The Role And Mechanism Of EMP1 Deficiency In Promoting Bladder Cancer Metastasis

BackgroundBladder cancer(Bca)is one of the most frequently diagnosed tumors worldwide and accounts for approximately 3% of new cancer cases and approximately 2.1% of deaths due to tumors each year.According to the latest tumor incidence and mortality statistics,Bca is the eighth and eleventh most prevalent tumor in the Chinese male population respectively,and has not improved significantly in the past 10 years.Bca is a heterogeneous epithelial malignancy characterized by progression along two distinct pathways.Most epithelial tumors are thought to progress along a single pathway from benign to malignant.However,clinical and pathological studies indicate that bladder cancer arises by at least two separate mechanisms.The non-muscle-invasive bladder carcinomas(NMIBCs)are generally considered less life-threatening,but they have a high risk of recurrence and progression,for which patients need lifelong surveillance imposing a heavy financial burden on them.The muscle-invasive Bca(MIBC)is the primary cause of mortality due to urothelial bladder cancer,and after metastasis,the 5-year survival rate significantly reduces to approximately 5.5%.Despite advances in clinical treatments,the prognosis of such patients remains challenging.Hence,exploring the possible mechanism of carcinogenesis and metastasis in bladder cancer is pivotal to developing new therapeutic strategies.Metastasis is the leading cause of death induced by bladder cancer.However,successfully full-blown metastasis is a challenging event.Cancer cells need to overcome multiple environmental hurdles during the metastatic cascade.The development of effective metastatic barriers and methods for reducing tumor migration and invasion has become important aspects of global metastasis research.Moreover,passing of metastasizing cells through the bloodstream is one of the most inefficient steps because of oxidative stress induced apoptosis and anoikis.A recent study further investigated the exact mechanisms through which oxidative stress clears metastasizing cells;they observed that ferroptosis,a mode of cell death caused by lipid oxidation,is how tumor cells get clear circulatory system.In addition,that metastasizing cancer cells adapt their metabolism during the metastatic cascade is also essential for cancer cells to successfully seed metastases from their primary site;Fatty acid metabolism,for example,has been reported involved in favoring many steps of metastatic cascade,including invading and migration,circulating and metastatic colonization.As a result,cell migration capability,resistance to ferroptosis,and fatty acid utilization are all factors that affect metastasis,on which this study will focus.PPARγ is a transcription factor known for its role in adipogenesis,fat metabolism and its functions in helping cells to resist oxidative and metabolic stress.Although it is also defined as a tumour suppressor in other types of tumors because of inhibiting cell growth and survival,accumulating studies reported that PPARγ might promote cancer progression by keeping redox balance and promoting cell survival.Furthermore,Katherine L et al.have reported PPARγ high expression in HCC and blocking the function of PPARγ leads to anoikis-mediated apoptosis by preventing adhesion.In bladder cancer research,PPARγtends to thought to act as a proto-oncogene and is involved in differentiation in the urothelium and has displayed an activation signature in the luminal subtype.The above results suggest that PPARγ may play a role in the progression of bladder cancer metastasis.However,there are no studies to confirm the effect of PPARγ on bladder cancer metastasis.Additionally,we discovered that membrane protein 1(EMP1)negatively regulates PPARγ,although it is unclear whether EMP1 will thus regulate metastasis of bladder cancer.EMP1 is a family member of epithelial membrane proteins(EMP1–EMP3).These proteins are hydrophobic and have four predicted transmembrane domains,consisting of two extracellular domains with N-glycosylation sites and two intracellular regions.Multiple studies involving EMPs and tumors have revealed their critical role in disease progression,and the exact role each EMPs plays is tumor-type specific.For example,EMP1 can enhance prostate cancer progression by activating a tyrosine kinase Src signaling cascade,and similar effects are also observed in other type of tumor,including breast cancer and colorectal adenocarcinoma.On the other hand,EMP1 has been reported to function as a TSG in nasopharyngeal and gastric cancers as it inhibits cell growth and metastasis by inducing apoptosis and blocking angiogenesis.The above results suggest that EMP1 has tumor type-dependent regulatory effects on metastasis in a variety of tumors,and there are no studies suggesting its role in bladder cancer metastasis.In conclusion,it is necessary to elaborate on the role of EMP1 in Bca