Merkel cell carcinoma-derived exosome-shuttle miR-375 induces fibroblast polarization by inhibition of RBPJ and p53.

Merkel cell carcinoma (MCC) is a highly invasive and metastatic skin cancer. While high expression of miR-375 is a characteristic of MCC, it seems not to contribute to the malignant phenotype of MCC cells. miR-375 enrichment in MCC-derived extracellular vesicles suggests its intercellular signaling function. Here, we demonstrate that horizontally transferred miR-375 causes fibroblast polarization toward cancer-associated fibroblasts (CAFs). The polarization is evidenced by phenotypic changes and induction of α-SMA, CXCL2, and IL-1ß. Fibroblast polarization is inhibited by specific antagomirs and mimicked by experimental miR-375 expression. Mechanistically, miR-375 downregulates RBPJ and p53, two key players regulating fibroblast polarization. In clinical MCC samples, in situ hybridization located miR-375 in CAFs, which correlated with high α-SMA protein and low RBPJ and TP53 expression; single-cell RNAseq revealed a disparate fibroblast polarization negatively correlating with p53 pathway-related gene expression. Thus, the functional role of miR-375 in MCC is to generate a pro-tumorigenic microenvironment by inducing fibroblast polarization.

Development of Human CBF1-Targeting Single-Stranded DNA Aptamers with Antiangiogenic Activity In Vitro.

C promoter binding factor 1 (CBF1) (alias RBPJ) is a critical transcription factor involved in Notch signaling. The activation of Notch signaling through CBF1 maintains the angiostatic state of endothelial cells suppressing angiogenesis, that is, the formation of new blood vessels. Vascular endothelial growth factor (VEGF) induces angiogenesis by promoting the proteasomal degradation of CBF1, in addition to endothelial cell proliferation. To date, angiogenic inhibitors targeting VEGF have been successfully used in clinics for cancer and age-related macular degeneration. Most antiangiogenic drugs, however, only target VEGF or VEGF receptors. In this study, to expand the repertoire of antiangiogenic therapeutics, we developed 15 single-stranded deoxyribonucleic acid (ssDNA) aptamers capable of binding to CBF1 with high affinity (Kd; 10-300 nM). To this end, systematic evolution of ligands by the exponential enrichment (SELEX) method was applied. One of the CBF1-binding ssDNA aptamers, Apt-3, inhibited angiogenesis through the activation of Notch signaling in vitro. We found that Apt-3 directly interacted with the LAG1 domain of CBF1. We suggest that the Apt-3 ssDNA aptamer may contribute to the development of a novel angiogenic inhibitor, which does not target VEGF.

Disruption of NOTCH signaling by a small molecule inhibitor of the transcription factor RBPJ.

NOTCH plays a pivotal role during normal development and in congenital disorders and cancer. γ-secretase inhibitors are commonly used to probe NOTCH function, but also block processing of numerous other proteins. We discovered a new class of small molecule inhibitor that disrupts the interaction between NOTCH and RBPJ, which is the main transcriptional effector of NOTCH signaling. RBPJ Inhibitor-1 (RIN1) also blocked the functional interaction of RBPJ with SHARP, a scaffold protein that forms a transcriptional repressor complex with RBPJ in the absence of NOTCH signaling. RIN1 induced changes in gene expression that resembled siRNA silencing of RBPJ rather than inhibition at the level of NOTCH itself. Consistent with disruption of NOTCH signaling, RIN1 inhibited the proliferation of hematologic cancer cell lines and promoted skeletal muscle differentiation from C2C12 myoblasts. Thus, RIN1 inhibits RBPJ in its repressing and activating contexts, and can be exploited for chemical biology and therapeutic applications.

Notch Signaling Regulates Differentiation and Steroidogenesis in Female Mouse Ovarian Granulosa Cells.

The Notch pathway is a highly conserved juxtacrine signaling mechanism that is important for many cellular processes during development, including differentiation and proliferation. Although Notch is important during ovarian follicle formation and early development, its functions during the gonadotropin-dependent stages of follicle development are largely unexplored. We observed positive regulation of Notch activity and expression of Notch ligands and receptors following activation of the luteinizing hormone-receptor in prepubertal mouse ovary. JAG1, the most abundantly expressed Notch ligand in mouse ovary, revealed a striking shift in localization from oocytes to somatic cells following hormone stimulation. Using primary cultures of granulosa cells, we investigated the functions of Jag1 using small interfering RNA knockdown. The loss of JAG1 led to suppression of granulosa cell differentiation as marked by reduced expression of enzymes and factors involved in steroid biosynthesis, and in steroid secretion. Jag1 knockdown also resulted in enhanced cell proliferation. These phenotypes were replicated, although less robustly, following knockdown of the obligate canonical Notch transcription factor RBPJ. Intracellular signaling analysis revealed increased activation of the mitogenic phosphatidylinositol 3-kinase/protein kinase B and mitogen-activated protein kinase/extracellular signal-regulated kinase pathways following Notch knockdown, with a mitogen-activated protein kinase kinase inhibitor blocking the enhanced proliferation observed in Jag1 knockdown granulosa cells. Activation of YB-1, a known regulator of granulosa cell differentiation genes, was suppressed by Jag1 knockdown. Overall, this study reveals a role of Notch signaling in promoting the differentiation of preovulatory granulosa cells, adding to the diverse functions of Notch in the mammalian ovary.

