Testis developmental related gene 1 regulates the chemosensitivity of seminoma TCam‐2 cells to cisplatin via autophagy

Abstract We previously identified testis developmental related gene 1 (TDRG1), a gene implicated in proliferation of TCam‐2 seminoma cells. Recent evidence has revealed that autophagy influences the chemosensitivity of cancer cells to chemotherapy. However, whether TDRG1 protein regulates autophagy in seminoma cells and influences their sensitivity to cis‐dichlorodiammine platinum (CDDP) remains unknown. In this study, we used TCam‐2 cells and male athymic BALB/c nude mice with xenografts of TCam‐2 cells to investigate autophagy, cell viability, apoptosis and the p110β/Rab5/Vps34 (PI3‐kinase Class III) pathway under the conditions of TDRG1 overexpression or knockdown and with or without CDDP treatment. We found that TDRG1 upregulation promoted autophagy in both TCam‐2 cells and seminoma xenografts via p110β/Rab5/Vps34 activation. Inhibition of autophagy reduced cell viability and promoted apoptosis during CDDP treatment of TCam‐2 cells. Similarly, TDRG1 knockdown inhibited autophagy, reduced cell viability and promoted apoptosis during CDDP treatment of TCam‐2 cells. TDRG1 knockdown inhibited tumour growth and promoted apoptosis in TCam‐2 cell xenografts, whereas TDRG1 overexpression had the opposite effect. According to these results, we propose that high expression of TDRG1 promotes autophagy through the p110β/Rab5/Vps34 pathway in TCam‐2 cells. TDRG1 overexpression promotes autophagy and leads to CDDP resistance, whereas TDRG1 knockdown inhibits autophagy and promotes chemosensitivity to CDDP both in vivo and in vitro. This study has uncovered a novel role of TDRG1 in reducing chemoresistance during CDDP treatment and provides potential therapeutic strategies for the treatment of human seminoma.


| INTRODUC TI ON
Testicular germ cell tumours (TGCT) is the most common malignancy in young men aged from 15 to 40 years, although it accounts for only 2% of all male malignancies. 1 Pure seminoma is a well-defined clinical and pathological entity, comprising 40%-60% of all TGCT, and its incidence been increasing worldwide for decades. 2 For patients with early-stage seminoma, including clinical Stage I and low-volume Stage II seminoma, inguinal orchiectomy followed by suitable radiotherapy is usually curative. 3,4 In patients with more advanced stages, cis-dichlorodiammine platinum (CDDP)-based chemotherapy regimens can achieve complete response rates of 70%-90%. 3,5,6 However, some patients with advanced-stage seminoma are resistant to CDDP chemotherapy, representing either refractory or relapse cases. [7][8][9] Thus, novel treatments to improve the overall chemosensitivity of seminoma are urgently needed.
Autophagy is an ancient and highly conserved process in which unneeded or damaged proteins, or other cytoplasmic components are transported to the lysosomal system to degrade. 10 As it was involved in many cellular processes, autophagy plays important roles in many kinds of diseases, including cancer. [11][12][13] But autophagy is a "double-edged sword" for cancer cells during tumour development. 14,15 Autophagy is thought to help prevent cancer initially; however, once a cancer is formed, autophagy activation may promote the growth and survival of tumour cell. 16,17 In chemotherapy, chemotherapeutic drugs kill cancer cells mainly by inducing apoptosis. However, the activation of autophagy and relevant pathways can inhibit apoptosis, which promotes resistance to chemotherapy. 18,19 Very recently, many studies have focused on inhibition of autophagy as a way to enhance chemosensitivity in mesothelioma, colorectal cancer, hepatocellular carcinoma, neuroblastoma and other kinds of cancers, [20][21][22][23] and have reported very good outcome. Therefore, autophagy could be a potential therapeutic target for improving the chemosensitivity of seminoma as well.
In metazoans, the initiation of autophagy is regulated by phosphoinositides, a product of phosphoinositide 3-kinases (PI3Ks).
According to sequence homology and substrate specificity, PI3K lipid kinases are divided into three classes (class I-III). 24,25 Class III PI3K converts phosphatidylinositol into phosphatidylinositol 3phosphate (PI(3)P) by binding catalytic subunit Vps34 to regulatory subunit Vps15, which is essential for the initiation of autophagy. [26][27][28] Recently, it was shown that the p110β (the catalytic subunit of class I PI3Ks) interacts with the small GTPase Rab5 to promote autophagy by maintaining Rab5 in the binding state of guanosine triphosphate (GTP) and improving the Rab5-Vps34 interaction. 29,30 Testis developmental related gene 1 (TDRG1) is a new gene identified by our group which is expressed in human testis and some higher non-human primates. Our previous study demonstrated that it encodes a 100-amino acid protein (TDRG1 protein) whose expression is enhanced in testicular seminoma than in normal testis. Moreover, we showed that TDRG1 facilitates the invasiveness and proliferation of seminoma cells by activating the PI3K/Akt signalling pathway, meaning that TDRG1 has a carcinogenic role in seminoma. 31 Although inhibition of autophagy can enhance chemosensitivity in many kinds of cancers, whether TDRG1 is involved in regulating the chemical sensitivity of CDDP through autophagy in seminoma remains unclear. In the present study, we investigated the effects of TDRG1 protein on tumour growth in vivo and on autophagy, cell viability, apoptosis and the p110β/Rab5/Vps34 (PI3-kinase Class III) pathway of seminoma TCam-2 cells in vitro, with or without CDDP treatment. Our results demonstrate that TDRG1 regulates the chemosensitivity of TCam-2 cells to CDDP via autophagy through a Class III PI3K.

