Role of SIRT1 in regulation of epithelial-to-mesenchymal transition in oral squamous cell carcinoma metastasis

Oral cancer is the sixth most common human cancer worldwide, and >90% of oral malignancies are squamous cell carcinomas [1]. Oral squamous cell carcinoma (OSCC) accounts for >95% of all head and neck cancers, and can develop from oral precancerous lesions such as leukoplakia and erythroplakia [1‐3]. The incidence of oral cancer in Taiwan has increased 30% during the last 5 years, and the overall mortality rate has increased 25%. Males aged 30–49 years have the highest rate of mortality due to oral cancer [4, 5]. More than 50,000 new cases of oral cancer are diagnosed annually, and the overall 5-year survival rate for OSCC patients during the last 2 decades has consistently remained between 34% and 62.7% [5‐7]. It was recently reported that the cervical lymph node is a critical prognostic indicator of the clinical course of OSCC, and that patients with cervical lymph node metastasis usually have lower survival rates [8‐10]. Similar to other cancers, oral cancer metastasis occurs after a localized tumor progresses to an advanced stage [11]. Therefore, an understanding of the molecular mechanism which regulates OSCC metastasis can provide information important for developing new drugs and guidelines for treating metastasized oral cancers. Cancer metastasis is accelerated by an epithelial to mesenchymal transition (EMT) process resulting in increased cell migration and invasion, cell-substrate adhesion, intravasation and extravasation, as well as increased cell survival. EMT plays an important role in cancer invasion and metastasis, during which epithelial cells lose their cell-adhesive properties, repress E-cadherin expression, and increase their levels of mobility, matrix metalloproteinases (MMPs), and expression of mesenchymal markers [12‐14]. E-cadherin is a cell-cell adhesion molecule expressed predominantly by epithelial cells. Reduction or loss of E-cadherin is considered a hallmark event of EMT, which initiates a series of signaling events and a major reorganization of the cell cytoskeleton [15, 16]. Concomitant with the loss of E-cadherin and actin reorganization, cells undergoing EMT acquire a mesenchymal phenotype that becomes apparent by the expression of mesenchymal cytoskeletal proteins such as vimentin, and increased deposition of extracellular matrix proteins by MMPs. These extracellular matrix components stimulate integrin signaling and facilitate cell migration [17, 18]. Furthermore, decreased expression of E-cadherin during EMT is accompanied by increased expression of N-cadherin, which renders the cell more motile and invasive [19‐21]. These different events result in a loss of apical-basal polarity, after which, the cells acquire a front-back polarity that allows them to migrate in a directional fashion. The increased MMP expression and activity allows the cells to degrade extracellular matrix proteins, permitting their delamination and escape from their epithelial components [22]. In cancer, epithelial tumor cells become more invasive after undergoing EMT, and enter the circulatory system through intravasation. This results in their dissemination to loci distal from the primary tumor. Hence, elucidating the molecular mechanism which regulates expression of E-cadherin, N-cadherin, and MMPs, has become pivotal for understanding cancer invasion and metastasis.

Sirtuins are nicotinamide adenine dinucleotide (NAD⁺)-dependent histone deacetylases [23]. Human homologues of the Sir2 gene are found in yeast, and are considered a critical link to longevity, as they prolong the cellular replication cycles of Saccbaromyces Cerevisiae and Caenorbabditis elegans[24, 25]. Several types of sirtuin enzymes have been identified (SIRT1-7), and their enzymatic activities are regulated by the ratio of NAD⁺ to NADH; high NAD⁺ levels activate sirtuin enzymes, and conversely, high NADH levels inhibit their activity [26]. Due to their abilities to deacetylate both histone and non-histone substrates, sirtuin enzymes have roles in regulating multiple cellular and physiological processes, including diabetes, inflammation, neurodegenerative diseases, stress responses, cell survival, metabolism, aging, and longevity [27‐30]. Sirtuin enzymes are widely expressed in normal tissues. SIRT1 localizes primarily in the nucleus, along with SIRT6 and SIRT7; whereas SIRT2 is in the cytoplasm, and SIRT3, SIRT4, and SIRT5 are localized in the mitochondria [31].

