DC-SCRIPT deficiency delays mouse mammary gland development and branching morphogenesis

Mammary glands are unique organs in which major adaptive changes occur in morphogenesis and development after birth. Breast cancer is the most common cancer and a major cause of mortality in females worldwide. We have previously identified the loss of expression of the transcription regulator DC-SCRIPT (Zfp366) as a prominent prognostic event in estrogen receptor positive breast cancer patients. DC-SCRIPT affects multiple transcriptional events in breast cancer cells, including estrogen and progesterone receptor-mediated transcription, and promotes CDKN2B-related cell cycle arrest. As loss of DC-SCRIPT expression appears an early event in breast cancer development, we here investigated the role of DC-SCRIPT in mammary gland development using wild-type and DC-SCRIPT knockout mice. Mice lacking DC-SCRIPT exhibited severe breeding problems and showed significant growth delay relative to littermate wild-type mice. Subsequent analysis revealed that DC-SCRIPT was expressed in mouse mammary epithelium and that DC-SCRIPT deficiency delayed mammary gland morphogenesis in vivo. Finally, analysis of 3D mammary gland organoid cultures confirmed that loss of DC-SCRIPT dramatically delayed mammary organoid branching in vitro. The study shows for the first time that DC-SCRIPT deficiency delays mammary gland morphogenesis in vivo and in vitro. These data define DC-SCRIPT as a novel modulator of mammary gland development

DC-SCRIPT is a novel regulator of the tumor suppressor gene CDKN2B and induces cell cycle arrest in ERα-positive breast cancer cells

Breast cancer is one of the most common causes of cancer-related deaths in women. The estrogen receptor (ERα) is well known for having growth promoting effects in breast cancer. Recently, we have identified DC-SCRIPT (ZNF366) as a co-suppressor of ERα and as a strong and independent prognostic marker in ESR1 (ERα gene)-positive breast cancer patients. In this study, we further investigated the molecular mechanism on how DC-SCRIPT inhibits breast cancer cell growth. DC-SCRIPT mRNA levels from 190 primary ESR1-positive breast tumors were related to global gene expression, followed by gene ontology and pathway analysis. The effect of DC-SCRIPT on breast cancer cell growth and cell cycle arrest was investigated using novel DC-SCRIPT-inducible MCF7 breast cancer cell lines. Genome-wide expression profiling of DC-SCRIPT-expressing MCF7 cells was performed to investigate the effect of DC-SCRIPT on cell cycle-related gene expression. Findings were validated by real-time PCR in a cohort of 1,132 ESR1-positive breast cancer patients. In the primary ESR1-positive breast tumors, DC-SCRIPT expression negatively correlated with several cell cycle gene ontologies and pathways. DC-SCRIPT expression strongly reduced breast cancer cell growth in vitro, breast tumor growth in vivo, and induced cell cycle arrest. In addition, in the presence of DC-SCRIPT, multiple cell cycles related genes were differentially expressed including the tumor suppressor gene CDKN2B. Moreover, in 1,132 primary ESR1-positive breast tumors, DC-SCRIPT expression also correlated with CDKN2B expression. Collectively, these data show that DC-SCRIPT acts as a novel regulator of CDKN2B and induces cell cycle arrest in ESR1-positive breast cancer cells

Molecular characterization of the murine homologue of the DC-derived protein DC-SCRIPT.

Contains fulltext : 49437.pdf ( ) (Open Access)Dendritic cell-specific transcript (DC-SCRIPT) is a putative DC zinc (Zn) finger-type transcription factor described recently in humans. Here, we illustrate that DC-SCRIPT is highly conserved in evolution and report the initial characterization of the murine ortholog of DC-SCRIPT, which is also preferentially expressed in DC as shown by real-time quantitative polymerase chain reaction, and its distribution resembles that of its human counterpart. Studies undertaken in human embryonic kidney 293 cells depict its nuclear localization and reveal that the Zn finger domain of the protein is mainly responsible for nuclear import. The human and the mouse genes are located in syntenic chromosomal regions and exhibit a similar genomic organization with numerous common transcription factor-binding sites in their promoter region, including sites for many factors implicated in haematopoiesis and DC biology, such as Gfi, GATA-1, Spi-B, and c-Rel. Taken together, these data show that DC-SCRIPT is well-conserved in evolution and that the mouse homologue is more than 80% homologous to the human protein. Therefore, mouse models can be used to elucidate the function of this novel DC marker

