Cancer cell-derived CD69 induced under lipid and oxygen starvation promotes ovarian cancer progression through fibronectin.

Cancer tissues generally have molecular oxygen and serum component deficiencies because of poor vascularization. Recently, we revealed that ICAM1 is strongly activated through lipophagy in ovarian clear cell carcinoma (CCC) cells in response to starvation of long-chain fatty acids and oxygen and confers resistance to apoptosis caused by these harsh conditions. CD69 is a glycoprotein that is synthesized in immune cells and is associated with their activation through cellular signaling pathways. However, the expression and function of CD69 in nonhematological cells is unclear. Here, we report that CD69 is induced in CCC cells as in ICAM1. Mass spectrometry analysis of phosphorylated peptides followed by pathway analysis revealed that CD69 augments CCC cell binding to fibronectin (FN) in association with the phosphorylation of multiple cellular signaling molecules including the focal adhesion pathway. Furthermore, CD69 synthesized in CCC cells could facilitate cell survival because the CD69-FN axis can induce epithelial-mesenchymal transition. Experiments with surgically removed tumor samples revealed that CD69 is predominantly expressed in CCC tumor cells compared with other histological subtypes of epithelial ovarian cancer. Overall, our data suggest that cancer cell-derived CD69 can contribute to CCC progression through FN.

Cholesterol Starvation and Hypoxia Activate the FVII Gene via the SREBP1-GILZ Pathway in Ovarian Cancer Cells to Produce Procoagulant Microvesicles.

Interaction between the transcription factors, hypoxia-inducible factor (HIF1α and HIF2α) and Sp1, mediates hypoxia-driven expression of FVII gene encoding coagulation factor VII (fVII) in ovarian clear cell carcinoma (CCC) cells. This mechanism is synergistically enhanced in response to serum starvation, a condition possibly associated with tumor hypoxia. This transcriptional response potentially results in venous thromboembolism, a common complication in cancer patients by producing procoagulant extracellular vesicles (EVs). However, which deficient serum factors are responsible for this characteristic transcriptional mechanism is unknown. Here, we report that cholesterol deficiency mediates synergistic FVII expression under serum starvation and hypoxia (SSH) via novel sterol regulatory element binding protein-1 (SREBP1)-driven mechanisms. Unlike conventional mechanisms, SREBP1 indirectly enhances FVII transcription through the induction of a new target, glucocorticoid-induced leucine zipper (GILZ) protein. GILZ expression induced in response to hypoxia by a HIF1α-dependent mechanism activates SREBP1 under SSH, suggesting reciprocal regulation between SREBP1 and GILZ. Furthermore, GILZ binds to the FVII locus. Xenograft tumor samples analyzed by chromatin immunoprecipitation confirmed that HIF1α-aryl hydrocarbon nuclear translocator and GILZ bind to the TSC22D3 (GILZ) and FVII gene loci, respectively, thereby potentially modulating chromatin function to augment FVII transcription. Thus, deficiency of both O2 and cholesterol, followed by interplay between HIFs, Sp1, and SREBP1-GILZ pathways synergistically induce fVII synthesis, resulting in the shedding of procoagulant EVs.

BRCA2 phosphorylated by PLK1 moves to the midbody to regulate cytokinesis mediated by nonmuscle myosin IIC.

Cytokinesis is the critical final step in cell division. BRCA2 disruption during cytokinesis is associated with chromosome instability, but mechanistic information is lacking that could be used to prevent cancer cell division. In this study, we report that BRCA2 phosphorylation by the mitotic polo-like kinase (PLK1) governs the localization of BRCA2 to the Flemming body at the central midbody, permitting an interaction with nonmuscle myosin IIC (NM-IIC). Formation of an NM-IIC ring-like structure at the Flemming body shows that the IIC-ring relies on its ATPase activity stimulated by interaction with BRCA2 and associated proteins. Notably, inhibiting this binding inactivated the ATPase activity, causing disassembly of the IIC-ring, defective formation of the midbody, and interruption of cytokinesis. An analysis of cancer-associated mutations in BRCA2 at the PLK1-binding site suggests that they may contribute to cytokinetic defects by altering BRCA2 localization. Our findings suggest that BRCA2-dependent IIC-ring formation is a critical step in proper formation of the midbody, offering an explanation for how chromosome instability may arise in breast cancer.

