Discrepancy of sensitivity to PLX in different melanoma cell lines
Vemurafenib (PLX4032) targets BRAF V600E-mutant melanoma. All three melanoma cell lines, A2058, A375, and SK-MEL-28, harbor BRAF mutation. To check the responses of melanoma cells to PLX treatment, cells were treated with PLX for 72 h. Cell viability was determined by MTT assay. The effect of PLX on the growth of melanoma cells at different concentrations is shown in Fig. 1b. SK-MEL-28 was most sensitive to PLX treatment, whereas A2058 was most resistant to PLX treatment. The IC50 of PLX of A2058, A375, and SK-MEL-28 cells was 10 μM, 1.0 μM, and 0.5 μM. The three cell lines, A2058, A375, and SK-MEL-28, were then constantly grown with the additional treatment of 10 μM, 1.0 μM, and 0.5 μM PLX. The cells were passaged twice every week for 1 year to induce BRAF inhibitor resistance and establish PLX-resistant (PLXr) cell lines. The vemurafenib-resistant cell lines were denoted as A2058PLXr, A375PLXr, and SK-MEL-28PLXr, respectively. The cell viability of PLX-resistant cell lines was determined by MTT assay (Fig. 1c). The IC50 of PLX of A2058 PLXr, A375 PLXr, and SK-MEL-28 PLXr were 15 μM, 10 μM, and 5 μM, respectively. The results showed that all three cell lines had developed resistance to BRAF inhibition with different levels of sensitivity.
ABCB5 was differentially expressed in BRAF inhibitor-resistant cell lines
ABCB5 is one of the most common genes that contribute to chemoresistance. To clarify whether ABCB5 was involved in PLX resistance in melanoma cells, the expression of ABCB5 mRNA was compared in PLX-resistant melanoma cell lines versus their parent melanoma cell lines by real-time PCR. The results showed that ABCB5 mRNA was upregulated in A2058 PLXr and SK-MEL-28 PLXr cells compared to A2058 and SK-MEL-28 cells (Fig. 2a); however, ABCB5 mRNA was downregulated in A375 PLXr cells compared to the A375 cells (Fig. 2a). We then examined the protein expression of ABCB5 by Western blotting. We also compared the expression of ABCB5 in the three parent cells that have BRAF mutation with that of another melanoma cell line, SK-MEL-2, which has wild-type BRAF expression. The results showed that SK-MEL-2 cells have a lower ABCB5 expression than the other three melanoma cell lines with BRAF mutation. ABCB5 protein level was upregulated in A2058 PLXr and SK-MEL-28 PLXr cells versus A2058 and SK-MEL-28 cells (Fig. 2b); however, ABCB5 protein level was downregulated in A375 PLXr cells versus A375 cells (Fig. 2b). The differential expression of ABCB5 protein was consistent with the differential expression of ABCB5 mRNA in the three PLX-resistant melanoma cell lines. These results suggest that not all the PLX-resistant melanoma cells have elevated ABCB5 expression. ABCB5 may be associated with resistance in some but not all BRAF inhibitor-resistant melanoma cell lines.
Differential expression of ABCB5 in TMZ-treated and BRAF inhibitor-treated melanoma cells
Unlike other chemotherapeutic reagents, PLX specifically targets BRAF mutation. We questioned whether there were different mechanisms between BRAF inhibitor-resistant cells and other chemoresistant cells. Temozolomide (TMZ) is another commonly used chemotherapeutic reagent in melanoma that has different mechanisms with PLX. According to our previous publication [13], human melanoma cells have variable sensitivity to TMZ in vitro. The IC50 of TMZ in SK-MEL-28, A2058, and A375 was 0.8 mM, 0.4 mM, and 0.4 mM. The dosages at IC50 of TMZ and PLX were used to treat in SK-MEL-28, A2058 and A375 cells. We compared the expression of ABCB5 in early passages of PLX-treated cells (passage 5, PLX P5) with that in early passages of TMZ-treated cells (passage 5, TMZ P5). The PCR results showed that ABCB5 mRNA had 4-fold increases in A2058 cells after TMZ treatment, whereas ABCB5 mRNA in SK-MEL-28 and A375 cells had 10-fold and 7-fold decreases after TMZ treatment (Fig. 3a). In passage 5 of PLX-treated cells, PCR results showed that ABCB5 mRNA increased significantly (14.8-fold) in A2058 cells. ABCB5 had marginal changes in SK-MEL-28 PLX P5 cells compared with SK-MEL-28 cells. ABCB5 had 4.7-fold decreases in A375 PLX P5 cells when compared with A375 cells (Fig. 3a). We also checked the ABCB5 protein expression in early passages of PLX-treated cells (passage 5, PLX P5) with that in early passages of TMZ-treated cells (passage 5, TMZ P5) by Western blotting. The results showed that the changes of the expression level of ABCB5 protein were mostly consistent with those of the ABCB5 mRNA, although the ratios were not exactly matched with the fold changes of the PCR (Fig. 3b). These results demonstrate that ABCB5 was differentially expressed in TMZ-treated and PLX-treated melanoma cells. ABCB5 might have different roles in response to different chemotherapeutic agents.
