Sa1804 Dysregulation of WNT5A/ROR2 Signaling Pathway Characterizes the Progression of...
Transcript of Sa1804 Dysregulation of WNT5A/ROR2 Signaling Pathway Characterizes the Progression of...
Sa1803
Dissecting the Oncogenic Activity of the Nuclear Receptor LRH-1 in the ColonJames R. Bayrer, Sridevi Mukkamala, Elena Sablin, Robert Fletterick
BACKGROUND. Colorectal cancers (CRC) account for nearly 10% of cancer deaths inindustrialized countries. Recent evidence points to a central role for the nuclear receptorLiver Receptor Homolog-1 (LRH-1) in intestinal tumorigenesis. Interaction of LRH-1 withthe Wnt/β-catenin pathway, highly active in a critical subpopulation of CRC cancer cells,underscores the importance of elucidating LRH-1's role in this disease. Reduction of LRH-1 diminishes tumor burden in murine models of CRC. The central role of receptor LRH-1in CRC pathogenesis and a wealth of structural data at atomic resolution make LRH-1 anattractive target. Methods: To evaluate the contributions of LRH-1 to CRC growth, weconstructed stably transduced CRC cell lines expressing Tet-inducible shRNA directed againstLRH-1. Cell growth of the LRH-1 knockdown lines was compared with growth of cellsexpressing a non-silencing control RNA. To explore alterations in gene expression due toLRH-1 suppression, we performed a microarray analysis of our knockdown cell lines.Additionally, we sought to develop and characterize specific, non-cytotoxic inhibitors ofLRH-1 function for use as both potential therapeutic agents and more broadly as researchtools to study organogenesis and tumorigenesis. We applied differential scanning fluorimetry(DSF), used to measure qualitative binding, to screen over 200 compounds for associationwith the LRH-1 ligand binding domain. Results: LRH-1 mRNA knockdown results in impairedcell proliferation. Cluster analysis of microarray gene expression demonstrates significantalterations in signal transduction, bile acid and cholesterol metabolism, and control ofapoptosis. Moreover, we discovered 9 unique compounds that interact with LRH-1, but notthe closely related receptor SF-1. We have further demonstrated that four of these compoundshave antiproliferative effects with IC50 values ranging from 8.6 to 30 μM. Conclusions:Silencing of LRH-1 expression in CRC cell lines leads to impaired cell growth, demonstratingthat LRH-1 is a viable target in CRC. LRH-1 may exert its effects via multiple signalingnetworks, with inhibition leading to cell cycle arrest or apoptosis. General features forbinding shared between all compounds include a linear array of hydrophobic groups withpolar atoms likely allowing for registration within the hormone pocket. Interestingly, twocompounds showing antiproliferative activity share a cholesterol motif which may be relatedto LRH-1's function in bile acid and cholesterol homeostasis. Support: K12 HD07222, F32CA163092, T32 DK007762
Sa1804
Dysregulation of WNT5A/ROR2 Signaling Pathway Characterizes theProgression of Barrett's-Associated Esophageal AdenocarcinomaOrestis Lyros, Tami Moore, Rituparna Medda, Linghui Nie, Mary F. Otterson, Ines Gockel,Hauke Lang, Reza Shaker, Parvaneh Rafiee
Introduction: The incidence of esophageal adenocarcinoma (EAC) has dramatically increasedover the last decade while its prognosis remains poor. The molecular mechanisms underlyingthe progression towards EAC remain elusive. Wnt5a is a non-transforming WNT familywith nulnown role in carcinogenesis. Wnt5a functions mainly via ROR2 receptor and exhibitstumor suppressor activities in cancers of thyroid, brain, breast and colorectal, but strangelyup-regulated in lung, stomach and prostate cancers. We investigated whether Wnt5a/ROR2signaling pathway play a role in EAC Methods: The use of human tissues and all experimentswere approved by the Institutional Review Board of the Medical College of Wisconsin.Wnt5aand ROR2 gene and protein expression were analyzed in esophageal specimens from patientswith Barrett's metaplasia (n=15), EAC patients (n=8) and healthy controls (n=10) as wellas esophageal cell lines by Real-time PCR, western blotting and immunohistochemistry.Esophageal adenocarcinoma cell line (OE33) was treated with human recombinant Wnt5a(hrWnt5a), then cell proliferation, survival and migration were assessed by [3H]-thymidineuptake assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assayand migration assay respectively. Activation of Wnt/β-catenin signaling was determined byTOP/FOPflash luciferase assay. ROR2 gene silencing was performed using siRNA withappropriate controls. Results: Both barrett's metaplasia and EAC exhibited low expressionof Wnt5a gene compared to healthy esophageal squamous mucosa. In contrast, in adenocarci-noma tissue ROR2 receptor was overexpressed compared to the corresponding proximalhealthy mucosa in EAC patients. In vitro, Wnt5a and ROR2 expression were in barrett'smetaplastic (CP-A) and OE33 cell lines compared to normal squamous esophageal cell lines(EPC2-hTERT). hrWnt5a dose and time dependently inhibited OE33 cell proliferation,survival and migration. Treatment with hrWnt5a decreased TOPflash activity in ROR2-wildtype OE33 cells, while it increased TOPflash activity in ROR2-knockdown OE33 cells.ROR2-knockdown resulted in decreased OE33 cell proliferation and migration. Conclusions:Early dysregulation of Wnt5a/ROR2 pathway is involved in the progression of EAC. Wnt5ademonstrates tumor suppression by inhibiting the Wnt/β-catenin signaling via ROR2 recep-tor. Loss of Wnt5a with simultaneous activation of Wnt5a-independent from ROR2 signalingmay contribute to the aggressive phenotype of EAC.
