Abstract
Breast cancer is the second leading cause of cancer death in women. Approximately 75% of invasive breast cancers are hormone receptor (HR) positive, and have the option to receive targeted endocrine therapy. However, a significant proportion of these patients will develop resistance to this treatment, through mechanisms that remain poorly understood.
Fibroblast Growth Factor Inducible 14 (Fn14), a tumour necrosis factor receptor family member, is expressed in 76% of invasive breast cancers, and not in normal mammary epithelium. Fn14 expression correlates with progression in HR-negative tumours, but the role of aberrant Fn14 expression in HR-positive disease is unclear.
We investigated a series of HR-positive breast cancer cell lines derived from MCF7 and T47D cells, engineered to stably overexpress Fn14. Fn14 expression status was first confirmed by western blotting, with negligible expression observed in wildtype, and empty vector controls. The 2D morphology of Fn14-positive cell lines appeared more mesenchymal, with frequent pseudopodia, compared to controls. MCF7 and T47D 3D-mammopsheres were smaller in size and more nodular in Fn14-positive cell lines compared to controls. Fn14-positive MCF7 cells did not show increased invasive capacity through Matrigel-Boyden chambers, which contrasts with increased invasion following transient Fn14 expression in MCF7 cells.
Fn14+ MCF7 cell lines proliferated in 2D culture at a consistently reduced rate in complete medium, compared to control cell lines. Similar to wildtype MCF7, Fn14-positive MCF7 cell lines were not able to proliferate in hormone-deprived medium, indicating forced Fn14 expression alone, is not sufficient to drive proliferation in MCF7 cell lines. All MCF7-derived cell lines could however, be stimulated to proliferate with 10 nM 17-beta-oestradiol (E2), however E2-stimulated growth of Fn14-positive MCF7 cell lines was less robust, suggesting Fn14 is disruptive to oestrogen signalling.
Receptor Tyrosine Kinase (RTK) signalling is antagonistic to oestrogen signalling in breast cancer, and has been linked with development of resistance to anti-oestrogens. We observed activation of ERK1/2 (one of 3 MAPK pathways driven by RTK signalling) in Fn14-positive T47D cells by western blotting. ERK1/2 did not however, appear activated by Fn14 in MCF7 cells. Finally, Fn14-positive MCF7 cell lines showed a modest increase in resistance to a dose escalation of 4-hydroxy tamoxifen.
Together our data suggest that aberrant overexpression of Fn14 in HR-positive breast cancer cell lines reduces their proliferative response to E2 and reduces their sensitivity to Tamoxifen. The mechanism by which this occurs may involve signalling antagonism through RTKs, and warrants further investigation.