I. Hepatocarcinogenesis by the Steroid Hormone

Research Program Division Tumor Cell Regulation Signaltransduction and Growth Control

Research Group Hormones and Signal Transduction (B0810) Head: PD Dr. rer. nat. Doris Mayer

Scientist: I. Hepatocarcinogenesis by the Steroid Dr. rer. nat. Bernd Schnarr (10/1997- 9/2000) Hormone Dehydroepiandrosterone Postgraduate and graduate students: D. Mayer, K. Kopplow Kathrin Kopplow ( - 12/2000) In cooperation with: Peter Bannasch, Christel Metzger, Dirk Martina Schmitt ( - 2/2000) Nehrbass, Cell Pathology, DKFZ; Axel Benner, Biostatistics, Kathrin Strunz ( - 12/2000) DKFZ; Julian Swierczynski, Medical University, Gdansk, Poland; Alexandra Dreuw (10/2000 - ) Gerhard Hobe, Hans-Knöll-Institute, Jena; Rolf Gebhardt, Institute for Biochemistry, University of Leipzig; Gayane Buniatian, Institute Visiting scientist: for Physiological Chemistry, University of Tübingen. Yun Niu (Tianjin, China, 4/2000 - ) Dehydroepiandrosterone, a steroid of the adrenal gland 90 Technical assistant: and a precursor in the biosynthesis of potent estrogens Jürgen Ohsam and androgens induces liver cancer in rats [1-5]. The tumour incidence is higher in females than in males which indicates a gender specific hormonal effect of DHEA re- The significance of steroid and peptide hormones for tu- lated to hepatocarcinogenesis. During the past years we mour initiation and progression in a number of organs and have studied the pathogenesis of hepatocellular carcino- tissues of various species including humans has been mas and biochemical alterations induced by DHEA in rat demonstrated by numerous experimental and epidemiolo- liver [3,6]. A number of different mechanisms associated gical studies. More recently different hormone-stimulated with DHEA-induced hepatocarcinogenesis could be eluci- signal transduction pathways have been partly unravelled dated. In this report only a few aspects on DHEA function and their components have been identified and characteri- will be briefly discussed. Other results are described in the sed. There is now increasing evidence of stimulatory or in- final report of the Division of Cell Pathology (C0100). hibitory interaction of different signalling pathways. The re- Alterations in cell cycle control by DHEA. search group ”Hormones and Signal Transduction” , which DHEA acts as a mitogen in liver, particularly of female rats. was established in October 1999, focuses their interest to This could be shown by BrdU-incorporation and by the study of the mechanisms of tumour induction and pro- immunohistochemical detection of proteins of the G1-S gression by steroid hormones in liver and breast epithelial phase of the cell cycle such as cyclins or proliferating cell cells as well as to hormone-controlled gene expression nuclear antigen (PCNA). It also causes a significant in- and the interaction of steroids with components of other crease in the size of hepatocytes [7] which is not due to cellular signal transduction pathways. polyploidy. Lipid peroxidation und protein carbonylation. DHEA induces a strong lipid peroxidation and, as a conse- quence, an increase in protein carbonylation in cellular membranes, particularly in mitochondria and microsomes. [1,3,8]. This increase in lipid peroxidation may be the con- sequence of induction by DHEA of microsomal cytochrome P450-dependent monooxygenases and of enzymes of the mitochondrial respiratory chain which are both sources of reactive oxygen species formation. Increased production of reactive oxygen species, increased lipid peroxidation and resulting hepatotoxic effects may be related to the car- cinogenic insult of DHEA. Proliferation of peroxisomes and mitochondria in rat liver. Livers of DHEA-treated rats show a strong increase in the number of peroxisomes and mitochondria [2]. It has been known for a long time that peroxisome proliferators act as hepatocarcinogens in rodents. However, our studies have clearly shown that hepatocellular carcinomas do not arise from liver areas with the strongest peroxisome proliferation but from focal lesions with abundant mitochondria [1,2,6] which questions the hypothesis that peroxisome proliferation is the cause for induction of hepatocarcinogenesis. Histochemical studies of enzymes of energy metabolism in the mitochondria-rich preneoplastic lesions and biochemi-

