CYP19A1 Is Regulated by BRD4 and Suppresses Castration-resistant Prostate Cancer Cell Invasion and Proliferation by Decreasing AR Expression

Abstract


Background
Prostate cancer (PCa) remains one of the most common carcinomas in elderly men.It is the second leading cause of cancer-related deaths in Western countries [1].There are various effective methods for treating PCa, including androgen deprivation therapy (ADT) [2].As the rst line therapy for treating PCa, ADT is effective at the primary stage [3].However, patients with PCa will inevitably develop castrationresistant prostate cancer (CRPC) after ADT treatment for less than 2 years [4].When patients relapse into the castration-resistant stage, the median survival rate is less than 20 months [5].Moreover, there is still a lack of effective methods for treating CRPC.
Cytochromes P450 (CYPs) are one of the largest and most diverse superfamily of enzymes [6].CYPs are involved in the metabolism of endogenous and exogenous substances, including drugs, and environmental compounds.As a terminal oxygenase, they are involved in sterol hormone in living organisms [7,8].The CYP19A1 gene is a member of the CYP family, which can encode aromatase, a key enzyme that can catalyzes the conversion of androgen to estrogen [9,10].Therefore, the expression level of CYP19A1 can affect the testosterone level and may be correlated with the occurrence of CRPC.
However, the role of CYP19A1 in CRPC is still unclear.
As a nuclear protein, BRD4 can bind to histone-acetylated lysine residues via its bromo-domains, and further recruit transcription factors to activate downstream gene expression [11,14].JQ1 is a BRD4 inhibitor, which has been developed to target BRD4 and play an anti-tumor role [12,14,15].The function of JQ1 in treating PCa has been demonstrated.However, the use of JQ1 in treating CRPC requires further investigation.
Androgen receptor (AR) is one of the key factors that promote CRPC [16,17].In addition, as the main component of androgen, testosterone can impact AR expression and cause CRPC [18].In the prostate, when AR binds to androgen, the AR-androgen complex translocates to the nucleus.Once in the nucleus, AR binds to androgen response elements upstream of target genes, leading to DNA transcription, and inducing prostate epithelial cell proliferation [17,19].Although the mechanisms of CRPC are still unclear, the abnormal ampli cation of AR by testosterone and structure changes are the main underlying causes of CRPC [20].
In this study, we found that the expression of CYP19A1 was lower in CRPC specimens than in normal tissues.CYP19A1 overexpression decreased CRPC cell invasion and proliferation.In addition, the expression of CYP19A1 directly modulated AR expression, affecting cell invasion and proliferation.CYP19A1 could suppress AR expression because it could metabolize testosterone.Moreover, the expression of CYP19A1 was increased when CRPC cells were subjected to JQ1 treatment or BRD4 knockdown.Therefore, CYP19A1 may be a potential therapeutic target for treating CRPC.BRD4-CYP19A1-AR may be an oncogenic pathway in CRPC development.

Bioinformatic analysis
starBase (http://starbase.sysu.edu.cn/starbase2/index) is a public database that provides gene expression data for different tumors based on The Cancer Genome Atlas (TCGA).UALCAN (http://ualcan.path.uab.edu/home) is another online web tool that contains sequence data from the TCGA database.The Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/)database is supported by the NCBI, which is used to collect gene expression chip data.We obtained GSE21034 from GEO database, which had gene chip data from both prostate cancer specimens and para-cancerous samples.The Chinese Prostate Cancer Genome and Epigenome Atlas (CPGEA) (http://www.cpgea.com/), is a database that collects the sequence data of prostate cancer patients in China.We downloaded the data and analyzed the gene expression using R software (R version 4.0.3).

Tissue samples
CRPC samples and para-cancerous samples were collected at Tongji Hospital, School of Medicine, Tongji University.The methods used for collecting the samples were approved by the Ethics Committee of Tongji Hospital, School of Medicine, Tongji University (SBKT-2021-220).Patients who provided the samples were familiar with the process of the experiment and gave informed consent.

