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. 2020 Nov 16;100(5):522–531. doi: 10.1177/0022034520972095

RGS12 Represses Oral Cancer via the Phosphorylation and SUMOylation of PTEN

C Fu 1,2,3,*, G Yuan 1,*, ST Yang 1, D Zhang 2,3, S Yang 1,4,5,✉
PMCID: PMC8060775  PMID: 33198557

Abstract

Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer characterized by aggressive local invasion and metastasis. The pathogenesis of OSCC is mainly due to the accumulation of genetic alterations in epithelial cells, but the underlying mechanism for its development remains unclear. Here, we found that the expression level of regulator of G protein signaling 12 (RGS12) was significantly reduced in human OSCC. To understand the role and mechanism of RGS12 in OSCC, we generated a novel RGS12 global knockout (CMVCre/+; RGS12fl/fl) mouse model by crossing RGS12fl/fl mice with CMV-Cre transgenic mice and then further induced the mice to develop OSCC by using 4-nitroquinoline 1-oxide (4NQO). Deletion of RGS12 exhibited aggressive OSCC in the tongue compared with the control RGS12fl/fl mice. Knockdown of RGS12 in OSCC cells significantly increased cell proliferation and migration. Mechanistically, we found that RGS12 associated with phosphatase and tension homolog (PTEN) via the PDZ domain to upregulate the phosphorylation and SUMOylation of PTEN and then correspondingly inactivated the AKT/mTOR signaling pathway. To test the potential therapeutic effect of RGS12 on OSCC, we overexpressed RGS12 in OSCC cells and found a significant inhibition of cancer cell proliferation and migration. Moreover, subcutaneous inoculation of RGS12-overexpressed OSCC cells in NOD scid mice showed a significant reduction in tumor formation. Our findings reveal that RGS12 is an essential tumor suppressor and highlights RGS12 as a potential therapeutic target and prognostic biomarker of OSCC.

Keywords: OSCC, head and neck neoplasms, 4-Nitroquinoline-1-oxide, PDZ domains, G-protein-coupled receptors, protein modifications

Introduction

Oral squamous cell carcinoma (OSCC) arises from the mucosa of the oral cavity and oropharynx, which is the most common form of head and neck cancer (HNC) representing the sixth most frequently occurring malignant tumor worldwide (Dixit et al. 2015; Luo et al. 2018). The prognosis of OSCC is poor due to the high occurrence of local invasion and metastasis as well as the lack of markers suitable for early detection (Ishida et al. 2017).

The aberrance of G proteins and G protein–coupled receptors (GPCRs) is highly involved in tumorigenesis (O’Hayre et al. 2013). Regulators of G protein signaling (RGS) proteins inhibit the active Gα by promoting the GTPase activity of this subunit to turn off GPCR signaling (Kehrl 2016). A number of RGS proteins were identified as differentially expressed genes in a variety of cancers such as prostate cancer, breast cancer, and leukemia (Yang et al. 2016). Several reports have shown that RGS proteins exhibit significant changes in expression or activity between malignant and noncancerous tissues (Hurst and Hooks 2009). Remarkably, RGS2 was involved in androgen-independent prostate cancer and acute myeloid leukemia. RGS5 was increased in hepatocellular carcinoma and the vasculature of renal cell carcinoma (Hurst and Hooks 2009). As the largest member of the RGS family, regulator of G protein signaling 12 (RGS12) has been reported to be lost in African American prostate cancer, but its role and mechanism in the regulation of cancer remain largely undefined (Wang et al. 2017).

