Aberrant activation of Notch signalling in human breast cancer

Like many developmental signalling pathways, the Notch pathway has been linked to the aetiology of several different human cancers. The development of focal adenocarcinomas in the murine mammary gland [1] and the transformation of both normal murine and human breast epithelial cell lines following Notch activation [1, 2] have long suggested that the pathway may play a role in human breast cancer. However, this question has received little attention.

Activation of the Notch pathway in human breast cancer cell lines and breast carcinoma samples was monitored by western blotting with an antibody that recognises the cleaved Notch1 intracellular domain which is produced during signalling. Regulation of apoptosis by Notch was studied in MCF 10A cells transformed by overexpressing the Notch1 intracellular domain. Apoptosis was triggered by treating cells with the kinase inhibitor staurosporine or the DNA damaging agents melphalan and mitoxantrone, and monitored by nuclear fragmentation or cleavage of caspase 3. Changes in the apoptotic machinery were examined by western blotting using a range of antibodies that recognise both total and phosphospecific forms of different components.

We will present data showing that Notch signalling is activated in a wide range of breast cancer cell lines and in a panel of 20 human breast carcinomas of different pathological grade and prognosis. In addition, we will demonstrate that sustained signalling is required to maintain the transformed phenotype of breast cancer cell lines, as its inhibition by expressing Numb, a natural inhibitor of the pathway, causes both MCF7 and MDA-MB-231 cells to adopt a normal phenotype. Our data with the normal breast epithelial cell line MCF 10A indicate that Notch signalling contributes to the transformed phenotype by inhibiting apoptosis. Activation of Notch signalling in these cells by overexpressing the Notch1 intracellular domain prevents apoptosis in response to growth factor withdrawal, removal from the extracellular matrix and DNA damage. Finally, we will provide evidence that the apoptosis resistance seen in Notch transformed MCF 10A cells is through the activation of the Akt survival pathway.

Altogether this suggests that targeting Notch signalling may be a novel therapeutic strategy for the treatment of breast cancer.