| Prostate cancer is the most prevalent cancer in men.Surgical and medical androgen deprivation therapy(ADT)remains the cornerstone for prostate cancer treatment,but relapse usually occurs.Immunotherapy is one of the most important anti-tumor treatments,which gets into spotlight over the past decade.Therefore,a proper combination therapy involoving ADT and immunotherpay could promote the immunotherapy-initiated anti-tumor immune response and suggest a new clinical solution for tumor control.Here,we used Myc-CaP,an established transplantable prostate tumor,to study the role of ADT in the regulation of immune response.Our previous work showed that the ADT by castration is initially effective.However,resistance eventually occurs.We combined immunotherapy with ADT in Myc-CaP model and observed that medical ADT with androgen receptor(AR)antagonists unexpectedly has a negative effect on the host immune response,compromising the potential synergistic effects of the combination therapy involving immunotherapy and conventional method.We used a herpes simplex virus-1 mouse model and the ovalbumin as model antigen to investigate the effect of AR antagonists on infectious disease processes and on adaptive immune response(including both humoral and cellular immune response).B16-human EGFRhigh cell line is also used to further study whether the non-steroidal AR antagonist,flutamide-induced immune-suppression depends on the AR pathway.We observed that AR antagonists themselves could broadly dysregulate the host immune response through an androgen-independent pathway.We observed that non-steroidal AR antagonists significantly inhibited IFN-γ production,whereas IFN-γ can be produced by either T cells or antigen-presenting cells(APCs),these responses in unfractionated splenocytes could reflect a direct effect on either T cells,APCs,or both.To determine which cells are directly responsible for this phenotype,we separately tested T cells,APC and B cells.Purified T cells exhibited diminished production of IFN-γ in response to non-steroidal AR antagonists with anti-CD3 and anti-CD28 stimulation.However,splenocytes taken from Rag1-/-mice,which contain only innate immune cells and no adaptive lymphocytes,were directly stimulated with LPS.In contrast to T cells,the AR antagonists showed no significant direct effects on the production of IFN-γ and TNF-α in Rag1-/-mice splenocytes,indicating that non-steroidal AR antagonists may not severely affect APCs function.Moreover,non-steroidal AR antagonists did not affect the B cell proliferation as well.Since it is reported that non-steroidal AR antagonists,including flutamide and enzalutamide,have an off-target mechanism through inhibiting GABA-A currents.AR antagonist-mediated T cell impairment may in some degree develop through a GABA-A receptor pathway on T cells.To further confirm our demonstration we used not only GABA antagonist bicuculline to reduce flutamide-mediated T cell suppression in vivo but also newly added a GABA-A receptor Cl-channel blocker picrotoxin to completely abolish the flutamide-induced IL-2 and IFN-in vitro.To determine the signaling pathway for mechanisms of suppression,we studied T cell receptor-induced activation of p38-MAPK,JNK,ERK1/2,and nuclear factor of activated T cells(NFAT)pathways in in vitro assays.We found that activation-associated phosphorylation of JNK,ERK,p38-MAPK,was not affected by flutamide,indicating that MAPK signaling pathway is normal in flutamide treated T cells.In contrast,NFAT c2 dephosphorylation,a critical step required for NFAT nuclear translocation was significantly delayed in flutamide treated T cells compared to anti-CD3,anti-CD28 stimulated wild type T cells.This demonstration is consistent with our observation that flutamide leads to IL-2 and IFN-γ reduction in activated T cells.To thoughtfully consider an alternative to medical ADT that does not suppress T cell response,we explored whether abiraterone,a steroidal compound that interferes androgen synthesis,could synergize with immunotherapy by avoiding off-target GABA-A effect of AR antagnists.We treated Myc-Cap tumor bearing mice with a new combination of abiraterone and CpG,comparing its anti-tumor effect with single treatment,and observed that the combination treatment significantly accelerated tumor regression at low dose levels and prolonged tumor-free survival at high dose levels.In conclusion,we have demonstrated that drug resistance might be associated with an off-target immune suppression by some AR antagnists through the GABA-A pathway.We therefore could develop an optimal combination therapy protocol to better control prostate tumor growth and increase survival. |
