Background: Lung adenocarcinoma is the most common type of lung cancer and one of the most lethal and prevalent cancers. Aberrant glycosylation was common and essential in tumorigenesis, with fucosylation as one of the most common types disrupted in cancers. However, it is still unknown whether genes involved in fucosylation are important for lung adenocarcinoma development and process. Methods: GMDS is involved in cellular fucosylation. Here we examined GMDS expression level at both mRNA and protein level in lung adenocarcinoma. The impact of GMDS knockdown on lung adenocarcinoma in vitro and in vivo was investigated. Transcriptome changes with GMDS knockdown in lung adenocarcinoma cells were also examined to provide insights into related molecular mechanisms. Results: GMDS expression is significantly upregulated in lung adenocarcinoma at both mRNA and protein levels. Lentivirus-mediated shRNA strategy inhibited GMDS expression efficiently in human lung adenocarcinoma cells A549 and H1299, and GMDS knockdown impaired cell proliferation, colony formation ability, induced cell cycle arrest, and apoptosis in both cell lines. Furthermore, GMDS knockdown inhibited tumorigenesis in a xenograft mice model of lung adenocarcinoma. Microarray analysis explored the GMDS-mediated molecular network and revealed that the CASP8- CDKN1A axis might be critical for lung adenocarcinoma development. Conclusions: These findings suggest that GMDS upregulation is critical for cell proliferation and survival in human lung adenocarcinoma and might serve as a potential biomarker for lung adenocarcinoma diagnosis and treatment. Keywords: Lung adenocarcinoma, GMDS, Cell proliferation, Survival, Tumorigenesis, Molecular network Background profiling of lung adenocarcinoma has been described and Lung cancer is one of the most lethal and prevalent human multiple oncogenic mutations have been identified, which cancers, and its five-year survival rate is currently less than might serve as markers for diagnosis and targeted therap- 20% [1, 2]. Lung cancer can be classified into non-small ies [5–7]. However, these results were far from sufficient, cell lung cancer (NSCLC) and small cell lung cancer and the diagnosis of many lung adenocarcinoma patients (SCLC), with NSCLC accounting for approximately 85% of remains poor, as most lung adenocarcinoma patients are all lung cancers . The most common subtype of NSCLC already at advanced or metastatic stages when first is lung adenocarcinoma, which accounts for approximately diagnosed owing to the lack of suitable diagnostic markers 40% of all lung cancers and leads to more than 500,000 [8, 9]. Thus, there is still an urgent need to explore the deaths each year . In recent years, with the development molecular pathways underlying lung adenocarcinoma of high-throughput sequencing technologies, molecular tumorigenesis and progression for insights into the identi- fication of appropriate markers, which will guide the development of novel diagnostic strategies and targeted * Correspondence: firstname.lastname@example.org Xing Wei and Kun Zhang contributed equally to this work. therapies in the future. Department of Lung Cancer, The Affiliated Hospital of Military Medical Glycosylation, a process of attaching saccharides to Sciences, The 307th Hospital of Chinese People’s Liberation Army, Beijing proteins, saccharides, or lipids, is an important post- 100071, China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wei et al. BMC Cancer (2018) 18:600 Page 2 of 14 translational modification involved in variant biological Methods physiological functions . Glycosylation defects have Tissue microarray and immunohistochemistry (IHC) for been linked to many pathophysiological processes, includ- GMDS ing inflammation, tumorigenesis, and cancer metastasis Tissue microarray (HLug-Ade150CS-01) containing 75 [11, 12]. Glycosylation can be divided into approximately pairs of formalin-fixed, Paraffin-embedded (FFPE) human 10 different kinds of oligosaccharide modifications accord- lung adenocarcinoma and adjacent normal sample were ing to the oligosaccharide structure, with fucosylation as obtained from Outdo Biotech Company (Shanghai, China). one of the most common types deregulated in cancers Detailed information about clinical parameters for these . In the process of cellular fucosylation, GDP-fucose patients was summarized in Table 1. Immunohistochemis- serves as the essential substrate, and its synthesis was try for GMDS protein expression status was carried out in driven by GDP-mannose-4,6-dehydratase (GMDS) and tissue microarray as follows: antigen unmasking was GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase performed using 10 mM sodium citrate buffer at 80 °C (FX) . As fucosylation changes have been linked to for 20 min. Tissue sections were then blocked in block- different cancers, and FX expression changes were also ing buffer for 60 min at 25 °C and further treated with observed in liver cancer cells, it is possible that GMDS GMDS antibody (Novus, NBP1–33424,1:100) at 4 °C might be involved in cancer development . GMDS for 12~ 16 h. After washing 2 times for 30 min using PBS mutations have been shown to be positively related to buffer, immunohistochemistry staining was performed colorectal cancer metastasis, and GMDS deficiency accounts using biotin-labeled secondary antibody, diaminobenzidine for tumor escape and resistance to cellular apoptosis in and counterstained with hematoxylin. Negative controls colon cancer cells [16–18]. were performed without primary antibody against GMDS. Here, we first analyzed gene expression profiling in 57 paired lung adenocarcinoma cases from The Cancer