Improving the efficacy of EGFR inhibitors by topical treatment of cutaneous squamous cell carcinoma with miR-634 ointment

For cutaneous squamous cell carcinoma (cSCC), topical treatment is an essential option for patients who are not candidates for or refuse surgery. Epidermal growth factor receptor (EGFR) plays a key role in the development of cSCC, but EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib, have shown only partial clinical benefit in this disease. Thus, there is an unmet need to develop novel strategies for improving the efficacy of TKIs in cSCC. We previously demonstrated that the tumor-suppressive microRNA (miRNA) miR-634 functions as a negative modulator of cytoprotective cancer cell survival processes and is a useful anticancer therapeutic agent. Here, we found that topical application of an ointment containing miR-634 inhibited in vivo tumor growth without toxicity in a cSCC xenograft mouse model and a DMBA/TPA-induced papilloma mouse model. Functional validation revealed that miR-634 overexpression reduced glutaminolysis by directly targeting ASCT2, a glutamine transporter. Furthermore, overexpression of miR-634 synergistically enhanced TKI-induced cytotoxicity by triggering severe energetic stress in vitro and in vivo. Thus, we propose that topical treatment with miR-634 ointment is a useful strategy for improving for EGFR TKI-based therapy for cSCC. We identified transcripts enriched in RNP immunoprecipitation (RIP) assays using an antibody against AGO2 in cells overexpressing miR-634 in A431 cells by microarray analysis (RIP-chip analysis). Cells were transfected with 10 nM miR-NC or miR-634 and then lysed after 24 h. The lysates were precleared and incubated with AGO2 antibody-immobilized beads or normal rabbit IgG antibody-immobilized beads for RIP. Each bead complex was washed, and RNA was isolated. RNA from AGO2-RIP (RIP) and total RNA (input) were labelled and hybridized on the Agilent 8 × 60K array. Each experiment was performed in duplicate. In both miR-NC-overexpressing cells and miR-634-overexpressing cells, the enrichment (RIP/Input) was calculated. Then, the fold change of enrichment (miR-634/miR-NC) was calculated.