Pharmacological Inhibition Of Notch Research Articles

Paired and solitary ionocytes in the zebrafish olfactory epithelium.

The sense of smell is generated by electrical currents that are influenced by the concentration of ions in olfactory sensory neurons and mucus. In contrast to the extensive morphological and molecular characterization of sensory neurons, there has been little description of the cells that control ion concentrations in the zebrafish olfactory system. Here, we report the molecular and ultrastructural characterization of zebrafish olfactory ionocytes. Transcriptome analysis suggests that the zebrafish olfactory epithelium contains at least three different ionocyte types, which resemble Na+/K+-ATPase-rich (NaR), Na+/Cl- cotransporter (NCC), and H+-ATPase-rich (HR) cells, responsible for calcium, chloride, and pH regulation, respectively, in the zebrafish skin. NaR-like and HR-like ionocytes are usually adjacent to one another, whereas NCC-like cells are usually solitary. The distinct subtypes are differentially distributed: NaR-like/HR-like cell pairs are found broadly within the olfactory epithelium, whereas NCC-like cells reside within the peripheral non-sensory multiciliated cell zone. Comparison of gene expression and serial-section electron microscopy analysis indicates that the NaR-like cells wrap around the HR-like cells and are connected to them by shallow tight junctions. The development of olfactory ionocyte subtypes is also differentially regulated, as pharmacological Notch inhibition leads to a loss of NaR-like and HR-like cells, but does not affect NCC-like ionocyte number. These results provide a molecular and anatomical characterization of olfactory ionocytes in a stenohaline freshwater teleost. The paired ionocytes suggest that both transcellular and paracellular transport regulate ion concentrations in the olfactory epithelium, while the solitary ionocytes may enable independent regulation of multiciliated cells.

Targeting Wnt Signaling via Notch in Intestinal Carcinogenesis.

Proliferation and differentiation of intestinal epithelial cells is assisted by highly specialized and well-regulated signaling cascades. The Wnt pathway, which is one of the fundamental pathways in the intestine, contributes to the organization of proliferative intestinal crypts by positioning and cycling of intestinal stem cells and their derivatives. The Wnt pathway promotes differentiation of intestinal secretory cell types along the crypt-plateau and crypt-villus axis. In contrast to the Wnt pathway, the intestinal Notch cascade participates in cellular differentiation and directs progenitor cells towards an absorptive fate with diminished numbers of Paneth and goblet cells. Opposing activities of Notch and Wnt signaling in the regulation of intestinal stem cells and the enterocytic cell fate have been elucidated recently. In fact, targeting Notch was able to overcome tumorigenesis of intestinal adenomas, prevented carcinogenesis, and counteracted Paneth cell death in the absence of caspase 8. At present, pharmacological Notch inhibition is considered as an interesting tool targeting the intrinsic Wnt pathway activities in intestinal non-neoplastic disease and carcinogenesis.

Impairment of oligodendrocyte lineages in spinal muscular atrophy model systems.

Survival motor neuron (SMN) deficiency indicates that various cellular processes are impaired in spinal muscular atrophy (SMA). Previous reports have shown that SMN deficiency causes motor neuron degeneration, whereas the numbers of astrocytes and microglia are significantly increased or activated in SMA model systems. Only a few groups have studied the role of oligodendrocyte (OL) lineages such as OL precursor cell and nerve/glial antigen 2 (NG2)-glia in SMA pathology. Our aim in this study was to investigate whether OL lineages are impaired in SMA model systems. We investigated the expression of myelin basic protein (MBP) and NG2, which are OL lineage markers, using SMNΔ7 mice (mSmn, SMN2, SMNΔ7) and cell cultures derived from induced pluripotent stem cells generated from SMA patients. We showed for the first time that the OL lineages, including NG2-positive OL precursor cells and MBP-positive myelinating OLs were impaired in SMNΔ7 mice and induced pluripotent stem cells derived from SMA patients. Notch was involved in the decline of NG2 expression in the spinal cord of SMNΔ7 mice. In addition, pharmacological Notch inhibition promoted MBP-positive OL differentiation in SMNΔ7 mice. These findings indicate that OL differentiation was impaired in SMA, which might be involved in the Notch dysregulation.

