Human Bone Marrow Mesenchymal Stem Cells Promote Gastric Cancer Growth via Regulating c-Myc

The clinical application of human bone marrow mesenchymal stem cells (hBM-MSCs) has generated a great deal of interest because of their potential use in regenerative medicine and tissue engineering. However, safety concerns over hBM-MSCs limit their clinical application. In this study, we observed that hBM-MSC-conditioned medium (hBM-MSC-CM) promotes gastric cancer development via upregulation of c-Myc. Our results showed that c-Myc was upregulated in MGC-803 and BGC-823 cells after hBM-MSC-CM treatment. Moreover, we found that the c-Myc inhibitor JQ1 and c-Myc siRNA decreased the expression of c-Myc in hBM-MSC-CM-treated tumor cells in vitro. Additionally, hBM-MSC-CM enhanced the migration and glucose uptake of gastric cancer cells. In vivo studies showed that JQ1 inhibited hBM-MSC-CM-induced gastric cancer growth. These results indicated that hBM-MSC-CM induced gastric cancer growth via upregulation of c-Myc, which may be a potential risk factor and/or a therapeutic target for clinical applications.


Introduction
Due to their multidirectional differentiation capacity, mesenchymal stem cells (MSCs) have been widely used in tissue engineering and cell replacement therapy [1]. However, studies have increasingly demonstrated that human bone marrow MSCs (hBM-MSCs) can promote tumor growth and metastasis [2,3], making safety of hBM-MSCs in clinical applications controversial.
Because of their promise for clinical applications, a variety of models have been used to prove the safety and effectiveness of hBM-MSCs [4,5]. Some previous clinical studies have concluded that hBM-MSCs do not pose an obvious risk of tumorigenesis when used to treat cartilage injuries or other diseases [6,7]. However, other studies have found that hBM-MSCs promote tumor proliferation, migration, and stemness in vitro and that hBM-MSCs promote tumor development in vivo [3,8,9]. Therefore, there is an urgent need to identify the factors from hBM-MSCs that promote tumor growth.
In this study, we found that hBM-MSC-CM caused gastric cancer cells to upregulate c-Myc expression, which is a well-known oncogene that is involved in tumor initiation and development. Abnormal c-Myc activation is responsible for a range of human cancers, including neuroblastoma [10], lung carcinoma [11], and gastric carcinoma [12]. By promoting c-Myc expression, hBM-MSC-CM increased the metabolism, migration, and proliferation of gastric cancer cells. Furthermore, we showed that the c-Myc inhibitor JQ1 inhibits the tumor-promoting effects of hBM-MSC-CM. Thus, we show that hBM-MSC-CM can upregulate c-Myc expression in gastric cancer cells, which may be a key factor in carcinogenesis and, therefore, a potential target for cancer prevention.

Xenograft Tumor
Model. This study and its consent procedure were approved by the local ethics committee of Jiangsu University (Jiangsu, China). BALB/c-nu/nu male mice (n = 30; aged 4-5 weeks) were purchased from SLAC Laboratory Animal (Shanghai, China) and maintained in pathogen-free conditions with sterilized chow and autoclaved water. The animals were randomly divided into five groups (n = 6 mice per group). MGC-803 cells were treated with either Dulbecco's modified Eagle's medium (DMEM; Gibco/Life Technologies, Carlsbad, CA, USA), dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA), hBM-MSC-conditioned media (hBM-MSC-CM), JQ1 (0.8 μM), or hBM-MSC-CM + JQ1 for 48 h. In the hBM-MSC-CM + JQ1 group, MGC-803 cells were pretreated with JQ1 for 4-6 h, washed three times with phosphate-buffered saline (PBS), and then treated with hBM-MSC-CM + JQ1. Then, subcutaneous xenografts were established by injection of 2 × 10 6 MGC-803 cells in 200 μL of PBS per mouse. Once tumors formed, tumor sizes were measured using Vernier calipers and tumor volumes were calculated using the following formula: tumor volume = length × width 2 /2. The experiment was stopped on day 30, when all mice were euthanized.

