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Supplementary Figures S1-S5. Supplementary Figure S1: Structures of Pt-DNA adducts. Figure S2: Histogram of HCT116 p53+/+ cells treated with oxaliplatin or cisplatin. Figure S3: (A) p53 and p21 induction by oxaliplatin. (B) Identification of the mitochondria isoform of dUTPase (DUT-M). Figure S4. Related to Figure 4 A-C. Pri-miR-34a induction (A), downregulation of DUT transcript (B) and changes in the amount of proteins (C) by oxaliplatin. Figure S5. Related to Figure 5E. (A) Ectopic expression of TK1-3xFLAG, (B) Histogram of HCT116 p53+/+ cells with transient TK1-3xFLAG expression and oxaliplatin treatment.

Trifluridine (FTD) is a key component of the novel oral antitumor drug TAS-102, which consists of FTD and a thymidine phosphorylase inhibitor. Like 5-fluoro-2′-deoxyuridine (FdUrd), a deoxynucleoside form of 5-fluorouracil metabolite, FTD is sequentially phosphorylated and not only inhibits thymidylate synthase activity, but is also incorporated into DNA. Although TAS-102 was effective for the treatment of refractory metastatic colorectal cancer in clinical trials, the mechanism of FTD-induced cytotoxicity is not completely understood. Here, we show that FTD as well as FdUrd induce transient phosphorylation of Chk1 at Ser345, and that this is followed by accumulation of p53 and p21 proteins in p53-proficient human cancer cell lines. In particular, FTD induced p53-dependent sustained arrest at G2 phase, which was associated with a proteasome-dependent decrease in the Cyclin B1 protein level and the suppression of CCNB1 and CDK1 gene expression. In addition, a p53-dependent increase in p21 protein was associated with an FTD-induced decrease in Cyclin B1 protein. Although numerous ssDNA and dsDNA breaks were induced by FdUrd, few DNA strand breaks were detected in FTD-treated HCT-116 cells despite massive FTD misincorporation into genomic DNA, suggesting that the antiproliferative effect of FTD is not due to the induction of DNA strand breaks. These distinctive effects of FTD provide insights into the cellular mechanism underlying its antitumor effect and may explain the clinical efficacy of TAS-102. Mol Cancer Ther; 14(4); 1004–13. ©2015 AACR.

Supplementary Figures S1-7. Supplementary Figure S1. The cellular response of RKO cells to IC50 concentration of FdUrd or FTD. Supplementary Figure S2. The cellular responses of A549 cells to IC50 concentration of FdUrd or FTD. Supplementary Figure S3. A549 cells either treated with siGL2 or siTP53 were cultured in the presence of IC50 concentration of FdUrd or FTD. Supplementary Figure S4. RKO (A) or A549 (B) cells were cultured in the presence of IC50 concentration of FdUrd or FTD. Supplementary Figure S5. Immunoflurorescent images of Cyclin B1 in exponentially growing (A) or FTD-treated (B,C) HCT-116 cells. Supplementary Figure S6. Immunoflurorescent images of Cyclin B1 in FTD-treated HCT-116 cells with or without MG132. Supplementary Figure S7. Representative images of alkaline or neutral comet assay in camptothecin-treated HCT-116 cells.

Chromatin dynamics mediated by post-translational modifications play a crucial role in cellular response to genotoxic stress for the maintenance of genome integrity. MDC1 is a pivotal chromatin adaptor in DNA damage response (DDR) and its methylation is essential to recruit repair factors at DNA double-strand break (DSB) sites, yet their precise molecular mechanisms remain elusive. Here we identified euchromatic histone-lysine N-methyltransferase 1 (EHMT1) and EHMT2 as novel regulators of MDC1, which is required for the accumulation of DDR factors e.g. 53BP1 and RAP80, at the DSB sites. MDC1 interacts mainly with EHMT1, which is facilitated by DNA damage-initiated ATM signalling, and EHMT2 dominantly modulates methylation of MDC1 lysine 45. This regulatory modification promotes the interaction between MDC1 and ATM to expand activated ATM on damaged chromatin and dysfunctional telomere. These findings identify EHMT1 and EHMT2 as DDR components, with implications for genome-integrity maintenance through proper dynamic methylation of MDC1.

Accumulating evidence indicates that the senescence-associated secretory phenotype (SASP) contributes to many aspects of physiology and disease. Thus, controlling the SASP will have tremendous impacts on our health. However, our understanding of SASP regulation is far from complete. Here, we show that cytoplasmic accumulation of nuclear DNA plays key roles in the onset of SASP. Although both DNase2 and TREX1 rapidly remove the cytoplasmic DNA fragments emanating from the nucleus in pre-senescent cells, the expression of these DNases is downregulated in senescent cells, resulting in the cytoplasmic accumulation of nuclear DNA. This causes the aberrant activation of cGAS-STING cytoplasmic DNA sensors, provoking SASP through induction of interferon-β. Notably, the blockage of this pathway prevents SASP in senescent hepatic stellate cells, accompanied by a decline of obesity-associated hepatocellular carcinoma development in mice. These findings provide valuable new insights into the roles and mechanisms of SASP and possibilities for their control., Activation of DNA damage response induces the acquisition of senescence-associated secretory phenotype (SASP) in senescent cells, but precise mechanisms remain unclear. Here, the authors show that the cytoplasmic accumulation of nuclear DNA activated cytoplasmic DNA sensors to provoke SASP.

