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The genome guardian p53 functions as a transcription factor that senses numerous cellular stresses and orchestrates the corresponding transcriptional events involved in determining various cellular outcomes, including cell cycle arrest, apoptosis, senescence, DNA repair, and metabolic regulation. In response to diverse stresses, p53 undergoes multiple posttranslational modifications (PTMs) that coordinate with intimate interdependencies to precisely modulate its diverse properties in given biological contexts. Notably, PTMs can recruit ‘reader’ proteins that exclusively recognize specific modifications and facilitate the functional readout of p53. Targeting PTM–reader interplay has been developing into a promising cancer therapeutic strategy. In this review, we summarize the advances in deciphering the ‘PTM codes’ of p53, focusing particularly on the mechanisms by which the specific reader proteins functionally decipher the information harbored within these PTMs of p53. We also highlight the potential applications of intervention with p53 PTM–reader interactions in cancer therapy and discuss perspectives on the ‘PTMomic’ study of p53 and other proteins.

Lung cancer is the leading cause of cancer-related death, and non-small cell lung cancer (NSCLC) accounts for almost 80-85% of all lung cancer cases. The transcriptional factor brachyury has been verified to promote tumor cells migrate, invade, and metastasis in various types of tumors, whereas divergent roles of brachyury on cell proliferation have been reported in several types of tumor cells. In this study, we attempted to explore the effect of brachyury on the cell cycle progression and proliferation capability of NSCLC cells. Firstly, we performed RNA-sequence and ChIP-sequence to explore underlying downstream pathways regulated by brachyury. Cell proliferation and colony formation assays were utilized to detect the effect of brachyury on the proliferation ability of two types of lung NSCLC cells: H460 and Calu-1, which represent different brachyury expression levels. Following cell cycle and cell apoptosis assays were used to investigate the mechanism by which brachyury promotes NSCLC grow and progression. RNA-sequence and ChIP-sequence (ChIP-seq) showed that one of the vital downstream pathways regulated by brachyury involves in cell cycle progression. Through cell proliferation assays and colony formation assays, we found that inhibition of brachyury could decrease the capability of proliferation in H460 cells. We also found that brachyury overexpression could prevent the transition from G0/G1 to S phase in Calu-1 cells, and brachyury knockdown could decrease the transition of G2/M phase in H460 cells. The cell apoptosis assays showed that inhibition of brachyury could promote apoptosis in H460 cells. In this study we demonstrate that brachyury and downstream target genes together involve in tumor cell cycle regulation by inducing accelerated transition through G2/M, promote tumor cell proliferation and inhibit apoptosis in lung NSCLC H460 cells. Targeting brachyury expression could be developed into a promising avenue for the prevention of lung cancer progression.

