Ikram El Zaoui1, Maya Bucher2, Donata Rimoldi3, Michael Nicolas4, Gurkan Kaya5, Rosanna Pescini Gobert6, Nicola Bedoni1, Ann Schalenbourg4, Ezziat Sakina4, Leonidas Zografos4, Serge Leyvraz7, Nicolo Riggi6, Carlo Rivolta1,8, Alexandre P Moulin4. 1. Department of Computational Biology, Unit of Medical Genetics, Lausanne University, Lausanne, Switzerland. 2. Dermatology Unit, CHUV, Lausanne University, Lausanne, Switzerland. 3. Ludwig Institute for Cancer Research, Epalinges, Switzerland. 4. Jules-Gonin Eye Hospital, Lausanne University, FAA, Lausanne, Switzerland. 5. Dermatology and Venerology Division, Dermatopathology Laboratory, Geneva University Hospital, Geneva, Switzerland. 6. Experimental Pathology, Lausanne University Pathology Institute, Lausanne, Switzerland. 7. Charité Cancer Comprehensive Center, Charité - Universitätsmedizin Berlin, Berlin, Germany. 8. Department of Genetics and Genome Biology, University of Leicester, Leicester, United Kingdom.
Abstract
Purpose: To analyze the activity of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinases/mechanistic target of rapamycin (PI3K/mTOR) pathways in benign and malignant conjunctival melanocytic proliferations and explore whether specific inhibitors can suppress growth of conjunctival melanoma (CJM) cells. Methods: The presence of a BRAF V600E mutation and activation of ERK, MEK, S6, and AKT were assessed with immunohistochemistry in 35 conjunctival nevi and 31 melanomas. Three CJM cell lines were used: CRMM1, carrying the BRAF V600E mutation; CRMM2, harboring the NRAS Q61L mutation; and T1527A, with a BRAF G466E mutation. WST-1 assays were performed with a BRAF inhibitor (vemurafenib), two MEK inhibitors (trametinib, selumetinib), a PI3K inhibitor (pictilisib), and a dual PI3K/mTOR inhibitor (dactolisib). The phosphorylation of ERK, MEK, and S6 were tested with western blots and apoptosis with cleaved caspase-3 immunostaining. Results: A BRAF V600E mutation was detected in 42.6% of nevi and in 35.5% of CJM. MEK and ERK activation were higher in CJM, occurring in 62.9% and 45.7% of the nevi and 90.3% and 96.8% of the CJM, respectively. There was also a significant increase in S6 activation in CJM (90.3%) compared with the nevi (20%). CRMM1 was sensitive to trametinib and the PI3K inhibitors but only marginally to vemurafenib. CRMM2 was moderately sensitive to pictilisib, whereas T1527A was resistant to all drugs tested. Conclusions: The MAPK pathway activity in CJM is increased, not only as a consequence of the BRAF V600E mutation. Targeted therapy may be useful for patients with CJM, especially those with activating BRAF mutations, whereas NRAS-mutated melanomas are relatively resistant.
Purpose: To analyze the activity of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinases/mechanistic target of rapamycin (PI3K/mTOR) pathways in benign and malignant conjunctival melanocytic proliferations and explore whether specific inhibitors can suppress growth of conjunctival melanoma (CJM) cells. Methods: The presence of a BRAFV600E mutation and activation of ERK, MEK, S6, and AKT were assessed with immunohistochemistry in 35 conjunctival nevi and 31 melanomas. Three CJM cell lines were used: CRMM1, carrying the BRAFV600E mutation; CRMM2, harboring the NRASQ61L mutation; and T1527A, with a BRAFG466E mutation. WST-1 assays were performed with a BRAF inhibitor (vemurafenib), two MEK inhibitors (trametinib, selumetinib), a PI3K inhibitor (pictilisib), and a dual PI3K/mTOR inhibitor (dactolisib). The phosphorylation of ERK, MEK, and S6 were tested with western blots and apoptosis with cleaved caspase-3 immunostaining. Results: A BRAFV600E mutation was detected in 42.6% of nevi and in 35.5% of CJM. MEK and ERK activation were higher in CJM, occurring in 62.9% and 45.7% of the nevi and 90.3% and 96.8% of the CJM, respectively. There was also a significant increase in S6 activation in CJM (90.3%) compared with the nevi (20%). CRMM1 was sensitive to trametinib and the PI3K inhibitors but only marginally to vemurafenib. CRMM2 was moderately sensitive to pictilisib, whereas T1527A was resistant to all drugs tested. Conclusions: The MAPK pathway activity in CJM is increased, not only as a consequence of the BRAFV600E mutation. Targeted therapy may be useful for patients with CJM, especially those with activating BRAF mutations, whereas NRAS-mutated melanomas are relatively resistant.
Authors: Jennifer Peil; Felix Bock; Friedemann Kiefer; Rebecca Schmidt; Ludwig M Heindl; Claus Cursiefen; Simona L Schlereth Journal: Int J Mol Sci Date: 2022-01-27 Impact factor: 5.923
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