Notch inhibition counteracts Paneth cell death in absence of caspase-8.

Opposing activities of Notch and Wnt signaling regulate mucosal barrier homeostasis and differentiation of intestinal epithelial cells. Specifically, Wnt activity is essential for differentiation of secretory cells including Wnt3-producing Paneth cells, whereas Notch signaling strongly promotes generation of absorptive cells. Loss of caspase-8 in intestinal epithelium (casp8∆int) is associated with fulminant epithelial necroptosis, severe Paneth cell death, secondary intestinal inflammation, and an increase in Notch activity. Here, we found that pharmacological Notch inhibition with dibenzazepine (DBZ) is able to essentially rescue the loss of Paneth cells, deescalate the inflammatory phenotype, and reduce intestinal permeability in casp8∆int mice. The secretory cell metaplasia in DBZ-treated casp8∆int animals is proliferative, indicating for Notch activities partially insensitive to gamma-secretase inhibition in a casp8∆int background. Our data suggest that casp8 acts in the intestinal Notch network.

Abstract P5-07-06: A novel non-canonical Notch1-IKKα-mTORC2-AKT pathway maintains survival in triple negative breast cancer cells and cancer stem-like cells

Abstract Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive breast cancers. TNBC patients have high risk of recurrence and metastasis, and current treatment options remain limited. There is strong evidence for the involvement of Notch signaling in TNBC and in breast cancer stem-like cells (CSCs). Notch1 is highly expressed in Basal-like 1 (BL1) and especially Mesenchymal-Stem-Like (MSL) TNBCs. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Treatment of TNBC with dual mTORC1/2 inhibitors leads to resistance through activation of Notch1. Expression of Notch1 protein correlates with pAKT and nuclear NF-κB in TNBC. Here, we demonstrate that Notch1 promotes cell survival in MDA-MB-231 cells, representative of MSL TNBC, in part by activating NF-κB. Notch activation by Jagged1-expressing stromal cells enhances transcription of the anti-apoptotic gene cIAP-2 (BIRC3), a known NF-κB target. This event is dependent on recruitment to the cIAP-2 promoter of NF-κB subunits, IKKα and Notch1. Short term exposure of MDA-MB-231 cells (MSL, PTEN wild-type), but not MDA-MB-468 cells (BL1, PTEN-null) to recombinant Jagged1 leads to rapid AKT phosphorylation. This is suppressed by dual mTORC1/2 inhibitors, AKT inhibitors and IKKα inhibitors but not Everolimus (mTORC1-selective inhibitor). Rapid AKT phosphorylation downstream of Notch1 requires mTORC2, PI3K and IKKα, and contributes to NF-κB activation. These observations support a model where canonical and non-canonical mechanisms downstream of Notch1 trigger rapid AKT phosphorylation and NF-κB activation in PTEN wild-type TNBC cells. Both arms of this pathway require IKKα. CSCs derived from MDA-MB-231 cells have increased Notch1, pAKT and pIKKα expression. Combined pharmacological inhibition of Notch and AKT or Notch and IKKα completely blocks secondary mammosphere formation. These data and published literature suggest that: 1) IKKα connects the Notch and mTORC2/AKT pathways in some TNBC subtypes; 2) IKKα is also required for nuclear Notch1-mediated NF-kB activation and may be a critical node in the Notch signaling network; 3) A feedback mechanism may exist in some TNBC cells between mTORC2/AKT and Notch1; 4) The non-canonical Notch-IKKα-AKT pathway has a potential therapeutic role in targeting CSCs of selected TNBC subtypes. Citation Format: Hossain F, Peng Y, Pannuti A, Backus K, Golde T, Osborne B, Miele L. A novel non-canonical Notch1-IKKα-mTORC2-AKT pathway maintains survival in triple negative breast cancer cells and cancer stem-like cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-07-06.

Notch signalling drives bone marrow stromal cell-mediated chemoresistance in acute myeloid leukemia.

