SHARP1 suppresses breast cancer metastasis by promoting degradation of hypoxia-inducible factors

The molecular determinants of malignant cell behaviours in breast cancer remain only partially understood. Here we show that SHARP1 (also known as BHLHE41 or DEC2) is a crucial regulator of the invasive and metastatic phenotype in triple-negative breast cancer (TNBC), one of the most aggressive types of breast cancer. SHARP1 is regulated by the p63 metastasis suppressor and inhibits TNBC aggressiveness through inhibition of hypoxia-inducible factor 1α (HIF-1α) and HIF-2α (HIFs). SHARP1 opposes HIF-dependent TNBC cell migration in vitro, and invasive or metastatic behaviours in vivo. SHARP1 is required, and sufficient, to limit expression of HIF-target genes. In primary TNBC, endogenous SHARP1 levels are inversely correlated with those of HIF targets. Mechanistically, SHARP1 binds to HIFs and promotes HIF proteasomal degradation by serving as the HIF-presenting factor to the proteasome. This process is independent of pVHL (von Hippel-Lindau tumour suppressor), hypoxia and the ubiquitination machinery. SHARP1 therefore determines the intrinsic instability of HIF proteins to act in parallel to, and cooperate with, oxygen levels. This work sheds light on the mechanisms and pathways by which TNBC acquires invasiveness and metastatic propensity.     

生命的低氧适应——2019年度诺贝尔生理学或医学奖成果解析

氧的利用和调节是高等生命赖以生存的基本条件,威廉·凯林、彼得·拉特克利夫和格雷格·塞门扎3位科学家因发现细胞感知和适应氧气供应的相关机制而获得了2019年度诺贝尔生理学或医学奖。他们发现低氧诱导因子1(hypoxia-inducible factors 1,HIF-1)广泛存在于急、慢性缺氧细胞中,是细胞适应低氧的重要转录因子。HIF-1水平受氧气含量的调节。高氧条件下,HIF-1被修饰进而降解;低氧条件下,HIF-1不被降解,并通过转录调节引起促红细胞生成素等低氧相关基因的表达。本文通过介绍HIF-1的发现和基本分子机制,探讨其在临床中的应用价值。     

低氧与表观遗传学互作的研究进展

表观遗传学是指基因组DNA序列不发生改变的情况下,基因表达水平发生变化从而导致的可遗传表型变化的现象.表观遗传可通过与低氧诱导因子(HIF)家族协同作用,以促使细胞适应低氧环境,从而参与到低氧应答的调控过程中.现就表观遗传学通过以下四个方面与低氧应答进行综述:1)VHL与PDH3调控HIF稳定性;2)通过影响HIF-1α共激活复合物的活性、HRE位点的修饰、HIF结合位点或附近区域的染色质活性,阻止HIF与HRE位点结合;3)组蛋白脱甲基酶对低氧应答相关基因的转录调控;4)低氧环境引起细胞内整体的组蛋白修饰程度和DNA甲基化水平改变.     

低氧诱导因子在疾病治疗中的新进展——2019年诺贝尔生理学或医学奖成果简析

 低氧诱导因子是一种异二聚体结构的DNA结合转录因子，它可以与特定的核辅因子结合，激活多种基因，在缺氧条件下优化氧的利用。美国癌症学家William G.Kaelin Jr、英国医学家Sir Peter J.Ratcliffe和美国医学家Gregg L.Semenza因发现了细胞如何感知和适应氧可用性，获得了2019年的诺贝尔生理学或医学奖，其中HIF发挥了重要的作用。介绍了HIF在肾性贫血、肿瘤、心血管疾病等治疗中的研究进展，探讨了其对人类健康的意义。     

