Norrin mediates tumor-promoting and -suppressive effects in glioblastoma via Notch and Wnt.

Glioblastoma multiforme (GBM) contains a subpopulation of cells, GBM stem cells (GSCs), that maintain the bulk tumor and represent a key therapeutic target. Norrin is a Wnt ligand that binds Frizzled class receptor 4 (FZD4) to activate canonical Wnt signaling. Although Norrin, encoded by NDP, has a well-described role in vascular development, its function in human tumorigenesis is largely unexplored. Here, we show that NDP expression is enriched in neurological cancers, including GBM, and its levels positively correlated with survival in a GBM subtype defined by low expression of ASCL1, a proneural factor. We investigated the function of Norrin and FZD4 in GSCs and found that it mediated opposing tumor-suppressive and -promoting effects on ASCL1lo and ASCL1hi GSCs. Consistent with a potential tumor-suppressive effect of Norrin suggested by the tumor outcome data, we found that Norrin signaling through FZD4 inhibited growth in ASCL1lo GSCs. In contrast, in ASCL1hi GSCs Norrin promoted Notch signaling, independently of WNT, to promote tumor progression. Forced ASCL1 expression reversed the tumor-suppressive effects of Norrin in ASCL1lo GSCs. Our results identify Norrin as a modulator of human brain cancer progression and reveal an unanticipated Notch-mediated function of Norrin in regulating cancer stem cell biology. This study identifies an unanticipated role of Norrin in human brain cancer progression. In addition, we provide preclinical evidence suggesting Norrin and canonical Wnt signaling as potential therapeutic targets for GBM subtype-restricted cancer stem cells.

Wnt and Notch signaling govern self-renewal and differentiation in a subset of human glioblastoma stem cells.

Developmental signal transduction pathways act diversely, with context-dependent roles across systems and disease types. Glioblastomas (GBMs), which are the poorest prognosis primary brain cancers, strongly resemble developmental systems, but these growth processes have not been exploited therapeutically, likely in part due to the extreme cellular and genetic heterogeneity observed in these tumors. The role of Wnt/ßcatenin signaling in GBM stem cell (GSC) renewal and fate decisions remains controversial. Here, we report context-specific actions of Wnt/ßcatenin signaling in directing cellular fate specification and renewal. A subset of primary GBM-derived stem cells requires Wnt proteins for self-renewal, and this subset specifically relies on Wnt/ßcatenin signaling for enhanced tumor burden in xenograft models. In an orthotopic Wnt reporter model, Wnthi GBM cells (which exhibit high levels of ßcatenin signaling) are a faster-cycling, highly self-renewing stem cell pool. In contrast, Wntlo cells (with low levels of signaling) are slower cycling and have decreased self-renewing potential. Dual inhibition of Wnt/ßcatenin and Notch signaling in GSCs that express high levels of the proneural transcription factor ASCL1 leads to robust neuronal differentiation and inhibits clonogenic potential. Our work identifies new contexts for Wnt modulation for targeting stem cell differentiation and self-renewal in GBM heterogeneity, which deserve further exploration therapeutically.

Paraneoplastic cerebellar degeneration secondary to ovarian carcinosarcoma: a cerebellar conundrum.

We present a case of an elderly female patient who presented with a 6-month history of progressive slurred speech, vertigo, unsteadiness and falls. She underwent an extensive battery of neurological and cardiovascular investigations, none of which demonstrated a diagnostic cause for her symptoms. She was referred to the stroke and neurology teams and was started on treatment for presumed anxiety. As her symptoms continued to progress, she was referred to the falls service. Following a multidisciplinary team discussion, she was reviewed by the consultant geriatrician who felt this may be due to a malignancy so the consultant geriatrician arranged blood testsand CT scan of her chest, abdomen and pelvis. These demonstrated a large left adnexal mass and a raised Ca-125 level. The patient was diagnosed with an ovarian tumour, which was treated surgically. A provisional diagnosis of paraneoplastic cerebellar degeneration, secondary to ovarian carcinosarcoma, was made.

Inhibition of Dopamine Receptor D4 Impedes Autophagic Flux, Proliferation, and Survival of Glioblastoma Stem Cells.

