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Downregulation of the Proton-Activated Cl- Channel TMEM206 Inhibits Malignant Properties of Human Osteosarcoma Cells

Transmembrane protein 206 (TMEM206), a proton-activated chloride channel, has been implicated in various biochemical processes, including bone metabolism, and has emerged as a novel cancer-related protein in multiple tumor types. However, its role in primary malignant bone tumors, particularly in osteosarcoma (OS), remains unclear. This study is aimed at exploring the effects of TMEM206 gene silencing on the proliferation, migration, invasion, and metastasis of human OS cells in vitro and in vivo using an shRNA-knockdown strategy. We found that TMEM206 is frequently overexpressed and that high levels of TMEM206 correlated with clinical stage and pulmonary metastasis in patients with OS. We provided evidence that TMEM206-silenced OS cancer cells exhibit decreased proliferation, migration, and invasion in vitro. Mechanistically, we identified β-catenin, a key member of Wnt/β-catenin signaling, as a downstream effector of TMEM206. TMEM206 silencing inhibits the Wnt/β-catenin signaling pathway in expression rescue experiments, confirming that TMEM206 silencing attenuates OS cell tumorigenic behavior, at least in part, via the β-catenin mediated downregulation of Wnt/β-catenin signaling. More importantly, TMEM206 knockdown-related phenotype changes were replicated in a xenograft nude mouse model where pulmonary metastases of OS cells were suppressed. Together, our results demonstrate that silencing TMEM206 negatively modulates the Wnt/β-catenin signaling pathway via β-catenin to suppress proliferation, migration, invasion, and metastasis in OS carcinogenesis, suggesting TMEM206 as a potential oncogenic biomarker and a potential target for OS treatment.


Introduction
Osteosarcoma (OS) is the most common primary aggressive and malignant tumor of the bone, with high morbidity and metastatic potential. Approximately 75% of OS patients are between 15 and 25 years of age, which heavily threatens the health of adolescents [1]. Although significant progress has been made in diagnostic and therapeutic methods, the primary treatment for OS mainly depends on surgery combined with adjuvant chemotherapy; however, the clinical outcome is unsatisfactory [2]. The five-year survival rate of osteosarcoma without metastasis is only 60-70% [3]. The poor outcome of OS therapy is mostly due to the high incidence of recurrence and metastasis. Metastasis is common in OS, and approximately 85% of metastases are located in lungs [4], which contributes to most patients' death. Thus, exploring efficient diagnostic biomarkers and therapeutic targets for HCC treat-ment could lead to novel intervention strategies for improving OS clinical outcomes.
Proliferation, invasion, and metastasis are the essential biological characteristics of OS [5] and are the root causes of tumor recurrence. When metastasis occurs, OS cells enter the peripheral vascular or lymphatic vascular system through normal tissues, invade the vascular system, stay in the target organ, and proliferate to form new lesions in the target organ, which fully demonstrates OS oncological characteristics [6]. Genetic changes are involved in tumor invasion and metastasis. Changes in these genes at the transcriptional and epigenetic levels are prerequisites for maintaining the malignant phenotype of tumor cells [6,7]. Studying the potential role and molecular mechanism of these genes will help clarify the characteristics of tumor invasion and metastasis and provide new targets for antitumor therapy.
Ion-activated chloride channels (IACC) are members of the transmembrane (TMEM) chloride channel family and are widely distributed in human tissues, including bone [8,9]. The TMEM family plays a vital role in the formation and osteoclastic processes of bone tissue [10,11]. At the same time, abnormal expression of the TMEM family plays an important role in the occurrence and development of various tumors, such as gastric cancer [12], breast cancer [13], head and neck cancer [14], and OS [15]. TMEM206, also known as PAC or PACC1, encodes the proton-activated chloride (Cl-) channel, and although it has been implicated in some tumors, its role in OS has not been explored [16,17]. A high incidence of OS during adolescence is accompanied by intensive bone development and bone remodeling, a continuous process that includes bone degradation, which depends on hydrogen ion translocation to solubilize bone minerals [18]. This hydrogen redistribution is supported by the parallel actions of a proton ATPase, and a chloride channel located in the osteoclast ruffled membrane [19]. These considerations hint that TMEM206 may also be closely related to OS occurrence, and its underlying mechanisms need to be explored.
In this study, we collected OS patient tissue samples to investigate whether TMEM206 was dysregulated in OS patients and correlated with prognosis. TMEM206 loss-of-function experiments in well characterized OS cell lines were then performed in vitro and tumor growth in vivo to confirm clinical observations. Our findings provide novel insights into the role of TMEM206 in OS cell proliferation, migration, invasion, and metastasis while shedding light on the potential molecular mechanisms.

