Date of Award

Summer 2017

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Dr Ann Hopkins

Funder/Sponsor

Breast Cancer Ireland

Keywords

Breast Cancer, JAM-A, HER2, Trastuzumab, Lapatinib, Resistance

Abstract

Junctional Adhesion Molecule-A (JAM-A) is an adhesion protein whose overexpression in breast tumour tissue has been associated with reduced breast cancer patient survival. It has also been shown that JAM-A expression correlates with that of the human epidermal growth factor receptor-2 (HER2), and that JAM-A regulates HER2 protein expression. HER2-targeted therapies, such as Trastuzumab and Lapatinib, have greatly improved patient outcomes. However, treatment efficacy is frequently limited by drug resistance. Hence, the discovery of new predictive markers and therapeutic targets for HER2-positive breast cancer patients is vital. Given the regulatory control of HER2 by JAM-A, we hypothesised that JAM-A might drive HER2- dependent stability or signalling and thus participate in resistance to HER2- targeted therapies in breast cancer models. RT-qPCR analysis in breast cell lines revealed that JAM-A regulates HER2 at a transcriptional level. Immunofluorescence and nuclear/extra-nuclear fractionation experiments suggested that this occurs via JAM-dependent sequestration/release of the HER2 transcriptional repressor ZONAB. Cell viability and western blot analyses revealed that JAM-A gene silencing or pharmacological antagonism was capable of re-sensitising Trastuzumab- and Lapatinib-resistant cell lines to the anti-proliferative effects of HER2-targeting drugs. Mechanistically, JAM-A silencing in drug-resistant cells reduced the expression of EGFR, a HER family member whose upregulation is associated with resistance to anti-HER2 therapies. Moreover, drug-resistant cells released a cleaved soluble form of JAM-A, in conjunction with increased expression of certain metalloproteases. Protease inhibition (and consequently JAM-A cleavage) re-sensitised drug-resistant cells to the anti-proliferative effects of HER2- targeted therapies. In parallel, treatment of cells with recombinant soluble JAM-A promoted cellular invasion in vitro and in a semi-in vivo model involving human tumour cell movement across a live chicken egg chorioallantoic membrane. Promisingly, cleaved soluble JAM-A was detectable in two pilot studies of serum samples from HER2-positive breast cancer patients, and its presence correlated with the development of resistance to anti-HER2 therapy.

Taken together, our data are consistent with a multi-faceted model whereby JAM-A expression participates in resistance to HER2-targeted therapies by stabilising transcription of the HER2 gene, promoting expression of other HER family members and enabling protease-driven JAM-A cleavage which in turn may promote invasive capacity. We suggest that soluble JAM-A holds potential as a biomarker of resistance to HER2-targeted therapies, and that co-targeting of JAM-A and HER2 may be a future therapeutic option for patients resistant to HER2-targeted therapies.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License.

File Size

23.4MB

Comments

A thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2017.

Available for download on Sunday, June 30, 2019

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