Roles of Polycomb group proteins Enhancer of zeste (E(z)) and Polycomb (Pc) during metamorphosis and larval leg regeneration in the flour beetle Tribolium castaneum.

Many organisms both undergo dramatic morphological changes during post-embryonic development and also regenerate lost structures, but the roles of epigenetic regulators in such processes are only beginning to be understood. In the present study, the functions of two histone modifiers were examined during metamorphosis and larval limb regeneration in the red flour beetle Tribolium castaneum. Polycomb (Pc), a member of Polycomb repressive complex 1 (PRC1), and Enhancer of zeste (E(z)), a member of Polycomb repressive complex 2 (PRC2), were silenced in larvae using RNA interference. In the absence of Pc, the head appendages of adults transformed into a leg-like morphology, and the legs and wings assumed a metathoracic identity, indicating that Pc acts to specify proper segmental identity. Similarly, silencing of E(z) led to homeotic transformation of legs and wings. Additional defects were also observed in limb patterning as well as eye morphogenesis, indicating that PcG proteins play critical roles in imaginal precursor cells. In addition, larval legs and antennae failed to re-differentiate when either Pc or E(z) was knocked down, indicating that histone modification is necessary for proper blastema growth and differentiation. These findings indicate that PcG proteins play extensive roles in postembryonic plasticity of imaginal precursor cells.

The prognostic significance of BMI1 expression in invasive breast cancer is dependent on its molecular subtypes.

PURPOSE: BMI1, which is a major component of the polycomb group complex 1, is an essential epigenetic repressor of multiple regulatory genes and has been identified as a cancer stem cell (CSC) marker in several cancers. However, its role in breast cancer (BC) remains to be defined. In this study, we have evaluated the prognostic significance of BMI1 among the different molecular subtypes and assessed its association with other breast CSC markers (BCSC). MATERIAL AND METHOD: BMI1 copy number and mRNA was assessed in large and well-characterised cohorts of early-stage BC patients [METABRIC (n = 1980) and the Bc-GenExMiner (n = 9616) databases]. BMI1 protein expression was assessed using tissue microarray and immunohistochemistry in a cohort of 870 invasive BC patients with long-term outcome data and the expression of a panel of BCSC markers was monitored. RESULT: BMI1 expression, prognostic significance and its association with BCSC markers were differed between molecular classes. In the luminal oestrogen receptor-positive (ER+) BC, BMI1 showed significantly higher expression compared to ER- tumours. BMI1 showed positive correlation with favourable prognostic features and it was negatively associated with the expression of key BCSC markers (ALDH1A1, CD24, CD44, CD133, SOX10 and SOX9). High expression of BMI1 was associated with longer breast cancer-specific survival (BCSS) independent of other prognostic variables. In the basal triple negative BC subtype, BMI1 expression showed positive association with CD133 and SOX10 and it was significantly associated with shorter BCSS. CONCLUSION: High BMI1 expression is associated with clinicopathological variables and outcome in BC. However, this association is dependent on the molecular subtypes. Further functional assessment to detect its underlying mechanistic roles in BC subtypes is warranted.

The expression and prognostic value of stem cell markers Bmi-1, HESC5:3, and HES77 in human papillomavirus-positive and -negative oropharyngeal squamous cell carcinoma.

Human papillomavirus is detected in over 50% of oropharyngeal squamous cell carcinomas. Human papillomavirus-positive oropharyngeal squamous cell carcinomas differ from human papillomavirus-negative tumors, and both expression patterns are classified as distinct entities. The Bmi-1 oncogene is a well-known member of the mammalian polycomb-group family. HESC5:3 and HES77 are newly developed monoclonal antibodies produced against undifferentiated embryonic stem cells. Our aim was to explore their roles in both human papillomavirus-positive and -negative oropharyngeal squamous cell carcinomas. Our cohort comprised 202 consecutive oropharyngeal squamous cell carcinoma patients diagnosed and treated with curative intent. We used tissue microarray tumor blocks to study the immunohistochemical expression of Bmi-1, HESC5:3, and HES77. We compared the expressions of these stem cell markers with p16 immunoexpression and human papillomavirus status, as well as with other characteristics of the tumor, and with patients' clinical data and follow-up data. Human papillomavirus- and p16-positive tumors expressed less Bmi-1 and more HESC5:3 than the negative tumors. HES77 expression was high in human papillomavirus-positive oropharyngeal squamous cell carcinoma, but it did not correlate with p16 positivity. In our multivariable model, Bmi-1 and HESC5:3 were still associated with human papillomavirus, but the association between human papillomavirus and HES77 remained absent. In conclusion, Bmi-1, HESC5:3, and HES77 may have a different role in human papillomavirus-positive and human papillomavirus-negative tumors. There was no correlation between Bmi-1, HESC5:3, and HES77 expression and survival.

