Abstract 3958: The phenotypic and signaling consequences of a novel aberrantly spliced transcript of fibroblast growth factor receptor 3 in hepatocellular carcinoma

Abstract

Aims: To identify the phenotypic and signaling consequences of a novel aberrantly spliced transcript of fibroblast growth factor receptor 3 (FGFR3) in hepatocellular carcinoma (HCC). Methods: The nested RT-PCR was employed to analyze the splicing of FGFR3 from 35 HCC cases and 11 cell lines. The bridging method was utilized to construct the sequence of FGFR3 Δ7-9 (deleting exon7, 8, and 9 from the full length of FGFR3 ORF). Based on the establishment of the FGFR3Δ7-9 lentiviral overexpress and knockdown models, the influence on bio-characters of HCC cell lines was inspected in vitro. The changes of tumorigenicity and metastases were examined quantitatively in vivo on nude mice using pathological study and Micro-PET/CT. Co-immunoprecipitation and surface plasmon resonance (SPR) binding analysis were employed to understand the ligand affinities between FGFR3Δ7-9 and FGFs. Interacting proteins and signal transduction pathway were detected by mass spectrometry and dual luciferase reporter assay. Results: FGFR3Δ7-9, lacking exons encoding the immunoglobulin-like domain III, was identified with high frequency in HCC. FGFR3Δ7-9 can apparently promote the proliferation, migration, and metastases of HCC cells both in vitro and in vivo. In contrast, knockdown of FGFR3Δ7-9 can restrict the abnormal malignancies of HCC cells. Co-immumoprecipation and SPR assay showed the binding affinity of FGFR3Δ7-9 to FGFs was significantly higher than that of wild type FGFR3 (P<0.01). More importantly, FGFR3Δ7-9 can be self-activated by self-dimerization and autophosphorylation even in the absence of ligand (FGFs). Consistent to the ligand-independence characteristic, FGFR3Δ7-9 demonstrated a stronger effect on inducing phosphorylation of ERK and AKT than wild type FGFR3, which may lead to abnormal down-stream PI3K/AKT/mTOR and ERK signal transductions. Furthermore, evidenced by tandem mass spectrum assay and Western blot, FGFR3Δ7-9 can up-regulate certain metastasis-related molecules: Snail and MMP-9. As a newly confirmed interaction protein to FGFR3Δ7-9, E-cadherin can be degraded via tyrosine phosphorylation, which may cause the abnormal cellular proliferation and migration consequently. Discussion/Conclusion: Dysregulation of mRNA splicing may generate an abnormal aberrant FGFR3 transcript, FGFR3Δ7-9. The joining of exon 6 to exon 10 in FGFR3Δ7-9 is in-frame, leading to the expression of a novel transmembrane form of FGFR3 containing an intact intracellular TK domain. As a ligand-independence or lowly ligand-dependent receptor, FGFR3Δ7-9 may function importantly in HCC tumorigenesis, proliferation, invasion and distant lung metastases. Altogether, our research strongly supports the idea that the dysregulation of FGFR3 expression by aberrant splicing of mRNA in a significant subset of HCC is an alternative pathway to neoplastic transformation. Note: This abstract was not presented at the meeting. Citation Format: Weihua Qiu, Weiping Yang, Xiaoqian Jing, Bingrui Wang, Xinyu Liu, Ding Ma, Helen Lin. The phenotypic and signaling consequences of a novel aberrantly spliced transcript of fibroblast growth factor receptor 3 in hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3958. doi:10.1158/1538-7445.AM2015-3958