SIRT1 is a class III histone deacetylase capable of deacetylating lysine residues on nuclear proteins, which is thought to affect their stability, transcriptional activity, and translocation. Recently, SIRT1-mediated deacetylation of nuclear proteins such as p53, FOXO, and Ku70 [32‐34], has been reported to promote cell survival. Roles for SIRT1 in skin, colon, breast, and lung cancers have been demonstrated through its affects on one or more of the aforementioned nuclear proteins [35‐39]. Additionally, SIRT1 can regulate vascular endothelial homeostasis by controlling angiogenesis and vascular function [40], and also regulates the transcription of numerous genes by interacting with transcription factors. For example, upon recruitment to chromatin by transcription factors, SIRT1 deacetylates histones to suppress gene transcription [28, 41, 42]. Despite evidence for SIRT1 involvement in a variety of cell regulatory and physiological processes, the role of SIRT1 in regulating oral cancer metastasis and EMT remains enigmatic. In this study, we investigated the involvement of SIRT1 in EMT as it occurs in oral cancer metastasis. We found that SIRT1 expression was substantially downregulated in OSCC cell lines, and was also widely attenuated in OSCC tumors as compared with expression in paired normal tissues. SIRT1 overexpression repressed the EMT process in oral cancers and blocked migration of OSCC cells in vitro. In contrast, knockdown of SIRT1 in oral cancer cells enhanced EMT and cancer metastasis in vitro. We also show that SIRT1 regulates expression of the epithelial marker E-cadherin, as well as the mesenchymal markers vimentin and N-cadherin. Moreover, we found that SIRT1 targets Smad4 to reduce EMT and MMP7 expression. Finally, we show that SIRT1 overexpression reduced the invasiveness and metastasis of oral cancer cells in immunodeficient mice. In summary, our data show that SIRT1 inhibited the EMT process in oral cancer by deacetylating Smad4 and repressing expression of MMP7. These results suggest a role for SIRT1 as a metastasis suppressor in oral cancer.

In this study, we demonstrated that SIRT1 suppresses the EMT process in oral squamous cell carcinoma cells by deacetylating Smad4 and repressing MMP7 expression. It was previously shown that SIRT1 helps regulate a variety of physiological processes by interacting with nuclear proteins [28, 52‐56]. Despite its implied involvement in a variety of physiological processes, the regulatory role of SIRT1 in oral cancer metastasis is poorly understood. In this study, we demonstrated for the first time that SIRT1 is a critical negative regulator of EMT and cell migration in vitro, and also of tumor metastasis in vivo (immunodeficient mouse model). Our studies showed that compared with expression in HOK cells, SIRT1 was overexpressed in both OSCC cell lines, and a similar result was found in an enzyme activity experiment. We also found that activation of SIRT1 in oral squamous cell carcinoma resulted in decreased cell migration and invasion. Therefore, we propose a molecular mechanism whereby SIRT1 regulates cell migration by interacting with and deacetylating TGF-β-inducing transcription factor Smad4 to suppress MMP7 expression. We found that increased levels of SIRT1 in oral squamous cell carcinoma tissue contributing to decreased Smad4 acetylation and repressed MMP7 activity. In addition, our findings revealed that an absence of SIRT1 led to Smad4 hyperacetylation, MMP7 hyperexpression, and degradation of E-cadherin on the cell surface. These events resulted in release of β-catenin from the E-cadherin-β-catenin complex junctions leading to the nucleus, and promoted metastasis of OSCC cells (Figure 9). In addition to the in vitro data showing that up-regulation of SIRT1 led to low cellular invasiveness and migratory abilities, SCID mice with SIRT-overexpressing OSCC cells showed significantly less lung metastasis compared to control mice. The EMT process represents the critical event in the transition from early stage to invasive carcinoma [11, 57], and E-cadherin downregulation is well associated with poor prognosis, lower survival, and higher rates of metastasis in OSCC patients [58‐61]. Our results showed that SIRT1 overexpression reduced oral cancer cell migration and metastasis, and these effects were largely independent of any general effects of SIRT1 on oral cancer growth and survival. Taken together, these data suggest that SIRT1 may prevent oral cancer metastasis by blocking the EMT process.

