Targeting invasive plasticity by inhibiting mitochondrial adaptations to matrix metalloproteinase loss

肿瘤细胞通过细胞外基质(ECM)的侵袭促进了肿瘤的局部和远处扩散， 这是癌症最致命的方面。细胞在不同的侵袭模式之间切换的能力， 当面临不同的物理或化学挑战时，称为可塑性或适应 无法开发抗侵入性疗法。识别有针对性的适应性反应以阻止入侵具有 由于缺乏识别、表征和测试密钥丢失的实验模型而受阻 促进可塑性的分子。为了满足这一关键需求，我们将重点放在矩阵上 金属蛋白酶(MMPs)，已成为广泛的临床试验的靶点，因为其强大的 与癌症的关系及其在降解细胞外基质中的作用。然而，抗基质金属蛋白酶的疗法一直无效， 很可能是因为侵入性的可塑性。为了识别和了解侵袭性细胞如何适应基质金属蛋白酶的丢失， 我们使用的是线虫体内锚定细胞(AC)入侵的模型。我们发现基因 去除MMPs会导致适应性侵袭反应，而不是ECM降解，AC 促进F-肌动蛋白聚合，强力穿透细胞外基质。使用基质金属蛋白酶为零的动物，我们进行了 第一个协同侵袭筛查，以确定促进适应性AC侵袭的基因，并确定 线粒体ATP/ADP转位酶，ANT-1.1是最强的候选基因。蚂蚁有多个 线粒体功能(ATP/ADP交换、有丝分裂、线粒体动力学)与ANT-1.1 蛋白质在AC线粒体中高度丰富，极化到ECM侵袭部位。ANT-1.1 在基质金属蛋白酶缺失的动物中，基因敲除可防止适应性F-肌动蛋白的形成，并抑制AC的侵袭。整体而言 本应用的目的是(目的1)阐明ANT-1.1如何促进基质金属蛋白酶后的适应性侵袭 在线虫中的丢失，以及(目标2)确定在4-D中是否同时丢失了基质金属蛋白酶和ANT活性 胶质母细胞瘤(GBM)器官型脑片模型阻断侵袭性活动。我们的中心假设是 了解Ant-1.1如何在线粒体中发挥作用以进行适应性侵袭将有助于靶向 ANTS和MMPs在临床相关脑片模型中的GBM侵袭。要了解如何 Ant-1.1促进适应性侵袭，将使用基因分析、荧光报告、代谢 生物传感器、细胞特定代谢分析和定量活细胞成像。然后我们将使用 定量共聚焦成像直接检测ANT和基质金属蛋白酶联合治疗基底膜的疗效 细胞入侵。我们希望确定ANT-1.1如何在线粒体内发挥作用，以促进适应 侵袭(可能通过多种功能)，并开发联合治疗方法 有效地阻断了GBM的入侵。这些贡献将是重要的，因为它们将揭示出 细胞在缺少MMPs的情况下自适应入侵，并建立一条可用于识别和 描述导致更有效的癌症治疗的协同侵袭靶点。

Tumor cell invasion through extracellular matrix (ECM) facilitates localized and distant cancer spread, which is the most lethal aspect of cancer. The ability of cells to switch between distinct invasive modes, termed plasticity or adaptation, when faced with varying physical or chemical challenges underlies the inability to develop anti-invasive therapies. Identifying targetable adaptive responses to halt invasion has been hindered by the lack of experimental models to identify, characterize, and test the loss of key molecules that facilitate plasticity. To address this critical need we have focused on matrix metalloproteinases (MMPs), which have been targeted in extensive clinical trials because of their strong association with cancer and role in degrading ECM. Anti-MMP therapies, however, have been ineffective, likely because of invasive plasticity. To identify and understand how invasive cells adapt to MMP loss, we are using the in vivo model of anchor cell (AC) invasion in C. elegans. We found that the genetic removal of MMPs results in an adaptive invasion response where instead of ECM degradation, the AC increases F-actin polymerization to forcefully penetrate ECM. Using MMP-null animals, we performed the first synergistic invasion screen to pinpoint genes that promote adaptive AC invasion and identified the mitochondrial ATP/ADP translocase, ant-1.1, as the strongest candidate. ANTs have multiple mitochondrial functions (ATP/ADP exchange, mitophagy, mitochondrial dynamics) and the ANT-1.1 protein is highly enriched in AC mitochondria that polarize to the site of ECM invasion. ANT-1.1 knockdown in MMP-null animals prevents adaptive F-actin formation and inhibits AC invasion. The overall objective of this application is to (Aim 1) elucidate how ant-1.1 promotes adaptive invasion after MMP loss in C. elegans, and (Aim 2) determine if the concurrent loss of MMP and ANT activity in a 4-D organotypic brain slice model of glioblastoma (GBM) blocks invasive activity. Our central hypothesis is that understanding how ANT-1.1 functions in mitochondrial for adaptive invasion will facilitate targeting ANTs along with MMPs in a clinically relevant brain slice model of GBM invasion. To understand how ANT-1.1 promotes adaptive invasion, will use genetic analysis, fluorescence reporters, metabolic biosensors, cell-specific metabolic analysis, and quantitative live-cell imaging. We will then use quantitative confocal imaging to directly test the efficacy of combined ANT and MMP therapies on GBM cell invasion. We expect to establish how ANT-1.1 functions within mitochondria to facilitate adaptive invasion (possibly through multiple functions) and to develop combined therapeutic approaches to effectively block GBM invasion. These contributions will be significant as they will reveal how invasive cells adaptively invade in the absence of MMPs and establish a pipeline that can be used to identify and characterize synergistic invasive targets resulting in more effective cancer therapies.