Mechanisms of cancer cell chemotaxis in perineural invasion

描述（由申请人提供）：选定的癌症侵入神经内部，周围和沿神经的能力是一个很好的描述事件，称为神经周围浸润（PNI）。PNI一再被证明是一种不良的病理发现，预示着更严重的局部疾病，更高的复发率和更差的生存。尽管广泛认识到PNI的临床意义，但其机制仍然知之甚少。我们最近证明了癌细胞趋化（外部梯度传感和定向迁移）对胶质源性神经营养因子（GDNF）的关键作用，以及RET酪氨酸激酶受体的激活是PNI的关键机制。趋化作用的机制先前主要在非恶性的盘状盘基骨柱（黏菌）和白细胞系统中被定义。然而，趋化机制在癌细胞中还没有很好地定义。有趣的是，与盘状骨和白细胞趋化相关的通路在癌症中经常是异常的。这些指标包括酪氨酸激酶受体活性、PI3K活性、Rho家族（Cdc42、Rac1、RhoA） GTPase和Ras GTPase活性。GDNF已被证明可以激活胰腺癌细胞中的这些因子。在PNI的临床重要背景下，我们将阐明这些因子的致癌激活与它们对GDNF的趋化反应之间的关系。我们的中心假设是，这些因子在某些癌症中的失调可能促进对GDNF的趋化性，从而导致PNI。我们将定义促进神经侵入性表型的致癌特征。我们将使用一系列技术来定义这些通路在PNI中的作用，这些技术包括：单细胞活延时成像，PNI的体外Matrigel模型（评估神经和癌细胞之间的相互作用），PNI的小鼠坐骨神经模型，以及手术切除的人类癌症的研究。这些方法的结合将使我们达到三个目标：1。确定癌细胞趋化GDNF的可能机制。我们将重点关注：(a) RET- gfr1受体表达和RET G681S多态性，(b) PI3K/PTEN活性和p1101 H1047R和E545K突变，(c) Rho家族（Cdc42, Rac1, RhoA）和Ras GTPase活性。2. 定义趋化机制在癌细胞PNI模型中的作用。通过体外和体内模型，我们将把Aim 1中因子的调节与PNI表型的变化联系起来。3. 验证已确定的人类癌症趋化机制。我们将使用手术标本的分子、病理和临床相关性来验证目标1和目标2中确定的因素。这一建议将使我们能够(1)确定肿瘤细胞趋化性在神经周围侵袭中的机制，(2)基于对癌细胞特征的评估来预测PNI行为，(3)通过趋化抑制确定新的治疗靶点，以及(4)引入一种新的治疗方法，针对癌症与微环境的相互作用来改变其行为，而不是针对其生存能力。

DESCRIPTION (provided by applicant): The ability of selected cancers to invade in, around, and along nerves is a well described event termed perineural invasion (PNI). PNI has repeatedly been shown to be an adverse pathologic finding, heralding more aggressive local disease, higher recurrence rates, and worsened survival. Despite widespread recognition of the clinical significance of PNI, its mechanisms remain poorly understood. We recently demonstrated the critical role of cancer cell chemotaxis (external gradient sensing and directional migration) towards glial-derived neurotrophic factor (GDNF) and activation of the RET tyrosine kinase receptor as a key mechanism for PNI. Mechanisms of chemotaxis have been previously defined primarily in the non-malignant systems of dictyostelium discoideum (slime mold) and leukocytes. However, chemotactic mechanisms have not been as well defined in cancer cells. Interestingly, pathways associated with chemotaxis in dictyostelium and leukocytes are frequently aberrant in cancer. These include tyrosine kinase receptor activity, PI3K activity, Rho family (Cdc42, Rac1, RhoA) GTPase and Ras GTPase activity. GDNF has been shown to activate each of these factors in pancreatic cancer cells. We will elucidate the relationship between oncogenic activation of these factors and their chemotactic response to GDNF, in the clinically important context of PNI. Our central hypothesis is that the dysregulation of these factors in some cancers may promote chemotaxis towards GDNF, leading to PNI. We will define the oncogenic characteristics that promote a neural invasive phenotype. We will define the roles of these pathways in PNI using a combination of techniques including: single cell live time-lapse imaging, an in vitro Matrigel model of PNI that assesses interactions between nerves and cancer cells, an in vivo murine sciatic nerve model of PNI, and studies of surgically resected human cancers. The combination of these methods will position us to achieve three Aims: 1. Identify putative mechanisms of cancer cell chemotaxis towards GDNF. We will focus on: (a) RET-GFR1 receptor expression, and the RET G681S polymorphism, (b) PI3K/PTEN activity and the p1101 H1047R and E545K mutations, (c) Rho family (Cdc42, Rac1, RhoA) and Ras GTPase activity. 2. Define the role of chemotactic mechanisms in models of cancer cell PNI. Using in vitro and in vivo models, we will correlate modulation of the factors in Aim 1 with changes in PNI phenotype. 3. Validate identified chemotactic mechanisms in human cancers. We will use surgical specimens for molecular, pathologic, and clinical correlations to validate the identified factors from Aims 1 & 2. This proposal will allow us to (1) define mechanisms of cancer cell chemotaxis in perineural invasion, (2) predict PNI behavior based on an assessment of cancer cell characteristics, (3) identify novel targets for therapy by chemotactic inhibition, and (4) introduce a novel therapeutic approach in targeting a cancer's interactions with its microenvironment to modify its behavior, rather than targeting its viability. PUBLIC HEALTH RELEVANCE: Some cancers have the ability to invade along nerves, an ominous event that causes nerve dysfunction and makes the cancer more difficult to eradicate. Our laboratory has determined that this process of perineural invasion is driven by the active migration of cancer cells towards a protein secreted by nerve cells in a process called chemotaxis. Our proposal will investigate how mechanisms of chemotaxis by cancer cells may specifically lead to perineural invasion. Results from this work may allow clinicians to identify patients with cancers that are likely to invade along nerves, and ultimately develop novel therapies to prevent this type of cancer progression.