Maelstrom Directs Myeloid-Derived Suppressor Cells to Promote Esophageal Squamous Cell Carcinoma Progression via Activation of the Akt1/RelA/IL8 Signaling Pathway.

Maelstrom directly and indirectly promotes growth of esophageal squamous cell carcinomas. MDSCs, recruited to the tumor's microenvironment through IL8 signaling, produce TGFβ that then regulates maelstrom expression in the tumor cells by inducing Smad2/Smad3 phosphorylation. Maelstrom (MAEL) is a novel cancer/testis-associated gene, which is not only expressed in the male testicular germ cells among human normal tissues, but is also aberrantly expressed in various cancer tissues. In our study, MAEL was characterized as a tumor-promoting gene and was significantly associated with esophageal squamous cell carcinoma (ESCC) recurrence and unfavorable prognosis. Kaplan–Meier analysis showed that patients with high MAEL expression had a shorter survival time. Functional experiments showed that MAEL promoted tumor cell growth and inhibited cell apoptosis. These results prompted us to investigate the factors affecting the tumorigenicity of MAEL. Further experimentation demonstrated that MAEL enhanced the expression of phosphorylated Akt1, with subsequent phosphorylation of nuclear factor kappa B (NF-κB) subunit RelA in tumor cells, and chemoattracted myeloid-derived suppressor cells (MDSCs) by upregulating interleukin-8 (IL8) to accelerate tumor progression in the tumor microenvironment. We also found that TGFβ secreted by MDSCs could upregulate MAEL by inducing Smad2/Smad3 phosphorylation. In summary, this study revealed a mechanism by which MAEL could upregulate IL8 through Akt1/RelA to direct MDSCs homing into the tumor, suggesting that MAEL could be an attractive therapeutic target and a prognostic marker against ESCC. Cancer Immunol Res; 6(10); 1246–59. ©2018 AACR.


Introduction
Maelstrom (MAEL) is a novel cancer/testis-associated gene, which is not only expressed in male testicular germ cells among human normal tissues, but is also aberrantly expressed in various cancer tissues (1)(2)(3). MAEL was initially identified in Drosophila and linked to spermatogenesis and meiosis (4). MAEL, comprising a MAEL-specific domain and a highmobility group domain, was considered to be indispensable for Piwi-mediated silencing (5,6). Researchers identified MAEL from 1q24, one of the most frequent chromosomal alterations in hepatocellular carcinoma (HCC; ref. 1), urothelial carcinoma of the bladder (UCB; ref. 7), and colorectal cancer (8). A growing body of evidence has shed considerable light on the important roles of MAEL in cancer progression (9,10). A previous study demonstrates that MAEL is an important player in the progression of HCC by activating Akt/GSK3b/Snail signaling (1). MAEL, regulated by miR-186, can promote tumor aggressiveness by epigenetically downregulating the metastasis suppressor 1 (MTSS1) gene through DNA methyltransferase (DNMT)3B in UCB (7). In colorectal cancer, MAEL can induce epithelial-mesenchymal transition (EMT) and stem cell properties to promote the progression of patients with colorectal cancer (8).
The tumor microenvironment (TME) plays a key role in tumor progression. The underlying mechanism by which MAEL regulates tumor progression in TME has not been elucidated. Myeloid-derived suppressor cells (MDSCs) have been extensively studied as one of the most important immunosuppressive cells in the TME (11,12). Previous studies reveal that interleukin-8 (IL8; CXCL8), a chemokine with tumorpromoting effects in the TME (13,14) and is functional on CXCR1 and CXCR2, can chemoattract MDSCs to the tumor site (15). MDSCs may drive tumor progression through secretion of cytokines and chemokines with protumorigenic functions, depending on the TME (16)(17)(18)(19). Given the immunosuppressive function of MDSCs, the IL8-MDSC axis offers an attractive immunotherapeutic target of cancer.
Esophageal squamous cell carcinoma (ESCC), the uppermost histologic subtype of esophageal cancer, is common in China (20). Despite improvements in treatment, the 5-year overall survival (OS) rate of patients is only 17% (20,21). Therefore, search for immunotherapy targets is of great importance. Nonetheless, potential oncogenic function of MAEL and corresponding molecular mechanisms in ESCC have not yet been addressed. In the present study, we found that MAEL was overexpressed in esophageal tumor tissues and was positively correlated with worse OS and progression-free survival (PFS). The molecular mechanisms underlying the important role that MAEL plays in regulating the TME of ESCC were elucidated. Overall, this study provides an explanation for ESCC tumorigenesis and progression with MAEL high expression.

