Download Research Group Hormones and Signal Transduction (B0810 / A105

Research Program B
Tumor Cell Regulation
B0810
Hormones and Signal Transduction
Research Group Hormones and Signal Transduction (B0810 / A105)
Head: Prof. Dr. rer. nat. Doris Mayer
I. The IGF / IGF-I receptor signalling pathway
Postgraduate and Graduate Students
Barbara De Servi (7/01 – 6/02) *
Alexandra Dreuw (- 5/01)
Stephanie Geiger (10/01 – 9/02)
Alexander Hermani (2/02 - ) *
Yongde Liao (10/01 - ) *
Senad Medunjanin (1/01 - )
a) in breast cancer cells
D. Mayer, A. Dreuw, S. Geiger, S. Medunjanin,
B. De Servi, G. Rincke, Y. Niu
Visiting Scientist
Yun Niu (Tianjin, China, - 5/01)
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Technical Assistants
Jürgen Ohsam ( - 4/01)
Gabriele Rincke (3/02 -)
* external funding
Hormones play an important role in the regulation of numerous cellular processes related to cell growth and differentiation. The significance of steroid and peptide hormones for tumour initiation and progression in a number
of organs and tissues of various species including humans has been documented by experimental and epidemiological studies. More recently various hormone-stimulated signal transduction pathways have been partly
unravelled and their components have been identified
and characterised. Although both signalling pathways result in expression alteration of target genes, the pathways
activated by steroid and peptide hormones show major
differences. Peptide hormones bind to receptors
localised in the plasma membrane and trigger a cascade
of phosphorylation reactions through protein kinases,
eventually resulting in activation of gene expression in
the nucleus. Steroid hormones bind to and activate
nuclear receptors which function as transcription factors.
Recent work has revealed manifold interactions between
both types of signalling pathways.
The research group “Hormones and Signal Transduction” focuse their interest on the study of the mechanisms
of tumour induction and progression by steroid hormones
in epithelial cells of the breast, prostate and liver, as well
as to hormone-controlled gene expression and the interaction of steroids with components of other cellular signal
transduction pathways.
The type I insulin-like growth factor receptor (IGF-IR) mediates the mitogenic, transforming, differentiating, and
antiapoptotic actions of the IGF ligands, IGF-1 and IGF-2.
In the mammary gland the IGF-IR has an important role in
the promotion of normal breast growth and development,
and in the initiation of breast cancer. We have recently reported that the IGF-IR and its substrate IRS-1 are highly
expressed in early stages of primary breast cancer. Moreover, the expression of both proteins correlates with the
expression of the estrogen receptor (ER), suggesting a
functional link between the ER and IGF systems. At late
malignant stages of breast cancer, IGF-IR and IRS-1, as
well as ER, were found to be significantly decreased
[Schnarr et al., Int. J. Cancer (Pred. Oncol.) 89, (2000)
506-513]. These findings suggest that the IGF-IR is required for initiation of breast cancer, whereas the acquisition of the malignant phenotype results from decreased
IGF-IR gene expression and action. In vitro studies in estrogen-dependent breast cancer cells have shown that the
expression of IGF-IR and IRS-1 is controlled by estradiol
(E2) [1].
In our attempt to unravel the molecular mechanisms involved in the control of IGF-IR and IRS-1 expression by E2
we started to investigate the effect of E2-treatment of estrogen-dependent breast cancer cells on the phosphorylation stage and activity of proteins of the IGF-IR pathway.
Stimulation of the IGF-IR by IGF results in
autophosphorylation on tyrosine residues which, in turn,
activates the intrinsic receptor tyrosine kinase activity towards other substrates. IRS-1 is the major substrate of the
IGF-IR and is tyrosine phosphorylated after IGF-IR stimulation. The phosphorylated IRS-1 represents a docking
protein for molecules mediating the activation of the
phosphatidylinositol-3-kinase (PI3K)/AKT/PKB pathway.
