Assessing Role Of Sphingomyelin-Biosynthetic Pathway In EGFR Localization And Function

Objectives: The epidermal growth factor receptor (EGFR) is highly expressed in human tumors, including 50% of oral squamous cell carcinomas (OSCCs), and contributes to the development of malignant tumors. Using cell culture and C. elegans model, studies in our lab identified fendiline and other acid sphingomyelinase (ASM) inhibitors as novel inhibitors of EGFR. The goal of this study was to determine if inhibiting other enzymes in the ceramide (CER)-sphingomyelin (SM) biosynthetic pathway with pharmacological agents or RNAi knockdown would alter EGFR localization and inhibit its function in the context of an entire organism. C. elegans expresses an EGFR homolog Let-23 and shows a well-characterized Let-23 gain-of-function multi vulva phenotype with the expression of constitutively active Let-23.

Methods: To determine the effects of pharmacological inhibition or genetic knock down of CER-SM pathway enzymes on Let-23 localization and function of wild type (WT) or constitutively active Let-23, Let-23-GFP- or constitutively active Let-23-expressing worms were exposed to drugs or RNAi for 48-72 hours, anesthetized, and imaged using a confocal microscope.

Results: As with fendiline, we showed that not only Let-23 was mislocalized in Let-23-GFP expressing worms treated with the CER-SM pathway inhibitors fumonisin B1, GT11, K1, 2OHOA and D609, but also the vulval precursor cell development was impaired. Similar results were obtained with gene knockdown of CER-SM pathway enzymes, although the effects were not as significant compared to drug treatment. Similarly, all drugs tested reverted the multi vulva phenotype of the constitutively active Let23-expressing worms, while only RNAi knockdown of sphk1 enzyme significantly reverted the multi-vulva phenotype.

Conclusions: Perturbation of the CER-SM pathway inhibits localization and function of over-expressed WT EGFR and constitutively active EGFR. These results suggest that CER-SM pathway inhibitors could potentially be used to treat EGFR overexpressing/mutant cancers, including OSCC.