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Flo thesisdefense.24th September.2013
- 2. 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
- 3. 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 PTC = 75-85% of all thyroid cancers UTC
- 4. 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, Columnar cell-, Solid-, Cribriform WHO Definition Histopathological Variants
- 6. Oncogenes Frequently Found in PTCs
- 7. PTC and Mice Mice PTC Mouse Models • Improve understanding of altered signaling pathways in carcinogenesis • Useful to develop new ways of cancer diagnosis and treatment • Existing models are transgenic and/or knock in PTC with BRAF – poor prognosis BRAF gene – strongest activator of MAPK pathway, frequently mutated T1799A transversion - glutamic acid substitution for valine (V600E) BRAFV600E PTCs– distinct aggressive clinicopathological features •More invasive •Advanced clinical stage at diagnosis • Higher recurrence rates • Refractory to RAI • Genetically similar to humans • Affordable, easy to maintain • Able to reproduce within 3 weeks • Short life span, cost effective BRAF
- 8. Tg-BRAFV600E Mouse Model of PTC Knauf, Mitsutake et al, Cancer Res 2005 bTg promoterbTg promoter BRAFV600EBRAFV600E Hypothyroid High Thyrotropin (TSH) ~500x (feedback mechanism) Role of elevated TSH needs to be elucidated
- 9. 9
- 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. What is unclear… WHERE IN THE CARCINOGENIC PROCESS DOES TSH ACT IN BRAFV600E -INDUCED PTC?
- 12. THE PURPOSE OF THIS STUDY WAS TO EXPLORE THE ROLE OF ELEVATED THYROTROPIN IN THYROID CANCER
- 13. 13
- 14. TSHR-KO Mice Marians, Ng et al, PNAS 2002 TSHR KO Severely hypothyroid Euthyroid on T4 supplemented diet WT
- 15. Tg-BRAFV600E Mouse Model of PTC Knauf, Mitsutake et al, Cancer Res 2005 bTg promoterbTg promoter BRAFV600EBRAFV600E High TSH (feedback mechanism) Role of elevated TSH in these models needs to be elucidated
- 16. 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 Cells: PC-BRAFV600E - 6 line Doxycycline inducible Group 1: BRAFwt /TshR+/- Group 2: BRAFwt /TshR-/- Group 3: Tg-BRAFV600E /TshR+/- Group 4: Tg-BRAFV600E /TshR-/-
- 17. Methods 1.Thyroid Histology and pathological scoring of lesions 2.Thyroid specific gene expression – qRT-PCR mRNA 3.Indices of apoptosis (Cleaved caspase-3), macrophage infiltration (F4/80) – Immunohistochemistry 5.Cell proliferation (Ki67) and Genomic instability (GIN) status – Immunoflourescence (53BP1, ϒH2AX) PC-BRAFV600E -6 cells (PCCL3) 1. Invasion assay 2. Immunoflourescence genomic instability (GIN) status Mice Cells
- 19. Mice Group 1: BRAFwt /TshR+/- Group 2: BRAFwt /TshR-/- Group 3: Tg-BRAFV600E /TshR+/- Group 4: Tg-BRAFV600E /TshR-/- ! " # " " $ ! % # $ ! % # $ ! % # $ ! % # & ' () *) & ' () + , - & ' () *) & ' () $ ! , - . /,012 4w – thyroid powder-supplemented diet (100ppm)
- 20. Gene Expression Levels Group 1: BRAFwt /TshR+/- Group 2: BRAFwt /TshR-/- Group 3: Tg-BRAFV600E /TshR+/- Group 4: Tg-BRAFV600E /TshR-/-
- 21. 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
- 22. 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
- 23. Pathological findings Group 3: Tg-BRAFV600E /TshR+/- P R R S T A B C FED
- 24. Histopathological scoring of thyroid lesions
- 25. Cleaved caspase-3 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
- 26. * 40 20 0 TSH Dox + + + + - - - - Numberofcells Cell invasiveness in PC-BRAFV600E – 6 cells PC-BRAFV600E cells: doxycycline-inducible BRAFV600E in rat thyroid PCCL3 cells
- 27. • Genomic instability (GIN) is a hallmark of cancer • Vital role in transformation and cancer progression • P53-binding protein 1 (53BP1), a DNA damage response (DDR) protein, and other related proteins (pATM, H2AX)γ form localized nuclear foci at sites of DNA double strand breaks (DSBs) • The induction of DSBs is a manifestation of GIN • 53BP1, H2AX, pATMγ ~ markers of GIN Cancer and genomic instability
- 28. 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 positive cells - 53BP1foci
- 29. 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, ϒ-H2AX score 12w 24w
- 30. 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
- 31. Lu et al., Endocrinology 2010 The FTC model TRβPV/PV mouse Thyrocytes TRβPV/PV TSHR-/- No TSH proliferation signaling Impaired growth (No thyroid cancer) Thyrocytes WT-PTU Thyrocytes TRβPV/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 -β FAK-p38 MAPK-MMP-9-signaling •PV-mediated -actin/ezrinβ •cytoskeletal remodeling WT TshR-/- TRβPV/PV /TshR-/-
- 32. 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-/-
- 33. Conclusion TSH signal important for: Progression Inducing genomic instability Preventing apoptosis MAPK FAFA FTCFTC PI3K-AKT PTCPTC Thyroid Cell Thyroid Cell TSH signaling : necessary for tumorigenesis?
