Cullins, critical members of the cullin-RING ubiquitin ligases (CRLs), are often aberrantly expressed in different cancers. However, the underlying mechanisms regarding aberrant expression of these cullins and the specific substrates of CRLs in different cancers are mostly unknown. Here, we demonstrate that overexpressed CUL4B in human osteosarcoma cells forms an E3 complex with DNA damage binding protein 1 (DDB1) and DDB1- and CUL4-associated factor 13 (DCAF13). In vitro and in vivo analyses indicated that the CRL4B^DCAF13 E3 ligase specifically recognized the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) for degradation, and disruption of this E3 ligase resulted in PTEN accumulation. Further analyses indicated that miR-300 directly targeted the 3′ UTR of CUL4B, and DNA hypermethylation of a CpG island in the miR-300 promoter region contributed to the downregulation of miR-300. Interestingly, ectopic expression of miR-300 or treatment with 5-AZA-2′-deoxycytidine, a DNA methylation inhibitor, decreased the stability of CRL4B^DCAF13 E3 ligase and reduced PTEN ubiquitination. By applying in vitro screening to identify small molecules that specifically inhibit CUL4B-DDB1 interaction, we found that TSC01131 could greatly inhibit osteosarcoma cell growth and could disrupt the stability of the CRL4B^DCAF13 E3 ligase. Collectively, our findings shed new light on the molecular mechanism of CUL4B function and might also provide a new avenue for osteosarcoma therapy.

Cullins是cullin-ring泛素连接酶（CRL）的关键成员，通常在不同的癌症中异常表达。但是，有关这些cullins和CRLs在不同癌症中的特定底物的异常表达的潜在机制大多是未知的。在这里，我们证明人类骨肉瘤细胞中过表达的CUL4B与DNA损伤结合蛋白1（DDB1）以及DDB1和CUL4相关因子13（DCAF13）形成E3复合体。体外和体内分析表明，CRL4B ^DCAF13E3连接酶特异性识别肿瘤抑制物PTEN（在10号染色体上缺失的磷酸酶和张力蛋白同源物）降解，并且这种E3连接酶的破坏导致PTEN积累。进一步的分析表明，miR-300直接靶向CUL4B的3'UTR ，并且miR-300启动子区域中CpG岛的DNA超甲基化有助于miR-300的下调。有趣的是，miR-300的异位表达或DNA甲基化抑制剂5-AZA-2'-脱氧胞苷的处理降低了CRL4B ^DCAF13 E3连接酶的稳定性，并降低了PTEN泛素化。通过体外应用通过筛选以鉴定特异性抑制CUL4B-DDB1相互作用的小分子，我们发现TSC01131可以极大地抑制骨肉瘤细胞的生长，并可能破坏CRL4B ^DCAF13 E3连接酶的稳定性。总的来说，我们的发现为CUL4B功能的分子机制提供了新的线索，也可能为骨肉瘤治疗提供新的途径。