Molecular Mechanisms of Bortezomib Resistant Adenocarcinoma Cells

Bortezomib (Velcade™) is a reversible proteasome inhibitor that is approved for the treatment of multiple myeloma (MM). Despite its demonstrated clinical success, some patients are deprived of treatment due to primary refractoriness or development of resistance during therapy. To investigate the role of the duration of proteasome inhibition in the anti-tumor response of bortezomib, we established clonal isolates of HT-29 adenocarcinoma cells adapted to continuous exposure of bortezomib. These cells were ∼30-fold resistant to bortezomib. Two novel and distinct mutations in the β5 subunit, Cys63Phe, located distal to the binding site in a helix critical for drug binding, and Arg24Cys, found in the propeptide region were found in all resistant clones. The latter mutation is a natural variant found to be elevated in frequency in patients with MM. Proteasome activity and levels of both the constitutive and immunoproteasome were increased in resistant cells, which correlated to an increase in subunit gene expression. These changes correlated with a more rapid recovery of proteasome activity following brief exposure to bortezomib. Increased recovery rate was not due to increased proteasome turnover as similar findings were seen in cells co-treated with cycloheximide. When we exposed resistant cells to the irreversible proteasome inhibitor carfilzomib we noted a slower rate of recovery of proteasome activity as compared to bortezomib in both parental and resistant cells. Importantly, carfilzomib maintained its cytotoxic potential in the bortezomib resistant cell lines. Therefore, resistance to bortezomib, can be overcome with irreversible inhibitors, suggesting prolonged proteasome inhibition induces a more potent anti-tumor response.

硼替佐米（Bortezomib，商品名Velcade™）是一种可逆性蛋白酶体抑制剂，已获批用于多发性骨髓瘤（multiple myeloma, MM）的治疗。尽管其临床疗效已得到证实，但部分患者因原发性难治或治疗过程中产生耐药性而无法接受该药物治疗。为探究蛋白酶体抑制时长在硼替佐米抗肿瘤应答中的作用，我们建立了可适配硼替佐米持续暴露的HT-29腺癌细胞克隆分离株。该细胞对硼替佐米的耐药性约为亲本细胞的30倍。在所有耐药克隆中，均检出β5亚基上的两处全新特异性突变：一处为位于结合位点远端、参与药物结合关键螺旋结构的Cys63Phe（半胱氨酸63→苯丙氨酸），另一处为位于前肽区域的Arg24Cys（精氨酸24→半胱氨酸）。其中后者为一种天然变异体，在多发性骨髓瘤患者中的检出频率有所升高。耐药细胞中，组成型蛋白酶体与免疫蛋白酶体（immunoproteasome）的活性及表达水平均有所上调，该变化与亚基基因表达的升高相关。上述变化与硼替佐米短暂暴露后蛋白酶体活性的更快恢复密切相关。恢复速率的提升并非由蛋白酶体周转增加所介导，因为在联合应用环己酰亚胺（cycloheximide）处理的细胞中仍可观察到类似现象。当我们将耐药细胞暴露于不可逆蛋白酶体抑制剂卡非佐米（carfilzomib）时，无论在亲本细胞还是耐药细胞中，其蛋白酶体活性的恢复速率均相较于硼替佐米处理组更慢。值得注意的是，卡非佐米在硼替佐米耐药细胞系中仍保留了其细胞毒性潜能。因此，硼替佐米的耐药性可通过不可逆蛋白酶体抑制剂予以克服，这提示延长蛋白酶体抑制时长可诱导更强效的抗肿瘤应答。