Date of Award
MD (Medical Doctor)
Dr Leonie Young
Breast Neoplasms, Drug Therapy, Drug Resistance, Neoplasms .
Breast cancer is the most commonly diagnosed malignancy in women in Ireland after non-melanoma skin cancer, with over 2,000 women diagnosed with the disease each year. It remains the leading cause of cancer-related mortalities in women, with an average annual rate of 644 deaths between 1994 and 2001. Although the mortality rate decreased between 1989 and 2006 by nearly 27%, it still remains one of the highest mortality rates in Europe. Targeted therapy against the key receptors identified to date as being integral to the development and proliferation of breast cancers has significantly improved the overall and disease-free survival rates for patients. However, innate and acquired resistance to these therapeutic agents, including tamoxifen and trastuzumab, results in tumour recurrence which is not susceptible to further standard therapeutic interventions. Resistance to tamoxifen can occur in up to one quarter of patients after ten years, while the majority of patients with metastatic disease will develop resistance to trastuzumab within one year of treatment.
Resistance to tamoxifen is thought to occur through a number of proposed mechanisms. One such proposal is the concept of crosstalk between ER and the growth factor receptor pathways including the EGFR/HER2 family of receptors. This can result in resistance developing through a number of different mechanisms including Src kinase activation of the metalloproteinases with resultant activation of the MAPK pathway as well as Akt upregulation through PI3K and ras activation of ERK1 and ERK2.
While extensive in vitro research has been performed to determine how resistance to trastuzumab develops, the exact mechanisms remain inconclusive. Hypotheses include alteration in the interaction between the receptor and the antibody, alternative signalling through other HER or alternate pathways or failure to activate immune-mediated mechanisms to destroy tumour cells. Understanding how resistance to trastuzumab develops would greatly improve the clinical prognosis for patients with HER2 expressing breast cancers, as it would enable clinicians to identify patients at risk of developing resistance and facilitate the tailoring of specific treatments to minimise the risk of such an outcome occurring.
Research has highlighted the complex interactions that occur at a molecular level within breast cancer cells. Receptor crosstalk has been implicated in the development of resistance to endocrine therapies such as tamoxifen. We hypothesise that resistance to trastuzumab can be mediated by crosstalk between the growth factor signalling pathway and the steroid hormone pathway resulting in an uncontrolled upregulation of the steroid hormone pathway following trastuzumab treatment.
The overall aim of this work is to further elucidate the interactions between the steroid hormone and growth factor signalling pathways in breast cancer, in particular, to determine the effect of trastuzumab treatment on the steroid hormone pathway. The first objective will be to assess the effect of trastuzumab treatment on this pathway at a molecular level in breast cancer cells, looking at its effect at both a protein level and a transcriptional level. Secondly, we will verify our in vitro findings in breast cancer tissue samples utilising a large tissue microarray, assessing the clinical significance of our proposed crosstalk hypothesis.
Protein expression levels of ER within the oestrogen sensitive MCF-7 cells was found to be increased following trastuzumab treatment while there was no effect observed in the oestrogen insensitive LCC-1 cells. Surprisingly, treatment of ERnegative SKBR-3 cells with trastuzumab resulted in the expression of ER. Luciferase assay and RT-PCR was performed to assess whether trastuzumab had an enhancing effect on the functional activity of the steroid hormone pathway within the cell model. pS2, a well established ER target gene, was utilised to demonstrate activity. Increased levels of pS2 was observed following trastuzumab treatment in both the MCF-7 and SKBR-3 cell lines, with no difference in the LCC-1 cells.
Methylation studies were performed to determine whether they played a role in the expression of ER within the ER-negative SKBR-3 cells following trastuzumab treatment. Treatment was found to result in the demethylation of five CpG islands within the promoter region of the ER gene within the cells, thereby demonstrating that trastuzumab can result in epigenetic modifications in certain ER-negative cells resulting in the potential expression of a functional ER.
Immunohistochemistry staining for pS2 of a tissue microarray of almost five hundred breast cancer specimens was performed. Survival analysis of HER2 positive patients who expressed pS2 showed that patients had a significantly increased rate of recurrence if they received trastuzumab compared to those who did not. Similarly, a small cohort of patients who received trastuzumab prior to surgical resection were found to have significantly stronger staining for pS2 in their post-trastuzumab treated specimens when compared to their pre-treatment biopsies.
Resistance to trastuzumab remains a significant factor in the development of tumour recurrence in HER2-expressing breast cancers, resulting in reduced overall and disease-free survival times. To date, the exact mechanism by which resistance can develop remains elusive. In this work, molecular studies have demonstrated crosstalk between the steroid hormone and growth factor receptor pathways, suggesting that resistance can develop through upregulation of the endocrine signalling pathway. Furthermore, trastuzumab was also found to result in the expression of a functional ER in ER-negative cells, providing a previously unconsidered potential therapeutic target for these cancers. Translational studies confirmed that the in vitro findings were replicable within a clinical cohort, affecting patient prognosis.
These findings constitute a minute fragment of the complex interactions between the steroid hormone and growth factor receptor pathways in breast cancer. The plasticity of breast cancer and its ability to overcome therapeutic interventions through receptor crosstalk as well as its capacity to alter receptor status demonstrates the complex molecular mechanisms involved in the disease process. Resistance to therapies is likely to present a significant challenge to both clinicians and academics for many years to come. However, translational research methods, as demonstrated within this body of work are essential to unravel our understanding of this complex disease.
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Solon G. Growth factor endocrine pathway crosstalk : can treatment with trastuzumab activate steroid signalling? [MD Thesis]. Dublin: Royal College of Surgeons in Ireland; 2012.