This Article  • Abstract  • PDF  • Reply to article  • Send to a friend  • Save in My Folder  • Save to citation manager  • Permissions  Citing Articles  • Citation map  • Citing articles on ISI (5)  • Contact me when this article is cited  Related Content  • Similar articles in this journal  Topic Collections  • Endocrine Disease of Head & Neck  • Endocrine Diseases  • Thyroid/ Parathyroid Diseases  • Alert me on articles by topic

Transforming Growth Factor Receptors and p27^kip in Thyroid Carcinoma

Carlos A. Muro-Cacho, MD, PhD; Teresita Muñoz-Antonia, PhD; Sandra Livingston; Douglas Klotch, MD

Arch Otolaryngol Head Neck Surg. 1999;125:76-81.

ABSTRACT
Objective  To investigate the role of cell cycle regulators in the pathogenesis of papillary carcinoma of the thyroid.

Design  Resistance to transforming growth factor –mediated inhibition is a well-known pathogenic mechanism in epithelial neoplasias. In a retrospective study, the expression of transforming growth factor receptors types I and II, cyclin D1, and the cyclin-dependent inhibitor p27^kip, was analyzed by immunohistochemistry. Results were interpreted in the context of clinicopathological data. Patient follow-up ranged from 1 to 18 years, with a mean of 4 years.

Materials  Twenty conventional primary papillary carcinomas and their metastases were selected according to current pathologic criteria. Nonconventional papillary carcinomas (eg, tall-cell, columnar) were excluded from the analysis.

Results  Cyclin D1 was expressed more intensely in the tumor than in adjacent nonneoplastic parenchyma. Within a given tumor, however, there was significant heterogeneity in expression intensity and percentage of positive cells, particularly in metastases. Type I receptors were strongly expressed in 90% of tumors, while 80% of the tumors revealed low to no expression of type II receptors. In 10% of tumors, type I receptors were absent and type II receptors expressed. Simultaneous absence of both receptors was not observed. While p27^kip was strongly expressed in nonneoplastic thyroid, it was not detected in any of the primary tumors or their metastases.

Conclusions  The results strongly suggest that functional abnormalities in type II receptors result in increased levels of cyclin D1 and down-regulation of p27^kip. This would maintain cells in a proliferative state and would promote tumor progression.

INTRODUCTION

Jump to Section  • Top  • Introduction  • Materials and methods  • Results  • Comment  • Author information  • References

IN RECENT YEARS, a large body of evidence has accumulated with regard to the central role played by cell cycle regulatory mechanisms in many cancer types.¹ The G1 phase of the cell cycle has proved to be a rate-limiting step beyond which cells are committed to division.² Progression through this G1 checkpoint depends on the availability of specific growth factors and can be blocked by negative regulators such as transforming growth factor (TGF). Current evidence indicates that the TGF receptors type I (TR-I) and type II (TR-II) are directly involved in TGF-mediated signal transduction. Signaling requires the formation of a heterotetramer containing 2 molecules each of TR-I and TR-II. A model for the mechanism of action of this complex has been proposed by Wrana and Attisano.³ According to this model, TR-II is constitutively autophosphorylated, and ligand binding results in recruitment and phosphorylation of TR-I. Kinase activity of TR-I would then be responsible for the phosphorylation of unknown downstream substrates and the generation of biological responses.

Many tumors become insensitive to the growth-inhibitory actions of TGF by down-regulating TR-II.^4-5 This decrease in TR-II expression correlates with tumorigenicity. For instance, the breast carcinoma cell line MCF7^(-) does not express TR-II, is resistant to TGF-mediated antiproliferative effects, and is tumorigenic. Transfection of this line with TR-II complementary DNA increases the number of surface TR-II, restores TGF sensitivity, and decreases tumorigenicity.⁶ Furthermore, TGF treatment of nontransformed epithelial cells blocks DNA synthesis, suppresses cyclin D1 messenger RNA (mRNA) synthesis and protein expression, and increases the expression of certain cyclin-dependent kinase inhibitors (p27^kip1, p15^ink4), resulting in G1 arrest.⁷

We have previously reported that cyclin D1 protein is overexpressed in thyroid papillary carcinomas and that the levels of cyclin D1 correlate with disease severity.⁸ In this article, we examine a mechanism for this cyclin D1 overexpression in thyroid papillary carcinomas, that is, the dysregulation of its expression by lack of TGF-mediated control. We report the absence of TR-II and p27^kip in primary papillary carcinomas of the thyroid and their metastases. These findings suggest a link between absence of TGF-mediated cell growth inhibition, abnormal G1 phase regulation, and thyroid oncogenesis.

