TFE3 Regulates the Function of the Autophagy-Lysosome Pathway to Drive the Invasion and Metastasis of Papillary Thyroid Carcinoma

Background Accumulating evidence shows that autophagy plays a vital role in tumor occurrence, development, and metastasis and even determines tumor prognosis. However, little is known about its role in papillary thyroid carcinoma (PTC) or the potentially oncogenic role of TFE3 in regulating the autophagy-lysosome system. Methods Immunohistochemistry and quantitative real-time PCR (qRT-PCR) were used to examine the expression of TFE3, P62/SQSTM1, and LC3 in PTC and paracancerous tissues. TFE3, P62/SQSTM1, LC3, cathepsin L (CTSL), and cathepsin B (CTSB) were evaluated using Western blot analysis. After inducing TFE3 overexpression by plasmid or TFE3 downregulation by small interfering RNA (siRNA) transfection, MTT, wound healing, and cell migration and invasion assays were used to verify the effects on invasion, migration, and the levels of autophagy-lysosome system-related proteins such as P62/SQSTM1, LC3, CTSL, and CTSB. Results TFE3 was overexpressed in PTC tissues compared with paracancerous tissues. Analysis of the clinicopathological characteristics of PTC patients showed that high TFE3 expression was significantly correlated with lymph node metastasis. TFE3 overexpression in the PTC cell lines KTC-1 and BCPAP promoted proliferation, invasion, and migration, while TFE3 knockdown had the opposite effects. Furthermore, we identified a positive relationship among the expression levels of TFE3, P62/SQSTM1, LC3, CTSL, and CTSB. We found that silencing TFE3 inhibited the expression of P62/SQSTM1, LC3, CTSL, and CTSB in PTC cells. However, TFE3 overexpression had the opposite effects. Conclusions The present study provided evidence for the underlying mechanisms by which TFE3 induces autophagy-lysosome system activity in PTC.


Introduction
Thyroid cancer (TC) is the most common endocrine malignancy [1], and its incidence has increased rapidly over the past decades [2]. Thyroid cancer can be classified as follicular thyroid cancer (FTC), papillary thyroid carcinoma (PTC), medullary thyroid carcinoma (MTC), or anaplastic thyroid carcinoma (ATC), and PTC accounts for approximately 80% of all thyroid carcinomas [3]. Although the prognosis of PTC is better than that of most other tumors, the prognosis of patients with tumors that are insensitive to traditional surgery and iodine chemotherapy is poor. Therefore, exploring new biomarkers is highly important for improving the diagnosis and treatment of PTC.
Transcription factor E3 (TFE3) is located on the short arm of X chromosome 11.22 and is a microphthalmia family member [4]. Recently, the MiT/TFE family was identified as a regulator of autophagy. Subsequent studies showed that TFE3 can bind to CLEAR elements, which are present in the promoter regions of many lysosomal genes, and regulate lysosomal biogenesis in several different cell types [5].
Autophagy is a highly evolutionarily conserved catabolic process essential for the maintenance of cell homeostasis and adaptation to various stress conditions [6]. Autophagy is primarily a response to microenvironmental stressors, such as hypoxia, nutrient deficiency, and the accumulation of reactive oxygen species (ROS). Dysfunctional autophagy contributes to many diseases, including cancer. Depending on the type and stage of the cancer, autophagy can play a positive or negative role in cancer development [7]. Thus, understanding the role of autophagy in PTC is crucial for identifying new therapeutic targets for PT. Gene set enrichment analysis (GSEA) was used to analyze the enrichment of autophagy-and lysosome-related biological functions in PTC. The expression of genes involved in autophagy and lysosome-related biological functions was upregulated in the PTC group.
In the present study, we investigated the expression of the autophagy regulator TFE3 in PTC and clarified its role as a potential target in PTC patients.