Table 1 Relationship between GMDS expression and clinical Genome Atlas (TCGA) database and found that GMDS pathological parameters (cases,%)by IHC staining expression was significantly upregulated in lung adenocar- Clinical pathological Case HSDL2 expression status P value cinoma tissues as compared to adjacent normal tissues. parameters number low high GMDS upregulation was further confirmed in specimens Ages(year) from lung adenocarcinoma patients using immunohisto- ≤ 65 111 53 58 0.080 chemistry. Then, two lung adenocarcinoma cell lines A549 and H1299 cells were chosen for further functional >65 54 18 36 analysis of GMDS. Lentiviral–mediated short hairpin Gender RNA (shRNA) specifically targeting human GMDS was Male 88 39 49 0.776 employed to inhibit GMDS expression in lung adenocar- Female 76 32 44 cinoma cells, and knockdown efficiency was confirmed at Tumor diameter(cm) both mRNA and protein level by quantitative real-time ≤ 5 137 62 75 0.203 polymerase chain reaction (qPCR) and western blot. GMDS knockdown in A549 cells and H1299 cells > 5 28 9 19 impaired cell proliferation and colony formation ability and Grades induced cell cycle arrest and apoptosis. Furthermore, nude I/II 126 54 72 0.936 mice tumorigenesis assays showed that GMDS knockdown III 39 17 22 inhibited tumor growth in vivo. To explore the molecular T staging mechanisms underlying GMDS-associated cellular pro- T1/T2 128 55 73 0.653 cesses, a microarray assay was used to investigate gene expression changes induced by GMDS knockdown, and T3/T4 37 16 21 potential targets of GMDS were further validated by N migration western blot. Taken together, our study revealed that Nx/N0 99 42 57 0.653 GMDS was upregulated in lung adenocarcinoma tissues, N1/N2/N3 63 29 34 which might serve as a biomarker for lung adenocarcin- M metastasis oma. We provided confidential evidence that GMDS M0 159 70 89 0.287 might serve as a tumor-promoting factor in lung adeno- carcinoma, which is in contrast to the reported anti-tumor M1 5 1 4 functions in colon cancers. It is necessary to further deter- TNM staging mine GMDS functions in other cancer types to provide a TNM1/2 112 51 61 0.580 comprehensive profile for GMDS in tumorigenesis and TNM3/4 49 20 29 progression. Wei et al. BMC Cancer (2018) 18:600 Page 3 of 14 GMDS expression status was determined by two patholo- Infections of human lung adenocarcinoma cells with gists blindly and independently with the following stan- lentivirus dards: intensity score was distinguished as score 3, strong Human lung adenocarcinoma cell line A549 cells and positive signal, 2, moderate positive signal, 1, weak positive H1299 cells were used for GMDS studies. In brief, cells signal and score 0, no staining signal; while positive rate were plated in 6-, 12- or 24-well plates according to wasscored as0,negative; 1, 1–25%; 2, 26–50%; 3, 51–75%; experiments and incubated in a 5% CO incubator at 4, 76–100%. The final immunoreactions score was quanti- 37 °C to achieve desired density. Then, lentivirus fied as GMDS immunoreactivity = intensity score*positive expressing either GMDS- or Scr-shRNA was added to rate. Samples with the final scores ≤2weredefined as the target plate (MOI = 5 and 5 ul lentivirus was used GMDS low status while others were considered as GMDS per well for 6-well plate). After culturing for another high status. Mann-Whitney U method was used for statis- 2–5 days, cellular infection efficiency was examined tical analysis. according to the percentage of GFP-positive cells observed using a fluorescence microscope. Expression profiles and clinical outcome analysis Fifty seven paired lung adenocarcinoma samples with RNA extraction, cDNA synthesis and real-time RNAseq data from TCGA was used for gene profiling quantitative PCR analysis. Expression profile of GSE31210 was downloaded A549 cells and H1299 cells infected with lentivirus and predictive value of GMDS with relapse-free survival expressing either Scr-shRNA or GMDS-shRNA were was analyzed with Pan Cancer Prognostics Database cultured for 5 days and then harvested for total RNA PROGgeneV2. extraction, reverse transcription and quantitative PCR. Briefly, Trizol reagent (Invitrogen, Carlsbad, CA, USA) was used for RNA purification. RNA was then quantified Cell culture using NanoDrop (Thermo, Rockford, IL, MA, USA) and Two human lung adenocarcinoma cell lines, namely the first-strand cDNA was produced using Oligo dT A549 cells and H1299 cells, were purchased from Cell primers (Sangon, Shanghai, China) and M-MLV reverse Bank of the Chinese Academy of Sciences (Shanghai, transcriptase (Promega, Madison, Wisconsin, USA). The China). A549 cells and H1299 cells were cultured in expression of GMDS and other genes was quantified with RPMI1640 medium containing 10% FBS and 1% antibi- SYBR mixture (Takara Biotechnology, Dalian, China) on a otics. These cell lines were cultured at 37 °C in a 5% Real-Time PCR machine TP800 (Takara Biotechnology, CO incubator. Dalian, China). Beacon designer 2 was used for primer design and these primers were synthesized by GeneChem (Shanghai, China). Lists of primers were as follows: Design of GMDS shRNA and lentivirus production GAPDH forward: 5’-TGACTTCAACAGCGACACC Short-hairpin RNA (shRNA) targeting human GMDS CA-3′; gene (sequence: 5’-CGTGAGGCGTATAATCTCTTT-3′) GAPDH reverse: 5’-CACCCTGTTGCTGTAGCCA was designed and oligonucleotides were synthesized by AA-3′; GeneChem (Shanghai, China).Thenoligonucleotides GMDS forward: 5′- TTTAATACGGGTCGAATTG were then annealed and inserted into a lentiviral vector AGCA-3′; pGCSIL-GFP with AgeI and EcoRI (both from NEB, GMDS reverse: 5′- TGAGATCGCCATAGTGCAA Ipswich, MA, USA). Lentivirus expressing GMDS