HJURP Research Articles

Holliday junction recognition protein promotes pancreatic cancer growth and metastasis via modulation of the MDM2/p53 signaling

Holliday junction recognition protein (HJURP) refers to a histone H3 chaperone that has been implicated in different kinds of malignancies. Yet, its character in pancreatic cancer remains unclear. The expression of HJURP was assessed in PDAC tissues by RT-qPCR, immunoblotting, and immunohistochemistry. HJURP-deficient or overexpressed PDAC cell lines were constructed, using shRNA or plasmids with HJURP insert. MTT, sphere formation assay, migration, and invasion assays were performed to evaluate the viability, proliferation, migration, and invasion of PDAC cells. We used xenograft mice models to assess the tumor growth and metastasis in vivo. RNA-seq was applicated in search of the potential downstream target of HJURP in PDAC and subsequent verification were fulfilled via multiple assays, including immunofluorescence. Additionally, chromatin immunoprecipitation and luciferase reporter assay were conducted to explore the potential regulation of MDM2 expression by HJURP through H3K4me2. In this current research, we found that the expression of HJURP in PDAC cells and tissue was significantly higher than those of adjacent normal tissue, and high HJURP expression predicted poor survival. HJURP significantly promoted the viability, sphere formation, migration, and invasion of PDAC cells in vitro, HJURP also facilitated tumor growth and metastasis in vivo. Mechanically, MDM2/p53 axis is critical for HJURP-mediated malignant behaviors in PDAC, and HJURP regulates MDM2 expression through H3K4me2. HJURP could serve as a promising biomarker, and target for PDAC prognosis and treatment.

Anti-cancer immunotherapy using cancer-derived multiple epitope-peptides cocktail vaccination clinical studies in patients with refractory/persistent disease of uterine cervical cancer and ovarian cancer [phase 2]

ABSTRACT We had conducted phase 1/2 studies of cancer vaccination therapy using neo-tumor antigens in patients with refractory/persistent cervical cancer (CC) and ovarian cancer (OC) to assess the feasibility and efficacy. Enrollees must be refractory/persistent disease for usual treatments with Human Leukocyte Antigen-A*0201 or A*2402. The targets were epitope peptides obtained from driver genes in surviving pathways as follows: for CC A*0201, peptides from Up Regulating Lung Cancer 10 gene (URLC10) and Hypoxia-inducible gene 2 (HIG-2) and for OC A*0201, HIG2, VEGFR (vascular epithelial growth factor receptor) 1 and 2 were used. For CC A*2402, Forkhead Box M1 (FOXM1), Maternal Embryonic Leucine zipper Kinase (MELK), and Holliday Junction Recognition Protein (HJURP) were used. For OC A*2402, cocktails of peptides from FOXM1, MELK, HJURP, VEGFR1, and VEGFR2 were used. Subcutaneous administration was performed with adjuvant weekly. The toxicity profiles and tumor-response were analyzed in eight-week interval. Sixty-six patients were accrued, and 64 were evaluable for adverse events (AEs), and 35 for response. AEs of G2/3 dermatologic reaction (DR) of injection site had been identified in 15.6% and no other severe AEs were detected. Response rate in OC and CC were 22.9% and 20%, respectively. Median overall survival showed longer in performance status (PS) 0 (versus PS1/2), in CRP negative (versus positive) and in DR positive (versus negative) such as 8.7 m versus 1.2 m (p < .001), 8.8 m versus 3.0 m (p < .05) and 10.2 m versus 1.2 m (p < .001), respectively. In conclusion, our vaccination therapy was feasible and effective in this cohort of patients.

Construction and analysis of circular RNA molecular regulatory networks in clear cell renal cell carcinoma.

