ABSTRACT
PURPOSE: Cancers have developed a number of strategies to escape immune responses including the differential expression of costimulatory molecules of the B7 family. B7-H3 and B7-H4 have recently been described in different tumor entities but the relevance for melanoma has not yet been studied so far. EXPERIMENTAL DESIGN: Using immunohistochemistry, B7-H3 and B7-H4 expression was studied on 29 melanoma lesions. Survival curves and log-rank tests were used to test the association of protein expression with survival. Cell lines were evaluated for B7-H3 and B7-H4 expression by PCR and flow cytometry. Functional T-cell-tumor coculture assays were carried out with in vitro generated tumor transfectants. RESULTS: B7-H3 and B7-H4 expression was detected in primary tumor lesions (29 of 29 and 28 of 29) and in metastases (28 of 29 and 26 of 29). The numbers of CD68(+) macrophages were significantly lower in patients with low B7-H4 expression, whereas CD8(+) T-cell infiltrates were independent of expression levels. Furthermore, a survival benefit for patients with B7-H4 low expressing melanoma was found, whereas B7-H3 was not associated with any clinical parameter. All 23 melanoma cell lines analyzed expressed B7-H3 and B7-H4 mRNA and protein, but B7-H4 was restricted to intracellular compartments. On silencing of B7-H3 by specific shRNA tumor-associated antigen-specific T cell responses were unaltered. Overexpression of B7-H4 on melanoma cells did not alter the cytotoxicity of different CD8(+) effector cells, but drastically inhibited cytokine production. CONCLUSIONS: Our study provides for the first time evidence of B7-H4 expression on melanoma cells as a mechanism controlling tumor immunity which is associated with patients' survival.
Subject(s)
B7-1 Antigen/genetics , Immunity, Innate/genetics , Melanoma/immunology , Melanoma/mortality , Skin Neoplasms/immunology , Skin Neoplasms/mortality , Adult , Aged , Aged, 80 and over , B7-1 Antigen/metabolism , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Melanoma/diagnosis , Melanoma/genetics , Middle Aged , Prognosis , Retrospective Studies , Skin Neoplasms/diagnosis , Skin Neoplasms/genetics , Survival Analysis , V-Set Domain-Containing T-Cell Activation Inhibitor 1ABSTRACT
ZDHHC9 (zinc finger, DHHC-type containing 9) is a protein acyl transferase involved in palmitoylation of basic signaling molecules. We found ZDHHC9 expression increased in hind leg muscles of newborn splay leg piglets. In order to elucidate the background of this increased expression we determined the structure of the porcine gene, including sequence variation, and analyzed the structure and expression of microRNAs potentially targeting the gene. We confirmed the expression results by RT Real-time PCR. The porcine ZDHHC9 gene has a similar structure to the human gene with two transcripts resulting in an identical protein. None of the 17 single nucleotide polymorphisms (SNPs) identified in the porcine gene affects the protein or putative microRNA binding sites, respectively. Two microRNAs (93 [minor] and 106b) were assayed in the muscles. Their expression variation proved to be independent from ZDHHC9 expression thus eliminating them as causally related to congenital splay leg.
Subject(s)
Acyltransferases/genetics , Hindlimb/metabolism , MicroRNAs/physiology , Muscle, Skeletal/metabolism , Swine Diseases/metabolism , Animals , Gene Expression Regulation , Hindlimb/pathology , Humans , MicroRNAs/genetics , Polymorphism, Single Nucleotide/genetics , Reverse Transcriptase Polymerase Chain Reaction , SwineABSTRACT
The congenital splay leg syndrome in piglets is characterized by a temporarily impaired functionality of the hind leg muscles immediately after birth. Etiology and pathogenetic mechanisms for the disease are still not well understood. We compared genome wide gene expression of three hind leg muscles (M. adductores, M. gracilis and M. sartorius) between affected piglets and their healthy littermates with the GeneChip Porcine Genome Array (Affymetrix) in order to identify candidate genes for the disease. Data analysis with standard algorithms revealed no significant differences between both groups. By application of an alternative approach, we identified 63 transcripts with differences in two muscles and 5 genes differing between the groups in three muscles. The expression of six selected genes (SQSTM1, SSRP1, DDIT4, ENAH, MAF, and PDK4) was investigated with SYBRGreen RT-Real time PCR. The differences obtained with the microarray analysis could be confirmed and demonstrate the validity of the alternative approach to microarray data analysis. Four genes with different expression levels in at least two muscles (SQSTM1, SSRP1, DDIT4, and MAF) are assigned to transcriptional cascades related to cell death and may thus indicate pathways for further investigations on congenital splay leg in piglets.