shRNA CT-3′; was produced as previously described . The Lentivector SKA1 forward: 5′- ATGAAGAAACGAAGGATAC Expression System (GeneChem, Shanghai, China) was used CAAAG-3′; for lentivirus expressing GMDS shRNA (GMDS-shRNA) SKA1 reverse: 5′- CCTCGGACCTCTGATAGCC-3′; or scrambled shRNA (Scr-shRNA, negative control, VEGFA forward: 5’-GCTTACTCTCACCTGCTTC sequence: 5’-TTCTCCGAACGTGTCACGT-3′). TG-3′; VEGFA reverse: 5′- GGCTGCTTCTTCCAACAAT GMDS expression analysis using TCGA database G-3′; Fifty seven paired lung adenocarcinoma samples which DDIT3 forward: 5′- CTTCTCTGGCTTGGCTGAC have been analyzed by RNA sequencing in TCGA data- TGA-3′; base were selected and transcriptome information was DDIT3 reverse: 5′- TGACTGGAATCTGGAGAGT downloaded. Gene expression profiling for these paired GAGG-3′; samples were analyzed by Log (lung adenocarcinoma MAD2L1 forward: 5′- GAGTCGGGACCACAGTTTA tissues/adjacent normal tissues). T-3′; Wei et al. BMC Cancer (2018) 18:600 Page 4 of 14 MAD2L1 reverse: 5′- TTTTGTAGGCCACCATGCT another 2 days, cells were re-seeded in 96-well plates A-3′; with 2000 cells/well in triplicate. After incubating for MAP3K7 forward: 5′- CCGGTGAGATGATCGAAGC another 24 h, cell growth was examined with Cellomics C-3′; ArrayScan VTI (Thermo, Rockford, IL, MA, USA) once a MAP3K7 reverse: 5′- GCCGAAGCTCTACAATAAA day for 5 days to produce cell growth curves. CGC-3′; CDKN1A forward: 5′- CTGTCACTGTCTTGTACCC MTT assay TTGT-3′; Lentiviral-infected A549 cells and H1299 cells were cultured CDKN1A reverse: 5′- AAATCTGTCATGCTGGTCT to reach logarithmic phase. Cells were then collected for cell GC-3′; number counting using a hemocytometer. Then cells were FAS forward: 5′- CTTCTTTTGCCAATTCCAC-3′; re-seeded into 96-well plates with 2000 cells/well in tripli- FAS reverse: 5′- CAGATAAATTTATTGCCACTG-3′; cate for further culturing. Cells were treated with 20 μLof CASP8 forward: 5′- TTTCTGCCTACAGGGTCAT MTT solution (5 mg/mL) per well and incubated for 4 h. GC-3′; Then culture medium replaced with150 μLof DMSO CASP8 reverse: 5′- TGTCCAACTTTCCTTCTCC for formazan dissolving. After incubating for 5–10 min, CA-3′; the absorbance at 490/570 nm was examined using a JUN forward: 5′- CGCCAAGAACTCGGACCTC-3′; microplate reader. JUN reverse: 5’-CCTCCTGCTCATCTGTCACG-3′; Relative gene expression was normalized to GAPDH, and data analysis was performed using the delta-delta CT Colony formation assay method. As described previously , Lentiviral-infected A549 cells and H1299 cells were cultured for 2 days and har- Immunoblotting vested in the logarithmic phase. After cell counting, cells A549 cells and H1299 cells infected with lentivirus were re-seeded into six-well plates with 800 cells/well in expressing either Scr-shRNA or GMDS-shRNA were triplicate and cultured at 37 °C for 2 weeks. Cells were cultured for 2 days for protein isolation. In brief, cells then fixed using paraformaldehyde for 30–60 min and were washed with PBS buffer and harvested with lysis stained with GIEMSA for 20 min. After washing with buffer (100 mM Tris-HCl, pH = 7.4 l 0.15 M NaCl; ddH O thoroughly, cell plate imaging was obtained with 5 mM EDTA, pH = 8.0; 1% Triton X100; 5 mM DTT; micropublisher 3.3RTV (Olympus) for the quantification 0.1 mM PMSF) to extract total proteins which were of cell colonies. quantified with BCA Protein Assay Kit (Pierce, Rockford, IL, USA). To perform western blot analysis, 20 μg protein Cell cycle analysis with flow cytometry samples were mixed with loading buffer. Then SDS-PAGE Cell cycle analysis was done as previously reported . electrophoresis and subsequent PVDF transmembrane Lentiviral-infected A549 cells and H1299 cells were cultured were performed (Amersham Biosciences, Pollards Wood, for48or72h.Cells were harvestedandfixedwithcold70% UK). Membrane was blocked with 5% milk dissolved in ethanol for about 1 h. After PBS washing, cells were incu- TBST buffer for 1 h and then incubated with primary bated with PI buffer (40 × PI stock (2 mg/ml), 100 × RNase antibodies overnight at 4 °C. Primary antibodies used stock (10 mg/ml) and 1 × PBS buffer at a dilution of here were as follows: Rabbit anti-GMDS, Novus Biological, 25:10:1000). FACS Calibur (Becton-Dickinson, San Jose, CA, NBP1–33424 (1:500); Rabbit anti-CDKN1A, Abcam, USA) was then used to analyze cell cycle status. More than ab7960 (1:500); Mouse anti-DDIT3, Abcam, ab11419 1×10 cells per sample were used for each experiment, and (1:1000); Rabbit anti-FAS, Abcam, ab82419 (1:1000); experiments were performed in triplicate. Rabbit anti-JUN, Abcam, ab32137 (1:1000); Rabbit anti-VEGFA, Abcam, ab183100 (1:500); mouse anti-Flag, Sigma, F1804 (1:1000); mouse anti-GAPDH, Santa-Cruz, Annexin V-APC assay for cell apoptosis analysis sc-32,233 (1:2000). After washing with TBST buffer for Annexin V-APC apoptosis detection kit (eBioscience, San three times, specific HRP conjugated secondary antibodies Diego, CA, USA) was used here. Initially, Lentiviral-infected from SantaCruzwereaddedand immunoactivitywas A549 cells and H1299 cells were cultured for 96 h. Cells detected with ECL-Plus kit (Amersham Biosciences, were subsequently harvested to produce cell suspensions 6 7 Pollards Wood, UK). containing 1 × 10 –1×10 /ml cells using staining buffer. Then, 5 μl annexin V-APC was added into 100 μl cell Cell proliferation analysis using Cellomics ArrayScan VTI suspensions and incubated at 25 °C for 10–15 min. Lentivirus expressing GMDS- or Scr-shRNA was FACS Calibur (Becton-Dickinson, San Jose, CA, USA) added used here for cell infection. After culturing for was used for cell apoptotic analysis. Wei et al. BMC Cancer (2018) 18:600 Page 5 of 14 Caspase3/7 activity analysis Statistical analysis Caspase-Glo® 