RGS12 is a multifunctional structural protein with multiple domains. The classical PDZ domain is located in the N-terminus of RGS12 gene, which is expressed in most tissues (Snow et al. 1998; Kimple et al. 2001). PDZ domains are the building blocks, which play essential roles in most aspects of cellular homoeostasis and tumor growth, development, and metastasis (Subbaiah et al. 2011). As protein-protein interaction modules, the PDZ domain binds to the extreme carboxy-terminal motif (usually with 3 or 4 amino acids) of target proteins (Snow et al. 1998). Notably, phosphatase and tension homolog (PTEN), as an important suppressor of human cancer (Milella et al. 2015), possesses a C-terminal PDZ domain-binding motif (PBM) that can be recognized and specifically bound by the PDZ domain from scaffolding and regulatory proteins (Lee et al. 1999). It has been demonstrated that certain PTEN-PDZ interactions may contribute to tumor suppression by stabilizing PTEN and targeting the specific plasma membrane regions (Sotelo et al. 2015). However, it remains unclear whether PTEN can be specifically recognized by the PDZ domain of RGS12. In addition, the findings from Rozengrut (2007) indicate that RGS proteins play a role in controlling cancer cell proliferation and/or migration via regulating the AKT/mTOR survival pathway. Further studies are needed to elucidate the role and mechanism of RGS12 in oral cancer.

As the most representative model, the 4-nitroquinoline 1-oxide (4NQO)–induced oral cancer mouse model is similar to human OSCC (Luo et al. 2018). In this study, we generated a novel RGS12 global knockout (CMVCre/+; RGS12fl/fl) mouse model by crossing RGS12fl/fl mice with CMV-Cre transgenic mice, then further induced the mice to develop OSCC by using 4NQO. By performing in vitro and in vivo studies, we found that RGS12 is an essential suppressor of oral cancer. RGS12 associates with PTEN via PDZ domain to control the phosphorylation and SUMOylation of PTEN and then inhibits AKT/mTOR pathways, which suppress the progenesis of OSCC. Thus, our results demonstrate that RGS12 acts as a potential therapeutic target and prognostic biomarker of OSCC.

Materials and Methods

The following methods are described in the Appendix: materials and reagents, methods for cell culture, human score methods of OSCC tissue samples (Bryne et al. 1992), generation of RGS12 global knockout mice (Yang et al. 2013), subcutaneous inoculation, histopathology, immunohistochemistry (IHC) and score of immunohistochemical staining, immunofluorescent (IF) staining, quantitative real-time polymerase chain reaction (PCR), Western blot (WB), co-immunoprecipitation (co-IP) and pull-down assay, plasmids construction and transfection (Yuan, Yang, Ng, et al. 2020), invasion assay, wound-healing assay, and WST-1 cell proliferation assay.

Human OSCC Tissue Samples

The samples of OSCC tissues and adjacent normal tongue (NAT) tissues (OR601b) (60 cases/60 cores) were obtained from US Biomax. All samples were divided into different TNM stages and pathology grades according to the manufacturer’s instructions and Bryne’s grading system (Bryne et al. 1992). See the Appendix Materials and Methods for the criteria for TNM staging and pathological grading.

Generation of RGS12 Global Knockout (CMVCre/+; RGS12fl/fl) Mouse Model

Methodologies for the generation of homozygous RGS12fl/fl mice on C57Bl/6 backgrounds have been previously described (Yang et al. 2013; Yuan, Yang, Liu, et al. 2020). To delete RGS12, we crossed RGS12fl/fl mice with CMV-Cre transgenic mice to generate CMVCre/+; RGS12fl/+ progeny, which were used for subsequent mating to produce heterozygous CMVCre/+; RGS12fl/fl mice. RGS12fl/fl mice were used as controls since the phenotype in RGS12fl/fl mice was indistinguishable from that in CMV-Cre mice.

Administration of 4NQO Model

Six-week-old CMVCre/+; RGS12fl/fl mice and RGS12fl/fl mice were used for the experimental group and the control group (n = 5/sex/group). Both male and female mice were used to include sex-related potential variations. The carcinogen 4NQO (Sigma) stock solution was prepared in propylene glycol at 5 mg/mL and stored at 4°C. The mice in experimental groups were administered drinking water with a final concentration of 100 µg/mL 4NQO for 16 wk and then changed to clean water for additional an 8 wk to observe the tongue tumorigenesis (Tang et al. 2004). All animal experiments were performed by following institutional standard operating protocols and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pennsylvania.

Statistical Analysis

All data are presented as mean ± standard deviation (SD). Two-tailed Student’s t test was used to evaluate numerical comparisons between 2 groups, and 1-way or 2-way analysis of variance (ANOVA) followed by Tukey post hoc test was used for comparison of multiple groups. P < 0.05 was considered statistically significant. The statistical analysis was evaluated by using GraphPad Prism 6.0 software (GraphPad Software).

