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Focuses on a study about the emerging roles of polycomb silencing in X-inactivation and stem cell maintenance that was presented at the 69th Cold Spring Harbor Symposia on Quantitative Biology in the U.S. in 2004. Search for mammalian polycomb response element and cellular memory modules; Implications of polycomb gene (PcG) for multilayered gene silencing; Connections among PcG regulation and cancer formation.

The initial dissemination of cancer cells from many primary tumors implies intravasation to lymphatic nodes or blood vessels. To investigate the mechanisms involved, we analyzed the expression of small non-coding RNAs in cutaneous squamous cell carcinoma (cSCC), a prevalent tumor that mainly spreads to lymph nodes. We report the reduced expression of small nucleolar RNAs in primary cSCCs that metastasized when compared to non-metastasizing cSCCs, and the progressive loss of DKC1 (dyskerin, which stabilizes the small nucleolar RNAs) along the metastasis. DKC1 depletion in cSCC cells triggered lipid metabolism by altering the mevalonate pathway and the acquisition of metastatic traits. Treatment of DKC1-depleted cells with simvastatin, an inhibitor of the mevalonate pathway, blocked the expression of proteins involved in the epithelial-to-mesenchymal transition. Consistently, the expression of the enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 1 was associated with pathological features of high metastatic risk in cSCC patients. Our data underpin the relevance of the mevalonate metabolism in metastatic dissemination and pave the possible incorporation of therapeutic approaches among the antineoplastic drugs used in routine patient care., (© 2023 Andrades et al.)

