Differentiation of tissue- and devlopment of tumor stem cells

肿瘤由终末分化细胞和干细胞样细胞组成。后者亚群被认为是驱动肿瘤生长和抵抗治疗的因素。然而，肿瘤干细胞很难识别。肿瘤干细胞的独特标记物尚未被描述，肿瘤干细胞的细胞起源仍不清楚。然而，组织干细胞可以通过表面标记物或转录因子如Oct4、nanog或Sox2的表达来识别。在此基础上，我们正在为组织干细胞中已知表达的这些因子开发荧光蛋白表达报告质粒，以鉴定上皮细胞、基质细胞和肿瘤细胞中的干细胞亚群。干细胞生活在一个特殊的微环境中，即干细胞生态位，它调节着干细胞的维持、增殖和分化。在干细胞行为调控中描述的信号分子包括Wnt、Notch、FGF-2和tgf - β。tgf - β通过Smad2/3、Smad4和MAPK等多种细胞内信号级联通路传递信号，并与PI3K/mTOR和BMP等其他信号级联通路广泛相互作用，参与调控干细胞和分化细胞的分化、存活、增殖和凋亡的网络。我们计划评估这些分子在干细胞维持中的作用，并且今年在实现这一目标方面取得了进展。异染色质调节TAZ是一种转录调节因子，已被证明通过控制Smad2/3-Smad4复合物响应tgf - β的核质穿梭，在维持人胚胎干细胞的多能性中发挥作用。我们已经证明，不活跃的核Smad2、3和4蛋白与异染色质结合，而大多数不活跃的smad4蛋白留在细胞质中。我们的研究表明，Smad3特异性结合人类染色体的中心粒、肠周粒和端粒区域，tgf - β处理导致Smad3与前者分离。我们计划利用184A1人乳腺上皮细胞进一步研究Smads在干细胞表观遗传调控中的作用。这些细胞产生乳腺球并进行不对称/对称分裂，表明未分化的184A1在干细胞中富集。我们计划分离对称分裂和非对称分裂产生的细胞，然后检查这两个群体的表观遗传修饰模式，以确定异染色质和/或常染色质结构中的关键修饰。我们还建立了条件Smad7缺失小鼠，以研究Smad7功能对干细胞维持的分子/生理机制。利用人乳腺癌细胞系，我们观察到有丝分裂肿瘤细胞亚群中tgf - β的不对称活化，这意味着tgf - β可能决定干细胞有丝分裂过程中子细胞的命运。我们正在与Ron McKay合作，利用皮质神经元干细胞（NSC）研究tgf - β对干细胞有丝分裂和分化的影响。我们已经证明，NSCs内源性表达活跃的tgf - β， tgf - β以浓度依赖的方式减少NSC死亡，而ALK5抑制剂SB431542增加细胞死亡。使用外源性tgf - β和小分子抑制剂来影响信号级联，我们正在使用McKay实验室建立并可用的高通量成像技术和延时成像技术研究tgf - β信号网络在NSC分化中的作用。我们的目标是确定tgf - β对干细胞分化的影响以及信号被激活的时间框架。我们还计划专门研究Smad2/Smad3在干细胞分化和维持中的作用。为此，我们在BALB/c和C57/b6背景下建立了Smad 3敲除菌落，获得了Smad 3敲除细胞和组织，并建立了神经干细胞培养。我们计划建立不同小鼠品系的神经干细胞生理学差异，以获得基线，然后确定Smad3野生型和敲除小鼠在大脑发育方面的差异。所有的方法都可以很容易地应用于其他类型的干细胞，只要这些干细胞被识别出来。利用与Jeff Green合作建立的p53/Smad4/E-Cadherin条件三敲除乳腺癌模型，我们正在研究Smad4在乳腺癌启动和干细胞维持中的作用。在这个模型系统中，我们发现这些基因的表达降低导致不同的生物学和组织学乳腺肿瘤类型。我们的工作还揭示了Smad4和E-Cadherin之间的显著遗传相互作用，这似乎导致Smad4抑制E-Cadherin介导的腺鳞状表型。类似的相互作用机制可能发生在人类乳腺癌的发展和谱系特异性中。我们还计划利用这些单敲除、双敲除和三重敲除小鼠的肿瘤组织，通过miRNA和mRNA表达分析来鉴定特定的靶分子。目前，我们已经在体外证明，从这些基因突变小鼠的肿瘤组织中获得的Sca-1+/Lin-乳腺癌细胞群增加了乳腺球的形成和侧群细胞的数量。这些茎样群体的致瘤性正在体外使用基质和软琼脂分析进行研究。在确认其致瘤活性后，我们将在体内进行验证。此外，利用六种彩色荧光染料，利用已知的干细胞标记物和/或从mRNA表达分析中获得的其他独特的细胞表面标记物，通过FACS分析鉴定癌症起始细胞/祖细胞正在进行中。最后，tgf - β通过改变细胞外基质的组成影响细胞生物学；一般来说，tgf - β通过增加基质胶原蛋白的产生来增加基质的刚性。进一步研究表明，干细胞分化受基质硬度本身的强烈影响。由于基质弹性一方面影响干细胞分化，另一方面参与乳腺癌的肿瘤发生和肿瘤进展，我们的目的是确定基质弹性是否通过改变肿瘤干细胞池的大小来影响肿瘤进展。细胞系的初步实验表明，随着基质密度的增加，乳腺上皮细胞确实表现出肿瘤细胞中常见的蛋白质表达改变。我们正在使用已经建立的MCF10A模型研究基质密度是否会增加上皮细胞的恶性肿瘤，以及这是否由于干细胞样细胞池的扩大。我们将进一步研究，如果组织干细胞样细胞生长在与组织密度不合适的基质上，是否能产生肿瘤细胞群。

Tumors are composed of terminally differentiated and stem like cells. The latter subpopulation is believed to drive tumor growth and resistance to therapy. However, tumor stem cells are difficult to identify. No unique marker for cancer stem cells has been described and the cell of origin of tumor stem cells remains unclear. Nevertheless, tissue stem cells can be identified by surface markers or by expression of transcription factors such as Oct4, nanog, or Sox2. Based on this, we are developing fluorescence protein expressing reporter plasmids for these factors known to be expressed in tissue stem cells in order to identify stem cell subpopulation in epithelial cells, stromal cells, and tumor cells. Stem cells reside in a specialized microenvironment, the stem cell niche, which regulates stem cell maintenance, proliferation, and differentiation. Signaling molecules described in the