Authors J. West and T. A. Munoz-Antonia have contributed equally to the preparation of this manuscript.
Transforming Growth Factor-β Type II Receptors and Smad Proteins in Follicular Thyroid Tumors †
Article first published online: 2 JAN 2009
Copyright © 2000 The Triological Society
Volume 110, Issue 8, pages 1323–1327, August 2000
How to Cite
West, J., Munoz-Antonia, T., G. Johnson, J., Klotch, D. and Muro-Cacho, C. A. (2000), Transforming Growth Factor-β Type II Receptors and Smad Proteins in Follicular Thyroid Tumors . The Laryngoscope, 110: 1323–1327. doi: 10.1097/00005537-200008000-00019
- Issue published online: 2 JAN 2009
- Article first published online: 2 JAN 2009
- Manuscript Accepted: 9 FEB 2000
- Transforming growth factor-β;
- Smad proteins;
- follicular thyroid tumors.
Objective Resistance to transforming growth factor (TGF)-β–mediated cell growth inhibition is a well-known pathogenic mechanism in epithelial neoplasia. TGF-β signaling requires normal function of downstream mediators such as TGF-β receptors (TβRs) and Smad proteins. The goal of this study is to investigate the expression of components of the TGF-β signaling pathway in follicular tumors of the thyroid.
Study Design Twenty follicular thyroid neoplasms were classified as adenomas (11) or minimally invasive follicular carcinomas (9) according to current pathological criteria. Protein expression was evaluated to identify differences between benign and malignant tumors that could be used as an adjunct to histopathological analysis.
Methods Paraffin-embedded tissue sections containing tumor and adjacent nonneoplastic parenchyma were analyzed by immunohistochemistry for the expression of TβR type II (TβR-II) and Smad2, Smad4, Smad6, and Smad7. Expression of each protein in the tumor was compared with that of the corresponding adjacent nonneoplastic thyroid parenchyma.
Results TβR-II expression was lost in 78% of the carcinomas. In the remaining 22%, TβR-II was preserved but Smad2 expression was lost. In all conventional adenomas, however, TβR-II expression was maintained. Furthermore, all tumors with normal expression of all proteins were adenomas.
Conclusions Downregulation of TβR-II is a consistent abnormality in follicular carcinomas and can be used to differentiate minimally invasive carcinomas from adenomas. Also, downregulation of Smad proteins is another mechanism by which carcinomas can become independent from TGF-β–mediated growth inhibition.
Thyroid carcinoma accounts for approximately 1.5% of all cancers in the United States, and follicular carcinoma (FC) represents 10% of all thyroid cancers. 1 Although some follicular carcinomas are obviously invasive and easy to diagnose, minimally invasive follicular carcinoma (MIFC) often presents as a nodule that is clinically indistinguishable from a variety of benign processes. 2 The distinction of MIFC from follicular adenoma requires the demonstration of capsular and vascular invasion. 2 Given the focal nature of this invasion, this differential diagnosis cannot be made by fine-needle aspiration and is often very difficult to make by histopathological assessment.
Transforming growth factor-β (TGF-β) belongs to an evolutionarily conserved superfamily of secreted factors that control cell fate by regulating proliferation, differentiation, motility, adhesion, and apoptosis. 3,4 TGF-β interacts with a complex of three types of cell surface receptors—TGF-β receptor types I, II, and III (TβR-I, TβR-II, and TβR-III). 5–7 TβR-III seems to modulate extracellular matrix composition, whereas TGF-β binding to TβR-II activates the TβR-I serine/threonine kinase and initiates a cascade of intracellular signals transduced by a family of related proteins known as Smad. 5,8,9 In normal epithelial cells, TGF-β causes reversible arrest in the mid-to-late G1 phase of the cell cycle. 4–6 Many malignant cells, however, become refractory to this antiproliferative effect, resulting in tumor development. 4,10–13 In some tumors this loss of negative regulation is accomplished by downregulation of TβR-II, indicating a direct relationship between levels of TβR-II, resistance to TGF-β, and oncogenesis. 14–24
We have previously reported that in papillary carcinoma of the thyroid, expression of TβR-II is markedly decreased. 25,26 In the present study, we have investigated the expression of TβR-II and Smad proteins in follicular neoplasms. We found that TβR-II is consistently absent in MIFC, whereas it is normally expressed in nonneoplastic thyroid tissue adjacent to the carcinoma and in all conventional adenomas examined. Furthermore, abnormalities in Smad protein expression are common in MIFC, whereas they are rarely seen in follicular adenomas. These results indicate that anomalies in the TGF-β signaling pathway play an important role in the oncogenesis of follicular carcinoma of the thyroid, and that expression of TβR-II is an excellent molecular marker to distinguish benign from malignant follicular tumors.