Both preclinical and clinical investigations suggest that Notch signalling is critical for the development of many cancers and for their response to chemotherapy. We previously showed that Notch inhibition abrogates stromal-induced chemoresistance in lymphoid neoplasms. However, the role of Notch in acute myeloid leukemia (AML) and its contribution to the crosstalk between leukemia cells and bone marrow stromal cells remain controversial. Thus, we evaluated the role of the Notch pathway in the proliferation, survival and chemoresistance of AML cells in co-culture with bone marrow mesenchymal stromal cells expanded from both healthy donors (hBM-MSCs) and AML patients (hBM-MSCs*). As compared to hBM-MSCs, hBM-MSCs* showed higher level of Notch1, Jagged1 as well as the main Notch target gene HES1. Notably, hBM-MSCs* induced expression and activation of Notch signalling in AML cells, supporting AML proliferation and being more efficientin inducing AML chemoresistance than hBM-MSCs*. Pharmacological inhibition of Notch using combinations of Notch receptor-blocking antibodies or gamma-secretase inhibitors (GSIs), in presence of chemotherapeutic agents, significant lowered the supportive effect of hBM-MSCs and hBM-MSCs* towards AML cells, by activating apoptotic cascade and reducing protein level of STAT3, AKT and NF-κB.These results suggest that Notch signalling inhibition, by overcoming the stromal-mediated promotion of chemoresistance,may represent a potential therapeutic targetnot only for lymphoid neoplasms, but also for AML.

Alterations in cellular metabolome after pharmacological inhibition of Notch in glioblastoma cells.

Notch signaling can promote tumorigenesis in the nervous system and plays important roles in stem-like cancer cells. However, little is known about how Notch inhibition might alter tumor metabolism, particularly in lesions arising in the brain. The gamma-secretase inhibitor MRK003 was used to treat glioblastoma neurospheres, and they were subdivided into sensitive and insensitive groups in terms of canonical Notch target response. Global metabolomes were then examined using proton magnetic resonance spectroscopy, and changes in intracellular concentration of various metabolites identified which correlate with Notch inhibition. Reductions in glutamate were verified by oxidation-based colorimetric assays. Interestingly, the alkylating chemotherapeutic agent temozolomide, the mTOR-inhibitor MLN0128, and the WNT inhibitor LGK974 did not reduce glutamate levels, suggesting that changes to this metabolite might reflect specific downstream effects of Notch blockade in gliomas rather than general sequelae of tumor growth inhibition. Global and targeted expression analyses revealed that multiple genes important in glutamate homeostasis, including glutaminase, are dysregulated after Notch inhibition. Treatment with an allosteric inhibitor of glutaminase, compound 968, could slow glioblastoma growth, and Notch inhibition may act at least in part by regulating glutaminase and glutamate.

A microRNA-mediated regulatory loop modulates NOTCH and MYC oncogenic signals in B- and T-cell malignancies.

Growing evidence suggests that microRNAs facilitate the cross-talk between transcriptional modules and signal transduction pathways. MYC and NOTCH1 contribute to the pathogenesis of lymphoid malignancies. NOTCH induces MYC, connecting two signaling programs that enhance oncogenicity. Here we show that this relationship is bidirectional and that MYC, via a microRNA intermediary, modulates NOTCH. MicroRNA-30a, a member of family of microRNAs that are transcriptionally suppressed by MYC, directly binds to and inhibits NOTCH1 and NOTCH2 expression. Using a murine model and genetically modified human cell lines, we confirmed that microRNA-30a influences NOTCH expression in a MYC-dependent fashion. In turn, through genetic modulation, we demonstrated that intracellular NOTCH1 and NOTCH2, by inducing MYC, suppressed microRNA-30a. Conversely, pharmacological inhibition of NOTCH decreased MYC expression, and ultimately de-repressedmicroRNA-30a. Examination of genetic models of gain and loss of microRNA-30a in diffuse large B-cell lymphoma (DLBCL) and T-acute lymphoblastic leukemia (T-ALL) cells suggested a tumor suppressive role for this microRNA. Finally, the activity of the microRNA-30a-NOTCH-MYC loop was validated in primary DLBCL and T-ALL samples. These data define the presence of a microRNA-mediated regulatory circuitry that may modulate the oncogenic signals originating from NOTCH and MYC.