Glioblastomas (GBM) grow in a rich neurochemical milieu, but the impact of neurochemicals on GBM growth is largely unexplored. We interrogated 680 neurochemical compounds in patient-derived GBM neural stem cells (GNS) to determine the effects on proliferation and survival. Compounds that modulate dopaminergic, serotonergic, and cholinergic signaling pathways selectively affected GNS growth. In particular, dopamine receptor D4 (DRD4) antagonists selectively inhibited GNS growth and promoted differentiation of normal neural stem cells. DRD4 antagonists inhibited the downstream effectors PDGFRß, ERK1/2, and mTOR and disrupted the autophagy-lysosomal pathway, leading to accumulation of autophagic vacuoles followed by G0/G1 arrest and apoptosis. These results demonstrate a role for neurochemical pathways in governing GBM stem cell proliferation and suggest therapeutic approaches for GBM.

GLP-1R agonists promote normal and neoplastic intestinal growth through mechanisms requiring Fgf7.

Glucagon-like peptide-1 (GLP-1) secreted from enteroendocrine L cells promotes nutrient disposal via the incretin effect. However, the majority of L cells are localized to the distal gut, suggesting additional biological roles for GLP-1. Here, we demonstrate that GLP-1 receptor (GLP-1R) signaling controls mucosal expansion of the small bowel (SB) and colon. These actions did not require the epidermal growth factor (EGF) or intestinal epithelial insulin-like growth factor (IGF1) receptors but were absent in Glp1r(-/-) mice. Polyp number and size were increased in SB of exendin-4-treated Apc(Min/+) mice, whereas polyp number was reduced in SB and colon of Glp1r(-/-):Apc(Min/+) mice. Exendin-4 increased fibroblast growth factor 7 (Fgf7) expression in colonic polyps of Apc(Min/+) mice and failed to increase intestinal growth in mice lacking Fgf7. Exogenous exendin-4 and Fgf7 regulated an overlapping set of genes important for intestinal growth. Thus, gain and loss of GLP-1R signaling regulates gut growth and intestinal tumorigenesis.

The intestinal epithelial insulin-like growth factor-1 receptor links glucagon-like peptide-2 action to gut barrier function.

Glucagon-like peptide-2 (GLP-2) is an intestinal growth-promoting hormone used to treat short bowel syndrome. GLP-2 promotes intestinal growth through a mechanism that involves both IGF-1 and the intestinal-epithelial IGF-1 receptor (IE-IGF-1R). GLP-2 also enhances intestinal barrier function, but through an unknown mechanism. We therefore hypothesized that GLP-2-enhanced barrier function requires the IE-IGF-1R and is mediated through alterations in expression and localization of tight junction proteins. Conditional IE-IGF-1R-null and control mice were treated with vehicle or degradation-resistant Gly(2)-GLP-2 for 10 days; some animals also received irinotecan to induce enteritis. Mice were then examined for gastrointestinal permeability to 4-kDa fluorescein isothiocyanate-dextran, jejunal resistance using Ussing chambers, tight junction structure by electron microscopy, and expression and localization of tight junction proteins by immunoblot and immunohistofluorescence, respectively. GLP-2 treatment decreased permeability to 4-kDa fluorescein isothiocyanate-dextran and increased jejunal resistance (P <.05-.01), effects that were lost in IE-IGF-1R-null mice. Electron microscopy did not reveal major structural changes in the tight junctions in any group of animals. However, the tight junctional proteins claudin-3 and -7 were upregulated by GLP-2 in control (P <.05-.01) but not null mice, whereas IE-IGF-1R deletion induced a shift in occludin localization from apical to intracellular domains; no changes were observed in expression or distribution of claudin-15 and zona occludins-1. Finally, in irinotecan-induced enteritis, GLP-2 normalized epithelial barrier function in control (P < .05) but not knockout animals. In conclusion, the effects of GLP-2 on intestinal barrier function are dependent on the IE-IGF-1R and involve modulation of key components of the tight junctional complex.

Loss of glucagon-like peptide-2-induced proliferation following intestinal epithelial insulin-like growth factor-1-receptor deletion.

