RESUMO
Neurofibromin 1 (NF1) is a tumor suppressor that has been previously reported to regulate RASMAPK signaling. The present study investigated the possible relationship between NF1 expression and antiEGFR 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, NF1related 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 cetuximabsensitive (NCIH508 and Caco2) or cetuximabresistant (KM12C and SM480). Western blotting revealed NF1 was highly expressed in cetuximabsensitive cell lines whilst there was little expression in their cetuximabresistant counterparts. Knocking down NF1 expression using small interfering RNA in the cetuximabsensitive 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 GTPaseactivating proteinrelated domain subunit rendered the cetuximabresistant cell lines, KM12C and SW480, more susceptible to cetuximabinduced apoptosis. RNA sequencing of 111 RAS and BRAFV600 wildtype tumor samples collected from cetuximabtreated patients with metastatic colorectal cancer revealed that the pretreatment 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 pretreatment samples, suggesting that exposure to the antiEGFR antibody may be associated with reduced NF1 expression levels. Nextgeneration 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 BRAFV600 wildtype tumors.