Structure-Function studies on the EGF/EGF receptor family
AWB, FW in collaboration with our Protein Biosensing Group, the Cell Biophysics Laboratory (LICR) , CSIRO and WEHI

The EGF receptor family consists of four distinct tyrosine kinase receptors, EGFR/HER/ErbB1, HER2/Neu/ErbB2, HER3/ErbB3, and HER4/ErbB4 which interact with at least 12 different ligands which can induce homo- or heterodimerization of the family members. Much remains to be determined about the basis of ligand affinity and specificity for the receptors, the basis of homo- and heterodimerization and how this leads to activation of the EGFR family tyrosine kinase domains by the ligands. We are currently pursuing answers to some of these questions by expressing recombinant forms of various EGF receptor family constructs and using a number of cell-biological and biophysical techniques to study them and their ligand interactions.

Epitope mapping of 806 and 175 antibodies
AWB, FW in collaboration with our Protein Biosensing Group; Terry Johns & Andrew Scott (Centre for Clinical Sciences, LICR); CSIRO and WEHI

The mAb 806 and mAb175 are monoclonal antibodies which act against a mutant form of the EGF receptor (de2-7 EGFR, or EGFRvIII). This receptor is frequently expressed in human malignant gliomas and has been reported in cancers of the lung, breast, and prostate. The antibodies react with only a small proportion of wild type EGF receptors on the surface of cells which over-express the EGFR, but both antibodies still possess significant antitumor activity against these cells. To gain a better understanding of the mechanism of action of this antibody, we are attempting to determine the epitope to which mAbs 806 and 175 bind. Preliminary results indicate that the epitope is exposed only in specific conformational states of the receptor. Our collaborative research suggest a mechanism of action for the inhibitory effects of the antibodies and helps elucidate the changes which occur when the EGFR undergoes a transition between the inactive and activated EGFR structures

Wnt ligands and their receptors: expression and role in colon cancer
FW, HHZ, AWB

The wnt family of ligands through their receptors (Frizzled and LRPs) regulate multiple intracellular pathways important in development, tissue homeostasis and cancer. As activators of the APC/catenin canonical pathway, wnt ligands and receptors are postulated to play a role in intestinal cancer; however the dearth of specific reagents to these molecules has hampered our understanding of their role in the physiology and pathology of the mammalian gut. We are now developing a panel of specific reagents and target cell lines to determine wnt ligand affinity and specificity for their cognate receptors, activation and downregulation of the ligand/receptor complexes and intracellular signalling complexes. These findings are crucial if we are to understand how wnt signalling is initiated, which proteins comprise the activated cell surface complexes and how tumorigenic activation can be reduced.

Analysis of the APC tumour suppressor protein function in colon cancer
MCF, JC, MC, KE, JJ-M, AWB in collaboration with Meredith Layton, JPRL (LICR)

Colon cancer affects more people in Australia than any other cancer. APC mutations can be inherited, but more than 80% of sporadic colon cancers carry truncating mutations in the tumour suppressor protein APC (adenomatous polyposis coli)1,2. APC mutations are thought to be an early event in a multistep process involving the successive acquisition of genetic mutations. This suggests a key role for APC in the maintenance of normal colonic cellular function, however, the precise mechanism of events arising from its loss of function that lead to the development of polyps and adenomas is not known. A well established role for APC is in the regulation of the Wnt signaling target -catenin. Recent studies demonstrate that APC is also involved in cytoskeletal regulation and is likely to play a role in cell migration, adhesion and differentiation. We have developed antibodies, recombinant proteins and cell lines for the study of different aspects of APC structure and function.

APC and cell adhesion
MCF, AWB in collaboration with Meredith Layton, JPRL (LICR)

We have evidence that the wild-type protein can influence cell adhesion. Restoration of full-length APC in colon cancer cells that normally contain only mutated APC results in enhanced cell adhesion. We are currently pursuing studies on these cells aimed at investigating the proteins associated with E-cadherin in the cells expressing full-length APC compared to cells with mutant APC using protein biochemistry and proteomics.

Subcellular location of APC and protein complex formation at specific subcellular locations
MCF, JC, AWB in collaboration with Meredith Layton, JPRL (LICR)

We believe that APC is a key mobile scaffold regulating cell adhesion and that its functions are intimately linked with its location and dynamic behaviour in the cell. The identification and analysis of proteins interacting with normal and defective forms of APC at specific subcellular locations will improve our understanding of its function in both normal and neoplastic colorectal cells. The results of this work will form the basis for developing improved strategies to eliminate colon cancer cells with defective APC.

Subcellular location of APC and axin in colon cancer cells
MCF, JC, JJ-M, AWB

Emerging studies of the subcellular localisation of APC have provided new insights into APC function in cytoskeletal organisation which may provide the key to its role as a tumour suppressor. The subcellular localisation and trafficking of APC are likely to be crucial determinants of the function of the molecule. We have generated antibodies to APC as well as fluorescent-fusion APC and axin proteins to study the localisation of APC in colon cancer cells and its dynamic behaviour in live cells.

Structure/function analysis of APC
MCF, AWB, KE in collaboration with Meredith Layton, JPRL (LICR) and Andrew Clayton, Cell Biophysics Laboratory (LICR)

APC is a large (2843 aa) protein with a modular structure containing a series of predicted protein-protein interaction domains. In colon cancers, APC protein is truncated losing a large part of the C-terminus of the protein. Even though APC is clearly an important molecule in colon cancer, little is known about its structure due to its large size and lack of homology with other proteins. This project aims to use classical structure/function analysis to characterise the shape and intramolecular interactions of the full-length and truncated APC protein and to translate these findings into a cellular context using sophisticated fluorescence microscopy techniques.

Intestinal Stem Cells: isolation and characterization.
YH, NS-S, AWB

The intestinal mucosa is a rapidly proliferating tissue. The production of all differentiated cell types in the intestinal crypts is maintained by the stem cells, which undergo self-renewal as well as differentiating to more mature cell types. Both of these characteristics are shared by tumor cells, raising the question of whether tumor cells arise from stem cells, or acquire “stem-cell like” properties in the process of transformation of committed progenitor cells. Thus it is important to gain some knowledge of the characteristics and growth requirements of colonic stem cells, particularly of the extracellular signals and/or the genetic mutations that favour self-renewal at the expense of differentiation. We are establishing systems for the isolation and “in vitro” enrichment of putative stem cells, their characterization using a panel of antigenic markers, and for the repopulation of depleted intestinal compartments with normal or genetically manipulated stem cells.

Novel cell-cycle inhibitory drugs: “in vitro” and “in vivo” anti-tumor effects
FW, AWB in collaboration with the Protein Biosensing Group (Ludwig) and the Department of Medicinal Chemistry (WEHI)

Chemotherapy is one of the major weapons in the fight against cancer. We are constantly searching for more effective anti-cancer molecules: some are targeted to specific pathways, shown to be upregulated in cancer (eg the Epidermal Growth Factor, PI-3-Kinase), some act in a more general way by preventing cell division in tumor cells. We are investigating a panel of novel drugs, designed at the Medicinal Chemistry department of the WEHI, to inhibit tumor cells. We are monitoring their effectiveness in “in vitro” killing of tumor cell lines, specificity for tumor subtype and ability to prevent the growth of tumor xenografts . These drugs exert their action by blocking cells in the G2/M phase of the cell cycle, where they become apoptotic and die. However the direct target of drug action is unknown. We are attempting to identify the site of action of the drug by monitoring drug binding to subcellular compartments by immunofluorescence microscopy; and to identify the target molecule using a combination of biosensor micro-affinity purification and proteomics .