Increasing evidence has indicated that circular (circ)RNAs participate in carcinogenesis; however, the specific regulatory mechanisms underlying the effects of circRNAs, microRNAs (miRNAs/miRs) and genes on the development of clear cell renal cell carcinoma (CCRCC) remain unclear. In the present study, RNA microarray data from CCRCC tissues and control samples were downloaded from the Gene Expression Omnibus and The Cancer Genome Atlas, in order to identify significantly dysregulated circRNAs, miRNAs and genes. The Cancer-Specific circRNA Database was used to explore the interactions between miRNAs and circRNAs, whereas TargetScan and miRDB were employed to predict the mRNA targets of miRNAs. Functional enrichment and prognostic analyses were conducted in R. The results revealed that 324 circRNAs were downregulated, whereas 218 circRNAs were upregulated in cancer. In addition, a circRNA-miRNA-mRNA interaction network was constructed. Gene Ontology analysis of the upregulated genes revealed that these genes were enriched in biological processes, including ‘flavonoid metabolic process’, ‘cellular glucuronidation’ and ‘T cell activation’. The downregulated genes were mainly enriched in biological processes, such as ‘nephron development’, ‘kidney development’ and ‘renal system development’. The hub genes, including membrane palmitoylated protein 7, aldehyde dehydrogenase 6 family member A1, transcription factor AP-2α, collagen type IV α 4 chain, nuclear receptor subfamily 3 group C member 2, plasminogen, Holliday junction recognition protein, claudin 10, kinesin family member 18B and thyroid hormone receptor β, and the hub miRNAs, including miR-21-3p, miR-155-3p, miR-144-3p, miR-142-5p, miR-875-3p, miR-885-3p, miR-3941, miR-224-3p, miR-584-3p and miR-138-1-3p, were significantly associated with CCRCC survival. In conclusion, these results suggested that the significantly dysregulated circRNAs, miRNAs and genes identified in this study may be considered potential biomarkers of the carcinogenesis of CCRCC and the survival of patients with this disease.

Upregulation of Holliday junction recognition protein predicts poor prognosis and biochemical recurrence in patients with prostate cancer.

Abnormal expression of Holliday junction recognition protein (HJURP) in several types of tumor cells plays a vital role in the formation and progression of tumors. Few studies have investigated the role of HJURP in prostate cancer (PCa). The aim of this study was to analyze the expression levels of HJURP in PCa and to establish the association with clinicopathological data. Reverse transcription quantitative polymerase chain reaction and immunohistochemical analysis were used to detect the expression levels of HJURP in benign and PCa prostate tissues. The Taylor dataset was statistically analyzed to determine if HJURP expression levels were associated with PCa clinicopathological data. HJURP was overexpressed in PCa tissues compared with benign prostate tissues. Statistical analysis of the Taylor dataset indicated that upregulation of HJURP was significantly associated with positive prostate-specific antigen (PSA) levels (P=0.004), high Gleason score (P=0.005), advanced pathological stage (P=0.007), metastasis (P<0.001) and PSA failure (P<0.001). Higher HJURP mRNA expression levels were significantly associated with shorter biochemical recurrence (BCR)-free survival (P<0.001). To the best of our knowledge, this study is the first report of HJURP upregulation in PCa tissues. Upregulation of HJURP may predict BCR-free survival and HJURP may be an oncogene that impacts the prognosis of patients with PCa.

HJURP knockdown disrupts clonogenic capacity and increases radiation-induced cell death of glioblastoma cells.

The Holliday Junction-Recognition Protein (HJURP) was reported as overexpressed in several cancers and also strongly correlated with poor prognosis of patients, especially in glioblastoma (GBM), the most common and deadly type of primary brain tumor. HJURP is responsible for loading the histone H3 variant-the Centromeric Protein A (CENP-A)-at the centromeres in a cell cycle-regulated manner, being required for proper chromosome segregation. Here we investigated HJURP association with survival and radioresistance of different GBM cell lines. HJURP knockdown compromised the clonogenic capacity and severely impaired survival of five distinct GBM cells, while nontumor astrocytes were not affected. U251MG cells showed a robust cell cycle arrest in G2/M phases followed by a drastic increment in cell death after HJURP silencing, while U138MG and U343MG cell lines presented augmented senescence with a comparable increase in cell death. Importantly, we verified that the impact on cell cycle dynamics and clonogenic survival were associated with loss CENP-A at the centromeres. Moreover, radiation resistance was also impacted by HJURP modulation in several GBM cell lines. U87MG, T98G, U138MG, and U343MG cells were all sensitized to ionizing radiation after HJURP reduction. These data reinforce the requirement of HJURP for proliferative capacity and radioresistance of tumor cells, underlining its potential as a promising therapeutic target for GBM.

