BackgroundWe are investigating the molecular basis of melanoma by defining genomic characteristics that correlate with tumour phenotype in a novel panel of metastatic melanoma cell lines. The aim of this study is to identify new prognostic markers and therapeutic targets that might aid clinical cancer diagnosis and management.Principal FindingsGlobal transcript profiling identified a signature featuring decreased expression of developmental and lineage specification genes including MITF, EDNRB, DCT, and TYR, and increased expression of genes involved in interaction with the extracellular environment, such as PLAUR, VCAN, and HIF1a. Migration assays showed that the gene signature correlated with the invasive potential of the cell lines, and external validation by using publicly available data indicated that tumours with the invasive gene signature were less melanocytic and may be more aggressive. The invasion signature could be detected in both primary and metastatic tumours suggesting that gene expression conferring increased invasive potential in melanoma may occur independently of tumour stage.ConclusionsOur data supports the hypothesis that differential developmental gene expression may drive invasive potential in metastatic melanoma, and that melanoma heterogeneity may be explained by the differing capacity of melanoma cells to both withstand decreased expression of lineage specification genes and to respond to the tumour microenvironment. The invasion signature may provide new possibilities for predicting which primary tumours are more likely to metastasize, and which metastatic tumours might show a more aggressive clinical course.

IntroductionMethotrexate (MTX) exerts at least part of its anti-inflammatory effects through adenosine receptors (ADOR). The aims of this study were to determine the expression of all four adenosine receptor genes (ADORA1, ADORA2A, ADORA2B, ADORA3 and ADORA3variant) in rheumatoid synovial tissue and any influence of MTX exposure on this expression. Furthermore, we investigated whether polymorphisms within ADORA3 were associated with response and/or adverse effects associated with MTX.MethodsAdenosine receptor gene expression was undertaken using PCR in 20 rheumatoid arthritis (RA) synovial samples. A separate cohort of 225 RA patients receiving MTX was genotyped for SNPs in the ADORA3 receptor gene. Double immunofluorescence was used to identify cells expressing ADOR protein.ResultsAll ADOR genes were expressed in all synovial samples. ADORA3 and A3variant were the dominant subtypes expressed irrespective of MTX therapy. Expression of ADORA2A and ADORA2B was increased in patients receiving MTX compared to those not receiving MTX. There was no association between the ADORA3 rs1544224 SNP and high and low disease activity or MTX-associated adverse effects. ADORA2B protein expression was most obvious in vascular endothelial cells whereas ADORA3 protein was more abundant and expressed by synovial fibroblasts.ConclusionsWe have shown that adenosine receptors are expressed in RA synovium. There is differential expression of receptors such that ADORA3 is expressed at significantly higher levels. This evidence demonstrates the potential for MTX to exert its anti-inflammatory effects at the primary site of pathology within the joints of patients with RA.

The interleukin (IL)-17/IL-23 axis is an important pro-inflammatory pathway in rheumatoid arthritis (RA). IL-23 maintains CD4 þ T-helper 17 (Th 17 ) cells, whereas IL-12 negates IL-17A production by promoting Th 1 -cell differentiation. We sought evidence for any effect of polymorphisms within the interleukin-23 receptor (IL-23R), IL-12 or IL-21 genes on serum cytokine concentrations in 81 patients with RA. Serum cytokines were measured using bead-based multiplex assays. Targeted cytokines were detected in up to 66% of samples. A subgroup of 48 patients had detectable serum IL-17A. Within this subgroup, patients, homozygous for the IL-23R rs11209026 major allele had significantly higher serum IL-17A concentrations compared with patients with the minor allele (394.51 ± 529.72 pg ml --1 vs 176.11 ± 277.32 pg ml --1 ; P ¼ 0.017). There was no significant difference in any of the cytokine concentrations examined in patients positive for the minor allele vs homozygosity for the major allele of IL-12B rs3213337, IL-12Bpro rs17860508 and IL-21 rs6822844. Our results suggest the IL-23R Arg381Gln substitution may influence serum IL-17A concentrations. In patients with the 381Gln allele higher IL-23 concentrations may be needed to produce similar IL-17A concentrations to those in patients with the 381Arg allele. This suggests altered IL-23R function in patients with the minor allele and warrants further functional studies.

