LUBAC promotes angiogenesis and lung tumorigenesis by ubiquitinating and antagonizing autophagic degradation of HIF1α.

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.

Novel role for caspase 1 inhibitor VX765 in suppressing NLRP3 inflammasome assembly and atherosclerosis via promoting mitophagy and efferocytosis.

Atherosclerosis is a maladaptive chronic inflammatory disease, which remains the leading cause of death worldwide. The NLRP3 inflammasome constitutes a major driver of atherosclerosis, yet the mechanism of action is poorly understood. Mitochondrial dysfunction is essential for NLRP3 inflammasome activation. However, whether activated NLRP3 inflammasome exacerbates mitochondrial dysfunction remains to be further elucidated. Herein, we sought to address these issues applying VX765, a well-established inhibitor of caspase 1. VX765 robustly restrains caspase 1-mediated interleukin-1ß production and gasdermin D processing. Our study assigned VX765 a novel role in antagonizing NLRP3 inflammasome assembly and activation. VX765 mitigates mitochondrial damage induced by activated NLRP3 inflammasome, as evidenced by decreased mitochondrial ROS production and cytosolic release of mitochondrial DNA. VX765 blunts caspase 1-dependent cleavage and promotes mitochondrial recruitment and phosphorylation of Parkin, a key mitophagy regulator. Functionally, VX765 facilitates mitophagy, efferocytosis and M2 polarization of macrophages. It also impedes foam cell formation, migration and pyroptosis of macrophages. VX765 boosts autophagy, promotes efferocytosis, and alleviates vascular inflammation and atherosclerosis in both ApoE-/- and Ldlr-/- mice. However, these effects of VX765 were abrogated upon ablation of Nlrp3 in ApoE-/- mice. This work provides mechanistic insights into NLRP3 inflammasome assembly and this inflammasome in dictating atherosclerosis. This study highlights that manipulation of caspase 1 paves a new avenue to treatment of atherosclerotic cardiovascular disease.

Inactivation of EGLN3 hydroxylase facilitates Erk3 degradation via autophagy and impedes lung cancer growth.

EGLN3 is critically important for growth of various cancers including lung cancer. However, virtually nothing is known about the role and mechanism for EGLN3 hydroxylase activity in cancers. EGLN3 catalyzes the hydroxylation of extracellular signal-regulated kinase 3 (Erk3), a potent driver of cancers. The role and mechanism for EGLN3-induced stabilization of Erk3 remain to be defined. Here, we show that Erk3 interacts with heat shock cognate protein of 70 kDa (HSC70) and lysosome-associated membrane protein type 2 A (LAMP2A), two core components of chaperone-mediated autophagy (CMA). As a consequence, Erk3 is degraded by the CMA-lysosome pathway. EGLN3-catalyzed hydroxylation antagonizes CMA-dependent destruction of Erk3. Mechanistically, hydroxylation blunts the interaction of Erk3 with LAMP2A, thereby blocking lysosomal decay of Erk3. EGLN3 inactivation inhibits macrophage migration, efferocytosis, and M2 polarization. Studies using EGLN3 catalytically inactive knock-in mice indicate that inactivation of EGLN3 hydroxylase in host cells ameliorates LLC cancer growth through reprogramming the tumor microenvironment (TME). Adoptive transfer of macrophages with inactivated EGLN3 restrains tumor growth by mounting anti-tumor immunity and restricting angiogenesis. Administration of EGLN3 hydroxylase pharmacologic inhibitor to mice bearing LLC carcinoma impedes cancer growth by targeting the TME. LLC cells harboring inactivated EGLN3 exhibit reduced tumor burden via mitigating immunosuppressive milieu and inducing cancer senescence. This study provides novel insights into the role of CMA in regulating Erk3 stability and the mechanism behind EGLN3-enhanced stability of Erk3. This work demonstrates that inactivation of EGLN3 in malignant and stromal cells suppresses tumor by orchestrating reciprocal interplays between cancer cells and the TME. This work sheds new light on the role and mechanism for EGLN3 catalytic activity in regulating cancer growth. Manipulating EGLN3 activity holds promise for cancer treatment.