Cleavage of RNA-binding protein HuR modulates c-Myc expression in OSCC

Objectives: A well-studied oncogene and RNA-binding protein associated with post-transcriptional regulation is the mRNA stability factor Hu antigen R (HuR, alias ELAVL1), which is highly abundant in many cancers, including oral squamous cell carcinoma (OSCC). Although significant progress has been made in understanding HuR regulation in gene expression, little is known about how HuR undergoes post-translational modifications and recruits target mRNAs during hypoxic stress. Here, we propose to define hypoxia-induced HuR overexpression and cleavage in the promotion of squamous cell transformation through post-transcriptional regulatory mechanisms.

Methods: HuR expression levels were estimated in surgically removed oral carcinomas by both Western blot and immunohistochemistry methods. Normal oral keratinocytes and oral cancer UM74B cells were used to study the HuR alterations during hypoxia. Western blot, Immunocytochemistry and RT-qPCR confirmed the over expression and cleavage of HuR in oral cancer tissues and cells. To assess the HuR role in mRNA stability, we knockdown HuR by siRNA and studied their associated mRNA stability by performing RT-qPCR.

Results: Our results indicate that during hypoxic stress, HuR was significantly overexpressed and undergoes caspase-dependent cleavage in OSCC cells. Unexpectedly, the HuR-cleavage product 1 (HuR-CP1) was found to strongly associate with the 3'-untranslated region (UTR) of c-myc mRNA and block mRNA translation. The binding efficiency of HuR to the 3'-UTR of c-myc mRNA was confirmed using ribonucleoprotein immunoprecipitation and site-directed mutagenesis at the AU-rich element sequences of the c-myc mRNA. Overexpression of a non-cleavable isoform, HuR-D226A, revealed a potent dominant-negative effect, repressing cleavage of endogenous HuR and promoting cell viability and proliferation. Surprisingly, under hypoxia, siRNA knockdown of HuR elevated c-Myc protein expression suggest that HuR act as a translational repressor.

Conclusions: These findings suggest an important role for HuR in hypoxia, and we may have revealed a novel post-transcriptional mechanism that controls c-Myc expression in oral cancer progression.