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Avaliação da expressão gênica de FOXE1 no câncer de tireoide
Filipe F. dos Santos¹; Erika Urbano de Lima²; Ileana GS Rubió¹.
¹Departamento de Ciências Biológicas. ²Pós-graduação de Biologia estrutural e funcional, UNIFESP.
INTRODUÇÃO
• 1.658.370 novos casos de câncer de tireoide (TC) e 589.430
mortes nos EUA em 2015 (Siegel et al., 2015);
• 9.200 novos casos de TC no Brasil em 2015 (INCA, 2014-2015);
•Taxa de incidência cresce cerca de 1% ao ano (INCA, 2014-2015);
•Cerca de 90% dos doentes apresentam baixo risco de morte
(Schlumberger, 1998);
•Prevalência em mulheres (3:1) e adultos (DeLellis et al., 2004).
RNA Proteína
Localização gene FOXE1
OBJETIVOS METODOLOGIA
JUSTIFICATIVA
Avaliar o perfil de expressão
gênica do RNA mensageiro
(mRNA) de FOXE1 em amostras
de tecidos tumorais e não
tumorais pareadas da glândula
tireoide.
1. Importante na formação da
glândula tireoide;
2. Já foi relacionado com outros
tumores, mas poucos
trabalhos em TC;
3. Sem consenso quanto ao seu
perfil de expressão;
4. É necessário compreender o
perfil de expressão de FOXE1
em TC para entender seu real
envolvimento nessa
patologia.
Amostras
• 10 amostras pareadas
(Tumor/Não Tumor) de
pacientes brasileiros
• Maioria PTC
• Mais 20 pares
Extração
• RNA total de tecido
• Invitrogen, Life
Technologies, Carlsbad,
CA
Quantificação
• Concentração
• Qualidade (260/280 ~
1,80-2,00)
cDNA
• Tratamento com DNAse
• Síntese de cDNA de FOXE1
(Super Script III, Random
Primers e Oligo (dT)16)
RT-PCR
• Estojo comercial SYBR
Green Master MIX (SYBR
Green I Dye)
• Gene endógeno S8
• Quantificação por ΔΔCT
Análise dos
dados
• Software
GraphPad Prism 5
• Dados dos tumores dos
pacientes
RESULTADOS/DISCUSSÃO
BIBLIOGRAFIA
1. DeLellis RA, Lloyd, R.V., Heitz, P.U., Eng, C. (2004) World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Endocrine Organs. Lyon, França: IARC Press.
2. INSTITUTO NACIONAL DO CÂNCER (INCA/MS) PRÓ-ONCO. Estimativas 2014, Disponível: http://www.inca.gov.br/estimativa/2014/sintese-de-resultados-comentarios.asp. (capturado em 31/05/2015).
3. Schlumberger MJ. Papillary and follicular thyroid carcinoma. The New England journal of medicine. 1998 Jan 29;338(5):297-306. PubMed PMID: 9445411.
4. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA: a cancer journal for clinicians. 2015 Jan-Feb;65(1):5-29. PubMed PMID: 25559415.
5. Abu-Khudir R, Magne F, Chanoine JP, Deal C, Van Vliet G, Deladoey J. Role for tissue-dependent methylation differences in the expression of FOXE1 in nontumoral thyroid glands. The Journal of clinical endocrinology and metabolism. 2014 Jun;99(6):E1120-9. PubMed PMID: 24646064.
6. Bell A, Bell D, Weber RS, El-Naggar AK. CpG island methylation profiling in human salivary gland adenoid cystic carcinoma. Cancer. 2011 Jul 1;117(13):2898-909. PubMed PMID: 21692051. Pubmed Central PMCID: 3123690.
7. Brancaccio A, Minichiello A, Grachtchouk M, Antonini D, Sheng H, Parlato R, et al. Requirement of the forkhead gene Foxe1, a target of sonic hedgehog signaling, in hair follicle morphogenesis. Human molecular genetics. 2004 Nov 1;13(21):2595-606. PubMed PMID: 15367491.
8. Bullock M, Duncan EL, O'Neill C, Tacon L, Sywak M, Sidhu S, et al. Association of FOXE1 polyalanine repeat region with papillary thyroid cancer. The Journal of clinical endocrinology and metabolism. 2012 Sep;97(9):E1814-9. PubMed PMID: 22736773.