Bmi1+ Progenitor Cell Dynamics in Murine Cornea During Homeostasis and Wound Healing.

The outermost layer of the eye, the cornea, is renewed continuously throughout life. Stem cells of the corneal epithelium reside in the limbus at the corneal periphery and ensure homeostasis of the central epithelium. However, in young mice, homeostasis relies on cells located in the basal layer of the central corneal epithelium. Here, we first studied corneal growth during the transition from newborn to adult and assessed Keratin 19 (Krt19) expression as a hallmark of corneal maturation. Next, we set out to identify a novel marker of murine corneal epithelial progenitor cells before, during and after maturation, and we found that Bmi1 is expressed in the basal epithelium of the central cornea and limbus. Furthermore, we demonstrated that Bmi1+ cells participated in tissue replenishment in the central cornea. These Bmi1+ cells did not maintain homeostasis of the cornea for more than 3 months, reflecting their status as progenitor rather than stem cells. Finally, after injury, Bmi1+ cells fueled homeostatic maintenance, whereas wound closure occurred via epithelial reorganization. Stem Cells 2018;36:562-573.

A cellular chemical probe targeting the chromodomains of Polycomb repressive complex 1.

We report the design and characterization of UNC3866, a potent antagonist of the methyllysine (Kme) reading function of the Polycomb CBX and CDY families of chromodomains. Polycomb CBX proteins regulate gene expression by targeting Polycomb repressive complex 1 (PRC1) to sites of H3K27me3 via their chromodomains. UNC3866 binds the chromodomains of CBX4 and CBX7 most potently, with a K(d) of ∼100 nM for each, and is 6- to 18-fold selective as compared to seven other CBX and CDY chromodomains while being highly selective over >250 other protein targets. X-ray crystallography revealed that UNC3866's interactions with the CBX chromodomains closely mimic those of the methylated H3 tail. UNC4195, a biotinylated derivative of UNC3866, was used to demonstrate that UNC3866 engages intact PRC1 and that EED incorporation into PRC1 is isoform dependent in PC3 prostate cancer cells. Finally, UNC3866 inhibits PC3 cell proliferation, consistent with the known ability of CBX7 overexpression to confer a growth advantage, whereas UNC4219, a methylated negative control compound, has negligible effects.

Transcriptional read-through is not sufficient to induce an epigenetic switch in the silencing activity of Polycomb response elements.

In Drosophila, Polycomb (PcG) and Trithorax (TrxG) group proteins are assembled on Polycomb response elements (PREs) to maintain tissue and stage-specific patterns of gene expression. Critical to coordinating gene expression with the process of differentiation, the activity of PREs can be switched "on" and "off." When on, the PRE imposes a silenced state on the genes in the same domain that is stably inherited through multiple rounds of cell division. When the PRE is switched off, the domain is in a state permissive for gene expression that can be stably inherited. Previous studies have suggested that a burst of transcription through a PRE sequence displaces PcG proteins and provides a universal mechanism for inducing a heritable switch in PRE activity from on to off; however, the evidence favoring this model is indirect. Here, we have directly tested the transcriptional read-through mechanism. Contrary to previous suggestions, we show that transcription through the PRE is not sufficient for inducing an epigenetic switch in PRE activity. In fact, even high levels of continuous transcription through a PRE fails to dislodge the PcG proteins, nor does it remove repressive histone marks. Our results indicate that other mechanisms involving adjacent DNA regulatory elements must be implicated in heritable switch of PRE activity.

Expression of stem cell factors in the adult sea cucumber digestive tube.

