Primary antibodies against human YAP, phospho-AKT (p-AKT), AKT, ERK, phospho-ERK (p-ERK), N-cadherin and E-cadherin were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against cyclin D1, MMP-2, MMP-9, and GAPDH were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The anti-tubulin and anti-AMOT antibodies were purchased from Abcam (Cambridge, UK). Rabbit polyclonal antibodies to human Bax and Bcl-2 were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Human CRC cell lines LoVo, SW620, HT29 and HCT116 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Normal human colon mucosal epithelium cell line NCM460 was ordered from the Incell Corporation, LLC (San Antonio, TX, USA). The CRC cells were all maintained in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin. The NCM460 cells were cultured in an M3F base medium containing 10% FBS. All cells were incubated at 37°C in a 5% CO₂ incubator (Life Technologies, Baltimore, MD, USA).

The knockdown and overexpression of AMOT in LoVo and HCT116 cells were performed using the lentiviral expression system provided by GeneChem Co., Ltd. (Shanghai, China). For AMOT overexpression and inhibition, the lentiviral vectors for human AMOT small hairpin RNA (shRNA) and vectors expressing AMOT (LV-AMOT) were constructed, packed and purified by GeneChem Co., Ltd. RNA interference sequences were used as follows: sh-AMOT-1, 5′-TGCAGAGATGGTGGAATAT-3′; sh-AMOT-2, 5′-ACACATCGAAATCCGAGAT-3′; the scrambled shRNA (sh-NC), 5′-TTCTCCGAATGTGTCACGT-3′. For transfection, cells were plated into 24-well plates. To increase the expression of AMOT, LoVo cells were transfected with LV-AMOT or control lentivirus (Lv-NC) in accordance with the manufacturer's instructions. The lentivirus encoding AMOT shRNA or sh-NC was introduced into HCT116 cells. Approximately 8 h later, cells were incubated with fresh DMEM. After a 48-h incubation, the stable clones were selected for treatment with puromycin for 3 weeks. The efficacy of the lentivirus transfection was evaluated by western blotting.

To silence the expression of YAP in LoVo cells, small interfering RNA (siRNA) targeting human YAP and scramble siRNA (siR-con) were designed and synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). The siRNA sequences were siR-YAP, 5′-GGUGAUACUAUCAACCAAATT-3′; siR-con, 5′-CCUACGCCACCAAUUUCGU-3′. After seeding into 6-well plates for 12 h, the cells were transfected with the siRNA duplexes (100 nM) aforementioned, using Lipofectamine 2000 (Invitrogen Life Technologies Carlsbad, CA, USA) according to the manufacturer's instructions. At 48 h post-transfection, the transfection efficiency was assessed by western blotting.

To quantify the mRNA levels of AMOT, total RNA from the indicated cells was extracted using RNAiso Plus (Takara Bio Inc., Otsu, Japan) according to the manufacturer's instructions. Then, the obtained RNA was reversely transcribed into the first strand cDNA using the High-Capacity cDNA Reverse Transcription kits (Applied Biosystems, Foster City, CA, USA). The specimens were then subjected to RT-PCR with the specific primers for AMOT (sense, 5′-CCAGAATATCCCTTCAAG-3′; antisense, 5′-GAGTTCCTGGCTGACAAT-3′). qRT-PCR was performed with a final volume of 20 µl according to the guidelines of the SYBR Premix Ex Taq™ II kit (Takara Bio Inc.). Human GAPDH was applied as the endogenous control. The relative expression of the target genes was calculated using the 2^−ΔΔCt method.

Cells from the aforementioned samples were lysed with RIPA buffer, and the extracted protein concentrations were quantified using the BCA protein assay kit (Beyotime Institute of Biotechnology, Haimen, China). Then, 30 µg of lysates was separated on SDS-PAGE and electroblotted onto PVDF membranes (Millipore, Bedford, MA, USA). After blocking the non-specific binding with 5% nonfat milk in TBST buffer, the membranes were incubated with the primary antibodies against human AMOT, PCNA, ERK, p-ERK, p-AKT, AKT, cyclin D1, Bcl-2, Bax, MMP-2, MMP-9, N-cadherin, E-cadherin and YAP at 37°C for 1 h. After 3 washes with TBST, a secondary antibody conjugated to horseradish peroxidase (HRP) was added for a further 1-h incubation. The immunoreactive bands were visualized using an ECL detection reagent (Beyotime Institute of Biotechnology). The band intensities were quantified by Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

