Autophagy Inhibition in BPH Epithelial Cells Promotes Stromal Proliferation via FGF6 Secretion Through the AKT/ERK Pathway

Autophagy Inhibition in BPH Epithelial Cells Promotes Stromal Proliferation via FGF6 Secretion Through the AKT/ERK Pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Autophagy Inhibition in BPH Epithelial Cells Promotes Stromal Proliferation via FGF6 Secretion Through the AKT/ERK Pathway DongDong Fan, Zhanliang Liu, Mingxin Jiang, Jie Ming, Song Jin, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9214223/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 19 You are reading this latest preprint version Abstract Benign prostatic hyperplasia (BPH) is a progressive disease in elderly men, mechanism of which has not been unknown. The aim of this study was to explore how autophagy inhibition in BPH epithelial cells influences Benign prostatic hyperplasia (BPH) progression. We found that the autophagy level in epithelial cells of stromal hyperplasia(SH) is markedly reduced,compared with glandular epithelial hyperplasia (EH), Paraffin-embedded prostate specimens were classified into two groups:stromal hyperplasia (SH)and glandular epithelial hyperplasia (EH),those proteins was evaluated via immunohistochemistry. Chloroquine (CQ) was employed to suppress autophagy of BPH-1 cells. Conditioned media (CM) were harvested and utilized for treating the WPMY-1 cell line and primary prostatic stromal cells (PPrSCs). CCK8 assays were employed to assess cells proliferation.qPCR and ELISA were utilized quantify cytokines and inflammatory factors. Treatment of WPMY-1 and PPrSC cells with CM from autophagy-inhibited BPH-1 cells significantly enhanced their proliferation. FGF6 levels were significantly elevated in CM from BPH-1 cells treated with CQ. Recombinant FGF6 protein stimulated proliferation in WPMY-1 and PPrSC cells and The Akt and ERK signaling pathways were activated following treatment with BPH-1 CM or recombinant FGF6, an effect that could be reversed by FGF6 neutralizing antibodies. The expression level of FGF6 in the SH group was significantly higher than that in the EH group, Meanwhile, in the SH group, the expression of FGF6 was negatively correlated with the expression of LC3.Autophagy suppression in prostate epithelial cells results in the secretion of substantial amounts of FGF6, that promote fibroblast proliferation and accelerating BPH progression. autophagy fibroblast growth factor 6 epithelial cell fibroblast proliferation benign prostatic hyperplasia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Benign prostatic hyperplasia (BPH) is a progressive condition predominantly affecting elderly men, yet the factors driving its acceleration remain largely enigmatic. Our prior research identified a subset of BPH samples characterized by aberrant stromal hyperproliferation and collagen deposition, and the level of autophagy in the epithelial cells of these tissues were significantly low. Recent advancements have underscored the pivotal role of autophagy in the development and progression of BPH, that is the most prevalent condition among elderly men[ 1 , 2 ], with an incidence rate of 30% in men over 40 years of age and exceeding 70% in those over 70 years[ 3 , 4 ]. BPH is frequently associated with lower urinary tract symptoms (LUTS), severely compromising quality of life due to dysuria, nocturia, urinary retention, and related complications[ 2 – 5 ]. The annual healthcare expenditure attributed to this condition is substantial, imposing a significant financial burden on both patients and society. As the global population ages, BPH is poised to become an increasingly critical public health concern. BPH is characterized by a chronic, progressive proliferation of prostatic glandular and stromal cells, with stromal cells playing a particularly pivotal role in disease progression[ 2 ]. However, the etiology of BPH remains poorly understood. In clinical practice, we have observed that young patients with BPH progression rapidly. Clinically, 8% of BPH patients exhibit rapid progression, necessitating surgical intervention before the age of 50[ 6 ]. However, the mechanisms of BPH progression remain unclear. Studying the mechanisms behind BPH progression in these patients may help us identify key breakthrough points in the advancement of BPH. To identify factors that drive BPH progression, our research team has investigated starting from patients with early-onset BPH[ 7 ]. Autophagy is a self-digestive process that is conserved in eukaryotic cells and responsible for maintaining cellular homeostasis through proteolysis. By this process, cells break down their own components in lysosomes. In recent years, research on autophagy in BPH has gained significant momentum, with accumulating evidence indicating its crucial role in the onset and progression of BPH. We observed that early-progressed BPH with prominent stromal hyperplasia was associated with autophagy suppression in the epithelial cells compared to those with prominent glandular epithelial hyperplasia. Recent studies have highlighted the significant role of autophagy in BPH development and progression[ 8 – 10 ]. However, the impact of autophagy inhibition in the BPH epithelium on disease progression remains unclear. Autophagy has been shown to regulate a wide array of proinflammatory cytokines, which can influence neighboring cells[ 11 – 15 ]. Among the various factors implicated in BPH progression, cytokines or growth factors involved in epithelial-mesenchymal interactions are particularly relevant[ 16 – 19 ]. Thus, we hypothesized that autophagy inhibition in BPH epithelial cells might affect stromal cells through the secretion of specific factors. Materials and methods Patient Specimens A total of 42 prostate tissue samples from BPH patients were collected and subsequently classified into two groups based on hematoxylin-eosin (HE) staining: those predominantly characterized by stromal hyperplasia (SH, n = 25) and those predominantly characterized by glandular epithelial hyperplasia (EH, n = 17). Patients with prostate cancer, prostatitis, or those receiving alpha-adrenergic receptor antagonists or 5α-reductase inhibitors were excluded. After obtaining informed consent from all participants and approval from the institutional review committee of Beijing Friendship Hospital, retrospective clinical data analysis and tissue collection were conducted. Cell Culture Huan primary prostatic stromal cells (PPrSCs) were isolated from BPH tissues obtained via transurethral resection of the prostate (TURP). Fresh prostate tissues were dissected into small fragments, and primary stromal cells were cultured as previously described.