3/7 Assay (Promega, Madison, Wisconsin, GraphPad Prism 6 was used for data analysis, and all USA) was used for Caspase3/7 activity analysis according experiments were done in triplicate. Data are shown as the to the manufacture’s manual. In brief, A549 cells and mean ± SEM of three independent experiments. Student’s H1299 cells infected with lentivirus expressing either two-tailed t-test was chosen for statistical analysis and Scr-shRNA or GMDS-shRNA were cultured for 3–5 days, P < 0.05 was considered statistically significant. For the After harvesting and cell counting using a haemocytom- analysis of the difference in GMDS expression between eter, cells were seeded into 96-well plates at a density of lung adenocarcinoma samples and adjacent normal sam- 1×10 cells/well. Then 100 μl Caspase-Glo reaction ples, Fisher’s exact test was used. buffer were added into cells per well and cell plate were shaken constantly at 300–500 rpm for 30 min. Then Results signals were quantified after cells were incubated at Upregulation of GMDS expression in human lung room time for 1–2h. adenocarcinoma To identify candidate genes involved in human lung Tumorigenicity in nude mice adenocarcinoma tumorigenesis, transcriptomes of 57 Nude mice experiments were approved by the Institutional paired lung adenocarcinoma tissues were selected from Animal Care and Use committee and all nude mice were TCGA database and gene profiling analysis were performed. feeded strictly following the institution guidelines. A lung It was shown that GMDS expression at mRNA level was adenocarcinoma xenograft model was established as significantly upregulated in lung adenocarcinoma tissues as follows: Lentiviral-infected H1299 cells were cultured compared to adjacent normal tissues (Fig. 1a). We then to reach logarithmic phase. After harvesting and cell examined the correlation between GMDS expression at counting, cells suspensions of 2 × 10 cells/ml were mRNA level and prognosis in one patient cohort (using prepared with PBS buffer. Then approximately 4 × 10 cells data set GSE31210), and revealed that higher GMDS were injected into nude mice subcutaneously. Then mice expression was correlated with poor prognosis of lung were divided into two groups: control group were injected adenocarcinoma patients (Fig. 1b). Without fresh specimens with H1299 cells infected with Scr-shRNA lentivirus and in hand, we further examined GMDS expressions using knockdown group injected with H1299 cells infected with immunohistochemistry with only one suitable antibody with GMDS-shRNA lentivirus. Tumour diameter in these nude tissue microarray and confirmed the upregulation of GMDS mice was examined every other day from the 10th day for expression at protein level in human lung adenocarcinoma, 7 times two times a week. Tumor weight was determined with GMDS protein density at 3.597 ± 1.908 in human lung at 29th day after killing nude mice. adenocarcinoma and 0.453 ± 1.119 in adjacent normal tis- sues (Fig. 1c-d). Then relationship between GMDS protein Microarray analysis for gene expression profiles in A549 cells level and clinicopathological parameters was analyzed. How- A549 cells infected with lentivirus expressing either ever, no obvious correlation was observed between GMDS Scr-shRNA (n =3) or GMDS-shRNA (n=3) were cultured and any clinicopathological parameters including gender, and then total RNA was extracted using Trizol reagents. age, tumor size and pathologic grades (Table 1). In addition, NanoDrop 2000 and Agilent Bioanalyzer 2100 were used for GMDS proteinexpressionincommonlungadenocarcin- RNA examination. For gene expression analysis, Affymetrix oma cell lines A549, H1299 and SPC-A-1 was examined. As human GeneChip primeview was used according to man- compared to normal cell lines BEAS-2B, MRC-5 and uals as described previously . In brief, GeneChip 3’ IVT HEK-293 cells, GMDS protein was significantly upregulated Expression Kit was used for first-strand complementary in A549 and H1299 cells (Fig. 1e), both of which were used DNA synthesis, double-stranded DNA template conversion, for subsequent functional analysis. It is possible that GMDS in vitro transcription for aRNA synthesis and labelling. might be involved in the early stage of lung adenocarcinoma Microarray hybridization, washing and staining were done development, so the impact of GMDS expression on using GeneChip Hybridization Wash and Stain Kit. Gene- cell proliferation and survival in lung adenocarcinoma Chip Scanner 3000 was used for array scanning to produce was examined in the following studies. raw data. Gene expression profiles in A549 cells infected with lentivirus expressing Scr-shRNA (n=3) or GMDS- Silencing of GMDS expression in human lung shRNA (n = 3) were analyzed to identify differentially adenocarcinoma cells with lentiviral-mediated shRNA expressed genes based on the following criteria: P value < strategy 0.05 andabsolutefold change>2. Pathwayenrichment To inhibit GMDS expression in human lung adenocarcin- and gene network analysis were done based on Ingenuity oma cells efficiently, RNA interference (RNAi) based on Pathway Analysis (IPA). Microarray data is accessible lentivirus was used for GMDS knockdown in two human through GEO series accession number GSE104123. lung adenocarcinoma cells A549 and H1299. A549 cells Wei et al. BMC Cancer (2018) 18:600 Page 6 of 14 Fig. 1 GMDS expression levels in human lung adenocarcinoma tissues and cells. a GMDS mRNA level in human lung adenocarcinoma tissues and adjacent normal tissues. Fifty seven paired lung adenocarcinoma samples in TCGA were used (**, p < 0.01). b Kaplan-Meier relapse-free survival curves in lung adenocarcinoma patients