Speakers - CWC 2023

Abstract

Introduction

Fibroblast growth factor receptor (FGFR) signaling is paramount in facilitating the physiological process, such as angiogenesis, cell differentiation, migration, proliferation, wound repair, and apoptosis. However, when deregulated, it can contribute to the progression of many cancers, including breast and cervical. Previous studies demonstrated a cleaved form of FGFR1 by Granzyme B to be localized to the nucleus of breast cancer (BCa) cell lines and act as a transcription factor. Interestingly, cancers with aberrant FGFRs face a major challenge which is the development of resistance against the FGFR Tyrosine Kinase inhibitors (TKI). Our aims are to (1) establish the involvement of FGFR signaling in cervical and breast cancer and (2) identify possible drug resistance (DR) mechanisms involved in human cervical cancer cell lines (HCCCLs) that are resistant to an FGFR inhibitor.

Methods

Activation of downstream signaling pathways, and/or FGFR subcellular localization, upon treatment with recombinant FGFs in the presence and absence of an FGFR inhibitor (PD173074) or MMP inhibitor (SB-3CT), was assessed with Western Blot (WB) and immunocytochemistry (ICC).  Transcriptomic analysis was performed to detect differentially expressed genes (DEG) between DR and wild-type HCCCLs (SiHa, HeLa, and Caski).  We have also transiently transfected MCF-7 and MDA-MB-231 human BCa cells with a pRT[TetOn]-FGFR1 construct, tagged on the N- and C- terminus with HA and FLAG respectively, to study the subcellular localization of FGFR1. Real-time PCR, WB, and ICC were used to validate transcriptome and assess FGFR1 overexpression and its subcellular localization. Cell proliferation, apoptosis, and cell migration were assessed using Incucyte Zoom, Annexin V Red Reagent, and WST-1.

Results

Activation of FGFR signaling enhanced metastatic behavior and promoted nuclear FGFR localization in breast and cervical cancer cell lines. These effects were abolished in the presence of PD173074 or SB-3CT inhibitors.  FGFR1 overexpression further supported the observation that cleaved FGFR can localize in the nucleus. DR HCCCLs were more proliferative and migratory and less apoptotic than the wild-type cells with and without PD173074. Interestingly, FGF treatment activated downstream signaling pathways, such as ERK, but co-treatment with PD173074 abolished the effect in both wild-type and DR cells. However, there was no difference in pAKT. Transcriptome analysis and subsequent validation revealed five upregulated (e.g., PLCB4) and nine downregulated (e.g., PLCB4) DEGs in DR HCCCL.

Conclusion

 FGFR activation promotes nuclear FGFR1 translocation, while MMP cleavage is a possible mechanism in BCa cells. Transcriptome analysis highlighted PHLDA1 and PLCB4 genes as a potential mechanism of drug resistance in CCCL.