Expression Of Notch Pathway Genes Research Articles

KrasG12D upregulates Notch signaling to induce gallbladder tumorigenesis in mice.

BackgroundKras mutations and increased Notch activation occur frequently in gallbladder cancer. However, their roles in gallbladder carcinogenesis have not been defined. This study was aimed at determining whether expression of mutant Kras was sufficient to induce gallbladder carcinoma and whether Notch deregulation played a role in this context.MethodsWe determined Cre recombination activity of Pdx1-Cre in the gallbladder using a reporter strain and examined gallbladder tumor development in the KrasLSL- G12D/+;Pdx1-Cre mice. We analyzed expression of Notch pathway genes in the mouse gallbladder by immunohistochemistry, quantitative RT-PCR, and Western blot analysis. We also determined the effect of Jag1 deletion on Kras-induced gallbladder tumor development.ResultsPdx1-Cre exhibits robust recombination activity in the gallbladder epithelium. KrasLSL-G12D/+;Pdx1-Cre mice form early onset adenoma in the gallbladder and adjacent biliary tract with complete penetrance, albeit short of invasive adenocarcinoma. KrasG12D upregulates expressions of Notch2, Notch3, Notch4, Jag1 and downstream target genes Hes1, Hey1 and Hey2, and deletion of Jag1 partially suppresses KrasG12D-induced adenoma development.ConclusionsKrasG12D induces gallbladder adenoma and Notch plays a key role in Kras-initiated gallbladder tumorigenesis.

Abstract 1332: NOTCH - HEY1 pathway induces EMT in head and neck squamous cell carcinoma

Abstract Background Head and neck squamous cell carcinoma (HNSCC) is noted to have a NOTCH mutation rate of 20% within the Cancer Genome Atlas (TCGA) dataset consistent with tumor suppressor gene status. However, NOTCH genes are also upregulated in wild type HNSCC and show signs of downstream activation in a substantial proportion of HNSCC. In this study, we aimed to further explore the potential for NOTCH pathways to be activated in HNSCC. Material and Methods mRNA sequence of TCGA data includes 520 HNSCC tumors and 46 normal tissues. We used 447 HNSCC excluding 73 tumors with NOTCH mutations. The activated groups of NOTCH downstream genes were defined as tumors with expression 1 standard deviation above the mean of normal tissue. We used four HNSCC cell lines (SKN3, Cal27, SCC61, SCC090) to ascertain the results of the TCGA analysis in vitro. Result In TCGA analysis, HEY1 activated HNSCC had significant more expression of NOTCH pathway genes. In particular NOTCH4 had the most significant correlation with HEY1 activation among NOTCH receptors. To confirm this result in vitro, HEY1 expression was compared between si-control and si-NOTCH4 of SKN3, Cal27, SCC61 and SCC090. As expected, HEY1 expression in all si-NOTCH4 treated cell lines were significant decreased. Next, we examined the relationship about NOTCH4 and an epithelial mesenchymal transition phenotype (EMT). Using TCGA data, we found that mesenchymal marker genes were significantly increased in high NOTCH4 group. In vitro experiments, si-NOTCH4 cells decreased mesenchymal marker gene expression such as Fibronectin and TWIST1. We also examined HEY1 function. Similar to NOTCH4, it was found that HEY1 is significantly related to EMT in silico and in vitro. Conclusion The NOTCH4-HEY1 pathway is significantly upregulated in HNSCC and induces EMT. Citation Format: Takahito Fukusumi, Theresa W. Guo, Shuling Ren, Akihiro Sakai, Mizuo Ando, Sunny Haft, Chao Liu, Joseph A. Califano. NOTCH - HEY1 pathway induces EMT in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1332. doi:10.1158/1538-7445.AM2017-1332

Abstract A10: Analysis of gene expression and DNA methylation profiles of Notch signaling pathway genes in human glioblastoma

