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

Hongsheng Lu Taizhou Central Hospital Chumeng Zhu Taizhou Central Hospital(Taizhou University Hospital) Yanyun Ruan Taizhou Central Hospital(Taizhou University Hospital) Lilong Fan Taizhou Central Hospital(Taizhou University Hospital) Kena Wei Taizhou Central Hospital(Taizhou University Hospital) Qi Chen Taizhou Central Hospital(Taizhou University Hospital) Qing Wei (  weiqing1971@tongji.edu.cn ) Taizhou Central Hospital


Introduction
Thyroid cancer (TC) is the most common endocrine malignancy [1]. The incidence of thyroid cancer is increasing rapidly over the past decades [2]. Thyroid cancer has been classi ed as follicular thyroid cancer (FTC), papillary thyroid cancer (PTC), medullary thyroid cancer(MTC) or anaplastic thyroid cancer (ATC), and PTC accounts for approximately 80% of all thyroid cancers [3]. Although the prognosis of PTC is a better than most other tumors, the prognosis is obviously poor when patients are insensitive to traditional surgery and iodine chemotherapy. Therefore, exploring new biomarkers is of great signi cance to improve the diagnosis and treatment of PTC TFE3 is located on the short arm of X chromosome 11.22, and is a member of the microphthalmia family [4]. Recently, the MiT / TFE family were identi ed as regulators of autophagy. Subsequent studies showed that TFE3 could bind to CLEAR elements, which present in the promoter regions of many lysosomal genes present in the promoter regions of many lysosomal genes, and regulate lysosomal biogenesis in several different cell types [5].
Autophagy is an evolutionary highly conservative catabolic process, which is essential for maintaining cell homeostasis and adapting to various stress situations [6]. Autophagy is primarily a response to the stress of the microenvironment, such as hypoxia, nutrient de ciency, and accumulation of reactive oxygen species (ROS). Dysfunctional autophagy contributes to many diseases, including cancer.
Depending on the type and stage of cancer, autophagy can play a positive or negative role in the development of cancer [7]. Thus, understanding the role of autophagy in PTC is crucial for identifying new targets for PTC therapy. Gene Set Enrichment Analysis (GSEA) was used to analyze the enrichment of autophagy and lysosome-related biological functions in PTC. The results showed that the expression of genes involved in autophagy and lysosome-related biological functions was upregulated in the PTC group.
In the present study, we have investigated the expression of the autophagy regulator TFE3 in PTC, and to clarify its role as a potential target in PTC patients.

Cell transfection
The overexpression of TFE3 was achieved by transfection of pcDNA3.1-TFE3. CDNA encoding full-length TFE3 was cloned into pcDNA vector which was sequence con rmed by sanger sequencing. siRNA targeted to TFE3 (si-TFE3) and the corresponding negative control (si-NC) were used for TFE3 knock down. The siRNA sequences were as follows: β-actin was used as an internal control. The relative quanti cation of each sample was calculated using 2 −ΔΔCt method.

Wound healing assay
Cells were cultured in 6-well plates at a density of 80%, then a 200ul pipette tip was used to create scratches on the cell culture plane, washed three times with phosphate buffer saline (PBS) and cultured in serum-free RPMI 1640 medium. Wounds were observed under the microscope and captured at 0 h and 24 h. The results were analyzed by Image J software (National Institutes of Health, Bethesda, MD, USA), and the rate of cell migration was determined as following: (diameter of original wound − diameter of wound at different times)/ diameter of original wound × 100%.

Cell migration and invasion assays
Cell invasion was performed using a trans well assay. Brie y, PTC cells were trypsinized and suspended in serum-free RPMI 1640 (100μl) and placed in the upper cavity of each Matrigel-coated Transwell insert (REF:3422, Corning, USA). The lower chambers were supplemented with complete culture medium (500μl). After 24 h incubation, cells in the upper chamber were transferred to the membrane of the lower chamber, which were xed with methanol and then stained with 0.1% crystal violet. The procedure of the migration experiment was the same as the invasion experiment except that no matrigel was required. Random elds were chosen for imaging, which were performed with Zeiss photomicroscope(Carl Zeiss Meditec, Dublin, CA, USA).

Immunohistochemistry
Tissue sections were depara nized with xylene, then hydrated with alcohol, and antigen retrieval was performed by heating 0.01 sodium citrate buffer in a microwave oven for 15 minutes. The sections were quenched with 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity. The nonspeci c binding was prevented by adding normal goat serum for blocking for 20 minutes. They were incubated with the primary antibody against TFE3 (1:500; Cell Signaling Technology, USA), P62/SQSTM1 (1:50; Proteintech, USA) and LC3 (1:500; Sigma-Aldrich, USA) for 1 h at room temperature, then added anti-rabbit or anti-mouse secondary antibodies (1:500; Pierce, Appleton, WI, USA)and incubated at room temperature for 1 h. After each treatment, washed 3 times with TBST for 5 minutes each time, and then developed with 3,3-diaminobenzidine. After the sections were counterstained with hematoxylin and differentiated with hydrochloric acid and alcohol, they were dehydrated, transparent, and xed. Random elds were chosen for imaging, which were performed with Zeiss photomicroscope (Carl Zeiss Meditec, Dublin, CA, USA).

