The Clinical Significance Of RSK2Expressed In Lung Adenocarcinoma And A Potential Therapeutic Target For Radiation Sensitization

Objective:The aim of this study is to investigate the expression and clinical significance of p90ribsomal S6kinase2(RSK2) in lung adenocarcinoma. We also investigated the effects ofionizing radiation on the expression and activity of RSK2in lung adenocarcinoma celllines and whether inhibition of RSK2activity could result in radiosensitization of lungadenocarcinoma cell lines. The role of RSK2in the radiosensitivity of lungadenocarcinoma was also determined.Methods:1. Immunohistochemical staining was performed to determine RSK2expression in172cases of non-small cell lung carcinoma (NSCLC), including26cases of squamous cellcarcinoma,10cases of large cell carcinoma and136cases of adenocarcinoma and20casesof non-neoplastic lung tissue away from the tumors. Clinical history of all the cases werecollected and analyzed.2. The cell cycle was synchronized by serum starvation or pretreatment withaphidicolin and the relationship between the RSK2expression and cell cycle in lungadenocarcinoma cell lines was determined.3. MTT assay was utilized to evaluate the impact of silencing of RSK2on theproliferation of lung adenocarcinoma cell, NCI-H1395. Western blot was applied to detectthe expression of cyclin B1and cyclin D1in the NCI-H1395cell line after the RSK2genesilencing.4. Western blot assay was performed to determine the expression of cyclin B1andRSK2, and the phosphorylation of RSK2in lung adenocarcinoma cell lines24hours afterexposed to X-ray irradiation.5. The specific inhibitor (BI-D1870) of RSK2was used half an hour prior to the radiation and the effects of RSK2inhibition on the cell cycle, colony formation, apoptosisas well as intracellular cyclin B1were examined24hours after lung adenocarcinoma cellswere exposed to irradiation.Results:1. There was no RSK2expression in the bronchiolar or alveolar epithelial cells in20cases of non-neoplastic lung tissue. The frequency of RSK2expression in lung squamouscell carcinoma, large cell carcinoma and adenocarcinoma was19.2%,50.0%,70.6%,respectively. RSK2was more frequently (p<0.05) expressed in non-squamous lung cellcarcinoma (adenocarcinoma and large cell carcinoma) than in squamous cell carcinomaand it was more frequently (p<0.05) detected in the tumors from women than men. RSK2expression in lung adenocarcinoma was also positively correlated to the tumor size and itwas more frequently detected in the tumors with≥3cm than in the tumors with <3cm.The RSK2expression was not correlated with the status of lymph nodes, distant metastasis,histological grading, visceral pleural involvement or clinical staging.2. Cell synchronization by serum starvation or pretreatment with aphidicolin showedthe expression of RSK2in lung adenocarcinoma was fluctuated with cell cycle and reachedthe peak level in G0/G1phase.3. The expression of cyclin B1and cyclin D1were inhibited by using siRNA tosilence the RSK2expression in the NCI-H1395cells. Silencing of RSK2also significantlydecreased the proliferation of NCI-H1395cells.4. After irradiation of the lung adenocarcinoma cells, the RSK2expression wassignificantly reduced and negatively correlated to the radiation dose. In contrast, the levelof phosphorylation of RSK2(P-RSK2), when radiation dosage≤2Gy, decreased slightly.With radiation dosage>2Gy, p-RSK2was increased in accordance with radiation dosageascension. When above4Gy, cyclin B1was apparently detected.5. G2/M arrest was induced in all four kinds of lung adenocarcinoma cell linesincluding LTEP-α-2, SPC-A-1, NCI-H1395and NCI-H1975after exposed to the X-ray,also this effect was enhanced if the RSK2activity was inhibited by5μM BI-D1870pretreatment for30min prior to the radiation. Inhibition of RSK2activity in lungadenocarcinoma cell also decreased the expression of cyclin B1and the ability of cellclone formation, but did not increase the cell apoptosis induced by the radiation. Conclusion:1. The frequency of RSK2expression was much higher in the lung large cellcarcinoma and adenocarcinoma than in the squamous cell carcinoma. These findingsindicate that RSK2is a valuable biomarker in distinguishing the lung squamous cellcarcinoma from non-squamous lung cell carcinoma in NSCLC.2. The RSK2expression was correlated to the tumor size of lung adenocarcinoma andthe tumors from female patients tended to be more frequently positive for RSK2. Therewas no correlation of RSK2expression with the status of lymph node, distant metastasis,degree of tumor differentiation, visceral pleural involvement or clinical staging.3. The expression of RSK2in lung adenocarcinoma cells was related to the cell cycle.And RSK2was involved in the proliferation of lung adenocarcinoma cells by regulatingboth cyclin B1and cyclin D1expression.4. RSK2was involved in the regulation of the radiosensitivity of the lungadenocarcinoma cell through cyclin B1. Inhibition of RSK2activity was able to increasethe radiosensitization of lung adenocarcinoma.