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P-081 Gpcr5a-knockout mouse: A novel model for spontaneous and tobacco carcinogen-enhanced lung adenocarcinoma
Posters/Basic science/Ceil and rnotecular biology ~P~
Validation of genes essoclafod with Achaets-scuts Homologue-1 that regulate the growth of pulmonary neuroendocrlne carcinomas In mice and men
I. Linnoila ~. H. KJmI . X. Wang I . E. Dakir I . F. DeMayo2. ~Nat/onal Cancer lnst]tute, N/H, Rock~lle, Maryland, USA, 2Baylor Collage or Medicine, Houston, Texas, USA Achaete-scute homologue-1 (ASH1). a proneural basic helix-loop-helix tTanschption factor, is essential for neuroendocdne (NE) cifferenfiafion in normal and neoplasfic lung Human ASH1 (hASH1) is highly e=pressed in lung NE ca~nomas such as small cell lung cancer (SCLC) and non SCLC with NE features (NSCLCNE). We have generated a bib'ansgenic mouse model Ibr NSCLCNE where oonstitutrve hASH1 express~on in lung adenocarc|nomas of CC10 SV40TAg leads to focal NE diff~rentiafion and enhanced tumor growth (Cancer Research 2000:60:4005-9). In the current study we wanted to identify c|fferenfially espressed genes asseclated with ASH1 which might be implicated in the aggressive growth of lung NE carcinomas resulting in shorter survival The median survival of CC10-SV40TAg mica was 130 days (n 33). whereas it was 75 days ~ r t h e bit]"ansgenic TAgfhASH1 mica (n 34) We used oligonucleofide microarray analysis to compare the gene profiles of turnor-conteining lungs obtained from 10 CC10-SV40TAg t]"ansgenic and ,5 TA~'hh,SH1 bit]"ansgenic mica We idenfified 393 genes whose e0¢pression was stalJstically cifferent Co < 0 0,5) and possibly related to hASH1 Changes of more than 2-fold were associated with ,57 genes We chose 14 candidate genes. which consisted of 3 genes whose expression was up~'egulated and 11 genes whose expression was down regulated in TAg/hASH1 mice. Their espression was verified by real~ime RT PCR and Western blot in mouse tumors and well charactertzed human lung cancer ~ell lines. In order to establish a functional link. hASH1 gene was knocked down by siRNAs in two human lung NE carcinoma cell lines (DMS,53 and NCI 727) that espress high levels of hASH1. Real~ime RTPCR and westem blot analysis revealed that about 50-80% of endogenous hASH1 was knocked down. which resulted in decreased cell proliferation and increased apoptosis in both cell lines In NCIJ,-1727calls this was associated with up-regulation of several of the candidate genes identified in the murine system In addition, we transfected hASH1 into three non-NE lung cancer call lines Interesfingly. this lead to decreased colony-formation and increased call migration Forced. stable expression of hASH1 in the NSCLC NCI-H441 cells resulted in marked down-regulafion of one of the candidate genes usually absent or low in SCLC cells. In summary, we have shown that consfitut]ve espression of hASH1 greatly enhances grew~ of lung NE carc|nomas, and downmgulat]on of hASH1 inhibits the growth of NE carcinoma cell lines. Using the murlne model of NSCLC NE we have detected and validated a number of different]ally espressed, hASH1 assec~ated genes which may impact various characterlst]os of neoplasfic growth. We conclude that through these genes hASH1 may influence a range of preperfies that are dependent on the cell type
~-o~T Prognostic exprseslon of a novel adaptor protsln XB130 In non-small cell lung cancer M Loby¢la ~.C Xhi ~.M Anraku ~.N Liu 3 . M Tsao 3'4.M Liu 1'2. S. KeshavJee ~'2. l Umverslty Health Network Toronto General Hospital,
