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  • br Discussion br AAMP is found in

    2020-08-12


    4. Discussion
    AAMP is found in many tumors which suggests it plays important role in cancers [6–8]. AAMP contains a repeated-WD40 domain that is correlated with functions such as apoptosis, autophagy, motility and growth. Therefore, it could be a multifunctional protein. However, nowadays we just have realized a little bit about it, such as inhibiting NF-κB signaling pathway [6] and promoting angiogenesis [31]. Its molecular mechanism in cancer UNC1999 remains largely unclear. In our study, we found that AAMP suppression inhibited proliferation in vitro in NSCLC cells. Moreover, heterogeneous tumors grow more slowly at the condition of AAMP silence in a xenograft mouse model. These data remind the importance of AAMP in tumorigenesis. Indeed, AAMP is a novel marker of lung cancer progression [13,14]. Because of its unique sequence, AAMP is likely to function through the interaction with other proteins. Accidentally, proteomic analysis showed that AAMP interacts with EGFR [23], which provides clues for mechanism research.
    There are > 58 encode transmembrane protein receptor tyrosine kinases (RTKs) in cells. Among them, epidermal growth factor receptor 
    (EGFR) was the first one identified and linked to cancer. Thus, it is the most intensively studied RTK all over the world. However, the me-chanism of EGFR activation is still not fully understood. EGFR functions as an oncogenic protein and is correlated with cell growth, differ-entiation and motility [16]. It exists as two forms, monomers and di-mers. When transformed from monomer state into dimer, EGFR is ac-tivated and autophosphorylated at tyrosine 1173, 1068 and 1086 sites to trigger downstream signal pathways, such as RAS-RAF-MEK-ERK, PI3K-AKT and PLCγ [32], which eventually promotes cell survival, proliferation and anti-apoptosis. In NSCLC cells, EGFR overexpression and continuous activation are widespread and provide momentum in tumorigenesis [33]. Meanwhile, tumor cells may benefit from dysre-gulation of EGFR to obtain drug resistance. Thus, how to block acti-vation of EGFR effectively is critical for NSCLC therapy. Nowadays, most studies of EGFR overactivity focus on its own mutations and overexpression and researches about accessory protein are rarely found.
    Here, we reported AAMP protein contributing to EGFR activation. We noted that AAMP silencing inhibited EGFR phosphorylation at tyrosine1173 site, which reminded us the important role of AAMP in EGFR activation. Co-IP experiments showed that AAMP interacted with EGFR and less dimers of EGFR were detected when AAMP was down-regulated. These results demonstrated that AAMP may be a helper in the process of dimerization to promote EGFR phosphorylation and ac-tivate downstream signaling pathways in NSCLC cells. Subsequent data revealed that AAMP inhibition suppressed phosphorylation of ERK1/2 and proliferation as expected.
    Oncogene-targeted therapy has always been favored and achieves better effects. Administration of EGFR tyrosine kinase inhibitors (TKIs) is wildly used in NSCLC treatment with a higher overall response rate, longer overall survival and lower incidence of adverse reactions relative
    to drug chemotherapy [34,35]. Unfortunately, almost all patients de-velop acquired resistance after EGFR TKI treatment for 1 year. Thus, combination is a better choice than EGFR TKI therapy alone. Currently, multiple combination treatments have developed, such as association of PD1 blockade with EGFR TKIs [36], Erlotinib plus Cabozantinib [37] and combination of EGFR TKIs with radiotherapy [38]. In our study, we found AAMP suppression elevated sensitivity to icotinib, the first gen-eration EGFR TKI, in drug-resistant H1975 cells. This means AAMP can both promote EGFR activation and improve its resistance to TKI. Fur-thermore, our results also showed that AAMP downregulation could improve apoptosis caused by doxorubicin in NSCLC cells. Those suggest that AAMP may play an important role in drug-resistance include EGFR TKI and chemotherapeutics. Therefore, it is significant to develop in-hibitors of AAMP or drugs downregulate it and combination of them with EGFR TKI or chemotherapy drugs may achieve extraordinary ef-fects.
    Taken together, our data demonstrate that loss of AAMP inhibits cell proliferation by impairing EGFR activation and enhances icotinib and doxorubicin sensitivity in EGFR mutation and wide-type NSCLC cells. In this study, we identified a regulator of EGFR dimerization and high-lighted a novel mechanism of EGFR regulation in NSCLC cells, sug-gesting that approaches to downregulate AAMP or interrupt the inter-action of AAMP and EGFR can enhance the efficacy of EGFR-targeted therapy in cancer treatment. Meanwhile, the downregulation of AAMP can also enhance the lethal effect of chemotherapy drugs. These find-ings may offer key insights into developing novel strategy to treat lung cancers.