MATERIALS AND METHODS

Jump to Section  • Top  • Introduction  • Materials and methods  • Results  • Comment  • Author information  • References

SELECTION OF CASES

Twenty conventional papillary carcinomas of the thyroid were selected from the tissue archives of the Pathology Department at the H. Lee Moffitt Cancer Center and Research Institute, Tampa, Fla. The diagnosis of papillary carcinoma was made, in hematoxylin-eosin–stained tissue sections, following standard criteria that included the presence of papillae, psammoma bodies, nuclear clearing, nuclear grooves, intranuclear pseudoinclusions, and fibrosis.⁹ Clinicopathological staging was done according to the recommendations of the American Joint Committee on Cancer¹⁰ as follows: T0, no evidence of primary tumor; T1, tumor measuring 1 cm or less; T2, tumor measuring more than 1 cm and less than 4 cm; T3, tumor measuring more than 4 cm; T4, tumor of any size extending beyond the thyroid capsule; N0, no regional lymph node metastasis; N1a, metastasis to ipsilateral cervical lymph nodes; N1b, metastasis to bilateral, midline, or contralateral lymph nodes; M0: no distant metastasis; M1, distant metastases; stage I, any T, any N, M0 in patients younger than 45 years, and T1 N0 M0 in patients older than 45 years; stage II, any T, any N, M1 in patients younger than 45 years, and T2 N0 M0 or T3 N0 M0 in patients older than 45 years; stage III, T4 N0 M0 and any T, N1, M0 in patients older than 45 years; and stage IV, any T, any N, M1 in patients older than 45 years. Tissue blocks containing both tumor and adjacent nonneoplastic thyroid parenchyma and blocks from regional lymph nodes containing metastatic disease were selected for immunohistochemical analysis in each of the 20 cases. The study followed institutional review board guidelines, without imposing added risk or morbidity to patients, and preserving patient confidentiality. All studies were performed at the Pathology Research Core Facility of the University of South Florida College of Medicine and H. Lee Moffitt Cancer Center and Research Institute.

IMMUNOHISTOCHEMISTRY

Sections (5 µm) from formalin-fixed, paraffin-embedded tissues were cut and placed in poly-L-lysine–coated slides. The slides were subjected to deparaffination in xylene and hydration, through a series of decreasing alcohol concentrations, following standard procedures. To allow penetration of reagents, tissue slides were digested with 50-mg/mL proteinase K in sodium chloride sodium phosphate buffer (pH 7.4) at 37°C. After 30 minutes, the slides were heated and extensively washed in sodium phosphate buffer solution (PBS) (pH 7.4) to inactivate the proteinase. Endogenous peroxidase was quenched with a 3% solution of hydrogen peroxide for 20 minutes at 37°C. The slides were washed in deionized water for 5 minutes. Antigen retrieval was performed by placing the slides in a clear plastic container with vented top containing 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" for 2 for 5 minutes. The slides were allowed to cool for 10 to 20 minutes, rinsed extensively in deionized water, placed in PBS for 5 minutes, and drained. Blocking serum was applied, and the slides were incubated in a humid chamber for 20 minutes at room temperature. After blotting, the following primary antibodies were applied at room temperature at a 1:100 dilution: anti-cyclin D1 (C-terminal) polyclonal antibody (Immunotech Inc, Westbrook, Me); anti–TR-II (C-terminal domain), anti–TR-I, and anti-p27^kip (Santa Cruz Biotechnology Inc, Santa Cruz, Calif). After 1 hour, the slides were rinsed in PBS for 5 minutes. For detection, the avidin-biotin complex method (Vectastain ABC Kit, Rabbit IgG, Elite series; Vector Laboratories Inc, Burlingame, Calif) was used following manufacturer's specifications. The biotinylated secondary antibody was applied for 20 minutes at room temperature in a humid chamber. At the end of this incubation, the slides were rinsed in PBS for 5 minutes, followed by the addition of the avidin-biotin complex. Slides were then incubated in a humid chamber for 30 minutes at room temperature and rinsed and placed in PBS for 5 minutes. Diaminobenzidine, prepared following manufacturer's specifications, was applied and color development monitored. When the desired intensity was reached (2-5 minutes), the slides were rinsed in water and counterstained with modified Mayer hematoxylin for 30 seconds. Slides were then washed in running water for 10 minutes, dehydrated, cleared, and mounted with resinous mounting medium. Antigen expression was classified as 0 (no expression), 1+ (<25% of cells), 2+ (25%-50% of cells), 3+ (50%-75% of cells), and 4+ (>75% of cells).