Lightening up a notch: Notch regulation of energy metabolism.

Inhibiting Notch signaling induces adipose browning, improves systemic glucose tolerance and insulin sensitivity, and suppresses weight gain in mice.

Inhibition of Notch signaling promotes browning of white adipose tissue and ameliorates obesity.

Beige adipocytes in white adipose tissue (WAT) are similar to classical brown adipocytes in that they can burn lipids to produce heat. Thus, an increase in beige adipocyte content in WAT browning would raise energy expenditure and reduce adiposity. Here we report that adipose-specific inactivation of Notch1 or its signaling mediator Rbpj in mice results in browning of WAT and elevated expression of uncoupling protein 1 (Ucp1), a key regulator of thermogenesis. Consequently, as compared to wild-type mice, Notch mutants exhibit elevated energy expenditure, better glucose tolerance and improved insulin sensitivity and are more resistant to high fat diet-induced obesity. By contrast, adipose-specific activation of Notch1 leads to the opposite phenotypes. At the molecular level, constitutive activation of Notch signaling inhibits, whereas Notch inhibition induces, Ppargc1a and Prdm16 transcription in white adipocytes. Notably, pharmacological inhibition of Notch signaling in obese mice ameliorates obesity, reduces blood glucose and increases Ucp1 expression in white fat. Therefore, Notch signaling may be therapeutically targeted to treat obesity and type 2 diabetes.

Dishevelled limits Notch signalling through inhibition of CSL.

Notch and Wnt are highly conserved signalling pathways that are used repeatedly throughout animal development to generate a diverse array of cell types. However, they often have opposing effects on cell-fate decisions with each pathway promoting an alternate outcome. Commonly, a cell receiving both signals exhibits only Wnt pathway activity. This suggests that Wnt inhibits Notch activity to promote a Wnt-ON/Notch-OFF output; but what might underpin this Notch regulation is not understood. Here, we show that Wnt acts via Dishevelled to inhibit Notch signalling, and that this crosstalk regulates cell-fate specification in vivo during Xenopus development. Mechanistically, Dishevelled binds and directly inhibits CSL transcription factors downstream of Notch receptors, reducing their activity. Furthermore, our data suggest that this crosstalk mechanism is conserved between vertebrate and invertebrate homologues. Thus, we identify a dual function for Dishevelled as an inhibitor of Notch signalling and an activator of the Wnt pathway that sharpens the distinction between opposing Wnt and Notch responses, allowing for robust cell-fate decisions.

BAZF, a novel component of cullin3-based E3 ligase complex, mediates VEGFR and Notch cross-signaling in angiogenesis.

Angiogenic homeostasis is maintained by a balance between vascular endothelial growth factor (VEGF) and Notch signaling in endothelial cells (ECs). We screened for molecules that might mediate the coupling of VEGF signal transduction with down-regulation of Notch signaling, and identified B-cell chronic lymphocytic leukemia/lymphoma6-associated zinc finger protein (BAZF). BAZF was induced by VEGF-A in ECs to bind to the Notch signaling factor C-promoter binding factor 1 (CBF1), and to promote the degradation of CBF1 through polyubiquitination in a CBF1-cullin3 (CUL3) E3 ligase complex. BAZF disruption in vivo decreased endothelial tip cell number and filopodia protrusion, and markedly abrogated vascular plexus formation in the mouse retina, overlapping the retinal phenotype seen in response to Notch activation. Further, impaired angiogenesis and capillary remodeling were observed in skin-wounded BAZF(-/-) mice. We therefore propose that BAZF supports angiogenic sprouting via BAZF-CUL3-based polyubiquitination-dependent degradation of CBF1 to down-regulate Notch signaling.

Wt1a, Foxc1a, and the Notch mediator Rbpj physically interact and regulate the formation of podocytes in zebrafish.

Podocytes help form the glomerular blood filtration barrier in the kidney and their injury or loss leads to renal disease. The Wilms' tumor suppressor-1 (Wt1) and the FoxC1/2 transcription factors, as well as Notch signaling, have been implicated as important regulators of podocyte fate. It is not known whether these factors work in parallel or sequentially on different gene targets, or as higher-order transcriptional complexes on common genes. Here, we use the zebrafish to demonstrate that embryos treated with morpholinos against wt1a, foxc1a, or the Notch transcriptional mediator rbpj develop fewer podocytes, as determined by wt1b, hey1 and nephrin expression, while embryos deficient in any two of these factors completely lack podocytes. From GST-pull-downs and co-immunoprecipitation experiments we show that Wt1a, Foxc1a, and Rbpj can physically interact with each other, whereas only Rbpj binds to the Notch intracellular domain (NICD). In transactivation assays, combinations of Wt1, FoxC1/2, and NICD synergistically induce the Hey1 promoter, and have additive or repressive effects on the Podocalyxin promoter, depending on dosage. Taken together, these data suggest that Wt1, FoxC1/2, and Notch signaling converge on common target genes where they physically interact to regulate a podocyte-specific gene program. These findings further our understanding of the transcriptional circuitry responsible for podocyte formation and differentiation during kidney development.

