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  • br mechanism is still unknown AAMP expression is

    2020-08-12


    mechanism is still unknown [12]. AAMP expression is increased in a number of cancer 145022-92-0 like invasive gastrointestinal and breast carci-noma cells; it is considered a marker of poor prognosis [13,14]. The epidermal growth factor receptor (EGFR), which is anchored in the cytoplasmic membrane, belongs to the ERBB family of receptor tyrosine kinases [15]. EGFR is widely expressed and plays a critical role in cell growth, proliferation and differentiation [16]. Dimerization is essential for EGFR activation in healthy cells. When activated, the COOH-terminal portion is phosphorylated at specific sites, such as 1173 tyrosine, which initiates intracellular signaling pathways [15,17]. As expected, dysregulated EGFR activation is found in multiple cancer cells, including lung cancer, indolent bronchioalveolar carcinoma and non–small cell lung cancer [18,19].
    Overexpression and mutations are the main causes of EGFR dysre-gulation. Its overexpression causes positive signal activated
    Abbreviations: NSCLC, non-small cell lung cancer cell; AAMP, Angio-associated migratory cell protein; EGFR, epidermal growth factor receptor; ERK1/2, extra-cellular regulated protein kinase; RTK, receptor tyrosine kinase; CCND, Cyclin-D; ACTB, β-actin; EGF, epidermal growth factor; SRB, sulforhodamine B; CTTM, cytoplasmic and transmembrane fragment of EGFR; ETTM, cellular and transmembrane fragment of EGFR; CT, cytoplasmic fragment of EGFR; CASP3, caspase3; CASP8, caspase8
    Corresponding authors at: Shandong University School of Life Sciences, Room N8-110, 72 Binhai Road, Qingdao 266237, China. E-mail addresses: [email protected] (X. Liu), [email protected] (L. Su).
    B
    A
    AAMP
    EGFR
    ACTB
    C
    EV AAMP
    AAMP
    ACTB
    E Calu-1
    siRNA ctrl
    AAMP siRNA-1
    AAMP siRNA-2 AAMP
    AAMP
    ACTB
    D
    AAMP
    ACTB
    Fig. 1. AAMP inhibition impaired cell proliferation in NSCLC cells. (A) Western blot of AAMP from six non-small cell lung cancer cells. (B) Calu-1 cells were treated with AAMP siRNAs for 24 h and then reseeded in 24-well plates. Cells were imaged at 24 h and 48 h with high connotation microscope. Cell number and proliferation rate were calculated using provided software. The error bars represent the SD (n = 4), *P < .05, **P < .01 and ***P < .001. (C) AAMP was overexpressed in A549 cells for 24 h. Cells were reseeded in 24-well plates and imaged at 24 h and 60 h with high connotation microscope. Cell number and proliferation rate were calculated using software. The error bars represent the SD (n = 4), *P < .05 and **P < .01. (D) AAMP was silenced in Calu-1 cells for 24 h then cultivated in 96-well plates. EdU assay was performed according to instructions. The error bars represent the SD (n = 5), ***P < .001. (E) Calu-1 was transfected with AAMP siRNAs and then cultivated in agar culture medium for 24 h. Cells were stained after 7 days and cell number was calculated by software. The error bars represent the SD (n = 3), **P < .01 and ***P < .0001. (F) Plasmid pcDNA3.1-AAMP and its empty control were transfected into H460 cells for 24 h. Cells were re-cultivated in agar culture medium for 7 days and cell number was calculated by software. The error bars represent the SD (n = 3), **P < .01.
    continuously, resulting in proliferation out of control and drug re-sistance. In some cases, EGFR silencing sensitized tumor cells to dox-orubicin-induced apoptosis [20]. Besides, EGFR activating mutations, including exon 19 detection an exon 21-point mutations, give the ability to escape negative regulation [21]. Because of the importance of EGFR, drugs targeted to mutational EGFR, such as gefitinib, erlotinib and icotinib, have been used for the treatment of lung cancers [19]. However, acquired resistance to these drugs was developed in long-term treatment. The specific mechanism of first-generation EGFR-TKIs resistance (e.g. gefitinib, erlotinib and icotinib) was a secondary T790M mutation acquisition [22]. The threonine to methionine mutation in-creases the affinity between ATP and EGFR in tyrosine kinase domain, thus repressing the binding of EGFR-TKIs to EGFR. The acquired re-sistance limits TKIs' long-term efficacy. Thus, it is significant to pursue strategies to overcome TKIs-resistance in NSCLC cells.
    It has been predicted by affinity capture MS that AAMP interacts with EGFR [23], but the significance of this combination is still un-known. In this study, we show AAMP interact with EGFR and active MAPK signal pathway by promoting EGFR dimerization, which en-hanced tumor cell proliferation and tumorigenesis. In addition, we also 
    find AAMP silencing enhances apoptosis caused by icotinib and dox-orubicin in EGFR mutation and wide-type NSCLC cells.