- 34. Acknowledgments Professor Shunichi Yamashita Ass. Professor Norisato Mitsutake Department of Radiation Medical Sciences Atomic Bomb Disease Institute Nagasaki University Dr Michiko Matsuse Dr Andrey Bychkov Dr Mami Nakahara Ms. Mika Shimamura Professor Yuji Nagayama Department of Molecular Medicine Atomic Bomb Disease Institute Nagasaki University Professor Masahiro Nakashima Department of Diagnostic Pathology Atomic Bomb Disease Institute Nagasaki University
Editor's Notes
- Good morning Professors, colleagues. Thank you for being here today. My thesis work focused on the role of Thyrotropin in Papillary Thyroid Cancer. Thyrotropin is the main regulator of thyroid cell differentiation and proliferation and this presentation is titled:
- Thyroid cancers are the most common endocrine neoplasm. They could be benign or malignant. Malignancies can be classified histologically based on the degree of differentiation. DTCs include Papillary TC Follicular TC - There is PDTC And undifferentiated ATC Rare form 5-10% of all TCs MTC ~ 4% arises from parafollicular C cells Of all these PTC is the most common accounting for 45-75% of all thyroid tumors. TRANSITION: OUR STUDY FOCUSED ON THE MOST PREVALENT THYROID CANCER - PTC
- The WHO defines PTC as a ….. The histopath. Variants are…..
- Microscopic features are the presence of papillae consisting of a Fibro-vascular central core lined by cell layers with characteristic papillary cytological features Also a follicular architecture that may be distorted to varying extents. Tall cells that are two times as high as they are wide Recently nuclear features have become the diagnostic hallmark of the tumor as described in the WHO definition – The nuclei in PTC are large, oval clear empty nuclei, nuclei with hypodense chromatin giving a ground glass appearance, grooves on the surface of the nuclei, intranuclear pseudo inclusions and crowded overlapping nuclei. Figures: Normal thyroid with follicular structure Follicle with overlapping nuclei Papillae lined by tall cells with overlapping nuclei High mag. Ground glass, pseudo inclusion TRANSITION: The molecular mechanisms involved in the initiation and progression of PTC are not fully understood. Psammoma bodies are associated with the papillary (nipple-like) histomorphology and are thought to arise from the infarction and calcification of papillae tips and (2) calcification of intralymphatic tumor thrombi.
- There are characteristic genetic alterations associated with the aetiology of PTC: These include Rearrangement during Transfection or RET/PTC rearrangements and activating point mutations of the RAS and BRAF family genes. These mutations all result in activation of the MAPK cascade that is critical for transformation of thyrocytes to PTC. However RET/PTC and Ras signal via other pathways also. This means that these mutations are mutually exclusive and rarely occur simultaneously. TRANSITION: Evidence shows that the most common event in thyroid carcinogenesis is a mutation in the BRAF gene BRAF The BRAF mutation results in constitutive activation of the Mitogen activated protein kinase pathway. (MAPK). In approximately 90% of all identified BRAF mutations, thymine is substituted with adenine at nucleotide 1799. This leads to valine (V) being substituted for by glutamate (E) at codon 600 (now referred to as V600E) In PTC, the frequency of BRAF V600E is relatively high, approximately 44 % (ranging from 29 % to 83 %). MAPK signaling drives malignant transformation therefore… BRAFV600E PTCs have distinct clinical and pathological features. They are more frequently invasive and present at an advanced stage at diagnosis (extrathyroidal extension, nodal metastases) They have higher rates of recurrence and are relatively refractory to radioiodine therapy. MICE Mice have been used to replicate the disease because they are genetically similar easy to maintain, they reproduce fast and don’t live long. PTC Mouse Models PTCs with BRAF mutation also have a higher disease specific mortality. Thus mouse models of the disease have been developed to help provide more understanding of the complex signaling pathways involved in the pathogenesis of PTC so as to develop better ways of diagnosis and treatment. These models are transgenic or knock in. Activation of MAPK = Regulates expression of several biomarkers- Which provide a promitogenic effect= Drives malignant transformation