Ewing sarcoma (EwS) is an aggressive tumor that affects adolescents and young adults. EwS is defined by a chromosomal translocation, EWSR1-FLI1 being the most common, that causes genome reprogramming through remodeling of enhancers. Here, we describe an unexpected function of RING1B, which is highly expressed in EwS. While retaining its repressive activity at Polycomb developmental regulated genes, RING1B colocalizes with EWSR1-FLI1 at active enhancers. We demonstrate that RING1B is necessary for the expression of key EWSR1-FLI1 targets by facilitating oncogene recruitment to their enhancers. Knockdown of RING1B impairs growth of tumor xenografts and expression of genes regulated by EWSR1-FLI1 bound enhancers. Pharmacological inhibition of AURKB with AZD1152 increases H2Aub levels causing down-regulation of RING1B/EWSR1-FLI1 common targets. Our findings demonstrate that RING1B is a critical modulator of EWSR1-FLI1-induced chromatin remodeling, and its inhibition is a potential therapeutic strategy for the treatment of these tumors., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Background : Keratinocytic epidermal naevi (KENs) are congenital benign skin mosaic lesions that share common mutations with some subsets of urothelial carcinomas. Moreover, several patients with extensive KEN who also developed urothelial carcinomas at young ages have been reported. Thus, patients with extensive KEN may harbour mosaic urothelial oncogenic mutations that would favour the early development of urothelial carcinomas. Methods: We selected five patients with extensive KEN involving the lower part of the back and performed a molecular characterisation of urothelial and cutaneous samples using a next-generation sequencing (NGS) custom panel targeting candidate oncogenic genes. Results: Mosaic pathogenic mutations were detected in KEN in all patients. In four out of five patients, mosaic pathogenic mutations in FGFR2 or HRAS were also detected in samples from the urothelial tract. Moreover, we report a patient who developed urothelial carcinomas at age 29 and harboured an HRAS G12S mutation both in skin and urothelial tumour samples. Conclusions: We conclude that patients with extensive KEN involving the lower part of the back frequently harbour oncogenic mutations in the urothelium that may induce the development of carcinomas. NGS panels can be considered as highly sensitive tools to identify this subgroup of patients, which might permit adoption of screening measures to detect malignant transformation at early stages., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Actinic keratosis is a common skin disease that may progress to invasive squamous cell carcinoma if left untreated. Ingenol mebutate has demonstrated efficacy in field treatment of actinic keratosis. However, molecular mechanisms on ingenol mebutate response are not yet fully understood. In this study, we evaluated the gene expression profiles of actinic keratosis lesions before and after treatment with ingenol mebutate using microarray technology. Actinic keratoses on face/scalp of 15 immunocompetent patients were identified and evaluated after treatment with topical ingenol mebutate gel 0.015%, applied once daily for 3 consecutive days. Diagnostic and clearance of lesions was determined by clinical, dermoscopic, and reflectance confocal microscopy criteria. Lesional and non-lesional skin biopsies were subjected to gene expression analysis profiled by Affymetrix microarray. Differentially expressed genes were identified, and enrichment analyses were performed using STRING database. At 8 weeks post-treatment, 60% of patients responded to ingenol mebutate therapy, achieving complete clearance in 40% of cases. A total of 128 differentially expressed genes were identified following treatment, and downregulated genes (114 of 128) revealed changes in pathways important to epidermal development, keratinocyte differentiation and cornification. In responder patients, 388 downregulated genes (of 450 differentially expressed genes) were also involved in development/differentiation of the epidermis, and immune system-related pathways, such as cytokine and interleukin signaling. Cluster analysis revealed two relevant clusters showing upregulated profile patterns in pre-treatment actinic keratoses of responders, as compared to non-responders. Again, differentially expressed genes were mainly associated with cornification, keratinization and keratinocyte differentiation. Overall, the present study provides insight into the gene expression profile of actinic keratoses after treatment with ingenol mebutate, as well as identification of genetic signatures that could predict treatment response., Competing Interests: Agustí Toll received research funds from LEO Pharma S.A. for this work. The other authors have indicated they have no financial relationships or conflicts of interest to disclose. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Although most cutaneous squamous cell carcinomas (cSCCs) develop from actinic keratoses (AKs), the key events in this evolution remain unclear. We have combined the results of different genomic and expression array platforms on matched concomitant samples of sun-exposed skin (SES), AK, and cSCC from 10 immunocompetent patients. Gene expression analysis and copy number alterations were assessed using GeneChip Human Gene 2.0 ST Array (Affymetrix, Santa Clara, CA) and CytoScan HD Cytogenetics Solution (Affymetrix) platforms, respectively. Integration of transcriptome and genome results was evaluated using the DR-Integrator tool. Additional studies (qPCR, immunohistochemistry, and Western blot) were performed for selected genes. FOSL1 and BNC1 encode transcription factors whose expression was increased in cSCC in the expression array and the qPCR. By immunohistochemistry, FOSL1 showed an intense staining at the invasive front of cSCC samples and BNC1 expression varied from a nuclear (SES) to a cytoplasmic location (cSCC). Western blot analyses confirmed the enhancement of FOSL1 and BNC1. In addition, the smallest overlapping regions (SORIs) of genomic imbalance involving at least three of the samples were selected. One of the SORIs was a deletion in the p24.1 band of chromosome 3, shared by seven of the cSCCs. A strong correlation in the integration analysis was found for NEK10, a gene contained in the previously mentioned SORI. Loss of NEK10 expression in cSCC was confirmed by immunohistochemistry and Western blot analyses. In addition, functional studies in NEK10 depleted cells were performed. In conclusion, we identified FOSL1 and BNC1, which could act as tumor drivers, and NEK10, which could function as a tumor suppressor, to be differentially expressed during cSCC development., (© 2018 Wiley Periodicals, Inc.)

Although desmoplasia has been associated with poor prognoses in cutaneous squamous cell carcinoma, little attention has been paid to the patterns of fibrosis. This study aimed to examine the different stromal fibrotic patterns as markers of metastatic risk. We performed a multicenter retrospective study that included 102 cutaneous squamous cell carcinomas (52 non-metastatic and 50 metastatic carcinomas). Clinical and histopathological data were registered. The fibrotic reaction pattern was classified as mature, intermediate or immature depending on the presence of keloid-like collagen and myxoid stroma. The immature pattern (areas characterized by myxoid changes with no inflammation) was observed in 18 samples and its presence was significantly associated with immunosuppression, budding, desmoplasia, perineural invasion, anatomic level, tumoural depth and metastatic risk in the multivariate analysis. Our findings suggest that the presence of an immature myxoid fibrotic pattern, which can be easily identified by routine hematoxylin-eosin staining, is strongly associated with metastatic risk.

