ABSTRACT
Infection with high-risk types of human papillomavirus (HPV) can cause the development of malignant tumors. To study mechanisms responsible for immune escape of tumor cells infected with HPV16, we previously used mouse oncogenic TC-1 cells producing HPV16 E6 and E7 oncoproteins to derive TC-1 clones resistant to immunization against E7. We have found immunoresistance of the clones to correlate with the point mutation in the E7 oncogene, which resulted in the N53S substitution in the immunodominant epitope RAHYNIVTF (aa 49-57). Here, we have shown that this mutation reduced stabilization of H-2D(b) molecules on RMA-S cells and eliminated immunogenicity of E7. The resistance of TC-1 clones was E7-specific as immunization against E6 inhibited tumor growth. Transduction of the TC-1/F9 clone carrying the mutated epitope with the wild-type E7 gene restored susceptibility to immunization against E7. Our results suggest that mutagenesis of tumor antigens can lead to the escape of malignant cells and should be considered in the development and evaluation of cancer immunotherapy.
Subject(s)
Immunodominant Epitopes/chemistry , Immunotherapy/methods , Neoplasms/immunology , Neoplasms/therapy , Oncogene Proteins, Viral/chemistry , Oncogene Proteins, Viral/genetics , Animals , Antigens, Neoplasm/chemistry , Epitopes/chemistry , Female , H-2 Antigens/chemistry , Histocompatibility Antigen H-2D , Humans , Mice , Mice, Inbred C57BL , Mutation , Neoplasms/metabolism , Oncogene Proteins/chemistry , Papillomavirus E7 Proteins , Point MutationABSTRACT
A better understanding of the molecular basis of tumor progression and invasion is needed to improve therapy for malignant tumors. Recently, we established a mouse metastatic MK16 model by transduction of secondary kidney cells with human papillomavirus type 16 (HPV16) E6 and E7 oncogenes and human H-ras activated by G12V mutation. In this study, we extended the model to MK16 cell lines derived from lung metastases and compared the oncogenicity of seven cell lines successively isolated from primary tumors or metastases. By observing the formation and growth of subcutaneous tumors and generation of lung metastasis, we showed a gradual increase in oncogenicity of MK16 cell lines. Interestingly, we demonstrated metastatic potential of MK16/A cells with low oncogenic potential in primary tumor development. To detect changes in gene expression associated with increasing oncogenicity of MK16 cell lines, we performed transcriptional profiling with the Atlas Plastic Mouse 5K microarray. We found that a substantial proportion of up-regulated genes encoded ribosomal proteins. Among the down-regulated genes, the highest number (n=10) belonged to a group coding for transcription factors. Expression of two of these, Pou3f2 and Gtl3, was reduced both in cells derived from primary tumors and those isolated from metastases. Furthermore, microarray hybridization suggested that the down-regulation of cyclin-dependent kinase inhibitors p16(Ink4a) and p57(Kip2) and up-regulation of A6 and A10 members of the S100 protein family might play a role in the increase of MK16 oncogenicity.
Subject(s)
Gene Expression Profiling , Neoplasms, Experimental/genetics , Animals , Cell Line, Transformed , Cell Transformation, Neoplastic/genetics , Cell Transformation, Viral/genetics , Disease Progression , Female , Genes, ras/genetics , Humans , Mice , Mice, Inbred C57BL , Neoplasm Transplantation , Neoplasms, Experimental/pathology , Oligonucleotide Array Sequence Analysis/methods , Oncogene Proteins, Viral/genetics , Papillomavirus E7 Proteins , Repressor Proteins/genetics , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction/methods , Tumor Cells, CulturedABSTRACT
Escape of tumor cells from the host immune system is probably the most difficult obstacle to overcome in attempts to enhance the efficacy of immunotherapy of tumors. To solve the problem, animal models with escape mechanisms found in human tumors are needed. We have already established cell lines with substantially reduced expression of MHC class I molecules that were derived from oncogenic TC-1 cells producing E6 and E7 oncoproteins of HPV16. In this study, we prepared other cell lines from TC-1-induced tumors formed infrequently in mice immunized against the E7 antigen. These clones differed in morphology and both in vitro and in vivo growth properties. After vaccination with the highly efficient anti-E7 DNA vaccine several clones appeared to be quite resistant to induced anti-tumor immunity. Detection of the production of the MHC class I molecules and B7.1 costimulatory molecule did not provide clues to understand the mechanism of immunoresistance of the clones. Therefore, we performed transcriptional profiling using Atlas Mouse Cancer 1.2 Array (BD Clontech). Among the genes differently expressed in the examined cell lines were those of three cytokines influencing immune cells: MCP-1, osteopontin, and midkine. Altered secretion of MCP-1 chemokine was verified by ELISA. In addition, expression of the E7 oncogene was reduced in all TC-1 clones. Most importantly, one of anchor amino acids in the immunodominant epitope of E7 was mutated in all immunoresistant clones. Such mutations of HPV oncogenes must be considered in therapeutic-vaccine development and evaluation.