BACKGROUND & AIMS: Glucagon-like peptide-2 (GLP-2) is an intestinal hormone that promotes growth of the gastrointestinal tract. Although insulin-like growth factor (IGF)-1 and the IGF-1 receptor (IGF-1R) are required for GLP-2-induced proliferation of crypt cells, little is known about localization of the IGF-1R which mediates the intestinotropic actions of GLP-2. METHODS: We examined intestinal growth and proliferative responses in mice with conditional deletion of IGF-1R from intestinal epithelial cells (IE-igf1rKO) after acute administration (30-90 min) of GLP-2, in response to 24-hour fasting and re-feeding (to induce GLP-2-dependent adaptation), and after chronic exposure (10 days) to GLP-2. RESULTS: IE-igf1rKO mice had normal small intestinal weight, morphometric parameters, proliferative indices, and distribution of differentiated epithelial cell lineages. Acute administration of GLP-2 increased nuclear translocation of ß-catenin in non-Paneth crypt cells and stimulated the crypt-cell proliferative marker c-Myc in control but not IE-igf1rKO mice. Small intestinal weight, crypt depth, villus height, and crypt-cell proliferation were decreased in control and IE-igf1rKO mice after 24-hour fasting. Although re-feeding control mice restored all of these parameters, re-fed IE-igf1rKO mice had reductions in adaptive regrowth of the villi and crypt-cell proliferation. Control mice that were given chronic GLP-2 had increases in small intestinal weight, mucosal cross-sectional area, crypt depth, villus height, and crypt-cell proliferation. However, the GLP-2-induced increase in crypt-cell proliferation was not observed in IE-igf1rKO mice, and growth of the crypt-villus axis was reduced. CONCLUSIONS: The proliferative responses of the intestinal epithelium to exogenous GLP-2 administration and conditions of GLP-2-dependent adaptive re-growth require the intestinal epithelial IGF-1R.

The "cryptic" mechanism of action of glucagon-like peptide-2.

Glucagon-like peptide-2 (GLP-2) is a peptide hormone with multiple beneficial effects on the intestine, including expansion of the mucosal surface area through stimulation of crypt cell proliferation, as well as enhancement of nutrient digestion and absorption. Recent advances in clinical trials involving GLP-2 necessitate elucidation of the exact signaling pathways by which GLP-2 acts. In particular, the GLP-2 receptor has been localized to several intestinal cell types that do not include the proliferating crypt cells, and the actions of GLP-2 have thus been linked to a complex network of indirect mediators that induce diverse signaling pathways. The intestinotropic actions of GLP-2 on the colon have been shown to be mediated through the actions of keratinocyte growth factor and insulin-like growth factor (IGF)-2, whereas small intestinal growth has been linked to IGF-1, IGF-2, and ErbB ligands, as well as the IGF-1 receptor and ErbB. The cellular source of these mediators remains unclear, but it likely includes the intestinal subepithelial myofibroblasts. Conversely, the anti-inflammatory and blood flow effects of GLP-2 are dependent on vasoactive intestinal polypeptide released from submucosal enteric neurons and nitric oxide, respectively. Finally, recent studies have suggested that GLP-2 not only modulates intestinal stem cell behavior but may also promote carcinogenesis in models of sporadic colon cancer. Further consideration of the molecular cross-talk and downstream signaling pathways mediating the intestinotropic effects of GLP-2 is clearly warranted.

Mechanism of action of glucagon-like peptide-2 to increase IGF-I mRNA in intestinal subepithelial fibroblasts.

IGF-I, a known secretory product of intestinal subepithelial myofibroblasts (ISEMFs), is essential for the intestinotropic effects of glucagon-like peptide-2 (GLP-2). Furthermore, GLP-2 increases IGF-I mRNA transcript levels in vitro in heterogeneous fetal rat intestinal cultures, as well as in vivo in the rodent small intestine. To determine the mechanism underlying the stimulatory effect of GLP-2 on intestinal IGF-I mRNA, murine ISEMF cells were placed into primary culture. Immunocytochemistry showed that the ISEMF cells appropriately expressed α-smooth muscle actin and vimentin but not desmin. The cells also expressed GLP-2 receptor and IGF-I mRNA transcripts. Treatment of ISEMF cells with (Gly2)GLP-2 induced IGF-I mRNA transcripts by up to 5-fold of basal levels after treatment with 10(-8) m GLP-2 for 2 h (P < 0.05) but did not increase transcript levels for other intestinal growth factors, such as ErbB family members. Immunoblot revealed a 1.6-fold increase in phospho (p)-Akt/total-(t)Akt with 10(-8) m GLP-2 treatment (P < 0.05) but no changes in cAMP, cAMP-dependent ß-galactosidase expression, pcAMP response element-binding protein/tcAMP response element-binding protein, pErk1/2/tErk1/2, or intracellular calcium. Furthermore, pretreatment of ISEMF cells with the phosphatidylinositol 3 kinase (PI3K) inhibitors, LY294002 and wortmannin, abrogated the IGF-I mRNA response to GLP-2, as did overexpression of kinase-dead Akt. The role of PI3K/Akt in GLP-2-induced IGF-I mRNA levels in the murine jejunum was also confirmed in vivo. These findings implicate the PI3K/Akt pathway in the stimulatory effects of GLP-2 to enhance intestinal IGF-I mRNA transcript levels and provide further evidence in support of a role for IGF-I produced by the ISEMF cells in the intestinotropic effects of GLP-2.