| Ethics statement
The Institutional Research Ethics Committee of Third Xiangya Hospital approved this study. Informed consent and all experiments were consistent with the principles of the Declaration of Helsinki, and all patients provided written-informed consent for the use of their tissue samples and records.

| Tissue and cell culture
Testicular seminoma tissues (n = 10) and normal testicular tissues
After cells reaching 70% confluence, the above vectors were transfected into TCam-2 cells by Lipofectamine 2000 (Invitrogen Life Technologies) following the instructions. GFP protein expressed by cells were imprinted using a laser confocal scanning microscope (Leica tcs-sp5) to check transfection efficiency. Meanwhile, the expression of PI3K/p110β and Rab5 was checked by Western blot.

| Autophagy flux assay
After different treatment, the autophagic flux in TCam-2 cells was determined by quantifying LC3-II with Western blot or immunofluorescence imaging. Briefly, TCam-2 cells were exposed to Baf A1 (Bafilomycin A1) in the last 6 hours before harvesting. After incubated in lysosomal inhibitor Baf A1 for 6 hours, the autophagy protein LC3-II accumulated and the amount depending on the autophagic flux.

| Vps34 kinase assay
Class III PI3K ELISA Kit (Echelon, Lot# K 3000) was used to check the Vps34 (PI3-Kinase Class III) activity. Firstly, the  and EDTA-free protease inhibitor cocktail. After centrifugation in 4°C for 10 minutes, the supernatants were isolated and incubated with GST-R5BD beads at 4°C for another 10 minutes, then boiled in 1× SDS sample buffer and subjected to Western blot. At the same time, the total Rab5 protein was also determined by Western blot.

| Cell viability assay
The MTT assay was performed to assess the cytotoxic effect of CDDP on TCam-2 cells with different autophagy and TDRG1 expression levels. According to our previous study, we have determined the IC25 DMSO was then added to each well and stirred gently to dissolve the purple-blue formazan crystals. Finally, the absorbance of each well at 570 nm (A570) was measured by Microplate Reader (Bio-tek elx-800).