Toronto, Ontario, Canada, 2Department of Surge~ University of Toronto, Toronto, Ontario, Canada: 3 Untverslty Health Network~Ontano Cancer lnst]tutelPdncess Margaret Hospital, Toronto, Ontario, Canada, ~Department ot LaDoratory Medicine and Pathot~etagy, Toronto, Ontario, Canada Purpose: Prognosis generafing measures for lung cancer such as hlstopathol ogy have proven to be disoppoint]ng predictors of disease progression and clinical outcome. It is hoped that the idenfifK:afion of new lung tumor biomarkers might enhance the effectiveness of current methods. XB130 is a novel adaptor protein located on chromosome 10q25.3 that has been idenfified in our laboratory. Our previous work has asseolated overespresslon of XB130 with decreased call growth These findings suggest that XB130 may play an important role in tumorigenesis Experimental Design: We analyzed X~130 mRNA ~pression from paraffinembedded samples obtained from biopsies and bronchoscopy by Northern as well as by QRT~CR (real-time quantitative reverse transchption polymerase chain reaction) All statJsfical analyses were per~rmed using SAS v9 1 Wilcoxon rank-sum test was used ~r compadsen of two independent sample groups. Cos preport]onal hazards regression was used for unvarlate and multivariate analyses. The relationship between X~130 and olinical parameters was tested by ;(2 test Five-year disaase-free survival was used to define the survival funo~ on Results: In our preliminary analysis we compared relative ,~3~130 mRNA e0¢pression levels in 12 adenocarcinema (ADC) and 8 squamoos cell carcinoma (SQCC) versus corresponcing benign lung parenohymal fissue, we found a significant downregulat]on of the XB130 gene in ADC (p= 00062) but not in SQCC (p = 0.389). To confirm our results on a larger scale, we further analyzed the express~on of XB130 in 130 lung cancer cases, which included 86 ADC samples as well as 40 SQCC. The overall expression was slgnificahtly lower in ADC versus SQCC. (P = 0.006). In addition. 18 normal specimens were used
$135
to define the normal XB130 expression level (mear'r~-1.645*SD) and thus split the cohort into low. normal, and high expression subgroups The ciseasa-free survival at ,5 years of high XB130 mF~IA level group was significantly better than these v~th low XB130 mRNA levels (x 2 test p= 0.048). A multrvarlate analysis revealed that high XB130 mRNA levels independently contnbuted to c|seas~freo survival. Conclusion: Based on our study ~ 1 3 0 expression could be a helpful marker in disfinguishing ADC from SQCC. Overall. XB130 espression profle may serve as a precictor of long-term survival of pafients with non-small cell lung cancer Further work is required to determine the clinical utility of XB130 in select]on of treatment and prognosis in lung cancer in humans
] P - ~ I ] GpcrSa-knockout mouse: A novel model for spontaneous and tobacco carclnogen-enhenced lung adenocarclnoma R Lotan T Men. J Fujimoto. Q Tao The Univsrsltyof Texas, M R Anderson
Cancer Center, Houston, Texas, USA Background: Retinoids (e.g.. all trans rebnoic acid. ATRA). play important physiological roles in lung embryonal development, maintenance of muco ciliary epithelial differenbation, and possibly also as endogenous inhibitor's of lung caranogenosis. Refinc~ds possess both chernoprevent]ve and therapeutic effects in animal models of lung carcinogenesis These ef~cts are mediated by acfivafion of nuclear retinoid receptors, which ere ligand
[ P ~ 2 ] Evaluation by ImmunolllstochemlslTy of EGF-R expression before and after chemotherapy In non small cell lung cancer P Lotheire ~ B Mar'dn2. A Meert2. V Ninane 3. J Soulier 2 ltns~tut Jules
Border, Department of surgical onco/ogy, Brussels, Belgium, 2lnstitut Jules Border, Department ot internal Medtcme, Brussels, Belgtum, 3 CHU St ,~erm, /~eumotagy, Brussels, Belgium Study purpose: In order to evaluate the Epidermal Growth Factor Receptor (EGF~q) expression in relafion with chemotherapy in non-small cell lung cancer (NSCLC). we analysed retrospectively pathology specimens obtained before and after chemotherapy near the same pafients. PaUsnts end methods: Specimens were obtained from 32 pafients between 1989 and 2004; in 19 cases, comparison of E G F R espress~on was performed between 2 biopsies (before and after chemotherapy) and in 13 cases. oompansen was performed on biopsy obtained before chemotherapy and on
