NUCKS promotes cell proliferation and suppresses autophagy through the mTOR-Beclin1 pathway in gastric cancer

The NUCKS antibody (12023–2-AP) was purchased from Proteintech (Wuhan, China). Polybrene (sc-134,220) was purchased from Santa Cruz Biotechnology (Shanghai, China). Antibodies against mammalian target of rapamycin (mTOR) (ab32028) and phosphor-S2448 mTOR (ab84400) were purchased from Abcam (Shanghai, China). The Cyclin Antibody Sampler Kit (no. 9869), apoptosis antibody (no. 5625, 9664, 3498), Phospho-p70 S6K antibody (no.9234) and Autophagy Antibody Sampler Kit (no.4445) were purchased from Cell Signaling Technology (Shanghai, China). The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (M5655), 5′-bromo-2-deoxyuridine (BrdU), and dimethyl sulfoxide (DMSO) (D5879) were obtained from Sigma-Aldrich. The tubulin antibody, 4′, 6-diamidino-2-phenylindole (DAPI) and 3, 3′-diaminobenzidine (DAB) were purchased from Beyotime (Shanghai, China). HRP goat anti-mouse and goat anti-rabbit antibodies were purchased from KPL, Inc. (Maryland, USA). Alexa Fluor 488 goat anti-rabbit IgG (H + L), Lipofectamine 2000, and puromycin (A1113803) were obtained from Life Technologies (Shanghai, China). The BrdU antibody (ab6326) was purchased from Abcam (Shanghai, China). All antibodies were diluted according to the instructions. Chloroquine and rapamycin were acquired from Must Bio-technology (Chengdu, China) and Beyotime (Shanghai, China), respectively.

All gastric cancer cell lines were purchased from ATCC (Rockville, MD, USA) and cultured in Roswell Park Memorial Institute-1640 medium supplemented with 10% FBS and 1% P/S. The 293FT cells were cultured in 293FT growth medium consisting of DMEM with 10% FBS, 1% P/S, 0.5 mg/mL G418, 4 mM L-glutamine, 0.1 mM nonessential amino acids, and 1 mM sodium pyruvate. The 293FT transfection medium, 293FT growth medium without P/S, and G418 were used in the present study. All cell lines were cultured at 37 °C under a humidified atmosphere with 5% CO₂. Growth media, FBS, antibiotics, and supplements were acquired from Thermo Fisher Scientific (Chengdu, China).

The negative control plasmid pLKO.1-shGFP was purchased from Addgene (Massachusetts, USA). The shNUCKS sequences (Table S1) were inserted into the pLKO.1 vector. As described previously, the cDNA sequence of human NUCKS was purchased from Youbio Biological Technology Co., Ltd. (Changsha, China), which was cloned into the pCDH-CMV-MCS-EF1-GFP-Puro vector provided by GeneCreate (Wuhan, China) [27]. The Beclin1 shRNA plasmid was purchased from Sigma-Aldrich (TRCN0000299864), and the plasmid GFP-LC3B [28] was a gift from Prof. Ning Gao at the Third Military Medical University, China. Lentiviruses were generated by co-transfecting the 293FT cell line with the packaging plasmids pLP1, pLP2, and pLP/VSVG and the corresponding shRNA plasmids. Lipofectamine 2000 was used for all transfections according to the manufacturer’s instructions. Virus-containing supernatants were harvested and used for cell infections at a final concentration of 4 mg/ mL polybrene. At 24 h after the final round of infection, the cells were cultured in the presence of 2 mg/mL puromycin, and the drug-resistant cells were selected and pooled for subsequent experiments.

Patient and gene expression data were downloaded from the TCGA: The Cancer Genome Atlas (https://​cancergenome.​nih.​gov) and UCSC Xena (https://​xena.​ucsc.​edu/​public/​). Kaplan-Meier survival analysis was conducted based on the high versus low NUCKS expression cutoff determined using the R packages.

To gain insight into the biological processes and signaling pathways associated with NUCKS expression in gastric cancer, GSEA was performed using the Broad Institute GSEA version 4.0 software. The TCGA database was downloaded from UCSC Xena (https://​xena.​ucsc.​edu/​public/​). The gene sets used for the enrichment analysis were downloaded from the Molecular Signatures Database (MsigDB, http://​software.​broadinstitute.​org/​gsea/​index.​jsp).

Total RNA was extracted from cells using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Then, 2 μg of RNA was reverse transcribed into cDNA for each sample. The mRNA expression was based on Ct values and was normalized to the values of glyceraldehyde-3-phosphate dehydrogenase as a control. The relevant primer sequences are presented in Supplementary Table S2.

