Molecular dissection of signaling pathways exerting bone anabolic and anti-tumor effects of physical stimuli in myeloma bone disease

多发性骨髓瘤（MM）是一种恶性浆细胞疾病，肿瘤细胞可诱发溶骨性骨病。虽然恶性肿瘤骨病的合成代谢治疗仍是一个讨论的问题，但一种预防或挽救骨质减少的非药物方法是使用控制良好的物理刺激。在临床前MM研究中，我们已经表明，机械负荷形式的物理刺激有希望保留骨骼的结果，甚至可以减轻肿瘤细胞的生长和传播。皮质骨细胞的转录组分析揭示了肿瘤细胞引起的一组细胞外基质相关基因的基因表达的实质性改变，这些基因在机械负荷下被拯救或刺激。细胞黏附是组织中机械载荷的重要组成部分，黏附机制的紧密性是通过载荷来增强的。在体外分析以不同力粘附于骨骼前体的MM细胞亚群时，我们发现在紧密粘附的MM细胞中有一组差异表达的基因。我们确定了体内和体外基因集之间的部分重叠，代表了一个令人信服的相互作用基因网络，其中候选基因属于三个集群：(1)骨骼发育，(2)脂质转运和(3)细胞外基质组织。此外，通过大量临床来源的RNAseq数据，我们发现该网络中主要候选蛋白（如低密度脂蛋白受体相关蛋白1 (LRP1)）的低表达与MM患者生存率降低有关。在这个项目中，我们将分子剖析粘附/机械转导和肿瘤归巢和扩散背后的网络，寻找有效类型的机械负荷和创新的药物靶点，以解决骨合成代谢和抗肿瘤作用。我们假设LRP1是协调机械反应性骨重塑和介导物理刺激对MM和骨细胞的拯救作用的枢纽的主要候选者。在MM存在的情况下，LRP1在骨中的表达减少，过去的研究显示其骨特异性敲除会严重破坏骨重塑。同样，Serpin H1是胶原折叠和完整性所必需的伴侣蛋白，介导MM细胞的紧密粘附并控制骨细胞的细胞外基质生成。这将在体外通过骨骼前体、骨细胞细胞系和MM细胞之间的相互作用实验，以及在体内通过使用全身低强度高频振动、MM细胞的基因工程和LRP1和Serpin H1信号的药理调节来调节粘附/负载条件的小鼠模型中得到证明。这将允许在机械负荷模型中识别目标，这些目标可以用于重建健康的骨组织，同时在微环境中赋予抗肿瘤作用。

Multiple myeloma (MM) is a malignant plasma cell disorder, in which tumor cells induce osteolytic bone disease. While anabolic treatment of bone disease in malignancies is still a matter of discussion, a non-pharmacological approach to prevent or rescue osteopenia is the use of well-controlled physical stimuli. In preclinical MM studies we have shown that physical stimuli in the form of mechanical loading have promising bone-sparing outcomes and even mitigate tumor cell growth and dissemination. Transcriptome profiling of cortical osteocytes revealed substantial alterations of gene expression caused by tumor cells in a set of extracellular matrix-related genes which were rescued or stimulated by mechanical loading. Cell adhesion is an important component of mechanical loading in tissue and the tightness of adhesion mechanisms is enforced by loading. While analyzing subpopulations of MM cells in vitro that adhered with different forces to skeletal precursors we found a characteristic set of differentially expressed genes in tightly adhering MM cells. We identified a partial overlap between the in vivo and in vitro gene sets representing a convincing interactive gene network in which candidates belong to three clusters of (1) skeletal development, (2) lipid transport and (3) extracellular matrix organization. In addition, we found that low expression of lead candidates from this network like the low-density lipoprotein receptor-related protein 1 (LRP1) is associated with worse MM patient survival using large cohorts of clinically derived RNAseq data. In this project we will molecularly dissect the networks behind adhesion / mechanotransduction and tumor homing and spread, searching for effective types of mechanical loading and innovative druggable targets that address the bone anabolic and anti-tumor effects. We hypothesize that LRP1 is a lead candidate for a hub orchestrating mechanoresponsive bone remodeling and mediating rescue effects of physical stimulation in MM and bone cells. LRP1 expression is diminished in bone in the presence of MM and its bone specific knockout was shown in the past to severely disrupt bone remodeling. Similarly, Serpin H1, a chaperone essential for collagen folding and integrity, mediates tight MM cell adhesion and controls extracellular matrix production in bone cells. It will be proven both in vitro by interaction experiments between skeletal precursors, osteocyte cell lines and MM cells and in vivo in respective mouse models where adhesion / loading conditions are modulated using whole-body low-magnitude high frequency vibration, genetic engineering of MM cells and pharmacological modulation of LRP1 and Serpin H1 signaling. This will allow for identifying targets in models of mechanical loading that can be addressed to reconstitute healthy bone tissue and at the same time impart anti-tumor effects in the microenvironment.