Protective Role Of Estrogen Receptor Alpha In Bladder Cancer Invasion

Bladder cancer (BCa) is the second most common malignancy tumor of the genitourinary system. The incidence of BCa increases with age among men and women. The disease hit about three times as many in men as women, including estimates of50,000men and17,000women in the United States in2012. Scientists pointed out that factors like greater exposure of men to cigarettes, industrial hazards or urinary infection had been responsible. However, even in the absence of these risk factors, the differences of gender-related incidence persisted. Some scientists have put forward new ideas recently that sex hormones and receptors might play a role, but solid evidence has been minimal until now.Part Ⅰ Estrogen receptor a (ERa) inhibits BCa cell invasionObjective:To study the relationship between ERa and BCa cell invasion.Methods:BCa cells were introduced either an ERa or empty vector plasmid. After5days puromycin selection, infection efficiency was tested by immunofluorescence(IF). Protein was collected both ERa and vector group to further confirm the efficiency by western blot. We also applied siRNA to knockdown ERa(shERa) in BCa647v cells. Invasion assay was performed using matrigel pre-coated transwell plates for24h by using three ERa negative cell line (T24, UMUC3and J82) and ERa positive cell line (647v).3D invasion assay was used to further confirm the BCa cell invasion ability.Results:1. Successfully established overexpress ERa and shERa BCa cells.2. We applied the transwell system to study ERa influence on BCa cell invasion. Addition of ERa in BCa T24cells led to suppress cell invasion. Similar results also obtained when we replaced T24cells with another two BCa UMUC3and J82cells. Otherwise, shERa in BCa647v cells led to enhance BCa cell invasion.3. Similar results were obtained when we applied2nd different invasion assay using3D invasion assay, showing adding ERa in UMUC3cells or knocking down ERa in647v cells led to suppress or enhance BCa cell invasion, respectively.Conclusions:Results from cell invasion assay and3D invasion assay clearly concluded that ERa might play protective roles to suppress BCa cell invasion. Part Ⅱ ERa inhibits BCa invasion in vivoObjective:To generate ERa knockout (ERaKO) mice and study the relationship between ERa and BCa invasion in vivo.Methods:To generate ERaKO mice, we mated floxed ERa homozygous male mice with CMV-Cre transgenic female mice to obtain the CMV-Cre/ERafl/+mice (F1). Floxed ERa homozygous male mice were then mated with female CMV-Cre/ERαfl/+mice (F1) to obtain ERαfl/+(wild type, WT) and CMV-Cre/ERafl/fl (ERaKO) mice (F2). Genotyping of tail snips to confirm the ERa gene deletion in the ERaKO mice. WT mice and ERaKO mice were fed0.05%N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) from6weeks to18weeks. Hematuria was tested at28and38weeks and bladders were harvested at30and40weeks in male and female, respectively. Invasive status was compared by Hematoxylin and Eosin (H&E) staining in these two groups. Survival rate was recorded for30and40weeks of males and females and presented by Kaplan-Meier survival curves.Results:1. We successfully generated ERaKO mice by mating floxed ERa male mice with female mice expressing Cre driven by the CMV promoter, and genotyping confirmed the ERa gene deletion in the ERaKO mice.2. ERaKO mice had more severe hematuria than those WT littermates. Importantly, Histological examination by H&E staining showed that ERaKO mice had more invasive tumor compared to WT mice. The invasion phenotype was characterized by cancer invasive to smooth muscle.3. Kaplan-Meier survival curves show that survival rate was lower in ERaKO mice compared with the WT mice.Conclusions:ERaKO mice developed more invasive tumor and predicted a worse prognosis than WT mice after induction of BCa by BBN.Part III ERa inhibits BCa invasion through downregulation of Insulin-like growth factors1(IGF1)/Matrix metalloproteinase2(MMP2) axisObjective:To reveal the mechanism of ERa suppressing BCa invasionMethods:1. To dissect the mechanism by which ERa was able to exert its effects, we first screened invasion related genes by qPCR array in UMUC3cells stably transfected with ERa or vector control and found IGF1and MMP2changes in the ERa positive cells.2. We then confirmed these initial screening results with qPCR analysis in T24and647v cells.3. UMUC3vector/ERa cells were treated with IGF1(lOng/ml,8h). After treatment, we collected protein from four groups (vec, ERa, vec+IGF1, ERa+IGF1) to test the MMP2expression by western blot and performed invasion assay in vec, ERa and ERa+IGF1groups and quantification.4. shERa647v cells were treated with MMP2inhibitor, ARP-100(50μM,12h). Invasion assays were performed in shGFP, shERa and shERa+ARP-100groups and quantification.5. The tumor tissues of WT and ERaKO mice were stained by immunohistochemistry (IHC) to confirm expression of ERa, IGF1, and MMP2.Results:1. The results of screening invasion related genes by qPCR in UMUC3cells showed decreased mRNA expression of IGF1and MMP2in the ERa positive cells.2. We then confirmed these initial screening results with qPCR analysis at mRNA level showing adding ERa into T24cells led to suppress IGF1and MMP2expression. As expected, knocking down ERa in647v cells resulted in increased IGF1and MMP2expression.3. Adding IGF1in UMUC3vector/ERa cells resulted in an increase of MMP2and showed a more invasive phenotype comparing with non-treated ERa expressed UMUC3cells.4. Decreasing MMP2expression with a specific MMP2inhibitor, ARP-100, led to a reversal of the shERa induction role in647v cell invasion.5. ERa was mainly detected in the WT mice with barely detectable ERa in ERaKO mice, which indicated that ERa was selectively ablated in ERaKO mice. In ERaKO mice, IGF1and MMP2were seen in a greater quantity and with stronger intensity than WT mice. Conclusions:Molecular mechanism dissection revealed ERa could inhibit BCa invasion through selectively downregulating IGF1to regulate MMP2.Part IV Therapeutic effect of ERa agonist propylpyrazole triol (PPT) in vitro and in vivoObjective:To study the therapeutic effect of ERa agonist PPT in vitro and in vivo.Methods:1. For in vitro part, the BCa647v cells were pre-treated for3days with the fresh10%charcoal dextran fetal bovine serum media (CD-FBS) and then DMSO or10nM ERa agonist PPT was added for24h incubation. Invasion assay was performed using matrigel pre-coated transwell plates for24h.2. In vivo, female B6mice (n=52) were treated by0.05%BBN from8weeks to20weeks.52mice were randomly divided into two groups and treated with DMSO/PPT since32weeks.50μl of DMSO/PPT (2x10-4M) were intraperitoncally injected for each mouse every the other day. All mice were euthanized at60days after DMSO/PPT treated and bladders were performed H&E staining to compare invasion status. Survival rate was recorded for60days after DMSO/PPT treated and presented by Kaplan-Meier survival curves.Results:1. In vitro, we found BCa647v cells treated with10nM PPT led to suppress BCa cell invasion.2. In vivo, we euthanized all the mice in the60th days after DMSO/PPT injection since hematuria appears severe. We found PPT group developed less invasive tumor. Kaplan-Meier survival curves showed the tendency that survival rate was lower in DMSO group compared with the PPT treated group (no significant differences).Conclusions:ERa agonist PPT can suppress BCa invasion in BCa647v cells and in BBN induced mouse model.