Human Thyroid Carcinoma Papillary, Follicular ...

Distinct Patterns of E-Cadherin CpG Island Methylation in Papillary, Follicular, Hurthle's Cell, and Poorly Differentiated Human Thyroid Carcinoma Jeremy R. Graff, Victoria E. Greenberg, James G. Herman, et al. Cancer Res 1998;58:2063-2066. Published online May 1, 1998.

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[CANCER RESEARCH 58. 2063-2066.

May 15. I998|

Advances in Brief

Distinct Patterns of E-Cadherin CpG Island Methylation in Papillary, Follicular, Hurthle's Cell, and Poorly Differentiated Human Thyroid Carcinoma1 Jeremy R. Graff,2 Victoria E. Greenberg, James G. Herman, William H. Westra, Erwin R. Boghaert, Kenneth B. Ain, Motoyasu Saji, Martha A. Zeiger, Stephen G. Zimmer, and Stephen B. Baylin The Oncology Center [J. R. G.. J. G. H., S. B. B.Â¡and Departments of Pathology Â¡V.E. G., E. R. B., S. G. Z.] and Surgery [M. S., M. A. Z.I. Johns Hopkins University School of Medicine, Baltimore, Maryland 21231: L P. Markey Cancer Center, Department of Microbntlogy and Immunology Â¡V.E. G., E. R. B.. S. G. Z/ and The Thvroid Cancer Research Laboratory, Veterans Affairs Medical Center and Department of Internal Medicine [K. B. A.J. University of Kentucky Medical Center. Lexington, Kentucky 40536

Abstract Expression of the invasion/metastasis suppressor, E-cadherin, is dimin ished or lost in thyroid carcinomas. Yet, mutational inactivation of I'.cadherin is rare. Herein, we show that this loss is associated with hypermethylation of the E-cadherin 5' CpG island in a panel of human thyroid cancer cell lines. This aberrant methylation is evident in 83% of papillary thyroid carcinoma, 11 % of follicular thyroid carcinoma, 40% of Hurthle's cell carcinoma, and 21% of poorly differentiated thyroid carcinomas. Contrary to previous reports, the majority of these poorly differentiated thyroid carcinomas express E-cadherin, but often within the cytoplasm rather than at the cell surface. Together, our data indicate that the invasion/metastasis suppressor function of E-cadherin is frequently com promised in human papillary, Hurthle's cell, and poorly differentiated thyroid carcinoma by epigenetic and biochemical events.

Introduction The Mr 120,000 E-cad3 protein complexes with a, ÃŸ,and y catenins to promote Ca2+-dependent, homotypic celiiceli adhesion and to establish normal epithelial tissue architecture ( 1). Disruption of the E-cadherin/catenin complex may facilitate tumor cell invasion (1). Indeed, reduced expression of the E-cad gene is common to many advanced stage, poorly differentiated carcinomas (1). However, mu tational inactivation of E-cad has been documented in only a few tumor types (2-4). Recent reports have now indicated that E-cad expression may be extinguished in association with aberrant CpG island hypermethylation (5-7), which may be coupled to alterations in transcription factor binding and chromatin conformation (5, 7-10). Immunohistochemical studies have revealed that the different histotypes of human thyroid carcinoma display distinct patterns of E-cad expression. FTCs generally retain the strong membranous E-cad stain ing seen in nonneoplastic thyroid tissue, whereas E-cad immunostaining is reduced and diffusely cytoplasmic in HTCs (11, 12). PTCs show a heterogeneous, "all-or-nothing" pattern of E-cad expression in adjacent cells. Finally, PDTCs, which include ATC and insular thy roid carcinomas, have greatly diminished E-cad expression (11, 12). Received 1/23/98; accepted 3/30/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 V. E. G., E. R. B.. and S. G. Z. were supported by the Medical Center Research Fund and the McDowell Cancer Network. L. P. Markey Cancer Center, University of Kentucky. J. R. G.. J. G. H.. and S. B. B. were supported by NIH Grant CA43318, Oncor Corpora tion, and the Valvano Foundation (to J. G. H.). S. B. B. and J. G. H. receive research funding and are entitled to sales royalties from Oncor, which is developing products related to research described in this report. The terms of this arrangement have been reviewed and approved by the Johns Hopkins University in accordance with its conflict of interest policies. 2 To whom requests for reprints should be addressed, at Johns Hopkins Oncology Center, 424 North Bond Street, Baltimore, MD 21231. 3 The abbreviations used are: E-cad. E-cadherin; FTC. follicular thyroid carcinoma; HTC. Hurthle's cell carcinoma; PTC, papillary thyroid carcinoma; PDTC. poorly differ entiated thyroid carcinoma; ATC, anaplastic thyroid carcinoma; MSP. methylalionspecific PCR.