// Ning Kon 1,* , Donglai Wang 1,* , Tongyuan Li 1 , Le Jiang 1 , Li Qiang 2,3,** and Wei Gu 1,3 1 Institute for Cancer Genetics, College of Physicians and Surgeons of Columbia University, New York, New York, USA 2 Naomi Berrie Diabetes Center, Department of Medicine, College of Physicians and Surgeons of Columbia University, New York, New York, USA 3 Department of Pathology and Cell Biology, College of Physicians and Surgeons of Columbia University, New York, New York, USA * These two authors contributed equally to this work ** This author is listed as a co-corresponding author Correspondence to: Wei Gu, email: // Keywords : p53; Mdmx; Mdm4; metabolism; adipose tissues Received : September 26, 2017 Accepted : December 08, 2017 Published : January 02, 2018 Abstract Although cell-cycle arrest, senescence and apoptosis remain as major canonical activities of p53 in tumor suppression, the emerging role of p53 in metabolism has been a topic of great interest. Nevertheless, it is not completely understood how p53-mediated metabolic activities are regulated in vivo and whether this part of the activities has an independent role beyond tumor suppression. Mdmx (also called Mdm4), like Mdm2, acts as a major suppressor of p53 but the embryonic lethality of mdmx-null mice creates difficulties to evaluate its physiological significance in metabolism. Here, we report that the embryonic lethality caused by the deficiency of mdmx , in contrast to the case for mdm2 , is fully rescued in the background of p53 3KR/3KR , an acetylation-defective mutant unable to induce cell-cycle arrest, senescence and apoptosis. p53 3KR/3KR /mdmx -/- mice are healthy but skinny without obvious developmental defects. p53 3KR/3KR /mdmx -/- mice are resistant to fat accumulation in adipose tissues upon high fat diet. Notably, the levels of p53 protein are only slightly increased and can be further induced upon DNA damage in p53 3KR/3KR /mdmx -/- mice, suggesting that Mdmx is only partially required for p53 degradation in vivo . Further analyses indicate that the anti-obesity phenotypes in p53 3KR/3KR /mdmx -/- mice are caused by activation of lipid oxidation and thermogenic programs in adipose tissues. These results demonstrate the specific effects of the p53/Mdmx axis in lipid metabolism and adipose tissue remodeling and reveal a surprising role of Mdmx inhibition in anti-obesity effects beyond, commonly expected, tumor suppression. Thus, our study has significant implications regarding Mdmx inhibitors in the treatment of obesity related diseases.

Acetylation of non-histone proteins plays important roles in regulating protein functions but the mechanisms of action are poorly understood. Our recent study uncovered a previously unknown mechanism by which C-terminal domain (CTD) acetylation of p53 serves as a “switch” to determine the interaction between a unique group of acidic domain-containing proteins and p53, as well as revealed that acidic domains may act as a novel class of “readers” for unacetylated p53. However, the properties of acidic domain “readers” are not well elucidated yet. Here, we identified that the charge effect between acidic domain “readers” and the p53 CTD is necessary for their interaction. Both the length and the amino acid composition of a given acidic domain contributed to its ability to recognize the p53 CTD. Finally, we summarized the characteristic features of our identified acidic domains, which would distinguish this kind of “readers” from other types of acidic amino acid-containing domains.

Background Denosumab is an inhibitor of receptor activator of NF-κB ligand (RANKL), which inhibits bone metastasis (BM) in breast cancer (BC), but does not completely control cancer cell BM in some BC patients. This study was designed to study whether denosumab inhibits human BC cells (MCF-7) cell line-induced spontaneous osteoclastogenesis via RANKL/metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/miR-124 axis. Methods We established a co-culture system of MCF-7-induced spontaneous osteoclastogenesis in RAW 264.7 cells, and denosumab is added into the co-culture system to inhibit RAW 264.7 cell differentiation into osteoclasts. Real-time PCR (RT-PCR), immunofluorescence and western blotting analysis were used to detect gene expression, while tartrate-resistant acid phosphatase (TRAP) staining was used to assess osteoclast formation. Results Denosumab inhibits MCF-7 cell line-induced spontaneous osteoclastogenesis, and the inhibition of denosumab was found to be more pronounced after MALAT1 downregulation and miR-124 overexpression. However, MALAT1 knockdown or miR-124 overexpression did not alter RANKL protein expression. Moreover, the dual luciferase gene reporter system showed that miR-124 targeted the inhibition of MALAT1, while si-MALAT1 upregulated miR-124 expression. miR-124-mimics were able to decrease the expression of Rab27a, IL-11, activated T-cell nuclear factor 1 (NFATc1) and TARP protein. Conclusions Denosumab inhibits MALAT1 expression by inhibiting RANKL, thereby upregulating miR-124 expression, which ultimately inhibits MCF-7 cell line-induced pseudo osteoclastogenesis.

