Estrogen (E) and epidermal growth factors (EGF) receptors were assayed in the liver of nine patients with hepatocellular carcinoma (HCC). Total E and nuclear E receptors were decreased significantly in neoplastic tissue as compared to the levels found in surrounding nonneoplastic tissue. The EGF receptor was decreased also in neoplastic tissue. On the basis of binding data, a decrease in the number but not in affinity of both the E and EGF receptors was found. © 1991 Plenum Publishing Corporation

A Francavilla

DR Twardtik

G Carpenter

H Shikata

JA Gustaffson

JA Gustafsson

JB Elder

JH Fallon

JM Kissane

JRC Sainsbury

K Westermark

LE King Jr

LE Porter

N Fausto

OH Lowry

PK Eagon

RB Dickson

S Ekberg

VR Mukku

PubMed

A. FRANCAVILLA

P. K. EAGON

A. AMORUSO

A. GIANGASPERO

M. S. ELM

L. GENNARI

V. MAZZAFERRO

G. COLELLA

D. H. VAN THIEL

T. E. STARZL

PANELLA, CARMINE DOMENICO MARIA

Archivio Istituzionale della Ricerca- Università degli Studi di Foggia

Role of estrogens and epidermal growth factor in hepatocellular carcinoma (HCC).

Crossref

Role of estrogens and epidermal growth factor in hepatocellular carcinoma (HCC)

Francavilla, A

Panella, C

Amoruso, A

Giangaspero, A

Gennari, L

Mazzaferro, V

Colella, G

Van Thiel, DH

Starzl, TE

Name not available

Role of estrogens and Epidermal Growth Factor in hepatocellular carcinoma (HCC)