Thyroid carcinomas, especially FTCs and PDTCs. which lack E-cad immunostaining, have a higher propensity to form distant mÃ©tastases (13), likely reflecting the invasion/metastasis-suppressing function of E-cad (14), and suggesting that E-cad expression may have prognostic significance (13, 15). The mechanism for the loss of E-cad in thyroid carcinoma does not involve irreversible genetic alterations (12). Instead, altered E-cad expression patterns in human thyroid carcinoma likely reflect transcriptional regulation or posttranslational modification (11, 12). Therefore, we have explored the possibility that E-cad expression may be epigenetically inactivated in human thyroid carcinoma. We exam ined methylation of the E-cad 5' CpG island by MSP in a panel of 13 human thyroid cancer cell lines and 43 primary thyroid tissue samples including PTCs, FTCs. HTCs, insular thyroid carcinomas, and ATCs. Our data reveal that the loss of E-cad expression in cell lines and primary carcinomas is associated with aberrant CpG island methyla tion, particularly in PTC and, occasionally, in PDTC. Our data also suggest that the intercellular adhesion function of E-cad may be frequently compromised in PDTCs as a consequence of aberrant cytoplasmic localization rather than decreased expression. Materials and Methods Cell Culture and Primary Tissue. All cell lines were maintained in RPMI 1640 supplemented with 10% fetal bovine serum. Cell lines KAK-1, KAT-10, KAT-7, KAT-4, and KAT-18 were derived as reported previously (16, 17). Dr. Guy J. F. Juillard supplied the cell lines NPA '87, WRO '82, DRO '90, MRO '87, and ARO '81 (18). Dr. Istvan Palyi provided cell line BHT-101 (19). and Dr. N. E. Heldin kindly provided C643 (20). Genomic DNA from cell lines and frozen PTC, FTC, and HTC tissue was extracted as described (7). DNA from the ATC and insular thyroid tumors was extracted from 10-/Miri paraffin sections as described (21). Methylation Analysis. Methylation of the E-cad 5' CpG island was ana lyzed by MSP after bisulfite treatment, as described (22). The primer sets used were described previously as island set 3. which spans the transcription start site of E-cad (23). Analysis of E-cad Protein Expression. Western blot analysis of thyroid tumor cell lines was performed as described (7) using an anti-E-cad antibody (Transduction Laboratories. Lexington. KY) at a 1:2000 dilution. Immunoperoxidase staining for E-cad expression in the PDTCs was performed on 5-fim paraffin sections using the same anti-E-cad antibody at a 1:100 dilution.

Results The expression of E-cad in cell lines derived from the major histotypes of human thyroid carcinoma was examined by Western blot analysis (Fig. IA). A cell line derived from a colloid goiter (KAT7; data summarized in Table 1), two PTC cell lines (NPA'87 and KAT 10), and two FTC cell lines (MRO'87 and KAK-1) expressed the Mr 120,000 E-cad protein. One cell line derived from a metastatic follic ular carcinoma (WRO'82) and four (DRO'90, C643, UHTU'74, and KAT 18) of seven ATC cell lines lacked detectable E-cad expression

2063


E-cad CpG ISLAND METHYLATION

BHT10I

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DRU'90 UHTU'7-t WRO'82

ARO'Hl

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Fig. 1. E-cad expression and CpG island methylation in human thyroid cancer cell lines. A. Western blot analysis for the expression of the M, 120.000 E-cad protein. The specific E-cad band is noted. Additional bands are nonspecific. Approximately 100 fig of total cellular protein was loaded per lane. B, the E-cad CpG island region and the region spanning the transcription start site that is amplified by the MSP primer sets used in this study. C representative MSP analyses of human thyroid cancer cell lines. MSP analyses for KAT-4 and BI1TI01 are not shown hut were identical to those of ARO'81.

