title: The Bag-1 inhibitor, Thio-2, reverses an atypical 3D morphology driven by Bag-1L overexpression in a MCF-10A model of ductal carcinoma in situ.
abstract: Mammary MCF-10A cells seeded on reconstituted basement membrane form spherical structures with a hollow central lumen, termed acini, which are a physiologically relevant model of mammary morphogenesis. Bcl-2-associated athanogene 1 (Bag-1) is a multifunctional protein overexpressed in breast cancer and ductal carcinoma in situ. When present in the nucleus Bag-1 is predictive of clinical outcome in breast cancer. Bag-1 exists as three main isoforms, which are produced by alternative translation initiation from a single mRNA. The long isoform of Bag-1, Bag-1L, contains a nuclear localisation sequence not present in the other isoforms. When present in the nucleus Bag-1L, but not the other Bag-1 isoforms, can interact with and modulate the activities of estrogen-, androgen- and vitamin D-receptors. Overexpression of Bag-1 mRNA in MCF-10A is known to produce acini with luminal filling reminiscent of ductal carcinoma in situ. As this mRNA predominantly overexpresses the short isoform of Bag-1, Bag-1S, we set out to examine whether the nuclear Bag-1L isoform is sufficient to drive premalignant change by developing a Bag-1L-overexpressing MCF-10A model. Two clones differentially overexpressing Bag-1L were grown in two-dimensional (2D) and three-dimensional (3D) cultures and compared with an established model of HER2-driven transformation. In 2D cultures, Bag-1L overexpression reduced proliferation but did not affect growth factor responsiveness or clonogenicity. Acini formed by Bag-1L-overexpressing cells exhibited reduced luminal clearing when compared with controls. An abnormal branching morphology was also observed which correlated with the level of Bag-1L overexpression, suggesting further malignant change. Treatment with Thio-2, a small-molecule inhibitor of Bag-1, reduced the level of branching. In summary, 3D cultures of MCF-10A mammary epithelial cells overexpressing Bag-1L demonstrate a premalignant phenotype with features of ductal carcinoma in situ. Using this model to test the small-molecule Bag-1 inhibitor, Thio-2, reveals its potential to reverse the atypical branched morphology of acini that characterizes this premalignant change.
authors.affiliation: Cancer Research UK Centre Cancer Sciences Unit, University of Southampton Faculty of Medicine, Southampton General Hospital, Southampton, UK.
authors.forename: E S
authors.initials: ES
authors.lastname: Papadakis
authors.affiliation: Cancer Research UK Centre Cancer Sciences Unit, University of Southampton Faculty of Medicine, Southampton General Hospital, Southampton, UK.
authors.forename: C R
authors.initials: CR
authors.lastname: Barker
authors.affiliation: Cancer Research UK Centre Cancer Sciences Unit, University of Southampton Faculty of Medicine, Southampton General Hospital, Southampton, UK.
authors.forename: H
authors.initials: H
authors.lastname: Syed
authors.affiliation: Cancer Research UK Centre Cancer Sciences Unit, University of Southampton Faculty of Medicine, Southampton General Hospital, Southampton, UK.
authors.forename: T
authors.initials: T
authors.lastname: Reeves
authors.affiliation: Institute of Pharmacy/Pharmacognosy, Center of Molecular Biosciences, University of Innsbruck, Innrain, Innsbruck, Austria.
authors.forename: S
authors.identifier: http://orcid.org/0000-0002-3731-7349
authors.initials: S
authors.lastname: Schwaiger
authors.affiliation: Institute of Pharmacy/Pharmacognosy, Center of Molecular Biosciences, University of Innsbruck, Innrain, Innsbruck, Austria.
authors.forename: H
authors.initials: H
authors.lastname: Stuppner
authors.affiliation: Daniel Swarovski Research Laboratory, Department of Visceral-, Transplant- and Thoracic Surgery, Innsbruck Medical University, Austria.
authors.forename: J
authors.identifier: http://orcid.org/0000-0002-0611-3837
authors.initials: J
authors.lastname: Troppmair
authors.affiliation: Cancer Research UK Centre Cancer Sciences Unit, University of Southampton Faculty of Medicine, Southampton General Hospital, Southampton, UK.
authors.forename: J P
authors.initials: JP
authors.lastname: Blaydes
authors.affiliation: Cancer Research UK Centre Cancer Sciences Unit, University of Southampton Faculty of Medicine, Southampton General Hospital, Southampton, UK.|University Hospital Southampton, University of Southampton Faculty of Medicine, Southampton General Hospital, Southampton, UK.
authors.forename: R I
authors.initials: RI
authors.lastname: Cutress