Programmed cell death ligand 1 (PD-L1) expression by tumor cells plays an important role in the inhibition of T cell-mediated immune response in cancer. PD-L1 expression by tumor cells has been linked to poor prognosis in a wide variety of cancers. However, PD-L1 expression in cutaneous squamous cell carcinoma (cSCC) has been scarcely studied, and its role as a prognosis biomarker remains controversial. The association of PD-L1 expression and the metastatic risk in a series of cSCC was assessed. PD-L1 and CD8 immunostainings of full excision sections of 99 primary tumors and 24 lymphatic metastases were semiquantitatively evaluated. Primary cSCCs were grouped according to the development of lymphatic metastatic spread [metastasizing squamous cell carcinoma (MSCC)] (n = 48) or the absence of progression [nonmetastasizing squamous cell carcinoma (NMSCC)] (n = 51). PD-L1-positive expression (cut off ≥1%) was found in 26% NMSCCs and in 50% MSCCs (P = 0.02). PD-L1 association with an increased metastatic risk was confirmed in the multivariate analysis (P < 0.05), along with the following features: recurrence, poor differentiation, and perineural invasion. Ninety percent of the metastases of PD-L1-positive tumors were also positive for PD-L1, displaying a trend toward a higher PD-L1 expression when compared with their primary tumors (P = 0.058). No significant differences in the peritumoral inflammatory infiltrate or in the expression of CD8 were found between metastasizing and nonmetastasizing primary tumors. Our results suggest that PD-L1 may play a relevant role in metastatic spread and may be a candidate prognostic biomarker in cSCC.

Cutaneous squamous cell carcinoma (cSCC) is the second most common malignancy in humans and approximately 5% metastasize, usually to regional lymph nodes. Epigenetic regulation of gene expression may allow tumoral cells to acquire new functions in order to escape from the primary tumor. The aim of this study was to investigate the expression and function of proteins of the Polycomb family of epigenetic regulators in the metastatic process of cSCC. A higher expression of RING1B and EZH2 was detected by immunohistochemistry in a series of primary cSCC tumors that metastasized (MSCCs) when compared with non-metastasizing cSCCs (non-MSCCs). Stable downregulation of RING1B and EZH2 in cSCC cells results in enhanced expression of inflammatory cytokines and activation of the NF-κB signaling pathway. Accordingly, non-MSCCs display higher levels of membranous pS176-inhibitor of NF-kB kinase, and their stroma is enriched in neutrophils and eosinophils when compared with MSCCs. In vitro, hematopoietic cells exhibit a substantial migratory response to supernatants from Polycomb-depleted cSCC cells. Altogether, these data indicate that RING1B and EZH2 repress the innate inflammatory cSCC function and impair tumor immunosurveillance and suggest that patients with high-risk cSCCs could benefit from clinical therapies addressed to harness the immune response.

Importance: Pigmented facial macules on photodamaged skin are a clinical, dermoscopic, and histopathologic challenge., Objectives: To clinically and dermoscopically characterize, by means of reflectance confocal microscopy (RCM), ambiguous pigmented facial macules and establish a correlation between RCM, histopathologic, and immunohistochemical findings., Design, Setting, and Participants: A prospective study of ambiguous pigmented facial macules on photodamaged skin was conducted in a tertiary referral center for dermatology between January 1, 2009, and December 31, 2015. Sixty-one patients with 63 ambiguous pigmented facial macules and 12 control photodamaged facial areas were included in the study. Melanocyte density in 1-mm basal layers was determined in skin biopsy specimens from all lesions stained with hematoxylin-eosin and immunohistochemical markers (melan-A, microphthalmia-associated transcription factor, and SRY-related HMG-box gene 10). Dermoscopic, RCM images, and histopathologic preparations were systematically evaluated for the presence of lentigo maligna (LM) criteria. Confocal evaluation was blinded to clinical and dermoscopic diagnosis. Sensitivity and specificity of RCM for LM diagnosis and κ value to establish correlations between dermoscopy, RCM, and histopathology were performed., Main Outcomes and Measures: Sensitivity and specificity of RCM for LM diagnosis., Results: Of the 61 patients included in the study, 31 (51%) were women; mean (SD) age was 71.8 (13.1) years. Twenty-four of the 63 (38%) lesions were diagnosed as LM or LM melanoma (LMM) and 39 (62%) as benign pigmented lesions. Reflectance confocal microscopy enhanced the diagnosis of pigmented facial macules with 91.7% sensitivity and 86.8% specificity. Multivariate analysis showed 2 dermoscopic and 2 confocal features associated with LM or LMM: (1) asymmetric follicular pigmentation and targetlike structures, and (2) round, large pagetoid cells and follicular localization of atypical cells, respectively. Continuous proliferation of atypical melanocytes was found in 21 (88%) LM or LMM and in 3 (77%) benign lesions. Asymmetric pigmented follicular openings by dermoscopy correlated with follicular localization of pagetoid cells by RCM (κ = 0.499, P < .001). The presence of 3 or more atypical cells at the dermal-epidermal junction (DEJ) by RCM correlated with hyperplasia of melanocytes in hematoxylin-eosin sections (κ = 0.422, P < .001)., Conclusions and Relevance: Reflectance confocal microscopy improves LM diagnosis in photodamaged skin with good histopathologic correlation although false-positive and false-negative cases exist. False-positives obtained with RCM in photodamaged skin are due to the presence of basal melanocyte hyperplasia and intraepidermal Langerhans cells. Histopathologic features of these lesions sometimes are not enough for a definite diagnosis and immunohistochemical studies may be required.