Cell
Culture. Healthy donor-derived bone marrow cells were collected at the Affiliated Hospital of Jiangsu University, and all protocols were approved by the local ethics committee of the Affiliated Hospital of Jiangsu University, China. Additionally, informed consent was received from all donors. The bone marrow cells were diluted in an equal volume of PBS, isolated with 1.077 g/mL Ficoll, and centrifuged at 800 ×g for 20 min. The cells were then rinsed with PBS and cultured in DMEM containing 10% fetal bovine serum (FBS; Gibco) at 37°C in a humidified incubator infused with 5% CO 2 ; adherent cells were collected after 5 days. The human gastric cancer cell lines MGC-803 and BGC-823 and normal line GES-1 were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Beijing, China) and were maintained in DMEM supplemented with 10% FBS at 37°C in 5% CO 2 .

Preparing hBM-MSC-CM and Coculture with Gastric
Cancer Cells. When hBM-MSCs had grown to 70% confluency, they were washed with PBS and incubated with fresh medium for 48 h. Then, the supernatant was collected, filtered through a 0.22 μm filter, and diluted at 1 : 1 with DMEM supplemented with 10% FBS (hBM-MSC-CM). MGC-803 and BGC-823 cells were washed with PBS and then treated with DMEM, DMSO, hBM-MSC-CM, JQ1 (0.8 μM), or hBM-MSC-CM + JQ1 at 37°C in 5% CO 2 for 48 h prior to being collected for use in subsequent experiments. In the experiment using c-Myc knockdown, the cells after c-Myc knockdown were cocultured with hBM-MSC-CM. After 48 h, the cells were collected by centrifugation (800 rpm/min) and 8 × 10 4 cells were seeded in the upper wells of a Transwell chamber in 200 μL of serumfree DMEM. The lower compartment of the chamber was filled with 500 μL DMEM supplemented with 10% FBS. The cells were incubated for 8 h, and after incubation, we used a cotton swab to remove the cells that did not migrate. Migrated cells were fixed with 4% formaldehyde for 30 min, stained with crystal violet, and then photographed. For quantitation, three random fields from each well were counted under a microscope (Ti-S; Nikon, Tokyo, Japan), and each experiment was independently repeated in triplicate. After 48 h, the cells were collected by centrifugation (800 rpm/min) and 1 × 10 6 cells/mL were seeded in 48-well plates and incubated for 8 h in DMEM. Then, a clinical chemistry analyzer (Xunda, XD811, Shanghai, China) and the hexokinase method were used to detect supernatant glucose concentrations.
2.6. Cell Viability Assay. We performed 3-[4,5-dimethylthiazol-2yl] diphenyltetrazolium bromide (MTT) assays to assess the half maximal inhibitory concentration (IC50) of JQ1. Cells were seeded into 96-well plates (Corning Inc.) at a density of 2 × 10 3 per well and incubated overnight at 37°C in 5% CO 2 . Then, the plates were incubated with DMSO or different JQ1 concentrations (0.4 and 0.8 μM) and cultured for 24, 48, or 72 h. MTT (5 mg/mL) was added to the cells for 4 h at 37°C. After 4 h, we added DMSO to terminate the reaction; results were determined by measuring optical density at 490 nm with a multiwell plate reader (FLx800, BioTek, Winooski, VT, USA). Each experiment was repeated three times. 2.11. Statistical Analysis. All data analyses were performed using GraphPad Prism 6 (Graph Software, La Jolla, CA, USA). Differences between groups were analyzed using oneway analysis of variance. The Kruskal-Wallis H test was used to analyze differences between in vivo tumor growths. A P value < 0.05 was considered statistically significant.

HBM-MSC-CM Increased c-Myc Expression in Gastric
Cancer Cells. The c-Myc oncogene has been reported to play important roles in gastric cancer; thus, we examined c-Myc levels in the gastric cancer cell lines MGC-803 and BGC-823 and normal line GES-1 with Western blot. GES-1 cells showed the lowest c-Myc expression, and the BGC-823 cells showed the highest c-Myc expression (Figure 1(a)). Next, we investigated whether treating the gastric cancer cells with hBM-MSC-CM for 48 h affected c-Myc protein levels. Compared with the untreated groups, c-Myc levels were increased in both MGC-803 and BGC-823 cells after hBM-MSC-CM treatment (Figure 1(b)). The upregulation of c-Myc expression in MGC-803 cells could maintain 12 hours after withdrawal of hBM-MSC-CM (Figure 1(c)). JQ1 has been shown to have antiproliferative effects in many cancers, primarily through inhibition of c-Myc [13]. MTT assays showed that 0.8 μM JQ1 for 72 h has significantly antiproliferative effects on MGC-803 (Figure 1(d)). Then, we used JQ1 at 0.8 μM concentration in the following experiment. Western blot analysis indicated that hBM-MSC-CM treatment can significantly increase the expression level of c-Myc protein, and the effect of hBM-MSC-CM upregulation of c-Myc in MGC-803 and BGC-823 cells can also be suppressed by JQ1 (Figures 1(e) and 1(f)).