Sample Collection.
Human OS tissue samples were collected from Renmin Hospital of Wuhan University, from November 2017 to June 2019, from patients aged 7-33 years (median, 18 years). There were 33 adjacent nontumor tissues and 48 OS samples. Grade of OS tissues were distinguished on the basis of Ennecking staging system during pathological examination. All participants have signed an informed consent form and been informed of all the surgery and experiment details in advance. Histologic sections were reviewed by two expert pathologists to verify the histologic diagnosis. We obtained written informed consent from all patients. The Ethics Committee of Renmin Hospital of Wuhan University approved the study protocol. Eighteen male BABL/c nude mice (6-8 weeks old) were purchased from the Wuhan University experimental animal center (Wuhan, China). All experimental procedures involving animals were strictly followed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by Animal Care and Use Committee of Renmin Hospital of Wuhan University. The human fetal osteoblastic cell line hFOB1. 19 and human OS cell lines SAOS2, U2OS, and MG63 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in Dulbecco's Modified Eagle Medium DMEM (Gibco, Carlsbad, CA, USA), and 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco, Grand Island, NY, USA) were added. Cells were cultured at 37°C with 5% CO2 in an incubator (Thermo Fisher Scientific, Waltham, MA, USA).

Western Blot
Assay. RIPA lysis buffer (Beyotime, PRC) was adopted to abstract the overall proteins as per supplier's guideline. Immunoblotting was used to identify the protein expressing levels herein as per the guideline provided in the previous study. Primary antibodies against TMEM206 (1 : 300, ab99055, Abcam, MA, USA), β-catenin (1 : 200, ab32572, Abcam), c-Myc (1 : 500, ab32072, Abcam), CyclinD1 (1 : 400, ab16663, Abcam), MMP7 (1 : 400, ab207299, Abcam), MMP9 (1 : 200, ab76003, Abcam), and GAPDH (1 : 500, ab9485, Abcam) were purchased as primary antibodies. HRP-predicted sheep antirabbit second antisubstances (1 : 5000) were acquired as well. Eventually, the visualization of the protein bands were realized via an ECL system, and grayscale results were determined via Image J soft. 2.4. Immunofluorescent Staining. The transfected U2OS cells were fixed with 4% paraformaldehyde for 30 min and rinsed thrice with PBS and. The cells were then permeabilized with PBS containing 0.5% Triton X-100 for 15 min. The TMEM206 antibody (HPA008540, Sigma, USA) was applied to the coverslip and incubated in a wet box overnight at 4°C. After washing three times with PBS, the fluorescent secondary antibody (1 : 500, Invitrogen, USA) was added and incubated for 1 h in the wet box at 25°C. Following three additional washes with PBS, the DAPI mounting solution (Vector, USA) was added to stain the nuclear compartment. The immunoreactive cultured cells were observed using a fluorescence microscope (Olympus, Japan).
2.5. CCK-8 Assay. CCK-8 detection (Bimake, Shanghai, China) uses a similar method, as described earlier. Transfected cells were inoculated into 96-well plates and incubated overnight. Add CCK-8 reagent 10 mL to each well and incubate at 37°C for 2 hours. The absorption value was measured at 450 nm with a microplate analyzer (Bio-Tek Instruments Inc., USA).
2.6. 5-Ethynyl-2 ′ -Deoxyuridine (EdU) Staining Assay. U2OS and MG63 cells (6 × 10 5 ) were inoculated into 96-well plates, and cell proliferation was assayed using an EdU kit (Roche, Basel, Switzerland) according to manufacturer's instructions. Hoechst 33342 was added to stain the nuclei. After a 30-   Oxidative Medicine and Cellular Longevity minute incubation in the dark, the cells were observed under a fluorescence microscope (Olympus, Japan).

Colony Formation Experiment.
Cells were collected during the exponential growth period, reinoculated on the plate, and cultured at 37°C for 6 days. After 6 days, the cells were washed with PBS, fixed with methanol, stained with crystal violet, and observed under the microscope.