Background: Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer and frequently progresses from an actinic keratosis (AK), a sun-induced keratinocyte intraepithelial neoplasia (KIN). Epigenetic mechanisms involved in the phenomenon of progression from AK to cSCC remain to be elicited., Methods: Expression of microRNAs in sun-exposed skin, AK and cSCC was analysed by Agilent microarrays. DNA methylation of miR-204 promoter was determined by bisulphite treatment and pyrosequencing. Identification of miR-204 targets and pathways was accomplished in HaCat cells. Immunofluorescence and immunohistochemistry were used to analyze STAT3 activation and PTPN11 expression in human biopsies., Results: cSCCs display a marked downregulation of miR-204 expression when compared to AK. DNA methylation of miR-204 promoter was identified as one of the repressive mechanisms that accounts for miR-204 silencing in cSCC. In HaCaT cells miR-204 inhibits STAT3 and favours the MAPK signaling pathway, likely acting through PTPN11, a nuclear tyrosine phosphatase that is a direct miR-204 target. In non-peritumoral AK lesions, activated STAT3, as detected by pY705-STAT3 immunofluorescence, is retained in the membrane and cytoplasm compartments, whereas AK lesions adjacent to cSCCs display activated STAT3 in the nuclei., Conclusions: Our data suggest that miR-204 may act as a "rheostat" that controls the signalling towards the MAPK pathway or the STAT3 pathway in the progression from AK to cSCC.

Purpose: Epithelial-to-mesenchymal transition is a phenomenon in epithelial tumors that involves loss of intercellular adhesion, mesenchymal phenotype acquisition and enhanced migratory potential. While the epithelial-to-mesenchymal transition process has been extensively linked to metastatic progression of squamous cell carcinoma, studies of the role of epithelial-to-mesenchymal transition in squamous cell carcinoma containing high risk human papillomaviruses are scarce. Moreover, to our knowledge epithelial-to-mesenchymal transition involvement in human penile squamous cell carcinoma, which can arise through transforming HPV infections or independently of HPV, has not been investigated. We evaluated the presence of epithelial-to-mesenchymal transition markers and their relationship to HPV in penile squamous cell carcinoma., Materials and Methods: We assessed the expression of E-cadherin, vimentin and the epithelial-to-mesenchymal transition related transcription factors Twist, Zeb1 and Snail by immunohistochemical staining in 64 penile squamous cell carcinoma cases. HPV was detected by polymerase chain reaction amplification., Results: Simultaneous loss of membranous E-cadherin expression and vimentin over expression were noted in 43.5% of penile squamous cell carcinoma cases. HPV was significantly associated with loss of membranous E-cadherin but not with epithelial-to-mesenchymal transition. Recurrence and mortality rates were significantly higher in cases showing epithelial-to-mesenchymal transition., Conclusions: Our findings indicate that in penile squamous cell carcinoma epithelial-to-mesenchymal transition is associated with poor prognosis but not with the presence of HPV., (Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

