82 { 1991) R7- R 12 0 1991 Elsevier Scientific Publishers Ireland, Ltd. ~303-7207/91/$~3.~~

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Human t~y~~tropi~ receptor gene: expression in thyroid tumors and correlation to markers of thyroid differentiation and d~differe~tiati~~ G. Brabant 2,1,C. Maenhaut ‘, J. Kiihrle 2, G. Scheumann j, H. Dralle 3, C. Hoang-Vu ‘, R-D. Hesch ‘, A. von zur Miihlen ‘, G. Vassart 1 and J.E. Dumont ' ~nst~tl~te of I~ter~~sc~~i~nu~ Rese~rc~t~ ~~~cul~ of Medick. and ~ep~r?~e~t~

Free &tit versity of

'

Erussels, Brrsssels, ~el~it~~

of ' ClinicalEndocr~~~~)l~&~umi .’ Swgety. ~~ed~~~~tiscjte~~c~tsc~tu~e ~a~~t~t,er~ Hunnor~er, F.R. G.

(Received 10 September 1991: accepted 1I September 1991)

Key words: Thyrotropin receptor; Thyrogiobuiin; Thyroid peroxidase: c-myc; @-Actin; mRNA expression; Thyroid carcinoma; Hypcrfunclioning adenoma

Human thyrotr~pin (TSH) receptor steady~state transcript levels were analyzed by Northern blot analysis in thyroids of patients with thyroid carcinoma, with hyperf~nctioning adenoma and in normal controls. In control tissue and benign tumors expression levels of TSH receptor mRNA were high whereas in anaplastic carcinomas no normal TSH receptor mRNA was detected. In papillary and follicular tumors it varied from normal to markedly reduced levels. Thyroid peroxidase (TPO) and thyrogiobulin (Tg) mRNA were strongly expressed in normal tissue and in hyperfunctioning adenomas but were completeIy lost in all anaplastic tumors. In papillary tumors expression of TPO and Tg mRNA varied from normal to a complete loss of expression of either TPO, Tg or both. Tg and TPO steady-state expression did not correlate to TSH receptor transcript levels. C-myc mRNA was highIy expressed in anaplastic carcinomas, very variable in normal controls and in differentiated thyroid tumors and low in hyper~nctioning adenomas. In summary, TSH receptor mRNA is persistently expressed in all differentiated thyroid tissues and tumors but lost in undifferentiated carcinomas. Its persistence far along the transformation pathway further supports the concept that this gene which inserts the thyrocytes in the physiological regulatory network is almost constitutively expressed in this cell.

Introduction Interaction of thyrotropin (TSH) with its specific receptor stimulates, via cyclic ASP-mediated pathways. thyroid function and the expression

Address for correspondence: G. Brabant, department of Clinical Endocrinology, Medizinische Hochschule Hannover, Konstanty Gutschowstr. 8, D-3000 Hannover 61, F.R.G.

of differentiation as assessed in culture, by iodide trapping, thyroglobulin (Tg) and thyroid peroxidase (TPO) gene expression, and in many species, including the human of growth (for review see ~aenhaut et al., 1990). Epidemiologica evidence supports a major role of TSH in the growth control of differentiated but not anaplastic thyroid carcinomas (Hay, 1990; Venkatesh et al., 1990). Comparing retrospectively the recurrence rate in patients, with or without thyroid hormone