Statistical analysis
All experimental procedures were repeated at least three times. Data were analyzed using SPSS 20.0 software (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 8.0 statistical software (GraphPad Software Inc, La Jolla, CA, USA). Expression association was analyzed with Spearman's correlation analysis.
Mann-Whitney U-test was used to compare mean values of more than two groups. All data were expressed as mean ± standard deviation (SD) , and P value < 0.05 indicated statistical signi cance.

Autophagy-lysosome is positively correlated with PTC progression
To explore the role of autophagy-lysosomes in the process of PTC, GSEA was used to analyze the enrichment of autophagy and lysosome-related biological functions in PTC. The results showed that the expression of genes involved in autophagy and lysosome-related biological functions was upregulated in the PTC group and the result of functional enrichment was signi cant (Fig. 1A, B, C, D and E, P<0.05). Consistent with the above results, immunohistochemistry assay indicated that the expression of LC3 and P62/SQSTM1 in PTC tissues were signi cantly higher than that in normal tissues ( Fig. 1F and G).

TFE3 expression is overexpressed in PTC
We examined the level of TFE3 in 78 pairs of PTC tissues and para-cancer tissues. The qRT-PCR showed that TFE3 expression was signi cantly increased in PTC tissues compared with para-cancer tissues ( Fig.   2A, B, and C, P<0.01). Then, we analyzed the correlation between clinicopathological characteristics and TFE3 expression in PTC patients, the result indicated that high expression of TFE3 was signi cantly correlated with lymph node metastasis ( Table 1, Fig. 2E, P<0.05). At the same time, immunohistochemistry assay con rmed that the expression of TFE3 in PTC tissues was higher than that in normal tissues (Fig. 2D).

TFE3 promotes the proliferation of PTC cells
Here, we detected the effect of differentially expressed TFE3 on PTC cell proliferation through MTT assay. As shown in Fig. 3C, compared with the negative control (si-NC), down-regulation of TFE3 expression signi cantly inhibited cell growth. In keeping with the results of the si-TFE3 group, the pcDNA3.1-TFE3 group demonstrated more obvious cell proliferation ability than pcDNA3.1 group (Fig. 3B, P<0.05). These data suggested that TFE3 promoted the proliferation in PTC cells. Assays were repeated in triplicate.

TFE3 accelerates PTC cell migration and invasion in vitro
To detect the role of TFE3 in PTC progression, we conducted a wound healing assay and transwell assay to examine its effects on migration and invasion. PTC cells were transfected with pcDNA3.1-TFE3 or si-TFE3 in 6-well plates. Firstly, the wound healing assay revealed that TFE3 transfection increased the migratory ability (Fig. 4A, B, C, and D, P<0.05). Then, by the transwell assay, the result surely manifested that, compared with si-NC group, the cell migration of PTC cells was signi cantly suppressed in si-TFE3 group, while the number of cells penetrating the lower surface in the pcDNA3.1-TFE3 group was more than that in the pcDNA3.1 group (Fig. 4E and F, P<0.05). Consistent with the results of the migration experiment, the invasion experiment showed that cell invasion was noticeably suppressed by TFE3 knockdown and enhanced by TFE3 overexpression (Fig. 4G and H, P<0.05). In conclusion, these results indicated that TFE3 might promote migration and invasion behavior in PTC cells. Assays were performed in triplicate.

TFE3 induces autophagy-lysosome in PTC cells
To determine whether autophagy-lysosome is related to TFE3, as shown in Fig. 5C, the expression of LC3 and P62/SQSTM1 in the pcDNA3.1-TFE3 group were higher than in the pcDNA3.1 group. Then, we detected the processing of LC3 I to LC3 II expression in BCPAP and KTC-1 cells transfected with pcDNA3.1-TFE3 or si-TFE3, which is a sign of autophagy [9].The results showed that TFE3 increased the ratio of LC3 /LC3 ( Fig. 5A and B, P<0.05). Next, we examined the level of P62/SQSTM1 protein.
P62/SQSTM1 is selectively incorporated into autophagosomes by simultaneously interacting with LC3 protein [10]. Compared with the pcDNA3.1 group, the pcDNA3.1-TFE3 group showed increased expression of P62/SQSTM1 ( Fig. 5A and B, P<0.05). Similarly, the si-TFE3 group showed a decrease in P62/SQSTM1 expression compared to the si-NC group. Through Q-PCR experiments, we found that the expression of LC3 and P62/SQSTM1 genes in PTC cells decreased after knocking down TFE3, and overexpression of TFE3, the expression of LC3 and P62/SQSTM1 in PTC cells was increased (Fig. 5C, P<0.05). The results above revealed that TFE3 might be able to promote autophagy-lysosome in PTC cells. Assays were performed in triplicate.

