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Golgi integral membrane protein 4 manipulates cellular proliferation, apoptosis and cell cycle in human head and neck cancer
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Xiaobo Cui, MD * 1, Dongxue Gao, MD1, Yunfei Bai, MD 1, Yaping Wang, MD 1, Boqian
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Wang, MD 1, Wei Wang, MD 1
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Inner Mongolia Medical University Affiliated Hospital, Hohhot, Inner Mongolia, People’s Republic of China
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*Corresponding Author: Xiaobo Cui,MD, Section of Inner Mongolia Medical University
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Affiliated Hospital Otorhinolaryngology,No.1,Tongdao North Street, Hohhot, 010050, Inner
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Mongolia, People’s Republic of China. Tel: +86 (0471) 3451386, Fax: +86 (0471) 3451386,
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Email:
[email protected]
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Summary statement: The incidence of global head and neck cancer has increased markedly
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in the last 10 years, and its prognosis is poor. Here we identify GOLIM4 as an oncogenic
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protein in head and neck cancer by participating in cell proliferation and apoptosis.
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Abbreviations: GOLIM4, Golgi integral membrane protein 4; STIM1, Stromal interaction
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molecule 1; BrdU, 5-Bromo-2'-Deoxyuridine; shRNA, short hairpin;
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Short title: GOLIM4 knockdown suppresses growth of head and neck cancer.
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Abstract
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The incidence of global head and neck cancer has increased markedly in the last 10 years, and
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its prognosis is poor, which seriously endangers people's life and health. At present, there are
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few studies on its pathogenesis. Golgi integral membrane protein 4 (GOLIM4) is a major
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member of the Golgi apparatus transporter complex, and its role in tumor is unclear. This
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study found that GOLIM4 was the key target protein downstream of STIM1, which can
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inhibit the proliferation of head and neck cancer cells FaDu (Human pharyngeal squamous
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carcinoma cell) and Tca-8113 (human tongue squamous carcinoma cell) with knockdown of
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GOLIM4 by lentivirus. And the decreased expression of GOLIM4 induced cellular apoptosis.
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Further experiments revealed that FaDu cell cycle progression was changed after GOLIM4
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silence, G1 phase arrest and the number of G2/M cells decreased significantly. It was also
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found that the cells in S phase decreased markedly after GOLIM4 was knockdown compared
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with the control group by BrdU incorporation experiment. In conclusion, we found that
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GOLIM4, as the target gene downstream of STIM1, inhibited the proliferation of head and
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neck cancer, promoted apoptosis and regulated cell cycle progression, and GOLIM4 is a novel
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oncogene in head and neck cancer and might help developing promising targeted therapies for
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head and neck cancer patients.
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Key words: GOLIM4, head and neck cancer, apoptosis, proliferation, cell cycle
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1. Introduction
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The incidence of global head and neck cancer has increased markedly in the last 10 years, and
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over 90% of head and neck cancer are squamous cell carcinoma. The prognosis of head and
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neck cancer is not optimistic, due to its rapid growth and infiltration. Because of the
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anatomical structure and the importance of the preservation of organ function, the treatment
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of head and neck cancer is complicated, the operation or nonoperative treatment not only
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affects the patient's survival, but also greatly affects the patient's quality of life. Postoperative
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recurrence and metastasis remained the main cause of death in patients with head and neck
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cancer，such as oral tongue squamous cell carcinoma (1). Therefore, it is urgent to study the
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pathogenesis and development of head and neck cancer.
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The imbalance of Ca2+ homeostasis plays an important role in the development of tumor (2).
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The dynamic equilibrium of cytoplasmic calcium concentration regulates several intracellular
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signaling pathways, which are related to the expression of many growth factors and cytokines.
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These affect many basic cellular functions, such as cell proliferation, migration,
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differentiation, apoptosis, and gene regulation (3). And store-operated Ca2+ entry (SOCE) (4)
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plays a key role in maintaining Ca2+ homeostasis.