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therapy following surgery, the risk is increased in patients with elevated TSH levels (Mazzaferri, 1981; Cady et al., 1983; Hay, 1990). Moreover. serum Tg, which is secreted by the majority of differentiated thyroid carcinomas in relapse, can be suppressed by thyroid hormones, most probably via a decrease in TSH (Schlumberger et al., 1986). However, Scatchard analysis of TSH receptors in patients with thyroid carcinomas revealed wide individual variations of TSH binding sites (Field et al., 1976; Carayon et al., 1980; Abe et al., 1981; Saltiel et al., 1981; Chang et al., 1988). The recent cloning of the human TSH receptor (Libert et al., 1989; Nagayama et al., 19X9; Mishrahi et al., 1990) allows us to invcstigate the expression of the TSH receptor gene in normal thyroid tissue in comparison to benign and malignant thyroid tumors and to correlate these findings with steady-state mRNA levels of thyroid differentiation markers such as Tg and TPO and a commonly used marker of proliferation and dedifferentiation, c-myc. Materials

and methods

Thyroid tissue was obtained from patients undergoing surgery for clinical indications. Only cancer tissue fragments macroscopically free of the tumor capsule were used for histological and gene expression studies. Specimens for the latter investigations were immediately frozen in liquid nitrogen and stored at - 80 ’ C. Histological diagnosis was obtained for all tissues. Details of the patients and the controls are given in Table 1. ‘Hot nodules’ as detected by thyroid scintiscan were well encapsulated lesions and can thcreforc be classified as hyperfunctional adenomas. This protocol was approved by the local Committees on Medical Ethics and all patients gave their written consent. Total RNAisolation and Northern blot analysis were carried out as described previously (Reuse et al., 1990). DNA probes for the human TSH receptor (2.4 kb BarnHI-XhoI insert of a pSVL construct (Libert et al., 1989)), for c-myc (1398 bp ClaI fragment of PKH47 human c-myc obtained from Dr. Saule), TPO (1900 bp EcoRI PBS fragment of hTP0 (Libert et al., 1987)), Tg (900 bp &I fragment pBR322 pHTg 1 (Brocas et al.,

1982)) and r-p-actin (1 kb h~1I bluescript fragment of @actin obtained from Dr. J.E. Leonard) were LY-j2 P-labelled by random priming extension to a specific activity of approximately 2 X 10” cpm/pg (Tg 10’ cpm/pg). The same filters were probed successively with TSHR, c-myc, TPO, pactin and Tg DNAs. Results Northern blots of total mRNA from 11 normal human thyroids (five obtained during surgery for hyperparathyroidism, six obtained as normal tissue in patients with thyroid tumors, see Table 1) revealed two major hTSHR transcripts of 4.6 and 4.4 kb and two minor transcripts of 1.9 and 1.1 kb with a low interindividual variability in the level of expression (Libert et al., 1989). The relative amounts of the various forms were proportional (Fig. 1). A comparable expression level of hTSHR mRNA was found in all patients with hyperfunctional adenomas (Fig. 2). In contrast, steady-state levels of hTSHR mRNA were not detectable in three out of four patients with anaplastic carcinomas and the only patient with detectable levels had evidence of an altered transcript showing only a single band around 4.5 kb (Fig. 2, confirmed by long-term exposure of the filter, data not shown). All 14 patients with papillary tumors had detectable levels of hTSHR mRNA. In four patients where tissue specimens of normal thyroid tissue were available for direct comparison TSHR mRNA was reduced in the tumor samples (Fig. 1). The patient with a follicular carcinoma exhibited low hTSHR mRNA expression levels compared to normal controls (Fig. 2). Comparable levels of hTSHR transcripts were found in three patients with hyperfunctional adenomas and in normal adjacent tissue. In normal thyroids as in hyperfunctional adenomas uniformly strong signals of TPO and Tg steady-state mRNA expression were detected whereas Tg and TPO expression was highly variable in patients with differentiated carcinomas and lost in all patients with undifferentiated carcinomas (Fig. 2). In papillary carcinomas normal expression of TPO (n = 5) and Tg (n = 5) mRNA was found as well as reduced levels (n = 6 for TPO or Tg respectively) or complete loss of ex-