Homeostatic cell turnover has been extensively characterized in mammals. In their adult tissues, lost or aging differentiated cells are replenished by a self-renewing cohort of stem cells. The stem cells have been particularly well studied in the intestine and are clearly identified by the expression of marker genes including Lgr5 and Bmi1. It is, however, unknown if the established principles of tissue renewal learned from mammals would be operating in non-mammalian systems. Here, we study homeostatic cell turnover in the sea cucumber digestive tube, the organ with high tissue plasticity even in adult animals. Both the luminal epithelium and mesothelium express orthologs of mammalian Lgr5 and Bmi1. However, unlike in mammals, there is no segregation of these positively labeled cells to specific regions in the luminal epithelium, where most of the cell proliferation would take place. In the mesothelium, the cells expressing the stem cell markers are tentatively identified as peritoneocytes. There are significant differences among the five anatomical gut regions in cell renewal dynamics and stem factor expression. The cloaca differs from the rest of the digestive tube as the region with the highest expression of the Lgr5 ortholog, lowest level of Bmi1 and the longest retention of BrdU-labeled cells.

MicroRNA-139-5p inhibits bladder cancer proliferation and self-renewal by targeting the Bmi1 oncogene.

MiR-139-5p has been reported to be overexpressed in many types of cancers, but its role in bladder cancer has not been elucidated yet. Here, we report that miR-139-5p functions as a tumor suppressor in bladder cancer and inhibits the cancer stem cell self-renewal by targeting Bmi1 directly. We found that miR-139-5p expression was significantly downregulated in the bladder cancer specimens compared with that in adjacent normal tissues. In vitro, restoration of miR-139-5p expression significantly inhibited the proliferation of bladder cancer cells. Mechanism analysis revealed that miR-139-5p could decrease Bmi1 protein levels by binding to the 3' untranslated region of Bmi1 messenger RNA. Stem cell-related proteins such as c-MYC, NANOG, OCT4, and KLF4 and signaling pathways such as Wnt signaling were suppressed by restoration of miR-139-5p in bladder cancer cells. In addition, miR-139-5p expression also blocked self-renewal of bladder cancer stem cells by inhibiting Bmi1. In summary, our study supports that miR-139-5p acts as a tumor suppressor in bladder cancer development and suppresses cancer stem cell property of bladder cancer. Our study also suggests that miR-139-5p has the potential to be used as a therapeutic molecule for bladder cancer treatment.

Spatiotemporal Patterns of RING1 Expression after Rat Spinal Cord Injury.

Ring finger protein 1 (RING1) is a RING domain characterized protein belonging to the RING finger family. It is an E3 ubiquitin-protein ligase that mediated monoubiquitination of histone H2A and the core component of PRC1 complex, which is the repressive multiprotein complex of Polycomb group (PcG). Previous studies showed the important tumorigenic role of RING1 via promoting cell proliferation and the crucial function in maintaining transcriptional program stability during development. However, its mechanism for spinal cord injury (SCI) is still unknown. In our research, we established an acute SCI model in adult rats and studied the expression and function profiles of RING1. RING1 protein level detected by western blot peaked at day 3 after trauma and then decreased gradually. Immunohistochemistry showed the increase of RING1 expression displayed in the white matter more obviously than in the gray matter. Furthermore, increased co-expression of RING1 and GFAP confirmed activated astrocytes in injured spinal cord via double immunofluorescence staining. Meanwhile, we also found the co-localization of PCNA, a famous marker of proliferative cells, with RING1 and GFAP, which indicated RING1 might play a role in astrocyte proliferation after SCI. In vitro studies, RING1 protein level in C6 cells increased after LPS challenge and RING1 was required for astrocyte proliferation and activation induced by LPS. In summary, we took a new insight into the function of RING1 in the cellular and molecular mechanism underlying the pathophysiology of SCI.

Epigenetic silencing of miR-218 by the lncRNA CCAT1, acting via BMI1, promotes an altered cell cycle transition in the malignant transformation of HBE cells induced by cigarette smoke extract.