Dendritic cells actively limit interleukin-10 production under inflammatory conditions via DC-SCRIPT and dual-specificity phosphatase 4

Dendritic cell (DC)-based immunotherapy makes use of the DC's ability to direct the adaptive immune response toward activation or inhibition. DCs perform this immune orchestration in part by secretion of selected cytokines. The most potent anti-inflammatory cytokine interleukin-10 (IL-10) is under tight regulation, as it needs to be predominantly expressed during the resolution phase of the immune response. Currently it is not clear whether there is active suppression of IL-10 by DCs at the initial pro-inflammatory stage of the immune response. Previously, knockdown of the DC-specific transcription factor DC-SCRIPT has been demonstrated to mediate an extensive increase in IL-10 production upon encounter with pro-inflammatory immune stimuli. Here, we explored how DC-SCRIPT contributes to IL-10 suppression under pro-inflammatory conditions by applying chromatin immunoprecipitation sequencing analysis of DC-SCRIPT and the epigenetic marks H3K4me3 and H3K27ac in human DCs. The data showed binding of DC-SCRIPT to a GA-rich motif at H3K27ac-marked genomic enhancers that associated with genes encoding MAPK dual-specificity phosphatases (DUSPs). Functional studies revealed that upon knockdown of DC-SCRIPT, human DCs express much less DUSP4 and exhibit increased phosphorylation of the three major MAPKs (ERK, JNK, and p38). Enhanced ERK signaling in DC-SCRIPT-knockdown-DCs led to higher production of IL-10, which was reverted by rescuing DUSP4 expression. Finally, DC-SCRIPT-knockdown-DCs induced less IFN-γ and increased IL-10 production in naïve T cells, indicative for a more anti-inflammatory phenotype. In conclusion, we have delineated a new mechanism by which DC-SCRIPT allows DCs to limit IL-10 production under inflammatory conditions and potentiate pro-inflammatory Th1 responses. These insights may be exploited to improve DC-based immunotherapies

Saponin-based adjuvants enhance antigen cross-presentation in human CD11c+ CD1c+ CD5− CD163+ conventional type 2 dendritic cells

Background Adjuvants are key for effective vaccination against cancer and chronic infectious diseases. Saponin-based adjuvants (SBAs) are unique among adjuvants in their ability to induce robust cell-mediated immune responses in addition to antibody responses. Recent preclinical studies revealed that SBAs induced cross-presentation and lipid bodies in otherwise poorly cross-presenting CD11b+ murine dendritic cells (DCs).Method Here, we investigated the response of human DC subsets to SBAs with RNA sequencing and pathway analyses, lipid body induction visualized by laser scanning microscopy, antigen translocation to the cytosol, and antigen cross-presentation to CD8+ T cells.Results RNA sequencing of SBA-treated conventional type 1 DC (cDC1) and type 2 DC (cDC2) subsets uncovered that SBAs upregulated lipid-related pathways in CD11c+ CD1c+ cDC2s, especially in the CD5− CD163+ CD14+ cDC2 subset. Moreover, SBAs induced lipid bodies and enhanced endosomal antigen translocation into the cytosol in this particular cDC2 subset. Finally, SBAs enhanced cross-presentation only in cDC2s, which requires the CD163+ CD14+ cDC2 subset.Conclusions These data thus identify the CD163+ CD14+ cDC2 subset as the main SBA-responsive DC subset in humans and imply new strategies to optimize the application of saponin-based adjuvants in a potent cancer vaccine

table_1_Dendritic Cells Actively Limit Interleukin-10 Production Under Inflammatory Conditions via DC-SCRIPT and Dual-Specificity Phosphatase 4.xlsx