Patients and tumor samples
A total of 120 paired tumor (typically 1 cm 3 ) and corresponding nontumor tissues (located more than 3 cm away from tumor tissues) were freshly obtained from ESCC patients (diagnosed in 2012-2014) at the department of Thoracic Surgery of the First Affiliated Hospital of Zhengzhou University (Zhengzhou, China). The tissues were washed with PBS and cut into small pieces. Some tissues were used for RNA exaction using TRIzol, according to standard protocol, and some tissues were processed by paraffin embedding and sectioned for IHC staining studies treated with 4% paraformaldehyde. No chemotherapy, radiotherapy, or other therapy was performed prior to entry into the study. Samples used in this study were approved by Zhengzhou University Medical Center Institutional Review Board protocols in accordance with Declaration of Helsinki. Written informed consent was obtained from each patient with available follow-up information prior to participation. All specimens were deidentified prior to analysis. The clinicopathologic features were analyzed according to age, gender, stage, differentiation, and lymph node metastasis, and patients were staged according to the tumor-node-metastasis staging system of the American Joint Committee on Cancer (AJCC, 6th edition).

ESCC cell lines and cell culture conditions
Seven ESCC cell lines (EC109, EC1, EC9706, TE1, TE7, KYSE450, and KYSE70) and one immortalized cell line (Het-1a) were purchased from Cellbank Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences, preserved in our laboratory, and frozen in liquid nitrogen until further use in our experiments. All ESCC cell lines were passed to the fifth or sixth generation and cultured in RMPI 1640 (SH30809.01, HyClone) supplemented with 10% fetal bovine serum (FBS, HyClone), penicillin (100 U/mL), and streptomycin (100 mg/mL) for 48 hours before used in experiments. Cells were maintained in a humidified incubator at 37 C in 5% CO 2. All cell lines were tested free for Mycoplasma. Cell lines were authenticated by STR profiling and stored in liquid nitrogen container when not in use.

RNA extraction and qRT-PCR
Detailed experiments were performed as described previously (21). Total RNA was extracted from ESCC cell lines and tissue specimens by TRIzol reagent (Invitrogen), according to the manufacturer's instructions. The first-strand cDNA was synthesized from 1 mg of total RNA using the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa). qRT-PCR was performed using SYBR Premix Ex Taq II (TaKaRa) in Agilent Mx3005P. Primer sequences (Sangon Biotech) for qRT-PCR were listed in Supplementary Table S1. GAPDH was used for normalization of data. The data were analyzed by 2 ÀDDCt .

Plasmid construction, transfection, and cell sorting
The stable overexpression of MAEL in TE7 cells and detailed experiments were performed as described previously (20). Briefly, full-length MAEL cDNA (Supplementary Table S2) was amplified by PCR using PrimeSTAR HS DNA polymerase (TaKaRa), and cloned into Xba1 and sal1 sites of pEGFP-N1 vector (Invitrogen), and then transfected into TE7 cells (MAEL-TE7) using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. Cells transfected with empty vector were used as controls (Vector-TE7). After 48 hours of transfection, the transfectants with mock vector and MAEL expression vector were sorted by MoFlo XDP (Beckman) based on the expression of green fluorescent protein (GFP). After several rounds of sorting, the purity of MAEL expression cells was over 95%.
Stable knockdown of MAEL in TE1 by short hairpin RNAs (shRNA) was achieved using the hU6-MCS-Ubiquitin-EGFP-IRES-puro vector plasmid purchased from GENE (GeneChem) and transfected into TE1 cells according to the manufacturer's instructions. Cells transfected with empty vector were used as controls. Stable knockdown of MAEL in TE1 cells and detailed experiments were performed as described previously (21). The purity was over 90%.