metastasis.ObjectiveThe aim of our study was to investigate the role of EMP1 and PPARγ in bladder cancer metastasis.In addition,we provide insight into the specific mechanisms through which EMP1 exerts its effects on the altered biological behavior of bladder cancer cells.We hope that the conclusion of our study will contribute more valuable information in bladder cancer treatment.Methods and results1.EMP1 is downregulated in bladder cancer.We first explored the endogenous EMP1 expression level from the perspective of pan-cancer by in silico study.Data from the TCGA databases indicated that low expression of EMP1 was detected in most types of cancers.Next,we evaluated EMP1 expression based on data acquired from NCBI GEO.All data indicated that low expression of EMP1 was detected in bladder cancers.By analyzing the expression of EMP1 in MIBC,we found that the expression of EMP1 was lower in the subgroups related to tumor progression.2.The deficiency of EMP1 promotes BCa cell proliferation and metastasisTo further explore the role of EMP1,a gain/loss-of-function strategy by lentivirus-mediated EMP1 gene overexpression/knockdown was conducted.The real-time cell analyzer(RTCA),colony formation assays and tumor xenograft models showed that the knockdown of the EMP1 promoted the proliferation of Bca cells.The wound healing and transwell assays showed that the knockdown of the EMP1 promoted the migration of Bca cells.By CCK-8 assasys,we found that the sensitivity of the Bca cell lines to Erastin was considerably increased with the knockdown of the EMP1.ROS probes combined with flow cytometry revealed that EMP1 deficiency significantly increased the resistance to oxidative stress in Bca cells.By intravenous injection of luciferase labeled tumor cells in immunocompromised mice,we affirmed that knockdown of EMP1 in Bca cells promoted its metastatic capacity significantly.3.EMP1 deficiency activates PPARγ signallingTo identify the mechanism by which EMP1 influences cell motility and redox homeostasis,we carried out RNA-seq to assay the transcriptomes of overexpressed EMP1 and EMP1-knockdown Bca cells.Gene set enrichment analysis(GSEA)was performed to investigate the downstream mechanism.We selected PPARγ pathway as a possible downstream pathway.Validation is then performed and data from q PCR,western blotting and Oil red O staining assay showed the increased level of reporter of the PPARγ pathway activation.Altogether,these results demonstrate that EMP1 knockdown activated the PPARγ pathway and increased the expression of PPARγ in Bca.4.PPARγ contributes significantly to EMP1 deficiency-induced promotion of migration and ferroptosis resistance via p FAK(Y397)and SLC7A11To evaluate the role of PPARγ in the EMP1 deficiency-driven progression of BCa cells,we knockdown PPARγ by si-RNA and found that the downregulation of PPARγ reduced all migration,ferroptosis resistance,and anti-oxidative stress ability conferred by knockdown of EMP1,indicating that EMP1 deficiency dependent tumor progression role is mediated by regulating PPARγ.Next,we explored the mechanisms through which PPARγ mediates these alterations in cell biological behavior.We identified the EMP1-PPARG-p FAK signaling axis and the EMP1-PPARG-SLC7A11 signaling axis as the mechanisms of PPARG regulation of bladder cancer cell migration and resistance to ferroptosis.5.PPARγ is involved in the regulation of migration and resistance to ferroptosis in bladder cancer cells by FABP4By transwell migration experiment,we found that PPARγ activation with Rosiglitazone further enhanced EMP1 deficiency-induced migratory ability.To ascertain wheter the enhancement of rosiglitazone may be conferred by FABP4,we knockdown FABP4 by si-RNA in EMP1 deficient,rosiglitazone treated tumor cells and found that all groups showed a decreased migration after the intervention of FABP4 compared to its control.we also found significantly increased intracellular ROS productions after si-FABP4 in these groups.In 5637 cells,FABP4 also plays a negative regulatory role.By interfering with FABP4 to affect the expression of PPARγ,cell migration ability and anti-ferroptosis ability were changed accordingly,indicating that PPARγ plays an important role in cell migration and ferroptosis resistance.ConclusionIn the current study,we provided evidence that EMP1 is downregulated in BCa.The deficiency of EMP1 promotes BCa cell migration and enhances its capacity to resist ferroptosis in vitro and cancer metastasis in vivo.Furthermore,we showed that EMP1deficiency-induced phenotype is mediated by PPARγ.By increasing the expression of p FAK(Y397)and SLC7A11,PPARγ promotes cancer cell migratory and anti-ferroptotic cell death capacities.Moreover,we found that inhibition of EMP1 expression sensitized cells to PPARγ’s ligand,which effect are metastatic phenotype promoted and could be reversed by FABP4 knockdown.Together,these findings reveal the unreported role of EMP1-mediated PPARγ in bladder cancer metastasis.