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In our previous study, we found that the expression of calcium channel protein STIM1
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(Stromal interaction molecule 1), a member of SOCE complex in head and neck cancer was
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significantly higher than that in the adjacent tissues. Furthermore, the expression of STIM1
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can be detected in FaDu, um-scc-17a and HEP-2 head and neck cancer cell lines. Knockdown
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of STIM1 dramatically inhibited the proliferation rate and the cloning ability of FaDu cells
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and the proportion of apoptotic cells increased. Moreover, silencing STIM1 significantly
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restrained tumor growth in nude mice (5-7). In order to further define the molecular
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mechanism of STIM1 in OSCC, we used gene microarray and bioinformatics analysis，and
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found that Golgi integral membrane protein 4 (GOLIM4) was the target of STIM1. GOLIM4
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is a component of the Golgi transport complex and plays an important role in the transport of
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Golgi proteins (8). Although it has been reported that the Golgi apparatus can be involved in
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the biological process of tumor, the functions of GOLIM4 in the development of
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tumorigenesis are not clear.
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In this study, we found that the STIM1-GOLIM4 signal axis played a pivotal role in head
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and neck cancer. Consistent with STIM1, the expression of GOLIM4 protein in head and
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neck cancer cells was high, and low GOLIM4 significantly inhibited the proliferation of head
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and neck cancer cell lines. Moreover, the low expression of GOLIM4 induced apoptosis and
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regulated cell cycle process, resulting in cellular G1-phase block, and the number of S-phase
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cells significantly reduced. This suggested that STIM1 can influence the proliferation,
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apoptosis and cell cycle progression of head and neck cancer by regulating the expression of
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GOLIM4.
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2. Materials and methods
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2.1 Cell lines and cell culture
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The cell lines FaDu (Human pharyngeal squamous carcinoma cell) and Tca-8113 (human
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tongue squamous carcinoma cell) were purchased from Cell Bank of the Chinese Academy of
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Sciences in Shanghai and cultured in DMEM medium (10-013-CVR, Corning) containing 10 %
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fetal bovine serum (VS500T,Ausbian), 100U/ml penicillin and 0.1 mg/ml streptomycin at 37℃
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in a humidified atmosphere containing 5% CO2.
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2.2 High-content screening (HCS) assay
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Cell viability was detected the by the Celigo™ cytometer (Nexcelom) using 96-well plates.
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FaDu and Tca-8113 cells were seeded 3,000 or 1500 cells per well with 100 μl culture
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medium. After 24 hours, cells plates were read once a day using Celigo™ cytometer to
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accurately calculate the number of green fluorescent cells from each scan of a plate for five
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consecutive days. Each experiment was analyzed in triplicate and at least three independent
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experiments were performed.
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2.3 Cell proliferation assay
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Cell proliferation was detected by 5-Bromo-2'-Deoxyuridine (BrdU) incorporation assay,
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measuring DNA synthesis using the BrdU Cell Proliferation ELISA kit (11647229001,
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Roche). Cells were seeded in 96-well plates at least for three independent experiments. The
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experiment was performed according to the manufacturer’s protocol. The number of
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proliferating cells is represented by the level of BrdU incorporation which directly correlates
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to the absorbance values and the OD was quantified at 450 nm with a reference wavelength of
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630 nm. 2.4 Relative quantitative PCR
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Cell total RNA was extracted using Trizol reagent (3101-100,Pufei Biotechnology) according
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to the manufacturer’s instructions. Trizol prepared RNA was reversely transcribed into cDNA
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using M-MLV Reverse Transcriptase (M1705,Promega). Based on SYBR Green PCR Master
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Mix (DRR041B,TaKaRa), relative quantitative PCR was subsequently performed on
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Lightcycler 480 Ⅱ (Applied Roche) using cDNA as template. GAPDH was used as a control
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for normalization. The relative GOLIM4 expression was normalized to GAPDH, and data
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analysis was conducted using the comparative CT method. The PCR primers used were as
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follows: GOLIM4 forward primer: 5’-AAACTCTATGCTCCCACCC-3’ , Reverse primer:
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5’-GCTGCTCTTTCCACTCCC-3’,
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5’-TGACTTCAACAGCGACACCCA-3’,
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CACCCTGTTGCTGTAGCCAAA-3’.
