应用剪切波弹性及光散射断层成像评价HIF-1α/TWIST1通路在乳腺癌新辅助化疗后耐药中的作用

The specific pathological response after neoadjuvant chemotherapy (NAC) is a crucial factor for predicting the clinical outcome. Exploring a reliable predictive modality and illustrating the molecular biology mechanism would provide an important reference for optimizing the individualized therapy of breast cancer. Our previous findings revealed the relationship between matrix stiffness of breast cancer evaluated by ultrasound elastography and pathological responses, breast cancers with resistance to NAC tend to have high stiffness readings, there is also a significantly negative correlation between matrix stiffness and oxygen saturation evaluated by diffuse optical tomography (DOT). Therefore, the applicant propose a novel working hypothesis "high matrix stiffness - high interstitial fluid pressure - tissue hypoperfusion hypoxia - HIF-1α/TWIST1 activation", the correlation between the expression of HIF-1α and TWIST1 of breast cancer tissue was also identified in applicant's previous study. Therefore, in the present study, we further investigate the relationship between matrix stiffness of breast cancer, oxygenation and HIF-1α/TWIST1 in breast cancer tissue of patients. In animal models, we study the decrease of matrix stiffness by hyaluronidase and inhibiting HIF-1α expression would reverse high matrix stiffness-mediated resistance to NAC, illuminating intermediate links between the high matrix stiffness and TWIST1-mediated resistance to NAC; in cellular models, the applicant study the molecular biological mechanisms of TWIST1/ITGB1 pathway mediated resistance to NAC.

乳腺癌新辅助化疗后病理反应性对于乳腺癌的预后至关重要，有针对性的预测模式并能在分子生物学机制中加以解释说明，将为优化乳腺癌个体化诊疗提供重要的依据。我们前期发现治疗前超声弹性成像评估的乳腺癌基质硬度越高，越易发生耐药，且乳腺癌硬度与含氧量成负相关。因此，本申请者建立了“高基质硬度-高间质渗透压-组织低灌注-乏氧-HIF-1α/TWIST1活化”这一全新工作假说，申请者前期发现HIF-1α和TWIST1的表达存在相关性。因此，申请者拟通过乳腺癌患者组织标本研究乳腺癌基质硬度、氧饱和度与HIF-1α/TWIST1表达的关联性；在裸鼠乳腺癌原位移植模型中，通过透明质酸酶降低乳腺癌基质硬度及抑制HIF-1α表达从而逆转高基质硬度介导新辅助化疗后耐药，同时在乳腺癌细胞模型中，研究TWIST1/ITGB1通路介导乳腺癌细胞对NAC药物耐药的多种分子生物学机制。

我们以肿瘤微环境对乳腺癌新辅助化疗病理反应性的影响为研究切入点，应用剪切波弹性及光学成像等超声成像技术验证了“高基质硬度-高间质渗透压-组织低灌注-缺氧”这一病理生理基础上，阐明了：1）HIF-1α，TWIST1和ITGB1与乳腺癌硬度及含氧程度均呈相关性，并通过多元回归模型的方式将Ki-67指数及TWIST1进行有机的结合，形成新的预测指数，确定HIF-1α，TWIST1和ITGB1三者及形成新的预测指数（predRCB）对乳腺癌新辅助化疗病理反应性有较高的预测性价值；2）我们观察了整合素β1（ITGB1）对三阴性乳腺癌（TNBC）的侵袭和转移的潜在影响，同时进一步探讨低氧诱导TNBC细胞对化疗耐药的潜在机制；3）通过探讨高硬度介导乳腺癌新辅助化疗耐药，我们证实了HIF-1α/TWIST1通路是高基质硬度所介导NAC后耐药的下游环节。TWIST1通过对ITGB1调控，促进了P-gp，BCRP及MRP1等耐药相关蛋白表达表达及促进自噬抑制凋亡，进而促进乳腺癌对新辅助化疗产生耐药的新机制；4）在裸鼠异体乳腺癌移植模型中，我们通过透明质酸酶降解透明质酸使乳腺癌基质硬度下降，进而抑制不同分子亚型的HIF-1α表达，并增加不同分子亚型乳腺癌对NAC的敏感性，降低乳腺癌耐药的发生；5）在前期研究基础上，我们将SWE及DOBI衍生出的诊断效能最佳的生物标志物（ΔEmean（t2）及BS2）与Ki-67进行相关性分析，明确无明显相关性，通过线性回归模型将诊断效能最佳的标志物与病理指标Ki-67相结合，采用多元线性回归模型将三种代表乳腺癌特征的指标进行有机地结合生成新的指数（predRCB），以predRCB为代表的评估三者结合后的预测性诊断效能优于单一指标，明确多种生物学标志物的结合较单一标志物对乳腺癌新辅助后病理反应性有更高的预测价值。基于上述研究成果，我们课题组已发表SCI收录文章四篇。