WNT-1 Signaling Inhibits Apoptosis by Activating β-Catenin/T Cell Factor–Mediated Transcription

Wnt signaling plays a critical role in development and oncogenesis. Although significant progress has been made in understanding the downstream signaling cascade of Wnt signaling, little is known regarding Wnt signaling modification of the cell death machinery. Given that numerous oncogenes transform cells by providing cell survival function, we hypothesized that Wnt signaling may inhibit apoptosis. Here, we report that cells expressing Wnt-1 were resistant to cancer therapy–mediated apoptosis. Wnt-1 signaling inhibited the cytochrome c release and the subsequent caspase-9 activation induced by chemotherapeutic drugs, including both vincristine and vinblastine. Furthermore, we found that Wnt-1–mediated cell survival was dependent on the activation of β-catenin/T cell factor (Tcf) transcription. Inhibition of β-catenin/Tcf transcription by expression of the dominant-negative mutant of Tcf-4 blocked Wnt-1–mediated cell survival and rendered cells sensitive to apoptotic stimuli. These results provide the first demonstration that Wnt-1 inhibits cancer therapy–mediated apoptosis and suggests that Wnt-1 may exhibit its oncogenic potential through a mechanism of anti-apoptosis

VEGFR-1 blockade disrupts peri-implantation decidual angiogenesis and macrophage recruitment

Background: Angiogenesis and macrophage recruitment to the uterus are key features of uterine decidualization; the progesterone-mediated uterine changes that allow for embryo implantation and initiation of pregnancy. In the current study, we characterized the expression of vascular endothelial growth factor receptor-1 (VEGFR-1) in macrophages and endothelial cells of the peri-implantation uterus and determined if VEGFR-1 function is required for decidual angiogenesis, macrophage recruitment, and/or the establishment of pregnancy. Methods: Expression of VEGFR-1 in uterine endothelial cells and macrophages was determined with immunohistochemistry. To assess the effect of continuous VEGFR-1 blockade, adult female mice were given VEGFR-1 blocking antibody, MF-1, every 3 days for 18 days. After 6 doses, females were mated and a final dose of MF-1 was given on embryonic day 3.5. Endothelial cells and macrophages were quantified on embryonic day 7.5. Pregnancy was analyzed on embryonic days 7.5 and 10.5. Results: F4/80+ macrophages are observed throughout the stroma and are abundant adjacent to the endometrial lumen and glands prior to embryo implantation and scatter throughout the decidua post implantation. VEGFR-1 expression is restricted to the uterine endothelial cells. F4/80+ macrophages were often found adjacent to VEGFR-1+ endothelial cells in the primary decidual zone. Continuous VEGFR-1 blockade correlates with a significant reduction in decidual vascular and macrophage density, but does not affect embryo implantation or maintenance of pregnancy up to embryonic day 10.5. Conclusions: We found that VEGFR-1 functions in both decidual angiogenesis and macrophage recruitment to the implantation site during pregnancy. VEGFR-1 is expressed by endothelial cells, however blocking VEGFR-1 function in endothelial cells results in reduced macrophage recruitment to the uterus. VEGFR-1 blockade did not compromise the establishment and/or maintenance of pregnancy

Inhibition of the Vascular Endothelial Cell (VE)-Specific Adhesion Molecule VE-Cadherin Blocks Gonadotropin-Dependent Folliculogenesis and Corpus Luteum Formation and Angiogenesis

