Cell Death and Disease (2017) 8, e2803 

Cytoplasmic RAP1 mediates cisplatin resistance of non-small cell lung cancer

Hongbiao Huang,Jinbao Liu


Cytotoxic chemotherapy agents (e.g., cisplatin) are the first-line drugs to treat non-small cell lung cancer (NSCLC) but NSCLC develops resistance to the agent, limiting therapeutic efficacy. Despite many approaches to identifying the underlying mechanism for cisplatin resistance, there remains a lack of effective targets in the population that resist cisplatin treatment. In this study, we sought to investigate the role of cytoplasmic RAP1, a previously identified positive regulator of NF-κB signaling, in the development of cisplatin resistance in NSCLC cells. We found that the expression of cytoplasmic RAP1 was significantly higher in high-grade NSCLC tissues than in low-grade NSCLC; compared with a normal pulmonary epithelial cell line, the A549 NSCLC cells exhibited more cytoplasmic RAP1 expression as well as increased NF-κB activity; cisplatin treatment resulted in a further increase of cytoplasmic RAP1 in A549 cells; overexpression of RAP1 desensitized the A549 cells to cisplatin, and conversely, RAP1 depletion in the NSCLC cells reduced their proliferation and increased their sensitivity to cisplatin, indicating that RAP1 is required for cell growth and has a key mediating role in the development of cisplatin resistance in NSCLC cells. The RAP1-mediated cisplatin resistance was associated with the activation of NF-κB signaling and the upregulation of the antiapoptosis factor BCL-2. Intriguingly, in the small portion of RAP1-depleted cells that survived cisplatin treatment, no induction of NF-κB activity and BCL-2 expression was observed. Furthermore, in established cisplatin-resistant A549 cells, RAP1 depletion caused BCL2 depletion, caspase activation and dramatic lethality to the cells. Hence, our results demonstrate that the cytoplasmic RAP1–NF-κB–BCL2 axis represents a key pathway to cisplatin resistance in NSCLC cells, identifying RAP1 as a marker and a potential therapeutic target for cisplatin resistance of NSCLC.It was previously reported that cytoplasmic RAP1 was detected in breast cancer tissue, and its pathology scores were positively correlated with the tumor grades.17 To test the hypothesis that cytoplasmic RAP1 is a biomarker for higher-grade NSCLC, we immunostained for RAP1 and quantified its cytoplasmic and nuclear expression in 93 lung adenocarcinoma and 75 lung squamous cell carcinoma tissues. The peri-tumoral normal tissue was used to reflect RAP1 expression in non-malignant cells (Supplementary Tables S1 and S2). Both cytoplasmic and nuclear RAP1 were increased in NSCLC tumors compared with normal tissues, but the difference in cytoplasmic RAP1 appears to be greater (Figures 1a, b, f, Supplementary Figures S1a and b). A higher level of cytoplasmic RAP1 expression was associated with a higher grade of NSCLCs (Figures 1c, d, Supplementary Figures S1c and d). Moreover, higher cytoplasmic RAP1 expression was associated with poorer prognosis of adenocarcinoma patients (Figure 1e; information of squamous cell carcinoma patients’ survival is unfortunately not available). These analyses identify cytoplasmic RAP1 as an indicator of high-grade NSCLC, suggesting that it may have a critical role in cancer progression.Acquisition, paraffin embedding, antibody staining and pathological scoring of lung adenocarcinoma and squamous cell carcinoma samples were performed by Shanghai Outdo Biotech Company (151 Libing Road, Shanghai, China, 201213). All specimens were handed anonymously following the company’s protocol and ethical standards. Paraffin-embedded sections were immunolabeled with anti-Rap1 antibody (Cell Signaling Technology, Danvers, MA, USA #5433). Positive staining and staining scores were determined by a pathologist in cancer cells and peri-tumoral normal tissues. RAP1 expression was calculated by multiplying the percentage of positivity with staining score in each sample. Representative images of different staining scores are shown in Supplementary Figure S1e.This work was supported by the National High Technology Research and Development Program of China (2006AA02Z4B5) and the National Natural Science Foundation of China (81272451/H1609, 81472762/H1609) (to JL) and the National Natural Science Foundation of China (81670156/H0812) (to HH).Supplementary Information accompanies this paper on Cell Death and Disease website (http://www.nature.com/cddis)Edited by A StephanouThe authors declare no conflict of interest.
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