Abstract In cancer, DNA methylation affects important signal transduction pathways leading to altered receptor function, disruption of normal cell-cell interaction, etc. Since methylation occurs at a very early stage, hypermethylated promoters hold great promise as biomarkers for early detection and an effective drug target for gene reactivation. The Notch signaling pathway is one such developmental pathway governing cell fate decisions. Dysregulated Notch signaling is found to have a prominent role in the development of various cancers. Glioblastoma is the most common primary brain tumor with a very poor prognosis. Therefore it is important to study genetic and epigenetic events leading to gliomagenesis and to guide new treatment strategies. The aim of this study was to detect Notch pathway genes potentially regulated by promoter methylation in human glioblastoma. We used real-time PCR and methylation-specific PCR to study gene expression and methylation status of seven Notch pathway genes (Notch1, Notch2, Notch3, Notch4, JAG1, JAG2 and DLL3) from human glioblastoma formalin fixed paraffin embedded sections. We identified Notch3 and JAG2 promoters as methylated and Notch4 with both methylated and unmethylated promoter. Despite methylation, Notch3 gene showed robust gene expression suggesting its partial dependency on promoter methylation and the presence of alternative regulatory mechanisms. However, low gene expression of JAG2 and the absence of Notch4 gene expression suggest a possibility of epigenetic silencing. This study for the first time provides gene expression and DNA methylation profiles of Notch pathway genes from glioblastoma patient samples. We have identified genes whose expression may be regulated by epigenetic mechanisms and thus can be used as markers that may guide treatment decisions. Note: This abstract was not presented at the conference. Citation Format: Madhuri G S Aithal, Rajeswari Narayanappa. Analysis of gene expression and DNA methylation profiles of Notch signaling pathway genes in human glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Improving Cancer Risk Prediction for Prevention and Early Detection; Nov 16-19, 2016; Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(5 Suppl):Abstract nr A10.

Exercise differentially affects metabolic functions and white adipose tissue in female letrozole- and dihydrotestosterone-induced mouse models of polycystic ovary syndrome

Here we hypothesized that exercise in dihydrotestosterone (DHT) or letrozole (LET)-induced polycystic ovary syndrome mouse models improves impaired insulin and glucose metabolism, adipose tissue morphology, and expression of genes related to adipogenesis, lipid metabolism, Notch pathway and browning in inguinal and mesenteric fat. DHT-exposed mice had increased body weight, increased number of large mesenteric adipocytes. LET-exposed mice displayed increased body weight and fat mass, decreased insulin sensitivity, increased frequency of small adipocytes and increased expression of genes related to lipolysis in mesenteric fat. In both models, exercise decreased fat mass and inguinal and mesenteric adipose tissue expression of Notch pathway genes, and restored altered mesenteric adipocytes morphology. In conclusion, exercise restored mesenteric adipocytes morphology in DHT- and LET-exposed mice, and insulin sensitivity and mesenteric expression of lipolysis-related genes in LET-exposed mice. Benefits could be explained by downregulation of Notch, and modulation of browning and lipolysis pathways in the adipose tissue.

EpCAM+ Liver Cancer Stem-Like Cells Exhibiting Autocrine Wnt Signaling Potentially Originate in Cirrhotic Patients.

Hepatocellular carcinoma (HCC) is believed to originate from cancer stem cells (CSCs). While epithelial cell adhesion molecule (EpCAM) is a marker of normal hepatic stem cells (HSCs), EpCAM+ cells from HCC behave like CSCs. Since HCC mostly develops on a cirrhotic background, we sought to determine whether CSC‐like EpCAM+ cells exist in patients with advanced cirrhosis. Both flow cytometry and immunohistochemistry showed that frequency of EpCAM+ cells in advanced cirrhosis was increased as compared to control. To determine whether increased EpCAM population in advanced cirrhosis harbors any CSC‐like cells, we compared molecular and functional features of EpCAM+ cells from advanced cirrhosis (Ep+CIR; n = 20) with EpCAM+ cells from both HCC (Ep+HCC; n = 20) and noncancerous/noncirrhotic (control) (Ep+NSC; n = 7) liver tissues. Ep+CIRs displayed similarity with Ep+HCC cells including upregulated expression of stemness and Notch pathway genes, enhanced self‐renewal in serial spheroid assay and generation of subcutaneous tumors in nonobese diabetic/severe combined immunodeficiency mice. Moreover, transcriptome and miRNome of Ep+CIRs appeared closer to that of Ep+HCC cells than Ep+NSCs. Interestingly, more than 50% micro RNAs (miRNAs) and transcripts specifically expressed in Ep+HCCs were also expressed in Ep+CIRs. However, none of Ep+NSC specific miRNAs and only 7% Ep+NSC specific transcripts were expressed in Ep+CIRs. Further, according to gene expression and in vitro Wnt inhibition analysis, autocrine Wnt signaling appeared to be a distinct feature of Ep+CIR and Ep+HCC cells, which was absent from Ep+NSCs. EpCAM+ cells in advanced cirrhosis possibly include a population of CSC‐like cells which can be explored for early diagnosis of HCC development. Stem Cells Translational Medicine 2017;6:807–818

Evaluation and selection of a non-PCR based technology for improved gene expression profiling from clinical formalin-fixed, paraffin-embedded samples.