9. Bychkov A, Saenko V, Nakashima M, Mitsutake N, Rogounovitch T, Nikitski A, et al. Patterns of FOXE1 expression in papillary thyroid carcinoma by immunohistochemistry. Thyroid : official journal of the American Thyroid Association. 2013 Jul;23(7):817-28. PubMed PMID: 23327367. Pubmed Central PMCID: 3704107.
10. Eichberger T, Regl G, Ikram MS, Neill GW, Philpott MP, Aberger F, et al. FOXE1, a new transcriptional target of GLI2 is expressed in human epidermis and basal cell carcinoma. The Journal of investigative dermatology. 2004 May;122(5):1180-7. PubMed PMID: 15140221.
11. Fan Y, Ding Z, Yang Z, Deng X, Kang J, Wu B, et al. Expression and clinical significance of FOXE1 in papillary thyroid carcinoma. Molecular medicine reports. 2013 Jul;8(1):123-7. PubMed PMID: 23715628.
12. Fernandez LP, Lopez-Marquez A, Martinez AM, Gomez-Lopez G, Santisteban P. New insights into FoxE1 functions: identification of direct FoxE1 targets in thyroid cells. PloS one. 2013;8(5):e62849. PubMed PMID: 23675434. Pubmed Central PMCID: 3652843.
13. He H, Li W, Liyanarachchi S, Jendrzejewski J, Srinivas M, Davuluri RV, et al. Genetic predisposition to papillary thyroid carcinoma: involvement of FOXE1, TSHR, and a novel lincRNA gene, PTCSC2. The Journal of clinical endocrinology and metabolism. 2015 Jan;100(1):E164-72. PubMed PMID: 25303483. Pubmed Central PMCID: 4283026.
14. Kallel R, Belguith-Maalej S, Akdi A, Mnif M, Charfeddine I, Galofre P, et al. Genetic investigation of FOXE1 polyalanine tract in thyroid diseases: new insight on the role of FOXE1 in thyroid carcinoma. Cancer biomarkers : section A of Disease markers. 2010;8(1):43-51. PubMed PMID: 21896990.
15. Martinez VD, Vucic EA, Pikor LA, Thu KL, Hubaux R, Lam WL. Frequent concerted genetic mechanisms disrupt multiple components of the NRF2 inhibitor KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex in thyroid cancer. Molecular cancer. 2013;12(1):124. PubMed PMID: 24138990. Pubmed Central PMCID: 4016213.
16. Nonaka D, Tang Y, Chiriboga L, Rivera M, Ghossein R. Diagnostic utility of thyroid transcription factors Pax8 and TTF-2 (FoxE1) in thyroid epithelial neoplasms. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc. 2008 Feb;21(2):192-200. PubMed PMID: 18084247.
17. Park E, Gong EY, Romanelli MG, Lee K. Suppression of estrogen receptor-alpha transactivation by thyroid transcription factor-2 in breast cancer cells. Biochemical and biophysical research communications. 2012 May 11;421(3):532-7. PubMed PMID: 22521644.
18. Sato N, Fukushima N, Maitra A, Matsubayashi H, Yeo CJ, Cameron JL, et al. Discovery of novel targets for aberrant methylation in pancreatic carcinoma using high-throughput microarrays. Cancer research. 2003 Jul 1;63(13):3735-42. PubMed PMID: 12839967.
19. Sequeira MJ, Morgan JM, Fuhrer D, Wheeler MH, Jasani B, Ludgate M. Thyroid transcription factor-2 gene expression in benign and malignant thyroid lesions. Thyroid : official journal of the American Thyroid Association. 2001 Nov;11(11):995-1001. PubMed PMID: 11762722.
20. Venza I, Visalli M, Tripodo B, De Grazia G, Loddo S, Teti D, et al. FOXE1 is a target for aberrant methylation in cutaneous squamous cell carcinoma. The British journal of dermatology. 2010 May;162(5):1093-7. PubMed PMID: 19845668.
