The androgen receptor (AR) is a transcription factor that drives the differentiation of prostate epithelium by regulating the expression of several hundred genes. Conversely, AR also plays a central role in prostate cancer (PCa) development, and it continues to be active in tumors that relapse after castration (castration-resistant prostate cancer, CRPC). The transactivation function of AR has been extensively studied, and AR can also function as a transcriptional repressor on a distinct set of genes, but the identity of the AR regulated genes that are critical for PCa remain unclear. Moreover, the extent to which AR acquires new functions during PCa development and progression remains to be determined. Recent studies have highlighted the central role of chromatin structure and histone posttranslational modifications in determining the spectrum of genes regulated by AR and all other transcription factors. While the role of DNA methylation in the epigenetic regulation of gene expression is well established, it is now appreciated that chromatin structure plays a central and dynamic role in the epigenetic regulation of gene expression. The focus of this review is on AR interactions with chromatin and how they regulate AR function in PCa development and progression.
Androgen receptor (AR); prostate cancer (PCa); transcription; epigenetics; histone methylation
Low-grade prostate cancers (Gleason pattern 3 [G3]) detected on needle biopsies are generally viewed as indolent and suitable for conservative management with only interval repeat biopsies to monitor by watchful wating. Higher grade tumors eventually emerge in 20–30% of these cases, but this process may only reflect incomplete sampling on the initial biopsy, such that it remains unknown how generally G3 tumors progress to higher grades. In this study, we examined a series of adjacent G3 and Gleason pattern 4 (G4) tumors in radical prostatectomy specimens and found that all were concordant for the TMPRSS2:ERG gene fusion. Using hybrid-capture and deep sequencing in four fusion-positive cases, we found that adjacent laser-capture microdissected G3 and G4 tumors had identical TMPRSS2:ERG fusion breakpoints, confirming their clonal origin. Two of these G3 tumors had deletion of a single PTEN gene that was also deleted in the adjacent G4, while the G4 tumors in two cases had additional PTEN losses. These findings establish that a subset of G3 tumors progress to G4, or emerge from a common precursor. Further, they show that G3 tumors which progress to G4 may have molecular features distinguishing them from G3 tumors that do not progress. Thus, determining the spectrum of these genetic or epigenetic features may allow for the identification of G3 tumors on needle biopsies that are truly indolent, versus those that have the potential to progress, or that may already be associated with a G4 tumor that was not sampled at the initial biopsy, therefore requiring more aggressive surveillance or intervention.
prostate cancer; Gleason pattern; progression; active surveillance; gene fusion
Loss of PTEN is a common event in many cancers and leads to hyperactivation of the PI 3-K/Akt signaling pathway. The mechanisms by which Akt isoforms mediate signaling to phenotypes associated with PTEN-inactivation in cancer have not been defined. Here we show that Akt2 is exclusively required for PTEN-deficient prostate tumor spheroid maintenance whereas Akt1 is dispensable. shRNA silencing of Akt2 but not Akt1 promotes regression of prostate cancer xenografts. Mechanistically, we show that Akt2 silencing up-regulates p21 and the pro-apoptotic protein Bax and downregulates the insulin-like growth factor receptor-1. We also show that p21 is an effector of Akt2 in mediating prostate tumor maintenance. Moreover, Akt2 is also exclusively required for the maintenance and survival of other PTEN-deficient solid tumors, including breast cancer and glioblastoma. These findings identify a specific function for Akt2 in mediating survival of PTEN-deficient tumors and provide a rationale for developing therapeutics targeting Akt2.