Trifluridine (FTD) is a key component of the novel oral antitumor drug TAS-102, which consists of FTD and a thymidine phosphorylase inhibitor. Like 5-fluoro-2′-deoxyuridine (FdUrd), a deoxynucleoside form of 5-fluorouracil metabolite, FTD is sequentially phosphorylated and not only inhibits thymidylate synthase activity, but is also incorporated into DNA. Although TAS-102 was effective for the treatment of refractory metastatic colorectal cancer in clinical trials, the mechanism of FTD-induced cytotoxicity is not completely understood. Here, we show that FTD as well as FdUrd induce transient phosphorylation of Chk1 at Ser345, and that this is followed by accumulation of p53 and p21 proteins in p53-proficient human cancer cell lines. In particular, FTD induced p53-dependent sustained arrest at G2 phase, which was associated with a proteasome-dependent decrease in the Cyclin B1 protein level and the suppression of CCNB1 and CDK1 gene expression. In addition, a p53-dependent increase in p21 protein was associated with an FTD-induced decrease in Cyclin B1 protein. Although numerous ssDNA and dsDNA breaks were induced by FdUrd, few DNA strand breaks were detected in FTD-treated HCT-116 cells despite massive FTD misincorporation into genomic DNA, suggesting that the antiproliferative effect of FTD is not due to the induction of DNA strand breaks. These distinctive effects of FTD provide insights into the cellular mechanism underlying its antitumor effect and may explain the clinical efficacy of TAS-102. Mol Cancer Ther; 14(4); 1004–13. ©2015 AACR.

p16Ink4a and p21Cip1/Waf1 act as tumour suppressors through induction of cellular senescence. However, senescence-independent roles of these CDK inhibitors are not well understood. Here, we report an unexpected function of p16Ink4 and p21Cip1/Waf1, namely, tumour promotion through chemotaxis. In monocytic myeloid-derived suppressor cells (Mo-MDSCs), p16Ink4 and p21Cip1/Waf1 are highly expressed and stimulate CX3CR1 chemokine receptor expression by preventing CDK-mediated phosphorylation and inactivation of SMAD3. Thus, deletion of p16 Ink4 and p21 Cip1/Waf1 reduces CX3CR1 expression, thereby inhibiting Mo-MDSC accumulation in tumours expressing CX3CL1 and suppressing the tumour progression in mice. Notably, blockade of the CX3CL1/CX3CR1 axis suppresses tumour growth, whereas inactivation of CDKs elicits the opposite effect. These findings reveal an unexpected function of p16 Ink4a and p21 Waf1/Cip1 and indicate that regulation of Mo-MDSCs chemotaxis is a valuable potential strategy for control of tumour development.

Cellular senescence prevents the proliferation of cells at risk for neoplastic transformation. However, the altered secretome of senescent cells can promote the growth of the surrounding cancer cells. Although extracellular vesicles (EVs) have emerged as new players in intercellular communication, their role in the function of senescent cell secretome has been largely unexplored. Here, we show that exosome-like small EVs (sEVs) are important mediators of the pro-tumorigenic function of senescent cells. sEV-associated EphA2 secreted from senescent cells binds to ephrin-A1, that is, highly expressed in several types of cancer cells and promotes cell proliferation through EphA2/ephrin-A1 reverse signalling. sEV sorting of EphA2 is increased in senescent cells because of its enhanced phosphorylation resulting from oxidative inactivation of PTP1B phosphatase. Our results demonstrate a novel mechanism of reactive oxygen species (ROS)-regulated cargo sorting into sEVs, which is critical for the potentially deleterious growth-promoting effect of the senescent cell secretome., Although senescent cell secretome can promote the growth of surrounding cancer cells, the role of extracellular vesicles in this process has not been well understood. Here the authors show that ROS increase the sorting of EphA2 into extracellular vesicles in senescent cells, which promotes proliferation of cancer cells.

Emerging evidence is revealing that exosomes contribute to many aspects of physiology and disease through intercellular communication. However, the biological roles of exosome secretion in exosome-secreting cells have remained largely unexplored. Here we show that exosome secretion plays a crucial role in maintaining cellular homeostasis in exosome-secreting cells. The inhibition of exosome secretion results in the accumulation of nuclear DNA in the cytoplasm, thereby causing the activation of cytoplasmic DNA sensing machinery. This event provokes the innate immune response, leading to reactive oxygen species (ROS)-dependent DNA damage response and thus induce senescence-like cell-cycle arrest or apoptosis in normal human cells. These results, in conjunction with observations that exosomes contain various lengths of chromosomal DNA fragments, indicate that exosome secretion maintains cellular homeostasis by removing harmful cytoplasmic DNA from cells. Together, these findings enhance our understanding of exosome biology, and provide valuable new insights into the control of cellular homeostasis.

p16Ink4a and p21Cip1/Waf1 act as tumour suppressors through induction of cellular senescence. However, senescence-independent roles of these CDK inhibitors are not well understood. Here, we report an unexpected function of p16Ink4 and p21Cip1/Waf1, namely, tumour promotion through chemotaxis. In monocytic myeloid-derived suppressor cells (Mo-MDSCs), p16Ink4 and p21Cip1/Waf1 are highly expressed and stimulate CX3CR1 chemokine receptor expression by preventing CDK-mediated phosphorylation and inactivation of SMAD3. Thus, deletion of p16 Ink4 and p21 Cip1/Waf1 reduces CX3CR1 expression, thereby inhibiting Mo-MDSC accumulation in tumours expressing CX3CL1 and suppressing the tumour progression in mice. Notably, blockade of the CX3CL1/CX3CR1 axis suppresses tumour growth, whereas inactivation of CDKs elicits the opposite effect. These findings reveal an unexpected function of p16 Ink4a and p21 Waf1/Cip1 and indicate that regulation of Mo-MDSCs chemotaxis is a valuable potential strategy for control of tumour development., Both p16Ink4a and p21Cip1/Waf1 are known oncosuppressors and have a role in senescence. Here, the authors show a pro-tumorigenic role for these two proteins: high expression in myeloid-derived suppressor cells stimulates their chemotactic function, favouring tumour progression.

