Blog

By Bo Zhan, Zhe Zhang, Chiyuan Piao, Xiao Dong, Yang Du, Chuize Kong, and Yuanjun Jiang

Excerpt from the article published in the Journal of Cellular and Molecular Medicine, 16 November 2021 https://doi.org/10.1111/jcmm.17047

Editor’s Highlights

• 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.
• Sigma receptors and their ligands function in processes beyond those related to cell differentiation and survival, including cholesterol metabolism, cancer cell growth, and invasion.
• TMEM97 was identified as a sigma-2 receptor ligand in 2017, which expanded the study of sigma receptor function.
• The sigma-2 receptor has been reported to be expressed in different types of tumors, including gastric, pancreatic, and breast tumors.
• Agonists and antagonists of the sigma-2 receptor play diverse roles in renal cancer.
• The Sigma-2 agonists play a significant role in the resensitization of chemoresistant tumors. 
• But also, a sigma-2 agonist might inhibit phosphorylation of constituents of the PI3K-AKT-mTOR signaling pathway, resulting in decreased proliferation, migration, and invasion of renal cancer in vitro and in vivo.

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.

1 INTRODUCTION

Renal cancer is a malignancy that originates in the renal tubular epithelial system and is one of the most common malignant tumours in the urinary system. Although surgical treatment is commonly performed, most patients still die of tumour recurrence and metastasis.¹ The sigma receptor was first reported in 1976, and there are two identified subtypes: sigma-1 and sigma-2.^2, 3 The sigma receptor has been of interest since its discovery due to its wide range of biological functions, and it is considered to play significant roles in regulating cell survival, morphology and differentiation.^4, 5 It has gradually become a target for cancer diagnosis and treatment.^6–9 The sigma-2 receptor is an 18–21 kD membrane protein,² and recent receptor-ligand binding assays have shown that it is highly 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.^16–19 The high-affinity sigma-2 receptor ligand PB28 (1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-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.

…

3 RESULTS

3.1 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, the proliferative ability of 786-O and ACHN cells was measured by the colony-forming assay, PB28 exerted a dose-dependent inhibitory effect on the viability of both cancer cell lines. Neither of the two cell lines was resistant to PB28 (786-O:p = 0.038, ACHN:p = 0.031). Sigma-2 receptor antagonist 1 was applied to treat 786-O and ACHN cells. Clonogenic assays were used to measure the impact of cell density on the colony-forming ability of 786-O and ACHN cells. After 8–10 days of incubation with sigma-2 receptor antagonist 1, colony formation was significantly augmented (Figure 1B) (786-O:p = 0.0249, ACHN:p = 0.0153). As for RCTA analysis, PB28 exhibited a dose-dependent inhibitory effect on the viability of 786-O and ACHN cells Figure 1C, p < 0.01). While 1000nM concentration of sigma-2 receptor antagonist 1 increased the proliferative activity of both 786-O and ACHN cells ( Figure 1D, p < 0.01).

3.2 PB28 Inhibited the Invasion and Migration of Renal Cancer Cells, while Sigma-2 Receptor Antagonist 1 Promoted These Activities

Tumour invasion is one of the main reasons for poor prognosis in patients with cancer. We investigated the potential role of PB28 and sigma-2 receptor antagonist 1 in modulating the invasion and migration abilities of renal cancer cells by the transwell invasion 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 (10 μM) of PB28 significantly inhibited the migration of 786-O cells and ACHN cells, respectively. Transwell invasion assays with Matrigel showed that the numbers of invading 786-O and ACHN cells were increased after treatment with 300 nM sigma-2 receptor antagonist 1 (Figure 3A). By contrast, sigma-2 receptor antagonist 1 significantly accelerated the migration of 786-O cells and ACHN cells at a low concentration (300 nM) (Figure 3B).

3.3 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.

3.4 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). Nevertheless, PB28 had no effect on the PI3K-AKT-mTOR signalling pathway in HK-2 cells (Figure 6D).