Notch inhibition allows oncogene-independent generation of iPS cells.

The reprogramming of somatic cells to pluripotency using defined transcription factors holds great promise for biomedicine. However, human reprogramming remains inefficient and relies either on the use of the potentially dangerous oncogenes KLF4 and CMYC or the genetic inhibition of the tumor suppressor gene p53. We hypothesized that inhibition of signal transduction pathways that promote differentiation of the target somatic cells during development might relieve the requirement for non-core pluripotency factors during iPSC reprogramming. Here, we show that inhibition of Notch significantly improves the efficiency of iPSC generation from mouse and human keratinocytes by suppressing p21 in a p53-independent manner and thereby enriching for undifferentiated cells capable of long-term self-renewal. Pharmacological inhibition of Notch enabled routine production of human iPSCs without KLF4 and CMYC while leaving p53 activity intact. Thus, restricting the development of somatic cells by altering intercellular communication enables the production of safer human iPSCs.

Hyper-Activation of Notch3 Amplifies the Proliferative Potential of Rhabdomyosarcoma Cells

Rhabdomyosarcoma (RMS) is a pediatric myogenic-derived soft tissue sarcoma that includes two major histopathological subtypes: embryonal and alveolar. The majority of alveolar RMS expresses PAX3-FOXO1 fusion oncoprotein, associated with the worst prognosis. RMS cells show myogenic markers expression but are unable to terminally differentiate. The Notch signaling pathway is a master player during myogenesis, with Notch1 activation sustaining myoblast expansion and Notch3 activation inhibiting myoblast fusion and differentiation. Accordingly, Notch1 signaling is up-regulated and activated in embryonal RMS samples and supports the proliferation of tumor cells. However, it is unable to control their differentiation properties. We previously reported that Notch3 is activated in RMS cell lines, of both alveolar and embryonal subtype, and acts by inhibiting differentiation. Moreover, Notch3 depletion reduces PAX3-FOXO1 alveolar RMS tumor growth in vivo. However, whether Notch3 activation also sustains the proliferation of RMS cells remained unclear. To address this question, we forced the expression of the activated form of Notch3, Notch3IC, in the RH30 and RH41 PAX3-FOXO1-positive alveolar and in the RD embryonal RMS cell lines and studied the proliferation of these cells. We show that, in all three cell lines tested, Notch3IC over-expression stimulates in vitro cell proliferation and prevents the effects of pharmacological Notch inhibition. Furthermore, Notch3IC further increases RH30 cell growth in vivo. Interestingly, knockdown of Notch canonical ligands JAG1 or DLL1 in RMS cell lines decreases Notch3 activity and reduces cell proliferation. Finally, the expression of Notch3IC and its target gene HES1 correlates with that of the proliferative marker Ki67 in a small cohort of primary PAX-FOXO1 alveolar RMS samples. These results strongly suggest that high levels of Notch3 activation increase the proliferative potential of RMS cells.

Renal tubular Notch signaling triggers a prosenescent state after acute kidney injury

The aging kidney has a diminished regenerative potential and an increased tendency to develop tubular atrophy and fibrosis after acute injury. In this study, we found that activation of tubular epithelial Notch1 signaling was prolonged in the aging kidney after ischemia/reperfusion (IR) damage. To analyze the consequences of sustained Notch activation, we generated mice with conditional inducible expression of Notch1 intracellular domain (NICD) in proximal tubules. NICD kidneys were analyzed 1 and 4 wk after renal IR. Conditional NICD expression was associated with aggravated tubular damage, a fibrotic phenotype, and the expression of cellular senescence markers p21 and p16(INK4a). In wild-type mice pharmacological inhibition of Notch using the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) improved tubulo-interstitial damage and antagonized the prosenescent pathway activation after IR. In vitro, activation of Notch signaling with delta-like-ligand-4 caused prosenescent changes in tubular cells while inhibition with DAPT attenuated these changes. In conclusion, our data suggest that sustained epithelial Notch activation after IR might contribute to the inferior outcome of old kidneys after injury. Sustained epithelial activation of Notch is associated with a prosenescent phenotype and maladaptive repair.