[ 20 , 21 ] PPrSCs were maintained in RPMI-1640 medium (SH30809.01B, HyClone, South Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, MA, USA) and 1% penicillin-streptomycin at 37°C in a humidified incubator with 5% CO2. After six passages, PPrSCs were utilized for subsequent experiments. The BPH-1 cell line was procured from Keygen Biotech (KG1008, Nanjing, China), and the BPH stromal cell line WPMY-1 was obtained from the American Type Culture Collection (ATCC). These cells were cultured in RPMI-1640 medium (01-100-1ACS, Biological Industries, Israel) containing 1% streptomycin, 1% penicillin, and 10% fetal bovine serum (04-001-1ACS, Biological Industries, Israel) at 37°C with 5% CO2. Reagents and Antibodies The following antibodies were employed for immunohistochemistry (IHC) and Western blot analysis: rabbit anti-Beclin 1 (ab62557; Abcam, Cambridge, UK), rabbit anti-PCNA (13110), and anti-Cyclin D1 (55506) (both from Cell Signaling Technology, Denver, MA), anti-FGF6 neutralizing antibody (ab89549; Abcam, Cambridge, UK), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (60004-1-Ig) and anti-β-tubulin (66240-1-Ig) (both from Proteintech Group Inc, Chicago, IL), anti-AKT (4691), anti-phospho-AKT (4060), anti-ERK (4695), and anti-phospho-ERK (4370) (all from Cell Signaling Technology, Denver, MA). Recombinant human (rh) FGF6 (100 − 30) was sourced from Perprotech, USA. FGF6 Polyclonal Antibody (PA5-112421)(from Invitrogen,USA). Immunohistochemistry The expression levels of LC3, p62 and FGF6 were analyzed by IHC in continuous paraffin sections. BPH tissues obtained from TURP were fixed in 4% formalin buffer at 4°C overnight, dehydrated, and embedded in paraffin. Sections were cut at 5 µm thickness. After dewaxing and hydration, endogenous peroxidase was inactivated with 3% hydrogen peroxide. The primary antibody was incubated overnight at 4°C. A biotinylated secondary antibody was applied for 30 minutes at room temperature, followed by visualization using the peroxidase substrate DAB kit. Image J was used to calculate mean MOD to evaluate protein expression of IHC. Cell Proliferation Assay WPMY-1 and PPrSC cells were seeded in 96-well plates (800 cells per well) and incubated overnight. The medium was then replaced with a 1:1 mixture of conditioned medium and fresh phenol red-free medium, supplemented with FGF6 or 10% FBS and anti-FGF6 neutralizing antibody. Cells were cultured for 6 days. Cell growth was assessed using the Cell Counting Kit-8 (CCK-8) (CK04; Dojindo Molecular Technologies, Tokyo, Japan) on days 0, 2, 4, and 6, with absorbance measured at 450 nm using a Varioskan Flash spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Quantitative Real-Time PCR (qRT-PCR) Total RNA was extracted from tissues using TRIzol reagent (Invitrogen, Big Island, New York), followed by reverse transcription using an iScript Reverse Transcription Kit (BioRad). qRT-PCR was performed on the Bio-Rad CFX96 system with SYBR green to quantify the mRNA expression levels of the target genes. Enzyme-Linked Immunosorbent Assay (ELISA) Conditioned medium (CM) collected from BPH-1 cells cultured or treated with chloroquine (CQ) for 4 days was used to measure FGF6 secretion using the FGF6 Quantikine ELISA Kit (P10767, Raybiotech, GA) according to the manufacturer's protocol. Western Blotting Assay Cells were lysed in RIPA buffer containing 1 mM PMSF, 0.5% phosphatase inhibitors, and 1% protease inhibitors (KGP250, Keygen Biotech, China). Proteins were transferred onto nitrocellulose membranes. The membranes were blocked with Tris-buffered saline containing 5% skim milk powder for 1 hour. Primary antibodies were incubated overnight at 4°C in TBS-T. After washing three times with TBS-T buffer, secondary antibodies (Invitrogen) were applied for 1 hour at room temperature. Signals were detected using a Western blotting chemiluminescence reagent (P90719, Millipore, MA, USA). Statistical Analysis Data are presented as the mean ± SEM from at least three independent experiments. Statistical analysis was performed using one-way analysis of variance with SPSS 24.0 (SPSS Inc., Chicago, IL). Statistical significance was defined as p < 0.05. Results Autophagy of Epithelial Cells in Stromal Hyperplasia BPH is Significantly Reduced We collected samples tissue of BPH during TURP and performed HE staining on them (Fig. 1 ). Subsequently, we found that the stromal cells proliferation showed significant in stromal hyperplasia(SH) BPH tissues. Therefore, we divided the tissues into stromal cells proliferation and epithelial proliferation. The expression of LC3 in epithelial cells was significantly diminished in the EH group compared to the SH group (p = 0.0119), while p62 exhibited the opposite trend (p = 0.0156), indicating that autophagy in epithelial cells associated with stromal hyperplasia is markedly reduced. (Fig. 2 A-B). Compared to the EH group, autophagy was significantly reduced in BPH epithelial cells in the SH group. These findings suggest that autophagy suppression in epithelial cells may be associated with BPH progression. Autophagy Inhibition in BPH Epithelial Cells Promotes the Proliferation of BPH Stromal Cells To investigate the impact of epithelial cell autophagy on stromal cells, BPH-1 cell lines were treated with varying concentrations of chloroquine (CQ, 1 nM and 10 nM) to induce autophagy suppression, and the conditioned medium (CM) was collected to treat WPMY-1 and PPrSC cells. CCK8 assays revealed that CM from BPH-1 cells treated with 10 nM CQ significantly enhanced stromal cell proliferation (Fig. 2 C). Besides, western blotting showed the expression of proliferation markers Cyclin D1 and PCNA were significantly increased in both stromal cell types following CM treatment (Fig. 2 D). To exclude the possibility that CQ directly promotes cell proliferation, BPH-1 and WPMY-1 cells were treated with 10 CQ. The results showed that 10nM CQ could inhibit the proliferation of both cell lines. (Fig. 2 E). Collectively, these findings indicate that a reduction in autophagy in BPH epithelial cells may contribute to the proliferation of prostate stromal cells through the secretion of specific cytokines. FGF6 Plays a Pivotal Role in Promoting BPH Stromal Cell Proliferation via Autophagy Inhibition in BPH Epithelial Cells To explore which factor promotes the proliferation of BPH stromal cells by conditioned media, we first used q-PCR to detect various chemokines, inflammatory factors and growth factors in CQ-treated BPH-1 cells and found that expression of fibroblast growth factor 6 (FGF6) was significantly increased (Fig. 3 A-C). We then used ELISA to assess expression of FGF6 in the conditioned medium and found it to be increased significantly (Fig. 3 D). To investigate whether FGF6 promotes BPH stromal cell proliferation, we added human recombinant FGF6 directly to WPMY-1 cells and PPrSC cells. CCK8 assays detected that the proliferation levels of WPMY-1 cells and PPrSC cells treated with FGF6 were significantly increased (Fig. 4 A). Based on Western blotting, Cyclin D1 and PCNA levels were significantly increased in the two kinds of cells (Fig. 4 B). We then treated WPMY-1 cells and PPrSC cells with/without the addition of FGF6 neutralizing antibodies to the conditioned medium and used CCK8 assays to detect proliferation. Proliferation of WPMY-1 and PPrSC cells was significantly inhibited after adding the FGF6 neutralizing antibody (Fig. 4 C). Western blotting showed that Cyclin D1 and PCNA protein levels were also significantly reduced (Fig. 4 D). The expression level of FGF6 in the SH group was significantly higher than that in the EH group as detected by immunohistochemistry(Fig. 