A. Francavilla

C. Panella

A. Amoruso

A. Giangaspero

L. Gennari

G. Colella

D. H. Van Thiel

T. E. Starzl

V. Mazzaferro

AIR Universita degli studi di Milano

D-Scholarship@Pitt

ail1~pllv" Diuaus and pci~ncrsI Vol. 36, No. 9 Ep~pltmb~r 199/1, pp. 1299-1302 CONFERENCE REPORT Role of Estrogens and Epidermal Growth Factor in Hepatocellular Carcinoma (RCC) ANTONIO FRANCAVILLA. MD. CARMINE PANELLA. MD. ANNACINZIA AMORUSO. MD. ANTONIO GIANGASPERO. MD. LEANDRO GENNARI. MD. VINCENZO MAZZAFERRO. MD. GIOV ANNI COLELLA. MD. DAVID H. V AN THIEL. MD. and THOMAS E. STARZL. MD. PhD Estrogen (E) and epidermal growth factors (EGF) receptors were assayed in the liver of nine patients with hepatocellular carcinoma (HCCl. Total E and nuclear E receptors were decreased significantlY in neoplasllc tissue as compared to the levels found in surrounding nonneoplastic tissue, The EGF receptor was decreased also in neoplastic tissue, On the basis of binding data. a decrease in the number but not in affinity of both the £ and EGF receptors was found. KEY WORDS: estrogen; epidermal growth factor receptors: hepatocellular carcinoma. Epidermal growth factor (EGF) is a polypeptide growth factor that was isolated originally from the submaxillary gland. It stimulates DNA synthesis in vitro in a variety of different cells (1-3). This effect is initiated by the interaction of EGF with a specific plasma membrane receptor (4). During the last five years. several lines of evidence have demonstrated that EGF receptor expression can be altered by hormones such as estrogen (E) (4), growth hormone (5). and thyroid stimulating hormone (TSH) (6) in target tissues for these hormones. The relationship between E and EGF has been studied extensively in normal uterus and in breast Manuscnpt received January 9. 1990: accepted September II. 1990. From the Departments of Medicine and Surgery. UniversIty Health Center of Pittsburgh. U niversllY of Pittsburgh. and the Veterans Administration Medical Center. Pittsburgh. Pennsylva-nia: and the Department of Gastroenterology. UniversllY of Bari. Italy and the Italian National Cancer (nslJlute. Milan. Italy :!0133. Presented at the Proceedings of the International Meeting on Nonnal and Neoplastic Growth In Hepatology. Ban. Italy. June 1989. Supponed by the Veterans Administration and Project Grant DK 29961 from the National Institutes of Health. Bethesda. Maryland. and grant 87/01291-44 from ConSiglio Nazlonale delle Ricerche. Italy. Address for repnnt requests: Dr. AntOniO FrancaVilla. Veter-ans AdmInIstratiOn Medical Center. UnIversity Drive C. Pitts-burgh. PennsylvanIa 15240, af~Dg/ff" DiuaUl and SClt'nal. "u/. 30. /Va. 91 St'p"ml1,. /991) cancer tissue (4-7). For example. it has been shown that E stimulates EGF receptor expression in uter-ine tissue obtained from immature rats (4). In con-trast, an inverse relationship exists between the EGF and E receptors content in breast cancer tissues (8). Recently it has been shown that the liver is also a target tissue for sex hormones (9-11). E receptors (ER) appear to play an important role in hepatic regeneration where a strong temporal rela-tionship between increased hepatic ON A synthesis and an increased activity and nuclear distribution of ER have been found. Because both ER and EGF are involved in hepatocyte proliferation. the pattern of E and EGF receptors in tissue samples obtained from patients with hepatocellular carcinoma (HCC) was studied. MATERIALS AND METHODS .Liver Specimens. Samples of HCC-containing and nor-ma! adjacent liver were collected from patients undergo-ing resection in Milan. Bari. and Pittsburgh. Both tumor and adjacent normal liver tissue were collected and their identities confirmed by histology. The samples were wrapped in aluminum foil. snap-frozen. and stored at -700 C until assayed for their content of E and EGF receptors, Prior studies using fresh and frozen liver have showed that both the E and EGF receptors are stable during frozen storage. 1299 OI6l-211t.'l1109OO-1299SOfI.S<W CI 1991 Plenum Publish,,,, C""""*,,,," FRANCA VILLA ET ,l.L 600 Total ER 300 K~ 20 Q) .... aJ > aJ E: 600 '0 300 ~ 600 u. 300 CytosOl ER Nuclear ER Fig l. Estrogen receptor actlvltv In HCC ( D) and surroundmg normal tissues I CJ). ER activity was measured m cytosollc (middle panell and nuclear Ilower panell fractions of HCC and normal liver tissue. Total receptor IIOP panel I content was calculated by adding the values for nuclear and cytosolic recep-tor. Materials. Radioactive [2.3.6.7-1H)estradiol ([lH)E.). 90 Cilmmol and [I2'I]EGF were purchased from New England Nuclear. The purity of the radiolabeled com-pounds was assessed periodically by thin-layer or column chromatography (12). Sources of other materials were as described previously (12-14), Binding Studies. The preparation of cytosolic. nuclear. and plasma membrane fractions was as described previ-ously (10. 