Although it has been previously demonstrated that administration of anti-vascular endothelial growth factor (VEGF) receptor-2 antibodies to hypophysectomized (Hx) mice during gonadotropin-stimulated folliculogenesis and luteogenesis inhibits angiogenesis in the developing follicle and corpus luteum (CL), it is unclear which of the many components of VEGF inhibition are important for the inhibitory effects on ovarian angiogenesis. To examine whether ovarian angiogenesis can be more specifically targeted, we administered an antibody to VE-cadherin (VE-C), an interendothelial adhesion molecule, to Hx mice during gonadotropin stimulation. In tumor models and in vivo and in vitro assays, the anti-VE-C antibody E4G10 has been shown to specifically inhibit angiogenesis, but VE-C has yet to be inhibited in the context of ovarian angiogenesis. In addition to studying the effect on neovascularization in the follicular and luteal phases, we also examined the effect of E4G10 on established vessels of the CL of pregnancy. The results demonstrate that E4G10 specifically blocks neovascularization in the follicular and luteal phases, causing an inhibition of preovulatory follicle and CL development, a decrease in the vascular area, and an inhibition of function demonstrated by reduced hormone levels. However, when administered during pregnancy, unlike anti-VEGF receptor-2 antibody, E4G10 is unable to cause disruption of the established vessels of the mature CL. These data demonstrate that E4G10 causes a specific inhibition of neovascularization in the ovary without destabilizing preexisting vasculature

Wnt signaling promotes oncogenic transformation by inhibiting c-Myc–induced apoptosis

Aberrant activation of the Wnt/β-catenin signaling pathway is associated with numerous human cancers and often correlates with the overexpression or amplification of the c-myc oncogene. Paradoxical to the cellular transformation potential of c-Myc is its ability to also induce apoptosis. Using an inducible c-MycER expression system, we found that Wnt/β-catenin signaling suppressed apoptosis by inhibiting c-Myc–induced release of cytochrome c and caspase activation. Both cyclooxygenase 2 and WISP-1 were identified as effectors of the Wnt-mediated antiapoptotic signal. Soft agar assays showed that neither c-Myc nor Wnt-1 alone was sufficient to induce cellular transformation, but that Wnt and c-Myc coordinated in inducing transformation. Furthermore, coexpression of Wnt-1 and c-Myc induced high-frequency and rapid tumor growth in nude mice. Extensive apoptotic bodies were characteristic of c-Myc–induced tumors, but not tumors induced by coactivation of c-Myc and Wnt-1, indicating that the antiapoptotic function of Wnt-1 plays a critical role in the synergetic action between c-Myc and Wnt-1. These results elucidate the molecular mechanisms by which Wnt/β-catenin inhibits apoptosis and provide new insight into Wnt signaling-mediated oncogenesis

Short-Term Administration of Antivascular Endothelial Growth Factor Antibody in the Late Follicular Phase Delays Follicular Development in the Rhesus Monkey

Indirect evidence in the nonhuman primate and human suggests that angiogenesis and regulators of angiogenesis such as vascular endothelial growth factor (VEGF) may play an active role in cyclic folliculogenesis. Indeed, the follicle selected for maturation and ovulation possesses a denser microvascular network, and VEGF messenger ribonucleic acid and its protein have been identified in granulosa cells of the developing follicle during the mid- and late follicular phases, with a more intense signal in the mature follicle. The objective of this study was to obtain direct evidence in the nonhuman primate for an active role of VEGF in follicular growth and maturation by studying the effect of VEGF-blocking antibodies in this process. After documenting two normal ovulatory cycles, female rhesus monkeys (n = 7) received iv injections of anti-VEGF antibodies (0.5 mg) twice on successive days in the late follicular phase. Three monkeys also received nonspecific goat IgG (0.5 mg) twice on successive days in the late follicular phase. Daily measurements of estradiol, progesterone, LH, and FSH were obtained during the two control cycles, the anti-VEGF treatment and posttreatment cycles, and the IgG treatment cycle. Anti-VEGF antibody administration significantly lengthened the follicular phase in six of seven monkeys to 17.8 ± 1.7 vs. 10.0 ± 0.7 and 9.8 ± 0.6 in control cycles and 10.7 ± 0.3 days (mean ± se) in IgG-treated cycles. The expected late follicular phase rise in estradiol, as documented in the control cycles (day 0, 96.1 ± 6.0; day 1, 125.5 ± 20.0; day 2, 165.5 ± 24.9; day 3, 183.8 ± 11.0 pg/mL), was interrupted by anti-VEGF antibody treatment (99.3 ± 5.0, day 0, preinjection control) to 63.3± 12.2 (day 1), 48.5 ± 8.7 (day 2), and 57.6 ± 9.0 (day 3). Mean FSH levels were significantly increased by day 2 of anti-VEGF antibody treatment. After a variable delay, estradiol concentrations increased to reach a preovulatory peak in all anti-VEGF-treated animals, followed by ovulation, normal luteal function, and a normal posttreatment cycle. The data clearly demonstrate that short-term inhibition of angiogenesis with an anti-VEGF-blocking antibody during the later growth phase of the dominant follicle interferes with normal follicular development. Persistence of estradiol secretion and delayed resumption of its rise also suggest recovery of the follicle. We conclude that the angiogenic regulator VEGF is a crucial component in the process of follicular growth in the primate

