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  • br Using plate clone assays to

    2020-07-29


    Using plate clone assays to investigate long-term proliferation of tumor cells, we found the number of tumor clusters decreased after AAMP was silenced in two weeks of culture (Fig. S2A, B and E). As expected, the number of tumor clusters increased after AAMP over-expression (Fig. S2C and D). Meanwhile, we found the ability of pro-liferation and clonogenicity positively correlated with the level of AAMP (Fig. S3A, B).
    We also conducted soft agar clone formation assays in vitro to in-vestigate cancer cell growth in a 3D environment in the absence or overexpression of AAMP. We found clone numbers were significantly reduced when AAMP was silenced in Calu-1 Galactose 1-phosphate  (Fig. 1E). However, more cell clones were detected after AAMP overexpression compared with the control cells (Fig. 1F). In summary, these data show AAMP promotes NSCLC cell proliferation and clonogenic ability in vitro. In-terestingly, we observed that the expression level of AAMP was also closely related to cell migration ability (Fig. S3C).
    3.2. Downregulation of AAMP suppressed H1792 xenograft growth in vivo
    We explored the role AAMP plays in tumor growth in vivo. In these experiments, H1792 pGIPZ-shAAMP-1 and pGIPZ-shAAMP-2 cells, with corresponding control cells pGIPZ-luc, were injected in the subcutis of
    A
    AAMP
    p-ERK
    ERK
    CCND1
    ACTB
    C
    AAMP
    ERK
    p-ERK
    ACTB
    B
    AAMP
    p-ERK
    ERK
    CCND1
    ACTB
    D
    AAMP
    EGFR
    ERK
    p-ERK
    ACTB
    B-NSG mice. The sizes of heterogeneous tumors were continuously measured until mice were sacrificed. The AAMP levels of the three H1792 cell lines are shown by western blot (Fig. 2A). Both H1792 pGIPZ-shAAMP-1 and pGIPZ-shAAMP-2 cell line xenografts grew more slowly compared with control group (Fig. 2A). In addition, the tumor weights were significantly less in H1792 pGIPZ-shAAMP xenografts compared with the control group (Fig. 2B). Western blot also showed that the phosphorylation of EGFR (Y1173) and ERK were lower in pGIPZ-shAAMP tumor tissues than that in controls (Fig. 2C). No sig-nificant difference in mouse weight was found among the three groups (Fig. S1E). These results demonstrate that AAMP promotes tumor cell growth in vitro and in vivo.
    3.3. AAMP promoted EGFR dimerization and phosphorylation at Tyr1173
    We wanted to understand how AAMP promotes cell proliferation and tumorigenesis and of other proteins participate in this process. Data suggest AAMP and EGFR interact with each other [23]. EGFR belongs to the HER family and plays a critical role in cell growth and proliferation [24]. We performed co-IP experiments to understand the relationship between these two proteins and found AAMP interacts with exogenous and endogenous EGFR in NSCLC cells (Fig. 3A and B). To locate the binding region, we divided EGFR into three fragments, including ex-tracellular and transmembrane fragment (ETTM), cytoplasmic and transmembrane fragment (CTTM) and cytoplasmic fragment (CT) (Fig. 3C). Co-IP results show AAMP interacts with the cytoplasmic part of EGFR (Fig. 3D).
    Next,we wonder the biological function of the combination. Given that protein dimerization is a precondition of EGFR activation [25], we asked whether AAMP would promote EGFR dimerization. Here, we 
    chose EGF as the agonist of EGFR. Calu-1 cells were transfected with the plasmid pcDNA3.1-EGFR-HA and pcDNA3.1-EGFR-FLAG in the pre-sence of AAMP knockdown. Co-IP results show that downregulation of AAMP inhibited EGFR dimerization (Fig. 3E). As reported, phosphor-ylation of EGFR is crucial for its activity, particularly at the Tyr1173 residue [26,27]. We questioned whether AAMP silencing would repress EGFR phosphorylation. Our experiments show reduction of AAMP in-hibited the phosphorylation of EGFR at Tyr1173 (Fig. 4A and B), and increased AAMP led to higher EGFR phosphorylation (Fig. 4C and D). Taken together, our data suggest that AAMP interacts with EGFR and promotes its phosphorylation by facilitating dimerization.
    3.4. AAMP activated downstream signaling pathway of EGFR
    As a receptor tyrosine kinase, EGFR initiates multiple signaling pathways [28]. Among these, the ERK1/2 MAPK pathway induces a wide array of physiological and pathological responses, such as cell growth and proliferation [29,30]. Given that AAMP interacts with EGFR and promotes its phosphorylation, we asked whether AAMP af-fects ERK1/2, the downstream signaling pathway of EGFR. Our data show ERK1/2 phosphorylation was reduced after AAMP knockdown (Fig. 5A) and increased after AAMP overexpression (Fig. 5B). To test whether the variation of p-ERK1/2 was influenced by AAMP expres-sion, we performed rescue experiments by AAMP inhibition with RNAi in Calu-1 and H1792 cells and expression was recovered by pcDNA3.1-AAMP plasmid transfection. Results show ERK1/2 phosphorylation was restored after AAMP expression was rescued (Fig. 5C).