Mutations in UVSSA cause UV-sensitive syndrome and impair RNA polymerase IIo processing in transcription-coupled nucleotide-excision repair.

UV-sensitive syndrome (UV(S)S) is a genodermatosis characterized by cutaneous photosensitivity without skin carcinoma. Despite mild clinical features, cells from individuals with UV(S)S, like Cockayne syndrome cells, are very UV sensitive and are deficient in transcription-coupled nucleotide-excision repair (TC-NER), which removes DNA damage in actively transcribed genes. Three of the seven known UV(S)S cases carry mutations in the Cockayne syndrome genes ERCC8 or ERCC6 (also known as CSA and CSB, respectively). The remaining four individuals with UVSS , one of whom is described for the first time here, formed a separate UV(S)S-A complementation group; however, the responsible gene was unknown. Using exome sequencing, we determine that mutations in the UVSSA gene (formerly known as KIAA1530) cause UV(S)S-A. The UVSSA protein interacts with TC-NER machinery and stabilizes the ERCC6 complex; it also facilitates ubiquitination of RNA polymerase IIo stalled at DNA damage sites. Our findings provide mechanistic insights into the processing of stalled RNA polymerase and explain the different clinical features across these TC-NER­deficient disorders.

BRCA2 and nucleophosmin coregulate centrosome amplification and form a complex with the Rho effector kinase ROCK2.

BRCA2 germline mutations account for the majority of heredity breast and ovarian cancer. Besides its role in DNA damage repair, BRCA2 also plays an important role in cytokinesis, transcription regulation, and cancer cell proliferation. Recently, we reported that BRCA2 localizes to centrosomes as well as nuclei and the dysfunction of BRCA2 in a centrosome causes abnormalities in cell division. Here, we identified a nucleolar phosphoprotein, nucleophosmin (NPM), as a novel BRCA2-associated protein. We also detected the binding of BRCA2 to ROCK2, an effector of Rho small GTPase. Because it is known that ROCK2 binds to NPM at centrosomes, these 3 proteins may form a complex. NPM-binding region was within amino acids 639-1,000 of BRCA2. Exogenous expression of this BRCA2 region resulted in aberrant centrosome amplification and a high frequency of multinucleated cells. Our results suggested that a complex consisting of BRCA2, NPM, and ROCK2 maintains the numerical integrity of centrosomes and accurate cell division and that dysfunction of this regulation might be involved in the tumorigenesis of breast cancer.

Three DNA polymerases, recruited by different mechanisms, carry out NER repair synthesis in human cells.

Nucleotide excision repair (NER) is the most versatile DNA repair system that deals with the major UV photoproducts in DNA, as well as many other DNA adducts. The early steps of NER are well understood, whereas the later steps of repair synthesis and ligation are not. In particular, which polymerases are definitely involved in repair synthesis and how they are recruited to the damaged sites has not yet been established. We report that, in human fibroblasts, approximately half of the repair synthesis requires both pol kappa and pol delta, and both polymerases can be recovered in the same repair complexes. Pol kappa is recruited to repair sites by ubiquitinated PCNA and XRCC1 and pol delta by the classical replication factor complex RFC1-RFC, together with a polymerase accessory factor, p66, and unmodified PCNA. The remaining repair synthesis is dependent on pol epsilon, recruitment of which is dependent on the alternative clamp loader CTF18-RFC.

PTIP promotes DNA double-strand break repair through homologous recombination.

PTIP (Pax2 transactivation domain-interacting protein) is a large nuclear protein containing six BRCT (BRCA1 C-Terminal) domains. PTIP is recruited to DNA-damage sites through its BRCT domains and thus has been implicated in the DNA damage response. To define the function of PTIP in DNA repair, we disrupted the PTIP gene of the chicken DT40 B cell line. PTIP mutant (PTIP(-/-/-) ) cells displayed phenotypes frequently observed in cells with defective homologous recombination (HR), i.e. a marked increase in the number of spontaneously arising DNA lesions as well as sensitivity to ionizing radiation (IR) and the topoisomerase I inhibitor, camptothecin. Accordingly, analysis of HR efficiency by using artificial recombination substrates showed that the HR efficiency was reduced in the PTIP-deficient chicken and the PTIP-depleted HeLa cells. As microarray analysis showed no apparent difference between wild-type and PTIP(-/-/-) cells in the expression of known HR factors, it is unlikely that this reduction in HR efficiency in PTIP-deficient cells is associated with PTIP's role in transcriptional regulation. We thus propose that PTIP promotes double-strand break repair through a direct role in HR.