B

E-cadherin 5' CpG island Map EMI

500

NPA87

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KAKI

KAT10

1500

KAT18

WRO82

DRO90

UMUMllMUMUMUMUMUM

Tahle I Summary of E-cad rnethylaltun in Intnuin thyroid carcinoma linesKAT7 Cell

typeColloid

lines that expressed E-cad showed no evidence for methylation at the

goiter No+ ++++ PTCPTCFollicular 10KAK-1MRO'87WRO'82DRO'90C643UHTU'74BHTIOIKAT4KAT AT No+ ++++ No+ ++++ adenomaFTCFollicular

transcription start site (Fig. 10, indicating a tight association between the methylation status of the island and the expression of E-cad. The only variant was the E-cad-negative ATC cell line, KAT 18. which shows only slight methylation around the transcription start site (Fig. 1C). Having established that cell lines lacking E-cad expression are densely methylated within the heart of the E-cad promoter-region CpG island, we next sought to determine whether methylation may also be evident in primary human thyroid carcinoma tissues. Our examination of 43 primary tissues revealed a distinct pattern for E-cad methylation that reflects much of the reported literature on E-cad immunostaining in human thyroid carcinoma. Methylation of the E-cad 5' CpG island was not evident in the normal thyroid samples

NoYesYesYesYes+ -1-+ + 4-

metastasisATCATCATCATCATCATCATCE-cad

No+ + -1-+ + NoYes+ + + + -t18ARO'81Cell

dataTumor

UHTU74

of this and other islands that corresponds best to transcriptional silencing (24). These cell lines were also methylated within the region at the 5' edge of the E-cad CpG island (data not shown). The eight cell

E-cadexpression methylation+

NPA'87K

NoPrimary

c643 UMUM

++++

tumor

typeColloid

analyzed (HC15N; Fig. 2 and data not shown). Methylation was apparent in 2 of 10 follicular and Hurthle's cell adenomas (Fig. 2,

goiter adenomaHurthle's Follicular adenomaFollicular

summarized in Table 1), suggesting that this epigenetic change may arise early during tumor development. Five of six primary PTCs showed clear evidence for methylation (Fig. 2, summarized in Table 1), consistent with the frequently reported loss of E-cad expression in PTC (11-13). In FTC, which infrequently shows decreased E-cad expression (11-13), only 3 of 12 tumors showed evidence for meth ylation (Fig. 2, summarized in Table 1), whereas 2 of 5 HTCs showed

carcinomaHTCPapillary carcinoma PDTCInsular carcinomaATCCarcinoma totalsE-cadmethylalion0/11/51/51/92/55/62/21/1011/32

(Fig. 1/4 and summarized in Table 1), consistent with previous reports documenting the absence of E-cad staining in ATCs (11-13). Sur prisingly, the remaining three ATC cell lines (ARO'81, BHT-101, and KAT4) showed significant expression of E-cad (Fig. \A; Table 1), suggesting that a subset of ATCs retain E-cad expression. Because loss of E-cad has been associated with dense hypermethylation of the 5' CpG island (5-7) and because irreversible genetic alterations of E-cad are rare in thyroid carcinoma (12), we next examined the methylation of the E-cad CpG island in these cell lines by MSP. Fig. IÃŸis a schematic of the E-cad gene 5' CpG island and

porci PDTC;PDTCJPDTC4PDTCSPDTCÃ’ I

M I

M I

M I

M I

M I

PDTC7

PDTCÂ«

PDTC9

PDTC10

PDTC1I

M

depicts the region analyzed by MSP, which spans the transcription start site of the gene. These analyses show an inverse relationship between E-cad expression and methylation of the 5' CpG island. Four