Formalin-fixed, paraffin-embedded (FFPE) clinical tissue samples have the potential to provide valuable gene-expression data for the development of cancer biomarkers. However, FFPE RNA is extensively fragmented, presenting a significant challenge for reliably detecting gene expression using traditional qPCR methods. We evaluated three novel methodologies along with the traditional qPCR method for their ability to detect Notch pathway gene expression in colorectal cancer FFPE tissue RNAs. We found that quantitative nuclease protection assay-detected gene expression in high-quality RNAs as sensitively as qPCR, and consistently detected mRNAs in highly fragmented FFPE tissue RNAs. Quantitative nuclease protection assay represents an improved methodology for detecting gene expression in FFPE tissue and has the potential to advance the development of cancer biomarkers.

Abstract PR08: Neuroblastoma is biphasic and includes classical neuroepithelial cells and chemoresistant mesenchymal cells

Abstract Introduction: Most high stage neuroblastoma initially respond to chemotherapy, but ultimately relapse as therapy-resistant tumor. The mechanisms driving relapse and resistance remain elusive. We investigated whether neuroblastoma tumors include phenotypically and functionally divergent subsets of tumor cells that may underlie its clinical plasticity. Experimental Procedures: Fresh tumor cells were cultured in neural stem cell medium and analyzed by FACS, whole genome sequencing, mRNA profiling, motility assays and chemo-sensitivity assays. Lentivirally transduced inducible gene constructs were used to test state-transitions. Immunohistochemistry was used to define cellular subtypes in tumors. Results: We observed that new neuroblastoma cell lines always include two phenotypically divergent cell types. Both types share the same genetic defects, but have highly divergent phenotypes. One cell type has a neuro-epithelial (NE) phenotype and expresses all classical and diagnostically used neuroblastoma markers. The other type has a mesenchymal (MES) character, lacks all neuroblastoma markers and is highly motile. At low frequency, both cell types can spontaneously transdifferentiate in vitro. Immunohistochemistry (IHC) of primary neuroblastoma detected a small fraction of MES cells in most tumors. To analyze the clinical relevance of MES-type cells, we investigated their sensitivity to chemotherapeutics used in neuroblastoma treatment. In four isogenic pairs, MES cells were more resistant to the drugs than their NE-type counterparts. We investigated whether the chemo-resistance of MES cells may operate in vivo. We analyzed a series of primary neuroblastoma tumors surgically removed immediately after chemotherapy. The viable cells in the post-therapy samples were strongly enriched in MES-type cells as compared to the pre-treatment tumors of the same patients. We also compared primary, pre-treatment tumors with relapses emerging 4-5 years later in the same patients. Most strikingly, also the relapsed neuroblastoma tumors were highly enriched for MES-type cells. As these data suggest a role for MES-type cells in development of therapy-resistant relapses, we analyzed their key regulatory pathways. mRNA profiling of isogenic MES-NE cell line pairs identified consistent mRNA expression differences between both phenotypes. Major signaling routes and transcription factors were highly differentially expressed. MES-type cells had high expression and activation of NOTCH pathway genes and expression of the homeobox gene PRRX1. Induced expression of NOTCH or PRRX1 transgenes in multiple NE-type cell lines converted them into MES-type cell lines, including chemo-resistance. Analysis of the changes in gene expression and activity downstream of NOTCH or PRRX1 allowed reconstruction of the molecular wiring of MES-type cells. This identified several drugable key-players, like MEK and PDGFRβ. Targeting of them with small-molecule inhibitors specifically killed MES cells in vitro. Conclusions: Our data suggest that neuroblastoma is a bi-phasic tumor. MES and NE cells have very different characteristics, but can transdifferentiate into each other. It is tempting to speculate that the MES- and NE-phenotypes recapitulate two developmental stages of neuroblasts: MES cells may correspond to the migrating cell type that has delaminated from the neural crest, while NE cells could correspond to the more differentiated cell in the target organs expressing markers of the adrenalin synthesis route. MES cells strongly accumulate after chemo-therapy and in relapses. They may survive classical therapy and over time seed relapses, that ultimately become heterogeneous again. Targeted elimination of MES cells with small molecule inhibitors shows how cells with a potential key role in relapse development are amenable to therapy. This abstract is also presented as Poster B30. Citation Format: Rogier Versteeg, Tim van Groningen, Bart A. Westerman, Jan J. Molenaar, Ellen M. Westerhout, Mohamed Hamdi, Godelieve A. Tytgat, Jan Koster, Johan van Nes. Neuroblastoma is biphasic and includes classical neuroepithelial cells and chemoresistant mesenchymal cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR08.