Migration and Invasion Assays.
Invasion assay was carried out using 24-well plates with the Matrigel coating (BD Bioscience, Franklin Lakes, NJ, USA) as previously described [20]. The migration assay was carried out without the Matrigel coating. Transfected cells were seeded in the upper chamber of a Transwell (BD Bioscience, USA) containing serum-free medium. DMEM or MEM medium containing 10% FBS was placed in the lower chamber. After incubation for 48 hr, migrated or invaded cells were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 20 min. Cells were imaged with a fluorescence microscope (Olympus, Japan).
2.9. Luciferase Reporter Assay. Reporter assays were performed with U2OS and MG63 cells, which were transfected with either wild type or mutant reporter plasmids containing the putative β-catenin binding site and cotransfected with the control (pcDNA-Con) or TMEM206 overexpression (pcDNA-TMEM206) plasmid. The resulting Firefly and Renilla luciferase activity were measured 48 h posttransfection using the Dual-Glo™ Luciferase Assay System, according to manufacturer's instructions (Promega, Wisconsin, USA).

Xenograft Mode. MG63 cells stably transfected with
TMEM206-shRNA or SC-shRNA (6 × 10 6 cells) were inoculated subcutaneously into the nude mice. Tumor volumes were measured every 7 days. After 21 days, mice were sacrificed, and tissues were collected for further analyses.
2.11. Statistical Analysis. Statistical analysis and graph making were performed using SPSS Software version 22.0 (IBM, USA) and GraphPad Prism Software version 7.01 (GraphPad Software Inc., USA), respectively. All data were expressed as mean ± standard deviation (SD) or mean ± standard error means (SEM). Comparison between two groups was determined by unpaired t test or one-way ANOVA test. P value < 0.05 was considered as statistically significant, which was displayed as * P < 0:05, * * P < 0:01, * * * P < 0:001, while the P value > 0.05 was displayed as not significant in all figures.

TMEM206 Is Highly Expressed in Human OS Tissues and Cell Lines and Predicts a Poor Prognosis.
We first set to explore whether TMEM206 was aberrantly expressed in OS tissues. Compared to paired adjacent normal tissues, the protein expression of TMEM206 was dramatically higher in tumor tissues (Figure 1(a), P < 0:001). The IHC results indicated that TMEM206 was aberrantly expressed in the advanced Enneckin stage (Figure 1

Silencing TMEM206 Inhibits Cell Proliferation and Colony Formation.
To elucidate the role of TMEM206 in OS, U2OS and MG63 cells were transfected with control scrambled shRNA or TMEM206-shRNA. Immunofluorescence staining indicated that TMEM206 expression was reduced after TMEM206-shRNA transfection (Figure 2(a)), and this knockdown strategy resulted in a significant decrease in TMEM206 protein expression both in U2OS and MG63 cells (Figure 2(b), P < 0:001). Next, we investigated whether TMEM206 regulates cell proliferation. EDU-staining and CCK-8 assays were used to evaluate the effects of silencing TMEM206 on cell proliferation. When TMEM206 was downregulated in OS cells, a significant decrease in proliferation rates was observed compared with SC-shRNA transfection The colony formation assay showed that TMEM206-shRNA-transfection displayed significantly fewer colonies than SC-shRNA-transfected U2OS and MG63 cells (Figure 2(e), P < 0:001). Collectively, these data suggest that TMEM206 plays an important role in OS cell growth in vitro.
3.3. TMEM206 Involved in the Regulation of OS Cell Migration and Invasion. Given that TMEM206 expression correlates with tumor stage, we investigated whether TMEM206 regulates OS cell migration and invasion, two critical features of malignant cell behavior. First, we analyzed the human OS tissues and found that the OS patients with metastasis showed much higher TMEM206 expression than the primary tumors without metastasis (Figure 3(a), P < 0:01). We then examined the role of TMEM206 in U2OS and MG63 cells on cell migration using a wound scratch assay. As illustrated in Figure 3(b), decreased expression of TMEM206 significantly inhibited wound closure compared with that in U2OS and MG63 cells transfected with SC-shRNA vector. To further identify these observations, we examined the effects of TMEM206 on OS cell migration and invasion using Transwell chambers with or without Matrigel. Consistently, OS cells derived from TMEM206-shRNA-transfection displayed a significantly lower migration and invasion ability than that derived from SC-shRNAtransfected cells (Figures 3(c) and 3(d), P < 0:001). Collectively, these data demonstrate that TMEM206 is involved in OS tumor metastasis in vivo and cell migration and invasion in vitro.