转录调节因子DEC1的研究进展

DEC1（Differentiated embryo-chondrocyte expressed gene1）是一个碱性螺旋一环一螺旋（bHLH）结构的转录因子,在软骨形成、神经发生、免疫应答、分子钟的调控、细胞分化、肿瘤的发生中起着重要的作用。DEC1还是一个缺氧调节基因,与肿瘤细胞在缺氧环境下存活密切相关,可调控肿瘤细胞生长、凋亡相关因子如缺氧诱导因子1α、转化生长因子-β、信号转导和转录激活因子、P53等的表达。DEC1还可通过调节同一家族的分子如DEC2的表达来调节细胞的功能。本文就其研究进展加以概述。     

细胞低氧感知与响应——2019年诺贝尔生理学或医学奖成果简析

 2019年诺贝尔生理学或医学奖颁给了William G.Kaelin、Jr、Sir Peter J.Ratcliffe、Gregg L.Semenza，以奖励他们在细胞低氧感知与适应的相关研究中所做出的开创性贡献。他们发现细胞利用了转录因子HIF-1的α亚基上的羟基化修饰来感知细胞内的氧水平，而低氧条件下未羟基化的HIF-1α会快速累积，并进入细胞核内调控上千个基因的转录。细胞低氧响应与多种人类疾病紧密相关，包括贫血症、红细胞增多症、心血管疾病、卒中以及癌症。回顾了细胞低氧响应信号通路的发现过程以及与低氧响应有关的人类疾病。     

低氧训练对大鼠骨骼肌HIF 1α基因表达的影响

探讨不同低氧训练模式对机体骨骼肌HIF 1α mRNA水平和蛋白水平表达的影响。6周龄SD雄性大鼠120只,经3周适应性训练和力竭实验筛选出90只,随机分成9组：常氧安静对照组、持续低氧安静组、间歇低氧安静组、低住低练耐力组、高住高练耐力组、高住低练耐力组、低住高练耐力组、高住高练后复氧训练组和高住低练后复氧训练组。采用常压低氧仓在13.6%的氧体积分数下（相当于海拔3?500?m的氧体积分数）进行低氧训练,根据血乳酸-速度曲线确定大鼠常氧训练的强度为35?m/min,低氧训练的强度为30?m/min。低氧训练持续时间为6周,每周训练5?d。其中,在第4周末进行运动能力测试,第5周末进行力竭测试,在第6周末的最后一次运动后休息48?h后处死,取材。采用实时荧光定量PCR、免疫组化、Western blot等技术测试大鼠骨骼肌HIF 1α mRNA水平和蛋白水平表达水平的变化,以进一步探讨低氧训练对骨骼肌HIF 1α表达的适应机制。结果显示,与低住低练组相比,高住高练组和高住低练骨骼肌HIF 1αmRNA表达有显著性升高(P＜0.05);和常氧安静对照组相比,高住高练骨骼肌HIF 1αmRNA表达升高105%,有非常显著性差异(P＜0.01);高住高练后复氧训练1周,大鼠骨骼肌HIF 1αmRNA表达有非常显著性降低(P＜0.01),回到常氧安静对照组水平; 高住低练后复氧训练1周,大鼠骨骼肌HIF 1αmRNA表达有显著性降低(P＜0.05),回到常氧安静对照组水平。可得结论：高住高练、高住低练和持续低氧安静组骨骼肌HIF 1α表达都明显增强,而低住低练和低住高练变化不大,复氧训练后回到常氧安静水平。HIF 1α表达与低氧的程度和时间有明显的依存关系。     

Lysyl oxidase is essential for hypoxia-induced metastasis

Metastasis is a multistep process responsible for most cancer deaths, and it can be influenced by both the immediate microenvironment (cell-cell or cell-matrix interactions) and the extended tumour microenvironment (for example vascularization). Hypoxia (low oxygen) is clinically associated with metastasis and poor patient outcome, although the underlying processes remain unclear. Microarray studies have shown the expression of lysyl oxidase (LOX) to be elevated in hypoxic human tumour cells. Paradoxically, LOX expression is associated with both tumour suppression and tumour progression, and its role in tumorigenesis seems dependent on cellular location, cell type and transformation status. Here we show that LOX expression is regulated by hypoxia-inducible factor (HIF) and is associated with hypoxia in human breast and head and neck tumours. Patients with high LOX-expressing tumours have poor distant metastasis-free and overall survivals. Inhibition of LOX eliminates metastasis in mice with orthotopically grown breast cancer tumours. Mechanistically, secreted LOX is responsible for the invasive properties of hypoxic human cancer cells through focal adhesion kinase activity and cell to matrix adhesion. Furthermore, LOX may be required to create a niche permissive for metastatic growth. Our findings indicate that LOX is essential for hypoxia-induced metastasis and is a good therapeutic target for preventing and treating metastases.     

