Sa1679 Effects of Urocortin 2 and Corticotropin-Releasing Hormone Receptor 2 in Colorectal Cancer...
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Sa1678 A Colon Cancer-Derived Mutant of KrüPpel-Like Factor 5 Inhibits Its Degradation by Glycogen Synthase Kinase 3β and the E3 Ubiquitin Ligase FBW7 Agnieszka Bialkowska, Yang Liu, Mandayam O. Nandan, Vincent W. Yang Background: Krüppel-like factor 5 (KLF5) is a zinc-finger transcription factor that is highly expressed in the crypt cells of the intestinal epithelia and plays a critical role in regulating proliferation of both normal intestinal epithelial cells and colorectal cancer cells. We previ- ously showed that KLF5 is a critical mediator for intestinal tumorigenesis resulted from either ApcMin mutation or oncogenic KRAS activation (McConnell et al. [2009] Cancer Res. 69:4125, Nandan et al. [2010] Molecular Cancer 9:63, respectively). KLF5 is regulated at the transcriptional, translational, and post-translational level. Previous studies indicate that the protein kinase GSK3β and E3 ubiquitin ligase FBW7 facilitate proteosomal degradation of KLF5 protein via phosphorylation (GSK3 β) and recognition (FBW7) of a phosphodegron sequence surrounding Ser303 in human KLF5. A recent genomic analysis of colorectal cancer tissues identified a somatic mutation (P301S) in KLF5 within the phosphodegron sequence (Sjöblom et al. [2006] Science 314:268). Aim: To investigate the impact of P301S mutation on KLF5 protein stability. Methods: Plasmids containing human wildtype (WT) and mutant (S303A, P301S, P301A, and P301E) KLF5 under the control of the CMV promoter were transiently transfected into HEK293 cells. The effect of FBW7 on the stability of KLF5 produced from the various constructs was investigated by cycloheximide chase and Western blot analysis. Transcriptional activity of various KLF5 constructs was examined by luciferase reporter assay with Cyclin D1 and CDC2 reporter plasmids. Results: All four KLF5 mutants examined are more resistant to FBW7-mediated proteosomal degradation as compared to WT KLF5. Proteasome inhibition using MG132 resulted in the accumulation of WT KLF5 but had little impact on P301S KLF5 protein levels. Co-immunoprecipitation experiments show that, in contrast to WT KLF5, P301S and S303A KLF5 do not interact with FBW7. Concomitantly, the P301S KLF5 mutant displayed reduced levels of phosphorylation at Ser303 in comparison with WT KLF5. Furthermore, luciferase assays using Cyclin D1 and CDC2 promoter constructs demonstrate significantly elevated transcriptional activity of P301S KLF5 as compared to WT KLF5. Conclusion: Our data indicate that residue 301 of KLF5 is critical for proper recognition of the phosphodegron sequence. Furthermore, we show that the colon cancer-derived P301S mutation in KLF5 inhibits FBW7 recognition of the phosphodegron, yielding a degradation-resistant KLF5 protein, elevated protein levels, and enhanced KLF5 transcriptional activity. These results suggest that the P301S mutation in KLF5 is potentially oncogenic in colon cancer. Sa1679 Effects of Urocortin 2 and Corticotropin-Releasing Hormone Receptor 2 in Colorectal Cancer Growth and Metastasis Jorge Rodriguez, Charalabos Pothoulakis, Stavroula Baritaki Background and aims: Colorectal cancer (CRC) represents the third most common form of cancer worldwide. Moreover, activation of inflammatory pathways participates in CRC growth. Corticotropin Releasing Hormone (CRH) family of peptides and their receptors CRHR1 and CRHR2 are expressed at the CNS and the periphery, including the intestine. We have shown that the CRH peptide family represents an important component of the intestinal inflammatory response. To-date there are no studies implicating the CRH family of peptides in CRC development and progression. Our goal was to identify a potential role of the CRH family members in CRC pathophysiology and explore the underlying molecular mechanisms. Methods: mRNA levels of CRH