Bromodomain and hedgehog pathway targets in small cell lung cancer

Small cell lung cancer (SCLC) is an extremely aggressive cancer that frequently recurs. Twenty-three human SCLC lines were selected representing varied Myc status. Gene expression of lung cancer, stem-like, hedgehog pathway, and notch pathway genes were determined by RT2-PCR array and Exon 1.0 ST array. Etoposide and topotecan concentration response was examined. The IC50's for etoposide and topotecan ranged over nearly 3 logs upon 96 hrs exposure to the drugs. Myc status, TOP2A, TOP2B and TOP1 mRNA expression or topoisomerase 1 and topoisomerase 2 protein did not account for the range in the sensitivity to the drugs. γ-secretase inhibitors, RO429097 and PF-03084014, had little activity in the SCLC lines over ranges covering the clinical Cmax concentrations. MYC amplified lines tended to be more sensitive to the bromodomain inhibitor JQ1. The Smo antagonists, erismodegib and vismodegib and the Gli antagonists, HPI1 and SEN-450 had a trend toward greater sensitivity of the MYC amplified line. Recurrent SCLC is among the most recalcitrant cancers and drug development efforts in this cancer are a high priority.

Inhibition of the NOTCH pathway using γ-secretase inhibitor RO4929097 has limited antitumor activity in established glial tumors.

Notch signaling is altered in many cancers. Our previous findings in primary pediatric ependymoma support a role for NOTCH in glial oncogenesis. The present study evaluates the γ-secretase inhibitor RO4929097 in glial tumor models. The expression of Notch pathway genes was evaluated using real-time RT-PCR in 21 ependymoma and glioma models. NOTCH1 mutations were analyzed by DNA sequencing. RO4929097 activity was evaluated in vitro and in vivo, as a single agent and in combination, in glioma and ependymoma models. Notch pathway genes are overexpressed in ependymomas and gliomas along with FBXW7 downregulation. NOTCH1 mutations in the TAD domain were observed in 20% (2/10) of ependymoma primary cultures. Blocking the Notch pathway with the γ-secretase inhibitor RO4929097 reduced cell density and viability in ependymoma short-term cultures. When combined with chemotherapeutic agents, RO4929097 enhanced temozolomide effects in ependymoma short-term cultures and potentiated the cytotoxicity of etoposide, cisplatinum, and temozolomide in glioma cells. RO4929097, in combined treatment with mTOR inhibition, potentiated cytotoxicity in vitro, but did not enhance antitumor effects in vivo. In contrast, RO4929097 enhanced irradiation effects in glioma and ependymoma xenografts and showed tumor growth inhibition in advanced-stage IGRG121 glioblastoma xenografts. RO4929097-mediated effects were independent of NOTCH1 mutation status or expression levels, but associated with low IL-6 levels. In established glial tumor models, NOTCH inhibition had limited effects as a single agent, but enhanced efficacy when combined with DNA-interfering agents. These preclinical data need to be considered for further clinical development of NOTCH inhibitors in glial tumors.

Abstract 3048: OMP-59R5 (Anti-Notch2/3) inhibits tumor growth and reduces cancer stem cell frequency in patient derived SCLC xenografts

Abstract Small cell lung cancer (SCLC) is a devastating disease with very poor prognosis and few treatment options. We have established patient derived xenografts from SCLC tumors and determined that Notch pathway target genes are up-regulated in this disease relative to normal lung tissues and relative to other tumor types. We examined the in vivo efficacy of OMP-59R5, an antibody that was initially identified by binding to Notch2 and was subsequently shown to block signaling of both human and mouse Notch2 and Notch3. We performed in vivo growth inhibition and tumorigenicity studies with this Notch2/3 antibody alone or in combination with chemotherapeutic agents in a panel of six patient-derived SCLC xenograft models. We found that OMP-59R5 treatment significantly reduced tumor recurrence in combination with chemotherapy in five of the six models. Through a serial transplantation study from a sensitive tumor, we found that OMP-59R5 in combination with chemotherapy (cisplatin plus irinotecan) profoundly reduced the cancer stem cell (CSC) frequency. In contrast, treatment with chemotherapeutic agents alone had the opposite effect and increased CSC frequency. In minimal residual disease models, we analyzed the ability of OMP-59R5 to delay tumor recurrence following chemotherapy-induced tumor regression. While chemotherapeutic agent-treated tumors re-grew rapidly following treatment termination, the growth of recurrent tumors was delayed by OMP-59R5. We also evaluated changes in gene expression post treatment and found that OMP-59R5 modulated the expression of Notch pathway, epithelial-to-mesenchymal transition, stromal and vasculature genes. Furthermore, we observed an increase in expression of neuroendocrine differentiation markers after OMP-59R5 treatment in sensitive tumors. Following up on these preclinical findings, OMP-59R5 is currently being evaluated for safety and efficacy in combination with cisplatin and etoposide for treatment of SCLC in an ongoing Phase 1b/2 clinical trial (PINNACLE). Citation Format: Marcus M. Fischer, Jalpa Shah, Jennifer Cain, Belinda Cancilla, James W. Evans, Christopher L. Murriel, Tracy Tang, Jie Wei, Wan-Ching Yen, Chun Zhang, Austin Gurney, John Lewicki, Ann M. Kapoun, Timothy Hoey. OMP-59R5 (Anti-Notch2/3) inhibits tumor growth and reduces cancer stem cell frequency in patient derived SCLC xenografts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3048. doi:10.1158/1538-7445.AM2014-3048

