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. 2013 Nov 5;2:e00969. doi: 10.7554/eLife.00969

Figure 6. Paradoxical ERK activation and JNK pathway inhibition make significant and separable contributions to BRAFi-induced growth.

(A) We envision two separable, parallel mechanisms by which PLX4720 and vemurafenib contribute to cSCC development. Drug-induced paradoxical ERK activation and inhibition of JNK signaling occur in parallel, but the former depends on intact CRAF. (B and C) Representative soft agar colonies of E1A and HRASG12V-transformed wild-type (WT) (B) and Craf−/− (C) MEFs, following exposure to 0.2 μM and 1.0 μM PLX4720 over 4–6 weeks show significant colony-forming advantages conferred by BRAFi. (D) The fold-change in colony counts of transformed wild-type (WT) (n = 22 replicates) and Craf−/− (n = 14 replicates) MEFs demonstrate a dose-dependent increase in colonies, particularly for WT MEFs. The difference between colony formation advantages conferred by 1.0 μM PLX4720 in WT vs Craf−/− MEFs was interpreted to reflect the contribution of paradoxical ERK signaling (red arrow), which depends upon Craf, and is 60% of the total effect (black arrow), with the remainder composed of other effects including JNK inhibition (blue arrow). All differences between each MEF population were significant (‘***’, p<0.001) (E) The fold-change in colony counts of transformed HaCaT cells with (‘TKD’) and without (‘SCR’) triple lentiviral shRNA knockdown of ZAK, MAP4K5, and MAP2K4, show significant differences between 1.0 μM PLX4720-treated and control-treated conditions (‘****’, p<10−10). Importantly, untreated TKD cells had a significant advantage over untreated SCR HaCaT cells (‘**’, p<0.01), which we interpreted to be the contribution of JNK signaling inhibition, of 17.6% (blue arrow). Drug-treated SCR and TKD cells both had a similar degree of total colony formation advantage (averaged as black arrow), as expected, since the TKD cells are not expected to have any additional suppression of JNK signaling in the presence of drug (‘NS’, p=0.17, Figure 3D). Therefore, the colony counts for these two distinct systems (D and E), when taken together, show that JNK pathway inhibition accounts for approximately 17.6–40% and paradoxical ERK activation accounts for approximately 60–82.4% of the total effects of PLX4720 on tumor growth.

DOI: http://dx.doi.org/10.7554/eLife.00969.022

Figure 6.

Figure 6—figure supplement 1. Paradoxical MEK and ERK activation require intact Craf.

Figure 6—figure supplement 1.

Wild-type (WT) and isogenic Craf−/− MEFs were retrovirally transduced with HRASG12V and adenovirus E1A thereby enabling anchorage-independent growth for soft agar assays. (A) WT MEFs exhibit paradoxical MEK and ERK activation, effects that are significantly reduced in Craf−/− MEFs, particularly for MEK activation. (B) HRASG12V–transformed HaCaT cells with (‘TKD’) and without (‘SCR’) triple knockdown of ZAK, MAP4K5, and MAP2K4 show equivalent paradoxical ERK activation. (C) Transformed WT and Craf−/− MEFs show equivalent expression of E1A (sc-25, Santa Cruz) and RAS (sc-32, Santa Cruz). (D) Transformed HaCaT cells show equivalent expression of RAS.
DOI: http://dx.doi.org/10.7554/eLife.00969.023
Figure 6—figure supplement 2. Dabrafenib fails to suppress apoptosis and phospho-JNK upregulation following UV irradiation at bioequivalent doses as compared to PLX4720.

Figure 6—figure supplement 2.

Based upon human pharmacokinetic data and in vitro experiments, dabrafenib and PLX4720 were compared in multiple settings at bioequivalent doses (0.05 μM and 1.0 μM, respectively). (A) Both BRAFi suppress the growth of A375 and WM35 BRAFV600E melanoma cell lines to the same degree at these doses. (B and C) At these doses, dabrafenib fails to suppress UV-induced apoptosis significantly in HaCaT and SRB1 cells. (D and E) Likewise, dabrafenib fails to suppress phospho-JNK induction, whereas PLX4720 potently suppresses phospho-JNK induction as shown earlier. (F) Dabrafenib inhibits ZAK kinase with an estimated IC50 of 28.92 ± 2.23 nM, with no significant inhibition of MAP4K5 or MKK4 up to 1 μM. At 0.01 μM of dabrafenib, the retained activity of ZAK kinase is over 64%.
DOI: http://dx.doi.org/10.7554/eLife.00969.024
Figure 6—figure supplement 3. Dabrafenib produces a colony formation advantage only in WT MEFs.

Figure 6—figure supplement 3.

At 0.05 μM, dabrafenib produce a significant growth advantage in E1A-HRASG12V- transformed WT MEFs. In E1A-HRASG12V-transformed Craf−/− MEFs, dabrafenib fails to confer a significant growth advantage, suggesting that in the absence of significant paradoxical ERK activation, dabrafenib does not have a relevant off-target effect that results in a growth advantage.
DOI: http://dx.doi.org/10.7554/eLife.00969.025