Cigarette smoking is the strongest risk factor for the development of lung cancer, the leading cause of cancer-related deaths. However, the molecular mechanisms leading to lung cancer are largely unknown. A long-noncoding RNA (lncRNA), CCAT1, regarded as cancer-associated, has been investigated extensively. Moreover, the molecular mechanisms of lncRNAs in regulation of microRNAs (miRNAs) induced by cigarette smoke remain unclear. In the present investigation, cigarette smoke extract (CSE) caused an altered cell cycle and increased CCAT1 levels and decreased miR-218 levels in human bronchial epithelial (HBE) cells. Depletion of CCAT1 attenuated the CSE-induced decreases of miR-218 levels, suggesting that miR-218 is negatively regulated by CCAT1 in HBE cells exposed to CSE. The CSE-induced increases of BMI1 levels and blocked by CCAT1 siRNA were attenuated by an miR-218 inhibitor. Moreover, in CSE-transformed HBE cells, the CSE-induced cell cycle changes and elevated neoplastic capacity were reversed by CCAT1 siRNA or BMI1 siRNA. This epigenetic silencing of miR-218 by CCAT1 induces an altered cell cycle transition through BMI1 and provides a new mechanism for CSE-induced lung carcinogenesis.

[PRODUCT OF THE BMI1--A KEY COMPONENT OF POLYCOMB--POSITIVELY REGULATES ADIPOCYTE DIFFERENTIATION OF MOUSE MESENCHYMAL STEM CELLS].

Bmil is a key component of Polycomb (PcG), which in mammals controls the basic functions of mammalian somatic stem cells (SSC) such as self-renewal and differentiation. Bmi1 supports SSC via transcriptional suppression of genes associated with cell cycle and differentiation. The most studied target genes of Bmi1 are the genes of Ink4 locus, CdkI p16(Ink4a) and p1(Arf), suppression of which due to activating mutations of the BMI1 results in formation of cancer stem cells (CSC) and carcinomas in various tissues. In contrast, inactivation of BMI1 results in cell cycle arrest and cell senescence. Although clinical phenomena of hypo- and hyperactivation of BMI1 are well known, its targets and mechanisms of regulation of tissue specific SSC are still obscure. The goal of this study was to evaluate the regulatory role of BMI1 in adipocyte differentiation (AD) of mouse mesenchymal stem cells (MSC). Induction of AD in mouse MSC of the C3H10T1/2 cell line was associated with an increase in the expression levels of BMI1, the genes of pRb family (RB, p130) and demethylase UTX, but not methyltransferase EZH2, whose products regulate the methylation levels of H3K27. It was observed earlier that H3K27me3 may play the role of the epigenetic switch by promoting AD of human MSC via activating expression of the PPARγ2, the master gene of AD (Hemming et al., 2014). Here we show that inactivation of BMI1 using specific siRNA slows and decreases the levels of AD, but does not abolish it. This is associated with a complete inhibition of the expression of adipogenic marker genes--PPARγ2, ADIPOQ and a decrease in the expression of RB, p130, but not UTX. The results obtained give evidence that the epigenetic mechanism regulating AD differentiation in mouse and human MSC is different.

The ZEB1 transcription factor acts in a negative feedback loop with miR200 downstream of Ras and Rb1 to regulate Bmi1 expression.

Ras mutations are frequent in cancer cells where they drive proliferation and resistance to apoptosis. However in primary cells, mutant Ras instead can cause oncogene-induced senescence, a tumor suppressor function linked to repression of the polycomb factor Bmi1, which normally regulates cell cycle inhibitory cyclin-dependent kinase inhibitors (cdki). It is unclear how Ras causes repression of Bmi1 in primary cells to suppress tumor formation while inducing the gene in cancer cells to drive tumor progression. Ras also induces the EMT transcription factor ZEB1 to trigger tumor invasion and metastasis. Beyond its well-documented role in EMT, ZEB1 is important for maintaining repression of cdki. Indeed, heterozygous mutation of ZEB1 is sufficient for elevated cdki expression, leading to premature senescence of primary cells. A similar phenotype is evident with Bmi1 mutation. We show that activation of Rb1 in response to mutant Ras causes dominant repression of ZEB1 in primary cells, but loss of the Rb1 pathway is a hallmark of cancer cells and in the absence of such Rb1 repression Ras induces ZEB1 in cancer cells. ZEB1 represses miR-200 in the context of a mutual repression loop. Because miR-200 represses Bmi1, induction of ZEB1 leads to induction of Bmi1. Rb1 pathway status then dictates the opposing effects of mutant Ras on the ZEB1-miR-200 loop in primary versus cancer cells. This loop not only triggers EMT, surprisingly we show it acts downstream of Ras to regulate Bmi1 expression and thus the critical decision between oncogene-induced senescence and tumor initiation.

