TFE3‐PD‐L1 axis is pivotal for sunitinib resistance in clear cell renal cell carcinoma

Abstract The microphthalmia of bHLH‐LZ transcription factor (MiT/TFE) family chromosomal translocation or overexpression is linked with a poor prognosis in clear cell renal cell carcinoma (ccRCC) with elevated recurrence and drug resistance, but the molecular mechanism is not fully understood. Here, we investigated whether the resistance to sunitinib (Sun), the standard treatment for metastatic ccRCC, is due to up‐regulation of programmed death ligand 1 (PD‐L1) by the transcription factor E3 (TFE3). In this study, we propose that TFE3 but not TFEB is essential for tumour survival which was associated with the poorer survival of cancer patients. We also found a positive correlation between TFE3 and PD‐L1 expression in ccRCC cells and tissues. Sun treatment led to enhanced TFE3 nuclear translocation and PD‐L1 expression. Finally, we observed the therapeutic benefit of Sun plus PD‐L1 inhibition which enhanced CD8+ cytolytic activity and thus tumour suppression in a xenografted mouse model. These data revealed that TFE3 is a potent tumour promoting gene and it mediates resistance to Sun by induction of PD‐L1 in ccRCC. Our data provide a strong rationale to apply Sun and PD‐L1 inhibition jointly as a novel immunotherapeutic approach for ccRCC treatment.

study, we propose that TFE3 but not TFEB is essential for tumour survival which was associated with the poorer survival of cancer patients. We also found a positive correlation between TFE3 and PD-L1 expression in ccRCC cells and tissues. Sun treatment led to enhanced TFE3 nuclear translocation and PD-L1 expression. Finally, we observed the therapeutic benefit of Sun plus PD-L1 inhibition which enhanced CD8+ cytolytic activity and thus tumour suppression in a xenografted mouse model. These data revealed that TFE3 is a potent tumour promoting gene and it mediates resistance to Sun by induction of PD-L1 in ccRCC. Our data provide a strong rationale to apply Sun and PD-L1 inhibition jointly as a novel immunotherapeutic approach for ccRCC treatment.

K E Y W O R D S
clear cell renal cell carcinoma, PD-L1, sunitinib, TFE3 and Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) could inhibit the proliferation and differentiation of immunocompetent cells and the recognition of tumour cells by tumour-infiltrating lymphocytes (TILs). Currently, PD-1/PD-L1 axis has attracted massive interest. 13 Blockade the PD-1/PD-L1 axis has been of benefit in the treatment of many different types of cancers including ccRCC. 14,15 TKI or ICI for the treatment of RCC has significantly improved the OS, PFS and durable responses in some patients. However, resistance and relapse are common; and only 15%-25% of patients exhibit clinical responses to checkpoint blocking when given as monotherapy. 16,17 So, innovative combinations of TKI with ICI are now part of the treatment strategy and have achieved exciting benefits according to the results of recently updated phase III trials. 10,18,19 However, the molecular mechanisms of these novel combinations need further investigations.
The MiT-TFE family of basic helix-loop-helix leucine-zipper transcription factors including TFEB, TFE3, TFEC and MITF play a major role as regulators of lysosome biogenesis, cellular energy homeostasis and immune responses; thus, they were originally described as oncogenes. 20 The expression of the TFEB and TFE3 and their activity are elevated in multiple types of human cancers and associated with enhanced proliferation and motility of these cancer cells. 21 Furthermore, TFEB or TFE3 fusion and overexpression caused by chromosomal translocation events is linked with a poor prognosis in a subset of RCC patients with elevated recurrence and metastasis. 22 But the molecular mechanism is not fully understood.
Recently, Zhang et al 23 reported that TFEB mediates immune evasion and resistance to mTOR inhibition of RCC via induction of PD-L1. These studies have shown that the MiT-TFE family plays an important role not only in the progression, but also chemotherapy resistance of RCC tumours.
In this study, we found that TFE3 is also a potent tumour promotor just like TFEB in ccRCC. Importantly, TFE3 but not TFEB is essential for the survival of tumour cells. TFE3 can also regulate PD-L1 expression in ccRCC cell lines and primary human ccRCC tumour tissues. We also found that Sun enhanced TFE3 nuclear translocation and PD-L1 expression. Combination of Sun with anti-PD-L1 enhanced the therapeutic efficacy in a mouse RCC xenograft model. Thus, our data provide rationale for the combined use of Sun and PD-L1 blockade as a potential therapeutic strategy to treat ccRCC.