Results

RGS12 Expression Level Significantly Decreases in Human OSCC Tissues in TNM Stage and Pathological Grade-Dependent Manner

To assess the expression of RGS12 in tongue mucosal epithelium and oral cancer tissues, human OSCC samples with different TNM stages and pathological grades and adjacent NAT samples were selected for IHC staining and scoring. The results indicated that RGS12 protein was highly expressed in the NAT mucosal epithelium but significantly decreased in OSCC samples (Fig. 1A). Moreover, with an increase in the TNM stage and pathological grade of the OSCC, the expression level of the RGS12 protein decreased (Fig. 1B), indicating a negative correlation between RGS12 and OSCC tumorigenesis.

Figure 1.

Figure 1.

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Decreasing of regulator of G protein signaling 12 (RGS12) promotes the development of oral squamous cell carcinoma (OSCC). (A) Representative images of immunohistochemistry (IHC) showed the expression of RGS12 protein in human OSCC tissues and adjacent normal tongue tissues (NAT). (B) RGS12 protein expression in human OSCC tissues with different TNM stages, different pathology grades, and quantitative analysis of the total RGS12 IHC score. Quantitative data are presented as mean ± SD. *P < 0.05, **P < 0.001, n = 5. (C) Representative images of the size of RGS12 global knockout (CMVCre/+;RGS12fl/fl) mice and control RGS12fl/fl mice with or without 4-nitroquinoline 1-oxide (4NQO) induction. (D) Macroscopic images of the tongue in female mice (n = 5, black arrows and white dot lines show the tumor area). (E) Hematoxylin and eosin staining of the tongue mucosa as depicted in panel D. The incidence of OSCC is shown in the right panel. (F) Left: representative images of immunofluorescence (IF) with cytokeratin 14 (CK14) antibody counterstained with DAPI. Scale bar: 100 μm. Right: quantitative analysis of mean fluorescence intensity of CK14. Quantitative data are presented as mean ± SD. ***P < 0.001 versus RGS12fl/fl mice after 4NQO induction, n = 5.

Deletion of RGS12 Significantly Promotes the Development of OSCC under 4NQO Induction

To understand the role of RGS12 in tumorigenesis in vivo, we generated RGS12 global knockout (CMVCre/+; RGS12fl/fl) mice and induced the mice to develop OSCC by oral administration of 4NQO drinking water (Fig. 1C). Deletion of RGS12 in CMVCre/+; RGS12fl/fl was confirmed by showing the deletion band in tongue genomic DNA (Appendix Fig. 1A) and significantly decreased RGS12 messenger RNA (mRNA) and protein levels (Appendix Fig. 1B–D). After 4NQO induction, the CMVCre/+; RGS12fl/fl mice showed an increase in the range of tongue lesions in both male and female mice (dotted line range) compared with the control mice (Fig. 1D and Appendix Fig. 2). Hematoxylin and eosin (H&E) staining results showed the increased tongue lesion range, the unclear boundary, the obvious epithelial dysplasia, a large number of aggregated cancer nests, and the severely destroyed basal and submucosal layers in the CMVCre/+; RGS12fl/fl mice. However, in the control mice, the localized lesion range and the relatively clear boundary without obvious epithelial dysplasia were observed (Fig. 1E and Appendix Fig. 2). Moreover, the CMVCre/+; RGS12fl/fl mice showed more incidence of invasive carcinoma under 4NQO treatment compared with control mice (Fig. 1E). The result of IF staining showed that the cytokeratin+ (CK14+) signal on the epithelial layer of the tongue mucosa was significantly enhanced and invaded into the muscular layer in 4NQO-treated CMVCre/+; RGS12fl/fl mice (Fig. 1F), suggesting that deletion of RGS12 significantly promotes the development of OSCC.

