Abstract
Breast cancer (BC) is a leading cause of global cancer mortality and the most commonly diagnosed malignancy among women in New Zealand. Tumor-specific overexpression of fibroblast growth factor-inducible 14 (Fn14) serves as a robust prognostic marker, correlating with invasive BC subtypes, reduced overall survival, and increase risk of metastasis. Fn14 plays a key role in tumour progression by promoting cell invasiveness and metastatic spread. While it’s function in malignancy is well explored, it’s mechanism of aberrant dysregulation remain poorly understood.
This study investigates genetic, epigenetic and transcriptional mechanisms of Fn14 dysregulation in BC cell lines, focusing on the proximal promoter region of Fn14. The mapping of potential regulatory factors was aided by publicly available data which suggested the association of Fn14 with established oncogenic signaling cascades and critical developmental pathways. Multisequence alignment of annotated whole genome sequences from BC cell lines with differential Fn14 expression, indicated sequence aberrations were not likely responsible for the dysregulation of Fn14. We performed bisulfite sequencing of the proximal promoter region of Fn14 in cell lines with differential Fn14 expression, identifying hypomethylation across a bioinformatically-predicted CpG island, and CpG sites indicated by publicly available Illumina 850K EPIC methylation array data as methylated in cells of non-epithelial origin.
Exploration of transcriptional mediators of Fn14 regulation in triple-negative BC (TNBC) cell lines MDA-MB-231 and BT-549 revealed that Fn14 protein expression was consistently modulatable by the removal of serum, causing robust 4.3-fold downregulation in BT-549 cells (p=0.007) and a modest 1.3-fold reduction in MDA-MB-231 cells (p=0.540). Serum reintroduction in TNBC cell lines resulted in de novo Fn14 protein re-expression within 24 hours, with BT-549 showing a 4.0-fold increase (p=0.048) and MDA-MB-231 showing a 1.3-fold increase (p=0.494). Epidermal growth factor (EGF) stimulated Fn14 protein expression 1.7-fold in BT-549 cells (p=0.049) and following serum withdrawal, suggesting that Receptor Tyrosine Kinase signaling, most likely through the RAS/MAPK and PI3K/AKT pathways, can lead to Fn14 transcriptional activation. Both TNBC cell lines showed a modest response in Fn14 expression following TGF-β stimulation in the absence of serum, with 1.3-fold increases observed in both BT-549 (p=0.208) and MDA-MB-231 (p=0.193) cells.
The relationship of TGF-β-mediated epithelial-mesenchymal transition (EMT) and Fn14 was explored using well-established epithelial cell lines from lung adenocarcinoma (A549) and murine mammary carcinoma (NMuMG). TGF-β stimulation induces a robust linear increase in Fn14 protein expression in both models, with a temporal correlation between Fn14 expression and EMT. These data support the potential for a role for Fn14 in TGF-β-mediated EMT.
These findings provide strong evidence for external growth factor-mediated control of cell signaling being the driving mode of Fn14 dysregulation in malignancy, warranting validation in models beyond BC cell lines in 2D culture. This research contributes to the growing field of clinically relevant biomarker studies and highlights the importance of dissecting molecular architecture in oncogenic signaling to aid translational research progress.