Src promotes castration-recurrent prostate cancer through androgen receptor-dependent canonical and non-canonical transcriptional signatures.

Progression of prostate cancer (PC) to castration-recurrent growth (CRPC) remains dependent on sustained expression and transcriptional activity of the androgen receptor (AR). A major mechanism contributing to CRPC progression is through the direct phosphorylation and activation of AR by Src-family (SFK) and ACK1 tyrosine kinases. However, the AR-dependent transcriptional networks activated by Src during CRPC progression have not been elucidated. Here, we show that activated Src (Src527F) induces androgen-independent growth in human LNCaP cells, concomitant with its ability to induce proliferation/survival genes normally induced by dihydrotestosterone (DHT) in androgen-dependent LNCaP and VCaP cells. Src induces additional gene signatures unique to CRPC cell lines, LNCaP-C4-2 and CWR22Rv1, and to CRPC LuCaP35.1 xenografts. By comparing the Src-induced AR-cistrome and/or transcriptome in LNCaP to those in CRPC and LuCaP35.1 tumors, we identified an 11-gene Src-regulated CRPC signature consisting of AR-dependent, AR binding site (ARBS)-associated genes whose expression is altered by DHT in LNCaP[Src527F] but not in LNCaP cells. The differential expression of a subset (DPP4, BCAT1, CNTNAP4, CDH3) correlates with earlier PC metastasis onset and poorer survival, with the expression of BCAT1 required for Src-induced androgen-independent proliferation. Lastly, Src enhances AR binding to non-canonical ARBS enriched for FOXO1, TOP2B and ZNF217 binding motifs; cooperative AR/TOP2B binding to a non-canonical ARBS was both Src- and DHT-sensitive and correlated with increased levels of Src-induced phosphotyrosyl-TOP2B. These data suggest that CRPC progression is facilitated via Src-induced sensitization of AR to intracrine androgen levels, resulting in the engagement of canonical and non-canonical ARBS-dependent gene signatures.

The immunogenicity of L1210 lymphoma clones correlates with their ability to function as antigen-presenting cells.

Major histocompatibility complex class II (MHCII) antigen expression is directly correlated with immunogenicity, and inversely correlated with tumorigenicity, in clones of the L1210 murine B lymphoma. Moreover, loss of MHCII expression on human diffuse large B-cell lymphoma is associated with dramatic decreases in patient survival. Thus, the role that MHCII antigens play in the progression of B-cell lymphomas is clinically important. In this study, we investigated the basis for the immunogenicity of MHCII(+) L1210 clones. Immunogenic, but not tumorigenic L1210 clones stimulated the proliferation of naïve T cells and their interleukin (IL)-2 production, which indicates that the immunogenic clones can function as antigen-presenting cells (APCs). However, subclonal variants of the immunogenic L1210 clones, which form tumours slowly in mice, could not activate T cells. The costimulatory molecules B7-1, B7-2 and CD40 were expressed on the immunogenic L1210 clones, but not the tumorigenic clones. Importantly, the tumour-forming subclonal variants expressed MHCII and B7-1, but lacked B7-2 and CD40. These results suggest that MHCII and B7-1 expression on L1210 cells is insufficient to activate naïve T cells, and, furthermore, loss of B7-2 and/or CD40 expression contributes to the decreased immunogenicity of L1210 subclones. Blocking B7-1 or B7-2 function on immunogenic L1210 cells reduced their capacity to activate naïve T cells. Furthermore, incubation of immunogenic L1210 cells with CD40 antibodies significantly enhanced APC function. Therefore, the immunogenicity of L1210 cells directly correlates (i) with their ability to stimulate naïve T cells, and (ii) with the concomitant expression of MHCII, B7-1, B7-2, and CD40.

Histone deacetylases inhibit IFN-gamma-inducible gene expression in mouse trophoblast cells.

