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Cell migration is essential for many biological processes, while abnormal cell migration is characteristic of cancer cells. Epithelial cells become motile by undergoing epithelial-to-mesenchymal transition (EMT), and mesenchymal cells increase migration speed by adopting amoeboid features. This review highlights how amoeboid behaviour is not merely a migration mode but rather a cellular state - within the EMT spectra - by which cancer cells survive, invade and colonise challenging microenvironments. Molecular biomarkers and physicochemical triggers associated with amoeboid behaviour are discussed, including an amoeboid associated tumour microenvironment. We reflect on how amoeboid characteristics support metastasis and how their liabilities could turn into therapeutic opportunities.

Metastasis involves dissemination of cancer cells away from a primary tumour and colonisation at distal sites. During this process, cancer cells must negotiate multiple physical constraints imposed by the microenvironment and tissue structure. The biophysical properties of the nucleus must be tuned since they pose a challenge to constrained migration. By analysing nuclear genes upregulated during the acquisition of metastatic potential, we discovered increased expression of the inner nuclear membrane protein LAP1 in metastatic melanoma cells and at the invasive fronts of human primary tumours and in metastases. Human cells express two LAP1 isoforms (LAP1B and LAP1C), which differ in their amino terminus. We found that whereas the longer isoform, LAP1B, binds more strongly to nuclear lamins and has restricted motility within the nuclear envelope, the shorter isoform, LAP1C, favours nuclear envelope blebbing and allows migration through constraints. We propose that LAP1 renders the nucleus plastic and enhances melanoma aggressiveness.

© The Author(s) 2021., [Background]: Metastasis is a hallmark of cancer and responsible for most cancer deaths. Migrastatics were defined as drugs interfering with all modes of cancer cell invasion and thus cancers’ ability to metastasise. First anti-metastatic treatments have recently been approved. [Methods]: We used bioinformatic analyses of publicly available melanoma databases. Experimentally, we performed in vitro target validation (including 2.5D cell morphology analysis and mass spectrometric analysis of RhoA binding partners), developed a new traceable spontaneously metastasising murine melanoma model for in vivo validation, and employed histology (haematoxylin/eosin and phospho-myosin II staining) to confirm drug action in harvested tumour tissues. [Results]: Unbiased and targeted bioinformatic analyses identified the Rho kinase (ROCK)-myosin II pathway and its various components as potentially relevant targets in melanoma. In vitro validation demonstrated redundancy of several RhoGEFs upstream of RhoA and confirmed ROCK as a druggable target downstream of RhoA. The anti-metastatic effects of two ROCK inhibitors were demonstrated through in vivo melanoma metastasis tracking and inhibitor effects also confirmed ex vivo by digital pathology. [Conclusions]: We proposed a migrastatic drug development pipeline. As part of the pipeline, we provide a new traceable spontaneous melanoma metastasis model for in vivo quantification of metastasis and anti-metastatic effects by non-invasive imaging., GOF’s lab was supported by Cancer Research UK [C48390/A21153], Worldwide Cancer Research [16-1153], and King’s Health Partners [King’s Medical Research Trust Joint Research Committee studentship to A.V.]. B.F. was supported by a King’s Health Partners studentship to V.S.M. and G.O.F. V.S.M.’s lab was supported by Cancer Research UK [C33043/A12065] and [C33043/A24478] (V.S.M., E.C.M., J.L.O., L.B. and GC), the Royal Society [RG110591] (V.S.M.), The Harry J. Lloyd Charitable Trust (J.L.O. and V.S.M.), the Barts Charity (V.S.M., J.L.O., O.M., I.R.H. and E.C.M.), the Fundacion Alfonso Martin Escudero and Marie Sklodowska-Curie Action [H2020-MSCA-IF-2014-EF-ST] (I.R.H.), and Fundacion Ramon Areces (E.C.M.). F.M. was supported by an MRC Career Development Award (MR/P009417/1). This work was further supported by the Department of Health (DoH) via the National Institute for Health Research (NIHR) Comprehensive Biomedical Research Centre award to King’s Health Partners, and the Wellcome/EPSRC Centre for Medical Engineering [WT203148/Z/16/Z]. Views expressed are those of the authors and not necessarily those of the NHS, NIHR or DoH.

