Mesenchymal stem cells recruited by castration-induced inflammation activation accelerate prostate cancer hormone resistance via chemokine ligand 5 secretion
Androgen deprivation (AD) as the first-line treatment for advanced prostate cancer (PCa) is insufficient for a long-term effect. Castration resistance remains the greatest obstacle in PCa clinical therapy. Mesenchymal stem cells (MSCs) can migrate into PCa tissues contributing to tumor progression, therefore, in this study we explored the effect of AD on MSC migration to PCa and elicited its importance for the emergence of castration resistance.
MSC migration assay was performed in several PCa cells (LNCaP, VCaP, and 22Rv1) using in-vivo and in-vitro approaches. Reactive oxygen species generation was evaluated by fluorescence assay. IL-1β was analyzed by immunohistochemistry, and neutralization experiments were conducted using neutralization antibody. Stem markers (CD133, CD44, and SOX2) were quantified by real-time PCR analysis. The concentration of chemokine ligand 5 was measured by enzyme-linked immunosorbent assay and small hairpin RNA was used for functional analyses.
AD could significantly contribute to PCa recruitment of MSCs in vivo and in vitro. AD-induced oxidative stress could promote the inflammatory response mediated by IL-1β secretion via activating the NF-κB signaling pathway. Moreover, N-acetylcysteine could significantly inhibit MSC recruitment to PCa sites when AD is performed. Furthermore, we found MSCs could increase stemness of PCa cells via promoting chemokine ligand 5 secretion in the AD condition, and consequently accelerate emergence of castration resistance.
Our results suggest that castration in clinical PCa therapy may elicit oxidative stress in tumor sites, resulting in increased MSC migration and in tumor cell growth in an androgen-independent manner. Blocking MSC migration to the tumor may provide a new potential target to suppress castration-resistant PCa emergence.
KeywordsMesenchymal stem cell Cancer stem cell Tumor microenvironment Oxidative stress Castration resistance
Androgen deprivation therapy
Chemokine ligand 5
Castration-resistant prostate cancer
Cancer stem cell
Dulbecco’s modified Eagle’s medium
Enzyme-linked immunosorbent assay
Fetal bovine serum
Green fluorescent protein
Mesenchymal stem cell
Prostate cancer stem cell
Reactive oxygen species
Real-time quantitative PCR
Small hairpin RNA
Prostate cancer (PCa) is the most commonly diagnosed cancer in men, and the second leading cause of male cancer-associated mortality in the United States . In China, PCa shows an increasing incidence and mortality in recent years . PCa progresses from prostatic intraepithelial neoplasia through locally invasive adenocarcinoma to hormone-resistant metastatic carcinoma. Early staged and localized PCa can be well controlled by prostatectomy or radiotherapy. For locally advanced and metastatic PCa, androgen deprivation therapy (ADT) is typically employed as the first-line treatment . However, most initially androgen-sensitive PCa would develop into castration-resistant prostate cancer (CRPC) after ADT within 12–18 months . So, the transition of androgen-dependent PCa cells to androgen-independent growth during ADT is the main obstacle for advanced PCa treatment.
Recently, considerable efforts toward elucidating the mechanisms of CRPC occurrence suggested that the tumor microenvironment (TME) may play a key role in response to ADT [3, 5, 6, 7]. The TME is constituted of tumor epithelial cells and stroma including mesenchymal stem cells (MSCs), also called multipotent mesenchymal stromal cells, inflammatory cells, and fibroblasts [8, 9]. Hypoxia in the TME is a cyclical event and perpetuates the inflammatory response by ensuring a constant production of angiogenic and inflammatory mediators . Hypoxic conditions result in reactive oxygen species (ROS) generation and oxidative stress, which can increase DNA damage in neighboring cells and lead to tissue damage . ROS also increase production of inflammatory mediators inducing inflammatory response in the TME . MSCs, as a vital component of the TME, are a subset of nonhematopoietic stem cells existing in the bone marrow . MSCs can be recruited from the bone marrow to areas of injured tissue and inflammation and then induce peripheral tolerance, promoting damaged cell survival . MSCs also have an innate tropism for tumor tissue in response to the inflammatory microenvironment present in malignant lesions  and contribute to various tumor progressions, including PCa [9, 16, 17]. Currently, many studies have shown that ADT increases tumor cell hypoxia in PCa [18, 19]. However, the alteration of MSC migration to PCa tissues and the inflammatory response induced by castration in the TME remain poorly understood.
In this study, we first investigated the contribution of androgen deprivation (AD) in MSC recruitment to PCa using in-vivo and in-vitro approaches, and revealed that castration-induced inflammation activation in response to oxidative stress could increase MSC recruitment to tumor sites. We also found that castration-induced cytokine secretion in MSCs increased the prostate cancer stem cell (PCSC) population, accelerating PCa growth transition from androgen dependent to castration resistant. Our results might represent new potential targets in the battle against advanced PCa.
