The Sigma‐2 Receptor/TMEM97 Agonist PB28 Suppresses Cell Proliferation and Invasion by Regulating the PI3K‐AKT‐mTOR Signalling Pathway in Renal Cancer

Abstract Sigma‐2 receptor/TMEM97 is overexpressed in many tumours, and sigma‐2 receptor ligands are under investigation for cancer therapy. We intended to evaluate the effect of PB28 on renal cancer in proliferation, migration and invasion in vitro and in vivo. Invasive renal cancer cell lines treated with PB28 (or sigma‐2 receptor antagonist 1) were subjected to cell proliferation, migration and invasion assays. The therapeutic effect of PB28 was performed on nude mice. Western blot for proteins in the PI3K‐AKT‐mTOR signalling pathway was conducted. A CCK‐8 assay was used to examine the effect of the combination of PB28 and cisplatin on renal cancer cells. Significant inhibitory effects were observed on proliferation, migration and invasion of 786‐O and ACHN cells after culturing with PB28. But, the outcomes of sigma‐2 receptor antagonist 1 presented the opposite tendency. PB28 significantly inhibited the proliferative and invasive ability of OS‐RC‐2 cells in vivo. Treatment resulted in decreased phosphorylation of constituents of the PI3K‐AKT‐mTOR pathway. The combination of PB28 and cisplatin showed enhanced efficacy in the inhibition of renal cancer cell proliferation. Taken together, PB28 inhibited the tumorigenic behaviours of renal cancer cells by regulating the PI3K‐AKT‐mTOR signalling pathway and was expected to be a sensitizer of cisplatin.

expressed in the liver and kidney of rabbits. 10 Overexpression of the sigma-2 receptor was also observed in the central nervous system and proliferative tumours. 6,11,12 At present, the sigma-2 receptor has emerged as potential treatment target in malignant tumours and neurological diseases. 7,13,14 Notwithstanding the increasing importance of the sigma-2 receptor in the treatment of diseases, the process of further understanding its biological functions has been greatly hindered because genes encoding the sigma-2 receptor have never been discovered. A breakthrough occurred in 2017, in which a study performed by Assaf Alo confirmed that TMEM97 encoded the sigma-2 receptor. 15 TMEM97 (also known as MAC30) is a member of the insulin-like growth factor family and is relevant to many biological functions in the human body. These processes are related to liver development and differentiation, cholesterol metabolism and cancer transformation. [16][17][18][19] The high-affinity sigma-2 receptor ligand PB28 (1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4tetrahydronaphthalen-1-yl)propyl]piperazine) has been recognized as a novel effective inhibitor of the proliferation of human breast cancer cell lines in vitro. 20,21 Furthermore, a recent study showed that PB28 could induce apoptosis by increasing the production of mitochondrial superoxide radicals in pancreatic cells and reducing tumour growth in vivo. 22 In addition, PB28 was suggested to reverse the resistance of breast cancer cells to doxorubicin by downregulating P-glycoprotein (P-gp) expression levels. 20 We suggest that the sigma-2 agonist PB28 could be a novel anticancer drug either as a monotherapy or in a combination regimen. Herein, we assessed the efficacy of PB28 and sigma-2 receptor antagonist 1 and a probable and unknown mechanism by which the sigma-2 receptor/TMEM97 affects renal cancer. In our current study, we investigated the ability of PB28 to inhibit cell proliferation, migration and invasion by decreasing phosphorylation of constituents of the PI3K-AKT-mTOR pathway. Moreover, the enhanced chemosensitivity of PB28-treated renal cancer cells to cisplatin was also studied.

| Proliferation Assay
Colony formation assay and RTCA (Real-time cell analysis) assay were used to detect the proliferation of renal cancer cells. Colony

| Invasion Assays
Transwell chambers were used to assess cell invasion. In all, 20 μl Afterwards, cells that invaded through to the lower chamber were stained with crystal violet solution for ten minutes. Cells in five random fields were counted under a light microscope (Olympus, Tokyo, Japan).