In early stages of metastasis malignant cells must acquire phenotypic changes to enhance their migratory behavior and their ability to breach the matrix surrounding tumors and blood vessel walls. Epigenetic regulation of gene expression allows the acquisition of these features that, once tumoral cells have escape from the primary tumor, can be reverted. Here we report that the expression of the Polycomb epigenetic repressor Ring1B is enhanced in tumoral cells that invade the stroma in human ductal breast carcinoma and its expression is coincident with that of Fak in these tumors. Ring1B knockdown in breast cancer cell lines revealed that Ring1B is required to sustain Fak expression in basal conditions as well as in Tgfβ-treated cells. Functionally, endogenous Ring1B is required for cell migration and invasion in vitro and for in vivo invasion of the mammary fat pad by tumoral cells. Finally we identify p63 as a target of Ring1B to regulate Fak expression: Ring1B depletion results in enhanced p63 expression, which in turns represses Fak expression. Importantly, Fak downregulation upon Ring1B depletion is dependent on p63 expression. Our findings provide new insights in the biology of the breast carcinoma and open new avenues for breast cancer prognosis and therapy.

Background: Cutaneous squamous cell carcinoma (cSCC) is the second most common malignancy in humans and approximately 5% metastasize, usually to regional lymph nodes. Epithelial to mesenchymal transition (EMT) is a process involving loss of intercellular adhesion, acquisition of a mesenchymal phenotype and enhanced migratory potential; epithelial markers, such as E-cadherin, are down-regulated and mesenchymal proteins (Vimentin), increased., Objective: To investigate the expression of EMT markers in metastatic SCC (MSCC) and their corresponding metastases, and to correlate them with clinico-pathological factors associated with an increased risk of metastasis., Methods: We performed a retrospective study that included 146 cSCC samples (51 primary non-metastatic, 56 primary metastatic, 39 lymphatic metastases). Immunohistochemistry for E-cadherin, Vimentin, Snail, beta-catenin, Twist, Zeb1 and Podoplanin was performed., Results: Loss of membranous E-cadherin was observed in 77% cSCCs, with no differences between MSCC and non-MSCC. Among the transcriptional factors controlling EMT, no significant Snail1 expression was detected. Twist, Zeb1, Vimentin, beta-catenin and Podoplanin were significantly overexpressed in MSCCs. Twist ectopic expression in SCC13 cells induced Zeb1, Vimentin and Podoplanin expression and E-cadherin delocalization. These changes resulted in a scattered migration pattern in vitro. Expression of EMT markers was decreased in the metastases when compared with the corresponding primary tumors., Conclusion: These results suggest that a partial EMT, characterized by the expression of Twist but without a total E-cadherin depletion, is involved in the acquisition of invasive traits by cSCC, but the process is downregulated in lymph node metastases., (Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Introduction: Deregulated expression/activation of transcription factors is a key event in the establishment and progression of human cancer. Furthermore, most oncogenic signaling pathways converge on sets of transcription factors that ultimately control gene expression patterns resulting in cancer development, progression, and metastasis., Methods: Ductal pancreatic adenocarcinoma (PDA) is the main type of pancreatic cancer and the fourth leading cause of cancer mortality in the Western world. The early stage of the disease is characterized by pancreatic intraepithelial neoplasia lesions bearing mutations in the K-RAS proto-oncogene, which progress to malignant PDA by accumulating additional mutations in the tumor suppressor gene CDKN2A (p16) and in SMAD4 and TP53 transcription factors. The involvement of other signaling pathways in PDA development and progression is an active area of research which may help to clarify the critical steps of this devastating disease., Results: In this regard, several in vitro and in vivo data have demonstrated the contribution of the transcription factor c-Myc to pancreatic carcinogenesis although the molecular mechanisms are poorly understood. c-Myc is a proto-oncogene which has a pivotal function in growth control, differentiation and apoptosis and is known to act as a downstream transcriptional effector of many signaling pathways involved in these processes. It is regulated at multiple levels and its abnormal expression contributes to the genesis of many human tumors., Conclusions: This review focuses on the role of c-Myc in pancreatic embryonic development and homeostasis as well as its involvement on pancreatic tumorigenesis. Evidences showing that c-Myc function is highly dose and cell context dependent, together with its recently demonstrated ability to reprogram somatic cells towards a pluripotent stem cell-like state, indicate that the role of c-Myc in pancreas pathophysiology might have been previously underscored.