Cell culture and drug treating
Prostate cell lines were purchased from the Chinese Academy of Science Cell Bank (Shanghai, China).The human normal prostate epithelial cell line RWPE-1 and human prostate cancer cells LNCaP, 22Rv1, and C4-2 were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma, Darmstadt, Germany ) with 10% fetal bovine serum (FBS) ( Gibco, Thermo Fisher Scienti c, Waltham, MA, USA ).The human prostate cancer cell VCaP was cultured in Dulbecco's modi ed Eagle's medium (DMEM) (Sigma) with 10% FBS.All cell lines were cultured in a humid environment with 5% CO 2 and 95% air at 37°C.JQ1 was purchased from SelleckChem (S7110).The prostate cancer cell lines were treated with different drug concentrations following the manufacturers' instructions.In addition, prostate cancer cells was cultured in culture medium without androgen.

Cell transfection and lentivirus production
Cell transfection assays were performed with Lipofectamine 2000 (Thermo Fisher Scienti c) and polyethylenimine (PEI) (Sigma-Aldrich, St. Louis, MO USA) following the manufacturer's instructions.The shRNA lentivirus was constructed for the knock-down of speci c gene.The shRNAs were purchased from Youze Biotechnology Company (Guangzhou, China).The lentivirus packaging plasmids PSPAX2 and PMD2.G, together with shRNAs for speci c genes, were transfected into 293 T cells with PEI.The culture medium was replaced after 24 h of transfection, and the medium was collected after another 48 h of culture.Then the medium was added to 22Rv1 and C4-2 cells.The shRNA sequence is shown in Table S1.

Cell invasion assay
After 48 h of transfection, the transfected 22Rv1 and C4-2 cells were digested and seeded into the upper chambers (N = 1*10 5 ) with a non-coated membrane and 200 µl 1640+ 2% FBS; 500 µl of 1640 medium +10% FBS was added to the lower chambers.After 48 h, cells on the upper lters were gently removed with a cotton swab.Cells that migrated to the lower chambers were xed with carbinol for 30 min.Then, the cells were washed three times to remove the carbinol.The chambers were stained by crystal violet for RNA extraction and qRT-PCR RNA was isolated from tissue samples and cell samples with TRIzol reagent (Sigma-Aldrich) according to the manufacturer's instructions.The RNA was reversed-transcribed to cDNA using a reverse transcription kit (Advantage® RT-for-PCR Kit, Takara Bio Inc., Kusatsu, Japan).qRT-PCR was performed using Applied Biosystems 7500 Sequence Detection System with qRT-PCR reagents and a kit (TB Green® Premix Ex Taq™ II, Takara Bio Inc.) according to the manufacturer's instructions.GAPDH was used as the normal control (NC).RNA expression was quanti ed according to the 2 −ΔΔCt method.The forward and reverse primer sequences are shown in Table S2.

Western blot
Total proteins from samples and cell lines were extracted with RIPA lysis buffer.Protein samples were treated with Dual Color Protein Loading Buffer (Thermo Fisher Scienti c).The proteins were separated on SDS-PAGE gels (7.5%, 10%), followed by transfer to nitrocellulose membranes (Merck KGaA, Darmstadt, Germany).Protein-Free Rapid Blocking Buffer (Thermo Fisher Scienti c) was used to block the membranes.Then, the membranes were incubated overnight at 4°C with primary antibodies against CYP19A1 (1:1000), AR (1:1000), BRD4 (1:1000), and GAPDH (1:1000) (Abcam UK, Cambridge, UK).On the next day, 1×TBST was used to wash the membranes three times (10 min, each).Then, the membranes were incubated at room temperature for 1 h with a matched secondary antibody (HRP-labeled Goat Anti-Human IgG (H+L), Beyotime Biotechnology, Shanghai, China).Lastly, the membranes were exposed to Xray irradiation.