21. Yu XM, Jaskula-Sztul R, Ahmed K, Harrison AD, Kunnimalaiyaan M, Chen H. Resveratrol induces differentiation markers expression in anaplastic thyroid carcinoma via activation of Notch1 signaling and suppresses cell growth. Molecular cancer therapeutics. 2013 Jul;12(7):1276-87. PubMed PMID: 23594881. Pubmed Central PMCID:
3707971.
22. Zhan M, Chen G, Pan CM, Gu ZH, Zhao SX, Liu W, et al. Genome-wide association study identifies a novel susceptibility gene for serum TSH levels in Chinese populations. Human molecular genetics. 2014 Oct 15;23(20):5505-17. PubMed PMID: 24852370.
23. Zhang P, Zuo H, Nakamura Y, Nakamura M, Wakasa T, Kakudo K. Immunohistochemical analysis of thyroid-specific transcription factors in thyroid tumors. Pathology international. 2006 May;56(5):240-5. PubMed PMID: 16669872.
24. Muro-Cacho CA, Ku NN. Tumors of the thyroid gland: histologic and cytologic features--part 2. Cancer control : journal of the Moffitt Cancer Center. 2000 Jul-Aug;7(4):377-85. PubMed PMID: 10895133.
25. Venza I, Visalli M, Teti D, Venza M. FOXE1 (forkhead box E1 (thyroid transcription factor 2)). Atlas of Genetics and Cytogenetics in Oncology and Haematology. 2011 (2).
REALIZAÇÃOAPOIO
Não há um perfil de expressão de FOXE1 no câncer de tireoide. Já teve
sua expressão explorada em outros tecidos.
Variação da expressão de FOXE1 entre os tumores da tireoide.
•A análise das primeiras amostras mostra variação do perfil de expressão gênica
de FOXE1 entre as amostras tumorais e não tumorais;
•Parece que o tecido Não-Tumoral apresenta maior expressão de FOXE1 em
relação ao tecido Tumoral;
•Maior número de amostras estão sendo investigadas;
•Serão comparados os dados clínicos do pacientes e as características
anatomopatológicas dos tumores com os valores de expressão de FOXE1.
Perfil de expressão para FOXE1 no câncer de tireoide.

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Poster IC Congresso UNIFESP Final

  • 1. Avaliação da expressão gênica de FOXE1 no câncer de tireoide Filipe F. dos Santos¹; Erika Urbano de Lima²; Ileana GS Rubió¹. ¹Departamento de Ciências Biológicas. ²Pós-graduação de Biologia estrutural e funcional, UNIFESP. INTRODUÇÃO • 1.658.370 novos casos de câncer de tireoide (TC) e 589.430 mortes nos EUA em 2015 (Siegel et al., 2015); • 9.200 novos casos de TC no Brasil em 2015 (INCA, 2014-2015); •Taxa de incidência cresce cerca de 1% ao ano (INCA, 2014-2015); •Cerca de 90% dos doentes apresentam baixo risco de morte (Schlumberger, 1998); •Prevalência em mulheres (3:1) e adultos (DeLellis et al., 2004). RNA Proteína Localização gene FOXE1 OBJETIVOS METODOLOGIA JUSTIFICATIVA Avaliar o perfil de expressão gênica do RNA mensageiro (mRNA) de FOXE1 em amostras de tecidos tumorais e não tumorais pareadas da glândula tireoide. 1. Importante na formação da glândula tireoide; 2. Já foi relacionado com outros tumores, mas poucos trabalhos em TC; 3. Sem consenso quanto ao seu perfil de expressão; 4. É necessário compreender o perfil de expressão de FOXE1 em TC para entender seu real envolvimento nessa patologia. Amostras • 10 amostras pareadas (Tumor/Não Tumor) de pacientes brasileiros • Maioria PTC • Mais 20 pares Extração • RNA total de tecido • Invitrogen, Life Technologies, Carlsbad, CA Quantificação • Concentração • Qualidade (260/280 ~ 1,80-2,00) cDNA • Tratamento com DNAse • Síntese de cDNA de FOXE1 (Super Script III, Random Primers e Oligo (dT)16) RT-PCR • Estojo comercial SYBR Green Master MIX (SYBR Green I Dye) • Gene endógeno S8 • Quantificação por ΔΔCT Análise dos dados • Software GraphPad Prism 5 • Dados dos tumores dos pacientes RESULTADOS/DISCUSSÃO BIBLIOGRAFIA 1. DeLellis RA, Lloyd, R.V., Heitz, P.U., Eng, C. (2004) World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Endocrine Organs. Lyon, França: IARC Press. 