Knockdown of RGS12 Promotes Cancer Cell Migration and Proliferation

To get further insight into the role RGS12 in squamous cell carcinoma, we first confirmed the RGS12 expression in vitro. By comparing the control cells (human umbilical vein endothelial cells [HUVECs]) and 2 squamous cell carcinoma cell lines (SCC4 and CAL27), we found that both the protein and mRNA expression of RGS12 were significantly lower (Fig. 2A, B) in SCC4 and CAL27 cells. To further determine the role of RGS12 in cancer cells, we transfected the SCC4 and CAL27 cells with scrambled short hairpin RNA (shRNA) control and short hairpin RGS12 (shRGS12) plasmids and confirmed the knockdown of RGS12 protein level by performing the Western blot (Fig. 2C). Matrigel invasion assays were performed in RGS12-silenced SCC4 and CAL27 cells in vitro (Fig. 2D). As shown in Fig. 2D, the number of invaded SCC4 and CAL27 cells was significantly increased in RGS12-silenced cells compared with that in the scrambled shRNA-transfected cells. To examine the effect of RGS12 on cell proliferation, the WST-1 assay was performed in SCC4 and CAL27 cells with and without RGS12 knockdown. The results showed that knockdown of RGS12 significantly increased cell proliferation compared with the scrambled shRNA control (Fig. 2E). All these data demonstrated that RGS12 inhibits the migration and proliferation of cancer cells.

Figure 2.

Figure 2.

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Knockdown of regulator of G protein signaling 12 (RGS12) promotes carcinoma cell migration and proliferation. (A) Left: immunoblot showing that the expression of RGS12 in human umbilical vein endothelial cells (HUVECs) and 2 squamous cell carcinoma cell lines (SCC4 and CAL27). Right: the relative levels of RGS12 (quantified with ImageJ software from the National Institutes of Health) as in panel A. A t test showed significant differences between the 2 groups, **P < 0.01 versus HUVECs. (B) The relative gene expression levels of RGS12. The t test showed significant differences between the 2 groups, **P < 0.01 versus HUVECs. (C) Biological effects in vitro of RGS12 silence. Western blots of lysates from SCC4 and CAL27 cells transfected with short hairpin RGS12 (shRGS12) plasmid (left). The quantitative analysis of Western blots is shown in the right panel. (D) Representative images of the Matrigel invasion assay. Quantitative analysis of SCC4 and CAL27 cell invasion is shown in the right panels. Quantitative data are presented as mean ± SD. ***P < 0.001, n = 3. (E) Proliferation was determined by WST-1 assay in SCC4 and CAL27 cells with or without RGS12 knockdown. Quantitative data are presented as mean ± SD. ***P < 0.001, n = 3.

RGS12 Associates with PTEN via the PDZ Domain

PTEN is an effective tumor suppressor, and the loss of PTEN activity results in cancer susceptibility and favors tumor progression (Lee et al. 2018). Wang et al. (2017) found that the knockdown of RGS12 increased the phosphorylation of AKT (S473) in PNT1A (PTEN wild-type) cells. To determine the mechanism by which RGS12 regulates PTEN, we explored the molecular structure of PTEN (Fig. 3A) and found that PTEN possesses a C-terminal 4 amino acid binding motif (ITKV) that can be recognized by a specific set of PDZ domains of some regulatory proteins (Hopkins et al. 2014). To verify whether PTEN is associated with RGS12, we first performed IF staining. The result showed that both RGS12 and PTEN were expressed in cytoplasm and nucleus, and they overlapped in the expression location (Fig. 3B). Co-IP experiment further demonstrated a strong affinity between RGS12 and PTEN (Fig. 3C). The N-terminal PDZ domain of RGS12 is a protein-protein interaction module that can bind with the 3 or 4 amino acid binding motifs at the extreme carboxy terminus of the target protein (Fig. 3D). Notably, after deletion of the PDZ domain of RGS12 (Fig. 3D), we found that RGS12 failed to bind with PTEN, further confirming that PTEN can be specifically recognized and bound by the PDZ domain of RGS12 (Fig. 3E). To further determine the function of RGS12ΔPDZ and RGS12ΔRGS, we first transfected the control plasmid pCMV, pCMV-RGS12ΔPDZ, pCMV-RGS12ΔRGS, and pCMV-RGS12 into SCC4 cells, respectively (Fig. 3F and Appendix Fig. 3). Then, we performed the cell invasion assay and proliferation assay. The results showed that RGS12ΔPDZ cannot inhibit the cancer cell migration and proliferation compared to the RGS12 OE group (Fig. 3F, G). However, RGS12ΔRGS can inhibit the migration and proliferation of cancer cells compared with control (Appendix Fig. 3). Thus, the PDZ domain of RGS12 may be a potential drug target in cancer studies.

