Molecular Basis of Slits as Tumor Suppressors Controlling CXCR4/SDF1 in Breast

描述(申请人提供)：癌症生物学的目标是了解发生在细胞中的遗传和表观遗传变化的进展，当它们成为肿瘤发生时。许多研究表明，CXCR4在引导转移的乳腺癌细胞到其配体SDF1(CXCL12)水平高的部位发挥关键作用。相比之下，尽管很大一部分(90%)的人类乳腺肿瘤在转化的早期阶段表现出不适当的CXCR4表达，但相对较少的研究探索了在病变发展早期解除这一途径的后果。CXCR4在疾病过程早期上调的观察结果，加上最近的研究表明，肿瘤相关成纤维细胞作为其配体SDF1的局部来源，加剧了人们对CXCR4可能在原发肿瘤生长中发挥关键作用的猜测。我们已经在Slit2和SLIT3或其Robo1受体功能丧失的乳腺增生性病变中发现了这种疾病特征，这给了我们在动物模型的综合生理学中理解CXCR4和SDF1在乳房转化早期阶段功能的独特机会。我们的初步数据表明，Sits或其Robo1受体功能缺失突变会导致组织结构丧失、增殖增加以及微环境的一系列变化，包括血管生成增加。根据我们的初步数据，该应用的总体假设是，乳房Sit/Robo1信号的丢失导致SDF1/CXCR4上调，这反过来又有助于上皮转化和肿瘤微环境的生成。为了解决这一假设，这项提议的具体目标有三个方面。在目标I中，我们建议通过使用获得和丧失功能的方法确定CXCR4在Slit2-/-、SLIT3-/-和Robo1-/-组织中的信号状态，来研究SDF1/CXCR4信号在早期病变发展中所起的作用。我们还将评估在人类乳腺肿瘤中Slits、Robo1和CXCR4之间是否存在类似的表达变化。在AIM II中，我们建议通过产生腺体来定义乳腺上皮和间质之间发生的串扰，在腺体中，Sit/Robo1信号在上皮或间质中选择性地被消除。在目标III中，我们研究了在没有Sit/Robo1信号的情况下出现的促血管生成环境。我们建议通过消除Robo1缺失背景中的血管内皮生长因子激活来单独阐明SDF1在促进新生血管生成中的作用。我们还建议通过在乳腺癌细胞中重新表达Slit2和SLIT3基因，并评估其对肿瘤生长和肿瘤血管生成的影响，来探索Slit2和SLIT3作为肿瘤抑制基因的作用。综上所述，我们已经确定SDF1/CXCR4是体内Sit/Robo1信号的关键下游调控靶点。我们建议阐明这个趋化素轴的错误调节如何协调细胞与其环境之间的不适当的相互作用，导致组织和周围微环境的转变。与公共卫生相关：乳腺癌的起源仍然难以捉摸。尽管乳腺癌被认为是一种可遗传的疾病，但据估计，在所有人类乳腺癌中，只有5%-10%与已知的基因突变有因果关系。识别其突变允许病变进展并变得恶性的基因对于识别潜在的治疗靶点至关重要。对于一些癌症，尤其是乳腺癌，一个在转移中具有既定作用的候选靶点是G蛋白偶联受体CXCR4。这个应用程序的目标是了解CXCR4如何以乳腺为模型系统促进肿瘤进展。

DESCRIPTION (provided by applicant): The goal of cancer biology is to understand the progression of genetic and epigenetic changes occurring in cells as they become tumorigenic. Many studies have demonstrated a critical role for CXCR4 in guiding metastasizing breast cancer cells to sites with high levels of its ligand SDF1 (CXCL12). In contrast, relatively few studies have explored the consequences of having this pathway deregulated early in lesion development, even though a large fraction (>90%) of human breast tumors display inappropriate CXCR4 expression at very early stages of transformation. The observation that CXCR4 is upregulated early in the disease process, combined with recent studies demonstrating that carcinoma associated fibroblasts serve as a local source of its ligand SDF1, have fueled speculation that CXCR4 may also play a pivotal role in primary tumor growth. We have identified such a disease signature in hyperplastic lesions of mammary glands harboring loss-of-function in Slit2 and Slit3 or its Robo1 receptor, giving us the unique opportunity to understand the function of CXCR4 and SDF1 in the early stages of breast transformation within the integrated physiology of an animal model. Our preliminary data demonstrate that loss-of-function mutations in Slits or their Robo1 receptor lead to loss of tissue organization, elevated proliferation and a host of changes in the microenvironment, including increased angiogenesis. Based on our preliminary data, the overall hypothesis of the application is that loss of SLIT/ROBO1 signaling in breast leads to upregulated SDF1/CXCR4, which, in turn, contributes to epithelial transformation and generation of the tumor microenvironment. To address this hypothesis, the Specific Aims of this proposal are three-fold. In Aim I, we propose to investigate the role SDF1/CXCR4 signaling plays in early lesion development by determining the signaling status of CXCR4 in Slit2-/-;Slit3-/- and Robo1-/- tissue using both gain- and loss-of-function approaches. We will also evaluate whether similar expression changes occur between Slits, Robo1 and Cxcr4 in human breast tumors. In Aim II, we propose to define the cross talk that occurs between mammary epithelia and stroma by generating glands in which SLIT/ROBO1 signaling is selectively eliminated in the epithelia or stroma. In Aim III, we investigate the pro-angiogenic environment that arises in the absence of SLIT/ROBO1 signaling. We propose to elucidate the role of SDF1, alone, in promoting neoangiogenesis by eliminating VEGF activation in the Robo1-null background. We also propose to explore the role of Slit2 and Slit3 as tumor suppressors by re-expressing the genes in breast cancer cells and evaluating the effects on tumor growth and tumor angiogenesis. In summary, we have identified SDF1/CXCR4 as key, downstream regulatory targets of SLIT/ROBO1 signaling in vivo. We propose to elucidate how misregulation of this chemokine axis orchestrates inappropriate interactions between cells and their environment, leading to transformation of the tissue and surrounding microenvironment. PUBLIC HEALTH RELEVANCE: The genesis of breast cancer has remained elusive. Even though it is considered a heritable disease, it is estimated that only 5-10% of all human breast cancers are causally linked to known genetic mutations. Identifying genes whose mutations allow a lesion to progress and become malignant is crucial for identifying potential therapeutic targets. For a number of cancers, prominently breast, a candidate target with an established role in metastasis is the G-protein coupled receptor CXCR4. The goal of this application is to understand how CXCR4 contributes to tumor progression using breast as a model system.