Receptor Tyrosine Kinases / János Szöllősi

Personal data: 

János Szöllősi
Ph.D., D.Sc.
E-mail: szollo[at]med.unideb.hu

http://www.biophys.dote.hu/cellanal

Research: 

The ErbB (HER) family of receptor tyrosine kinases fulfills important roles in differentiation, apoptosis and in the regulation of physiological and pathological cell proliferation. The family consists of four members, ErbB1 (also known as epidermal growth factor receptor), and ErbB2-4. Ligand binding initiates the formation of receptor homo- and heterodimers, or the rearrangement of pre-existing receptor associations leading to phosphorylation of tyrosine residues and subsequent boosting of secondary signal routes. ErbBs are overexpressed in a wide range of human cancers. The first humanized monoclonal antibody against solid tumors was trastuzumab (TR), a “smart” drug targeting ErbB2. The palette has now extended to over 20 anti-ErbB antibodies, many already in clinical trials, and several small molecule ErbB kinase inhibitors. Unfortunately, resistance issues are also wide-spread, including resistance to TR. Protein interactions are key elements in all signaling pathways including that of ErbB kinases; in addition to the 4 members of the ErbB family, other proteins (cell surface mucins, integrins, CD44) are also involved in ErbB protein clusters. These accessory proteins endow the ErbB axis with an enhanced signaling potential, and perceivably also contribute to therapy resistance.

We have shown that association of ErbB2 is organized on two levels. In addition to the well-known direct association (e.g. dimers), large-scale clusters are also formed containing ~1000 ErbB2/cluster. These clusters colocalize with lipid rafts, and their size increases upon receptor stimulation. We have shown that raft localization of ErbB2 is important for its function, and also the action of anti-ErbB2 antibodies. Furthermore, a flow cytometric, fluorescence polarization-based model established that ErbB1 and ErbB2 behave in a fundamentally different way: whereas huge ErbB2 clusters serve as a reservoir of inactive coreceptors and dissociate upon stimulation, small ErbB1 homoclusters join to form higher-order oligomers after ligand binding. Towards the role of accessory proteins, we have established that β1-integrins associate with ErbB2, and that both of them colocalize with lipid rafts. The association is broken up by crosslinking either entity, but, surprisingly, integrins still appear to compete for ErbB molecules, and shift the emphasis from ErbB homoassociation to integrin-ErbB heteroassociation, with a concomitant shift of signaling towards Akt-mediated cell survival. Such studies have been made possible by innovative application of microscopic fluorescence resonance energy transfer experiments, whereby multiple pairwise molecular interactions could be quantitated in a correlated manner, on a pixel-by-pixel basis. Other accessory molecules involved in tumor resistance to anti-ErbB2 antibody therapy have also been highlighted. In the TR resistant breast cancer cell line JIMT-1, the mucin MUC4 was overexpressed, and its level was inversely correlated with the TR-binding capacity of single cells. Knockdown of MUC4 by RNAi, as well as conjugating TR to paramagnetic microbeads increased the binding of TR, substantiating that MUC4 likely inhibits TR binding by steric hindrance. Inhibiting the synthesis of the CD44-ligand hyaluronan by 4-methylumbelliferon (4-MU) also lead to enhanced binding of TR to ErbB2, and increased ErbB2 down-regulation. As an unexpected finding, the in vitro resistant JIMT-1 cells were eliminated in TR-mediated ADCC when introduced into SCID mice, but only at small tumor sizes or in the form of disseminated tumor cells. This ADCC-based inhibitory effect of TR was significantly increased by 4-MU, promoting the idea that massive extracellular matrices can contribute to resistance against therapeutic antibodies. The data point to the importance of the CD44-hyaluronan pathway in the escape of tumour cells from receptor-oriented therapy and the hitherto overlooked, but potentially important ADCC-mediating effect of ErbB-targeting antibodies that can be enhanced by reducing the ECM. Additionally, it also became clear that both TR, and pertuzumab, an antibody which blocks ErbB dimerization, can exerted a beneficial reducing effect on CD44 shedding and high cellular motility that are evoked by local EGF or heregulin stimuli either in vitro or in vivo.

The key concept continues to be the use of high resolution biophysical methods to investigate systems generated by molecular and cell biological tools (tumor cell lines sensitive and resistant to specific treatments, receptors of adjustable expression levels, fluorescent fusion protein, circulating tumor cells) in order to reveal the hierarchical levels of molecular interactions, correlate them with cellular responses to treatments targeting cell proliferation / survival, and provide proof of concept using in vivo models. The results are expected to provide fundamental information on molecular interactions and expression patterns of ErbB kinases, and the predictive power thereof regarding proliferative and metastatic capabilities and amenability as molecular targets.

Photos: 

 

Figure 1.

Triple colocalization of ErbB2, β1 integrin and lipid rafts (A–D). Confocal images taken from the surface of an N-87 cell showing the distribution of (A) ErbB2, (B) β1 integrin and (C) lipid rafts. The three channels are overlaid in image (D) showing high overlap between membrane areas rich in these three components.

 

 

 

 

 

 

 

Figure 2.