Tissue Samples.
Seventy-eight pairs of PTC tissues and paracancerous tissues were acquired from patients who underwent surgical resection at Taizhou Central Hospital between March 2017 and January 2019. Patients with other tumors or a history of other tumors were excluded from this study. The patients were not given any chemotherapy or radiotherapy. The PTC population comprised 56 women and 22 men ranging in age from 18-79 years. All tissue samples were snap frozen in liquid nitrogen and quickly stored at -80°C. The clinicopathological characteristics of the PTC patients are listed in Table 1. The clinical stage, tumor stage, and lymph node stage of the patients are listed according to the tumor-node-metastasis (TNM) classification [8]. In addition, this research was approved by the Ethics Committee of Taizhou Central Hospital (Taizhou, China). Formal written approval was obtained from each patient. , hereafter referred to as complete culture medium. Cells were incubated at 37°C in a humidified atmosphere with 5% CO 2 and were passaged routinely.
2.5. Wound Healing Assay. Cells were cultured in 6-well plates at a density of 80%, and a 200 μl pipette tip was then used to create scratches on the surface of the cultured cells. Cells were  3 Analytical Cellular Pathology washed three times with phosphate-buffered saline (PBS) and cultured in serum-free RPMI 1640 medium. Wounds were observed under a microscope, and images were acquired at 0 h and 24 h. The results were analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA), and the rate of cell migration was determined as follows: ððdiameter of original wound − diameter of wound at different time pointsÞ/diameter of original woundÞ × 100%: 2.6. Cell Migration and Invasion Assays. Cell invasion was performed using a Transwell assay. In brief, PTC cells were trypsinized, suspended in serum-free RPMI 1640 medium (100 μl), and seeded in the upper chamber of each Matrigelcoated Transwell insert (REF: 3422, Corning, USA). Complete culture medium (500 μl) was added to the lower chambers. After 24 h of incubation, cells in the upper chamber had traversed to the bottom surface of the membrane, which was fixed with methanol and then stained with 0.1% crystal violet. The migration assay was conducted with the same procedure used for the invasion assay except that the membrane was not coated with Matrigel. Random fields were selected for imaging with a Zeiss photomicroscope (Carl Zeiss Meditec, Dublin, CA, USA).

Analytical Cellular Pathology
± standard deviation (SD) values, and a P value < 0.05 indicates statistical significance.

Autophagy-Lysosome System Activity Is Positively
Correlated with PTC Progression. To explore the role of the autophagy-lysosome system in the process of PTC, GSEA was used to analyze the enrichment of autophagy-and lysosome-related biological functions in PT. The expression of genes involved in autophagy-and lysosome-related biological functions was upregulated in the PTC group, and the functional enrichment analysis showed significant differences (Figures 1(a)-1(e), P < 0:05). Consistent with the above results, the results of immunohistochemical assays indicated that the expression levels of LC3 and P62/SQSTM1 in PTC tissues were significantly higher than those in paracancerous tissues (Figures 1(f) and 1(g)).
3.2. TFE3 Is Overexpressed in PTC Tissues and Cells. We examined the level of TFE3 in 78 pairs of PTC tissues and paracancerous tissues. The qRT-PCR results showed that TFE3 expression was significantly increased in PTC tissues compared with paracancerous tissues (Figures 2(a)-2(c), P < 0:01). Then, we analyzed the correlations between clinicopathological characteristics and TFE3 expression in PTC patients. The results indicated that high expression of TFE3 was significantly correlated with lymph node metastasis (Table 1, Figure 2(e), P < 0:05 ). Moreover, the results of immunohistochemical assays confirmed that the expression of TFE3 in PTC tissues was higher than that in paracancerous tissues (Figure 2(d)). We examined the level of TFE3 in Nthy-ori 3-1, KTC-1, and BCPAP. The qRT-PCR results showed that TFE3 expression was significantly increased in KTC-1 and BCPAP compared with Nthyori 3-1 cells (Figure 3(a), P < 0:01).

TFE3 Promotes the Proliferation of PTC Cells.
Here, we detected the effect of differential TFE3 expression on PTC cell proliferation through an MTT assay. As shown in Figure 3(d), downregulation of TFE3 expression significantly inhibited cell growth compared with that of si-NC-transfected cells. Consistent with the results in the si-TFE3 group, the pcDNA3.1-TFE3 group demonstrated a much higher cell proliferation ability than the pcDNA3.1 group (Figure 3(c), P < 0:05). These data suggested that TFE3 promoted the proliferation of PTC cells. The assays were repeated in triplicate.