In multicellular organisms differentiated cells must maintain their cellular memory, which will be faithfully inherited and maintained by their progeny. In addition, these specialized cells are exposed to specific environmental and cell-intrinsic signals and will have to appropriately respond to them. Some of these stimuli lead to changes in a subset of genes or to a genome-wide reprogramming of the cells that will remain after stimuli removal and, in some instances, will be inherited by the daughter cells. The molecular substrate that integrates cellular memory and plasticity is the chromatin, a complex of DNA and histones unique to eukaryotes. The nucleosome is the fundamental unit of the chromatin and nucleosomal organization defines different chromatin conformations. Chromatin regulators affect chromatin conformation and accessibility by covalently modifying the DNA or the histones, substituting histone variants, remodeling the nucleosome position or modulating chromatin looping and folding. These regulators frequently act in multiprotein complexes and highly specific interplays among chromatin marks and different chromatin regulators allow a remarkable array of possibilities. Therefore, chromatin regulator nets act to propagate the conformation of different chromatin regions through DNA replication and mitosis, and to remodel the chromatin fiber to regulate the accessibility of the DNA to transcription factors and to the transcription and repair machineries. Here, the state-of-the-art of the best-known chromatin regulators is reviewed.

Chronic pancreatitis and pancreatic ductal adenocarcinoma (PDAC) are associated with major changes in cell differentiation. These changes may be at the basis of the increased risk for PDAC among patients with chronic pancreatitis. Polycomb proteins are epigenetic silencers expressed in adult stem cells; up-regulation of Polycomb proteins has been reported to occur in a variety of solid tumours such as colon and breast cancer. We hypothesized that Polycomb might play a role in preneoplastic states in the pancreas and in tumour development/progression. To test these ideas, we determined the expression of PRC1 complex proteins (Bmi1 and Ring1b) during pancreatic development and in pancreatic tissue from mouse models of disease: acute and chronic pancreatic injury, duct ligation, and in K-Ras(G12V) conditional knock-in and caerulein-treated K-Ras(G12V) mice. The study was extended to human pancreatic tissue samples. To obtain mechanistic insights, Bmi1 expression in cells undergoing in vitro exocrine cell metaplasia and the effects of Bmi1 depletion in an acinar cancer cell line were studied. We found that Bmi1 and Ring1B are expressed in pancreatic exocrine precursor cells during early development and in ductal and islet cells-but not acinar cells-in the adult pancreas. Bmi1 expression was induced in acinar cells during acute injury, in acinar-ductal metaplastic lesions, as well as in pancreatic intraepithelial neoplasia (PanIN) and PDAC. In contrast, Ring1B expression was only significantly and persistently up-regulated in high-grade PanINs and in PDAC. Bmi1 knockdown in cultured acinar tumour cells led to changes in the expression of various digestive enzymes. Our results suggest that Bmi1 and Ring1B are modulated in pancreatic diseases and could contribute differently to tumour development., (2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Pancreatic cancer has a very poor prognosis, in part due to its diagnosis at late stages of the disease and to limited response to chemotherapy and radiotherapy. The vast majority of pancreatic cancers are classified as pancreatic ductal adenocarcinomas (PDACs). Despite advances in knowledge on the cellular origin of PDAC or the involvement of signal transduction pathways therein, many questions remain unanswered. In this review, we summarize recent findings and current hypotheses regarding these two questions. Since pancreatitis is a risk factor for human PDAC, and the latter proceeds with an intense fibrotic reaction, we also analyze the role of the stroma in PDAC progression. An improved understanding of these key aspects for PDAC ontogeny will open new avenues for tumor prevention, early detection, and improved therapy., (Copyright 2008 S. Karger AG, Basel.)

Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that is involved in modulating chromatin structure, regulation of gene expression, and sensing DNA damage. Here, we report that PARP-1 enzymatic activity is inhibited by macroH2A, a vertebrate histone H2A variant that is enriched on facultative heterochromatin. MacroH2A family members have a large C-terminal non-histone domain (NHD) and H2A-like histone domain. MacroH2A1.2 and PARP-1 interact in vivo and in vitro via the NHD. The NHD of each macroH2A family member was sufficient to inhibit PARP-1 enzymatic activity in vitro. The NHD of macroH2A1.2 was a mixed inhibitor of PARP-1 catalytic activity, with affects on both catalytic activity and the substrate binding affinity of PARP-1. Depletion of PARP-1 by RNA interference caused reactivation of a reporter gene on the inactive X chromosome, demonstrating that PARP-1 participates in the maintenance of silencing. These results suggest that one function of macroH2A in gene silencing is to inhibit PARP-1 enzymatic activity, and this may affect PARP-1 association with chromatin.

Polycomb group (PcG) proteins are epigenetic chromatin modifiers involved in heritable gene repression. Two main PcG complexes have been characterized. Polycomb repressive complex 2 (PRC2) is thought to be involved in the initiation of gene silencing, whereas Polycomb repressive complex 1 (PRC1) is implicated in the stable maintenance of gene repression. Here, we investigate the kinetic properties of the binding of one of the PRC1 core components, BMI1, with PcG bodies. PcG bodies are unique nuclear structures located on regions of pericentric heterochromatin, found to be the site of accumulation of PcG complexes in different cell lines. We report the presence of at least two kinetically different pools of BMI1, a highly dynamic and a less dynamic fraction, which may reflect BMI1 pools with different binding capacities to these stable heterochromatin domains. Interestingly, PRC2 members EED and EZH2 appear to be essential for BMI1 recruitment to the PcG bodies. Furthermore, we demonstrate that the maintenance DNA methyltransferase DNMT1 is necessary for proper PcG body assembly independent of DNMT-associated histone deacetylase activity. Together, these results provide new insights in the mechanism for regulation of chromatin silencing by PcG proteins and suggest a highly regulated recruitment of PRC1 to chromatin.

X inactivation involves the stable silencing of one of the two X chromosomes in XX female mammals. Initiation of this process occurs during early development and involves Xist (X-inactive-specific transcript) RNA coating and the recruitment of Polycomb repressive complex (PRC) 2 and PRC1 proteins. This recruitment results in an inactive state that is initially labile but is further locked in by epigenetic marks such as DNA methylation, histone hypoacetylation, and MACROH2A deposition. Here, we report that the E3 ubiquitin ligase consisting of SPOP and CULLIN3 is able to ubiquitinate the Polycomb group protein BMI1 and the variant histone MACROH2A. We find that in addition to MACROH2A, PRC1 is recruited to the inactivated X chromosome in somatic cells in a highly dynamic, cell cycle-regulated manner. Importantly, RNAi-mediated knock-down of CULLIN3 or SPOP results in loss of MACROH2A1 from the inactivated X chromosome (Xi), leading to reactivation of the Xi in the presence of inhibitors of DNA methylation and histone deacetylation. Likewise, Xi reactivation is also seen on MacroH2A1 RNAi under these conditions. Hence, we propose that the PRC1 complex is involved in the maintenance of X chromosome inactivation in somatic cells. We further demonstrate that MACROH2A1 deposition is regulated by the CULLIN3/SPOP ligase complex and is actively involved in stable X inactivation, likely through the formation of an additional layer of epigenetic silencing.

Neurofibromin (NF1) (the product of Nf1 gene) is a large cytosolic protein known as a negative regulator of Ras. A fragment of some 400 residues located at the center of the NF1 GAP-Related Domain (NF1-GRD) has strong identity with other molecules of the GAP family, which comprises, among others, the mammalian proteins NF1 and p120GAP, and the yeast proteins IRA1 and IRA2. GAP family members are known by their ability to promote the GTPase activity of Ras proteins, facilitating the transit of those proteins to their inactive state. Recent findings (Tong et al., 2002, Nat Neurosci 5:95-96) indicate that NF1 may be involved in the regulation of adenyl cyclase activity. Our results show that NF1-GRD cooperates with Ras in the anchorage-independent growth capacity of Ras-expressing fibroblasts, without affecting: (i) their ability to grow in low serum, (ii) their cellular adhesion capability, or (iii) the expression of key proteins involved in cell-cell and cell-matrix interactions. On the other hand, NF1 overexpression induces an increase in the expression levels of the focal adhesion kinase (FAK), and specific changes in the activation status of the mitogen-activated protein kinases (MAPKs). These results suggest the existence of a Ras-independent NF1-dependent pathway able to modify the levels of expression of FAK and the levels of activation of MAPKs. Because FAK and many proteins recently found to bind NF1 have a role in the cytoskeleton, this pathway may involve rearrangement of cytoskeletal components that facilitate anchorage independence., (Copyright 2003 Wiley-Liss, Inc.)