LoVo cells from the different treatments were harvested and lysed on ice for 20 min with lysis buffer, followed by centrifugation. For immunoprecipitation, 10 µg of anti-YAP antibodies were pre-treated with 50 µl protein A-Sepharose beads (Invitrogen Life Technologies) in 500 µl of lysis buffer at 4°C for 15 min. Then, the aforementioned mixture was added into the lysates at a volume of 500 µl for a 1-h incubation. After washing with ice-cold lysis buffer, the formed immunocomplex was eluted by heating for 5 min at 95°C. The immunoprecipitated proteins were separated by SDS-PAGE, then subjected to immunoblotting with the specific antibodies against AMOT.

Briefly, cells were seeded into a 96-well plate and incubated for 1–4 days after transfection. Then, the culture medium was removed and replaced with fresh medium including 5 mg/ml MTT (Sigma-Aldrich). After further incubation for 5 h at 37°C, the remaining supernatant was discarded and 200 µl of DMSO was introduced to dissolve the formed crystal formazan. The color reaction was detected by measuring the absorbance at 570 nm with an enzyme immunoassay analyzer (Bio-Rad Laboratories, Inc.).

The cell apoptosis ratio was evaluated using an Annexin V-FITC/PI Apoptosis Detection kit (Beyotime Institute of Biotechnology). At 48 h after transfection, cells were collected by trypsinization and suspended in 500 µl of binding buffer. Then, 10 µl Annexin V-FITC and 5 µl PI (Sigma-Aldrich) were added for a 15-min incubation at room temperature in the dark. The samples were subjected to flow cytometry (FACScan; BD Biosciences, San Jose, CA, USA) in a device equipped with CellQuest software to assess the apoptotic cells.

Cell invasion and migration assays were performed with or without Matrigel-coated inserts in the Transwell chambers (Millipore) according to the manufacturer's instructions. After treatment with the indicated conditions, cells in 200 µl serum-free medium were seeded into the upper chamber. The medium including 10% FBS was used as a chemoattractant to add into the lower chamber. Approximately 24 h later, the non-invading or non-migrated cells were removed by scrapping with a cotton swab. After fixation with 100% methanol, the invasive or migratory cells were stained with 0.5% crystal violet (Sigma-Aldrich) for 15 min at room temperature. The number of cells that had migrated or invaded through the membrane was quantified by counting the cells from at least three random fields with an inverted microscope.

Data were obtained from at least three independent experiments and are presented as the mean ± SD. All data analysis was carried out using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). The statistical comparisons among the groups were evaluated using a Student's t-test and ANOVA. P<0.05 was defined as statistically significant.

In order to explore the function of AMOT in the progression of CRC, a preliminary cohort study was performed in a series of CRC cell lines. In contrast to the NCM460 normal colon epithelium cell line, the mRNA levels of AMOT were markedly higher in the four examined CRC cell lines (Fig. 1A). Concomitantly, western blotting showed the notable increase of AMOT protein levels in CRC cells when compared to NCM460 cells (Fig. 1B).

To dissect the effect of AMOT in CRC cells, AMOT protein levels were effectively increased in LoVo cells by LV-AMOT transfection (Fig. 2A). An MTT assay corroborated that overexpression of AMOT markedly increased the proliferation rate of the LoVo cells (Fig. 2B), concomitant with a similar increase in the expression of the PCNA protein, a common marker for cell proliferation (Fig. 2C). To further evaluate the role of AMOT overexpression on cell apoptosis, 5-fluorouracil (5-FU) was added to induce low basal apoptosis levels. As shown in Fig. 2D, AMOT upregulation obviously suppressed 5-FU-induced cell apoptosis from 29.87 to 16.94%. To better investigate the function of AMOT in CRC cell growth, we constructed the stably AMOT-silenced cell line HCT116 by LV-AMOT shRNA transfection (Fig. 2E). In contrast, knockdown of AMOT expression in HCT116 cells markedly dampened the cell proliferation ability (Fig. 2F), accompanied by a corresponding decrease in PCNA expression (Fig. 2G). Compared to the control or sh-NC groups, AMOT silencing markedly increased the apoptotic ratio to 15.87% (Fig. 2H). Collectively, these results indicate that AMOT promotes CRC cell growth and protects CRC cells against apoptosis.