IHC and evaluation of IHC staining
Detailed IHC and evaluation of IHC staining experiments were performed as described previously (22). The paraffinembedded ESCC tissues and their paired marginal tissues were detected for the expression of IL8 (1:1,000; Abcam) and MAEL (1:800; Abcam) by IHC. Sections were treated with 3% hydrogen peroxide (H 2 O 2 ) and 5% bovine serum albumin and incubated overnight with primary antibodies at 4 C. After incubation with horseradish peroxidase-conjugated secondary antibody for 1 hour at 37 C, sections were washed and counterstained with hematoxylin.

Cell proliferation assay
The cell growth rate was assessed by the CCK-8 (Dojindo) kit at a density of 1 Â 10 3 cells per well in 100 mL RMPI 1640 (SH30809.01, HyClone). Cells were seeded in 96-well plates. After every 24 hours for 3 days, cells were stained with 10 mL of CCK-8 reagent at 37 C for 1 hour, and the absorbance was measured at 450 nm (MULTISKANMK3, Thermo Scientific). The number of viable cells was determined by OD value. The results are expressed as the mean AE SEM of 3 independent experiments.

Apoptosis assay
After 48 hours of transfection with Vector-TE7 (Vec) and MAEL-TE7 (MAEL), shControl-TE1 (shC) and shMAEL-TE1 (shM) cells were harvested and washed with ice-cold PBS twice. Next, the cells were collected and suspended in the 200 mL Annexin V-binding buffer (BioLegend) to a final concentration of 10 6 cells/mL. Cells were incubated with 1 mL AlexaFluor647 Annexin V (BioLegend) for 15 minutes at 4 C in the dark, and then propidium iodide (PI; Sigma) was added. Samples were immediately analyzed by flow cytometry (BD FACSCanto II).

Three-dimensional (3D) culture
Detailed 3D culture experiments were performed as described previously (23). Briefly, before seeding into 3D fibrin gels, Vector-TE7 (Vec) and MAEL-TE7 (MAEL) were maintained in RPMI-1640 culture medium containing 10% FBS and antibiotics, and cell density was adjusted to 10 4 cells/mL. Fibrinogen was diluted into 2 mg/mL with T7 buffer (pH 7.4, 50 mmol/L Tris, 150 mmol/L NaCl). Fibrinogen and the cell solution were mixed at a ratio of 1:1, resulting in 1 mg/mL fibrinogen and 5,000 cells/mL in the mixture. Cell/fibrinogen mixture (250 mL) was seeded into each well of a 24-well plate and mixed well with preadded 5 mL thrombin (0.1 U/mL). The plate was then moved into a 37 C cell culture incubator and incubated for 10 minutes. Finally, 1 mL RPMI-1640 medium was added.

Multiplex bead-based assay
The Human Chemokine and Cytokine detection kit uses multiplex bead-based assay panel provided by BioLegend LEGENDplex (#740001). Supernatants were collected from the same quantity of Vector-TE7 (Vec), MAEL-TE7 (MAEL), shControl-TE1 (shC), and shMAEL-TE1 (shM) cells after culturing for 48 hours and were centrifuged prior to analysis. Results were normalized to standards provided by the BioLegend LEGENDplex. Detailed experiments were performed according to the standard protocol.

Transwell assay
The chemotactic migration of PMN-MDSCs was evaluated in 24-well plates with 5-mm pore size polycarbonate filters (Corning Inc.). Freshly isolated PMN-MDSCs from patient peripheral blood were seeded onto the upper chamber. Tumor cells or anti-IL8 neutralizing antibodies (50 mg/mL; Abcam, #ab18672) were added to the lower chamber. PMN-MDSCs that had migrated into the lower chamber were counted after 24 hours.

Animal model
All Half of the mice in each group were treated as follows: mice were inspected every other 2 days, and tumor growth was evaluated by measuring length and width of tumor mass with calipers. At day 6, mice with detectable tumors were injected 2.0 Â 10 7 PMN-MDSCs (i.v.). Reparixin (20 mg) was administered subcutaneously daily from days 6 to 24. At day 24, mice were sacrificed to obtain tumor tissues used for detection of the tumor weight. Tumor volumes were calculated using the formula: (length Â width 2 )/2. The remaining half of the mice in each group were handled as follows: reparixin (20 mg) was administered subcutaneously daily from days 18 to 24. At day 23, mice were injected 2.0 Â 10 7 PMN-MDSCs (i.v.), and at day 24, mice were sacrificed to obtain tumor tissues that were used to assess the percentage of PMN-MDSCs recruited to tumor site by flow cytometry.