HJURP Promotes Epithelial-to-Mesenchymal Transition via Upregulating SPHK1 in Hepatocellular Carcinoma

Holliday Junction Recognition Protein (HJURP) is involved in various cancers including hepatocellular carcinoma (HCC). Current studies have showed that HJURP is correlated with HCC proliferation. However, the role of HJURP in HCC Epithelial-to-Mesenchymal Transition remains unclear. In this study, we found that HJURP knockdown significantly reduced the migration and invasion abilities of HCC cells both in vivo and in vitro by interacting with Sphingosine kinase1 (SPHK1). Conversely, HJURP overexpression enhanced these biological abilities. Moreover, high HJURP expression is related to poor prognosis of HCC patients. In conclusion, HJURP plays an important role in tumor metastasis by upregulating SPHK1. And high HJURP expression may predict a lower disease-free survival rate and higher possibility of microvascular invasion in HCC patients.

Holliday junction recognition protein interacts with and specifies the centromeric assembly of CENP-T

The centromere is an evolutionarily conserved eukaryotic protein machinery essential for precision segregation of the parental genome into two daughter cells during mitosis. Centromere protein A (CENP-A) organizes the functional centromere via a constitutive centromere-associated network composing the CENP-T complex. However, how CENP-T assembles onto the centromere remains elusive. Here we show that CENP-T binds directly to Holliday junction recognition protein (HJURP), an evolutionarily conserved chaperone involved in loading CENP-A. The binding interface of HJURP was mapped to the C terminus of CENP-T. Depletion of HJURP by CRISPR-elicited knockout minimized recruitment of CENP-T to the centromere, indicating the importance of HJURP in CEPN-T loading. Our immunofluorescence analyses indicate that HJURP recruits CENP-T to the centromere in S/G2 phase during the cell division cycle. Significantly, the HJURP binding-deficient mutant CENP-T6L failed to locate to the centromere. Importantly, CENP-T insufficiency resulted in chromosome misalignment, in particular chromosomes 15 and 18. Taken together, these data define a novel molecular mechanism underlying the assembly of CENP-T onto the centromere by a temporally regulated HJURP-CENP-T interaction.

Anti-cancer Immunotherapy Epitope-peptides Vaccination in Patients with Refractory/Persistent Disease of Cervical Cancer and Ovarian Cancer (Phase 1 Studies)