Sa1805
An mTOR-Sirtuin Axis in the Intestinal Cell Differentiation NetworkQingding Wang, Yuning Zhou, Heidi L. Weiss, B. Mark Evers
The intestinal mucosa undergoes a continual process of proliferation, differentiation, andapoptosis, which is regulated by multiple signaling pathways. The human sirtuin proteins(Sirt1-7) possess a unique NAD-dependent protein deacetylase activity and are involved inthe regulation of metabolism, differentiation and cell survival. Previously, we have shownthat the PI3K/Akt/mTOR pathway plays a critical role in intestinal homeostasis. However,the downstream targets mediating the effects of mTOR in intestinal cells are largely undefined.The purpose of this study was to investigate: i) the regulation of sirtuin expression andactivity by mTOR, and ii) the role of sirtuin proteins in intestinal cell differentiation.METHODS. Differentiation was induced by treatment of HT29 and Caco-2 human coloncancer cells with sodium butyrate (NaBT) and assessed by measurement of intestinal alkalinephosphatase (IAP) activity, an enterocyte differentiation marker. HEK293 (human embryonic
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kidney cells) and Caco-2 cells were transfected with constructs overexpressing Myc-rictor;Myc-raptor; FLAG-tagged Sirt proteins 1, 2, 6; control siRNA or siRNA targeting Sirt1, Sirt2,Sirt6, mTOR, rictor, and raptor. The expression of Sirt1, Sirt2, Sirt6, acetylated histone H3K9, and cell cycle-dependent kinase inhibitors p21waf1 and p27kip1 was analyzed byWestern blot and real time RT-PCR, respectively. The interaction between sirtuin proteinsand mTOR was investigated by immunoprecipitation followed by Western blotting. Sirt1,Sirt2, and Sirt6 expression was analyzed by immunohistochemistry in normal human colonand small bowel mucosa. RESULTS. We found that mTOR inhibits Sirt1, Sirt2, and Sirt6protein expression and deacetylase activity without affecting mRNA expression, suggestinga posttranscriptional regulation of sirtuin proteins by mTOR signaling. In addition, we showthat mTOR directly interacts with Sirt2 and Sirt6. Furthermore, Sirt1, Sirt2, and Sirt6 areexpressed in the mucosa of the intestine with the most pronounced staining near the epithelialsurface. Inhibition or knockdown of Sirt1, Sirt2, and Sirt6 attenuated NaBT-mediated induc-tion of IAP activity and p21waf1 and p27kip1 protein expression; overexpression of Sirt1,Sirt2, and Sirt6 enhanced NaBT-increased p21waf1 and p27kip1 protein expression. CON-CLUSIONS. We provide evidence showing that sirtuin proteins contribute to intestinal celldifferentiation. Importantly, our results suggest that mTOR regulation of intestinal celldifferentiation may be through inhibition of sirtuin signaling.
