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 (DCIS). 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 oestrogen, androgen, and vitamin D receptors, Overexpression of Bag-1 mRNA in MCF-10A, is known to produce acini with luminal filling reminiscent of DCIS. As this mRNA predominantly overexpresses the short isoform of Bag-1, Bag-1S, we set out to examine whether the nuclear Bag-1 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 2D and 3D cultures and compared to an established model of HER2-driven transformation. In 2D cultures, Bag-1L overexpression reduced proliferation but did not affect insulin responsiveness or clonogenicity. Acini formed by Bag-1L-overexpressing cells exhibited reduced luminal clearing when compared to 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 DCIS. Using this model to test the small molecule Bag-1 inhibitor, Thio-2, reveals its potential to reverse the atypical branched morphology of acini which characterises this premalignant change.

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Identifiers

ORCID for C.R. Barker: orcid.org/0000-0001-8263-4151

ORCID for J.P. Blaydes: orcid.org/0000-0001-8525-0209

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