Jingchen Lu

Targeting EBV-LMP1 DNAzyme enhances radiosensitivity of nasopharyngeal carcinoma cells by inhibiting telomerase activity

The latent membrane protein 1 (LMP1), which is encoded by the Epstein–Barr virus (EBV), has been suggested to be one of the major oncogenic factors in nasopharyngeal carcinoma (NPC). In previous studies, we experimentally demonstrated that downregulation of LMP1 expression by targeting EBV-LMP1 DNAzyme (Dz1) could increase the radiosensitivity of NPC. However, how Dz1 contributes to the radiosensitivity in NPC is still not clear. In the present study, we confirmed that Dz1 decreases LMP1 expression and downregulates the expression of the catalytic subunit of telomerase (hTERT), both at the protein and mRNA levels (P < 0.01), and therefore, consequently inhibits telomerase activity (P < 0.05) in LMP1-positive NPC cells. We also observed that LMP1 mediated Akt phosphorylation is involved in the regulation of hTERT expression and phosphorylation. After LMP1 and hTERT expression was silenced by Dz1 and hTERT-targeted siRNA, respectively, the radiosensitivity increased in CNE1-LMP1 cells (P < 0.05). The inhibition was more significant after Dz1 treatment was combined with siRNA (P < 0.01). We concluded that hTERT expression and phosphorylation could be regulated by LMP1 through the Akt pathway, and Dz1 enhances radiosensitivity of LMP1-positive NPC cells by inhibiting telomerase activity.

EBV-LMP1 suppresses the DNA damage response through DNA-PK/AMPK signaling to promote radioresistance in nasopharyngeal carcinoma.

We conducted this research to explore the role of latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus (EBV) in modulating the DNA damage response (DDR) and its regulatory mechanisms in radioresistance. Our results revealed that LMP1 repressed the repair of DNA double strand breaks (DSBs) by inhibiting DNA-dependent protein kinase (DNA-PK) phosphorylation and activity. Moreover, LMP1 reduced the phosphorylation of AMP-activated protein kinase (AMPK) and changed its subcellular location after irradiation, which appeared to occur through a disruption of the physical interaction between AMPK and DNA-PK. The decrease in AMPK activity was associated with LMP1-mediated glycolysis and resistance to apoptosis induced by irradiation. The reactivation of AMPK significantly promoted radiosensitivity both in vivo and in vitro. The AMPKα (Thr172) reduction was associated with a poorer clinical outcome of radiation therapy in NPC patients. Our data revealed a new mechanism of LMP1-mediated radioresistance and provided a mechanistic rationale in support of the use of AMPK activators for facilitating NPC radiotherapy.

Neoalbaconol inhibits angiogenesis and tumor growth by suppressing EGFR-mediated VEGF production

Neoalbaconol, derived from Albatrellus confluens, shows anti‐cancer activities in the previously study, but its role in angiogenesis is unknown. Here, we determined whether neoalbaconol could attenuate angiogenesis and how does it occur. Data demonstrated that neoalbaconol could inhibit the proliferation of breast cancer cells and induce apoptosis. Also, neoalbaconol suppressed vascular endothelial growth factor (VEGF)‐induced human umbilical vascular endothelial cells (HUVECs) proliferation, migration, invasion, and capillary‐like tube formation in vitro and reduced tumor angiogenesis in vivo. VEGF receptor activation and the downstream signal transduction cascades activation were inhibited by neoalbaconol. Additionally, neoalbaconol blocked EGFR‐mediated VEGF production. EGFR overexpression reversed the neoalbaconol‐induced VEGF reduction, confirming the importance of the EGFR inhibition in anti‐angiogenesis of neoalbaconol. Furthermore, neoalbaconol inhibited tumor growth and tumor angiogenesis in a breast cancer xenograft model in vivo. Taken together, these results indicate that neoalbaconol could inhibit tumor angiogenesis and growth through direct suppression effects on vascular endothelial cells and reduction of proangiogenic factors in cancer cells.