Interestingly, our results differed from previous reports which indicated that SIRT1 serves as a positive regulator of epithelial-mesenchymal transition, the metastatic growth of prostate cancer cells [62], and is associated with malignancy in chronic myelogenous leukemia [63]. Additionally SIRT1 involvement has also been suggested in epigenetic silencing of DNA-hypermethylated tumor suppressor genes in breast cancer cells [64]. Recently, SIRT1 has been shown to be an important target of miR-200 in regulating breast cancer cell migration [65, 66]. Additionally, SIRT1 is highly expressed in various cancers [35, 67] such as prostate cancer, and high levels of SIRT1 expression are associated with a poor prognosis in lung cancer, breast cancer, gastric carcinomas, and B-cell lymphoma [68‐71]. In prostate cancer, SIRT1 was shown to enhance cell migration and metastasis by cooperating with EMT transcription factor ZEB1 to suppress E-cadherin transcription. In lung cancer, the SIRT1 activator compound 1720 was shown to increase lung metastasis of implanted breast cancer cells, suggesting SIRT1 as a potential target for suppressing metastasis to the lung [72]. Moreover, miR-200 negatively regulated SIRT1 expression and inhibited the EMT process in normal mouse mammary epithelial cells [65]. However, the role of SIRT1 in tumorigenesis remains controversial, and may depend on the tumor type. A recent report showed that enhanced SIRT1 expression in a β-catenin-dependent mouse model of colon cancer inhibited intestinal-tumor formation, thereby indicating that the effects of SIRT1 might vary in different tumor models, and depend on the presence of appropriate downstream targets [73]. Moreover, SIRT1 was shown to protect against gut carcinomas in APCmin mice [73], as well as inhibit tumorigenesis in p53^+/− mice [74, 75]. Wang et al. found that Sirt1^+/−; p53^+/− mice develop tumors in multiple tissues, and activation of SIRT1 by resveratrol reduces tumorigenesis. Moreover, several independent investigations have found reduced levels of SIRT1 in Sirt1^+/−; p53^+/− mice as compared to normal controls, and suggested SIRT1 as an important antagonist of EMT in various types of cancer cells [76‐78]. In lung cancer, SIRT1 down-regulation by hypoxia in a SUMOylation-dependent manner promotes EMT, and eventually leads to tumor metastasis. This result supports the hypotheses that SIRT1 activation ameliorates lung cancer metastasis in vitro and in vivo by blocking the entry of pre-cancerous cells into EMT [76]. Additionally, SIRT1 has been shown to suppress the EMT process in metastasizing breast cancer cells, and the development of fibrosis in organs following their implantation into nude mice. A reduction in SIRT1 levels was shown to promote the metastasis of breast epithelial cells in an orthotopic model of breast cancer, as well as increase the motility of the epithelial cells [78]. Furthermore, while EMT can be induced in both breast and kidney epithelial cells in vitro, this induction is repressed by SIRT1 [78]. A previous study found that both miR-520c and miR-373 suppressed SIRT1 mRNA translation, leading to activation of the Ras/Raf/MEK/Erk pathway. Moreover, NF-κB increased MMP9 expression and enhanced the migration of fibrosarcoma cells [79]. Our data builds upon the results in these previous studies by further verifying SIRT1 as a critical regulator of cancer progression, and an important target for prevention or possible treatment of cancer metastasis.