- BRAF gene is a the strongest activator of MAPK pathway which is critical for thyrocytes transformation to PTC It is the most frequently mutated human oncogene in the kinase superfamily of genes. Most common mutation is the T1799A transversion resulting in the substitution of glutamic acid for valine V600E have distinct clinical and pathological features. They are more frequently invasive and present at an advanced stage at diagnosis (extrathyroidal extension, nodal metastases) . They have higher rates of recurrence and are relatively refractory to radioiodine therapy. Mice have been used to replicate the disease because they are genetically similar easy to maintain , they reproduce fast and don’t live long. PTCs with BRAF mutation also have a higher disease specific mortality. Thus mouse models of the disease have been developed to help provide more understanding of the complex signaling pathways involved in the pathogenesis of PTC so as to develop better ways of diagnosis and treatment. These models are transgenic or knock in. TRANSITION: A classic example is the Tg-BRAFV600E mouse model. BRAF Generally PTC has excellent prognosis with treatment The problem is that TCs with BRAF mutation have a poor prognosis. The BRAF mutation results in constitutive activation of the Mitogen activated protein kinase pathway. (MAPK). In approximately 90% of all identified BRAF mutations , thymine is substituted with adenine at nucleotide 1799. This leads to valine (V) being substituted for by glutamate (E) at codon 600 (referred to as V600E) In PTC, the frequency of BRAF V600E is relatively high, approximately 44 % (ranging from 29 % to 83 %). MAPK signaling drives malignant transformation therefore… BRAFV600E PTCs have distinct clinical and pathological features. They are more frequently invasive and present at an advanced stage at diagnosis (extrathyroidal extension, nodal metastases) They have higher rates of recurrence and are relatively refractory to radioiodine therapy. MICE Mice have been used to replicate the disease because they are genetically similar easy to maintain, they reproduce fast and don’t live long. PTC Mouse Models PTCs with BRAF mutation also have a higher disease specific mortality. Thus mouse models of the disease have been developed to help provide more understanding of the complex signaling pathways involved in the pathogenesis of PTC so as to develop better ways of diagnosis and treatment. These models are transgenic or knock in. Activation of MAPK = Regulates expression of several biomarkers- Which provide a promitogenic effect= Drives malignant transformation TRANSITION: A classic example is the Tg-BRAFV600E mouse model.
- The Tg BRAFV600E model of PTC… expresses the transgene BRAFV600E under the bovine Thyroglobulin promoter Tg which drives thyroid specific expression of the oncogene. In the WT mice thyroid macro and microscopic structure was normal. In the mutant mice thyroid cells expressing the oncogene were transformed and progressed to Invasive PTCS with high penetrance. However these mice had growth retardation and were hypothyroid with consequently high TSH levels ~ 500x that of their WT littermates. These tumors showed characteristic features of aggressive human PTCs with distortion of the normal follicular architecture, papillae with tall cells and other nuclear features. (it has been shown that over expression of oncogenic activators of MAPK impair thyroid hormone synthesis in vivo) as a result Thyroid hormone , Thyroglobulin and other thyroid specific genes were down regulated (TPO). And so TSH was elevated due to the normal feedback compensatory mechanism. The elevated TSH is significant because other mouse models of thyroid cancer show high TSH levels also. TRANSITION: Several epidemiological studies have been carried out about the role of TSH in thyroid carcinogenesis
- And so…What we do know from Epidemiological studies is that … In benign thyroid nodular disease the risk of malignancy increases with serum TSH level even when TSH level is normal. Also higher TSH levels are associated with advanced stage Furthermore higher TSH is also associated with ETE independent of age but not with tumor size and distant metastasis
- What we do not know is where exactly in the ……
- This study was designed using two strains of mice to assess the role of TSH in BRAFV600E induced thyroid carcinogenesis. TSHR-ko mice were developed to investigate the role of TSHR in thyroid cell function. This is an illustration of the targeting vector used to generate the TSHR ko mice by homologous recombination. Thyroids from the Wt mice were normal. TSHR ko thyroids showed few follicles and nuclear changes.
- The second strain is the classic Tg-BRAF model I described earlier.