| Western blotting
For Western blotting (WB), the mouse xenograft tumours and TCam-2 cells were lysed in lysis buffer containing protease inhibitors for 30 minutes. After centrifugation, the supernatants were isolated and protein concentration was determined using bicinchoninic acid assay (Beyotime Biotechnology). An equivalent amount of protein (20 μg) of each sample was separated by 10% SDS-PAGE and then transferred to PVDF membranes. Then blocked the membranes with 5% non-fat dry milk containing 0.1% Tween-20 in Tris-buffered saline (TBS-T) at room temperature for 1 hour and then incubated with primary antibodies (Table 1) at 4°C overnight. After incubated in secondary antibody, immunoreactivity was detected by the enhanced chemiluminescence method (Thermo Scientific).

| Immunofluorescence staining
For immunofluorescence staining, after 72 hours of different treatment, cells were fixed in cold methanol for 5 minutes and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 8 minutes at room temperature. Then, cells were blocked with 3% BSA in PBS for 1 hour and incubated with primary antibody against LC3-II followed with secondary antibody (anti-rabbit IgG labelled with Alexa 555).
Then stained the nucleus with DAPI. Finally, cells were observed by confocal microscope.

| Xenografts
Twenty-four 4-5 weeks old male athymic BALB/c nude mice were purchased from the Institute of Experimental Animals of Central South University. All mice were free to get standard laboratory mouse food and water. After one week be kept in a sterile environment, TCam-2 cells (1 × 10 6 suspended in 200 μL medium) with siRNA control, TDRG1-overexpressing or TDRG1 knockdown were randomly injected into groin area subcutaneously. After one week, mice were divided into four groups (six mice per group) based on the level of TDRG1 expression. Meanwhile, 3-MA (3 mg/kg, i.p., once per week for 4 weeks) and CDDP (3 mg/kg, i.p., once per week for 4 weeks) were managed as followed: CDDP Control group, CDDP + TDRG1 overexpression group, CDDP + TDRG1 knockdown group, CDDP + 3-MA group. The mice were followed up to 31 days then sacrificed, then the tumours were removed.
The tumour volume was calculated according to the formula length × width 2 × 0.5.

| Immunohistochemistry analysis
Tumour sections of xenografts were deparaffinized in xylene, then rehydrated in ethanol and finally rehydrated in doubledistilled water. The sections were then placed in 0.01 mol/L citrate buffer (pH 6.0), and microwave heated for 20 minutes to retrieval antigen. The sections were blocked with 3% goat serum for 60 minutes and then incubated with anti-Ki67 at 4°C overnight. The primary antibody was observed by light microscopy with SP method following the instructions (Maixin Biotechnology). According to the intensity and proportion of immunoreactive cells, protein expression levels were classified semi-quantitatively.

| Statistical analysis
All experiments were repeated in triplicate and all data were presented as the average of three independent experiments. Data were analysed with Prism 5 (GraphPad Software) and presented as mean ± standard deviation (SD). Student's t test was used to analyse the statistical significance of the difference between the two groups. One-way ANOVA was performed for statistical significance among multiple groups.

| Establishment of seminoma TCam-2 cells with TDRG1, PI3K/p110β or Rab5 knockdown and TDRG1 overexpression
In our previous study, we established TDRG1 knockdown and overexpressing seminoma TCam-2 cell lines. 32 In the present study, we generated PI3K/p110β or Rab5 siRNA stably transfected TCam-2 cells. Successful transfection was confirmed by GFP expression using fluorescence microscopy ( Figure S1). Moreover, Western blot demonstrated that siRNA against PI3K/p110β or Rab5 was efficient and specific in knocking down the respective gene at the protein level ( Figure S1). Thus, cell models for PI3K/p110β and Rab5 knockdown were set up successfully.
To examined whether TDRG1 promotes autophagy of TCam-2 cells, expression level of LC3-II in TCam-2 cells and TCam-2 cells with TDRG1 knocked down or overexpressed was compared. By immunofluorescence imaging, the fluorescence intensity of LC3-II was increased in TDRG1-overexpressing TCam-2 cells and decreased in TDRG1 knockdown ( Figure 1B). We also applied the lysosomal inhibitor Baf A1 to obtain a more accurate measure of the level of autophagy. First, we examined the response of TCam-2 cells to a range of Baf A1 concentrations ( Figure S2) and found 200 nmol/L to be the optimal concentration for lysosomal inhibition. Western blot analysis further confirmed that overexpression of TDRG1 promotes  After adding Baf A1, the LC3-II/GAPDH ratio was 0.59 ± 0.03 in TCam-2 control cells, increased to 0.85 ± 0.03 in TDRG1-overexpressing cells (P < .01) and decreased to 0.42 ± 0.01 in TDRG1 knockdown cells (P < .01).