The serine/threonine kinase AKT is a central component of
the pathway which controls the activity of numerous downstream molecules. Activation of AKT results in inhibition of
apoptosis and inactivation of proteins that inhibit cell cycle
progression. In vitro studies suggest that E2 interacts with
this signalling pathway at different levels. While IGF-IR
and IRS-1 are not phosphorylated by short-term E2-treatment, AKT/PKB is phosphorylated and activated in estrogen-dependent breast cancer cells under the influence of
E2. Using appropriate inhibitors (e.g. wortmannin, an inhibitor of PI3K) it could be shown that PI3K is involved in
E2-stimulation of AKT-phosphorylation. The (enzymatic)
reaction that activates PI3K has not yet been clearly identified. It is possible that activation of the tyrosine kinase csrc which is tyrosine-phosphorylated after stimulation of
MCF-7 cells with E2 is involved in activation of PI3K. Inhibition of Src by a specific inhibitor resulted in a 30% inhibition of E2-induced cell proliferation. This agrees with find-
DKFZ 2003: Research Report 2001/2002
Research Program B
Tumor Cell Regulation
ings on estradiol-dependent gene expression which was
inhibited by 30% in cells stably transfected with a dominant negative src-mutant [2] or with a dominant negative
AKT mutant. It is concluded that E2-treatment of estrogendependent breast cancer cells may activate the PI3K-AKT
pathway.
b) in prostate cancer cells
Y. Liao, A. Hermani, D. Mayer
In cooperation with Dr. Peter Angel, Division of Signal Transduction and Growth Control, DKFZ; Dr. Rainer Grobholz, Dept. Pathology, University Hospital Mannheim, Professor Dr. Dr. Ulrich
Abel, Dept. of Medical Biometrics, Heidelberg University
Additional funding by Tumorzentrum Heidelberg/Mannheim
Insulin-like growth factors are important growth factors in
the development of prostate cancer and its progression.
The biological activity of IGFs is mediated via the IGF-I receptor signalling pathway, which involves activation of the
protein kinase AKT/PKB, and is regulated by inhibitory
IGF-binding proteins (IGFBP). Overexpression of related
proteins or changes which raise the balance of IGF/IGF-IR
activity versus IGFBP function can potentially contribute to
prostate carcinogenesis.
Expression of AKT, IGF1, IGF2 and IGFBP3 proteins, investigated by immunohistochemistry in human prostate
cancer specimens, was correlated with clinicopathological
parameters [Gleason score, pathological tumour stage
(pT) and preoperative serum level of prostate specific antigen (PSA)]. The immunoreaction was investigated separately in benign prostate tissue, PIN lesions (prostatic
intraepithelial neoplasia) and prostate cancer tissue according to intensity and extent (fraction of positive cells)
and according to an immunoreactivity score = staining intensity × extent. AKT, IGF1 and IGF2 proteins were found
overexpressed in prostate cancer tissues compared to
benign prostate tissues with respect to the intensity, frac-
IGF1/2
PSA
IGFBP3
IGF-IR
IRS1
PI3K
Cell membrane
AKT
Growth
stimulation
Inhibition of
apoptosis
Fig. 1. IGF-signaling pathway in prostate cancer. IGF-1, IGF-2
and AKT are overexpressed in prostate cancer whereas expression of the regulatory / inhibitory IGF-binding protein-3 (IGFBP3)
remains unchanged compared to benign tissue. Proteolysis of
IGFBP3 by prostate specific antigen (PSA) may result in increase
of free IGFs. It is suggested that overexpression of components
of the IGF-signaling pathway results in growth stimulation and inhibition of apoptosis in prostate cancer cells.
B0810
Hormones and Signal Transduction
tion of positive cells and score of the immunoreactivity.
