Forschungs-AG Brockhoff

Klinik für Frauenheilkunde und Geburtshilfe
Lehrstuhl der Universität Regensburg
Forschungs-AG Brockhoff

(last update: May 2010)

Research Group
PD Dr. rer. nat. Gero Brockhoff

Contact c/o:
Institut für Pathologie
Universität Regensburg
Franz-Josef-Strauß-Allee 11
93053 Regensburg
Tel: 0941/944-6607
FAX: 0941/944-6602
gero.brockhoff@klinik.uni-regensburg.de

Quelle:
www.hybridmedicalanimation.com

Importance of ErbB-Receptor-Tyrosine-Kinases

We are working on both functional and quantitative analysis of ErbB-Receptor-Tyrosine-Kinases (RTK). They are typically located in the cell membrane of numerous (epithelial) tissues and regulate and control cellular and tissue development and differentiation. In tumor tissues however they contribute to carcinogenesis, tumor progression and metastasis. ErbB-RTK function is extensively regulated on a posttranslational level by receptor interaction, cross-phosphorylation, internalization, degradation and recycling. On the cellular level RTK show a pleiotropic impact on cell proliferation, growth, migration, adhesion, survival, and death. A typical feature of the four cognate receptors EGFR, ErbB2, ErbB3, and ErbB4 (synonyms are HER1-4) is their capacity to interact upon ligand activation (typically growth factor binding) amongst each other and with other membrane located molecules as well (lateral or horizontal signal transduction). As a result of ErbB receptor interaction and activation intracellular signal transduction is triggered (vertical signaling) causing activation of a ligand-receptor dependent cellular response (e. g. stimulation of proliferation). Although the EGFR and the ErbB2 receptor are utilized as targets for antigen specific therapies the ErbB-RTK represent a complex functional unit which is incompletely understood und subject of our research.

Background

Humanized and chimeric antibodies as well as enzyme specific kinase inhibitors represent target specific therapeutics used in a variety of cancer treatment. The antibody Trastuzumab (Herceptin®) and the tyrosine kinase inhibitor Lapatinib (Tyverb®) target individual members of the ErbB-RTK which are, in contrast to normal tissue, frequently overexpressed in breast cancer cells, hence represent hyperactive oncoproteins. In absence of ErbB receptors (e. g. in triple negative breast cancer) other receptors like GnRH-R or GHRH-R can potentially be targeted by using specific peptide agonists or antagonists (e. g Cetrorelix). Overall target specific therapeutics are expected to (specifically) inhibit tumor cell growth / proliferation or even to induce tumor cell death (apoptosis).

Tumor cells however are frequently insensitive to target specific therapies or develop reduced responsiveness during therapy. The cellular mechanisms causing therapy resistance are incompletely understood and are subject of investigation in our research group.

Goals

Identification of molecular mechanisms involved in responsiveness and resistance to target specific treatments of breast cancer cells
Identification of predictive markers and signatures, responsible for responsiveness to target specific therapies
Evaluation of efficiency of combined target specific tumor cell treatments

In a number of integrative subprojects we perform quantitative / descriptive as as well as functional analysis e. g…

Fluorescence-in-situ-Hybridization (FISH) in primary tissues and cell lines
Multiparametric immunophenotyping of ErbB receptor coexprssion profiles
Quantitative expression analysis of ErbB receptors and respective splice variants
Quantitative analysis of ErbB receptor interaction (homo- and heterodimerization) and (cross-)activation
Analysis of ErbB receptor dependent intracellular signaling
Analysis of treatment dependent cellular response (proliferation, survival, apoptosis, etc.)

A selection of applied techniques

Multiparametric flow cytometry (8- and 12-color instruments; FACSCanto-II, LSR-II, both BD Biosciences)
Multichannel pseudo-confocal (3D) fluorescence mikroscopy (AxioImager Z1 equipped with Apotome, Axiovision image analysis software, Zeiss)
Quantitative (high throughput) PCR (LightCycler 480, Roche)
Cell culture, Immunohistochemistry, Western-Blotting a.o.m.
In-vivo studies based on nude and humanized mice

A selection of methods

Flow cytometry phenotyping of breast cancer cells
Quantification of receptor interactionen via Fluorescence-Resonance-Energy-Transfer (FRET)
Static und dynamic proliferation assessment via flow cytometry, analysis of apoptosis and cell viability
Fluorescence-in-situ-Hybridization (FISH) in primary tumor tissues
Biochemical und biomolecular analysis of receptor (co-)expression und activation
Analysis of lateral und transversal signal transduction
Analysis of tumor growth and dissemination in in nude and humanized mice

AxioImager Z1 (Carl Zeiss)

Diagram of (Fluorescence)-in-situ-Hybridization

Microscopic images of FISH specimens without (left) and with (right) Apotome technology. The image in the middle shows structured illumination based on grid projection

Example of her2 gene amplification (green signals) and low grade polysomy (red signals: CEN 17)

Principle of a (flow cytometric) analysis of receptor interaction by quantification of FRET

Dynamic proliferation assessment via flow cytometry for e. g. quantification of quiescent cells (Ko = control; Herc = Herceptin treated breast cancer cells)