Naslov
Differences in molecular and cellular characteristics between BRAF V600E-mutated vemurafenib-resistant and sensitive colon cancer cell lines

Autori
Grbčić, Petra ; Kraljević Pavelić, Sandra ; Sedić, Mirela

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
15th Central European Oncology Congress / - , 2019

Skup
15th Central European Oncology Congress (CEOC 2019)

Mjesto i datum
Opatija, Hrvatska, 26.06.2019. - 29.06.2019

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
colorectal cancer ; BRAF mutation ; vemurafenib ; chemoresistance ; sphingolipids

Sažetak
Patients with colorectal carcinoma (CRC) harbouring BRAF V600E mutation (7-10%) have poor response to standard treatment regimes. Vemurafenib, a potent and selective BRAF kinase inhibitor approved for treatment of metastatic melanoma with BRAF V600E mutation, has brought hope for metastatic CRC patients with BRAF V600E mutation. However, in these patients, monotherapy with vemurafenib showed limited clinical efficacy and induced rapid development of chemoresistance. In this study, we have developed vemurafenib- resistant colon cancer cell line carrying BRAF V600E mutation (RKO-R) by exposing RKO cells to successively increased concentrations of vemurafenib in the period of 6 months until stable resistance to clinically relevant doses of vemurafenib (11.52 μM) has been reached. Acquired resistance was confirmed by the MTT cell proliferation assay showing higher IC 50 value for resistant cells (29.76 μM) in comparison to parental (sensitive) RKO cells (3 μM). Distinct morphological features of vemurafenib-resistant cells were observed including the formation of pseudopodia and spindle-like shape. Woundhealing assay revealed increased migratory ability of resistant cells in comparison to parental cells, whereas clonogenic potency was more pronounced in parental line as early as 10 days of incubation. These findings were additionally corroborated by the transwell migration and invasion assays. Further molecular validation of this newly developed pre-clinical model of vemurafenib resistance in colon cancer cells harbouring BRAF V600E mutation was carried out by Western blot analysis demonstrating the upregulation of PI3K/Akt and RAF/MEK/ERK signalling pathways, standard biomarkers of resistance to vemurafenib. To further characterise vemurafenib resistance at molecular level, we set out to investigate the possible involvement of metabolism and signalling of simple sphingolipid species in the development of resistance to vemurafenib in BRAF-mutant colon cancer cells. Results obtained by Western blot analysis clearly showed several differentially expressed enzymes between vemurafenib-resistant and sensitive cells which regulate sphingolipid metabolism including acid ceramidase (ASAH1), ceramide synthases 2 and 6 (Lass2 and 6), neutral sphingomyelinases 1 and 2 (Nsmase 1 and 2) and sphingosine kinases 1 and 2 (SphK1 and 2). These findings point to altered ceramide and sphingosine-1-phosphate metabolism in BRAF V600E colon cancer cells with acquired resistance to vemurafenib and support further studies to investigate the potential of targeting sphingolipid metabolism as a novel strategy to counteract vemurafenib resistance in BRAF-mutant CRC.

Izvorni jezik
Engleski

Znanstvena područja
Interdisciplinarne prirodne znanosti, Biotehnologija u biomedicini (prirodno područje, biomedicina i zdravstvo, biotehničko područje)

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