Cten Is Targeted by Kras Signalling to Regulate Cell Motility in the Colon and Pancreas

CTEN/TNS4 is an oncogene in colorectal cancer (CRC) which enhances cell motility although the mechanism of Cten regulation is unknown. We found an association between high Cten expression and KRAS/BRAF mutation in a series of CRC cell lines (p = 0.03) and hypothesised that Kras may regulate Cten. To test this, Kras was knocked-down (using small interfering (si)RNA) in CRC cell lines SW620 and DLD1 (high Cten expressors and mutant for KRAS). In each cell line, Kras knockdown was mirrored by down-regulation of Cten Since Kras signals through Braf, we tested the effect of Kras knockdown in CRC cell line Colo205 (which shows high Cten expression and is mutant for BRAF but wild type for KRAS). Cten levels were unaffected by Kras knockdown whilst Braf knockdown resulted in reduced Cten expression suggesting that Kras signals via Braf to regulate Cten. Quantification of Cten mRNA and protein analysis following proteasome inhibition suggested that regulation was of Cten transcription. Kras knockdown inhibited cell motility. To test whether this could be mediated through Cten, SW620 cells were co-transfected with Kras specific siRNAs and a Cten expression vector. Restoring Cten expression was able to restore cell motility despite Kras knockdown (transwell migration and wounding assay, p<0.001 for both). Since KRAS is mutated in many cancers, we investigated whether this relationship could be demonstrated in other tumour models. The experiments were repeated in the pancreatic cancer cell lines Colo357 & PSN-1(both high Cten expressors and mutant for KRAS). In both cell lines, Kras was shown to regulate Cten and forced expression of Cten was able to rescue loss of cell motility following Kras knockdown in PSN-1 (transwell migration assay, p<0.001). We conclude that, in the colon and pancreas, Cten is a downstream target of Kras and may be a mechanism through which Kras regulates of cell motility.

CTEN/TNS4 是结直肠癌（colorectal cancer, CRC）中的致癌基因，尽管Cten的调控机制尚未明确，但其可增强细胞运动能力。我们在一系列结直肠癌细胞系中发现，Cten高表达与KRAS/BRAF突变存在显著相关性（p=0.03），据此推测Kras可能对Cten存在调控作用。为验证该推测，我们在高表达Cten且携带KRAS突变的结直肠癌细胞系SW620与DLD1中，采用小干扰RNA（small interfering RNA, siRNA）敲低Kras，结果显示两种细胞系中的Cten表达均出现下调。由于Kras信号通路依赖Braf介导，我们进一步在Colo205结直肠癌细胞系（该细胞系高表达Cten，携带BRAF突变但KRAS为野生型）中测试了Kras敲低的效应，结果发现Kras敲低对Cten水平无影响，而Braf敲低可降低Cten的表达，这提示Kras通过Braf信号通路调控Cten。对Cten的mRNA定量分析及蛋白酶体抑制后的蛋白检测结果表明，该调控作用发生在Cten的转录层面。Kras敲低可抑制细胞运动能力。为验证该效应是否由Cten介导，我们将Kras特异性siRNA与Cten过表达载体共转染至SW620细胞中，结果显示，尽管Kras被敲低，但恢复Cten的表达可挽救细胞运动能力（Transwell迁移实验与划痕愈合实验均显示p<0.001）。鉴于KRAS在多种癌症中存在突变，我们进一步探究该调控关系是否可在其他肿瘤模型中得到验证。我们在胰腺癌细胞系Colo357与PSN-1（两种细胞系均高表达Cten且携带KRAS突变）中重复了上述实验，结果显示两种细胞系中Kras均可调控Cten的表达；在PSN-1细胞中，恢复Cten的表达可挽救Kras敲低后导致的细胞运动能力下降（Transwell迁移实验p<0.001）。综上，我们认为在结直肠癌与胰腺癌中，Cten是Kras的下游靶基因，且这可能是Kras调控细胞运动能力的潜在分子机制。