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pression (n = 3). The relative expression of TPO and Tg was not parallel, showing complete loss of one mRNA or the other or both (Fig. 2, patients 6, 7, 12, and 17). In the four patients in which normal and tumor tissue could be compared TPO and Tg mRNAs were reduced in the tumor tissue. No clear relationship in the steady-state mRNA levels of TPO and Tg with hTSHR was found in patients with papillary tumors. A greatly reduced but positive signal for both differentia-

TABLE

I

CLINICAL Classification differentiated Patient

tion parameters was visible after 2 weeks of exposure in the patient with follicular carcinoma. C-myc expression varied considerably even in normal tissues. Very low transcript levels were found in three hyperfunctional adenomas. One patient with a hyperfunctional adenoma and two patients with papillary carcinomas showed clearly higher expression of c-myc mRNA in the adjacent normal tissue than in the tumor (Fig. 1, patients 13 and 18; Fig. 2, patient 21). In the

DATA

OF PATIENTS

WITH

THYROID

CARCINOMAS

AND CONTROLS

of tumor differentiation according to a subjective classification by the pathologist: undifferentiated (-1 to well (+ + + 1. TSH serum levels refer to preoperative serum levels. Patients were not treated with thyroxine. Age (years)

Sex

Histology

Tissue source

Tumor size (0 in cm)

TSH (mU/II < 0.03 nd. n.d. < 0.03

84 59 64 72

ate ate ate ate

Ln. Thyroid Thyroid Thyroid

51

ftc

Thyroid

67 82 77

ptc/atc

Thyroid Local Thyroid

4 Rec. 2

Ptc Ptc nt

Grade of differentiation

++ + ++ t

n.d. 0.6 n.d.

78

F

Ptc nt

Thyroid

5

10 I1 I2 I3

47 29 62 34

F M M M

Ptc Ptc Ptc Ptc nt

L.n. Thyroid Thyroid Thyroid

Rec. 4 5 5

+ + ++ ++

32.3 I.6 1.5 1.5

I4 I5 16 17 I8

76 79 84 16 50

F F M F F

Ptc Ptc Ptc Ptc Ptc nt

L.n. Local L.n. Thyroid Thyroid

Rec. Rec. Rec. I 4

++ + + ++ +

I.5 < 0.03 n.d. I.6 0.9

1’)

67

F

Ptc

Ln.

Rec.

+

< 0.03

20

83

F

21

69

M

22

85

F

ha nt ha nt ha

23-27

22-67

4xF/ 1xM

0.2

nt

ate = anaplastic carcinoma; ftc = follicular carcinoma; ptc = papillary carcinoma; mas; Local = local recurrence; L.n. = tissue from lymph node; Rec. = recurrence;

nt = normal thyroid; ha = hyperfunctional n.d. = not determined.

adeno-

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uniformly high c-myc anaplastic carcinomas, mRNA expression was found (Fig. 2). No apparent correlation was detected between c-myc transcript levels in papillary carcinomas and hTSHR expression. Acridine orange staining was used to normalize the amount of total RNA (10 pg) loaded onto the filters. Initially it was planned to utilize /?actin as an additional means to quantify mRNA. However, in contrast to acridine orange staining p-actin mRNA expression levels (Figs. 1 and 2) a high interindividual variation was observed in

--p * TSHR

Q

TF’O

_

Tg

eBU 0CL 617

c-myc

i h 7

I

z

0

12

5

L)

TG 2

3

41

m

E

_c 21

Fig. 2. hTSHR (arrows indicate 4.6 and 4.4 kb), TPO (arrows indicate 4.0 and 3.2 kb), Tg (arrow indicates 8.5 kb), c-myc (arrow indicates 2.4 kb) and p-actin (arrow indicates 2.0 kb) mRNA levels in patients with papillary thyroid carcinoma (ptc), a patient with a follicular thyroid carcinoma (ftc). patients with anaplastic thyroid carcinomas (ate) and a patient with a hyperfunctional adenomas (ha) with the adjacent normal tissue (nt). Numbers below the lanes refer to patient numbers in Table 1.