Effect of hBM-MSC-CM on Cell Cycle Progression and
Apoptosis. Cell cycle analysis revealed that JQ1 treatment increased the percentage of G1-phase cells from 51.42% to 61.49%, while the fraction of S-phase cells decreased from 30.31% to 22.3%, which were not statistically significant changes (Figure 3(a)). Additionally, hBM-MSC-CM had no effect on the cyclin-D1 expression or the apoptosis rates of MGC-803 cells (Figures 3(b) and 3(c)); however, JQ1 inhibited cyclin-D1 expression.

Discussion
Previous studies have suggested that MSCs are closely associated with tumor progression and growth [3]; thus, there are  legitimate safety concerns that limit their clinical application. Our previous study revealed that MSCs promote tumor growth in vivo, which suggested that these cells secrete mitogenic paracrine factors [8,9]. Jung et al. demonstrated that the MSCs recruited by tumors facilitate metastasis [14], and Li et al. revealed that gastric cancer-derived MSCs enhanced the proliferation and migration of gastric cancer cell lines [15]. Djouad et al. reported that in vivo tumor growth was increased when melanoma cells were coinjected with MSCs [16]. Interestingly, Karnoub et al. found that in situ breast cancer was unchanged in the absence of MSCs; however, the migratory ability of tumor cells was significantly increased when exogenous MSCs were added [17]. In this study, we chose MGC-803 and BGC-823 cells as representative cell lines to further study the mechanism through which hBM-MSC-CM promotes tumor growth. We explored the potential risks of using hBM-MSCs in clinical applications. Although the tumor-promoting activity of patient-derived hBM-MSCs may be more dramatic, patient-derived hBM-MSCs could not be used in clinical applications; thus, we used healthy donor-derived hBM-MSCs. Our preliminary studies showed that the mitogenic activity of hBM-MSCs on tumor cells was primarily through paracrine signaling [8]. So, we subsequently focused on the potential factors from hBM-MSCs that promoted cancer progression, as these comprised the molecular signature of potential targets that could allow their clinical application.
c-Myc is a protooncogene that is mutated in approximately 20% of human cancers and has particularly important functions in gastric carcinogenesis [18]. In the present study, we found that c-Myc expression was increased in hBM-MSC-CM-treated gastric cancer cells, suggesting that hBM-MSC-CM may promote tumor growth by upregulating c-Myc. Moreover, c-Myc has been shown to maintain normal adult stem cells and tumor stem cells [19].  [21,22]. Other studies have reported that a critical mechanism of JQ1 in suppressing tumor growth is its ability to block c-Myc expression [23][24][25][26]. We found that JQ1 inhibits the expression of c-Myc. Moreover, hBM-MSC-CM promoted glucose uptake and cell migration via upregulating c-Myc in gastric cancer, both of which were suppressed by JQ1. Finally, in vivo studies showed that JQ1 inhibited the increased growth of gastric cancer xenografts that was induced by hBM-MSC-CM. Clinical trials have proven that hBM-MSCs do not undergo tumorigenesis [4,6]. But tumor occurrence is the result of variety of tumor-promoting factors. However, BM-MSCs are immunosuppressive, and factors that affect the immune system should be a concern with regard to tumorigenesis [27,28]. In the present study, we found that hBM-MSC-CM can promote tumor cell proliferation, migration, and glucose uptake via upregulating c-Myc. Although there are no reports of hBM-MSCs causing tumor formation, their potential to promote tumorigenesis is worthy of concern for potential applications in tissue engineering.

Conclusions
hBM-MSC-CM promotes gastric cancer cell proliferation by upregulating c-Myc. c-Myc inhibitors may be effective at preventing the protumor effects of hBM-MSC-CM and, therefore, could solve the clinical safety issues of hBM-MSCs.

Data Availability
The data used to support the findings of this study are included within the article.

Ethical Approval
This study and the consent procedure were approved by the local ethics committee of Jiangsu University, China. The procedure for deriving bone marrow cells from healthy donors was approved by the local ethics committee of the Affiliated Hospital of Jiangsu University, China. Informed consent was given by all donors.