Exogenous testosterone culture and test
Testosterone was purchased from Beyotime (Beyotime Biotechnology, China) and added (1 nmol/ml) to the culture medium.At 0, 2, 48, and 72 h of culture, the testosterone in the cells was analyzed by using the Testosterone ELISA Kit ((Beyotime Biotechnology, Shanghai, China) S according to the manufacturer's instructions.The absorbance was measured at 450 nm using a multi-mode reader (LD942, Beijing, China).

Statistical analysis
The data represent the results of at least three independent experiments.The results are shown as the mean ± standard deviation (SD).Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test for multiple comparisons or Student's t-test for comparison between two groups.A P value less than 0.05 was considered statistically signi cant.

CYP19A1 downregulation in PCa and CRPC specimens
To examine the expression of CYP19A1 in PCa, we analyzed the CPGEA, GEO, and TCGA databases.We found that the mRNA level of CYP19A1 was signi cantly downregulated in Chinese patients (Figure 1A).The result was similar for GSE21034, which included 130 tumor and 18 normal sample tissues (Figure 1B).Similar results were also obtained using the TCGA database.We found that CYP19A1 was downregulated in PCa samples compared with normal samples in the starBase and UALCAN databases (Figure 1C-D).Next, to investigate the expression of CYP19A1 in CRPC specimens, we collected both CRPC tissue samples and para-cancerous normal tissues at our hospital.Similar to the results using the GEO, CPGEA, and TCGA, the expression of CYP19A1 was downregulated at both the mRNA and protein levels (Figure 1E-F).In addition, we determined the expression of CYP19A1 in the normal prostate epithelial cell line RWPE-1 and PCa cell lines.We found that CYP19A1 was downregulated in PCa cell compared with RWPE-1.In addition, CYP19A1 was lower in non-androgen-dependent PCa cells (Figure 1G-H).The results demonstrated that CYP19A1 was downregulated in PCa and CRPC samples.

Effect of CYP19A1 on CRPC cell invasion and proliferation in vivo and vitro
As CYP19A1 was downregulated in PCa and CRPC specimens, we hypothesized that CYP19A1 may prevent CRPC occurrence.To verify our hypothesis, we constructed a CYP19A1 overexpression (OE) plasmid and CYP19A1 knockdown (shCYP19A1) lentivirus.We transfected the OE plasmid and shCYP19A1 lentivirus into 22Rv1 and C4-2 prostate cancer cells, which are CRPC cell lines that can grow without androgen [21,22].After transfection, the effect of transfection and the function of CYP19A1 in cell invasion and proliferation ability were examined.We found that after transfection with the OE plasmid, the expression of CYP19A1 was upregulated at both the mRNA and protein levels in 22Rv1 and C4-2 cells (Figure 2A-B).On the other hands, after transfection with the shCYP19A1 lentivirus, the expression of CYP19A1 was markedly downregulated in 22Rv1 and C4-2 cell at both mRNA and protein level (Figure 2C-D).As expected, CYP19A1 could affect cell invasion and proliferation.When transfected with CYP19A1 OE plasmid, invasion ability was decreased for both 22Rv1 and C4-2 cells.However, after transfection with the CYP19A1 knockdown lentivirus, the invasion ability of 22Rv1 and C4-2 cells was increased (Figure 2E-F).In addition, cell growth was slower after 22Rv1 and C4-2 cells were transfected with the CYP19A1 OE plasmid.On the other hand, when transfected with the shCYP19A1 lentivirus, the cells showed faster growth (Figure 2G-H).We injected stable shCYP19A1 and shControl 22Rv1 cells subcutaneously into NOD-SCID mice.Based on the in vivo results, shCYP19A1 increased tumor size and tumor growth in vivo (Figure 2I-K).Collectively the results indicated that CYP19A1 could suppress CRPC cell invasion and proliferation.

