Citations

Abstract Activation of the Sonic hedgehog (Shh) pathway and increased expression of Gli1 play an important role in proliferation and transformation of granule cell progenitors (GCP) in the developing cerebellum. Medulloblastomas arising from cerebellar GCPs are frequently driven by Shh pathway–activating mutations; however, molecular mechanisms of Shh pathway dysregulation and transformation of neural progenitors remain poorly defined. We report that the transcription factor and oncogene Snail1 (Sna1) is directly induced by Shh pathway activity in GCPs, murine medulloblastomas, and human medulloblastoma cells. Enforced expression of Sna1 was sufficient to induce GCPs and medulloblastoma cell proliferation in the absence of Shh/Gli1 exposure. In addition, enforced expression of Sna1 increased transformation of medulloblastoma cells in vitro and in vivo. Analysis of potential Sna1 targets in neural cells revealed a novel Sna1 target, N-Myc, a transcription factor known to play a role in Shh-mediated GCP proliferation and medulloblastoma formation. We found that Sna1 directly induced transcription of N-Myc in human medulloblastoma cells and that depletion of N-Myc ablated the Sna1-induced proliferation and transformation. Taken together, these results provide further insight into the mechanism of Shh-induced transformation of neural progenitor cells and suggest that induction of Sna1 may serve to amplify the oncogenic potential of Shh pathway activation through N-Myc induction. Cancer Res; 71(15); 5336–45. ©2011 AACR.

Abstract Activation of the Sonic Hedgehog (SHH) pathway and increased expression of the Gli1 transcription factor have been implicated in the tumorigenesis of both sporadic and syndromic medulloblastoma, a malignant pediatric brain tumor that arises from cerebellar progenitor cells. While SHH pathway activity is required for normal neural stem cell proliferation, molecular mechanisms of SHH pathway dysregulation and transformation of neural precursor cells remain poorly defined. In epithelial cells, Snail1 was found to be induced by Gli1 expression and is believed to promote Gli1-mediated transformation (Li et al. 2006), however the role of Snail1 in neural cells is not well understood. We have identified Snail1 as a putative target of Gli1 in neural stem cells. We hypothesize that Snail1 participates in transduction of SHH/Gli1 signaling in the nervous system and plays a role in proliferation of neuronal progenitor cells in the cerebellum. Mice haploinsufficient for Patched (Ptc) have constitutive activation of the SHH pathway and a subset develop medulloblastoma. Quantitative RT-PCR analyses of mRNA isolated from early postnatal untransformed cerebella show that Snail1 mRNA is increased in vivo in Ptc+/- mice versus wild-type (WT) siblings.. Enforced expression of Gli1 in cultured WT murine granule precursor cells (GPCs) induced Snail1 mRNA.. Enforced expression of Snail1 was also sufficient to induce proliferation of GCPs in the absence of SHH/Gli1 exposure. Given that activation of SHH/Gli1-pathway induces proliferation of GPCs and is associated with medulloblastoma formation, we hypothesized that Snail1 may play a role in the proliferation and transformation of neural progenitor cells. We have found that the stem-like cells generated from medulloblastomas in Ptc+/- mice express higher levels of Snail mRNA than tumor parenchyma. Quantitative RT-PCR analysis demonstrates that Snail1 mRNA is expressed at baseline and is dramatically induced in human medulloblastoma cell lines after exposure to SHH. Enforced expression of Snail1 further increased proliferation of medulloblastoma cells as demonstrated by EdU incorporation. Analysis of potential Snail1 targets in human medulloblastoma cells reveals that β-catenin and N-Myc protein are induced by Snail1 overexpression. N-Myc is induced by SHH in GPCs (Kenney et al. 2003) and is known to induce proliferation in GPCs during both normal cerebellar development and medulloblastoma formation. Together these results give further insight into the mechanism of SHH-induced proliferation of neural progenitor and suggest that Snail1 may contribute to malignant transformation of GCPs through N-Myc. These data establish Snail1 as a target of SHH/Gli1 pathway activity in both normal and neoplastic cells derived from cerebellum. Ongoing experiments will further elucidate how this novel proliferative process affects transformation of neural cells. 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 7.

A retrospective review identified 99 adolescents (79% female) referred to a tertiary care center to evaluate the relationship between symptoms of orthostatic intolerance and chronic pain. Regression analysis indicated that functional disability was strongly associated with pain intensity (P < .001) and depression (P = .024). The association between functional disability and number of symptoms of orthostatic intolerance trended toward significance (P = .057). Meeting a threshold heart rate increment of 30 beats per minute on head-up tilt was not associated with functional disability (P = .188). Separate regression analysis of female patients showed similar results to the full sample but with a stronger relationship between depression and functional disability and a weaker relationship between heart increment and functional disability. In this sample of adolescents with symptoms of orthostatic intolerance and chronic pain, pain intensity and depression were associated with functional status, but postural tachycardia was not. Further research is needed to clarify potential gender differences.