Statistical analysis revealed a significant increase in the
expression of AKT, IGF1 and IGF2 in tumours compared
to benign tissue with a clear increase from benign tissue
over PIN lesions to tumour and with tumour progression,
whereas IGFBP3 expression was similar in all tissue
types. The expression of AKT in prostate cancer, regarding the intensity, and the expression of IGF1 and IGF2, regarding the intensity, fraction of positive cells and score of
the immunoreactivity were positively correlated to high
preoperative PSA serum levels. Moreover, high expression of IGF2 in prostate cancer, with respect to fraction of
positive cells and score of the immunoreactivity, was associated with tumour progression as indicated by
Gleason score. IGFBP3 expression was not correlated
with any of the clinicopathological parameters. It is concluded that the IGF/IGFBP balance is altered in prostate
cancer (Fig. 1). This alteration is characterized by a significant overexpression of IGF1 and IGF2, whereas
IGFBP3 expression is unchanged in tumour compared to
benign prostate tissue. AKT/PKB, a central element of the
IGF/IGF-IR/AKT signalling pathway is also overexpressed
in prostate cancer. These data provide evidence that the
IGF signalling pathway plays an important role in the initiation and progression of human prostate cancer. Further investigations on this pathway may illustrate the implications in prognosis and treatment of prostate cancer
[Liao, Y. Thesis, 2003].
II. Dehydroepiandrosterone effects on cell
proliferation
K. Kopplow, K. Forstner, M. Schmitt, D. Mayer
In cooperation with Professor Dr. Peter Bannasch, DKFZ, Axel
Benner, Central Unit Biostatistics, DKFZ; Dr. Klaus Klinga, Hormone Laboratory, Heidelberg University; Professor Dr. Julian
Swierczynski, Department of Biochemistry, Medical University,
Gdansk, Poland; Professor Dr. Robert Morfin, Laboratoire de
Biotechnologie, Conservatoire National des Arts et Métiers, Paris,
France
a) in liver
Dehydroepiandrosterone (DHEA), the main adrenal steroid in humans and a precursor in the biosynthesis of potent androgens and estrogens, acts as a hepatocarcinogen in rats [3, 4]. Neoplasms emerge from a glycogenotic /
amphophilic / basophilic preneoplastic cell lineage (Fig. 2).
A higher female tumour incidence suggests a relevant influence of sex hormones. DHEA enhances hepatocarcinogenesis induced by N-nitrosomorpholine (NNM) which is
characterised by the glycogenotic / basophilic cell lineage.
The tumour promoting effect is related to an additional amphophilic / basophilic preneoplastic lesion sequence and
to faster proliferation of the basophilic preneoplastic lesions. Nevertheless, hepatocellular carcinomas provided
under DHEA treatment are growing more slowly and seem
to have a less malignant phenotype compared to tumours
induced by NNM only. Further, DHEA treatment reduces
growth and generation of glycogen storage foci (GSF) in
initial NNM treated rats. Thus, DHEA treatment results in
both, a growth stimulation (promotion) of the late baso-
DKFZ 2003: Research Report 2001/2002
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Research Program B
Tumor Cell Regulation
IC
GSF
Publications (* = external co-author)
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AMPF
BCF
BCF
Tumor
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B0810
Hormones and Signal Transduction
Fig. 2. Model suggesting the sequence of lesions in rat liver
treated with N-Nitrosomorpholine and DHEA. Black arrows: sequence of lesions induced by NNM; open arrows: effect of DHEA
on sequence of lesions. IC, initiated cell; GSF, glycogen storage
focus; BCF, basophilic cell focus; AMPF, amphophilic cell focus.
AMPF can rise from GSF by DHEA-treatment. It is not yet clear
whether AMPF can emerge directly from initiated cells.
philic lesion type with an additional amphophilic lesion
sequence, and in a growth inhibition of early
preneoplastic lesions, addressing especially GSF [5].