An oxygen-regulated switch in the protein synthesis machinery

Protein synthesis involves the translation of ribonucleic acid information into proteins, the building blocks of life. The initial step of protein synthesis is the binding of the eukaryotic translation initiation factor 4E (eIF4E) to the 7-methylguanosine (m(7)-GpppG) 5'?cap of messenger RNAs. Low oxygen tension (hypoxia) represses cap-mediated translation by sequestering eIF4E through mammalian target of rapamycin (mTOR)-dependent mechanisms. Although the internal ribosome entry site is an alternative translation initiation mechanism, this pathway alone cannot account for the translational capacity of hypoxic cells. This raises a fundamental question in biology as to how proteins are synthesized in periods of oxygen scarcity and eIF4E inhibition. Here we describe an oxygen-regulated translation initiation complex that mediates selective cap-dependent protein synthesis. We show that hypoxia stimulates the formation of a complex that includes the oxygen-regulated hypoxia-inducible factor 2α (HIF-2α), the RNA-binding protein RBM4 and the cap-binding eIF4E2, an eIF4E homologue. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis identified an RNA hypoxia response element (rHRE) that recruits this complex to a wide array of mRNAs, including that encoding the epidermal growth factor receptor. Once assembled at the rHRE, the HIF-2α-RBM4-eIF4E2 complex captures the 5'?cap and targets mRNAs to polysomes for active translation, thereby evading hypoxia-induced repression of protein synthesis. These findings demonstrate that cells have evolved a program by which oxygen tension switches the basic translation initiation machinery.     

HIF-1-modified BMSCs improve migration and reduce neuronal apoptosis after stroke in rats

Bone marrow-derived mesenchymal stem cells (BMSCs) have been demonstrated ameliorating neurologic deficits after stroke. Hypoxia-inducible factor-1 (HIF-1), the key regulator of cellular responses to low oxygen concentration, can activate multiple genes involving in crucial aspects for neurologic recovery. In this study, we present that rat BMSCs overexpression of HIF-1 α showed higher expression of HIF-1 target genes in HIF-1-BMSCs, including CXCR4, EPO, and VEGF. BMSCs-mHIF-1α also exhibited an enhanced mobility towards the ischemic area within rat brain. Neural cell apoptosis in ischemic brain shown less severe in rats transplanted with HIF-1-BMSCs. Furthermore, the number of cells expressing neural progenitor markers PAX6 and DCX were increased in BMSCs-mHIF-1α-transplanted rats. These results suggest that HIF-1α in BMSCs reduces neuronal apoptosis and promotes neurogenesis after stroke in rats.     

Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia

Acetyl coenzyme A (AcCoA) is the central biosynthetic precursor for fatty-acid synthesis and protein acetylation. In the conventional view of mammalian cell metabolism, AcCoA is primarily generated from glucose-derived pyruvate through the citrate shuttle and ATP citrate lyase in the cytosol. However, proliferating cells that exhibit aerobic glycolysis and those exposed to hypoxia convert glucose to lactate at near-stoichiometric levels, directing glucose carbon away from the tricarboxylic acid cycle and fatty-acid synthesis. Although glutamine is consumed at levels exceeding that required for nitrogen biosynthesis, the regulation and use of glutamine metabolism in hypoxic cells is not well understood. Here we show that human cells use reductive metabolism of α-ketoglutarate to synthesize AcCoA for lipid synthesis. This isocitrate dehydrogenase-1 (IDH1)-dependent pathway is active in most cell lines under normal culture conditions, but cells grown under hypoxia rely almost exclusively on the reductive carboxylation of glutamine-derived α-ketoglutarate for de novo lipogenesis. Furthermore, renal cell lines deficient in the von Hippel-Lindau tumour suppressor protein preferentially use reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels. These results identify a critical role for oxygen in regulating carbon use to produce AcCoA and support lipid synthesis in mammalian cells.     