- Tg-Braf mice were crossed with TSHr KO mice (heterozygous) The offspring were genotyped and the pups of these 4 genotypes were selected …. BRAF wild type with and without intact TSH signaling and mutant BRAF with and without intact TSH signaling. TSHR is in blue , BRAF is in red. They were sacrificed at 12 and 24 weeks then analyzed for abnormalities in thyroid histology, levels of gene expression and evidence of genomic instability. We also replicated these experiments in a doxycycline inducible thyroid cancer cell line.
- We performed histopathological analysis on the thyroids of these mice. And assessed the levels of expression of thyroid specific genes by quantitative real time reverse transcriptase PCR. We also performed IHC staining using antibodies for detection of Cleaved caspase 3 as an index of apoptosis And for F4/80 which is an index of macrophage infiltration. The F4/80 antigen is expressed on the surface of macrophage cells which are known to Cell proliferation and GIN status were also assessed by IF staining for 53BP1 and ϒH2AX We wanted to reproduce these experiments in vitro using the PC-BRAFV600E-6 cell line derived from PCCL3 cells. These are a clonal rat thyroid cell line that expresses BRAFV600E on induction by doxycycline. We examined the effect of TSH on cell invasiveness and GIN status.
- Regarding the body weight… At 5w grp 2 and 4 mice with TSHR ko (no TSH signaling) were smaller than g1 and 3 likely due to hypothyroidism. At 4 weeks supplemented diet containing desiccated thyroid powder was introduced and these differences between the groups were absent until 12w.
- In our model the activity of the TG promoter which drives expression of BRAFV600E is dependent on TSH stimulation in vitro. However previous studies have shown that Tg is expressed in thyroids of TshR ko mice. So we analyzed BRAF expression levels. Our results show that BRAF was correctly transcribed with comparable expression in g3 and g4 at 12 w irrespective of TSH signaling. We also analyzed the expression levels of the thyroid specific genes NIS and thyroglobulin In the absence of TSH signaling, NIS expression was markedly down regulated in the TSHR ko groups 2 and 4 while Thyroglobulin expression was decreased in group 3 as well as g2 and 4. (Dedifferentiating effect on BRAFV600E on expression of Tg – strong)
- The thyroid weight to body weight ratio was the highest in group 3 as compared to the other groups (Fig. 2A). However, there was no significant difference between groups 1, 2, and 4. TRANSITION: This clearly demonstrates the influence of TSH signaling on BRAFV600E induced thyroid tumors.
- These are Representative low-power images of thyroid sections from each group. Thyroid from group 1 were of normal size and structure. In group 2 the thyroids had fewer follicles with some adipocytes. In group 3, the glands were markedly enlarged and no follicles were observed. In group 4 the normal follicular architecture was completely disrupted also similar to G3 but the gland was much smaller and weight was much lower. There were no metastases in G3 and 4.
- Next we performed histopathological analyses of thyroids from groups 3 and 4 mice. These are representative thyroids from G3 mice expressing mutant BRAF with intact TSH signaling. A)This shows the tumor completely distorting normal thyroid follicular structure. B) Shows a papillary growth projecting into the lumen of a follicle C) Shows tall cells lining papillary structures D) Solid pattern replacing normal thyroid architecture E) This is Capsular invasion showing the remnant of the capsule F) Nuclear clearing was also observed. TRANSITION: These features are similar to features of aggressive human PTC.
- This table summarizes the results of histopathological scoring of the lesions observed in G3 and 4. Most mice in G3 and 4 developed neoplasia similar to human PTCs, (column 2) including charactersistice nuclear features such as nuclear clearing (column 3). Another aggressive feature is the solid pattern, which in our study was seen more frequently in G3 than G4. Capsular invasion, another aggressive feature was only observed in G3 also. G4 showed no features of ETE or capsular invasion. Papillary pattern observed more in G4 than 3 ; at 12w (data not shown) was replaced by solid pattern at 24w in G4. Overall the aggressiveness score for G3 was higher than that of G4 and this difference was statistically significant. TRANSITION: Therefore these data suggest that TSH signaling results in a more aggressive phenotype in BRAFV600E induced PTCs shown by the differences observed between groups 3 and 4.
- In order to examine the influence of apoptosis on this difference between G3 and 4, we performed IHC for CC3. Programmed cell death or Apoptosis is mediated by specific proteases , (cysteinyl-aspartic acid proteases) or caspases. CC3 is is a fragment of activated caspase 3 and is a measure of the extent of apoptosis. The 1st figure shows the %age of positively stained cells higher in G4 than in G3 at both 12 and 24 weeks, this was statistical significance in 12w. Not significant in 24w just tendency Macrophage… Recently it was demonstrated that TAMs have a role in the progression of BRAFV600E induced thyroid cancer particularly progression to PDTC. We test this possibility we also performed IHC using antibodies against the F4/80 antigen, a well-characterized membrane protein and marker for macrophages. However there was no difference between the groups TRANSITION: Thus we see that TSH prevents apoptosis but not macrophage infiltration.