The bone marrow has been long known to host a unique environment amenable to colonization by metastasizing tumor cells. Yet, the underlying molecular interactions which give rise to the high incidence of bone metastasis (BM) in breast cancer patients have long remained uncharacterized. In our study, in vitro and in vivo assays demonstrated that Brachyury (Bry) could promote breast cancer BM. Bry drives epithelial–mesenchymal transition (EMT) and promotes breast cancer aggressiveness. As an EMT driver, SOX5 involves in breast cancer metastasis and the specific function in BM. Chromatin immunoprecipitation (ChIP) assays revealed SOX5 is a direct downstream target gene of Bry. ChIP analysis and reporter assays identified two Bry-binding motifs; one consistent with the classic conserved binding sequence and the other a new motif sequence. This study demonstrates for the first time that Bry promotes breast cancer cells BM through activating SOX5. In clinical practice, targeting the Bry-Sox5-EMT pathway is evolving into a promising avenue for the prevention of bone metastatic relapse, therapeutic resistance and other aspects of breast cancer progression.Brachyury directly regulates the expression of SOX5 by binding to two motifs in its promoter region. The Bry-SOX5-EMT pathway may represent a potential target to develop treatments to prevent and treat bone metastasis from breast cancer.

Although lysine acetylation is now recognized as a general protein modification for both histones and non-histone proteins, the mechanisms of acetylation-mediated actions are not completely understood. Acetylation of the C-terminal domain (CTD) of p53 (also known as TP53) was an early example of non-histone protein acetylation and its precise role remains unclear. Lysine acetylation often creates binding sites for bromodomain-containing 'reader' proteins. Here we use a proteomic screen to identify the oncoprotein SET as a major cellular factor whose binding with p53 is dependent on CTD acetylation status. SET profoundly inhibits p53 transcriptional activity in unstressed cells, but SET-mediated repression is abolished by stress-induced acetylation of p53 CTD. Moreover, loss of the interaction with SET activates p53, resulting in tumour regression in mouse xenograft models. Notably, the acidic domain of SET acts as a 'reader' for the unacetylated CTD of p53 and this mechanism of acetylation-dependent regulation is widespread in nature. For example, acetylation of p53 also modulates its interactions with similar acidic domains found in other p53 regulators including VPRBP (also known as DCAF1), DAXX and PELP1 (refs. 7, 8, 9), and computational analysis of the proteome has identified numerous proteins with the potential to serve as acidic domain readers and lysine-rich ligands. Unlike bromodomain readers, which preferentially bind the acetylated forms of their cognate ligands, the acidic domain readers specifically recognize the unacetylated forms of their ligands. Finally, the acetylation-dependent regulation of p53 was further validated in vivo by using a knock-in mouse model expressing an acetylation-mimicking form of p53. These results reveal that acidic-domain-containing factors act as a class of acetylation-dependent regulators by targeting p53 and, potentially, other proteins.

Macroautophagy is an evolutionarily conserved cellular process involved in the clearance of proteins and organelles. Although the autophagy regulation machinery has been widely studied, the key epigenetic control of autophagy process still remains unknown. Here we report that the methyltransferase EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit) epigenetically represses several negative regulators of the MTOR (mechanistic target of rapamycin [serine/threonine kinase]) pathway, such as TSC2, RHOA, DEPTOR, FKBP11, RGS16 and GPI. EZH2 was recruited to these genes promoters via MTA2 (metastasis associated 1 family, member 2), a component of the nucleosome remodeling and histone deacetylase (NuRD) complex. MTA2 was identified as a new chromatin binding protein whose association with chromatin facilitated the subsequent recruitment of EZH2 to silenced targeted genes, especially TSC2. Downregulation of TSC2 (tuberous sclerosis 2) by EZH2 elicited MTOR activation, which in turn modulated subsequent MTOR pathway-related events, including inhibition of autophagy. In human colorectal carcinoma (CRC) tissues, the expression of MTA2 and EZH2 correlated negatively with expression of TSC2, which reveals a novel link among epigenetic regulation, the MTOR pathway, autophagy induction, and tumorigenesis.

