Foxp3 Expression In Melanoma Cells As A Novel Mechanism Of Immune Escape

Malignant melanoma, a great number arising from epidermis melanocyte of the nevus and stains, is the most invasive and deadly form of skin cancer at present. The increase of the attack rate of melanoma is the fast in all malignant tumor, and the annual incremental rate is about 3-5%. Although the attack rate of melanoma of Nordic is far more than the else Races, the attack rate of China, which is low in the past, is increasing rapidly for the past few years. Now, the attack rate of part of China, Beijing and Shanghai, is almost one per hundred thousand, and the age of onset is becoming younger. Because of being largely refractory to existing therapies, metastatic melanoma has a very poor prognosis and the 5-year survival rate is less than 5%. Cancer vaccines and adoptive immune cells transfer have been shown to increase the frequency of tumor antigen-specific T cells, but these approaches only cause clinical tumor regression in a few patients. These observations indicate that tumor resistance to immune destruction may dominate in individual patients, in which CD4+CD25+T regulatory cells (Tregs) play a key role in maintenance the tumor resistance to immune destruction. The immune system is subject to many levels of control, and the Tregs make a major contribution to such regulation. Tregs not only prevent autoimmunity to maintain the immunotolerance, but also have a negative effect on health by down-regulating beneficial immune responses, such as those mounted against tumors. Tregs are characterized by constitutive expression of CD25 and the Foxp3. Tregs control the properties of other immune cells by a cell contact-dependent mechanism and secreting suppressive cytokines such as TGF-βand IL-10. Furthermore, several reports have documented the presence of Treg within human tumor tissues, the frequency of which may suppress the anti-tumor attack and negatively correlate with survival. A combination of depletion or attenuation of Tregs and concomitant stimulation of effector T cells may provoke effective anti-tumor attack and produce tumor regression.Foxp3 is the only marker of Treg, which seems to act as a"master switch"for the development and function of Treg. Mice and human lacking functional expression of Foxp3 completely lack Treg and develop severe autoimmunity. Moreover, ectopic expression of Foxp3 in conventional T cells endows them with the part or all phenotype and function of Treg. Until recently, Foxp3 expression has been thought to be restricted to the T-cell lineage. In the present study, however, a report has provided evidence that Foxp3 is expressed in pancreatic carcinoma cells and the pancreatic carcinoma cells prevent the immune destroy by mimicking Treg function. We propose that Foxp3 may be expressed in melanoma cells and participate in maintenance the tumor resistance to immune attack.We have done some experiments to identify our hypothesis. Objective: To identify whether Foxp3 is expressed in melanoma cells and participate in maintenance the tumor resistance to immune attack. Methods: Detection of Foxp3 mRNA is performed using RT-PCR in melanoma cell lines and melanocytes, while protein expression is assessed by western blot and flow cytometry. The subcellular distribution of the Foxp3 molecule is assessed by confocal microscopy. Sections of resected human melanoma and nevus specimens are stained with anti human Foxp3 antibody. The Foxp3 staining intensity of the melanoma was assessed to analyze the relationship between expression of Foxp3 and Ki67. The contrast study is performed between the Foxp3 silenced and overexpressed melanoma cells. We have detected the expression of the immune inhibitory molecules via realtime RT-PCR, western blot and ELISA. Electron microscope is used to observe the cell morphous and cellular organ. MTT assay and flow cytometry is separately used to evaluate the proliferation and cell cycles. Coculture with mixed peripheral blood lymphocytes or T cell is used to assess the inhibiting proliferation effect of the Foxp3 silenced and overexpressed melanoma cells. Result: The Foxp3 mRNA and protein is detected in melanoma cell lines, but not melanocytes. The subcellular distribution of the Foxp3 molecule is confirmed in the nucleus and cytoplasm of the melanoma cell. Immunohistochemistry analysis of melanoma and nevus tissues reveal that subtotal of melanoma displayed nucleus and cytoplasm Foxp3 staining, but not nevus. And the Foxp3 staining intensity is significantly associated with Ki67 staining intensity. The contrast study, performed between the Foxp3 silenced and overexpressed melanoma cells, has revealed that the proliferation of the latter is down-regulated. Realtime RT-PCR and Western blot showes that the expression of the immune inhibitory molecules, such as B7-H1, TGF-β1 and TGF-β2, are up-regulated in the Foxp3 overexpressed melanoma cells. ELISA assay also find that TGF-β1 and TGF-β2 are higher in supernatant of culture medium of the Foxp3 overexpressed melanoma cells. Cocultured with the Foxp3 overexpressed melanoma cells, the proliferation of lymphocytes is down-regulated. The results of the Foxp3 silenced melanoma cells are contrary. Conclusion: Foxp3 is expressed in melanoma cell lines and melanoma tissues, but not in melanocytes and nevus. The overexpression of Foxp3 endows melanoma cells the up-regulatory expression of immune suppressive molecules, and the stronger capability to repress the cell proliferation of lymphocytes.To sum up, we could hypothesis that melanoma cells expressing Foxp3 may represent a novel mechanism by which melanoma cells suppress the immune system to escape destruction. Here is just the beginning of Foxp3 function exploration in melanoma,and it need further study. And perhaps, these findings could provide a target and some effective strategies to clinical therapeutics.