Bile acid and inflammation activate gastric cardia stem cells in a mouse model of barrett-like metaplasia

Esophageal adenocarcinoma (EAC) arises from Barrett esophagus (BE), intestinal-like columnar metaplasia linked to reflux esophagitis. In a transgenic mouse model of BE, esophageal overexpression of interleukin-1β phenocopies human pathology with evolution of esophagitis, Barrett-like metaplasia and EAC. Histopathology and gene signatures closely resembled human BE, with upregulation of TFF2, Bmp4, Cdx2, Notch1, and IL-6. The development of BE and EAC was accelerated by exposure to bile acids and/or nitrosamines, and inhibited by IL-6 deficiency. Lgr5+ gastric cardia stem cells present in BE were able to lineage trace the early BE lesion. Our data suggest that BE and EAC arise from gastric progenitors due to a tumor-promoting IL-1β-IL-6 signaling cascade and Dll1-dependent Notch signaling. © 2012 Elsevier Inc

Transcription factor TFCP2L1 patterns cells in the mouse kidney collecting ducts

Although most nephron segments contain one type of epithelial cell, the collecting ducts consists of at least two: intercalated (IC) and principal (PC) cells, which regulate acid-base and salt-water homeostasis, respectively. In adult kidneys, these cells are organized in rosettes suggesting functional interactions. Genetic studies in mouse revealed that transcription factor Tfcp2l1 coordinates IC and PC development. Tfcp2l1 induces the expression of IC specific genes, including specific H(+)-ATPase subunits and Jag1. Jag1 in turn, initiates Notch signaling in PCs but inhibits Notch signaling in ICs. Tfcp2l1 inactivation deletes ICs, whereas Jag1 inactivation results in the forfeiture of discrete IC and PC identities. Thus, Tfcp2l1 is a critical regulator of IC-PC patterning, acting cell-autonomously in ICs, and non-cell-autonomously in PCs. As a result, Tfcp2l1 regulates the diversification of cell types which is the central characteristic of 'salt and pepper' epithelia and distinguishes the collecting duct from all other nephron segments

Neural innervation stimulates splenic TFF2 to arrest myeloid cell expansion and cancer

CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) expand in the spleen during cancer and promote progression through suppression of cytotoxic T cells. An anti-inflammatory reflex arc involving the vagus nerve and memory T cells is necessary for resolution of acute inflammation. Failure of this neural circuit could promote procarcinogenic inflammation and altered tumour immunity. Here we show that splenic TFF2, a secreted anti-inflammatory peptide, is released by vagally modulated memory T cells to suppress the expansion of MDSCs through CXCR4. Splenic denervation interrupts the anti-inflammatory neural arc, resulting in the expansion of MDSCs and colorectal cancer. Deletion of Tff2 recapitulates splenic denervation to promote carcinogenesis. Colorectal carcinogenesis could be suppressed through transgenic overexpression of TFF2, adenoviral transfer of TFF2 or transplantation of TFF2-expressing bone marrow. TFF2 is important to the anti-inflammatory reflex arc and plays an essential role in arresting MDSC proliferation. TFF2 offers a potential approach to prevent and to treat cancer

Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2) Functions to Promote Uterine Decidual Angiogenesis during Early Pregnancy in the Mouse

