Our laboratory aims to improve our understanding of colon cancer biology and to use this knowledge to improve treatments for colon cancer patients.

Research focus: Epithelial Biochemistry Laboratory

Cancers which arise from epithelial cells (carcinomas) account for more than three-quarters of all new cancer cases. The focus of the Epithelial Biochemistry Laboratory is on the biology and biochemistry of normal and tumorous epithelial cells from the colon. Our research involves five major themes:

1) The regulatory mechanisms that govern cell production and differentiation in normal tissue homeostasis and in colon cancer. These include growth factor/receptor interactions, with emphasis on the wnt/frizzled and notch systems, and intracellular signalling pathways, such as axin/apc and catenin/cadherins. An understanding of these pathways and their constituent proteins will further enable us to develop more effective therapies against colon cancer.

2) Role of notch signalling in colon biology.

3) The isolation and characterization of mesenchymal stem cells and intestinal stem cells. The intestinal stem cells are the precursors from which all other intestinal cell types emerge. Cancer cells have many stem-cell characteristics: an understanding of the origin and biology of the normal and cancer stem cells will greatly assist in defining the regulatory mechanisms perturbed in colon cancer.

4) The role of phosphatidyl inositol phosphates (PIPs) in maintaining colon cancer cells. We are analysing the proteins associated with PIP, PIP2 and PIP3 in colon cell lines with or without PI3 kinase mutations.

5) The identification and testing of potential anti-cancer agents. These studies comprise the testing of novel anti-cancer drugs on tumour cell lines in vitro and in xenograft models, and the identification of their target molecules by immunofluorescence, affinity isolation and proteomics.

Wnt ligands and their receptors: expression and role in colon cancer
FW, HHZ
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/ 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 pathways. 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.

Role of CRC stem cell-specific  molecules in the maintenance of the tumor phenotype.
FW, HHZ
Intestinal tumors arise from the stem cell compartment through a series of mutations which allow the cells to escape the normal physiological controls. Colorectal cancer (CRC) stem cells share many characteristics with the normal intestinal stem cells, such as self-replication and the generation of more differentiated cell types. Accordingly, many of the cell lines derived from human colorectal cancer maintain many stem-cell characteristics and express molecules, such as LGR5, PHLDA-1, CD133, CD44 and Msi-1, which are normally restricted to intestinal stem cells and their early progeny.  These antigens have been very useful as markers for the identification of normal and cancer stem cells; however very little is known on the functional role these proteins in the maintenance of the cancer stem cell phenotype. Using well-defined human CRC cell lines, we are currently modulating the expression of these stem-cell markers by knockdown (RNAi) and inducible overexpression. These experiments allow us to define the cellular parameters which are regulated by these genes, expand our understanding of ‘stemness’ and of the factors that control the balance between self-renewal and differentiation, and may lead to the identification of stem-cell specific therapeutic targets.

Analysis of the APC tumour suppressor protein function in colon cancer
MCF, JC, MC, KE
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). 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 signalling target b-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.

Structure/function analysis of APC
AWB, MCF, KE
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.

Investigation of phosphorylated b-catenin
MCF, JC, AWB
The current paradigm for how truncation of APC results in initiation of tumourigenesis is through failure to regulate the cellular concentration of b-catenin by proteasome-mediated degradation within a complex known as the ‘destruction complex’. The critical step in b-catenin regulation is the phosphorylation of serine and threonine residues in the N-terminus of b-catenin, which creates a recognition sequence for an E3 ligase resulting in its ubiquitination.  In addition, mutations in b-catenin, which also prevent its phosphorylation, occur frequently in CRC and several other malignancies. Dysregulation of phosphorylation of b-catenin is proposed to result in its accumulation and subsequent activation of Wnt/b-catenin-target genes, which are important in gut development and in driving colorectal tumourigenesis. We have recently shown that the axin destruction complex is localised to cytoplasmic puncta and that formation of axin-puncta is required for b-catenin degradation.  We have recently examined sub-populations of phosphorylated b-catenin in a number of cell types.  The subcellular distribution of phosphorylated b-catenin is different in colon cancer cells with mutated APC.  We hypothesise that there are populations of N-terminally phosphorylated b-catenin that are not part of the destruction complex.  We are pursuing these studies to explore the function(s) of subpopulations of phosphorylated b-catenin. 

Notch signalling in colonic cells
SPW, HHZ, FW, AWB
The self renewal, proliferation and differentiation of colonic crypt cells are influenced by wnt, notch and tyrosine kinase signalling systems. We are investigating the interactions between these signalling systems in crypt cells, colon cancer cell lines and mesenchymal cells expressing recombinant forms of frizzled and notch receptors. We are studying the biochemistry of jagged and delta interactions with notch and the activation of the signalling processes. Our colon cancer cell lines appear to have significantly different levels of notch activation; we are investigating the biological consequences of notch and/or wnt activation and the relationship of these effects to the different oncogenic mutations in each cell line.

Intestinal Stem Cells: isolation and characterization
YH, SC, FW, 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 tumour cells, raising the question of whether tumour 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.

Development of therapeutic strategies targeted to CRC stem cells
SC, AWB, FW
Cancer stem cells share many of the characteristics of normal intestinal  stem cells, and are believed to be responsible for cancer relapses after apparent remission from disease through their resistance to chemo- and radio-therapy We aim  to develop effective therapeutic strategies able to specifically target these cells in human colorectal cancer. We are establishing optimal ‘in vitro’ culture conditions for the CRC stem cells isolated from human primary tumors; these cultures will be used to expand the cancer stem cell population, to assess their tumor inititating capacity, and to determine their sensitivity to a panel of chemotherapeutic drugs and signalling pathway inhibitors.

Phosphatidyl Inositol phosphate (PIP) associated proteins and colon cancer
BC, MC, NC, MP with Andrew Holmes (Bio21 Institute)
PIPs are important components of the plasma membrane and are associated with several signal transduction systems in cancer biology. In recent years we have used synthetic PIP analogues to identify PIP binding proteins in colon cancer cell lines. These cell lines have different combinations of tumour suppressor and oncogenic mutations, often leading to significant perturbations in the levels and distribution of PI(3,5)P2, PI(4,5)P2 and PI(3,4,5)P3. Using affinity capture and proteomics we have identified proteins which bind to the different PIPs as a result of oncogenic or growth factor stimulation. We are investigating the role of specific PIP binding proteins in maintaining the transformed state in colon cancer and extending our analyses to study the role of phosphoinositol polyphosphates in colon cancer.

Novel cell-cycle inhibitory drugs: in vitro and in vivo anti-tumour effects
FW, JW, BC
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 unregulated in cancer (eg the Epidermal Growth Factor, PI-3-Kinase), some act in a more general way by preventing cell division in tumour cells. We are investigating a panel of novel drugs, designed at the Medicinal Chemistry department of the WEHI, to inhibit tumour cells. We are monitoring their effectiveness in “in vitro” killing of tumour cell lines, specificity for tumour subtype and ability to prevent the growth of tumour 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.