regulation of stem cell behavior include Wnt, Notch, FGF-2, and TGF-beta. TGF-beta signals through several intracellular signaling cascades such as the Smad2/3, Smad4 and MAPK pathways, and extensively interacts with other signaling cascades such as PI3K/mTOR and BMP, and as such participates in a network that regulates differentiation, survival, proliferation and apoptosis of stem cells and differentiated cells. We plan to assess the role of these molecules in stem cell maintenance and have made strides this year in achieving this end. Heterochromatin Regulation TAZ, a transcriptional regulator, has been shown to play a role in the maintenance of pluripotency of human embryonic stem cells by controlling nucleocytoplasmic shuttling of Smad2/3-Smad4 complexes in response to TGF-beta. We have shown that inactive nuclear Smad2, 3 and 4 proteins bind to heterochromatin while the majority of inactive Smads remain in the cytoplasm. Our studies indicate that Smad3 binds specifically to centeromeric, pericenteromeric and telomeric regions of human chromosomes and that TGF-beta treatment leads to dissociation of smad3 from the two former. We plan to further investigate the role of Smads on epigenetic regulation of stem cells using 184A1 human mammary epithelial cells. These cells generate mammospheres and undergo asymmetric/symmetric division suggesting that undifferentiated 184A1 are enriched in stem cells. We plan to isolate the cells generated by symmetric and by asymmetric division and will then examine the epigenetic modification patterns in both populations to identify critical modifications in the heterochromatin and/or euchromatin structure. We have also generated the conditional Smad7 null mice to investigate the molecular/physiological mechanism of Smad7 function on stem cell maintenance. Asymmetric Cell Division Using a human breast cancer cell line we observed asymmetric activation of TGF-beta in a subset of mitotic tumor cells, implying that TGF-beta may determine the fate of daughter cells during stem cell mitosis. We are employing cortical neuronal stem cells (NSC) to investigate the influence of TGF-beta on stem cell mitosis and differentiation in collaboration with Ron McKay. We have demonstrated that NSCs endogenously express active TGF-beta, and that TGF-beta decreases NSC death in a concentration dependent fashion, while the ALK5 inhibitor, SB431542, increased cell death. Using exogenous TGF-beta and small molecule inhibitors to influence signaling cascades, we are investigating the role of the TGF-beta signaling network on NSC differentiation using high throughput imaging techniques and time lapse imaging which are established and available in the McKay lab. We aim to identify the effects of TGF-beta on stem cell differentiation and the time frame in which signaling is activated. We also plan to specifically investigate the role of Smad2/Smad3 in stem cell differentiation and maintenance. To do so we have established Smad 3 knockout colonies on BALB/c and C57/b6 backgrounds, procured Smad 3 knockout