| Cell culture and reagents
786-O, A498, TK-10 ccRCC cells, Renca mouse RCC cell and HepG2 liver adenocarcinoma cell (the Cell Bank of the Chinese Academy of Sciences) were cultured in RPMI-1640 medium supplemented with 10% foetal bovine serum (HyClone) and 100 U/mL penicillin and 100 g/ mL streptomycin. All these cells were routinely cultured in 5% CO 2 at 37°C. After chemical treatments, cells were collected for Western blots or other assays. Sunitinib malate (#S1042) was purchased from Selleck.

| Western blots analysis and antibody
Cells or tumour tissues were washed with ice-cold PBS and lysed in RIPA lysis buffer containing a fresh protease and phosphate inhibitor mixture (50 mg/mL aprotinin, 0.5 mmol/L phenylmethanesulfonyl fluoride, 1 mmol/L sodium orthovanadate, 10 mmol/L sodium fluoride and 10 mmol/L β-glycerolphosphate). Cell lysates were then

TA B L E 1 Primer
F I G U R E 1 TFE3 but not TFEB affect cell proliferation of ccRCC cells. A, The relationship between TFE3/TFEB and patient prognosis in ccRCC was analysed in data from Kaplan-Meier plotter database. B, The expression of TFE3 and TFEB in ccRCC specimens and RCC cells were analysed in data from TCGA and CCLE database. C, The expression of TFE3 and TFEB in ccRCC specimens was analysed by qPCR. D, siRNA knockdown of TFE3 and TFEB was analysed by qPCR. E, siRNA knockdown of TFE3 and TFEB was detected by Western blots. F, Cell viability was analysed using a xCELLigence RTCADP instrument. G, siRNA knockdown of TFE3 and TFEB was performed, and the cell proliferation was analysed by morphology. H, siRNA knockdown of TFE3 and TFEB was performed, and the cell proliferation was analysed by EdU. I, siRNA knockdown of TFE3 and TFEB was performed, and the cell proliferation was analysed by clone formation. Data are mean ± SD, *P < .05, **P < .01 and ***P < .001

| Real-time quantitative PCR
Total RNAs was extracted using an RNAiso plus kit (TaKaRa).
Complementary DNA was synthesized through reverse transcription using ReverTra Ace qPCR RT Kit (TOYOBO). Quantitative PCR analysis of cDNA was performed with SYBRGreen reaction master mix on a Real-time PCR System (Eppendorf International). Target mRNA levels were normalized to the level obtained for GAPDH.
Changes in transcript level were calculated using DD△Ct method.
The primers used in this experiment were listed in Table 1.

| xCELLigence
Experiments were carried out using the RTCADP instrument (Roche) which was placed in a humidified incubator maintained at 37°C with 5% CO 2 . For time-dependent cell response profiling, 10 000 cells/ well were added to 16-well E-Plates. The electronic sensors provided a continuous and quantitative measurement of cell index in each well.
Cell index is a quantitative measure of cell number present in a well, that is lower cell index reflects fewer cells are attached to the electrodes. The E-Plate 16 was monitored over the time frame indicated.

| ethynyl-2'-deoxyuridine (EdU) incorporation assay
EdU cell proliferation kit (17-10527) was purchased from Millipore. Pretreatment with siRNA, the cells were incubated 16 hours at 37°C in complete media supplemented with 10 μmol/L EdU. After washing in PBS, the cells were fixed and permeabilized. Reaction cocktail and DAPI (Beyotime) were then added. The fluorescence change of cells was detected with flow cytometry or microscope.

| Microscopy
To measure TFE3 nuclear translocation, cells following TFE3-GFP transfection and Sun (5 µmol/L) treatments were incubated with DAPI for 10 minutes. The cells were then washed with PBS, and nuclear translocation fluorescence was measured using confocal microscopy (Carl Zeiss). as a mean score that considers both the intensity of the staining and a positive reaction.

| Flow cytometry
The PD-L1 expression in the cells was determined using flow cytometry. Cells following various treatments were collected by centrifugation.
After two washes with ice-cold PBS, cells were stained with antibod-

| Luciferase reporter assay
Luciferase reporter assay was identified as reported previously 23 and modified slightly. The PD-L1 promoter sequence (−250 to