Introduction and characterization of a polymerase-dead point mutation into the POLK gene in vertebrates.

The chicken DT40 cell line is widely used for gene knock-outs. We attempted to introduce a polymerase-dead point mutation into Polkappa, a polymerase for translesion DNA synthesis, taking advantage of the highly efficient targeted integration in DT40 cells. The resulting cells (REV3(-/-)POLK(/)(pol-dead)) proliferated with the same kinetics as the parental REV3(-/-) cells. Though the mock-treated REV3(-/-)POLK(/)(mock) cells showed the same sensitivity as the parental REV3(-/-) cells to methyl methanesulfonate, the REV3(-/-)POLK(/)(pol-dead) cells demonstrated the same sensitivity as the REV3(-/-)POLK(/-) double knock-out cells. This implies that the presence of the polymerase-dead Polkappa does not interfere with other polymerases repairing monoalkylation damage.

Genetic evidence for single-strand lesions initiating Nbs1-dependent homologous recombination in diversification of Ig v in chicken B lymphocytes.

Homologous recombination (HR) is initiated by DNA double-strand breaks (DSB). However, it remains unclear whether single-strand lesions also initiate HR in genomic DNA. Chicken B lymphocytes diversify their Immunoglobulin (Ig) V genes through HR (Ig gene conversion) and non-templated hypermutation. Both types of Ig V diversification are initiated by AID-dependent abasic-site formation. Abasic sites stall replication, resulting in the formation of single-stranded gaps. These gaps can be filled by error-prone DNA polymerases, resulting in hypermutation. However, it is unclear whether these single-strand gaps can also initiate Ig gene conversion without being first converted to DSBs. The Mre11-Rad50-Nbs1 (MRN) complex, which produces 3' single-strand overhangs, promotes the initiation of DSB-induced HR in yeast. We show that a DT40 line expressing only a truncated form of Nbs1 (Nbs1(p70)) exhibits defective HR-dependent DSB repair, and a significant reduction in the rate--though not the fidelity--of Ig gene conversion. Interestingly, this defective gene conversion was restored to wild type levels by overproduction of Escherichia coli SbcB, a 3' to 5' single-strand-specific exonuclease, without affecting DSB repair. Conversely, overexpression of chicken Exo1 increased the efficiency of DSB-induced gene-targeting more than 10-fold, with no effect on Ig gene conversion. These results suggest that Ig gene conversion may be initiated by single-strand gaps rather than by DSBs, and, like SbcB, the MRN complex in DT40 may convert AID-induced lesions into single-strand gaps suitable for triggering HR. In summary, Ig gene conversion and hypermutation may share a common substrate-single-stranded gaps. Genetic analysis of the two types of Ig V diversification in DT40 provides a unique opportunity to gain insight into the molecular mechanisms underlying the filling of gaps that arise as a consequence of replication blocks at abasic sites, by HR and error-prone polymerases.

Novel BRCA2-interacting protein BJ-HCC-20A inhibits the induction of apoptosis in response to DNA damage.

The major hereditary breast cancer susceptibility gene BRCA2 is associated with familial breast and ovarian cancer. BRCA2 plays a role in DNA repair, transcription, cell cycle regulation, maintenance of genomic stability in response to DNA damage, centrosome regulation, and cytokinesis. To further understand the function of BRCA2, we used a yeast two-hybrid method and identified a novel BRCA2-interacting protein, BJ-HCC-20A, which is reported to be a potential cancer-testis antigen. We confirmed the interaction between endogenous BJ-HCC-20A and BRCA2 in mammalian cells, and showed that BJ-HCC-20A interacts with a portion of the highly conserved region of BRCA2 in various mammals, and M phase-specific phosphorylation of the binding region of BRCA2 modulates BJ-HCC-20A binding. Overexpression of BJ-HCC-20A increases cell growth, and downregulation of endogenous BJ-HCC-20A expression using small interfering RNA suppresses cell growth and leads to the induction of apoptosis. Importantly, the BJ-HCC-20A mRNA level is downregulated by adriamycin (ADR)-induced DNA damage and depletion of BJ-HCC-20A expression by small interfering RNA promotes the reduction of BRCA2 expression and enhances cell apoptosis in response to DNA damage. Additionally, the recovery of BJ-HCC-20A expression in ADR-induced DNA damage inhibits ADR-induced apoptosis. The data suggest that BJ-HCC-20A promotes cell growth and may regulate the induction of cell apoptosis in response to DNA damage in cooperation with BRCA2 in an M phase-dependent manner. Therefore, we speculate that targeting BJ-HCC-20A may aid in the treatment of breast tumors.

