Transforming growth factor-α in normal and neoplastic human endocrine tissues

Transforming Growth Factor-a in Normal and Neoplastic Human Endocrine Tissues DAVID K. DRIMAN, MBCHB, MICHAEL S KOBRIN, PHD, JEFFREY E. KUDLOW, MD, AND SYLVIA L. ASA, MD, PHD Transforming

growth

factor-a

(TGF-(Y) is a polypeptide

tor that binds to the epidermal to stimulate in tumor gression

It has been believed

to play a role

by inducing

notype; overexpression of TGF-a

been documented

the reversible

transformed

of TGF-a! may be important

via autocrine

Expression

stimulation

and oncogene

and the epidermal

in several nontumorous

forming

in normal

and neoplastic

overexpression.

thyroid,

pancreatic

parathyroid,

adrenal

islets. Immunoreactivity

and malignant gliocytomas. showed

to localize

endocrine

pheochromocytomas,

hypothalamus,

and medulla,

greater

suggest that in endocrine marker

1365. Copyright

neogan-

and adrenal cortical carcinomas

immunoreactivity

than their

between

stage, or hormone

degree

content.

These

normal of reacresults

tissues, TGF-(Y is unlikely to prove useful

but that the growth

both normal physiology

and

in most benign

tract. Hypothalamic

but there was no correlation

tivity and tumor grade, as a tumor

TGF-

tissues. Trans-

tumors of these tissues, as well as in endocrine

consistently

counterpart,

cortex

was detected

plasms of the lung and gastrointestinal

has

tissues and in a variety

growth factor-a was found in nontumorous

pituitav,

phe-

for tumor pro-

growth factor receptor

of tumors. This study used immunohistochemistry a expression

fac-

and is thought

cell proliferation.

initiation

growth

growth factor receptor

factor

and tumorigenesis.

li 1992 by W.B. Saunders

geststhatan increased

level of the receptor for TGF-cu may provide a proliferative signal to the cell. Transforming growth factor-0 has been identified in embryonic tissues “-” and in several normal human tissues, ’ ‘-” as well as in a variety of neoplastic tissues and tumor cell lines.“‘-“” The protean presence of TGFLYsuggests that in addition to its possible role in neoplastic transformation, this factor may play a role in normal physiology. To further define the localization of TGF-LU. we studied its immunoreactivity in normal endocrine tissues as well as in a variety of endocrine tumors using immunohistochemical analysis; previous studies have indicated that localization by our immunohistochemical technique correlates well with the presence of TGF-cr and its mRNA expression.- ““-2?The results of our current study indicate that TGF-a is widely distributed in both normal and neoplastic endocrine tissues.

may play a role in

HUM PATHOL ‘23:1360Company

MATERIALS

AND METHODS

Case Material Transforming growth factor-a (TGF-(u), one of the large group of polypeptide growth factors, was first discovered as a substance secreted by murine sarcoma \Tirus-transformed cells.’ It was shown to reversibly transform immortalized normal rat kidney fibroblasts in the presence of transformin~,~owtli factor-P, an essentially unrelated growth factor.- Transforming growth factor(Yshares extensive amino acid sequence honiology with epidermal growth factor (EGF). binds to the EGF receptor,“.’ and has biologic functions similar to EGF.‘-’ Interest in TGF-cu has revolved around its postulated role in neoplaslic transformalion.“~’ It is thought that it most likely acts via autocrine stimulation in which a cell produces a growth factor for which it also bears receptors. This may confer growth advantages on such cells by making them less dependent on the environment provided by other cells and by allowing amplification of growth responses.” The importance of autocrine growth stimulation to the neoplastic transformation of cell lines has been deInonstr~lted.!‘~“’ Homology between the EGF receptor and the oncogene protein V-rlh-B” also sug-

hlatetiaf was t-etrieved in the form of fot.tll~tlitt-fisetf, fairafin-embedded tissue from both the surgicxf pathlop XKI autopsy fifes of St Mic~haef’s Hospital. All tissues had IXTII fixed in 10% ldfer-ed formalin and etnf~eclclecl in pardfin. FOI tissue attti tmttor classification, sections were stained with hematos~fin-eosin and a \xriery ol~inttttuttohistoc~ie~~ii~~if stains. In some casex elect]-ott titiux~~copy was used to verify diagtioses. Selcctioti of matctiaf for tfiis stud! w;ib tmsed f)t-itttarily 011 dia~tiohis, arid ittclutled tutiiors and ~1on~umorous tissue front pa’tents of vxiotts ages arid hotIt sexes to obtain apputptiatt ttutttl~et-s of each tissitr and 1tmior tyfw.