Figure 3.

Figure 3.

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Regulator of G protein signaling 12 (RGS12) associates with phosphatase and tension homolog (PTEN) through the PDZ domain. (A) PTEN consists of a phosphatase (PHOS) domain, a C2 domain containing SUMO sites, a tail domain containing PHOS sites, and a C-terminal PDZ domain-binding motif (PBM) containing 4 amino acids (ITKV). (B) Representative images of immunofluorescence (IF) with RGS12 antibody and PTEN antibody, counterstained with DAPI. IF showed that PTEN and RGS12 were colocalized and expressed in both cytoplasm and nucleus. (C) Co-immunoprecipitation (co-IP) showed that RGS12 associates with PTEN. (D) RGS12 is multidomain, including a PDZ (PSD-95/Dlg/ZO-1) domain, a phosphotyrosine-binding (PTB) domain, a RGS domain, a pair of Ras-binding domains (RBDs), and a single Gai/o-Loco (GoLoco) motif. The RGS12ΔPDZ mutant showed the deletion of the PDZ domain located on the N-terminal containing 77 amino acid residues. (E) Pull-down experiments showed that RGS12 could not bind to PTEN after deletion of the PDZ domain. (F) Representative images of the Matrigel invasion assay. The SCC4 cells were transfected with pCMV, pCMV-RGS12ΔPDZ, and pCMV-RGS12 plasmids with Fugene HD transfection reagent for 24 h. The cells were then seeded in Transwell plates for 16 h. Quantitative analysis of cell invasion is shown in the right panels. Note that overexpression of RGS12ΔPDZ cannot inhibit the cell invasion compared with RGS12. (G) Proliferation was determined by WST-1 assay. The SCC4 cells were treated as depicted in panel F. Quantitative data are presented as mean ± SD. **P < 0.001, ***P < 0.001, n = 3.

RGS12 Regulates PTEN Phosephorylation and SUMOylation

PTEN is the target of multiple modes of posttranslational modification, including phosphorylation, ubiquitination, and SUMOylation (Wang and Jiang 2008). The phosphorylation of PTEN negatively regulates the function of PI3K signaling (Vazquez et al. 2000). SUMO1 modification of PTEN regulates tumorigenesis, and nuclear SUMO-PTEN is required for the repair of DNA double-strand breaks (Huang et al. 2012; Bassi et al. 2013). To further characterize the underlying mechanism that RGS12 negatively regulates OSCC tumorigenesis, we first tested whether RGS12 affects posttranslational modification of PTEN.

SCC4 cells were transfected with control pCMV, pCMV-RGS12, and shRGS12 plasmids and analyzed for the protein level of p-PTEN and total PTEN by Western blot. The results showed that p-PTEN was increased in the RGS12 OE group while decreased in the RGS12 knockdown group (Fig. 4A, B), suggesting that RGS12 regulates p-PTEN in cancer cells. To further assess how RGS12 regulates the SUMOylation of PTEN, the IP and immunoblotting were performed. The results showed that the SUMOylation (SUMO1-PTEN) in SCC4 and 293T cells was increased after overexpression of RGS12 (Fig. 4C, D and Appendix Fig. 4). Furthermore, the AKT/mTOR signaling pathway plays a key role in tumorigenesis, which acts downstream of PTEN (Gao et al. 2016; Cavazzoni et al. 2017). Our results showed that the levels of the phosphorylated RAC-α serine/threonine-protein kinase (AKT) and phosphorylated mechanistic target of rapamycin (mTOR) were decreased after overexpression of RGS12 (Fig. 4E, F). Thus, our data demonstrated that RGS12 negatively regulates OSCC tumorigenesis through controlling PTEN-related AKT/mTOR signaling pathway. Bassi et al. (2013) have shown that SUMOylation is important for PTEN nuclear localization and DNA double-strand break repair based on homologous recombination. To verify whether RGS12 affects SUMO1-PTEN associated DNA double-strand break repair, the γH2AX level was analyzed by performing IF staining. The results showed that the γH2AX level significantly decreased after RGS12 overexpression compared to the control (Fig. 4G and Appendix Fig. 5). Hence, our findings indicated that RGS12 upregulated p-PTEN and SUMO1-PTEN to inhibit OSCC.

