Targeting of Phosphoinositide Signaling in Cell Migration and Tumor Progression

描述（由申请人提供）：肿瘤转移是乳腺癌患者中大多数治疗失败的原因。在上皮来源的癌症中，上皮细胞极性的丧失和转化为迁移表型是癌细胞侵袭的关键步骤。癌细胞的内渗似乎依赖于表皮生长因子（EGF）刺激的趋化性。趋化和侵袭依赖于与细胞外基质的动态粘附和肌动蛋白细胞骨架的重塑。磷脂酰肌醇信号在这些过程中起着关键作用，但其潜在的机制还不清楚。I型γ磷脂酰肌醇-4-磷酸5-激酶（PIPKIgamma）在细胞内以时间和空间方式合成磷脂酰肌醇-4，5-二磷酸（PIP 2）。假设：PIPKI γ亚型通过其表达、靶向和EGF刺激的调节产生信使PIP 2，其调节局灶性粘连和调节趋化性的囊泡运输的空间和时间组装。PIPKI γ在调节迁移中的作用是肿瘤转移的关键。具体目的：（1）研究PIPKIgamma在生长因子刺激的定向迁移和入侵中的机制作用。将表征导致PIPKI γ磷酸化的EGF信号传导机制，以及PIP 2产生在调节PIPKI γ和其他蛋白质在粘着斑处的酪氨酸磷酸化中的作用。将探讨PIPK γ参与EGF刺激的趋化性和侵袭的潜在机制，重点是粘着斑（FA）和囊泡运输的动态组装。GFP/RFP-粘着斑蛋白将用于确定PIPKIgamma在EGF刺激的细胞中粘着斑动力学中的作用。(2)两个新的选择性剪接PIPKI γ亚型，表达独特的C-末端延伸的特点。将定义PIPKI γ剪接变体相互作用伴侣、细胞内靶向和调节。将研究这些PIPKI γ同种型在细胞迁移中所起的作用。(3)将使用小鼠模型来定义PIPKI γ剪接同种型在乳腺癌浸润和转移中的作用。(4)乳腺肿瘤中PIPKI γ含量和亚型表达的变化将使用大的且充分表征的乳腺肿瘤组织微阵列进行研究。我们将研究乳腺肿瘤中PIPKIgamma表达水平的变化，并将其与其他信号分子和生物标志物以及患者结局相关联。这一目标将是一项长期研究的开始，该研究将PIPKI γ表达的变化与上皮来源的肿瘤联系起来。这种方法可能会产生重要的信息，这将有助于确定我们的细胞和分子生物学研究的潜在机制。此外，我们的机制研究将转化为对乳腺癌细胞侵袭性的更好理解。

DESCRIPTION (provided by applicant): Tumor metastasis is responsible for most treatment failures in breast cancer patients. In cancers of epithelial origin, loss of epithelial cell polarity and transformation into a migratory phenotype are key steps in invasion of cancer cells. Intravasation of cancer cells appears to be dependent upon epidermal growth factor (EGF)-stimulated chemotaxis. Chemotaxis and invasion depends upon dynamic adhesion to extracellular matrix and actin cytoskeleton remodeling. Phosphoinositide signaling plays a key role in these processes, but the underlying mechanisms are poorly defined. Type Igamma phosphatidylinositol-4-phosphate 5-kinase (PIPKIgamma) synthesizes phosphatidylinositol-4,5- bisphosphate (PIP2) in a temporal and spatial fashion within cells. Hypothesis: PIPKIgamma isoforms, via their expression, targeting, and regulation by EGF-stimulation, generate the messenger PIP2, which modulate the spatial and temporal assembly of focal adhesions and vesicular trafficking that regulates chemotaxis. The role of PIPKIgamma in regulating migration is key in the metastasis of tumors. Specific Aims: (1) The mechanistic role of PIPKIgamma in growth factor-stimulated directional migration and invasion will be investigated. EGF signaling mechanisms leading to phosphorylation of PIPKIgamma will be characterized as will the role of PIP2 production in regulating tyrosine phosphorylation of PIPKIgamma and other proteins at focal adhesions. The underlying mechanism for PIPKgamma participation in EGF- stimulated chemotaxis and invasion will be explored, with an emphasis on the dynamic assembly of focal adhesions (FA) and vesicular trafficking. GFP/RFP-focal adhesion proteins will be used to define the role of PIPKIgamma in focal adhesions dynamics in EGF-stimulated cells. (2) Two new alternatively spliced PIPKIgamma isoforms that express unique C-terminal extensions will be characterized. PIPKIgamma splice variants interacting partners, intracellular targeting, and regulation will be defined. The roles played by these PIPKIgamma isoforms in cell migration will be investigated. (3) The role of PIPKIgamma splice isoforms in breast cancer intravasation and metastasis will be defined using a mouse model. (4) Changes in PIPKIgamma content and isoform expression in breast tumors will be studied using a large and well characterized breast tumor tissue microarray. We will investigate changes in PIPKIgamma expression levels in breast tumors and correlate this with other signaling molecules and biomarkers and with patient outcomes. This Aim will be the beginning of a long-term study to relate changes in PIPKIgamma expression with tumors of epithelial origin. This approach may yield important information, which will help to define the underlying mechanisms for our cell and molecular biology studies. In addition, our mechanistic studies will be translated into a greater understanding of breast cancer cell invasiveness.