6 )༎These results indicated that FGF6 played a pivotal role in promoting the proliferation of BPH stromal cells by reducing the autophagy of BPH epithelial cells. Autophagy Inhibition in BPH Epithelial Cells Promotes Stromal Cell Proliferation via the AKT/ERK Pathway To explore the mechanism by which reducing autophagy in BPH epithelial cells promotes the proliferation of BPH stromal cells, we focused on the AKT/ERK signaling pathway. Western blotting was used to detect the expression levels of AKT, p-AKT, ERK and p-ERK proteins in WPMY-1 and PPrSC cells at different time points after conditioned medium treatment. We found that the levels of P-AKT and P-ERK increased significantly after conditioned culture treatment (Fig. 5 A). Then, we used Western blotting to detect the expression levels of AKT, p-AKT, ERK and p-ERK proteins in WPMY-1 and PPrSC cells at different time points after FGF6 treatment and found that levels of P-AKT and P-ERK also increased significantly after FGF6 treatment (Fig. 5 B). Western blotting was also applied to detect the expression levels of AKT, p-AKT, ERK and p-ERK proteins in WPMY-1 and PPrSC cells at different time points after treatment with conditioned medium and FGF6 neutralizing antibodies, and the levels of P-AKT and P-ERK were not significantly increased after the addition of FGF6 neutralizing antibodies (Fig. 5 C). These results suggested that reducing autophagy in BPH epithelial cells promoted the proliferation of BPH stromal cells via AKT/ERK signaling. Discussion Using clinical samples and in vitro experiment, this study demonstrated that Inhibition of autophagy in BPH epithelial cells can promote stromal cell proliferation by secreting FGF6, and this proliferation occurs through the AKT/ERK signaling pathway༎These research results may help clinicians in providing new treatment options for BPH. Targeting autophagy or FGF6 may represent a potential therapeutic strategy for BPH.For examples, Promoting the increase of autophagy in BPH epithelial cells or targeting the inhibition of the AKT/ERK signaling pathway may be potential approaches for treating the interstitial hyperplasia subgroup of BPH in the future. Numerous factors contribute to BPH progression, including hormones and inflammation. Recent study has found that in hypoxia, activation of the HIF-1α/VEGF pathway plays a crucial role in regulating cell proliferation in a BPH stromal cell line[ 22 ]. Some studies have increasingly highlighted the role of autophagy in BPH progression[ 23 – 26 ]. Autophagy is a cellular homeostatic mechanism characterized by cyclic degradation. It plays an essential role in maintaining cellular quality and survival by eliminating dysfunctional cellular components. In recent years, more and more studies have proven that autophagy plays a crucial role in benign prostatic hyperplasia. However, the role of autophagy in benign prostatic hyperplasia remains controversial. Some researchers believe that the continuous progression of BPH is due to upregulation of autophagy༎Other researchers have found that autophagy in BPH tissue cells is reduced under both basal and induced conditions[ 27 – 31 ]. Numerous studies have implicated autophagy in BPH occurrence and progression [ 8 – 10 ]. Hong et al. demonstrated that chronological ageing attenuates autophagic flux in C57BL/6 murine prostates and WPMY-1 stromal cells, thereby accelerating hyperplastic progression[ 32 ]. Zhang and colleagues reported that hypoxia amplifies autophagy in WPMY-1 cells, fuelling their proliferative capacity[ 33 ]. Jiang et al. revealed that inflammatory insults suppress autophagy in BPH-1 epithelial cells and in benign human prostate tissue, fostering aberrant growth[ 34 ]. De Nunzio et al. established that adiposity-driven metabolic stress curtails autophagy and consequently exacerbates benign prostatic hyperplasia[ 35 ]. Liu et al. delineated how supraphysiological androgen exposure dampens autophagy in prostate epithelial cells from castrated rats, instigating unchecked proliferation[ 36 ]. Similarly, Kim et al. showed that oestrogen excess attenuates autophagic activity in BPH-1 cells, thereby promoting their clonal expansion[ 37 ]. In this study, autophagy was found to be suppressed in BPH epithelial cells in clinical samples. Subsequent in vitro experiments explored the impact of epithelial cell autophagy suppression on BPH progression. Previous reports have shown that cells with altered autophagy can influence neighboring cells through the secretion of various growth factors[ 11 , 14 , 15 ]. We all know that the fibroblast growth factor family plays a crucial role in cell growth and proliferation. Meanwhile, Fibroblast growth factors (FGFs) and FGF receptors (FGFRs) play an important role in the maintenance of tissue homeostasis and the development and differentiation of prostate tissue through epithelial-stromal interactions[ 38 ]. In this study, conditioned medium from epithelial cells with autophagy suppression promoted stromal cell proliferation, with FGF6 identified as the key factor. FGF6 has been implicated in promoting cell proliferation, particularly in stromal cells[ 39 – 42 ]. This study confirmed FGF6's role in proliferation using recombinant FGF6 and neutralizing antibodies. Then, the deeper mechanism by which autophagy inhibition in BPH epithelial cells promotes stromal cell proliferation was explored. We focused on the AKT/ERK signaling pathway, which has been reported to be closely related to proliferation and growth[ 43 – 45 ]. For example, FGF6 promotes proliferation and is also related to the AKT/ERK signaling pathway[ 46 – 50 ]. We detected changes in AKT, p-AKT, ERK1/2 and P-ERK1/2 by Western blotting and verified their role. The results suggested that autophagy inhibition in BPH epithelial cells promoted the proliferation of BPH stromal cells by secreting FGF6 through the AKT/ERK pathway. This study's strengths include its novel exploration of the impact of epithelial cell autophagy on BPH stromal cell proliferation. However, limitations exist. First, the sample size is relatively small, necessitating larger studies for validation. Second, animal experiments are required to further corroborate these findings. In the following research, we will conduct a more in-depth exploration of how autophagy inhibition leads to the secretion of FGF6 by epithelial cells, as well as the specific signaling pathways through which FGF6 promotes the proliferation of stromal cells. Conclusions This study demonstrates that autophagy is suppressed in BPH epithelial cells in the stromal hyperplasia group. Autophagy inhibition in BPH epithelial cells promotes stromal cell proliferation via FGF6 secretion through the AKT/ERK pathway (Fig. 7 ). Enhancing epithelial cell autophagy and targeting FGF6 may emerge as promising therapeutic strategies for BPH in the future. Declarations Author Contributions Song Jin and Yinong Niu conceived and designed the study. Zhanliang Liu and Mingxin Jiang provided study materials and methods. Jie Ming conducted data analysis, Dongdong Fan collected and assembled data, wrote the manuscript, and provided final approval. Funding This study was supported by the National Natural Science Foundation of China (grant numbers: 82000718 and 82170783) and the Youth Talent Cultivation Program of Beijing (grant number: QML20230904). Data Availability Data may be made available upon request. 