1. 1. Thyrotropin signaling confers more aggressive features on BRAFV600E -induced thyroid tumors in a mouse model of papillary thyroid cancer Florence Orim Department of Radiation Medical Sciences Atomic Bomb Disease Institute Nagasaki University Thesis Defense September 2013
2. 2. Thyroid Cancer Histotypes Papillary thyroid carcinoma (PTC) Follicular thyroid carcinoma (FTC) Poorly differentiated thyroid carcinoma (PDTC) Anaplastic thyroid carcinoma (ATC) Medullary thyroid carcinoma (MTC) DTC
3. 3. Papillary Thyroid Carcinoma (PTC) Malignant epithelial tumor showing evidence of follicular cell differentiation and distinctive nuclear features Classic papillary carcinoma Papillary microcarcinoma Follicular variant (FVPTC) Oncocytic-, Clear cell- , Diffuse sclerosing, Tall cell, Columar cell-, Solid-, Cribriform WHO Definition Histopathological Variants
4. 4. Papillary Carcinoma Papillae Fibro-vascular central core lined by cell layers with characteristic papillary cytological features Follicular architecture the follicular variant of PTC exists in a completely follicular tumor pattern Tall cells Nuclear Features Nuclear grooves Overlapping nuclei Orphan Annie Ground glass appearance Intranuclear inclusion Mitoses are rare Psammoma bodies DD calcification Microscopy
5. 5. Papillary Carcinoma Normal follicles Papillae with overlapping nuclei Follicle with overlapping nuclei
6. 6. Oncogenes Frequently Found in PTCs
7. 7. BRAF Mutations Matsuse, Mitsutake et al, Int J Cancer 2013 V600delinsYM
8. 8. PTC and Mice BRAF Why Mice? PTC Mouse Models Useful to develop new ways of cancer diagnosis and treatment Improve understanding of altered signaling pathways in carcinogenesis Existing models are transgenic and / or knock in BRAFV600E - aggressive clinicopathological features Advanced clinical stage at diagnosis (extrathyroidal extension, nodal metastases) High recurrence Genetically similar to human beings Affordable, easy to maintain Able to reproduce within 3 weeks Short life span, small size, cost effective
9. 9. Tg-BRAFV600E Mouse Model of PTC Knauf, Mitsutake et al, Cancer Res 2005 bTg promoterbTg promoter BRAFV600EBRAFV600E Have high TSH (feedback mechanism) Role of elevated TSH in these models needs to be elucidated
10. 10. What is known The risk of malignancy in a thyroid nodule increases with serum TSH level even within the normal range. Boelaert et al, JCEM 2006 Higher TSH level is associated with advanced stage. Haymart et al, JCEM 2008 Independent of age, it is associated with extrathyroidal extension but not with tumor size and metastasis. Haymart et al, Clin Endocrinol 2009
11. 11. What is unclear WHERE IN THE CARCINOGENIC PROCESS DOES TSH ACT IN BRAFV600E INDUCED PTC? WHAT IS THE ROLE OF TSH IN THYROID CARCINOGENESIS?
12. 12. Breeding Scheme Sacrifice Age : 12 weeks 24 weeks Experimental Design Tg-BRAFV600E TshR-/- Genotyping ~ 0.5cm Tail for DNA extraction, PCR Analyses Mouse specimens: thyroid, serum Cells: PC-BRAFV600E - 6 line Group 1: BRAFwt /TshR+/- Group 2: BRAFwt /TshR-/- Group 3: Tg-BRAFV600E /TshR+/- Group 4: Tg-BRAFV600E /TshR-/-
13. 13. Tg-BRAFV600E Mouse Model of PTC Knauf, Mitsutake et al, Cancer Res 2005 bTg promoterbTg promoter BRAFV600EBRAFV600E Have high TSH (feedback) Role of elevated TSH in these models needs to be elucidated
14. 14. TSHR-KO Mice Marians, Ng et al, PNAS 2002 WT TSHR-KO
15. 15. Methods 1.Serum 2.Thyroid Histology 3.Genes qRT-PCR mRNA expression 4.Immunohistochemistry for indices of apoptosis, macrophage infiltration 5.Immunoflourescence Genomic instability (GIN) status PC-BRAFV600E -6 cells (PCCL3) 1.Invasion assay 2.Immunoflourescence genomic instability (GIN) status Mice Cells
16. 16. Mice Group 1: BRAFwt /TshR+/- Group 2: BRAFwt /TshR-/- Group 3: Tg-BRAFV600E /TshR+/- Group 4: Tg-BRAFV600E /TshR-/- ! " # " " $ ! % # $ ! % # $ ! % # $ ! % # & ' () *) & ' () + , - & ' () *) & ' () $ ! , - . /,012
17. 17. Gene Expression Levels Group 1 BRAFwt /TshR+/- Group 2 BRAFwt /TshR-/- Group 3 BRAFV600E /TshR+/- Group 4 BRAFV600E /TshR-/-