With the evolution of new genomic sequencing technologies an important amount of genomic data has been provided. As a consequence of this, many gene polymorphisms have been shown to be significantly associated with different disorders. Many strategies have been implemented to reveal the role of having more than one allele at a specific locus and their involvement in the illnesses. Site-directed mutagenesis is one of the most common strategies to understand the regulatory regions of genes and the relationship between the protein structure and its function. Here, we describe the analysis of lymphotoxin alpha expression in human retina and the generation of expression vectors to functional characterization of polymorphisms in the tumor necrosis factor locus using pCEFL-Flag expression vector and transfection assays in COS-1 cell line.

Salvador Pastor-Idoate,1,2 Irene Rodríguez-Hernández,2,3 Jimena Rojas,1 Lucia Gonzalez-Buendia,1 Santiago Delgado-Tirado,1,4 Jose Carlos López,1 Rogelio González-Sarmiento,2,3 Jose C Pastor1,4 1IOBA Eye Institute, University of Valladolid, Valladolid, 2Molecular Medicine Unit, Department of Medicine, 3Molecular and Cellular Cancer Biology Institute, High Council of Scientific Research, Biomedical Research Institute of Salamanca, University of Salamanca, Salamanca, 4Department of Ophthalmology, Hospital Clínico Universitario, Valladolid, Spain Purpose: The objective of this study is to determine the expression and localization of lymphotoxin alpha (LTA) in human retinas and the functionality of one of its polymorphisms rs2229094 (C13R) (T>C), previously associated with proliferative vitreoretinopathy (PVR) development.Materials and methods: Total RNA from three healthy human retinas were extracted and subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis, using flanking primers of LTA cDNA. In addition, three human eyes with retinal detachment (RD) and three healthy control eyes were subjected to immunohistochemistry (IHC) with a specific antibody against LTA. The functionality of T and C alleles was assessed by using pCEFL-Flag expression vector and transient transfection assays in COS-1 cell line. In addition, expression analysis by RT-PCR, Western blot and subcellular localization of both alleles and by immunofluorescence assay was performed.Results: RT-PCR analysis revealed no significant levels of messenger RNA (mRNA) LTA in healthy human retinas. Sequential IHC staining showed differences between healthy human and RD retinas. No differences in mRNA and protein expression levels and in subcellular localization between both alleles were found. Both alleles were located in the cytoplasm of COS-1 cells.Conclusion: Although results suggest lack of functionality, the differences found in IHC study and its strong association with PVR and its relationship with tumor necrosis factor locus, warrant further studies and could justify the use of this polymorphism as a valid biomarker to identify high-risk patients to develop PVR after RD. Keywords: proliferative vitreoretinopathy, lymphotoxin alpha, tumor necrosis factor alpha, inflammation, cytokines, polymorphism

The PPARγ coactivator 1 alpha (PGC1α) is a prostate tumor suppressor that controls the balance between anabolism and catabolism. PGC1A downregulation in prostate cancer is causally associated with the development of metastasis. Here we show that the transcriptional complex formed by PGC1α and estrogen-related receptor 1 alpha (ERRα) controls the aggressive properties of prostate cancer cells. PGC1α expression significantly decreased migration and invasion of various prostate cancer cell lines. This phenotype was consistent with remarkable cytoskeletal remodeling and inhibition of integrin alpha 1 and beta 4 expression, both in vitro and in vivo. CRISPR/Cas9-based deletion of ERRα suppressed PGC1α regulation of cytoskeletal organization and invasiveness. Mechanistically, PGC1α expression decreased MYC levels and activity prior to inhibition of invasiveness. In addition, PGC1α and ERRα associated at the MYC promoter, supporting the inhibitory activity PGC1α. The inverse correlation between PGC1α–ERRα activity and MYC levels was corroborated in multiple prostate cancer datasets. Altogether, these results support that PGC1α–ERRα functions as a tumor-suppressive transcriptional complex through the regulation of metabolic and signaling events. Significance: These findings describe how downregulation of the prostate tumor suppressor PGC1 drives invasiveness and migration of prostate cancer cells.

