TGF-betas in breast cancer progression

在2016财年，我们继续使用我们开发的一种新的功能成像方法来研究转化生长因子-β在调节癌症干细胞(CSC)隔间中的作用，该方法允许实时和原位显示这一少数细胞群体。我们的慢病毒CSC报道使用了一种合成启动子，其中荧光蛋白的表达是由干细胞主转录因子Oct4和Sox2驱动的。使用这种方法，我们已经开发出方法，允许在3D培养系统和体外肺切片模型中扩展单个CSCs的细胞命运图。我们现在正在研究转化生长因子-β超家族成员对乳腺癌模型系统中CSC增殖、存活和运动的影响，该模型系统显示了对转化生长因子-β的抑癌或促进展反应。在异种移植乳腺癌模型中，转化生长因子-β作为肿瘤抑制因子发挥作用，我们已经鉴定出具有高内源性转化生长因子-β信号的CSCs亚群，其固有的增殖能力低于其他CSCs，提示转化生长因子-β在维持部分CSCs处于静止状态方面可能起重要作用。外源性转化生长因子-β选择性地抑制CSC室内不对称的自我更新有丝分裂，并特异性地抑制CSCs通过基底膜的侵袭。与这些观察一致的是，体内用转化生长因子-β中和抗体治疗增加了两个高分化乳腺癌移植模型的CSC含量。在更具侵袭性的晚期疾病模型中，平行工作正在进行中。我们与AECOM的Condeelis实验室进行了重大合作，使用活体视频显微镜实时观察CSCs在小鼠异种或同基因移植乳腺癌模型的原发肿瘤和转移肺中的定位和行为。了解CSCs在体内是如何调控的，对于开发更有效的癌症治疗方法至关重要，因为这些细胞在很大程度上对现有的治疗方法具有抵抗力。我们正在使用集成的基因组方法，包括RNA-Seq和ATAC-Seq，以解决在疾病过程的早期和晚期，转化生长因子-β对CSC和非CSC间隔的不同影响的分子机制。最后，我们还使用我们的癌症干细胞报告开发了一种中到高通量的药物筛选，以测试NCI天然产品存储库中选择性抑制癌症干细胞的新型药物。

In FY16, we have continued to address the role of TGF-beta in regulating the cancer stem cell (CSC) compartment using a novel functional imaging approach that we developed to allow visualization of this minority cell population in real time and in situ. Our lentiviral-based CSC reporter uses a synthetic promoter in which expression of a fluorescent protein is driven by the stem cell master transcription factors Oct4 and Sox2. Using this approach, we have developed methods that allow extended cell fate mapping of individual CSCs in 3D culture systems and in lung slice models ex vivo. We are now addressing the effect of TGF-beta superfamily members on CSC proliferation, survival and motility in breast cancer model systems that show either tumor suppressor or pro-progression responses to TGF-beta. Using a xenograft breast cancer model in which TGF-beta functions as a tumor suppressor, we have identified a subpopulation of CSCs with high endogenous TGF-beta signaling that were intrinsically less proliferative than the other CSCs, suggesting that TGF-beta may be important in maintaining a fraction of CSCs in the quiescent state. Exogenous TGF-beta selectively inhibited asymmetric self-renewing mitoses in the CSC compartment, and specifically inhibited the invasion of CSCs through basement membrane. Consistent with these observations, in vivo treatment with TGF-beta neutralizing antibodies increased the CSC content of two xenograft models of well-differentiated breast cancer. Parallel work is ongoing in models of more aggressive advanced disease. We have a major collaboration with the Condeelis Lab at AECOM to use intravital videomicroscopy to observe the localization and behavior of CSCs in real time in the primary tumor and metastatic lungs of mouse xenograft or syngeneic allograft breast cancer models. Understanding how CSCs are regulated in vivo will be critical to development of more effective cancer therapies, as these cells are largely resistant to existing therapeutic approaches. We are employing integrated genomic approaches including RNA-Seq and ATAC-Seq to address the molecular mechanisms underlying differential effects of TGF-beta on CSC and non-CSC compartments at early and late stages of the disease process. Finally we are also using our cancer stem cell reporter to develop a medium-to-high throughput drug screen to test the NCI Natural Product Repository for novel agents that selectively inhibit the cancer stem cell.