The epithelial-to-mesenchymal transition (EMT) is a crucial program for the invasion and metastasis of epithelial tumors that involves loss of cell–cell adhesion and increased cell mobility; however, mechanisms underlying this transition are not fully elucidated. Here, we propose a novel mechanism through which the nicotinamide adenine dinucleotide-dependent histone deacetylase SIRT1 regulates EMT in prostate cancer cells through cooperation with the EMT inducing transcription factor ZEB1. We found that forced expression of SIRT1 in non-transformed PZ-HPV-7 prostate epithelial cells disrupts the epithelial morphology concomitant with decreased expression of the epithelial marker, E-cadherin, and increased expression of mesenchymal markers. In contrast, silencing SIRT1 in metastatic prostate tumor cells restores cell–cell adhesion and induces a shift toward an epithelial morphology concomitant with increased expression of E-cadherin and decreased expression of mesenchymal markers. We also found that SIRT1 has a physiologically relevant role in endogenous EMT induced by EGF signaling in prostate cancer cells. We propose that the regulation of EMT by SIRT1 involves modulation of, and cooperation with, the EMT inducing transcription factor ZEB1. Specifically, we show that SIRT1 silencing reduces expression of ZEB1 and that SIRT1 is recruited to the E-cadherin proximal promoter by ZEB1 to deacetylate histone H3 and to reduce binding of RNA polymerase II, ultimately suppressing E-cadherin transcription. We thus identify a necessary role for ZEB1 in SIRT1-mediated EMT. Finally, we show that reduction of SIRT1 decreases prostate cancer cell migration in vitro and metastasis in vivo in immunodeficient mice, which is largely independent of any general effects of SIRT1 on prostate cancer growth and survival. We therefore identify SIRT1 as a positive regulator of EMT and metastatic growth of prostate cancer cells and our findings implicate overexpressed SIRT1 as a potential therapeutic target to reverse EMT and to prevent prostate cancer progression.

Recently reported lines of Th9 cells, producing IL-9 and IL-10, were generated by polarization with IL-4 and TGF-β and activation with Abs against CD3 and CD28. In this paper, we analyzed features of Th9 lines similarly polarized but activated by the “natural mode” (i.e., exposure of CD4 cells to their target Ag, hen egg lysozyme [HEL] and APCs). Main observations are the following: 1) both IL-9 and IL-10 were expressed by the line cells, but with strikingly different kinetics, with IL-9 being produced rapidly, reaching a peak on day 3 in culture and declining sharply thereafter, whereas IL-10 production increased gradually, resembling IL-4 and IL-17 production by their corresponding lineage cells; 2) reactivation of Th9, following expansion, triggered faster and higher production of both IL-9 and IL-10; 3) incubating Th9 cells in polarizing media specific for other phenotypes stimulated moderate levels of phenotype switching to Th1 or Th17 but a massive switching to Th2; 4) Th9 cells induced moderate inflammation in HEL-expressing recipient eyes but only when producing high levels of IL-9; and 5) IL-9–producing donor cells were detected in the blood of Th9 recipients but not in their inflamed eyes, suggesting that similar to findings in culture, exposure to HEL in these eyes arrested the IL-9 production in Th9 cells. Collectively, these data provide new information concerning Th9 cells and reveal their uniqueness, in particular with regard to the unusual production kinetics of IL-9 and the short retention of these cells in affected target tissues.