Wnt/β-Catenin Signaling Upregulated in OS and
Interacted with TMEM206. Considering that Wnt/β-catenin signaling plays a critical role in the progression of tumor disease, we sought to determine the expression pattern of Wnt/β-catenin signaling in OS tissues. The IHC results indicated that βcatenin was aberrant increasingly expressed with advanced Enneckin stage (Figure 4(a)), and WB results further confirmed this observation (Figure 4(b), P < 0:05). The correlation between the TMEM206 and β-catenin reminds us that there is a interaction mechanism among them.
A dual-luciferase reporter analysis was performed to prove the interaction between TMEM206 and β-catenin. The results showed that overexpression of TMEM206 significantly promoted the activity of firefly luciferase that carried wild-type but not mutant TCF-binding sites both in U2OS and MG63 cells (Figure 4(c), P < 0:01), which indicated that β-catenin is indeed a downstream target of TMEM206. In addition, by using immunofluorescence staining, we also observed that upregulation of TMEM206 expression noticeably promoted the β-catenin accumulation in MG63 cells (Figure 4(d)).

10
Oxidative Medicine and Cellular Longevity whether β-catenin is required for the observed phenotypes induced by TMEM206 knockdown in OS cells, we then conducted the following rescue experiments: first, the Wnt/βcatenin signaling activator lithium chloride (LiCl) was added to the TMEM206-shRNA-transfected U2OS and MG63 cells to rescue the function of Wnt/β-catenin signaling. As expected, the decreased migration and invasion of MG63 cells transfected with TMEM206-shRNA was reupregulated after LiCl administration (Figure 6(a), P < 0:001). To further confirm our hypothesis, we constructed TMEM206 overexpressing MG63 cells, and the β-catenin inhibitor IWP-2 was added to observe its effect. Transfection of the plasmid containing TMEM206 promoted the migration and invasion of MG63 cells, and this promotive effect was abolished by IWP-2 treatment ( Figure 6(b), P < 0:001). Taken together, these data revealed that the defects in proliferation and migration induced by TMEM206 knockdown in OS cells were at least in part via the β-catenin-mediated downregulation of the Wnt/β-catenin signaling pathway.