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2.5 Western blot analysis
GAPDH Reverse
forward primer:
primer: 5’-
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Cell lysates were prepared with RIPA lysis buffer (P0013B,Beyotime) at 4℃. Protein
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concentrations were measured by BCA Protein Assay Kit (P0010S,Beyotime). Proteins were
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fractionated using SDS-polyacrylamide gel electrophoresis and then electroblotted onto 0.45
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μm PVDF membranes and then blocked in 5% non-fat milk dissolved in TBST for 1 hour at
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room temperature. The membranes were incubated with primary antibodies overnight, washed
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in TBST for 10 min three times, and then incubated with the HRP-conjugated secondary
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antibody. Primary antibodies against GAPDH (sc-32233,Santa Cruz) were purchased from
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Santa Cruz Biotechnology. The antibodies against GOLIM4 was purchased from Abnova
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(PAB28477). The antibodies against MDM2 (ab38618), CDK6 (ab151247) and GADD45
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(ab180768) were purchased from Abcam. Secondary antibody goat anti-rabbit IgG-HRP
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(sc-2005,Santa Cruz) were obtained from Santa Cruz Biotechnology. The blots were detected
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using Pierce™ ECL Western Blotting Substrate kit (M3121,Thermo Scientific).
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2.6 RNA Interference
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The lentivirus-mediated short hairpin RNA (shRNA) expressing vector was constructed and
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synthesized from Genechem Technology (Shanghai, China). The sequences of shRNA
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targeting genes were showed in Table 1. Cells were seeded at ~50% confluence and infected
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at a multiplicity of infection (MOI) of 50 and 5 μg/ml polybrene. 48 h after infection, the cells
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were cultured in DMEM containing puromycin (2 μg/ml) to obtain the stably infected cells.
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2.7 Cell apoptosis measurement
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Cell apoptosis was evaluated by Annexin V Apoptosis Detection Kit APC (88-8007, BD
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Bioscience). After 4 days lentivirus infection, FaDu and Tca-8113 cells were harvested and
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stained according to manufacturer’s protocol. Data acquisition and analysis were performed in
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a Guava easyCyte HT (Millipore) and data were analyzed and calculated using FlowJo
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software.
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2.8 Cell cycle analysis
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Cell cycle assay was determined by propidium iodide (P4170,Sigma) staining using a flow
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cytometer. In brief, cells infected with control lentivirus or GOILM4-shRNA lentivirus were
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seeded in 6-well plates and culture to 80% confluence, then harvested by trypsinization and
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fixed with 70% cold ethanol at 4℃ for 6 hours to overnight. Then, cells were incubated with
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10 mg/ml RNase (EN0531,Fermentas) for 30 min at room temperature. DNA distribution was
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analyzed by FACS on a Guava easyCyte HT (Millipore) cytometry and data were analyzed
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and calculated using FlowJo software.
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2.9 Statistical analysis
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The statistical analyses were performed using SPSS 16.0 software (SPSS, Inc., Chicago,
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IL,U.S.A.). Eachexperimentis conducted at least three independent repeats. Student’s t-tests
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were used to analyze the differences between two groups. A probability value of less than
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0.05 was considered significant.