Early aggressive metastasis of osteosarcoma (OS) leads to rapid progression and poor prognosis. Increasing evidence has demonstrated that long non-coding RNAs (lncRNAs) could serve as crucial regulators to modulate tumour metastasis. In this study, we reported the critical role of lncRNA TUG1 in determining OS metastasis. TUG1 was significantly upregulated in OS tissues and associated with tumour size, distant metastasis, TNM stage, and overall and recurrence-free survival, which further indicated poor prognosis. Furthermore, CAFs-derived TGF-β could upregulate TUG1 expression, and the crosstalk between CAFs and OS cells induced TUG1 to promote OS cell metastasis. Dysregulated TUG1 expression could act as an miRNA “sponge” to competitively protect the HIF-1α mRNA 3′UTR from miR-143-5p. Our study emphasised the effects of TUG1 in OS and demonstrated a novel axis by which TUG1 regulated OS cell metastasis, angiogenesis, and proliferation in vivo and in vitro. Collectively, TUG1 might be a prognostic indicator for OS and could be a therapeutic target for OS.

Our previous study showed that the oncoprotein SET acts as a new reader of unacetylated p53 for transcriptional repression. To further elucidate the physiological significance of SET in vivo, we generated set knockout mice. Set knockout mice died during embryonic development between day 11.5 and day 12.5 post coitum, exhibiting cardiac edema and open neural tube, among other developmental defects. Further analyses revealed that loss of SET leads to upregulation of p53 target genes including p21 and puma without any obvious effect on p53 stability in set knockout embryos. Notably, the developmental defects of set knockout mice were significantly, but nonetheless partially, rescued by concomitant deletion of p53. The failure to obtain fully live set/p53 double knockout mice suggested that p53-independent targets of SET also contribute to the embryonic lethality of set knockout mice. Indeed, we found that FOXO1 acts as an important target of SET and that SET-mediated regulation of FOXO1 is also acetylation-dependent. Taken together, these data underscore the importance of SET oncoprotein during embryonic development and reveal both of the p53-dependent and the p53-independent functions of SET in vivo.

PR-SET7 (also named SET8 or KMT5a) is a sole lysine methyltransferase that catalyzes monomethylation of histone H4 lysine 20 (H4K20me1) in higher eukaryotes. The abundance of PR-SET7 is dynamically mediated by the distinct E3 ubiquitin ligases in different cell cycle phases. PR-SET7 is closely related to the regulation of cell proliferation, and the H4K20me1 catalyzed by PR-SET7 has been implicated in regulating the diverse biological processes, including DNA replication, chromosome condensation and the activation of DNA replication checkpoints. Loss of PR-SET7 results in mas-sive DNA damage, cell cycle arrest and induction of apoptosis. In addition, PR-SET7 involves in regulating the transcrip-tion of several genes, such as ERa, Wnt and p53. PR-SET7 is also essential for individual development and participates in the formation of genomic imprinting. Moreover, PR-SET7 has been reported to promote tumorigenesis and metastasis, sug-gesting that PR-SET7 may be a potential target for cancer treatment. In this review, we focus on analyzing the structure of PR-SET7 and factors influencing histone modification on regulation of PR-SET7, and discuss the mechanisms by which PR-SET7 modulates cell-cycle progression, gene transcription, individual development and tumorigenesis.

Numerous studies indicate that Sirtuin 1 (SIRT1), a mammalian nicotinamide adenine dinucleotide (NAD + )-dependent histone deacetylase (HDAC), plays a crucial role in p53-mediated stress responses by deacetylating p53. Nevertheless, the acetylation levels of p53 are dramatically increased upon DNA damage, and it is not well understood how the SIRT1–p53 interaction is regulated during the stress responses. Here, we identified Set7/9 as a unique regulator of SIRT1. SIRT1 interacts with Set7/9 both in vitro and in vivo. In response to DNA damage in human cells, the interaction between Set7/9 and SIRT1 is significantly enhanced and coincident with an increase in p53 acetylation levels. Importantly, the interaction of SIRT1 and p53 is strongly suppressed in the presence of Set7/9. Consequently, SIRT1-mediated deacetylation of p53 is abrogated by Set7/9, and p53-mediated transactivation is increased during the DNA damage response. Of note, whereas SIRT1 can be methylated at multiple sites within its N terminus by Set7/9, a methylation-defective mutant of SIRT1 still retains its ability to inhibit p53 activity. Taken together, our results reveal that Set7/9 is a critical regulator of the SIRT1-p53 interaction and suggest that Set7/9 can modulate p53 function indirectly in addition to acting through a methylation-dependent mechanism.