TCGA database analysis
The expression of MAEL and IL8 of 190 esophageal cancer cases were obtained from The Cancer Genome Atlas in 2016 (TCGA; https://cancergenome.nih.gov/). All the cases were esophageal cancer patients without any treatment.

Statistical analysis
A paired t test was used to compare MAEL and IL8 expression between ESCC tissues and nontumor tissues, using GraphPad Prism 5 software (GraphPad Software). The Pearson c 2 test analyzed the association between MAEL expression and clinicopathologic features, and MAEL and IL8. Photoshop software was used to detect the gray value. OS and disease-free survival (DFS) curves were evaluated by the Kaplan-Meier method and log-rank tests. Results were considered statistically significant when P < 0.05.
To study the potential clinical relevance of MAEL expression in patients with ESCC, we performed IHC on tissue sections, and selected representative IHC images are shown in Fig. 1C. We found that MAEL was expressed in tumor tissues and was absent or weakly stained in marginal tissues. Kaplan-Meier analysis revealed that higher MAEL expression was associated with lower OS and poorer disease-free survival (DFS) rates of patients with ESCC (Fig. 1D). Our results also revealed that MAEL expression was upregulated with increased tumor stage (P ¼ 0.0398), lymph node metastasis (P ¼ 0.0076), differentiation (P ¼ 0.0378), and tumor size (P ¼ 0.0338), whereas MAEL expression had no significant correlation with patient age or gender (Supplementary  Table S3). Collectively, MAEL was considered as an independent prognostic marker for ESCC.

Overexpression or knockdown of MAEL influences the tumorigenesis of ESCC cell lines
To evaluate the oncogenic function of MAEL, we transfected a gene construct expressing GFP-MAEL into TE7. After several rounds of sorting based on GFP expression, stable MAELoverexpressing TE7 cells were obtained, and the purity was over 97% (Supplementary Fig. S1A). The efficiency of MAEL upregulation at the mRNA and protein levels was respectively detected by qRT-PCR ( Fig. 2A) and Western blot (Fig. 2B), and the gray value was summarized by histogram ( Supplementary  Fig. S1B). Ectopic expression of MAEL was shown to promote cell growth (Fig. 2C) by CCK-8 assay and inhibit cell apoptosis ( Supplementary Fig. S1C) by flow cytometry assay. The colony formation assay yielded a higher number and larger colonies in MAEL-transfected cells than in control cells (Fig. 2D). We also found that sphere-forming ability of MAEL-overexpressing cells was significantly increased compared with control cells (Fig. 2E). We further investigated whether MAEL could increase the stemness of ESCC cells and found that MAEL could upregulate many stemness-associated genes (Oct4, NANOG, and NOTCH1) and surface antigen CD133, which is associated with cancer stem cells (Fig. 2F).
To validate the effect of MAEL on tumor growth in vivo, a xenograft tumor mouse model was established by subcutaneously injecting MAEL-overexpressing and control cells into the left dorsal flank of mice. We found that volumes and weights of tumors were significantly greater in MAEL-overexpressing cells than in controls (Fig. 2G). IHC staining also confirmed that expression of MAEL in MAEL-transfected cells was higher compared with control cells in xenograft tumors (Fig. 2H). Lump images of xenograft tumors formed in nude mice injected with MAEL-and vector-transfected cells are shown in Supplementary Fig. S1D.
In order to demonstrate tumorigenic function, MAEL was silenced by shRNA. The sorting purity of transfected cells was over 90% (Supplementary Fig. S1E). The efficiency of MAEL downregulation at the mRNA and protein levels was respectively detected by qRT-PCR (Fig. 3A) and Western blot (Fig. 3B), and the gray value was calculated by histogram ( Supplementary Fig. S1F). MAEL knockdown could inhibit cell growth (Fig. 3C) by CCK-8 assay and promote cell apoptosis ( Supplementary Fig. S1G) by flow cytometry assay. A colony formation assay yielded a lower number and smaller colonies in MAEL-silenced cells than in control cells (Fig. 3D). The sphere-forming ability of MAEL-silenced cells was significantly decreased compared with control cells (Fig. 3E). MAEL knockdown also downregulated stemness-associated genes (Oct4, NANOG, and NOTCH1) and surface antigen CD133 (Fig. 3F).
We found that volumes and weights of tumors were significantly smaller and lighter in MAEL-silenced cells than in control cells in vivo (Fig. 3G). Representative images from IHC staining also confirmed that MAEL expression was significantly decreased in MAEL-silenced cells compared with control cells (Fig. 3H). Images of the xenograft tumors formed in nude mice injected with MAEL-silenced and control cells are displayed in Supplementary  Fig. S1H. Overall, MAEL overexpression or knockdown could promote or inhibit cell growth, self-renewal ability, and stemness

