Background: Radiation-induced cancer (RIC) is a late complication in patients who have been treated for nasopharyngeal carcinoma (NPC). The comparison of index anatomic location, index histological type, and survival of RIC in patients with NPC after different radiotherapy modalities (intensity-modulated radiotherapy [IMRT], 3-dimensional conformal radiotherapy [3D-CRT], and conventional 2D radiotherapy) is currently unavailable. Methodology: A total of 38,565 patients with NPC who received curative-intent radiotherapy at Sun Yat-sen University Cancer Center between January 1986 and December 2017 were reviewed. A total of 141 patients who developed RIC and fulfilled the study criteria were included. Categorical variables were compared by the chi(2) test or Fisher's exact test. Kaplan-Meier curves were used to evaluate overall survival. Cox proportional hazards models were used to examine the independent significance of RIC treatment. Results: Among IMRT, 3D-CRT, and conventional 2D radiotherapy, the incidence of mandible RIC was higher in patients who received 3D-CRT (0.07%) than in those who received IMRT (0%). The proportion of mandible RICs was higher in patients who received 3D-CRT (16.667%) than in those who received IMRT (0%) and conventional 2D radiotherapy (3.529%). Regarding the histological type, the incidence of squamous cell carcinoma (SCC) was higher in patients who received conventional 2D radiotherapy (0.266%) than in those who received 3D-CRT (0.175%); patients who received IMRT had a higher proportion of SCC than those who received 3D-CRT/conventional 2D radiotherapy (86.4% vs. 41.7% vs. 74.2%); the incidence of sarcoma was higher in patients who received 3D-CRT (0.175%) than in those who received IMRT (0.025%); and the proportion of sarcoma was higher in patients who received 3D-CRT (41.667%) than in those who received IMRT (6.818%) and conventional 2D radiotherapy (17.647%). Patients who received surgery for RICs had better survival than those who received no surgery (64.49 vs. 12.42 months). In the univariate and multivariate analyses, surgery was an independent prognostic factor for overall survival. Conclusions: Our results have implications for long-term follow-up of RIC, multidisciplinary management, and patient counseling of RIC after nasopharyngeal carcinoma treatment by treating clinicians.

The build-in hypoxia-driven dual reporter 9HRE-HSV1-TKeGFP hypoxia research model does not modify intrinsic radiosensitivity of SUNE1 cells under the condition of normoxia or hypoxia, suggesting it be used as preclinical model to explore the therapeutic efficacy of various treatment modalities to overcome hypoxia by fluorescence or nuclear imaging techniques.

Tumor hypoxia, a common phenomenon in solid neoplasms has been associated with aggressive malignant phenotypes and resistance to chemo- and radiotherapy. The purpose of the study was to investigate the effect of carbogen on oxygenation status in tumoral and muscular tissues by direct probe pO2 measurement, to compare the difference of hypoxia biodistribution by imaging exogenous and endogenous hypoxia markers and to explore potential hypoxia-modifying modality to enhance the effect of radiotherapy. BALB/c-nu/nu nude mice were used as tumor-bearing host. pO2 measurements in tumoral and muscular tissues were initiated at the time of probe insertion and the values were recorded continuously for at least 60 min using the fiber-optic oxygen-sensing device (OxyLiteÔ Oxford Optronix, UK) when the tumor-bearing host was under anesthesia which was accomplished with a gas mixture containing 1.0% isoflurane balanced by either air (21% O2) or carbogen (95% O2 + 5% CO2). Image study with CA IX and exogenous hypoxia markers including pimonidazole and EF5 was used to evaluate the effect of spatial biodistribution when animals were treated with air- and carbogen-breathing. Hypoxia in tumor is heterogeneously observed in solid tumors when animal breathing air by using probe pO2 measurement or image study, whereas it ameliorated when breathing carbogen. 4 patterns of pO2 response in tumor can be subcategorized as: i) immediate response, ii) moderate immediate response, iii) slow response and iv) no response when animal was switched from air to carbogen breathing; while 2 patterns, i.e., immediate or no response in muscular tissue are observed. pO2 values in tumoral tissue may remain at relatively higher level at 30 or 60 minutes when the breathing gas was switched from carbogen to air than that of the baseline at initial air breathing; while pO2 value in muscular tissue at air breathing switched from carbogen returned to the same level of baseline at initial air breathing. Tumor hypoxia existed heterogeneously within tumor tissues thus could be subcategorized into 4 patterns according to the pO2 response to carbogen breathing. The observation that pO2 value may remain at relatively high level when air breathing was switched from carbogen in comparison to the baseline at initial air breathing may provide a potential modality that radiation therapy may benefit immediately after carbogen-breathing by improved oxygen status in tumor but not in muscular tissue thus may enhance the therapeutic index. Further research should be merited.