Fusion of the androgen receptor-regulated (AR-regulated) TMPRSS2 gene with ERG in prostate cancer (PCa) causes androgen-stimulated overexpression of ERG, an ETS transcription factor, but critical downstream effectors of ERG-mediating PCa development remain to be established. Expression of the SOX9 transcription factor correlated with TMPRSS2:ERG fusion in 3 independent PCa cohorts, and ERG-dependent expression of SOX9 was confirmed by RNAi in the fusion-positive VCaP cell line. SOX9 has been shown to mediate ductal morphogenesis in fetal prostate and maintain stem/progenitor cell pools in multiple adult tissues, and has also been linked to PCa and other cancers. SOX9 overexpression resulted in neoplasia in murine prostate and stimulated tumor invasion, similarly to ERG. Moreover, SOX9 depletion in VCaP cells markedly impaired invasion and growth in vitro and in vivo, establishing SOX9 as a critical downstream effector of ERG. Finally, we found that ERG regulated SOX9 indirectly by opening a cryptic AR-regulated enhancer in the SOX9 gene. Together, these results demonstrate that ERG redirects AR to a set of genes including SOX9 that are not normally androgen stimulated, and identify SOX9 as a critical downstream effector of ERG in TMPRSS2:ERG fusion–positive PCa.
Androgen receptor (AR) is reactivated in castration resistant prostate cancer (CRPC) through mechanisms including marked increases in AR gene expression. We identify an enhancer in the AR second intron contributing to increased AR expression at low androgen levels in CRPC. Moreover, at increased androgen levels the AR binds this site and represses AR gene expression through recruitment of lysine specific demethylase 1 (LSD1) and H3K4me1,2 demethylation. AR similarly represses expression of multiple genes mediating androgen synthesis, DNA synthesis and proliferation, while stimulating genes mediating lipid and protein biosynthesis. Androgen levels in CRPC appear adequate to stimulate AR activity on enhancer elements, but not suppressor elements, resulting in increased expression of AR and AR repressed genes that contribute to cellular proliferation.
prostate cancer; androgen receptor; androgen deprivation therapy; H3K4 methylation; LSD1
The androgen receptor (AR) is critical for the normal development of prostate and for its differentiated functions. The consistent expression of AR in prostate cancer (PCa), and its continued activity in PCa that relapse after androgen deprivation therapy (castration-resistant prostate cancer (CRPC)), indicate that at least a subset of these genes are also critical for PCa development and progression. This review addressed AR regulated genes that may be critical for PCa, and how AR may acquire new functions during PCa development and progression.
It appears clear that androgen receptor (AR) regulated expression of the TMPRSS2:ERG fusion gene plays an early role in prostate cancer (PC) development or progression, but the extent to which TMPRSS2:ERG is downregulated in response to androgen deprivation therapy and whether AR reactivates TMPRSS2:ERG expression in castration resistant prostate cancer (CRPC) have not been determined. We show that ERG message levels in TMPRSS2:ERG fusion positive CRPC are comparable to the levels in fusion gene positive primary PC, consistent with the conclusion TMPRSS2:ERG expression is reactivated by AR in CRPC. To further assess whether TMPRSS2:ERG expression is initially downregulated in response to androgen deprivation therapy, we examined VCaP cells, which express the TMPRSS2:ERG fusion gene, and xenografts. ERG message and protein rapidly declined in response to removal of androgen in vitro and castration in vivo. Moreover, as observed in the clinical samples, ERG expression was fully restored in the VCaP xenografts that relapsed after castration, coincident with AR reactivation. AR reactivation in the relapsed xenografts was also associated with marked increases in mRNA encoding AR and androgen synthetic enzymes. These results demonstrate that expression of TMPRSS2:ERG, similarly to other AR regulated genes, is restored in CRPC and may contribute to tumor progression.
prostate cancer; androgen receptor; ERG; TMPRSS2; androgen deprivation therapy
Androgen receptor (AR) recruitment of transcriptional corepressors NCoR and SMRT can be enhanced by antagonists such as mifepristone. This study shows that enhanced NCoR binding to the mifepristone liganded AR is mediated by the NCoR C-terminal N1 CoRNR box, and that this selectivity is due to charged residues unique to the C-terminal CoRNR boxes of NCoR and SMRT. Significantly, these residues are on a helical face adjacent to oppositely charged residues in helix 4 of the AR ligand-binding domain (LBD). Mutagenesis of these AR residues in helix 4, as well as mutation of lysine 720 in helix 3 (predicted to interact with the CoRNR box), markedly impaired AR recruitment of NCoR, indicating that N1 CoRNR box binding is being stabilized by these ionic interactions in the AR LBD coactivator/corepressor binding site. Finally, results using a helix 12 deleted AR indicate that mifepristone induces allosteric changes in addition to helix 12 displacement that are critical for NCoR binding. These findings demonstrate that AR antagonists can enhance corepressor recruitment by stabilizing a distinct antagonist conformation of the AR coactivator/corepressor binding site, and support the development of additional antagonists that may be able to further enhance AR recruitment of corepressors.
