In the present study, we demonstrated in functional studies that restoration of TCF21 expression in ACC cells results in reduction of migration and invasion in vitro. These results reinforce the tumor suppressor function of TCF21 in ACC, in accordance with our studies that demonstrated that TCF21 is markedly downregulated in adult ACCs compared with adenomas and normal tissue [12, 14]. The tumor suppressor function of TCF21 is also demonstrated in different tumors such as lung, colorectal and breast cancer, where the activation of TCF21 expression reduced cell growth, EMT and suppress migration and invasion [9, 28, 29]. In addition, downregulation of TCF21 has associated with promoter hypermethylation in different tumors [17, 18]. Here, we reported that TCF21 promoter hypermethylation is involved in the repressed expression of TCF21 observed in ACC cells. Furthermore, the hypermethylation condition of TCF21 was reverted with the 5-Aza treatment in a dose and time dependent manner. This result suggest that the promoter methylation is an important mechanism of epigenetic control of TCF21 expression in ACC, and it can be reverted at least in vitro. In other human tumors, hypermethylation is described as the mechanism preponderant for silencing TCF21 expression [9, 17, 29].
In order to study the biological function of TCF21 in ACC cells, SW13 and H295R cells, we used two different technologies to increase the TCF21 expression, the CRISPR/dCas9/TCF21 system and transfection with pCMVMyc-Pod1, respectively. Regardless of the TCF21 expression levels obtained, the ability of migration and invasion were greatly inhibited in the TCF21 upregulated cells in comparison to negative control cells. In addition, and also important, the downregulation of TCF21 in an adrenocortical adenoma cell culture, enable the migration and invasive ability of tumor adrenocortical cells when compared to control cells. Together, these results suggest a role of TCF21 in ACC metastasis, and reveal that the presence of TCF21 expression may be a good marker for prognosis of metastasis.
We further investigate the potential molecular mechanism regulated by TCF21 in adrenocortical tumor cells. The MMPs are a family of zinc-dependent endoproteases involved in tissue remodeling and degradation of various proteins in the extracellular matrix. Despite performing important extracellular actions several MMPs are known to function intracellularly in diverse tissue . Alterations in MMP expression occur in normal biological processes such as cell proliferation, migration, and differentiation and have also been implicated in tumor progression and invasiveness. MMPs have been examined as potential therapeutic targets in various disorders as well as cancer . In our study, the presence of TCF21 in ACC cells induced the increased of anti-invasive effectors, KISS-1, MMP-8 and TIMP-1 expression whereas the pro-invasive, MMP-9, MMP-14, MMP-2 and VIM were downregulated in ACC cells.
Arab and coworkers  found that TCF21 directly bind the promoter of KISS-1 gene, known as metastasis inhibition gene in a number of tissues, to enhance its expression in melanomas. In renal cancer, the expression of KISS-1 was downregulated with TCF21 gene silencing . More recently, TCF21 was found to induce KISS-1 and reduce MMPs expression through the PI3K/Akt pathway in colorectal cancer . In our study, KISS-1 was also upregulated significantly for TCF21 in ACC cells as well MMP-8 and TIMP-1. The anti-tumor properties of MMP-8 were first demonstrated in MMP-8-deficient mice . The absence of MMP-8 strongly increases the incidence of skin tumors in these mice. Also, the overexpression of MMP-8 in metastatic breast cancer cells reduces their metastatic potential, and higher MMP-8 expression is correlated with the lower incidence of lymph node metastasis and good prognosis . These results indicate that MMP-8 is a tumor-protective factor, but as far as we know, there is no reporter on the action of TCF21 in the regulation of MMP-8 expression.