| Wound Healing Assay
Cells were seeded in a 24-well plate and incubated at 37°C until they reached confluence. Then, the monolayer was scratched using a plastic tip and then washed two times with PBS to remove the cell fragments. Then, the cells were incubated with culture medium in the presence or absence of PB28 (10 µM) /sigma-2 receptor antagonist 1 (300 nM). Microscopy images of the scratches were taken immediately after the scratch (zero hour) and at 24 h. Cell migration ability was assayed using ImageJ software.

| Statistical Methods
SPSS software for Windows 17.0 (SPSS Inc., Chicago, USA) was used to conduct statistical analyses. Student's t test was used to analyse differences in cell invasion and migratory. Analysis of variance of repeated measures was used to evaluate the RTCA results and the tumour growth curves in nude mice. One-way ANOVA analysis was performed on the colony formation assay. P values < 0.05 were considered significantly.

| PB28 Inhibited the Proliferation of Renal Cancer Cells while Sigma-2 Receptor Antagonist 1 Induced the Proliferation of Renal Cancer Cells
We detected the effect of PB28 on the proliferation of 786-O and ACHN cells. As shown in Figure 1A  assay with Matrigel and the wound healing assay, respectively. We observed that PB28 (5 μM) significantly inhibited the invasion of ACHN cells by more than 50%, and the invasion of 786-O cells was also inhibited after PB28 (5 μM) treatment for 48 h (Figure 2A). Furthermore, as shown in Figure 2B, the in vitro migration assays showed that a low dose (

| PB28 inhibited OS-RC-2 cell proliferation and invasion in vivo
We further investigated the effect of PB28 in vivo. OS-RC-2 cells were injected into the armpit (four mice per group) or the caudal vein (three mice per group) of nude mice. We found that tumours derived from PB28 treatment group grew at a slower rate (p < 0.0001) and were lighter than tumours derived from control group (p = 0.00286), and Ki-67 presented a weaker staining (Figure 4). Furthermore, not surprisingly, images of gross specimens and HE staining, PB28 significantly reduced the number and volume of lung metastatic nodules (p < 0.0001) ( Figure 5). The above results were consistent with the findings in vitro, implicating TMEM97 as a candidate tumour suppressor in renal cancer.

| PB28 Suppressed the PI3K-AKT-mTOR Signalling Pathway
According to the GESA bioinformatics results, we predicted that TMEM97 was positively associated with the PI3K-AKT-mTOR signalling pathway in renal cancer ( Figure 6A). Then, Western blotting analysis was used to examine the expression of proteins related to this signalling pathway after PB28 treatment. The results showed that the expression of likely constituents in the PI3K-AKT-mTOR signalling pathway (PI3K-110α, p-mTOR and p-AKT) was significantly inhibited by PB28 (AKT and mTOR were not influenced) in

786-O and ACHN cells, and the expression of EMT-related proteins
(N-cadherin and Vimentin) showed the same trend ( Figure 6B, 6C).

| PB28 Increased the Sensitivity of Renal Cancer Cells to Cisplatin
As multidrug resistance is common in human renal cancer, the effects of PB28 and cisplatin on renal cancer cells were investigated to study the potential implication of TMEM97 in drug sensitivity and in vitro chemoresistance. We examined three renal cancer cell lines,