Reconstitution of Golgi disassembly by mitotic Xenopus egg extract in semi-intact MDCK cells.

Semi-intact cells are cells with plasma membranes that have been permeabilized by bacterial pore-forming toxins or surfactants. The addition of mitotic Xenopus egg extract to semi-intact cells can reconstitute a number of intracellular events that occur specifically at the onset of mitosis. In this chapter, we describe methods for reconstituting the disassembly of the Golgi apparatus by introducing mitotic Xenopus egg extract into semi-intact Mardin-Darby canine kidney (MDCK) cells. The Golgi apparatus was visualized in the cells by expression of green fluorescence protein (GFP)-tagged galactosyltransferase, a marker of trans-Golgi cisternae. Xenopus egg extracts arrested at mitosis or interphase were then prepared and added to the semi-intact MDCK cells. Disassembly of the Golgi apparatus was induced by mitotic Xenopus egg extract. This system can be used not only to elucidate the factors that are involved in the reconstitution process, but also to dissect the process into several elementary steps morphologically and biochemically.

Critical roles for polymerase zeta in cellular tolerance to nitric oxide-induced DNA damage.

Nitric oxide (NO), a signal transmitter involved in inflammation and regulation of smooth muscle and neurons, seems to cause mutagenesis, but its mechanisms have remained elusive. To gain an insight into NO-induced genotoxicity, we analyzed the effect of NO on a panel of chicken DT40 clones deficient in DNA repair pathways, including base and nucleotide excision repair, double-strand break repair, and translesion DNA synthesis (TLS). Our results show that cells deficient in Rev1 and Rev3, a subunit essential for DNA polymerase zeta (Polzeta), are hypersensitive to killing by two chemical NO donors, spermine NONOate and S-nitroso-N-acetyl-penicillamine. Mitotic chromosomal analysis indicates that the hypersensitivity is caused by a significant increase in the level of induced chromosomal breaks. The data reveal the critical role of TLS polymerases in cellular tolerance to NO-induced DNA damage and suggest the contribution of these error-prone polymerases to accumulation of single base substitutions.

Involvement of vertebrate Polkappa in translesion DNA synthesis across DNA monoalkylation damage.

DNA lesions that escape excision repair pathways can cause arrested DNA replication. This replication block can be processed by translesion DNA synthesis (TLS), which is carried out by a number of specialized DNA polymerases. A sequential lesion bypass model has been proposed; one of the lesion-specific polymerases inserts nucleotide(s) opposite the damaged template, followed by extension from the inserted nucleotide by the same or another polymerase. Polzeta and Polkappa have been proposed as candidates for executing the extension step in eukaryotic cells. We previously disrupted separately Rev3, the catalytic subunit of Polzeta, and Polkappa in chicken B lymphocyte DT40 cells. We found that each cell line showed significant UV sensitivity, implying that both contribute to UV radiation damage repair. In the present studies we generated REV3(-/-)POLK(/-) double knock-out cells to determine whether they participate in the same or different pathways. The double mutant was viable and proliferated with the same kinetics as parental REV3(-/-) cells. The cells showed the same sensitivity as REV3(-/-) cells to UV, ionizing radiation, and chemical cross-linking agents. In contrast, they were more sensitive than REV3(-/-) cells to monofunctional alkylating agents, even though POLK(/-) cells barely exhibited increased sensitivity to those. Moreover Polk-deficient mouse embryonic stem and fibroblast cells, both of which have previously been shown to be sensitive to UV radiation, also showed moderate sensitivity to methyl methanesulfonate, a monofunctional alkylating agent. These data imply that Polkappa has a function in TLS past alkylated base adducts as well as UV radiation DNA damage in vertebrates.