androgen receptor; prostate cancer; corepressor; antagonist; mifepristone
The majority of prostate cancers (PCa) express high levels of androgen receptor (AR) and are dependent for their growth on testosterone produced by the testes, which is reduced in the prostate to the higher affinity ligand 5α-dihydrotestosterone (DHT). PCa growth can be suppressed by androgen deprivation therapy, which involves removal of testicular androgens (surgical or medical castration) or treatment with an AR antagonist (or a combination of both), but patients invariably relapse with tumors that have been termed castration recurrent/resistant PCa (CRPC). Importantly, AR transcriptional activity becomes reactivated at this CRPC stage of the disease and remains essential for tumor growth. The objective of this review is to outline one clinically important mechanism contributing to this AR reactivation, which is increased intratumoral synthesis of testosterone and DHT from weak androgens produced by the adrenal glands and possibly de novo from cholesterol. Early studies showed that a substantial fraction of CRPC patients responded to adrenalectomy or medical suppression of adrenal androgen synthesis using agents such as ketoconazole (CYP17A1 inhibitor), and a recent phase III study of a more potent and selective CYP17A1 inhibitor (abiraterone) has demonstrated an improvement in survival. With the pending FDA approval of abiraterone for CRPC, defining the molecular mechanisms contributing to CYP17A1 inhibitor resistance/relapse and AR reactivation is now critical to build on these advances.
The nonreceptor tyrosine kinase BMX (bone marrow tyrosine kinase gene on chromosome X) is abundant in various cell types and activated downstream of phosphatidylinositol-3 kinase (PI3K) and the kinase Src, but its substrates are unknown. Positional scanning peptide library screening revealed a marked preference for a priming phosphorylated tyrosine (pY) in the −1 position, indicating that BMX substrates may include multiple tyrosine kinases that are fully activated by pYpY sites in the kinase domain. BMX phosphorylated focal adhesion kinase (FAK) at Tyr577 subsequent to its Src-mediated phosphorylation at Tyr576. Loss of BMX by RNA interference or by genetic deletion in mouse embryonic fibroblasts (MEFs) markedly impaired FAK activity. Phosphorylation of the insulin receptor in the kinase domain at Tyr1189 and Tyr1190, as well as Tyr1185, and downstream phosphorylation of the kinase AKT at Thr308 were similarly impaired by BMX deficiency. However, insulin-induced phosphorylation of AKT at Ser473 was not impaired in Bmx knockout MEFs or liver tissue from Bmx knockout mice, which also showed increased insulin-stimulated glucose uptake, possibly because of decreased abundance of the phosphatase PHLPP (PH domain leucine-rich repeat protein phosphatase). Thus, by identifying the pYpY motif as a substrate for BMX, our findings suggest that BMX functions as a central regulator among multiple signaling pathways mediated by tyrosine kinases.
Epigenetic regulators represent a promising new class of therapeutic targets for cancer. Enhancer of zeste homolog 2 (EZH2), a subunit of Polycomb repressive complex 2 (PRC2), silences gene expression via its histone methyltransferase activity. Here we report that the oncogenic function of EZH2 in castration-resistant prostate cancer (CRPC) is independent of its role as a transcriptional repressor. Instead, it involves the ability of EZH2 to act as a co-activator for critical transcription factors including the androgen receptor (AR). This functional switch is dependent on phosphorylation of EZH2, and requires an intact methyltransferase domain. Hence, targeting the non-PRC2 function of EZH2 may have significant therapeutic efficacy for treating metastatic, hormone-refractory prostate cancer.