of ESCC cells, and tumor formation and development in vivo,
indicating that MAEL had a strong oncogenic function in the progression of ESCC.

IL8, regulated by MAEL, positively correlated with patient prognosis
Accumulating evidence suggests that TME plays a crucial role in promoting the development of solid cancers (11,26). In TME, cytokines and chemokines relevant to tumor progression are thought to be key tumor-promoting factors. In the present study, we detected 10 cytokines and 13 chemokines using a human multiplex bead-based kit and found that only IL8 had a significant and contrary change in MAEL-overexpressing and MAEL-silenced cells at the protein level (Fig. 4A). The expression of 14 chemokines was detected by qRT-PCR, represented as heat map (Fig. 4B). The results demonstrated that expression of CCL5, CCL22, CXCL5, and IL8 was significantly upregulated in MAEL-transfected cells compared with control cells (Fig. 4C), whereas the expression of CCL5, CXCL5, CXCL6, and IL8 was downregulated in MAEL-silenced cells (Fig. 4D). Only IL8 was significantly changed among different groups by various methods (Fig. 4E). In concordance with multicytokine and qRT-PCR analysis, ELISAs also demonstrated that MAELoverexpressed cells induced a significant upregulation of IL8 in parallel with a significant downregulation of IL8 in MAELsilenced cells (Fig. 4F). Thus, IL8 could be a downstream target of MAEL.
The expression of IL8 was positively correlated with MAEL at the mRNA level ( Supplementary Fig. S2A). TCGA database (N ¼ 190) also demonstrated a significant positive correlation between MAEL and IL8 (Supplementary Fig. S2B). To investigate the potential clinical relevance of IL8 in patients with ESCC, by qRT-PCR, we found that the expression of IL8 was higher in 90 esophageal tumor tissues than in nontumor tissues ( Supplementary Fig. S2C). Representative IHC images showed that expression of IL8 protein was significantly higher in tumor tissues compared with matched marginal tissues (Fig. 4G). Kaplan-Meier analysis revealed that higher IL8 expression was associated with poorer OS and DFS rates of 64 ESCC patients (Fig. 4H). These results suggest that IL8 could be regulated by MAEL in TME in ESCC.