15). The methods for the estrogen receptor assay (10) and the EGF binding assay have been de· scnbed previously (15). Other Methods. Protein concentrations were deter-mmed by the method of Lowry et al (16). DNA concen-trations of homogenates and the nuclear preparations were determined by the method of l(jssane and Robins (17). Statistical analyses were performed using the Hew-lett-Packard 9815S. The radioactivity content of tritiated samples was determined using a Packard TriCarb 4530 with an automatic dpm option. ACS scintillant (Amer-sham. was used for single-phase counting of tritium-containing samples. RESULTS The clinical characteristics of the patients studied have been reported in an earlier paper (18). Figure I shows the E receptor activity which was quanti-tated in both the cytosolic and nuclear fraction as well as in whole tissue. The results are expressed as femtomoles E bound per gram wet liver. The bind-ing capacity of the cytosolic ER was different between nonneoplastic and neoplastic tissue. A 1300 -o '-a. Ol E ..... 10 o E Fig 1. EGF receptor activity in plasma membranes derived from HCC E~F and normal liver specimens (0). trend for an increase in cytosolic E receptors con-tent in cancerous tissue was evident. The nuclear ER content was significantly lower in neoplastic than in nonneoplastic tissue from the same patient (P < 0.05). Additionally total ER content was reduced in the neoplastic tissue as compared to the nonneoplastic tissue (P < 0.025). Figure 2 shows the EGF binding of plasma mem-branes isolated from cancerous and surrounding normal hepatic tissue. The values are expressed as femtomoles EGF bound per milligram of membrane protein. It can be seen that hepatic membranes isolated from neoplastic tissue have a lower content of EGF receptor than do membranes isolated from nonneoplastic tissue. The binding affinity data dem-onstrate that the KD values were similar for normal and neoplastic tissue. demonstrating that the ob-served reduction in binding is a result of a reduction in number. not affinity. of the receptor. DISCUSSION Several lines of evidence have demonstrated a direct relationship between E and EGF receptors. First. the administration of estrogen increases the EGF receptor content in the uterus of immature female rats (4). Second. a similar effect has been reponed also in normal breast tissue (7). Recently it has been demonstrated that E may playa role in the regulation of hepatic regeneration in that there is a strong temporal relationship between hepatic DNA synthesis and the nuclear ER content of regenerat-ing liver. In this same model. a reduction in the EGF receptor binding capacity of the liver has been shown to correlate with the increase in EGF recep-tors. These results have been extended recently in aiI~:lffvr Diseas" and ScirflcrI. Vol. )6. No.9 (SrplrmHr 1991/ ESTROGENS AND EGF IN HCC an in vitro model where hepatocytes treated with estrogen have been shown to lose their capacity for EGF-induced stimulation of proliferation (19). These data suggest the existence of a relationship between E receptors. EGF receptors. and tissue growth. Several additional lines of evidence indi-cate that EGF is involved in proliferation of both normal and neoplastic hepatic tissue. Recently it has become clear that EGF is synthesized at several sites in the body (20-22) and that EGF functions more in a paracrine rather than endocrine manner. Considering these facts. it is possible that the reg-ulation of EGF expression in a particular tissue such as the liver might be under endocrine control and help to explain the relationships between TSH and the EGF receptor production in the thyroid. estrogen and the EGF receptor in the uterus. and growth hormone as well as estrogens and the EGF receptor in the liver. The usual relationship in normal tissue between E and EGF may be altered in neoplastic tissue. In fact. Sainsbury et al (8) recently have reported a significant inverse relationship exists between the activity of the EGF receptor and the E receptor in primary breast cancer·tissue. Moreover. EGF re-ceptor was found in tissues with a greater frequency of metastases. presumably having a greater prolif-erative activity but were E receptor-negative. The results reported here demonstrate that the usual relationship between E receptors and EGF recep-tors existing in nonnal tissue is altered in neoplastic hepatic tissue. particularly when there is an overall reduction in the number of EGF receptors (Figure 2). It is significant that this reduction in EGF recep-tor content in HCC is proportional to the reduction in ER found in the same tissue and correlates best with a reduction in nuclear E receptor content. It is well known that the nuclear E receptor number is the most biologicaUy imponant fraction of the total E receptor content of hepatic tissue. The number receptor fraction controls the synthetic activity specific for E (23. 24). In relation to these data it is interesting to note that a reduction in EGF receptor content occurs also in regenerating rat liver but is coupled to an increase in the number of nuclear E receptors. These data demonstrate that in HCC tissue. a reduction in EGF receptor content that is not coupled with an increase in E receptor content occurs. The reasons for and importance of this unusual relationship between E and EGF receptor content in HCC is not known. It may be that. while DilrtJlwt DiJtauJ aNi Scitll",J. Vol. J6. No.9 IStpltmbtr 19911 regenerating rat liver is characterized by rapid pro-liferation. the growth is tightly regulated and is transient: no permanent growth-controlling alter-ations in the tissue have occurred and the tissue becomes normal once appropriate growth has con-cluded. In the case of HCC. the proliferative activ-ity is not regulated by the normal regulatory signals. It is also possible that in neoplastic tissue (25. 