2. INSTITUTO NACIONAL DO CÂNCER (INCA/MS) PRÓ-ONCO. Estimativas 2014, Disponível: http://www.inca.gov.br/estimativa/2014/sintese-de-resultados-comentarios.asp. (capturado em 31/05/2015). 3. Schlumberger MJ. Papillary and follicular thyroid carcinoma. The New England journal of medicine. 1998 Jan 29;338(5):297-306. PubMed PMID: 9445411. 4. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA: a cancer journal for clinicians. 2015 Jan-Feb;65(1):5-29. PubMed PMID: 25559415. 5. Abu-Khudir R, Magne F, Chanoine JP, Deal C, Van Vliet G, Deladoey J. Role for tissue-dependent methylation differences in the expression of FOXE1 in nontumoral thyroid glands. The Journal of clinical endocrinology and metabolism. 2014 Jun;99(6):E1120-9. PubMed PMID: 24646064. 6. Bell A, Bell D, Weber RS, El-Naggar AK. CpG island methylation profiling in human salivary gland adenoid cystic carcinoma. Cancer. 2011 Jul 1;117(13):2898-909. PubMed PMID: 21692051. Pubmed Central PMCID: 3123690. 7. Brancaccio A, Minichiello A, Grachtchouk M, Antonini D, Sheng H, Parlato R, et al. Requirement of the forkhead gene Foxe1, a target of sonic hedgehog signaling, in hair follicle morphogenesis. Human molecular genetics. 2004 Nov 1;13(21):2595-606. PubMed PMID: 15367491. 8. Bullock M, Duncan EL, O'Neill C, Tacon L, Sywak M, Sidhu S, et al. Association of FOXE1 polyalanine repeat region with papillary thyroid cancer. The Journal of clinical endocrinology and metabolism. 2012 Sep;97(9):E1814-9. PubMed PMID: 22736773. 9. Bychkov A, Saenko V, Nakashima M, Mitsutake N, Rogounovitch T, Nikitski A, et al. Patterns of FOXE1 expression in papillary thyroid carcinoma by immunohistochemistry. Thyroid : official journal of the American Thyroid Association. 2013 Jul;23(7):817-28. PubMed PMID: 23327367. Pubmed Central PMCID: 3704107. 10. Eichberger T, Regl G, Ikram MS, Neill GW, Philpott MP, Aberger F, et al. FOXE1, a new transcriptional target of GLI2 is expressed in human epidermis and basal cell carcinoma. The Journal of investigative dermatology. 2004 May;122(5):1180-7. PubMed PMID: 15140221. 11. Fan Y, Ding Z, Yang Z, Deng X, Kang J, Wu B, et al. Expression and clinical significance of FOXE1 in papillary thyroid carcinoma. Molecular medicine reports. 2013 Jul;8(1):123-7. PubMed PMID: 23715628. 12. Fernandez LP, Lopez-Marquez A, Martinez AM, Gomez-Lopez G, Santisteban P. New insights into FoxE1 functions: identification of direct FoxE1 targets in thyroid cells. PloS one. 2013;8(5):e62849. PubMed PMID: 23675434. Pubmed Central PMCID: 3652843. 13. He H, Li W, Liyanarachchi S, Jendrzejewski J, Srinivas M, Davuluri RV, et al. Genetic predisposition to papillary thyroid carcinoma: involvement of FOXE1, TSHR, and a novel lincRNA gene, PTCSC2. The Journal of clinical endocrinology and metabolism. 2015 Jan;100(1):E164-72. PubMed PMID: 25303483. Pubmed Central PMCID: 4283026. 14. Kallel R, Belguith-Maalej S, Akdi A, Mnif M, Charfeddine I, Galofre P, et al. Genetic investigation of FOXE1 polyalanine tract in thyroid diseases: new insight on the role of FOXE1 in thyroid carcinoma. Cancer biomarkers : section A of Disease markers. 