stratified by GMDS expression level (p = 0.013). c Quantified GMDS protein level in 75 paired lung adenocarcinoma samples examined by Immunohistochemistry (**, p < 0.01). d Representative images of GMDS IHC staining in human lung adenocarcinoma and adjacent normal tissues. (Magnification, left is × 20, right is × 100). e GMDS protein level in different cell lines including BEAS-2B (1), MRC-5 (2), HEK-293 (3), A549 (4), H1299 (5), SPC-A-1 (6). A59, H1299 and SPC-A-1 are all human lung adenocarcinoma cell lines and H1299 cells were infected with lentivirus expressing expression at mRNA level in A549 and H1299 cell lines either scrambled shRNA (Scr-shRNA) or human GMDS- infected with lentivirus expressing GMDS-shRNA as specific shRNA (GMDS-shRNA). Lentivirus used here compared to control group, respectively (Fig. 2a). contained GFP expression cassette, which served as label- Furthermore, knockdown efficiency of GMDS shRNA ing marker for infection efficiency and cells with infection at protein level was determined in both A549 and efficiency > 80% were used for subsequent functional H1299 cell lines using western blot assay and GMDS analysis. Knockdown efficiency of GMDS shRNA was protein level reduced significantly in cells infected with examined using quantitative real-time PCR. It was shown lentivirus expressing GMDS-shRNA as compared to that approximately 70 and 80% reduction of GMDS control group (Fig. 2b). Wei et al. BMC Cancer (2018) 18:600 Page 7 of 14 Fig. 2 Impaired cell proliferation in human lung adenocarcinoma cell lines with GMDS knockdown via Cellomics ArrayScan VTI. a GMDS mRNA level in A549 cells and H1299 cells infected with lentivirus expressing either Scr-shRNA or GMDS-shRNA examined by quantitative real-time PCR (normalized to GAPDH mRNA). Data shown here was one out of three independent experiments (**, p < 0.01). b Relative GMDS protein level in A549 cells and H1299 cells infected with lentivirus expressing either Scr-shRNA or GMDS-shRNA examined by western blot. GAPDH protein was used as internal control. c-d. Representative microscope pictures of A549 cells (c) and H1299 cells (d) infected with lentivirus expressing either Scr-shRNA or GMDS-shRNA at different time points. e-f. Proliferation profiling of A549 cells (e) and H1299 cells (f) infected with lentivirus expressing either Scr-shRNA or GMDS-shRNA for continuous 5 days examined by Cellomics ArrayScan VTI. Histogram shown here was relative fold changes of cell numbers compared to Day 1 and representing the mean ± SEM of three independent experiments (**, p < 0.01) Impairment of cell proliferation ability by GMDS of GMDS on cell growth of lung adenocarcinoma. After knockdown in human lung adenocarcinoma cells culturing for 24 h, cell numbers were quantified using Two human lung adenocarcinoma cell lines A549 and the high-content screening system Cellomics ArrayScan H1299 were infected with lentivirus expressing either VTI every day for continuous 5 days (Representative Scr-shRNA or GMDS-shRNA to investigate the impact images for A549 and H1299 cells shown in Fig. 2c-d). Wei et al. BMC Cancer (2018) 18:600 Page 8 of 14 Results showed that impaired cell proliferation was obvi- knockdown led to impaired cell proliferation ability in ous in both cell lines at Day 2 and the inhibitory impact A549 and H1299 cell lines. of GMDS knockdown on cell proliferation was more obvious in the following 3 days (Fig. 2e-f). Inhibition of colony formation by GMDS knockdown in As unlimited cell proliferation ability was the key human lung adenocarcinoma cells feature of tumor initiation and development, we further Colony formation assay was used here to examine the determined the influence of GMDS knockdown on cell impact of GMDS knockdown on colony formation ability proliferation ability in both lung adenocarcinoma cell in human lung adenocarcinoma cells A549 and H1299. lines A549 and H1299 with MTT assay for continuous After culturing for 14 days, the colony formation ability 5 days. In consistent with results obtained with Cellomics was inhibited significantly in both A549 and H1299 cells ArrayScan VTI assay, MTT assay also revealed that GMDS infected with GMDS-shRNA as compared to cells infected knockdown led to impaired cell proliferation ability in both with Scr-shRNA (Fig. 3c-d). The average colony number cell lines (Fig. 3a-b). In A549 cells, impaired cell prolifera- reduced to 75 from 167 in A549 cells while the average tion was observed at Day 2 and the inhibitory effect was number reduced to 95 from 185 in H1299 cells. more obvious in the following 3 days (Fig. 3a) while in H1299 cells, impaired cell proliferation was observed at Cell cycle arrest induced by GMDS knockdown in human Day 3 and the inhibitory effect was still obvious in the lung adenocarcinoma cells following 2 days (Fig. 3b). Taken together, both Cellomics Cell proliferation and colony formation ability was ArrayScan VTI and MTT assay confirmed that GMDS closely associated with cell cycle progression, so it is Fig. 3 Impaired cell proliferation colony formation in human lung adenocarcinoma cell lines with GMDS knockdown. a-b Cell proliferation profiling in A549 cells (a) and H1299 cells (b) infected with lentivirus expressing either Scr-shRNA or GMDS-shRNA for continuous 5 days analyzed by MTT assay. Data shown here was relative fold changes of absorbance at OD490 compared to Day 1 and representing the mean ± SEM of three independent experiments (**, p < 0.01). c-d Colony formation in A549 cells (c) and H1299 cells (d) infected with lentivirus expressing either Scr-shRNA or GMDS-shRNA. Histogram shown here was average colony numbers and representing the mean ± SEM of three