Hypoxia-inducible factor 1 (HIF1) is critically important for driving angiogenesis and tumorigenesis. Linear ubiquitin chain assembly complex (LUBAC), the only known ubiquitin ligase capable of catalyzing protein linear ubiquitination to date, is implicated in cell signaling and associated with cancers. However, the role and mechanism of LUBAC in regulating the expression and function of HIF1α, the labile subunit of HIF1, remain to be elucidated. Herein we showed that LUBAC increases HIF1α protein expression in cultured cells and tissues of human lung cancer and enhances HIF1α DNA-binding and transcriptional activities, which are dependent upon LUBAC enzymatic activity. Mechanistically, LUBAC increases HIF1α stability through antagonizing HIF1α decay by the chaperone-mediated autophagy (CMA)-lysosome pathway, thereby potentiating HIF1α activity. We further demonstrated that HIF1α selectively interacts with HOIP (the catalytic subunit of LUBAC) primarily in the cytoplasm. LUBAC catalyzes linear ubiquitination of HIF1α at lysine 362. Linear ubiquitination shields HIF1α from interacting with heat-shock cognate protein of 70 kDa and lysosome-associated membrane protein type 2 A, two components of CMA. Consequently, linear ubiquitination confers protection against CMA-mediated destruction of HIF1α, increasing HIF1α stability and activity. We found that prolyl hydroxylation is not a perquisite for LUBAC’s effects on HIF1α. Functionally, LUBAC facilitates proliferation, clonogenic formation, invasion and migration of lung cancer cells. LUBAC also boosts angiogenesis and exacerbates lung cancer growth in mice, which are greatly compromised by inhibition of HIF1α. This work provides novel mechanistic insights into the role of LUBAC in regulating HIF1α homeostasis, tumor angiogenesis and tumorigenesis of lung cancer, making LUBAC an attractive therapeutic target for cancers.

缺氧诱导因子 1 (HIF1) 对于驱动血管生成和肿瘤发生至关重要。线性泛素链组装复合物 (LUBAC) 是迄今为止唯一已知的能够催化蛋白质线性泛素化的泛素连接酶，参与细胞信号传导并与癌症相关。然而，LUBAC 在调节 HIF1α（HIF1 不稳定亚基）的表达和功能中的作用和机制仍有待阐明。在此，我们表明，LUBAC 增加了人肺癌培养细胞和组织中 HIF1α 蛋白的表达，并增强了 HIF1α DNA 结合和转录活性，这取决于 LUBAC 酶活性。从机制上讲，LUBAC 通过分子伴侣介导的自噬 (CMA)-溶酶体途径拮抗 HIF1α 衰变，从而提高 HIF1α 稳定性，从而增强 HIF1α 活性。我们进一步证明，HIF1α 主要在细胞质中选择性地与 HOIP（LUBAC 的催化亚基）相互作用。LUBAC 催化 HIF1α 在赖氨酸 362 处的线性泛素化。线性泛素化可防止 HIF1α 与 70 kDa 的热休克同源蛋白和溶酶体相关膜蛋白 2 A 型（CMA 的两个成分）相互作用。因此，线性泛素化可防止 CMA 介导的 HIF1α 破坏，从而增加 HIF1α 的稳定性和活性。我们发现脯氨酰羟基化并不是 LUBAC 对 HIF1α 影响的必要条件。从功能上讲，LUBAC 促进肺癌细胞的增殖、克隆形成、侵袭和迁移。LUBAC 还能促进小鼠的血管生成并加剧肺癌的生长，而抑制 HIF1α 会极大地损害肺癌的生长。这项工作为 LUBAC 在调节 HIF1α 稳态、肿瘤血管生成和肺癌肿瘤发生中的作用提供了新的机制见解，使 LUBAC 成为有吸引力的癌症治疗靶点。