RESULTS

Jump to Section  • Top  • Introduction  • Materials and methods  • Results  • Comment  • Author information  • References

A summary of the data for all 20 papillary carcinomas of the thyroid examined is presented in Table 1. Cases are listed according to disease stage and increasing patient age at the time of initial clinical presentation. The intensity of immunohistochemical expression is listed independently, in each case, for normal thyroid, tumor, and metastasis.

View this table: [in this window] [in a new window] Expression of TR-I, TR-II, p27kip, and Cyclin D1 in Papillary Carcinoma of the Thyroid*

CYCLIN D1 EXPRESSION

To examine the relationship between overexpression of cyclin D1 and the status of TR-II in thyroid papillary carcinomas, 20 primary tumors, their metastases, and corresponding normal tissue counterparts were subjected to immunohistochemical analysis. In agreement with our previous results in the thyroid, and those of others in other tumors,^11-12 cyclin D1 expression was observed both in the nucleus and in the cytoplasm (data not shown). As reported previously,⁸ intensity of expression of cyclin D1 was higher in tumors than in adjacent normal thyroid. Within a given tumor, however, there was significant heterogeneity in expression intensity and percentage of positive cells, particularly in the metastasis. Southern blot analysis of thyroid papillary carcinomas overexpressing cyclin D1 protein did not reveal an increased number of copies or other alterations in the cyclin D1 gene (data not shown). This is in agreement with the results of Lazzereschi et al,¹³ who used the cyclin D1 gene to normalize their Southern blots, and did not observe genetic alterations.

TGF RECEPTORS EXPRESSION

Since increase in cyclin D1 expression could be due to a defect in signaling through TGF receptors,¹⁴ expression of TR-I and TR-II was evaluated in the same 20 thyroid papillary carcinomas. While both receptors were strongly expressed in normal thyroid tissue (Figure 1, A, and Figure 2, A) and TR-I was strongly expressed in the vast majority of tumors (Figure 1, A) and their metastases (Figure 1, B), 80% of the primary tumors (Figures 2, A and B) and the vast majority of the metastases (Figure 2, C) revealed low to no expression of TR-II. Of the 4 cases (20%) expressing TR-II, 3 also showed receptor expression in the metastases. However, in 1 case, the primary tumor expressed TR-II but the metastasis did not. In only 2 tumors (10%), TR-I was absent and TR-II expressed. In no case was the simultaneous absence of both receptors detected, indicating that redundancy is not necessary to escape growth inhibition control. These results are in agreement with those previously reported by Lazzereschi et al¹³ who reported decreased levels of TR-II in nonpapillary thyroid carcinomas, and suggest that escape from TGF antiproliferative control might be a generalized mechanism in thyroid cancer.

View larger version (264K): [in this window] [in a new window] Figure 1. Immunohistochemical detection of transforming growth factor receptor type I in papillary carcinoma (x250). A, Intense cytoplasmic expression is observed in nonneoplastic thyroid parenchyma (upper left) and papillary carcinoma (lower right). B, Strong receptor expression is seen in lymph node metastasis (right).