Figure 4.

Figure 4.

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Regulator of G protein signaling 12 (RGS12) upregulates phosphorylation and SUMOylation of phosphatase and tension homolog (PTEN). (A) RGS12 regulates the phosphorylation of PTEN. The SCC4 cells were transfected with control plasmid (pCMV), pCMV-RGS12, and short hairpin RGS12 (shRGS12) plasmids. The immunoblotting showed the protein level of RGS12, p-PTEN, PTEN, and α-tubulin. α-Tubulin was used as an internal control. (B) Quantitative analysis of p-PTEN. The ratio of p-PTEN and PTEN from panel A was calculated. *P < 0.05 and **P < 0.01 versus control (n = 3). (C) Immunoprecipitation (IP) and immunoblotting showed SUMOylation of PTEN (SUMO1-PTEN) expression with or without overexpression of RGS12. SCC4 cells were overexpressed with pCMV-RGS12 (RGS12OE) or pCMV (control) for 24 h. Lysates as an input were immunoblotted with anti-PTEN and anti–β-actin antibodies. Lysates were immunoprecipitated with PTEN antibody and then immunoblotted with SUMO1 antibody. After stripping, the same membrane was detected with PTEN antibody. (D) Quantitative analysis of panel C showed the SUMO1-PTEN protein expression. The data are expressed as mean ± SD. Note that SUMO1-PTEN was increased after overexpression of RGS12. (E) Immunoblots show overexpression of RGS12 (RGS12 OE) in SCC4 cells decreased the p-AKT and p-mTOR expression. (F) Quantitative analysis of panel E shows the relative p-AKT and p-mTOR expression. A t test shows significant differences between the 2 groups, **P < 0.01 versus control (n = 3). (G) Representative images of immunofluorescence (IF) with γ-H2AX antibody counterstained with DAPI. Images of γ-H2AX immunofluorescence are displayed in green. Images of the DAPI-stained nucleus are shown on the left, displayed in blue. Bright areas correspond to nuclear regions with increased DNA presence a reflection of densely packaged heterochromatin. Overlay images are shown on the right. Scale bar: 20 μm. The number of γ-H2AX positive SCC4 cells was significantly reduced after overexpression of RGS12. Quantitative analysis of the percentage of γ-H2AX–positive SCC4 cells is shown on the right. Quantitative data are presented as mean ± SD. ***P < 0.001, n = 3.

Ectopic Expression of RGS12 Inhibits Oral Squamous Cell Carcinoma

To test the potential therapeutic effect of RGS12 on OSCC, we stably transfected pCMV (control) and pCMV-RGS12 (RGS12 OE) in SCC4 cells. Stable overexpression of RGS12 was confirmed by Western blot (Fig. 5A). The results from the Matrigel invasion assay showed that stable overexpression of RGS12 reduced more than 50% of invaded cells (Fig. 5B) and significantly inhibited the recovery of wound area (45% recovery) following 48 h postscratch (Fig. 5C). In addition, the WST-1 assay revealed that stable overexpression of RGS12 significantly inhibited cell proliferation (Fig. 5D). To further test the therapeutic effect of RGS12 in vivo, SCC4 cells with stable RGS12 overexpression were subcutaneously injected into the hind leg of the immunodeficient NOD scid mice to detect tumor formation. The result showed a significant decrease in tumor volume and weight after overexpression of RGS12 compared to control groups (Fig. 5E). We then harvested the tumors and confirmed the RGS12 level by performing real-time PCR. The results showed RGS12 mRNA level in tumors of RGS12 OE group was still higher than that of the control group (Fig. 5F). All these data demonstrated that overexpression of RGS12 can inhibit OSCC progression, suggesting that RGS12 is a potential therapeutic target for OSCC.

Figure 5.

Figure 5.