Previous studies have identified a novel oncogene, rgr, which has homology to the guanine nucleotide exchange factor (GEF) Ral guanine dissociation stimulator (RALGDS). To determine the mechanism of activation of rgr, the wild-type form was isolated. rgr is expressed physiologically at very low levels, due, at least in part, to a long 5'-untranslated region that contains eight AUGs, which inhibit translation of the main open reading frame. When these regulatory sequences are removed, the wild-type gene is expressed at high levels. An investigation of how this GEF could transform cells showed that RGR interacts with RAS, supporting its involvement as a RAS-GEF. Because RAL is localized mainly to the Golgi, the expression of the RGR protein was identified in RK13 cells, a cell line that expresses endogenous rgr. RGR localizes to endomembranes. To determine its location upon transformation, a green fluorescent protein-RGR fusion protein was used to track the movement of RGR. Increasing amounts of expression result in enhanced localization of RGR to the plasma membrane. These results indicate that rgr is activated when its tight translational controls are eliminated and increased expression allows its relocation to the plasma membrane, where efficient activation of RAS occurs.

In this study, we show that reactive oxygen species production induced by tumour necrosis factor alpha (TNF-alpha) in L929 cells was associated with a decrease in the steady-state mRNA levels of the mitochondrial transcript ATPase 6-8. Simultaneously, the transcript levels of two nuclear-encoded glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphofructokinase, were increased. These changes were associated with decreased protein levels of the ATPase subunit a (encoded by the mitochondrial ATPase 6 gene) and cytochrome c oxidase subunit II, and increased protein levels of phosphofructokinase. Since TNF-alpha had no effect on the amount of mitochondrial DNA, the results suggested that TNF-alpha acted at the transcriptional and/or post-transcriptional level. Reactive oxygen species scavengers, such as butylated hydroxianisole and butylated hydroxytoluene, blocked the production of free radicals, prevented the down-regulation of ATPase 6-8 transcripts, preserved the protein levels of ATPase subunit a and cytochrome c oxidase subunit II, and attenuated the cytotoxic response to TNF-alpha, indicating a direct link between these two phenomena.

Background/aims: The toxic oil syndrome appeared in Spain in 1981 as a result of ingestion of rapeseed oil denatured with aniline. Some patients developed scleroderma-like skin lesions and liver cirrhosis. Mechanisms of these fibrotic lesions are not known. The present study was designed to investigate the effect of toxic oils on collagen metabolism., Methods: We measured the relative rate of collagen production, absolute rate of collagen synthesis, production, secretion, and degradation, proline transport, steady-state levels of procollagen alpha 1(l)-messenger RNA (mRNA) in cultured fat-storing cells, and chloramphenicol acetyltransferase activity in transfected cells., Results: Toxic oils increased collagen synthesis, procollagen alpha 1(l)-mRNA levels, and chloramphenicol acetyltransferase activity in cultured fat-storing cells. Effect on collagen production correlated with lipid peroxide content in oils. Cycloheximide, alpha-tocopherol, and methylene blue prevented the increase in procollagen alpha 1(l)-mRNA. Oleylanilide and linoleylanilide, markers for toxic oils, reproduced the stimulatory effects of toxic oils on collagen production and procollagen alpha 1(l)-mRNA., Conclusions: Toxic oils increased collagen synthesis by acting on the promoter of procollagen alpha 1(l) gene, probably through lipid peroxides derived from acylanilides. We suggest that toxic oil may have stimulated procollagen gene expression through the formation of adducts of aldehydes with some transcription factor.