Tumor suppressive function of CYP19A1 via decreased AR expression
AR is well known to play an oncogenic role in the occurrence of PCa and even CRPC.Moreover, AR plays an essential role in the resistance to anti-androgen therapy.As an important member in the androgen metabolic pathway, AR is essential for the development of PCa and CRPC [23][24][25].As mentioned above, CYP19A1 can encode aromatase, which can catalyze the conversion of androgen to estrogen.Therefore, CYP19A1 may regulate the expression of AR by metabolizing testosterone.We hypothesized that there may be a correlation between CYP19A1 and AR.
To investigate our hypothesis, we observed the expression of AR after 22Rv1 and C4-2 prostate cancer cells were transfected with the CYP19A1 OE (oeCYP19A1) plasmid or CYP19A1 knock-down (shCYP19A1) lentivirus.After transfection with the oeCYP19A1 plasmids, the expression of AR in 22Rv1 and C4-2 cells was decreased at both the mRNA and protein levels (Figure 3A-B).We found that the expression of AR was increased following CYP19A1 knockdown (Figure 3C-D).The results indicated that CYP19A1 could affect the expression of AR.Subsequently, we investigated whether CYP19A1 suppresses cell invasion and proliferation by regulating AR.We constructed a shAR lentivirus and found that the shRNA lentivirus can inhibit the expression of AR at both the mRNA and protein levels truly (Figure 3E-F).Following CYP19A1 knockdown, 22Rv1 and C4-2 cell invasion was increased as shown in the results above.Likewise, cell invasion ability was markedly reduced after the shAR lentivirus transfected into 22Rv1 and C4-2 cells.However, there was no difference between shCYP19A1+shAR and shAR for both 22Rv1 and C4-2 cells (Figure 3G-H).Similar results were also obtained for cell proliferation.In comparison with NC cells, shCYP19A1-transfected 22Rv1 and C4-2 cells grew faster.With shAR transfection, the proliferative ability of 22Rv1 and C4-2 cells was markedly decreased compared with that of NC cells.However, the proliferative ability of CRPC cells was not signi cantly different between shCYP19A1+shAR and shAR (Figure 3I-J).
Taken together, the results demonstrated that CYP19A1 could affect cell invasion and proliferation by targeting AR.

CYP19A1 mediated androgen-dependent regulation of AR expression
The main function of CYP19A1 in the human body is to metabolize androstenedione and testosterone.The level of testosterone can affect the expression level of AR [26].Therefore, we hypothesized that the regulation of AR by CYP19A1 attributed to the metabolism of testosterone.To investigate the hypothesis, we added exogenous testosterone to the culture medium without androgen.
First, we measured the testosterone level in 22Rv1 and C4-2 cells after transfection with the CYP19A1 OE (oeCYP19A1) plasmids and shCYP19A1 lentivirus.We added exogenous testosterone to the culture medium of the oeCYP19A1 group and oeControl group for both CRPC cell lines.After 48 h of culture, the level of testosterone was markedly lower in the cytoplasm of the oeCYP19A1 group compared with the oeControl group (Figure 4A-B).Following CYP19A1 knock-down by shCYP19A1 lentivirus, the level of testosterone was markedly increased (Figure 4C-D).Subsequently, we determined whether the testosterone level would change with the addition of exogenous testosterone to the culture medium of the oeCYP19A1 group.We found although CYP19A1could decrease the level of testosterone, the level of testosterone in the cytoplasm was not signi cantly different when exogenous testosterone was added in the culture medium (Figure 4E-F).To determine whether the change in AR expression in CRPC cells following CYP19A1 level alteration is associated with a change in testosterone level, we measured CYP19A1, and AR expression at both the mRNA and protein levels after exogenous testosterone was added in the oeCYP19A1 group.We found that testosterone increased the expression of CYP19A1, and AR at both the mRNA and protein levels.An increase in the level of testosterone reduced the effect of CYP19A1 on AR (Figure 4G-J).However, when we added dihydrotestosterone (DHT), a compound that cannot be metabolized by CYP19A1, to the culture medium, CYP19A1 did not affect AR expression (Figure 4K-L).
Taken together, the results showed that CYP19A1 could regulate the expression of AR by metabolizing testosterone in vitro.

