Glover Sarah, Delaney Melissa, Dematte Cecile, Kornberg Lori, Frasco Melissa, Tran-Son-Tay Roger, Benya Richard V
Department of Medicine, University of Illinois at Chicago and Chicago Veterans Administration Medical Center (West Side Division), Chicago, Illinois 60612, USA.
J Cell Physiol. 2004 Apr;199(1):77-88. doi: 10.1002/jcp.10456.
We have proposed that gastrin-releasing peptide (GRP) and its receptor (GRP-R) are morphogens that when aberrantly re-expressed in colon cancer promote tumor cell differentiation and retard metastasis. Because circumstantial evidence suggested that these properties were mediated via focal adhesion kinase (FAK), the purpose of this study was to elucidate the role of GRP-induced activation of this enzyme on properties fundamental to metastasis including cell attachment, motility, and deformability. To do this, we studied 293 cells, a non-malignant epithelial cell line that we show expresses GRP and GRPR. To dissect out the role of FAK, 293 cells were modified to inducibly express the dominant negative enzyme FAK-related non-kinase (FRNK) under control of a Tet-On (i.e., doxycycline-sensitive) promoter. Under serum-free conditions, GRP acting in an autocrine manner caused FAK to be phosphorylated at Y397; and this could be completely inhibited either by incubating with the specific GRP-R antagonist D-Phe(6)(bombesin) methyl ester, or by upregulating FRNK using doxycycline. To measure cell attachment, we designed a cone-plate viscometer that recorded the shear stress required to detach cells from their underlying matrix. To assess motility, confluent cells were wounded and behavior assessed by time-lapse photography. To measure deformability, we recorded the ability of cells to be completely drawn into a micropipette <50% the size of the non-deformed cell. Control 293 cells adhered more avidly to their underlying matrix, rapidly remodeled wounded tissues without any increase in overall proliferation, and were less distensible than cells treated with antagonist or doxycycline. Thus, these findings suggest that expression of GRP/GRPR in cancer inhibits metastasis by enhancing cell attachment to the matrix, regulating motility in the context of remodeling, and decreasing deformability.
我们提出胃泌素释放肽（GRP）及其受体（GRP-R）是形态发生素，当它们在结肠癌中异常重新表达时，会促进肿瘤细胞分化并延缓转移。由于间接证据表明这些特性是通过粘着斑激酶（FAK）介导的，因此本研究的目的是阐明GRP诱导的该酶激活对转移基本特性（包括细胞附着、运动性和可变形性）的作用。为此，我们研究了293细胞，这是一种非恶性上皮细胞系，我们发现它表达GRP和GRPR。为了剖析FAK的作用，对293细胞进行改造，使其在Tet-On（即强力霉素敏感）启动子的控制下可诱导表达显性负性酶FAK相关非激酶（FRNK）。在无血清条件下，以自分泌方式作用的GRP使FAK在Y397处磷酸化；这可以通过与特异性GRP-R拮抗剂D-苯丙氨酸（6）（蛙皮素）甲酯孵育，或通过使用强力霉素上调FRNK来完全抑制。为了测量细胞附着，我们设计了一种锥板粘度计，记录将细胞从其下层基质上分离所需的剪切应力。为了评估运动性，对汇合的细胞进行划痕，并用延时摄影评估其行为。为了测量可变形性，我们记录了细胞被完全吸入尺寸小于未变形细胞50%的微量移液器的能力。对照293细胞更紧密地附着于其下层基质，能迅速重塑受损组织且总体增殖无任何增加，并且比用拮抗剂或强力霉素处理的细胞更不易伸展。因此，这些发现表明癌症中GRP/GRPR的表达通过增强细胞与基质的附着、在重塑过程中调节运动性以及降低可变形性来抑制转移。