Preclinical activity of the novel B-cell-specific Moloney murine leukemia virus integration site 1 inhibitor PTC-209 in acute myeloid leukemia: Implications for leukemia therapy.

Curing patients with acute myeloid leukemia (AML) remains a therapeutic challenge. The polycomb complex protein B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is required for the self-renewal and maintenance of leukemia stem cells. We investigated the prognostic significance of BMI-1 in AML and the effects of a novel small molecule selective inhibitor of BMI-1, PTC-209. BMI-1 protein expression was determined in 511 newly diagnosed AML patients together with 207 other proteins using reverse-phase protein array technology. Patients with unfavorable cytogenetics according to Southwest Oncology Group criteria had higher levels of BMI-1 compared to those with favorable (P = 0.0006) or intermediate cytogenetics (P = 0.0061), and patients with higher levels of BMI-1 had worse overall survival (55.3 weeks vs. 42.8 weeks, P = 0.046). Treatment with PTC-209 reduced protein level of BMI-1 and its downstream target mono-ubiquitinated histone H2A and triggered several molecular events consistent with the induction of apoptosis, this is, loss of mitochondrial membrane potential, caspase-3 cleavage, BAX activation, and phosphatidylserine externalization. PTC-209 induced apoptosis in patient-derived CD34(+)CD38(low/-) AML cells and, less prominently, in CD34(-) differentiated AML cells. BMI-1 reduction by PTC-209 directly correlated with apoptosis induction in CD34(+) primary AML cells (r = 0.71, P = 0.022). However, basal BMI-1 expression was not a determinant of AML sensitivity. BMI-1 inhibition, which targets a primitive AML cell population, might offer a novel therapeutic strategy for AML.

The role of polycomb group protein Bmi-1 and Notch4 in breast cancer stem cell inhibition by benzyl isothiocyanate.

We showed previously that garden cress constituent benzyl isothiocyanate (BITC) inhibits self-renewal of breast cancer stem cells (bCSC) in vitro and in vivo. The present study offers novel insights into the mechanism by which BITC inhibits bCSC. Flow cytometry and mammosphere assay were performed to quantify bCSC fraction. Protein expression was determined by western blotting. Apoptosis was assessed by flow cytometry using Annexin V-propidium iodide method. Cell migration was determined by Boyden chamber assay. BITC treatment resulted in a marked decrease in protein level of polycomb group protein B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1) in cultured human breast cancer cells (MCF-7, SUM159, MDA-MB-231, and MDA-MB-361) and MDA-MB-231 xenografts in vivo. Overexpression (MCF-7) or knockdown (SUM159, and MDA-MB-231) of Bmi-1 protein had no meaningful impact on the BITC's ability to inhibit cell viability and cell migration and/or induce apoptosis. On the other hand, inhibition of bCSC markers (aldehyde dehydrogenase 1 activity and mammosphere frequency) resulting from BITC exposure was significantly altered by Bmi-1 overexpression and knockdown. BITC was previously shown to cause activation of Notch1, Notch2, and Notch4 in association with induction of γ-secretase complex component Nicastrin, which are also implicated in maintenance of cancer stemness. BITC-mediated inhibition of bCSC was augmented by knockdown of Notch4 and Nicastrin, but not by RNA interference of Notch1 or Notch2. The present study highlights important roles for Bmi-1 and Notch4 in BITC-mediated suppression of bCSC.

Upregulated expression of polycomb protein Ring1 contributes to poor prognosis and accelerated proliferation in human hepatocellular carcinoma.