Enhanced blood vessel (BV) formation is thought to drive tumor growth through elevated nutrient delivery. However, this observation has overlooked potential roles for mural cells in directly affecting tumor growth independent of BV function. Here we provide clinical data correlating high percentages of mural-β3-integrin-negative tumor BVs with increased tumor sizes but no effect on BV numbers. Mural-β3-integrin loss also enhances tumor growth in implanted and autochthonous mouse tumor models with no detectable effects on BV numbers or function. At a molecular level, mural-cell β3-integrin loss enhances signaling via FAK-p-HGFR-p-Akt-p-p65, driving CXCL1, CCL2, and TIMP-1 production. In particular, mural-cell-derived CCL2 stimulates tumor cell MEK1-ERK1/2-ROCK2-dependent signaling and enhances tumor cell survival and tumor growth. Overall, our data indicate that mural cells can control tumor growth via paracrine signals regulated by β3-integrin, providing a previously unrecognized mechanism of cancer growth control.

Summary Despite substantial clinical benefit of targeted and immune checkpoint blockade-based therapies in melanoma, resistance inevitably develops. We show cytoskeletal remodeling and changes in expression and activity of ROCK-myosin II pathway during acquisition of resistance to MAPK inhibitors. MAPK regulates myosin II activity, but after initial therapy response, drug-resistant clones restore myosin II activity to increase survival. High ROCK-myosin II activity correlates with aggressiveness, identifying targeted therapy- and immunotherapy-resistant melanomas. Survival of resistant cells is myosin II dependent, regardless of the therapy. ROCK-myosin II ablation specifically kills resistant cells via intrinsic lethal reactive oxygen species and unresolved DNA damage and limits extrinsic myeloid and lymphoid immunosuppression. Efficacy of targeted therapies and immunotherapies can be improved by combination with ROCK inhibitors., Graphical Abstract, Highlights • Therapy-resistant melanoma cells restore myosin II activity to increase survival • High myosin II activity identifies targeted and immunotherapy-resistant melanomas • ROCK-myosin II inhibition increases ROS-DNA damage and decreases PD-L1 and Tregs • ROCK inhibition enhances efficacy of MAPK inhibitors and immunotherapies, Orgaz et al. show that myosin II activity increases during melanoma adaptation to MAPK pathway inhibition. ROCK-myosin II signaling supports survival of resistant melanoma cells and promotes immunosuppression. ROCK inhibitors improve the efficacy of MAPK inhibitors and immunotherapies in melanoma models.

Melanoma is a malignant tumor derived from melanocytes. Once disseminated, it is usually highly resistant to chemotherapy and is associated with poor prognosis. We have recently reported that T-type calcium channels (TTCCs) are overexpressed in melanoma cells and play an important role in melanoma progression. Importantly, TTCC pharmacological blockers reduce proliferation and deregulate autophagy leading to apoptosis. Here, we analyze the role of autophagy during migration/invasion of melanoma cells. TTCC Cav3.1 and LC3-II proteins are highly expressed in BRAFV600E compared with NRAS mutant melanomas, both in cell lines and biopsies. Chloroquine, pharmacological blockade, or gene silencing of TTCCs inhibit the autophagic flux and impair the migration and invasion capabilities, specifically in BRAFV600E melanoma cells. Snail1 plays an important role in motility and invasion of melanoma cells. We show that Snail1 is strongly expressed in BRAFV600E melanoma cells and patient biopsies, and its expression decreases when autophagy is blocked. These results demonstrate a role of Snail1 during BRAFV600E melanoma progression and strongly suggest that targeting macroautophagy and, particularly TTCCs, might be a good therapeutic strategy to inhibit metastasis of the most common melanoma type (BRAFV600E). Supported by grants from ISCIII/FEDER “Una manera de hacer Europa” (FIS-PI1200260 and PI1500711 to RMM, PI1301980 to JH and CIBERONC-CB16/12/00231 to XMG), Fundació la Marató de TV3 (FMTV 201331-30 to SP, -31 to RMM, and -32 to AF), Generalitat de Catalunya (2014/SGR138 to XMG); and Cancer Research UK grants C33043/A12065 and C33043/A24478 (VSM, JLO). OM holds a predoctoral fellowship from IRBLleida/Diputació de Lleida and CB a predoctoral fellowship from University of Lleida. AM holds a postdoctoral fellowship from AECC Scientific Foundation. IRH is supported by Marie Sklodowska-Curie Action (H2020-MSCA-IF-2014-EF-ST). Tumor samples were obtained with the support of Xarxa de Bancs de Tumors de Catalunya sponsored by Pla Director d'Oncologia de Catalunya (XBTC) and IRBLleida Biobank (B.0000682) and PLATAFORMA BIOBANCOS (PT17/0015/0027).