Cell lines and culture
Human prostatic carcinoma cell lines, including LNCaP, VCaP, and 22Rv1 cells, and bone marrow-derived MSCs were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai Institute of Cell Biology, Shanghai, China. LNCaP and 22Rv1 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), while VCaP cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS. MSCs were cultured in MSC basal medium (all from Invitrogen, Carlsbad, CA, USA). MSCs were transfected with the adenoviral vector GFP-mock (Invitrogen). After transfection for about 48 h, MSCs-GFP were collected for further experiments. All cells were cultured at 37 °C in a 5% CO2 humidified atmosphere. AD was performed using charcoal-stripped medium for cell culture as described previously .
In-vivo xenograft experiment
Nude mice, 6–8 weeks old, were obtained from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences, Shanghai, China, and housed in pathogen-free conditions. All aspects of the animal care and experimental procedures were in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Chinese Academy of Sciences’ Committee on Animals. PCa cells were prepared as single-cell suspensions (1 × 106 cells in 200 μl PBS) and subcutaneously administered in the armpit area of nude mice. When tumors grew to approximately 200 mm3 in size, the nude mice were castrated via scrotal incision under methoxyflurane anesthesia. The mice (n = 6 per group) were then injected with the green fluorescent protein (GFP)-labeled MSCs (MSCs-GFP) through the tail vein every 3 days. Mice were examined every day and tumor growth was evaluated by measuring the length and width of the tumor mass. All tumor-bearing mice survived until they were sacrificed at the end of the experiment; tumors were then removed and dissected quickly for frozen section preparation, or were stored at − 80 °C.
Oxidative stress parameter estimation
The frozen tumor samples were thawed and homogenized on ice. PCa cells (3 × 103 cells) were plated in 96-well plates and incubated with charcoal-stripped medium with or without 10 nM of dihydrotestosterone for 48 h. Then, 5 mM of CM-H2DCFDA (Invitrogen) was added for 30 min in the dark. Intracellular ROS levels were measured as the mean fluorescence intensity (arbitrary units) according to the manufacturer’s instructions. The 4-HNE adduct was estimated in tumor homogenates using an OxiSelect™ HNE ELISA Kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer’s instructions.
The chemotactic effect of PCa cells on MSCs was assayed utilizing 24-well (8-mm pore size) Transwell plates (Cell Biolabs), while six-well (0.4-mm pore size) Transwell plates were used for coculture. The plate was incubated at 37 °C in a humidified atmosphere containing 5% CO2 for 48–72 h. For the migration assay, cells invaded through pores to the lower surface were stained with crystal violet dye and the positively stained cells were counted in six random fields under a microscope.
Real-time quantitative PCR
To quantify mRNA expression of CCL5 and stem cell markers (CD133, CD44, and SOX2) , total RNA was isolated using Trizol reagent (Invitrogen) and cDNA synthesis was performed using the Prime Script RT reagent Kit (Takara, Kyoto, Japan) according to the manufacturer’s specifications. Quantitative PCR was performed using the SYBR Green PCR Kit (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer’s instructions. β-actin was used as an internal control for RNA integrity and loading normalization.
Western blot analysis
Tissues were lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) with 1 mM PMSF. An equal amount of proteins was separated by SDS-PAGE and transferred to nitrocellulose membrane. The membranes were then washed, blocked, and incubated with specific primary antibodies against IκBα, p-IκBα (both from Cell Signaling Technology, USA), CD133, GFP, and β-actin (all from Abcam, Cambridge, MA, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (HuaAn Biotech, Hangzhou, China). Signals were visualized by chemiluminescent detection (Beyotime).
LNCaP xenografted tumors were sectioned (4 μm thick) and mounted on glass slides, then stained with Meyer’s hematoxylin and eosin (H&E), and immunostained with primary antibodies for IL-1β (Invitrogen). After glass slides were mounted, each sample was observed at a 200× magnification of the microscopic field. For human PCa immunohistochemistry evaluation, tissue sections were immunostained with primary antibodies for SSEA-4 (eBioscience, San Diego, CA, USA), 4-HNE (Abcam), and IL-1β (Invitrogen). Each sample was observed at a 400× magnification of the microscopic field in 10 randomly selected areas. The intensity and extent of staining were evaluated by score, assigning from 0 to 3 where 0 = none, 1 = weak, 2 = intermediate, and 3 = strong. Final scores were computed using a composite of intensity scores multiplied by the extent of staining score. A score of 1–4 was assessed as low expression and 6–9 as high expression.