Similar to other cancers, oral cancer metastasis requires degradation of the extracellular matrix via increased expression of matrix metalloproteinases (MMPs). For example, MMP2, 7, and 9 are overexpressed in oral carcinoma tissue [45, 80, 81]. Importantly, MMP7 expression is most pronounced at the invasive front of tumors [45, 82, 83], has been reported as an independent prognostic factor which closely correlates with clinical stage, tumor size, lymph node metastasis, and poor survival of oral cancer patients. Our data also showed that MMP7 expression levels and activity were significantly decreased in OSCC cells overexpressing SIRT1 (Figure 4). Additionally, we found that SIRT1 knockdown OSCC cells showed increased MMP7 secretion and expression. We examined the interaction between SIRT1 and MMP7 in SIRT1 knockdown OSCC cells by immunoprecipitation, and found no direct interaction of SIRT1 with MMP7 (data not shown). A previous study showed that MMP7 was not required for malignant cell invasion in Smad4-deficient adenocarcinomas [84]. Kitamoto et al. found that MMP7 was required for tumor formation, but not for the invasion of the colon cancer cells in which Smad4-dependent TGF-β family signaling had been blocked. Smad4 is indispensable for EMT, and RNA interference-mediated knockdown of Smad4 expression results in preserved E-cadherin expression [20, 85‐87]. Additionally, Kume et al. [48] showed that in a mesangial kidney cell line, SIRT1 directly interacted and deacetylated the negative regulator of TGF-β signaling, Smad7, to destabilize the protein. Recently, numerous studies have revealed that TGF-β stimulates the EMT process in certain epithelial cells [22]. TGF-β drives cancer progression by inducing EMT, during which, epithelial cells acquire a mesenchymal phenotype, leading to their enhanced motility and invasiveness. TGF-β signaling directly activates the expression of EMT transcription factors, including δ EF1/ZEB1, SIP1/ZEB2, and Snail/SNAI1, which are induced by TGF-β-Smad signaling and play critical roles in TGF-β-induced EMT [88‐90]. TGF-β also binds to type II and type I transmembrane kinase receptors, TβRII and TβRI. Following ligand binding, TβRII phosphorylates TβRI, which activates Smad2 and Smad3. These two activated Smad proteins then combine with one Smad4 molecule to form trimeric Smad complexes that translocate into the nucleus and regulate the expression of target genes involved in the EMT process. For example, an active complex formed by Smad3/Smad4 and Snail can bind to the regulatory promoter sequences of genes encoding the epithelial junction proteins E-cadherin and occluding, leading to TGF-β-induced repression of their expression [91]. E-cadherin downregulation decreases the strength of cellular adhesion within a tissue, resulting in increased cellular motility [92]. Furthermore, decreased E cadherin expression during the EMT process is accompanied by increased expression of N-cadherin, which renders a cell more motile and invasive [19‐21]. Additionally, TGF-β regulates the expression and activity of extracellular proteases such as matrix metalloproteinases, which allow cells to degrade extracellular matrix proteins and increase their migratory and invasive behaviors. [92‐94]. In cancer, epithelial tumor cells become more invasive after undergoing EMT, and access the circulatory system through intravasation, resulting in their dissemination to loci distal from the primary tumor. In our current study, we postulated that the effects of SIRT1 on MMP7 might manifest via its interaction with the TGF-β-activated transcription factor, Smad4. Our data revealed that MMP7 expression levels and activity were significantly decreased in the Smad4 knockdown OSCC cells (Figure 6). Additionally, we used immunoprecipitation techniques to confirm the occurrence of interactions between SIRT1, Smad4, and MMP7 in OSCC cells. Interestingly, SIRT1 was shown to directly interact with Smad4 in vivo, but did not interact with MMP7 protein (Figure 5 and Additional file 1). We also showed that overexpression of SIRT1 repressed TGF-β-induced MMP7 expression by deacetylating Smad4, which becomes hyperexpressed and hyperacetylated under conditions of TGF-β stimulation (Figure 5D, 7A and Additional file 2). SIRT1 was shown to affect Smad4 transcriptional activity by deacetylation, and inhibition of Smad4 function repressed TGF-β-induced EMT. These observations clearly show that SIRT1 might influence MMP7 expression, secretion, and activity; and subsequently, cell migration, invasion, and metastasis through Smad4 deacetylation. Furthermore, we also showed that SIRT1-overexpressing cells inhibited MMP7 secretion and increased E-cadherin accumulation, leading to suppression of cellular invasion and migration. Our results indicate that MMPs can mediate both the EMT process [50] and cell metastasis [95], as well as cause nuclear translocation of β-catenin [50, 95] by proteolytic cleavage and release of E-cadherin from the cell surface. It is therefore interesting to speculate that SIRT1 maybe lead to repression of a second pathway involved in EMT, such as the Wnt signaling pathway.

In conclusion, our study identified SIRT1 as a novel metastatic suppressor which acts through deacetylation of TGF-β-activated transcription factor Smad4 to suppress the effect of TGF-β signaling on MMP7 transcription, leading to reduced migration and metastasis of OSCC cells. SIRT1 shows potential for serving as a predictor and biomarker for metastasis, and up-regulation of SIRT1 is a potentially useful therapeutic strategy for inhibiting the metastasis of oral cancers.