cells and tissues and established a culture of neural stem cells. We plan to establish differences in neural stem cell physiology for different mouse strains in order to obtain a baseline and then to identify differences in brain development between Smad3 wildtype and knockout mice. All methods can be readily applied to other types of stem cells as soon as those are identified. Cancer Initiation and Stem Cell Maintenance Using a a p53/Smad4/E-Cadherin conditional triple knockout breast cancer model established in collaboration with Jeff Green, we are investigating the role of Smad 4 in breast cancer initiation and stem cell maintenance. In this model system, we found that the decreased expression in each of these genes led to distinct biologic and histologic mammary tumor types. Our work also revealed a significant genetic interaction between Smad4 and E-Cadherin that appears to result in suppression of an E-Cadherin mediated adenosquamous phenotype by Smad4. Similar interactive mechanisms may occur in the development and lineage specificity of human breast cancer. We also plan to identify specific target molecules by miRNA and mRNA expression analysis using tumor tissue from these single, double, and triple knockout mice. Currently we have shown in vitro that population of Sca-1+/Lin- breast cancer cells obtained from tumor tissues of those genetically mutated mice have increased mammosphere formation and increased numbers of side population cells. The tumorigenecity of these stem-like populations is under investigation using matrigel and soft agar analysis in vitro. After confirming the tumorigenic activities, we will validate them in vivo. Furthermore, identification of cancer initiating/progenitor cells by FACS analysis using six color fluorochromes with known stem cell markers and/or other unique cell surface markers obtained from mRNA expression analysis is underway. Matrix Elasticity Finally, TGF-beta influences cell biology by altering the composition of the extracellular matrix; generally, TGF-beta increases matrix rigidity via increased stromal collagen production. It has further been shown that stem cell differentiation is strongly influenced by the matrix rigidity itself. Since matrix elasticity influences stem cell differentiation on one hand, and is involved in tumorigenesis and tumor progression in breast cancer on the other, we aim to identify whether matrix elasticity influences the tumor progression by altering the size of the tumor stem cell pool. Preliminary experiments using cell lines indicate that with increasing matrix density breast epithelial cells indeed show alterations of protein expression that are typically found in tumor cells. We are investigating whether matrix density increases malignancy in epithelial cells using the already established MCF10A model, and if this is due to the expansion of a stem cell like cell pool. We will furthermore investigate if tissue stem cell like cells can give rise to tumor cell populations if grown on a matrix of inappropriate density for the tissue.