- We supported these in-vivo findings by performing a cell invasion assay in vitro using a doxycycline inducible BRAFV600E rat thyroid cell line. The PC-BRAFV600E-6 cell line expresses BRAFV600E on treatment with Doxycycline. Cells were starved of TSH for 7 days and then cultured on Matrigel coated transwell inserts with or without dox and in the presence or absence of TSH. Cells that invaded the matrix were counted. This figure shows that BRAF actually induced cell invasiveness and that this invasiveness remarkably increased in the presence of TSH. TRANSITION: This demonstrates that BRAFV600E and TSH both cooperate to induce invasiveness in thyroid cells and that TSH enhances cell invasiveness.
- I’d like to briefly explain the relationship between cancer and GIN. Genomic instability (GIN) is a hallmark of cancers it plays a crucial role in the progression of thyroid tumors To explore the mechanisms by which TSH signaling confers more aggressive features on thyroid tumor cells, we focused on genomic instability. p53-binding protein 1 (53BP1) is a DNA damage response protein which rapidly localizes at the site of DNA double strand breaks (DSB) together with phosphorylated ATM and many other related proteins such as gH2AX DNA damage response is a way of keeping the genome stable thus defective DNA damage response can induce GIN. Therefore 53BP1 γH2AX, pATM, are considered as markers of genomic instability.
- To explore the influence of TSH signaling and BRAFV600E on tumor growth/induction of aggressive features, we performed dual-label immunoflourescence for 53BP1 and Ki67. These are representative merged images from each group. Group 3 and 4 showed the highest frequency of 53BP1 focus positive and Ki67 positive cells. yellow arrowheads show 53BP1 and white Ki67.
- The percentage of 53BP1 focus-positive cells was calculated and compared between groups 3 and 4. the frequency in group 3 was significantly higher than that in group 4 in 24-week male and 12-week female mice. The frequency in 24w female mice was also significantly higher but did not attain statistical significance. Also in G1 mice the percentage was higher at 24 weeks and was overall higher in female than in male mice. We went further to confirm these findings regarding GIN by using another marker of DNA damage phosphorylated H2AX at ser-139. gH2AX. The frequency of g-H2AX foci in group 3 was significantly higher than that in group 4 in all age/sex pairs. These findings were replicated in vitro with PC-BRAFV600E-6 cells. Showing that TSH induces GIN
- The Ki67 proliferative index was low in G1 and 2 mice. In contrast, BRAFV600E remarkably increased the frequency of Ki67 positive cells in G3 and 4, but with no statistical significance in the presence or absence of TSH. These data demonstrate that TSH signaling induces GIN in this model of PTC. TRANSITION: A few thyroid cancer models have been designed to investigate the role of TSH.
- The FTC model is one of two mouse models designed to investigate the role of TSH signaling in TC. Using the thyroid hormone-β receptor (TRβ) mutant mice which develop tumors and have elevated TSH. When TSH signaling was removed by crossing with TSHr ko mice, there was no cancer. Only TRβPV/PV mice developed metastatic FTC. PV acted through other mechanisms… Thus, growth stimulated by TSH is a required but not sufficient for metastatic cancer to occur. Additional genetic alterations (such as PV), are required drive the progression to invasive and metastatic cancers
- The other PTC model is the LSLBRAF/TPO-Cre model which develops classic infiltrative PTCs by 5w. Elimination of TSH signal by crossing with TSHR ko mice resulted in benign tumors with no nuclear features of PTC with a much milder phenotype. This model is very similar to ours thyroid cells lacking TshR can be transformed by Braf after a longer latency and are phenotypically less aggressive.
- From the previous FTC model, tumorigenesis in thyrocytes is usually induced by activation of the PI3K – AKT pathway and TSH signaling seems to be necessary for tumorigenesis. Suggesting that TSH is required for Tumorigenesis. But in our PTC model, tumorigenesis is dependent on MAPK signaling rather than TSH. Because our data show that TSH is crucial in progression of the carcinogenic process. Also for Induction of GIN and for prevention of apopotisis.
- Thank you for your attention. I would like particularly to thank Professor Yamashita for this opportunity. My thanks also to go to the professors who reviewed this work. To Prof Mitsutake for his support and guidance. And to all professors and members of Genken, thank you so very much.