Based on the pro-growth role of AMOT in CRC cell growth, we further defined its effects on CRC cell metastatic potential by detecting cell invasion, migration and EMT. As shown in Fig. 3A, LV-AMOT-treated LoVo cells exhibited a statistically significant increase in cell invasion, compared with the control groups. Furthermore, an increase in AMOT markedly enhanced the number of LoVo cells migrating through the membrane (Fig. 3B). EMT is widely accepted as a major contributor to cancer cell metastasis. As expected, AMOT overexpression also reduced the expression of epithelial marker E-cadherin and increased the expression of mesenchymal marker N-cadherin (Fig. 3C). To further corroborate the aforementioned effects, the expression of AMOT was knocked down by LV-AMOT shRNA treatment. Blocking AMOT expression significantly antagonized the invasion ability of HCT116 cells (Fig. 3D). Simultaneously, the number of migrating HCT116 cells was also notably attenuated when cells were transfected with LV-AMOT shRNA (Fig. 3E). Notably, downregulation of AMOT inhibited EMT by increasing the expression levels of E-cadherin and decreasing the levels of N-cadherin (Fig. 3F).

AMOT has been reported to interact with YAP to stimulate its activity (7). Upon dephosphorylation, YAP is activated and then translocated to the nucleus to exert its oncogenic potential by activating downstream signaling (12). We further clarified the relationship between AMOT and YAP in CRC. In HCT116 cells, an antibody against YAP efficiently knocked down AMOT (Fig. 4A). To explore the effect of AMOT on YAP activity, the expression levels of phosphorylated YAP (p-YAP) were analyzed. As shown in Fig. 4B, p-YAP levels were mitigated in AMOT-overexpressing HCT116 cells (Fig. 4B). Moreover, upregulation of AMOT reduced the expression of cytoplasmic YAP (Fig. 4C). Notably, treatment with LV-AMOT significantly increased the relative expression of nuclear YAP (Fig. 4D). These data indicated that AMOT induces the activity of YAP in CRC cells.

To elucidate the underlying molecular mechanism involved in the AMOT-induced oncogenic effect in HCT116 cells, we explored the role of YAP during this process. Western blotting illustrated the knockdown of YAP in HCT116 cells transfected with YAP siRNA (Fig. 5A). A function assay validated that silencing of YAP markedly antagonized the pro-proliferation effect of AMOT overexpression in HCT116 cells (Fig. 5B). Furthermore, AMOT upregulation notably enhanced cell resistance to 5-FU-induced apoptosis, which was obviously attenuated following YAP downregulation (Fig. 5C). Concomitantly, knockdown of YAP significantly attenuated the invasion (Fig. 5D) and migration (Fig. 5E) of HCT116 cells triggered by AMOT upregulation. Collectively, these results revealed that AMOT may promote CRC cell growth, invasion and migration mainly through YAP.

Abnormal activation of the ERK and PI3K/AKT pathways has been reported in various carcinomas and they play critical roles in carcinogenesis by regulating cell proliferation, invasion and migration (13–16). To further elucidate the mechanism involved in AMOT-mediated oncogenic potential, both of these two pathways were explored further. In line with our hypothesis, overexpression of AMOT markedly induced the expression of p-ERK and p-AKT, but not the total ERK and AKT (Fig. 5F). A previous study confirmed that YAP can transcriptionally activate the PI3K/AKT and ERK signaling pathways in human cutaneous squamous cell cancer (12). Further analysis of the mechanism demonstrated that silencing of YAP expression markedly attenuated AMOT-induced expression of p-ERK and p-AKT (Fig. 5G), accompanied by the decrease in the subsequent expression of cyclin D1, Bcl-2 and the increase in the expression of Bax (Fig. 5H). Additionally, the downstream increases in MMP-2 and MMP-9 levels in AMOT-elevated cells were obviously attenuated after YAP siRNA transfection (Fig. 5I). Consistently, YAP knockdown also inhibited AMOT-induced EMT by increasing E-cadherin expression and suppressing N-cadherin levels. All of these results implied that AMOT could induce the activation of the YAP-ERK/PI3K-AKT signaling pathway.