SIRT1 is an NAD+-dependent deacetylase in mammalian cells and plays important roles in senescence, cell death and tumorigenesis. To clarify the regulation of SIRT1 expression and activity is crucial for scientists to understand the biological function of SIRT1. This review mainly focuses on the mechanisms by which SIRT1 expression and activity are regulated.

During past decades, acetylation has emerged as a general post-translational modification that is widespread and distributed on lysine residues of histones and nonhistone proteins. Lysine acetylation has been suggested to create a platform for the recruitment of bromodomaincontaining proteins that serve as “readers” to decode information within the acetylated lysine residues [1]. However, the precise mechanism of acetylation-mediated regulatory effects is still not fully understood. p53 is the first identified non-histone protein modified by lysine acetylation; p53 acetylation regulates p53-dependent gene-specific transcription during stress responses [2]. Our recent study focusing on acetylation of the p53 C-terminal domain (CTD) revealed the acidic domain-containing proteins as novel “converse readers” of protein acetylation [3]. Unlike bromodomain “readers” that preferentially bind with acetylated ligands, the acidic domain “readers” specifically recognize the unacetylated forms of their ligands. By performing an in vitro proteomic screen, we identified the oncoprotein SET as a major binding partner of p53 when the CTD is unacetylated; conversely, this p53-SET interaction was completely abolished by CTD acetylation. Of note, this acetylation-mediated regulation was unique, as other modifications on the same lysine residues including methylation, ubiquitination, sumoylation and neddylation, had no obvious effect on p53-SET interaction. Functionally, SET negatively regulated p53 transactivity by acting as a co-repressor inhibiting p300/CBP-mediated H3K18 and H3K27 acetylation on p53 target promoters. Depletion of endogenous SET in p53-wildtype cells significantly suppressed xenograft tumor formation, indicating that p53-SET interplay is crucial for p53-mediated tumor growth repression. During DNA damage response, p53 is stabilized and CTD acetylation is highly induced; the p53-SET interaction in both soluble and chromatinbound fractions was severely disrupted, releasing p53 in an activated status. This observation was further proved in physiological conditions by using a CTD acetylationmimicking mouse model (p53KQ/KQ) where p53 failed to interact with SET. More importantly, p53KQ/KQ mice exhibited neonatal lethality due to p53 hyperactivity in multiple tissues, consistent with the notion that CTD acetylation activates p53. SET consists of an N-terminal dimerization domain, a middle “earmuff” domain and a C-terminal acidic domain (AD). Biochemical analysis indicated that the p53 CTD directly interacted with the SET AD. The p53 CTD is a lysine-rich domain, which harbors a positive charge. Conversely, the SET AD is enriched with acidic amino acids, exhibiting a highly negative charge. Therefore, we postulated that p53-SET interaction is mediated by a charge effect between the CTD and the AD. Upon acetylation, the positive charge of the CTD is neutralized, resulting in a dissociation of the p53-SET complex due to disruption of the charge effect between the CTD and the AD. Bioinformatic analysis of human proteomics revealed that numerous proteins contain acidic domains or lysinerich domains; our additional binding assays suggested a general mechanism that the acidic domain acts as a “reader” of unacetylated lysine residues and acetylation acts as a “switch” to determine the interaction between acidic domain-containing proteins and their lysine-rich domain-containing ligands. This mechanism was further illustrated by the observation that other acidic domain-containing factors of p53 including VPRBP, DAXX and PELP1, also interacted with the p53 CTD through their acidic domains; of note, their interactions were completely blocked by CTD acetylation. More importantly, all these proteins lost their interactions with p53 in p53KQ/KQ mouse model, Editorial