Discussion
Osteosarcoma (OS), the most common malignant bone tumor in children and adolescents, is characterized by a high degree of malignancy and high occurrence of lung metastasis [2]. The lack of efficient intervention approaches contributes to OS unsatisfactory clinical outcomes, which is a leading cause of cancer mortality worldwide. Exploring the molecular mechanisms and identifying therapeutic targets of OS has always been a hot topic in OS research. Although a growing body of evidence suggests that the TMEM family plays crucial roles in tumorigenesis and the progression of various malignant tumors, its role in OS cancer development is not fully understood. In the current report, we present evidence to demonstrate TMEM206 role in OS development and its potential regulatory pathway. Taking advantage of clinical specimens and wellcharacterized OS cell lines, we sought to establish whether TMEM206 is associated with OS pathogenesis. TMEM206 was consistently found to be upregulated in clinical tumor samples and OS cell lines, and the levels of TMEM206 positively correlated with clinical stages in OS patients, which demonstrates that TMEM206 overexpression might drive OS progression. Similar to our results, the expression of TMEM206 positively correlated with the prognosis of HCC [17] and promoted colorectal cancer malignancy [21]. Consistently, our findings showed that TMEM206 expression is upregulated in highly malignant invasive OS cell lines, such as MG63, suggesting a link between the molecular levels of TMEM206 and OS clinical outcomes. We further constructed shRNA vectors and utilized a set of well-characterized OS cell lines to elucidate the role of TMEM206 in cell proliferation, migration, invasion, and metastasis.
Tumor biological functional experiments in OS cells showed that TMEM206 downregulation influences OS cell 11 Oxidative Medicine and Cellular Longevity proliferation, colony formation, migration, and invasive capacity, the most important features of malignant cell behavior. Consistent with our present data, Zhao et al. found that knockdown or upregulation silencing of TMEM206 inhibited or promoted the proliferation of colorectal cancer cells [21], which supports our hypothesis that TMEM206 is an oncogenic gene and may be a promising therapeutic target. The underlying mechanism of TMEM206 involvement in OS tumorigenicity remains elusive. We then attempted to elucidate the underlying mechanisms by which TMEM206 functions in OS. Zhao et al. found that TMEM206 regulates the phosphorylation of AKT and its downstream signaling pathway [21]. In the present study, the Wnt/β-catenin signaling pathway was regulated by TMEM206 in OS cells. TMEM206 knockdown significantly altered the expression levels of Wnt/β-catenin signaling pathway markers in OS cells dual-luciferase reporter analysis showing that TMEM206 could directly interact with β-catenin. The results of rescue experiments further proved that the defects in proliferation and migration induced by TMEM206 knockdown in OS cells were, at least partly, due to the downregulation of the Wnt/β-catenin signaling pathway. Along the same line of arguments, Jiang et al. reported that TMEM48 could promote cervical cancer invasion and metastasis in vitro and in vivo via activating the Wnt/β-catenin pathway [22]. Moreover, TMEM168 knockdown could also inhibit glioblastoma cell proliferation via suppression of the Wnt/β-catenin pathway [23]. The canonical Wnt/β-catenin pathway is crucial for bone morphogenesis and bone regeneration [24], and various Wnt antagonists have been tested to suppress the proliferation of OS cells in vitro and OS tumor growth in vivo [25]. TMEM206 knockdown mechanism of action on OS inhibition was determined by inhibiting the activation of the Wnt signaling pathway.
Based on the above evidence, we speculate that targeting TMEM206 might be a useful approach to repress the proliferation, migration, and metastasis of OS in vivo. Therefore, we utilized a xenograft model to determine the effect of silencing TMEM206 on OS tumor growth in vivo. Inhibition  Figure 6: Wnt/β-catenin signaling is involved in TMEM206-mediated OS-metabolic properties. (a) Treatment with Wnt/β-catenin signaling activator LiCl recued the effect of TMEM206-shRNA in OS cell migration and invasion ( * * * P < 0:001 compared with SC-shRNA, ### P < 0:001 compared with TMEM206-shRNA, respectively). Scale bar: 100 μm. (b) Treatment with Wnt/β-catenin signaling pathway inhibitor IWP-2 reversed the TMEM206-overexpression mediate migration and invasion promotion ( * * * P < 0:001 compared with pcDNA-Con, ### P < 0:001 compared with pcDNA-TMEM206, respectively). Scale bar: 100 μm. The data are represented as means ± SD from 3 replicate experiments.  13 Oxidative Medicine and Cellular Longevity expression of TMEM206 remarkably suppressed tumor growth, as reflected by the tumor volume accompanied by decreased immunohistochemical reactivity of Ki-67 and CD31. The lung is the most common site of distant metastasis of OS [26]. Our patient results showed that there was more TMEM206 expression in these patients with lung metastasis. Interestingly, after silencing the TMEM206 of the tumor in situ, significantly fewer metastatic foci in the lungs were observed. OS invasion and metastasis require complicated biological processes involving various molecules. The Wnt/β-catenin pathways, MMP7 and MMP9, have been regarded as metastasis-related genes, which play a critical role in OS invasion, metastasis, and tumor cell angiogenesis [27,28]. The decreased tumor growth and metastasis in vivo could be explained by the inhibition of the Wnt/βcatenin pathway. Taken together, our results collectively indicate that blocking the TMEM206 could inhibit OS. Nevertheless, although the results are inspiring, the antitumor effects of the TMEM206 target should be investigated in patients in future clinical studies.
In summary, our findings are the first to show that TMEM206 is increased in human OS tissues and may represent a novel biomarker of OS. Silencing TMEM206 impedes the proliferation and invasion of OS cells in vitro and in vivo by downregulating Wnt/β-catenin signaling. These findings provide sufficient in vitro and in vivo evidence of the anticancer effects and tumor inhibitory role of TMEM206 inhibition in OS and confirm TMEM206 as a key regulator of Wnt/β-catenin signaling. Our study highlights a critical role for the TMEM206/Wnt/β-catenin signaling axis in OS progression and suggests that TMEM206 is a potential therapeutic target for OS diagnosis and therapy.

Data Availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.