Department of Clinical Chemistry, Medicum, Helsinki University Hospital, University of Helsinki, Finland.
Minerva Foundation Institute for Medical Research, Helsinki, Finland.
Mol Oncol. 2018 Feb;12(2):224-238. doi: 10.1002/1878-0261.12160. Epub 2017 Dec 27.
The mitogen-activated protein kinase (MAPK) pathway plays a central role in colorectal cancers (CRC). In particular, BRAF V600E-mutant tumors, which represent around 10% of CRCs, are refractory to current therapies. Overexpression and secretion of serine peptidase inhibitor Kazal type 1 (SPINK1) are observed in around 50% of CRCs, and its serum level can be used as a biomarker for poor prognosis. Utilizing a recently developed extendable blocking probe assay, we analyzed the BRAF mutation status in a CRC patient cohort (N = 571) using tissue-derived RNA as the starting material. From the same RNA samples, we measured the relative SPINK1 expression levels using a quantitative real-time PCR method. Expression of mutant BRAF V600E correlated with poor prognosis, as did low expression of SPINK1 mRNA. Further, BRAF V600E correlated negatively with SPINK1 levels. In order to investigate the effect of MAPK pathway-targeted therapies on SPINK1 secretion, we conducted in vitro studies using both wild-type and V600E CRC cell lines. BRAF inhibitor vemurafenib, and subsequent MAPK pathway inhibitors trametinib and SCH772984, significantly increased SPINK1 secretion in V600E CRC cell lines Colo205 and HT-29 with a concomitant decrease in trypsin-1 and -2 secretion. Notably, no SPINK1 increase or trypsin-1 decrease was observed in BRAF wild-type CRC cell line Caco-2 in response to MAPK pathway inhibitors. In further mechanistic studies, we observed that only trametinib was able to diminish completely both MEK and ERK phosphorylation in the V600E CRC cells. Furthermore, the key regulator of integrated stress response, activating transcription factor 4 (ATF-4), was downregulated both at mRNA and at protein level in response to trametinib treatment. In conclusion, these data suggest that sustained inhibition of not only MAPK pathway activation, but also ATF-4 and trypsin, might be beneficial in the therapy of BRAF V600E-mutant CRC and that SPINK1 levels may serve as an indicator of therapy response.
丝氨酸蛋白酶抑制剂 Kazal 型 1（SPINK1）的过表达和分泌在大约 50%的 CRC 中观察到，其血清水平可作为预后不良的生物标志物。利用最近开发的可扩展阻断探针检测，我们分析了组织衍生 RNA 作为起始材料的 571 例 CRC 患者队列中的 BRAF 突变状态。从相同的 RNA 样本中，我们使用定量实时 PCR 方法测量了相对的 SPINK1 表达水平。突变型 BRAF V600E 的表达与预后不良相关，SPINK1 mRNA 的低表达也是如此。此外，BRAF V600E 与 SPINK1 水平呈负相关。为了研究 MAPK 通路靶向治疗对 SPINK1 分泌的影响，我们使用野生型和 V600E CRC 细胞系进行了体外研究。BRAF 抑制剂 vemurafenib，以及随后的 MAPK 通路抑制剂 trametinib 和 SCH772984，显著增加了 Colo205 和 HT-29 这两种 V600E CRC 细胞系中 SPINK1 的分泌，同时降低了胰蛋白酶-1 和 -2 的分泌。值得注意的是，MAPK 通路抑制剂对 BRAF 野生型 CRC 细胞系 Caco-2 没有观察到 SPINK1 增加或胰蛋白酶-1 减少。在进一步的机制研究中，我们观察到只有 trametinib 能够完全减少 V600E CRC 细胞中 MEK 和 ERK 的磷酸化。此外，应激反应整合关键调节因子激活转录因子 4（ATF-4），在 trametinib 治疗后，其 mRNA 和蛋白水平均下调。总之，这些数据表明，不仅持续抑制 MAPK 通路的激活，而且持续抑制 ATF-4 和胰蛋白酶，可能有益于 BRAF V600E 突变型 CRC 的治疗，并且 SPINK1 水平可能作为治疗反应的指标。