| TDRG1 promotes autophagy through activation of p110β/Rab5/Vps34 (PI3-Kinase Class III)
To gain insight into the mechanism underlying the autophagy triggered by TDRG1, we focused on the p110β/Rab5/Vps34 We tested this hypothesis by using TCam-2 cells with p110β or Rab5 knockdown. As showed in Figure 2C B, Vps34 activity was measured by analysing PI(3)P production by the ELISA described in Section 2. C, Protein expression of TDRG1, p110β, Rab5-GTP and LC3-II (untreated or treated with 200 nmol/L Baf A1 for 6 h) was measured by protein gel blotting 72 h after transfection with siRNA to block the expression of p110β and Rab5 or treatment with 3-MA (autophagy inhibitor). GAPDH served as the internal control. D, Vps34 activity was measured by analysing PI(3)P production by the ELISA described in Section 2. (Data from three independent experiments, error bars represent SD, *P < .05, **P < .01, ***P < .001) to 0.04 ± 0.01 (P < .01) without Baf A1 and to 0.11 ± 0.02 (P < .001) with Baf A1. These results indicate that TDRG1 cannot activate Vps34 without Rab5, which means that TDRG1 activates Vps34 through Rab5. When we used 3-MA, an inhibitor of Vps34, the concentration of PI(3)P decreased to 28.76 ± 1.36 pmol (P < .001) and the LC3-II/GAPDH ratio decreased to 0.02 ± 0.01 (P < .001) without Baf A1 and to 0.05 ± 0.01 (P < .001) with Baf A1. These results confirm that TDRG1 promotes autophagy through p110β/Rab5/Vps34 (PI3-Kinase Class III) activation.

| TDRG1 and autophagy regulate cell viability and apoptosis in response to CDDP treatment in TCam-2 cells
Based on our previous study, 32  Based on these data, we assume that in the case of CDDP(IC25) treatment, TDRG1 overexpression attenuates the chemosensitivity of TCam-2 cells to CDDP, and TDRG1 knockdown or autophagy inhibition enhances the chemosensitivity. The promoting effect of TDRG1 knockdown and autophagy inhibition is similar; and, finally, the effect on apoptosis and proliferation is close to that exerted by CDDP(IC50).

| TDRG1 regulates the chemosensitivity of TCam-2 cells to CDDP via autophagy
As TDRG1 can regulate autophagy, and because autophagy activation may be an important factor in promoting resistance to chemotherapy, 18,19 we next sought to determine whether autophagy is involved. To do this, we examined the expression levels of autophagic biomarkers in TCam-2 cells in which TDRG1 was either knocked down or overexpressed. By IF microscopy, the fluorescence intensity of LC3-II was increased in TCam-2 cells overexpressing TDRG1, and it was decreased in TDRG1 knockdown cells and in cells treated with 3-MA and CDDP(IC25); in contrast, there were no significant differences between the NC, CDDP(IC25) and CDDP(IC50) groups ( Figure 4A). Western blot measurements further confirmed these findings ( Figure 4B) 0.29 ± 0.02 in TCam-2 cells, increased to 0.66 ± 0.04 in TDRG1-overexpressing cells (P < .01) and decreased to 0.04 ± 0.01 in TDRG1 knockdown cells (P < .001; Figure 4B). The level of p110β expression in TCam-2 cells treated with CDDP(IC25) was similar to that in cells without CDDP treatment, which indicates that TDRG1 regulates autophagy through p110β/Rab5/Vps34 (PI3-Kinase Class III) independent of CDDP treatment.
As caspase-3 is a key effector in apoptosis, we next examined the expression of activated caspase-3. As shown in Figure 4B, the ratio of activated caspase-3 to GAPDH was very low (around 0.16 ± 0.01) in the NC group, increased to 0.28 ± 0.03 (P < .05) in CDDP(IC25) group and to 0.39 ± 0.03 (P < .01) in the CDDP(IC50) group ( Figure 4B). This result indicates that CDDP can increase the level of apoptosis of TCam-2 cells in a concentration-dependent manner. After incubation in CDDP(IC25), the ratio of activated caspase-3 to GAPDH decreased to 0.06 ± 0.01(P < .01) in the TDRG1-overexpressing group and increased to 0.45 ± 0.01 (P < .01) in the TDRG1 knockdown group ( Figure 4B). A similar result was observed in the 3-MA-treated group, where the ratio increased to 0.74 ± 0.08 (P < .001). As caspase-3 is very important in the autophagy-associated apoptosis pathway, these results indicate that TDRG1 regulates apoptosis through its effects on autophagy.