View larger version (286K): [in this window] [in a new window] Figure 2. Immunohistochemical detection of transforming growth factor receptor type II in papillary carcinoma (x250). A, Receptor expressed in nonneoplastic thyroid parenchyma (upper) and markedly reduced in adjacent papillary carcinoma (lower). No expression was observed in central areas of the tumor (B) and lymph node metastasis (C).

p27^kip EXPRESSION

In proliferating cells, p27^kip is bound to active cyclin-cdk4 complexes. Stimulation of TGF induces a redistribution of p27^kip from cyclin D-cdk4 to cyclin E-cdk2, resulting in inhibition of cdk-2.^{7, 15} To investigate the role played by p27^kip in papillary carcinoma, we have analyzed its expression by immunohistochemistry. As it has been reported in other tumor types,¹⁶ the location of p27^kip, in thyroid papillary carcinoma, is predominantly nuclear. While p27^kip is strongly expressed in normal thyroid, p27^kip was not detected in any of the primary tumors or their metastases (Figure 3). These results are in agreement with previous reports in other neoplasias where p27^kip expression was decreased or absent.¹⁶

View larger version (304K): [in this window] [in a new window] Figure 3. Immunohistochemical detection of p27kip in papillary carcinoma (x100). A, Nuclear expression is observed in nonneoplastic thyroid parenchyma (left). No expression is observed in papillary carcinoma (right). B, No expression is observed in the lymph node metastasis (lower half).

COMMENT

Jump to Section  • Top  • Introduction  • Materials and methods  • Results  • Comment  • Author information  • References

Cell cycle regulatory mechanisms play a fundamental role in oncogenesis. Transition through the different phases of the cycle is the result of the sequential activation and deactivation of a family of cyclin-dependent kinases (CDKs). Enzymatic activity of CDK is regulated at 3 different levels: (1) binding to cyclin proteins, (2) activation of the cyclin-CDK complex by subunit phosphorylation, and (3) CDK inhibition.¹⁷ During the G1 phase, a critical checkpoint of the cell cycle, D-type cyclins (D1, D2, and D3) bind to cdk4 and/or cdk6, and phosphorylate the Rb protein, resulting in release of the E2F family of transcriptional activators and entry into the S-phase.¹⁸ Cells that overexpress cyclin D1 have a shortened G1 phase, are less dependent on growth factors, and are able to traverse the G0/G1 and/or the G1/S transition under conditions that limit the growth of normal cells.¹⁹ Overexpression of cyclin D1 through a variety of mechanisms has been reported in a variety of neoplasias.²⁰ We have previously shown that cyclin D1 is overexpressed in papillary carcinoma of the thyroid, and that the levels of cyclin D1 correlate with disease severity.⁸ Our preliminary Southern blot and Northern blot analyses, and those of others,¹³ have failed to reveal gene amplifications, translocations, or increased mRNA transcription (data not shown). Here we have explored posttranslational mechanisms that may explain the overexpression of cyclin D1 in thyroid papillary carcinomas and its role in thyroid oncogenesis.

Transforming growth factor is an important modulator of cellular proliferation and a potent growth inhibitor of many epithelial cells. It exerts its action at the G1 phase via specific TGF receptors (TR). Despite normal levels of TGF, many tumors become insensitive to TGF-mediated, growth-inhibitory effects through abnormal regulation of TR.^{4, 14, 21} Loss of this negative regulation has been implicated as a major contributor to the development of epithelial tumors. Transforming growth factor blocks G1 progression by specifically suppressing cyclin D1 mRNA and protein expression, without affecting the levels of other D-type cyclins (D2 and D3).²⁰ Furthermore, in the absence of TGF function, cyclin D-cdk4 complexes serve both as a p27^kip reservoir and as down-regulators of its activity, allowing G1 progression.^{4, 7} The activities of the cyclin-CDK complexes are negatively regulated by CDK inhibitors that, in response to a variety of growth-modulating signals, physically associate with and inhibit the activity of CDKs.^{17, 22} The role played in cell cycle regulation by p27^kip, as G1 inhibitor, has been highlighted in recent years. The p27^kip protein, a CDK inhibitor expressed in most cells, is present in maximal amounts during the quiescent (G0) and prereplicative (G1) phases, and decreases as cells are induced to enter the cell cycle.^{7, 23} Mutations in the p27^kip gene and alterations of the p27^kip locus, in chromosome 12p13, have been found in a small number of tumors.¹⁴ Expression of p27^kip has been shown to have antiproliferative activity, in vivo, in a variety of tissues while absence or decreased expression of p27^kip leads to hyperplasia and has been observed in both benign and malignant neoplasias.¹⁶

