Mo1624 Mechanical Induction of Epithelial to Mesenchymal Transition in Colon Cancer Cells
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AGA Abstracts Barretts cancers develop the same mutational spectrum as the originating dysplasia. However, recent work has found that different areas of oesophageal epithelium may in fact develop mutations independently resulting in a mutationally diverse environment(2), and that such diversity itself predisposes to cancer development(3). This raises the question of whether competition between cells of different clonal origin is a relevant mechanism causing progres- sion. In this context it is unclear how a Barretts lesion composed of multiple distinct clones develops into cancer. It may be that only a single clone forms the cancer, and originates from a polyclonal milieu. Aim: To investigate whether early Barretts adenocarcinomas share the same mutational spectrum as their surrounding dysplasia. Methods: Crypt by crypt laser capture microdissection was performed on 7 resected oesophageal adenocarcinomas with known somatic mutations and also on the associated surrounding high grade dysplasia. The dissected material underwent PCR sequencing for the mutation(s) identified in the specimen (TP53, CDKN2A or K-RAS). Results: In 6/7 patients the cancer contained the same somatic mutation throughout the entire cancer. The neighbouring dysplasia however displayed crypts containing a mutation, adjacent to wild-type crypts. In one patient 3 mutations were found in the tumour suppressor gene CDKN2A. The dysplasia contained a mutation in p.A148T and p.R128W as well as p.A148T alone and p.T137A alone. The cancer contained a mutation p.A148T at alone. Conclusion: Early Barretts adenocarcinoma arises as a clonal outgrowth from polyclonal dysplasia This favours competitive models of cancer progression within field cancerization. References 1. Galipeau PC, Prevo LJ, Sanchez CA, Longton GM, Reid BJ. Clonal expansion and loss of heterozygosity at chromosomes 9p and 17p in premalignant esophageal (Barretts) tissue. J Natl Cancer Inst. 1999 ;91(24):2087-2095. 2. Leedham SJ, Preston SL, McDonald SAC, Elia G, Bhandari P, Poller D, et al. Individual crypt genetic heterogeneity and the origin of metaplastic glandular epithelium in human Barretts oeso- phagus. Gut. 2008 ;57(8):1041-1048. 3. Maley CC, Galipeau PC, Finley JC, Wongsurawat VJ, Li X, Sanchez CA, et al. Genetic clonal diversity predicts progression to esophageal adenocarcinoma. Nat Genet. 2006 ;38(4):468-473. Mo1624 Mechanical Induction of Epithelial to Mesenchymal Transition in Colon Cancer Cells Sarah C. Glover, Shruthi Bharadwaj Introduction: Epithelial to Mesenchymal Transition (EMT) is characterized by cytoskeletal rearrangement to aid in the movement of cells through a 3D extra-cellular matrix (ECM). During EMT, the cellular movement is transformed from proteolysis dependent movement to an amoeboid movement, in which cells do not use proteases to traverse through ECM. EMT is also involved in the key steps of tumor metastasis and several other GI disorders, where the ECM is disrupted. Since ECM directly influences EMT, we hypothesize that the surface topography of ECM also plays a role in inducing EMT. Methods: In order to test our hypothesis, time-lapse imaging was performed using Nikon Biostation IM wherein SW620 cells were plated and observed over a period of 96 hours on micro-patterned scaffolds mimicking normal (pit) and tumorogenic (post) tissue. Standard molecular biology techniques were used to measure proliferation and G/F actin ratios. SW620 cell motility on micro-patterned substrate was assessed by evaluating the mean square displacement of cells over a period of 96 hours. SW620 cells were also stained with epithelial marker E-Cadherin and mesenchymal markers Snail and Vimentin to analyze EMT. Results: Cells plated on posts had a ‘rounder' morphology whereas cells plated on ‘pits' showed a more elongated morphology. A 2-fold decrease in SW620 cell proliferation was also noted when cells were plated on post micro-topography. G/F actin ratio for SW620 cells plated on post micro- topography was 2.5 fold higher than the ratio for cells plated on either planar or pit topographies. SW620 cells were immotile when plated on planar or pit topographies. However, when plated on posts, motility of SW620 cells increased by 11%. Confined diffusion of SW620 cells was up-regulated with a 10-fold increase in the diffusion coefficient when plated on post microtopography and a 5-fold increase when plated on pit microtopogra- phy. Up-regulation