Emerging evidence is revealing that exosomes contribute to many aspects of physiology and disease through intercellular communication. However, the biological roles of exosome secretion in exosome-secreting cells have remained largely unexplored. Here we show that exosome secretion plays a crucial role in maintaining cellular homeostasis in exosome-secreting cells. The inhibition of exosome secretion results in the accumulation of nuclear DNA in the cytoplasm, thereby causing the activation of cytoplasmic DNA sensing machinery. This event provokes the innate immune response, leading to reactive oxygen species (ROS)-dependent DNA damage response and thus induce senescence-like cell-cycle arrest or apoptosis in normal human cells. These results, in conjunction with observations that exosomes contain various lengths of chromosomal DNA fragments, indicate that exosome secretion maintains cellular homeostasis by removing harmful cytoplasmic DNA from cells. Together, these findings enhance our understanding of exosome biology, and provide valuable new insights into the control of cellular homeostasis., The role of exosomes in intercellular communication is well established, however less in known about the biological roles of exosome secretion in exosome-secreting cells. Here the authors show that exosome secretion controls cellular homeostasis in exosome-secreting cells by removing harmful cytoplasmic DNA from cells.

Chromatin ubiquitylation flanking DNA double-strand breaks (DSBs), mediated by RNF8 and RNF168 ubiquitin ligases, orchestrates a two-branch pathway, recruiting repair factors 53BP1 or the RAP80-BRCA1 complex. We report that human demethylase JMJD1C regulates the RAP80-BRCA1 branch of this DNA-damage response (DDR) pathway. JMJD1C was stabilized by interaction with RNF8, was recruited to DSBs, and was required for local ubiquitylations and recruitment of RAP80-BRCA1 but not 53BP1. JMJD1C bound to RNF8 and MDC1, and demethylated MDC1 at Lys45, thereby promoting MDC1-RNF8 interaction, RNF8-dependent MDC1 ubiquitylation and recruitment of RAP80-BRCA1 to polyubiquitylated MDC1. Furthermore, JMJD1C restricted formation of RAD51 repair foci, and JMJD1C depletion caused resistance to ionizing radiation and PARP inhibitors, phenotypes relevant to aberrant loss of JMJD1C in subsets of breast carcinomas. These findings identify JMJD1C as a DDR component, with implications for genome-integrity maintenance, tumorigenesis and cancer treatment.

Genotoxic insults, such as ionizing radiation (IR), cause DNA damage that evokes a multifaceted cellular DNA damage response (DDR). DNA damage signaling events that control protein activity, subcellular localization, DNA binding, protein-protein interactions, etc. rely heavily on time-dependent posttranslational modifications (PTMs). To complement our previous analysis of IR-induced temporal dynamics of nuclear phosphoproteome, we now identify a range of human nuclear proteins that are dynamically regulated by acetylation, and predominantly deacetylation, during IR-induced DDR by using mass spectrometry-based proteomic approaches. Apart from cataloging acetylation sites through SILAC proteomic analyses before IR and at 5 and 60 min after IR exposure of U2OS cells, we report that: (1) key components of the transcriptional machinery, such as EP300 and CREBBP, are dynamically acetylated; (2) that nuclear acetyltransferases themselves are regulated, not on the protein abundance level, but by (de)acetylation; and (3) that the recently reported p53 co-activator and methyltransferase MLL3 is acetylated on five lysines during the DDR. For selected examples, protein immunoprecipitation and immunoblotting were used to assess lysine acetylation status and thereby validate the mass spectrometry data. We thus present evidence that nuclear proteins, including those known to regulate cellular functions via epigenetic modifications of histones, are regulated by (de)acetylation in a timely manner upon cell's exposure to genotoxic insults. Overall, these results present a resource of temporal profiles of a spectrum of protein acetylation sites during DDR and provide further insights into the highly dynamic nature of regulatory PTMs that help orchestrate the maintenance of genome integrity.

The three-dimensional context of endogenous chromosomal regions may contribute to the regulation of gene clusters by influencing interactions between transcriptional regulatory elements. In this study, we investigated the effects of tumor necrosis factor (TNF) signaling on spatiotemporal enhancer-promoter interactions in the human tumor necrosis factor (TNF)/lymphotoxin (LT) gene locus, mediated by CCCTC-binding factor (CTCF)-dependent chromatin insulators. The cytokine genes LTα, TNF, and LTβ are differentially regulated by NF-κB signaling in inflammatory and oncogenic responses. We identified at least four CTCF-enriched sites with enhancer-blocking activities and a TNF-responsive TE2 enhancer in the TNF/LT locus. One of the CTCF-enriched sites is located between the early-inducible LTα/TNF promoters and the late-inducible LTβ promoter. Depletion of CTCF reduced TNF expression and accelerated LTβ induction. After TNF stimulation, via intrachromosomal dynamics, these insulators mediated interactions between the enhancer and the LTα/TNF promoters, followed by interaction with the LTβ promoter. These results suggest that insulators mediate the spatiotemporal control of enhancer-promoter associations in the TNF/LT gene cluster.