independent experiments (**, p < 0.01) Wei et al. BMC Cancer (2018) 18:600 Page 9 of 14 necessary to examine the influence of GMDS knockdown fluorescence-activated cell sorting (FACS). It was shown on cell cycle process to elucidate underlying mechanisms that cell cycle arrest was induced by GMDS knockdown in for GMDS-mediated cell growth abnormalities. Human both cell lines at 72 h after lentiviral infection. In A549 lung adenocarcinoma cell lines A549 and H1299 were cells, GMDS knockdown led to a significant G1 arrest with infected with lentivirus expressing either Scr-shRNA or a parallel G2/M-phase reduction and cell percentage at GMDS-shRNA and cell cycle distribution was analyzed G0/G1 phase, S phase and G2/M phase in cells infected using propidium iodide (PI) staining in combined with with GMDS-shRNA as compared to Scr-shRNA was 55.8% Fig. 4 Abnormal Cell cycle process and cell apoptosis in human lung adenocarcinoma cell lines with GMDS knockdown. a-b Cell cycle distribution in A549 cells (a) and H1299 cells (b) infected with lentivirus expressing either Scr-shRNA or GMDS-shRNA. Both cells infected with lentivirus expressing either Scr-shRNA or GMDS-shRNA were cultured for 72 h. After propidium iodide (PI) staining, cell cycle distribution was analyzed with flow cytometry. The graph represents the mean ± SEM of cell proportion in the G1 phase, S phase and G2/M phase from three independent experiments (*, p < 0.05; **, p < 0.01). c-d Cell apoptosis in A549 cells (c) and H1299 cells (d) infected with lentivirus expressing either Scr-shRNA or GMDS-shRNA. Both cells infected with lentivirus expressing either Scr-shRNA or GMDS-shRNA were cultured for 4 days and cell apoptosis was analyzed using ANNEXIN-V assay in combined with flow cytometry. Data shown are the mean ± SEM of cell percentage in apoptosis from three independent experiments (**, p < 0.01) Wei et al. BMC Cancer (2018) 18:600 Page 10 of 14 vs 58.2, 30.7% vs 31.1, and 14.4% vs 10.6%, respectively In vivo tumorigenicity was impaired in the xenograft mice (Fig. 4a); while in H1299 cells, GMDS knockdown led to a model of lung adenocarcinoma by GMDS knockdown significant G1 arrest with a parallel S and G2/M-phase Our studies in two human lung adenocarcinoma cells A549 reduction and cell percentage at G0/G1 phase, S phase and H1299 cells confirmed that GMDS was important for and G2/M phase in cells infected with GMDS-shRNA tumorigenesis of lung adenocarcinoma in vitro. However, in as compared to Scr-shRNA was 28% vs 46.1, 59.4% vs vivo evidence for the involvement of GMDS in tumorigen- 50.8, and 12.6% vs 3.1%, respectively (Fig. 4b). Furthermore, esis of lung adenocarcinoma remained elusive. Here we similar cell cycle arrest was also induced by GMDS knock- established xenograft mice model of lung adenocarcinoma down in both cell lines at 48 h after lentiviral infection with subcutaneously injection of H1299 cells infected with (Additional file 1: Figure S1a-b). These results implies that lentivirus expressing either Scr-shRNA or GMDS-shRNA GMDS participates in cell cycle regulation in human lung into nude mice. These mice were fed for 29 days for tumor adenocarcinoma. volume and weight analysis. As shown in Fig. 5a,Reduction of tumor size was obvious in nude mice injected with Induction of cell apoptosis by GMDS knockdown in cells infected with lentivirus expressing GMDS-shRNA human lung adenocarcinoma cells as compared to cells infected with lentivirus expressing Evasion of apoptosis is another hallmark of cancer, and Scr-shRNA. Then the tumor volume was quantified and would also promote cell growth and proliferation, so it confirmed that GMDS knockdown inhibited tumor growth is important to examine the cell apoptotic status in at all 5 measured time points (Fig. 5b). What’s more, tumor human lung adenocarcinoma cells infected with lentivirus weight was significantly lower in nude mice injected with expressing GMDS-shRNA. Cell apoptosis was analyzed GMDS-shRNA cells (Fig. 5c) as compared to nude mice using annexin V-APC assay in combined with FACS tech- injected with Scr-shRNA cells, as the mean weight was nology in A549 and H1299 cells infected with lentivirus reduced from 0.46 g to 0.08 g. expressing either Scr-shRNA or GMDS-shRNA after culturing for 4 days. In A549 and H1299 cells infected Global gene expression changes in human lung with lentivirus expressing Scr-shRNA, apoptosis was adenocarcinoma cells with GMDS knockdown observed in about 4% cells while in cells infected with To elucidate the underlying molecular mechanisms of lentivirus expressing GMDS-shRNA, percentage of cell the tumor suppressive roles of GMDS knockdown in apoptosis reached ~ 23% in A549 cells and ~ 15% in human lung adenocarcinoma cells, microarray analysis H1299 cells (Fig. 4c-d). was performed to examine the gene expression profiling Fig. 5 Tumorigenesis was inhibited by GMDS knockdown in xenograft mice model of lung adenocarcinoma. a Representative tumor pictures from nude mice injected subcutaneously with lung adenocarcinoma cell line H1299 cells infected with either Scr-shRNA or GMDS-shRNA. Tumor volume was examined from the 10th day after H1299 cell injections for 5 times with a frequency of 2 times a week. Nude mice were killed at the 29th day to check tumor weight. b Tumor volume in nude mice injected subcutaneously with lung adenocarcinoma cell line H1299 cells infected with either Scr-shRNA or GMDS-shRNA. Data shown here is the mean ± SEM of tumor volume from 10 nude mice in each group (**, p < 0.01). c Tumor weight in nude mice injected subcutaneously with lung adenocarcinoma cell line H1299 cells infected with either Scr-shRNA or GMDS-shRNA. Data shown