Despite the improvement of treatments, refractory or chemotherapy resistant ovarian and cervical cancers have been still incurable. In such tumors, the actionable salvage gene-pathways of up-regulating lung cancer 10 (URLC10), hypoxia inducible factor (HIF) and its core protein HIG2- tumor growth factor beta (TGF beta)- the Caenorhabditis elegans SMA (small worm phenotype) and Drosophila Mothers Against Decapentaplegic (SMAD), maternal embryonic leucine zipper kinase (MELK)- forkhead box M1 (FOXM1) which induces and stimulates stathmin concerning cell (vascular endothelial cell and tumor cell) migration and counter pathway of P53, and holliday junction recognition protein (HJURP)-histone H3-like centromeric protein A (CEMPA)-Histone, which play important roles in tumor proliferation, metastasis and cell cycling. They had been shifted from original driver gene such as Ras-MAPK or PIK3CA-mTOR. Furthermore, tumor specific micro-environmental factors such as vascular endothelial growth factor (VEGF) receptors facilitate tumor new-angiogenesis, invasion and metastasis, as well. We found human leukocyte antigen (HLA)-A*2402 and 0201 restricted epitope neo-antigens or, epitope peptides of VEGF receptor 1 and 2, using micro-cDNA assay form clinical samples. The peptides consisted in nine to eleven mer peptides, which were presented by HLA (major histocompatibility 1) on cell membrane. We administered the multiple peptides subcutaneously as vaccination and it activated intrinsic cell immune system of cytotoxic T cell (CTL). We conducted a phase 1/2 study of those peptides vaccine (PV) cocktails to elucidate their toxicity profiles and efficacy from 4 June 2010 to Jan 2013 for phase 1 studies, and subsequently continued phase 2 studies at outpatient’s clinic of our hospital. PV were administered at a dose of 1mg of each peptide with MONTANIDE*ISA51 (SEPPIC Co. Ltd, France). Enrollees were obtained written informed consent after our IRB approval on 3 June 2010. In results, no major adverse events were seen except dermatologic reactions at injection site. One patient showed complete response, two showed partial response and 10 showed stable disease out of 22 evaluable patients. Median overall survival was 5 months and 9 months in HLA-A2402 and 0201 group, respectively. In conclusion, these findings suggest the peptides cocktail vaccines were safe and applicable for advanced/recurrent OC.

Nuclear Actin Polymerized by mDia2 Confines Centromere Movement during CENP-A Loading

SummaryCentromeres are specialized chromosomal regions epigenetically defined by the histone H3 variant centromere protein A (CENP-A). CENP-A needs to be replenished in every cell cycle, but how new CENP-A is stably incorporated into centromeric chromatin remains unclear. We have discovered that a cytoskeletal protein, diaphanous formin mDia2, is essential for the stable incorporation of new CENP-A proteins into centromeric nucleosomes. Here we report that mDia2-mediated formation of dynamic and short nuclear actin filaments in G1 nucleus is required to maintain CENP-A levels at the centromere. Importantly, mDia2 and nuclear actin are required for constrained centromere movement during CENP-A loading, and depleting nuclear actin or MgcRacGAP, which lies upstream of mDia2, extends centromeric association of the CENP-A loading chaperone Holliday junction recognition protein (HJURP). Our findings thus suggest that nuclear actin polymerized by mDia2 contributes to the physical confinement of G1 centromeres so that HJURP-mediated CENP-A loading reactions can be productive, and centromere's epigenetic identity can be stably maintained.

Phase I Study of Multiple Epitope Peptide Vaccination in Patients With Recurrent or Persistent Cervical Cancer

Cancer immunotherapy has now been established as a leading standard therapeutic option in a subset of patients with cancer. In this study, we conducted a phase I dose-escalation trial using a mixture of 5 peptides to vaccinate cervical cancer patients with HLA-A*2402. The primary endpoints were safety and determination of a recommended vaccine dose, and the secondary endpoints were evaluations of immunologic responses and clinical efficacy. All patients had recurrent or persistent disease and had failed to respond to or were intolerant to prior standard chemotherapy. Peptides derived from forkhead box protein M1 (FOXM1), maternal embryonic leucine zipper kinase (MELK), Holliday junction-recognition protein, and vascular endothelial growth factor receptors 1 and 2 were administered to 9 patients in a 3 patient-cohort design, with doses of 0.5, 1, or 2 mg of each of the individual peptides in a mixture with incomplete Freund's adjuvant. The major adverse events were anemia and injection site reactions, which were seen in 77.8% (7/9) and 66.7% (6/9) of patients, respectively. Grade 3 anemia was observed in 1 patient. No dose-limiting toxicity of the vaccine was observed. Seven (78%) patients achieved stable disease, and the median progression-free survival was 3.3 months (102 d). Interferon-γ enzyme-linked immunospot assays for each of the 5 antigens showed that 8 (89%) and 7 (78%) patients had high T-cell responses to FOXM1 and MELK, respectively. In conclusion, we demonstrated that this 5-peptide vaccine was tolerable, and that FOXM1 and MELK could be promising targets for immunotherapy in patients with cervical cancer.