Previous research reported that chemotherapy leads to the selection of ABCB5-expression cells [11]. We sought to identify what changes occur in ABCB5 within early passages of PLX-resistant cells as compared with the established PLX-resistant cell lines. We collected early passages of PLX cell lines and compared the ABCB5 expression in early passages of PLX-resistant cells with the established PLX-resistant cells by real-time PCR. As shown in Fig. 3a, ABCB5 did not have significant changes in early passages of SK-MEL-28 PLX P5 cells, but ABCB5 had a considerable 4.7-fold increase in established SK-MEL-28 PLXr cells. In A2058 cells, ABCB5 had a drastic 14.8-fold increase in early passages of A2058 PLX P5 cells. In contrast, ABCB5 mRNA had a 1.8-fold increase in established A2058 PLXr cells. In A375 cells, ABCB5 was decreased at the early time point of PLX treatment, showing 2.4-fold downregulation in A375 PLX P5 cells versus A375 cells. The level of ABCB5 remained low (− 8 fold) in A375 PLXr cells. The changes of ABCB5 protein expression were similar as we observed in the PCR. These results showed that ABCB5 expression fluctuated in early passages of PLX-resistant cells and the established PLX-resistant cells. Based on these results, one may conclude that ABCB5 could respond differently during the course of chemotherapeutic treatment.
Upregulation of ABCB5 in BRAF inhibitor-resistant melanoma cell lines was associated with upregulation of p-ERK
Reactivation of the extracellular signal-regulated kinase (RAF-ERK) pathway and activation of the PI3K pathway were two major mechanisms identified in BRAF resistance [14,15,16,17]. We questioned whether the differential expression of ABCB5 in BRAF inhibitor-resistant cell lines had any connection with the expression of ERK, p-ERK, Akt, and p-Akt status in those cells. Western blotting showed that ERK expression did not have significant changes in all three types of BRAF inhibitor-resistant cells versus non-resistant cells. In A2058 PLXr and SK-MEL-28 PLXr cells in which ABCB5 was overexpressed, p-ERK expression was also increased. On the other hand, in A375 PLXr cells in which ABCB5 expression decreased, p-ERK level was also reduced (Fig. 3b). Akt was downregulated in all three types of BRAF inhibitor-resistant cells versus non-resistant cells. Nevertheless, p-Akt was upregulated in all three types of BRAF inhibitor-resistant cells versus non-resistant cells (Fig. 3b). In SK-MEL-2 cells with wild-type BRAF, the ERK expression had no significant difference with the other three BRAF mutation cells. However, the Akt level was much lower in SK-MEL-2 cells compared with the other three BRAF mutation cells (Fig. 2b). These results indicated that upregulation of ABCB5 in BRAF inhibitor-resistant cell lines might be associated with the activation of p-ERK status.
Akt was upregulated in early passages of TMZ-treated and BRAF inhibitor-treated cells
As we noted above, ABCB5 was differentially expressed in early passages of TMZ-treated and BRAF inhibitor-treated melanoma cells. We questioned whether there were any differences in the expression level of ERK and Akt in melanoma cells in response to different chemotherapeutic agents. Western blotting showed that ERK level nearly had no changes in early passages of either TMZ-treated or PLX-treated melanoma cells (Fig. 3b). However, unlike the downregulation of Akt in A2058 PLXr, A375 PLXr, and SK-MEL-28 PLXr cells versus their parent cells, Akt was increased in early passages of both TMZ-treated and PLX-treated melanoma cells. In contrast, Akt levels were decreased in all three BRAF inhibitor-resistant melanoma cells (Fig. 3b). These results suggested that Akt expression first increased as an early response to PLX treatment and then decreased as a late response to PLX treatment.
Knockdown of ABCB5 did not re-sensitize A2058 PLXr, SK-MEL-28 PLXr or A375 cells to PLX treatment
A375 parent cells had the highest level of ABCB5 expression among all the cells tested in this study. The expression of ABCB5 increased in A2058 PLXr and SK-MEL-28 PLXr cells versus A2058 and SK-MEL-28 cells. We questioned whether knockdown of ABCB5 might re-sensitize A2058 PLXr, SK-MEL-28 PLXr, and A375 cells to PLX treatment. A375, A2058 PLXr, and SK-MEL-28 PLXr cells were transfected with ABCB5 siRNA and then subjected to analysis of cell viability and a cytotoxicity assay. There were no significant changes of cell viability after PLX treatment in A2058 PLXr (Fig. 4a), SK-MEL-28 PLXr (Fig. 4b), and A375 cells (Fig. 4c) after control siRNA and ABCB5 siRNA transfection (Fig. 4d and e). These results showed that, even though ABCB5 was highly expressed in A375, A2058PLXr, and SK-MEL-28PLXr BRAF inhibitor-resistant cells, knockdown of ABCB5 did not re-sensitize these cells to PLX treatment. ABCB5 upregulation may not be the key player to BRAF inhibitor-resistance in A375, A2058PLXr, and SK-MEL-28PLXr cells.
Inhibition of p-ERK re-sensitized SK-MEL-28PLXr and A2058PLXr cells to PLX treatment
Since p-ERK were upregulated after SK-MEL-28 and A2058 had developed resistance to BRAF inhibitor PLX, we were interested in knowing whether inhibition of p-ERK can re-sensitize SK-MEL28PLXr and A2058 PLXr cells to PLX treatment. We used PD98059, a selective p-ERK inhibitor, to treat these two PLX-resistant cell lines. Our initial data showed that PD98059 does not cause significant cell death at the dose of 2.5uM (data not shown). We used this dose to test the cell viability after PLX treatment. The results showed that inhibition of p-ERK can re-sensitize SK-MEL-28PLXr and A2058PLXr cells to PLX treatment (Fig. 5a). We then checked whether knockdown of ABCB5 in combination with inhibition of p-ERK could have a synergistic effect on re-sensitizing SK-MEL-28PLXr and A2058PLXr cells to PLX treatment. The results showed that knockdown of ABCB5 in combination with inhibition of p-ERK can also re-sensitize PLXr cells to PLX treatment, but they did not have a synergistic effect as compared to inhibition of p-ERK alone (Fig.5b). These results suggested that inhibition of p- ERK may play a more important role than the repression of ABCB5 in PLX resistance in these two melanoma cell lines.