Background MMP2 has been shown to play an important role in cancer cell invasion and the expression of MMP2 is associated with the poor prognosis of prostate cancer; however, the mechanism of MMP2 expression is largely unknown. SIRT1 is a nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase (class III HDAC) that has recently been shown to have implications in regulating cancer cell growth and apoptosis. The purpose of this study is to determine the role of SIRT1 in regulating MMP2 expression and tumor invasion in prostate cancer cells. Methods The interfering RNAi was used to knockdown SIRT1 from prostate cancer cells. Immunoblots, RT-PCR, zymographic assays, co-immunoprecipitation analysis and transwell assays were used to examine the effects of SIRT1 silencing on MMP2 expression and activity, on SIRT1 and MMP2 interaction, and on prostate cancer cell invasion. The immuno-histochemical assay was performed to study SIRT1 expression in prostate cancer tissues. Results We show that SIRT1 associates and deacetylates MMP2 and SIRT1 regulates MMP2 expression by controlling MMP2 protein stability through the proteosomal pathway. Thus, we demonstrated a novel mechanism in that MMP2 expression can be regulated at the posttranslational level by SIRT1. Furthermore, we determined that SIRT1 inhibition reduced prostate cancer cell invasion and SIRT1 is highly expressed in advanced prostate cancer tissues. Conclusions SIRT1 is an important regulator of MMP2 expression, activity, and prostate cancer cell invasion. Overexpressed SIRT1 in advanced prostate cancer may play an important role in prostate cancer progression.

To understand the role of clathrin-mediated endocytosis in the internalization of normal cellular prion protein (PrPc) in neuronal cells, N2a cells were depleted of clathrin by RNA interference. PrPc internalization via the constitutive endocytic pathway in the absence of Cu2+ and the stimulated pathway in the presence of Cu2+ were measured in both control and clathrin-depleted cells. Depletion of clathrin had almost no effect on the internalization of PrPc either in the presence or absence of Cu2+, in contrast to the marked reduction observed in transferrin uptake. By contrast, the internalization of PrPc was inhibited by the raft-disrupting drugs filipin and nystatin, and by the dominant-negative dynamin-1 mutant dynamin-1 K44A, both in the presence and absence of Cu2+. The internalized PrPc was found to colocalize with cargo that traffic in the Arf6 pathway and in large vacuoles in cells expressing the Arf6 dominant-active mutant. These results show that PrPc is internalized in a clathrin-independent pathway that is associated with Arf6.

SummarySynthetic oligodeoxynucleotides (ODN) expressing 'suppressive' TTAGGG motifs down-regulate a variety of proinflammatory and T helper type 1 (Th1)-mediated pathological immune responses. The ability of the archetypal suppressive ODN A151 to inhibit ocular inflammation was examined in two murine models: experimental autoimmune uveitis, induced by immunization with a retinal antigen, interphotoreceptor retinoid-binding protein (IRBP) and adoptively transferred ocular inflammation, induced by transferring Th1 cells specific to hen egg lysozyme (HEL) into recipient mice that express HEL in their eyes. A151 treatment suppressed the inflammation in both models. In addition, A151 inhibited IRBP-specific cytokine production and lymphocyte proliferation in mice immunized with IRBP. These findings suggest that suppressive ODN affects both afferent and efferent limbs of the immunopathogenic process and may be of use in the treatment of autoimmune ocular inflammation.

Subpopulations of pathogenic or nonpathogenic Th17 cells were reported to develop when pre-sensitized CD4 cells were activated with their target Ag during polarization by either IL-23 or IL-6 and TGF-β, respectively. Here, we generated two Th17 subpopulations by using a system in which naïve CD4 cells from TCR transgenic mice specific to hen egg lysozyme (HEL) are polarized with IL-6/TGF-β and, concurrently, are activated either with HEL presented by APC, or with anti-CD3/CD28 Abs. Only the former cells were pathogenic, inducing inflammation in eyes expressing HEL. Naïve CD4 cells activated by the anti-CD3/CD28 Abs acquired pathogenicity, however, when co-cultured with HEL/APC. Importantly, however, the naïve CD4 cells did not acquire pathogenicity when co-cultured with APC stimulated with LPS, or when separated from the HEL presenting cells by a semi-permeable membrane. Unlike with pre-sensitized Th17, soluble IL-23 does not participate in pathogenicity acquisition by naïve CD4 cells: no pathogenicity was induced by adding IL-23 to cultures activated with anti-CD3/CD28 Abs. Furthermore, Abs against IL-23 or IL-23R did not inhibit acquisition of pathogenicity in cultures of naïve CD4 cells activated by HEL/APC. Our data thus show that, unlike pre-sensitized CD4 cells, naïve CD4 polarized toward Th17 phenotype acquire pathogenicity only by direct interaction with APC presenting the Ag, with no apparent involvement of soluble IL-23. We suggest that the Th17 lymphocytes derived from naive CD4 cells participate in pathogenic and other immune processes, along with the IL-23 dependent Th17 cells.