Lgr5-Positive Supporting Cells Generate New Hair Cells in the Postnatal Cochlea

SummaryThe prevalence of hearing loss after damage to the mammalian cochlea has been thought to be due to a lack of spontaneous regeneration of hair cells, the primary receptor cells for sound. Here, we show that supporting cells, which surround hair cells in the normal cochlear epithelium, differentiate into new hair cells in the neonatal mouse following ototoxic damage. Using lineage tracing, we show that new hair cells, predominantly outer hair cells, arise from Lgr5-expressing inner pillar and third Deiters cells and that new hair cell generation is increased by pharmacological inhibition of Notch. These data suggest that the neonatal mammalian cochlea has some capacity for hair cell regeneration following damage alone and that Lgr5-positive cells act as hair cell progenitors in the cochlea.

Notch signaling and new therapeutic options in liver disease

Notch signaling is a crucial determinant of cell fate decision during development and disease in several organs. Notch effects are strictly dependent on the cellular context in which it is activated. In the liver, Notch signaling is involved in biliary tree development and tubulogenesis. Recent advances have shed light on Notch as a critical player in liver regeneration and repair, as well as in liver metabolism and inflammation and cancer. Notch signaling is finely regulated at several levels. The complexity of the pathway provides several possible targets for development of therapeutic agents able to inhibit Notch. Recent reports have shown that persistent activation of Notch signaling is associated with liver malignancies, particularly hepatocellular with stem cell features and cholangiocarcinoma. These novel findings suggest that interfering with the aberrant activation of the Notch pathway may have therapeutic relevance. However, further studies are needed to clarify the mechanisms regulating physiologic and pathologic Notch activation in the adult liver, to better understand the mechanistic role(s) of Notch in liver diseases and to develop safe and specific therapeutic agents.

The importance of Notch signaling in peripheral T-cell lymphomas

Peripheral T-cell lymphomas (PTLs) represent an area of high medical need. Previously, we demonstrated high expression of Notch, a known oncogene, in primary cutaneous anaplastic large cell lymphoma (ALCL). In this study, we performed immunohistochemical staining for Notch1 in lymph nodes from PTL not otherwise specified (PTL-NOS) and systemic ALCL (ALK+ and ALK−) and report a similar distribution among the three subgroups: Negative, moderate and strong expression was, respectively, 18%, 27% and 55% for PTL-NOS (33 cases), 20%, 0% and 80% for ALCL ALK+ (10 cases) and 45%, 22% and 33% for ALCL ALK− (nine cases) (p > 0.05). In the ALK+ ALCL cell line, Karpas-299, pharmacological inhibition of Notch with γ-secretase inhibitor (GSI) I was far more potent than with GSI IX, XX and XXI with regard to cell viability and apoptosis. In conclusion, PTL tumor cells have prominent Notch1 expression and treatment with Notch inhibitors has cytotoxic effects.

Combined pharmacological inhibition of Notch and JAK/STAT pathways effectively suppresses conversion from acinar-ductal metaplasia to pancreatic ductal adenocarcinoma and is superior to monotherapy

Combined pharmacological inhibition of Notch and JAK/STAT pathways effectively suppresses conversion from acinar-ductal metaplasia to pancreatic ductal adenocarcinoma and is superior to monotherapy

Angiotensin II Contributes to Renal Fibrosis Independently of Notch Pathway Activation

