Abstract
In vitro cell cultures are frequently used to define the molecular background of drug resistance. The majority of currently available data have been obtained from 2D in vitro cultures, however, 3D cell culture systems (spheroids) are more likely to behave similarly to in vivo conditions. Our major aim was to compare the gene expression signature of 2D and 3D cultured BRAFV600E mutant melanoma cell lines. We successfully developed BRAF-drug resistant cell lines from paired primary/metastatic melanoma cell lines in both 2D and 3D in vitro cultures. Using Affymetrix Human Gene 1.0 ST arrays, we determined the gene expression pattern of all cell lines. Our analysis revealed 1049 genes (562 upregulated and 487 downregulated) that were differentially expressed between drug-sensitive cells grown under different cell cultures. Pathway analysis showed that the differently expressed genes were mainly associated with the cell cycle, p53, and other cancer-related pathways. The number of upregulated genes (72 genes) was remarkably fewer when comparing the resistant adherent cells to cells that grow in 3D, and were associated with cell adhesion molecules and IGF1R signalling. Only 1% of the upregulated and 5.6% of the downregulated genes were commonly altered between the sensitive and the resistant spheroids. Interestingly, we found several genes (BNIP3, RING1 and ABHD4) with inverse expression signature between sensitive and resistant spheroids, which are involved in anoikis resistance and cell cycle regulation. In summary, our study highlights gene expression alterations that might help to understand the development of acquired resistance in melanoma cells in tumour tissue.
Highlights
Melanoma is the most serious type of skin cancer, which develops from pigment-producing cells known as melanocytes [1]
The relevance of using 3D, in addition to monolayer cell cultures was evaluated for breast cancer drug sensitivity and resistance by Breslin et al, and they concluded that the biological information represented by 3D and 2D cell cultures is substantially different [30]. They described that 3D cell cultures demonstrate higher innate resistance to anticancer drugs compared to the adherent cell cultures. It was just recently reported by Ryabaya et al [31] that binimetinib (MEK inhibitor) combined with metformin is a promising therapy against melanoma and described that this combination of drugs has a synergistic effect on melanoma cells
Our data provide the first insight on differently expressed genes that might be involved in 3D spheroid formation in BRAF inhibitor (BRAFi) sensitive and resistant melanoma cells
Summary
Melanoma is the most serious type of skin cancer, which develops from pigment-producing cells known as melanocytes [1]. 40–60% of melanomas harbour an activating mutation in the BRAF oncogene. In association with elevated EGFR expression [6]. Molnar et al found that high levels of EGFR are associated with a lower sensitivity against BRAF- and EGFR inhibitors and cells with high EGFR expression show significantly lower sensitivity to vemurafenib treatment and represents higher Erk activation [6]. It was proven that EGFR inhibition enhanced the antitumor effect of vemurafenib in BRAF-mutant human melanoma [7]. BRAF and MITF amplifications as well as PTEN loss are responsible for resistance to targeted therapies [8]. Amplification of MITF was found in BRAF/MEK inhibitor resistant tumours [9], which is probably associated with growth advantage when the MAPK pathway is inhibited [10]
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