Essential role for centromeric factors following p53 loss and oncogenic transformation.

In mammals, centromere definition involves the histone variant CENP-A (centromere protein A), deposited by its chaperone, HJURP (Holliday junction recognition protein). Alterations in this process impair chromosome segregation and genome stability, which are also compromised by p53 inactivation in cancer. Here we found that CENP-A and HJURP are transcriptionally up-regulated in p53-null human tumors. Using an established mouse embryonic fibroblast (MEF) model combining p53 inactivation with E1A or HRas-V12 oncogene expression, we reproduced a similar up-regulation of HJURP and CENP-A. We delineate functional CDE/CHR motifs within the Hjurp and Cenpa promoters and demonstrate their roles in p53-mediated repression. To assess the importance of HJURP up-regulation in transformed murine and human cells, we used a CRISPR/Cas9 approach. Remarkably, depletion of HJURP leads to distinct outcomes depending on their p53 status. Functional p53 elicits a cell cycle arrest response, whereas, in p53-null transformed cells, the absence of arrest enables the loss of HJURP to induce severe aneuploidy and, ultimately, apoptotic cell death. We thus tested the impact of HJURP depletion in pre-established allograft tumors in mice and revealed a major block of tumor progression in vivo. We discuss a model in which an "epigenetic addiction" to the HJURP chaperone represents an Achilles' heel in p53-deficient transformed cells.

Silencing of HJURP induces dysregulation of cell cycle and ROS metabolism in bladder cancer cells via PPARγ-SIRT1 feedback loop.

Holliday Junction Recognition Protein (HJURP) is a centromeric histone chaperone involving in de novo histone H3 variant CenH3 (CENP-A) recruitment. Our transcriptome and in vivo study revealed that HJURP is significantly upregulated in bladder cancer (BCa) tissues at both mRNA and protein levels. Knockdown of HJURP inhibited proliferation and viability of BCa cell lines revealed by CCK-8, colony formation and Ki-67-staining assays, and induced apoptosis and reactive oxygen species (ROS) production, as well as triggered cell cycle arrest at G0/G1 phase possibly via loss of CENP-A. Interestingly, in the HJURP-reduced BCa cells the levels of PPARγ and acetylated-p53 were increased, while the ratio of phosphorylated/total SIRT1 protein was decreased. Moreover, after treatment of the BCa cells using PPARγ antagonist (GW9662) and SIRT1 agonist (resveratrol, RSV) respectively, thee phenotypes of cell cycle arrest, increased ROS production and inhibited proliferation rate were all rescued. Taken together, our results suggested that HJURP might regulate proliferation and apoptosis via the PPARγ-SIRT1 negative feedback loop in BCa cells.

Differential Binding Partners of the Mis18α/β YIPPEE Domains Regulate Mis18 Complex Recruitment to Centromeres.

The Mis18 complex specifies the site of new CENP-A nucleosome assembly by recruiting the CENP-A-specific assembly factor HJURP (Holliday junction recognition protein). The human Mis18 complex consists of Mis18α, Mis18β, and Mis18 binding protein 1 (Mis18BP1/hsKNL2). Although Mis18α and Mis18β are highly homologous proteins, we find that their conserved YIPPEE domains mediate distinct interactions that are essential to link new CENP-A deposition to existing centromeres. We find that Mis18α directly interacts with the N terminus of Mis18BP1, whereas Mis18β directly interacts with CENP-C during G1 phase, revealing that these proteins have evolved to serve distinct functions in centromeres of higher eukaryotes. The N terminus of Mis18BP1, containing both the Mis18α and CENP-C binding domains, is necessary and sufficient for centromeric localization. Therefore, the Mis18 complex contains dual CENP-C recognition motifs that are combinatorially required to generate robust centromeric localization that leads to CENP-A deposition.