| TDRG1 regulates seminoma growth via autophagy in response to CDDP in vivo
Xenograft tumour model in male BALB/c nude mice was established to investigate the effect of TDRG1 on the chemosensitivity of seminoma cells to CDDP through autophagy in vivo. As shown in Figure 5A, after four cycles of CDDP(IC25) treatment, the mean volume of tumours with TDRG1 overexpression was significantly bigger and TDRG1 knockdown or 3-MA treatment obviously smaller than control.
To check the molecular mechanism, we performed Western blot analyses to check the autophagy and a related apoptotic pathway.

| D ISCUSS I ON
Chemotherapy is widely used for treatment to many kinds of cancers, including seminoma. Indeed, CDDP is one of the most commonly used anti-cancer agents in seminoma patients due to the high sensitivity. 34,35 Most seminoma patients, even those in later clinical stages, could have a good outcome with chemotherapy based on CDDP. However, patients who are not sensitive to CDDP experience pain and inevitably succumb to the disease. 7,8,36 Although the mechanisms that confer resistance or sensitivity of seminoma cells to CDDP are unclear, several have been postulated. 37,38 The TDRG1 gene was involved in one of the mechanisms. In our model, we hypothesized that H19 long non-coding RNA promotes the expression of TDRG1 by sequestering miRNA-106b-5p. 39 In turn, up-regulated TDRG1 promotes resistance to CDDP through PI3K/Akt/mTOR signalling and the mitochondria-mediated apoptotic pathway. 32 In the present study, we aimed to investigate whether TDRG1 protein regulates autophagy and chemosensitivity to CDDP in seminoma cells.
Autophagy is an important cellular process for maintaining proper cell function and homeostasis in response to various stress conditions. We examined the expression of TDRG1 and the level of autophagy in both normal testicular tissues and seminoma. The data indicated that the expression levels of LC-3II, an autophagy marker, and TDRG1 were higher in seminoma than in normal tissue, demonstrate a potential relationship between TDRG1 and autophagy in seminoma. Furthermore, we confirmed that TDRG1 overexpression up-regulates and TDRG1 knockdown down-regulates the level of autophagy in TCam-2 cells. These results indicate that TDRG1 upregulation in seminoma is likely a key driver of the high level of autophagy in this kind of tumour.
To further investigate the mechanism by which TDRG1 regu- The p110β-Rab5 interaction protects Rab5-GTP and increases the amount of activated Rab5, then promotes Rab5 interactions with Vps34, which finally induces autophagy. 29,30 In this study, we found that the expression of p110β and Rab5, the activity of Vps34, and the autophagy flux were all increased in

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest.

AUTH O R S ' CO NTR I B UTI O N S
DP and YT designed the experiments. DP, JW, XJ, YX and YD performed the experiments and collected the data. DP, JW and YG analysis and interpretation of the data. RK provided the TCam-2 cells.
DP, JY and YT were the major contributors in writing the manuscript.
All authors read and approved the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.