Genes involved in the transport, metabolism, and mechanism of action of MTX are expressed in rheumatoid joint synovium. These data provide evidence that MTX has the potential to be polyglutamated within the joint. The higher expression of FPGS compared to GGH in synovial tissue might favor production of long-chain MTX polyglutamates. Thus MTX has the potential to exert its therapeutic effects at the primary site of the inflammatory process in RA.

Abstract. Genomic imprinting confers allele-specific expression in less than 1% of genes, in a parent-of-origin specific fashion. In humans and mice the Peg1/Mest gene (Mest) is maternally repressed, and paternally expressed. Mest is expressed in embryogenic mesoderm-derived tissues and in adult brain, and paternal mutations in Mest lead to growth retardation and defective maternal behaviour. Despite our current understanding of mechanisms associated with the establishment of imprinting of Mest and other imprinted genes, it is unclear to what extent Mest imprinting needs to be maintained in adult tissues. Aberrations of imprinting are known to occur in certain rare syndromes, and involve either inherited mutations, or constitutive epigenetic alterations occurring soon after fertilization. Imprinting abnormalities may also occur in the aging somatic tissues of adult individuals. Here we report an occurrence of post-embryonic somatic variability of Mest allelic expression in a colony of mice where heterozygotes at the imprinted Mest locus for a mutation inherited from the father spontaneously expressed the normally silenced allele from the mother. In addition, a newly acquired ability to overcome the deficit in maternal reproductive behaviour had occurred in the mutant mice, but this appeared not to be directly linked to the Mest mutation. Our results suggest that at least one allele of Mest expression is required in the somatic tissues of adult individuals and that under certain conditions (such as in the presence of a Mest insertional mutation or in an altered genetic background), somatically acquired alterations of allelic expression at the Mest locus may occur. Key words: Adult brain expression, Epigenetic, Imprinting, Maternal behaviour, Somatic expression, Peg1/Mest (J. Reprod. Dev. 58: [490][491][492][493][494][495][496][497][498][499][500] 2012) I n mammals the mesoderm-specific locus, Paternally expressed gene 1 (Peg1/Mest; hereafter called Mest), transcribes a maternally imprinted gene expressed in the mesoderm lineage during embryogenesis, and in adult tissues, such as brain [1]. The precise function of Mest is unknown, although its gene product has similarity to the α/β hydrolase family of proteins [2]. Knock out studies in mice show that Mest is important for proper tissue growth and adult maternal reproductive behaviour [3]. Mest +/tm1Lef heterozygous mutant mice carrying a non-functional paternally inherited mutant allele (denoted here as Mest +/-) display retarded growth, and in females a reduction of their reproductive fitness caused by failure to nurture or care for pups [3], whereas conversely heterozygous mutant mice with the maternally inherited mutant allele Mest tm1Lef/+ (denoted here as Mest -/+ ) are normal, because Mest is a paternally expressed gene.While reproductive fitness is an important trait for species survival, relatively little is known about the role of genomic imprinting in reproductive fitness. Stochastic errors of imprinting or other spontaneous imprinting epimutations, s...