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Regulator of G protein signaling 12 (RGS12) is a potential therapeutic target for oral squamous cell carcinoma. (A) Western blots of lysates from SCC4 cells stably transfected with pCMV-RGS12 plasmid. (B) Representative images of the Matrigel invasion assay. The SCC4 cells were treated as depicted in panel A. Quantitative analysis of invasion is shown on the right. Data are presented as mean ± SD. ***P < 0.001, n = 3. (C) Representative images of wound-healing assays performed on SCC4 cells with or without stable RGS12 overexpression at 0, 24, and 48 h postscratch. Cell migration was assessed by the percent recovery of wound area. Scale bar: 50 μm. Quantitative analysis of cell migration is shown on the right. **P < 0.01, ***P < 0.001, n = 3. (D) Proliferation was determined by WST-1 assay in SCC4 cells with or without stable RGS12 overexpression. Quantitative data are presented as mean ± SD. **P < 0.01, ***P < 0.001, n = 3. (E) Subcutaneous tumorigenic images in NOD scid mice. SCC4 cells with or without stable RGS12 overexpression were subcutaneously injected into the immunodeficient (NOD scid) mice. All tumors were harvested after 4 wk to detect tumor volume (mm3) and weight (g) (right panel). Quantitative data are presented as mean ± SD. **P < 0.01, ***P < 0.001, n = 5. (F) Subcutaneous tumors from panel E were harvested and the messenger RNA of RGS12 were analyzed by real-time polymerase chain reaction. Note the RGS12 expression was higher in RGS12 OE subcutaneous tumors compared with control. Quantitative data are presented as mean ± SD. ***P < 0.001, n = 3.

Discussion

Recent studies have shown that different RGS proteins play specific roles in diverse cancer types (Hurst and Hooks 2009). As the largest member of the RGS family, RGS12 has been reported to be lost selectively in African American prostate cancer (Wang et al. 2017). More important, based on the proteomics database of the Human Protein Atlas (https://www.proteinatlas.org) from HNC patients, the 5-y survival rate of patients with high RGS12 expression is significantly better than that of patients with low expression. In our study, by performing an IHC score analysis of RGS12 expression in human OSCC tissues, we found a significant reduction of RGS12 expression in those OSCC tissues. Further analysis showed that the reduction in RGS12 expression was in the TNM stage and pathological grade-dependent manner, suggesting that RGS12 may serve as a biomarker for OSCC diagnosis, treatment, and prognosis. The pathogenesis of OSCC is mainly due to the accumulation of multiple genetic mutations regulated by genetic predisposition. Our in vivo and in vitro data thus demonstrated that RGS12 is an oral cancer suppressor.

The 4NQO-induced OSCC model is a classical method for studying oral cancer. Vitale-Cross et al. (2009) compared different 4NQO-induced OSCC models, wherein Young (2008) reported that some of the C57Bl/6 mice failed to develop OSCC when a 50-µg/mL dose of 4NQO was given in the mouse drinking water and stimulated even up to 32 wk. In contrast, Tang et al. (2004) reported a 100% success rate of OSCC generation in C57BL/6 mice by giving a 100-µg/mL dose of 4NQO in the drinking water and stimulated for 16 wk, followed by 8 to 16 wk of normal drinking water, suggesting the importance of 4NQO dosage in generating the OSCC model (Vitale-Cross et al. 2009; Wang et al. 2019). To ensure the creation of the OSCC model with a higher success rate in the C57BL/6 mouse strain, we followed the method by Tang et al. (2004) and obtained a 100% success rate of OSCC generation in the control and RGS12 mutant mice, featuring multiple lesions, papillomas, and carcinomas over the surface of the tongue (Tang et al. 2004). Therefore, our data indicate that this is an optimal method for the generation of OSCC and is an ideal model to disclose different progression of carcinoma in the 4NQO-treated mice.

