br APPBP interacts with microtubules
APPBP2 interacts with microtubules and is functionally associated with beta-amyloid precursor protein (APP) transport and/or processing . Microtubules participate in the formation of the spindle during cell division (mitosis) responsible for cell proliferation. APP is a membrane protein expressed in many tissues and has been implicated as a regula-tor of synapse formation , neural plasticity , and iron export . Interestingly, studies have suggested that APP controls cells viabil-ity, proliferation, migration, and aggressiveness in numerous types of cancers [23–26]. Based on these findings, we hypothesized that
Fig. 2. APPBP2 silencing suppressed proliferation and induced apoptosis in NSCLC cells.(a-d) The immunostaining results of BrdU indicates the proliferation of A549 cells (a,b) and H1299 cells (c,d) treated with sh-NC (a,c) or sh-APPBP2 (b,d). Blue signal represents DAPI. (e) The percentages of BrdU+DAPI+cells in DAPI+ cells. 189 cells, 221 cells, 135 cells, and 250 cells were counted from left to right column, respectively. (f) Effects of APPBP2 silencing on proliferation of A549 and H1299 stable cells by MTT assay (n = 3). (g,h) Effects of APPBP2 silencing on the colony formation of H549 (g) and H1299(h) stable cells, respectively. (i) The statistical results of colony formation of NSCLC stable cells, the colon numbers are 220, 130, 296, and 136 from the left to right column, respectively. (j) Knockdown of APPBP2 increased cell apoptosis. Cell death was determined by Annexin V and flow cytometric analysis. (k) The BYL719 of A549 (or H1299) stable cells was analysed by propidium iodide staining and flow cytometry. (l–n) APPBP2 silencing inhibited tumour growth of A549 stable cells in vivo. (l) Tumour pictures from mice treated with sh-NC (upper) and sh-APPBP2 (lower). (m) Growth curve of tumour volume was measured on indicated days and (n) tumour weight at the end of experiment. (o-
Fig. 3. APPBP2 silencing inhibits cells migration and invasiveness in NSCLC. (a,b) Effects of APPBP2 silencing on migration and invasion of A549 stable cells evaluated by transwell assays, n
= 3. (c,d) Effects of APPBP2 silencing on the migration and invasion of H1299 cells evaluated by transwell assays, n = 3. (e) Effects of APPBP2 silencing on migration of A549 stable cells
APPBP2 could participate in the oncogenesis of cancers through modu-lating microtubules and/or APP. Actually, the oncogenic roles of APPBP2 in numerous types of cancer has been reported [5–8,27]. However, the roles of APPBP2 in NSCLC are still poorly understood.
Molecular biology studies have demonstrated that cancers are a complexity of multi-stage biological process with the involvement of various factors. This includes the activation of oncogene and the inacti-vation of tumour suppressor gene, leading to functionally relevant mo-lecular aberrations. In the current study, the investigators compared the expression of APPBP2 mRNA between paired tumour and the adjacent normal tissues in two NSCLC cohorts, discovering a significantly ele-vated expression of APPBP2 in tumours relative to normal tissues. This result was confirmed in the samples of NSCLC and paired adjacent
normal tissues. Similar to our findings in NSCLC, overexpression/ampli-fication of the APPBP2 gene has been reported in other cancers, such as breast cancer , ovarian clear cell adenocarcinomas , desmoplastic medulloblastomas , andneuroblastomas . Taken together, these findings indicate that APPBP2 correlates with the oncogenesis of can-cers, including NSCLC. Uncontrolled proliferation and invasiveness are the key hallmarks of cancer cells and are the leading cause of NSCLC-related mortality. There-fore, the investigators undertook this study to shed light on the associ-ation of APPBP2 with the malignant phenotype of NSCLC cells. First, the investigators demonstrated that APPBP2 silencing suppressed cell proliferation and viability while inducing apoptosis in NSCLC cells. Next, the investigators confirmed that knockdown of APPBP2 inhibited
Fig. 4. APPBP2 regulated the expression of PPM1D and SPOP in NSCLC tissues and cells. (a) The heatmap of gene expression profiling organized by APPBP2 expression level by reanalysing the TCGA data. Samples were derived from human LUAD patients. Red circles indicate the genes to be studied. (b,c) PPM1D mRNA level (b) and SPOP mRNA level (c) had a positive linear relationship with the expression of APPBP2. (d) Immunostaining of PPM1D (brown colour) of human LUAD tissues and adjacent normal tissues. (e) Immunostaining of SPOP (brown colour) of human LUAD tissues and adjacent normal tissues. DAPI in (d) and (e) is indicated as blue colour. (f) PPM1D mRNA and SPOP mRNA expression level of human LUAD tissues and adjacent normal tissues were measured by RT-PCR. Yellow colour and cyan colour represent normal and tumour samples, respectively. n = 3. (g) RT-PCR results showed PPM1D mRNA and SPOP mRNA expression level in APPBP2 silenced cells of A549 or H1299 (n = 3). (h) PPM1D and SPOP were examined by western blotting on APPBP2 silenced A549 and H1299 stable cells. GAPDH acts as the internal standard. (i) co-IP between the APPBP2-flag and PPM1D or SPOP on A549 or H1299 cell contexts. (j) co-IP between PPM1D-His and APPBP2 or SPOP on A549 or H1299 cell contexts. Data are presented as mean ± SEM. (Two-tailed t-test for group comparison) 0.01 b *p b 0.05, 0.001 b **p b 0.01, ***p b 0.001 for indicated comparison. Scale bars: 50 μm.