Upregulation of TCF21 inhibits migration of adrenocortical carcinoma cells

Background Adrenocortical carcinomas (ACC) are rare and aggressive cancer. Our previous study has revealed that the transcription factor 21, TCF21, is downregulated in ACC and regulates steroidogenic factor 1 (SF-1) binding to the SF-1 E-box promoter. In addition, it could be found that TCF21 is a predictor of overall survival (OS) in adult carcinomas. Methods In this study, it was investigated the correlation between TCF21 expression and the promoter methylation status in adrenocortical tumor cells, carcinomas and adenoma. The biological function and potential molecular mechanism of TCF21 restoration in migration and invasion of ACC cells was examined. Results We could be demonstrated a negative correlation between the level of TCF21 expression and methylation of its promoter in adenoma and carcinoma cells indicating the epigenetic control of TCF21 expression. It was also demonstrated that the expression of TCF21 inhibits migration and invasion in the ACC cell line, H295R cells, using plasmid transfection to express TCF21. Furthermore, it could be investigated the TCF21 function as tumor suppressor probably through Kisspeptin 1 (KISS-1) expression and epithelial–mesenchymal transition (EMT) reversion, as well as the modulation of several metalloproteinases in ACC cells. Conclusions Our results suggest that enhancement of TCF21 expression levels may be a potential strategy to revert invasive abilities in adrenocortical carcinomas. Supplementary Information The online version contains supplementary material available at 10.1007/s12672-021-00417-6.


Introduction
Adrenocortical tumors are usually incidentally diagnosed in 6-7% of the population [1]. Most of them are benign and non-functioning classified as adenomas (ACA), requiring only clinical monitoring [2]. In contrast, adrenocortical carcinomas (ACC) are rare with incidence of 1-2 cases per million and are aggressive and metastatic tumors [3]. Transcription factor 21 (TCF21) also known as capsulin, epicardin, and Pod1 belongs to the basic helix-loop-helix (bHLH) family and locates at chromosome 6q23-q24 [4][5][6]. The TCF21 encodes a transcription factor which binds DNA as a heterodimer through the E-box CANNTG sequence. The normal function of TCF21 is to promote the mesenchymal-epithelial transition (MET) during the development and differentiation process [7,8]. The downregulation of TCF21 reverts the MET Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s12672-021-00417- 6. process favoring migration and tumor invasion as reported in colorectal tumors, esophageal squamous cell carcinoma and urogenital cancers [9][10][11]. Thus, it could be reported that TCF21 is downregulated in ACC and regulates the steroidogenic factor 1 (SF-1) and StAR protein (steroidogenic acute regulatory protein) binding to the SF-1 E-box promoter in adrenocortical tumor and normal adrenal cells [12,13]. In addition, it could be found that TCF21 is a predictor of overall survival (OS) in adult carcinomas [14]. The epigenetically inactivation of TCF21 is associated with regulation of epithelial-mesenchymal transition (EMT), invasion, metastasis, cell cycle, and autophagy in different tumors, and might be an important role in tumor development [15,16]. However, no studies have reported on the role of TCF21 in migration and invasion of ACC, and the molecular mechanism is unknown. One of common mechanism in many human tumors is the hypermethylation-mediated silenced expression of TCF21, which has not yet been studied in adrenocortical tumors [17,18]. In the present study, it was investigated the correlation between TCF21 expression and the promoter methylation status in adrenocortical tumor cells, carcinoma and adenoma cells, and the biological function and potential molecular mechanism of TCF21 in motility of ACC cells. Our results demonstrated that there is a negative correlation between the level of TCF21 expression and methylation of its promoter. Upregulation of TCF21 in vitro inhibited migration and invasion functions expressively, which could act partly through involvement of EMT reversion and Kisspeptin 1 (KISS-1) and metalloproteinases (MMPs) expression.

Tumor cell cultures
Human adrenocortical carcinoma cell line NCI-H295R [19] obtained from American Type Culture Collection (ATCC) was used. H295R were cultured in Gibco RPMI medium with 2% Fetal Bovine Serum (FBS) and 1% Insulin Transferrin-Selenium (ITS). Secondary cell culture ACC-T36 was obtained from adult patient diagnosed with ACC as described in [12]. ACAPed-T7 pediatric secondary cell culture were obtained from a functioning adrenocortical adenoma as described in Almeida et al. [20]. Both secondary cell culture was maintained in Dulbecco's Modified Eagle Medium (DMEM) with 10% FBS and used until the fourth or sixth passage. All cultures were maintained at 37 °C in a 95% air 5% CO 2 and humidified environment. All cell cultures were regularly authenticated by STR DNA profiling analysis, and mycoplasma contamination was excluded by using qPCR Mycoplasma test kit (PanReac AppliChem, ITW Reagents, Illinois, Chicago, USA).