Upregulation of CYP19A1 by suppressing BRD4 and modulating the sensitivity of the CRPC cell response to JQ1 treatment
The BRD4 protein has been found to play an important role in the occurrence of PCa.JQ1 as a BRD4 inhibitor may be used to treat PCa.However, the mechanism of JQ1 in the treatment of PCa requires further investigation.In a previous study, researchers found that some anti-cancer compounds can collectively affect the function of BRD4 and aromatase [27].BRD4 is involved in the regulation of downstream target gene expression [15].Therefore, we hypothesized that CYP19A1 may be a downstream target regulated by BRD4.
First, we examined the expression of BRD4 using the TCGA database.We found that BRD4 was upregulated in tumor tissues compared with normal tissues (Figure 5A).Then, we examined the correlation between BRD4 and CYP19A1 for GSE21034.We found that there was a negative correlation between BRD4 and CYP19A1(R = -0.32,P = 6.0e-05) (Figure 5B).To further con rmed the correlation between BDR4 and CYP19A1, we purchased a BRD4 inhibitor (JQ1) and constructed a shBRD4 lentivirus.We found that after 22Rv1 and C4-2 cells were treated with JQ1, the expression of CYP19A1 was increased at both the mRNA and protein levels; simultaneously, the expression of AR was decreased.In addition, the expression level of CYP19A1 was increased with a higher concentration of JQ1 (Figure 5C-F).BRD4 knockdown in 22Rv1 and C4-2 cells transfected with the shBRD4 lentivirus showed that the expression of CYP19A1 was increased at the both mRNA and protein levels (Figure 5G-I).Furthermore, when CYP19A1 overexpression or downregulated, the expression of BRD4 did not change (Figure S1A-D).The results indicated that BRD4 may be an oncogenic protein that contributes to PCa, and it may play a potential role in suppressing the expression of CYP19A1.Finally, we investigated whether the expression of CYP19A1 modulates the effect of JQ1.We treated shCYP19A1-transfected CRPC cells and NC cells with different concentrations of JQ1.We found that when CYP19A1 was downregulated, the cells were more resistant to JQ1.The IC50 value of JQ1 was increased from 8.92 µM to 16.74 µM for 22Rv1 cells and from 7.62µM to 14.86µM for C4-2 cells (Figure 5L-M).These results demonstrated that CYP19A1 could be regulated by BRD4, which could affect the sensitivity of CRPC cells to treatment with JQ1.
Taken together, CYP19A1 could be downregulated by BRD4, and its function could be reversed by JQ1.In addition, CYP19A1 could suppress CRPC cell invasion and proliferation by regulating AR (Figure 5N).