The development of stomach cancer is closely associated with chronic inflammation, and the Wnt/beta-catenin signaling pathway is activated in most cases of this cancer. High-mobility group A (HMGA) proteins are oncogenic chromatin factors that are primarily expressed not only in undifferentiated tissues but also in various tumors. Here we report that HMGA1 is induced by the Wnt/beta-catenin pathway and maintains proliferation of gastric cancer cells. Specific knockdown of HMGA1 resulted in marked reduction of cell growth. The loss of beta-catenin or its downstream c-myc decreased HMGA1 expression, whereas Wnt3a treatment increased HMGA1 and c-myc transcripts. Furthermore, Wnt3a-induced expression of HMGA1 was inhibited by c-myc knockdown, suggesting that HMGA1 is a downstream target of the Wnt/beta-catenin pathway. Enhanced expression of HMGA1 coexisted with the nuclear accumulation of beta-catenin in about 30% of gastric cancer tissues. To visualize the expression of HMGA1 in vivo, transgenic mice expressing endogenous HMGA1 fused to enhanced green fluorescent protein were generated and then crossed with K19-Wnt1/C2mE mice, which develop gastric tumors through activation of both the Wnt and prostaglandin E2 pathways. Expression of HMGA1-enhanced green fluorescent protein was normally detected in the forestomach, along the upper border of the glandular stomach, but its expression was also up-regulated in cancerous glandular stomach. These data suggest that HMGA1 is involved in proliferation and gastric tumor formation via the Wnt/beta-catenin pathway.

Pancreatic cancer is a highly aggressive malignancy due to elevated mitotic activities and epithelial-mesenchymal transition (EMT). Oncogenic RAS and transforming growth factor-beta signaling are implicated in these malignant features. The mechanisms that underlie EMT need to be addressed since it promotes tissue invasion and metastasis. The high-mobility group A protein 2 (HMGA2) is a non-histone chromatin factor that is primarily expressed in undifferentiated tissues and tumors of mesenchymal origin. However, its role in EMT in pancreatic cancer is largely unknown. Here we report that HMGA2 is involved in EMT maintenance in human pancreatic cancer cells. Specific knockdown of HMGA2 inhibited cell proliferation, leading to an epithelial-state transition that restores cell-cell contact due to E-cadherin up-regulation. Consistently, an inverse correlation between HMGA2-positive cells and E-cadherin-positive cells was found in cancer tissues. Inhibition of the RAS/MEK pathway also induced an epithelial transition, together with HMGA2 down-regulation. Transcriptional repressors of the E-cadherin gene, such as SNAIL, decreased after HMGA2 knockdown since HMGA2 directly activated the SNAlL gene promoter. The decrease of SNAIL after RAS/MEK inhibition was suppressed by HMGA2 overexpression. Further, let-7 microRNA-mediated HMGA2 down-regulation had no effect on the prevention of the transformed phenotype in these cells. These data shed light on the importance of HMGA2 in reversibly maintaining EMT, suggesting that HMGA2 is a potential therapeutic target for the treatment of pancreatic cancer.

Polycomb group (PcG) proteins form two distinct complexes, PRC1 and PRC2, to regulate developmental target genes by maintaining the epigenetic state in cells. PRC2 methylates histone H3 at lysine 27 (H3K27), and PRC1 then recognizes methyl-H3K27 to form repressive chromatin. However, it remains unknown how PcG proteins maintain stable and plastic chromatin during cell division. Here we report that PcG-associated chromatin is reproduced in the G(1) phase in post-mitotic cells and is required for subsequent S phase progression. In dividing cells, H3K27 trimethylation (H3K27Me(3)) marked mitotic chromosome arms where PRC2 (Suz12 and Ezh2) co-existed, whereas PRC1 (Bmi1 and Pc2) appeared in distinct foci in the pericentromeric regions. As each PRC complex was increasingly assembled from mitosis to G(1) phase, PRC1 formed H3K27Me(3)-based chromatin intensively during middle and late G(1) phase; this chromatin was highly resistant to in situ nuclease treatment. Thus, the transition from mitosis to G(1) phase is crucial for PcG-mediated chromatin inheritance. Knockdown of Suz12 markedly reduced the amount of H3K27Me(3) on mitotic chromosomes, and as a consequence, PRC1 foci were not fully transmitted to post-mitotic daughter cells. S phase progression was markedly delayed in these Suz12-knockdown cells. The fact that PcG-associated chromatin is reproduced during post-mitotic G(1) phase suggests the possibility that PcG proteins enable their target chromatin to be remodeled in response to stimuli in the G(1) phase.

