Abstract
Breast cancer is the leading cause of cancer-associated death in women worldwide (1), largely due to metastatic disease progression. Fibroblast Growth Factor Inducible 14 (Fn14) is a type 1 transmembrane receptor and member of the tumour necrosis factor superfamily. In 75 – 87% of invasive breast adenocarcinomas, Fn14 is aberrantly overexpressed in the ductal epithelial cells but not in normal breast epithelium (2,3). Fn14 protein is known to be elevated in HER2-positive breast cancer (2). However, its association with other breast cancer subtypes and clinicopathological factors has not been thoroughly characterised. Fn14 is known to drive cancer cell migration and invasion through NF-kB signalling and the Rho GTPase, RAC1. However, the impact of elevated Fn14 on signalling pathways in breast cancer biologic contexts has not been comprehensively studied.
In order to characterise the clinical context of Fn14 within breast cancer, four publically available breast cancer transcriptome datasets containing clinical annotation information were used for analysis. The association between Fn14 mRNA expression with basic clinicopathological factors, receptor subtypes (ER/PR/HER2 by IHC), and PAM50 intrinsic subtypes were determined by constructing violin plots and testing for differences between subgroups. Next, the ability of Fn14 mRNA expression tertiles to stratify breast cancer patients into additional metastatic risk groups was conducted using Kaplan Meier analysis. Finally, the independent ability of Fn14 mRNA expression tertiles to estimate distant metastasis-free survival (DMFS) with respect to other clinicopathological factors was evaluated using a multivariate Cox proportional hazards (Cox PH) model. Fn14 CRISPR knockout (KO) isogenic cell lines were created in MDA-MB-231 triple-negative breast cancer (TNBC) cells so that the transcriptomes could be compared via RNA sequencing (RNAseq) and differential expression analysis (using EdgeR). This facilitated the discovery of novel Fn14-mediated pathways, which were subsequently validated in MDA-MB-231 cells in vitro.
Fn14 showed significantly elevated mRNA expression in HER2+, TNBC, HER2-enriched, and Basal-like subtypes, while expression was lowest in Luminal and hormone receptor-positive (HR+) subtypes. Fn14 mRNA expression tertiles in Kaplan Meier plots significantly stratified patients into additional metastatic risk groups in HR-/HER2+ and TNBC subtypes. In multivariate Cox PH analyses, a one tertile increase in Fn14 mRNA expression was significantly associated with a 34% increase risk in distant metastasis when all other factors (age, histologic grade, lymph node status, tumour size, endocrine therapy, and chemotherapy) were held constant (HR = 1.34, CI [1.10-1.65], FDR-adjusted P-value = 0.022).
To discover novel candidate Fn14-mediated signalling pathways, stable Fn14 KO derivatives of MDA-MB-231 cells (Fn14-sgRNA clones) were analysed by RNAseq and differential expression analysis. Fn14-sgRNA clones showed a change in gene expression in gene sets involved in Epithelial to Mesenchymal Transition (EMT), NF-kB signalling, cell adhesion, migration, invasion, and KRAS-mediated MAPK upregulation. To validate the effect of Fn14 KO on adhesion, migration, and invasion, in vitro assays were conducted on Fn14-sgRNA clones compared to Fn14 wild type controls. Unexpectedly, Fn14-sgRNA clones showed significantly elevated adhesion, migration, and invasion in 2D assays compared to control clones. Previous experiments in MDA-MB-231 wild type cells with transient Fn14 knockdown (by siRNA) show a reduction in invasive capacity (2). It was, therefore, hypothesised that complete loss of Fn14 triggered a critical shift in the default signalling mechanism to that driven by the oncogenic KRAS (G13D) mutation present in MDA-MB-231 cells. Though, further experiments are required to fully explore this finding. The Enrichment of differentially expressed genes, within the EMT gene set, in Fn14-sgRNA clones was a novel finding that has not been reported in the literature. Therefore, the association between Fn14 and EMT was investigated in A549 lung cancer and NMuMG murine breast cancer cell lines. Unlike MDA-MB-231 cells, these are well-established models of TGF-β-inducible EMT. In both cell lines, Fn14 expression (at the mRNA and protein level) correlated with loss of E-cadherin and increased Vimentin after TGF-β treatment. Though, further experiments are required to evaluate whether Fn14 is critical for TGF-β induced EMT.
In conclusion, aberrant Fn14 expression was shown to have particular significance in HER2+ and TNBC receptor subtypes and is a significant independent factor in predicting DMFS in breast cancer. Furthermore, we have confirmed Fn14 to have a role in the regulation of NF-kB signalling, invasion, migration, and EMT. Complete removal of Fn14 expression by Fn14 gene knockout resulted in increased migration and invasion in MDA-MB-231 cells, which coincided with an increase in KRAS-mediated MAPK signalling. These data suggest that future targeted monotherapy approaches, which inhibit Fn14 signalling in TNBC tumours harbouring oncogenic KRAS mutations, should be avoided.