The PI3K/AKT/c‐MYC Axis Promotes the Acquisition of Cancer Stem‐Like Features in Esophageal Squamous Cell Carcinoma

The importance of intratumoral heterogeneity has been highlighted by the identification and characterization of cancer stem cells (CSCs). Based on the differential responsiveness to a Sox2 reporter, SRR2, we had found a novel dichotomy in esophageal squamous cell carcinoma (ESCC) cells, with reporter‐responsive (RR) cells showing more CSC‐like features than reporter‐unresponsive (RU) cells. Specifically, RR cells exhibited significantly higher tumorsphere formation capacity, proportions of CD44High cells, chemoresistance to cisplatin, and tumorigenic potential in vivo. H2O2, a potent inducer of oxidative stress and reactive oxygen species, was found to induce a conversion from RU to RR cells; importantly, converted RR cells acquired CSC‐like features. The PI3K/AKT/c‐MYC signalling axis is important in this context, since pharmacologic blockade of PI3K‐AKT or siRNA knockdown of c‐MYC effectively inhibited the RR phenotype and its associated CSC‐like features, as well as the H2O2‐induced RU/RR conversion. In a cohort of 188 ESCC patient samples, we found a significant correlation between strong c‐MYC expression and a short overall survival (p = .009). In conclusion, we have described a novel intratumoral heterogeneity in ESCC. The identification of the PI3K/AKT/c‐MYC axis as a driver of CSC‐like features carries therapeutic implications. Stem Cells 2016;34:2040–2051 Oxidative stress induced by H2O2 promotes RU/RR conversion and the acquisition of CSC‐like properties. (A) Reactive oxygen species (ROS) induced by H2O2 (500 μM for 24 hours) was measured by flow cytometry. (B) H2O2‐induced RU/RR conversion in KYSE150‐RU cells, as indicated by the increase of GFP‐positive cells and luciferase activity. Representative flow cytometry data at Day 4 was shown in the left panel. Note that cells grown at a 100% confluence were used in this assay. (C) Flow cytometry was performed to measure GFP expression and ROS levels to determine the effect of N‐Acetyl‐Cysteine (NAC), an antioxidant agent, on oxidative stress‐induced RU/RR conversion. Median ROS values are indicated by bidirectional arrows. (D) Tumorsphere formation assay was used to evaluate H2O2‐induced CSC‐like properties. The RU cells used in this assay were treated with different concentrations of H2O2 for four cycles, each cycle includes a 24 hours H2O2 treatment and a recovery phase for 3 days. Images were taken at ×100 magnification. (E) RU cells were treated with 300 μM H2O2 for 48 hours or left untreated, a