MATERIALS AND METHODS
Selection of Cases
A total of 20 follicular neoplasms were selected from the tissue archives of the pathology departments at the H. Lee Moffitt Cancer Center and the James A. Haley Veterans Administration Hospital (University of South Florida, Tampa, FL) from 1987 to 1998. The tumors were classified as follicular adenoma (11 cases) or minimally invasive carcinoma (9 cases) according to strict histopathological criteria 2 (Table I). Four adenomas were classified as oncocytic. Patients had not previously received any form of systemic or local therapy. Paraffin-embedded tissue sections containing tumor and nonneoplastic thyroid tissue in the same slide were selected for immunohistochemical studies.
MIFC = minimally invasive follicular carcinoma; N = non-neoplastic adjacent tissue; T = tumor; 0 = negative; 1+ = positive in < 25% of cells; 2+ = positive in 25% to 50% of cells; 3+ = positive in > 50% of cells.
Five-micrometer sections from formalin-fixed, paraffin-embedded tissues were cut and placed in poly-l-lysine–coated slides. Slides were subjected to deparaffinization in xylene and hydration through a series of decreasing alcohol concentrations following standard procedures. Endogenous peroxidase was quenched with a 3% solution of H2O2 for 20 minutes at 37°C. Slides were then washed in deionized water twice for 5 minutes. Antigen retrieval was performed by placing slides in citrate buffer (0.1 mol/L citric acid, 4.5 mL; 0.1 mol/L sodium citrate, 21.5 mL; deionized water, 225 mL) in a microwave oven set (on “high 2”) for 5 minutes. Slides were allowed to cool for 20 minutes, rinsed in deionized water, placed in phosphate-buffered saline (PBS) for 5 minutes, and drained. Blocking serum was applied, and slides were incubated in a humid chamber for 20 minutes at room temperature. After blotting, primary antibodies were applied for 1 hour at room temperature, at the dilutions indicated below. Slides were rinsed and placed in PBS for 5 minutes. For signal detection, the Vectastain ABC Kit Rabbit Immunoglobulin G (IgG) Elite series (Vector Laboratories, Burlingame, CA) was used as indicated by supplier. The biotinylated secondary antibody was applied for 20 minutes at room temperature in a humid chamber. Slides were rinsed and placed on PBS for 5 minutes. The avidin-biotin complex (ABC) was added and slides were incubated in a humid chamber for 30 minutes at room temperature, rinsed, and placed in PBS for 5 minutes. Diaminobenzidine (DAB) was prepared following package instructions and applied, and color was developed until desired intensity was reached (2–5 min). Slides were rinsed in tap water and counterstained with modified Mayer's hematoxylin for 30 seconds. Slides were washed in running water for 10 minutes, dehydrated, cleared, and mounted with resinous mounting medium.
The following primary antibodies were used at the indicated dilutions: Smad2 at 1:25 and Smad4 at 1:50 (Upstate Biotechnologies, Lake Placid, NY); Smad6 at 1:50 (Zymed Laboratories, San Francisco, CA); Smad7 at 1:50 (Santa Cruz Laboratories, Santa Cruz, CA); cyclin D1 at 1:400 (Coulter-Immunotech, Miami, FL); and TGFBrII-TMA (Teresita Munoz-Antonia) at 1:50. The location of all proteins was predominantly cytoplasmic. Protein expression was classified as 0 (no expression), 1+ (<25% of cells), 2+ (25%–50% of cells), or 3+ (>50% of cells).
Minimally Invasive Follicular Carcinoma
Results for minimally invasive follicular carcinoma are shown in Figure 1 (right panel). A total of nine cases satisfied strict histopathological criteria for MIFC. 2 In seven of these nine cases (78%), TβR-II was undetectable (Fig. 1, right panel, A2). Five of these seven (71%) lacked Smad2 (Fig. 1, right panel, B2), and two of these five (40%) also lacked Smad4 (Fig. 1, right panel, C, upper right). In the two cases (22%) with preserved TβR-II, Smad2 was undetectable. Therefore all MIFCs were missing at least one essential component of the activating (downstream) TGF-β pathway. One component was missing in four (36%), two components in five (56%) and all three components in two (22%) cases (Table I). However, results were different for proteins of the inhibitory component of the pathway. Whereas Smad7 was present in all cases at levels similar to adjacent nonneoplastic tissue, Smad6 was overexpressed in all cases (Fig. 1, right panel, D, right).
