Expression Of CHMP4C In Radiation - Induced Non - Small Cell Lung Cancer And Its Mechanism

BackgroundHuman lung cancer is highly invasive and most malignant among human tumors, which is classified into non-small cell lung cancer and small cell lung cancer. Non-small cell lung cancer accounts for about 85% of lung cancers and the current therapeutic strategy includes surgical resection together with radiation and/or chemo-treatment. Radiation resistance is the severe outcome of lung cancer radiotherapy and therefore poses a critical barrier for the radio-therapeutic effect. Hence, how to avoid the radiation resistance and to improve its treatment efficiency is a major challenge. A recent study found that in the cell division, there is a very important cytokinetic abscission checkpoint regulated by CHMP4C, which is a subunit of ESCRT-III, controls abscission time to coordinate midbody resolution and prevents accumulation of DNA damage. CHMP4C can check the abscission timing through AuroraB-directed phosphorylation. CHMP4C has lower expression in normal tissues and high expression in cancers. Overall, despite CHMP4C is involved in abscission checkpoint and autophagy, how it serves its function in DNA damage response as well as in lung cancer formation still remains unclear.ObjectiveTo demonstrate that the subunit of ESCRT-III CHMP4C is involved in cellular radiation reactions and cellular sensitivity to radiation. To investigate CHMP4C expression upon IR and the function of CHMP4C on IR induced celluar response. To reveal the relationship between CHMP4C and p53 or Aurora B signaling pathway in mediating radiation resistance. To stablishe a new function of CHMP4C in radiation resistance, which will offer a potential strategy for non-small cell lung cancer by disrupting CHMP4C.Methods1. The function of CHMP4C on cell viability, cell apoptosis and cell cycle in A549 cells. A549 cells were depleted of CHMP4C for 48 h by siRNA. Cell viability was measured by alamarBlue reagent. Caspase 3/7 activity was tested by Caspase-GloR 3/7 reagent. Cell cycle and cell apoptosis was measured by flow cytometry. Cell cycle relevant protein was analyzed by western bolt.2. Regulation effect of AuroraB on CHMP4C. To verify whether Aurora B regulates CHMP4C, we silenced or overexpressed Aurora B in A549 and H1299. CHMP4C and Aurora B protein expression were checked by Western blot. CHMP4C and Aurora B mRNA expression were checked by realtime-PCR. The A549 and H1299 cells were treated with AZD1152-HQPA to inhibit AuroraB activity, and then CHMP4C phosphorylation was examined by Western blot.3. Radiation induces CHMP4C and Aurora B expression. A549 and H1299 cells were y-irradiated with 2 Gy, CHMP4C expression and phosphorylation was checked by western bolt at 4,8,24,48h after IR. cells were y-irradiated with 2,4, and 6 Gy, Aurora B expression, CHMP4C expression and phosphorylation was checked by western bolt at 24h after IR. A549 cells were depleted of p53 by siRNA,24h after IR, CHMP4C or p53 expression were detected by Western blot.4. The function of CHMP4C or Aurora B on radiosensitivity in A549 and H1299. CHMP4C or Aurora B knockdown/overexpression and/or IR (0,2,4,6 Gy) in A549 and H1299 cells, colony formation assays were employed to detect if CHMP4C or Aurora B acts in cell radioresistance of A549 and HI299.5. The function of CHMP4C on cell cycle and cell apoptosis upon IR. A549 and H1299 cells were depleted of CHMP4C or p21 for 24 h by siRNA.24h after IR with 2 Gy, cell cycle were analyzed on flow cytometer,and CHMP4C or p21 expression were detected by Western blot. A549 cells was depleted of CHMP4C,24h after IR with 6 Gy, cell apoptosis was analyzed on flow cytometer.6. The influence of CHMP4C on DNA damage repair. To analyze if CHMP4C is immediately involved in DNA damage repair signaling. The yH2AX and 53BP1 foci assembly in A549 cells with or without CHMP4C silencing were examined by fluorescence microscopy.Results1. The function of CHMP4C on cell viability, cell apoptosis and cell cycle in A549 cells. CHMP4C knockdown caused lagging S phase in cell cycle through enhancing the phosphorylation of Rb and cdc2, raising the expression of Cyclin B1, and suppressing the activation of Cyclin A. Meanwhile, CHMP4C deletion depressed cell survival via decreasing cell viability and increasing caspase 3/7 activity.2. CHMP4C acts downstream of AuroraB. CHMP4C knockdown has no effect on Aurora B protein level, whereas AuroraB silencing inhibits CHMP4C protein level indicating that Aurora B acts upstream of CHMP4C. Moreover, AuroraB overexpression leads to increased CHMP4C phosphorylation and inhibiting AuroraB kinase activity using phosphorylation inhibitor AZD1152-HQPA reduces CHMP4C phosphorylation. The CHMP4C mRNA level was unchanged following AuroraB deletion.3. Radiation induces CHMP4C expression and phosphorylation. CHMP4C protein increases after irradiation and peaks at 24 h in A549 but not in H1299 cells. However, the level of CHMP4C phosphorylation and AuroraB expression both increase with IR in both A549 and H1299. CHMP4C protein did not increase after irradiation in p53-cleaned A549 cells.4. CHMP4C or AuroraB deletion increased radiosistivity. Colony formation assays showed that CHMP4C knockdown decreases cell survival compared to control with IR. Similarly, the result was also found in Aurora B clearing cells. Inversely, CHMP4C or Aurora B expression can increase cell growth upon irradiation.5. CHMP4C silencing delayed S-phase of the cell cycle and increased cell apoptosis with irradiation. CHMP4C could impact S phase exit. When A549 and H1299 cells were treated with 2 Gy of radiation, G2/M transition was severely blocked and S-phase was retarded, whereas combined radiation and CHMP4C knockdown only results in S-phase delay. In the meantime, similar results were found in p21-cleaned A549 cells. The double siRNA experiments show that both CHMP4C and p21 depletion exerts an additive effect in the S phase exit. In addition, CHMP4C knockdown has no influence on p21 expression and p21 depletion cannot effect CHMP4C expression. CHMP4C silencing increases cell apoptosis with irradiation. Combined CHMP4C silencing and irradiation produce more evident apoptosis than single treatment.6. CHMP4C reduction suppressed IR-induced yH2AX and 53BP1 foci formation. during 4 Gy of irradiation, CHMP4C reduction decreased the intensity and number of γH2AX and 53BPlfoci compared with the normal CHMP4C, proving that CHMP4C repression indeed represses recruitment of γH2AX,53BP1 and other related proteins to the DNA lesions.Conclusion1. CHMP4C functioned on proliferation in A549 cells.2. CHMP4C disruption sensitizes NSCLC to irradiation.3. The close correlation between CHMP4C and AuroraB signaling pathway in mediating radiation resistance is not p53 dependent.