| DISCUSS ION
Renal cancer is a malignancy that increases the burden of human cancers and has a poor prognosis rate, and there are difficulties in the clinical management of distant metastasis. 23 Sigma receptors and their ligands function in processes beyond those related to cell differentiation and survival, including cholesterol metabolism, cancer cell growth and invasion. [17][18][19] Furthermore, TMEM97 was identified as a sigma-2 receptor ligand in 2017, which expanded the study of sigma receptor function. 15 It was reported that TMEM97 has been confirmed to be a factor associated with cancer progression, 18,19,23 but the related regulatory signalling pathways were not further studied. To this end, we investigated the mechanism of TMEM97 in renal cancer.
First, the sigma-2 receptor has been reported to be expressed in different types of tumours, including gastric, pancreatic and breast tumours. [18][19][20] In parallel, exogenous sigma-2 receptor ligands have been confirmed as antitumour therapies. 7-9,24 Among many sigma-2 receptor ligands, PB28 (as well as agonist) has exhibited exciting outcomes in breast and pancreatic cancer therapy. 20 Third, we predicted probable signalling pathway correlations with TMEM97 in renal cancer by studying bioinformatics analysis.
The GSEA results suggested that TMEM97 is positively correlated with the PI3K-AKT-mTOR signalling pathway in renal cancer. Coutte F I G U R E 4 Representative images of tumours and tissue slices of nude mice. Tumours derived from EG (experimental group (i.p. with PB28)) grew at a slower rate and were smaller in size than tumours derived from CG (control group (i.p. with saline solution)). The volumes of the tumours on the flanks of the mice were recorded 1, 3, 5, 7 and 9 days after the injection of PB28. Tumours derived from control group grew at a faster rate (p < 0.0001) and were larger in volume (p = 0.00286) than tumours derived from experimental group on all days assessed. Ki-67 (brown staining) localized to the nuclear in tumour tissues derived from OS-RC-2 cells. Furthermore, Ki-67 presented a stronger staining in the control group compared with the experimental group and TMEM97 (brown staining) mainly localized to the cytoplasm in tumour tissues F I G U R E 5 Representative images of nude mice, lung metastases and H&E staining slices. All mice were sacrificed followed a four-week treatment of PB28. The number and volume of lung metastatic nodules (red arrows) in experimental group (EG) were significantly decreased compared to the control group (CG) (black arrows) (p < 0.0001) L et al. demonstrated that the PI3K/AKT pathway is highly activated in renal cell cancer. 26 Therefore, we evaluated the expression of proteins involved in the PI3K-AKT-mTOR signalling pathway and EMT in cells treated with PB28. We found that PI3K-110α, p-mTOR and p-AKT, N-cadherin, and Vimentin levels were decreased after PB28 was added, indicating that PB28 binding to its receptor TMEM97 could inhibit the subunit of PI3K, phosphorylation of AKT and mTOR (AKT and mTOR were not influenced) to suppress renal cancer cell growth and metastasis. However, the changes mentioned above had not happened in HK-2 cells. We suspected that TMEM97 take effect on pathological or physiological status of renal via different signalling pathways.
Finally, because renal tumours are not very sensitive to chemotherapeutic drugs and the incidence of multidrug resistance has skyrocketed in recent years, 27,28 new chemical agents and combined chemotherapy regimens are urgently needed. Sigma-2 agonists could modulate the expression of MDR-1 and reduce the expression of P-gp in numerous types of tumour cells, which provided the F I G U R E 6 PB28 regulated the expression of proteins in the PI3K-AKT-mTOR signalling pathway and EMT. A Results from GSb EA bioinformatics analysis. B-D Western blotting analysis of 786-O, ACHN and HK-2 cells treated with various concentrations PB28. The protein levels of PI3K-110α, p-mTOR and p-AKT, mTOR, AKT, N-cadherin and Vimentin were analysed, and β-actin was measured to indicate protein loading. The ratios of target genes to β-actin expression were determined in basal cells and were set at 1 F I G U R E 7 PB28 fortified the sensitivity of renal cancer cells to cisplatin. The CCK-8 assay was performed to measure the change in the IC50 value of cisplatin in renal cancer cells with or without PB28. The IC50 of cisplatin in Caki-1 cells decreased from 17.1 μg ml −1 to 6.86 μg ml −1 when PB28 was added (p = 0.0047) and that in ACHN cells decreased from 30.54 μg ml −1 to 17.28 μg ml −1 (p = 0.0039) first evidence of this type of drug reversal of multidrug resistance in 1997. 29  in a significant decrease in the IC50 value stated above. Our findings were consistent with the hypothesis that sigma-2 agonists can enhance chemotherapy efficacy in renal cancer cells and identified PB28 as not only a therapeutic agent but also a novel sensitizer of cisplatin in renal cancer.

| CON CLUS ION
In conclusion, PB28 binding to TMEM97 might inhibit phosphorylation of constituents of the PI3K-AKT-mTOR signalling pathway, resulting in decreased proliferation, migration and invasion of renal cancer in vitro and in vivo. In parallel, PB28 increased the sensitivity of renal cancer cell lines to cisplatin.

ACK N OWLED G EM ENTS
The language of our manuscript had been polished by Nature Publishing Group Language Editing (NPG Language Editing).

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