Trophoblast cells are the first cells to differentiate from the developing mammalian embryo, and they subsequently form the blastocyst-derived component of the placenta. IFN-gamma plays critical roles in activating innate and adaptive immunity, as well as apoptosis. In mice, IFN-gamma is produced in the pregnant uterus, and is essential for formation of the decidual layer of the placenta and remodeling of the uterine vasculature. Responses of mouse trophoblast cells to IFN-gamma appear to be selective, for IFN-gamma activates MHC class I expression and enhances phagocytosis, but fails to activate either MHC class II expression or apoptosis in these cells. To investigate the molecular basis for the selective IFN-gamma responsiveness of mouse trophoblast cells, IFN-gamma-inducible gene expression was examined in the trophoblast cell lines SM9 and M-11, trophoblast stem cells, and trophoblast stem cell-derived giant cells. IFN-gamma-inducible expression of multiple genes, including IFN regulatory factor-1 (IRF-1), was significantly reduced in trophoblast cells compared with fibroblast cells. Decreased IRF-1 mRNA expression in trophoblast cells was due to a reduced rate of IRF-1 transcription relative to fibroblast cells. However, no impairment of STAT-1 tyrosine phosphorylation or DNA-binding capacity was observed in IFN-gamma-treated mouse trophoblast cells. Importantly, histone deacetylase (HDAC) inhibitors significantly enhanced IFN-gamma-inducible gene expression in trophoblast cells, but not fibroblasts. Our collective studies demonstrate that IFN-gamma-inducible gene expression is repressed in mouse trophoblast cells by HDACs. We propose that HDAC-mediated inhibition of IFN-gamma-inducible gene expression in mouse trophoblast cells may contribute to successful pregnancy by preventing activation of IFN-gamma responses that might otherwise facilitate the destruction of the placenta.

Dampening of IFN-gamma-inducible gene expression in human choriocarcinoma cells is due to phosphatase-mediated inhibition of the JAK/STAT-1 pathway.

Trophoblast cells (TBCs) form the blastocyst-derived component of the placenta and play essential roles in fetal maintenance. The proinflammatory cytokine IFN-gamma plays a central role in activating cellular immunity, controlling cell proliferation, and inducing apoptosis. IFN-gamma is secreted by uterine NK cells in the placenta during pregnancy and in mice is required for proper formation of the decidual layer and remodeling of the uterine vasculature. Despite the presence of IFN-gamma in the placenta, TBCs do not express either MHC class Ia or class II Ags, and are resistant to IFN-gamma-mediated apoptosis. In this study, we demonstrate that IFN-gamma-induced expression of multiple genes is significantly reduced in human trophoblast-derived choriocarcinoma cells relative to HeLa epithelial or fibroblast cells. These results prompted us to investigate the integrity of the JAK/STAT-1 pathway in these cells. Choriocarcinoma cells and HeLa cells express comparable levels of the IFN-gamma receptor. However, tyrosine phosphorylation of JAK-2 is compromised in IFN-gamma-treated choriocarcinoma cells. Moreover, phosphorylation of STAT-1 at tyrosine 701 is substantially reduced in both IFN-gamma-treated human choriocarcinoma and primary TBCs compared with HeLa cells or primary foreskin fibroblasts. A corresponding reduction of both IFN regulatory factor 1 mRNA and protein expression was observed in IFN-gamma-treated TBCs. Treatment of choriocarcinoma cells with the tyrosine phosphatase inhibitor pervanadate significantly enhanced IFN-gamma-inducible JAK and STAT-1 tyrosine phosphorylation and select IFN-gamma-inducible gene expression. We propose that phosphatase-mediated suppression of IFN-gamma signaling in TBCs contributes to fetal maintenance by inhibiting expression of genes that could be detrimental to successful pregnancy.

Regulation of major histocompatibility complex class II gene expression in trophoblast cells.

Trophoblast cells are unique because they are one of the few mammalian cell types that do not express major histocompatibility complex (MHC) class II antigens, either constitutively or after exposure to IFN-gamma. The absence of MHC class II antigen expression on trophoblast cells has been postulated to be one of the essential mechanisms by which the semi-allogeneic fetus evades immune rejection reactions by the maternal immune system. Consistent with this hypothesis, trophoblast cells from the placentas of women suffering from chronic inflammation of unknown etiology and spontaneous recurrent miscarriages have been reported to aberrantly express MHC class II antigens. The lack of MHC class II antigen expression on trophoblast cells is due to silencing of expression of the class II transactivator (CIITA), a transacting factor that is essential for constitutive and IFN-gamma-inducible MHC class II gene transcription. Transfection of trophoblast cells with CIITA expression vectors activates both MHC class II and class Ia antigen expression, which confers on trophoblast cells both the ability to activate helper T cells, and sensitivity to lysis by cytotoxic T lymphocytes. Collectively, these studies strongly suggest that stringent silencing of CIITA (and therefore MHC class II) gene expression in trophoblast cells is critical for the prevention of immune rejection responses against the fetus by the maternal immune system. The focus of this review is to summarize studies examining the novel mechanisms by which CIITA is silenced in trophoblast cells. The elucidation of the silencing of CIITA in trophoblast cells may shed light on how the semi-allogeneic fetus evades immune rejection by the maternal immune system during pregnancy.