Somites Without a Clock

The formation of body segments (somites) in vertebrate embryos is accompanied by molecular oscillations (segmentation clock). Interaction of this oscillator with a wave traveling along the body axis (the clock-and-wavefront model) is generally believed to control somite number, size, and axial identity. Here we show that a clock-and-wavefront mechanism is unnecessary for somite formation. Non-somite mesoderm treated with Noggin generates many somites that form simultaneously, without cyclic expression of Notch-pathway genes, yet have normal size, shape, and fate. These somites have axial identity: The Hox code is fixed independently of somite fate. However, these somites are not subdivided into rostral and caudal halves, which is necessary for neural segmentation. We propose that somites are self-organizing structures whose size and shape is controlled by local cell-cell interactions.

Notch1-Induced Brain Tumor Models the Sonic Hedgehog Subgroup of Human Medulloblastoma

While activation of the Notch pathway is observed in many human cancers, it is unknown whether elevated Notch1 expression is sufficient to initiate tumorigenesis in most tissues. To test the oncogenic potential of Notch1 in solid tumors, we expressed an activated form of Notch1 (N1ICD) in the developing mouse brain. N1ICD;hGFAP-cre mice were viable but developed severe ataxia and seizures, and died by weaning age. Analysis of transgenic embryo brains revealed that N1ICD expression induced p53-dependent apoptosis. When apoptosis was blocked by genetic deletion of p53, 30% to 40% of N1ICD;GFAP-cre;p53(+/-) and N1ICD;GFAP-cre;p53(-/-) mice developed spontaneous medulloblastomas. Interestingly, N1ICD-induced medulloblastomas most closely resembled the sonic hedgehog subgroup of human medulloblastoma at the molecular level. Surprisingly, N1ICD-induced tumors do not maintain high levels of the Notch pathway gene expression, except for Notch2, showing that initiating oncogenic events may not be decipherable by analyzing growing tumors in some cases. In summary, this study shows that Notch1 has an oncogenic potential in the brain when combined with other oncogenic hits, such as p53 loss, and provides a novel mouse model of medulloblastoma. Cancer Res; 73(17); 5381-90. ©2013 AACR.

FGF Signaling Expands Embryonic Cortical Surface Area by Regulating Notch-Dependent Neurogenesis

The processes regulating cortical surface area expansion during development and evolution are unknown. We show that loss of function of all fibroblast growth factor receptors (FgfRs) expressed at the earliest stages of cortical development causes severe deficits in surface area growth by embryonic day 12.5 (E12.5) in the mouse. In FgfR mutants, accelerated production of neurons led to severe loss of radial progenitors and premature termination of neurogenesis. Nevertheless, these mutants showed remarkably little change in cortical layer structure. Birth-dating experiments indicated that a greater proportion of layer fates was generated during early neurogenic stages, revealing that FgfR activity normally slows the temporal progression of cortical layer fates. Electroporation of a dominant-negative FgfR at E11.5 increased cortical neurogenesis in normal mice--an effect that was blocked by simultaneous activation of the Notch pathway. Together with changes in the expression of Notch pathway genes in FgfR mutant embryos, these findings indicate that Notch lies downstream of FgfR signaling in the same pathway regulating cortical neurogenesis and begin to establish a mechanism for regulating cortical surface expansion.