2010;8(1):43-51. PubMed PMID: 21896990. 15. Martinez VD, Vucic EA, Pikor LA, Thu KL, Hubaux R, Lam WL. Frequent concerted genetic mechanisms disrupt multiple components of the NRF2 inhibitor KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex in thyroid cancer. Molecular cancer. 2013;12(1):124. PubMed PMID: 24138990. Pubmed Central PMCID: 4016213. 16. Nonaka D, Tang Y, Chiriboga L, Rivera M, Ghossein R. Diagnostic utility of thyroid transcription factors Pax8 and TTF-2 (FoxE1) in thyroid epithelial neoplasms. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc. 2008 Feb;21(2):192-200. PubMed PMID: 18084247. 17. Park E, Gong EY, Romanelli MG, Lee K. Suppression of estrogen receptor-alpha transactivation by thyroid transcription factor-2 in breast cancer cells. Biochemical and biophysical research communications. 2012 May 11;421(3):532-7. PubMed PMID: 22521644. 18. Sato N, Fukushima N, Maitra A, Matsubayashi H, Yeo CJ, Cameron JL, et al. Discovery of novel targets for aberrant methylation in pancreatic carcinoma using high-throughput microarrays. Cancer research. 2003 Jul 1;63(13):3735-42. PubMed PMID: 12839967. 19. Sequeira MJ, Morgan JM, Fuhrer D, Wheeler MH, Jasani B, Ludgate M. Thyroid transcription factor-2 gene expression in benign and malignant thyroid lesions. Thyroid : official journal of the American Thyroid Association. 2001 Nov;11(11):995-1001. PubMed PMID: 11762722. 20. Venza I, Visalli M, Tripodo B, De Grazia G, Loddo S, Teti D, et al. FOXE1 is a target for aberrant methylation in cutaneous squamous cell carcinoma. The British journal of dermatology. 2010 May;162(5):1093-7. PubMed PMID: 19845668. 21. Yu XM, Jaskula-Sztul R, Ahmed K, Harrison AD, Kunnimalaiyaan M, Chen H. Resveratrol induces differentiation markers expression in anaplastic thyroid carcinoma via activation of Notch1 signaling and suppresses cell growth. Molecular cancer therapeutics. 2013 Jul;12(7):1276-87. PubMed PMID: 23594881. Pubmed Central PMCID: 3707971. 22. Zhan M, Chen G, Pan CM, Gu ZH, Zhao SX, Liu W, et al. Genome-wide association study identifies a novel susceptibility gene for serum TSH levels in Chinese populations. Human molecular genetics. 2014 Oct 15;23(20):5505-17. PubMed PMID: 24852370. 23. Zhang P, Zuo H, Nakamura Y, Nakamura M, Wakasa T, Kakudo K. Immunohistochemical analysis of thyroid-specific transcription factors in thyroid tumors. Pathology international. 2006 May;56(5):240-5. PubMed PMID: 16669872. 24. Muro-Cacho CA, Ku NN. Tumors of the thyroid gland: histologic and cytologic features--part 2. Cancer control : journal of the Moffitt Cancer Center. 2000 Jul-Aug;7(4):377-85. PubMed PMID: 10895133. 25. Venza I, Visalli M, Teti D, Venza M. FOXE1 (forkhead box E1 (thyroid transcription factor 2)). Atlas of Genetics and Cytogenetics in Oncology and Haematology. 2011 (2). REALIZAÇÃOAPOIO Não há um perfil de expressão de FOXE1 no câncer de tireoide. Já teve sua expressão explorada em outros tecidos. Variação da expressão de FOXE1 entre os tumores da tireoide. •A análise das primeiras amostras mostra variação do perfil de expressão gênica de FOXE1 entre as amostras tumorais e não tumorais; •Parece que o tecido Não-Tumoral apresenta maior expressão de FOXE1 em relação ao tecido Tumoral; •Maior número de amostras estão sendo investigadas; •Serão comparados os dados clínicos do pacientes e as características anatomopatológicas dos tumores com os valores de expressão de FOXE1. Perfil de expressão para FOXE1 no câncer de tireoide.