Results for conventional adenoma are shown in Figure 1, left panel). A total of 11 cases satisfied the histopathological criteria for follicular adenoma. 2 Of these 11 cases, 7 (64%) were classified as “conventional” and 4 (36%) were composed of oncocytes in more than 75% of their volume and were classified as “oncocytic adenomas” (Table I). TβR-II expression was preserved in the seven (64%) conventional adenomas (Fig. 1, left panel, A, lower right). However, in three (43%) of these adenomas Smad2 was undetectable, and in two of these three (75%) Smad4 was also undetectable. Therefore one component of the activating pathway was missing in one (11%) of the seven conventional adenomas and two components were missing in two (22%). No significant changes were observed in Smad7 and Smad6 (Fig. 1, left panel, D2).
Of the four oncocytic adenomas, two (50%) lacked TβR-II expression, one (25%) lacked TβR-II and Smad2, and one (25%) lacked only Smad4. Smad6 was mildly overexpressed in three (75%). No significant alterations were found in Smad7 (Table I).
The distinction of follicular adenoma and minimally invasive follicular carcinoma requires histological demonstration of invasion of the full thickness of the tumor capsule and/or invasion of venous-caliber extracapsular vessels, preferably with tumor cells attached to the vessel wall. 2 Because of the focal nature of this invasion, extensive sampling of the tissue is often required for its demonstration. Occasionally, strict criteria are not fulfilled and the distinction of adenoma from carcinoma cannot be made. In these cases, molecular markers differentially expressed in these neoplasms would facilitate this differential diagnosis. Furthermore, little is known about the mechanisms responsible for neoplastic transformation in these tumors.
Proteins that regulate the cell cycle have been shown to be good molecular predictors of tumor behavior in many tumor types. 25–31 In normal epithelial cells, TGF-β causes reversible arrest in the mid-to-late G1 phase of the cell cycle. 4–6 Many malignant cells, however, become refractory to this antiproliferative effect via downregulation of TβR-II, indicating a direct relationship between levels of TβR-II, resistance to TGF-β1, and oncogenesis. 14–20 Furthermore, abnormalities of Smad protein expression and function, primarily Smad4, have been shown in several malignancies, including colon and pancreatic carcinoma. 32–35
We have previously shown that the TGF-β signaling pathway is aberrantly regulated in papillary carcinomas of the thyroid. 21,22 This suggests that the TGF-β pathway plays an important role in thyroid oncogenesis and that protein members of this cascade could provide diagnostic and prognostic information.
In the present study we have observed downregulation of TβR-II in 78% of carcinomas and in 50% of oncocytic adenomas. In all conventional adenomas, TβR-II expression was preserved. Downregulation of Smad2 and Smad4 occurs in both benign and malignant tumors. Simultaneous downregulation of all three proteins, however, was observed only in carcinomas (22%). The expression of Smad7 was minimally altered in both benign and malignant tumors. Interestingly, however, overexpression of Smad6 was consistently observed in carcinomas and in the majority of oncocytic adenomas. Although the number of oncocytic tumors examined is small, their profile is somewhat intermediate between adenomas and carcinomas. This may explain the more unpredictable behavior of oncocytic tumors. 2 Some adenomas lacked Smad2 or Smad4, or both, and two of the carcinomas lacked only Smad2. Because TβR-II is preserved in these tumors, other proteins, such as Smad3, may compensate for the function loss attributable to downregulation of Smads. However, how the signal is transmitted in the absence of Smad4 is unclear. 28,31
In general, the profile of the vast majority of carcinomas is as follows: 1) downregulation of TβR-II, 2) downregulation of one or two of the activating Smads, and 3) overexpression of Smad6. The profile of the majority of conventional adenomas is as follows: 1) normal expression of TβR-II, 2) variable downregulation of activating Smads, and 3) normal expression of Smad6. Since no recurrences or metastases have been reported in any of these tumors to date, correlation of these abnormalities with clinical behavior cannot be performed at this time.
The present study strongly suggests that expression of members of the TGF-β pathway could be used as an adjunct to histopathological assessment in the differential diagnosis of benign, non-oncocytic, adenomas from minimally invasive follicular carcinomas. Also, anomalies in the TGF-β signaling pathway seem to play an important role in the oncogenesis of follicular carcinoma of the thyroid.
- 2.Tumors of the thyroid gland. In: Atlas of Tumor Pathology [series 3, fascicle 5]. Washington, DC: Armed Forces Institute of Pathology, 1992., , .
- 22.Transforming growth factor β resistance in human small cell lung cancer cell lines: transcriptional inactivation of the type II receptor. Proc Am Assoc Cancer Res 1998; 39:250–255., , , .