e

normal showed 24

,“-I_..^^ .“,

25

26

27

18

9

13

8

Fig. 1. hTSHR (arrows indicate 4.6 and 4.4 kb), TPO (arrows indicate 4.0 and 3.2 kb), Tg (arrow indicates 8.5 kb), c-myc (arrow indicates 2.4 kb) and p-actin (arrow indicates 2.0 kb) mRNA levels in patients with papillary thyroid carcinomas (ptc) and adjacent normal thyroid tissue (nt) as well as in unrelated normal controls (nt). Numbers below the lanes refer to patient numbers in Table 1.

thyroid increased

tissue. All anaplastic tumors p-actin transcript levels.

Discussion The present data show that steady-state levels of mRNA expression of the TSH receptor gene may serve as an index of cell differentiation. hTSHR mRNA was comparable to the normal tissue in hyperfunctional adenomas but was lost or altered in anaplastic tumors and uniformly reduced in four patients with papillary cancers

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where normal tissue was available for direct comparison. This suggests some degree of down-regulation associated with the neoplastic alteration. A similar lowered TSHR gene expression has been recently found by Berlingieri et al. (1990) in oncogene-transfected rat thyroid cells showing correlation between transformation, loss of TSH-dependent growth and loss of TSHR gene expression. hTSHR transcripts are detected in all patients with differentiated tumors suggesting that disappearance of the TSH receptor occurs late in the disease. Our data on TSHR mRNA content support and explain the clinical paradigm of TSH suppressive therapy by thyroid hormones in differentiated thyroid neoplasms and explain the clinical ineffectiveness of this treatment in anaplastic carcinomas (Mazzaferri, 1981; Cady et al., 1983; Hay, 1990). Our results complement previous indirect methods such as the measurement of labelled TSH binding to thyroid cells, the response of thyroid membrane adenylate cyclase to TSH, the in vivo Tg secretion and radioiodine uptake by tumor tissue or the therapeutic response of Tg secretion to thyroid hormones. Scatchard plot analysis of labelled TSH binding and the TSH stimulation of adenylate cyclase support the variability of hTSHR mRNA levels found in our study with a loss of binding sites and a diminished stimulation of cyclic AMP accumulation by TSH in dedifferentiated tumors (Sand et al., 1976; Field et al., 1977; Carayon et al., 1980; Abe et al., 1981). The proposed correlation between the aggressiveness of the tumor and the decrease in cyclicAMP response to TSH (Siperstein et ai., 1988) is, however, not supported by our results on the expression of hTSHR transcripts but caution is needed to interpret mRNA 1eveIs in terms of protein concentrations. Our data demonstrate that markers of differentiation such as TPO and Tg transcripts are no longer detectable in the most dedifferentiated tumors. This fits with previous investigations (van Herle et al., 1975; Pacini et al., 1980; Berge-LeFranc et al., 1985) and with recent observations of an inactivation of transacting factors required for the expression of the Tg gene when thyroid cells are transformed with Kirsten murine sarcoma virus (Awedimento et al., 1988). Our findings on a variable combination