Retinoblastoma protein (RB) acts as a tumor suppressor in many tissue types, by promoting cell arrest via E2F-mediated transcriptional repression. In addition to the aberrant forms of the RB gene found in different types of cancers, many viral oncoproteins including the simian virus 40 large T antigen target RB. However, cellular factors that inhibit RB function remain to be elucidated. Here, we report that RB interacts with the high mobility group protein A1 (HMGA1), a-non-histone architectural chromatin factor that is frequently overexpressed in cancer cells. HMGA1 binds the small pocket domain of RB, and competes with HDAC1. Subsequently, overexpression of HMGA1 abolishes the inhibitory effect of RB on E2F-activated transcription from the cyclin E promoter. Under serum starvation, T98G cells had been previously shown to be arrested in the G0 phase in an RB-mediated manner. The G0 phase was characterized by growth arrest and low levels of transcription, together with the hypophosphorylation of RB and the downregulation of HMGA1. In contrast, such serum-depleted G0 arrest was abrogated in T98G cells overexpressing HMGA1. The overexpressed HMGA1 was found to form complexes with cellular RB, suggesting that downregulation of HMGA1 is required for G0 arrest. There were no phenotypic changes in HMGA1-expressing T98G cells in the presence of serum, but the persistent expression of HMGA1 under serum starvation caused various nuclear abnormalities, which were similarly induced in T antigen-expressing T98G cells. Our present findings indicate that overexpression of HMGA1 disturbs RB-mediated cell arrest, suggesting a negative control of RB by HMGA1.

Methylated DNA binding domain (MBD) proteins and Polycomb group (PcG) proteins maintain epigenetic silencing of transcriptional activity. We report that the DNA methylation-mediated repressor MBD1 interacts with Ring1b and hPc2, the major components of Polycomb repressive complex 1. The cysteine-rich CXXC domains of MBD1 bound to Ring1b and the chromodomain of hPc2. Chromatin immunoprecipitation analysis revealed that MBD1 and hPc2 were present in silenced Homeobox A (HOXA) genes which could be reactivated by knockdown of either MBD1 or hPc2, suggesting that MBD1 and hPc2 cooperate for transcriptional repression of HOXA genes. In the nuclei of HeLa cells, MBD1 existed in close association with these PcG proteins in some heterochromatin foci, whereas an MBD1 mutant lacking the CXXC domains or an hPc2 mutant lacking the chromodomain lost this colocalization in foci. Use of the DNA demethylating agent 5-azadeoxycytidine abolished the formation of MBD1 foci but not PcG foci. Knockdown of MBD1 by small interfering RNAs did not affect the foci containing hPc2 and Ring1b, whereas the MBD1 foci were not influenced by knockdown of hPc2. These indicate that the heterochromatin foci showing MBD1 and hPc2 colocalization arise through the interaction of MBD1 and hPc2 and that the foci of MBD1 are separable from those of the PcG proteins per se. Our present findings suggest that MBD1 and PcG proteins have overlapping roles in epigenetic gene silencing and heterochromatin foci formation through their interactions.

Temporal regulation of microtubule dynamics is essential for proper progression of mitosis and control of microtubule plus-end tracking proteins by phosphorylation is an essential component of this regulation. Here we show that Aurora B and CDK1 phosphorylate microtubule end-binding protein 2 (EB2) at multiple sites within the amino terminus and a cluster of serine/threonine residues in the linker connecting the calponin homology and end-binding homology domains. EB2 phosphorylation, which is strictly associated with mitotic entry and progression, reduces the binding affinity of EB2 for microtubules. Expression of non-phosphorylatable EB2 induces stable kinetochore microtubule dynamics and delays formation of bipolar metaphase plates in a microtubule binding-dependent manner, and leads to aneuploidy even in unperturbed mitosis. We propose that Aurora B and CDK1 temporally regulate the binding affinity of EB2 for microtubules, thereby ensuring kinetochore microtubule dynamics, proper mitotic progression and genome stability., Temporal regulation of microtubule dynamics in mitosis can be achieved by phosphorylation of microtubule plus-end proteins. Here, the authors show that Aurora B and CDK1 phosphorylate EB2, which changes microtubule binding affinity and controls kinetochore microtubule dynamics and genome stability.

Topoisomerase IIβ-binding protein 1 (TOPBP1) participates in DNA replication and DNA damage response; however, its role in DNA repair and relevance for human cancer remain unclear. Here, through an unbiased small interfering RNA screen, we identified and validated TOPBP1 as a novel determinant whose loss sensitized human cells to olaparib, an inhibitor of poly(ADP-ribose) polymerase. We show that TOPBP1 acts in homologous recombination (HR) repair, impacts olaparib response, and exhibits aberrant patterns in subsets of human ovarian carcinomas. TOPBP1 depletion abrogated RAD51 loading to chromatin and formation of RAD51 foci, but without affecting the upstream HR steps of DNA end resection and RPA loading. Furthermore, TOPBP1 BRCT domains 7/8 are essential for RAD51 foci formation. Mechanistically, TOPBP1 physically binds PLK1 and promotes PLK1 kinase–mediated phosphorylation of RAD51 at serine 14, a modification required for RAD51 recruitment to chromatin. Overall, our results provide mechanistic insights into TOPBP1’s role in HR, with potential clinical implications for cancer treatment.

Sox2 is a member of the high mobility group (HMG) domain DNA-binding proteins for transcriptional control and chromatin architecture. The HMG domain of Sox2 binds the DNA to facilitate transactivation by the cooperative transcription factors such as Oct3/4. We report that mouse Sox2 is modified by SUMO at lysine 247. Substitution of the target lysine to arginine lost the sumoylation but little affected transcriptional potential or nuclear localization of Sox2. By contrast with the unmodified form, Sox2 fused to SUMO-1 did not augment transcription via the Fgf4 enhancer in the presence of Oct3/4. Further, SUMO-1-conjugated Sox2 at the lysine 247 or at the carboxyl terminus reduced the binding to the Fgf4 enhancer. These indicate that Sox2 sumoylation negatively regulates its transcriptional role through impairing the DNA binding.