Abstract 618: Anti-tumor effects of Notch pathway activation in Gastrointestinal Stromal Tumors cells

Abstract Gastrointestinal Stromal Tumors (GISTs) are the most common sarcomas of the GI tract and harbor a gain-of-function mutation of the KIT receptor tyrosine kinase. The introduction of imatinib, a specific inhibitor of KIT, has significantly extended survival of many GIST patients. Unfortunately, patients inevitably develop resistance, prompting interest in understanding GIST biology and developing new therapeutic options. Notch signaling may act as an oncogene or a tumor suppressor depending cellular context. In neuroendocrine tumors Notch signaling acts constantly as a tumor suppressor. Additionally, histone deacetylase inhibitors (HDACi) were recently shown to induce Notch signaling in neuroendocrine neoplasms. Given that GIST is of neuroectodermal origin, we hypothesized that inhibition of growth in GIST cells by HDAC inhibitors is mediated by activation of Notch signaling. To characterize Notch signaling in GIST, we assessed the expression of Notch-1, and its effector human hairy enhancer of split-1 (HES1) in human GIST cell lines (GIST-T1 and GIST 882) by quantitative RT-PCR. To determine the activity of Notch signaling in GIST, we assayed the effect of Notch activation in GIST cells by transduction with retroviral vectors to overexpress active forms of Notch1 and HES1 in GIST cells. The proportion of GFP-positive cells in unselected culture was assessed to determine the transgene's effect on cell growth. To study the effect of HDAC inhibition, we quantified viability, caspase activity, cell cycle analysis and expression of Notch pathway genes after HDAC inhibition with suberoylanilide hydroxamic acid (SAHA). Notch signaling genes (Notch1, HES1) were weakly expressed at the basal level in cells compared to other sarcomas. Notch signaling with a constitutively active, truncated form of the receptor (ICN-1) and HES1 potently induced growth arrest in GIST cell lines. The proportion of ICN-1 GFP+ cells dramatically declined after transduction compared to GFP- cells, (decrease of 69% at day 7 and 78% at day 9 after transduction). Vector empty-transduced GFP+ cells gave no change in the proportion of positive cells. SAHA caused dose dependent decrease in viability, activation of caspase 3 and 7, increase of sub-G1 population (6 fold increase at 1µM, p<0.01), as well as upregulation of Notch-1 expression (5 fold at 1µM, p<0.01). Retroviral silencing of downstream targets of Notch (dnHes-1 and dnMastermind [dnMAM]) partially rescued GIST-T1 cells from the inhibitory effects of SAHA (61% v 105%, p<0.05 for dnHes-1 and 61 v 87%, p<0.01 for dnMAM after 2 days of treatment with SAHA 0.6 µM). These results suggest that Notch signaling is a tumor suppressor in GIST cells and the activation of Notch signaling by HDAC inhibitors may represent a new strategy for treating GIST patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 618. doi:10.1158/1538-7445.AM2011-618

Abstract 5190: Biomarkers of sensitivity to PF-03084014, a γ-secretase Inhibitor, in a preclinical model of colorectal cancer

Abstract Purpose: To evaluate the efficacy of PF-03084014, a γ-secretase inhibitor, in a preclinical model of colorectal cancer (CRC) and to identify biomarkers that predicts sensitivity to the compound. Experimental Design: Fourteen patient-derived CRC explants were treated with PF-03084014 (150mg/kg/day- BID) for 28 days. Gene expression of the Notch pathway was evaluated at baseline and day 28 using RT-PCR. Additionally, baseline gene expression profiles were determined using gene array. An Aldefluor assay (stem cell technologies) was used to determine the effect of treatment on tumor initiating cells. Fluorescence in situ hybridization (FISH) was used to determine Notch 1 gene copy number. Results: Three out of the fourteen explants showed sensitivity (cytostatic effects) to PF-03084014. Treatment reduced Notch pathway gene expression (Hes-1, Notch 1, Notch 3, and Jag1) in the sensitive tumors when evaluated by RT-PCR. Baseline gene array data showed an increase in IL-8 gene expression in resistant tumors and pathway analysis demonstrated an increase in focal adhesion, Jack-STAT, and MAPK signaling pathways in resistant explants. Evaluation of the ALDH+ cell population after 28 days of treatment showed that PF-03084014 reduced the ALDH+ cell population in the most sensitive explant (CRC001) when compared to the vehicle group. Evaluation of Notch 1 gene copy number in four CRC explants by FISH showed increased gene copy number in two sensitive explants (CRC027 and CRC001) and one intermediate explant (CRC036) and no gain in gene copy number in one resistant explant (CRC042). Conclusion: Treatment with PF-03084014 significantly reduced tumor growth in three CRC explants. In addition, treatment inhibited expression of the Notch pathway and reduced the tumorigenic ALDH+ population. These results indicate that a subset of patients may derive benefit from treatment with PF-03084014 and biomarkers could be identified that predict sensitivity to the compound. Acknowledgements: This work was supported by a Pfizer IIR grant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5190. doi:10.1158/1538-7445.AM2011-5190