Purpose To compare the distribution of a p53 gene polymorphism among European subjects undergoing primary retinal detachment (RD) surgery in relation to the development of proliferative vitreoretinopathy (PVR). Design Case-controlled gene association study conducted as a component of the Retina 4 Project (a European multicenter study). Participants and Controls Five hundred fifty DNA samples, 134 with PVR secondary to primary RD and 416 with RD without PVR. Methods The p53 codon 72 polymorphism (rs1042522) was analyzed using allele-specific primer polymerase chain reaction. Proportions of genotypes and the proline (Pro-P) homozygote groups between subsamples from different countries were analyzed in 2 phases. In the first, subsamples from Spain and Portugal were analyzed. After significant results were found, samples from the United Kingdom (UK) and The Netherlands were analyzed (second phase). Genotypic and allelic frequencies were compared between cases and controls in the global sample. Main Outcome Measures Single significant associations with PVR. Results A significant difference ( P P P = 0.07). All control samples were in Hardy-Weinberg equilibrium. Considering the entire sample, significant differences were found in genotype frequencies between cases (RR, 30.59%; RP, 43.28%; PP, 26.11% [R = Arg; P=Pro]) and controls (RR, 39.66%; RP, 52.64%; PP, 7.69%) and in Pro homozygote carriers between controls (Pro homozygote 95% CI, 18.67–33.52) and cases (Pro homozygote 95% CI, 5.1–10.2). Conclusions Results indicate that the Pro variant of p53 codon 72 polymorphism is associated with a higher risk of PVR developing after a primary RD. Further studies are necessary to understand the role of this polymorphism in the development of PVR. Financial Disclosure(s) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

B cells participate in immune surveillance in human circulation and tissues, including tumors such as melanoma. By contrast, the role of humoral responses in cutaneous immunity is underappreciated. We report circulating skin-homing CD22+CLA+B cells in healthy volunteers and melanoma patients (n = 73) and CD22+ cells in melanoma and normal skin samples (n = 189). Normal and malignant skin featured mature IgG and CD22 mRNA, alongside mRNA for the transiently-expressed enzyme Activation-induced cytidine Deaminase (AID). Gene expression analyses of publically-available data (n = 234 GEO, n = 384 TCGA) confirmed heightened humoral responses (CD20, CD22, AID) in melanoma. Analyses of 51 melanoma-associated and 29 normal skin-derived IgG sequence repertoires revealed lower IgG1/IgGtotal representation compared with antibodies from circulating B cells. Consistent with AID, comparable somatic hypermutation frequencies and class-switching indicated affinity-matured antibodies in normal and malignant skin. A melanoma-associated antibody subset featured shorter complementarity-determining (CDR3) regions relative to those from circulating B cells. Clonal amplification in melanoma-associated antibodies and homology modeling indicated differential potential antigen recognition profiles between normal skin and melanoma sequences, suggesting distinct antibody repertoires. Evidence for IgG-expressing B cells, class switching and antibody maturation in normal and malignant skin and clonally-expanded antibodies in melanoma, support the involvement of mature B cells in cutaneous immunity.