of Vimentin and Snail was also observed in cells plated on post micro- topography . Conclusion: The data suggests that tumorogenic micro-topography induces the transition from epithelial to mesenchymal state. The decreased cell proliferation caused by EMT may reduce the potency of chemotherapy thereby increasing cancer recurrence. Further studies, especially in an In-Vivo model, are required to fully understand the implica- tions of EMT in colon cancer and other GI disorders. Mo1625 Co-Targeting of the PI3K and RAS Pathways for the Treatment of Carcinoid Tumors Joseph Valentino, Jing Li, Ji Tae Kim, Jun Song, W. C. Mustain, Heidi L. Weiss, Lowell Anthony, Courtney M. Townsend, B. Mark Evers Introduction: Carcinoid tumors are frequently resistant to traditional chemotherapeutic agents, and their propensity to secrete bioactive products associated with carcinoid syndrome further complicates management. The PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways have been recognized as integral oncogenic pathways in a number of human malignancies; however, their role in carcinoid tumors is not well defined. It is known, however, that PI3K inhibition alone results in increased neurotensin (NT) secretion from carcinoid cells. The purpose of this study was to clarify the role these pathways play in carcinoid cell proliferation, apoptosis, and secretion as well as to determine the effects of combined inhibition on carcinoid tumors. Methods: BON (human pancreatic carcinoid) and UMC-11 and NCI- H727 (lung carcinoid) cells were initially treated with increasing doses of the PI3K inhibitors PIK-75, BKM120, or the dual PI3K/mTOR inhibitor, BEZ235, either alone or in combination with a MEK inhibitor. Cell proliferation was evaluated using a cell viability analyzer and apoptosis quantified using DNA-fragmentation ELISA. Secretion was measured by neuroten- sin EIA. Effects on phosphorylation of downstream target proteins (AKT, ERK, p70S6K, S6, and 4E-BP1) were determined by western blot. Results: Treatment with PI3K inhibitors alone resulted in a dose dependent decrease in cell proliferation and an increase in apoptosis, but increased phosphorylation of ERK through a negative feedback loop. When PI3K inhibi- tion was combined with the MEK inhibitor PD0325901, there was a significant decrease in cell viability (p <0.001-0.013) and increase in apoptosis (p < 0.001 - 0.005) both relative S-644 AGA Abstracts to control and to individual treatments. Additionally, the enhanced NT secretion noted with PIK-75 treatment was blocked by co-treatment with a MEK inhibitor. Western blot analysis demonstrated decreased phosphorylation of the downstream targets p70S6K, S6, and 4E- BP1 with increasing doses of PI3K inhibitors as well as with combination treatment. Conclu- sions: PI3K inhibition is a promising therapeutic option for the treatment of carcinoid tumors; however, this may cause increased secretion of bioactive products, potentially resulting in additional adverse effects in the clinical setting. Combined PI3K and MEK inhibition not only augments anti-neoplastic effects but also prevents increased peptide secretion. This combined approach may offer an optimal strategy for the management of carcinoid tumors. Mo1626 PI3K P110α/AKT Signaling Negatively Regulates Neurotensin Secretion Through Interference of α-Tubulin Acetylation Jing Li, Jun Song, Margaret G. Cassidy, Garretson Epperly, Heidi L. Weiss, Courtney M. Townsend, B. Mark Evers Neurotensin (NT), a gut peptide localized to enteroendocrine N cells, plays an important role in intestinal secretion, motility and gut growth. The class IA PI3K family, comprised of p110 catalytic (α, β and δ) and p85 regulatory (α and β) subunits, represents a critical signaling pathway for numerous cell functions including gene expression and secretion. We previously demonstrated that mTOR, an important downstream PI3K/Akt effector, negatively regulates NT gene expression and NT secretion. Surprisingly, p110α also negatively regulates NT secretion but not through the regulation of NT mRNA. The purpose of the present study was to determine the mechanisms underlying the effect of PI3K/Akt signaling on NT secretion. METHODS. BON and QGP-1 (both are pancreatic endocrine cell lines which produce and secrete NT peptide) were used. i) To determine whether p110α regulates NT granule trafficking, BON cells stably expressing GFP-NT were established; the accumulation of GFP- NT on the plasma membrane was monitored in live cells, in the presence or absence of PIK-75, by TIRF microscopy; ii) to