lmmunohistochemistry Itntttuttos~aittittg was pet-l’ormed ott 1 10 6 Fttt-t1tic.k SC’Ctiotts usittg the a~idirt-l,iorilt-~er-osiclasc c-otttplex tec.httiqitc and ;I \~eff-characteri~e~l tttottoclottal atttihodv to ~I‘(;F-tu. R/IF!I, as previoitsly descrihecl.“~‘tx The difutiott ;)f pt.itttat-y antiserm~t was I:20 in 0. 1 tnof/I, Tris -0.9% Na(:l bttffrr, pH 7.6. and [he drtr;~tioti of exposure was 24 hours at -4°C:. ‘I‘hr ittttitutiolo~ic reactiott was visuafi~etl I,v the dctc>ction of fxx~‘ositlasc activitk uaittg ;L solution of ~~.~l’~di;ttttittot~cll/idillc ~ittd hydrogen fwroxide. The speciticily of ittttttttttos~;tirtillg was bet-ifietl 1,) t-epl:tc.ittg the tttotto~lottal anrihodv witfi normal tnottw ascites. ivith itt~related tttcmoclot~al airilx~dics of the sxtte stibtyfw (ittcfudiIts lo\+ and hi~gh tttolecwlatweight wtokeratinz. cpitllclial tnctitbt-atic atitigeti. ttc.ut.ofilat,tcnts, and s(.\.ctd Icrtkowtc titat-krt-s), arid with MF-!I absor-lwtl with 10 pg/tttl, svttthrticc.;t~t)os~l-tet-tttitt~t~ 7.(GF-tu prptitic (Pcttitisula I .;ihot~;i~orics. Bcfmont,

(:.A).

estat~li5fi (,o-lo~;tliz~irioti in tissue5 (.otttprisittg tttttlliplc, ct-11lyc5, c~ottsr~~~livr srctiotts ofhtmian pituirmy and fxttt~rcas \vvt‘~ (,ac-f1 slaittctl with tllc ;ttttiser’ttttt to T(;F-tu atttl ati attti‘I’0

1360

TGFw

IN HUMAN ENDOCRINE

TISSUES

(Driman et al)

FIGURE 1. Tumor cells of a hypothalamic

gangliocytoma demonstrate intense cytoplasmic positivity for TGF-c\: nerve fibers in the neuropil also demonstrate immunoreactivity. (Avidinbiotin-peroxidase complex technique; magnification a 185.1

predoniiiiant in the lateral wiugs. (k)r-tic otrophs \\cre less frequently and less intensely posiliv~; c,ortic-otrophs present in posterior lobes. rrpreseuting txtsophil invasion, also contained focal T(;F-cu rractivi(y. In the posterior lobe focal staining of pituicyteh and dilated ncrvc terminals known as Herring bodies was Iloted. The 12 pituitarv adeiioiiias studied includrd sis sparxly granulated growth horriiorie-proc~li~iii~ tumults. one’ proktillonla. two cx)rticotroph adt-nomas producing A(:TH, two null cull adenonm. and OIIC oncocvtoma. One growth horrlloilc-pl-od~l~iri~ ademma and ‘the null cell adeiioinas were not iiiimunoI.eacti\e. (Iells of the other uiiir adenoiiias contained weak to riiotler.~~tr cytoplasmic imniunopositi~it\;.

Thyroid Follicular epithelial cells of’ normal I hyroid glands showed variable cytoplasmic posili\;i[y. which tended co thyhave a granular quality. Two of three h~~pq~lastic roid uodules, all cases of follicular (three) aud HiirNc cell (three) adenornas (Fig 2). and all cxx~s of papillar)

1361

HUMAN PATHOLOGY

Volume 23, No. 12 (December

1992)

showed the strongest staining. The nontunlorous adrenal medulla stained positively in some cases, but the results were inconsistent. Of six cortical aclenomas. two were associated with Cushing’s syndrome and four with Conn’s syndrome; all demonstrated weak positive immunostaining, whereas all five carcinomas examined showed stronger staining. The positivity did not correlate with the cytoplasmic density of tumor cells; clear cells and compact cells were both positive. The greatest degree of immunoreactivity was seen in pheochromocytomas, which stained uniformly and intensely (Fig 3). In all the positive cells staining was cytoplasmic ancl granular. Pancreas Cells of all hormone-producing types in normal islets of Langerhans showed diffuse, moderate to strong, cytoplasmic positivity. Immunoreactivity was more intense in islets than in scattered positive ductal epithelial cells, and the acinar parenchyma was negative. Seven of eight pancreatic endocrine tumors of various hormonal profiles were immunoreactive (Fig 4). Of these,