Implantation of an embryo induces rapid proliferation and differentiation of uterine stromal cells, forming a new structure, the decidua. One salient feature of decidua formation is a marked increase in maternal angiogenesis. Vascular endothelial growth factor (VEGF)-dependent pathways are active in the ovary, uterus, and embryo, and inactivation of VEGF function in any of these structures might prevent normal pregnancy development. We hypothesized that decidual angiogenesis is regulated by VEGF acting through specific VEGF receptors (VEGFRs). To test this hypothesis, we developed a murine pregnancy model in which systemic administration of a receptor-blocking antibody would act specifically on uterine angiogenesis and not on ovarian or embryonic angiogenesis. In our model, ovarian function was replaced with exogenous progesterone, and blocking antibodies were administered prior to embryonic expression of VEGFRs. After administration of a single dose of the anti-VEGFR-2 antibody during the peri-implantation period, no embryos were detected on embryonic d 10.5. The pregnancy was disrupted because of a significant reduction in decidual angiogenesis, which under physiological conditions peaks on embryonic d 5.5 and 6.5. Inactivation of VEGFR-3 reduced angiogenesis in the primary decidual zone, whereas administration of VEGFR-1 blocking antibodies had no effect. Pregnancy was not disrupted after administration of anti-VEGFR-3 or anti-VEGFR-1 antibodies. Thus, the VEGF/VEGFR-2 pathway plays a key role in the maintenance of early pregnancy through its regulation of peri-implantation angiogenesis in the uterine decidua. This newly formed decidual vasculature serves as the first exchange apparatus for the developing embryo until the placenta becomes functionally active

Intraovarian regulation of gonadotropin-dependent folliculogenesis depends on notch receptor signaling pathways not involving Delta-like ligand 4 (Dll4)

Background: In-situ hybridisation studies demonstrate that Notch receptors and ligands are expressed in granulosa cells (GCs) and in the theca layer vasculature of growing follicles. Notch signaling involves cell-to-cell interaction mediated by transmembrane receptors and ligands. This signaling pathway may represent a novel intraovarian regulator of gonadotropin-dependent follicular development to the preovulatory stage. We hypothesized that blocking Notch pathways would disrupt follicular maturation in the mouse ovary. Methods: Hypophysectomized CD21 female mice were administered pregnant mare serum gonadotropin (PMSG) for 3 days to stimulate follicular development. In one experiment, a pan-notch inhibitor, compound E, was initiated 2 days prior to and throughout stimulation (n = 10), while in a second experiment, a humanized phage Dll4 blocking antibody, YW152F, was used (n = 5). After sacrifice, ovarian histology, serum estradiol levels and uterine weights were compared to controls. The ovarian morphology was evaluated with hematoxylin/eosin staining and immunohistochemistry was performed for Notch1, Notch2, Notch3, Notch4, Jagged1, Dll4, platelet endothelial cell adhesion molecule (PECAM) and alpha-smooth muscle actin (α-SMA) detection. Results: We localized specific Notch ligands and receptors in the following structures: Dll4 is specific to theca layer endothelial cells (ECs); Notch1/Notch4 and Jagged1 are expressed in theca layer ECs and vascular smooth muscle cells (VSMCs), whereas Notch3 is restricted to VSMCs; Notch2 is expressed mostly on GCs of small follicles. Administration of a pan-Notch inhibitor, compound E, inhibits follicular development to the preovulatory stage (8.5 preovulatory follicles in treatment vs. 3.4 preovulatory follicles in control, p < 0.01; average number per ovary) with significant secondary effects on ovarian and uterine weight and estradiol secretion in a setting of uninhibited vascular proliferation, but disorganized appearance of ECs and VSMCs. Inhibition of endothelial Notch1 function through the inactivation of its ligand Dll4 with the blocking antibody YW152F induces mild disorganisation of follicular vasculature, but has no significant effect on gonadotropin-dependent folliculogenesis. Conclusions: Our experiments suggest that the complete blockage of the Notch signaling pathway with compound E impairs folliculogenesis and induces disruption of gonadotropin stimulated angiogenesis. It seems the mechanism involves Notch1 and Notch3, specifically, causing the improper assembly of ECs and VSMCs in the theca layer, although the potential role of non-angiogenic Notch signaling, such as Jagged2 to Notch2 in GCs, remains to be elucidated