乳腺癌细胞诱导间质成纤维细胞活化及PTEN失活分子机制

Co-evolution of cancer cells and stromal fibroblasts plays a pivotal role in breast cancer initiation, invasion and metastasis. We and other groups have demonstrated up-regulation of IL-6 in different subtypes of breast cancer, indicating that this multifunctional cytokine might be an important factor in mediating the sophisticated interaction between cancer cells and stromal fibroblasts. Moreover, non-cell-autonomous functions of stromal PTEN have been reported to suppress breast cancer development upon activation of ErbB2 in epithelial compartment. However, how breast cancer cells induce stromal fibroblast activation and PTEN inactivation is still obscure. We propose that breast cancer cells induce stromal PTEN loss and reprogram stromal fibroblasts to become activated stroma(tumor-promoting) via releasing soluble factors (such as IL-6) and activating downstream signaling pathways to destabilize PTEN in fibroblasts. Using human breast cancer cell lines and normal breast fibroblasts, orthotopic animal model, clinical samples with innovative molecular and cellular techniques as well as gene expression profiling, we will validate this hypothesis with the following specific aims: 1) Identify soluble factors from breast cancer cells that mediate stromal PTEN loss and activate stromal fibroblasts; 2) Determine signaling pathways involved in down-regulating stromal PTEN and reprogramming stromal fibroblasts via using IL-6/JAK/STAT3 pathway as a paradigm; 3) Determine the role of PTEN destabilizers in reprogramming stromal fibroblasts; 4) Investigate the potential reversion of activated fibroblasts by targeting JAK/STAT3 pathway in vivo , and evaluate the clinical relevance of PTEN destabilizers and stromal activation. This study will provide exciting new biological insights on co-evolution of cancer cells and stromal fibroblasts in breast cancer, and pave the novel avenues for precision cancer medicine in breast cancer.

癌细胞与间质成纤维细胞的共进化在乳腺癌演进中发挥重要作用，但乳腺癌细胞如何诱导间质细胞PTEN失活及成纤维细胞活化，目前未完全清楚。我们前期研究结果提示乳腺癌细胞可能通过分泌各种细胞因子如IL-6等，激活成纤维细胞中相关信号通路，上调PTEN负调控因子而使PTEN失活，从而激活成纤维细胞。借助分子/细胞生物学及基因表达谱分析新技术，我们将从IL-6入手，研究以下内容：1）鉴定不同亚型乳腺癌细胞中诱导间质成纤维细胞PTEN失活的关键因子；2）确定乳腺癌间质成纤维细胞中参与调控PTEN活性的关键信号通路；3）确定调控乳腺癌间质成纤维细胞PTEN稳定性的关键分子；4）探讨靶向IL-6/JAK/STAT3通路联合抑制乳腺癌细胞及活化成纤维细胞功能的可行方案，同时明确PTEN调控分子的临床相关性。本研究将初步阐明乳腺癌细胞激活间质成纤维细胞的分子机制，为乳腺癌精准治疗开辟新道路。

癌细胞与间质成纤维细胞共进化是促进乳腺癌发生、耐药、免疫逃逸、复发转移的重要因素，但乳腺癌细胞如何激活间质成纤维细胞？间质成纤维细胞又如何反过来改变乳腺癌细胞的免疫功能等？目前具体分子机制尚未完全清楚。本课题中我们通过体外共培养系统、分子生物学等高通量技术和动物模型探讨乳腺癌细胞与间质成纤维细胞之间的共进化分子机制。我们发现不同亚型乳腺癌的细胞因子和趋化因子谱存在很大差异，其中三阴性乳腺癌（TNBC）不仅表达高水平的细胞因子、趋化因子和免疫抑制因子，而且种类繁多。TNBC条件培养基能激活正常乳腺成纤维细胞并获得癌相关成纤维细胞表型（CAFs）,活化成纤维细胞下调表达PTEN及RB1，而且TNBC来源的癌相关成纤维细胞（CAFs）PTEN蛋白水平明显下降，敲减CAF中UBE2K基因能上调PTEN表达。另外，活化成纤维细胞上调表达一些转录因子，且与趋化因子受体激活相关。我们进一步发现活化成纤维细胞亦表达特定的细胞因子和趋化因子谱，如CXCL1, CXCL2, CXCL5, CXCL8, CSF2, CSF3, CCL2, CCL5等；这些活化成纤维细胞条件培养基能促进TNBC癌细胞中上调表达PD-L1和IDO1。而且浸润性乳腺癌间质成纤维细胞过表达TNC、α-SMA 、CD29和PDGFα等。此外，我们还发现耐药乳腺癌细胞伴有细胞骨架重排并伴有相关蛋白Rho（Rac1, CDC42）功能下降。更重要的是，我们不仅成功制备PD-L1杂交瘤，且获得高纯度符合免疫组化要求的PD-L1单克隆抗体，而且通过筛选合成了一类新型含氚的微管调节分子，AQ-4。该分子能靶向微管聚合作用，有效阻断微管的动态聚合，从而使细胞周期阻滞并诱导凋亡。这些结果将为进一步阐明乳腺癌与间质共进化机制及其靶向治疗提供重要基础，本项目部分工作仍在继续进行中。