Validation of genes essoclafod with Achaets-scuts Homologue-1 that regulate the growth of pulmonary neuroendocrlne carcinomas In mice and men
I. Linnoila ~. H. KJmI . X. Wang I . E. Dakir I . F. DeMayo2. ~Nat/onal Cancer lnst]tute, N/H, Rock~lle, Maryland, USA, 2Baylor Collage or Medicine, Houston, Texas, USA Achaete-scute homologue-1 (ASH1). a proneural basic helix-loop-helix tTanschption factor, is essential for neuroendocdne (NE) cifferenfiafion in normal and neoplasfic lung Human ASH1 (hASH1) is highly e=pressed in lung NE ca~nomas such as small cell lung cancer (SCLC) and non SCLC with NE features (NSCLCNE). We have generated a bib'ansgenic mouse model Ibr NSCLCNE where oonstitutrve hASH1 express~on in lung adenocarc|nomas of CC10 SV40TAg leads to focal NE diff~rentiafion and enhanced tumor growth (Cancer Research 2000:60:4005-9). In the current study we wanted to identify c|fferenfially espressed genes asseclated with ASH1 which might be implicated in the aggressive growth of lung NE carcinomas resulting in shorter survival The median survival of CC10-SV40TAg mica was 130 days (n 33). whereas it was 75 days ~ r t h e bit]"ansgenic TAgfhASH1 mica (n 34) We used oligonucleofide microarray analysis to compare the gene profiles of turnor-conteining lungs obtained from 10 CC10-SV40TAg t]"ansgenic and ,5 TA~'hh,SH1 bit]"ansgenic mica We idenfified 393 genes whose e0¢pression was stalJstically cifferent Co < 0 0,5) and possibly related to hASH1 Changes of more than 2-fold were associated with ,57 genes We chose 14 candidate genes. which consisted of 3 genes whose expression was up~'egulated and 11 genes whose expression was down regulated in TAg/hASH1 mice. Their espression was verified by real~ime RT PCR and Western blot in mouse tumors and well charactertzed human lung cancer ~ell lines. In order to establish a functional link. hASH1 gene was knocked down by siRNAs in two human lung NE carcinoma cell lines (DMS,53 and NCI 727) that espress high levels of hASH1. Real~ime RTPCR and westem blot analysis revealed that about 50-80% of endogenous hASH1 was knocked down. which resulted in decreased cell proliferation and increased apoptosis in both cell lines In NCIJ,-1727calls this was associated with up-regulation of several of the candidate genes identified in the murine system In addition, we transfected hASH1 into three non-NE lung cancer call lines Interesfingly. this lead to decreased colony-formation and increased call migration Forced. stable expression of hASH1 in the NSCLC NCI-H441 cells resulted in marked down-regulafion of one of the candidate genes usually absent or low in SCLC cells. In summary, we have shown that consfitut]ve espression of hASH1 greatly enhances grew~ of lung NE carc|nomas, and downmgulat]on of hASH1 inhibits the growth of NE carcinoma cell lines. Using the murlne model of NSCLC NE we have detected and validated a number of different]ally espressed, hASH1 assec~ated genes which may impact various characterlst]os of neoplasfic growth. We conclude that through these genes hASH1 may influence a range of preperfies that are dependent on the cell type
~-o~T Prognostic exprseslon of a novel adaptor protsln XB130 In non-small cell lung cancer M Loby¢la ~.C Xhi ~.M Anraku ~.N Liu 3 . M Tsao 3'4.M Liu 1'2. S. KeshavJee ~'2. l Umverslty Health Network Toronto General Hospital,