Fig. 2. MSP analysis for methylation of the E-cad CpG island primary follicular adenoma (FA), papillary (PTC), follicular (FTC), Hurthle's cell adenoma (HA) and

of the five cell lines lacking E-cad expression were heavily methyl ated around the transcription start site of E-cad (Fig. 1Q, the portion

carcinoma (HTC), adjacent normal (HCN). or poorly differentiated thyroid carcinomas (PDTC). E-cad expression for PDTC2 is depicted in Fig. 3. B and E. PDTC3 is depicted in A and D. and PDTC8 is depicted in C and F.

2064


E-cad CpG ISLAND METHYLATION

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Fig. 3. E-cad immunostaining in PDTCs. A, a representative ATC sample (PDTC3) with normal memhranous staining for E-cad. B. a typical area of greatly diminished H-cad staining in an ATC sample (PDTC2) with heterogeneous loss of E-cad staining. C, a typical example of diffuse cyloplasmic E-cad staining (PDTCS). D. E. and F, 2x magnifications of A, B, and C, respectively.

methylation (Fig. 2, Table 1). However, Â¡nPDTC, which reportedly shows little to no E-cad immunostaining ( 11-13), only 3 of 14 showed

ations of E-cad are rare in human thyroid carcinoma ( 12). Our current data provide the first evidence that the loss of E-cad expression in

evidence for methylation (Fig. 2 and summarized in Table 1). Two of these three were classified as insular thyroid carcinomas (Fig. 2 and summarized in Table 1). PDTCs generally have diminished E-cad expression (11-13) but show no evidence for irreversible genetic alterations in E-cad (12) and only infrequent involvement of promoter-region methylation (Fig. 2 and Table 1). These results suggest that E-cad expression and function may be disrupted in PDTC as a consequence of aberrant cell signaling events. To ensure that our PDTC samples reflect the loss of E-cad expression documented previously for PDTC (11-13), we evaluated E-cad expression by immunohistochemistry in 12 PDTCs. The majority of these samples expressed E-cad in a mixed pattern and generally with less intensity than adjacent normal tissue (exemplified by Fig. 3, A, C, D. and F). The three samples with methylation (one ATC and two insular carci nomas) showed heterogeneous, patchy loss of E-cad immuno staining (such pockets of loss are exemplified by Fig. 3, B and E). Four of the remaining 10 ATCs retained distinct membranous staining for E-cad, whereas 6 of these ATCs showed predomi nantly diffuse, cytoplasmic staining for E-cad (exemplified in Fig. 3, C and F). Such altered subcellular distribution of E-cad is unlikely to be a consequence of E-cad mutation because E-cad-

primary human thyroid carcinoma is associated with methylation of the E-cad 5' CpG island, as has been documented in primary breast,

herin mutations in thyroid cancer are rare (12). Furthermore, no mutations were detected in the E-cad-expressing ATC cell line, ARO'81 (data not shown). Discussion E-cad expression is decreased or lost frequently in PTC and PDTC and infrequently in FTC (11-13). Such loss in PTC and PDTC may be an important prognostic indicator, perhaps reflecting a capacity to metastasize to distant sites (13, 15). Yet, irreversible genetic alter

