PTEN expression is lost in many cancers, and even small changes in PTEN activity affect susceptibility and prognosis in a range of highly aggressive malignancies, such as melanoma and triple-negative breast cancer (TNBC). Loss of PTEN expression occurs via multiple mechanisms, including mutation, transcriptional repression and epigenetic silencing. Transcriptional repression of PTEN contributes to resistance to inhibitors used in the clinic, such as B-Raf inhibitors in BRAF mutant melanoma. We aimed to activate PTEN expression using the CRISPR system, specifically dead (d) Cas9 fused to the transactivator VP64-p65-Rta (VPR). dCas9-VPR was directed to the PTEN proximal promoter by single-guide RNAs (sgRNAs), in cancer cells that exhibited low levels of PTEN expression. The dCas9-VPR system increased PTEN expression in melanoma and TNBC cell lines, without transcriptional regulation at predicted off-target sgRNA binding sites. PTEN activation significantly repressed downstream oncogenic pathways, including AKT, mTOR, and MAPK signaling. BRAF V600E mutant melanoma cells transduced with dCas9-VPR displayed reduced migration, as well as diminished colony formation in the presence of B-Raf inhibitors, PI3K/mTOR inhibitors, and with combined PI3K/mTOR and B-Raf inhibition. CRISPR-mediated targeted activation of PTEN may provide an alternative therapeutic approach for highly aggressive cancers that are refractory to current treatments.

在许多癌症中，PTEN表达丧失，甚至PTEN活性的微小变化也会影响一系列高度侵袭性恶性肿瘤（例如黑素瘤和三阴性乳腺癌（TNBC））的易感性和预后。PTEN表达的丧失是通过多种机制发生的，包括突变，转录抑制和表观遗传沉默。PTEN的转录抑制作用有助于抵抗临床中使用的抑制剂，例如BRAF突变型黑色素瘤中的B-Raf抑制剂。我们旨在使用CRISPR系统激活PTEN表达，特别是将死（d）Cas9与反式激活因子VP64-p65-Rta（VPR）融合。dCas9-VPR被定向到PTEN在低PTEN表达水平的癌细胞中，通过单向导RNA（sgRNA）的近端启动子。dCas9-VPR系统增加了黑色素瘤和TNBC细胞系中PTEN的表达，而没有预测的脱靶sgRNA结合位点的转录调控。PTEN激活显着抑制下游致癌途径，包括AKT，mTOR和MAPK信号传导。dCas9-VPR转导的BRAF V600E突变型黑素瘤细胞显示出减少的迁移，并且在存在B-Raf抑制剂，PI3K / mTOR抑制剂以及PI3K / mTOR和B-Raf联合抑制的情况下，菌落形成减少。CRISPR介导的PTEN靶向激活 可以为当前治疗难以治愈的高度侵袭性癌症提供替代治疗方法。