Life in the crypt: a role for glucagon-like peptide-2?

The epithelial layer of the intestinal tract serves as a model to study the mechanisms regulating tissue renewal. Central to this process is the intestinal stem cell and, thus, both the intrinsic and extrinsic factors that modulate the function of these cells must be understood. Amongst the intrinsic regulators, both the canonical wnt and bone morphogenic protein (bmp) signaling pathways have been shown to be essential determinants of stem cell dynamics and intestinal homeostasis. The intestinotrophic hormone, glucagon-like peptide-2 (GLP-2), has also recently been demonstrated to exert a variety of effects on the intestinal crypt cells, including enhancement of the putative stem cell marker, musashi-1, as well as stimulating intestinal proliferation. As the GLP-2 receptor is not expressed by the crypt cells, these actions have been hypothesized to be mediated indirectly, through other gut peptides and/or growth factors. Of these, recent studies have demonstrated a requirement for insulin-like growth factor-1 in the proliferative effects of GLP-2, through a pathway that involves activation of the canonical wnt signaling pathway. This extrinsic pathway represents a novel mechanism by which intestinal stem cell dynamics may be regulated.

Glucagon-like peptide-2 activates beta-catenin signaling in the mouse intestinal crypt: role of insulin-like growth factor-I.

Chronic administration of glucagon-like peptide-2 (GLP-2) induces intestinal growth and crypt cell proliferation through an indirect mechanism requiring IGF-I. However, the intracellular pathways through which IGF-I mediates GLP-2-induced epithelial tropic signaling remain undefined. Because beta-catenin and Akt are important regulators of crypt cell proliferation, we hypothesized that GLP-2 activates these signaling pathways through an IGF-I-dependent mechanism. In this study, fasted mice were administered Gly(2)-GLP-2 or LR(3)-IGF-I (positive control) for 0.5-4 h. Nuclear translocation of beta-catenin in non-Paneth crypt cells was assessed by immunohistochemistry and expression of its downstream proliferative markers, c-myc and Sox9, by quantitative RT-PCR. Akt phosphorylation and activation of its targets, glycogen synthase kinase-3beta and caspase-3, were determined by Western blot. IGF-I receptor (IGF-IR) and IGF-I signaling were blocked by preadministration of NVP-AEW541 and through the use of IGF-I knockout mice, respectively. We found that GLP-2 increased beta-catenin nuclear translocation in non-Paneth crypt cells by 72 +/- 17% (P < 0.05) and increased mucosal c-myc and Sox9 mRNA expression by 90 +/- 20 and 376 +/- 170%, respectively (P < 0.05-0.01), with similar results observed with IGF-I. This effect of GLP-2 was prevented by blocking the IGF-IR as well as ablation of IGF-I signaling. GLP-2 also produced a time- and dose-dependent activation of Akt in the intestinal mucosa (P < 0.01), most notably in the epithelium. This action was reduced by IGF-IR inhibition but not IGF-I knockout. We concluded that acute administration of GLP-2 activates beta-catenin and proliferative signaling in non-Paneth murine intestinal crypt cells as well as Akt signaling in the mucosa. However, IGF-I is required only for the GLP-2-induced alterations in beta-catenin.

Gynecologic cancer treatment and the impact of cancer-related infertility.

OBJECTIVE: The objective of this study was to empirically assess the emotional and sexual impact of cancer-related infertility in women with a history of gynecologic cancer. METHOD: Women with a history of gynecologic cancer were approached during their gynecologic oncology clinic appointment; they were provided a description of the study and asked to participate. All participants completed a one-time self-report survey. We present data acquired via the following methods: Center for Epidemiologic Studies-Depression Scale (CES-D), Impact of Events Scale (IES), Modified Inventory of Traumatic Grief (M-ITG), Female Sexual Function Index (FSFI), and the Menopausal Symptom Checklist. RESULTS: The study sample consisted of 20 women, ages 27 to 49 years (mean, approximately 40 years), who had undergone treatment for cervical (40%), ovarian (20%), or uterine (40%) cancer. Forty percent of the sample reported depressive symptoms as measured by the CES-D, with 35% of the women experiencing moderate to severe levels of distress as measured by the IES. The women in this sample experienced dissatisfaction with their overall sex lives (67%), pain during vaginal penetration (62%), and low levels of sexual desire (56%). CONCLUSIONS: The preliminary findings of this study indicated that feelings of depression, grief, stress, and sexual dysfunction are being experienced by women with a history of gynecologic cancer who have lost their fertility as a result of their cancer treatment.