Ring finger protein 1 (Ring1) have recently been reported to be closely related to aggressive tumor features in multiple cancer types, including prostate cancer, non-small-cell lung cancer, and bladder cancer. However, the role of Ring1 in human hepatocarcinogenesis remains unclear. In this study, we aimed at investigating the latent role of Ring1 in hepatocellular carcinoma (HCC) development. The expression of Ring1 was evaluated using Western blot analysis in 8 paired fresh HCC tissues and immunohistochemistry on 98 paraffin-embedded sections from 2005 to 2008. Moreover, RNA interference, CCK-8, colony formation, and flow-cytometry analyses were performed to investigate the role of Ring1 in the regulation of HCC cell proliferation. Compared with adjacent normal tissues, the level of Ring1 was significantly increased in HCC specimens. High expression of Ring1 was associated with histological grade (P = 0.011) and tumor size (P = 0.004), and Ring1 expression was positively related with the proliferation marker Ki-67 (P < 0.001). Moreover, knocking down Ring1 induced growth impairment and G1/S cell cycle arrest in HCC cells. Kaplan-Meier survival curves showed that high expression of Ring1 indicated poor prognosis of HCC (P = 0.03). On the basis of these results, we proposed that the expression of Ring1 protein may be a novel indicator of HCC prognosis.

Polygala tenuifolia polysaccharide (PTP) inhibits cell proliferation by repressing Bmi-1 expression and downregulating telomerase activity.

In our previous study, we isolated a homogeneous polysaccharide (PTP) with antitumor activity from the roots of Polygala tenuifolia. In view of the close correlation between Bmi-1 expression and progression of ovarian cancer, we intend to elucidate the mechanism of its activity by determining the Bmi-1 expression and the telomerase activity in human ovarian carcinoma OVCAR-3 cells following treatment with PTP at three concentrations of 0.5, 1, and 2 mg/mL for 48 h. MTT and colony-forming assays revealed that PTP had a significant inhibitory effect on the cell growth and colony formation of OVCAR-3 cells. Furthermore, Western blot and real-time PCR analysis showed that PTP inhibited Bmi-1 both in protein and transcript levels. Besides, the telomerase activity in OVCAR-3 cells was also downregulated after PTP treatment for 48 h. Taken together, the inhibitory effect of PTP on the cell growth was at least in part mediated via the downregulation of Bmi-1 expression and the telomerase activity in OVCAR-3 cells, and PTP might be a new candidate for chemotherapeutic agent against human ovarian cancer.

Bmi-1 is essential for the oncogenic potential in CD133(+) human laryngeal cancer cells.

It has been hypothesized that cancer stem cells (CSCs) are a principal culprit of tumor initiation, invasion, metastasis, and treatment resistance. Previous studies have confirmed that cancer stem cells can be detected in laryngeal carcinoma. This study aimed to evaluate whether population of CD133(+) cells that existed in primary human laryngeal carcinoma have characteristic of CSCs with enhanced capacity of proliferation and invasion, and to understand whether and how Bmi-1 implicated in self-renewal and tumorigenesis. We clarified the tumorigenic potential of CD133 sorted populations of cancer cells derived from primary human laryngeal tumor sample. After fluorescence activated cell sorting, real-time polymerase chain reaction (PCR) and western blot confirmed Bmi-1 was differentially expressed in CD133 sorted laryngeal tumor cells. Bmi-1 was knocked down, and proliferation, colony formation, invasion, cell cycle assay, and apoptosis assays were performed, and the impact on Bmi-1 pathway was evaluated. It was found that CD133(+) cells existed in primary human laryngeal tumor with enhanced capacity of proliferation and invasion. Bmi-1, implicated in self-renewal and tumorigenesis, was coexpressed with the CD133. Furthermore, knockdown of Bmi-1 expression in CD133(+) cells led to inhibition of cell growth, colony formation, cell invasion in vitro, and tumorigenesis in vivo, through up-regulation of p16(INK4A) and p14(ARF). Our data indicate that Bmi-1 expression is central to the tumorigenicity of CD133(+) cells, which functions as a pleiotropic regulator that maintains the viability and proliferative capacity of human laryngeal tumor. It negatively regulates the transcription of the downstream INK4a/ARF gene and inhibits expression of P16(ink4a)/P14(ARF), so as to maintain the high ability of proliferation and differentiation in laryngeal cancer stem cells.

Expression of chromobox homolog 7 (CBX7) is associated with poor prognosis in ovarian clear cell adenocarcinoma via TRAIL-induced apoptotic pathway regulation.