症例は40歳,女性で,食欲低下と腹部膨満感があるため近医で腹部CT検査を受けたところ,腹腔内に巨大腫瘤を指摘され当院を紹介された.胃,大腸内視鏡検査では,圧排所見のみで粘膜面には異常を認めなかった.腹部エコー, CTおよびMRI検査において,左上腹部に比較的血流の少ない20cm大の充実性腫瘤を認め,膵腫瘍を疑い手術を施行した.腫瘤は18×22×7cmの線維性腫瘍で,横行結腸間膜と一塊となり,胃,膵臓を巻き込むように増殖していた.表面は平滑,硬さは弾性硬であり割面は白色調であった.病理組織学的,免疫組織学的に,横行結腸間膜発生と考えられる腸間膜線維腫症と診断された.本症は家族性大腸線腫症や開腹手術の既往のある患者に発生することが多く,それらを伴わない単独発生は稀と考え報告した.

慢性膵炎の経過観察中膵内に腫瘤を認め, F-18-fluorodeoxyglucose positron emission tomography (FDG-PET)にて高集積を示し,膵癌との鑑別が困難であった1例を経験したので報告する.症例は76歳男性,食思不振と体重減少を主訴に来院し,慢性膵炎急性増悪の診断にて経過観察としていた.経過中CT, MRIおよび超音波検査にて膵体部に腫瘤を検出し,腫瘤形成性膵炎と膵癌との鑑別目的でFDG-PETを施行した.同部に異常集積(standardized uptake value: SUV=5.1)を認め,さらに遅延相で増強(SUV=5.4)した.膵癌が強く疑われ,膵体尾部脾合併切除術を施行した.病理組織所見では膵の慢性および急性炎症像に一部膵管上皮の過形成性変化あり,悪性所見は認めなかった. FDG-PETは腫瘤形成性膵炎と膵癌との鑑別に有用であるが,両者の集積レベルにはオーバーラップがあり,今後とも症例を重ね評価法の工夫が必要である.

The methyl–CpG dinucleotide containing a symmetrical 5-methylcytosine (mC) is involved in gene regulation and genome stability. We report here that methylation-mediated transcriptional repressor methylated DNA-binding domain 1 (MBD1) interacts with methylpurine–DNA glycosylase (MPG), which excises damaged bases from substrate DNA. MPG itself actively represses transcription and has a synergistic effect on gene silencing together with MBD1. Chromatin immunoprecipitation analysis reveals the molecular movement of MBD1 and MPG in vivo : ( i ) The MBD1–MPG complex normally exists on the methylated gene promoter; ( ii ) treatment of cells with alkylating agent methylmethanesulfonate (MMS) induces the dissociation of MBD1 from the methylated promoter, and MPG is located on both methylated and unmethylated promoters; and ( iii ) after completion of the repair, the MBD1–MPG complex is restored on the methylated promoter. Mobility-shift and structural analyses show that the MBD of MBD1 binds a methyl–CpG pair (mCpG × mCpG) but not the methyl–CpG pair containing a single 7-methylguanine (N) (mCpG × mCpN) that is known as one of the major lesions caused by MMS. We further demonstrate that knockdown of MBD1 by specific small interfering RNAs significantly increases cell sensitivity to MMS. These data suggest that MBD1 cooperates with MPG for transcriptional repression and DNA repair. We hypothesize that MBD1 functions as a reservoir for MPG and senses the base damage in chromatin.

DNA methylation is involved in a variety of genome functions, including gene control and chromatin dynamics. MBD1 is a transcriptional regulator through the cooperation of a methyl-CpG binding domain, cysteine-rich CXXC domains, and a transcriptional repression domain. A yeast two-hybrid screen was performed to investigate the role of MBD1 in methylation-based transcriptional repression. We report a mediator, MBD1-containing chromatin-associated factor (MCAF), that interacts with the transcriptional repression domain of MBD1. MCAF harbors two conserved domains that allow it to interact with MBD1 and enhancer-like transactivator Sp1. MCAF possesses a coactivator-like activity, and it seems to facilitate Sp1-mediated transcription. In contrast, the MBD1-MCAF complex blocks transcription through affecting Sp1 on methylated promoter regions. These data provide a mechanistic basis for direct inhibition of gene expression via methylation-dependent and histone deacetylation-resistant processes.

十二指腸乳頭部癌に対する幽門輪温存膵頭十二指腸切除後5年目に発生した残膵癌に対し残膵全摘を行い根治切除をしえた1 例を経験したので報告する. 患者は75 歳の女性で, 1994年9月十二指腸乳頭部癌に対して幽門輪温存膵頭十二指腸切除術を施行し, 膵断端は組織学的に癌浸潤陰性であり, 膵胃吻合により再建した. 術後経過良好であったが5年後の1999年11月検診目的で施行した胃内視鏡検査で胃体中部後壁の膵胃吻合部に中心陥凹を有する隆起性病変を認め生検ではtubular adenocarcinomaであった. 隆起の主座は残膵吻合部にあったので残膵癌と判断し残膵全摘, 胃幽門側切除, 摘脾を行い胃空腸吻合で再建した. 最終病理組織診断は膵乳頭腺癌で (ts4, s0, rpo, pv0, a0, t1b, n0, stage II) であった. 十二指腸乳頭部癌切除術後の残膵癌の切除報告は極めてまれであり, 根治切除ができた貴重な症例と考えられるので重複癌や転移再発癌との鑑別も含めて報告する.

