Pak1 - PIN Pathway in Breast Cancer Progression

描述(申请人提供)：乳腺癌是美国最常见的恶性肿瘤之一，影响到每九名女性中的一名。乳腺癌进展为更恶性行为的分子机制目前尚不完全清楚，据信涉及由生长因子及其受体激活的信号通路的解除调控。例如，EGF配体家族过度表达或过度激活人表皮生长因子受体经常与乳腺癌的侵袭性临床病程、降低无病生存率和转移有关。尽管关于生长因子及其受体的信息有了显著的增长，但对于这些细胞表面启动的通路的下游靶点(S)调控乳腺癌进展的机制的了解仍不清楚。如下所述，我们最近的工作表明，生长因子刺激p21激活的激酶(PAC1)是一种丝氨酸/苏氨酸激酶，在细胞的运动、侵袭和存活中发挥着重要作用，所有这些都是正常乳腺发育和肿瘤形成所必需的。我们的初步研究首次发现PIN是Pak1和生长因子信号转导的生理底物，PIN1-PIN通路可能与乳腺癌向更具侵袭性的表型发展密切相关。在这里，我们打算研究PIN参与提高细胞存活率、解除调控的G1-S转变和乳腺癌细胞致瘤表型发展的分子机制。最近的研究表明，动力蛋白轻链-8(DLCs)/一氧化氮合酶抑制蛋白(PIN)是一种新的Pak1介导性蛋白，生长因子和PACK1促进内源性PK1和PIN的相互作用，PIN是PK1及其上游激活物如hereglin和EGF的生理底物，PIN在MCF10AT进展模型中表达上调，在MMTVHER2小鼠和人的乳腺肿瘤中，PIN过表达促进生长刺激、锚定非依赖性和细胞周期进展，PK1信号是PIN表达所必需的且充分的，Pak1信号也调节其底物的表达。我们的工作假设是“解除对PIN活性的调控刺激PIN的表达和磷酸化，从而有助于提高细胞存活率、细胞周期进展和锚定独立性，以及乳腺肿瘤细胞的肿瘤发生；PIN的这些表型效应可能受PIN的Ser 88磷酸化所控制。”为了解决这些假说，我们的具体目标是确定：(1)PK1-PIN相互作用的功能意义和PIN在乳腺癌细胞中表达的调控机制；(2)PIN及其Ser88磷酸化对与乳腺癌细胞进展相关的表型变化的影响；(3)PIN转基因表达对乳腺癌细胞生物学、发育和早期肿瘤形成的影响；以及(4)PIN和PK1在乳腺癌和浸润性乳腺癌住院患者多步发病过程中的表达特点和意义。这些研究将独一无二地确定PIN及其上游PK1激酶调控乳腺癌细胞存活、有丝分裂和肿瘤发生的机制。我们提出的研究具有重要意义，因为这项研究可能通过确定PIN作为关键的调节结节，为在识别新的分子靶点、检测、预防和治疗乳腺癌方面取得新的进展奠定基础。

DESCRIPTION (provided by applicant): Breast cancer is one of the most common malignancies in the United States, affecting one in nine women. The molecular mechanisms underlying the progression of breast cancer to more malignant behavior are not completely understood at the present time and are believed to involve deregulation of signaling pathways activated by growth factors and their rece!btors. For example, overexpression or overactivation of the human epidermal growth factor receptors by EGF family of ligands is frequently associated with an aggressive clinical course, decreased disease-free survival, and metastasis in human breast cancer. Despite the remarkable growth of information about growth factors and its receptors, the progress in understanding the mechanism by which downstream target(s) of these cell surface-initiated pathways regulate breast cancer progression remains elusive. As described below, our recent work indicate that growth factor-stimulation of p21-activated kinase (Pak1), a serine/threonine kinase, plays a significant role in the cell motility, invasiveness and survival, all of which are required for both normal mammary gland development and tumor formation. Our preliminary studies have discovered for the first time that PIN is a physiological substrate of Pak1 and of growth factor signaling and that Pak1-PIN pathway may be closely involved in breast cancer progression to more invasive phenotypes. Here we propose to investigate the molecular mechanism by which PIN participates in the development of enhanced cell survival, deregulated G1-S transition and tumorigenic phenotypes in breast cancer cells. Recent studies suggest that dynein light chain-8 (DLCS)/Protein Inhibitor of NOS (PIN) is a novel Pak1-intercating protein, growth factors and Pakl promotes interaction of endogenous Pakl and PIN, PIN is a physiologic substrate of Pak1 and its upstream activators such as heregulin and EGF, PIN is upregulated in MCF10AT progression model, and in mammary tumors from MMTVHER2 mice and human, PIN overexpression promotes growth-stimulation, anchorage-independence, and cell cycle progression, and Pak1 signaling is required and sufficient for PIN expression, and Pak1 signaling also regulates the expression of its substrate. Our working hypothesis is that "deregulation of Pak1 activity stimulates the expression and phosphorylation of PIN, and consequently, contributes to an enhanced cell survival, cell cycle progression and anchorage-independence, and tumorigenesis of breast tumor cells; these phenotypic effects of PIN might be controlled by Ser 88 phosphorylation of PIN by Pak1 ." To address these hypotheses, our specific aims are to determine: (1) The functional significance of Pak1-PIN interaction and the mechanism of Pak1 regulation of PIN expression in breast cancer cells; (2) The influence of PIN and its Ser 88 phosphorylation on the phenotypic changes associated with the progression of breast cancer cells; (3) The influence of PIN transgene expression on the biology, development and the early stages of tumorigenesis in mammary gland; and (4) The expression characteristics and significance of PIN and Pak1 during multi-step pathogenesis of breast carcinoma and in-patients with invasive breast cancer. These studies will uniquely define the mechanisms through which PIN and its upstream Pak1 kinase regulate survival, mitogenesis and tumorigenesis of breast cancer cells. Our proposed research is significant as this research might form the basis for new advances in identifying novel molecular targets, detecting, preventing and treating breast cancer, by identifying PIN as a key regulatory nodule.