Hypoxia and TGF-β Drive Breast Cancer Bone Metastases through Parallel Signaling Pathways in Tumor Cells and the Bone Microenvironment

BackgroundMost patients with advanced breast cancer develop bone metastases, which cause pain, hypercalcemia, fractures, nerve compression and paralysis. Chemotherapy causes further bone loss, and bone-specific treatments are only palliative. Multiple tumor-secreted factors act on the bone microenvironment to drive a feed-forward cycle of tumor growth. Effective treatment requires inhibiting upstream regulators of groups of prometastatic factors. Two central regulators are hypoxia and transforming growth factor (TGF)- β. We asked whether hypoxia (via HIF-1α) and TGF-β signaling promote bone metastases independently or synergistically, and we tested molecular versus pharmacological inhibition strategies in an animal model.
Methodology/Principal FindingsWe analyzed interactions between HIF-1α and TGF-β pathways in MDA-MB-231 breast cancer cells. Only vascular endothelial growth factor (VEGF) and the CXC chemokine receptor 4 (CXCR4), of 16 genes tested, were additively increased by both TGF-β and hypoxia, with effects on the proximal promoters. We inhibited HIF-1α and TGF-β pathways in tumor cells by shRNA and dominant negative receptor approaches. Inhibition of either pathway decreased bone metastasis, with no further effect of double blockade. We tested pharmacologic inhibitors of the pathways, which target both the tumor and the bone microenvironment. Unlike molecular blockade, combined drug treatment decreased bone metastases more than either alone, with effects on bone to decrease osteoclastic bone resorption and increase osteoblast activity, in addition to actions on tumor cells.
Conclusions/SignificanceHypoxia and TGF-β signaling in parallel drive tumor bone metastases and regulate a common set of tumor genes. In contrast, small molecule inhibitors, by acting on both tumor cells and the bone microenvironment, additively decrease tumor burden, while improving skeletal quality. Our studies suggest that inhibitors of HIF-1α and TGF-β may improve treatment of bone metastases and increase survival.

【背景】多数晚期乳腺癌患者会发生骨转移（bone metastases），可引发疼痛、高钙血症（hypercalcemia）、骨折、神经压迫（nerve compression）乃至瘫痪。化疗会进一步加剧骨丢失，而现有的骨靶向治疗仅能起到姑息作用。多种肿瘤分泌因子可作用于骨微环境（bone microenvironment），进而形成驱动肿瘤生长的前馈循环（feed-forward cycle）。目前有效的治疗手段需要靶向调控促转移因子（prometastatic factors）群的上游调控因子（upstream regulators）。其中两个核心调控因子为缺氧（hypoxia）与转化生长因子β（transforming growth factor-β, TGF-β）。本研究旨在探讨缺氧（通过缺氧诱导因子1α（hypoxia-inducible factor 1α, HIF-1α）介导）与TGF-β信号通路是否独立或协同促进骨转移，并在动物模型（animal model）中验证分子层面与药理学层面的阻断策略的效果。

【方法与主要结果】我们在MDA-MB-231乳腺癌细胞（MDA-MB-231 breast cancer cells）中分析了HIF-1α与TGF-β通路的相互作用。在检测的16个基因中，仅血管内皮生长因子（vascular endothelial growth factor, VEGF）与CXC趋化因子受体4（CXC chemokine receptor 4, CXCR4）可被TGF-β与缺氧叠加性上调，且该调控作用发生于近端启动子（proximal promoters）区域。我们通过短发夹RNA（short hairpin RNA, shRNA）与显性负性受体（dominant negative receptor）技术在肿瘤细胞中阻断HIF-1α与TGF-β通路。单独阻断任一通路即可抑制骨转移，联合双重阻断并未产生额外效果。我们还测试了可同时靶向肿瘤与骨微环境的通路药理学抑制剂（pharmacologic inhibitors）。与分子层面的阻断不同，联合药物治疗相较于单一药物治疗可更有效地抑制骨转移，其作用除了靶向肿瘤细胞外，还可作用于骨骼，减少破骨性骨吸收（osteoclastic bone resorption）并增强成骨细胞活性（osteoblast activity）。

【结论与意义】缺氧与TGF-β信号通路可协同驱动肿瘤骨转移，并调控一组共同的肿瘤基因。与之相反，小分子抑制剂（small molecule inhibitors）可同时作用于肿瘤细胞与骨微环境，叠加性降低肿瘤负荷（tumor burden）并改善骨骼健康状况。本研究提示，HIF-1α与TGF-β抑制剂或可改善骨转移的治疗效果并提高患者生存率。