| TFE3 but not TFEB affects cell proliferation of ccRCC cells
The levels of TFEB and TFE3 are elevated in multiple types of human cancers and have been linked with both occurrence and poor prognosis. [20][21][22]24 Recent study claimed that TFEB has little effect on RCC proliferation. 23 To clarify the role of TFEB and TFE3 in ccRCC, we first analysed the publicly available Kaplan-Meier plotter and The Cancer Genome Atlas (TCGA) database on the expression of TFEB and TFE3. As shown in Figure 1A, TFE3 but not TFEB was negatively correlated with the survival of patients in clear cell RCC.
We also found that the basal expression of TFE3 is higher than that of TFEB in ccRCC specimens and tumour cell lines by TCGA and Cancer Cell Line Encyclopedia (CCLE) database ( Figure 1B). To further validate the database information, we examined 30 clinical renal clear cell carcinoma samples by qPCR and confirmed that TFE3 expression is higher ( Figure 1C). To determine whether this difference defines the proliferative advantage of ccRCC, we tried to knockdown TFEB and TFE3 in 786-O cells ( Figure 1D,E). As a result, knockdown of TFEB did not affect cell proliferation. This is consistent with previous report. 23 But interestingly, knockdown of TFE3 significantly inhibited cell proliferation ( Figure 1F). These results were further validated by EdU staining and clone formation assay ( Figure 1G-I). In addition, we also knocked down TFEB and TFE3 in hepatocellular carcinoma cells and got similar results ( Figure S1A-D). This indicated that TFE3 can be at least another potent tumour promotor beyond TFEB in specific tumour types such as ccRCC.

| TFE3 mediates immune evasion by positively regulation the expression of PD-L1 in ccRCC cells and ccRCC patients
Recent study showed that TFEB can mediate immune evasion by positively regulation the expression of PD-L1 in RCC. 23  down-regulated accordingly ( Figure S2A). These results were further confirmed by Western blots, immunofluorescence and flow cytometry ( Figure 2B-E). Furthermore, TFE3 overexpression can significantly enhance luciferase activity driven by the PD-L1 promoter ( Figure 2F). We also found that expression of TFE3 but not TFEB was positively correlated with the levels of PD-L1 in RCC tumour cell lines by CCLE database ( Figure 2G and S2B). Then, we choose TFE3 for further study. We next evaluated whether the level of TFE3 correlated with PD-L1 expression in primary ccRCC patients. Within individual tumours, PD-L1 staining showed heterogeneous expression, which can be readily differentiated into PD-L1 − and PD-L1 + areas. Higher expression and enhanced nuclear localizations of TFE3 were seen in the PD-L1 + regions ( Figure 3A,B). These results were further confirmed by Western blots (Figure 3C,D). Together, these findings demonstrate that TFE3 can positively regulate PD-L1 expression.

| Sunitinib enhances PD-L1 expression via activation of TFE3 in ccRCC cells
Sunitinib is an oral TKI that is currently registered for the treatment of advanced or metastatic RCC. 8,9 Despite its initial excitement for the treatment of ccRCC, Sun rarely achieved complete responses and most patients ultimately developed resistance to Sun therapy, and the mechanism of resistance is not fully understood yet. Recent study reported that Sun increased PD-L1 expression in liver tumour cells. 25 Therefore, we proposed that the tolerance induced by Sun can also be related to the up-regulation of PD-L1 expression in ccRCC cells. As shown in Figure 4A

| Anti-PD-L1 immunotherapy enhances the response to sunitinib in RCC
We then asked whether combined use of PD-L1 antibody could po-  Figure 5E). The combination treatment also resulted in increased survival ( Figure 5F). Together, these data demonstrated that the combined use of Sun and anti-PD-L1 can be a novel immunotherapeutic approach for ccRCC treatment. Sunitinib is an oral TKI that is currently registered for the treatment of advanced or metastatic RCC, gastrointestinal stromal tumour and neuroendocrine tumour. 8,26,27 Despite the early success of Sun on the treatment for ccRCC, most patients ultimately developed resistance whose mechanism is not fully understood. In fact, the resistance has been largely attributed to the derailing of intracellular signalling pathways, but less on the immune microenviromment. 8,9 In this study, we demonstrated that Sun led to enhanced nucleus translocation of TFE3 in RCC cells, which subsequently induces PD-L1 expression. Furthermore, combination of Sun and anti-PD-L1 enhanced the cytotoxic functions of tumour-infiltrating CTL and therapeutic efficacy in a mouse RCC xenograft model.

| D ISCUSS I ON
In summary, it is indicated that TFE3, like TFEB, is also a potent tumour promotor based on its significant proliferative effect. And more importantly, it mediates PD-L1 up-regulation, which can ultimately attenuate Sun therapeutic efficacy via tumour-associated immune-suppression. By emphasizing on the pivotal role of TFE3, our data provide a valuable rational for the application of chemoimmunotherapy on the RCC patients.

ACK N OWLED G EM ENTS
This work was supported by the Shandong Key Research and Development Program, China (2019GSF108263).

CO N FLI C T O F I NTE R E S T
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and/or analysed during the current study are available from the corresponding author on reasonable request.