determine the involvement of α-tubulin acetylation, which stabilizes microtubules (MTs) and speeds up vesicle transport, α-tubulin acetylation was observed by both immunofluorescence confocal microscopy and western blot using acetylated α-tubulin specific antibody. In addition, cells were treated with either taxol (an MT stabilization reagent) or nocodazole (an MT destabilization reagent) or transfected with active (K40Q) or inactive (K40R) α-tubulin plasmids and NT secretion measured; iii) to determine the involvement of HDAC6, a class IIb histone deacetylase that has been implicated in downregulation of α-tubulin acetylation and upregulation of Akt phosporylation, cells were transfected with HDAC6 and NT secretion measured. RESULTS. i) PIK-75 significantly increased GFP-NT granules docked on the plasma membrane, suggesting the regulation of p110α for NT granule trafficking; ii) α-tubulin acetylation was increased by PIK-75. Taxol treatment, but not nocodazole, increased α-tubulin acetylation and PIK-75-stimulated NT secretion. Overexpression of K40Q, but not K40R, led to a further increase of PIK-75- enhanced NT secretion; iii) consistently, HDAC6 overexpression decreased α-tubulin acetylation and NT secretion but increased Akt phosphorylation. CONCLUSIONS. p110α/ Akt signaling negatively regulates NT peptide release through the inhibition of α-tubulin acetylation. HDAC6, an α-tubulin deacetylase, negatively regulates α-tubulin acetylation and inhibits NT release enhanced by inhibition of p110α/Akt signaling. These findings show, for the first time, the importance of PI3K/Akt signaling and its interaction with MTs and HDAC6 in the regulation of granule trafficking and intestinal peptide release. Mo1627 Transcriptional Factor Etv1 Activates MAPK Signaling via Up-Regulation of PDGFRA Kohei Funasaka, Ryoji Miyahara, Kazuhiro Furukawa, Keiichi Sakamaki, Hidezumi Tatematsu, Issei Tsurudome, Fumiko Yamamoto, Ippei Matsuzaki, Eizaburo Ohno, Hiroki Kawashima, Akihiro Itoh, Naoki Ohmiya, Yoshiki Hirooka, Osamu Watanabe, Osamu Maeda, Takafumi Ando, Takeshi Senga, Hidemi Goto Background: Accumulating evidence has revealed that c-kit is crucial for the development of GISTs; however, active c-kit alone is not sufficient for GIST formation. Recent study has shown that ETV1, a transcriptional factor, co-operates with c-kit to promote cancer cell proliferation In Vitro and In Vivo. To gain further insight about the molecular pathogenesis of GIST, we examined changes in signal transduction and gene expression by c-kit and ETV1 expression. In addition, we performed gene expression analysis using clinical samples of GIST. Methods: To establish cells that constitutively expressed wild type c-kit, mutant c-kit (deletion of exon11 557-558) and ETV1, we infected recombinant retrovirus to human fibroblast cell line, KMST-6. We established 6 cell lines, including KIT-Wt, KIT-Mut, ETV1, KIT-Mut+ETV1, KIT-Wt+ETV, and control. mRNA level of c-kit, PDGFRA and ETV1 was examined by real-time PCR, and activation of signal molecules was determined by immunob- lot using phospho-specific antibodies. Forty six samples of GIST were obtained by surgery or EUS-FNA, mRNA level of c-kit, PDGFRA and ETV1 were analyzed. Result: Expression of neither wild type nor mutant c-kit affected expression of ETV1. Interestingly, ETV1 expression in the presence of mutant c-kit increased level of endogenous c-kit around two- folds. Expression of ETV1 in both KIT-Wt and KIT-Mut cells significantly induced expression of PDGFRA. Regarding changes in signal transduction, remarkable increase of endogenous c-kit phosphorylation and elevation of MAPK phosphorylation was observed in KIT-Mut cells. On the other hands, AKT and Stat was not activated in KIT-Mut cells. Expression of ETV1 in KIT-Wt cells induced phosphorylation of MAPK similar to the level of KIT-Mut cells. ETV1 induced expression of PDGFRA, which subsequently activated MAPK. Analysis of clinical samples showed that mRNA level of mutant c-kit was considerably higher than that of wild type c-kit. PDGRFRA mutation was observed in some clinical samples, but did not affect level of mRNA. Expression of ETV1 was correlated with expression of both c-kit and PDGFRA, but expression of c-kit and PDGFRA was not. Conclusion: Our experiments suggest that ETV1 induces expression of PDGFRA and activates MAPK signal pathways. The analysis using clinical samples showed the relationship between ETV1 and PDGFRA expression. These results show a novel mechanism of ETV1 for the development and progression of GISTs.
