MAP3K11 is required for serum-stimulated cell proliferation and for mitogen and cytokine activation of p38, ERK, and JNK1. MAP3K11 also plays a role in mitogenstimulated phosphorylation and activation of BRAF, without phosphorylating BRAF directly. Thus, MAP3K11 functions as a node in the mitogen and stress signaling pathways. We have previously shown that activation of the MAP kinase pathway correlates with prostate cancer progression in a variety of settings and determined that stress kinase signaling regulates AR Ser 650 phosphorylation. In this study, we confirmed that stress kinase signaling regulates AR Ser 650 phosphorylation; knockdown of MAP3K11 stoichiometrically decreased PMA-induced AR Ser 650 phosphorylation. Modulation of Ser 650 phosphorylation may be regulating AR transcriptional activity of the AR target genes that were altered upon MAP3K11 knockdown, including TMPRSS2, SGK, ORM1, DKK and FST. We also found that the castration-resistant prostate cancer AR regulated M-phase genes CDC20, CDK1, and UBE2C, were decreased in response to MAP3K11 knockdown, although the decrease in transcription of these genes may reflect the inhibition of growth triggered by MAP3K11 knockdown and not represent altered AR transcriptional activity. Our screen also identified other stress kinases, including MAP3K7, MAP4K3, and MAPKAPK5, which underscores the critical nature of stress kinase signaling in regulating prostate cancer cell growth. DGK catalyzes the phosphorylation of DAG by converting it to PA, thereby exchanging one second messenger for Epoxomicin another and activating protein kinase C. There is increasing evidence suggesting that DGKD is involved in regulating DAG and PA levels in response to various growth factors and hormones. DGKD was reported to interact with RACK1, a protein that we had previously demonstrated as an AR interacting protein that regulates AR phosphorylation and transcriptional activity. Thus, DGKD may contribute to AR regulation through RACK1. However, knockdown of DGKD did not have a significant effect on AR transcriptional activity. Previous research has shown that in the absence of DGKD, EGFR signaling is decreased because both expression and kinase activity are inhibited. This effect on EGFR is a result of a decrease in a deubiquitinase, USP-8, and therefore increased ubiquitination and degradation of the EGFR. Growth factor signaling is a known regulator of prostate cancer cell growth. It is therefore possible that the growth effect that corresponds with DGKD knockdown is the result of altered receptor tyrosine kinase signaling. ICK is a serine/threonine kinase containing a dual phosphorylation site found in mitogen-activating protein kinases whose activity is regulated by cell cycle-related kinase and human protein phosphatase 5. ICK is related to male germ cell-associated protein kinase. MAK is an AR coregulator that directly binds the AR in co-immunoprecipitation experiments and enhances AR-dependent transcription in a kinasedependent manner. Inhibition of MAK with either RNAi or a kinase-dead form decreased LNCaP cell growth.

This suggests that therapeutic strategies targeting kinase cascades can overcome the compensatory signaling mechanisms that limit the effectiveness of androgen ablation. In order to identify the signaling pathways that regulate prostate cancer cell growth, we screened a panel of WZ8040 shRNAs that target the human kinome against LNCaP prostate cancer cells grown in the presence and absence of androgen. We searched for kinases that had general growth effects and kinases that compensated for androgen ablation. The screen identified multiple shRNA clones against gene targets that regulate both androgen sensitivity and cell growth. We report here the results of our screen and the detailed evaluation of a subset of kinases identified as regulators of prostate cancer cell growth. As an intermediate step to examining the activation state of the kinases, we examined kinase message levels in the Oncomine database. We found that in at least two independent studies the mRNA levels for the six kinases increased either when primary prostate cancer is compared to normal prostate or when metastatic prostate cancer is compared to primary disease or normal prostate. We validated the growth effect and knockdown of our six selected kinases using the CyQuant Assay, which measures DNA content as a surrogate for cell number, and used this technique to also extend our analysis to the castration-resistant cell line, C4-2B. The cells were transduced with lentiviral particles expressing two shRNAs specific for each kinase of interest or pLKO empty vector control in the presence or absence of androgen. As observed in Figure 2, growth was decreased in both cell lines in response to each shRNA. In general, kinase knockdown inhibited growth in the presence and absence of androgen. Furthermore, kinase knockdown affected growth equivalently in both the androgen-dependent LNCaP and castration-resistant C4-2B cell line. qPCR was used to determine kinase knockdown by shRNA in LNCaP and C4-2B cells. Hormone was added at various concentrations and RNA was isolated at 2 and 24 hours following hormone treatment. These hormone treatments were the same as those used to assess the effect of kinase knockdown on AR transcriptional activity, which is described below and presented in Table 1. Each kinase was knocked down in both cell lines with two different shRNAs and compared to the pLKO empty vector control. We did not observe an effect of hormone dose on the efficiency of kinase knockdown; thus, the data shown in Figure 3 are the qPCR values averaged across biological replicates and hormone concentrations for each shRNA or the pLKO control at 24 hours following hormone stimulation. The shRNA viruses elicit greater than 50% knockdown of the target kinase mRNA as compared to pLKO, with most knockdowns greater than 70% at both time points and in both cell lines. There was some differential knockdown of kinase mRNA by shRNA, which may account for the differential knockdown of growth.