Divergence patterns of genic copy number variation in natural populations of the house mouse (Mus musculus domesticus) reveal three conserved genes with major population-specific expansions.

Copy number variation represents a major source of genetic divergence, yet the evolutionary dynamics of genic copy number variation in natural populations during differentiation and adaptation remain unclear. We applied a read depth approach to genome resequencing data to detect copy number variants (CNVs) ≥1 kb in wild-caught mice belonging to four populations of Mus musculus domesticus. We complemented the bioinformatics analyses with experimental validation using droplet digital PCR. The specific focus of our analysis is CNVs that include complete genes, as these CNVs could be expected to contribute most directly to evolutionary divergence. In total, 1863 transcription units appear to be completely encompassed within CNVs in at least one individual when compared to the reference assembly. Further, 179 of these CNVs show population-specific copy number differences, and 325 are subject to complete deletion in multiple individuals. Among the most copy-number variable genes are three highly conserved genes that encode the splicing factor CWC22, the spindle protein SFI1, and the Holliday junction recognition protein HJURP. These genes exhibit population-specific expansion patterns that suggest involvement in local adaptations. We found that genes that overlap with large segmental duplications are generally more copy-number variable. These genes encode proteins that are relevant for environmental and behavioral interactions, such as vomeronasal and olfactory receptors, as well as major urinary proteins and several proteins of unknown function. The overall analysis shows that genic CNVs contribute more to population differentiation in mice than in humans and may promote and speed up population divergence.

Cdk1: conductor of CENP-A symphony orchestra

The inheritance of human genome through mitosis is precisely regulated by a series of tightly orchestrated events such as chromosome condensation, bi-orientated spindle formation, chromosome congression, segregation and cytokinesis. Chromosome movements during mitosis are governed by dynamic interactions of spindle microtubules with a specialized chromosome domain called centromere. Centromere provides the platform for the assembly of a protein machine named kinetochore that orchestrates accurate interaction between chromosomes and spindle microtubules during cell division. In eukaryotic organisms, the centromere is epigenetically specified by nucleosomes containing a unique histone H3 variant CENP-A, with the exception of budding yeast [1]. CENP-A orthologues present in all known eukaryotes. It is the most important epigenetic marker of centromere [2] and is required for the correct localization of most centromereand kinetochorerelated proteins [2]. CENP-A is the epigenetic marker to maintain and propagate the centromere identity, which assembles into a nucleosome together with histones H4, H2A and H2B without any known specific selection of DNA sequence [1, 2]. Centromeric chromatin is interspersed with CENP-A-nucleosomes and histone H3-containing nucleosomes, and it forms a repressive heterochromatin structure through the concerted action of various epigenetic mechanisms including histone methylation [2]. The plasticity of centromeric chromatin is crucial for the assembly of a large protein machine kinetochore that links chromosome to the spindle microtubule for mitotic segregation. Interestingly, recruitment of new CENP-A to centromere is not contemporaneous with replication of centromeric DNA in S phase. Rather, it is restricted to a brief interval in G1 immediately following mitosis in human cells [3] and slightly earlier in anaphase in the rapidly dividing Drosophila syncytial embryo. The assembly of new CENP-A nucleosomes in early G1 is dependent on CENP-C, Mis18 complex and HJURP (Holliday junction recognition protein). Among these proteins, HJURP is of great importance for CENP-A deposition as it is a CENPA-specific histone chaperone. HJURP interacts directly with CENP-A and histone H4 and recruits CENP-A to the centromere in a cell cycle-dependent manner. HJURP contains an approximately 80-amino acid CENP-A-binding domain (CBD) at the N terminus that is essential and sufficient for binding CENP-A. In addition, CENP-A contains a typical C-terminal histone fold domain, known as the centromere targeting domain (CATD), is required for interaction with HJURP [4]. In yeast, the HJURP homolog Scm3 is required for the deposition of centromeric nucleosomes. Down-regulation of HJURP impairs the deposition of newly synthesized CENP-A in mammalian cells. As a molecular chaperone of CENP-A, HJURP forms prenucleosomal complex with CENP-A/H4 heterodimer via its highly conserved Scm3 domain [4]. Recent studies show that mitotic master kinase Cdk1 phosphorylates HJURP at its C-terminal domain by which negatively regulates HJURP interaction with Mis18b and subsequent inhibits CENP-A loading in early mitosis when Cdk1 activity is high [5]. In fact, inhibition of Cdk1 resulted in premature C. Jin X. Yao (&) Anhui Key Laboratory for Cellular Dynamics & Chemical Biology, University of Science and Technology of China, Hefei 230027, China e-mail: yaoxb@ustc.edu.cn