A subset of human CD4−CD8− T cells that expresses an invariant Vα24-JαQ T cell receptor (TCR)-α chain, paired predominantly with Vβ11, has been identified. A series of these Vα24 Vβ11 clones were shown to have TCR-β CDR3 diversity and express the natural killer (NK) locus–encoded C-type lectins NKR-P1A, CD94, and CD69. However, in contrast to NK cells, they did not express killer inhibitory receptors, CD16, CD56, or CD57. All invariant Vα24+ clones recognized the MHC class I–like CD16 molecule and discriminated between CD1d and other closely related human CD1 proteins, indicating that recognition was TCR-mediated. Recognition was not dependent upon an endosomal targeting motif in the cytoplasmic tail of CD1d. Upon activation by anti-CD3 or CD1d, the clones produced both Th1 and Th2 cytokines. These results demonstrate that human invariant Vα24+ CD4−CD8− T cells, and presumably the homologous murine NK1+ T cell population, are CD1d reactive and functionally distinct from NK cells. The conservation of this cell population and of the CD1d ligand across species indicates an important immunological function.
Relapse of castration-resistant prostate cancer (CRPC) that occurs after androgen deprivation therapy of primary prostate cancer can be mediated by reactivation of the androgen receptor (AR). One important mechanism mediating this AR reactivation is intratumoral conversion of the weak adrenal androgens DHEA and androstenedione into the AR ligands testosterone and dihydrotestosterone (DHT). DHEA and androstenedione are synthesized by the adrenals through the sequential actions of the cytochrome P450 enzymes CYP11A1 and CYP17A1, so that CYP17A1 inhibitors such as abiraterone are effective therapies for CRPC. However, the significance of intratumoral CYP17A1 and de novo androgen synthesis from cholesterol in CRPC, and the mechanisms contributing to CYP17A1 inhibitor resistance/relapse, remain to be determined. We report that AR activity in castration-resistant VCaP tumor xenografts can be restored through CYP17A1-dependent de novo androgen synthesis, and that abiraterone treatment of these xenografts imposes selective pressure for increased intratumoral expression of CYP17A1, thereby generating a mechanism for development of resistance to CYP17A1 inhibitors. Supporting the clinical relevance of this mechanism, we found that intratumoral expression of CYP17A1 was markedly increased in tumor biopsies from CRPC patients after CYP17A1 inhibitor therapy. We further show that CRPC cells expressing a progesterone responsive T877A mutant AR are not CYP17A1 dependent, but that AR activity in these cells is still steroid dependent and mediated by upstream CYP11A1 dependent intraturmoral pregnenolone/progesterone synthesis. Together, our results indicate that CRPCs resistant to CYP17A1 inhibition may remain steroid dependent and therefore responsive to therapies that can further suppress de novo intratumoral steroid synthesis.
prostate cancer; androgen receptor; CYP17A1; steroid synthesis; androgen deprivation therapy
Invariant natural killer T-cells (‘iNKT’) are the best-known CD1d-restricted T-cells, with recently-defined roles in controlling adaptive immunity. CD1d-restricted T-cells can rapidly produce large amounts of Th1 and/or Th2//Treg/Th17-type cytokines, thereby regulating immunity. iNKT can stimulate potent anti-tumor immune responses via production of Th1 cytokines, direct cytotoxicity, and activation of effectors. However, Th2//Treg-type iNKT can inhibit anti-tumor activity. Furthermore, iNKT are decreased and/or reversibly functionally impaired in many advanced cancers. In some cases, CD1d-restricted T-cell cancer defects can be traced to CD1d+ tumor interactions, since hematopoietic, prostate, and some other tumors can express CD1d. Ligand and IL-12 can reverse iNKT defects and therapeutic opportunities exist in correcting such defects alone and in combination. Early stage clinical trials have shown potential for reconstitution of iNKT IFN-gamma responses and evidence of activity in a subset of patients, with rational new approaches to capitalize on this progress ongoing, as will be discussed here.