family members were monitored by qRT-PCR in 46 and 56 normal colon and CRC human samples, and in CRC cell lines (HT-29, DLD1, SW403, SW480, SW620, HCT-116, and Caco2). All cell lines were lentivirally transduced for CRHR2 expression (CRHR2+). Parental and CRHR2+ cells were tested for mRNA expres- sion of the CRHR2 specific ligand urocortin-2 (UCN2), as well as IL-6 and IL-6R using qRT-PCR. Tumor progression was assessed by proliferation, invasion and migration assays. Single cell colony formation was assessed by soft agar assays. Expression of pSTAT3 and EMT markers was determined by western blot. Results: CRC biopsies and cell lines showed significantly downregulated CRHR2 and increased UCN2 mRNA expression, compared to controls (4-fold each). Four CRHR2+ CRC clones had significantly reduced endogenous UCN2 (2-10 fold) and pro-inflammatory cytokine mRNA levels including IL-6 (2-20 fold) and its receptor IL-6R (1.5-5 fold). These effects were more profound in the highly metastatic CRC cell lines SW620 and HCT-116 at baseline and after exogenous administration of UCN2. CRHR2+ clones with downregulated IL-6R levels showed significantly reduced pSTAT3 levels (2-10 fold) compared to parental cells, and associated with decreased cell proliferation, colony formation, invasion and migration at baseline and in response to IL-6. These responses were reversed by the selective CRHR2 antagonist, astressin 2B, indicating that they were CRHR2 specific. The EMT markers Snail and RKIP were also significantly dysregulated in CRHR2+ clones, suggesting EMT as a downstream target of CRHR2 signaling in CRC. Conclusions: This is the first demonstration supporting a protective role of CRHR2 in preventing CRC growth rates and aggressiveness via inhibition of Stat3 signaling pathway. Furthermore, monitoring CRHR2 and Ucn2 levels in CRC tissues may benefit early diagnosis of CRC metastasis, and might serve as a novel drug target for prevention and treatment of advanced stages of CRC. Supported by NIH PO-1 DK33506 (DK), P50 DK064539 (CP) and T32 5T32HD007512-15 (JR) grants. Sa1680 Identification of a Tight Junction/Focal Adhesion Signaling Complex: BVES and Bcar3 Shenika Poindexter, Wei Ning, Bobak Parang, Vishruth K. Reddy, Richard Near, Adam Lerner, Christopher Williams Blood Vessel Epicardial Substance (BVES) -a tight junction associated protein- is silenced via promoter hypermethylation in all stages of colon cancer. Interference with BVES function in human corneal epithelial cells promotes increased migration, decreased trans-epithelial S-281 AGA Abstracts resistance, and reduced expression of epithelial markers including E-cadherin and Zo-1: characteristic for epithelial to mesenchymal transition (EMT). In contrast, expression of BVES in mesenchymal-like (Lim 2405 and SW620) colorectal cancer cell lines results in mesenchymal to epithelial transition (MET). These data suggest that BVES loss in cancer may contribute to cancer progression via promotion of mesenchymal states. We have deter- mined that BVES regulates RhoA signaling, but incompletely understand how BVES modulates these networks. Therefore, identifying BVES interacting proteins is imperative to mapping BVES regulated intracellular signaling pathways. Thus, we employed a yeast two-hybrid (Y2-H) screen with the intracellular domain of BVES (aa115-360) as bait to probe a placental library. This screen yielded 67 interacting proteins. One of which was Breast Cancer Anti- estrogen Resistance 3 (BCAR3), a known GEF with oncogenic activity that has been shown to interact with RhoA. BCAR3 modulates signaling via complex formation with p130Cas, a focal adhesion protein. We hypothesized that BCAR3 regulates BVES dependent RhoA activity in colon cancer. We confirmed the interaction via directed Y2-H and co-IP. To determine the functional significance of this interaction, we used complimentary approaches of overex- pression and knockdown of BCAR3 in BVES-expressing CACO2 and non-BVES- expressing