Notch target gene deregulation and maintenance of the leukemogenic phenotype do not require RBP-J kappa in Ikaros null mice.

Ikaros and Notch are transcriptional regulators essential for normal T cell development. Aberrant activation of Notch target genes is observed in Ikaros-deficient thymocytes as well as leukemia cell lines. However, it is not known whether Notch deregulation plays a preferential or obligatory role in the leukemia that arise in Ikaros null (Ik(-/-)) mice. To answer this question, the expression of the DNA-binding Notch target gene activator RBP-Jkappa was abrogated in Ik(-/-) double-positive thymocytes. This was accomplished through conditional inactivation using CD4-Cre transgenic mice containing floxed RBP-Jkappa alleles (RBPJ(fl/fl)). Ik(-/-) x RBPJ(fl/fl) x CD4-Cre(+) transgenic mice develop clonal T cell populations in the thymus that escape to the periphery, with similar kinetics and penetrance as their CD4-Cre(-) counterparts. The clonal populations do not display increased RBP-Jkappa expression compared with nontransformed thymocytes, suggesting there is no selection for clones that have not fully deleted RBP-Jkappa. However, RBPJ-deficient clonal populations do not expand as aggressively as their RBPJ-sufficient counterparts, suggesting a qualitative role for deregulated Notch target gene activation in the leukemogenic process. Finally, these studies show that RBP-Jkappa plays no role in Notch target gene repression in double-positive thymocytes but rather that it is Ikaros that is required for the repression of these genes at this critical stage of T cell development.

Inhibition of Notch pathway prevents osteosarcoma growth by cell cycle regulation.

The study shows constitutive activation of the Notch pathway in various types of malignancies. However, it remains unclear how the Notch pathway is involved in the pathogenesis of osteosarcoma. We investigated the expression of the Notch pathway molecules in osteosarcoma biopsy specimens and examined the effect of Notch pathway inhibition. Real-time PCR revealed overexpression of Notch2, Jagged1, HEY1, and HEY2. On the other hand, Notch1 and DLL1 were downregulated in biopsy specimens. Notch pathway inhibition using gamma-secretase inhibitor and CBF1 siRNA slowed the growth of osteosarcomas in vitro. In addition, gamma-secretase inhibitor-treated xenograft models exhibited significantly slower osteosarcoma growth. Cell cycle analysis revealed that gamma-secretase inhibitor promoted G1 arrest. Real-time PCR and western blot revealed that gamma-secretase inhibitor reduced the expression of accelerators of the cell cycle, including cyclin D1, cyclin E1, E2, and SKP2. On the other hand, p21(cip1) protein, a cell cycle suppressor, was upregulated by gamma-secretase inhibitor treatment. These findings suggest that inhibition of Notch pathway suppresses osteosarcoma growth by regulation of cell cycle regulator expression and that the inactivation of the Notch pathway may be a useful approach to the treatment of patients with osteosarcoma.

NF-kappaB serves as a cellular sensor of Kaposi's sarcoma-associated herpesvirus latency and negatively regulates K-Rta by antagonizing the RBP-Jkappa coactivator.

Successful viral replication is dependent on a conducive cellular environment; thus, viruses must be sensitive to the state of their host cells. We examined the idea that an interplay between viral and cellular regulatory factors determines the switch from Kaposi's sarcoma-associated herpesvirus (KSHV) latency to lytic replication. The immediate-early gene product K-Rta is the first viral protein expressed and an essential factor in reactivation; accordingly, this viral protein is in a key position to serve as a viral sensor of cellular physiology. Our approach aimed to define a host transcription factor, i.e., host sensor, which modulates K-Rta activity on viral promoters. To this end, we developed a panel of reporter plasmids containing all 83 putative viral promoters for a comprehensive survey of the response to both K-Rta and cellular transcription factors. Interestingly, members of the NF-kappaB family were shown to be strong negative regulators of K-Rta transactivation for all but two viral promoters (Ori-RNA and K12). Recruitment of K-Rta to the ORF57 and K-bZIP promoters, but not the K12 promoter, was significantly impaired when NF-kappaB expression was induced. Many K-Rta-responsive promoters modulated by NF-kappaB contain the sequence of the RBP-Jkappa binding site, a major coactivator which anchors K-Rta to target promoters via consensus motifs which overlap with that of NF-kappaB. Gel shift assays demonstrated that NF-kappaB inhibits the binding of RBP-Jkappa and forms a complex with RBP-Jkappa. Our results support a model in which a balance between K-Rta/RBP-Jkappa and NF-kappaB activities determines KSHV reactivation. An important feature of this model is that the interplay between RBP-Jkappa and NF-kappaB on viral promoters controls viral gene expression mediated by K-Rta.