are the mean ± SEM of tumor weight from 10 nude mice in each group (**, p < 0.01) Wei et al. BMC Cancer (2018) 18:600 Page 11 of 14 of A549 cells infected with lentivirus expressing either examine the impact of GMDS knockdown on the status of Scr-shRNA or GMDS-shRNA. A total of 739 genes CDKN1A-associated pathways including factors CDKN1A, showing significantly expression changes were detected, CASP8, MAP3K7, FAS, JUN, DDIT3, VEGFA, SKA1 and with P < 0.05 and > 1.5 absolute value of fold change, MAD2L1. It was shown that GMDS knockdown induced including 316 upregulated genes and 423 downregulated the expression of CDKN1A, CASP8, MAP3K7 and FAS genes (Fig. 6a). Then, disease and functional analysis with while inhibited JUN, DDIT3, VEGFA, SKA1 and MAD2L1 Ingenuity Pathway Analysis (IPA) revealed that cell cycle at mRNA level (Fig. 6b). Furthermore, at the protein level and cellular growth and proliferation were enriched in CDKN1A and FAS were also enhanced while VEGFA, GMDS-regulated gene sets in human lung adenocarcinoma DDIT3 and JUN were inhibited by GMDS knockdown cells. Furthermore, upstream analysis with IPA revealed (Fig. 6c). Out of these GMDS-regulated genes, it was that GMDS knockdown stimulated CDKN1A (p21) and noticed that cell death and survival was the most downstream pathways significantly (P= 1.1E-07). Then affected biological process, so we further performed real-time PCR and western blot were performed to caspase3/7 analysis to examine the impact of GMDS Fig. 6 Widespread changes of gene expressions and critical pathways in human lung adenocarcinoma cells A549 with GMDS knockdown. a Heatmap containing 739 differentially expressed genes in human lung adenocarcinoma cell line A549 infected with lentivirus expressing either Scr-shRNA (purple) or GMDS-shRNA (red) with the criteria P <0.05 and▏fold change▏ > 1.5. Genes and samples were listed in rows and columns, respectively. A colour standard for normalized expression data was shown at the bottom of the microarray heatmap (green represents downregulated genes while red represents upregulated genes). b Gene expression changes identified in microarray were confirmed using real-time quantitative PCR for selected genes CASP8, MAP3K7, CDKN1A, FAS, JUN, DDIT3, VEGFA, SKA1 and MAD2L1 in human lung adenocarcinoma cell line A549 infected with lentivirus expressing either Scr-shRNA or GMDS-shRNA. Histogram shown here was one out of three independent experiments (p<0.01) and normalized to GAPDH. c Protein level of FAS, VEGFA, DDIT3, JUN and CDKN1A in human human lung adenocarcinoma cell line A549 infected with lentivirus expressing either Scr-shRNA or GMDS-shRNA. GAPDH was used as internal control. d Caspase3/7 activity analysis in A549 cells and H1299 cells infected with lentivirus expressing either Scr-shRNA or GMDS-shRNA. Data shown are the mean ± SEM of caspase3/7 activity from three independent experiments (**, p < 0.01) Wei et al. BMC Cancer (2018) 18:600 Page 12 of 14 knockdown on caspase-mediated cell apoptosis process. It proteins and can be classified into N-glycans or O-glycans was shown that GMDS knockdown enhanced caspase3/7 depending on the covalently attachment patterns of gly- activity significantly in both A549 cells and H1299 cells cans via either nitrogen or oxygen linkages, respectively (Fig. 6d). Furthermore, IPA network analysis showed . In terms of the complexity, glycosylation exceeds that CASP8-CDKN1A axis was at the core of GMDS- most of other PTMs as it involves the linkage of divergent associated gene interaction network, emphasizing the carbohydrates ranging from monosaccharide to oligosac- core of CDKN1A in GMDS-mediated lung adenocar- charides and could occur in at least 9 of the 20 amino cinoma growth (Fig. 7). acids, so it is not surprising that glycosylation was essential for many biological processes and its abnor- Discussion malities account for many human diseases including Glycosylation is an important post-translational modifi- cancer [10, 23]. Indeed, glycosylation alterations in cation occurred in more than half of all known human tumor cells influence cell growth and survival, tumor Fig. 7 Modulation of CDKN1A-CDK1-mediated gene interaction network in human lung adenocarcinoma cell line A5492 cells after GMDS knockdown. Genes represented in green were downregulated while genes in red were upregulated in A549 cells after GMDS knockdown. Solid line represented direct interactions while dotted line represented indirect interactions. The meaning of abbreviations in the figure: A, activation; B, binding; C, causation/leads to; CO, correlation; CC, chemical-chemical interaction; CP, chemical-protein interaction; E, expression; EC, enzyme catalysis; I, inhibition; L, molecular cleavage; LO, localization; M, biochemical modification; miT, microRNA targeting; MB, group/complex membership; nTRR, non-targeting RNA-RNA interaction; P, phosphorylation/dephosphorylation; PD, protein-DNA binding; PP, protein-protein binding; PR, protein-RNA binding; PY, processing yields; RB, regulation of binding; RE, reaction; RR, RNA-RNA binding; T, transcription; TR, translocation; UB, ubiquitination Wei et al. BMC Cancer (2018) 18:600 Page 13 of 14 cell invasion and metastasis, tumor angiogenesis and pathways were activated in cells treated with GMDS- cell-microenvironment interactions [24–26]. In lung shRNA by IPA analysis. Further network analysis revealed cancer and especially lung adenocarcinoma, the involve- that CASP8-CDKN1A axis was at the core of GMDS-me- ment of glycosylation abnormalities in tumorigenesis has diated gene expression dataset. Both confirmed that been established previously [27, 28]. However, studies on CDKN1A