PMN-MDSCs were chemotactically recruited by IL8
Multiple mechanisms of immune suppression can arise in the disease state to facilitate the progression of cancer (15,27). Among them, tumor recruitment of MDSCs represents one major impediment to effective antitumor immune responses. According to previous studies, MDSCs can be recruited by IL8, and cell supernatants of advanced cancer patients are usually rich in IL8 commonly produced by malignant cells (28). Therefore, we performed a classic transwell migration assays to verify the recruitment of PMN-MDSCs in patient PBMCs by IL8. Representative flow cytometry gating of PMN-MDSCs (Lin À HLA-DR À CD33 þ CD15 þ CD14 À CD11b þ ) is shown in Figure 5A. We found that the numbers of MDSCs chemoattracted by cell supernatant were higher in shControl (shC) than sh-MAEL (shM) cells, and that in MAEL-overexpressed than in control cells (Fig. 5B). Migration of PMN-MDSCs could be blocked by IL8-neutralizing antibody (Fig. 5B). A positive correlation between the percentage of PMN-MDSCs and IL8 mRNA in ESCC tissues (N ¼ 11) was found ( Supplementary  Fig. S2D). PMN-MDSCs were subjected to a suppression assay. These cells inhibited T-cell proliferation, shown by a reduced proliferation index in T cells (Supplementary Fig. S2E) and decreased expression of perforin and granzyme B secreted by effector T cells (Supplementary Fig. S2F-S2G). We then detected whether CXCR1, the chemokine receptor for IL8, was expressed on esophageal cancer cells and PMN-MDSCs and found that the expression of CXCR1 was respectively 16.20% AE 2.889% (N ¼ 6) on esophageal cancer cells ( Supplementary  Fig. S2H) and 70.17% AE 4.540% (N ¼ 10) on PMN-MDSCs ( Supplementary Fig. S2I), indicating that the expression of CXCR1 on PMN-MDSCs was significantly higher compared with that on esophageal cancer cells.
Reparixin, a described pharmacologic inhibitor of CXCR1 and CXCR2, has been shown to decrease the recruitment of MDSCs in vivo (15,29,30). To further investigate the effect of IL8 on PMN-MDSCs in vivo, a further study was designed as described in Fig. 5C. We found that volumes and weights of tumors were significantly greater in the shC þ PMN-MDSCs group (compared with the shC group) and in the MAEL þ PMN-MDSCs group (compared with the MAEL group), which could be counteracted by downregulating MAEL or using reparixin ( Fig. 5D and E). Images of the xenograft tumors formed in nude mice are shown in Supplementary Fig. S3A and S3B. As shown in Fig. 5F and G, we found that the percentage of PMN-MDSCs chemoattracted to the tumor site was significantly upregulated in the shC þ PMN-MDSCs group compared with the shC group and also in the MAEL þ PMN-MDSCs group compared with the MAEL group, and the effect could be counteracted by downregulating MAEL or using reparixin. From these results, we could infer that IL8, regulated by MAEL, chemoattracted PMN-MDSCs to tumor site to promote cell growth, which could be blocked by downregulating MAEL or IL8-neutralizing antibody in vitro or reparixin in vivo.

MAEL upregulated IL8 by activating the Akt1/RelA signaling pathway
Liu and colleagues demonstrate that MAEL can activate the Akt/GSK-3b/Snail signaling pathway in HCC (1), which prompted us to investigate whether MAEL-regulated IL8 expression is mediated by Akt1 protein in ESCC cells. As anticipated, Western blot results demonstrated that expression of phosphorylated Akt1 was substantially enhanced in MAELoverexpressed compared with control cells (Fig. 6A), and reduced expression of phosphorylated Akt1 was observed in MAEL-silenced cells (Fig. 6B). The most common upstream target of chemokines is the nuclear transcription factor RelA, and it has been reported that RelA can upregulate IL8 (31-33).
As we expected, expression of phosphorylated RelA and IL8 was upregulated in MAEL-overexpressed compared with control cells (Fig. 6A), and opposite results were observed in MAEL-silenced cells (Fig. 6B). To further demonstrate that MAEL-regulated IL8 was through activation of the Akt1/RelA signaling pathway, tumor cells were treated with protein (PI3K, Akt1, and RelA) inhibitors. In line with previous reports, we revealed that PI3K inhibitor (LY) could downregulate phosphorylated Akt1, phosphorylated RelA, and IL8 expression. An Akt1 inhibitor (AZD) could also downregulate phosphorylated Akt1, phosphorylated RelA, and IL8 expression. The RelA inhibitor (QNZ (EVP)) could downregulate phosphorylated RelA and IL8 expression ( Fig. 6A and B). ELISAs also demonstrated that expression of IL8 in the supernatants from tumor cells was significantly downregulated when cells were treated with either LY, AZD, or QNZ (EVP; Fig. 6A and B). By gray value analysis, we found that these results were statistically significant ( Supplementary Fig. S4A and S4B). qRT-PCR assay demonstrated that total AKT1, RELA, and IL8 expression was significantly changed when MAEL was overexpressed or knockdown, or when cells were treated with either LY, AZD, or QNZ (EVP; Supplementary Fig. S4C and S4D). Therefore, we could infer that the oncogenic function of MAEL is to upregulate IL8 for the recruitment of PMN-MDSCs via activation of the Akt1/RelA signaling pathway.