Transcript of Mo1624 Mechanical Induction of Epithelial to Mesenchymal Transition in Colon Cancer Cells
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sBarretts cancers develop the same mutational spectrum as the originating dysplasia. However,recent work has found that different areas of oesophageal epithelium may in fact developmutations independently resulting in a mutationally diverse environment(2), and that suchdiversity itself predisposes to cancer development(3). This raises the question of whethercompetition between cells of different clonal origin is a relevant mechanism causing progres-sion. In this context it is unclear how a Barretts lesion composed of multiple distinct clonesdevelops into cancer. It may be that only a single clone forms the cancer, and originatesfrom a polyclonal milieu. Aim: To investigate whether early Barretts adenocarcinomas sharethe same mutational spectrum as their surrounding dysplasia. Methods: Crypt by crypt lasercapture microdissection was performed on 7 resected oesophageal adenocarcinomas withknown somatic mutations and also on the associated surrounding high grade dysplasia. Thedissected material underwent PCR sequencing for the mutation(s) identified in the specimen(TP53, CDKN2A or K-RAS). Results: In 6/7 patients the cancer contained the same somaticmutation throughout the entire cancer. The neighbouring dysplasia however displayed cryptscontaining a mutation, adjacent to wild-type crypts. In one patient 3 mutations were foundin the tumour suppressor gene CDKN2A. The dysplasia contained a mutation in p.A148Tand p.R128W as well as p.A148T alone and p.T137A alone. The cancer contained a mutationp.A148T at alone. Conclusion: Early Barretts adenocarcinoma arises as a clonal outgrowthfrom polyclonal dysplasia This favours competitive models of cancer progression withinfield cancerization. References 1. Galipeau PC, Prevo LJ, Sanchez CA, Longton GM, ReidBJ. Clonal expansion and loss of heterozygosity at chromosomes 9p and 17p in premalignantesophageal (Barretts) tissue. J Natl Cancer Inst. 1999 ;91(24):2087-2095. 2. Leedham SJ,Preston SL, McDonald SAC, Elia G, Bhandari P, Poller D, et al. Individual crypt geneticheterogeneity and the origin of metaplastic glandular epithelium in human Barretts oeso-phagus. Gut. 2008 ;57(8):1041-1048. 3. Maley CC, Galipeau PC, Finley JC, WongsurawatVJ, Li X, Sanchez CA, et al. Genetic clonal diversity predicts progression to esophagealadenocarcinoma. Nat Genet. 2006 ;38(4):468-473.
Mo1624
Mechanical Induction of Epithelial to Mesenchymal Transition in ColonCancer CellsSarah C. Glover, Shruthi Bharadwaj
Introduction: Epithelial to Mesenchymal Transition (EMT) is characterized by cytoskeletalrearrangement to aid in the movement of cells through a 3D extra-cellular matrix (ECM).During EMT, the cellular movement is transformed from proteolysis dependent movementto an amoeboid movement, in which cells do not use proteases to traverse through ECM.EMT is also involved in the key steps of tumor metastasis and several other GI disorders,where the ECM is disrupted. Since ECM directly influences EMT, we hypothesize that thesurface topography of ECM also plays a role in inducing EMT. Methods: In order to testour hypothesis, time-lapse imaging was performed using Nikon Biostation IM whereinSW620 cells were plated and observed over a period of 96 hours on micro-patternedscaffolds mimicking normal (pit) and tumorogenic (post) tissue. Standard molecular biologytechniques were used to measure proliferation and G/F actin ratios. SW620 cell motility onmicro-patterned substrate was assessed by evaluating the mean square displacement of cellsover a period of 96 hours. SW620 cells were also stained with epithelial marker E-Cadherinand mesenchymal markers Snail and Vimentin to analyze EMT. Results: Cells plated onposts had a ‘rounder' morphology whereas cells plated on ‘pits' showed a more elongatedmorphology. A 2-fold decrease in SW620 cell proliferation was also noted when cells wereplated on post micro-topography. G/F actin ratio for SW620 cells plated on post micro-topography was 2.5 fold higher than the ratio for cells plated on either planar or pittopographies. SW620 cells were immotile when plated on planar or pit topographies.However, when plated on posts, motility of SW620 cells increased by 11%. Confineddiffusion of SW620 cells was up-regulated with a 10-fold increase in the diffusion coefficientwhen plated on post microtopography and a 5-fold increase when plated on pit microtopogra-phy. Up-regulation of Vimentin and Snail was also observed in cells plated on post micro-topography . Conclusion: The data suggests that tumorogenic micro-topography inducesthe transition from epithelial to mesenchymal state. The decreased cell proliferation causedby EMT may reduce the potency of chemotherapy thereby increasing cancer recurrence.Further studies, especially in an In-Vivo model, are required to fully understand the implica-tions of EMT in colon cancer and other GI disorders.
