Expression of neurofibromin 1 in colorectal cancer and cetuximab resistance

Abstract

Neurofibromin 1 (NF1) is a tumor suppressor that has been previously reported to regulate RAS-MAPK signaling. The present study investigated the possible relationship between NF1 expression and anti-EGFR antibody (cetuximab) sensitivity in colorectal cancer cell lines. In addition, primary or metastatic colorectal cancer samples from patients treated with cetuximab were assessed for the association of cetuximab sensitivity. The quantities of the NF1 transcript, NF1-related pathway enrichment and NF1 mutation profile were measured and investigated using RNA sequencing and targeted DNA sequencing. Based on growth inhibition and colony formation assay results, cell lines were designated to be cetuximab-sensitive (NCI-H508 and Caco2) or cetuximab-resistant (KM12C and SM480). Western blotting revealed NF1 was highly expressed in cetuximab-sensitive cell lines whilst there was little expression in their cetuximab-resistant counterparts. Knocking down NF1 expression using small interfering RNA in the cetuximab-sensitive cell lines enhanced the phosphorylation of MEK and ERK according to western blotting. NF1 knockdown also reduced apoptosis, as observed by the decreased number of apoptotic bodies by DAPI nuclear staining and reduced cleavage of caspase and poly-(ADP ribose) polymerase. NF1 overexpression by transfection with GTPase-activating protein-related domain subunit rendered the cetuximab-resistant cell lines, KM12C and SW480, more susceptible to cetuximab-induced apoptosis. RNA sequencing of 111 RAS and BRAF(V600) wild-type tumor samples collected from cetuximab-treated patients with metastatic colorectal cancer revealed that the pre-treatment NF1 expression levels were not associated with the cetuximab response. However, tumor samples obtained after cetuximab treatment displayed slightly lower NF1 transcript levels compared with those in the pre-treatment samples, suggesting that exposure to the anti-EGFR antibody may be associated with reduced NF1 expression levels. Next-generation sequencing revealed that the frequency of inactivating mutations in NF1 were rare (1.8%) in patients with colorectal cancer and were not associated with the protein expression levels of NF1 except for in a small number of cases (0.5%), where the biallelic inactivation of NF1 was observed. To conclude, the present study showed that modification of NF1 expression can affect sensitivity to cetuximab in colorectal cancer cell lines, though a limitation exists in terms of its potential application as a biomarker for RAS and BRAF (V600) wild-type tumors.