Cells were lysed in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% SDS with a complete protease inhibitor cocktail (Roche) and phosphatase inhibitors (Sigma-Aldrich). The proteins in lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membrane. SDS-PAGE gels were calibrated using Magic Mark XP Western Standard (Invitrogen). After being blocked with skim milk for 2 h, the membrane sections were incubated with the primary antibody and the corresponding secondary antibody. Primary antibodies were used at a dilution according to the instructions, while secondary antibodies were used at a dilution of 1:5000. Finally, bound antibodies were detected by chemiluminescence using the ECL Prime Western blotting detection system (GE Healthcare), and images were analyzed with a Lumino Imager (LAS- 4000 mini; Fuji Film Inc.).

The human paraffin embedded stomach tissue array (ST812) was purchased from US Biomax (Rockville, MD) though Shanxi Alenabio Biotechnology ltd. (Shanxi, China). As the manufacturer’s suggested protocols, the array was incubated with NUCKS antibodies at 4 °C overnight, followed by incubation with HRP-conjugated secondary antibodies at room temperature for 3 h. Staining was visualized using DAB, and tissues were counterstained with hematoxylin. Information about age, gender and tumor stages of donors is available on the website https://​www.​biomax.​us. The ethics for collecting human tissue samples and the informed consent of patients have been verified by US Biomax, lnc.

Cell growth curves were obtained using the MTT assay. To assess cell viability, cells were seeded at a density of 8 × 10²/per well in 200 mL of medium in 96-well plates. The MTT assay was performed at the indicated time points from day 0 to 5, according to the manufacturer’s instructions. All experiments were independently repeated three times.

For BrdU staining, 1 × 10⁴ cells were cultured on coverslips in 24-well plates. Then, the cells were incubated with 10 μg/ml BrdU for 30 min, washed with 1 × PBS and fixed with 4% paraformaldehyde for 15 min. Then, samples were blocked with 5% goat serum for 2 h before being incubated with a primary antibody against BrdU for 1 h, followed by Alexa Fluor® 594 secondary antibody. DAPI was used for nuclear staining, and the percentage of BrdU incorporation was calculated from at least 10 randomly selected fields.

For cell cycle analysis, 1 × 10⁶ cells were harvested into 60 mm plates and fixed in ice-cold 70% ethanol overnight at 4 °C, incubated with propidium iodide, and then analyzed using flow cytometry (BD Biosciences, San Jose, CA, USA) with Cell Quest (BD Biosciences, San Jose, CA, USA).

For the in vitro soft agar assays, growth medium with 0.6% agarose was added to 6-well plates to form the lower layer, while growth medium with 0.3% agarose and 7 × 10² cells was added to form the upper layer. Colonies were imaged and recorded after 2–3 weeks of growth. Subcutaneous xenograft were used for the in vivo study. The lateral backside of NOD/SCID female mice was subcutaneously injected with 2 ~ 3 × 10⁶ cells. Cells in which NUCKS was stably silenced were implanted into the right side of each mouse, while control cells were implanted into the left side. Calipers were used to measure the tumor volume, which was calculated using the formula volume = (π/6) × length × width². After 4 weeks of growth, the mice were euthanized, and tumors were removed and weighed. At the endpoint, the original tumors were imaged, and fixed in neutral buffered formalin, and embedded in paraffin. Hematoxylin and eosin (H&E) staining and immunohistochemical analysis were performed for histopathological evaluations of the tissues. All mice were raised and monitored under SPF conditions. Animal welfare and experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of Southwest University.

For immunofluorescence staining, 2 × 10⁴ cells were seeded on coverslips in 24-well plates and cultured for 24 h. The cells were then washed with PBS, fixed in 4% paraformaldehyde for 15 min at room temperature, and then permeabilized using 0.1% Triton X-100 for 10 min. Coverslips were blocked in 10% goat serum for 1 h, after which the cells were incubated with primary antibodies against microtubule-associated protein light chain 3 beta (LC3B) before being incubated with Alexa Fluor 488 goat anti-rabbit IgG (H + L) as the secondary antibody. Finally, DAPI was used for nuclear staining.