Discussion
PTC is the most common endocrine malignancy, ranked 5th in female tumor incidence [2]. Currently, the treatment of PTC 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 chemotherapy resistance in refractory PTC lead to a decrease in the survival rate of patients with PTC [12].
TFE3, which belongs to the MiT/TFE family, is a regulator of autophagy and lysosomal biogenesis [13]. It was rst discovered in 2009 that the MiT / TFE family could regulate most lysosomal genes (including promoters encoding hydrolases and lysosomal-related proteins) [14]. Importantly, under stress conditions, complex interactions between MiT / TFE family-dependent autophagy homeostasis pathways and apoptotic processes may occur in cancer cells, which ultimately determine their fate between cell death or survival [15]. TFE3 simultaneously regulates autophagy induction, lysosomal biogenesis, oxidative metabolism, and oxidative stress, making it play an important role in determining cell fate [16]. Notably, under nonstress conditions, TFE3 interacts with 110 14-3-3 proteins and remains in the cytoplasm. At this time, TFE3 is phosphorylated at Ser321. when affected by stress, TFE3 is dephosphorylated and TFE3/14-3-3 complex is dissociated, while TFE3 is transferred from the cytoplasm to the nucleus to promote autophagy and lysosome biogenesis [5]. TFE3 was found to be fused with papillary renal cell carcinoma (PRCC) 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 enhances melatonin-induced apoptosis in tongue squamous cell carcinoma. Furthermore, increased TFE3 expression in RCC was associated with poor PFS [19]. In accordance with these results, we found that TFE3 increased PTC cell proliferation, invasion, and decreased apoptosis.
M. Anselmier, a French physiologist, rst used the term "autophagy" in a short article describing the effects of fasting on mice published in 1859 [20]. Autophagy, a highly conserved protein degradation pathway from yeast to humans, is essential for clearing protein aggregates and misfolded proteins from healthy cells. Under stress conditions, cells produce a multitude of damaged proteins or organelles, and a double membrane will be produced in the cytoplasm to swallow defective or toxic molecules and organelles to form autophagosomes. Then, autophagosomes fuse with lysosomes and release lysosomal acid enzymes in the vesicles to decompose toxic molecules and other substances, and the resulting products re-synthesize new proteins or organelles [21].The whole process of autophagy involves a variety of evolutionary conserved genes, namely autophagy-related genes (ATGs) [22]. According to the different ways of transporting cellular material to the lysosome, autophagy can be divided into macro autophagy, micro autophagy, and molecular chaperone-mediated autophagy (CMA) [23]. Previous studies demonstrated that autophagy is an important participant in the pathogenesis of many diseases including cancer [24,25]. Genome-wide association studies found that ATG5 is associated with systemic lupus erythematosus (SLE) in Chinese, 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 cases of PTC and 18 normal samples were selected from the TCGA database for analyzing the mechanism of PTC. Based on GSEA the enrichment of autophagy and lysosomal related biological functions involved in LC3 and P62/SQSTM1 in PTC data was analyzed the results showed autophagy lysosome was positively correlated with thyroid cancer progression. LC3 and P62/SQSTM1 have been widely reported as indicators of autophagy [28,29]. Meanwhile, TFE3 has been identi ed as a regulator of autophagy by previous studies [16]. Based on bioinformatics analysis and GSEA data, we furthered validated it in tissue and cells in vitro. We found that TFE3 was signi cantly higher in 78 PTC tissues than in para-cancer tissues. High expression levels of TFE3 was closely associated with lymph node metastasis. Then, we conducted function assays in KTC-1 and BCPAP cell lines. The results showed that TFE3 enhanced the proliferation, invasion, and migration of PTC cells by regulating autophagylysosome, 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 ratio of LC3 /LC3 . Therefore, we hypothesized that TFE3 might positively regulate autophagy lysosome in PTC.
However, the underlying mechanisms of TFE3-mediated autophagy-lysosome and PTC remain unclear. Recent studies have linked the accumulation of ROS to TFE3 activation in the prognosis of cancer [30]. The potential mechanisms of autophagy-lysosome and TFE3 and PTC require deeper research.

Conclusions
In summary, we detected the expression of TFE3 and its malignant characteristics in PTC. The present study revealed that TFE3 promotes PTC progression through autophagy-lysosome. Moreover, TFE3 as a sensitive marker for lymph node metastasis might be a novel diagnostic and therapeutic target for PTC.