A phospho-proteomic study of cetuximab resistance in KRAS/NRAS/BRAFV600 wild-type colorectal cancer,Cellular Oncology

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A phospho-proteomic study of cetuximab resistance in KRAS/NRAS/BRAFV600 wild-type colorectal cancer
Cellular Oncology ( IF 6.6 ) Pub Date : 2021-08-30 , DOI: 10.1007/s13402-021-00628-7
Alexandros Georgiou _{1,

2} , Adam Stewart ₁ , Lisa Pickard ₁ , Steven R. Whittaker ₁ , Udai Banerji _{1,

3} , Georgios Vlachogiannis ₄ , Nicola Valeri _{2,

4} , David Cunningham ₂

Affiliation

Purpose

We hypothesised that plasticity in signal transduction may be a mechanism of drug resistance and tested this hypothesis in the setting of cetuximab resistance in patients with KRAS/NRAS/BRAF^V600 wild-type colorectal cancer (CRC).

Methods

A multiplex antibody-based platform was used to study simultaneous changes in signal transduction of 55 phospho-proteins in 12 KRAS/NRAS/BRAF^V600 wild-type CRC cell lines (6 cetuximab sensitive versus 6 cetuximab resistant) following 1 and 4 h in vitro cetuximab exposure. We validated our results in CRC patient samples (n = 4) using ex vivo exposure to cetuximab in KRAS/NRAS/BRAF^V600 cells that were immunomagnetically separated from the serous effusions of patients with known cetuximab resistance.

Results

Differences in levels of phospho-proteins in cetuximab sensitive and resistant cell lines included reductions in phospho-RPS6 and phospho-PRAS40 in cetuximab sensitive, but not cetuximab resistant cell lines at 1 and 4 h, respectively. In addition, phospho-AKT levels were found to be elevated in 3/4 patient samples following ex vivo incubation with cetuximab for 1 h. We further explored these findings by studying the effects of combinations of cetuximab and two PI3K pathway inhibitors in 3 cetuximab resistant cell lines. The addition of PI3K pathway inhibitors to cetuximab led to a significantly higher reduction in colony formation capacity compared to cetuximab alone.

Conclusion

Our findings suggest activation of the PI3K pathway as a mechanism of cetuximab resistance in KRAS/NRAS/BRAF^V600 wild-type CRC.

中文翻译：

KRAS/NRAS/BRAFV600野生型结直肠癌西妥昔单抗耐药的磷酸化蛋白质组学研究

目的

我们假设信号转导的可塑性可能是耐药性的一种机制，并在KRAS/NRAS/BRAF ^V600野生型结直肠癌 (CRC)患者西妥昔单抗耐药的情况下测试了这一假设。

方法

基于多重抗体的平台用于研究 12 种KRAS/NRAS/BRAF ^V600野生型 CRC 细胞系（6 种西妥昔单抗敏感对 6 种西妥昔单抗耐药）中 55 种磷蛋白的信号转导在体外 1 小时和 4 小时后的同步变化西妥昔单抗暴露。我们在 CRC 患者样本（n = 4）中验证了我们的结果，在KRAS/NRAS/BRAF ^V600细胞中体外暴露于西妥昔单抗，这些细胞与已知西妥昔单抗耐药的患者的浆液性渗出液进行了免疫磁性分离。

结果

西妥昔单抗敏感细胞系和耐药细胞系中磷蛋白水平的差异包括分别在 1 小时和 4 小时时西妥昔单抗敏感细胞系中磷酸化 RPS6 和磷酸化 PRAS40 的减少，但西妥昔单抗耐药细胞系中的磷酸化 RPS6 和磷酸化 PRAS40 不存在。此外，在与西妥昔单抗离体孵育 1 小时后，发现 3/4 患者样本中的磷酸化 AKT 水平升高。我们通过研究西妥昔单抗和两种 PI3K 通路抑制剂组合在 3 个西妥昔单抗耐药细胞系中的作用，进一步探索了这些发现。与单独使用西妥昔单抗相比，向西妥昔单抗添加 PI3K 通路抑制剂导致集落形成能力显着降低。