DKFZ 2001: Research Report 1999/2000 Research Program Division Tumor Cell Regulation Signaltransduction and Growth Control cal studies of DHEA-treated liver revealed a characteristic and in 21 control tissues by immunohistochemistry. In ad- pattern of metabolic alterations indicating a thyroid hor- dition, cell proliferation (percentage of Ki67+ nuclei) and mone-like effect of DHEA on the liver of both genders. The estrogen receptor (ER) expression were determined. IGF- molecular mechanism underlying this thyromimetic effect IR, IRS-1 and IR were found expressed mainly in epithelial of a steroid hormone remains to be clarified. It may be cells. IRS-1 and IGF-IR were expressed at high levels in noteworthy in this context that the thyroid hormone recep- control tissues and in well and moderately differentiated tor belongs to the steroid hormone receptor family and carcinomas but at low levels in poorly differentiated breast may form heterodimers with estrogen and androgen re- cancers. IR expression did not show a significant correla- ceptors. tion with the differentiation grade of the tissues investi- gated. Statistical analysis demonstrated that down-regu- Phenotypic modulation of preneoplastic liver lation of IGF-IR and IRS-1 correlated better with tumour lesions by DHEA. progression than reduction of ER expression or increase in Administration of DHEA to rats previously treated with a ni- cell proliferation, IGF-IR showing the best correlation fol- trosamine to induce hepatocarcinogenesis results in an in- lowed by IRS-1 and, less significant, ER or Ki67. Our find- crease of the number of neoplasms in the liver (tumour 91 ings clearly show that progression of breast cancer is ac- promotion). Surprisingly, it also results in the modulation of companied by a reduction of IGF-IR/ IRS-1 expression and the lesions to a less malignant phenotype [1-6]. This mo- that IGF-IR/ IRS-1 expression inversely correlates with dulation is accompanied by the down-regulation of pro- high proliferation rate in dedifferentiated breast cancers. teins of the insulin / IGF-I signal transduction pathway, The strong correlation of IGF-IR and IRS-1 down-regula- such as insulin receptor substrate-1 (IRS-1) [9]. The reduction with tumour progression suggests the use of IGF-IR tion in the expression of proteins of the insulin signalling and IRS-1 as a novel set of marker proteins for tumour pathway by DHEA may be explained by an interaction of grading [11]. peptide and steroid hormone signalling pathways. Androgenic effect of DHEA. While a thyromimetic effect of DHEA was observed in the III. DHEA- effect on Breast Cancer Cells livers of both genders, an androgenic effect was observed D. Mayer, B. Schnarr, M. Schmitt, J. Ohsam only in females. This androgenic effect was detected when In cooperation with R. Morfin, Conservatoire National des Arts et the expression and distribution of glutamine synthetase Métiers, Paris, France; K. Klinga, University Hospital of Gynaeco- was studied. This enzyme is expressed exclusively in peri- logy and Obstetrics, Heidelberg; A. Benner, Biostatistics, DKFZ venular hepatocytes. In male rats glutamine synthetase is DHEA serves as precursor for the biosynthesis of estro- detected immunohistochemically in about three cell layers gens and androgens in peripheral organs in vivo. It has around the central terminal vein and in one cell layer in fe- been reported from epidemiological studies that post- males [10]. During DHEA-treatment the expression pattern menopausal women with high DHEA blood-levels have an in the liver of female rats changed significantly and be- increased risk for breast cancer. The compound is accu- came similar to that observed in males as determined by mulated in breast tissue and breast cancer from the blood. combination of morphometric and immunohistochemical We were interested to clarify whether DHEA is a mitogen methods. In males no effect of DHEA on glutamine syn- for estrogen-dependent breast cancer cells, and if so thetase expression pattern was observed. In this context it whether it required conversion to estrogens in order to may be of interest that DHEA induces a significantly higher stimulate cell proliferation and estrogen-dependent gene incidence of hepatocellular carcinomas in female rats as expression. After incubation of cells with 100 nM DHEA for compared to males [1]. It remains to be clarified whether four days, estradiol was present in the medium at a con- the hepatocarcinogenic effect of DHEA is due to its andro- centration of ~200 pM. Other compounds identified were genic properties. testosterone and estrone. Significant stimulation of cell proliferation by 1 nM estradiol and 100 nM DHEA was ob- II. The Insulin / IGF-IR Signalling Pathway in served after 38 h and 4 days of incubation, respectively, Human Breast Cancer indicating the necessity of DHEA conversion. DHEA doses ³10 nM induced estrogen-dependent gene expression in D. Mayer, B. Schnarr, K. Strunz, J. Ohsam MCF-7 cells transfected with a luciferase reporter gene un- In cooperation with J. Wacker, University Hospital of Gynaecology der the control of the estrogen response element. DHEA- and Obstetrics, Heidelberg; A. Benner, Biostatistics, DKFZ dependent stimulation of proliferation and luciferase induc- The ligands, receptors and related signalling proteins of tion could be inhibited by the anti-estrogens ICI182,780 the insulin-like growth factor family are involved in the and tamoxifen, respectively, and by the aromatase inhibi- regulation of breast cancer cell growth. Human breast tor 4-hydroxyandrostenedione. An androgenic effect of carcinomas have been shown to produce IGF-I and IGF-II DHEA on proliferation and gene expression of MCF-7 cells which were suggested to stimulate the proliferation of the was not observed. We conclude that DHEA is converted to cancer cells in a paracrine or autocrine manner. We inves- estrogens, particularly estradiol, in breast cancer cells, and tigated the expression pattern of insulin-like growth factor-I that this conversion is required to exert a mitogenic re- receptor (IGF-IR), insulin receptor (IR) and insulin receptor sponse [12,13]. substrate-1 (IRS-1), a core downstream signalling protein, in 69 primary breast cancer specimens of different grades