Discussion
PCa is one of the most common malignant tumors in elderly men.In the United States, the incidence rate of PCa is nearly 20%.It is also the second leading causing of death from cancer in men [1].In China, PCa is a serious health problem among men.According to the data from the National Cancer Center of China, the incidence and mortality rates of PCa have greatly increased [28].Androgen plays an important role in the occurrence of PCa and thus is a target in ADT.ADT is a rst line treatment for PCa [3].However, after a treatment period of 18-24 months, PCa patients will inevitably relapse into the castration-resistant stage [4].Patients with CRPC typically have a median survival time of no more than 2 years [5].Thus far, there is no effective method for treating CRPC, and the mechanism of CRPC is still unclear.
The androgen metabolic pathway is important in androgen synthesis and metabolism.It has been demonstrated that the abnormal function of the androgen metabolic pathway plays key role in the occurrence of CRPC [29].The androgen metabolic pathway consists of several components, which include cytochromes P450 (CYPs) [30].CYPs are one of the largest and most diverse superfamily of enzymes [6].CYPs are involved in the process of metabolizing various substances.As a terminal oxygenase, they are involved in sterol hormone synthesis in living organisms [7,8].The CYP19A1 gene is a member of CYPs, which can encode aromatase, a key enzyme that catalyzes the conversion of androgen to estrogen [9,10].Therefore, the higher the expression of CYP19A1, the lower the androgen level in serum.In addition, CYP19A1 has been demonstrated to play an important role in the occurrence of breast cancer and even affect patients' survival [31].Various drugs can inhibit the function of CYP19A1, such as Letrozole, have been used in clinical settings to treat breast cancer [32].PCa and breast cancer share many similarities; for example, they are both hormone-dependent neoplasms.In addition, CYP19A1 can metabolize testosterone to estrogen.Therefore, CYP19A1 may be correlated with the occurrence of CRPC and may be a potential therapeutic target for treating CRPC.
AR binds to androgen and has been found to play an important role in both hormone-dependent PCa and CRPC occurrence and development [33].AR is a member of the nuclear steroid receptor superfamily of transcription factors.It is located at Xq11-12 and contains eight exons that encode a protein of ~919 amino acids [34][35][36][37].Since 1995, there is a consensus that AR can regulate multiple cellular events such as proliferation, apoptosis, migration, invasion, and differentiation.AR mutation and ampli cation occur in PCa and CRPC.Moreover, AR expression changes in PCa with lymph node, visceral, and bone metastases [38,39].Apart from primary PCa, AR is also highly expressed and transcriptionally active in CRPC [25].Given its involvement, AR is a target in enzalutamide treatment [40].A series of studies with vitro models similarly showed increased AR expression and restoration of AR activity in tumors that relapsed after castration, and RNA interference and related approaches established that AR was required for growth in these CRPC models [41][42][43][44][45].
BRD4 has been reported to be overexpressed in various tumors including CRPC [11][12][13].As a carcinogenic factor, BRD4 can promote breast cancer cell invasion and migration by targeting the Jagged1/Notch1 pathway [13].Increased BRD4 expression is related to lung cancer lymph node metastasis, and the mechanism may involve the binding of BRD4 to RelA, an important component of the NF-κB complex, then leading to in ammatory responses [46].In addition, BRD4 can regulate the transcription of the oncoprotein c-Myc and in uence the downstream gene expression [15].JQ1, a BET inhibitor, has been demonstrated to function by targeting BRD4, which is a transcription factor that belongs to the BET family and has been reported to be a novel and important oncogenic protein in human prostate cancer [47,48].Moreover, JQ1 disrupts the BRD4-acetylated lysine interaction, further suppressing the transcriptional activity of BRD4 [15].Here, we found that both BRD4 and JQ1 could affect the expression of CYP19A1, which indicating that CYP19A1 may be a potential downstream gene of BRD4.
In this study, we found that the expression of CYP19A1 was lower in CRPC samples.In addition, CYP19A1 affected CRPC cell invasion and proliferation, which may be attributed to the regulation of AR by CYP19A1.Moreover, as CYP19A1 could metabolize testosterone, it could affect AR expression.We found that CYP19A1 expression could be in uenced by BRD4, and it could modulate the effect of JQ1 in CRPC.The results suggest that CYP19A1 may be a potential target for treating CRPC, especially when using BET inhibitors.

Conclusion
In conclusion, CYP19A1 may be one of the causes for the occurrence of CRPC.CYP19A1 may affect CRPC cell invasion and proliferation.In CRPC cells, CYP19A1 may target AR and may be regulated by BRD4.The expression of CYP19A1 may in uence cell sensitivity to BET inhibitors.Therefore, the BRD4-

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