Mo1625
Co-Targeting of the PI3K and RAS Pathways for the Treatment of CarcinoidTumorsJoseph Valentino, Jing Li, Ji Tae Kim, Jun Song, W. C. Mustain, Heidi L. Weiss, LowellAnthony, Courtney M. Townsend, B. Mark Evers
Introduction: Carcinoid tumors are frequently resistant to traditional chemotherapeuticagents, and their propensity to secrete bioactive products associated with carcinoid syndromefurther complicates management. The PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathwayshave been recognized as integral oncogenic pathways in a number of human malignancies;however, their role in carcinoid tumors is not well defined. It is known, however, that PI3Kinhibition alone results in increased neurotensin (NT) secretion from carcinoid cells. Thepurpose of this study was to clarify the role these pathways play in carcinoid cell proliferation,apoptosis, and secretion as well as to determine the effects of combined inhibition oncarcinoid tumors. Methods: BON (human pancreatic carcinoid) and UMC-11 and NCI-H727 (lung carcinoid) cells were initially treated with increasing doses of the PI3K inhibitorsPIK-75, BKM120, or the dual PI3K/mTOR inhibitor, BEZ235, either alone or in combinationwith a MEK inhibitor. Cell proliferation was evaluated using a cell viability analyzer andapoptosis quantified using DNA-fragmentation ELISA. Secretion was measured by neuroten-sin EIA. Effects on phosphorylation of downstream target proteins (AKT, ERK, p70S6K, S6,and 4E-BP1) were determined by western blot. Results: Treatment with PI3K inhibitorsalone resulted in a dose dependent decrease in cell proliferation and an increase in apoptosis,but increased phosphorylation of ERK through a negative feedback loop. When PI3K inhibi-tion was combined with the MEK inhibitor PD0325901, there was a significant decrease incell viability (p <0.001-0.013) and increase in apoptosis (p < 0.001 - 0.005) both relative
S-644AGA Abstracts
to control and to individual treatments. Additionally, the enhanced NT secretion noted withPIK-75 treatment was blocked by co-treatment with a MEK inhibitor. Western blot analysisdemonstrated decreased phosphorylation of the downstream targets p70S6K, S6, and 4E-BP1 with increasing doses of PI3K inhibitors as well as with combination treatment. Conclu-sions: PI3K inhibition is a promising therapeutic option for the treatment of carcinoidtumors; however, this may cause increased secretion of bioactive products, potentiallyresulting in additional adverse effects in the clinical setting. Combined PI3K and MEKinhibition not only augments anti-neoplastic effects but also prevents increased peptidesecretion. This combined approach may offer an optimal strategy for the management ofcarcinoid tumors.
Mo1626
PI3K P110α/AKT Signaling Negatively Regulates Neurotensin SecretionThrough Interference of α-Tubulin AcetylationJing Li, Jun Song, Margaret G. Cassidy, Garretson Epperly, Heidi L. Weiss, Courtney M.Townsend, B. Mark Evers
Neurotensin (NT), a gut peptide localized to enteroendocrine N cells, plays an importantrole in intestinal secretion, motility and gut growth. The class IA PI3K family, comprisedof p110 catalytic (α, β and δ) and p85 regulatory (α and β) subunits, represents a criticalsignaling pathway for numerous cell functions including gene expression and secretion. Wepreviously demonstrated that mTOR, an important downstream PI3K/Akt effector, negativelyregulates NT gene expression and NT secretion. Surprisingly, p110α also negatively regulatesNT secretion but not through the regulation of NT mRNA. The purpose of the present studywas to determine themechanisms underlying the effect of PI3K/Akt signaling on NT secretion.METHODS. BON and QGP-1 (both are pancreatic endocrine cell lines which produce andsecrete NT peptide) were used. i) To determine whether p110α regulates NT granuletrafficking, BON cells stably expressing GFP-NT were established; the accumulation of GFP-NT on the plasma membrane was monitored in live cells, in the presence or absence ofPIK-75, by TIRF microscopy; ii) to determine the involvement of α-tubulin acetylation,which stabilizes microtubules (MTs) and speeds up vesicle transport, α-tubulin acetylationwas observed by both immunofluorescence confocal microscopy and western blot usingacetylated