SW620 cells and screened for changes in BVES-dependent phenotypes. Lentiviral mediated transduction of these lines with WT BCAR3 and the BCAR3/p130Cas uncoupling mutant, R743A, produced no morphological change in the CACO2 cells but promoted membrane ruffling in SW620; in contrast, BCAR3 knockdown resulted in epithelial-like appearance suggesting that BCAR3 is downstream of BVES. We next developed a BVES inducible SW620 cell line that stably overexpresses BCAR3 and performed cell attachment assays. Following induction of BVES, there was a trend toward increased cell attachment to the culture dish at Day 11 as compared to non-induced SW620s (P=.076). Additionally, there was an increased number of detached cells in non-BVES induced SW620 cells at Day 11(P=.02), suggesting that BVES may be important in regulating adhesion. We have identified BCAR3 as a BVES interacting protein. BCAR3 appears to antagonize BVES by promoting EMT. This interaction potentially reveals a unique tight junction to focal adhesion signaling pathway important in regulating EMT. Clarifying the role of BVES and BCAR3 in EMT may lead to therapeutic strategies targeting this interaction in colon cancer. Sa1681 Inhibition of the Prostaglandin EP1 Receptor Suppresses Colon Cancer Cell Invasion and Metastasis. Grace P. O'Callaghan, Michaela Reagan, Ludmila M. Flores, Yong Zhang, Aldo Roccaro, Fergus Shanahan, Irene Ghobrial, Aileen Houston Background: The role of cyclooxygenase-2 (COX-2) and its major metabolite prostaglandin E2 (PGE 2 ) in colon carcinogenesis is well established. PGE 2 signals through four different receptor subtypes, EP1 - EP4. Elucidation of the role of these receptors in colon tumorigenesis may identify novel targets for therapy, potentially circumventing the side-effects associated with COX inhibition, whilst retaining the anti-cancer properties. We have recently shown that blocking EP1 receptor signalling in a subcutaneous model of colon tumorigenesis reduced tumor growth in vivo. However, whether the EP1 receptor plays a role in colon cancer metastasis is unknown. Aim: To investigate the role of the EP1 receptor in promoting migration, invasion and metastasis of colon cancer cells. Methods: Colon tumor cells (SW480, HT29 and CT26 cells) were treated with an EP1 receptor specific antagonist (ONO-8713), or were stably transfected (CT26 and HT29 cells) with short hairpin RNA (shRNA) targeted against the EP1 receptor (EP1 shRNA) or with scrambled control (Scr shRNA). Changes in cell proliferation, migration and invasion were investigated in vitro. HT29 transfected clones were tagged with a Green Fluorescent Protein (GFP) label for subsequent in vivo studies. 3 x 10 6 cells were injected into the tail vein of SCID-bg mice and homing of the cells to the brain assessed at day 24 by in vivo confocal microscopy of the mouse skull. After sacrifice, the number of GFP-positive tumors in the liver, kidney and lung was determined. Results: Suppression of the EP1 receptor had no effect on cell proliferation in vitro. In contrast, both migration and invasion of colon tumor cells was significantly reduced by EP1 receptor antagonism and following suppression of EP1 receptor expression. Moreover, suppression of the EP1 receptor in vivo blocked homing of the tumor cells to the brain. Tumor cells were detected in the skull of mice injected with HT29 Scr shRNA cells at day 24, but not in the skull of mice inoculated with HT29 EP1 shRNA cells. Metastasis of the tumor cells to other organs was also affected by suppression of the EP1 receptor. Conclusion: Suppression of the EP1 receptor reduces colon tumor cell migration and metastasis in vitro and in vivo. Unravelling the mechanisms involving the EP1 receptor in colon cancer metastasis may aid in the search for new therapeutic targets against this malignant disease. AGA Abstracts
Transcript of Sa1679 Effects of Urocortin 2 and Corticotropin-Releasing Hormone Receptor 2 in Colorectal Cancer...