TFE3 Accelerates PTC Cell Migration and Invasion In Vitro.
To determine the role of TFE3 in PTC progression, we conducted wound healing and Transwell assays to examine its effects on migration and invasion. PTC cells were transfected with pcDNA3.1-TFE3 or si-TFE3 in 6-well plates. First, the results of the wound healing assay revealed that TFE3 transfection increased the migration ability of PTC cells (Figures 4(a)-4(d), P < 0:05). In addition, the Transwell assay results showed that the migration of PTC cells was significantly suppressed in the si-TFE3 group compared with the si-NC group, while more cells penetrated the lower surface of the membrane in the pcDNA3.1-TFE3 group than in the pcDNA3.1 group (Figures 4(e) and 4(f), P < 0:05). Consistent with the migration assay results, the invasion assay results showed that cell invasion was noticeably suppressed by TFE3 knockdown and enhanced by TFE3 overexpression (Figures 4(g) and 4(h), P < 0:05). In conclusion, these results indicated that TFE3 might promote migratory and invasive behaviors in PTC cells. The assays were performed in triplicate.

TFE3
Induces Autophagy-Lysosome System Activity in PTC Cells. We sought to determine whether autophagylysosome system activity is related to TFE3. As shown in Figure 5(c), the expression levels of LC3 and P62/SQSTM1 in the pcDNA3.1-TFE3 group were higher than those in the pcDNA3.1 group. Then, we detected the conversion of LC3 I to LC3 II, which is an indicator of autophagy, in BCPAP and KTC-1 cells transfected with pcDNA3.1-TFE3 or si-TFE3 [9]. TFE3 increased the LC3 (Figures 5(a) and 5(b), P < 0:05). Next, we examined the protein level of P62/SQSTM1. P62/SQSTM1 is selectively incorporated into autophagosomes by simultaneously interacting with the LC3 protein [10]. Compared with the pcDNA3.1 group, the pcDNA3.1-TFE3 group showed increased expression of P62/SQSTM1 (Figures 5(a) and 5(b)). Similarly, compared to the si-NC group, the si-TFE3 group showed decreased P62/SQSTM1 expression. Through qRT-PCR experiments, we found that the mRNA expression levels of LC3 and P62/SQSTM1 in PTC cells were decreased after TFE3 knockdown and increased after TFE3 overexpression ( Figure 5(c), P < 0:05). To better explore autophagy in PTC cells, we measured the activity of lysosomal enzymes (CTSB and CTSL). Compared with the pcDNA3.1 group, the pcDNA3.1-TFE3 group showed increased activity of CTSB and CTSL ( Figure 5(d)). Similarly, the activity of CTSB and CTSL in the si-NC group was higher than that in the si-TFE3 group ( Figure 5(d)). The above results revealed that TFE3 might be able to promote autophagy-lysosome system activity in PTC cells. The assays were performed in triplicate.