Abstract Preclinical investigations have identified insulin-like growth factor (IGF) signaling as a key mechanism for cancer growth and resistance to clinically useful therapies in multiple tumor types including breast cancer. Thus, agents targeting and blocking IGF signaling have promise in the treatment of solid tumors. To identify possible mechanisms of resistance to blocking the IGF pathway, we generated a cell line that was resistant to the IGF-1R/InsR benzimidazole inhibitors, BMS-554417 and BMS-536924, and compared expression profiles of the parental and resistant cells lines using Affymetrix GeneChip Human Genome U133 arrays. Compared with MCF-7 cells, breast cancer resistance protein (BCRP) expression was increased 9-fold in MCF-7R4, which was confirmed by immunoblotting and was highly statistically significant (P = 7.13E-09). BCRP was also upregulated in an independently derived resistant cell line, MCF-7 924R. MCF-7R4 cells had significantly lower intracellular accumulation of BMS-536924 compared with MCF-7 cells. Expression of BCRP in MCF-7 cells was sufficient to reduce sensitivity to BMS-536924. Furthermore, knockdown of BCRP in MCF-7R4 cells resensitized cells to BMS-536924. Four cell lines selected for resistance to the pyrrolotriazine IGF-1R/InsR inhibitor, BMS-754807, did not have upregulation of BCRP. These data suggest that benzimidazole IGF-1R/InsR inhibitors may select for upregulation and be effluxed by the ATP-binding cassette transporter, BCRP, contributing to resistance. However, pyrrolotriazine IGF-1R/InsR inhibitors do not appear to be affected by this resistance mechanism. Mol Cancer Ther; 10(1); 117–25. ©2011 AACR.

Abstract Abstract #2149 Background: Inhibitors of the insulin-like growth factor 1 receptor (IGF-1R) are currently undergoing clinical testing. Preclinical investigations have identified IGF-1 signaling as a key mechanism for breast cancer growth and resistance to clinically useful therapies, including tamoxifen and trastuzumab. Thus, agents targeting IGF-1R have promise in the treatment of breast cancer. Determining mechanisms that can confer resistance to these agents may aid their clinical development.&#x2028; Methods: To understand factors may be important in predicting sensitivity to targeting the IGF-1 signaling pathway, we developed a cell line (MCF-7R4) that is resistant to BMS-554417, a small molecule, dual-kinase inhibitor of IGF-1R and insulin receptor (InsR). Compared with the parental MCF-7 cells, MCF-7R4 cells are 40- to 50-fold resistant to BMS-554417 and cross-resistant to the similar compound BMS-536924. The expression profiles of MCF-7R4 and that of MCF-7 were compared using Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays. Intracellular concentrations of BMS-536924 were examined by reverse phase high performance liquid chromatography. BMS-536924 cellular accumulation in vitro was visualized by fluorescence microscopy using a DAPI filter set. MCF-7 cells stably transfected with either the empty mammalian expression vector pcDNA (MCF-EV) or full length BCRP (MCF-BCRP) were examined for sensitivity to BMS 536924 by MTS assays.&#x2028; Results: Compared to MCF-7 cells, BCRP expression was increased 9-fold in MCF-7R4, which was highly statistically significant by t-test (p= 7.13E-09). Little change was observed in other ABC transporter proteins, including ABCB1. No change was observed in IGF-1R or InsR expression. BCRP overexpression in MCF-7R4 cells was confirmed by western blotting. MCF-7R4 cells had significantly lower intracellular accumulation of BMS-536924 compared to MCF-7 cells. Confirming these results, MCF-BCRP cells were significantly less sensitive to the cytoxic effects of BMS-536924 cells than MCF-EV cells.&#x2028; Conclusions: BCRP expression was stimulated by prolonged exposure of MCF-7 cells to BMS-554417. Upregulation of BCRP is one of the most significant changes observed in MCF-7R4 cells in comparison to parental cells. BCRP expression decreased cellular exposure to BMS-536924 and was sufficient to confer resistance. These data suggest that BSM-536924 is a substrate for BCRP-mediated efflux. Expression of BCRP may be important in de novo and acquired resistance to benzimidazole –based inhibitors of IGF-1R/InsR. Supported in part by the Mayo Clinic Breast SPORE (CA116201-03), NIH K12 (CA090628-05) and the Mayo Clinic Cancer Center (CA15083). Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2149.

Paracrine signaling between cholangiocytes and stromal cells regulates biliary remodeling. Cholangiocytes have neuroepithelial characteristics and serotonin receptor agonists inhibit their growth, but whether they are capable of serotonin biosynthesis is unknown. We hypothesized that cholangiocytes synthesize serotonin and that cross talk between liver myofibroblasts (MF) and cholangiocytes regulates this process to influence biliary remodeling. Transwell cultures of cholangiocytes ± MF, and tryptophan hydroxylase-2 knockin (TPH2KI) mice with an inactivating mutation of the neuronal tryptophan hydroxylase (TPH) isoform, TPH2, were evaluated. Results in the cell culture models confirm that cholangiocytes have serotonin receptors and demonstrate for the first time that these cells express TPH2 and produce serotonin, which autoinhibits their growth but stimulates MF production of TGF-β1. Increased TGF-β1, in turn, counteracts autocrine inhibition of cholangiocyte growth by repressing cholangiocyte TPH2 expression. Studies of TPH2KI mice confirm that TPH2-mediated production of serotonin plays an important role in remodeling damaged bile ducts because mice with decreased TPH2 function have reduced biliary serotonin levels and exhibit excessive cholangiocyte proliferation, accumulation of aberrant ductules and liver progenitors, and increased liver fibrosis after bile duct ligation. This new evidence that cholangiocytes express the so-called neuronal isoform of TPH, synthesize serotonin de novo, and deploy serotonin as an autocrine/paracrine signal to regulate regeneration of the biliary tree complements earlier work that revealed that passive release of serotonin from platelets stimulates hepatocyte proliferation. Given the prevalent use of serotonin-modulating drugs, these findings have potentially important implications for recovery from various types of liver damage.