br APPBP knockdown suppresses cells proliferation but induce
3.2. APPBP2-knockdown suppresses BYL-719 proliferation, but induces apopto-sis in NSCLC in vitro
As unlimited growth is one of the key hallmarks of cancers , we attempted to assess the influence of APPBP2 on cell proliferation and ap-optosis in NSCLC. First, lentivirus mediated sh-APPBP2 was applied to si-lence the APPBP2 expression in both A549 and H1299 cells, with a scrambled shRNA (sh-NC) as the negative control (Fig. S1a). Following the transfection of cells, qPCR and western blotting assays were per-formed to examine the endogenous APPBP2 expression. The results showed that the expression levels of APPBP2 dropped by about 70% (Fig. S1b-d).
= 3; Fig. 2c,d,e) cells. This was consistent with the results from MTT (Fig. 2f) and colony formation assays (Fig. 2 g–i). Flow cytometry assays revealed that cell apoptosis was dramatically enhanced in two cell lines transfected with sh-APPBP2 compared with sh-NC (Fig. 2j and Fig. S2a, b), but no significant differences were observed on cell cycles (Fig. 2k and Fig. S2c, d).
3.3. Silencing APPBP2 reduces NSCLC growth in vivo
Xenografts are models of cancer where the tissue or cell lines are im-planted into an immunodeficient or humanized mouse, which are used to create an environment that allows for the natural growth of cancer. To assess the action of APPBP2 on NSCLC growth in vivo, we constructed xenograft tumours of A549 and H1299 cells transfected with sh-APPBP2 and sh-NC respectively, followed by the investigation of tumour growth. Interestingly, we found that silencing APPBP2 reduced both the size (Fig. 2m, p) and weight (Fig. 2n, q) of xenografts.
3.4. APPBP2 silencing inhibits cell migration and invasiveness in NSCLC
Metastasis is the leading cause of NSCLC-related mortality, with about 90% of NSCLC patients succumbing to metastasis as opposed to their primary tumours .To elucidate the molecular mechanism
(s) underlying NSCLC metastasis, wound healing and transwell assays were performed to assess the roles of APPBP2 in the migration and inva-siveness of NSCLC cells. Transwell assays showed that the invasiveness of both A549 and H1299 cells was substantially suppressed by APPBP2 silencing (Fig. 3a–d). Similarly, wound healing assays suggested that the APPBP2 knockdown obviously impaired the migratory activity in both cell lines (Fig. 3e, f).
3.5. APPBP2-knockdown reduced the expression of PPM1D and SPOP in NSCLC
To clarify the molecular mechanism underlying the action of APPBP2 in NSCLC, the investigators analysed a heatmap of potential correlations which synchronized well with APPBP2 expression in human NSCLC samples from the TCGA cohort (TCGA-LUAD). The heatmap revealed
several cancerous genes (Fig. 4a), among which the expression of PPM1D and SPOP may be highly correlated with the APPBP2 levels in the TCGA-LUAD cohort (spearman ρ = 0·65 for PPM1D and 0·54 for SPOP; Fig. 4b,c). The investigators performed western blotting assays to explore the correlations of APPBP2 with PPM1D and SPOP in ten paired NSCLC tissues and found that three proteins of APPBP2, PPM1D, and SPOP were all enhanced in tumours relative to normal tissues (Fig. S3a,b). This was consistent with the results from the immunohisto-chemistry and qPCR assays (Fig. 4d–f). Moreover, the investigators found that APPBP2-knockdown significantly reduced the expression of PPM1D and SPOP in both A549 and H1299 cells, as determined by qPCR and western-blot assays (Fig. 4g, h).
3.6. Interaction of APPBP2 with PPM1D and SPOP in NSCLC
Co-immunoprecipitation (co-IP) is a popular technique to identify physiologically relevant protein–protein interactions by using target protein-specific antibodies to indirectly capture proteins that are bound to a specific target protein. To assess the protein interactions among APPBP2, PPM1D, and SPOP, co-IP experiments were performed on NSCLC cells using antibodies anti-Flag (fusion with APPBP2) as well as anti-His (fusion with PPM1D) and revealed that APPBP2 could bind with PPM1D (but not SPOP) in both A549 and H1299 cell contexts (Fig. 4i, j).
3.6.1. Overexpression of PPM1D and SPOP attenuate APPBP2-knockdown inhibition of cell proliferation and invasiveness in NSCLC
To evaluate the mediation of PPM1D and SPOP on the effect of APPBP2 on NSCLC, plasmids containing PPM1D and SPOP expression cassette were transfected into APPBP2-silenced A549 and H1299 cells to enhance the expression of PPM1D and SPOP. This was followed by the examination of cell proliferation, apoptosis, migration, and invasive-ness using colony formation, MTT, flow cytometry and transwell assays. APPBP2-knockdown inhibition of biological behaviours in NSCLC cells was strongly attenuated by the overexpression of PPM1D and SPOP. (Figs. 5, 6 and Fig. S3c–e).
Lung cancer remains the leading cause of cancer related deaths worldwide and targeted therapies are showing great promise in effec-tively treating certain patients [18,19]. Therefore, new targeted molecu-lar therapeutic strategies for lung cancer are needed to aid patients with this disease. Characterizing and targeting the functionally-relevant mo-lecular aberrations in lung cancer helps to identify new approaches to managing this disease. The investigators report on the oncogenic role of APPBP2 in NSCLC.