Inhibition than CIT shRNA-1, which parallels the effect on kinase mRNA knockdown, where CIT shRNA-2 reduced CIT mRNA levels more than CIT shRNA-1. However, the parallels Kinase Inhibitor Library between growth inhibition and mRNA knockdown are not evident for all kinases targeted. In order to determine if the inhibition of growth induced by kinase knockdown was specific to prostate cancer cells, we measured the growth of LHS and MCF10A cells in response to shRNA targeting the seven kinases. LHS cells are nontumorigenic immortalized human prostate epithelial cells generated by ectopic expression of SV40 large, small T antigen, and human telomerase. MCF10A is a non-tumorigenic, spontaneously immortalized breast epithelial cell line. In general, shRNA directed against the kinases had minimal effect on LHS and MCF10A cell growth, suggesting selectivity towards prostate cancer cells. The knockdown of kinase message in LHS and MCF10A cells was variable. In LHS cells, MAP3K11 was effectively knocked down and PSKH1; however the knockdown was inefficient for the other kinases. In MCF10A cells, CIT was inhibited and PSKH1, DGKD, and GALK2 were each inhibited by approximately 50%. The inability to inhibit kinase expression to a similar extent as in LNCaP, C4-2B, and CWR22Rv1 cells, complicates interpreting the importance of these kinases in normal cell growth and survival. However, all six kinases were knocked down in at least one of the normal cell lines tested. Thus, these results are consistent with there being selectivity for targeting these kinases in cancer cells over normal cells. Since the AR is a major regulator of prostate cancer cell growth, we wanted to determine if any of the six selected kinases might affect growth through regulating the AR transcriptome. To examine the effect of kinase knockdown on AR target gene transcription, qPCR was used to measure transcript levels of two AR target genes, TMPRSS2 and SGK, in LNCaP and C4-2B cells with two independent shRNAs used to inhibit kinase expression. We examined transcription of these genes at 2 and 24 hours to evaluate the effect of kinase knockdown on the immediate-early response and steady-state levels of AR transcriptional activity. Statistical analysis indicates that there was no effect of hormone dose on the ability of kinase knockdown to affect AR transcription; kinase knockdown altered transcription equivalently, or had no effect, at each androgen dose. Maintenance of androgen induction in pLKO was observed in all analyzed experiments. Reported in Table 1 are the statistically significant changes in AR transcription of TMPRSS2 and SGK in response to kinase knockdown by two independent shRNAs at 2 and 24 hours post three different androgen dose treatments. Both shRNAs had to alter gene transcription significantly in the same direction for reporting in the table. There was no consistent decrease in AR transcriptional activity in response to knockdown of the six kinases across both cell lines, AR target genes examined, and the two time points tested.