prostate, and hepatocellular carcinoma (6, 7). Dense methylation within the heart of the E-cad 5' CpG island is tightly associated with the lack of E-cad expression in a panel of 13 human thyroid cell lines. Furthermore, methylation is quite frequent in primary PTC (80%), which reportedly shows heterogeneously reduced E-cad expression (11, 12). Methylation is also apparent in 40% of HTC, which often shows reduced E-cad staining intensity (11). Methylation is infrequent in FTC (11%), which generally retains strong homogeneous surface expression of E-cad, and in ATC (10%), which reportedly shows greatly reduced E-cad expression (11-13). Contrary to these reports, the majority of our ATC samples actually express E-cad, although the diffuse cytoplasmic E-cad immunolocalization in many of these ATCs suggests that the intercellular adhesion function of E-cad may be compromised. The patterns of E-cad methylation, coupled with the patterns of E-cad immunostaining, suggest distinct differences for the role of E-cad in the malignant progression of PTC, FTC, and PDTC. PTC shows the greatest incidence for methylation and a well-described heterogeneous loss of E-cad expression, suggesting that methylation is associated with the inactivation of E-cad in this histotype of thyroid carcinoma. Similarly, a significant fraction of HTCs also show E-cad CpG island methylation and generally display decreased E-cad im munostaining (11). By contrast, FTC generally retains strong surface expression of E-cad and rarely demonstrates detectable E-cad meth ylation, suggesting that loss of E-cad does not play a role in the malignant progression of FTC. Finally, our data suggest that E-cad function is frequently disrupted in PDTC, related either to methylation-associated silencing or to the inappropriate localization of E-cad within the cytoplasm.

2065


h-cad CpG ISLAND METHYLATION

Cytoplasmic localization of E-cad has been documented previously in human breast carcinoma (25), a small fraction of PDTCs and HTCs (11, 12), and a high proportion of human pancreatic adenocarcinomas (26), a tumor type that shows no evidence for E-cad CpG island methylation.4 The biochemical mechanism responsible for this cytoplasmic translocation is unclear. However, it is interesting that pan creatic adenocarcinoma and anaplastic thyroid carcinoma share a significant propensity for mutational activation of the ras oncogene (27). It is plausible that aberrant cell signaling events, like activation of the ra.v-signaling cascade, could induce the cytoplasmic transloca tion of E-cad in these tumors. Indeed, activation of the src oncogene, which can signal through ras. changes the distribution of E-cadherin from normal surface expression to a more diffuse, cellular pattern of expression (28). Additionally, activation of protein kinase C, which may be involved in ra.v-mediated cell signaling, can also elicit cyto plasmic translocation of E-cadherin (29). Finally, overexpression of the Wnt oncogene in HC11 cells elicits a similar cytoplasmic redis tribution of E-cad (30). Hence, multiple signaling cascades may impact upon the subcellular distribution and function of E-cad. In deed, expression of the ras oncogene in MDCK cells induces invasiveness as a function of reduced E-cad-mediated celiiceli adhesion (14, 31). In these cells, E-cad-mediated celiiceli adhesion is restored, and invasion is inhibited by activation of Tiaml-Rac signaling, which antagonizes the oncogenic ras signal in those MDCK cells (32). In summary, our data demonstrate that silencing of the E-cadherin gene is frequently associated with dense methylation of the E-cad 5' CpG island in cultured cell lines of human thyroid cancer. Further more, our analysis of E-cad CpG island methylation, in conjunction with reports on E-cad expression, suggest that methylation-associated silencing of E-cad is primarily important in PTCs, HTCs, and a subset of PDTCs, but not FTCs. Furthermore, our present data clearly show that the intercellular adhesion function of E-cad may be frequently disrupted in PDTCs as a consequence of a cytoplasmic redistribution of E-cad. Together, these data suggest that the invasion/metastasis suppressor function of E-cadherin may be frequently compromised in human thyroid carcinoma by epigenetic and biochemical events, rather than irreversible mutational events. Acknowledgments We thank Dr. Douglas Ball for helpful discussion and Dr. Joe Gray and his laboratory for helpful discussion and sequencing of the E-cadherin coding region in the cell line ARO'81. We also thank Dr. Guy F. Juillard (Dept. of Radiation Oncology, UCLA School of Medicine). Dr. Istvan Palyi (National Institute of Oncology. Budapest, Hungary), and Dr. Nils-Erik Heldin (Uppsala University, Uppsala, Sweden) for generously providing various thyroid tumor cell lines. We also thank Tammy Means for assistance in assembling the manuscript.

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