To investigate whether NUCKS is a novel biomarker for diagnosing gastric cancer, we first performed immunohistochemical staining on primary tissue microarray samples from gastric cancer patients. The results showed that NUCKS expression levels were significantly increased in the malignant gastric tumor compared to those observed in adjacent normal tissues (Fig. 1a). Then we confirmed the status of NUCKS as a putative amplified target in gastric cancer by evaluating data from the Cancer Genome Atlas (TCGA) and observing a significant difference in NUCKS levels in the tumors and adjacent normal tissues (p < 0.05; Fig. 1b). A Kaplan-Meier analysis of overall patient survival was performed to show that NUCKS was highly expressed in 223 of 393 cases of gastric cancer, and high expression was significantly correlated with reduced patient survival in the TCGA dataset (TCGA samples-478), whereas low NUCKS expression was correlated with good overall survival (p < 0.01; Fig. 1c). Similarly, high NUCKS expression was shown to be a factor for poor patient prognosis in datasets from other clinical databases, such as TCGA samples (407; Fig. 1d), TCGA samples (450; Fig. S1A), OncoLnc STAD samples (378; Fig. S1B). In addition, univariate cox regression analysis results showed that age, depth of invasion, lymph node metastasis, Lauren’s histological type and NUCKS expression were significantly interrelated with overall survival (Table 1). Furthermore, multivariate cox regression analysis confirmed age (P = 0.011), depth of invasion (P = 0.018), and lymph node metastasis (P < 0.001) as independent prognostic factors of the overall survival of patients with gastric cancer (Fig. 1e). Subsequently, we examined NUCKS expression in several gastric cancer cell lines (HGC-27, SGC-7901, and MKN-45) and normal stomach cells (GES-1) and observed that NUCKS was commonly expressed in all cell lines through both quantitative reverse transcription PCR assays and Western blot analyses. The cell line GES-1, which is a normal stomach cell line, exhibited relatively lower levels of NUCKS expression (Fig. 1f and g). Taken together, these results demonstrated that NUCKS could be a meaningful prognostic marker and may play an oncogenic role in tumor development.

Next, we knocked down NUCKS in two gastric cancer cell lines, HGC-27 and SGC-7901, by independently transducing three short hairpin RNA (shRNA) sequences, shNUCKS#1, #2 and #3. Western blot and qRT–PCR assay results showed that shNUCKS#1 and #2 most successfully knocked down NUCKS expression, whereas shNUCKS#3 exhibited a relatively lower efficiency in both HGC-27 and SGC-7901 (Fig. 2a). We then investigated cell viability after knocking down NUCKS in the two cell lines using shNUCKS#1 and #2 respectively. MTT assay results demonstrated that the shNUCKS groups resulted in a significant decrease cell growth (Fig. 2b). The 5′-bromo-2-deoxyuridine (BrdU) assay results consistently showed that the BrdU-positive rates in shNUCKS groups were much lower than those observed in the corresponding control groups (Fig. 2c). Then, we examined the cell cycle distribution of NUCKS knockdown and control cells by flow cytometry and observed that NUCKS knockdown induced cell-cycle arrest at S phase (Fig. 2d). To confirm the results, we measured the expression of some cyclins and CDKs, which can promote cells to pass the S-phase checkpoints and observed that the levels of CDK2, Cyclin E2 expression were decreased but that of p21 was increased following NUCKS knockdown (Fig. 2e). Taken together, these results indicated that NUCKS silencing can reduce cell proliferation and induce the cell-cycle arrest of gastric cancer cells.

To further confirm the effect of NUCKS on cell proliferation, we overexpressed NUCKS in shNUCKS#1 knockdown cells. The overexpression efficiency was examined at the mRNA level, and the results showed that NUCKS was successfully overexpressed after being knocked down by shNUCKS#1(Fig. 3a). To investigate the influence of NUCKS overexpression on cell growth and proliferation, MTT and BrdU staining assays were performed. The results demonstrated that the restoration of NUCKS after knocking it down rescued cell growth and proliferation (Fig. 3b and c). Furthermore, we measured the expression of CDK2 and Cyclin E2, which can promote cells to go through the G1/S checkpoint by Western blot analysis. The data showed that the restoration of NUCKS rescued CDK2, Cyclin E2 and p21 expression (Fig. 3d). Taken together, these findings suggest that NUCKS is indispensable for gastric cancer cells growth and proliferation, and regulates cell cycle progression by downregulating the CCNE-CDK2 complex.

