Background
Gastric cancer (GC) is the third most common cause of cancer-related deaths worldwide [
1]. Despite advances in diagnostic and therapeutic strategies, the clinical outcomes and prognosis of patients with GC remain unsatisfactory. Many studies have shown that dysregulated expression and accumulation of relevant genes are crucial for the development and occurrence of gastric cancer [
2]. Thus, targeting these molecules has become crucial for tumor therapy in recent years. For example, cetuximab, the first monoclonal antibody that targets the epidermal growth factor receptor (EGFR) [
3], is entering the clinical application stage, but the therapeutic effect has not reached the expected standard [
4]. VEGF is another important target for GC treatment [
5]. After treatment with monoclonal antibodies against VEGF, such as bevacizumab, patient prognosis did not reach statistical significance, and patients suffered strongly adverse reactions [
6]. Therefore, elucidating novel functions of relevant genes that contribute to the pathogenesis of GC is crucial for the development of new effective therapies.
CSCs are a small subpopulation of quiescent cells with self-renewal abilities and pluripotency that can drive tumor initiation and cause relapses [
7]. CSCs generate the bulk of tumors via their self-renewal and their ability to differentiate into multiple cellular subtypes [
8]. Moreover, these CSCs acquire multidrug resistance, thus protecting themselves from most traditional chemotherapeutic agents. As a result, this small subpopulation of persistent cells forms more aggressive and chemoresistant tumors, resulting in the failure of cancer therapy [
9]. Thus, identifying and targeting these rare cancer cells are believed to be important for understanding the etiology of cancer and developing a novel therapeutic strategy for cancer therapy [
10].
The development of therapeutic strategies that target these tumor-initiating CSCs mainly relies on the use of cell surface markers to discriminate and identify CSCs [
11]. For instance, leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), also known as G-protein coupled receptor 49 (GPR49), is a well-characterized stem cell surface marker that is expressed in several tissues/organs, including the stomach, small intestine, colon, and liver [
12]. Accumulating evidence has demonstrated that LGR5 is a marker of resident adult epithelial stem cells at the gland base and that LGR5 + cells are multipotent stem cells that are responsible for the long-term renewal of the gastric epithelium or small intestine villus [
13]. Moreover, LGR5 was recently reported to be highly upregulated in gastroenterological carcinoma [
14]; the selective ablation of LGR5 + CSCs led to tumor regression, and targeting LGR5 + human colon CSCs enhanced the effects of chemotherapy [
15]. Although it plays important regulatory effects in stem cells, LGR5 is highly expressed in both normal stem cells [
16] and CSCs, which makes it difficult to distinguish between normal stem cells and CSCs in cancer-targeted therapy. Furthermore, LGR5 is also highly expressed in CSCs from tumors of various origins [
17]. Thus, the identification of novel surface markers that can specifically identify and characterize CSCs from tumors of specific origins remains a challenge for understanding tumor biology and developing CSC-based therapeutic strategies.
Here, we examined single-cell transcriptome profiles of paired gastric mucosa tissues and gastric tumor tissues, and we identified and characterized a novel surface marker, namely, AQP5, that is more highly expressed in GC-CSCs than in gastric cancer epithelial cells and gastric mucosa stem cells (GM-SCs). The biological functions of AQP5 in gastric carcinogenesis were genetically assessed in several in vitro and in vivo models. Moreover, we demonstrated that AQP5 functions synergistically with LGR5 to determine the fates of GC-CSCs. Mechanistically, mass spectrometry combined with integrative analysis revealed that AQP5 enhances autophagy in GC-CSCs by interacting with the E3 ligase TRIM21 and promoting the ubiquitination of the key autophagy protein ULK1. Thus, our study identified a specific GC-CSC surface marker, AQP5, with biological, mechanistic, and clinical impacts on human gastric cancer. These findings highlight the importance of AQP5 in tumor biology, adding an important layer to the connection between AQP5 and gastric carcinogenesis, which can be translated into novel targeted therapies.
