Maroni Paola, Matteucci Emanuela, Drago Lorenzo, Banfi Giuseppe, Bendinelli Paola, Desiderio Maria Alfonsina
Istituto Ortopedico Galeazzi, IRCCS, Milano, Italy.
Dipartiimento di Scienze Biomediche per la Salute, Molecular Pathology Laboratory, Università degli Studi di Milano, Milano, Italy.
Exp Cell Res. 2015 Jan 15;330(2):287-299. doi: 10.1016/j.yexcr.2014.10.004. Epub 2014 Oct 19.
The present study deals with the molecular mechanisms involved in the regulation of E-cadherin expression under hypoxia, because the adjustment of the amount of E-cadherin due to physical stimuli of the microenvironment might influence the colonization of metastasis to skeleton. We analyzed the effect of 1% oxygen tension, that is similar to that encountered in the bone marrow by metastatic cells spreading from breast carcinoma. The purpose was to evaluate the hypoxia-orchestrated control of E-cadherin transactivation via hypoxia inducible factor-1 (HIF-1) and peroxisome proliferator activated receptor-γ (PPARγ), and the involvement of Hippo pathway members, as regulators of transcription factors. To give a translational significance to the study, we took into consideration human pair-matched ductal breast carcinoma and bone metastasis: E-cadherin and Wwox were expressed in bone metastasis but not in breast carcinoma, while HIF-1α and TAZ seemed localized principally in nuclei of metastasis and were found in all cell compartments of breast carcinoma. A close examination of the regulatory mechanisms underlying E-cadherin expression in bone metastasis was done in 1833 clone derived from MDA-MB231 cells. Hypoxia induced E-cadherin only in 1833 clone, but not in parental cells, through HIF-1 and PPARγ activities, while Wwox decreased. Since Wwox was highly expressed in bone metastasis, the effect of ectopic Wwox was evaluated, and we showed E-cadherin transactivation and enhanced invasiveness in WWOX transfected 1833 cells. Also, hypoxia was additive with ectopic Wwox remarkably enhancing HIF-1α nuclear shuttle and accumulation due to the lengthening of the half-life of HIF-1α protein; under this experimental condition HIF-1α appeared as a slower migrated band compared with control, in agreement with the phosphorylation state. The in vitro data strongly supported the almost exclusive presence of HIF-1α in nuclei of human-bone metastasis. Thus, we identified Wwox as a novel molecule in the HIF-1α-HDM2 regulatory loop, necessary for the dynamic regulation of the HIF-1α amount, and we suggested that the reduction of endogenous Wwox free pool under hypoxia might also be due to the interaction with HDM2, sequestering the E3 ubiquitin ligase. We highlighted the importance of nuclear HIF-1α in the biology of metastasis for the mesenchymal-epithelial transition: this phenotype was regulated by Wwox plus hypoxia through E-cadherin target gene, playing a pivotal role in bone metastasis colonization.
本研究探讨了缺氧条件下E-钙黏蛋白表达调控所涉及的分子机制，因为微环境的物理刺激导致的E-钙黏蛋白量的调节可能会影响转移灶在骨骼中的定植。我们分析了1%氧张力的影响，这与乳腺癌转移细胞在骨髓中遇到的氧张力相似。目的是评估通过缺氧诱导因子-1（HIF-1）和过氧化物酶体增殖物激活受体-γ（PPARγ）对E-钙黏蛋白反式激活的缺氧调控，以及作为转录因子调节因子的Hippo信号通路成员的参与情况。为了赋予该研究转化意义，我们考虑了人配对的导管乳腺癌和骨转移灶：E-钙黏蛋白和WWOX在骨转移灶中表达，但在乳腺癌中不表达，而HIF-1α和TAZ似乎主要定位于转移灶的细胞核中，在乳腺癌的所有细胞区室中均有发现。在源自MDA-MB231细胞的1833克隆中，对骨转移灶中E-钙黏蛋白表达的调控机制进行了仔细研究。缺氧仅通过HIF-1和PPARγ的活性在1833克隆中诱导E-钙黏蛋白表达，而在亲本细胞中则不诱导，同时WWOX表达降低。由于WWOX在骨转移灶中高表达，因此评估了异位表达WWOX的作用，我们发现WWOX转染的1833细胞中E-钙黏蛋白反式激活且侵袭性增强。此外，缺氧与异位表达的WWOX具有累加效应，由于HIF-1α蛋白半衰期延长，显著增强了HIF-1α的核穿梭和积累；在这种实验条件下，与对照相比，HIF-1α呈现出迁移较慢的条带，这与磷酸化状态一致。体外数据有力地支持了HIF-1α几乎仅存在于人类骨转移灶的细胞核中。因此，我们确定WWOX是HIF-1α-HDM2调节环中的一个新分子，是动态调节HIF-1α量所必需的，并且我们认为缺氧条件下内源性WWOX游离池的减少也可能是由于与HDM2相互作用，隔离了E3泛素连接酶。我们强调了核HIF-1α在间充质-上皮转化的转移生物学中的重要性：这种表型由WWOX加缺氧通过E-钙黏蛋白靶基因调节，在骨转移灶定植中起关键作用。
