Tetraspanins CD151, a transmembrane 4 superfamily protein, has been identified participating in the initiation of a variety of cancers. However, the precise function of CD151 in non-small cell lung cancer (NSCLC) remains unclear. Here, we addressed the pro-tumoral role of CD151 in NSCLC by targeting EGFR/ErbB2 which favors tumor proliferation, migration and invasion.

First, the mRNA expression levels of CD151 in NSCLC tissues and cell lines were measured by RT-PCR. Meanwhile, CD151 and its associated proteins were analyzed by western blotting. The expression levels of CD151 in NSCLC samples and its paired adjacent lung tissues were then verified by Immunohistochemistry. The protein interactions are evaluated by co-immunoprecipitation. Flow cytometry was applied to cell cycle analysis. CCK-8, EdU Incorporation, and clonogenic assays were used to analyze cell viability. Wound healing, transwell migration, and matrigel invasion assays were utilized to assess the motility of tumor cells. To investigate the role of CD151 in vivo, lung carcinoma xenograft mouse model was applied.

High CD151 expression was identified in NSCLC tissues and cell lines, and its high expression was significantly associated with poor prognosis of NSCLC patients. Further, knockdown of CD151 in vitro inhibited tumor proliferation, migration, and invasion. Besides, inoculation of nude mice with CD151-overexpressing tumor cells exhibited substantial tumor proliferation compared to that in control mice which inoculated with vector-transfected tumor cells. Noteworthy, we found that overexpression of CD151 conferred cell migration and invasion by interacting with integrins. We next sought to demonstrate that CD151 regulated downstream signaling pathways via activation of EGFR/ErbB2 in NSCLC cells. Therefore, we infer that CD151 probably affects the sensitivity of NSCLC in response to anti-cancer drugs.

Based on these results, we demonstrated a new mechanism of CD151-mediated tumor progression by targeting EGFR/ErbB2 signaling pathway, by which CD151 promotes NSCLC proliferation, migration, and invasion, which may considered as a potential target of NSCLC treatment.

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Published in Journal of Experimental & Clinical Cancer Research, v. 40, article no. 192.

© The Author(s) 2021

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This work was supported by grants from National Natural Science Foundation of China (81702870 and 81802885 and 82073213), Jiangsu Provincial Medical Youth Talent (No. QNRC2016746), the Suzhou Gusu Medical Youth Talent (GSWS2020016), Science and Technology Plan Project of Suzhou (No. SYS201749), Suzhou Key Laboratory for Respiratory Medicine (No. SZS201617), Clinical Medical Center of Suzhou (No.Szzx201502), Jiangsu Provincial Key Medical Discipline (No. ZDXKB2016007), Medical and health science and technology project in Zhejiang Province (2019ZD024), Natural science research program of Huai’an city (No. HAB201813).

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Additional file 1: Table S1. Demographic and clinical characteristics and levels of CD151 protein expression in NSCLC tissue.

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Additional file 2: Table S2. Demographic and clinical characteristics and levels of CD151 mRNA expression in NSCLC tissue.

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Additional file 3: Figure S1. Inhibition of NSCLC cell cycle by CD151 knockout. Flow cytometry analysis of NSCLC cell lines (sh-CD151 cells vs. sh-NC cells). Cells were harvested at 72 h and stained with propidium iodide. The percentage of cells in each cell cycle phase is shown in each panel, in which the values represent the mean ± SEM of three measurements.

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Additional file 4: Figure S2. Antibody map of the proteome profilerTMarrays. Adapted from the Proteome Profiler Array protocol.

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Additional file 5: Figure S3. Human Soluble Receptor Antibody array. a-b Human Soluble Receptor Antibody array in common analytes array analysis of stable A549 cells in which CD151 either silenced or overexpressed.

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Additional file 6: Table S3. The results of Proteome Profiler Array-Human Soluble Receptor Array Non-hematopoietic panel and Common Analytes panel. (Part N and Part C).

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Additional file 7: Figure S4. CD151 was correlated with integrin α3/α6/β1 mRNA level in lung cancer. a-c Data obtained from TCGA database (https://portal.gdc.cancer.gov/) were analysed to explore the correlation between CD151 and integrin α3/α6/β1 mRNA levels in 103 normal tissues and 999 NSCLC tissues. d-f Data obtained from CCLE database (https://portals.broadinstitute.org/ccle) were analysed to explore the correlation between CD151 and integrin α3/α6/β1 mRNA levels in 188 lung cancer cell lines. g Total protein were extracted from several cell lines, and the expression of EGFR/ErbB2 and integrins was measured by western blotting, respectively. h Co-immunoprecipitation of CD151 and integrinβ1are shown. Protein were immunoprecipitated and detected from lysates of A549 and H1299 cells using a specific monoclonal antibody.

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Additional file 8: Figure S5. Antibody map of the tyrosine kinase arrays. Adapted from the RayBio Human RTK Phosphorylation Antibody Array protocol.

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Additional file 9: Table S4. List of signal densities of human RTK phosphorylation Antibody Array.

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Additional file 10: Figure S6. Overexpression of CD151reduced the sensitivity of NSCLC cells to gefitinib, lapatinib and vs6063. The A549 and H1299stable cell lines were transfected with10 μM gefitinib (a&d), 20 μM lapatinib (b&e) or 5μM vs6063(c&f) for 48 h, respectively. After theaforementioned treatments, cell viability was assessed using CCK-8assays. The data shown represent the mean ± SD values of fourreplicate experiments. All the data were obtained from three independentexperiments and are shown as the mean ± SD values. *P<0.05; **P<0.01; ***P<0.001

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Additional file 11: Figure S7. More sensitive to gefitinib and vs6063 after CD151 knockdown in NSCLC cells. a, b Effects of VS6063 and Gefitinib on cell cycle of vector and sh-CD151 cells. c, d The viability of vector and sh-CD151 cells after treated with VS6063 and Gefitinib was determined by CCK8 assay.