PMN-MDSCs secreted TGFb to upregulate MAEL through activating Smad2/Smad3
It has been reported that MDSCs supernatant could upregulate another cancer-testis antigen (CTA), MAGE-A4, in lung cancer (34). In accordance with previous studies, we found that MDSC supernatants could significantly upregulate MAEL, activate EMT, and upregulate stemness-associated genes in esophageal cancer cell lines (Fig. 7A). To demonstrate which cytokines in MDSC supernatants exerted this effect, we detected expression of several more common factors secreted by PMN-MDSCs, such as IL10, IL6, IL8, TGFb, and TNFa by ELISA (35,36), and we found that TGFb, IL10, and IL6 was high ( Supplementary Fig. S4E). It has been reported that TGFb (37), IL10 (38), and IL6 (39) can activate EMT. In order to further confirm the cytokines in PMN-MDSC supernatants that could upregulate MAEL, we treated TE7 cells with TGFb, IL10, and IL6 and detected the expression of MAEL, EMT markers, and stemness-associated genes. We found that only TGFb could upregulate MAEL (Fig. 7B) and that the expression of TGFb was positively correlated with MAEL at the mRNA level ( Supplementary Fig. S4F). These results demonstrated that TGFb secreted by PMN-MDSCs could upregulate MAEL.
Regarding the TGFb/Smad signaling pathway, studies have revealed that TGFb and TGFbR1 binding complexes can promote translocation of Smad complexes to the nucleus (40). To further verify whether TGFb-modulated MAEL was also mediated by Smad proteins, we tested expression of several proteins involved in the TGFb/Smad pathway by Western blot. In our results, when TE7 and TE1 cells were treated with TGFb, the expression of pSmad2, pSmad3, and MAEL was significantly upregulated (Fig. 7C and D). As expected, opposite expression of these proteins was detected when TE7 and TE1 were treated with TGFbR1 inhibitor (SB; Fig. 7C and D). By gray value analysis, the results were statistically significant ( Supplementary Fig. S4G-S4H). These results demonstrated