18. 18. Thyroid Sections Group 1: BRAFwt /TshR+/- Group 2: BRAFwt /TshR-/- Group 3: Tg-BRAFV600E /TshR+/- Group 4: Tg-BRAFV600E /TshR-/- Group 1 Group 2 Group 3 Group 4
19. 19. Thyroid Weights Group 1: BRAFwt /TshR+/- Group 2: BRAFwt /TshR-/- Group 3: Tg-BRAFV600E /TshR+/- Group 4: Tg-BRAFV600E /TshR-/- M a le 1.2 1.0 0.8 0.6 0.4 0.2 0.0 1 2 3 4 * Thyroidweight(mg)/BW(g) F e m a le 2 3 41 1.0 0.8 0.6 0.4 0.2 0.0 Thyroidweight(mg)/BW(g) 1.0 0.8 0.6 0.4 0.2 0.0 1 2 3 4 Thyroidweight(mg)/BW(g) 0.8 0.6 0.4 0.2 0.0 1 2 3 4 Thyroidweight(mg)/BW(g) * * * * * * * * * * * 12w24w
20. 20. Pathological findings Group 3: Tg-BRAFV600E /TshR+/- P R R S T A B C FED
21. 21. Histopathological scoring of thyroid lesions
22. 22. * 40 20 0 TSH Dox + + + + - - - - Numberofcells Cell invasiveness in PC-BRAFV600E cells PC-BRAFV600E cells: doxycycline-inducible BRAFV600E in rat thyroid PCCL3 cells
23. 23. Cleaved caspase-3 F4/80 Group 1: BRAFwt /TshR+/- Group 2: BRAFwt /TshR-/- Group 3: Tg-BRAFV600E /TshR+/- Group 4: Tg-BRAFV600E /TshR-/- ! " " # $%& '()(*+ $, + --' . / . / . / . / 0 ! $1 ! / $1 0 ! $1 ! / $1 2 3 -+ 4 + 5 3 -+ 0 " 6 6 7-+ 3*+ 8$,3 '% 3 '+ 9. ! " ! " ! " ! " # $ %& $ " %& # $ %& $ " %& ' ( )* + * , ( )* - # - $ - . % /0 123 24* %5 *)) 1 + "67- %8, ( 59 0/ :( ;* < Immunohistochemistry Macrophage infiltration indexApoptotic index
24. 24. Impaired DNA damage response (DDR) can result in genomic instability (GIN) GIN leads to transformation and cancer progression P53-binding protein 1 (53BP1), a DDR protein, forms localized nuclear foci at sites of DNA double strand breaks (DSBs) Presence of 53BP1 foci considered cytologic marker reflecting GIN Cancer and genomic instability
25. 25. Foci formation of P-53-binding protein 1(53BP1) in thyroid tumors: Activation of genomic instability during thyroid carcinogenesis Nakashima et al, Int. J Cancer 2008 Co-staining of Ki67/53BP1 was found in ATC cells
26. 26. 1 2 3 4 Group 1: BRAFwt /TshR+/- Group 2: BRAFwt /TshR-/- Group 3: Tg-BRAFV600E /TshR+/- Group 4: Tg-BRAFV600E /TshR-/- Ki67/53BP1 co-staining - Ki-67 foci - 53BP1foci
27. 27. Group 1: BRAFwt /TshR+/- Group 2: BRAFwt /TshR-/- Group 3: Tg-BRAFV600E /TshR+/- Group 4: Tg-BRAFV600E /TshR-/- Proliferative index Ki67 score ! " #$%& '( )#*#+,&- ,..) $ / 0 1 ' &2 &1 31 4 ' &2 &1 31 4 ' &5 &1 31 6 7 0 / 4 1 ' &2 &1 31 4 ' &2 &1 31 4 ' &5 &1 31 6 ' &2 &1 31 4 ' &2 &1 31 4 ' &5 &1 31 6' &2 &1 31 6 ' &2 &1 31 6 ' &5 &1 31 6 4 0 7 / 4 0 7 / 4 0 7 / 4 0 7 / 8 9., :, ;9 ., ! "# $% &'( )# *#+ ,&- , ..) 4 0 &< 0 / &< $ / 0 1 7 0 / 4 1
28. 28. DNA damage responses Group 1: BRAFwt /TshR+/- Group 2: BRAFwt /TshR-/- Group 3: Tg-BRAFV600E /TshR+/- Group 4: Tg-BRAFV600E /TshR-/- PC-BRAFV600E cells !"#$ %&'"() *+ , - . / 0 "1 "/ 2/ ' 0 "1 "/ 2/ ' 0 "3 "/ 2/ # , - . / , - . / 0 "3 "/ 2/ # 0 "1 "/ 2/ ' 0 "1 "/ 2/ # 0 "3 "/ 2/ # 0 "1 "/ 2/ # 0 "3 "/ 2/ # . , - / !"#$ %&'"() *+ 0 "1 "/ 2/ # 0 "1 "/ 2/ #0 "1 "/ 2/ / ' ' . $ - ' . $ -' . $ - ' . $ - 45 67 879 567 53BP1, -H2AX
29. 29. Lu et al, Endocrinology 2010 The FTC model TRPV/PV mouse Thyrocytes TRPV/PV TSHR-/- No TSH proliferation signaling Impaired growth (No thyroid cancer) Thyrocytes WT-PTU Thyrocytes TRPV/PV TSH proliferation signaling TSH proliferation signaling PV-activated proliferation via PI3K-AKT signaling Aberrant growth (No metastatic thyroid cancer) Severely Aberrant growth Increased cell invasion and migration Metastatic thyroid cancer PV-activated intergrin/TGF - FAK-p38 MAPK-MMP-9-signaling PV-mediated -actin/ezrin cytoskeletal remodeling WT TshR-/- TRPV/PV /TshR-/-
30. 30. Franco et al, PNAS 2011 The other PTC model Benign tumor no nuclear features of PTC LSL-BrafV600E /TPO-Cre/TshR-/- WT LSL-BrafV600E / TPO-Cre LSL-BrafV600E / TPO-Cre/TshR-/-
31. 31. MAPK TSH signal important for: Progression Inducing genomic instability Preventing apoptosis Summary TSH signaling : necessary for tumorigenesis? FAFA FTCFTCPI3K-AKT PTCPTC Thyroid Cell Thyroid Cell
32. 32. Acknowledgments Department of Radiation Medical Sciences Atomic Bomb Disease Institute Nagasaki University Graduate School of Biomedical Sciences Japan THANKYOU