α-tubulin specific antibody. In addition, cells were treated with either taxol (anMT stabilization reagent) or nocodazole (an MT destabilization reagent) or transfected withactive (K40Q) or inactive (K40R) α-tubulin plasmids and NT secretion measured; iii) todetermine the involvement of HDAC6, a class IIb histone deacetylase that has been implicatedin downregulation of α-tubulin acetylation and upregulation of Akt phosporylation, cellswere transfected with HDAC6 and NT secretion measured. RESULTS. i) PIK-75 significantlyincreased GFP-NT granules docked on the plasma membrane, suggesting the regulation ofp110α for NT granule trafficking; ii) α-tubulin acetylation was increased by PIK-75. Taxoltreatment, but not nocodazole, increased α-tubulin acetylation and PIK-75-stimulated NTsecretion. Overexpression of K40Q, but not K40R, led to a further increase of PIK-75-enhanced NT secretion; iii) consistently, HDAC6 overexpression decreased α-tubulinacetylation and NT secretion but increased Akt phosphorylation. CONCLUSIONS. p110α/Akt signaling negatively regulates NT peptide release through the inhibition of α-tubulinacetylation. HDAC6, an α-tubulin deacetylase, negatively regulates α-tubulin acetylationand inhibits NT release enhanced by inhibition of p110α/Akt signaling. These findingsshow, for the first time, the importance of PI3K/Akt signaling and its interaction with MTsand HDAC6 in the regulation of granule trafficking and intestinal peptide release.
Mo1627
Transcriptional Factor Etv1 Activates MAPK Signaling via Up-Regulation ofPDGFRAKohei Funasaka, Ryoji Miyahara, Kazuhiro Furukawa, Keiichi Sakamaki, HidezumiTatematsu, Issei Tsurudome, Fumiko Yamamoto, Ippei Matsuzaki, Eizaburo Ohno, HirokiKawashima, Akihiro Itoh, Naoki Ohmiya, Yoshiki Hirooka, Osamu Watanabe, OsamuMaeda, Takafumi Ando, Takeshi Senga, Hidemi Goto
Background: Accumulating evidence has revealed that c-kit is crucial for the developmentof GISTs; however, active c-kit alone is not sufficient for GIST formation. Recent study hasshown that ETV1, a transcriptional factor, co-operates with c-kit to promote cancer cellproliferation In Vitro and In Vivo. To gain further insight about the molecular pathogenesisof GIST, we examined changes in signal transduction and gene expression by c-kit andETV1 expression. In addition, we performed gene expression analysis using clinical samplesof GIST. Methods: To establish cells that constitutively expressed wild type c-kit, mutantc-kit (deletion of exon11 557-558) and ETV1, we infected recombinant retrovirus to humanfibroblast cell line, KMST-6. We established 6 cell lines, including KIT-Wt, KIT-Mut, ETV1,KIT-Mut+ETV1, KIT-Wt+ETV, and control. mRNA level of c-kit, PDGFRA and ETV1 wasexamined by real-time PCR, and activation of signal molecules was determined by immunob-lot using phospho-specific antibodies. Forty six samples of GIST were obtained by surgeryor EUS-FNA, mRNA level of c-kit, PDGFRA and ETV1 were analyzed. Result: Expressionof neither wild type nor mutant c-kit affected expression of ETV1. Interestingly, ETV1expression in the presence of mutant c-kit increased level of endogenous c-kit around two-folds. Expression of ETV1 in both KIT-Wt and KIT-Mut cells significantly induced expressionof PDGFRA. Regarding changes in signal transduction, remarkable increase of endogenousc-kit phosphorylation and elevation of MAPK phosphorylation was observed in KIT-Mutcells. On the other hands, AKT and Stat was not activated in KIT-Mut cells. Expression ofETV1 in KIT-Wt cells induced phosphorylation of MAPK similar to the level of KIT-Mutcells. ETV1 induced expression of PDGFRA, which subsequently activated MAPK. Analysisof clinical samples showed that mRNA level of mutant c-kit was considerably higher thanthat of wild type c-kit. PDGRFRA mutation was observed in some clinical samples, but didnot affect level of mRNA. Expression of ETV1 was correlated with expression of both c-kitand PDGFRA, but expression of c-kit and PDGFRA was not. Conclusion: Our experimentssuggest that ETV1 induces expression of PDGFRA and activates MAPK signal pathways.The analysis using clinical samples showed the relationship between ETV1 and PDGFRAexpression. These results show a novel mechanism of ETV1 for the development andprogression of GISTs.