Ovarian cancer is the most lethal gynecologic malignancy, and clear cell adenocarcinoma of the ovary (OCCA), in particular, has a relatively poor prognosis among the ovarian cancer subtypes because of its high chemoresistance. Chromobox (CBX) 7 is a polycomb repressive complex 1 component that prolongs the lifespan of normal human cells by downregulating the INK4a/ARF expression which promotes cell-cycle progression. However, recent reports studying the relationship between CBX7 expression and patient survival have differed regarding the tumor cell origins, and the precise role of CBX7 in human carcinomas remains obscure. In this study, we analyzed CBX7 expression by immunohistochemistry in 81 OCCA patients and evaluated its association with their clinical outcomes. Both the overall and progression-free survival rates of the CBX7-positive patients were significantly shorter than those of the CBX7-negative patients (p < 0.05). CBX7 knockdown experiments using two OCCA cell lines, TOV21G and KOC-7C, revealed that cell viability was significantly reduced compared to the control cells (p < 0.001). Expression microarray analysis revealed that apoptosis-related genes, particularly tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), were significantly upregulated in CBX7 knockdown cells (p < 0.01). We further confirmed that CBX7 knockdown resulted in TRAIL-induced apoptosis in the OCCA cells. Thus, in this study, we showed for the first time that CBX7 was associated with a decreased OCCA prognosis. We also successfully demonstrated that the TRAIL pathway is a novel target for CBX7 expression modulation in these cells, and therapeutic agents utilizing the TRAIL pathway may be particularly effective for targeted OCCA therapy.

Expression and clinicopathological significance of Mel-18 mRNA in colorectal cancer.

Mel-18 is a member of the polycomb group (PcG) of proteins, which are chromatin regulatory factors that play an important role in oncogenesis. This study was designed to investigate the clinical and prognostic significance of Mel-18 in colorectal cancer (CRC) patients. For this purpose, expression of Mel-18 mRNA was evaluated in 82 primary CRC and paired noncancerous mucosa samples by qRT-PCR and Western blotting. We found that overall Mel-18 mRNA expression in the CRC tissue was significantly lower than in the noncancerous mucosal tissue (p = 0.007, Wilcoxon matched-pairs signed-ranks test). Mel-18 was conversely correlated with the pathological classifications (p = 0.003 for T, p < 0.001 for N, and p = 0.015 for M classifications, respectively) and clinical AJCC stage (p < 0.001). Furthermore, CRC patients with a higher level of Mel-18 showed prolonged disease-free survivals (DFS) (p < 0.001). In multivariate analysis, the diminished Mel-18 expression may be a risk factor for the patients' 3-year DFS (HR = 1.895; 95 % CI 1.032, 3.477; p = 0.039). It was therefore concluded that the lower Mel-18 expression might contribute to the CRC development/progression.

Polycomb chromobox (Cbx) 7 modulates activation-induced CD4+ T cell apoptosis.

CD4(+) T cell polarization plays a critical role in a number of immune disorders; the pathogenesis is unclear. Chromobox homolog 7 (Cbx7) is involved in the gene transcription of several cell types. This study aims to investigate the mechanism by which Cbx7 modulates the CD4(+) T cell polarization. Expression of Cbx7 was assessed by quantitative RT-PCR and Western blotting. Apoptosis of CD4(+) T cell was analyzed by flow cytometry. The FasL promoter methylation was evaluated by the methylation specific PCR. The results showed that CD4(+) CD25(-) T cells express Cbx7 that was increased significantly after activation by exposing to anti-CD3/CD28 Ab, but suppressed by exposing to specific antigens. More apoptotic cells were detected in CD4(+) T cells with the Cbx7 gene knockdown. Exposure to insulin-like growth factor-1 up regulated the expression of Cbx7 in CD4(+) T cells. After antigen-specific TCR activation, Cbx7-deficient CD4(+) T cells expressed more FasL and showed the FasL gene promoter hyper demethylation than wild CD4(+) T cells. In addition, CD4(+) T cells with overexpression of Cbx7 showed lower levels of FasL gene promoter demethylation. We conclude that CD4(+) T cells express Cbx7; the latter prevents FasL expression and the activation-induced CD4(+) T cell apoptosis.