STUDY DESIGN The histological comparative study was performed on chordoma and notochordal cell rests (NCRs). OBJECTIVE To understand the histological similarity and homology of chordoma and NCRs, further supplying direct evidence of chordoma origin from NCRs. SUMMARY OF BACKGROUND DATA Although many studies supported the hypothesis that chordoma arise from NCRs, there has been little direct evidence reported to date. Of the base of our previous study, we conducted a comparative histological study among NCRs coexisting in chordoma, fetal NCRs, and chordoma tumor components. METHODS Thirty fetal nucleus pulposus and 46 chordoma specimens were harvested, and classic chordoma tumor markers and brachyury expression levels were investigated through immunohistochemical method. RESULTS The fetal NCRs existed in the form of clusters in the center of nucleus pulposus

Suppressor of variegation 3–9 homolog 1 (SUV39H1), a histone methyltransferase, catalyzes histone 3 lysine 9 trimethylation and is involved in heterochromatin organization and genome stability. However, the mechanism for regulation of the enzymatic activity of SUV39H1 in cancer cells is not yet well known. In this study, we identified SET domain-containing protein 7 (SET7/9), a protein methyltransferase, as a unique regulator of SUV39H1 activity. In response to treatment with adriamycin, a DNA damage inducer, SET7/9 interacted with SUV39H1 in vivo, and a GST pull-down assay confirmed that the chromodomain-containing region of SUV39H1 bound to SET7/9. Western blot using antibodies specific for antimethylated SUV39H1 and mass spectrometry demonstrated that SUV39H1 was specifically methylated at lysines 105 and 123 by SET7/9. Although the half-life and localization of methylated SUV39H1 were not noticeably changed, the methyltransferase activity of SUV39H1 was dramatically down-regulated when SUV39H1 was methylated by SET7/9. Consequently, H3K9 trimethylation in the heterochromatin decreased significantly, which, in turn, led to a significant increase in the expression of satellite 2 ( Sat2 ) and α-satellite ( α-Sat ), indicators of heterochromatin relaxation. Furthermore, a micrococcal nuclease sensitivity assay and an immunofluorescence assay demonstrated that methylation of SUV39H1 facilitated genome instability and ultimately inhibited cell proliferation. Together, our data reveal a unique interplay between SET7/9 and SUV39H1—two histone methyltransferases—that results in heterochromatin relaxation and genome instability in response to DNA damage in cancer cells.

Forkhead box O (FOXO) transcriptional protein family members, including FOXO1 and FOXO3, are involved in the modulation of autophagy. However, whether there is redundancy between FOXO1 and FOXO3 in the ability to induce autophagy remains unclear. In this study, we showed that FOXO3 induced a transcription-dependent autophagy, and FOXO1 was required for this process. Overexpression of wild-type FOXO3 (WT) or FOXO3 (3A), which harbors alanine mutations at residues Thr32, Ser253 and Ser315, but not transcription-inactive FOXO3 (∆DB3A), significantly induced autophagy in the human embryonic kidney cell line HEK293T and mouse embryonic fibroblast (MEF) cell lines. Interestingly, depletion of FOXO1 by siRNA attenuated FOXO3-induced autophagy. Our data also showed that FOXO3 overexpression did not increase the expression of FOXO1 at the protein level, although FOXO3 was capable of binding the promoter region of FOXO1 and inducing an increase in the transcription of FOXO1 mRNA. Furthermore, our results showed that FOXO3 promoted the translocation of FOXO1 from the nucleus to the cytoplasm, resulting in an increase in FOXO1-induced autophagy. Moreover, our results supported a mechanism whereby FOXO3 dramatically increased the expression of the class I PtdIns3K catalytic subunit PIK3CA, leading to an increase in AKT1 activity, which resulted in the phosphorylation and nuclear export of FOXO1. To the best of our knowledge, our data are the first to suggest that FOXO1 plays a central role in FOXO3-induced autophagy.

