Targeting of Phosphoinositide Signaling in Cell Migration and Tumor Progression

描述(申请人提供)：肿瘤转移是乳腺癌患者治疗失败的主要原因。在上皮起源的癌症中，上皮细胞极性的丧失和向迁移表型的转变是癌细胞侵袭的关键步骤。癌细胞的血管内转移似乎依赖于表皮生长因子(EGF)刺激的趋化作用。趋化和侵袭依赖于与细胞外基质的动态黏附和肌动蛋白细胞骨架的重塑。肌醇磷脂信号在这些过程中起着关键作用，但其潜在的机制尚不清楚。IGamm型磷脂酰肌醇-4-磷酸5-激酶(PIPKIGamma)在细胞内以时间和空间的方式合成磷脂酰肌醇-4，5-二磷酸(PIP2)。假设：PIPKIGamma异构体通过其表达、靶向和受EGF刺激的调节，产生信使PIP2，该信使调节焦点粘连和囊泡运输的时空组装，从而调节趋化作用。PIPKIGamma在调节肿瘤转移中的作用是关键。具体目标：(1)研究PIPKIGamma在生长因子刺激的定向迁移和侵袭中的作用机制。将描述导致PIPKIGamma磷酸化的EGF信号机制，以及PIP2产生在调节PIPKIGamma和其他粘连蛋白的酪氨酸磷酸化中的作用。我们将探讨PIPKGamma参与EGF刺激的趋化和侵袭的潜在机制，重点是焦点黏附(FA)和囊泡运输的动态组装。GFP/RFP焦点黏附蛋白将被用来确定PIPKIGamma在EGF刺激的细胞中的焦点黏附动力学中的作用。(2)两个表达独特C-末端延伸的新的选择性剪接的PIPKIGamma异构体将被表征。将定义PIPKIGamma剪接变体相互作用的伙伴、细胞内靶向和调控。这些PIPKIGamma亚型在细胞迁移中所起的作用将被研究。(3)PIPKIGamma剪接异构体在乳腺癌侵袭和转移中的作用将通过小鼠模型来确定。(4)将利用一个大型的、特征良好的乳腺肿瘤组织芯片来研究乳腺肿瘤中PIPKIGamma含量和异构体表达的变化。我们将研究PIPKIGamma在乳腺肿瘤中表达水平的变化，并将其与其他信号分子和生物标记物以及患者预后相关联。这一目标将是一项长期研究的开始，该研究旨在将PIPKIGamma表达的变化与上皮性肿瘤联系起来。这种方法可能会产生重要的信息，这将有助于定义我们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.