUCE knockout (LKO) mouse model, we found that BRUCE deficiency, in conjunction with DEN exposure, induced hepatic fibrosis in both premalignant as well as malignant stages, thus recapitulating the chronic fibrosis background often observed in HCC patients. Activated in fibrosis and HCC, ß-catenin activity depends on its stabilization and subsequent translocation to the nucleus. Interestingly, we observed that livers from BRUCE KO mice demonstrated an increased nuclear accumulation and elevated activity of ß-catenin in the three stages of carcinogenesis: Pre-malignancy, tumor initiation, and HCC. This suggests that BRUCE negatively regulates ß-catenin activity during liver disease progression. ß-catenin can be activated by phosphorylation by protein kinases, such as protein kinase A (PKA), which phosphorylates it at Ser-675 (pSer-675-ß-catenin). Mechanistically, BRUCE and PKA were colocalized in the cytoplasm of hepatocytes where PKA activity is maintained at the basal level. However, in BRUCE deficient mouse livers or a human liver cancer cell line, both PKA activity and pSer-675-ß-catenin levels were observed to be elevated. CONCLUSION: Our data support a "BRUCE-PKA-ß-catenin" signaling axis in the mouse liver. The BRUCE interaction with PKA in hepatocytes suppresses PKA-dependent phosphorylation and activation of ß-catenin. This study implicates BRUCE as a novel negative regulator of both PKA and ß-catenin in chronic liver disease progression. Furthermore, BRUCE-liver specific KO mice serve as a promising model for understanding hepatic fibrosis and HCC in patients with aberrant activation of PKA and ß-catenin.

BRUCE preserves genomic stability in the male germline of mice.

BRUCE is a DNA damage response protein that promotes the activation of ATM and ATR for homologous recombination (HR) repair in somatic cells, making BRUCE a key protector of genomic stability. Preservation of genomic stability in the germline is essential for the maintenance of species. Here, we show that BRUCE is required for the preservation of genomic stability in the male germline of mice, specifically in spermatogonia and spermatocytes. Conditional knockout of Bruce in the male germline leads to profound defects in spermatogenesis, including impaired maintenance of spermatogonia and increased chromosomal anomalies during meiosis. Bruce-deficient pachytene spermatocytes frequently displayed persistent DNA breaks. Homologous synapsis was impaired, and nonhomologous associations and rearrangements were apparent in up to 10% of Bruce-deficient spermatocytes. Genomic instability was apparent in the form of chromosomal fragmentation, translocations, and synapsed quadrivalents and hexavalents. In addition, unsynapsed regions of rearranged autosomes were devoid of ATM and ATR signaling, suggesting an impairment in the ATM- and ATR-dependent DNA damage response of meiotic HR. Taken together, our study unveils crucial functions for BRUCE in the maintenance of spermatogonia and in the regulation of meiotic HR-functions that preserve the genomic stability of the male germline.

Loss of BRUCE reduces cellular energy level and induces autophagy by driving activation of the AMPK-ULK1 autophagic initiating axis.

Autophagy is an intracellular catabolic system. It delivers cellular components to lysosomes for degradation and supplies nutrients that promote cell survival under stress conditions. Although much is known regarding starvation-induced autophagy, the regulation of autophagy by cellular energy level is less clear. BRUCE is an ubiquitin conjugase and ligase with multi-functionality. It has been reported that depletion of BRUCE inhibits starvation-induced autophagy by blockage of the fusion step. Herein we report a new function for BRUCE in the dual regulation of autophagy and cellular energy. Depletion of BRUCE alone (without starvation) in human osteosarcoma U2OS cells elevated autophagic activity as indicted by the increased LC3B-II protein and its autophagic puncta as well as further increase of both by chloroquine treatment. Such elevation results from enhanced induction of autophagy since the numbers of both autophagosomes and autolysosomes were increased, and recruitment of ATG16L onto the initiating membrane structure phagophores was increased. This concept is further supported by elevated lysosomal enzyme activities. In contrast to starvation-induced autophagy, BRUCE depletion did not block fusion of autophagosomes with lysosomes as indicated by increased lysosomal cleavage of the GFP-LC3 fusion protein. Mechanistically, BRUCE depletion lowered the cellular energy level as indicated by both a higher ratio of AMP/ATP and the subsequent activation of the cellular energy sensor AMPK (pThr-172). The lower energy status co-occurred with AMPK-specific phosphorylation and activation of the autophagy initiating kinase ULK1 (pSer-555). Interestingly, the higher autophagic activity by BRUCE depletion is coupled with enhanced cisplatin resistance in human ovarian cancer PEO4 cells. Taken together, BRUCE depletion promotes induction of autophagy by lowering cellular energy and activating the AMPK-ULK1-autophagy axis, which could contribute to ovarian cancer chemo-resistance. This study establishes a BRUCE-AMPK-ULK1 axis in the regulation of energy metabolism and autophagy, as well as provides insights into cancer chemo-resistance.

