Date of Award

2010

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Dr. Leonie Young

Keywords

Breast Neoplasms

Abstract

Background

Progression of breast cancer despite hormonal and biological treatments continues to pose a therapeutic challenge. Receptor crosstalk is a resistant mechanism that can occur between the estrogen receptor (ER) and the receptor tyrosine kinases, such as HER2, in response to inhibition by anti-endocrine treatments. Briefly, tumours can overactivate growth factor signalling cascades and give rise to an endocrine independent tumour. Wehypothesized that treatment with tyrosine kinase receptor inhibitors (TKIs), trastuzumab (Herceptin®) and lapatinib (Tyverb®), could induce similar receptor crosstalk by overactivating endocrine-driven tumour growth.

Methods

Proteomic analysis (LC-Mass Spectrometry) was used to assess proteins that interact with the proto-oncogene, c-Myc, that are involved in growth factor driven tumorigenesis and invasion. We compared c-Myc associated proteins in the ER-overexpressing MCF7 cell line and the ER and HER2-positive, endocrine-independent LCCl cells. With our findings, we proceeded to better elucidate the function and importance of these interactions both in vitro and by analysis of our clinical breast cancer patient cohort. Finally, we utilized protein macroarray technology to uncover autoantibodies in HER2-positive breast cancer patients that could potentially serve as biomarkers to idenbfy patients developing tumour resistance to trastuzumab, as well as other roles.

Results

LC- Mass Spectrometry revealed a novel interaction between the proto-oncogene c-Myc and the corepressor, SMRT in endocrine sensitive MCF7 cell line, but not in the LCCl cell line. We hypothesized that SMRT may be silencing the oncogenic effects of c-Myc in the endocrine dependent tumour model. Coirnmunoprecipitation revealed this interaction was lost when cells were treated with TKIs, thus liberating c-Myc from SMRT repression and enhancing c-Myc transcriptional activity as reflected by increased expression of the downstream target gene, survivin. Furthermore, the endocrine pathway became overactive with recruitment of the coactivator, CBP to ERα and increased levels of PS2 reflecting ER activity. Clinically, in our HER2-overexpressingearly, node-negative breast cancer patient population who received trastuzumab, thosewho also overexpressed ER were noted to have an increased risk of recurrence (18% vs10%). When anti-estrogen treatments were used in combination with TKI treatments, a number of in vitro observations were made. The dual treatment had a superior inhibitory effect upon cell growth, c-Myc recruited SMRT once again silencing its transcriptional ability and CBP / ERα interaction was reduced.

PEX protein macroarray comparative analysis of HER2-positive and HER-negative patients revealed a list of autoantibodies that can be detected in patient sera. These may be of benefit as biomarkers in the future management of breast cancer patients. The ER coactivator, Ada3, detected in HER2-overexpressing patient sera did not associated with the larger patient group and is unlikely to be useful as a marker of receptor crosstalk or tumour recurrence. Further work on the other identified autoantibodies may uncover more promising proteins.

Conclusion

This work reveals the possibility of bidirectional receptor crosstalk between the growth factor and endocrine pathways in breast cancer. It also suggests a role for the protooncogene, c-Myc and the coregulatory proteins, SMRT and CBP in this relationship. This has clear implications for the future treatment of breast cancer and indicated thatpatients, who overexpress ER and HER2, may require simultaneous treatment regimes with anti-estrogen and anti-growth factor therapies. It also raises the pressing need to identify the tumours that are more likely to progress despite TKI therapy by discovering biomarkers that can aid clinical and radiological investigations.

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Comments

A thesis submitted to the Royal College of Surgeons in Ireland for the degree of Doctor of Philosophy from the National University of Ireland, 2010.