cytokines; tumor immunity; CD1; CD1d-reactive T cells; iNKT; NKT
Androgen deprivation is still the standard systemic therapy for metastatic prostate cancer (PCa), but patients invariably relapse with a more aggressive form of PCa termed hormone refractory, androgen independent, or castration resistant PCa (CRPC). Significantly, the androgen receptor (AR) is expressed at high levels in most cases of CRPC, and these tumors resume their expression of multiple AR-regulated genes, indicating that AR transcriptional activity becomes reactivated at this stage of the disease. The molecular basis for this AR reactivation remains unclear, but possible mechanisms include increased AR expression, AR mutations that enhance activation by weak androgens and AR antagonists, increased expression of transcriptional coactivator proteins, and activation of signal transduction pathways that can enhance AR responses to low levels of androgens. Recent data indicate that CRPC cells may also carry out intracellular synthesis of testosterone and DHT from weak adrenal androgens and may be able to synthesize androgens from cholesterol. These mechanisms that appear to contribute to AR reactivation after castration are further outlined in this review.
androgen receptor; prostate cancer; testosterone; androgen; androgen deprivation therapy; AR antagonist
The purine nucleoside adenosine is an important anti-inflammatory molecule, inhibiting a variety of immune cells by adenosine receptor-mediated mechanisms. Invariant natural killer T (iNKT) cells recognize glycolipids presented on CD1d molecules and produce vigorous amounts of cytokines upon activation, hence regulating immune reactions. The mechanisms polarizing their cytokine pattern are elusive. Previous studies demonstrated that adenosine can suppress IFN-γ production by iNKT cells.
We describe the expression of all four known adenosine receptors A1R, A2aR, A2bR, and A3R, on mouse iNKT cells. We show that IL-4 production in primary mouse iNKT cells and a human iNKT line is efficiently inhibited by A2aR blockade with an inverse relation to IL-4. These data are supported by A2aR-deficient mice, which exhibit largely decreased levels of IL-4, IL-10 and TGF-β concomitantly with an increase of IFN-γ upon α-GalCer administration in vivo. While A2aR inhibits other lymphocyte populations, A2aR is required for the secretion of IL-4 and IL-10 by iNKT cells. These data suggest adenosine:A2aR-mediated mechanisms can control the cytokine secretion pattern of iNKT cells.
NKT cells; Cellular activation; Immune regulation
CD1d-restricted ‘NKT’ rapidly stimulate innate and adaptive immunity through production of Th1 and/or Th2 cytokines and induction of CD1d+ antigen-presenting cell (APC) maturation. However, therapeutic exploitation of NKT has been hampered by their paucity and defects in human disease. NKT:APC interactions can be modeled by direct stimulation of human APC through CD1d in vitro. We have now found that direct ligation with multiple CD1d mAbs also stimulated bioactive IL-12 release from CD1d+ but not CD1d KO murine splenocytes in vitro. Moreover, all CD1d mAbs tested also induced IL-12 as well as both IFN-γ and IFN-α in vivo from CD1d+ but not CD1d-deficient recipients. Unlike IFN-γ, CD1d-induced IFN-α was at least partially dependent on invariant NKT. Optimal resistance to infection with picornavirus encephalomyocarditis virus (EMCV) is known to require CD1d-dependent APC IL-12-induced IFN-γ as well as IFN-α. CD1d ligation in vivo enhanced systemic IL-12, IFN-γ, and IFN-α, and was protective against infection by EMCV, suggesting an alternative interpretation for previous results involving CD1d ‘blocking’ in other systems. Such protective responses, including elevations in Th1 cytokines, were also seen with CD1d FAb’2s in vivo, while an IgM mAb (with presumably minimal tissue penetration) was comparably effective at protection in vivo as well as cytokine induction both in vivo and in vitro. Although presumably acting immediately ‘downstream’, CD1d mAbs were protective later during infection than iNKT agonist α-galactosylceramide. These data indicate that NKT can be bypassed with CD1d-mediated induction of robust Th1 immunity, which may have therapeutic potential both directly and as adjuvant.
antibodies; cell activation; cytokines; NKT cells; viral infection
Genetic rearrangement of TMPRSS2 regulatory sequences and coding sequences of the ERG gene has been detected in nearly half of prostate cancers. Quantitative assays to detect such TMPRSS2-ERG gene fusion have been limited to real time PCR techniques that rely on reverse transcriptase-based amplification. We sought to develop a novel assay that uses branched DNA (bDNA) technology to measure TMPRSS2-ERG fusion.