The marked decrease in expression of TR-II and the absence of p27^kip found in the present study indicate that the mechanisms responsible for regulation of the G1 phase of the cell cycle are severely impaired in papillary carcinoma of the thyroid. Similar findings have been described in intestinal adenomas, where lack of TR-II has been observed concomitantly to increased expression of cyclin D1.²⁰ Transforming growth factor receptor type I, the other component of the receptor heterodimer, is, however, readily detected both in normal thyroid cells and in papillary carcinoma. Our preliminary studies (unpublished results) have revealed point mutations in the TR-II promoter in a small group of these papillary carcinomas. These mutations are accompanied by lack of TR-II mRNA transcription. This suggests that, in the majority of papillary carcinomas, a selective abnormality in the function of the TR-II^{5, 24} may be primarily responsible for the lack of inhibitory control mediated by TGF, which is expressed and secreted by normal thyroid cells.²⁵ We postulate that, in papillary carcinoma, abnormal expression of TR-II results in lack of TGF-mediated inhibitory function, increased expression of cyclin D1, and down-regulation of p27^kip. This would maintain cells in a proliferative state, promoting tumor progression.

AUTHOR INFORMATION

Jump to Section  • Top  • Introduction  • Materials and methods  • Results  • Comment  • Author information  • References

Accepted for publication July 27, 1998.

This work was sponsored in part by the American Cancer Society, Atlanta, Ga (Institutional Research Grant 202) (project 6115-107 LO).

Presented as an abstract at the 40th Annual Meeting of the American Society for Head and Neck Surgery, Palm Beach, Fla, May 14, 1998.

Reprints: Carlos A. Muro-Cacho, MD, PhD, Pathology Department, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Dr, Tampa, FL 33612 (e-mail: murocacho{at}moffitt.usf.edu).

From the Departments of Pathology (Dr Muro-Cacho and Ms Livingston) and Biochemistry and Molecular Biology (Dr Muñoz-Antonia), and Division of Otolaryngology (Dr Klotch), H. Lee Moffitt Cancer Center and Research Institute and University of South Florida College of Medicine, Tampa.

REFERENCES

Jump to Section  • Top  • Introduction  • Materials and methods  • Results  • Comment  • Author information  • References