EBV-LMP1-targeted DNAzyme induces DNA damage and causes cell cycle arrest in LMP1-positive nasopharyngeal carcinoma cells.

This study aimed to determine the molecular mechanisms underlying the effect of the LMP1-targeted DNAzyme 1 (DZ1) on cell cycle progression in nasopharyngeal carcinoma (NPC) cells. We showed that the active DZ1 inhibited the expression of latent membrane protein 1 (LMP1) and induced a G1 phase arrest. In addition, this cell cycle deregulation was shown to be accompanied by upregulation of the DNA damage marker γ-H2AX, downregulation of the DNA damage response factor p-p53-Ser15 and cell proliferation inhibition. To investigate what affected the cell cycle progression, we examined the expression of two checkpoint-related cyclins and cyclin-dependent kinases (CDKs). We found a decrease of cyclin D1 and cyclin E protein levels at 24 h from the DZ1 treatment. Moreover, we observed inhibition of CDK4 activity and decreased cyclin D1 expression in the complexes immunoprecipitated with CDK4 antibody. We also found a reduction in cdc2 phosphorylation at Thr161 which partially stands for the cdc2 kinase activity in DZ1-treated CNE1-LMP1 cells, although the downregulation of LMP1 expression had no effect on the cyclin B1 and cdc2 expression. Further, we analyzed changes in cdc2 kinase activity induced by DZ1 and found that the downregulation of the LMP1 expression resulted in a 5-fold reduction in cdc2 kinase activity in CNE1-LMP1. The data suggest that the downregulation of the LMP1 expression by DZ1 was able to induce DNA damage, which then further inhibited the cell proliferation and resulted in malfunction of cell cycle checkpoints that led to G1 phase arrest and the decrease in number of cells in G2/M phase.

A new functional IDH2 genetic variant is associated with the risk of lung cancer.

Recently, mutations in isocitrate dehydrogenase 1/2 (IDH1/2) were discovered in 70% of low‐grade glioma and secondary glioblastoma multiforme. The discovery of an oncogenic function and the identification of onco‐metabolites of IDH1/2 support new roles for metabolism in cancer. For example, some evidence indicates that IDH2 might also exhibit oncogenic functions by promoting cellular metabolism and cancer cell growth. We examined the association between IDH2 rs11540478 and lung cancer risk in 262 lung cancer patient cases and 602 healthy control subjects and also investigated the biological function of rs11540478 in vivo. We found that a higher risk was observed in lung cancer patient carriers of rs11540478 TT and CT compared with CC carriers (OR = 1.44; 95%CI = 1.04–2.00; P = 0.03). The frequency of IDH2 rs11540478 TT and CT carriers was decreased in healthy individuals between the ages of 50–77 compared to those aged 30–49 (OR = 0.67; 95%CI = 0.47–0.96; P = 0.03). Functional analysis showed the effect of rs11540478 on IDH2 expression and lung cancer cell viability, with higher IDH2 expression and cell viability among T allele compared with C allele. IDH2 mRNA was higher in peripheral blood lymphocytes from lung cancer patients compared to healthy subjects. Herein, for the first time we identified IDH2 rs11540478 as a new susceptibility locus for lung cancer. The effect of rs11540478 on mRNA expression of IDH2 and lung cancer cell viability might provide new insight for the genetic basis of lung cancer.

Targeting CPT1A-mediated fatty acid oxidation sensitizes nasopharyngeal carcinoma to radiation therapy