Branched DNA probes were designed to detect TMPRSS2-ERG gene fusion in prostate cancer cell lines. Non-quantitative, nested reverse transcription (RT)-PCR and fluorescence in situ hybridization (FISH) were used to ascertain TMPRSS2-ERG gene fusion status in prostate tissues.
The branched DNA assay detected TMPRSS2-ERG gene fusion from less than 200 picogram of prostate cancer RNA, whereas more than 600 picogram of RNA was required for fusion gene detection by one step real time RT-PCR. In evaluation of clinical prostatectomy specimens, the branched DNA assay showed concordant detectable fusion signal in all 9 clinical samples that had fusion detected by nested RT-PCR or FISH. Moreover, branched DNA detected gene fusion in 2 of 16 prostate cancer tissue specimens that was not detected by FISH nor nested RT-PCR.
Our findings demonstrate a branched DNA assay that is effective for detection of TMPRSS2-ERG gene fusion in prostate cancer clinical specimens, thus providing an alternative method to ascertain the TMPRSS2-ERG gene fusion in human prostate cancer tissue.
branched DNA; prostate cancer; ERG; biomarker
CD1d-restricted invariant NKT (iNKT) cells are important immunoregulatory cells in antitumor immune responses. However, the quantitative and qualitative defects of iNKT cells in advanced multiple myeloma (MM) hampered their antitumor effects. Therefore, the development of functional iNKT cells may provide a novel strategy for the immunotherapy in MM treatment.
We activated and expanded iNKT cells from MM patients with α-galactosylceramide(α-GalCer)-pulsed-dendritic cells (DCs), characterized their antitumor effects by the cytokine production profile and cytotoxicity against MM cells, and explored the effects of immunomodulatory drug lenalidomide on these iNKT cells. We also investigated the expression of CD1d by primary MM cells and its function to activate iNKT cells.
We established highly purified functional iNKT cell lines from newly diagnosed and advanced MM patients. These CD1d-restricted iNKT cell lines produced high level of antitumor Th1 cytokine in response to α-GalCer-pulsed-primary MM cells, CD1d-transfected MM1S cell line or DCs. Moreover, MM iNKT cell lines displayed strong cytotoxicity against α-GalCer-pulsed-primary MM cells. Importantly, lenalidomide further augmented the Th1-polarization by iNKT cell lines via the increased Th1 cytokine production and the reduced Th2 cytokine production. We also demonstrated that CD1d was expressed in primary MM cells at mRNA and protein levels from the majority of MM patients, but not in normal plasma cells and MM cell lines, and CD1d+ primary MM cells presented antigens to activate iNKT cell lines.
Taken together, our results provide the pre-clinical evidence for the iNKT cells-mediated immunotherapy and a rationale for their use in combination with lenalidomide in MM treatment.
iNKT cells; multiple myeloma; lenalidomide; immunotherapy
CD1d-restricted invariant NKT (iNKT) cells play important regulatory roles in various immune responses, including antitumor immune responses. Previous studies have demonstrated quantitative and qualitative defects in iNKT cells of cancer patients, and these defects are clinically relevant as they are associated with poor prognosis. In this study we demonstrate that defects in the iNKT cell population can, at least in part, be attributed to defective interactions between iNKT cells and CD1d-expressing circulating myeloid dendritic cells (mDC), as mDC of patients with advanced melanoma and renal cell cancer reduced the activation and Th1 cytokine production of healthy donor-derived iNKT cells. Interestingly, this reduced activation of iNKT cells was restricted to patients with low circulating iNKT cell numbers and could be reversed by IL-12 and in part by the neutralization of TGF-β, but it was further reduced by the neutralization of IL-10 in vitro. Additional experiments revealed discordant roles for TGF-β and IL-10 on human iNKT cells, because TGF-β suppressed iNKT cell activation and proliferation and IFN-γ production while IL-10 was identified as a cytokine involved in stimulating the activation and expansion of iNKT cells that could subsequently suppress NK cell and T cell responses.
