VEGF signaling in Mammary Tumorigenesis

该提案将检验一个意想不到的假设，即 α6β4 整合素（称为“β4”）有助于 通过非细胞自主机制来控制乳腺肿瘤的发生和转移。这个假设 源自乳腺癌干细胞 (CSC) 中 β4 表达较低或不表达的观察结果，但 它有助于乳腺癌的形成和转移。该提案的一个主要目标是 协调这些不一致的观察结果并了解 β4 如何促进肿瘤形成和转移。 有趣的是，三阴性乳腺癌（TNBC）包含一个独特的细胞亚群，其特征是 β4 整合素 (β4high) 的高表达与 CSC (β4-/low/CD24low/CD44high) 不同，表现出基础 上皮分化且缺乏干细胞特性。这些观察结果表明 β4 的抑制是 CSC 功能所必需的。事实上，我们出乎意料地观察到，CSC 中的 β4 表达促进了其 分化并抑制其自我更新。第一个具体目标将研究其机制 这一现象的基础，它将重点关注令人兴奋的可能性，即它涉及 β4 的能力 调节粘着斑和细胞骨架张力，导致干细胞特性减弱。这一目标将 还测试了细胞骨架张力的改变在促进分化中具有因果作用的可能性 CSC 的数量。 第二个具体目标将评估β4high亚群发挥非细胞自主性的假设 CSC 的监管。换句话说，β4high 亚群的一个关键功能是扩大 CSC 群。 更具体地说，有人提出这个独特的亚群与 CSC 参与旁分泌信号传导 由甲状旁腺激素相关蛋白（PTHrP）介导，并且该信号传导有助于扩张 CSC 的数量。该假设基于 β4 调节 PTHrP 表达以及 β4high 亚群和 CSC 在乳腺肿瘤中非常接近。此外，我们的初步数据表明 β4high 亚种群有助于维持自我更新和生存。这些观察结果提供了解释 β4 整合素如何在 CSC 中不表达的情况下促进肿瘤的发生和转移。 最终的具体目标是在体内研究CSCs与β4high细胞之间的关系并评估其作用 对肿瘤发生和转移的相对贡献。第一组实验将检验两者是否 使用转基因模型有效肿瘤形成和转移需要 CSC 和 β4high 细胞 TNBC。第二个子目标是更深入地探讨 CSC 和 β4high 细胞之间的关系 评估 CSC 体内分化程度并评估其与 β4high 细胞的关联 利基市场。使用报告构建体来标记这些不同的群体将有助于实现这些目标。这个 方法将使我们能够解决几个关键问题，包括 CSC (GFP) 分化的频率 体内，“生态位”中 β4high 和 CSC 的关联以及“生态位”中 CSC 和 β4high 细胞的相对频率 转移。

This proposal will examine the unanticipated hypothesis that the α6β4 integrin (referred to as `β4') contributes to the initiation and metastasis of breast tumors by a non-cell autonomous mechanism. This hypothesis derives from the observations that the expression of β4 is low or absent in breast cancer stem cells (CSCs) but that it contributes to the formation and metastasis of breast cancers. A major goal of this proposal is to reconcile these discrepant observations and understand how β4 contributes to tumor formation and metastasis. Interestingly, triple-negative breast cancers (TNBCs) contain a distinct sub-population of cells characterized by high expression of the β4 integrin (β4high) that is distinct from CSCs (β4-/low/CD24low/CD44high), exhibits basal epithelial differentiation and lacks stem cell properties. These observations indicate that repression of β4 is necessary for CSC function. In fact, we observed unexpectedly that β4 expression in CSCs promotes their differentiation and inhibits their self-renewal. The first specific aim will investigate the mechanism that underlies this phenomenon and it will focus on the exciting possibility that it involves the ability of β4 to modulate focal adhesions and cytoskeletal tension resulting in diminished stem cell properties. This aim will also test the possibility that alterations in cytoskeletal tension have a causal role in promoting the differentiation of CSCs. The second specific aim will evaluate the hypothesis that the β4high sub-population exerts non-cell autonomous regulation of CSCs. In other terms, a key function of the β4high subpopulation is to expand the CSC population. More specifically, it is proposed that this distinct sub-population engages in paracrine signaling with CSCs mediated by parathyroid hormone-related protein (PTHrP) and that this signaling contributes to the expansion of CSCs. This hypothesis is based on the observations that β4 regulates PTHrP expression and that the β4high sub-population and CSCs are in proximity in breast tumors. Also, our preliminary data indicate that the β4high sub-population helps to maintain self-renewal and survival. These observations provide an explanation for how the β4 integrin contributes to tumor initiation and metastasis without being expressed in CSCs. The final specific aim will study the relationship between CSCs and β4high cells in vivo and evaluate their relative contribution to tumor initiation and metastasis. The first set of experiments will examine whether both CSCs and β4high cells are required for efficient tumor formation and metastasis using a transgenic model of TNBC. The second sub-aim probe more deeply into the relationship between CSCs and β4high cells by evaluating the extent to which CSCs differentiate in vivo and assessing their association with β4high cells in niches. These goals will be facilitated by the use of reporter constructs to tag these distinct populations. This approach will allow us to address several key issues including the frequency that CSCs (GFP+) differentiate in vivo, the association of β4high and CSCs in `niches' and the relative frequency of CSCs and β4high cells in metastases.