Methylation analysis of TCF21 promoter
The bisulfite conversion was performed using EZ DNA Methylation Gold kit (Zymo Research Cat No. D5005), according to the manufacturer's protocol. As a control, a Human Methylated & Non-methylated DNA Set kit (D5014-1-Zymo Research) following the Polymerase Chain Reaction (PCR) conditions was used, according to the manufacturer. A regulatory region of the TCF21 promoter was selected [21,22] and a sequence of 600 bp just before the start of the transcription site (TSS) was used as showed in Table 1S. DNA samples were amplified by conventional PCR using the Hot-Start Platinum DNA Polymerases enzyme (Thermo Fisher Scientific) according to the manufacturer's protocol, using the primers 5ʹ-TCA CCA TAA AGA TTC TAG GAA GCA -3ʹ and 5ʹ-GAG CGA GCG GCT GAG GAA T-3ʹ for control DNA, and 5ʹ-TTA TTA TAA AGA TTT TAG GAAGT-3ʹ and 5ʹ-GAG YGA GYG TTG AGG AAT -3ʹ for bisulfite-converted DNA. Primers were selected using the tool "bisulfite primer seeker" from Zymo Research ® (https:// www. zymor esear ch. eu/ bisul fite-primer-seeker). PCR reaction was analyzed on an agarose gel (1%) by electrophoresis and the product was purified using Exonuclease 1 (Applied Biosystems) and Shrimp Alkaline Phosphatase-SAP (Applied Biosystems), according to the manufacturer. For Sanger sequencing, the primers 5ʹ-TCA CCA TAA AGA TTC TAG GAA GCA -3ʹ for control DNA, and 5ʹ-TTA TTA TAA AGA TTT TAG GAA GTA -3ʹ for bisulfite-converted DNA were used. The reaction was prepared using the BigDye ® Terminator v3.1 Cycle Sequencing Kit, performed in the ABI 3730 DNA Analyzer (Life Technologies, Applied Biosystems). The data were analyzed by DNA Sequence Assembler ® software comparing sequenced bases without and with the bisulfite-conversion. The control was performed using the database with the sequence of the TCF21 gene (ENSG00000118526; r = 6: 133,889,138-133,895,553) GeneCards ® The Human Gene Database.

qRT-PCR
2.0-8.0 × 10 5 cells were seeded and after 48 h total RNA was extracted using Trizol ® (Invitrogen) as described by the manufacturer. The RNA integrity and its concentration were evaluated by agarose gel electrophoresis (2%) and spectrometry (NanoDrop 2000c, Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was generated from 1 µg of RNA using M-MLV Reverse Transcriptase (Invitrogen, USA). The qRT-PCR was performed using the 7500 Real Time PCR System Sequencer (Applied Biosystems, Foster City, CA, USA) and Sybr Green reagents (Applied Biosystems) and primers (Table 1). A cycle threshold (CT) value was selected in the linear range of amplification for each sample in triplicate and was normalized by endogenous control genes GUSB (human β-glucuronidase) or β-actin. The relative expression levels were calculated using the 2 −ΔΔCt method [23], where ΔΔCt is the difference between ΔCt value of a given sample and the ΔCt of commercial normal human adrenal pool (NA) (BioChain, USA). The data from three different experiments were presented as the mean ± SD.

Statistical analysis
Data were analyzed using unpaired t test or Analysis of Variance (ANOVA), when indicated, by GraphPad Prism 8 software.
The results were considered statistically significant when p < 0.05.

Negative correlation between the level of expression of TCF21 and methylation of its promoter
Analysis of TCF21 expression in different adrenocortical tumor cell cultures (Fig. 1A) showed lower mRNA expression of TCF21 in human ACC cells; H295R cells and ACC-T36 cells when compared to cell culture from pediatric adenoma, ACAPed-T7 and human normal adrenal pool (NA), which was used as reference. These results of TCF21 expression level in vitro are in agreement with previous study in a cohort of adrenocortical tumor samples from patients [12,14,25]. Sequencing for analysis of methylation of the TCF21 promoter in ACC cell cultures, H295R and ACC-T36 cells, showed 70% and 58% methylation of TCF21 promoter, respectively, whereas in ACAPed-T7 was found 8% methylation (Fig. 1B). The Pearson's correlation coefficient of TCF21 promoter methylation in different cell types was negative (r = − 0.9984) in agreement with the level of mRNA TCF21 expression (Fig. 1C). To confirm the regulation of TCF21 by promoter methylation, H295R cells were treated with demethylating agent 5-aza-2-deoxycytidine (5-aza). 100 µM of 5-aza for 48 h increased the relative expression of TCF21 mRNA level reversing the hypermethylation condition of the TCF21 promoter ( Figure 1S). 1 3

Upregulation of TCF21 reduces ACC cell migration and invasion
To examine the effect of TCF21 on migration and invasion, Transwell assays were performed using H295R/TCF21 and the corresponding control cells (Fig. 3). The results indicated that the number of migrated and invaded cells was lower in H295R/TCF21 than control cells, indicating that the increase of TCF21 has a negative effect on the migration and invasion of ACC cells in vitro.