FIGURE 2. The tumor cells of a Hijrthle cell adenoma of thyroid stained strongly for TGF-n in a cytoplasmic distribution. (Avidinbiotin-peroxidase complex technique; magnification x185.)

(six), follicular (six), H”urt hl e cell (two), medullary (five), and insular (one) carcinomas were positive. Staining was cytoplasmic and diffuse, and varied both in the number of positive cells and in the degree of positivity within each of the morphologic categories. In a case of de Quervain’s thyroiditis found incidentally in association with a neoplasm, follicular epithelial cells in foci of granulomatous inflammation were strongly immunoreactive, with less intense staining in adjacent. less-inflamed epithelium. Parathyroid Only occasional parenchymal cells of normal parathyroid glands were positive; these appeared to be chief cells. One hyperplastic gland and one parathyroid carcinoma showed no immunostaining. Positive cells were generally weak and focal in three hyperplastic giands, one adenoma, and one carcinoma. No correlation between cell type and immunoreactivity was observed. Adrenal

FIGURE 3. The cells of a pheochromocytoma are strongly immunoreactive for TGF-u. The surrounding stroma does not stain. (Avidin-biotin-peroxidase complex technique; magnification X75.)

Most cells of all three layers of the adrenal cortex exhibited weak immunoreactivity. The Lana fasciculata 1362

TGF-n

IN HUMAN ENDOCRINE

TISSUES

(Driman et al)

DISCUSSION

FIGURE 4. An endocrine tumor of the pancreas contains intense cytoplasmic staining for TGF-a. as do scattered endocrine cells dispersed in the surrounding parenchyma; the exocrine pancreas is inegative. (Avidin-biotin-peroxidase complex technique; magnification * 185.)

thwc

wcw

known

10 Iw malignant.

rrac.~ic.ity

Positiw

did ilot correlate with known biologic oi with prptid~. producJion. Gastrointestinal

iiimwno-

behavior

Tract

khrii ga.\troinlestiIial endocrine tulliors arising the duodenum, ileum. and appendix were studied; lrast four were known to be malignant at the time thv 5tutl\ ‘rcii showed cytoplasniic iiiiirluiioI”)sitivity, whit h vilrictl iii nunih~r 0I‘ cells stained and degree rrxikit\

in at of of

Lung Foul- 01’tiw puln~onar~ endocrine tumors show4 k;irialAe c\:toplasinic positivit\;. Of these, one wits a socallrd carcinoid. 011~ was an atypical carcinoid. and two were cudoc rine cCuxinomas. one of which was classified as a small c-ell(.arcinoma. One carcinoid tumor did not 41din. 1363

