Overcoming resistance to anti-VEGF therapy via epigenetic regulation of BARD1 [SKOV3 BARD1 siRNA-RNASeq]

Despite widespread use of anti-vascular endothelial growth factor (VEGF) antibody (AVA) for cancer therapy, most patients develop progressive disease after initial response. Therefore, novel strategies are needed to understand and overcome AVA resistance. Here, we identify an unexpected role of BRCA1-associated RING domain 1 (BARD1) in overcoming adaptive resistance to AVA. We investigated the effects of epigenetic modulation on cellular response to AVA; in particular, the molecular effects of both global and targeted DNA methylation. Using in vitro assays, BARD1 was identified as being specifically involved in angiogenesis. Sequential treatment with azacytidine overcame AVA therapy resistance in cancer models in vivo. We further demonstrated that specifically targeting BARD1 with either small-interfering RNA or CpG-targeted demethylation reduced tumor growth when combined with AVA therapy (compared with AVA alone) in mouse models of ovarian cancer. These results identify a previously unrecognized role for BARD1 in tumor angiogenesis and show that targeted restoration of BARD1 can enhance the efficacy of AVA therapy in cancer models. Overall design: To understand the effect of BARD1 silencing on tumor angiogenesis pathways, we performed RNA sequencing of BARD1-silenced SKOV3ip1 cells. We transfected SKOV3ip1 cells with BARD1 siRNA or control siRNA for 48h, then subjected to RNA isolation follewed by RNAsequencing.

尽管抗血管内皮生长因子（vascular endothelial growth factor, VEGF）抗体（AVA）在癌症治疗中广泛应用，但多数患者在初始应答后仍会出现疾病进展。因此，亟需新的策略以解析并克服AVA耐药性。本研究揭示了BRCA1相关环指结构域1（BRCA1-associated RING domain 1, BARD1）在克服AVA适应性耐药中的未曾预见的作用。我们探究了表观遗传调控对细胞应答AVA的影响，特别是全局及靶向DNA甲基化的分子效应。通过体外实验，我们发现BARD1特异性参与血管生成过程。阿扎胞苷（azacytidine）序贯给药可在体内癌症模型中克服AVA治疗耐药性。我们进一步证实，在卵巢癌小鼠模型中，联合使用小干扰RNA（small-interfering RNA, siRNA）靶向抑制BARD1或CpG靶向去甲基化，并配合AVA治疗，相较于单独使用AVA，可显著抑制肿瘤生长。上述结果表明，BARD1在肿瘤血管生成中具有此前未被认知的作用，且靶向恢复BARD1表达可增强AVA治疗在癌症模型中的疗效。总体实验设计：为解析BARD1沉默对肿瘤血管生成通路的影响，我们对BARD1沉默的SKOV3ip1细胞进行了RNA测序（RNA sequencing）。将SKOV3ip1细胞用BARD1小干扰RNA或对照小干扰RNA转染48小时后，提取RNA并进行RNA测序。