<p>Dendritic cell (DC)-based immunotherapy makes use of the DC’s ability to direct the adaptive immune response toward activation or inhibition. DCs perform this immune orchestration in part by secretion of selected cytokines. The most potent anti-inflammatory cytokine interleukin-10 (IL-10) is under tight regulation, as it needs to be predominantly expressed during the resolution phase of the immune response. Currently it is not clear whether there is active suppression of IL-10 by DCs at the initial pro-inflammatory stage of the immune response. Previously, knockdown of the DC-specific transcription factor DC-SCRIPT has been demonstrated to mediate an extensive increase in IL-10 production upon encounter with pro-inflammatory immune stimuli. Here, we explored how DC-SCRIPT contributes to IL-10 suppression under pro-inflammatory conditions by applying chromatin immunoprecipitation sequencing analysis of DC-SCRIPT and the epigenetic marks H3K4me3 and H3K27ac in human DCs. The data showed binding of DC-SCRIPT to a GA-rich motif at H3K27ac-marked genomic enhancers that associated with genes encoding MAPK dual-specificity phosphatases (DUSPs). Functional studies revealed that upon knockdown of DC-SCRIPT, human DCs express much less DUSP4 and exhibit increased phosphorylation of the three major MAPKs (ERK, JNK, and p38). Enhanced ERK signaling in DC-SCRIPT-knockdown-DCs led to higher production of IL-10, which was reverted by rescuing DUSP4 expression. Finally, DC-SCRIPT-knockdown-DCs induced less IFN-γ and increased IL-10 production in naïve T cells, indicative for a more anti-inflammatory phenotype. In conclusion, we have delineated a new mechanism by which DC-SCRIPT allows DCs to limit IL-10 production under inflammatory conditions and potentiate pro-inflammatory Th1 responses. These insights may be exploited to improve DC-based immunotherapies.</p

image_3_Dendritic Cells Actively Limit Interleukin-10 Production Under Inflammatory Conditions via DC-SCRIPT and Dual-Specificity Phosphatase 4.PDF

<p>Dendritic cell (DC)-based immunotherapy makes use of the DC’s ability to direct the adaptive immune response toward activation or inhibition. DCs perform this immune orchestration in part by secretion of selected cytokines. The most potent anti-inflammatory cytokine interleukin-10 (IL-10) is under tight regulation, as it needs to be predominantly expressed during the resolution phase of the immune response. Currently it is not clear whether there is active suppression of IL-10 by DCs at the initial pro-inflammatory stage of the immune response. Previously, knockdown of the DC-specific transcription factor DC-SCRIPT has been demonstrated to mediate an extensive increase in IL-10 production upon encounter with pro-inflammatory immune stimuli. Here, we explored how DC-SCRIPT contributes to IL-10 suppression under pro-inflammatory conditions by applying chromatin immunoprecipitation sequencing analysis of DC-SCRIPT and the epigenetic marks H3K4me3 and H3K27ac in human DCs. The data showed binding of DC-SCRIPT to a GA-rich motif at H3K27ac-marked genomic enhancers that associated with genes encoding MAPK dual-specificity phosphatases (DUSPs). Functional studies revealed that upon knockdown of DC-SCRIPT, human DCs express much less DUSP4 and exhibit increased phosphorylation of the three major MAPKs (ERK, JNK, and p38). Enhanced ERK signaling in DC-SCRIPT-knockdown-DCs led to higher production of IL-10, which was reverted by rescuing DUSP4 expression. Finally, DC-SCRIPT-knockdown-DCs induced less IFN-γ and increased IL-10 production in naïve T cells, indicative for a more anti-inflammatory phenotype. In conclusion, we have delineated a new mechanism by which DC-SCRIPT allows DCs to limit IL-10 production under inflammatory conditions and potentiate pro-inflammatory Th1 responses. These insights may be exploited to improve DC-based immunotherapies.</p