Introduction
Gastric carcinoma (GC) is a major malignancy with high global incidence and mortality rates [
1,
2]. China in particular and East Asia in general account for ≤ 44% of all new GC diagnoses worldwide [
2]. When GC is detected early, its 5-year survival rate is > 90% [
3]. East Asia, particularly China, is disproportionately burdened with this neoplastic disease, contributing nearly 44% of all novel diagnoses on a global scale [
4]. Chemotherapy and targeted treatment modalities have only limited efficacy in advanced GC cases [
5,
6]. Hence, innovative timely prophylactic and therapeutic approaches and interventions for GC are urgently required.
Members of the heterogeneous transmembrane protein (TMEMs) family are embedded within the lipid bilayers [
7,
8] of cell and organellar membranes [
9]. They promote angiogenesis [
10], regulate endoplasmic reticulum (ER) stress [
11,
12], maintain mitochondrial function [
13,
14], facilitate protein glycosylation [
15], control epidermal keratinization [
16], and modulate smooth muscle contraction [
17].
The multifunctional TMEM176B is a key member of the TMEM family. It was found to be upregulated in a rat model of allograft tolerance and designated as
tolerance-related and induced transcript (TORID). Other functions of TMEM176B have since been identified [
18]. TMEM176B forms a co-polymer with TMEM176A, retards dendritic cell (DC) maturation, and may regulate DCs overall [
19]. TMEMs are also associated with antigen delivery in DCs and the modulation of DC-mediated immune responses [
20,
21]. TMEM176B bolsters antitumor immunity and synergistically increases immune checkpoint blockade efficacy by activating inflammasomes [
22]. TMEM176B promotes triple-negative breast cancer progression by regulating the Akt/mTOR signaling pathway [
23]. TMEM176B expression in melanoma indicates a favorable prognosis. Thus, TMEM176B could serve as both a diagnostic and a prognostic marker for certain malignancies [
24]. It may also be a melanoma immunotherapy target as it regulates CD8 + T cells. However, the mechanisms of TMEM176B in the onset and progression of GC remain to be clarified.
Phosphatidylinositol 3-kinases (PI3Ks) are crucial coordinators of intracellular signalling in response to the extracellular stimulators. Hyperactivation of PI3K signalling cascades is one among the most ordinary events in human cancers [
25]. As an intracellular protein complex, rapamycin complex 1mTORC1 consists of five components, namely, mTOR, RAPTOR, DEPTOR, mLST8 and PRAS40 [
26]. mTORC1 can integrate signals from growth factors and nutrients to control biosynthesis, including protein, lipid and nucleic acid synthesis [
27]. Phosphatidylinositol 3-kinases (PI3Ks) are crucial coordinators of intracellular signalling in response to the extracellular stimulators. Hyperactivation of PI3K signalling cascades is one among the most ordinary events in human cancers [
28]. Asparagine synthetase (ASNS) is a key enzyme in amino acid (AA) metabolism [
29,
30]. It generates asparagine from glutamine-derived nitrogen and aspartate [
31] and is implicated in lung, ovarian, and gastric cancer progression [
32‐
34].
The present study aimed to elucidate the molecular mechanisms of TMEM176B and identify novel therapeutic targets and optimize treatment strategies for gastric cancer.
Materials and methods
Cell culture
The gastric epithelial cell lines GES-1, MGC803, HGC27, AGS, and SGC7901 were sourced from GeneChem (Shanghai, China) and cultivated in RPMI-1640 medium (Corning Life Sciences, Corning, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Clark Bioscience, Richmond, VA, USA) and 1% (w/v) penicillin plus 1% (w/v) streptomycin (HyClone Laboratories, Logan, UT, USA). All cell lines were maintained at 37 ℃ under a humidified 5% CO2 atmosphere.
Quantitative real-time polymerase chain reaction (qRT‒PCR)
Total RNA was extracted from GC cells and tissues with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) per the manufacturer’s instructions [
35]. The RNA was then reverse-transcribed to cDNA and the latter was subjected to qRT-PCR according to standard procedures. The primer sequences used in this study are shown in Additional file
1: Table S1.
Western blotting
The proteins from the GC cells and tissues were extracted with M-per protein lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with protease and phosphatase inhibitors (BBI Life Sciences Corporation, Shanghai, China) and subjected to western blotting [
36] with the primary antibodies anti-GAPDH (No 7074 T; Cell Signaling Technology (CST), Danvers, MA, USA), anti-TMEM176B (No. PHC0758; Abmart, Wuhan, China), anti-ASNS (No. R22614; ZenBio, Chengdu, China), anti-AKT (No. R23411; ZenBio), anti-p-AKT (No. R381555; ZenBio), anti-PI3K (No. R381092; ZenBio), anti-p-PI3K (No. 310164; ZenBio), and anti-PI3K (No. R381092; ZenBio). The mTOR Substrates Antibody Sampler Kit (No. 9862 T) was sourced from CST.
