Mechanistic Study of Irradiated Dying NPC Cells Promoting the Metastatic Ability of Surviving NPC Cells After Radiotherapy

Abstract

<h3>Purpose/Objective(s)</h3> Distant metastasis remained the major cause of mortality in nasopharyngeal carcinoma (NPC) receiving radiotherapy despite favorable locoregional control in the past decades. Tumor microenvironment plays a critical role in tumor progression by secreting factors that regulate cancer cell metastasis. However, the underlying mechanisms remain elusive. The aim of this study was to elucidate the role and mechanism of necrotic or apoptotic NPC cells killed by radiotherapy in distant metastasis. We hypothesize that necrotic or apoptotic NPC cells killed by radiotherapy could release metastasis-stimulating signals to the microenvironment and promote the metastatic ability of the surviving cancer cells. <h3>Materials/Methods</h3> Based on the co-culture system, cell migration assay and cell invasion assay were used to analyze the effect of lethally irradiated NPC CNE1 cell line on migration and invasion abilities of radioresistant NPC cell line CNE1-IR. Subsequently, ITRAQ was used to screen the key factors in this process, and ELISA, qRT-PCR and Western blot were used to detect the expression of slit2 in lethally irradiated CNE1 cells. CNE1-IR cells were infected with Slit2 shRNA lentivirus (LV3-shSlit2) or LV3-NC. Cell migration and invasion assay as well as tumor metastasis experiment in vivo were used to examine the role of Slit2 in promoting metastasis of NPC cell lines. Lastly, Cell migration, invasion assay and Co-IP assay were used to explore which Robo receptors was involved in Slit2 mediated metastasis of NPC cell lines. <h3>Results</h3> The migration and invasion abilities of CNE1-IR cells were significantly promoted (<i>P</i> < 0.01) when CNE1-IR was co-cultured with lethally irradiated CNE1 cells. ITRAQ found that the expression of Slit2 was upregulated in the supernatant of lethally irradiated CNE1 cells. ELISA assay confirmed that Slit2 in the supernatant of lethally irradiated CNE1 cells was significantly higher than control cells. qRT-PCR analysis and Western blot analysis also showed that the expressions of Slit2 was significantly overexpressed in irradiated NPC cells compared with unirradiated control CNE1 cells. Knockdown of Slit2 significantly suppressed lung and liver metastasis in vivo. The expressions of both Robo1 and Robo2 could be detected in CNE1 and CNE1-IR. Subsequently, addition of rSlit2 could enhance cell migration ability. In contrast, Robo1-depleted cells showed decreased migration ability and did not respond to rSlit2. When rSlit2 was added to CNE1-IR/shRobo2 cells, the number of trans-membrance CNE-IR cells was increased by about 26% compared with CNE1-IR/NC cells. Co-IP assay with anti-Robo1 antibody indicated that Robo1 was immunoprecipitated with Slit2, indicating the interaction of Robo1 and Slit2. <h3>Conclusion</h3> Lethally irradiated NPC cells could promote migration and invasion of radioresistant NPC cell lines. Slit2, a secreted factor released from irradiated dying NPC cells could promote metastasis of living/resistant NPC cells via Robo1 receptor.