26). the EGF receptors are saturated by other growth factors that bind to the receptor such as alpha-TGF. a newly characterized growth substance. which also stimulates cell proliferation via an interaction with the EGF receptor. REFERENCES I. Carpenter G: Epidennal growth factor. Handb Exp Phanna· col 57:90-126. 1981 :. Carpenter G. Cohen S: Human epidennal growth factor and the proliferation of human fibroblasts. J Cell Physiol 88:227-237. 1976 3. King LE Jr. Carpenter GF: BiochemIStry and physiology of the skin. In Epidennal Growth Factor. Ed. I. LA Goldsmith (ed). New York. Oxford University Press. 1983. pp 269-281 4. Mukku VR. Stancel GM: Regulation of epidennal growth factor receptor by estrogen. J Bioi Chem 260:9820-9824. 1985 5. Ekberg S. Carlsson L. Carlsson B. Billig H. Jansson 10: Plasma growth honnone pattern regulates epidermal growth factor (EGF) receptor messenger ribonucleic acid levels and EGFbinding in rat liver. Endocrinology 125:2158-2167. 1989 6. Westennark K. Westennark K. Karlsson FA. Ericson LE: Location of epidennal growth factor receptors on procine thyroid follicle cells and receptror regulation by thyrotropin. Endocrinology 118: 1040. 1986 7. Dickson RB. Lippman ME: Estrogenic regulation of growth and polypeptide growth factor secretIon in human breast carcinoma. Endocrinol Rev 8:29-43. 1987 8. Sainsbury JRC. Sherbet GV. Famdon JR. Harris AL: Epi· dennal·growth-factor receptors and oestrogen receptors in human breast cancer. Lancet 1:364-366. 1985 9. Eagon PK. Poner LE. Francavilla A. Dileo A. Van Thiel OH: Estrogen and androgen receptors in liver: their role in liver disease and regeneration. Semin Liver Dis 5:59-69. 1985 10. Francavilla A. Dileo A. Eagon PK. WU S-Q. Ove P. Van Thiel DH. Starzl TE: Regenerating rat liver: Correlations between estrogen receptor localization and DNA synthesis. Gastroenterology 86: 1410-1416. 1984 11. Francavilla A. Eagon PK. Dileo A. Polimeno L. Panella C. Aquilino AM. lngrosse M. Van Thiel DH. Starzi TE: Sex honnone related functions in regeneratmg male rat liver. Gastroenterology 91: 1263-1270. 1986 12. Eagon PK. Fisher SE. lmhotl AF. Porter LE. Stewan RR. Van Thiel DH. Lester R: Estrogen·binding protems of male rat liver: Influences of honnonal changes. Arch Biochem Biophys 201:486-499. 1980 1301 13. Poner LE. Elm MS. Van Thiel DH. Dugas ~CK Eagon PK: Charactenzation and quantitation of human hepatic estrogen receptor. Gastroenterology 84:704-712. 1983 14. Eagon PK. Seguiti SM. Rogerson BJ. ~qulre TF. Poner LE. Seeley DH: Androgen receptor In rat liver: character· Ization and separation from a male· speCific estrogen binding protein. Arch Biochem Biophys 268:161-175.1989 15. Francavilla A. Ove P. Polimeno L. Sciascia C. Coetzee M. Pellici R. Todo S. Kam I. Stanl TE: Different response to epidermal growth factor bf hepatocytes 10 cultures isolated from male or female rat liver: Inhibitor elIect of estrogen on binding and mitogenic elIect of epidermal growth factor. Gastroenterology 93:597-605. 1987 16. Lowry OH. Rosebrough NJ. Farr AL. Randall RJ: Protein measurement with the Folin-phenol reagent. J Bioi Chem 193:265-275. 1951 17. Kissane JM. Robins E: The ftuorometnc measurement of dexoynbonuc1eic acid in am mal tissues With speCific refer-ence to the central nervous system. J BioI Chem 233: 184-188. 1958 18. Eagon PK. FrancaVIlla A. DiLeo A. Elm MS. Gennan L. \1azzaierro V. Colella G. Van Thiel DH. Starzl TE: Quan-tltaUon oi estrogen and androgen receptors In hepatocellular carcinoma and adjacent normal human liver. Dig Dis Sci (in press) 19. FrancaVIlla A. Polimeno A. DiLeo A. Barone M. Ove P. Coetzee M. Eagon P. Makowka L. AmbrOSinO G. Mazza-1302 FRANCA VILLA ET AL ferro V. Starzl TE: The elIect of estrogen and tamoxlfen on hepatocyte proliferation In vivo and in I·j/m. Hepatology 4:614-620. 1989 ::0. Elder JB. Williams G. Lacey E. Gregory H: Cellular local-Ization of human urogestrone/epldermal growth factor. ;'ia-ture 271:464-467. 1978 11. Fallon JH. Loughlin SEe Morrison RS. Bradshaw RA: Epidennal growth factor immunoreactive material in the central nervous system: location and development. Science (Washington. DC) 224: 1107-1109. 1984 21. Shikata H. Utsumi N. Hiramatsu M. MinamI N. Nemoto N. Skidata T: Immunohistochemical localization of nerve growth factor and epidermal growth factor in guinea pig prostate gland. Histochemistry 80:411-413. 1984 23. Gustafsson JA. Mode A. Norstedt G. et aI: The hypothalam-opituitary-Iiver axis: A new hormonal system in control of hepatic sterOId and drug metabolism. Biochem Action Horm 7:47-89. 1980 24. GustatIson JA. Mode A. Norstedt G. Skett P: Sex steroid induced changes In hepatic enzymes. Annu Rev Physiol 45:51-60. 1983 2.5. Twardtik DR. Sherwin SA. Ranchalis J. Todaro GJ: Trans-forming growth factors In the uterus of nonnal. pregnant and tumor-bearing humans. J Nat! Cancer Inst 69:793-798. 1982 26. Fausto N. Mead JE: Biology of disease. Regulation of liver growth: Protoncogenes and transforming growth factors. Lab Invest 60:4-13. 1989 DiltrSlIYr aisras~s and pci~"usK Vol. 36. No. 9 {ptpl~m~ /99/j 

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Role of estrogens and epidermal growth factor in hepatocellular carcinoma (HCC)

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