Tg-BRAF transgenic mice

Molecular dissection of papillary thyroid cancer progression to poorly differentiated carcinoma in Tg-BRAF transgenic mice: Evidence for epithelial-mesenchymal transition and identification of candidate pathways.

Abstract

Mice (Tg-BRAF) with thyroid-specific expression of oncogenic BRAF (BRAFV600E) develop papillary thyroid cancers (PTC ) by 3 weeks of age. At 5 months >90% of PTCs are locally invasive, and ~50% have well-defined foci of poorly differentiated carcinoma (PDTC). To investigate the PTC-PDTC progression in Tg-BRAF mice, we performed a microarray analysis using RNA prepared from cells collected by laser capture microdissection from paired samples of PDTC and well-differentiated PTC from the same animal. Analysis of 8 paired samples hybridized to a Operon microarray with a 35473 oligo density found 98 genes with consistent expression changes between PTC and PDTC in at least 7 of the 8 paired samples. EASE analysis indicated that genes involved in cell adhesion and intracellular junctions were significantly represented, with changes consistent with an epithelial-mesenchymal transition (EMT). Decreased expression of E-cadherin and desmocollin 2 and increased expression of procollagen and vimentin, all of which are hallmarks of EMT, were observed in at least 7 of 8 PDTC foci. The upregulation of vimentin in PDTC foci was confirmed by IHC. There were no consistent expression changes in LEF/TCF or in the snail family, suggesting that the Wnt and sonic hedgehog pathways are not involved in the induction of EMT, or in progression to PDTC. By contrast, increased expression of PDGF-B and/or D was found in all 8 PDTC foci. As TGFβI expression is increased in the thyroids of the Tg-BRAF mice, these data are consistent with a role for a TGFβ-activated autocrine loop involving PDGF in EMT. Decreased E-cadherin has also been observed in human BRAFV600E positive anaplastic carcinomas, suggesting that thyroid cancer progression in humans may also involve EMT. Pathways regulating this transition may be of biological and therapeutic interest.

INTRODUCTION

The BRAFT1799A mutation is the most common genetic change in PTC. It is not found in any other form of well-differentiated follicular neoplasm (1). BRAF mutations can occur early in development of PTC, as they are present in microscopic PTCs (2). Most (2,3), but not all (4), studies show that PTCs with BRAF mutations present more often with extrathyroidal invasion and at a more advanced stage. Tall cell variant PTCs, regarded as more aggressive, have a very high prevalence of BRAF mutation (2). Undifferentiated or anaplastic carcinomas arising from preexisting PTCs have a significant prevalence of BRAF mutations, whereas those arising from preexisting follicular carcinoma do not (2,5). These data show that BRAF mutations may be an alternative tumor-initiating event in PTC, and that PTCs with this genotype likely carry a worse prognosis. The role of oncogenic BRAF as a tumor-initiating event has been confirmed in mice with overexpression of B-RafV600E targeted to thyroid cells by means of the thyroglobulin (Tg) gene promoter (6). Tg-BRAFV600E mice develop PTCs with high penetrance early in life, and progress to dedifferentiation, capsular and microvascular invasion, confirming many of the features found in the human tumors.

MATERIALS AND METHODS

Experimental animals:

Creation and initial characterization of the Tg-BRAF2 (mice with thyroid-specific expression of BRAFV600E) have been described (6). Mice were house in . All described procedures were approved by the institutional animal committee.

Thyroid collection and laser capture.

Animals were euthanized with CO2 and thyroids collected and immediately frozen in OTC. Frozen sections were stained with H&E and examined by pathologist (YN) for WD and PD cancer. Twelve serial sections (7.5 mM) were taken from regions found to have a PD focus. The serial sections were stained with HistoGeneTM LCM Frozen Section Staining Kit (Arcturus Bioscience, Inc., Mountain View, CA) and cells from PD focus and a representative region of WD cancer were isolated using the artsus Arcturus PixCell II laser capture microscope System. RNA was isolated from the laser captured cells using PicoPureTM RNA Isolation Kit Kit (Arcturus Bioscience, Inc., Mountain View, CA) and then subjected to 2 round mRNA amplification using the messageAMP RNA amplification kit (Ambion, Austin, TX).

Microarray analysis.