Transforming growth factor-o is \videly distributed in both normal and neoplast ic endocrii I(‘ tissues. We have demonstrated. using an ii1direc.t immunoperosidase technique, that TGF-(U is present in nontulixn-ous hypothalamus. pituitary, thyroid, parathvroid, adrenal cortex, adrenal medulla. and pancreatic islets. Endocrine tumors arising in each of these tissues were immunoreactive for TGF-cu. as were endocrine ltimior\ of the lung and gastrointestinal tract. Transforming growth factor-cu has been detected previously in embryonic tissues. “-“’ in other normal tissues, 17.!!!I ;md in a variety of neoplastic tissues and tunlor cell lines.“-“!’ Its widespread presence suggests ;I role in normal physiology. However, most interest has been directed at its putative role in neoplastit transformation.‘.’ rrransformation by several oncogenes has been shown to result in TGF-(U gene expression’ and it has been suggested that TGF-LU interacts with (ells secreting it in an autocrine fashion.“’ Autocrine stimulation bestows growth advantages on transformed cells, allowing proliferation to proceed more readil!,. This is the first study of which WC art aware that has examined the presence of T(;F-tu esclusi\ely in a variety of human endocrine tissues. ‘I‘ransforniing growth factor-a immunoreactivity has bxn investigated in the bovine anterior pituitary gland,“‘,“’ where reactivitv was localized in lactotrophs and not in corticotrophs or in cells producing glycoprorein hormones. In this study of huiman tissues TGF-a expression was detected in all adenohypophysial cell types. Lt’hile this ma) reflect the fact that the bovine pit&al-v studies did not include neoplasms. we found immunc;rr;ictivily of all cell types in nontunx)rous human pituitarv. indicating that this probahl\ reflects a species diHrrc*nce. Some of the growth hormone-producing adPnomas were associated with hypothalamic gangliocytonias, a relationship previously described”‘; 11has beeii sugge5led that procluction of growth hormone-releasing factor bv these hypothalamic tmnors plays a pathogenetic. I-C& in the formation of’ the pituital:y, adenonlas. WC detected strong T(;F-tu iiiiiiiunopositl\.it~~ in iieui (mb 01 the tivpothalamic g;mgliocytonias, raising the possibilitv thin production of this substance by hypothalamic neurons n1q affect ~~clenohypophysi~tl cell l,i~oliferation. It is known to Ix inm)lved in the release of luteinking hormane-relatirig hornione in the dox4opiiig h, pothalaniu~.“~ Epidermal growth factor rec’eptc brs have been localized only. in adenohyI>ot)h~si;ll c-ells containing the glycoprotein hornlones thyroid-stirllula~ing hormone, f’ollicle-stimulating hormone, and IutciniAng hornlone”; thus, I‘GF-cu may not play a significan1 role iii the association between these two tumor 1ypt’x. In addition, a recent stud\ has shown that TGF-tu is in fact a growthinhibiting fac’tor for the GH.I<:, rat pituitarv cell line. I3 Tl~erefore. it is possible that TGF-LU src,retio;i bv growlh Ilormoi~e-I”‘oduciilg adenomas plays a role other than that of growth stimulation. Further htutlies arc needed to clarif!, the role of TGF-LU iI1 tile various adenohypol)hysial c.ell t\~pcs.

HUMAN PATHOLOGY

Volume 23, No. 12 (December

All normal and neoplastic thyroid follicular epithelium was immunoreactive for TGF-cu, with no significant difference in reactivity between tumorous and nontumorous tissue. Inflamed follicular epithelium, however, was more strongly immunopositive than uninflamed epithelium in one specimen. Epidermal growth factor receptors have been localized on human thyroid cells.“-‘7 The affinity of these receptors for EGF is lower in differentiated thyroid carcinomas than in adenomas and nonneoplastic thyroids,‘X and there is a close relationship between the binding affinity of the EGF receptor and thyroid cell growth.‘“.‘” It also has been shown that thyroid hormone (T3) decreases the expression of EGF receptors on thyroid follicular cells.3” Although a recent study has demonstrated that papillary carcinomas co-express TCF-CY and EGF receptor mRNA at higher levels than nontumorous thyroid tissue,“’ the present study

does

not

demonstrate

a significant

difference

in

TGF-a reactivity between normal and neoplastic follicular epithelium. This implies that while EGF binding to its receptor is likely important in modulating thyroid cell growth, binding of TGF-cv to the same receptor could have other eff‘ects. However, increased responsiveness to TGF-a may be a function, not of increased endogenous expression of the factor, but of increased numbers of EGF receptors, increased affinity of the receptors for TGF-LU. or increased signal transduction across the receptor complex.’ In most tissues examined in the present study tumors stained more strongly than the nontumorous counterparts. This was most evident in pheochromocytomas, which were strongly reactive, and to a lesser extent in adrenocortical carcinomas. Nontumorous adrenal cortex and medulla showed weak immunopositivi’y: All other tumors showed variability in immunoreactlvlty; in none of the tumors was there a correlation between degree of reactivity and tumor grade or stage. Other studies attempting to correlate TGF-a expression in tumors with pathologic stage and prognosis have produced mixed results.““.‘” The status of EGF receptors in many of the tissues studied here is not known with certainty. One group failed to localize EGF receptors in thyroid or pancreatic islets,“’ while another failed to localize the receptors in thyroid, pituitary, pancreatic islets, or adrenal gland.“’ Others have shown EGF receptors to be present in thyroid tissue-“‘-“8 and pituitary.“” Of interest, pheochromocytoma cells in the rat have been shown to have cell surface EGF receptors, with EGF activity acting as a mitogen for these cells.“” It has been demonstrated that small cell lung cancer cells possess no cell membrane EGF receptors due to lack of EGF receptor gene expression, the intact gene being present in these cells.‘” We detected TGF-cr expression in four of five endocrine lung tumors, one of which was a small cell carcinoma. Interestingly, it has been shown that a certain TGF-CYsecreting melanoma cell line shows no EGF receptor expression, even under conditions that prevent receptor downregulation and degradation.“’ This suggests that in such cells, the lack of cell surface EGF receptors is independent of TGF-CY secretion. 1364

