The Hiram C. Polk, Jr MD Department of Surgery, University of Louisville School of Medicine, Louisville, KY, 40292, USA.
BMC Cancer. 2018 Jun 22;18(1):675. doi: 10.1186/s12885-018-4583-3.
More than 50% of metastatic melanoma patients have a specific mutation in the serine/threonine kinase BRAF. This results in constitutive activation of the RAS-RAF-MEK-ERK-MAP kinase pathway, which causes uncontrolled cell growth. Vemurafenib (PLX4032) is an oral chemotherapeutic agent that targets the specific mutation V600E in the BRAF protein. Initial response rates are high in patients with BRAF mutant melanoma treated with a BRAF inhibitor such as vemurafenib, but resistance nearly always develops and disease progression ensues. There are several different mechanisms by which melanoma develops BRAF inhibitor resistance. One potential component of resistance is increased drug efflux. Overexpressed ABCB5 (ATP-binding cassette transporter, subfamily B, member 5) has been shown to efflux anti-cancer drugs from cancer cells. The purpose of this study is to determine whether ABCB5 is highly expressed in BRAF inhibitor-resistant melanoma cells and to evaluate whether ABCB5 is involved in the development of resistance to BRAF inhibitors in cutaneous melanoma.
We established three BRAF inhibitor-resistant melanoma cell lines with BRAF mutation. The expression level of ABCB5 in PLX-resistant cell lines was checked by real-time PCR and Western blot analysis. SK-MEL-2 melanoma cells with wild-type BRAF were used for comparison. The association of different levels of ABCB5 with the changes of ERK, p-ERK, Akt and p-Akt was also assessed by Western blotting. Re-sensitization of melanoma cells to PLX was tested by p-ERK inhibitor PD58059 and ABCB5 knockdown by ABCB5 siRNA, respectively.
We showed that ABCB5 was overexpressed in SK-MEL-28PLXr and A2058PLXr cells but not in A375PLXr cells. ABCB5 overexpression is associated with activation of p-ERK status but not Akt. Inhibition of p-ERK re-sensitized SK-MEL-28PLXr and A2058PLXr cells to PLX treatment, but knockdown of ABCB5 did not re-sensitize A2058 PLXr and SK-MEL-28 PLXr cells to PLX treatment.
These results confirm that, even though ABCB5 was overexpressed in SK-MEL-28 and A2058 melanoma cells that develop resistance to BRAF inhibitors, ABCB5 may not be a major targetable contributor to BRAF resistance. p-ERK inhibition may play important roles in BRAF resistance in these two melanoma cell lines.
超过 50%的转移性黑色素瘤患者存在丝氨酸/苏氨酸激酶 BRAF 的特定突变。这导致 RAS-RAF-MEK-ERK-MAP 激酶通路的组成性激活，从而导致不受控制的细胞生长。威罗非尼（PLX4032）是一种针对 BRAF 蛋白中特定突变 V600E 的口服化疗药物。用 BRAF 抑制剂（如威罗非尼）治疗 BRAF 突变黑色素瘤患者，初始反应率很高，但几乎总是会出现耐药性，随后疾病会进展。黑色素瘤产生 BRAF 抑制剂耐药性有几种不同的机制。耐药性的一个潜在组成部分是药物外排增加。过度表达的 ABCB5（ATP 结合盒转运蛋白，亚家族 B，成员 5）已被证明可将抗癌药物从癌细胞中排出。本研究的目的是确定 ABCB5 是否在 BRAF 抑制剂耐药性黑色素瘤细胞中高度表达，并评估 ABCB5 是否参与黑色素瘤对 BRAF 抑制剂的耐药性发展。
我们建立了具有 BRAF 突变的三种 BRAF 抑制剂耐药性黑色素瘤细胞系。通过实时 PCR 和 Western blot 分析检查 PLX 耐药细胞系中 ABCB5 的表达水平。使用具有野生型 BRAF 的 SK-MEL-2 黑色素瘤细胞作为对照。还通过 Western blot 评估不同水平的 ABCB5 与 ERK、p-ERK、Akt 和 p-Akt 变化之间的关联。通过 p-ERK 抑制剂 PD58059 和 ABCB5 siRNA 分别使黑色素瘤细胞对 PLX 重新敏感，以测试 PLX 的重新敏感性。
我们表明，ABCB5 在 SK-MEL-28PLXr 和 A2058PLXr 细胞中过度表达，但在 A375PLXr 细胞中不表达。ABCB5 的过表达与 p-ERK 状态的激活有关，但与 Akt 无关。抑制 p-ERK 可使 SK-MEL-28PLXr 和 A2058PLXr 细胞对 PLX 治疗重新敏感，但 ABCB5 的敲低不能使 A2058PLXr 和 SK-MEL-28PLXr 细胞对 PLX 治疗重新敏感。
这些结果证实，尽管 SK-MEL-28 和 A2058 黑色素瘤细胞在 BRAF 抑制剂耐药性方面过度表达 ABCB5，但 ABCB5 可能不是 BRAF 耐药性的主要可靶向贡献者。p-ERK 抑制可能在这两种黑色素瘤细胞系的 BRAF 耐药性中发挥重要作用。