Discussion
Esophageal cancer ranks as the sixth leading cause of cancerrelated death and the eighth most commonly diagnosed malignant disease in China (41,42). Although profited from the development of diagnostic techniques and therapeutic modalities, the prognosis of ESCC has been poor. Up to now, the exact cellular and molecular mechanisms leading to ESCC have not been systematically evaluated. Thus, there is urgency to find new treatment targets for ESCC, especially molecularly targeted therapies.
MAEL, as a novel CTA, was identified as a potential therapeutic target of human HCC (1), UCB (7), and colorectal cancer (8). However, tumorigenic function and the underlying mechanisms of MAEL in ESCC have remained unclear thus far. In our study, we found that expression of MAEL, correlating with tumor stage, differentiation, and lymph node metastasis, was higher in tumor tissues compared with nontumor tissues. MAEL could promote cell growth, colony formation, and stemness in vitro and led to a significant increase in tumor volumes and weights of mice in vivo. Taken together, MAEL plays an important role in esophageal cancer progression and could be used as an effective therapeutic target.
Some studies have shown that some oncogenes, such as IL33, can target cancer stem cell genes to directly accelerate colon cancer cell growth and facilitate macrophage recruitment to indirectly promote the progression of colon cancer (43). Twist1's role in directly promoting the development of gastric cancer and indirectly driving the transdifferentiation of quiescent fibroblasts into CAFs in the TME (44,45) has also been demonstrated. Studies investigating MAEL have focused on studying its tumorigenicity (1,7), with little attention paid to its possible role in the TME. In order to investigate whether MAEL also plays a certain role in regulating the TME in ESCC, the potential mechanisms of MAEL need to be further explored. An increasing number of studies have shown that chemokines (46,47) or cytokines (48) can promote tumor development. Based on these results, we wanted to explore whether MAEL was also associated with chemokines or cytokines in the TME. Using multicytokine, qRT-PCR, and ELISA, we detected the expression of chemokines and cytokines and found that only IL8 was significantly changed. We further observed a significant positive correlation between the expression of MAEL and IL8 and that IL8 was a downstream target of MAEL.
Some researchers indicate that IL8 can promote tumor cell invasion and migration by directly interacting with CXCR1/2 of ESCC cells (49). Other researchers have found that IL8 can recruit MDSCs through their expression of CXCR1/2 to further facilitate the growth of tumor cells in the TME (15,50,51). In our study, we found that the expression of CXCR1 on PMN-MDSCs was significantly higher compared with that on esophageal cancer cells, indicating that IL8 mainly played a role in recruiting PMN-MDSCs, which was significantly attenuated by IL8-neutralizing antibody in vitro and reparixin in vivo. These results demonstrated that MAEL regulated the development of ESCC via regulation of IL8, recruiting MDSCs to tumor sites.
Nonetheless, the molecular mechanisms of MAEL-regulated IL8 have not been elucidated. In HCC (1), MAEL can promote tumor cell invasion and migration via activation of Akt/GSK3b/Snail signaling. Therefore, we hypothesized that MAEL could regulate IL8 via Akt1 in ESCC as well. In this study, we found that expression of phosphorylated Akt1 increased when MAEL was overexpressed, and opposite results were observed when MAEL was silenced. The expression of IL8 was decreased when cells were treated with LY or AZD. Many studies have demonstrated that the most common upstream target of chemokines is RelA (13,52,53), and RelA can regulate IL8 (13,32). These results prompted us to investigate whether RelA could regulate IL8 in ESCC. Our data showed that IL8 was significantly decreased after tumor cell treatment with QNZ (EVP), and phosphorylated RelA increased or decreased accordingly when MAEL was overexpressed or silenced. We further explored whether Akt1 could regulate RelA in ESCC. In line with previous studies (54,55), we found that inhibiting Akt1 in transfected cells by LY or AZD suppressed activation of the RelA signaling pathway, indicating that MAEL could regulate IL8, which recruited PMN-MDSCs to the tumor site to promote tumor progression via activation of Akt1/RelA signaling.
Shi and colleagues demonstrated that MAGE-A4, a CTA, could be regulated by MDSC supernatants (34), and Sampson and colleagues demonstrated TGFb could upregulate another A and B, The expression of Akt1, pAkt1, RelA, pRelA, and IL8 was detected by Western blot and ELISAs between MAEL and Vec cells, and between shC and shM cells when cells were treated with PI3K inhibitor (LY), Akt inhibitor (AZD), and RelA inhibitor (QNZ (EVP)). Ã , P < 0.05; ÃÃ , P < 0.01; ÃÃÃ , P < 0.001 (Student t test). Three independent experiments were performed, and results are summarized as mean AE SEM.
CTA, GAGEC1 (3). In our study, we found that MDSC supernatants also upregulated MAEL. These results prompted us to further investigate which cytokines in MDSC supernatants, such as TGFb, IL6, IL10 or IL8, might influence expression of MAEL. Cytokines found to be produced were added to tumor cells to detect the expression of MAEL. Our data indicated that only TGFb could upregulate MAEL. Accumulating evidence has revealed that blocking TGFb/Smad signaling is a promising cancer therapy (56,57). These results led us to investigate whether TGFb-triggered MAEL upregulation could be Smad dependent. Our results demonstrated that the expression of MAEL and phosphorylated Smad2/Smad3 were considerably upregulated in TGFb-treated cells, and when TGFb inhibitor was added, the effects were reversed. These results indicated that TGFb induced upregulation of MAEL via activation of Smad2/Smad3. In summary, MAEL played an important role in the progression of ESCC via activation of Akt1/RelA/IL8 signaling to recruit PMN-MDSCs to tumor sites, and TGFb secreted by PMN-MDSCs could induce Smad2/Smad3 phosphorylation to upregulate MAEL to further promote tumor progression in patients with ESCC.

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.

Disclaimer
Pupu Li, Xinfeng Chen, Guohui Qin, and Yi Zhang had full access to all data and take responsibility for its integrity and accuracy of the data analysis.