of Tg and TPO mRNA expression in papillary tumors showing high Tg mRNA associated with low TPO transcripts and vice versa are in close accordance to previous studies correlating radioiodine uptake in recurrent disease following total thyroidectomy and Tg serum levels fDralle et al., 1985). The differentiai inactivation of Tg and TPO genes in individual papillary tumors fits well also with the reported differences in the kinetics and requirements for induction of the two genes by TSH (Gerard et al., 1989). Obviously, as for experimental tumors there is no unique pathway leading to a fully dediffercntiated phenotype. Such a biochemical diversity may explain the very different progression of apparently similar tumors. This may reflect the activation of oncogenes at different levels of the signal transduction cascades leading to differential inactivation of TPO and Tg transcription. Such dependency on the activation of intracellular signal transduction cascades has been supported by studies of our group on Tg and recently on TSH receptor gene expression in primary dog thyroid culture where a decrease of message levels was observed following stimulation by EGF and TPA and decreased transcription of TPO gene after EGF treatment (Gerard et al., 1989; Pohl et al., 1990; Maenhaut et al., in press). Recent evidence suggests that accumulation of the c-myc mRNA is increased in association with stimuli leading to thyrocyte dedifferentiation whereas TSH, maintaining differentiation, only leads to a temporary increase followed by a rapid down-regulation (Reuse et al., 1990). These findings are in agreement with previous observations and our data in benign and malignant thyroid neoplasms where steady-state c+?ryc mRNA levels were discussed to be correlated to a dedifferentiation of the tumor (Yamashita et al., 1986; Burman et al., 1987; Aasland et al., 1988; Terrier et al., 1988). However, the high transcript levels found in normal tissue adjacent to hyperfunctional adenomas or malignant tumors warrant further investigation on a possible stimulation 01 c-rnyc transcription by local, potentiahy tumor derived, growth factors. Markers of thyroid differentiation and dedifferentiation as in this study may be of help in the follow-up of patients with thyroid cancer. Their

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use may allow to define patterns of alteration which indicate the prognosis better than the currently available criteria. Such investigation will hopefully provide clues for the treatment of these tumors. With regard to TSH receptor mRNA expression the present results show that this expression is retained much further along the pathway of transformation than the expression of function related genes such as TPO or Tg. This confirms that the expression of this gene is very robust and little regulated, i.e. almost constitutive in the thyrocyte (Maenhaut et al., in press). This makes sense physiologicaIIy as this receptor is the address of the cell without which the thyrocyte is no longer integrated in the physiological regulatory network. Acknowledgements

The tissue for the study of hyperfunctional adenomas was kindly provided by J. van Sande. This work was supported in part by Deutsche Krebshilfe, from the Minis&e de la Politique Scientifique, the Fonds de la Recherche Scientifique Medicale, the Fonds Cancerologique de la Caisse d’Epargne, and I’Association Belge contre ie Cancer. C.M. is Aspirant of the Fonds National de la Recherche Scientifique. References Aasland, R., Lillehaug, J.R., Male, R.. Josendal. O., Vdrhaug, J.E. and Kleppe, K. (1988) Br. J. Cancer 57, 358-363. Abe, Y., Ichikawa, Y., Muraki, T.. Ito, K. and Homma. M. C1981 f J. Clin. Endocrinol. Metab. 52, 23-28. Awedimento, V.E.. Musti, A., Fusco. A., Bonapace, M.J. and Di Laura. R. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1744- 1748. Berge-LeFranc. J.L.. Cartouzou, G., de Mica. C., Fragu, P. and Lissitzky, S. (1985) Cancer 56, 345-350. Berlingieri. M.T., Akamizu, T.. Fusco. A., Grieco, M.. Colletta. G.. Cirafici, A.M. et al. (1990) Biochem. Biophys. Res. Commun. 173, 172-178. Brocas. H.. Christophe, D.. Pohl, V. and Vassart, G. (1982) FEBS Lett. 137, 189-193. Burman. K.D.. Djuh, Y.Y., La Rocca, R.V., Nunes, M.E., D-Avis. J.C., Nicholson, D.E. et al. (1987) Horm. Metab. Res. Suppl. 17, 63-65. Carayon, P.. Thomas-Morvan. C.. Castanas, E. and Tubiana, M. (1980) J. Clin. Endocrinol. Metab. 51, 915-920.