Sa1678
A Colon Cancer-Derived Mutant of KrüPpel-Like Factor 5 Inhibits ItsDegradation by Glycogen Synthase Kinase 3β and the E3 Ubiquitin LigaseFBW7Agnieszka Bialkowska, Yang Liu, Mandayam O. Nandan, Vincent W. Yang
Background: Krüppel-like factor 5 (KLF5) is a zinc-finger transcription factor that is highlyexpressed in the crypt cells of the intestinal epithelia and plays a critical role in regulatingproliferation of both normal intestinal epithelial cells and colorectal cancer cells. We previ-ously showed that KLF5 is a critical mediator for intestinal tumorigenesis resulted fromeither ApcMin mutation or oncogenic KRAS activation (McConnell et al. [2009] Cancer Res.69:4125, Nandan et al. [2010] Molecular Cancer 9:63, respectively). KLF5 is regulated atthe transcriptional, translational, and post-translational level. Previous studies indicate thatthe protein kinase GSK3β and E3 ubiquitin ligase FBW7 facilitate proteosomal degradationof KLF5 protein via phosphorylation (GSK3β) and recognition (FBW7) of a phosphodegronsequence surrounding Ser303 in human KLF5. A recent genomic analysis of colorectal cancertissues identified a somatic mutation (P301S) in KLF5 within the phosphodegron sequence(Sjöblom et al. [2006] Science 314:268). Aim: To investigate the impact of P301S mutationon KLF5 protein stability. Methods: Plasmids containing human wildtype (WT) and mutant(S303A, P301S, P301A, and P301E) KLF5 under the control of the CMV promoter weretransiently transfected into HEK293 cells. The effect of FBW7 on the stability of KLF5produced from the various constructs was investigated by cycloheximide chase and Westernblot analysis. Transcriptional activity of various KLF5 constructs was examined by luciferasereporter assay with Cyclin D1 and CDC2 reporter plasmids. Results: All four KLF5 mutantsexamined are more resistant to FBW7-mediated proteosomal degradation as compared toWT KLF5. Proteasome inhibition using MG132 resulted in the accumulation of WT KLF5but had little impact on P301S KLF5 protein levels. Co-immunoprecipitation experimentsshow that, in contrast to WT KLF5, P301S and S303A KLF5 do not interact with FBW7.Concomitantly, the P301S KLF5 mutant displayed reduced levels of phosphorylation atSer303 in comparison with WT KLF5. Furthermore, luciferase assays using Cyclin D1 andCDC2 promoter constructs demonstrate significantly elevated transcriptional activity ofP301S KLF5 as compared to WT KLF5. Conclusion: Our data indicate that residue 301 ofKLF5 is critical for proper recognition of the phosphodegron sequence. Furthermore, weshow that the colon cancer-derived P301S mutation in KLF5 inhibits FBW7 recognition ofthe phosphodegron, yielding a degradation-resistant KLF5 protein, elevated protein levels,and enhanced KLF5 transcriptional activity. These results suggest that the P301S mutationin KLF5 is potentially oncogenic in colon cancer.
