The Role of PI3K Beta in Breast Cancer Metastasis

肿瘤转移是乳腺癌死亡的主要原因。以前的研究已经表明 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， 促进继发部位的侵袭、内渗、外渗和肿瘤生长。这种旁分泌信号 依赖于基质细胞产生的生长因子、细胞因子和趋化因子的相互产生， 细胞和肿瘤细胞，其中许多通过G蛋白偶联受体（GPCR）发出信号。我们现在提出 大量的初步数据显示GPCR信号传导至PI 3 K β对于肿瘤细胞侵袭是至关重要的， 内渗和外渗。重要的是，GPCR信号传导至PI 3 K β的缺失具有更严重的表型， 在体内对肿瘤内渗和外渗的抑制作用比激酶活性的丧失更明显，这表明抑制 p110β-Gβγ结合可能为乳腺癌的预防提供一种新的治疗方法 转移本研究的目的是通过体外实验和免疫组化方法，检测PI 3 K β在乳腺癌转移中的作用。 体内方法。第一个目的包括机制研究，以评估PI 3 K β在形成中的作用。 侵袭伪足，其允许肿瘤细胞侵入周围组织。我们将重点讨论两种p110β的模型 在侵袭伪足成熟中的功能：（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）作为整联蛋白信号传导的调节剂，其对于 侵袭足成熟和MMP分泌。目的2研究了p110β如何整合来自 GPCR、RTKs和Rac 1，并检测Rac 1信号转导至PI 3 K β在乳腺癌转移中的作用。在 特别地，我们发现p110β中Gβγ结合位点的突变对Rac 1GTP结合没有影响， 体外p110β激活，但在细胞中通过组成型活性Rac 1阻断PI 3 K β激活。同样地， 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激活PI 3 K β。我们将探讨两种假设来解释这些数据：第一，Rac与 细胞中的PI 3 K β需要将PI 3 K β靶向细胞膜，其次，Gβγ或Gβγ激活PI 3 K β， SH 2介导的相互作用使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PI 3 K β的作用 在乳腺癌转移中使用已建立的遗传小鼠模型MMTV-PyMT， 乳腺上皮性肿瘤具有高转移率，并具有完整的免疫系统。我们将PyMT小鼠 表达p110β GPCR非偶联突变体的敲入小鼠，以明确确定 肿瘤进展和转移中GPCR信号转导至PI 3 K β。总之，这些研究将导致重要的 对PI 3 K β基础生物学的新见解，以及这种复杂信号酶在乳腺癌中的作用 转移

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 Rac1 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.