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Regulation of skeletal muscle stem cells by fibroblast growth factors. Dev Dyn. 2017; 246(5): 359-67. https://doi.org/10.1002/dvdy.24495. Zofkie W, Southard S M, Braun T, et al.Fibroblast growth factor 6 regulates sizing of the muscle stem cell pool. Stem Cell Reports. 2021;16(12): 2913-27. https://doi.org/10.1016/j.stemcr.2021.10.006. LabrecqueM P, Brown L G,Coleman I M,et al.Targeting the fibroblast growth factor pathw-ay in molecular subtypes of castration-resistant prostate cancer.Prostate2024;84:100-10.https://doi.org/10.1002/pros.24630. Bollenbecker S, Barnes J W, Krick S. Fibroblast Growth Factor Signaling in Development and Disease. Int J Mol Sci. 2023; 24(11) :9734. https://doi.org/10.3390/ijms24119734. Xie Y,Su N,Yang J,et al.FGF/FGFR signaling in health and disease.Signal Transduct Target Ther.2020;5(1):81.https://doi.org/10.1038/s41392-020-00222-7. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9214223","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":631855306,"identity":"c011e9a4-29b9-4158-9272-8305802c205a","order_by":0,"name":"DongDong Fan","email":"","orcid":"","institution":"Department of Urology,Beijing Friendship Hospital,Capital Medical University,","correspondingAuthor":false,"prefix":"","firstName":"DongDong","middleName":"","lastName":"Fan","suffix":""},{"id":631855307,"identity":"82575348-fe9e-4823-9ed9-63c4262c7635","order_by":1,"name":"Zhanliang Liu","email":"","orcid":"","institution":"Department of Urology,Beijing Friendship Hospital,Capital Medical University,","correspondingAuthor":false,"prefix":"","firstName":"Zhanliang","middleName":"","lastName":"Liu","suffix":""},{"id":631855308,"identity":"51cf7f14-1ffd-4904-bdc7-debedd5a7f0b","order_by":2,"name":"Mingxin Jiang","email":"","orcid":"","institution":"Department of Urology,Beijing Friendship Hospital,Capital Medical University,","correspondingAuthor":false,"prefix":"","firstName":"Mingxin","middleName":"","lastName":"Jiang","suffix":""},{"id":631855309,"identity":"f9593636-b1e0-4e4d-ae0d-884e7713683c","order_by":3,"name":"Jie Ming","email":"","orcid":"","institution":"Department of Urology,Beijing Friendship Hospital,Capital Medical University,","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Ming","suffix":""},{"id":631855310,"identity":"18c07bcc-0adc-44e3-8231-25e962ba7549","order_by":4,"name":"Song Jin","email":"","orcid":"","institution":"Department of Urology,Beijing Friendship Hospital,Capital Medical University,","correspondingAuthor":false,"prefix":"","firstName":"Song","middleName":"","lastName":"Jin","suffix":""},{"id":631855311,"identity":"124ff768-b127-49ee-a412-6eb2bdaa0a43","order_by":5,"name":"Yinong Niu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIie3QoQ7CMBCA4SZLirmAPcJSBC9QsmQKzXO0pmokyAoEC2QTwLsgkSNLpgoaWR4Bh4OiIXQ4RL9U9k+vR0gQ/CHaWVWV4BOgnTy3Qi/8SRcaaa1WrAt1za1p/AnDbDS2pk4YTlX/uo5aDAaGoiwiWSCkWi4p6ZUb4fnLrnEJlcVgm17kISZoTnvPK2flEpBFbFxiKOE48ySYpS55nSyduwlbJQkXhicUlSLtkteShRaMuiWjMA14/zIsV9Xxzh8wLPP8dtcL1it335M38Nv1IAiC4KMnWI1Lk3AYZugAAAAASUVORK5CYII=","orcid":"","institution":"Department of Urology,Beijing Friendship Hospital,Capital Medical University,","correspondingAuthor":true,"prefix":"","firstName":"Yinong","middleName":"","lastName":"Niu","suffix":""}],"badges":[],"createdAt":"2026-03-24 16:08:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9214223/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9214223/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108209388,"identity":"e273d149-9f49-47ac-accf-00509be7cb77","added_by":"auto","created_at":"2026-04-30 13:31:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1096721,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrouping method of prostatic hyperplasia tissue. \u003c/strong\u003eUnder low-power microscopy, the whole samples were divided into the epithelial hyperplasia (EH) and the stromal hyperplasia of benign prostatic hyperplasia tissue based on hematoxylin-eosin (HE) staining,8cases of that was display by microscopy and images acquisition (100x).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9214223/v1/b938e050a4ae5b86b332bb3e.png"},{"id":108491556,"identity":"53aaf63b-d470-4ac2-aa2b-ec9638af8c3c","added_by":"auto","created_at":"2026-05-05 09:54:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":609957,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAutophagy inhibition in BPH epithelial cells and conditioned media of BPH-1 cells with inhibited autophagy promoted the proliferation of BPH stromal cells. \u003c/strong\u003e(A,B) Representative immunohistochemical images of LC3 and p62 expression level in BPH group tissues with the obvious glandular epithelium hyperplasia(EH) and the obvious stromal hyperplasia(SH). Light microscopy and image acquisition were performed, and ImageJ software was used for semi-quantitative analysis of specific protein expression.(C) CCK8 assays showed the proliferation of WPMY-1 and PPrSC cells treated with conditioned medium of BPH-1 cells with inhibited autophagy. (D) Western blotting detected Cyclin D1 and PCNA expression in WPMY-1 and PPrSC cells treated with conditioned medium of BPH-1 cells with inhibited autophagy. (E) CCK8 assays showed the proliferation of WPMY-1 and PPrSC cells treated with CQ.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9214223/v1/322d7b06ba58a2285f88dfcf.png"},{"id":108491563,"identity":"e591c8cc-6429-4938-a606-440c2db34524","added_by":"auto","created_at":"2026-05-05 09:54:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":166980,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytokine changes in conditional medium of BPH-1 cells with inhibited autophagy.\u003c/strong\u003e (A-C) qPCR was used to detect cytokines in conditioned medium in BPH-1 cells with inhibited autophagy. (D) ELISA assay to detect FGF6 expression in conditioned medium in BPH-1 cells with inhibited autophagy.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9214223/v1/7163d49d0add205a2696cdb8.png"},{"id":108209391,"identity":"fb04e077-cfcd-47b9-8b05-42f477d8fbc1","added_by":"auto","created_at":"2026-04-30 13:31:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":200289,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFGF6 plays an important role in promoting the proliferation of BPH stromal cells by conditioned medium. \u003c/strong\u003e(A) CCK8 assays showed the proliferation of WPMY-1 and PPrSC cells treated with FGF6. (B) Western blotting detected Cyclin D1 and PCNA expression in WPMY-1 and PPrSC cells treated with FGF6. (C)CCK8 assays showed the proliferation of WPMY-1 and PPrSC cells treated with conditioned medium with/without the anti-FGF6 neutralizing antibody. (D) Western blotting detected Cyclin D1 and PCNA expression in WPMY-1 and PPrSC cells treated with conditioned medium with/without the anti-FGF6 neutralizing antibody.