Sa1806
Upregulation of Importins Is a Novel Key Mechanism for Aberrant, IncreasedVEGF Expression in Colon Cancer Cells and Sustained Proliferation of TheseCells via an Autocrine PathwayAmrita Ahluwalia, Michael K. Jones, Andrzej S. Tarnawski
Background & Aims: Colorectal cancer (CRC) cells have increased expression of VEGF vs.normal colonic epithelial cells, which do not express VEGF. However, the mechanismsunderlying this aberrant expression of VEGF are not explained. Transcriptional activationof the VEGF gene requires nuclear transport of transcription factors into the CRC cells.Importins can mediate in some cells (e.g., HeLa) the nuclear transport of transcription factorsbut whether this paradigm applies to CRC cells is unknown. We hypothesized that importin-α is upregulated in CRC cells and facilitates nuclear transport of transcription factors thatactivate the VEGF gene promoter, and is the underlying mechanism for increased VEGFexpression in these cells and CRC cell proliferation. Methods: We used: (1) human CRCcell lines - HCT116 & HT29 and (2) normal colonic epithelial cells - NCM356 & NCM460cultured in nutrient media in the presence or absence of exogenous VEGF. Importin-αexpression in cultured CRC cells using specific siRNA. Studies: 1) cell proliferation byBrdU assay; 2) apoptosis by TUNEL staining; 3) importin-α and VEGF mRNA and proteinexpression by Real-Time RT-PCR and Western blotting, respectively. Results: 1) CRC celllines exhibit increased expression of VEGF (6- to10-fold; p<0.001) and importin-α protein(2- to 3-fold; p<0.05) vs. normal epithelial cells; 2) Silencing of importins -α3 and -α7 inCRC cells reduced: a) VEGF expression by > 2.2-fold (both p<0.001), and b) these cells'proliferation by 2.3- to 4.8-fold (p<0.01); 3) Treatment with exogenous VEGF partly reversedthe inhibition of cell proliferation caused by importin-α silencing. Conclusions: 1) Importin-α is increased in CRC cells; 2) Importin-α, mediates aberrant, increased VEGF expressionin CRC cells and CRC cell proliferation; 3) VEGF secreted by CRC cells increases thesecells' proliferation directly via an autocrine pathway; 4) These studies suggest a novel roleof VEGF in CRC cell proliferation independent of its role in stimulating angiogenesis.
Sa1807
Fibroblast-Secreted Activin a Inhibits Invasion and Angiogenesis inEsophageal Squamous Cell CarcinomaHolli A. Loomans, Gregoire F. Le Bras, Claudia D. Andl
The progression of cancer is greatly dependent on signaling from the tumor microenviron-ment. However, few candidate pathways have been identified to regulate crosstalk betweenthe epithelial cells and the stroma. TGFβ, which acts in a cell type and context specificmanner, is known to mediate stromal crosstalk and activation of fibroblasts, epithelial-mesenchymal-transition, as well as the induction of angiogenesis in esophageal cancer. Wetested the hypothesis if Activin A (INHBA), a member of the TGFβ superfamily, functionsin a similar way and is a novel regulator of epithelial-mesenchymal crosstalk. To investigatethe functional consequences of fibroblast-derived INHBA on epithelial cell invasion, weestablished a fibroblast cell line stably overexpressing INHBA (FEF-INHBA) and examinedtheir function in three-dimensional organotypic reconstruct cultures (OTC). Interestingly,epithelial cell invasion into the underlying matrix was completely suppressed in OTC cultureswith embedded FEF-INHBA. The autocrine signaling effects of INHBA on fibroblasts includedreduced proliferation and decreased expression of MMP2, αSMA, vimentin, N-cadherin, αvintegrin, podoplanin and fibronectin. These observations were in contrast to stimulation offibroblasts with INHBA on plastic, which does not allow for crosstalk with extracellularmatrix and epithelial cells. We further, analyzed the ability of FEF-INHBA to induce branchingof endothelial cells and could show that INHBA prevents angiogenesis in vitro. Takentogether, these results denote an exclusive role of INHBA signaling in epithelial-mesenchymalcrosstalk and the inhibition of tumor microenvironment activation.
Sa1808
Hoxa Cluster Expression Along the Gastrointestinal TractVincent T. Janmaat, Auke Verhaar, Marco J. Bruno, Manon C. Spaander, Maikel P.Peppelenbosch
Background: Various gastrointestinal (GI) pathologies, such as Crohn's disease and ulcerativecolitis, show a preference for specific locations along the GI tract. However, the mechanismssensing positional information and mediating the resulting location-specific gene expressionremain poorly understood. Hox genes encode transcriptional regulatory proteins that controlorganogenesis, maintain tissue homeostasis and are key drivers of developmental processes.Hox genes are grouped in clusters (a, b, c and d). The 3' to 5' sequence of the Hox genes
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