Showdown with multi-molecular complexes by tsFRET Disregulation of signal transduction processes starting from assemblies of cell surface molecules contributes to the pathogenesis of various cancers. Interactions between members of multi-molecular signaling complexes are therefore of particular interest. A new, innovative experimental set-up termed two-sided FRET (tsFRET) allows two pairwise interactions of three arbitrarily chosen molecules.

 

 

 

 

 

 

Figure 3.

Stimulation of ErbB2 by trastuzumab-derivatized paramagnetic microbeads (Green) YFP-fused ErbB2 on a cluster of carcinoma cells; (White) reflection image of beads; (Red) phosphorylated ErbB2 exclusively under the beads; (overlay) specific stimulation where beads dragged into the membrane overcome steric hindrance.

 

 

 

 

 

 

 

Figure 4.

ErbB2 - β1-integrin heteroassociation increased in focal adhesions Z-section of adherent cells labeled for ErbB2 (A) and β1-integrin (B). Pseudocolor fluorescence resonance energy transfer (FRET) map indicates higher degree of molecular interactions at the adhesion surface.

 

 

 

 

 

Figure 5.

Detecting circulating and disseminated tumor cells from the blood and bone marrow of mice xenografted with human breast carcinoma Tumor cells of human breast cancer origin in the blood (buffy coat, A-D) and bone marrow smear of the mouse (E-H) are identified based on their MHC-I positivity (A, E, green), ErbB1 (B, F, red) and ErbB2 (C, G, blue) expression. Overlay (D, H) shows these cells among the numerous cells of mouse origin. 

 

 

 

 

 

Figure 6.

Down-regulation of ErbB2 membrane expression by trastuzumab on JIMT-1 breast tumor cells xenografted into mice Immunofluorescent labeling of frozen sections indicates decreased membrane levels of ErbB2 (B) upon long term trastuzumab treatment of the mice as compared to saline control (A).

 

 

 

 

 

 

 

 

Figure 7. 4-MU, an inhibitor of hyaluronan synthase, increases the binding of trastuzumab to JIMT-1 xenografts Frozen sections from the tumors double labeled for trastuzumab (green) and ErbB2 (red) indicate that inhibition of hyaluronic acid synthesis by 4-methylumbelliferone (4-MU) improves trastuzumab binding in vivo. Contour plots show how added 4-MU (red plot) shifts the emphasis to highly TR positive pixels, which at the same time exhibit reduced ErbB2 expression, as compared to TR alone (black contours).

 

Publications: 

Szöllősi J, Balázs M, Feuerstein BG, Benz CC, Waldman FM. ERBB-2 (HER2/neu) gene copy number, p185HER-2 overexpression, and intratumor heterogeneity in human breast cancer. Cancer Res 1995;55:5400-7.

Nagy P, Jenei A, Kirsch AK, Szöllősi J, Damjanovich S, Jovin TM. Activation-dependent clustering of the erbB2 receptor tyrosine kinase detected by scanning near-field optical microscopy. J Cell Sci 1999;112 ( Pt 11):1733-41.

Nagy P, Vereb G, Sebestyén Z, Horváth G, Lockett SJ, Damjanovich S, Park JW, Jovin TM, Szöllősi J. Lipid rafts and the local density of ErbB proteins influence the biological role of homo- and heteroassociations of ErbB2. J Cell Science 2002;115:4251-4262.

Vereb G, Szöllősi J, Matkó J, Nagy P, Farkas T, Vigh L, Mátyus L, Waldmann TA, Damjanovich S. Dynamic, yet structured: The cell membrane three decades after the Singer-Nicolson model. Proc Natl Acad Sci U S A 2003;100:8053-8058.

Mocanu M, Fazekas Z, Petrás M, Nagy P, Sebestyén Z, Isola J, Tímár J, Park JW, Vereb G, Szöllősi J. Associations of ErbB2, beta1-integrin and lipid rafts on Herceptin (Trastuzumab) resistant and sensitive tumor cell lines. Cancer Lett 2005;227:201-212.

Pályi-Krekk Z, Barok M, Kovács T, Saya H, Nagano O, Szöllősi J, Nagy P. EGFR and ErbB2 are functionally coupled to CD44 and regulate shedding, internalization and motogenic effect of CD44. Cancer Lett 2008;263:231-42.

Barok M, Isola J, Pályi-Krekk Z, Nagy P, Juhász I, Vereb G, sr., Kauraniemi P, Kapanen A, Tanner M, Vereb G, Szöllősi J. Trastuzumab causes antibody-dependent cellular cytotoxicity-mediated growth inhibition of submacroscopic JIMT-1 breast cancer xenografts despite intrinsic drug resistance. Mol Cancer Ther 2007;6:2065-72.

Fazekas Z, Petrás M, Fábián Á, Pályi-Krekk Z, Nagy P, Damjanovich S, Vereb G, Szöllősi J. Two-sided fluorescence resonance energy transfer for assessing molecular interactions of up to three distinct species in confocal microscopy. Cytometry A 2008;73:209-19.

Szabó A, Horváth G, Szöllősi J, Nagy P. Quantitative characterization of the large-scale association of ErbB1 and ErbB2 by flow cytometric homo-FRET measurements. Biophys J 2008;95:2086-96.

Fábián A, Barok M, Vereb G, Szöllősi J. Die hard: are cancer stem cells the Bruce Willises of tumor biology? Cytometry A 2009;75:67-74.