Background Oestrogen receptor-positive (ER+) breast cancer is commonly treated using endocrine therapies such as aromatase inhibitors which block synthesis of oestradiol, but the influence of this therapy on the immune composition of breast tumours has not been fully explored. Previous findings suggest that tumour infiltrating lymphocytes and immune-related gene expression may be altered by treatment with aromatase inhibitors. However, whether these changes are a direct result of impacts on the host immune system or mediated through tumour cells is not known. We aimed to investigate the effect of oestrogen deprivation on the expression of chemokines and immune infiltration in vitro and in an ER+ immunocompetent mouse model. Methods RT-qPCR and a bead-based Bioplex system were used to investigate the expression of chemokines in MCF-7 breast cancer cells deprived of oestrogen. A migration assay and flow cytometry were used to measure the migration of human peripheral blood mononuclear cells (PBMCs) to MCF-7 cells grown without the main biologically active oestrogen, oestradiol. Using flow cytometry and immunohistochemistry, we examined the immune cell infiltrate into tumours created by injecting SSM3 ER+ breast cancer cells into wild-type, immunocompetent 129/SvEv mice. Results This study demonstrates that oestrogen deprivation increases breast cancer secretion of TNF, CCL5, IL-6, IL-8, and CCL22 and alters total human peripheral blood mononuclear cell migration in an in vitro assay. Oestrogen deprivation of breast cancer cells increases migration of CD4+ T cells and decreases migration of CD11c+ and CD14+ PBMC towards cancer cells. PBMC migration towards breast cancer cells can be reduced by treatment with the non-steroidal anti-inflammatory drugs, aspirin and celecoxib. Treatment with endocrine therapy using the aromatase inhibitor letrozole increases CD4+ T cell infiltration into ER+ breast cancer tumours in immune competent mice. Conclusions These results suggest that anti-oestrogen treatment of ER+ breast cancer cells can alter cytokine production and immune cells in the area surrounding the cancer cells. These findings may have implications for the combination and timing of anti-oestrogen therapies with other therapies.

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Background: Previous analysis has revealed that high pre-treatment expression of an inflammatory gene expression signature and lymphocytic infiltration are correlated with poor response to neoadjuvant aromatase inhibitor (AI) treatment1. Upon treatment with AIs, inflammatory gene expression also increases1. These changes are also seen in vitro where an increase in chemokine secretion has been observed upon oestrogen deprivation, suggesting that treatment with anti-oestrogen therapy may modulate the immune response. In addition, non-steroidal anti-inflammatory drug (NSAID) intake is associated with a significantly decreased risk of disease recurrence and breast cancer related death. In this study we set out to investigate the immunological response to AI treatment alone and in combination with NSAID treatment in an animal model of estrogen receptor positive (ER+ve) breast cancer. Methods: A syngeneic immunocompetent murine ER+ breast tumour model was used in which SSM3 ER+ BrCa cells were injected into the mammary pad of wild-type 129SvEv mice. Once tumours reached a size of 49 mm2, mice were ovariectomised and treated with combinations of letrozole, celecoxib, aspirin and vehicle control. Tumours were measured for 25 days following treatment and tumours were analysed using flow cytometry and immunohistochemistry. Results: Treatment with letrozole alone and letrozole + celecoxib resulted in a significant reduction tumour size after 25 days of treatment (p<0.001). Examination of the immune cell infiltrate of these tumours using flow cytometry determined that combination letrozole and celecoxib resulted in decreased numbers of CD11b+ cells compared to vehicle treated tumours (p = 0.02), and to celecoxib only treated tumours (p = 0.008). Working within recommended guidelines for assessing stromal TILs in H&E sections in a larger cohort (n=51), we determined that the percentage of stromal TILs increased in all treatment groups compared to the pre-treatment biopsy, and that the combination treatment had lower levels of TILs compared to vehicle treated tumours (p = 0.02). Conclusions: We conclude that treatment of ER+ BrCa with letrozole induces an immune response in the tumour microenvironment which can be modulated by the addition of an NSAID. Our findings suggest that modulation of the immune response to endocrine therapy may have the potential to enhance response to treatment. References: 1. Dunbier et al., 2013. Clin Cancer Res. 19:2775-86. Citation Format: Anita K Dunbier, Jody A Hazlett, Katelyn Powell, Ginny Niemi, Roslyn Kemp. Investigation of combination treatment an aromatase inhibitor and anti-inflammatory treatment in a model of oestrogen receptor positive breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-03-07.