The mouse 70-mer oligonucleotide library version ??? consists of ??? optimized oligos (Qiagen) and was arrayed and printed as previously outlined. The complete gene lists can be viewed at ???. Fluorescence-labeled cDNAs were synthesized from amplied RNA using an indirect amino allyl labeling method via an oligo(dT)-primed, reverse transcriptase reaction. The cDNAs were labeledAmplified RNA was with monofunctional reactive cyanine-3 and cyanine-5 dyes (Cy3 and Cy5; Amersham, Piscataway, NJ). Pairwise hybridizations were done between labeled cDNAs corresponding to unstimulated versus doxycycline-treated cells for each of the cell lines and time points. In addition, to increase the statistical power of the experiment, paired hybridizations were done to compare expression between cell lines at the same time points before or after oncoprotein activation. Details of hybridization and washing conditions can be found at ???. Imaging and data generation were carried out using a GenePix 4000A and GenePix 4000B (Axon Instruments, Union City, CA) and associated software from Axon Instruments, Inc. (Foster City, CA). The microarray slides were scanned with dual lasers with wavelength frequencies to excite Cy3 and Cy5 fluorescence emittance. Images were captured in JPEG and TIFF files, and DNA spots were captured by the adaptive circle segmentation method. Information extraction for a given spot is based on the median value for the signal pixels minus the median value for the background pixels to produce a gene set data file for all the DNA spots. The Cy3 and Cy5 fluorescence signal intensities were normalized. Data normalization was done in two steps for each microarray separately (19-21). First, background-adjusted intensities were log transformed, and the differences (R) and averages (A) of logtransformed values were calculated as R = log2(X1) log2(X2) and A = [log2(X1) + log2(X2)] / 2, where X1 and X2 denote the Cy5 and Cy3 intensities after subtracting local backgrounds, respectively. Second, data centering was done by fitting the array-specific local regression model of R as a function of A. The difference between the observed log-ratio and the corresponding fitted value represented the normalized log-transformed gene expression ratio. The statistical analysis was done for each gene separately by fitting a mixed-effects linear model . Assumptions about model variables are the same as described in reference (7), with array effects assumed to be random and treatment and dye effects assumed to be fixed. Statistical significance of differential expression was assessed by calculating Ps and adjusting for multiple hypotheses testing by calculating false discovery rates (8). Estimates of fold change were also calculated. Data normalization and statistical analyses were done using SAS statistical software package (SAS Institute, Inc., Cary, NC). Gene annotation was supplemented with human and mouse homologues for unknown oligos.

Immunohistochemistry:

Animals were euthanized with CO2 and thyroids collected and immediately placed in 4% PFA. After 24 hours they thyroids were placed in 70% ethanol and embedded in. ??? was ? . Serial sections from region found to contain a PD focus were ??? and incubated with indicated antidbodies. Immunoreactive was detected by incubating with and ?

RESULTS

Gene expression profile of WD and PD thyroid cancers from Tg-BRAF mice: Tg-BRAF2 mice develop PTC by 3 weeks of age and by 12 weeks of the animals had locally invasive PTC and approximately 50% had focal areas of PDTC (6). The PDTC were identified by a solid growth pattern containing spindle-shaped cells (Fig 1A). Additional confirmation that the foci were PDTC was provided by an increased number of mitotic (Fig 1B) and Ki67 positive cells (Fig 1C) as well as the presence of necrotic/apoptosis cells (Fig 1C). To identify gene expression changes involved in the transition from the WD PTC to the PDTC we used LCM to isolate cells from from 8 unique poorly differentiated foci and a representative area of WD PTC from the same Tg-BRAF2 mice. RNA was isolated from the laser captured cells, amplified, labeled with Cy5 or Cy3 and hybridized to operon ??? chip. This identified ??? genes with significant expression changes (p<0.05, FDR<0.1) and of these ??? had an expression changes that was greater 1.5 fold. There were ??? genes products that decreased and ??? that increased. To identify signaling pathways that may mediate or contribute to these expression changes we used EASE analysis to compare our data set to the Gene Ontology and KEGG databases. This found that genes involved in tight junction, ?? and where significantly represented in the PDTC data set. Closer examination indicated that that genes involved in tight juntction and cell contact were decreased, while the intermediate filament gene increased in expression (Table 2). These changes are consistent with an EMT. To confirm cells in the PDTC had undergone EMT a second set of thyroids from Tg-BRAF2 animal were IHC stained for E-cadherin and vimentin, hall marks of EMT. pathways

REFERENCES

1. Kimura ET, Nikiforova MN, Zhu Z, Knauf JA, Nikiforov YE, Fagin JA. High Prevalence of BRAF Mutations in Thyroid Cancer: Genetic Evidence for Constitutive Activation of the RET/PTC-RAS-BRAF Signaling Pathway in Papillary Thyroid Carcinoma. Cancer Res 2003;63:1454-7.

2. Nikiforova MN, Kimura ET, Gandhi M, et al. BRAF Mutations in Thyroid Tumors Are Restricted to Papillary Carcinomas and Anaplastic or Poorly Differentiated Carcinomas Arising from Papillary Carcinomas. J Clin Endocrinol Metab 2003;88:5399-404.

3. Xing M, Westra WH, Tufano RP, et al. BRAF Mutation Predicts a Poorer Clinical Prognosis for Papillary Thyroid Cancer. J Clin Endocrinol Metab 2005;.

4. Puxeddu E, Moretti S, Elisei R, et al. BRAF(V599E) mutation is the leading genetic event in adult sporadic papillary thyroid carcinomas. J Clin Endocrinol Metab 2004;89:2414-20.

5. Namba H, Nakashima M, Hayashi T, et al. Clinical implication of hot spot BRAF mutation, V599E, in papillary thyroid cancers. J Clin Endocrinol Metab 2003;88:4393-7.

6. Knauf JA, Ma X, Smith EP, et al. Targeted expression of BRAFV600E in thyroid cells of transgenic mice results in papillary thyroid cancers that undergo dedifferentiation. Cancer Res 2005;65:4238-45.

7. Wolfinger RD, Gibson G, Wolfinger ED, et al. Assessing gene significance from cDNA microarray expression data via mixed models. J Comput Biol 2001;8:625-37.

8. Reiner A, Yekutieli D, Benjamini Y. Identifying differentially expressed genes using false discovery rate controlling procedures. Bioinformatics 2003;19:368-75.