All of the experiments were repeated at least three times. Final data were expressed as mean ± standard deviation (SD). Student’s t test was performed to compare between mean values of two groups using GraphPad Prism 5. Clinically, statistical analysis was performed using SPSS 22.0. Differences between categorical variables were assessed by the chi-square test or Fisher’s exact test. A value of at least p < 0.05 was considered statistically significant.
Castration increases MSC recruitment and oxidative stress in PCa
AD activates inflammatory response mediated by NF-κB signaling
Oxidative stress suppression inhibits MSC recruitment via reducing inflammation activation in PCa
MSCs increase PCa stemness in AD to accelerate CRPC progression
MSCs induce PCa androgen-independent growth depending on chemokine ligand 5 secretion
Previous studies have proved that, after an initial response, PCa cells can adapt to AD via altering the local TME [3, 5]. Therefore, the effect of AD on tumor inflammatory microenvironment alteration and MSC recruitment in PCa tissues attracted our attention. In the current study, we evaluated the influence of AD on the migration of MSCs to the prostate TME. We found that PCa suffering AD could recruit more MSCs to PCa sites in both in-vivo and in-vitro experiments (Figs. 1c and 2a–c).
The TME has been reported to perform critical roles for cancer progression. The interaction between epithelial cells and stromal cells in the TME ranges from supporting tumor cell proliferation to inducing tumorigenesis and metastasis [28, 29, 30]. Evidence has suggested that infiltrating macrophage and cancer cell interaction in the prostate TME can promote castration resistance via a nuclear receptor derepression pathway . Prostate fibroblasts were also demonstrated to facilitate PCa development of castration resistance and metastatic potential . As we know, MSCs have been implicated in tumorigenesis through multiple mechanisms, including promoting proliferation, angiogenesis, and metastasis, in addition to the generation of an immunosuppressive microenvironment assisting tumor escape from immunosurveillance [32, 33, 34]. However, the role of MSCs in development of CRPC during ADT remains poorly clarified. It has been reported that MSCs can secrete a large number of cytokines to affect cancer stem cell (CSC) formation and mediate tumor development [35, 36]. CSCs refer to a subset of tumor cells that has the ability to self-renew and differentiate, continually sustaining tumorigenesis, progression, and metastasis . They are also thought to be the main force behind resistance to chemotherapy and radiotherapy [38, 39]. So, we speculated that CSCs induced by MSCs might play a key role in emergence of castration resistance. In the present study, we validated that MSCs increased the population and self-renewal ability of the original PCSCs via a large amount of CCL5 secretion in AD, accelerating the progression to CRPC (Figs. 6 and 7).
Nowadays, although encouraging new drugs like abiraterone and enzalutamide have been developed recently, CRPC is still incurable by current treatment strategies. We think that blocking MSC migration to PCa might be an effective therapy during progression to castration resistance. So, the mechanism for increased MSC migration to PCa when AD is performed must be elucidated to find the successful target. In the present study, in-vivo and in-vitro studies confirmed that AD could significantly induce oxidative stress and inflammation activation mediated by NF-κB signaling, promoting MSC migration to PCa sites (Figs. 3 and 4). Furthermore, NAC administration could effectively alleviate MSC migration to PCa when AD is performed, and, as expected, PCa tumor growth was also inhibited in castrated mice with MSC treatment (Fig. 5).
In summary, our results reveal that castration could effectively contribute to PCa recruitment of MSCs. The data also show that this effect is dependent on inflammation activation mediated by castration-induced oxidative stress via activating the NF-κB signaling pathway. Antioxidant application could significantly inhibit MSC recruitment to PCa sites when AD is performed. Furthermore, we found MSCs could increase the stemness of PCa cells via promoting chemokine ligand 5 secretion in AD, and consequently accelerate emergence of castration resistance.
Our current study showed that castration could induce PCa oxidative stress and then increase the inflammatory response, contributing to MSCs recruited to PCa sites. In AD, MSCs could promote PCa castration resistance via secreting chemokine ligand 5 and increasing the PCSC population. According to our study, MSCs may be involved in the pathogenesis of CRPC in human PCa, indicating that castration at the same time as targeting oxidative stress using an antioxidant to block PCa recruitment of MSCs would be a new potential treatment strategy to prevent the progression to CRPC.
This work was supported by National Natural Science Foundation of China (Grant No. 81660424) and the Guangxi Natural Science Foundation for Young Scientists (Grant No. 2016GXNSFBA380139).
Availability of data and materials
Please contact the corresponding author for data requests.
YY, JC, XY, and ZH conceived the study and drafted the manuscript. YY, QZ, CM, and RL performed the experiments. YY, YL, and HZ gathered and analyzed the data. All authors reviewed and approved the final manuscript.
All aspects of the animal experimental procedures were approved by the Chinese Academy of Sciences’ Committee on Animals.
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The authors declare that they have no competing interests.
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