Excessive MET signaling causes acquired resistance and addiction to MET inhibitors in the MKN45 gastric cancer cell line
Excessive MET signaling causes acquired resistance and addiction to MET inhibitors in the MKN45 gastric cancer cell line
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DOI：
10.1007/s10637-013-9959-2
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发表时间：
2013-10-01
影响因子：
3.4
通讯作者：
Minami, Hironobu
中科院分区：
文献类型：
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作者：
Funakoshi, Yohei;Mukohara, Toru;Minami, Hironobu
MET酪氨酸激酶抑制剂（MET-TKI）的临床疗效受到获得性耐药的阻碍，这对药物发现构成了障碍。为了阐明MET-TKI获得性耐药的机制，我们通过将MET扩增的胃癌细胞系MKN 45连续暴露于MET-TKI、PHA 665752（MKN 45-PR）和GSK 1363089（MKN 45-GR）来建立耐药模型。与MKN 45细胞相比，MKN 45-PR和MKN 45-GR中MET的基线表达和磷酸化水平升高，与亲本细胞相比，需要更高浓度的MET-TKI来抑制MET磷酸化。在耐药细胞中观察到先前与MET-TKI耐药相关的MET改变，包括与MKN 45细胞相比在两种耐药细胞系中观察到的MET拷贝数升高，以及在MKN 45-PR细胞中检测到的Y1230 H突变。值得注意的是，在不存在MET-TKI的情况下，抗性品系的生长较低，表明对抑制剂“成瘾”。虽然MKN 45-PR细胞在不存在PHA 665752的情况下表现出更高的S期分数，但溴脱氧尿苷（BrdU）摄取是相同的。与MKN 45细胞相比，MKN 45-PR中ATR、Chk 1和p53和p21（waf 1/Cip 1）表达的基线磷酸化水平更高，并且水平降低至PHA 665752处理后未处理的MKN 45细胞中观察到的水平。此外，靶向敲低MET增强了MKN 45-PR细胞的生长。这些发现表明，MET的改变导致获得性MET-TKI耐药，可能导致过量的MET信号传导，随后的复制应激和DNA损伤反应，以及在不存在MET-TKI的情况下的S期内阻滞。因此，部分MET抑制对于耐药细胞增殖是必要的，我们将这种现象称为MET-TKI“成瘾”。
The clinical efficacy of MET tyrosine kinase inhibitors (MET-TKIs) is hindered by the emergence of acquired resistance, presenting an obstacle to drug discovery. To clarify the mechanisms underlying acquired resistance to MET-TKIs, we established resistance models by continuous exposure of the MET-amplified gastric cancer cell line MKN45 to MET-TKIs, PHA665752 (MKN45-PR) and GSK1363089 (MKN45-GR). Baseline expression and phosphorylation of MET were elevated in MKN45-PR and MKN45-GR compared to MKN45 cells, and higher concentrations of MET-TKIs were required to inhibit MET phosphorylation compared to parental cells. Alterations in MET previously associated with resistance to MET-TKIs were observed in resistant cells, including elevated MET copy number, observed in both resistant lines compared to MKN45 cells, and the Y1230H mutation, detected in MKN45-PR cells. Notably, the growth of resistant lines was lower in the absence of MET-TKIs, suggesting "addiction" to inhibitors. While MKN45-PR cells exhibited a higher S-phase fraction in the absence of PHA665752, bromodeoxyuridine (BrdU) uptake was identical. Baseline phosphorylation of ATR, Chk1 and p53 and p21(waf1/Cip1) expression was higher in MKN45-PR compared to MKN45 cells, and levels were reduced to those observed in untreated MKN45 cells following PHA665752 treatment. Furthermore, targeted knockdown of MET enhanced the growth of MKN45-PR cells. These findings suggest that alterations in MET leading to acquired MET-TKI resistance, may cause excessive MET signaling, subsequent replication stress and DNA damage response, and intra-S-phase arrest in the absence of MET-TKIs. Thus, partial MET inhibition is necessary for resistant cells to proliferate, a phenomenon we refer to as MET-TKI "addiction".