Discussion
PTC is the most common endocrine malignancy and is the 5th most common female tumor [2]. Currently, PTC treatment is based on 131 I radiotherapy and chemotherapy combined with thyroid-stimulating hormone (TSH) suppression therapy after surgical resection [11]. However, tumor recurrence and metastasis and the presence of chemotherapeutic resistance in refractory PTC lead to decreased survival rates of patients with PTC [12].
TFE3, which belongs to the MiT/TFE family, is a regulator of autophagy and lysosomal biogenesis [13]. In 2009, the MiT/TFE family was initially discovered to be able to regulate most lysosomal genes (including promoters encoding hydrolases and lysosomal proteins) [14]. Importantly, under stress conditions, complex interactions between MiT/TFE family-dependent autophagic homeostasis pathways and apoptotic processes may occur in cancer cells, and these interactions ultimately determine the fate of these cells in relation to cell death or survival [15]. TFE3 simultaneously regulates autophagy induction, lysosomal biogenesis, oxidative metabolism, and oxidative stress and thus plays an  Analytical Cellular Pathology important role in determining cell fate [16]. Notably, under nonstress conditions, TFE3 interacts with 110 14-3-3 proteins, remains in the cytoplasm, and is phosphorylated at Ser321. In contrast, under stress conditions, TFE3 is dephosphorylated, the TFE3/14-3-3 complex dissociates, and TFE3 translocates from the cytoplasm to the nucleus to promote autophagy and lysosome biogenesis [5]. The TFE3 gene was found to be fused with the papillary renal cell carcinoma (PRCC) gene on chromosome 1q21.2 [PRCC-TFE3 t(X;1)(p11.2;q21)] [17]. Moreover, Fan et al. [18] found that inhibiting MT2-TFE3-dependent autophagy enhanced melatonin-induced apoptosis in tongue squamous cell carcinoma. Furthermore, in renal cell carcinoma (RCC), increased TFE3 expression was associated with poor progression-free survival (PFS) [19]. Consistent with these results, we found that TFE3 increased the proliferation and invasion of PTC cells and decreased their apoptosis. M. Anselmier, a French physiologist, first used the term "autophagy" in a short article published in 1859 describing the effects of fasting on mice [20]. Autophagy, a protein degradation pathway that is highly conserved from yeast to humans, is essential for clearing protein aggregates and 9 Analytical Cellular Pathology misfolded proteins from healthy cells. Under stress conditions, cells produce many damaged proteins or organelles, and double-membrane vesicles reform in the cytoplasm to engulf defective or toxic molecules and organelles to subsequently form autophagosomes. Then, autophagosomes fuse with lysosomes and release lysosomal acid lipase in the vesicles to degrade toxic molecules and other substances, and the resulting products are used to resynthesize new proteins or organelles [21]. The whole process of autophagy involves a variety of evolutionarily conserved genes, namely, autophagy-related genes (ATGs) [22]. Autophagy can be classified as macroautophagy, microautophagy, and molecular chaperone-mediated autophagy (CMA) according to the different ways of transporting cellular material to lysosomes [23]. Previous studies demonstrated that autophagy is an important participant in the pathogenesis of many diseases, including cancer [24,25]. In addition, genome-wide association studies showed that ATG5 is associated with systemic lupus erythematosus (SLE) in Chinese individuals, indicating that autophagy may be related to the pathogenesis of SLE [26]. Zou et al. [27] found that suppressing autophagy can enhance the chemotherapeutic effects of paclitaxel in cervical cancer cells. However, the relationship between PTC and autophagy has not been fully elucidated.
In this study, 90 samples of PTC tissue and 18 samples of paracancerous tissue were selected from the TCGA database to analyze the mechanism of PTC. The enrichment of autophagy-and lysosome-related biological functions involved in LC3 and P62/SQSTM1 in PTC data was analyzed by GSEA, which showed that autophagy-lysosome system activity was positively correlated with thyroid cancer progression. LC3 and P62/SQSTM1 have been widely reported as indicators of autophagy [28,29]. CTSB and CTSL are lysosomal acid cysteine proteases that modulate autophagy processes [30]. In addition, TFE3 was identified as a regulator of autophagy in previous studies [16]. Based on bioinformatic analysis and GSEA data, we further validated this role in tissues and cells in vitro. We found that the TFE3 level was significantly higher in a set of 78 PTC tissues than in the paired paracancerous tissues. High expression levels of TFE3 were closely associated with lymph node metastasis. Then, we conducted functional assays in KTC-1 and BCPAP cells and found that TFE3 enhanced the proliferation, invasion, and migration of PTC cells by regulating the autophagy-lysosome system, suggesting that TFE3 is a potential sensitive marker in PTC. In this study, we found that autophagy was induced by TFE3, as evidenced by the upregulation of P62/SQSTM1 protein expression and the increased LC3II/LC3I ratio. Therefore, we hypothesized that TFE3 positively regulates the autophagy-lysosome system in PTC.
However, the mechanisms underlying TFE3-mediated autophagy-lysosome system activity and PTC remain unclear. Recent studies have linked the accumulation of ROS to TFE3 activation in cancer prognosis [31]. The potential mechanisms underlying the links among autophagy-lysosome system activity, TFE3, and PTC require additional in-depth research. The study showed that the high expression of TFE3 was significantly correlated with lymph node metastasis, which reflected the possible relationship between lymph node metas-tasis and autophagy-lysosome levels in PTC patients. Whether autophagy-lysosome-related markers can be used as biomarkers for diagnosis of thyroid papillary carcinoma needs further study.

Conclusions
In summary, we evaluated the expression of TFE3 and its role in malignant characteristics in PTC, showing that TFE3 promotes PTC progression by regulating the autophagy-lysosome system.

Data Availability
Data is available on request.

Ethical Approval
This study was approved by the Ethics Committee of Taizhou Central Hospital (2019-035).

Consent
Written informed consent was obtained from each individual in the study.