Stress Induced Overexpression of Stanniocalcin 2 (STC2) Plays Key Role in Radiation Resistance and Metastasis of Nasopharyngeal Carcinomas

Abstract

Stanniocalcin2 (STC2) expression is up-regulated by multiple stress conditions including hypoxia, nutrient depletion and radiation, and overexpression of STC2 correlates with tumor progression and poor prognosis. Particularly, it has been demonstrated that overexpression of STC2 in nasopharyngeal carcinomas (NPC) positively correlates with radiation resistance and metastasis, two major clinical obstacles to further improvement of NPC management. However, a contributing role of STC2 overexpression in radiation resistance and metastasis of NPC has not been established. We hypothesize that stress-induced overexpression of STC2 plays a key role in radiation resistance and metastasis of NPC. This study has two objectives: 1) to define the importance of STC2 overexpression in radiation resistance in NPC; and 2) to determine if targeting STC2 can be explored as a strategy to sensitize NPC to radiation. Radiation resistant CNE2 cell line was used as a model system. CRISPR/Cas9-based genome editing was used to create STC2 knockout lines (CNE2-STC2-KO). The genomic status of KO lines was confirmed by T7E1 assays. The STC2 mRNA and protein levels of parental and KO cell lines were examined using quantitative reverse transcription polymerase chain reaction (PCR) and Western blotting analysis, respectively. NPC cells were radiated with an ELEKTA accelerator (6MVx, Sweden) at distance of 100 cm at 285cGy/min. Cell survival was assayed by flow cytometric analysis, TrypanBlue staining and clonogenic analysis. Cell cycle distribution was analyzed by flow cytometry, and invasion and migration abilities were gauged with Transwell/MatriGel-based assays. SPSS 17.0 was used for statistical analysis, and p<0.05 was set as significant. CRISP/Cas9 construct was made and transfected into CNE2 cells to create CNE2-KO lines. The KO lines were verified at genomic, mRNA and protein levels. Compared with the parental radiation resistant CNE2 line, the STC2-KO lines show increased radiation sensitivity under either normoxic or hypoxic stress. In response to radiation, STC2-KO cells manifest decreased invasion and migration ability. Cell cycle analyses reveal that loss of STC2 significantly increases NPC cells at G2/M phase post radiation, consistent with an enhanced cytotoxicity. These data demonstrate that elevated expression of STC2 under various stress conditions promotes NPC resistance to radiation, facilitates cell survival and metastasis. Targeting STC2 function may provide a novel strategy to effectively overcome radiation resistance and to prevent metastasis of NPC.