Methods
Patients
GCs and GMs were obtained from patients with gastric cancer who underwent surgery at the Affiliated Hospital of Jining Medical University. GC patients had primary, nonmetastatic gastric tumors and had not received radiotherapy or chemotherapy prior to surgery.
Identification of cell types
Based on the single-cell reference expression quantitative public dataset, SingleR was used to calculate the correlation between the expression profiles of the cells to be identified and the reference dataset, and the cell type was determined according to the Spearman correlation coefficient.
Screening of differentially expressed genes
The FindMarkers function in the Seurat package was used to identify differentially expressed genes, and significantly differentially expressed genes were identified based on the criteria of p value less than 0.05 and differential fold change greater than 2.5.
Cell culture and transfection
The AGS (RRID: CVCL_0139) and HGC-27 (RRID: CVCL_1279) gastric cancer (GC) cell lines and the HEK 293 T (RRID: CVCL_D585) cell line were purchased from Procell. The cells were grown in DF-12 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco). The cells were incubated at 37 °C in a humidified atmosphere with 5% CO2.
The cells were transfected with pSLenti-AQP5 and control vector according to the manufacturer's instructions. Stably transfected cell lines were obtained after selection with 1.5 μg/mL puroMycin (Gibco) for 6 days. The control and AQP5/LGR5-specific shRNA sequences are listed in Supplementary Table
4. Transfection of ULK1/TRIM21/Ubiquitin/k63R-Ubiquitin plasmid and control empty vector was performed using Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer's instructions. Transfection of TRIM21/ATG7/UBB/UBC siRNA and negative control siRNA (NC) was performed using Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer's instructions. The corresponding sequences are shown in Supplementary Table
4. The cells were harvested 48–72 h post-transfection for various assays.
Discussion
In the present study, we identified and validated AQP5 as a novel specific surface marker of GC-CSCs by analyzing a cell atlas of GCs and GMs. To this end, we performed single-cell sequencing to probe the key marker genes of each cell cluster. By comparison with non-GC-CSCs, we found that AQP5 was a novel specific surface marker of GC-CSCs. At the functional level, we demonstrate that AQP5 promotes the self-renewal and tumorigenesis of GC-CSCs. Interestingly, AQP5 complements LGR5 to promote the tumorigenesis of GC-CSCs. The results suggest that CSCs express their own specific marker that reflects their own tumor origin, highlighting the importance of AQP5 as a specific marker of gastric cancer that could be targeted by potential novel therapeutic strategies. First, this study sheds new light on the novel biological functions of the membrane protein AQP5. Although previous studies reported AQP5 as a marker that is enriched in mouse and human adult pyloric stem cells [
25], its biological functions in CSCs, especially in GC-CSCs, remain unknown. Our findings demonstrate that AQP5 is highly expressed in GCs and is clinically correlated with the progression of gastric cancer. The oncogenic role of AQP5 was functionally validated in several in vitro and in vivo experimental models. Downregulation of AQP5 markedly suppresses cell growth and tumor growth in cultured GC-CSCs and xenograft mouse models. By comparison with non-GC-CSCs, we identified and verified AQP5 as a novel specific marker of GC-CSCs, and AQP5 is co-expressed with the canonical stem cell markers LGR5. Functionally, we demonstrate that AQP5 promotes the self-renewal and tumorigenicity of GC-CSCs. Thus, the results consistently point to the notion that the AQP5 + cell compartment is an important tumor-initiating population.