Chemokine receptor CXCR4 downregulated by von Hippel-Lindau tumour suppressor pVHL

Organ-specific metastasis is governed, in part, by interactions between chemokine receptors on cancer cells and matching chemokines in target organs. For example, malignant breast cancer cells express the chemokine receptor CXCR4 and commonly metastasize to organs that are an abundant source of the CXCR4-specific ligand stromal cell-derived factor-1alpha (ref. 1). It is still uncertain how an evolving tumour cell is reprogrammed to express CXCR4, thus implementing the tendency to metastasize to specific organs. Here we show that the von Hippel-Lindau tumour suppressor protein pVHL negatively regulates CXCR4 expression owing to its capacity to target hypoxia-inducible factor (HIF) for degradation under normoxic conditions. This process is suppressed under hypoxic conditions, resulting in HIF-dependent CXCR4 activation. An analysis of clear cell renal carcinoma that manifests mutation of the VHL gene in most cases revealed an association of strong CXCR4 expression with poor tumour-specific survival. These results suggest a mechanism for CXCR4 activation during tumour cell evolution and imply that VHL inactivation acquired by incipient tumour cells early in tumorigenesis confers not only a selective survival advantage but also the tendency to home to selected organs.     

SUMO-1在ApoE-/-小鼠PM2.5暴露中调控血管HIF-1α/VEGF信号通路的研究

本文研究了ApoE-/-小鼠经PM2.5暴露后,其主动脉弓中SUMO-1、HIF-1及其靶基因血管内皮生长因子（VEGF）表达变化规律及HIF-1的SUMO化修饰情况。利用气管滴注方法将颗粒物滴入ApoE-/-小鼠,利用Western blot、QPCR检测小鼠主动脉中SUMO-1、HIF-1、VEGF的表达情况和CO-IP法检测SUMO-1及HIF-1共修饰情况。 结果显示： PM2.5暴露后,小鼠主动脉SUMO-1、HIF-1、VEGF的表达上调。CO-IP 显示：对照组SUMO-1没有对HIF-1α发生SUMO化修饰,PM2.5暴露后SUMO-1对HIF-1α产生了明显的SUMO化修饰。SUMO化的HIF-1与HIF-1α、HIF-1、VEGF蛋白均呈正相关。 PM2.5暴露可能通过上调细胞中SUMO-1的表达,稳定HIF-1α或者上调HIF-1的表达,进而影响VEGF的表达。     

Modulation of A-type potassium channels by a family of calcium sensors

In the brain and heart, rapidly inactivating (A-type) voltage-gated potassium (Kv) currents operate at subthreshold membrane potentials to control the excitability of neurons and cardiac myocytes. Although pore-forming alpha-subunits of the Kv4, or Shal-related, channel family form A-type currents in heterologous cells, these differ significantly from native A-type currents. Here we describe three Kv channel-interacting proteins (KChIPs) that bind to the cytoplasmic amino termini of Kv4 alpha-subunits. We find that expression of KChIP and Kv4 together reconstitutes several features of native A-type currents by modulating the density, inactivation kinetics and rate of recovery from inactivation of Kv4 channels in heterologous cells. All three KChIPs co-localize and co-immunoprecipitate with brain Kv4 alpha-subunits, and are thus integral components of native Kv4 channel complexes. The KChIPs have four EF-hand-like domains and bind calcium ions. As the activity and density of neuronal A-type currents tightly control responses to excitatory synaptic inputs, these KChIPs may regulate A-type currents, and hence neuronal excitability, in response to changes in intracellular calcium.