Abstract

As a key process within the tissue microenvironment, integrin signaling can influence cell functional responses to growth factor stimuli. We show here that clustering of integrin α5ß1 at the plasma membrane of colorectal cancer-derived epithelial cells modulates their ability to respond to stimulation by receptor tyrosine kinase (RTK)-activating growth factors EGF, NRG and HGF, through GSK3-mediated suppression of Akt pathway. We observed that integrin α5ß1 is lost from the membrane of poorly organized human colorectal tumors and that treatment with the integrin-clustering antibody P4G11 is sufficient to induce polarity in a mouse tumor xenograft model. While adding RTK growth factors (EGF, NRG and HGF) to polarized colorectal cancer cells induced invasion and loss of monolayer formation in 2D and 3D, this pathological behavior could be blocked by P4G11. Phosphorylation of ErbB family members as well as MET following EGF, NRG and HGF treatment was diminished in cells pretreated with P4G11. Focusing on EGFR, we found that blockade of integrin α5ß1 increased EGFR phosphorylation. Since activity of multiple downstream kinase pathways were altered by these various treatments, we employed computational machine learning techniques to ascertain the most important effects. Partial least-squares discriminant analysis identified GSK3 as a major regulator of EGFR pathway activities influenced by integrin α5ß1. Moreover, we used partial correlation analysis to examine signaling pathway crosstalk downstream of EGF stimulation and found that integrin α5ß1 acts as a negative regulator of the AKT signaling cascade downstream of EGFR, with GSK3 acting as a key mediator. We experimentally validated these computational inferences by confirming that blockade of GSK3 activity is sufficient to induce loss of polarity and increase of oncogenic signaling in the colonic epithelial cells.

中文翻译：

---

细胞表面整合素 α5ß1 聚集通过糖原合酶激酶 3 负调节结直肠癌细胞中的受体酪氨酸激酶信号传导

摘要

作为组织微环境中的一个关键过程，整合素信号可以影响细胞对生长因子刺激的功能反应。我们在这里展示了整合素 α5ß1 在结直肠癌衍生上皮细胞的质膜上的聚集，通过 GSK3 介导的 Akt 抑制来调节它们对受体酪氨酸激酶 (RTK) 激活生长因子 EGF、NRG 和 HGF 刺激的反应能力途径。我们观察到整合素 α5ß1 从组织不良的人类结肠直肠肿瘤的膜上丢失，并且用整合素聚集抗体 P4G11 治疗足以在小鼠肿瘤异种移植模型中诱导极性。在向极化的结直肠癌细胞中添加 RTK 生长因子（EGF、NRG 和 HGF）时，会在 2D 和 3D 中诱导侵袭和单层形成的丧失，这种病理行为可以被 P4G11 阻断。在用 P4G11 预处理的细胞中，EGF、NRG 和 HGF 处理后 ErbB 家族成员以及 MET 的磷酸化降低。专注于 EGFR，我们发现整合素 α5ß1 的阻断增加了 EGFR 磷酸化。由于这些不同的处理改变了多个下游激酶途径的活性，我们采用计算机机器学习技术来确定最重要的影响。偏最小二乘判别分析确定 GSK3 是受整合素 α5ß1 影响的 EGFR 通路活性的主要调节因子。此外，我们使用偏相关分析来检查 EGF 刺激下游的信号通路串扰，发现整合素 α5ß1 是 EGFR 下游 AKT 信号级联的负调节剂，GSK3 作为关键调解人。我们通过确认 GSK3 活性的阻断足以诱导结肠上皮细胞中的极性丧失和致癌信号传导的增加，通过实验验证了这些计算推论。