The BRUCE-ATR Signaling Axis Is Required for Accurate DNA Replication and Suppression of Liver Cancer Development.

Replication fork stability during DNA replication is vital for maintenance of genomic stability and suppression of cancer development in mammals. ATR (ataxia-telangiectasia mutated [ATM] and RAD3-related) is a master regulatory kinase that activates the replication stress response to overcome replication barriers. Although many downstream effectors of ATR have been established, the upstream regulators of ATR and the effect of such regulation on liver cancer remain unclear. The ubiquitin conjugase BRUCE (BIR Repeat containing Ubiquitin-Conjugating Enzyme) is a guardian of chromosome integrity and activator of ATM signaling, which promotes DNA double-strand break repair through homologous recombination. Here we demonstrate the functions for BRUCE in ATR activation in vitro and liver tumor suppression in vivo. BRUCE is recruited to induced DNA damage sites. Depletion of BRUCE inhibited multiple ATR-dependent signaling events during replication stress, including activation of ATR itself, phosphorylation of its downstream targets CHK1 and RPA, and the mono-ubiquitination of FANCD2. Consequently, BRUCE deficiency resulted in stalled DNA replication forks and increased firing of new replication origins. The in vivo impact of BRUCE loss on liver tumorigenesis was determined using the hepatocellular carcinoma model induced by genotoxin diethylnitrosamine. Liver-specific knockout of murine Bruce impaired ATR activation and exacerbated inflammation, fibrosis and hepatocellular carcinoma, which exhibited a trabecular architecture, closely resembling human hepatocellular carcinoma (HCC). In humans, the clinical relevance of BRUCE down-regulation in liver disease was found in hepatitis, cirrhosis, and HCC specimens, and deleterious somatic mutations of the Bruce gene was found in human hepatocellular carcinoma in the Cancer Genome Atlas database. Conclusion: These findings establish a BRUCE-ATR signaling axis in accurate DNA replication and suppression of liver cancer in mice and humans and provides a clinically relevant HCC mouse model.

The UBC Domain Is Required for BRUCE to Promote BRIT1/MCPH1 Function in DSB Signaling and Repair Post Formation of BRUCE-USP8-BRIT1 Complex.

BRUCE is implicated in the regulation of DNA double-strand break response to preserve genome stability. It acts as a scaffold to tether USP8 and BRIT1, together they form a nuclear BRUCE-USP8-BRIT1 complex, where BRUCE holds K63-ubiquitinated BRIT1 from access to DSB in unstressed cells. Following DSB induction, BRUCE promotes USP8 mediated deubiquitination of BRIT1, a prerequisite for BRIT1 to be released from the complex and recruited to DSB by binding to γ-H2AX. BRUCE contains UBC and BIR domains, but neither is required for the scaffolding function of BRUCE mentioned above. Therefore, it remains to be determined whether they are required for BRUCE in DSB response. Here we show that the UBC domain, not the BIR domain, is required for BRUCE to promote DNA repair at a step post the formation of BRUCE-USP8-BRIT1 complex. Mutation or deletion of the BRUCE UBC domain did not disrupt the BRUCE-USP8-BRIT1 complex, but impaired deubiquitination and consequent recruitment of BRIT1 to DSB. This leads to impaired chromatin relaxation, decreased accumulation of MDC1, NBS1, pATM and RAD51 at DSB, and compromised homologous recombination repair of DNA DSB. These results demonstrate that in addition to the scaffolding function in complex formation, BRUCE has an E3 ligase function to promote BRIT1 deubiquitination by USP8 leading to accumulation of BRIT1 at DNA double-strand break. These data support a crucial role for BRUCE UBC activity in the early stage of DSB response.

Knockdown of the Inhibitor of Apoptosis BRUCE Sensitizes Resistant Breast Cancer Cells to Chemotherapeutic Agents.