DKFZ 2001: Research Report 1999/2000 Research Program Division Tumor Cell Regulation Signaltransduction and Growth Control

Publications (* = external coauthor) [1] Mayer, D., Metzger, C., Nehrbass, D., Bannasch, P.: Hepato- carcinogenesis by dehydroepiandrosterone. Current Topics in Ste- roid Research 1, 135-144 (1998) [2] Bannasch, P., Metzger, C., Mayer, D.: Hepatocarcinogenesis by dehydroepiandrosterone. I. Sequential cellular changes during neoplastic develoment. In: Dehydroepiandrosterone (DHEA). Bio- chemical, physiological and clinical aspects (W. Regelson, M. Kalimi, eds.) Walter de Gruyter, Berlin, pp. 237-249 (2000) [3] Mayer, D., Metzger, C., Bannasch, P.: Hepatocarcinogenesis by dehydroepiandrosterone. II. Biochemical and molecular changes during neoplastic development. In: Dehydroepiandro- sterone (DHEA). Biochemical, physiological and clinical aspects (W. Regelson, M. Kalimi, eds.) Walter de Gruyter, Berlin, pp. 251- 260 (2000) 92 [4] Mayer, D., Metzger, C., Nehrbass, D., Bannasch, P.: Charac- teristics of dehydroepiandrosterone-induced hepatocarcinogenesis in the rat. In: Hormonal Carcinogenesis III (Eds. Li, J.J., J.R. Daling, S.A. Li) Springer-Verlag, New York, pp. 477-483 (2000) [5] *Hobe, G., *Hillesheim, H.-G., *Schön, R., *Undisz, K., *Valentin, U., *Reddersen, G., *Ritter, P., Bannasch, P., Mayer, D.: Studies on the metabolism of DHEA in rats and mice. In: Dehydroepiandrosterone (DHEA). Biochemical, physiological and clinical aspects (W. Regelson, M. Kalimi, eds.) Walter de Gruyter, Berlin, pp. 343-362 (2000) [6] Mayer, D., Metzger, C., Leonetti, P., Beier, K.*, Benner, A., Bannasch, P.: Differential expression of key enzymes of energy metabolism in preneoplastic and neoplastic rat liver lesions induced by N-nitrosomorpholine and dehydroepiandrosterone. Int. J. Cancer 79, 232-240 (1998) [7] Kopplow, K., Mayer, D.: Dehydroepiandrosterone induces both cell proliferation and increase in cell size in rat liver. J. Cancer Res. Clin. Oncol. 125, S31 (1999) [8] Swierczynski, J. and Mayer, D.: Vitamin E prevents induction of carbonyl group formation in microsomal protein by dehydroepiandrosterone. Nutrition and Cancer 32, 101-106 (1998) [9] Nehrbass, D., Klimek, F., Bannasch, P., Mayer, D.: Insulin receptor substrate-1 is overexpressed in glycogenotic but not in am- phophilic preneoplastic hepatic foci induced in rats by N- nitrosomorpholine and dehydroepiandrosterone. Cancer Letters 140, 75-79 (1999) [10] Mayer, D., *Buniatian, G., Metzger, C., Bannasch, P., *Gebhardt, R.: Dehydroepiandrosterone increases the zone of glutamine synthetase positive hepatocytes in female rat liver: a putative androgenic effect. Histochem. Cell Biol. 111, 375-380 (1999) [11] Schnarr, B., Strunz, K., Ohsam, J., Benner, A., *Wacker, J., Mayer, D.: Downregulation of insulin-like growth factor-1 receptor and insulin receptor substrate-1 in advanced human breast cancer. Int. J. Cancer (Pred. Oncol.) 89, 506-513 (2000) [12] Schmitt, M., *Klinga, K., Mayer, D.: Dehydroepiandrosterone (DHEA) stimulates proliferation of MCF-7 cells after being me- tabolized to estrogens. Proc. Am. Assoc. Cancer Res. 41, 139 (2000) [13] Schmitt, M., *Klinga, K., Schnarr, B., *Morfin, R., Mayer, D.: Dehydroepiandrosterone stimulates proliferation and gene expression in breast cancer cells after conversion to estradiol. Mol. Cell. Endocrinol. 173, 1-13 (2001)

DKFZ 2001: Research Report 1999/2000