Patient samples and follow-up
One hundred and seven formalin-fixed, paraffin-embedded GC and 22 adjunct normal tissues were curated from The First Affiliated Hospital of Anhui Medical University between October 2012 and December 2013. None of the patients with GC had undergone preoperative chemotherapy or radiotherapy, and all of them were followed up for 8–71 mo. The clinicopathological data and staging based on the American Joint Committee on Cancer (AJCC) v. 8 system are displayed in Table
1. Ethical approval of the research protocol was obtained from the Ethics Association of Anhui Medical University, and all eligible trial participants provided written informed consent.
Table 1
Correlation of TMEM176B expression with clinicopathologic parameters in GC patients
Gender | 0.045 | 0.833 |
Male | 71 | 39 | 32 |
Female | 36 | 19 | 17 |
Age(years) | 0.448 | 0.503 |
< 61 | 43 | 25 | 18 |
≥ 61 | 64 | 33 | 31 |
Tumor location | 0.045 | 0.833 |
Upper | 36 | 19 | 17 |
Middle + lower | 71 | 39 | 32 |
Tumor size(cm) | 9.696 | 0.002a |
< 6 | 48 | 34 | 14 |
≥ 6 | 59 | 24 | 35 |
Depth of invasion | 5.313 | 0.021a |
T1 + T2 | 29 | 21 | 8 |
T3 + T4 | 78 | 37 | 41 |
Lymph node metastasis | 4.584 | 0.032a |
Absent | 23 | 17 | 6 |
Present | 84 | 41 | 43 |
Differentiation | 7.313 | 0.007a |
Well + moderate | 41 | 29 | 12 |
Poor | 66 | 29 | 37 |
TNM stage | 5.388 | 0.020a |
I + II | 34 | 24 | 10 |
III + IV | 73 | 34 | 39 |
Immunohistochemical (IHC) staining
Tissue microarrays (TMAs) were subjected to IHC staining per established protocols [
37] to quantify TMEM176B expression. For this purpose, anti-TMEM176B (No. PHC0758; 1:1,000; Abmart) was used. Staining intensity ranged from 0 (none) to 3 (strong). The staining area ranged from 0 (none) to 4 (76–100%). Assessments were made independently by two expert pathologists. The staining score was the product of staining intensity and staining area. Values ≥ 5 and those in the range of 0–4 indicated high and low TMEM176B expression, respectively.
Cell lentivirus infection
Three small hairpin RNAs (shRNAs) and a TMEM176B overexpression lentivirus were acquired from GeneChem. GC cells were seeded in a 12-well plate and transfected with the lentivirus at Multiplicity of Infection (MOI) = 10 per the manufacturer’s guidelines. Successful transfection was confirmed by using a medium supplemented with 2 mg/mL puromycin, and the transfected cells were maintained in a medium supplemented with 1 mg/mL puromycin. The shRNA sequences used were as follows:
sh#1: 5ʹ-GUAGGUCUUCGAAACUUGUTT-3ʹ
sh#2: 5ʹ-GCAGGCUUUGCUACAGCUATT-3ʹ.
GC cells were inoculated into a six-well plate at a density of 800/well. The medium was replenished every 3rd day. Starting at day 7, the cultures were inspected for clonal growth and the culture was terminated as soon as clones could be detected with the unaided eye. The cells were then fixed with 4% (v/v) paraformaldehyde (PFA) and stained with 0.1% (w/v) crystal violet. The clones in each well were counted and the stained cells were air-dried and imaged.
EdU assay
The present assay was conducted using an EdU Kit (No. C0078S; Beyotime Biotechnology, Shanghai, China). Sterile slides were set in 12-well plates, seeded with cells, and incubated overnight to the optimal density. The cells were then exposed to 2X EdU solution, rinsed with phosphate-buffered saline (PBS) plus 3% (v/v) bovine serum albumin (BSA), permeabilized with 0.3% (v/v) Triton X-100, and rinsed. A Click reaction solution was added to the 12-well plate and the cells were incubated in it for 30 min. The nuclei were then stained with Hoechst 33,342 for 10 min. The cells were subjected to an anti-quenching agent and examined under a microscope (Leica Microsystems, Wetzlar, Germany).
Transwell assay
Transwell assay was performed as described previously [
38]. For invasion assay, GC cells in log phase were incubated into the Transwell chamber (Corning Life Sciences, Corning, NY, USA) at the optimal density in serum-free medium for 24 h. Matrigel (BD Biosciences, Shanghai, China) was diluted and uniformly applied to the upper Transwell chamber and the treated cells were incubated at 37 ℃ for 5 h. A cell suspension containing 1 × 10
8 cells and serum-rich medium were introduced into the upper- and lower Transwell chambers, respectively. After a predetermined incubation period, the Transwell chambers were treated with 4% (v/v) PFA, stained with 0.1% (w/v) crystal violet, and examined and imaged under a microscope (Leica Microsystems). The migration assay does not use Matrigel and the rest of the steps are the same as the invasion assay. The incubation time was 28 h for HGC27 cells, 24 h for SGC7901 cells, and 18 h for AGS cells.