Mitotic Regulator Mis18β Interacts with and Specifies the Centromeric Assembly of Molecular Chaperone Holliday Junction Recognition Protein (HJURP)

The centromere is essential for precise and equal segregation of the parental genome into two daughter cells during mitosis. CENP-A is a unique histone H3 variant conserved in eukaryotic centromeres. The assembly of CENP-A to the centromere is mediated by Holliday junction recognition protein (HJURP) in early G1 phase. However, it remains elusive how HJURP governs CENP-A incorporation into the centromere. Here we show that human HJURP directly binds to Mis18β, a component of the Mis18 complex conserved in the eukaryotic kingdom. A minimal region of HJURP for Mis18β binding was mapped to residues 437-460. Depletion of Mis18β by RNA interference dramatically impaired HJURP recruitment to the centromere, indicating the importance of Mis18β in HJURP loading. Interestingly, phosphorylation of HJURP by CDK1 weakens its interaction with Mis18β, consistent with the notion that assembly of CENP-A to the centromere is achieved after mitosis. Taken together, these data define a novel molecular mechanism underlying the temporal regulation of CENP-A incorporation into the centromere by accurate Mis18β-HJURP interaction.

New biomarkers to predict the evolution of in situ breast cancers.

Background. Genomic studies have shown that gene expression profiles are similar in in situ (CIS) and invasive breast cancers, suggesting that several biofunctional modifications of the transformation process occur before or during the development of CIS lesion. Methods. We investigated 3 biomarkers in 44 patients with CIS: TG2 (transglutaminase 2), HJURP (Holliday junction recognition protein), and HIF-1α (hypoxia inducible factor-1 alpha). Results. TG2 was more highly expressed than the other two markers and significantly more so in stromal than in tumor cells. HIF-1α evaluation showed a higher expression in both tumor and stromal cells in patients with relapsed G3 tumors, indicating a potential role of this marker in CIS evolution. A greater than sevenfold higher risk of relapse (P = 0.050) was observed in patients highly expressing HJURP in stroma and a tenfold higher recurrence risk (P = 0.026) was seen in those with a higher stromal HIF-1α expression. An important increase in risk accuracy (AUC 0.80) was obtained when HIF-1α and HJURP were evaluated together. Conclusions. Despite the limited number of relapsed patients, we formulated some hypotheses on the factors responsible for malignant evolution and recurrence which are now being tested in a large case series with a longer follow-up.

