The physiologic relevance of the IFN-γ–dependent JAK/STAT pathway was characterized by the functional analysis of JAK/STAT knockout mice and has been linked to anti-tumor responses (23
). IFN-γ can directly act on tumor cells by exerting antiproliferative, proapoptotic, and antiangiogenic effects (24
). STATs and JAKs are thought to play a role in promoting these IFN-γ effects on tumor cells, and defects of the JAK/STAT signal transduction intermediates have been associated with an IFN-γ–resistant phenotype in lung carcinoma and melanoma cells (3
). This might provide tumor cells with a selective growth advantage. Indeed, more than 30% of human tumors exhibited unresponsiveness or reduced sensitivity to IFN-γ associated with tumor progression. The variable IFN-γ responsiveness of melanoma cells could be (i) associated with a lower capacity of IFN-γ to induce JAK/STAT (12
) or (ii) mediated by downstream components or an additional signaling pathway (28
) or (iii) due to lack of STAT1 phosphorylation and epigenetic silencing of the IRF1 transactivation (14
Although abnormalities of HLA class I APM components represent one major mechanism of tumor cells to evade immune surveillance, there exist only limited information about the role of deficient IFN signal transduction in the regulation of these immunomodulatory molecules. Therefore, the constitutive and IFN-γ–inducible expression pattern of several components of the HLA class I APM and the IFN signal transduction pathway was determined in a number of melanoma cells. With the exception of Colo 857 cells, the other 7 melanoma cell lines analyzed constitutively expressed heterogeneous levels of JAK2 whereas the other IFN signal transduction molecules were constitutively expressed and upregulated by IFN-γ in these tumor cells. Although genetic abnormalities of the IFN-γ signal pathway such as mutations, deletions, and recombinations have been described in tumors of distinct origin (29
), no structural alterations in these molecules have been yet reported in melanoma. In this context, it is noteworthy that resistance of melanoma cells to IFN-α is due to multiple defects in the type I IFN signal transduction pathway including lack of Tyk2 (tyrosine kinase 2; ref. 30
Our results showed that loss of JAK2 expression in the melanoma cell line Colo 857 was caused by a deletion of the JAK2 gene, which is accompanied by a defective IFN-γ signaling and lack of the IFN-γ–mediated HLA class I inducibility. Even more important, impaired JAK2 expression significantly downregulated the constitutive mRNA and protein levels of HLA class I APM components despite a functional APM pathway, thereby providing a selective advantage to tumors. However, this was neither mediated by loss of the IFN-γ R, as Colo 857 cells express this receptor, nor by abnormalities of components of the HLA class I APM. The latter was confirmed by the induction of HLA class I APM molecules, which was accompanied by an upregulation of HLA class I surface expression in these cells on TNF-α and IFN-α treatment, respectively. In addition, JAK2 is required for IFN-γ–induced growth inhibition, as Colo 857 cells lacking JAK2 were not growth inhibited by IFN-γ in contrast to JAK2+ melanoma cells such as Colo 794. This loss of growth-restraining functions might influence tumor progression of JAK2− cells, which will be investigated in future studies.
To confirm the importance of a functional IFN-γ signaling for constitutive HLA class I APM component expression, JAK2 expression was restored in the JAK2−
melanoma cells Colo 857 by stable transfection by using a JAK2-specific expression vector. JAK2
gene transfer into JAK2−
Colo 857 cells increased the constitutive HLA class I APM component and surface antigen expression. Furthermore, functional JAK2 restored IFN-γ inducibility of HLA class I APM components. Thus, there exists a direct link between abnormalities of HLA class I antigen processing and presentation molecules and impaired JAK2 function. These might also result in reduced CTL sensitivity but increased susceptibility to natural killer cell–mediated lysis. Although a positive correlation between JAK2 and HLA class I antigens has been proven in this study for the first time, a recent publication has shown an improved patient survival when tumors expressed high MHC class I and STAT1 levels in association with a broad T-cell infiltrate (31
). Furthermore, loss of STAT1 signaling has been shown to be associated with a higher incidence of tumors in mice (23
). These results strengthen our hypothesis of an important role of a functional IFN-γ signal cascade for the immunosurveillance of tumor cells.
Owing to the complexity of the IFN-γ signal transduction pathway, a comprehensive explanation how and at which level other elements of the IFN-γ system besides JAK2 and STAT1 modulate HLA class I APM component expression is still awaiting. Because JAK2 is a key regulator of IFN-γ responses and is induced by other growth factors and DNA damage, tumors acquiring resistance to IFN-γ by dysregulation or structural alterations of JAK2 might evade the immunosurveillance leading to tumor progression; vice versa, an impaired IFN-γ signaling in association with a reduced HLA class I APM component expression pattern suggests that defects in the IFN-γ cascade might play a crucial role in the malignant transformation process and might be involved in the frequent development of immune escape phenotypes caused by HLA class I APM component abnormalities. This is further supported by cDNA microarray analysis of different tumors showing an altered expression of IFN signaling molecules. In conclusion, the present study identified for the first time that a deletion of JAK2 in melanoma cells or inhibition of JAK2 signaling caused an impaired MHC class I APM component expression, suggesting that abnormalities of the JAK/STAT pathway might play an important role during development of melanoma. In addition, these results underscore the biological significance of JAK2 for IFN-γ treatment-independent immunosurveillance and provide evidence that defects in JAK2 might represent a novel immune escape mechanism and a potential target in combination with T-cell–based therapies. Further experiments are currently been carried out to validate these data in a large series of tumor lesions by using a tissue microarray and associate them with the survival of melanoma patients.