Sensory neuron differentiation is regulated by notch signaling in the trigeminal placode

Trigeminal sensory neurons develop from the neural crest and neurogenic placodes, and have been studied as a principal model of sensory neuron formation. While the Notch pathway has been extensively characterized in central nervous system development and other developmental processes, it has not been well characterized in sensory neurogenesis. Here we studied the functional role of Notch signaling in the trigeminal ophthalmic (opV) placode, a prime model of sensory neurogenesis. To establish a good spatiotemporal description of Notch pathway genes in the chick trigeminal placode, a stage-specific expression analysis was conducted, showing that expression of most Notch pathway genes and effectors are expressed in the placode, with expression primarily being confined to ectodermal cells. Expression was highest at stages of peak neuronal differentiation. To test the function of Notch signaling in opV placode cell differentiation, Notch receptor cleavage was blocked using the gamma-secretase inhibitor, DAPT, or signaling was activated by misexpression of the Notch intracellular domain (NICD). Notch activation resulted in a significant reduction in sensory neurogenesis. Cells remained in the ectoderm and did not differentiate. Expression of the opV specification marker Pax3 was also lost in targeted cells. DAPT exposure resulted in a dramatic increase in neurogenesis without increasing proliferation, where many differentiated cells were found in the mesenchyme and, surprisingly, within the ectoderm. This is the first result clearly showing prolific neuronal differentiation in the ectoderm of the trigeminal placodes after experimental manipulation of a molecular signaling pathway, thus identifying Notch signaling as a primary regulator of the sensory neuron fate in the opV placode.

Abstract B41: Identification of a time- and dose-responsive transcriptional signature of Notch pathway inhibition in plucked human hair follicles following exposure to the gamma-secretase inhibitor MK-0752

Abstract Introduction: Gamma-secretase inhibitors (GSIs) inhibit Notch pathway signaling and have potential as cancer therapeutics. Clinical development of GSIs has been complicated by mechanism-related goblet cell hyperplasia and dose-limiting diarrhea. A non-invasive biomarker of Notch pathway inhibition would facilitate the identification of a well-tolerated dose/schedule with maximal biological effect. In this study, a transcriptional biomarker of Notch pathway inhibition was developed from whole genome profiling of human plucked hair follicles (PHFs) for use in demonstrating target engagement and defining a PK/PD relationship. Procedures: This randomized, placebo-controlled, 3-period crossover study evaluated the effects of 350mg or 1000mg of the oral GSI MK-0752 on PHF gene expression in 30 healthy male subjects. Pooled PHFs, single anagen hairs, and whole blood samples were collected at various timepoints for pharmacokinetic and pharmacodynamic (mRNA profiling) analysis. Results: There was a significant decrease in a pre-specified 15-gene Notch signature score (NSS) at 8.5, 28.5, 48 and 96 hours in response to either 350mg or 1000mg MK-0752, compared to placebo, at the nominal 1-sided 0.05 level. The maximum response in pooled PHFs was seen following administration 1000mg MK-0752, which resulted in a significant (p <0.001) decrease from baseline in the NSS at 8.5 hours post-treatment, with an observed effect size of 2.34. A smaller, but significant, decrease from baseline in the NSS was seen in single anagen hairs at 8.5h post-treatment for 1000mg MK-0752 compared to placebo (effect size 1.40, p<0.001). Changes in the hair follicle NSS were driven primarily by 5 Notch pathway genes. A linear concentration-effect relationship was shown to be consistent across both dose levels using PHFs. In addition to Notch pathway changes, transcriptional signatures of PI3K signaling and cell proliferation were shown to be suppressed at 8.5h and 28.5h post-dose, respectively. Treatment with MK-0752 also resulted in various late (48h post-dose) transcriptional changes in hair follicles including the upregulation of other signaling pathways including Wnt and receptor tyrosine kinases. Conclusions: The results of this study demonstrate the development of a compound-specific biomarker in a novel surrogate tissue. This is the first known report of whole genome mRNA profiling of human hair follicles in response to a GSI, and demonstrates that PHFs contain cells with intact Notch signaling pathways responsive to MK-0752. A 1000mg dose of MK-0752, which is smaller than the clinically-tolerated dose of 1800mg/week, decreases expression of canonical Notch pathway genes for up to 96 hours. In addition, treatment with a GSI results in various biological changes in PHFs, including early downregulation of PI3K pathway and proliferation genes, and a late (48h) upregulation of other signaling pathways that are potentially relevant to cancer cell proliferation. This novel plucked hair follicle biomarker provides important insights into the effect of GSIs on cell signaling, and also facilitates the development of gamma secretase targeted drugs for oncology indications. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B41.