Not only does 5-aza-2'-deoxycytidine (5-aza-CdR) induce the reexpression of silenced genes through the demethylation of CpG islands, but it increases the expression of unmethylated genes. However, the mechanism by which 5-aza-CdR activates the expression of genes is not completely understood. Here, we report that the pRb pocket proteins pRb, p107, and p130 were degraded in various cancer cell lines in response to 5-aza-CdR treatment, and this effect was dependent on the proteasome pathway. Mouse double minute 2 (MDM2) played a critical role in this 5-aza-CdR-induced degradation of pRb. Furthermore, PP2A phosphatase-induced MDM2 dephosphorylation at S260 was found to be essential for MDM2 binding to pRb in the presence of 5-aza-CdR. pRb degradation resulted in the significant reexpression of several genes, including methylated CDKN2A, RASFF1A, and unmethylated CDKN2D. Finally, knockdown of pRb pocket proteins by either RNAi or 5-aza-CdR treatment induced a significant decrease in the recruitment of SUV39H1 and an increase in the enrichment of KDM3B and KDM4A to histones around the promoter of RASFF1A and thus reduced H3K9 di- and trimethylation, by which RASFF1A expression is activated. Our data reveal a novel mechanism by which 5-aza-CdR induces the expression of both methylated and unmethylated genes by degrading pRb pocket proteins.

Autophagy is characterized by the sequestration of bulk cytoplasm, including damaged proteins and organelles, and delivery of the cargo to lysosomes for degradation. Although the autophagic pathway is also linked to tumour suppression activity, the mechanism is not yet clear. Here we report that cytosolic FoxO1, a forkhead O family protein, is a mediator of autophagy. Endogenous FoxO1 was required for autophagy in human cancer cell lines in response to oxidative stress or serum starvation, but this process was independent of the transcriptional activity of FoxO1. In response to stress, FoxO1 was acetylated by dissociation from sirtuin-2 (SIRT2), a NAD(+)-dependent histone deacetylase, and the acetylated FoxO1 bound to Atg7, an E1-like protein, to influence the autophagic process leading to cell death. This FoxO1-modulated cell death is associated with tumour suppressor activity in human colon tumours and a xenograft mouse model. Our finding links the anti-neoplastic activity of FoxO1 and the process of autophagy.

5-Aza-2'-deoxycytidine (5-aza-CdR) is used extensively as a demethylating agent and acts in concert with histone deacetylase inhibitors (HDACI) to induce apoptosis or inhibition of cell proliferation in human cancer cells. Whether the action of 5-aza-CdR in this synergistic effect results from demethylation by this agent is not yet clear. In this study we found that inhibition of cell proliferation was not observed when cells with knockdown of DNA methyltransferase 1 (DNMT1), or double knock down of DNMT1-DNMT3A or DNMT1-DNMT3B were treated with HDACI, implying that the demethylating function of 5-aza-CdR may be not involved in this synergistic effect. Further study showed that there was a causal relationship between 5-aza-CdR induced DNA damage and the amount of [(3)H]-5-aza-CdR incorporated in DNA. However, incorporated [(3)H]-5-aza-CdR gradually decreased when cells were incubated in [(3)H]-5-aza-CdR free medium, indicating that 5-aza-CdR, which is an abnormal base, may be excluded by the cell repair system. It was of interest that HDACI significantly postponed the removal of the incorporated [(3)H]-5-aza-CdR from DNA. Moreover, HDAC inhibitor showed selective synergy with nucleoside analog-induced DNA damage to inhibit cell proliferation, but showed no such effect with other DNA damage stresses such as gamma-ray and UV, etoposide or cisplatin. This study demonstrates that HDACI synergistically inhibits cell proliferation with nucleoside analogs by suppressing removal of incorporated harmful nucleotide analogs from DNA.