Flow cytometry
Flow cytometry was performed as described previously [
39]. This assay was performed with an Annexin V-FITC/PI Apoptosis Kit (Yeason Biotechnology, Shanghai, China). 2 × 10
5 HGC27, SGC7901, or AGS GC cells were cultured overnight in 12-well plates, respectively. GC cell suspensions were washed with PBS, resuspended at a 1:1 (v/v) ratio in binding buffer, stained with Annexin V-FITC (fluoroisothiocyanate) and PI (propidium iodide) reagents, and incubated in the dark for 15 min. Apoptosis was detected by CytoFLEX flow cytometry (Beckman Coulter, Brea, CA, USA).
Xenograft mouse model
BALB/C nude mice were procured from GemPharmatech, Jiangsu, China and acclimated under specific pathogen-free (SPF) conditions for 4 weeks. Each mouse was subcutaneously injected in the left axilla with 5 × 106 enzymatically digested GC cells in PBS suspension. Throughout the experiment, the mice had ad libitum food and water access and their body weight and tumor dimensions were recorded every 3 day. Mice exhibiting ≥ 20% weight loss and/or tumor diameter > 1.5 cm were humanely euthanized.
Statistical analysis
All data were subjected to Student’s t-test or one-way analysis of variance (ANOVA) and otherwise processed with SPSS v. 22.0 (SPSS, Inc., Chicago, IL, USA), GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA, USA), and R v. 4.1.2. p < 0.05, p < 0.01, and p < 0.001 indicated statistical significance.
Discussion
Advances have been made in reducing the incidence and mortality of gastric cancer (GC) in recent years. Nevertheless, there are few efficacious treatment options for this disease [
41]. Hence, novel therapeutic targets are required for GC. Transmembrane protein 176B (TMEM176B) plays multiple complex roles in oncogenesis. Its overexpression drives breast cancer progression [
23] while its knockdown enhances the efficacy of anticancer immune checkpoint inhibitors [
22]. Moreover, its upregulation is paradoxically associated with improved melanoma prognosis. However, our in vitro and in vivo experiments revealed that TMEM176B significantly promotes GC progression. Patients with GC and low TMEM176B expression levels have relatively improved survival. Thus, TMEM176B is a potential therapeutic target for this disease.
Here, qRT-PCR and WB demonstrated that TMEM176B was overexpressed in GC cell lines and tissue samples (Fig.
1). TMEM176B knockdown inhibited proliferation, migration, and invasion (Fig.
2A) but promoted apoptosis (Fig.
2B–G and
3) in HGC27 and SGC7901 cell lines. Conversely, TMEM176B overexpression in AGS cell lines (Fig.
4A) had the opposite effects (Fig.
4B–I). Taken together, the foregoing data indicate that TMEM176B drives progression in GC cell lines.
We used bioinformatics analyses to clarify the mechanisms by which TMEM176B drives GC progression. Pathway enrichment analyses disclosed that the PI3K-Akt signaling pathway is strongly associated with TMEM176B (Fig.
5A, B). PI3K-Akt signaling plays crucial roles in a wide range of cancers [
28,
42‐
45]. We used WB to evaluate the expression levels of PI3K-Akt signaling pathway proteins in GC cell lines subjected to TMEM176B knockdown and overexpression. TMEM176B knockdown and overexpression substantially decreased and increased PI3K-Akt phosphorylation, respectively (Fig.
5C). Downstream of the PI3K-Akt signaling pathway, mTOR regulates tumor growth, metabolism, immunity, and other processes [
46]. P70S6K and 4EBP1 are essential components of mTOR [
47]. Phosphorylation of the aforementioned mTOR proteins decreased and increased in response to TMEM176B knockdown and overexpression, respectively (Fig.
5D). In many cancers, mTOR is activated and controls cell growth and metabolism. The mTOR signaling pathway regulates AA, glucose, nucleotide, fatty acid (FA), and lipid metabolism [
48]. An earlier study showed that mTOR is closely associated with ASNS [
40]. We discovered that TMEM176B induces ASNS by inhibiting its ubiquitination degradation (Fig.
5D–F).
The addition of the mTOR inhibitor rapamycin in the presence of TMEM176B overexpression (Fig.
6A) partially weakened the potentiating effect of TMEM176B overexpression on GC cells (Fig.
6B–I). The preceding findings suggest that TMEM176B drives GC progression via the PI3K-Akt-mTOR signaling pathway. The results of our mouse tumor xenograft model supported this theory (Fig.
7).
Our survival and clinicopathological analyses established that TMEM176B expression is closely associated with the clinicopathology of patients with GC and confirmed that TMEM176B upregulation indicates poor prognosis (Fig.
8).
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