was the core of GMDS-mediated lung adenocar- genes responsible for glycosylation dysfunctions in lung cinoma progression. It has been reported that CDKN1A adenocarcinoma are still limited. Here we focused on was involved in G1 phase of cell cycle process, cell prolifer- GMDS, a gene involved in glycosylation, and confirmed ation and apoptosis [33, 34], which is in accordance with its tumor-promoting role in lung adenocarcinoma in vitro the roles of GMDS in cell proliferation, apoptosis and cell and in vivo. cycle, as GMDS knockdown in lung adenocarcinoma cells GMDS is an important enzyme involved in guanosine led to impaired cell proliferation, enhanced cellular apop- diphosphate (GDP)-fucose synthesis and GDP-fucose is tosis and cell cycle retardation at G1 phase revealed in this the donor substrate of fucosylation, one of the most study. Indeed, GMDS knockdown induced the expression common type of cancer-associated glycosylation alterations of CASP8 and CDKN1A, which might be the underlying . Fucosylation abnormalities have been observed in molecular mechanisms for the observation that GMDS many cancer types including colorectal cancer, hepato- knockdown induced cell cycle arrest and cellular apoptosis. cellular carcinoma and papillary carcinoma of the thyroid [15, 30–32]. As a critical enzyme in fucosylation, GMDS Conclusions deregulation was also detected in colorectal cancer and Taken together, this study provided valuable insights into GMDS dysfunction led to tumor escape and resistance to the molecular mechanisms underlying GMDS-regulated cell cellular apoptosis in colorectal cancer cells [16–18]. How- growth in lung adenocarcinoma. Microarray and subsequent ever, these studies were just performed at cell level and network analysis are instructive for functional analysis of the expression status of GMDS in clinical caner specimens GMDS in different types of cancer. GMDS upregulation in has not been examined. What’s more, roles of GMDS in lung adenocarcinoma implies a potential biomarker and lung adenocarcinoma have not been described previously, targets for lung adenocarcinoma diagnosis and treatment. so according to our knowledge, this study was the first In the future, establishment of GMDS-CDKN1A axis and to systematically analyze the functional impact and mo- functional investigations would provide more valuable lecular mechanisms of GMDS in lung adenocarcinoma insights into lung adenocarcinoma. in vitro and in vivo. We first examined GMDS expression at mRNA level in human lung adenocarcinoma using transcriptome data of 57 paired human lung adenocarcin- Additional file oma tissues from TCGA database and showed that GMDS was upregulated in human lung adenocarcinoma as com- Additional file 1: Figure S1. Cell cycle arrest in human lung adenocarcinoma cell lines with GMDS knockdown 48 h after lentiviral pared to adjacent normal tissue. We further examined infection. a-b. Cell cycle distribution in A549 cells (a) and H1299 cells (b) GMDS protein density using tissue microarray in paired infected with lentivirus expressing either Scr-shRNA or GMDS-shRNA. Both cells human lung adenocarcinoma samples and confirmed the infected with lentivirus expressing either Scr-shRNA or GMDS-shRNA were cultured for 48 h. After propidium iodide (PI) staining, cell cycle distribution was upregulation of GMDS in human lung adenocarcinoma. analyzed with flow cytometry. The graph represents the mean ± SEM of cell However, no significant correlation was observed between proportion in the G1 phase, S phase and G2/M phase from three independent GMDS expression and any clinical pathological parame- experiments (*, p < 0.05; **, p < 0.01). (DOCX 151 kb) ters, which suggests that GMDS might be involved in the early stage of lung adenocarcinoma development. Functional Abbreviations importance of GMDS in tumorigenesis was confirmed as it FACS: Fluorescence-activated cell sorting; FX: GDP-4-keto-6-deoxymannose- was shown that GMDS knockdown led to delayed cell 3,5-epimerase-4-reductase; GMDS: GDP-mannose-4,6-dehydratase; NSCLC: Non-small cell lung cancer; PI: Propidium iodide; RNAi: RNA proliferation, impaired colony formation ability, cell interference; SCLC: Small cell lung cancer; shRNA: Short hairpin RNA; cycle arrest and increased apoptosis. Xenograft tumor TCGA: The Cancer Genome Atlas mouse model experiments further revealed that GMDS knockdown inhibited tumor growth in vivo. Taken together, Acknowledgements these results confirmed that GMDS is involved in tumori- We thank GeneChem (Shanghai, China) for the Ingenuity Pathway Analysis (IPA) used in this study. genesis of lung adenocarcinoma. In accord with the tumor-promoting roles of GMDS in lung adenocarcinoma described above, gene expression Funding This research was supported by National Natural Science Foundation of profiling analysis with microarray showed that genes essen- China (Grant number 81572266). The funder had no role in the design of the tially for cell survival and proliferation were regulated by study and collection, analysis and interpretation of data and in writing the GMDS. We revealed that CDKN1A (p21) and downstream manuscript. Wei et al. BMC Cancer (2018) 18:600 Page 14 of 14 Availability of data and materials 10. Ohtsubo K, Marth JD. Glycosylation in cellular mechanisms of health and All data and materials on which the conclusions of this study rely are disease. Cell. 2006;126(5):855–67. displayed in the paper. Microarray data is accessible through GEO series 11. Pinho SS, Reis CA. Glycosylation in cancer: mechanisms and clinical accession number GSE104123. implications. 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Published: May 29, 2018