DHEA inhibits not only growth of NNM-induced glycogenotic liver lesions and neoplasias but also the growth of
physiologically proliferating liver tissue, e.g. compensatory
proliferation after partial hepatectomy [6]. This might be
explained by a DHEA related cellular metabolism [7;
Mayer et al., Int. J. Cancer (Pred. Oncol.), 79 (1998) 232240], which is characterized by significant energy consumption. Additionally, DHEA treatment of rats resulted in
alterations of cytokine and growth factor levels, e.g. of IL-6
and IGF-1 in rat liver, that might contribute to this growth
inhibition as well [5, 6].
[1] Dreuw, A. (2001) Wirkung von IGF-I und Estradiol auf die
Tyrosin-Phosphorylierung des Insulin-like Growth Factor-I
Rezeptors und auf die estragonabhängige Genexpression in
Mammakarzinomzellen. Diplomarbeit, Biologische Fakultät,
Universität Heidelberg
[2] Geiger, S. (2002) Phosphorylation and activation of pp60Src in
estrogen-dependent breast cancer cells. Diplomarbeit,
Mathematisch-Naturwissenschaftliche Fakultät I, HumboldtUniversität Berlin.
[3] Mayer, D. (2001) Hormonal and carcinogenic effects of
dehydroepiandrosterone on liver.
In: Surgical Pathology, Update 2001, 18th European Congress of
Pathology (Eds. S. Hauptmann, M. Dietel, M. Sobrinho-Simões),
ABW Wissenschaftsverlag Berlin, pp. 363-367.
[4] Mayer, D. (2002) DHEA effects on liver. In: DHEA and the
Brain (R. Morfin, ed.), Volume 1, Chapter 5, Harwood Academic
Publisher, pp. 81-100.
[5] Mayer, D., Forstner, K., Kopplow, K. (2003) Induction and
modulation of hepatic preneoplasia and neoplasia in the rat by
dehydroepiandrosterone. Toxicol. Pathol. 31, 1-10.
[6] Kopplow, K. (2002) Untersuchung des Einflusses von
Dehydroepiandrosteron auf die Proliferationsaktivität von
Hepatozyten und die Expression von Zellzyklusparametern in der
Rattenleber. Dissertation Dr. rer. nat., NaturwissenschaftlichMathematische Gesamtfakultät, Universität Heidelberg
[7] *Swierczynski, J., *Slominska, E., *Smolenski, R.T., Mayer, D.
(2001) Increase in NAD but not ATP and GTP concentrations in
rat liver by dehydroepiandrosterone feeding. Pol. J. Pharmacol.
53, 125-130.
[8] Schmitt, M., *Klinga, K., Schnarr, B., *Morfin, R., Mayer, D.
(2001) Dehydroepiandrosterone stimulates proliferation and gene
expression in breast cancer cells after conversion to estradiol.
Mol. Cell. Endocrinol. 173, 1-13.
b) in breast cancer cells
DHEA serum levels in humans are high in young adults
and decrease continuously with age. DHEA replacement
in the elderly has been reported to result in increased wellbeing. For this reason DHEA has been suggested for use
as anti-aging drug.
Epidemiological studies have shown that postmenopausal
women with high DHEA-plasma levels have an increased
risk to develop breast cancer. In vivo, DHEA can be accumulated in normal breast tissue and in breast cancer from
the blood. In vitro, DHEA stimulates proliferation of MCF-7
cells, an estrogen-dependent cell line derived from a human breast carcinoma. We could show that growth stimulation is not a direct effect of DHEA but due to the conversion of DHEA to significant amounts of estradiol in MCF-7
cells. Inhibition of aromatase activity prevented growth
stimulation by DHEA. It could further be documented that
DHEA acts exclusively as estrogen in MCF-7 cells. The
cells did not respond to androgenic DHEA-derivatives [8].
We conclude from our results that DHEA acts as an estrogen and represents a mitogen to estrogen-dependent
breast cancer cells. Therefore, DHEA replacement in postmenopausal women should be avoided because of potential increase in breast cancer risk.
DKFZ 2003: Research Report 2001/2002