Chang, T.C.. Kuo. S.H.. Liaw. K.Y., Chang, C.C. and Chen. F.W. (1988) Clin. Endocrinol. 29; 477-484. Chirgwin, M.. Przybyla. A.E.. MacDonald, R.J. and Rutter, W.Y. (1979) Biochemistry 18. 5294-5300. Dralle, H.. Schwarzrock, R.. Lang. W., BBcker, W., Ziegler. H., Schriider, S. et al. (1985) Acta Endocrinol. 108, 504510. Field. J.B., Bloom. Ct.. Chou, M.C.Y., Kerins, M.E., Larsen, P.R.. Kotani, M. et al. (1978) J. Olin. Endocrinol. Metab. 47. 1052-58. Gerard, C., Lefort, A., Christophe, D.. Libert, F., van Sande, J. et al. (1989) Mol. Endocrinol. 3, 2110-2118. Hay. I.D. (1990) Endocrinol. Metab. Clin. N. Am. 19. 545-576. Libert. F.. Ruel, J.. Ludgate, M., Swillens, S., Alexander, N., Vassart, G. et al. (19871 EMBO J. 6, 4193-4196. Libert, F., Lefort, A.. Gerard, C.. Parmentier, M.. Perret, J.. Ludgdte, M. et al. (1989) Biochem. Biophys. Res. Commutt. 165, 1250-1255. Maenhaut, C., Lefort, A.. Libert, F.. Parmentier, Rasp& E.. Roger, P.P. et al. (1990) Horm. Metab. Res. Suppl. 23. 51-61. Maenhaut. C., Brabant, G., Vassar& G. and Dumont, J.E. (in press) In vitro and in vivo regulation of thyrotropin mRNA levels in dog and human thyroid cells. J. Biol. Chem. Mazzaferri, E. (1981) Annu. Rev. Med. 32, 73-91. McMaster. G.K. and Carmichaei, G.C. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4835548323. Mishrahi, M.. Loosfeldt, H., Atger, M., Sar, S., GuichonMantel, A. and Milgrom, E. (1990) Biochem. Biophys. Res. Commun. 166, 3944403. Nagayama. Y., Kaufman, K.D., Seto, P. and Rapoport, B. 1198Yf Biochem. Biophys. Res. Commun. 165, 1184-l 190. Pacini, F., Pinchera. A., Giani, C.. Grasso, L., Doveri. F. and Baschieri, L. (1980) J. End[~crinol. Invest. 3, 283-292. Pohl, V.. Roger. P.. Cristophe. D.. Pattyn, C., Vassart, G. and Dumont, J.E. (1990) J. Cell Biol. 111, 663-672. Reuse. S., Maenhaut, C. and Dumont, J.E. (1990) Exp. Cell Res. 189, 33-40. Saltiel. A.R., Powell-Jones, C.H.J.. Thomas, C.G. and Nayfeh, S.N. (19811 Cancer Res. 41, 2360-2365. Sand, G., Jortay, A., Pochet. R. and Dumont, J.E. (1976) Eur. J. Cancer 12, 447-453. Schlumber~er, M., Tubiana, M., de Vanthaire, F., Hill, C.. Gardet. P.. Travagli, J.P. et al. (1986) J. Clin. Endocrinol. Metab. 63, 960-965. Siperstein, A.E., Zeng, OH., Gum, ET., Levin, K.E. and Clark, O.H. (1988) World J. Surg. 12, 528-533. Terrier, P., Sheng, Z.M., Schlumberger, M., Tubiana, M., Caillou, B., Travagli, J.P. et al. (1988) Br. J. Cancer 57, 43-41. Thomas. P.S. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 52015205. Venkatesh, Y.S.S., Ordonez, N.G., Schultz, P.N., Hickey, R.C., Goepfert, H. and Samaan. N.A. (1990) Cancer 66.321-330. Yamashita, S., Ong. J.. Fagin, J.A. and Melmed, S. (1986) J. Clin. Endocrinol. Metab. 63, 1170-l 173.

Human thyrotropin receptor gene: expression in thyroid tumors and correlation to markers of thyroid differentiation and dedifferentiation.

Human thyrotropin (TSH) receptor steady-state transcript levels were analyzed by Northern blot analysis in thyroids of patients with thyroid carcinoma...
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