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9214223/v1/c75bab3419cd1365f375d19c.png"},{"id":108492090,"identity":"5ca8b2bd-129e-4259-ae19-a33bb83d4269","added_by":"auto","created_at":"2026-05-05 09:56:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":208590,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAutophagy inhibition in BPH epithelial cells promoted the proliferation of BPH stromal cells via the AKT/ERK pathway. \u003c/strong\u003e(A) Western blotting detected the expression of AKT, p-AKT, ERK1/2 and p-ERK1/2 in WPMY-1 and PPrSC cells treated with conditioned medium. (B) Western blotting detected the expression of AKT, p-AKT, ERK1/2 and p-ERK1/2 in WPMY-1 and PPrSC cells treated with FGF6. (C) Western blotting detected the expression of AKT, p-AKT, ERK1/2 and p-ERK1/2 in WPMY-1 and PPrSC cells treated with conditioned medium with/without the anti-FGF6 neutralizing antibody.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9214223/v1/80edc2a231dbfe083a4524a2.png"},{"id":108209393,"identity":"2e585f6c-4021-43fe-bb4d-c407566cf15e","added_by":"auto","created_at":"2026-04-30 13:31:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":722968,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression level of FGF6 in the SH group was significantly higher than that in the EH group\u003c/strong\u003e．(A)Representative immunohistochemical images of FGF6 expression level in BPH group tissues with the glandular epithelium hyperplasia (EH) and the stromal hyperplasia (SH). (B) Moreover, in the stromal hyperplasia (SH), the expression of FGF6 was negatively correlated with that of LC3.Light microscopy and image acquisition were performed, and ImageJ software was used for semi-quantitative analysis of specific protein expression.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9214223/v1/d5319de3edab9fd52473c9d1.png"},{"id":108209395,"identity":"adc9eb7b-91dd-4964-a93f-57036f84877c","added_by":"auto","created_at":"2026-04-30 13:31:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":362552,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of autophagy in BPH epithelial cells can promote stromal cell proliferation by secreting FGF6, and this proliferation occurs through the AKT/ERK signaling pathway\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9214223/v1/6c2aea01c5c7fbeb97d64db0.png"},{"id":108976419,"identity":"8f28e309-7eb8-43fc-b11e-d7e2b54099a1","added_by":"auto","created_at":"2026-05-11 11:17:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3486708,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9214223/v1/90a8f598-4137-407d-a2f3-ef1a3f3c0276.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Autophagy Inhibition in BPH Epithelial Cells Promotes Stromal Proliferation via FGF6 Secretion Through the AKT/ERK Pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBenign prostatic hyperplasia (BPH) is a progressive condition predominantly affecting elderly men, yet the factors driving its acceleration remain largely enigmatic. Our prior research identified a subset of BPH samples characterized by aberrant stromal hyperproliferation and collagen deposition, and the level of autophagy in the epithelial cells of these tissues were significantly low. Recent advancements have underscored the pivotal role of autophagy in the development and progression of BPH, that is the most prevalent condition among elderly men[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], with an incidence rate of 30% in men over 40 years of age and exceeding 70% in those over 70 years[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. BPH is frequently associated with lower urinary tract symptoms (LUTS), severely compromising quality of life due to dysuria, nocturia, urinary retention, and related complications[\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The annual healthcare expenditure attributed to this condition is substantial, imposing a significant financial burden on both patients and society. As the global population ages, BPH is poised to become an increasingly critical public health concern.\u003c/p\u003e \u003cp\u003eBPH is characterized by a chronic, progressive proliferation of prostatic glandular and stromal cells, with stromal cells playing a particularly pivotal role in disease progression[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, the etiology of BPH remains poorly understood. In clinical practice, we have observed that young patients with BPH progression rapidly. Clinically, 8% of BPH patients exhibit rapid progression, necessitating surgical intervention before the age of 50[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, the mechanisms of BPH progression remain unclear. Studying the mechanisms behind BPH progression in these patients may help us identify key breakthrough points in the advancement of BPH. To identify factors that drive BPH progression, our research team has investigated starting from patients with early-onset BPH[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Autophagy is a self-digestive process that is conserved in eukaryotic cells and responsible for maintaining cellular homeostasis through proteolysis. By this process, cells break down their own components in lysosomes. In recent years, research on autophagy in BPH has gained significant momentum, with accumulating evidence indicating its crucial role in the onset and progression of BPH.\u003c/p\u003e \u003cp\u003eWe observed that early-progressed BPH with prominent stromal hyperplasia was associated with autophagy suppression in the epithelial cells compared to those with prominent glandular epithelial hyperplasia. Recent studies have highlighted the significant role of autophagy in BPH development and progression[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, the impact of autophagy inhibition in the BPH epithelium on disease progression remains unclear. Autophagy has been shown to regulate a wide array of proinflammatory cytokines, which can influence neighboring cells[\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Among the various factors implicated in BPH progression, cytokines or growth factors involved in epithelial-mesenchymal interactions are particularly relevant[\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Thus, we hypothesized that autophagy inhibition in BPH epithelial cells might affect stromal cells through the secretion of specific factors.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatient Specimens\u003c/h2\u003e \u003cp\u003eA total of 42 prostate tissue samples from BPH patients were collected and subsequently classified into two groups based on hematoxylin-eosin (HE) staining: those predominantly characterized by stromal hyperplasia (SH, n\u0026thinsp;=\u0026thinsp;25) and those predominantly characterized by glandular epithelial hyperplasia (EH, n\u0026thinsp;=\u0026thinsp;17). Patients with prostate cancer, prostatitis, or those receiving alpha-adrenergic receptor antagonists or 5α-reductase inhibitors were excluded. After obtaining informed consent from all participants and approval from the institutional review committee of Beijing Friendship Hospital, retrospective clinical data analysis and tissue collection were conducted.