Down-regulation of the Notch Pathway in Human Airway Epithelium in Association with Smoking and Chronic Obstructive Pulmonary Disease

The airway epithelium of smokers is subject to a variety of mechanisms of injury with consequent modulation of epithelial regeneration and disordered differentiation. Several signaling pathways, including the Notch pathway, control epithelial differentiation in lung morphogenesis, but little is known about the role of these pathways in adults. We tested the hypotheses that Notch-related genes are expressed in the normal nonsmoker small airway epithelium of human adults, and that Notch-related gene expression is down-regulated in healthy smokers and smokers with chronic obstructive pulmonary disease (COPD). We used microarray technology to evaluate the expression of 55 Notch-related genes in the small airway epithelium of nonsmokers. We used TaqMan quantitative polymerase chain reaction (PCR) to confirm the expression of key genes and we used immunohistochemistry to assess the expression of Notch-related proteins in the airway epithelium. Changes in expression of Notch genes in healthy smokers and smokers with COPD compared with nonsmokers were evaluated by PCR. Microarray analysis demonstrated that 45 of 55 Notch-related genes are expressed in the small airway epithelium of adults. TaqMan PCR confirmed the expression of key genes with highest expression of the ligand DLL1, the receptor NOTCH2, and the downstream effector HES1. Immunohistochemistry demonstrated the expression of Jag1, Notch2, Hes1, and Hes5 in airway epithelium. Several Notch ligands, receptors, and downstream effector genes were down-regulated in smokers, with more genes down-regulated in smokers with COPD than in healthy smokers. These observations are consistent with the hypothesis that the Notch pathway likely plays a role in the human adult airway epithelium, with down-regulation of Notch pathway gene expression in association with smoking and COPD.

Select γ-Secretase Inhibitors Induce Apoptosis in Pre-B ALL Cells and Disrupt the Balance Between Constitutive Notch Signaling and Repression.

Several γ secretase inhibitors (GSIs) were tested for the ability to induce apoptosis in precursor B acute lymphoblastic leukemia (pre-B ALL) cells. Of five GSI's tested, treatment with two compounds resulted in effective killing of both pre-B lymphoblasts and cells from multiple pre-B ALL lines. Since Notch receptors represent an important group of γ secretase targets, we evaluated expression and activation status of Notch receptors in CD19+ lymphoblasts from pediatric pre-B ALL patients, as well as cultured pre-B ALL cells. We found that, unlike T-ALL where activating mutations are common, pre-B ALL cells appear to drive constitutive Notch signaling through autocrine signals. Blasts from 11 patients expressed 3 Notch receptors and multiple Notch counter-ligands. Expression of Notch pathway genes was also confirmed by microarray analysis of genes expressed in 207 children with high risk B precursor ALL. GSI treatment of pre-B ALL cells led to dephosphorylation of AKT and Foxo3, Bim expression and caspase activation. GSI treatment also blocked cleavage of Notch 1 and 2 to their active forms and inhibited expression of Notch targets, Hey2 and Myc. In contrast, increased expression of Hes1 and Hey1 was correlated with GSI-induced loss of the co-repressor, SMRT. GSI treatment appears to induce precursor B cell death by disrupting the balance between constitutive Notch signaling and repression.

Transcriptome Analysis of the Normal Human Mammary Cell Commitment and Differentiation Process

Mature mammary epithelial cells are generated from undifferentiated precursors through a hierarchical process, but the molecular mechanisms involved, particularly in the human mammary gland, are poorly understood. To address this issue, we isolated highly purified subpopulations of primitive bipotent and committed luminal progenitor cells as well as mature luminal and myoepithelial cells from normal human mammary tissue and compared their transcriptomes obtained using three different methods. Elements unique to each subset of mammary cells were identified, and changes that accompany their differentiation in vivo were shown to be recapitulated in vitro. These include a stage-specific change in NOTCH pathway gene expression during the commitment of bipotent progenitors to the luminal lineage. Functional studies further showed NOTCH3 signaling to be critical for this differentiation event to occur in vitro. Taken together, these findings provide an initial foundation for future delineation of mechanisms that perturb primitive human mammary cell growth and differentiation.