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3. Results 3.1 GOLIM4 is elevated in head and neck cancer
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To identify the genes which regulated by STIM1 that affect the growth, apoptosis and cell
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cycle of head and neck cancer cells, we silenced STIM1 in FaDu cells (Human pharyngeal
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squamous carcinoma cell) and found 20 candidate genes significantly down-regulated. Then
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we used lentivirus to knockdown these 20 candidate genes in FaDu cells (Table 1), and tested
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the effect of candidate genes on cell proliferation. We found that knockdown of GOLIM4 and
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DLGAP5 could significantly inhibit proliferation of FaDu cells (Figure 1A). The fluorescence
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intensity of cells knockdown of GOLIM4 was observed under microscope, and it was found
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that the fluorescence intensity of cells knockdown of GOLIM4 decreased significantly
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compared with the negative control group (Figure 1B). And analysis the number of cells also
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found that knockdown of GOLIM4 significantly inhibited the growth of FaDu cells (Figure
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1C). Furthermore, we compared the expressions of GOLIM4 in 44 normal tissues and 521
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head and neck squamous cell carcinoma from TCGA (The Cancer Genome Atlas) database,
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and found that the expression of GOLIM4 was significantly higher in tumor tissues (Figure
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1D). In addition, we also found a positive correlation between the expression of GOLIM4 and
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STIM1 in head and neck tumor tissues (Figure 1E). 3.2 Knockdown of GOLIM4 inhibits head and neck cancer cell viability
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In order to further clarify the effect of GOLIM4 on cell viability, we first examined the
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expression of GOLIM4 at RNA and protein levels with lentivirus infection, and found that the
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knockdown efficiency reached more than 60% (Figure 2A-2D). Then, we use Celigo
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experiment to detect the effect of GOLIM4 on the two head and neck cancer cell lines FaDu
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cells and Tca-8113 cells (human tongue squamous carcinoma cell), According to the
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fluorescence intensity, the group that knockdown of GOLIM4 had lower active cells than the
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control group since day 4 (Figure 2E), and that the number of active cells decreased
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significantly (Figure 2F). It is proved that GOLIM4 can maintain cell proliferation activity,
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the low expression of GOLIM4 can inhibit the growth of head and neck cancer cells. 3.3 GOLIM4 affects the cell cycle progression of head and neck cancer cells
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As GOLIM4 regulating cell proliferation activity, we wanted to explore whether GOLIM4
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affects the cell cycle of head and neck cancer cells. Therefore, we used the flow cytometry to
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detect the changes in the cell cycle of FaDu and Tca-8113 cells after knockdown of GOLIM4
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(Figure 3A and 3C). We found that the percentage of cells in G1-phase was increased by
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GOLIM4 knockdown, whereas the cells in the G2/M phase were significantly reduced (Figure
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3B and 3D). The results showed that low expression of GOLIM4 could cause G1-phase arrest,
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suggesting that the inhibitory effect of shGOLIM4 on cell growth may be caused by cell cycle
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block in G1 phase. Then we detected the expression of three key genes of p53 pathway
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MDM2, CDK6 and GADD45A when knockdown of GOLIM, and found that MDM2, CDK6
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were decreased while GADD45A was increased (Figure S1A). This is consistent with the
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effect of STIM1 on head and neck cancer cell cycle.
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3.4 Decreased GOLIM4 expression results in reduced S-phase cells
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In addition, we further applied BrdU incorporation assay to test the effect of GOLIM4
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expression on the number of S-phase cells. It was found that knockdown of GOLIM4 in FaDu
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and Tca-8113 cells could significantly reduce the number of S-phase cells (Figures 4A and
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4B). This result implicates that GOLIM4 can not only lead to G1 phase arrest, but also
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significantly keep cells from entering into S phase, thus regulating the cell proliferation. 3.5 Low expression of GOLIM4 induces apoptosis of head and neck cancer cells
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Finally, we also used flow cytometry to detect the effect of GOLIM4 expression on cellular
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apoptosis (Figure 5A and 5C). Compared with the control group, the proportion of annexin V
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positive cells in GOLIM4 knockdown group was increased significantly (Figure 5B and 5D),
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which indicated that the number of early-stage apoptotic cells elevated by GOLIM4
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knockdown. Therefore, GOLIM4 can maintain cell viability by reducing apoptosis of head
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and neck cancer cells.
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4. Discussion
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Our research further revealed the molecular mechanism of STIM1 in head and neck cancer,
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which regulated cell proliferation, apoptosis and cell cycle, and found GOLIM4 was the target
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gene downstream of STIM1. Results showed that knockdown of GOLIM4 could inhibit cell
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proliferation, promote apoptosis, and change cell cycle progression. These results revealed the
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mechanism of STIM1-GOLIM4 signaling pathway in the development of head and neck
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cancer, and expounded the regulation network of endoplasmic reticulum-Golgi interaction in
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the formation of head and neck cancer.
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Some studies have found that the Golgi apparatus can participate in the process of tumor
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biology. For example, Golgi (Golgi protein, GOLPH2) participates in the development of
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hepatocellular carcinoma and is considered to be a potential indicator of liver cancer serum (9,
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10). Golgi protein GOLM1 is related to the development of prostate cancer (11).
