Why do most c-erbB-2/HER-2-positive breast cancer patients fail to respond to Herceptin?

Herceptin is active in a subset of patients over-expressing the epidermal growth factor receptor (EGFR) c-erbB-2 (HER2) but it is not possible to predict which individuals will respond. Several molecular hypotheses have been proposed for how Herceptin causes tumour regression: one is that the antibody binds to HER2 and causes it to be internalised into breast cancers cells, where it is either degraded or locates to a compartment in which it can no longer signal (or signal in the same way).

The present research aims to explore possible molecular and cellular mechanisms involved in resistance of Herceptin. We are also interested in identifying whether inhibiting other pathways (such as signalling via HER3) would increase the number of patients who show a response. We have created a plasmid containing c-erbB-2 fused to Yellow Fluorescent Protein (c-erbB-2-YFP) and an epidermal growth factor receptor fused to Green Fluorescent Protein (EGFR-GFP). The correct sequence was obtained for both of these and we showed that they react with specific antibodies using western blotting. We have established a system in which we can express c-erbB2-YFP with or without coexpression of the EGFR labelled (or not) with GFP and add Herceptin chemically coupled to the red fluorescent compound Alexa Fluor 568 to see if there is an effect in cell trafficking. We have made a monoclonal antibody called SGP1 that recognises the extracellular domain of HER3 receptor [1] and we would like to see whether addition of a HER3-specific monoclonal antibody to Herceptin will increase its anticancer activity. If so, SGP1 antibody could be humanised and then both coadministrated with Herceptin in clinical trials.

Both constructs c-erbB-2-YFP and EGFR-GFP were used to transiently transfect COS-7 cells to determine their biosynthesis and transport to the cell surface. Time-course studies using low-light fluorescent microscopy have shown that both receptors are found on the surface of cells between 18 and 24 hours post transfection. We have chemically labelled Herceptin immunoglobulin (Genentech Inc., South San Francisco, CA, USA) with Alexa Fluor 568 (Invitrogen Molecular Probes, Inc., CA, USA) and have shown that this binds only to cells expressing the c-erbB2-YFP receptor. The specificity is notably high in relation to previous experience with similar antibodies as no signal has been seen on any untransfected cells of several types. Treatment of cells at 37°C with Herceptin-568 for increasing time periods up to 48 hours was performed. Most of the effects of the drug on receptor localisation were seen in the first four hours and so in future experiments we employed this timeframe.

These studies indicate that Herceptin applied to cells expressing only c-erbB-YFP induces receptor internalisation into a compartment apparently just under the surface of the plasma cell membrane, supporting the observations of Austin and colleagues [2] who explored this by electron microscopy. Addition of Herceptin-568 to cells expressing the EGFR gave no binding as expected. However, cotransfection of c-erbB-2 (unlabelled) with EGFR tagged to GFP gave the unexpected result that the EGFR was internalised over about 1 hour (significantly slower than the effect of adding EGFR-Alexafluor). Preliminary results to determine the effect of the antibody SGP1 on the c-erbB-3 receptor have shown induced phosphorylation of a 60 kDa protein that is probably Shc, which already has been identified as one of the main second messenger proteins recruited by HER3. However, further studies are needed to fully characterise this protein.

The results from the present work have shown that both constructs can be expressed in mammalian cells and that receptor trafficking can be observed and evaluated using two-colour digital fluorescent microscopy. In addition we have fluorescently labelled Herceptin, and its ability to bind c-erbB-2 is retained. We showed that cotransfection with c-erbB-2-YFP and EGFR labelled (or not) with GFP and addition of the labelled Herceptin is affected by the presence of EGFR. Our preliminary results using monoclonal antibody SGP1 have shown that the presence of HER3 receptor can affect the extent of downregulation. It may be that multiple targeting of the HER-family receptors will help to increase the number of patients that respond to the therapy.