Recent studies have described that the Notch signaling pathway is activated in a wide range of renal diseases. Angiotensin II (AngII) plays a key role in the progression of kidney diseases. AngII contributes to renal fibrosis by upregulation of profibrotic factors, induction of epithelial mesenchymal transition and accumulation of extracellular matrix proteins. In cultured human tubular epithelial cells the Notch activation by transforming growth factor-β1 (TGF-β1) has been involved in epithelial mesenchymal transition. AngII mimics many profibrotic actions of TGF-β1. For these reasons, our aim was to investigate whether AngII could regulate the Notch/Jagged system in the kidney, and its potential role in AngII-induced responses. In cultured human tubular epithelial cells, TGF-β1, but not AngII, increased the Notch pathway-related gene expression, Jagged-1 synthesis, and caused nuclear translocation of the activated Notch. In podocytes and renal fibroblasts, AngII did not modulate the Notch pathway. In tubular epithelial cells, pharmacological Notch inhibition did not modify AngII-induced changes in epithelial mesenchymal markers, profibrotic factors and extracellular matrix proteins. Systemic infusion of AngII into rats for 2 weeks caused tubulointerstitial fibrosis, but did not upregulate renal expression of activated Notch-1 or Jagged-1, as observed in spontaneously hypertensive rats. Moreover, the Notch/Jagged system was not modulated by AngII type I receptor blockade in the model of unilateral ureteral obstruction in mice. These data clearly indicate that AngII does not regulate the Notch/Jagged signaling system in the kidney, in vivo and in vitro. Our findings showing that the Notch pathway is not involved in AngII-induced fibrosis could provide important information to understand the complex role of Notch system in the regulation of renal regeneration vs damage progression.

Notch1 Deficiency Results in Decreased Inflammation during Wound Healing and Regulates Vascular Endothelial Growth Factor Receptor-1 and Inflammatory Cytokine Expression in Macrophages

We investigated whether Notch signaling plays a role in regulating macrophage responses to inflammation. In a wound healing assay, macrophage recruitment was decreased in Notch1(+/-) mice, and the wounds were characterized by decreased TNF-α expression. As wound healing progressed, Notch1(+/-) wounds had increased vascularization and collagen deposition compared with wild-type wounds. In mice with myeloid-specific Notch1 deletion, wounds had decreased macrophage recruitment as well as decreased TNF-α expression, indicating the specific role of Notch1 in the inflammatory response in these cells. In vitro, we found that vascular endothelial growth factor receptor-1 (VEGFR-1) was upregulated in macrophages in response to LPS/IFN-γ and that this upregulation depended on Notch signaling. Furthermore, macrophages from Notch1(+/-) mice had decreased expression of VEGFR-1 compared with macrophages from wild-type mice, whereas VEGFR-1 expression in Notch4(-/-) macrophages was normal. Inhibition of Notch signaling decreased induction of the inflammatory cytokines IL-6, IL-12, CXCL10, MCP-1, monokine induced by IFN-γ, and TNF-α in macrophages in response to LPS/IFN-γ. Additionally, macrophages from Notch1(+/-) mice demonstrated decreased induction of IL-6, IL-12, and TNF-α in response to stimulation compared with wild-type mice. Thus, both pharmacological inhibition of Notch and genetic analysis demonstrate that Notch1 regulates VEGFR-1 and cytokine expression in macrophages. We have also established that Notch1 is important for the inflammatory response during wound healing in mice.

Notch signaling specifies prosensory domains via lateral induction in the developing mammalian inner ear

During inner ear morphogenesis, the process of prosensory specification defines the specific regions of the otic epithelium that will give rise to the six separate inner ear organs essential for hearing and balance. The mechanism of prosensory specification is not fully understood, but there is evidence that the Notch intercellular signaling pathway plays a critical role. The Notch ligand Jagged1 (Jag1) is expressed in the prosensory domains, and mutation of Jag1 impairs sensory formation. Furthermore, pharmacological inhibition of Notch in vitro during prosensory specification disrupts the prosensory process. Additionally, activation of Notch by cDNA electroporation in chick otocysts results in formation of ectopic sensory patches. Here we test whether Notch activity is sufficient for prosensory specification in the mouse, using a Cre-/loxP approach to conditionally activate the Notch pathway in nonsensory regions of the inner ear epithelia during different stages of otic vesicle morphogenesis. We find that broad ectopic activation of Notch at very early developmental stages causes induction of prosensory markers throughout the entire otic epithelium. At later stages of development, activation of Notch in nonsensory regions leads to induction of sensory patches that later differentiate to form complete ectopic sensory structures. Activation of Notch in isolated nonsensory cells results in lateral induction of Jag1 expression in neighboring cells and spreading of prosensory specification to the adjacent cells through an intercellular mechanism. These results support a model where activation of Notch and propagation through lateral induction promote prosensory character in specific regions of the developing otocyst.