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Phosphatidylinositol 4-phosphate can regulate the adhesion of tumor cells thus influence the
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metastasis of breast cancer (12), and it is reported that the polarization of the Golgi body also
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participates in the metastasis of breast cancer (13). In addition, the Golgi apparatus also
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participate in colorectal cancer by controlling the sensitivity of mTOR to amino acids, and is
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closely related to the prognosis of colorectal cancer (14). The difference of Golgi network
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also determines the secretory phenotype of different tumors (15).
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GOLIM4,identified as a Golgi protein, is a 130kD membrane protein that are integrated
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into the cis/medial Golgi apparatus, which is a kind II Golgi protein.GOLIM4 is a Golgi
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protein that circulates between Golgi apparatus and endosomes, and its shuttle is pH-sensitive.
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GOLIM4 is considered being a homologous with the membrane proteins of the Golgi
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apparatus of rat. Although located in the early Golgi apparatus, it has advanced Golgi
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apparatus modification function (16).The ST-1 of Shiga toxin can directly combine with
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GOLIM4, so that the ST-1 is free from lysosome degradation, the increase of Mn can
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downregulate the expression of GOLIM4, reduce the toxicity of Shiga toxin(17).The
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increased, non-toxic dose of Mn protects the ST-1-induced cell death of Shigella by lowering
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the GOLIM4. Mn is combined with GOLIM4 in the Golgi matrix, causing the GOLIM4 to
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form an oligomer, and then the complex is transferred to lysosomal degradation (18). These
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suggest that GOLIM4 may be associated with cell death, but the role of GOLIM4 in tumors
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has not been reported. The Golgi apparatus can also coordinate with the endoplasmic
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reticulum to regulate the biological behavior of the tumor (19). Although the function of
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Golgi apparatus in the development of multiple tumors has been reported, the function of
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Golgi and ER complex proteins and their interactions with endoplasmic reticulum in head and
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neck cancer is unclear. Here, we showed that GOLIM4 regulated the proliferation, apoptosis
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and cell cycle progression of head and neck cancer. We first clarified the functions of
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GOLIM4 in tumors and the roles of Golgi-endoplasmic reticulum interaction in head and
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neck cancer, but the interaction of STIM1 and the expression of GOLIM4 as well as the role
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of GOLIM4 in the formation of head and neck cancer needed further study.
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In summary, we have revealed that STIM1-GOLIM4 influence the development of head
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and neck cancer through the regulation of cell proliferation, apoptosis and cell cycle, and
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found that endoplasmic reticulum and Golgi apparatus interaction was important in the
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occurrence of head and neck cancer, suggesting that the endoplasmic reticulum and Golgi
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apparatus maybe the cut-in point targeted head and neck cancer cells. It provides a new
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potential target for clinical treatment of head and neck cancer.
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5. Funding
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This work received financial support from the Natural Science Foundation of Inner
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Mongolia (Grant No.2017MS0839) and Inner Mongolia Medical University Affiliated
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Hospital Major Research Projects Funded Project (Grant No.NYFY ZD 016).
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6. Declaration of interest None
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7. Author contribution
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Xiaobo Cui conceived the study, carried out the experimental design and data interpretation,
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and prepared and revised the manuscript. Dengxue Gao and Yunfei Bai performed most of the
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experiments. Yunfei Bai performed the HCS assay. Yaping Wang performed the western blot.
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Wei Wang and Boqian Wang performed statistical analysis.
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Figure legends
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Fig.1 GOLIM4 is decreased when knockdown of STIM1.
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A. Cell proliferation was measured after knockdown of 20 candidate genes in FaDu cells.
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B. The representative images of FaDu cells that infected with negative control lenti virus
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(shCtrl-EGFP) and shGOLIM4-EGFP lentivirus. Green fluorescence showed the viable cells.
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C. The growth curves of the corresponding negative control group (shCtrl) and shGOLIM4
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group in the FaDu cells as described in (A).
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D. The expression of GOLIM4 in head and neck cancer tissues(n=521) and normal donors
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(n=44) from TCGA database.
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E．The correlation analysis of GOLIM4 and STIM1 in head and neck cancer tissues from
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TCGA database. Pearson Correlation Coefficient was used, P
