The Role of PI3Kbeta in Breast Cancer Metastasis

 描述（由申请人提供）：肿瘤转移是人乳腺癌死亡的主要原因。既往研究表明，乳腺癌转移是由肿瘤细胞和基质细胞之间的旁分泌信号驱动的，旁分泌信号促进肿瘤在继发部位的侵袭、内渗、外渗和肿瘤生长。这种旁分泌信号传导依赖于基质细胞和肿瘤细胞产生的生长因子、细胞因子和趋化因子的相互产生，其中许多通过G蛋白偶联受体（GPCR）进行信号传导。我们现在提供了大量的初步数据，表明GPCR信号转导至PI 3 K β对于肿瘤细胞侵袭、内渗和外渗至关重要。重要的是，GPCR信号转导至PI 3 K β的损失在体内肿瘤内渗和外渗中具有比激酶活性损失更严重的表型，这表明抑制p110β-Gβγ结合可能提供 预防乳腺癌转移的替代治疗方法。该提案使用体外和体内方法来检查PI 3 K β在乳腺癌转移中的作用。第一个目的包括机制研究，以评估PI 3 K β在侵袭伪足形成中的作用，侵袭伪足允许肿瘤细胞侵入周围组织。我们将关注p110β在侵袭伪足成熟中的两种功能模型：（a）作为PI[3，4，5]P3的局部来源，其代谢为PI[3，4]P2募集关键的侵袭伪足蛋白Tks 5;和（B）作为整合素信号传导的调节剂，其对侵袭伪足成熟和MMP分泌很重要。目的2研究p110β如何整合GPCR、RTK和Rac 1的上游信号，并探讨Rac 1信号通路对PI 3 K β在乳腺癌转移中的作用。特别地，我们发现p110β中Gβγ结合位点的突变在体外对Rac 1GTP结合和p110β的激活没有影响，但在细胞中阻断组成型活性Ra 1对PI 3 K β的激活。类似地，阻断p85调节亚基与酪氨酸磷酸化蛋白结合的抑制剂也抑制CA-Rac对PI 3 K β的激活。我们将探讨两个假设来解释这些数据：第一，Rac与细胞中PI 3 K β的结合需要靶向 第二，Gβγ或SH 2介导的相互作用激活PI 3 K β使PI 3 K β对Rac敏感。我们还将直接测试Rac与p110β结合的作用， 在乳腺癌转移中使用体外和体内异种移植模型。最后，我们将研究 使用已建立的遗传小鼠模型MMTV-PyMT研究PI 3 K β在乳腺癌转移中的作用，该模型产生具有高转移率的乳腺上皮肿瘤，并具有完整的免疫系统。我们将PyMT小鼠与表达p110β GPCR非偶联突变体的敲入小鼠杂交，以明确确定GPCR信号传导至PI 3 K β在肿瘤进展和转移中的作用。总而言之，这些研究将为PI 3 K β的基础生物学以及这种复杂的信号传导酶在乳腺癌转移中的作用提供重要的新见解。

 DESCRIPTION (provided by applicant): Tumor metastasis is the major cause of mortality in human breast cancer. Previous studies have shown that breast cancer metastasis is driven by paracrine signaling between tumor cells and stromal cells, which promotes invasion, intravasation, extravasation and tumor growth at secondary sites. This paracrine signaling is dependent on the reciprocal production of growth factors, cytokines and chemokines produced by stromal cells and tumor cells, many of which signal via G-protein-coupled receptors (GPCRs). We now present extensive preliminary data showing that GPCR signaling to PI3Kβ is critical for tumor cell invasion, intravasation and extravasation. Importantly, loss of GPCR signaling to PI3Kβ has a more severe phenotype on tumor intravasation and extravasation in vivo than loss of kinase activity, suggesting that inhibition of p110β-Gβγ binding might provide an alternative therapeutic approach for the prevention of breast cancer metastasis. This proposal examines the role of PI3Kβ in breast cancer metastasis, using both in vitro and in vivo approaches. The first aim comprises mechanistic studies to evaluate the role of PI3Kβ in the formation of invadopodia, which allow tumor cells to invade into surrounding tissue. We will focus on two models of p110β function in invadopodia maturation: (a) as a local source of PI[3,4,5]P3, whose metabolism to PI[3,4]P2 recruits the critical invadopodia protein Tks5; and (b) as a regulator of integrin signaling, which is important for invadopodia maturation and MMP secretion. Aim 2 examines how p110β integrates upstream signals from GPCRs, RTKs and Rac1, and examines the role of Rac1 signaling to PI3Kβ in breast cancer metastasis. In particular, we find that mutation of the Gβγ binding site in p110β has no effect on Rac1GTP binding and activation of p110β in vitro, but blocks PI3Kβ activation by constitutively active Ra1 in cells. Similarly, inhibitors that block the binding of the p85 regulatory subunit to tyrosine-phosphorylated proteins also inhibit PI3Kβ activation by CA-Rac. We will explore two hypotheses to explain these data: first, that Rac binding to PI3Kβ in cells requires the targeting of PI3Kβ to the membrane, and second, that activation of PI3Kβ by Gβγ or SH2-mediated interactions sensitizes PI3Kβ to Rac. We will also directly test the role of Rac binding to p110β in breast cancer metastasis using in vitro and in vivo xenograft models. Finally, we will study the role of PI3Kβ in breast cancer metastasis using an established genetic mouse model, MMTV-PyMT, which develops mammary epithelial tumors with high penetrance and has an intact immune system. We will cross PyMT mice to a knock-in mouse expressing the GPCR-uncoupled mutant of p110β, to definitively establish the role of GPCR signaling to PI3Kβ in tumor progression and metastasis. Altogether, these studies will lead to important new insights into the basic biology of PI3Kβ, and the role of this complex signaling enzyme in breast cancer metastasis.