One reason why cancers invade the surrounding tissues and spread to distant organs (metastasis) is because they can evade or have lost the ability to respond to normal control mechanisms. EphB2 is a protein which is part of a normal growth control mechanism in the colon and most colon cancers lose the EphB2 protein as they progress to an invasive/metastatic type. Restoring EphB2 expression to colon cancer cell lines retards their ability to grow as tumours. This study investigates the mechanism by which EphB2 inhibits tumour growth by studying the effects restoring EphB2 expression to colon cancer cells in culture and will also determine the cause of EphB2 induced growth retardation in tumours.

Updated Report - 1 July 2008
EphB2/ephrinB interaction as a growth regulator of colon cancer

Dr. P.V.Senior & Prof. S. T. F. Chan

Lay Title

Investigation of how Eph B2 inhibits the growth of Colon Cancer

Introduction

Colon cancer arises in the epithelial lining of the colon. Whilst surgical removal can be curative once the tumour has spread (metastasised) to other organs mortality is high.

One factor in the progression of cancer is the loss of response to normal control systems.

EphB2 is a receptor is expressed on dividing normal colonic epithelium but is lost when they stop dividing. Interaction of EphB2 with ephrins expressed on other cells ensure these dividing cells are contained in their correct location. EphB2 expression is turned off in a high proportion of colon cancers and this correlates with poorer prognosis. The loss of EphB2 appears to remove a restraint on growth and is important in the progression of colon cancer.

The aim of this project is to study the mechanism by which EphB2 inhibits cancer growth. To this end we have used an EphB2 negative colon cancer cell line and introduced an inducible EphB2 construct which allows us to regulate the expression of EphB2.

Objectives and progress to date

Objective

Observe the effects of interaction of EphB2 expressing tumour cells with its ligands (Ephrin B1, B2 & B3) on their growth and behaviour in vitro.

Progress

1. We have demonstrated that EphB2 expression has a profound effect on the tumour cells ability to degrade extracellular matrix and also to migrate in response to chemical attractant. Even low amounts of EphB2 expression can reduce migration 5-10 fold.
   
2. A number of experiments have been completed showing that expression of EphB2 has little direct effect on cell growth rate in culture. Treatment of EphB2 expressing cells with ephrin B1 similarly has no effect on growth rate. This is consistent with most other studies which suggest the Eph/Ephrin system does not directly regulate cell proliferation.
   
3. We have also shown in preliminary experiments that EphB2 expression with or without ephrin stimulation has no effect on the response to apoptosis inducers such as the drug etoposide.
   
4. We have created cells with inducible EphB2 expression using another colon cancer cell line (SW480) to repeat the above studies.
   
5. We have created a cell line which constitutively express ephrinB1 on the cell membrane for use in co-culture experiments to determine the effect of EphB2/ephrin interaction. These cells are also tagged with a LacZ gene which means they can be stained blue to distinguish them from the EphB2 expressing cells.

Objective

Determine the reason for the retarded growth of in vivo tumours derived from EphB2 expressing cells.

Progress

Analysis of experimental tumours with low, moderate and high levels of EphB2 suggest that growth is retarded in high EphB2 expressing tumours due to a reduction in the proportion of dividing cells. Conversely the level of cell loss due to apoptosis is similar in all three groups.

Using immunocytochemistry we have detected the presence of ephrins on stromal cells surrounding areas of tumour suggesting the potential for EphB2/ephrin interaction.

Ongoing and future studies

The negative effect of EphB2 expression on invasion and migration is an important finding. We are interested in determining the effect of EphB2 expression on the levels of genes known to be important in migration and invasion.

EphB2 stimulation in vivo occurs when a cell bearing an ephrin (such as ephrinB1) on its surface interacts with a EphB2 receptor expressing cell. Using a soluble form of ephrin is much less effective in activating EphB2. To overcome this we are:

1. Attempting to create a cell line which has inducible expression of a form of EphB2 which has been mutated to be constitutively active (i.e. permanently switched on). Thus we can study the effect of EphB2 activation in the absence of ephrin stimulation.
   
2. Use cells which express ephrins on their surface in co-culture experiments to study the effect of cell-cell interaction.

In culture EphB2/ephrin interaction does not have an effect on cell growth but in vivo we see reduced growth of EphB2 expressing tumours. This suggests an indirect mechanism is at work. One possibility is reduced vascularisation, this would constrain tumour growth. We will use immunocytochemistry to assess the vascular status of tumours.