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. 2008 Sep;7(9):2876-83.
doi: 10.1158/1535-7163.MCT-08-0431.

Increased cyclin D1 expression can mediate BRAF inhibitor resistance in BRAF V600E-mutated melanomas

Keiran S M Smalley  1 , Mercedes Lioni, Maurizia Dalla Palma, Min Xiao, Brijal Desai, Suzanne Egyhazi, Johan Hansson, Hong Wu, Alastair J King, Patricia Van Belle, David E Elder, Keith T Flaherty, Meenhard Herlyn, Katherine L Nathanson
Affiliations

Increased cyclin D1 expression can mediate BRAF inhibitor resistance in BRAF V600E-mutated melanomas

Keiran S M Smalley et al. Mol Cancer Ther. 2008 Sep.

Abstract

Recent studies have shown that there is a considerable heterogeneity in the response of melanoma cell lines to MEK and BRAF inhibitors. In the current study, we address whether dysregulation of cyclin-dependent kinase 4 (CDK4) and/or cyclin D1 contribute to the BRAF inhibitor resistance of melanoma cells. Mutational screening identified a panel of melanoma cell lines that harbored both a BRAF V600E mutation and a CDK4 mutation: K22Q (1205Lu), R24C (WM39, WM46, and SK-Mel-28), and R24L (WM902B). Pharmacologic studies showed that the presence of a CDK4 mutation did not alter the sensitivity of these cell lines to the BRAF inhibitor. The only cell line with significant BRAF inhibitor resistance was found to harbor both a CDK4 mutation and a CCND1 amplification. Array comparative genomic hybridization analysis showed that CCND1 was amplified in 17% of BRAF V600E-mutated human metastatic melanoma samples, indicating the clinical relevance of this finding. As the levels of CCND1 amplification in cell lines are lower than those seen in clinical specimens, we overexpressed cyclin D1 alone and in the presence of CDK4 in a drug-sensitive melanoma line. Cyclin D1 overexpression alone increased resistance and this was enhanced when cyclin D1 and CDK4 were concurrently overexpressed. In conclusion, increased levels of cyclin D1, resulting from genomic amplification, may contribute to the BRAF inhibitor resistance of BRAF V600E-mutated melanomas, particularly when found in the context of a CDK4 mutation/overexpression.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest: A.J. King is an employee of GlaxoSmithKline. All other authors report no conflicts of interest.

Figures

Figure 1
Figure 1
Identification of CDK4 mutations in BRAF V600E – mutated human melanoma cell lines. DNA sequences of CDK4 in a wild-type melanoma cell line (WM983) and R24C CDK4-mutated melanoma cell lines (WM39, WM46, and SK-Mel-28), and an R24L-mutated melanoma cell line (WM902B).
Figure 2
Figure 2
Melanoma cell lines with CDK4 mutations are not resistant to SB590885. A, melanoma cell lines with a CDK4 mutation (WM39, WM46, SK-Mel-28, WM902B, WM793, and 1205Lu: red, open symbols) and melanoma lines without CDK4 mutations (WM983, WM164, and 451Lu; blue, closed symbols) were treated with increasing concentrations of SB590885 (1 nmol/L – 10 μmol/L) for 72 h before being treated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Absorbance was read at 540 nm and expressed as a percentage of control absorbance. Points, mean of three independent experiments; bars, SE. B, expression of phosphorylated ERK and actin expression in a panel of melanoma cell lines. C, SB590885 reduces phosphorylated ERK levels in the WM39 and 451Lu cell lines. Cells were treated with increasing concentrations of SB590885 for 1 h, after which, protein was extracted and probed for phosphorylated ERK expression (pERK), equal protein loading was confirmed by levels of total ERK (ERK). D, SB590885 induces a similar level of cell cycle arrest in CDK4-mutated (1205Lu) and CDK4 wild-type (WM983) cell lines. Cells were treated with 1 μmol/L for 24 h prior to fixation, propidium iodide staining, and resolution by flow cytometry.
Figure 3
Figure 3
Identification of human melanoma samples and a cell line with high levels of cyclin D1 (CCND1) amplification. A, DNA copy number profiles of chromosome 11 for melanoma tumor samples TB2668F1, TB2673F1, and the melanoma cell line WM39. Arrow, copy number amplification of the BAC on which CCND1 is located. Log2 ratio for each of the genomic clones is plotted according to chromosome position using a “moving average smoother” with a three-clone window. B, the WM39 cell line shows increased expression of cyclin D1 compared with two SB590885-sensitive melanoma cell lines (451Lu and WM983). For comparison, the 451Lu cell line infected with an adenovirus encoding for cyclin D1 (451Lu/CCND1) is also shown. Western blot of cyclin D1 expression, blots were stripped and re-probed for actin to show equal protein loading.
Figure 4
Figure 4
Overexpression of cyclin D1 reduces sensitivity to the BRAF inhibitor SB590885. A, infection of 451Lu melanoma cells with either virus for cyclin D1 or for CDK4. Proteins were extracted from green fluorescent protein (GFP, control), cyclin D1 (CycD1), and CDK4-infected 451Lu melanoma cells before being resolved by Western blotting. Blots were stripped and re-probed for actin to show equal protein loading. B, overexpression of cyclin D1 alone and in combination with CDK4 reduces the growth-inhibitory effect of SB590885. Cells that were infected with the green fluorescent protein virus, virus encoding for cyclin D1, virus for CDK4 or the two viruses encoding for CDK4 and cyclin D1 before being treated with SB590885 (300 nmol/L) for 72 h and analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Data shows the mean inhibition of cell growth following drug treatment relative to untreated controls (*, P < 0.05 relative to growth inhibition in cells transfected with viral control). C, cell cycle profiles of cells treated with SB590885 that overexpress CDK4 or CDK4/cyclin D1. Cells were infected with virus as described in B before being treated with SB590885 (3 μmol/L) for 24 h. After this time, cells were fixed, harvested, and stained with propidium iodide before being analyzed by flow cytometry.
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