The epithelial-to-mesenchymal transition (EMT) is a crucial program for initiating invasion and metastasis in many epithelial tumors. EMT is characterized by loss of cell adhesion and increased cell mobility; however, the mechanism underlying this transition is not fully understood. Here we identified SIRT1, a class III histone deacetylase, as a critical regulator of EMT. We show that forced expression of SIRT1 disrupts the epithelial morphology in non-transformed prostate epithelial cells, concomitant with a decrease of epithelial marker E-cadherin, and an increase of mesenchymal protein N-cadherin, and fibronection expression, indicating EMT. In contrast, silencing SIRT1 expression in metastatic prostate tumor cells restores cell to cell adhesion and induces changes in morphology characteristic of epithelial cells, concomitant with an increase of E-cadherin and a decrease of N-cadherin and fibronection expressions. In addition, we show that SIRT1 represses the activity of the E-cadherin proximal promoter with an E-box dependent mechanism, indicating a connection between SIRT1 and the zinc-finger EMT transcription factor, which needs the E-box to suppress E-cadherin transcription. Moreover, we performed ChIP assays to show that SIRT1 and ZEB1 bind to the same DNA fragment on the E-cadherin promoter, and SIRT1 occupancy in the E-cadherin promoter requires ZEB1. In addition, we show that acetylated H3K9 levels were inversely related to occupancy of SIRT1 and ZEB1. Finally, our results show that SIRT1 knockdown reduces prostate cancer cell migration and invasion. Thus, in this study we identified SIRT1 as an important regulator of EMT and demonstrated a new transcriptional mechanism regulating E-cadherin expression by the cooperation of SIRT1 with ZEB1. These findings suggest that overexpressed SIRT1 in prostate cancer cells may be targeted to reverse EMT, and SIRT1 could be a potential target in the treatment of prostate cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3417. doi:10.1158/1538-7445.AM2011-3417

Th17 cells play a major role in immunopathogenic processes. McGeachy et al, (Nat. Immunol., 2007) showed that two distinct subsets of Th17 were generated from activated CD4 cells when different polarization modes were used, with only one subset being encephalitogenic. Here, we analyzed the biological features of Th17 generated by the same polarization mechanism, i.e., incubation with IL-6 and TGF-b, but by two different modes of stimulation. Naïve CD4 cells, collected from TCR transgenic mice specific against hen egg lysozyme (HEL), were activated by either HEL and APC (“HA”), or by plate-bound antibodies against CD3/CD28 (“PbAb”). PbAb Th17 produced moderately higher levels of IL-17 than HA Th17, but the latter cells released profoundly higher levels of IL-22. Interestingly, Th17 cells of the two subsets produced similar levels of IL-10, shown by both analysis of the culture supernatants and intracellular staining. Importantly, when adoptively transferred into recipients expressing HEL in their lens, only HA Th17 cells induced ocular inflammation in recipients, while no disease was detected in eyes of PbAb cell recipients. It is also of note that the two Th17 subsets differed in their profile of chemokine transcripts, resembling the two different profiles recorded by McGeachy et al with the pathogenic and non-pathogenic subsets. Thus, the pathogenic or non-pathogenic Th17 subsets are generated in our system, without pre-activation, by two stimulation modes.