Class II transactivator (CIITA) promoter methylation does not correlate with silencing of CIITA transcription in trophoblasts.

Trophoblast cells are unique because they do not express major histocompatibility complex (MHC) class II antigens, either constitutively or after exposure to interferon-gamma (IFN-gamma). The absence of MHC class II antigens on trophoblasts is thought to play a critical role in preventing rejection of the fetus by the maternal immune system. The inability of trophoblasts to express MHC class II genes is primarily due to lack of the class II transactivator (CIITA), a transacting factor that is required for constitutive and IFN-gamma-inducible MHC class II transcription. We, therefore, investigated the silencing of CIITA expression in trophoblasts. In transient transfection assays, transcription from the IFN-gamma-responsive CIITA type IV promoter was upregulated by IFN-gamma in trophoblasts, which suggests that CIITA is silenced by an epigenetic mechanism in these cells. Polymerase chain reaction analysis demonstrated that the CIITA type IV promoter is methylated in both the human choriocarcinoma cell lines JEG-3 and Jar and in 2fTGH fibrosarcoma cells, which are IFN-gamma inducible for CIITA. Conversely, methylation of the CIITA type IV promoter was not observed in human primary cytotrophoblasts isolated from term placentae or in mouse or rat trophoblast cell lines. Simultaneous treatment with IFN-gamma and the histone deacetylase inhibitor trichostatin A weakly activated CIITA transcription in mouse trophoblasts. Stable hybrids between human choriocarcinoma and fibrosarcoma cells and between mouse trophoblasts and fibroblasts expressed CIITA following treatment with IFN-gamma. These results suggest that silencing of CIITA transcription is recessive in trophoblasts and involves an epigenetic mechanism other than promoter methylation. The fact that CIITA is expressed in the stable hybrids implies that trophoblasts may be missing a factor that regulates chromatin structure at the CIITA promoter.

DNA alkylating agents alleviate silencing of class II transactivator gene expression in L1210 lymphoma cells.

MHC class II (Ia) Ag expression is inversely correlated with tumorigenicity and directly correlated with immunogenicity in clones of the mouse L1210 lymphoma (1 ). Understanding the mechanisms by which class II Ag expression is regulated in L1210 lymphoma may facilitate the development of immunotherapeutic approaches for the treatment of some types of lymphoma and leukemia. This study demonstrates that the variation in MHC class II Ag expression among clones of L1210 lymphoma is due to differences in the expression of the class II transactivator (CIITA). Analysis of stable hybrids suggests that CIITA expression is repressed by a dominant mechanism in class II-negative L1210 clones. DNA-alkylating agents such as ethyl methanesulfonate and the chemotherapeutic drug melphalan activate CIITA and class II expression in class II negative L1210 cells, and this effect appears to be restricted to transformed cell lines derived from the early stages of B cell ontogeny. Transient transfection assays demonstrated that the CIITA type III promoter is active in class II(-) L1210 cells, despite the fact that the endogenous gene is not expressed, which suggests that these cells have all of the transacting factors necessary for CIITA transcription. An inverse correlation between methylation of the CIITA transcriptional regulatory region and CIITA expression was observed among L1210 clones. Furthermore, 5-azacytidine treatment activated CIITA expression in class II-negative L1210 cells. Collectively, our results suggest that 1) CIITA gene expression is repressed in class II(-) L1210 cells by methylation of the CIITA upstream regulatory region, and 2) treatment with DNA-alkylating agents overcomes methylation-based silencing of the CIITA gene in L1210 cells.