Nasopharyngeal carcinoma (NPC) has a particularly high prevalence in southern China, southeastern Asia and northern Africa. Radiation resistance remains a serious obstacle to successful treatment in NPC. This study aimed to explore the metabolic feature of radiation-resistant NPC cells and identify new molecular-targeted agents to improve the therapeutic effects of radiotherapy in NPC. Methods: Radiation-responsive and radiation-resistant NPC cells were used as the model system in vitro and in vivo. Metabolomics approach was used to illustrate the global metabolic changes. 13C isotopomer tracing experiment and Seahorse XF analysis were undertaken to determine the activity of fatty acid oxidation (FAO). qRT-PCR was performed to evaluate the expression of essential FAO genes including CPT1A. NPC tumor tissue microarray was used to investigate the prognostic role of CPT1A. Either RNA interference or pharmacological blockade by Etomoxir were used to inhibit CPT1A. Radiation resistance was evaluated by colony formation assay. Mitochondrial membrane potential, apoptosis and neutral lipid content were measured by flow cytometry analysis using JC-1, Annexin V and LipidTOX Red probe respectively. Molecular markers of mitochondrial apoptosis were detected by western blot. Xenografts were treated with Etomoxir, radiation, or a combination of Etomoxir and radiation. Mitochondrial apoptosis and lipid droplets content of tumor tissues were detected by cleaved caspase 9 and Oil Red O staining respectively. Liquid chromatography coupled with tandem mass spectrometry approach was used to identify CPT1A-binding proteins. The interaction of CPT1A and Rab14 were detected by immunoprecipitation, immunofluorescence and in situ proximity ligation analysis. Fragment docking and direct coupling combined computational protein-protein interaction prediction method were used to predict the binding interface. Fatty acid trafficking was measured by pulse-chase assay using BODIPY C16 and MitoTracker Red probe. Results: FAO was active in radiation-resistant NPC cells, and the rate-limiting enzyme of FAO, carnitine palmitoyl transferase 1 A (CPT1A), was consistently up-regulated in these cells. The protein level of CPT1A was significantly associated with poor overall survival of NPC patients following radiotherapy. Inhibition of CPT1A re-sensitized NPC cells to radiation therapy by activating mitochondrial apoptosis both in vitro and in vivo. In addition, we identified Rab14 as a novel CPT1A binding protein. The CPT1A-Rab14 interaction facilitated fatty acid trafficking from lipid droplets to mitochondria, which decreased radiation-induced lipid accumulation and maximized ATP production. Knockdown of Rab14 attenuated CPT1A-mediated fatty acid trafficking and radiation resistance. Conclusion: An active FAO is a vital signature of NPC radiation resistance. Targeting CPT1A could be a beneficial regimen to improve the therapeutic effects of radiotherapy in NPC patients. Importantly, the CPT1A-Rab14 interaction plays roles in CPT1A-mediated radiation resistance by facilitating fatty acid trafficking. This interaction could be an attractive interface for the discovery of novel CPT1A inhibitors.

Wild-type IDH2 promotes the Warburg effect and tumor growth through HIF1α in lung cancer

Hotspot mutations of isocitrate dehydrogenase 1 and 2 (IDH1/2) have been studied in several cancers. However, the function of wild-type IDH2 in lung cancer and the mechanism of its contribution to growth of cancer cells remain unknown. Here, we explored the role and mechanism of wild-type IDH2 in promoting growth of lung cancer. Methods: Information regarding genomic and clinical application focusing on IDH2 in cancer was examined in several databases of more than 1,000 tumor samples. IDH2 expression was assessed by immunohistochemistry in tissues from lung cancer patients. The biological functions of IDH2 were evaluated by using cell-based assays and in vivo xenograft mouse models. Results: Here we reported that wild-type IDH2 is up-regulated and is an indicator of poor survival in lung cancer and several other cancers. Targeting IDH2 with shRNA resulted in decreased HIF1α expression, leading to the attenuation of lung cancer cell proliferation and tumor growth. Treatment of lung cancer cells with AGI-6780 (a small molecule inhibitor of IDH2), PX-478 (an inhibitor of HIF1α) or incubation with octyl-α-KG inhibited lung cancer cell proliferation. Conclusion: IDH2 promotes the Warburg effect and lung cancer cell growth, which is mediated through HIF1α activation followed by decreased α-KG. Therefore, IDH2 could possibly serve as a novel therapeutic target for lung cancer.

WITHDRAWN: miR-504 affects the radio-resistance in nasopharyngeal carcinoma by down-regulating the expression of nuclear respiratory factor 1

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