1992)

Our results suggest that TGF-(Y is not a valuable tumor marker in the neoplasms studied. This differs from other tumors, such as lung adenocarcinoma, in which high levels of TGF-cu portend a worse prognosis,” but is consistent with the findings in the gastrointestinal tract.““,“” Immunoreactivity in certain endocrine tumors that is stronger than that in the nonneoplastic counterparts suggests that there may be a role for TGF-(r in abnormal cell roliferation in endocrine tissues, as there . is in the gut.5 g Given the suggested role of the growth factor in neoplastic transformation via autocrine stimulation, it would be instructive to study definitively the co-expression of TGF-a and EGF receptors in these tissues. Demonstration of co-expression of the factor and its receptor would provide important information. Furthermore, Northern blot analysis and/or in situ hybridization studies to detect TGF-CY mRNA would be useful to assess active synthesis of this growth factor. Ackr~owledgment. The authors wish to thank Nahid Nelson and Gail Robertson-Davies for technical assistance, Colette Drvodelic for typing the manuscript, and Mark Moreland for help in preparing the photographs.

REFERENCES 1. DeLarco JE. Todaro GJ: Growth factors lrom murine sarcoma virus-transformed cells. Proc Nat1 Acad Sci USA 75:4001-4005. 1078 2. Dervnck R: Tramfur-ming growth fxtor (Y.Cell 51:59Y-595, 1988 3. Marquardt H, Hunkapiller MW. Hood LE, et al: Transforming Cgrowth factors produced by retrovirus-transformed rodent fibroblasts and human melanoma cells: Amino acid sequence homology with epidermal growth factor. Proc Nat1 Acad Sci USA 80:4684-4688. 1983 4. Marquardt H. Hunkapiller MW. Hood I.E. et al: Kat transfi)rming growth factor type I: Structure and relation to epidermal growth factor. Science 223: 107!&1082, 1984 5. Elizalde PV. Charreau EH: a-Transforming growth factor like activities and bifunctional regulators of cell growth in human malignant neoplasms. Cancer Invest 8:365-37-l. 1990 6. Sandgren EP. l.uetteke NC. Palmiter RD. et al: Overexpression of TGFa in transgenic mice: Induction of epithelial hyperplasia, pancreatic metaplasia and carcinoma of the breast. Cell 6 1 : 1 12 I - 1 13.5. I WI 7. Kosenrhal A. 1,indquist PB, Bringman TS. et al: Expression in rat fibroblasts of a human transforming growth factor-cu cDNA results in transformation. Cell 46:301-309. 1986 8. Cross M, Dexter TM: Growth factors in development. transfol-mation. and tumorigenesis. Cell 64:271-280. I991 9. Heldin (Z-H, Westermark B: Growth factors as transforming I”-oteins. Eur J Bioc-hem 184:487-496. 1989 10. Lang RA. Burgess AW: Autorrine growth lactors and tunourigenic transformation. Immunol Today 1 1:244-249. 1990 11. Gullick WJ, Berger MS. Bennett PLP. et al: Expression of the c-&B-:! protein in normal and transformed cells. Int J Cancel 40946-254, 1987 12. Matrisian I,M, Pathaak M, Magun BE: Identification of an epidermal growth factor-related transforming growth factor from rat fetuses. Biochem Biophys Res Commun 107:761-769. 1982 13. Han VKM, Hunter ES III, Pratt KM, et al: Expression of rat tl-ansforming growth factor- alpha mRNA during development occurs predominantly in the maternal decidua. Mel (:ell Biol 7:2335-9843. 1987 14. Stromberg K. Pigott DA, Kanchalis JE, et al: Human term placenta contains transforming growth factors. Rio&em Biophys Res Commun 106:354-36 1, 1982 15. Twardzik DR: Differential expression of transforming growth factor-u during prenatal development of the mouse. Cancer Res 45: 54 13-54 16, 1985

TGF-LU IN HUMAN ENDOCRINE

1365

TISSUES

(Driman et al)