Abstract 14: Targeting Notch in malignant brain tumors: Crosstalk with Hedgehog as a potential mechanism of treatment resistance

Abstract Developmental signaling cascades such as Notch, Hedgehog, and Wnt are increasingly being investigated as novel therapeutic targets in malignant brain tumors. These pathways are thought to play particularly important roles in stem-like cancer cells, which seem resistant to standard chemo- and radiation therapies. Indeed, inhibiting Notch signaling in medulloblastoma or glioblastoma (GBM) cultures decreases the proportion of stem-like cells. However, we thought that other developmental stem cell maintenance pathways might compensate for the pharmacological inhibition of Notch signaling, and therefore investigated Hedgehog and Wnt. Interestingly, we found that levels of the Hedgehog targets Gli1 and Patched1B increased almost two-fold in response to pharmacological inhibition of Notch in neurosphere lines derived from malignant gliomas. The Wnt target Axin2 was also upregulated in some lines, but in a less reproducible fashion. We hypothesized that there might be direct transcriptional regulation of Hedgehog targets by the Notch pathway, and used chromatin immunoprecipitation to evaluate whether the repressive factor Hes1 bound to the first intron of Gli1. We found that Hes1 does bind to multiple N-boxes in this intron, consistent with the concept that it acts as a transcriptional repressor for Gli1. Together, our data suggest a negative feedback loop between Notch and Hedgehog by which GBMs could escape from pharmacological Notch inhibition by up-regulating Hedgehog signaling. We also investigated whether targeting both Notch and Hedgehog signaling simultaneously would kill brain tumor cells more effectively than as monotherapies. As we hypothesized, while pharmacological inhibition of Notch (using gamma-secretase inhibitor) or Hedgehog (using cyclopamine) signaling alone inhibited the growth and clonogenicity of GBM neurosphere cell lines in a dose-dependent fashion, co-treatment resulted in significantly more robust inhibition of cell growth and colony-forming ability. Moreover, when neurosphere lines capable of forming intracranial tumors were pretreated with one or both pathway inhibitors before injection into mice, co-treatment significantly prolonged survival. This is consistent with our model that inhibiting both pathways more effectively kills the xenograft-initiating cells within the culture. We next investigated whether these findings applied to primary human GBMs and found that freshly cultured neurospheres were highly susceptible to co-treatment but more refractory to monotherapy. Our findings suggest that targeting multiple developmental pathways may be necessary to fully ablate the stem-like population in malignant brain tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 14.

Loss of negative regulation by Numb over Notch is relevant to human breast carcinogenesis

The biological antagonism between Notch and Numb controls the proliferative/differentiative balance in development and homeostasis. Although altered Notch signaling has been linked to human diseases, including cancer, evidence for a substantial involvement of Notch in human tumors has remained elusive. Here, we show that Numb-mediated control on Notch signaling is lost in ∼50% of human mammary carcinomas, due to specific Numb ubiquitination and proteasomal degradation. Mechanistically, Numb operates as an oncosuppressor, as its ectopic expression in Numb-negative, but not in Numb-positive, tumor cells inhibits proliferation. Increased Notch signaling is observed in Numb-negative tumors, but reverts to basal levels after enforced expression of Numb. Conversely, Numb silencing increases Notch signaling in normal breast cells and in Numb-positive breast tumors. Finally, growth suppression of Numb-negative, but not Numb-positive, breast tumors can be achieved by pharmacological inhibition of Notch. Thus, the Numb/Notch biological antagonism is relevant to the homeostasis of the normal mammary parenchyma and its subversion contributes to human mammary carcinogenesis.