A first demonstration that type-I IFN may be sensed by Vc9Vd2 Tcells was reported by Kunzmann et al., showing an increase of CD69 after IFN-a treatment. We LY2109761 confirmed this observation and showed the ability of IFN-a to increase Vc9Vd2 T-cell response to PhAgs stimulation in terms of IFN-c production both in HD and in HCV-infected patients. In particular, the significant impairment of Vc9Vd2 T-cells in HCVinfected patients did not allow to obtain their complete restoration by IFN-a. Nevertheless, individual relative impact of PhAg/IFN-a co-stimulation was found much higher in HCV patients, due to the very low level of responsiveness to PhAgs. Thus, the possibility to restore IFN-c production in vivo by combining standard IFN-a treatment and PhAg stimulation may have a positive impact on HCV inhibition. Indeed several reports show that IFN-a and IFNc may synergistically inhibit HCV replication in vitro and this effect is also reported for other viruses. Nevertheless, a study aimed to evaluate the antiviral impact of PhAg/IFN-a combination is ongoing and may validate new combined treatment strategies. Interestingly, PhAg-activated Vc9Vd2 Tcells are able not only to produce IFN-c but also to deploy many different response pathways, such as DC activation, and neutrophils recruitment/activation, thus improving the overall protective immune response capability. Noteworthy, IFNa effect on PhAg/response was found also in vivo in pre-clinical trials on non-human primates, inducing an increase in IFN-c amount in animals sera. A time-course study of in vivo IFN-a treatment on Vc9Vd2 T-cell responsiveness to PhAg in HCVinfected patients is currently in progress. About possible mechanisms mediating this improvement, we found that IFN-a acts by increasing IFN-c-mRNA persistence, that may result in increased IFN-c translation levels. Similar observations were reported on NK cells, as IFN-c production after IL-12 and IL-18 stimulation was regulated by mechanisms involving IFN-c-mRNA stabilization. Indeed, mRNA stabilization is now considered as one of the main post-transcriptional control mechanisms responsible for the initiation and resolution of inflammation. In recent years, a new attention on new direct antiviral drugs for chronic HCV infection is growing. The definition of other combined immunomodulating approaches may contribute to optimize the antiviral response. In this context Vc9Vd2 T-cells may represent a good target of immunomodulating strategies for their ability to be easily activated in vivo by PhAgs without HLA restriction and to orchestrate a complex network of antiviral and immunomodulating activities. We show here for the first time that IFN-a, currently used in standard therapy, is able to improve Vc9Vd2 T-cell responsiveness in HCV patients. This, and the finding that IFN-c can act synergistically with IFN-a to inhibit HCV replication, strengthen the rational for testing combined standard antiviral and immunostimulating therapeutical strategies. To this aim, future in vivo studies on HCV-infected non-human primates aimed to define the antiviral capability of the combined treatment are necessary both to assess safety and antiviral effectiveness of this combined approach, and to disclose the cellular/molecular mechanisms involved. Wnt/b-catenin signalling pathway is critical for early and late embryonic development and it plays important roles during tumorigenesis.

For example, Drosophila GRH regulates the levels of genes encoding enzymes involved in cuticle melanization and chitin metabolism, cell adhesion proteins, and protein components of the cuticle. In mice, Grhl3 regulates the levels of genes that encode structural-barrier proteins in keratinocytes and the enzymes that crosslink such proteins, as well as cell-adhesion proteins and proteins that modulate the lipid composition of the epidermis. We propose that the original functions of Grainy head-like proteins in the opisthokont last common ancestor predisposed GRH-like proteins to regulate many aspects of extracellular-barrier formation and wound healing in early animals, as well as to evolve the related ability of regulating cellcell adhesion genes in many epithelial tissues. In the metazoan lineage, many types of epidermal barriers have evolved over time, including epithelia with chitin-based extracellular barriers, and it is interesting that chitin is one of the few extracellular structural biopolymers common to both fungi and animals. While chitin synthase itself does not appear to be regulated by GRH-like proteins in any system yet studied, it appears that GRH and GRH-like proteins of the CP2 superfamily regulate the expression of many genes involved in the formation and remodeling of chitin-based barriers, at least in Neurospora and Drosophila. It is also intriguing that chitinase 1 in Neurospora and chitinase 3 in Drosophila both appear to be strongly regulated by GRHL and GRH, respectively, consistent with an ancestral transcriptional control of chitinase expression by GRH-like proteins in the opisthokont last common ancestor. We believe it is possible that components of the ancestral opisthokont cell wall were repurposed during the evolution of chitin-based apical extracellular barriers in some basal multicellular animals, with GRH proteins maintaining a role in barrier formation and remodeling during the process. A similar process may have occurred during the evolution of multicellular volvocine algae, as it has been proposed that the outer cell wall of unicellular algae evolved to become part of the apical extracellular barrier of multicellular algae. This would have been Bortezomib independent of control by CP2 superfamily proteins, as sequenced genomes in the algal lineage do not encode recognizable members of this superfamily. The evolution of multicellularity in fungi was presumably less complicated than in metazoans, as one can invoke incomplete cell division creating syncytial colonies of fungi. In this evolutionary scenario, the conservation of ancestral GRHL function with respect to barrier formation and remodeling would be straightforward, as the cell walls of the unicellular opisthokont last common ancestor and extant multicellular fungi would be very similar in structure and function. In addition to the greatly lowered expression of the chitinase 1 gene, we also found evidence that Neurospora GRHL plays a role in the expression of enzymes involved in the synthesis and remodeling of another key biopolymer of the fungal cell wall – beta-1,3-glucan. GRHL may turn out to have a more general role in promoting cell wall development, although we were unable to uncover phenotypic evidence for this, despite testing the growth of grhl mutant strains under several conditions shown elsewhere to inhibit the growth of S. cerevisiae strains with compromised cell walls.