Tumor hypoxia has been associated with malignant phenotype and resistance to chemo- and radio-therapy. The purpose is to research tumor hypoxia by establishing a preclinical model of nasopharyngeal cancer (NPC) with hypoxia-driven gene expression, comparing the biodistribution between exogenous hypoxia tracer and endogenous hypoxia gene expressions and investigating effect of synthetic lethality by irradiation combined with chemotherapeutic agent or PI3K/mTOR inhibitor. SUNE1 cells, originated from an NPC patient were transfected with plasmid containing a hypoxia-inducible dual reporter herpes simplex virus type 1 thymidine kinase and enhanced green fluorescence protein (HSV1-TKeGFP) under control of 9 repeats of hypoxia response element (9x HRE) from human EGFR. Single cloned cells were obtained by repetitive selection with G418 and repeated live cell FACS sorting gated for hypoxia-induced eGFP expression and in vitro radioactive substrate trapping assays with 14C-FIAU. MicroPET imaging study, immunofluorescence on tumor hypoxia and immunohistochemical (IHC) analysis of xenografts were made for comparison of exogenous hypoxia tracer and endogenous hypoxia gene expressions. This hypoxia research model was used in vitro and in vivo to research the effect of synthetic lethality by applying cisplatin, inhibitor targeted on PI3K/mTOR in combination with irradiation including foci formation, clonogenic survival formation and tumor growth delay. We have established and characterized a novel model for studying and imaging of hypoxia-induced gene expression. A hypoxia-inducible dual reporter HSV1-TKeGFP, under the control of 9xHRE, was stably transfected into SUNE1 cells. Selected clones were further enriched by repeated live cell sorting gated for hypoxia-induced eGFP expression. Fluorescent microscopy, and radioactive substrate trapping assays showed strong hypoxia-induced expression of eGFP and HSV1-tk enzyme in SUNE1/9xHRE cells. Detailed spatial distributions in tumor sections were obtained and compared for following hypoxia-associated biomarkers in SUNE1/9xHRE xenograft: Hoechst (blood perfusion), pimonidazole (exogenous biomarker), eGFP and CA9 (endogenous biomarkers). Intratumoral distributions of eGFP, pimonidazole, and CA9 colocalized in the same areas but not in well-perfused regions that were positive for Hoechst and anti-CD31 (blood vessels). The effect of synthetic lethality was observed when SUNE1/9xHRE-TKeGFP cells or xenografts are exposed to cisplatin or PI3K/mTOR inhibitor and irradiation. In enabling the detection of hypoxia-induced molecular events and mapping their distribution in vivo including serial noninvasive PET imaging, and multiple variable analysis with immunohistochemistry assay, this human xenograft model provides a valuable tool for investigating tumor hypoxia and exploring therapeutic efficacy of different treatment modalities to overcome hypoxia.

To investigate the mechanism of NU7441, a highly selective DNA-PK inhibitor in NHEJ-competent mouse embryonic fibroblast (MEF) cells and its NHEJ-deficient cells and explore the feasibility of its application to sensitize nasopharyngeal carcinoma (NPC) SUNE1 cells to ionizing radiation (IR). We generated MEF, DNA-PKcs-/- and Ku70-/- MEF cells. Clonogenic survival assays, flow cytometry and immunoblot were performed to study the effect of NU7441 on survival, cell cycle, and DNA repair. gH2AX was associated with phospho-Ataxia-telangiectasia mutated kinase (Ser1987) and phospho-checkpoint effector kinase 1 (Ser345) foci for arrest of cell cycle through G2/M phase. Inhibition of DNA-PKcs by NU7441 profoundly radiosensitized wild-type MEF cells and SUNE-1 cells, but not DNA-PKcs-/-and Ku70-/-MEF cells confirming that its potentiation was due to DNA-PK inhibition. NU7441 significantly suppressed radiation-induced DSB repair and greatly prolonged G2/M phase arrest post-IR. The effect was associated with activation of cell cycle checkpoints. DSBs were introduced and cell cycle checkpoints were recruited after irradiation in NPC SUNE-1 cells. These results provide evidence of the efficacy of combined treatment of DNA-PK inhibitor and IR in NPC cells and reveal a mechanism that inhibition of DNA-PK sensitizes cells to irradiation through unrepaired and lethal DNA damage, cell cycle arrest and activation of cell cycle checkpoints, justifying further development of DNA-PK inhibitors in cancer therapy.

Radiotherapy is the standard therapy for nasopharyngeal carcinoma (NPC); however, radioresistance can hinder successful treatment. Here we report that microRNA (miR)-24 acts as a tumor suppressor and radiosensitizer in NPC cells and xenografts by targeting Jab1/CSN5. Although accumulating evidence has shown that Jab1/CSN5 functions as an oncoprotein in human cancers, its regulation through miRs has not been described. In this study, we found that Jab1/CSN5 functioned in a manner opposite to that of miR-24 in NPC tumorigenesis and radioresistance. We demonstrated that miR-24 inhibits Jab1/CSN5 translation via direct binding to its 3' untranslated region (3'UTR) and 5'UTR, leading to tumor growth inhibition, and sensitizes NPC tumors to radiation in vivo. Furthermore, silencing Jab1/CSN5 phenocopied the function of miR-24 in NPC cells after ionizing radiation treatment, resulting in increased apoptosis. Finally, we analyzed 50 paired samples of primary and matched recurrent NPC tissues from 25 NPC patients and subjected them to high-throughput genomic quantitative nuclease protection assay for quantifying simultaneously miR and mRNA levels. Our results showed that miR-24 levels were significantly decreased in recurrent NPC and that levels of Jab1/CSN5, as its target, were higher than those in primary NPC. Together, our findings indicate that miR-24 inhibits NPC tumor growth and increases NPC radiosensitivity by directly regulating Jab1/CSN5 and that both miR-24 and Jab1/CSN5 can serve as prognostic markers for NPC recurrence; this, in turn, may provide a promising therapeutic strategy for reversing NPC radioresistance.