The evolution of prostate cancer from an androgen-dependent state (ADPCa) to one that is androgen-independent (AIPCa) marks its lethal progression. The androgen receptor (AR) is essential in both, though its function in AIPCa is poorly understood. We have defined the direct AR-dependent target genes in both AIPCa and ADPCa by generating AR-dependent gene expression profiles and AR cistromes. In contrast to ADPCa, AR selectively up-regulates M-phase cell cycle genes in AIPCa including UBE2C, a gene that inactivates the M-phase checkpoint. Selective epigenetic marks and collaborating transcription factor occupancy at UBE2C enhancers leads to increased AR recruitment and UBE2C over-expression in AIPCa cell lines and clinical cases. Silencing of UBE2C blocks AIPCa but not ADPCa growth. Thus the role of AR in AIPCa is not to direct the androgen-dependent gene expression program without androgen, but rather to execute a distinct program resulting in androgen-independent growth.
Numerical and functional defects of invariant natural killer T cells (iNKT) have been documented in human and mouse cancers, resulting in a defect in IFN production in several malignancies. iNKT cells recognize glycolipids presented on CD1d molecules by dendritic and related cells, leading to their activation and thereby regulating immune reactions. Activated iNKT cells cytokine secretion and cytotoxicity can inhibit existing and spontaneous tumor growth, progression, and metastasis. We have identified functional iNKT cell defects in the murine TRAMP prostate cancer model. We found that iNKT cells show the ability to migrate into TRAMP prostate tumors. This infiltration was mediated through CCL2: CCR5 chemokine: receptor interaction. Prostate tumor cells expressing CD1d partially activated iNKT cells, as appreciated by up-regulation of CD25, PD-1 and the IL-12R. However, despite inducing up-regulation of these activation markers and, hence, delivering positive signals, prostate tumor cells inhibited the IL-12-induced STAT4 phosphorylation in a cell-cell contact dependent but CD1d-independent manner. Consequently, tumor cells did not induce secretion of IFNγ by iNKT cells. Blocking the inhibitory Ly49 receptor on iNKT cells in the presence of α-GalCer restored their IFNγ production in vivo and in vitro. However, Ly49 blockade alone was not sufficient. Importantly, this defect could be also be reversed into vigorous secretion of IFNγ by the addition of both IL-12 and the exogenous CD1d ligand alpha-galactosylceramide, but not by IL-12 alone, both in vivo and in vitro. These data underscore the potential to optimize iNKT-based therapeutic approaches.
A significant fraction of CD1d-restricted T cells express an invariant T cell receptor (TCR) α-chain. These highly conserved ‘iNKT’ populations are important regulators of a wide spectrum of immune responses. The ability to directly identify and manipulate iNKT is essential to understanding their function and to exploit their therapeutic potential. To that end, we sought monoclonal and polyclonal antibodies specific for iNKT by immunizing CD1d KO mice, which lack iNKT, with a cyclic peptide modeled after the TCR-α CDR3 loop. One monoclonal antibody (mAb; 6B11) was specific for cloned and primary human but not rodent iNKT and the human invariant TCR-α, as shown by transfection and reactivity with human invariant TCR-α transgenic T cells ex vivo and in situ. 6B11 was utilized to identify, purify, and expand iNKT from an otherwise minor component of human peripheral blood lymphocytes and to specifically identify human iNKT in tissue. Thus, we report a novel and general strategy for the generation of monoclonal antibodies specific for the CDR3 loop encoded by the TCR of interest. Specifically, an anti-Vα24Jα18 CDR3 loop clonotypic TCR mAb is available for the enumeration and therapeutic manipulation of human and non-human primate iNKT populations.
anti-TCR; CD161; clonotypic; cyclic peptide; IL-4; invariant; NKT
Irreversible HER/erbB inhibitors selectively inhibit HER-family kinases by targeting a unique cysteine residue located within the ATP-binding pocket. Sequence alignment reveals that this rare cysteine is also present in ten other protein kinases including all five Tec-family members. We demonstrate that the Tec-family kinase Bmx is potently inhibited by irreversible modification at Cys496 by clinical stage EGFR-inhibitors such as CI-1033. This cross-reactivity may have significant clinical implications.