1. Hartwell LH. Cell cycle control and cancer. Science. 1994;266:1821-1828. FREE FULL TEXT 2. Paulovich AG, Toczyski DP, Hartwell LH. When checkpoints fail. Cell. 1997;88:315-321. FULL TEXT | ISI | PUBMED 3. Wrana JL, Attisano L. MAD-related proteins in TGF-beta signaling. Trends Genet. 1996;12:493-496. FULL TEXT | ISI | PUBMED 4. Moses HL, Yang EY, Pietenpol JA. TGF stimulation and inhibition of cell proliferation: new mechanistic insights. Cell. 1990;60:245-247. 5. Fynan TM, Reiss M. Resistance to inhibition of cell growth by TGF- and its role in oncogenesis. Crit Rev Oncog. 1993;4:493-540. ISI | PUBMED 6. Geiser AG, Burmester JK, Webbink R, Roberts AB, Sporn MB. Inhibition of growth by transforming growth factor- following fusion of two non-responsive human carcinoma cell lines. J Biol Chem. 1992;267:2588-2593. FREE FULL TEXT 7. Polyak K, Kato JY, Solomon MJ, et al. p27^kip, a cyclin-CDK inhibitor, links TGF- and contact inhibition to cell cycle arrest. Genes Dev. 1994;8:9-22. FREE FULL TEXT 8. Muro-Cacho CA, Holt T, Mora L, Livingston S, Klotch D. Cyclin D1 expression as a prognostic marker in papillary carcinoma of the thyroid. Otolaryngol Head Neck Surg. In press. 9. Carcangiu ML, Zampi G, Rupi A, Castagnoli A, Rosai J. Papillary carcinoma of the thyroid: a clinico-pathologic study of 241 cases treated at the University of Florence, Italy. Cancer. 1985;55:805-828. FULL TEXT | ISI | PUBMED 10. Beahrs OH, Henson DE, Hulter RVP, Kennedy BJ. Manual for Staging of Cancer: American Joint Committee on Cancer. 4th ed. Philadelphia, Pa: JB Lippincott Co; 1992. 11. Baldin V, Lucas J, Marcote MJ, Pagano M, Draetta G. Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes Dev. 1993;7:812-816. FREE FULL TEXT 12. Shinozaki H, Ozawa S, Ando N, et al. Cyclin D1 amplification as a new predictive classification for squamous cell carcinoma of the esophagus, adding gene information. Clin Cancer Res. 1996;2:1155-1161. ABSTRACT 13. Lazzereschi D, Mincione G, Coppa A, et al. Oncogenes and antioncogenes involved in human thyroid carcinogenesis. Exp Clin Cancer Res. 1997;3:325-332. 14. Weiser R, Attisano L, Wrana JL, Massague J. Signaling activity of TGF- type II receptors lacking specific domains in the cytoplasm region. Mol Cell Biol. 1993;13:7239-7247. FREE FULL TEXT 15. Ravitz MJ, Yan S, Herr KD, Wenner CE. TGF beta induced activation of cyclin E-cdk2 kinase and down-regulation of p27^kip1 in C3H 10 T1/2 mouse fibroblasts. Cancer Res. 1995;55:1413-1416. FREE FULL TEXT 16. Lloyd RV, Jin L, Ishida N, et al. Mice lacking p27^kip1 display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors. Cell. 1996;85:707-720. FULL TEXT | ISI | PUBMED 17. Grana X, Reddy EP. Cell cycle control in mammalian cells: role of cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and cyclin-dependent kinase inhibitors (CKIs). EMBO. 1995;11:211-219. 18. Bates S, Bonetta L, MacAllan D, et al. cdk6 (PLSTIRE) and cdk4 (PSK-J3) are a distinct subset of the cyclin-dependent kinases that associate with cyclin D1. Oncogene. 1994;9:71-79. ISI | PUBMED 19. Stillman B. Cell cycle control and DNA replication. Science. 1996;274:1659-1664. FREE FULL TEXT 20. Motokura T. Cyclin D and oncogenesis. Curr Biol. 1993;3:5-9. 21. Markovits S, Wang L, Myeroff R, Parsons L, Sun J. Inactivation of the type II TGF- receptor in colon cancer with microsatellite instability. Science. 1995;268:1336-1338. FREE FULL TEXT 22. Lew DJ, Kornbluth S. Regulatory roles of cyclin-dependent kinase phosphorylation in cell cycle control. Curr Opin Cell Biol. 1996;8:795-804. FULL TEXT | ISI | PUBMED 23. Kiyokawa H, Kinerman RD, Manova-Todorova M, et al. Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27^kip1. Cell. 1996;85:721-732. FULL TEXT | ISI | PUBMED 24. Muñoz-Antonia T, Li XC, Jackson R, Reiss M, Antonia SJ. A mutation in the TGF- type II receptor gene promoter associated with loss of gene expression. Cancer Res. 1996;56:4831-4835. FREE FULL TEXT 25. Ohta K, Pang XP, Berg L, Hershman JM. Antitumor actions of cytokines on new human papillary thyroid carcinoma cell lines. J Clin Endocrinol Metab. 1996;81:2607-2612. ABSTRACT