Dimerization of the CENP-A assembly factor HJURP is required for centromeric nucleosome deposition

The epigenetic mark of the centromere is thought to be a unique centromeric nucleosome that contains the histone H3 variant, centromere protein-A (CENP-A). The deposition of new centromeric nucleosomes requires the CENP-A-specific chromatin assembly factor HJURP (Holliday junction recognition protein). Crystallographic and biochemical data demonstrate that the Scm3-like domain of HJURP binds a single CENP-A-histone H4 heterodimer. However, several lines of evidence suggest that HJURP forms an octameric CENP-A nucleosome. How an octameric CENP-A nucleosome forms from individual CENP-A/histone H4 heterodimers is unknown. Here, we show that HJURP forms a homodimer through its C-terminal domain that includes the second HJURP_C domain. HJURP exists as a dimer in the soluble preassembly complex and at chromatin when new CENP-A is deposited. Dimerization of HJURP is essential for the deposition of new CENP-A nucleosomes. The recruitment of HJURP to centromeres occurs independent of dimerization and CENP-A binding. These data provide a mechanism whereby the CENP-A pre-nucleosomal complex achieves assembly of the octameric CENP-A nucleosome through the dimerization of the CENP-A chaperone HJURP.

A phase I/II study of multiple peptides cocktail vaccine for advanced/recurrent ovarian cancer.

TPS3116 Background: Despite improvement in chemotherapy including the molecular targeting agents, advanced or recurrent ovarian cancer (A/ROC) are still incurable. Some tumor associated proteins, hypoxia-inducible protein 2 (HIG2), and tumor related vascular endothelial growth factor receptor 1,2 (VEGFR1,VEGFR2) were found to be candidates as new targets, and their epitope peptides have been shown to have the ability to induce specific cytotoxic T lymphocyte ( CTL) responses. We conducted a phase I/II study of these peptides cocktail vaccine (PCV) in patients with A/ROC in order to evaluate toxicity, immunological response, and tumor response. Methods: 23 patients positive for human leukocyte antigen (HLA)-A2402 (A24) and 15 patients with HLA-A0201(A02) were enrolled in this study. Enrollment is still on going up to 30 evaluable patients in each group. PCV for A24 comprises FOXM1(Forkhead Box M1), MELK(maternal embryonic leucine zipper kinase), HJURP(Holliday junction recognition protein), VEGFR1 and VEGFR2. As for A02- PCV comprises HIG2, VEGFR1and VEGFR2. Cocktails were made at a dose of 1mg of each peptide with GMP grade-adjuvant, MONTANIDE ISA51. Vaccination schedule included weekly subcutaneous administration for first 12 weeks, then bi-weekly administration for next 16 weeks, then further vaccination was done monthly for 8 times and 3-6 months as a maintenance step according to patient’s need. Pre- and post-vaccination blood samples were obtained from the patients for toxicity assessment and immunological evaluation. Clinical responses were evaluated every three months by RECIST v 1.1. Results: PCV were generally well tolerated with no major adverse events, and most of the patients developed specific CTL responses. One patient showed complete response, two showed partial response and 10 showed stable disease in 22 evaluable patients. At the time of the analysis, median overall survival was 5 months (from 30 to 623 days) and 9 months(from 54 to 921 days),in A24 and A02, respectively. 11 patients remained alive with median follow up of 9 months. Conclusions: These findings suggest these peptides cocktail vaccines are safe and applicable for advanced/recurrent ovarian cancer. Clinical trial information: UMIN000003862, UMIN000003860.

HJURP Regulates Cellular Senescence in Human Fibroblasts and Endothelial Cells Via a p53-Dependent Pathway

Holliday junction recognition protein (HJURP), a centromere protein-A (CENP-A) histone chaperone, mediates centromere-specific assembly of CENP-A nucleosome, contributing to high-fidelity chromosome segregation during cell division. However, the role of HJURP in cellular senescence of human primary cells remains unclear. We found that the expression levels of HJURP decreased in human dermal fibroblasts and umbilical vein endothelial cells in replicative or premature senescence. Ectopic expression of HJURP in senescent cells partially overcame cell senescence. Conversely, downregulation of HJURP in young cells led to premature senescence. p53 knockdown, but not p16 knockdown, abolished senescence phenotypes caused by HJURP reduction. These data suggest that HJURP plays an important role in the regulation of cellular senescence through a p53-dependent pathway and might contribute to tissue or organismal aging and protection of cellular transformation.