Toronto, Ontario, Canada, 2Department of Surge~ University of Toronto, Toronto, Ontario, Canada: 3 Untverslty Health Network~Ontano Cancer lnst]tutelPdncess Margaret Hospital, Toronto, Ontario, Canada, ~Department ot LaDoratory Medicine and Pathot~etagy, Toronto, Ontario, Canada Purpose: Prognosis generafing measures for lung cancer such as hlstopathol ogy have proven to be disoppoint]ng predictors of disease progression and clinical outcome. It is hoped that the idenfifK:afion of new lung tumor biomarkers might enhance the effectiveness of current methods. XB130 is a novel adaptor protein located on chromosome 10q25.3 that has been idenfified in our laboratory. Our previous work has asseolated overespresslon of XB130 with decreased call growth These findings suggest that XB130 may play an important role in tumorigenesis Experimental Design: We analyzed X~130 mRNA ~pression from paraffinembedded samples obtained from biopsies and bronchoscopy by Northern as well as by QRT~CR (real-time quantitative reverse transchption polymerase chain reaction) All statJsfical analyses were per~rmed using SAS v9 1 Wilcoxon rank-sum test was used ~r compadsen of two independent sample groups. Cos preport]onal hazards regression was used for unvarlate and multivariate analyses. The relationship between X~130 and olinical parameters was tested by ;(2 test Five-year disaase-free survival was used to define the survival funo~ on Results: In our preliminary analysis we compared relative ,~3~130 mRNA e0¢pression levels in 12 adenocarcinema (ADC) and 8 squamoos cell carcinoma (SQCC) versus corresponcing benign lung parenohymal fissue, we found a significant downregulat]on of the XB130 gene in ADC (p= 00062) but not in SQCC (p = 0.389). To confirm our results on a larger scale, we further analyzed the express~on of XB130 in 130 lung cancer cases, which included 86 ADC samples as well as 40 SQCC. The overall expression was slgnificahtly lower in ADC versus SQCC. (P = 0.006). In addition. 18 normal specimens were used
$135
to define the normal XB130 expression level (mear'r~-1.645*SD) and thus split the cohort into low. normal, and high expression subgroups The ciseasa-free survival at ,5 years of high XB130 mF~IA level group was significantly better than these v~th low XB130 mRNA levels (x 2 test p= 0.048). A multrvarlate analysis revealed that high XB130 mRNA levels independently contnbuted to c|seas~freo survival. Conclusion: Based on our study ~ 1 3 0 expression could be a helpful marker in disfinguishing ADC from SQCC. Overall. XB130 espression profle may serve as a precictor of long-term survival of pafients with non-small cell lung cancer Further work is required to determine the clinical utility of XB130 in select]on of treatment and prognosis in lung cancer in humans
] P - ~ I ] GpcrSa-knockout mouse: A novel model for spontaneous and tobacco carclnogen-enhenced lung adenocarclnoma R Lotan T Men. J Fujimoto. Q Tao The Univsrsltyof Texas, M R Anderson
Cancer Center, Houston, Texas, USA Background: Retinoids (e.g.. all trans rebnoic acid. ATRA). play important physiological roles in lung embryonal development, maintenance of muco ciliary epithelial differenbation, and possibly also as endogenous inhibitor's of lung caranogenosis. Refinc~ds possess both chernoprevent]ve and therapeutic effects in animal models of lung carcinogenesis These ef~cts are mediated by acfivafion of nuclear retinoid receptors, which ere ligand
[ P ~ 2 ] Evaluation by ImmunolllstochemlslTy of EGF-R expression before and after chemotherapy In non small cell lung cancer P Lotheire ~ B Mar'dn2. A Meert2. V Ninane 3. J Soulier 2 ltns~tut Jules
Border, Department of surgical onco/ogy, Brussels, Belgium, 2lnstitut Jules Border, Department ot internal Medtcme, Brussels, Belgtum, 3 CHU St ,~erm, /~eumotagy, Brussels, Belgium Study purpose: In order to evaluate the Epidermal Growth Factor Receptor (EGF~q) expression in relafion with chemotherapy in non-small cell lung cancer (NSCLC). we analysed retrospectively pathology specimens obtained before and after chemotherapy near the same pafients. PaUsnts end methods: Specimens were obtained from 32 pafients between 1989 and 2004; in 19 cases, comparison of E G F R espress~on was performed between 2 biopsies (before and after chemotherapy) and in 13 cases. oompansen was performed on biopsy obtained before chemotherapy and on