RGS12 is a multidomain protein with the classical PDZ (PSD-95/Dlg/ZO-1) domain in the N-terminus (Snow et al. 1998; Kimple et al. 2001). More than 250 proteins containing the PDZ domain have been reported in the human proteome. As so many proteins possess multiple PDZ domains, their potential combinations with PBM-containing proteins appear to be enormous. However, the recognition of the PDZ domain is highly specific (Subbaiah et al. 2011). Traditionally, PDZ domains have been classified as I-V classes depending on the consensus sequences of PBMs. Studies have been reported that the RGS12 N-terminal PDZ domain belongs to class I PDZ sequence (STTL), which is commonly thought to be specifically recognized and bound by C-terminal polypeptide class I PBM (Romero et al. 2011; Subbaiah et al. 2011). Coincidentally, the last 4 amino acids at the C-terminus of the PTEN protein (residues Ile400-Thr401-Lys402-Val403-COOH; ITKV) constitute a PBM for class I PDZ domain (Valiente et al. 2005). In this study, we identified the highly specific association between RGS12 and PTEN via the PDZ domain and that RGS12 activates PTEN through both phosphorylation and SUMOylation. Moreover, SUMO-PTEN is essential for tumorigenesis (Li et al. 2013) and is also important for DNA double-strand break repair based on homologous recombination (Bassi et al. 2013; Jamaspishvili et al. 2018). Consistently, we revealed a significant decrease in γ-H2AX after RGS12 overexpression, indicating that RGS12 associates with PTEN to enhance the ability of DNA double-strand break repair in the nucleus. Our results also showed that RGS12 can enhance PTEN-related AKT/mTOR signaling to regulate cell migration and proliferation. More important, we found that continuous overexpression of RGS12 in tumor cells can effectively inhibit tumor proliferation and invasion, suggesting that exogenous administration of RGS12 may be a potential treatment strategy for oral cancer. Most interestingly, we found that the overexpression of RGS12 inhibited cancer cell proliferation and invasion, which were disrupted by PDZ domain mutation but not by RGS domain mutation. These results suggested that the PDZ domain plays an important role in the regulation of PTEN and downstream signaling, while the RGS domain of RGS12 may be not mainly involved in PTEN regulation or tumorigenesis. Given RGS proteins can inhibit G protein signaling through the RGS domain, which interacts with the Gi and/or Gaq subunits and accelerates their GTP hydrolysis (Berman et al. 1996; Tesmer et al. 1997), we cannot exclude the regulation of RGS12 in G protein since GPCRs regulate many aspects of tumorigenesis. Thus, future study will focus on the exploration of the upstream target proteins of RGS12.

In summary, RGS12 is an important factor in the development of oral cancer, mainly regulating tumor proliferation, migration, and invasion. RGS12 associates and activates PTEN through its functional PDZ domain. In addition, RGS12 regulates the PTEN-related AKT/mTOR signaling and DNA repair via phosphorylation and SUMOylation of PTEN to inhibit OSCC tumorigenesis. Overexpression of RGS12 can inhibit the occurrence and development of OSCC. Thus, our studies indicate that RGS12 is an essential tumor suppressor and highlight RGS12 as a potential therapeutic target in OSCC.

Author Contributions

C. Fu, G. Yuan, S. Yang, contributed to conception, design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript; S.T. Yang, contributed to conception and data acquisition, drafted and critically revised the manuscript; D. Zhang, contributed to conception and data analysis, drafted and critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.

Supplemental Material

DS_10.1177_0022034520972095 – Supplemental material for RGS12 Represses Oral Cancer via the Phosphorylation and SUMOylation of PTEN
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Supplemental material, DS_10.1177_0022034520972095 for RGS12 Represses Oral Cancer via the Phosphorylation and SUMOylation of PTEN by C. Fu, G. Yuan, S.T. Yang, D. Zhang and S. Yang in Journal of Dental Research

Footnotes

A supplemental appendix to this article is available online.

Declaration of Conflicting Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding: The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants from the National Institutes of Health (NIH): National Institute of Dental and Craniofacial Research (DE023105), National Institute on Aging (AG048388), and National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR066101) to S. Yang.

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Supplementary Materials

DS_10.1177_0022034520972095 – Supplemental material for RGS12 Represses Oral Cancer via the Phosphorylation and SUMOylation of PTEN
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Supplemental material, DS_10.1177_0022034520972095 for RGS12 Represses Oral Cancer via the Phosphorylation and SUMOylation of PTEN by C. Fu, G. Yuan, S.T. Yang, D. Zhang and S. Yang in Journal of Dental Research

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