Second, the present study suggests that AQP5 complements LGR5 and synergistically promotes the tumorigenesis of GC-CSCs. LGR5 is a well-characterized stem cell marker that is expressed in several tissues/organs, including the small intestine, colon, and liver. LGR5 + stem cells are involved in the process of oncogenesis, acting as tumor-initiating cells of intestinal cancer and fueling tumor growth [
26]. Here, we demonstrate that AQP5 is specifically expressed in GC-CSCs and is involved in the regulation of these cells by LGR5. Our results indicate that co-knockdown of AQP5 and LGR5 substantially attenuates the self-renewal and tumorigenicity of GC-CSCs compared to knockdown of AQP5 or LGR5 alone. These results suggest that AQP5 coordinates with LGR5 to promote tumorigenesis through an unknown mechanism. Previous studies have shown that AQP5 + cells act as cells of origin for tumors [
25]. Thus, we hypothesized that the AQP5 + /LGR5 + stem cell bank is the origin of gastric cancer. It is worth emphasizing that LGR5 and AQP5 are expressed in the same cells in both GCs and GMs. Interestingly, the expression of AQP5 in GC-CSCs is much higher than that in GM-CSCs. Cancer stem cell markers, such as LGR5 and CD133, are not able to distinguish CSCs from normal tissue stem cells. However, AQP5 can clearly distinguish these cell populations, suggesting that AQP5 is a more suitable target for the treatment of gastric cancer than LGR5.
Third, the results demonstrate that AQP5 is specially expressed in GC-CSCs rather than CSCs from tumors of other origins, which allows us to propose a novel concept that specific surface markers can identify the CSCs from tumors of individual origins, unlike conventional CSC markers. We found the following: 1. AQP5 is highly expressed in tissues from gastric cancer patients; 2. AQP5 is specifically expressed in GC-CSCs rather than GM-SCs or CSCs from tumors of other origins; and 3. AQP5 promotes the self-renewal and tumorigenicity of GC-CSCs. Thus, we propose that AQP5 is a specific marker for GC-CSCs. Our future study will focus on the identification of specific markers of CSCs from tumors of other origins.
Fourth, the present study reveals a previously unknown mechanism by which AQP5 regulates the autophagy and stemness of GC-CSCs. Autophagy is necessary to maintain the stemness of CSCs in various tumor types, and another aquaporin family member, AQP3, which has been shown to facilitate chemoresistance by stimulating autophagy [
22]. Thus, we postulated that AQP5 may exert biological effects on GC-CSCs through autophagy. Indeed, AQP5 promotes autophagy in GC-CSCs, and knockdown of the key autophagy protein ATG7 or treatment with the autophagy inhibitor CQ reversed this effect. When we explored the mechanisms by which AQP5 affects the autophagy and stemness of GC-CSCs, we found the involvement of TRIM21. We revealed that AQP5 recruits TRIM21 to the key autophagy protein ULK1 and induces the ubiquitination of ULK1, thus activating autophagy and enhancing the stemness of GC-CSCs. This notion is supported by three lines of experimental evidence: (i) AQP5 directly binds to TRIM21 and ULK1; (ii) knockdown of TRIM21 reduces the interaction of AQP5 and ULK1 and reverses the activation of autophagy and self-renewal capacity induced by AQP5; and (iii) blocking the interaction between AQP5 and TRIM21 reverses the activation of autophagy and self-renewal capacity induced by AQP5. In accordance with our study, it has been reported that TRIM21 can act as an autophagy receptor, recruit and organize key components of the autophagic machinery, including ULK1, BECLIN1, and ATG16L1 [
27]. AQP5 is embedded in the lipid bilayer of the cytoplasmic membrane and forms a tetramer. Due to the unique structure of AQP5, it plays an important role in the transmembrane transport of water and small molecular compounds [
28]. Previous studies have reported that AQP5 cooperates with the calcium channel TRPV4 to regulate cell volume [
29]. Moreover, AQP5 has also been shown to interact with the Na + /K + transporter ATP1A2 and the H + transporter ATP6V0A1 on the plasma membrane to regulate cells[
30]. In addition, AQP5 can also interact with WNT2 [
31] and PIP [
32], however the specific mechanism has not been elucidated. Furthermore, we found that AQP5 can bind to TRIM21. Altogether, AQP5 can not only function as a channel protein but also involve in the ubiquitination modification via binding to E3 enzyme regulatory proteins.
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