Platinum-based chemotherapeutic drugs are widely used as components of combination chemotherapy in the treatment of cancer. One such drug, oxaliplatin, exerts a synergistic effect against advanced colorectal cancer in combination with 5-fluorouracil (5-FU) and leucovorin. In the p53-proficient colorectal cancer cell line HCT116, oxaliplatin represses the expression of deoxyuridine triphosphatase (dUTPase), a ubiquitous pyrophosphatase that catalyzes the hydrolysis of dUTP to dUMP and inhibits dUTP-mediated cytotoxicity. However, the underlying mechanism of this activity has not been completely elucidated, and it remains unclear whether factors other than downregulation of dUTPase contribute to the synergistic effect of 5-FU and oxaliplatin. In this study, we found that oxaliplatin and dachplatin, platinum-based drugs containing the 1,2-diaminocyclohexane (DACH) carrier ligand, repressed the expression of nuclear isoform of dUTPase (DUT-N), whereas cisplatin and carboplatin did not. Oxaliplatin induced early p53 accumulation, upregulation of primary miR-34a transcript expression, and subsequent downregulation of E2F3 and E2F1. Nutlin-3a, which activates p53 nongenotoxically, had similar effects. Introduction of miR-34a mimic also repressed E2F1 and DUT-N expression, indicating that this miRNA plays a causative role. In addition to DUT-N, oxaliplatin repressed, in a p53-dependent manner, the expression of genes encoding enzymes involved in thymidylate biosynthesis. Consequently, oxaliplatin significantly decreased the level of dTTP in the dNTP pool in a p53-dependent manner. These data indicate that the DACH carrier ligand in oxaliplatin triggers signaling via the p53–miR-34a–E2F axis, leading to transcriptional regulation that ultimately results in accumulation of dUTP and reduced dTTP biosynthesis, potentially enhancing 5-FU cytotoxicity. Mol Cancer Ther; 14(10); 2332–42. ©2015 AACR.

High‐mobility group A1 (Hmga1) protein is an architectural chromatin factor, and aberrant Hmga1 expression in mice causes hematopoietic malignancies with defects in cellular differentiation. However, the functional involvement of Hmga1 in hematopoietic development and leukemic cells remains to be elucidated. Using Hmga1‐green fluorescent protein (GFP) knock‐in mice that endogenously express an Hmga1‐GFP fusion protein, we examined Hmga1 expression in undifferentiated and differentiated populations of hematopoietic cells. During early T cell development in the thymus, Hmga1 is highly expressed in CD4/CD8‐double negative (DN) cells and is transiently downregulated in CD4/CD8‐double positive (DP) cells. Consistently, Hmga1 directly binds to cis‐regulatory elements in the CD4/CD8 loci and the heterochromatin foci in DN‐stage cells, but not in DP cells. Interestingly, CD4/CD8 expression in DN‐stage leukemic cells is induced by inhibition of Hmga1 binding to nuclear DNA or RNA interference‐mediated Hmga1 knockdown. In addition, Hmga1‐depleted leukemic T cells markedly diminish proliferation, with transcriptional activation of cyclin‐dependent kinase inhibitor genes as a direct target of Hmga1. The data in the present study reveal a role of Hmga1 in transcriptional silencing in T cell lineages and leukemic cells. (Cancer Sci 2012; 103: 439–447)

Telomerase maintains telomere length and is implicated in senescence and immortalization of mammalian cells. Two essential components for this enzyme are telomerase reverse transcriptase (TERT) and the telomerase RNA component (encoded by the TERC gene). These telomerase subunit genes are known to be mainly expressed by specificity protein 1 (Sp1). MBD1-containing chromatin-associated factor 1 (MCAF1), also known as ATFa-associated modulator (AM) and activating transcription factor 7-interacting protein (ATF7IP), mediates gene regulation, although the precise function of MCAF1 remains to be elucidated. Here, we report that MCAF1 is involved in Sp1-dependent maintenance of telomerase activity in cancer cells. Two evolutionarily conserved domains of MCAF1 directly interact with Sp1 and the general transcriptional apparatus. Selective depletion of MCAF1 or Sp1 down-regulates TERT and TERC genes in cultured cells, which results in decreased telomerase activity. The transcriptionally active form of RNA polymerase II and the general transcription factor ERCC3 decreased in the TERT promoter under the loss of MCAF1 or Sp1. Consistently, MCAF1 is found to be frequently overexpressed in naturally occurring cancers that originate in different tissues. Our data suggest that transcriptional function of MCAF1 facilitates telomerase expression by Sp1, which may be a common mechanism in proliferative cancer cells.