Poor prognosis after ischemic stroke or intracerebral hemorrhage is linked to a particular polymorphism in the human gene encoding p53., The functional outcome after stroke is unpredictable; it is not accurately predicted by clinical pictures upon hospital admission. The presence of apoptotic neurons in the ischemic penumbra and perihematoma area may account for poor prognosis, but whether the highly variable stroke outcome reflects differences in genetic susceptibility to apoptosis is elusive. The p53 tumor suppressor protein, an important transcriptional regulator of apoptosis, naturally occurs in humans in two variants with single nucleotide polymorphisms resulting in Arg or Pro at residue 72. We show that poor functional outcome after either ischemic or hemorrhagic stroke was linked to the Arg/Arg genotype. This genotype was also associated with early neurological deterioration in ischemic stroke and with increased residual cavity volume in intracerebral hemorrhage. In primary cultured neurons, Arg72-p53, but not Pro72-p53, interacted directly with mitochondrial Bcl-xL and activated the intrinsic apoptotic pathway, increasing vulnerability to ischemia-induced apoptotic cell death. These results suggest that the Tp53 Arg/Arg genotype governs neuronal vulnerability to apoptosis and can be considered as a genetic marker predicting poor functional outcome after stroke.

Epithelial to mesenchymal transition is a common event during tumour dissemination. However, direct epithelial to amoeboid transition has not been characterized to date. Here we provide evidence that cells from hepatocellular carcinoma (HCC), a highly metastatic cancer, undergo epithelial to amoeboid transition in physiological environments, such as organoids or three-dimensional complex matrices. Furthermore, the NADPH oxidase NOX4 inhibits this transition and therefore suppresses efficient amoeboid bleb-based invasion. Moreover, NOX4 expression is associated with E-cadherin levels and inversely correlated with invasive features. NOX4 is necessary to maintain parenchymal structures, increase cell–cell and cell-to-matrix adhesion, and impair actomyosin contractility and amoeboid invasion. Importantly, NOX4 gene deletions are frequent in HCC patients, correlating with higher tumour grade. Contrary to that observed in mesenchymal cell types, here NOX4 suppresses Rho and Cdc42 GTPase expression and downstream actomyosin contractility. In HCC patients, NOX4 expression inversely correlates with RhoC and Cdc42 levels. Moreover, low expression of NOX4 combined with high expression of either RhoC or Cdc42 is associated with worse prognosis. Therefore, loss of NOX4 increases actomyosin levels and favours an epithelial to amoeboid transition contributing to tumour aggressiveness.

Genetics on PVR Study Group: et al., Meeting Presentation: EVER Annual Meeting, Nice, October 2014 as a poster and rapid paper., [Purpose]: To compare the distribution of BCL-2 -938C>A (rs2279115) and BAX -248G>A (rs4645878) genotypes among European subjects undergoing rhegmatogenous retinal detachment (RRD) surgery in relation to the further development of proliferative vitreoretinopathy (PVR). [Methods]: A case-control gene association study, as a part of Retina 4 project, was designed. rs2279115 and rs4645878 polymorphisms were analysed in 555 samples from patients with RRD (134 with PVR secondary to surgery). Proportions of genotypes and AA homozygous groups of BCL-2 and BAX polymorphisms between subsamples were analysed in two phases. Genotypic and allelic frequencies were compared in global sample and in subsamples. [Results]: BAX: Differences were observed in the genotype frequencies and in AA carriers between controls and cases in the global series. The odds ratio (OR) of A carriers in the global sample was 1.7 (95% CI: 1.23-2.51). Proportions of genotypes in Spain + Portugal were significant different. The OR of A carriers from Spain and Portugal was 1.8 (95% CI: 1.11-2.95). BCL-2: No significant differences were observed in genotype frequencies. However, proportions of genotypes in Spain + Portugal were significant. A protective effect (OR: 0.6 95% CI: 0.43-0.96) was found in A carriers from Spain and Portugal. [Conclusions]: Results suggest that A allele of rs4645878 could be a biomarker of high risk of developing PVR in patients undergoing RD surgery. The possible role of BCL-2 (inhibitor of necroptosis pathway) as a possible new target in PVR prophylaxis should be investigated., The authors acknowledge (a proportion of their) financial support from the Department of Health through the award made by the National Institute for Health Research to Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology for a Specialist Biomedical Research Centre for Ophthalmology. The views expressed in this publication are those of the authors and not necessarily those of the Department of Health. This research was partially funded by SAF 2007-66394, FIS PI10/00219 and Group of Excellence Grant (GR15) from Junta de Castilla y León. The funding organization had no role in the design or conduct of this research.