To elucidate the molecular mechanisms that underlie the observed NUCKS silencing-mediate inhibition on cell growth and proliferation, we first assessed whether NUCKS silencing could induce gastric cancer cell apoptosis by Western blot analyses. Western blot results showed the apoptosis proteins activated in gastric cancer cells after down-regulating NUCKS (Fig. 4a). This results are consistent with the reported role of NUCKS in the induction of apoptosis in gastric cancer cells [29]. The light microscope images showed that cells changed morphology and displayed high number of vacuoles after silencing NUCKS (Fig. 4b). Therefore, further experiments were performed to investigate the effect of NUCKS on autophagy status in gastric cancer cells. As LC3B is a specific marker of autophagy initiation, we transiently transfected gastric cancer cells with a GFP-LC3B plasmid and examined the cells under a fluorescence microscope. Interestingly, the shNUCKS#1 group showed a punctate pattern of GFP-LC3B fluorescence that indicated the recruitment of LC3B-II to autophagosomes and the formation of autophagic vacuoles. This result differed from the diffuse LC3B-associated green fluorescence observed in the control group. In contrast, in NUCKS-restored gastric cancer cells, this punctate pattern of GFP-LC3B fluorescence was significantly decreased compared to that observed in the shNUCKS#1/GFP group (Fig. 4c). To further confirm our findings, immunofluorescence analysis was performed to assess the conversion of LC3B-I to LC3B-II and the progression of autophagy. As predicted, the formation of LC3B-positive puncta markedly increased after NUCKS silencing and decreased after NUCKS rescue (Fig. 4d). These results are in agreement with the biochemical findings, as demonstrated by the increased conversion of LC3B and decreased levels of p62 (Fig. 4e). To distinguish whether the increase in LC3-II was due to autophagy induction or a block in downstream steps, LC3 turnover assays were performed. As shown in Fig. 4f, using CQ, an inhibitor of autophagic flow, we demonstrated that autophagy was induced by NUCKS knockdown. Taken together, our results demonstrated that NUCKS silencing induces autophagy in gastric cancer cells.

To investigate the role of NUCKS in gastric cancer progression, GSEA analyses were used to evaluate the potential pathways through which NUCKS functions. We observed that NUCKS was negatively associated with the autophagy process (Fig. 5a), and positively associated with the mTOR signaling pathway (Fig. 5b). To test whether downregulation of NUCKS silencing induces autophagy through the mTOR pathway, the mTOR and downstream protein expression was assessed by Western blot analysis. As shown in Fig. 5c, NUCKS knockdown obviously upregulated Beclin1 levels and downregulated those of phosphorylated mTOR (Ser-2448) and total mTOR level compared to that observed in the control group. In contrast, the levels of these proteins in the NUCKS-restored groups were all significantly rescued compared with that observed their corresponding control groups (Fig. 5c), and the downregulation of mTOR levels was also confirmed by qRT–PCR assays (Fig. 5d). To further to confirm the above results, we knocked down Beclin1 in NUCKS-silenced HGC-27 and SGC-7901 cells and observed that the recruitment of LC3B-II was also blocked (Fig. 5e). Then, we performed a rescue experiment and inhibited the mTOR pathway using rapamycin, a mTOR inhibitor. Compared with that observed in the control groups, rapamycin treatment partly reversed the effect of NUCKS overexpression on the rescue of proteins in the mTOR signaling pathway (Fig. 5f). Taken together, these data suggest that autophagy activation resulting from NUCKS silencing is mainly dependent on mTOR-Beclin1 pathway activation.

Subsequently, we examined the clonogenic abilities of the two cell lines after NUCKS knockdown using soft agar assays in vitro. As shown in Fig. 6a, smaller and fewer colonies were observed for the NUCKS knockdown cells compared with that observed in the controls. However, larger and greater numbers of colonies were observed for the NUCKS-overexpressed cells, compared with that observed using the control cells (Fig. 6a). Subcutaneous xenograft experiments with NOD/SCID mice were carried out and the results showed that the tumors formed by the NUCKS-knockdown SGC-7901 cells grew much slower, while those formed by the NUCKS-overexpressed SGC-7901 cells grew much faster (Fig. 6b). At the termination of the experiment, the mice were sacrificed, and the tumors were excised. The weights of tumors formed by NUCKS-knockdown cells were lower than those observed in the control group, while those formed by the NUCKS-overexpressed SGC-7901 cells were higher (Fig. 6c). These results indicated that NUCKS can promote the tumor growth of gastric cancer cells. To determine whether NUCKS enhances the tumor progression of gastric cancer cells by promoting cell proliferation, NUCKS and Ki-67 expression were examined in the tumor xenografts tissues by IHC analysis. As shown in Fig. 6d, the NUCKS and Ki-67 expression in the tumor tissues formed by the NUCKS-knockdown SGC-7901 cells was decreased compared with that observed in the control cells. In contrast, NUCKS and Ki-67 levels were also rescued in the tumor samples in the NUCKS-overexpressed SGC-7901 cells (Fig. 6d). Taken together, these results indicated that NUCKS most likely enhanced the tumor progression of gastric cancer cells by promoting cell growth and proliferation.