Background: TAS-102 is a combination of an antineoplastic thymidine-based nucleoside analogue, trifluridine (FTD), and a thymidine phosphorylase inhibitor, tipiracil (TPI), at a molar ratio of 1:0.5. FTD is the active antitumor component of TAS-102, and its triphosphate form is incorporated into the DNA in tumor cells. TPI, a potent inhibitor of the FTD-degrading enzyme thymidine phosphorylase, maintains adequate plasma concentrations of orally administered FTD, thereby potentiating its antitumor activity. In an international multicenter randomized double blind Phase III study (RECOURSE), TAS-102 administration resulted in a significant improvement in overall survival and progression-free survival and a favorable safety profile compared to placebo treatment in patients with metastatic colorectal cancer refractory to standard chemotherapies. In this study, we investigated the mechanism of FTD-induced cytotoxicity in colorectal cancer and other cell lines. Method: The colorectal cancer cell lines RKO and HCT-116, the subline HCT-116 p53−/−, and the lung adenocarcinoma cell line A549 were used. Cells were treated with FTD or FdUrd using the IC50 values for 12 to 72 h. Molecules involved in DNA damage and the cell cycle were evaluated by western blot and RT-PCR. Cell cycle analysis and DNA strand-breaks were evaluated by flow cytometry and alkaline- and neutral-comet assays. Results: FTD and FdUrd activated similar DNA damage response pathways via transient phosphorylation of Chk1 at Ser345, followed by accumulation of p53 and p21 in HCT-116 cells. A similar biochemical response was observed using the IC50 concentration of FdUrd or FTD in RKO and A549 cells. FTD repressed G2 to M phase progression and the cells accumulated in G2 phase 72 h after treatment, with a 35% and 29% decrease in the CDK1 and CCNB1 mRNA levels compared to the control. The G2 phase arrest by FTD was commonly induced in each p53 proficient cell lines (HCT-116, RKO, and A549). In contrast, FTD-induced G2 phase arrest was not observed when p53 expression was suppressed. Furthermore, the p53-dependent G2 phase arrest by FTD was associated with the downregulation of Cyclin B1, both through its transcriptional repression and proteasomal degradation. Although FdUrd induced numerous single- and double-strand DNA breaks, FTD did not induce detectable DNA strand breaks. FTD was massively incorporated into DNA (41.4 pmol/μg DNA), whereas less FdUrd was incorporated (0.6 pmol/μg DNA) 72 h after treatment. Conclusion: Our analyses revealed that FTD induced sustained G2 phase arrest and few DNA strand breaks, in spite of massive incorporation into DNA. These results suggest that the mechanism of FTD action is unique at the cellular level, resulting in the clinical efficacy of TAS-102. Citation Format: Kazuaki Matsuoka, Makoto Iimori, Shinichiro Niimi, Hiroshi Tsukihara, Sugiko Watanabe, Shinichi Kiyonari, Mamoru Kiniwa, Koji Ando, Eriko Tokunaga, Hiroshi Saeki, Eiji Oki, Hiroyuki Kitao, Yoshihiko Maehara. Trifluridine induces p53-dependent sustained G2 phase arrest with its massive misincorporation into DNA and few DNA strand breaks. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2556. doi:10.1158/1538-7445.AM2015-2556

DNA methylation cooperates with methylation at lysine 9 of histone H3 (H3-K9), a modified histone molecule that is targeted by heterochromatin protein 1, to form a transcriptionally silent chromatin. Methyl CpG-binding protein MBD1 recognizes methylated CpG dinucleotide and recruits H3-K9 methyltransferases such as SETDB1 to genomic regions. Here we show that MBD1-containing chromatin-associated factor (MCAF) 1, also known as the human homologue of murine ATFa-associated modulator (AM), is required for transcriptional repression and heterochromatin formation by MBD1, together with the involvement of SETDB1. Moreover, the amino acid sequence of MCAF1 shows similarity to a number of sequences of the MCAF/AM-related proteins, resulting in the identification of a new member of the protein family, termed MCAF2. Immunoprecipitation and in vitro binding analyses reveal that both MCAF proteins interact with MBD1, SETDB1, and Sp1 via two evolutionarily conserved distinct domains. Furthermore, MCAF1 enhances transcriptional repression by MBD1 together with SETDB1, and exogenous expression of MCAF2 partly compensates for the repressive activity in MCAF1 knockdown HeLa cells. The expression of MBD1 mutant, which lacks interaction with MCAF proteins, perturbs heterochromatin protein 1-enriched heterochromatin formation at the MBD1-containing chromosomal loci. These data suggest that MBD1·MCAF1·SETDB1 complex facilitates the formation of heterochromatic domains, emphasizing the role of MCAF/AM family proteins in epigenetic control.

Cytosine methylation and posttranslational modifications of the amino termini of the core histones in the nucleosome provide epigenetic codes for genome regulation. In the nucleus, not only is the DNA methylated, but the methylated DNA is also interpreted by methyl-CpG binding domain (MBD) proteins. MBD1 possesses an MBD involved in mediating DNA methylation-dependent transcriptional repression. The MBD of MBD1 binds a symmetrically methylated CpG sequence, but the precise roles of this domain have not been investigated. In addition, little is understood about the state of histone modifications within MBD1-containing heterochromatin on methylated gene promoters. Here we show that histone H3 methylase Suv39h1 and the methyl lysine-binding protein HP1 directly interact with MBD of MBD1 in vitro and in cells. Suv39h1 was found to enhance MBD1-mediated transcriptional repression via MBD but not via the C-terminal transcriptional repression domain of MBD1. Furthermore, MBD1 links to histone deacetylases through Suv39h1, resulting in methylation and deacetylation of histones for gene inactivation. These data indicate that MBD1 may tether the Suv39h1-HP1 complex to methylated DNA regions, suggesting the presence of a pathway from DNA methylation to the modifications of histones for epigenetic gene regulation.