Tissue Inhibitors of Metalloproteinases (TIMPs) participate in controlling the local activities of MMPs in tissues. TIMPs are smaller, 22–30 kDa, and capable of binding and inactivating MMPs. Four TIMPs have been identified (TIMP-1 to TIMP4), that form noncovalent bonds with the latent and active forms of MMPs. Overexpression of TIMP-1, TIMP-2, and TIMP-3 reduces tumor growth . Although TIMPs inhibit MMPS and have an antitumoral effect, they are also involved in the activation of MMPs, thus promoting tumor progression . The TCF21 knockdown in visceral derived adipose stem cells was showed to suppress the expression of TIMP-1 in the remodeling of the extracellular matrix of adipose tissue . Our data also showed downregulation of MMP pro-invasive MMPs, MMP-9, MMP-14 and MMP-2. MMP-9 or gelatinase B is a type IV collagenase produced by a variety of cells. It inhibits or stimulates the process of degradation of extracellular matrix during tissue remodeling which is essential for tumor invasion and metastasis. The MMP-9 enzyme is secreted as pro-MMP-9 which is an inactive form of MMP-9 and it is activated by various activators including MMP-2 and MMP-3 . In addition, the MMP-9 and MMP-8 serum levels were evaluated in patients with adrenal tumors prior and after surgery. Increased MMP-9 and MMP-8 levels were noted in patients with ACA and ACC prior surgery. After surgery, MMP-8 and MMP-9 levels decreased significantly in patients with ACC whereas in patients with ACA the decreased was not statistically significant. However, no correlation between the levels of evaluated MMPs and tumor sizes were observed . In other study, serum MMP-9 levels were used as diagnostic tool in determining the functioning status of benign adrenal tumors. The results suggested that MMP-9 may be useful in differentiating benign subclinical functioning adrenal tumors from benign nonfunctioning adrenal tumors . TCF21 was overexpressed in SMMC-7721 hepatocellular carcinoma cell line and showed raised of KISS-1 and p53, and downregulated MMP-9 proteins and inhibition of migration ability .
The MMP-2 is a type IV collagenase or gelatinase A. Indeed, the MMP-2 is ubiquitous in many cells and tissues and is involved in different processes such as angiogenesis, tissue repair, and inflammation. The proMMP-2 is recruited to the cell surface and undergoes autocatalytic cleavage at the cell surface. The imbalance of MMP-2 and its inhibitors TIMP-1 contribute to tumor invasion and metastasis, and tumor progression . The involvement of MMP-2 in cancer has been studied in different malignancies . Investigation of 50 ACC and 50 ACA by immunohistochemical showed that MMP-2 was detected in 74% of ACC and 2% in ACA. In addition, strong MMP-2 expression was recognized as an unfavorable prognostic factor for ACC . The MMP-2 inhibition by presence of TCF21 in ACC cells was also reported in colon rectal and ovarian cancer cells [34, 45], showing the TCF21 properties in decrease cell invasion of different types of tumors through inhibition of MMP-9 and MMP-2.
The MMP-14 was found to be highly expressed in different cancers. Thus, MMP-14 expression promotes migration, invasion and metastasis of tumor cells in vitro and in vivo . The MMP-14 is a membrane MMP that its primary role is Extracellular Matrix (ECM) degradation, in fact, it is located at the leading edge of migrating cells. A critical catalytic domain was identified for the enhancement of cellular invasion by regulating cleavage of pro-MMP-2 to active MMP-2 . Indeed, the MMP-14 are inhibited by TIMPs, RECK (GPI-anchored glycoprotein), chondroitin/heparan sulfate proteoglycans and the keratan sulfate Lumican . Our data showed that MMP-14 was efficiently inhibited by TCF21 in ACC cells, by a mechanism not yet known but together with KISS-1, TIMP-1 and MMPs, MMP-14 inhibits the motility capacity of ACC cells.
In the present study, we found that the methylation is an important and reversible mechanism of epigenetic control of TCF21 expression in ACC, and that overexpression of TCF21 inhibits migration, and invasion of ACC cells. Furthermore, the expression of MMP-8, TIMP-1 and KISS were increased while MMP-9, MMP-14 and MMP-2 were decreased.