Purpose To compare the distribution of BAX G(-248)A and BCL-2 C(-938)A genotypes among European subjects undergoing rhegmatogenous retinal detachment surgery in relation to further development of proliferative vitreoretinopathy (PVR). Methods A case-control gene association study as part of the Retina 4 Project was designed. Two promoter single nucleotide polymorphisms (rs2279115 and rs4645878) were analysed by TaqMan 5'exonuclease allelic discrimination assay, using a StepOne® system in 134 samples from patients with PVR and 421 without PVR. Proportions of genotypes and AA homozygote groups of these polymorphisms were analysed. Genotypic and allelic frequencies were compared in global sample and in sub-samples. Results BAX gene: In the comparison of proportions of genotypes in Spain, Spain+Portugal and in the global sample, significant differences were found. The odds ratio (OR) for A carriers in Spain and Spain+Portugal was 1.8, and 1.7 in the global sample. BCL-2 gene: Significant differences were observed regarding proportions of genotypes in Spain+Portugal. Furthermore, a protective effect was found in the analysis of A carriers from Spain+Portugal with an OR of 0.6. Conclusion The A allele of BAX is associated with a higher risk of developing PVR, suggesting that a down-regulation in the apoptosis pathway could be an important key in PVR pathogenesis. Additionally, the role of BCL-2 gene (inhibitor of necroptosis pathway) is proposed as a possible new target in PVR prophylaxis.

[Background]: Glioblastoma is the most common and aggressive primary brain tumor in adults. Despite several factors such as ionizing radiation exposure or rare genetic syndromes have been associated with the development of glioblastoma, no underlying cause has been identified for the majority of cases. We thus aimed to investigate the role of DNA repair polymorphisms in modulating glioblastoma risk. [Methods]: Genotypic and allelic frequencies of seven common polymorphisms in DNA repair genes involved in nucleotide excision repair (ERCC1 rs11615, ERCC2 rs13181, ERCC6 rs4253079), base excision repair (APEX1 rs1130409, XRCC1 rs25487), double-strand break repair (XRCC3 rs861539) and mismatch repair (MLH1 rs1800734) pathways were analyzed in 115 glioblastoma patients and 200 healthy controls. Haplotype analysis was also performed for ERCC1 rs11615 and ERCC2 rs13181 polymorphisms, located on the same chromosomal region (19q13.32). [Results]: Our results indicated that carriers of the ERCC2 Gln/Gln genotype were associated with a lower glioblastoma risk (OR = 0.32, 95% CI 0.12-0.89; P = 0.028), whereas carriers of the MLH1 AA genotype were associated with an increased risk of glioblastoma (OR = 3.14, 95% CI 1.09-9.06; P = 0.034). Furthermore, the haplotype containing the C allele of ERCC2 rs13181 polymorphism and the T allele of ERCC1 rs11615 polymorphism was significantly associated with a protective effect of developing glioblastoma (OR = 0.34, 95% CI 0.16-0.71; P = 0.004). [Conclusions]: These results pointed out that MLH1 rs1800734 and ERCC2 rs13181 polymorphisms might constitute glioblastoma susceptibility factors, and also suggested that the chromosomal region 19q could be important in glioblastoma pathogenesis., This work was supported by Fondo de Investigacion Sanitaria (FIS PI 10/00219), Instituto de Estudios de Ciencias de la Salud de Castilla y Leon IECSCYL and Junta de Castilla y Leon y Fondo Social Europeo (Orden EDU/330/2008).

