Project description:The generation of highly-reactive T lymphocytes against tumor-associated antigens remains an obstacle for effective adoptive immunotherapy of cancer. Recently, we found that microRNA-181a (miR-181a) acts as an intrinsic modulator of T cell antigen sensitivity in a transgenic T cell line in vitro. This molecule is part of a growing family of evolutionarily selected small interfering RNA that control gene expression at the posttranscriptional level by targeting messenger RNA (mRNA) for degradation or translational repression. Here, we explored the use of miR-181a to improve anti-tumor T cell responsiveness against weakly immunogenic tumor-associated antigens. We found that genetic engineering of T lymphocytes with miR-181a dramatically augmented the function of poorly responsive human tumor-infiltrating lymphocytes and TCR-engineered peripheral blood lymphocytes, resulting in potent anti-tumor reactivity. Furthermore, in a mouse model, miR-181a increased the function of self/tumor-specific CD8+ T cells enabling effective tumor destruction in the absence of vaccination or exogenous cytokines that were otherwise essential requirements and thus represents a significant advance over previous approaches. Although miRNAs have been previously shown to play critical functional roles in the development and function of vertebrate immune systems and pathogenesis of cancer, this report comprises the first use of a miRNA gene as tool in the treatment of disease. Keywords: cellular modification design For the 6 mouse samples the Operon Array-Ready Oligo Set (AROSâ¢) V 4.0 contains 35,852 longmer probes representing 25,000 genes and about 38,000 gene transcripts and also includes 380 controls platform was used and each sample was hybridized in duplicate. For the 6 human samples, the human cDNA microarray platform was used which consist of 17 k cDNA array included a combination from a RG_HsKG_031901 7 k clone set and 10,000 clones from the RG_Hs_seq_ver_070700 40 k clone set (Research Genetics, Huntsville, AL). The cDNA clones include 12,072 uniquely named genes, 875 duplicates of named genes.

Project description:Vitamin D may have anti-tumorigenic actions by influencing the gene expression profile of target tissues, which possess vitamin D receptors. We used a more physiological in vitro model, represented by short-term culture of breast cancer tissue slices, to study the transcriptional effects of a near physiological concentration of calcitriol. Methods. Breast cancer fragments were sliced and maintained in culture for 24 hours in the presence or absence (control) of calcitriol 0.5nM or 100nM (called physiological or supra-physiological concentrations). Five and 16 samples were included in a test or validation group and gene expression was analyzed by microarray (using SAM paired analysis) or qPCR, respectively. Results. Nine genes were regulated by calcitriol 0.5nM including CYP24A1, DPP4, EFTUD1 (FDR£0.01), which were up-modulated. However, using a less stringent FDR value (0.25) 61 genes were differentially expressed. Among the down-regulated transcripts were members of the MHC class II complex (HLA-DPA1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3) and IgG binding (FCGR2A, FCGR2B, FCGR2C). There was an enrichment of genes presenting transcription factor binding sites for vitamin D, among the up-regulated genes and for interferon regulatory factor IRF1, among the down-regulated genes. Analyzing calcitriol supra-physiological effects, a more impressive transcriptional modulation was identified and 136 and 60 genes (FDR 0.1) were more and less expressed in treated samples. Up-regulated genes were involved in vitamin metabolic process, regulation of leukocyte-mediated immunity and positive regulation of alpha-beta T cell activation. Many of the induced genes were already reported as vitamin D responsive genes, including CD14, which was also up-regulated in another set of 16 samples. Genes modulated by both calcitriol concentrations were CYP24A1, DPP4, CA2 (these three in both sets of samples), EFTUD1, TKTL1, KCNK3. Conclusion. Small increments in calcitriol concentration, within the physiological range, for a relatively short period of time may exert transcriptional effects in breast cancer samples. Further studies employing physiological concentrations of vitamin D for longer periods of time may help to elucidate the hormone effects in breast cancer treatment and prevention. Breast cancer fragments were sliced and maintained in culture for 24 hours in the presence or absence (control) of calcitriol 0.5nM or 100nM (called physiological or supra-physiological concentrations). Five samples were included and were categorized according to treatment in three groups: control (A), 1,25(OH)2D3 0.5nM (B) and 1,25(OH)2D3 100nM (C).Total RNA was isolated using RNeasy kit and than was carried out according to microarray Affymetrix protocol. Data was then assessed with GeneSpring X software for background correction, normalization and summarization of raw data (CEL files) using the Robust Multi-Array Average (RMA). Five samples were included in a test and gene expression was analyzed by microarray (using SAM paired analysis). To establish a differential gene expression profile between vitamin D treated and untreated samples, SAM two class paired, provided on MEV (MultiExperiment Viewer – Boston, MA, USA) was used. Unsupervised hierarchical clustering based on Euclidean distance and average linkage was used to verify association patterns. The reliability of the clustering was assessed by the Bootstrap technique using MEV (MultiExperiment Viewer – Boston, MA, USA). Samples (16) were included in a validation group and was analyzed by qPCR.

Project description:Vitamin D may have anti-tumorigenic actions by influencing the gene expression profile of target tissues, which possess vitamin D receptors. We used a more physiological in vitro model, represented by short-term culture of breast cancer tissue slices, to study the transcriptional effects of a near physiological concentration of calcitriol. Methods. Breast cancer fragments were sliced and maintained in culture for 24 hours in the presence or absence (control) of calcitriol 0.5nM or 100nM (called physiological or supra-physiological concentrations). Five and 16 samples were included in a test or validation group and gene expression was analyzed by microarray (using SAM paired analysis) or qPCR, respectively. Results. Nine genes were regulated by calcitriol 0.5nM including CYP24A1, DPP4, EFTUD1 (FDR£0.01), which were up-modulated. However, using a less stringent FDR value (0.25) 61 genes were differentially expressed. Among the down-regulated transcripts were members of the MHC class II complex (HLA-DPA1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3) and IgG binding (FCGR2A, FCGR2B, FCGR2C). There was an enrichment of genes presenting transcription factor binding sites for vitamin D, among the up-regulated genes and for interferon regulatory factor IRF1, among the down-regulated genes. Analyzing calcitriol supra-physiological effects, a more impressive transcriptional modulation was identified and 136 and 60 genes (FDR 0.1) were more and less expressed in treated samples. Up-regulated genes were involved in vitamin metabolic process, regulation of leukocyte-mediated immunity and positive regulation of alpha-beta T cell activation. Many of the induced genes were already reported as vitamin D responsive genes, including CD14, which was also up-regulated in another set of 16 samples. Genes modulated by both calcitriol concentrations were CYP24A1, DPP4, CA2 (these three in both sets of samples), EFTUD1, TKTL1, KCNK3. Conclusion. Small increments in calcitriol concentration, within the physiological range, for a relatively short period of time may exert transcriptional effects in breast cancer samples. Further studies employing physiological concentrations of vitamin D for longer periods of time may help to elucidate the hormone effects in breast cancer treatment and prevention.

Dataset Information

Therapeutic Use of Vitamin C in Cancer: Physiological Considerations.

Publications

Therapeutic Use of Vitamin C in Cancer: Physiological Considerations.

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