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell Culture\u003c/h3\u003e\n\u003cp\u003eHuan primary prostatic stromal cells (PPrSCs) were isolated from BPH tissues obtained via transurethral resection of the prostate (TURP). Fresh prostate tissues were dissected into small fragments, and primary stromal cells were cultured as previously described.[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] PPrSCs were maintained in RPMI-1640 medium (SH30809.01B, HyClone, South Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, MA, USA) and 1% penicillin-streptomycin at 37\u0026deg;C in a humidified incubator with 5% CO2. After six passages, PPrSCs were utilized for subsequent experiments. The BPH-1 cell line was procured from Keygen Biotech (KG1008, Nanjing, China), and the BPH stromal cell line WPMY-1 was obtained from the American Type Culture Collection (ATCC). These cells were cultured in RPMI-1640 medium (01-100-1ACS, Biological Industries, Israel) containing 1% streptomycin, 1% penicillin, and 10% fetal bovine serum (04-001-1ACS, Biological Industries, Israel) at 37\u0026deg;C with 5% CO2.\u003c/p\u003e\n\u003ch3\u003eReagents and Antibodies\u003c/h3\u003e\n\u003cp\u003eThe following antibodies were employed for immunohistochemistry (IHC) and Western blot analysis: rabbit anti-Beclin 1 (ab62557; Abcam, Cambridge, UK), rabbit anti-PCNA (13110), and anti-Cyclin D1 (55506) (both from Cell Signaling Technology, Denver, MA), anti-FGF6 neutralizing antibody (ab89549; Abcam, Cambridge, UK), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (60004-1-Ig) and anti-β-tubulin (66240-1-Ig) (both from Proteintech Group Inc, Chicago, IL), anti-AKT (4691), anti-phospho-AKT (4060), anti-ERK (4695), and anti-phospho-ERK (4370) (all from Cell Signaling Technology, Denver, MA). Recombinant human (rh) FGF6 (100\u0026thinsp;\u0026minus;\u0026thinsp;30) was sourced from Perprotech, USA. FGF6 Polyclonal Antibody (PA5-112421)(from Invitrogen,USA).\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eThe expression levels of LC3, p62 and FGF6 were analyzed by IHC in continuous paraffin sections. BPH tissues obtained from TURP were fixed in 4% formalin buffer at 4\u0026deg;C overnight, dehydrated, and embedded in paraffin. Sections were cut at 5 \u0026micro;m thickness. After dewaxing and hydration, endogenous peroxidase was inactivated with 3% hydrogen peroxide. The primary antibody was incubated overnight at 4\u0026deg;C. A biotinylated secondary antibody was applied for 30 minutes at room temperature, followed by visualization using the peroxidase substrate DAB kit. Image J was used to calculate mean MOD to evaluate protein expression of IHC.\u003c/p\u003e\n\u003ch3\u003eCell Proliferation Assay\u003c/h3\u003e\n\u003cp\u003eWPMY-1 and PPrSC cells were seeded in 96-well plates (800 cells per well) and incubated overnight. The medium was then replaced with a 1:1 mixture of conditioned medium and fresh phenol red-free medium, supplemented with FGF6 or 10% FBS and anti-FGF6 neutralizing antibody. Cells were cultured for 6 days. Cell growth was assessed using the Cell Counting Kit-8 (CCK-8) (CK04; Dojindo Molecular Technologies, Tokyo, Japan) on days 0, 2, 4, and 6, with absorbance measured at 450 nm using a Varioskan Flash spectrophotometer (Thermo Fisher Scientific, Waltham, MA).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative Real-Time PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from tissues using TRIzol reagent (Invitrogen, Big Island, New York), followed by reverse transcription using an iScript Reverse Transcription Kit (BioRad). qRT-PCR was performed on the Bio-Rad CFX96 system with SYBR green to quantify the mRNA expression levels of the target genes.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEnzyme-Linked Immunosorbent Assay (ELISA)\u003c/h3\u003e\n\u003cp\u003eConditioned medium (CM) collected from BPH-1 cells cultured or treated with chloroquine (CQ) for 4 days was used to measure FGF6 secretion using the FGF6 Quantikine ELISA Kit (P10767, Raybiotech, GA) according to the manufacturer's protocol.\u003c/p\u003e\n\u003ch3\u003eWestern Blotting Assay\u003c/h3\u003e\n\u003cp\u003eCells were lysed in RIPA buffer containing 1 mM PMSF, 0.5% phosphatase inhibitors, and 1% protease inhibitors (KGP250, Keygen Biotech, China). Proteins were transferred onto nitrocellulose membranes. The membranes were blocked with Tris-buffered saline containing 5% skim milk powder for 1 hour. Primary antibodies were incubated overnight at 4\u0026deg;C in TBS-T. After washing three times with TBS-T buffer, secondary antibodies (Invitrogen) were applied for 1 hour at room temperature. Signals were detected using a Western blotting chemiluminescence reagent (P90719, Millipore, MA, USA).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM from at least three independent experiments. Statistical analysis was performed using one-way analysis of variance with SPSS 24.0 (SPSS Inc., Chicago, IL). Statistical significance was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAutophagy of Epithelial Cells in Stromal Hyperplasia BPH is Significantly Reduced\u003c/h2\u003e \u003cp\u003eWe collected samples tissue of BPH during TURP and performed HE staining on them (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Subsequently, we found that the stromal cells proliferation showed significant in stromal hyperplasia(SH) BPH tissues. Therefore, we divided the tissues into stromal cells proliferation and epithelial proliferation. The expression of LC3 in epithelial cells was significantly diminished in the EH group compared to the SH group (p\u0026thinsp;=\u0026thinsp;0.0119), while p62 exhibited the opposite trend (p\u0026thinsp;=\u0026thinsp;0.0156), indicating that autophagy in epithelial cells associated with stromal hyperplasia is markedly reduced. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). Compared to the EH group, autophagy was significantly reduced in BPH epithelial cells in the SH group. These findings suggest that autophagy suppression in epithelial cells may be associated with BPH progression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAutophagy Inhibition in BPH Epithelial Cells Promotes the Proliferation of BPH Stromal Cells\u003c/h2\u003e \u003cp\u003eTo investigate the impact of epithelial cell autophagy on stromal cells, BPH-1 cell lines were treated with varying concentrations of chloroquine (CQ, 1 nM and 10 nM) to induce autophagy suppression, and the conditioned medium (CM) was collected to treat WPMY-1 and PPrSC cells. CCK8 assays revealed that CM from BPH-1 cells treated with 10 nM CQ significantly enhanced stromal cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Besides, western blotting showed the expression of proliferation markers Cyclin D1 and PCNA were significantly increased in both stromal cell types following CM treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eTo exclude the possibility that CQ directly promotes cell proliferation, BPH-1 and WPMY-1 cells were treated with 10 CQ. The results showed that 10nM CQ could inhibit the proliferation of both cell lines. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Collectively, these findings indicate that a reduction in autophagy in BPH epithelial cells may contribute to the proliferation of prostate stromal cells through the secretion of specific cytokines.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFGF6 Plays a Pivotal Role in Promoting BPH Stromal Cell Proliferation via Autophagy Inhibition in BPH Epithelial Cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo explore which factor promotes the proliferation of BPH stromal cells by conditioned media, we first used q-PCR to detect various chemokines, inflammatory factors and growth factors in CQ-treated BPH-1 cells and found that expression of fibroblast growth factor 6 (FGF6) was significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). We then used ELISA to assess expression of FGF6 in the conditioned medium and found it to be increased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eTo investigate whether FGF6 promotes BPH stromal cell proliferation, we added human recombinant FGF6 directly to WPMY-1 cells and PPrSC cells. CCK8 assays detected that the proliferation levels of WPMY-1 cells and PPrSC cells treated with FGF6 were significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Based on Western blotting, Cyclin D1 and PCNA levels were significantly increased in the two kinds of cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). We then treated WPMY-1 cells and PPrSC cells with/without the addition of FGF6 neutralizing antibodies to the conditioned medium and used CCK8 assays to detect proliferation. Proliferation of WPMY-1 and PPrSC cells was significantly inhibited after adding the FGF6 neutralizing antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Western blotting showed that Cyclin D1 and PCNA protein levels were also significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The expression level of FGF6 in the SH group was significantly higher than that in the EH group as detected by immunohistochemistry(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e)༎These results indicated that FGF6 played a pivotal role in promoting the proliferation of BPH stromal cells by reducing the autophagy of BPH epithelial cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAutophagy Inhibition in BPH Epithelial Cells Promotes Stromal Cell Proliferation via the AKT/ERK Pathway\u003c/h2\u003e \u003cp\u003eTo explore the mechanism by which reducing autophagy in BPH epithelial cells promotes the proliferation of BPH stromal cells, we focused on the AKT/ERK signaling pathway. Western blotting was used to detect the expression levels of AKT, p-AKT, ERK and p-ERK proteins in WPMY-1 and PPrSC cells at different time points after conditioned medium treatment. We found that the levels of P-AKT and P-ERK increased significantly after conditioned culture treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Then, we used Western blotting to detect the expression levels of AKT, p-AKT, ERK and p-ERK proteins in WPMY-1 and PPrSC cells at different time points after FGF6 treatment and found that levels of P-AKT and P-ERK also increased significantly after FGF6 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Western blotting was also applied to detect the expression levels of AKT, p-AKT, ERK and p-ERK proteins in WPMY-1 and PPrSC cells at different time points after treatment with conditioned medium and FGF6 neutralizing antibodies, and the levels of P-AKT and P-ERK were not significantly increased after the addition of FGF6 neutralizing antibodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These results suggested that reducing autophagy in BPH epithelial cells promoted the proliferation of BPH stromal cells via AKT/ERK signaling.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eUsing clinical samples and in vitro experiment, this study demonstrated that Inhibition of autophagy in BPH epithelial cells can promote stromal cell proliferation by secreting FGF6, and this proliferation occurs through the AKT/ERK signaling pathway༎These research results may help clinicians in providing new treatment options for BPH. Targeting autophagy or FGF6 may represent a potential therapeutic strategy for BPH.For examples, Promoting the increase of autophagy in BPH epithelial cells or targeting the inhibition of the AKT/ERK signaling pathway may be potential approaches for treating the interstitial hyperplasia subgroup of BPH in the future.\u003c/p\u003e \u003cp\u003eNumerous factors contribute to BPH progression, including hormones and inflammation. Recent study has found that in hypoxia, activation of the HIF-1α/VEGF pathway plays a crucial role in regulating cell proliferation in a BPH stromal cell line[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Some studies have increasingly highlighted the role of autophagy in BPH progression[\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAutophagy is a cellular homeostatic mechanism characterized by cyclic degradation. It plays an essential role in maintaining cellular quality and survival by eliminating dysfunctional cellular components. In recent years, more and more studies have proven that autophagy plays a crucial role in benign prostatic hyperplasia. However, the role of autophagy in benign prostatic hyperplasia remains controversial. Some researchers believe that the continuous progression of BPH is due to upregulation of autophagy༎Other researchers have found that autophagy in BPH tissue cells is reduced under both basal and induced conditions[\u003cspan additionalcitationids=\"CR28 CR29 CR30\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Numerous studies have implicated autophagy in BPH occurrence and progression [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Hong et al. demonstrated that chronological ageing attenuates autophagic flux in C57BL/6 murine prostates and WPMY-1 stromal cells, thereby accelerating hyperplastic progression[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Zhang and colleagues reported that hypoxia amplifies autophagy in WPMY-1 cells, fuelling their proliferative capacity[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Jiang et al. revealed that inflammatory insults suppress autophagy in BPH-1 epithelial cells and in benign human prostate tissue, fostering aberrant growth[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. De Nunzio et al. established that adiposity-driven metabolic stress curtails autophagy and consequently exacerbates benign prostatic hyperplasia[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Liu et al. delineated how supraphysiological androgen exposure dampens autophagy in prostate epithelial cells from castrated rats, instigating unchecked proliferation[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Similarly, Kim et al. showed that oestrogen excess attenuates autophagic activity in BPH-1 cells, thereby promoting their clonal expansion[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In this study, autophagy was found to be suppressed in BPH epithelial cells in clinical samples. Subsequent in vitro experiments explored the impact of epithelial cell autophagy suppression on BPH progression.