Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA.
Oncogene. 2011 Feb 3;30(5):561-74. doi: 10.1038/onc.2010.430. Epub 2010 Oct 18.
KRAS mutation is a predictive biomarker for resistance to cetuximab (Erbitux) in metastatic colorectal cancer (mCRC). This study sought to determine if KRAS mutant CRC lines could be sensitized to cetuximab using dasatinib (BMS-354825, Sprycel), a potent, orally bioavailable inhibitor of several tyrosine kinases, including the Src family kinases (SFKs). We analyzed 16 CRC lines for: (1) KRAS mutation status, (2) dependence on mutant KRAS signaling and (3) expression level of epidermal growth factor receptor (EGFR) and SFKs. From these analyses, we selected three KRAS mutant (LS180, LoVo and HCT116) cell lines and two KRAS wild-type cell lines (SW48 and CaCo2). In vitro, using poly-D-lysine/laminin plates, KRAS mutant cell lines were resistant to cetuximab, whereas KRAS wild-type lines showed sensitivity to cetuximab. Treatment with cetuximab and dasatinib showed a greater antiproliferative effect on KRAS mutant lines when compared with either agent alone in vitro and in vivo. To investigate potential mechanisms for this antiproliferative response in the combinatorial therapy, we performed Human Phospho-Kinase Antibody Array analysis, measuring the relative phosphorylation levels of 39 intracellular proteins in untreated, cetuximab, dasatinib or the combinatorial treatment in the KRAS mutant lines LS180, LoVo and HCT116 cells. The results of this experiment showed a decrease in a broad spectrum of kinases centered on the β-catenin pathway, the mitogen-activated protein kinase (MAPK) pathway, AKT/mammalian target of rapamycin (mTOR) pathway and the family of signal transducers and activators of transcription (STATs) when compared with the untreated control or monotherapy treatments. Next, we analyzed tumor growth with cetuximab, dasatinib or their combination in vivo. KRAS mutant xenografts showed resistance to cetuximab therapy, whereas KRAS wild type demonstrated an antitumor response when treated with cetuximab. KRAS mutant tumors exhibited minimal response to dasatinib monotherapy. However, as in vitro, KRAS mutant lines exhibited a response to the combination of cetuximab and dasatinib. Combinatorial treatment of KRAS mutant xenografts resulted in decreased cell proliferation, as measured by Ki67, and higher rates of apoptosis, as measured by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). The data presented in this study indicate that dasatinib can sensitize KRAS mutant CRC tumors to cetuximab and may do so by altering the activity of several key signaling pathways. Furthermore, these results suggest that signaling via EGFR and SFKs may be necessary for cell proliferation and survival of KRAS mutant CRC tumors. These data strengthen the rationale for clinical trials combining cetuximab and dasatinib in the KRAS mutant CRC genetic setting.
KRAS 突变是结直肠癌（mCRC）对西妥昔单抗（Erbitux）耐药的预测性生物标志物。本研究旨在探讨使用达沙替尼（BMS-354825，Sprycel），一种有效的、口服生物利用的多种酪氨酸激酶抑制剂，包括Src 家族激酶（SFKs），是否可以使 KRAS 突变的 CRC 细胞系对西妥昔单抗敏感。我们分析了 16 种 CRC 细胞系：（1）KRAS 突变状态，（2）对突变型 KRAS 信号的依赖性，（3）表皮生长因子受体（EGFR）和 SFKs 的表达水平。从这些分析中，我们选择了三个 KRAS 突变（LS180、LoVo 和 HCT116）细胞系和两个 KRAS 野生型细胞系（SW48 和 CaCo2）。在体外，使用多聚-D-赖氨酸/层粘连蛋白板，KRAS 突变细胞系对西妥昔单抗具有耐药性，而 KRAS 野生型细胞系对西妥昔单抗敏感。与单独使用任何一种药物相比，西妥昔单抗和达沙替尼联合治疗在体外和体内对 KRAS 突变细胞系具有更强的抗增殖作用。为了研究组合治疗中这种增殖抑制反应的潜在机制，我们对 LS180、LoVo 和 HCT116 细胞系中未处理、西妥昔单抗、达沙替尼或联合治疗的 39 种细胞内蛋白的相对磷酸化水平进行了人磷酸化激酶抗体阵列分析。该实验的结果表明，与未处理对照或单药治疗相比，以 β-连环蛋白通路、丝裂原活化蛋白激酶（MAPK）通路、AKT/哺乳动物雷帕霉素靶蛋白（mTOR）通路和信号转导和转录激活因子（STATs）家族为中心的广泛的激酶活性降低。接下来，我们分析了体内西妥昔单抗、达沙替尼或它们联合应用对肿瘤生长的影响。KRAS 突变的异种移植瘤对西妥昔单抗治疗具有耐药性，而 KRAS 野生型对西妥昔单抗治疗表现出抗肿瘤反应。KRAS 突变肿瘤对达沙替尼单药治疗反应极小。然而，与体外结果一样，KRAS 突变细胞系对西妥昔单抗和达沙替尼的联合治疗有反应。KRAS 突变异种移植瘤的联合治疗导致 Ki67 测量的细胞增殖减少，以及 TUNEL（末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记）测量的细胞凋亡增加。本研究中的数据表明，达沙替尼可以使 KRAS 突变的 CRC 肿瘤对西妥昔单抗敏感，其作用机制可能是改变几种关键信号通路的活性。此外，这些结果表明，EGFR 和 SFKs 信号可能是 KRAS 突变 CRC 肿瘤增殖和存活所必需的。这些数据为 KRAS 突变 CRC 遗传背景下联合使用西妥昔单抗和达沙替尼的临床试验提供了更有力的依据。
