The role of the Edar signalling pathway in mammary gland development and tumourigenesis

UoM administered thesis: Phd

  • Authors:
  • Rebecca Williams

Abstract

Edar is a member of the death receptor subfamily of TNF receptors that plays an important role in the development of ectodermal appendages. Mutations in this signalling pathway cause the developmental disorder hypohidrotic ectodermal dysplasia (HED). HED has a similar phenotype in a number of mammals, and includes improper development of ectodermal appendages such as hair, teeth and glands. Edar signalling has not previously been linked with tumourigenesis, however, increased NFĸB signalling has been associated with a number of cancers, including breast cancer, and is known to be downstream of the Edar pathway. In addition, Edar signalling is important in mammary gland development, and enhanced Edar signalling in mice has been associated with precocious elongation and branching of mammary gland ducts. Recent research has shown that overexpression of Edar in Edartg951/951 mice leads to the development of mammary gland tumours, particularly in multiparous mice. These tumours were ER negative and displayed squamous metaplasia. EDAR expression was also found to be upregulated in a subset of human metaplastic breast cancers, suggesting that activation of Edar signalling might play a role in the aetiology of these tumours. In this investigation, immunohistochemical staining and qRT-PCR was used to show that the Edartg951/951 mammary gland tumours had high levels of Wnt and Edar signalling. Using DNA sequencing and Western blot assays, we showed that the tumours in Edartg951/951 mice all contained truncations in β-catenin, specifically deletions of exon 3 of the β-catenin gene. This mutation was found to be somatic, and specific to the mammary gland. The Edartg951/951 mice were found to have an increased number of dying cells in the lumen of the mammary gland during involution day 2, and were susceptible to mastitis during early involution. We have also shown using immunohistochemistry that the Edartg951/951 mammary glands with mastitis stained positively for phospho γ-H2A.X, with high levels of cytoplasmic β-catenin in the affected cells. The majority of Edartg951/951 mammary glands with mastitis showed an increase in mAID expression using qRT-PCR analysis, and a Western blot assay showed that the majority contain a truncated form of β catenin. These findings suggest that the mastitis may have generated a mutagenic environment, able to promote tumourigenesis via expression of mAID. Finally, a soft agar assay showed that Edar expression was transforming in EpH4 cells.

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Original languageEnglish
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Award date31 Dec 2016