\u003c/p\u003e \u003cp\u003ePrevious reports have shown that cells with altered autophagy can influence neighboring cells through the secretion of various growth factors[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. We all know that the fibroblast growth factor family plays a crucial role in cell growth and proliferation. Meanwhile, Fibroblast growth factors (FGFs) and FGF receptors (FGFRs) play an important role in the maintenance of tissue homeostasis and the development and differentiation of prostate tissue through epithelial-stromal interactions[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In this study, conditioned medium from epithelial cells with autophagy suppression promoted stromal cell proliferation, with FGF6 identified as the key factor. FGF6 has been implicated in promoting cell proliferation, particularly in stromal cells[\u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. This study confirmed FGF6's role in proliferation using recombinant FGF6 and neutralizing antibodies.\u003c/p\u003e \u003cp\u003eThen, the deeper mechanism by which autophagy inhibition in BPH epithelial cells promotes stromal cell proliferation was explored. We focused on the AKT/ERK signaling pathway, which has been reported to be closely related to proliferation and growth[\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. For example, FGF6 promotes proliferation and is also related to the AKT/ERK signaling pathway[\u003cspan additionalcitationids=\"CR47 CR48 CR49\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. We detected changes in AKT, p-AKT, ERK1/2 and P-ERK1/2 by Western blotting and verified their role. The results suggested that autophagy inhibition in BPH epithelial cells promoted the proliferation of BPH stromal cells by secreting FGF6 through the AKT/ERK pathway.\u003c/p\u003e \u003cp\u003eThis study's strengths include its novel exploration of the impact of epithelial cell autophagy on BPH stromal cell proliferation. However, limitations exist. First, the sample size is relatively small, necessitating larger studies for validation. Second, animal experiments are required to further corroborate these findings.\u003c/p\u003e \u003cp\u003eIn the following research, we will conduct a more in-depth exploration of how autophagy inhibition leads to the secretion of FGF6 by epithelial cells, as well as the specific signaling pathways through which FGF6 promotes the proliferation of stromal cells.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrates that autophagy is suppressed in BPH epithelial cells in the stromal hyperplasia group. Autophagy inhibition in BPH epithelial cells promotes stromal cell proliferation via FGF6 secretion through the AKT/ERK pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Enhancing epithelial cell autophagy and targeting FGF6 may emerge as promising therapeutic strategies for BPH in the future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSong Jin and Yinong Niu conceived and designed the study. Zhanliang Liu and Mingxin Jiang provided study materials and methods. Jie Ming conducted data analysis, Dongdong Fan collected and assembled data, wrote the manuscript, and provided final approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (grant numbers: 82000718 and 82170783) and the Youth Talent Cultivation Program of Beijing (grant number: QML20230904).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData may be made available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval declaration and consent to partricipate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics Committee of Beijing Friendship Hospital approved this study (2024-P2-465-01), and written informed consent was obtained from each participating patient.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eVuichoud C and KR Loughlin.Benign prostatic hyperplasia:epidemiology,economics and evaluation. 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Stem Cell Reports. 2021;16(12): 2913-27. https://doi.org/10.1016/j.stemcr.2021.10.006.\u003c/li\u003e\n \u003cli\u003eLabrecqueM P, Brown L G,Coleman I M,et al.Targeting the fibroblast growth factor pathw-ay in molecular subtypes of castration-resistant prostate cancer.Prostate2024;84:100-10.https://doi.org/10.1002/pros.24630.\u003c/li\u003e\n \u003cli\u003eBollenbecker S, Barnes J W, Krick S. Fibroblast Growth Factor Signaling in Development and Disease. Int J Mol Sci. 2023; 24(11) :9734. https://doi.org/10.3390/ijms24119734.\u003c/li\u003e\n \u003cli\u003eXie Y,Su N,Yang J,et al.FGF/FGFR signaling in health and disease.Signal Transduct Target Ther.2020;5(1):81.https://doi.org/10.1038/s41392-020-00222-7.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-urology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"buro","sideBox":"Learn more about [BMC Urology](http://bmcurol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/buro/default.aspx","title":"BMC Urology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"autophagy, fibroblast growth factor 6, epithelial cell, fibroblast proliferation, benign prostatic hyperplasia","lastPublishedDoi":"10.21203/rs.3.rs-9214223/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9214223/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBenign prostatic hyperplasia (BPH) is a progressive disease in elderly men, mechanism of which has not been unknown. The aim of this study was to explore how autophagy inhibition in BPH epithelial cells influences Benign prostatic hyperplasia (BPH) progression. We found that the autophagy level in epithelial cells of stromal hyperplasia(SH) is markedly reduced,compared with glandular epithelial hyperplasia (EH), Paraffin-embedded prostate specimens were classified into two groups:stromal hyperplasia (SH)and glandular epithelial hyperplasia (EH),those proteins was evaluated via immunohistochemistry. Chloroquine (CQ) was employed to suppress autophagy of BPH-1 cells. Conditioned media (CM) were harvested and utilized for treating the WPMY-1 cell line and primary prostatic stromal cells (PPrSCs). CCK8 assays were employed to assess cells proliferation.qPCR and ELISA were utilized quantify cytokines and inflammatory factors. Treatment of WPMY-1 and PPrSC cells with CM from autophagy-inhibited BPH-1 cells significantly enhanced their proliferation. FGF6 levels were significantly elevated in CM from BPH-1 cells treated with CQ. Recombinant FGF6 protein stimulated proliferation in WPMY-1 and PPrSC cells and The Akt and ERK signaling pathways were activated following treatment with BPH-1 CM or recombinant FGF6, an effect that could be reversed by FGF6 neutralizing antibodies. The expression level of FGF6 in the SH group was significantly higher than that in the EH group, Meanwhile, in the SH group, the expression of FGF6 was negatively correlated with the expression of LC3.Autophagy suppression in prostate epithelial cells results in the secretion of substantial amounts of FGF6, that promote fibroblast proliferation and accelerating BPH progression.\u003c/p\u003e","manuscriptTitle":"Autophagy Inhibition in BPH Epithelial Cells Promotes Stromal Proliferation via FGF6 Secretion Through the AKT/ERK Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-30 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