BACKGROUND AND OBJECTIVES: Management of patients with breast cancer often fails because of inherent or acquired resistance to chemotherapy. BRUCE (BIR repeat containing ubiquitin-conjugating enzyme) is a member of the inhibitor of apoptosis protein (IAP) family. It has various cellular functions including suppression of apoptosis and promotion of cytokinesis. Furthermore, it pays a critical role in promotion of DNA damage repair and preservation of genome stability, a new function recently reported by our group. Although BRUCE is expressed in breast cancer cell lines, its expression in human primary breast tumors and its contribution to chemoresistance in breast cancers has not been explored. Chemotherapeutic drugs are used in the treatment of breast cancer patients. However, they are not effective to all patients and patients often develop resistance. Consequently we explored if BRUCE protein level, as judged by immunohistochemistry (IHC), is higher in primary breast tumors than normal breast tissue. We also examined if depletion of BRUCE, using a lentiviral shRNA approach, enhances cell sensitivity to multiple chemotherapeutic agents, including cisplatin, an agent that induces DNA damage by generating DNA cross-links, and taxol, a microtubule stabilizer and mitotic inhibitor. The reason for including these two chemotherapeutic agents in this study is that they hit two essential cellular processes of DNA repair and cytokinesis in which BRUCE plays critical roles. RESULTS AND METHODS: IHC analysis of BRUCE revealed significantly higher levels of BRUCE in primary breast tumors than normal breast tissue. Knockdown of BRUCE protein expression by lentiviral shRNA resulted in increased sensitivity to cisplatin in the resistant breast cancer MDB-MD-231 cell line. Moreover, depletion of BRUCE in this cell line achieved a more profound level of cell killing when coupled to low doses of cisplatin and taxol combined, rather than either drug used alone. CONCLUSIONS: Our data suggest that elevated protein levels of BRUCE in breast tumors may contribute to chemoresistance in breast cancer patients. In support of this suggestion, our data demonstrate that a reduction in BRUCE expression in breast cancer cell lines increases the toxicity of several chemotherapeutic agents. In all likelihood, the contribution of increased BRUCE levels to chemoresistance are likely due to its roles in suppression of apoptosis, promotion of cytokinesis and facilitation of DNA damage repair. These observations suggest that therapeutic suppression of BRUCE could improve chemosensitivity in chemo-resistant breast cancer patients. Therefore, future development of effective inhibitors of BRUCE could benefit patients with high BRUCE expression and chemoresistance.

BRUCE regulates DNA double-strand break response by promoting USP8 deubiquitination of BRIT1.

The DNA damage response (DDR) is crucial for genomic integrity. BRIT1 (breast cancer susceptibility gene C terminus-repeat inhibitor of human telomerase repeat transcriptase expression), a tumor suppressor and early DDR factor, is recruited to DNA double-strand breaks (DSBs) by phosphorylated H2A histone family, member X (γ-H2AX), where it promotes chromatin relaxation by recruiting the switch/sucrose nonfermentable (SWI-SNF) chromatin remodeler to facilitate DDR. However, regulation of BRIT1 recruitment is not fully understood. The baculovirus IAP repeat (BIR)-containing ubiquitin-conjugating enzyme (BRUCE) is an inhibitor of apoptosis protein (IAP). Here, we report a non-IAP function of BRUCE in the regulation of the BRIT1-SWI-SNF DSB-response pathway and genomic stability. We demonstrate that BRIT1 is K63 ubiquitinated in unstimulated cells and that deubiquitination of BRIT1 is a prerequisite for its recruitment to DSB sites by γ-H2AX. We show mechanistically that BRUCE acts as a scaffold, bridging the ubiquitin-specific peptidase 8 (USP8) and BRIT1 in a complex to coordinate USP8-catalyzed deubiquitination of BRIT1. Loss of BRUCE or USP8 impairs BRIT1 deubiquitination, BRIT1 binding with γ-H2AX, the formation of BRIT1 DNA damage foci, and chromatin relaxation. Moreover, BRUCE-depleted cells display reduced homologous recombination repair, and BRUCE-mutant mice exhibit repair defects and genomic instability. These findings identify BRUCE and USP8 as two hitherto uncharacterized critical DDR regulators and uncover a deubiquitination regulation of BRIT1 assembly at damaged chromatin for efficient DDR and genomic stability.

OsHUS1 facilitates accurate meiotic recombination in rice.