3.5 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, 786-O, Caki-1 and ACHN. The cells were incubated with cisplatin in the presence or absence of PB28 for 24 h. Cell viability was evaluated by a CCK-8 assay, and the half-maximal inhibitory concentration (IC50) of cisplatin (with or without PB28) was also examined. The IC50 values of cisplatin were as follows: 786-O, 2.387 μg ml⁻¹ (the result was not presented); Caki-1, 17.1 μg ml⁻¹; and ACHN, 30.54 μg ml⁻¹. We speculated that 786-O cells were more sensitive to cisplatin and that Caki-1 and ACHN cells were resistant to cisplatin. Therefore, we selected Caki-1 and ACHN cells for the following experiment. We added a low concentration of PB28 (Caki-1: 10 μM, ACHN: 20 μM) to the medium of the two cell lines. After a 24 h incubation, the CCK-8 results showed a sharp drop in the IC50 value of cisplatin in Caki-1 (6.86 μg ml⁻¹) and ACHN (17.28 μg ml⁻¹) cells (Figure 7). We hypothesized that PB28 could enhance cisplatin efficacy in renal cancer cells with few side effects.

4 DISCUSSION

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.²³Sigma receptors and their ligands function in processes beyond those related to cell differentiation and survival, including cholesterol metabolism, cancer cell growth and invasion.^17–19 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.^18–20 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, 22 Based on our experimental results, 786-O and ACHN cells cultured with PB28 exhibited a reduction in proliferation, migration and invasion compared with those in control cells. Therefore, we hypothesized that TMEM97 plays a vital role in the development of renal cancer, PB28 might serve as a new therapeutical agent for renal cancer treatment. Then, sigma-2 receptor antagonist 1 was added to cultured renal cancer cells. According to the matrigel and migration experiments, the results indicated that sigma-2 receptor antagonist 1 promoted the invasion and migration of both 786-O and ACHN cells. Colony formation assay and RTCA assay were also selected to determine the effect of sigma-2 receptor antagonist 1 on renal cancer cells. Then, encouraging results were observed: the proliferative ability of 786-O and ACHN cells increased significantly with sigma-2 receptor antagonist 1 treatment. Therefore, the proliferative, migratory and invasive abilities of renal cancer cells showed an increasing trend when sigma-2 receptor antagonist 1 was added to the cells. According to the results of above experiments, agonist and antagonist of sigma-2 receptor play diverse roles in renal cancer. We speculated that TMEM97 might act as a tumour suppressor during the development of renal cancer, and PB28 had a therapeutic effect on it.

Second, we had confirmed an effective treatment of PB28 in vitro on renal cancer cells. Meanwhile, a nude mice experiment also came to a similar conclusion. Both the volume of subcutaneous tumours and the number (scope) of lung metastasis were significantly decreased by PB28 in vivo. Taken together, in vitro and in vivo experiment results suggested a latent vital role of PB28 in renal cancer therapy. However, the mechanism was unknown. A study by Pati ML, et al. suggests that the mitochondrial superoxide pathway is activated by PB28 in the treatment of pancreatic tumours.²² Furthermore, PB28 induces P-gp down-modulation through the activation of the caspases enrolled in both extrinsic and intrinsic apoptotic pathways. It can be used for treatment of multiple myeloma.²⁵ Therefore, PB28 acted as an anticancer agent could induce cell death by different mechanisms. How about renal cancer?

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 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 first evidence of this type of drug reversal of multidrug resistance in 1997.²⁹ Furthermore, Francesco Berardi et al. demonstrated that PB28 reversed doxorubicin chemoresistance in breast cancer cells in a P-gp-dependent manner.²¹The above results indicated that sigma-2 agonists play a significant role in the resensitization of chemoresistant tumours. Hence, we chose three renal cancer cell lines, 786-O, Caki-1 and ACHN, to examine the IC50 value of cisplatin. As expected, we observed that Caki-1 and ACHN cells were resistant to cisplatin, with IC50 values of 17.1 μg ml⁻¹ and 30.54 μg ml⁻¹, respectively. Combined treatment comprising cisplatin and PB28 resulted 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.

5 CONCLUSION

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.