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., Proliferative vitreoretinopathy (PVR) is still the major cause of failure in retinal detachment (RD) surgery. It is believed that down-regulation in the p53 pathway could be an important key in PVR pathogenesis. The purpose was to evaluate the impact of T309G MDM2 polymorphism (rs2279744) in PVR. Distribution of T309G MDM2 genotypes among European subjects undergoing RD surgery was evaluated. Proportions of genotypes between subsamples from different countries were analyzed. Also, a genetic interaction between rs2279744 in MDM2 and rs1042522 in p53 gene was analyzed. Significant differences were observed comparing MDM2 genotype frequencies at position 309 of intron 1 between cases (GG: 21.6%, TG: 54.5%, TT: 23.8%) and controls (GG: 7.3%, TG: 43.9%, TT: 48.7%). The proportions of genotypes between sub-samples from different countries showed a significant difference. Distribution of GG genotype revealed differences in Spain (35.1-53.0)/(22.6-32.9), Portugal (39.0-74.4)/(21.4-38.9), Netherlands (40.6-66.3)/(25.3-38.8) and UK (37.5-62.4)/(23.3-34.2). The OR of G carriers in the global sample was 5.9 (95% CI: 3.2 to 11.2). The OR of G carriers from Spain and Portugal was 5.4 (95% CI: 2.2-12.7), whereas in the UK and the Netherlands was 7.3 (95% CI: 2.8-19.1). Results indicate that the G allele of rs2279744 is associated with a higher risk of developing PVR in patients undergoing a RD surgery. Further studies are necessary to understand the role of this SNP in the development of PVR., This work was supported by SAF 2007-66394, FIS PI10/00219 and Group of Excellence Grant (GR15) from Junta de Castilla y León.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., Malignant astrocytomas are the most aggressive primary brain tumors with a poor prognosis despite optimal treatment. Dysfunction of mismatch repair (MMR) system accelerates the accumulation of mutations throughout the genome causing uncontrolled cell growth. The aim of this study was to characterize the MMR system defects that could be involved in malignant astrocytoma pathogenesis. We analyzed protein expression and promoter methylation of MLH1, MSH2 and MSH6 as well as microsatellite instability (MSI) and MMR gene mutations in a set of 96 low- and high-grade astrocytomas. Forty-one astrocytomas failed to express at least one MMR protein. Loss of MSH2 expression was more frequent in low-grade astrocytomas. Loss of MLH1 expression was associated with MLH1 promoter hypermethylation and MLH1 -93G>A promoter polymorphism. However, MSI was not related with MMR protein expression and only 5% of tumors were MSI-High. Furthermore, the incidence of tumors carrying germline mutations in MMR genes was low and only one glioblastoma was associated with Lynch syndrome. Interestingly, survival analysis identified that tumors lacking MSH6 expression presented longer overall survival in high-grade astrocytoma patients treated only with radiotherapy while MSH6 expression did not modify the prognosis of those patients treated with both radiotherapy and chemotherapy. Our findings suggest that MMR system alterations are a frequent event in malignant astrocytomas and might help to define a subgroup of patients with different outcome., This work was supported by Fondo de Investigación Sanitaria (FIS PI 10/00219), Instituto de Estudios de Ciencias de la Salud de Castilla y León IECSCYL and Junta de Castilla y León y Fondo Social Europeo (Orden EDU/330/2008).