Meiotic recombination normally takes place between allelic sequences on homologs. This process can also occur between non-allelic homologous sequences. Such ectopic interaction events can lead to chromosome rearrangements and are normally avoided. However, much remains unknown about how these ectopic interaction events are sensed and eliminated. In this study, using a screen in rice, we characterized a homolog of HUS1 and explored its function in meiotic recombination. In Oshus1 mutants, in conjunction with nearly normal homologous pairing and synapsis, vigorous, aberrant ectopic interactions occurred between nonhomologous chromosomes, leading to multivalent formation and subsequent chromosome fragmentation. These ectopic interactions relied on programmed meiotic double strand breaks and were formed in a manner independent of the OsMER3-mediated interference-sensitive crossover pathway. Although early homologous recombination events occurred normally, the number of interference-sensitive crossovers was reduced in the absence of OsHUS1. Together, our results indicate that OsHUS1 might be involved in regulating ectopic interactions during meiosis, probably by forming the canonical RAD9-RAD1-HUS1 (9-1-1) complex.

The role of OsCOM1 in homologous chromosome synapsis and recombination in rice meiosis.

COM1/SAE2 is a highly conserved gene from yeast to higher eukaryotes. Its orthologs, known to cooperate with the MRX complex (Mre11/Rad50/Xrs2), are required for meiotic DNA double-strand break (DSB) ends resection and specific mitotic DSB repair events. Here, the rice (Oryza sativa, 2n = 2x = 24) COM1/SAE2 homolog was identified through positional cloning, termed OsCOM1. Four independent mutants of OsCOM1 were isolated and characterized. In Oscom1 mutants, synaptonemal complex (SC) formation, homologous pairing and recombination were severely inhibited, whereas aberrant non-homologous chromosome entanglements occurred constantly. Several key meiotic proteins, including ZEP1 and OsMER3, were not loaded normally onto chromosomes in Oscom1 mutants, whereas the localization of OsREC8, PAIR2 and PAIR3 seemed to be normal. Moreover, OsCOM1 was loaded normally onto meiotic chromosomes in Osrec8, zep1 and Osmer3 mutants, but could not be properly loaded in Osam1, pair2 and OsSPO11-1(RNAi) plants. These results provide direct evidence for the functions of OsCOM1 in promoting homologous synapsis and recombination in rice meiosis.

OsREC8 is essential for chromatid cohesion and metaphase I monopolar orientation in rice meiosis.

The successful transmission of chromosomes during mitosis and meiosis relies on the establishment and subsequent release of cohesion between replicated chromatids. Cohesion is mediated by a four-subunit structural maintenance of chromosome complex, called cohesins. REC8 is a key component of this meiotic cohesion complex in most model organisms studied to date. Here, we isolated and dissected the functions of OsREC8, a rice (Oryza sativa) REC8 homolog, using two null Osrec8 mutants. We showed that OsREC8 encodes a protein that localized to meiotic chromosomes from approximately meiotic interphase to metaphase I. Homologous pairing and telomere bouquet formation were abnormal in Osrec8 meiocytes. Furthermore, fluorescent in situ hybridization experiments on Osrec8 meiocytes demonstrated that the mutation eliminated meiotic centromeric cohesion completely during prophase I and also led to the bipolar orientation of the kinetochores during the first meiotic division and accordingly resulted in premature separation of sister chromatid during meiosis I. Immunolocalization analyses revealed that the loading of PAIR2, PAIR3, OsMER3, and ZEP1 all depended on OsREC8. By contrast, the presence of the OsREC8 signal in pair2, pair3, Osmer3, and zep1 mutants indicated that the loading of OsREC8 did not rely on these four proteins. These results suggest that OsREC8 has several essential roles in the meiotic processes.

OsAM1 is required for leptotene-zygotene transition in rice.

The events occurring at the onset of meiosis have not been fully elucidated. In the present study, OsAM1 was identified in rice (Oryza sativa L.) by map-based cloning. OsAM1, a homolog of Arabidopsis SWI1 and maize AM1, encodes a protein with a coiled-coil domain in its central region. In the Osam1 mutant, pollen mother cells are arrested at leptotene, showing that OsAM1 is required for the leptotene-zygotene transition. Immunocytological analysis revealed that OsAM1 exists as foci in early prophase I meiocytes. Very faint OsREC8 foci persisted in the Osam1 mutant, indicating that OsAM1 is not required for the initial meiotic recruitment of OsREC8. In the absence of OsAM1, many other critical meiotic components, including PAIR2, ZEP1 and OsMER3, could not be correctly installed onto chromosomes. In contrast, in pair2, Osmer3 and zep1 mutants, OsAM1 could be loaded normally, suggesting that OsAM1 plays a fundamental role in building the proper chromosome structure at the beginning of meiosis.