Inhibition of TCF21 expression promotes ACA cell migration and invasion
To confirm the involvement of TCF21 in adrenocortical tumor mobility, TCF21 expression was efficiently inhibit in ACAPed-T7 cells by siRNATCF21 as showed in Fig. 2B. Migration and invasion assays using ACAPed-T7siRNATCF21, and respective control cells, showed that inhibition of TCF21 enable the migration and invasion of adrenocortical cells when compared to control cells (Fig. 4). Taken together, these data indicate that adrenocortical tumor cells become less likely to migrate upon presence of TCF21 expression. The results were expressed as the mean ± SD. Statistical significance was assessed by unpaired t test

TCF21 increased the expression of anti-invasive effectors and downregulate pro-invasive effectors in ACC cells
To explore the potential molecular mechanism of TCF21 in cellular mobility of adrenocortical carcinoma cells, we analyzed the effect of TCF21 on the expression of metalloproteinases MMP-2, MMP-8, MMP-9, MMP-14, metalloproteinase inhibitor 1 precursor TIMP-1, KISS-1 and Vimentin (VIM) in H295R/TCF21 cells and respective control. We observed significant increase in the mRNA level and protein expression of MMP-8, and mRNA level of TIMP-1 and metastasissuppressor KISS in H295R/TCF21 cells, in relation to control cells (Fig. 5A-C). In contrast, pro-invasive effectors, MMP-9 mRNA and protein, MMP-14, MMP-2 and VIM mRNA were remarkably downregulated in H295R/TCF21 cells, although the decrease of VIM protein has not been observed (Fig. 6A-D). To confirm the involvement of MMP-8 and MMP-9 in the motility of ACTs, the mRNA and protein expression of both MMPs was evaluated when TCF21 was inhibited in ACAPed-T7siRNATCF21 cells (Fig. 7). Results showed that the inhibition of TCF21 inhibited the mRNA level and protein expression of MMP-8 (Fig. 7A), in contrast to the increased of mRNA level and protein expression of MMP-9 (Fig. 7B).

MMP-8 and MMP-9 are important factors for the motility of adrenocortical tumors
To explore the effect of MMP-8 and MMP-9, both representatives of families of inhibitors and inducers of cell motility [26], in the migration and invasion of adrenocortical tumors, H295R cells were transfected with pCMVMMP-8 and pCMVMMM9 (Figs. 8 and 9). After MMP-8 and MMP-9 overexpression (Figs. 8A and 9A), MMP-8 decreased, and MMP-9 increased the  (Fig. 10A) resulted in a gain in the motility of these cells (Fig. 10B). Together, we showed that MMP-8 and MMP-9 play an important role in adrenocortical tumor cell motility, which may suggest a role in the metastatic process.

Discussion
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 [9,27,28]. 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, NCI-H295R and ACC-T36. Furthermore, the hypermethylation condition of TCF21 was reverted in H295R cell line 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 H295R cells, we used transfection with pCMVMyc-Pod1 as an experimental method necessary to study the role of TCF21. 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 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 [30]. 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 [26]. 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 [29] 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 The results were expressed as the mean ± SD. Statistical significance was assessed by unpaired t test was downregulated with TCF21 gene silencing [31]. More recently, TCF21 was found to induce KISS-1 and reduce MMPs expression through the PI3K/Akt pathway in colorectal cancer [32]. 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 [33]. 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 [34]. Thus, it was proposed that MMP-8 is a tumor protective factor, but as far as we know, there is no report of the action of TCF21 in regulating 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 [35]. Although TIMPs inhibit MMPS and have an antitumoral effect, they are also involved in the activation of MMPs, thus promoting tumor progression [36]. 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 [37]. 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 [38]. 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 [39]. 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 [39]. 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 [40]. The results were expressed as the mean ± SD. Statistical significance was assessed by unpaired t test 1 3 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 [30]. The involvement of MMP-2 in cancer has been studied in different malignancies [29]. 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 [41]. The MMP-2 inhibition by presence of TCF21 in ACC cells was also reported in colon rectal and ovarian cancer cells [32,42], showing the TCF21 properties in decrease cell invasion of different types of tumors through inhibition of MMP-9 and MMP-2.