RNA splicing factors and long noncoding RNA reciprocally regulate EGFRvIII expression in human cancer cells

Abstract Background Dysregulated epidermal growth factor receptor variant III (EGFRvIII) contributes to tumor progression in several human cancers, including oral squamous cell carcinoma (OSCC) and glioblastoma multiforme. Although genomic alterations are frequently associated with EGFRvIII expression, the contribution of environmental carcinogens and RNA-based regulatory mechanisms to its expression remains poorly understood and was investigated in this study. Methods Chronic exposure models of the tobacco-specific nitrosamine nicotine-derived nitrosamine ketone (NNK) and the areca nut alkaloid arecoline were established in dysplastic oral keratinocytes. Expression and splicing patterns of EGFR isoforms were analyzed using molecular assays and long-read sequencing. Functional assays, RNA pull-down, AGO2 immunoprecipitation, MS2-based RNA affinity purification, and reporter assays were performed to investigate the roles of splicing factors, long noncoding RNAs (lncRNAs), and microRNAs in EGFRvIII regulation. Results Carcinogen exposure induced sustained EGFR and EGFRvIII expression, increased the levels of exon 1–8 junction-containing transcripts, and expanded EGFR isoform diversity. These changes were accompanied by upregulation of splicing regulators, particularly SRSF1. Transcriptomic and functional analyses identified the lncRNA RP11-30P6.6 as a carcinogen-responsive factor that promoted cell proliferation and migration and sustained increased EGFRvIII expression. RNA affinity-based assays showed preferential recovery of SRSF1 and EGFRvIII-related RNA in RP11-30P6.6 pull-down fractions, consistent with its involvement in EGFRvIII-associated RNA regulation. Functional and AGO2 immunoprecipitation analyses were consistent with a competing endogenous RNA (ceRNA)-like relationship in which RP11-30P6.6 may attenuate repression by candidate microRNAs that showed differential regulatory effects on EGFRvIII relative to WT-EGFR under the conditions tested. Perturbation of SRSF1, RP11-30P6.6 , or candidate microRNAs attenuated EGFRvIII expression, while functional modulation of these regulatory components was associated with altered tumor cell growth and migration. Conclusion The study findings support a model in which environmental carcinogens reprogram RNA splicing and noncoding RNA networks to sustain EGFRvIII expression. SRSF1, RP11-30P6.6 , and microRNAs may contribute to the post-transcriptional regulation of EGFRvIII expression and may provide potential targets for EGFRvIII-associated cancers.