Antibodies have been detected against known melanoma-associated antigens in patients with malignant disease. While much is known about cancer-specific T cell responses, the nature of cancer-specific B cells and their antibody repertoires are less well understood. We investigated circulating skin-homing B cells (CD19+CD22+CLA+) in healthy volunteers (n = 24) and melanoma patients (n = 49) and examined the presence of CD22+ B cells in melanoma lesions (n = 173) and normal skins tissue (n = 16). We detected mature IgG mRNA in 13 of 27 normal skin and 9 of 21 cutaneous melanoma samples and we detected mRNA for the enzyme Activation-induced cytidine deaminase (AID). IgG variable heavy chain sequences from melanomas (n = 51) and normal skins (n = 29) featured lower IgG1/IgGtotal subclass representation compared with circulating B cell antibody sequences (n = 36). The presence of affinity-matured cutaneous antibody repertoires in malignant skin was supported by evidence of somatic hypermutation, class-switching and B cell clonal family trees in melanoma lesions and a subset of melanoma-associated clones featuring shorter CDR3 region lengths relative to circulating B cell sequences. Homology modelling also indicated differential putative binding site patterns in melanoma compared to normal skin-resident antibodies, suggesting distinct melanoma-associated repertoires and potentially, antigen recognition profiles. These findings support the presence of a mature tumor-resident B cell compartment with characteristics distinct to that of B cells in normal skin and the circulation. Citation Format: Louise Saul, Kristina M. Ilieva, Heather J. Bax, Panagiotis Karagiannis, Isabel Correa, Irene Rodriguez-Hernandez, Debra H. Josephs, Isabella Tosi, Isioma U. Egbuniwe, Sara Lombardi, Silvia Crescioli, Carl Hobbs, Federica Villanova, Anthony Cheung, Jenny LC Geh, Ciaran Healy, Mark Harries, Victoria Sanz-Moreno, David Fear, James F. Spicer, Katie E. Lacy, Frank O. Nestle, Sophia N. Karagiannis. IgG antibody switching and clonal expansion in melanoma and normal skin microenvironments [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A116.

Background: Abnormal cell migration and invasion underlie metastasis, and actomyosin contractility is a key regulator of tumor invasion. The links between cancer migratory behavior and DNA damage are poorly understood. Methods: Using 3D collagen systems to recapitulate melanoma extracellular matrix, we analyzed the relationship between the actomyosin cytoskeleton of migrating cells and DNA damage. We used multiple melanoma cell lines and microarray analysis to study changes in gene expression and in vivo intravital imaging (n = 7 mice per condition) to understand how DNA damage impacts invasive behavior. We used Protein Tissue Microarrays (n = 164 melanomas) and patient databases (n = 354 melanoma samples) to investigate the associations between markers of DNA damage and actomyosin cytoskeletal features. Data were analyzed with Student's and multiple t tests, Mann-Whitney's test, one-way analysis of variance, and Pearson correlation. All statistical tests were two-sided. Results: Melanoma cells with low levels of Rho-ROCK–driven actomyosin are subjected to oxidative stress-dependent DNA damage and ATM-mediated p53 protein stabilization. This results in a specific transcriptional signature enriched in DNA damage/oxidative stress responsive genes, including Tumor Protein p53 Inducible Protein 3 (TP53I3 or PIG3). PIG3, which functions in DNA damage repair, uses an unexpected catalytic mechanism to suppress Rho-ROCK activity and impair tumor invasion in vivo. This regulation was suppressed by antioxidants. Furthermore, PIG3 levels decreased while ROCK1/2 levels increased in human metastatic melanomas (ROCK1 vs PIG3; r = -0.2261, P < .0001; ROCK2 vs PIG3: r = -0.1381, P = .0093).Conclusions: The results suggest using Rho-kinase inhibitors to reactivate the p53-PIG3 axis as a novel therapeutic strategy; we suggest that the use of antioxidants in melanoma should be very carefully evaluated

1. Instituto de Estudios de Salud de Castilla y Leon (IESCYL)-HUSAL, Spain 2. Departamento de Medicina, Universidad de Salamanca, Spain 3. Departamento de Neurocirugia Universidad de Salamanca, Spain 4. Departamento de Anatomia Patologica, Hospital Universitario de Salamanca, Spain 5. Centro de Investigacion del Cancer, Spain 6. Unidad de Animales Geneticamente Modificados, Universidad de Salamamca, Spain