Sa1679
Effects of Urocortin 2 and Corticotropin-Releasing Hormone Receptor 2 inColorectal Cancer Growth and MetastasisJorge Rodriguez, Charalabos Pothoulakis, Stavroula Baritaki
Background and aims: Colorectal cancer (CRC) represents the third most common form ofcancer worldwide. Moreover, activation of inflammatory pathways participates in CRCgrowth. Corticotropin Releasing Hormone (CRH) family of peptides and their receptorsCRHR1 and CRHR2 are expressed at the CNS and the periphery, including the intestine.We have shown that the CRH peptide family represents an important component of theintestinal inflammatory response. To-date there are no studies implicating the CRH familyof peptides in CRC development and progression. Our goal was to identify a potential roleof the CRH family members in CRC pathophysiology and explore the underlying molecularmechanisms. Methods: mRNA levels of CRH family members were monitored by qRT-PCRin 46 and 56 normal colon and CRC human samples, and in CRC cell lines (HT-29, DLD1,SW403, SW480, SW620, HCT-116, and Caco2). All cell lines were lentivirally transducedfor CRHR2 expression (CRHR2+). Parental and CRHR2+ cells were tested for mRNA expres-sion of the CRHR2 specific ligand urocortin-2 (UCN2), as well as IL-6 and IL-6R usingqRT-PCR. Tumor progression was assessed by proliferation, invasion and migration assays.Single cell colony formation was assessed by soft agar assays. Expression of pSTAT3 andEMT markers was determined by western blot. Results: CRC biopsies and cell lines showedsignificantly downregulated CRHR2 and increased UCN2 mRNA expression, compared tocontrols (4-fold each). Four CRHR2+ CRC clones had significantly reduced endogenousUCN2 (2-10 fold) and pro-inflammatory cytokine mRNA levels including IL-6 (2-20 fold)and its receptor IL-6R (1.5-5 fold). These effects were more profound in the highly metastaticCRC cell lines SW620 and HCT-116 at baseline and after exogenous administration of UCN2.CRHR2+ clones with downregulated IL-6R levels showed significantly reduced pSTAT3 levels(2-10 fold) compared to parental cells, and associated with decreased cell proliferation,colony formation, invasion and migration at baseline and in response to IL-6. These responseswere reversed by the selective CRHR2 antagonist, astressin 2B, indicating that they wereCRHR2 specific. The EMT markers Snail and RKIP were also significantly dysregulated inCRHR2+ clones, suggesting EMT as a downstream target of CRHR2 signaling in CRC.Conclusions: This is the first demonstration supporting a protective role of CRHR2 inpreventing CRC growth rates and aggressiveness via inhibition of Stat3 signaling pathway.Furthermore, monitoring CRHR2 and Ucn2 levels in CRC tissues may benefit early diagnosisof CRC metastasis, and might serve as a novel drug target for prevention and treatment ofadvanced stages of CRC. Supported by NIH PO-1 DK33506 (DK), P50 DK064539 (CP)and T32 5T32HD007512-15 (JR) grants.
Sa1680
Identification of a Tight Junction/Focal Adhesion Signaling Complex: BVESand Bcar3Shenika Poindexter, Wei Ning, Bobak Parang, Vishruth K. Reddy, Richard Near, AdamLerner, Christopher Williams
Blood Vessel Epicardial Substance (BVES) -a tight junction associated protein- is silencedvia promoter hypermethylation in all stages of colon cancer. Interference with BVES functionin human corneal epithelial cells promotes increased migration, decreased trans-epithelial
S-281 AGA Abstracts
resistance, and reduced expression of epithelial markers including E-cadherin and Zo-1:characteristic for epithelial to mesenchymal transition (EMT). In contrast, expression ofBVES in mesenchymal-like (Lim 2405 and SW620) colorectal cancer cell lines results inmesenchymal to epithelial transition (MET). These data suggest that BVES loss in cancermay contribute to cancer progression via promotion of mesenchymal states. We have deter-mined that BVES regulates RhoA signaling, but incompletely understand howBVESmodulatesthese networks. Therefore, identifying BVES interacting proteins is imperative to mappingBVES regulated intracellular signaling pathways. Thus, we employed a yeast two-hybrid(Y2-H) screen with the intracellular domain of BVES (aa115-360) as bait to probe a placentallibrary. This screen yielded 67 interacting proteins. One of which was Breast Cancer Anti-estrogen Resistance 3 (BCAR3), a known GEF with oncogenic activity that has been shownto interact with RhoA. BCAR3 modulates signaling via complex formation with p130Cas, afocal adhesion protein.We hypothesized that BCAR3 regulates BVES dependent RhoA activityin colon cancer. We confirmed the interaction via directed Y2-H and co-IP. To determinethe functional significance of this interaction, we used complimentary approaches of overex-pression and knockdown of BCAR3 in BVES-expressing CACO2 and non-BVES- expressingSW620 cells and screened for changes in BVES-dependent phenotypes. Lentiviral mediatedtransduction of these lines with WT BCAR3 and the BCAR3/p130Cas uncoupling mutant,R743A, produced no morphological change in the CACO2 cells but promoted membraneruffling in SW620; in contrast, BCAR3 knockdown resulted in epithelial-like appearancesuggesting that BCAR3 is downstream of BVES. We next developed a BVES inducible SW620cell line that stably overexpresses BCAR3 and performed cell attachment assays. Followinginduction of BVES, there was a trend toward increased cell attachment to the culture dishat Day 11 as compared to non-induced SW620s (P=.076). Additionally, there was anincreased number of detached cells in non-BVES induced SW620 cells at Day 11(P=.02),suggesting that BVES may be important in regulating adhesion. We have identified BCAR3as a BVES interacting protein. BCAR3 appears to antagonize BVES by promoting EMT. Thisinteraction potentially reveals a unique tight junction to focal adhesion signaling pathwayimportant in regulating EMT. Clarifying the role of BVES and BCAR3 in EMT may lead totherapeutic strategies targeting this interaction in colon cancer.
Sa1681
Inhibition of the Prostaglandin EP1 Receptor Suppresses Colon Cancer CellInvasion and Metastasis.Grace P. O'Callaghan, Michaela Reagan, Ludmila M. Flores, Yong Zhang, Aldo Roccaro,Fergus Shanahan, Irene Ghobrial, Aileen Houston
Background: The role of cyclooxygenase-2 (COX-2) and its major metabolite prostaglandinE2 (PGE2) in colon carcinogenesis is well established. PGE2 signals through four differentreceptor subtypes, EP1 - EP4. Elucidation of the role of these receptors in colon tumorigenesismay identify novel targets for therapy, potentially circumventing the side-effects associatedwith COX inhibition, whilst retaining the anti-cancer properties. We have recently shownthat blocking EP1 receptor signalling in a subcutaneous model of colon tumorigenesisreduced tumor growth in vivo. However, whether the EP1 receptor plays a role in coloncancer metastasis is unknown. Aim: To investigate the role of the EP1 receptor in promotingmigration, invasion andmetastasis of colon cancer cells. Methods:Colon tumor cells (SW480,HT29 and CT26 cells) were treated with an EP1 receptor specific antagonist (ONO-8713),or were stably transfected (CT26 and HT29 cells) with short hairpin RNA (shRNA) targetedagainst the EP1 receptor (EP1 shRNA) or with scrambled control (Scr shRNA). Changes incell proliferation, migration and invasion were investigated in vitro. HT29 transfected cloneswere tagged with a Green Fluorescent Protein (GFP) label for subsequent in vivo studies. 3x 106 cells were injected into the tail vein of SCID-bg mice and homing of the cells to thebrain assessed at day 24 by in vivo confocal microscopy of the mouse skull. After sacrifice,the number of GFP-positive tumors in the liver, kidney and lung was determined. Results:Suppression of the EP1 receptor had no effect on cell proliferation in vitro. In contrast, bothmigration and invasion of colon tumor cells was significantly reduced by EP1 receptorantagonism and following suppression of EP1 receptor expression. Moreover, suppressionof the EP1 receptor in vivo blocked homing of the tumor cells to the brain. Tumor cellswere detected in the skull of mice injected with HT29Scr shRNA cells at day 24, but not inthe skull of mice inoculated with HT29EP1 shRNA cells. Metastasis of the tumor cells to otherorgans was also affected by suppression of the EP1 receptor. Conclusion: Suppression ofthe EP1 receptor reduces colon tumor cell migration and metastasis in vitro and in vivo.Unravelling the mechanisms involving the EP1 receptor in colon cancer metastasis may aidin the search for new therapeutic targets against this malignant disease.
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