P76 Brk expression may affect the differentiation status of breast cancer cells

S39 P76

Brk expression may affect the differentiation status of breast cancer cells

AJ Harvey

, CJ Pennington

, DR Edwards

, SA Eccles

, MR Crompton

1

Biosciences Brunel University, Uxbridge, UK;

School of Biological Sciences, University of East Anglia, Norwich, UK;

3

Cancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, UK;

School of Biological Sciences, Royal Holloway, University of London, Egham, UK

Breast Cancer Res 2008, 10(Suppl 2):P76 (doi: 10.1186/bcr 1960) The breast tumour kinase Brk (PTK6) is found in over two-thirds of breast cancer cell lines and tumours but is not expressed in normal mammary cells. Brk has previously been shown to play a role in regulating proliferation in breast tumour cells [1]. However, in vivo, the site of Brk expression in normal tissues is restricted to nonproliferating cells that are undergoing terminal differentiation such as those in the gut or the skin [2,3]. This led us to hypothesise that Brk expression in breast tumours could be reflective of a differentiation phenotype, especially as a previous study had shown that involucrin, a marker of terminal keratinocyte differentiation, was expressed in a subset of tumours [4]. We therefore examined involucrin expression in breast tumour cells lines and patient biopsy samples. In addition we investigated whether inducers of differentiation in keratinocytes such as prolonged culture in suspension or vitamin D3 treatment could also affect differentiation of breast tumour cells.

We found that the expression of Brk in cultured cell lines correlated with involucrin expression. In addition the change in Brk expression, as a result of culture conditions, was accompanied by a change in involucrin levels. Moreover, treatment with vitamin D3 resulted in a decrease in cell numbers in the Brk-positive cell lines relative to the control treatments. The Brk-negative cell line was unaffected by vitamin D3 treatment.

These data suggest that Brk and involucrin may be coregulated and that inducers of differentiation such as vitamin D3 could be considered potential therapeutic strategies.

Acknowledgements Funded by Breast Cancer Campaign and Brunel University.

References

1. Harvey AJ, Crompton MR: Use of RNA interference to vali- date Brk as a novel therapeutic target in breast cancer:

Brk promotes breast carcinoma cell proliferation. Onco- gene 2003, 22:5006-5010.

2. Vasioukhin V, Serfas MS, Siyanova EY, Polonskaia M, Costi- gan VJ, Liu B, Thomason A, Tyner AL: A novel intracellular epithelial cell tyrosine kinase is expressed in the skin and gastrointestinal tract. Oncogene 1995, 10:349-357.

3. Llor X, Serfas MS, Bie W, Vasioukhin V, Polonskaia M, Derry J, Abbott CM, Tyner AL: BRK/Sik expression in the gastroin- testinal tract and in colon tumors. Clin Cancer Res 1999, 5:

1767-1777.

4. Tsuda H, Sakamaki C, Fukutomi T, Hirohashi S: Squamoid features and expression of involucrin in primary breast carcinoma associated with high histological grade, tumour cell necrosis and recurrence sites. Br J Cancer 1997, 75:1519-1524.

P77

C35 overexpression defines subsets of human breast cancer and its immunoreceptor tyrosine-based activation motif represents a novel treatment target

E Katz

, D Faratian

, JMS Bartlett

, K MacLeod

, H Pedersen

, A Larionov

, EM Smith

, AP Howell

, JM Dixon

, EE Evans

, SP Langdon

, DJ Harrison

1

Cancer Research Centre, University of Edinburgh, UK;

Vaccinex Inc., Rochester, NY, USA

Breast Cancer Res 2008, 10(Suppl 2):P77 (doi: 10.1186/bcr 1961) C35 is a protein overexpressed in invasive breast cancer. The C35 gene is located on chromosome 17, next to ERBB2/HER2. C35 encodes a canonical immunoreceptor tyrosine-based activation motif (ITAM) sequence. ITAM-containing proteins have key signalling roles in the hematopoietic system and in oncogenic retroviruses. The ITAM interacts with Syk kinase, which mediates downstream signalling events.

C35-overexpressing breast tumours were found to be of two subsets. In one subset, C35 is coexpressed with HER2. The second subset is found within the basal-like carcinoma group. In order to evaluate the therapeutic potential of targeting C35 ITAM/Syk signalling, we utilised 3D cell cultures. Transformed cell lines act in a manner resembling their in vivo behaviour when grown in 3D cultures, on reconstituted basement membrane. Using this method, C35-expressing cells formed enlarged structures in both an ITAM- dependent and Syk-dependent manner. Furthermore, BT474 cells coexpressing C35 and HER2 formed more normal 3D structures when treated with a combination of Herceptin and Syk inhibitors.

P78

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

ML Murphy, SKW Chan, WJ Gullick

Department of Biosciences, University of Kent, Canterbury, UK Breast Cancer Res 2008, 10(Suppl 2):P78 (doi: 10.1186/bcr 1962) Background 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

Available online http://breast-cancer-research.com/supplements/10/S2