Bone Metastasis Of Prostate Cancer Cells Research Articles

Long non\u2010coding RNA NORAD promotes the prostate cancer cell extracellular vesicle release via microRNA-541-3p-regulated PKM2 to induce bone metastasis of prostate cancer

BackgroundBone metastasis is the leading cause of mortality and reduced quality of life in patients with metastatic prostate cancer (PCa). Long non-coding RNA activated by DNA damage (NORAD) has been observed to have an abnormal expression in various cancers. This article aimed to explore the molecular mechanism underlying the regulatory role of NORAD in bone metastasis of PCa.MethodsNORAD expression in clinical PCa tissues and cell lines was detected with the application of qRT-PCR. Cancer cells were then transfected with plasmids expressing NORAD, after which Transwell assay and CCK-8 assay were carried out to detect proliferation, migration, and bone metastasis of PCa. NORAD downstream target molecules were screened through bioinformatics analysis, followed by further verification using dual luciferase assay. Extracellular vesicles (EVs) were labeled with PKH67 and interacted with bone marrow stromal cells. The gain- and loss-function method was applied to determine the internalization and secretion of PCa cells-derived EVs under the intervention of downstream target molecules or NORAD.ResultsPCa tissues and cell lines were observed to have a high expression of NORAD, particularly in tissues with bone metastasis. NORAD knockdown resulted in reduced secretion and internalization of EVs, and suppressed proliferation, migration, and bone metastasis of PCa cells. It was indicated that NORAD interacted with miR-541-3p, leading to the upregulation of PKM2. Forced expression of PKM2 promoted the transfer of PKH67-labeled EVs to bone marrow stromal cells.ConclusionsNORAD might serve as a ceRNA of miR-541-3p to promote PKM2 expression, thereby enhancing the development of bone metastasis in PCa by promoting internalization and transfer of EVs of cancer cells, providing an insight into a novel treatment for the disorder.

Ectopic Expression of miR-532-3p Suppresses Bone Metastasis of Prostate Cancer Cells via Inactivating NF-κB Signaling

miR-532-3p is a widely documented microRNA (miRNA) involved in multifaceted processes of cancer tumorigenesis and metastasis. However, the clinical significance and biological functions of miR-532-3p in bone metastasis of prostate cancer (PCa) remain largely unknown. Herein, we report that miR-532-3p was downregulated in PCa tissues with bone metastasis, and downexpression of miR-532-3p was significantly associated with Gleason grade and serum prostate-specific antigen (PSA) levels and predicted poor bone metastasis-free survival in PCa patients. Upregulating miR-532-3p inhibited invasion and migration abilities of PCa cells in vitro, while silencing miR-532-3p yielded an opposite effect on invasion and migration abilities of PCa cells. Importantly, upregulating miR-532-3p repressed bone metastasis of PCa cells in vivo. Our results further demonstrated that overexpression of miR-532-3p inhibited activation of nuclear facto κB (NF-κB) signaling via simultaneously targeting tumor necrosis factor receptor-associated factor 1 (TRAF1), TRAF2, and TRAF4, which further promoted invasion, migration, and bone metastasis of PCa cells. Therefore, our findings reveal a novel mechanism contributing to the sustained activity of NF-κB signaling underlying the bone metastasis of PCa.

MiR-204-5p Represses Bone Metastasis via Inactivating NF-κB Signaling in Prostate Cancer

The prime issue derived from prostate cancer (PCa) is its high prevalence to metastasize to bone. MicroRNA-204-5p (miR-204-5p) has been reported to be involved in the development and metastasis in a variety of cancers. However, the clinical significance and biological functions of miR-204-5p in bone metastasis of PCa are still not reported yet. In this study, we find that miR-204-5p expression is reduced in PCa tissues and serum sample with bone metastasis compared with that in PCa tissues and serum sample without bone metastasis, which is associated with advanced clinicopathological characteristics and poor bone metastasis-free survival in PCa patients. Moreover, upregulation of miR-204-5p inhibits the migration and invasion of PCa cells in vitro, and importantly, upregulating miR-204-5p represses bone metastasis of PCa cells in vivo. Our results further demonstrated that miR-204-5p suppresses invasion, migration, and bone metastasis of PCa cells via inactivating nuclear factor κB (NF-κB) signaling by simultaneously targeting TRAF1, TAB3, and MAP3K3. In clinical PCa samples, miR-204-5p expression negatively correlates with TRAF1, TAB3, and MAP3K3 expression and NF-κB signaling activity. Therefore, our findings reveal a new mechanism underpinning the bone metastasis of PCa, as well as provide evidence that miR-204-5p might serve as a novel serum biomarker in bone metastasis of PCa.This study identifies a novel functional role of miR-204-5p in bone metastasis of prostate cancer and supports the potential clinical value of miR-204-5p as a serum biomarker in bone metastasis of PCa.

Transcriptional downregulation of miR-133b by REST promotes prostate cancer metastasis to bone via activating TGF-\u03b2 signaling

High avidity of bone metastasis is an important characteristic in prostate cancer (PCa). Downexpression of miR-133b has been reported to be implicated in the development, progression and recurrence in PCa. However, clinical significance and biological roles of miR-133b in bone metastasis of PCa remain unclear. Here we report that miR-133b is downregulated in PCa tissues and further decreased in bone metastatic PCa tissues. Downexpression of miR-133b positively correlates with advanced clinicopathological characteristics and shorter bone metastasis-free survival in PCa patients. Upregulating miR-133b inhibits invasion, migration in vitro and bone metastasis in vivo in PCa cells. Mechanistically, we find that miR-133b suppresses activity of TGF-β signaling via directly targeting TGF-β receptor I and II, which further inhibits bone metastasis of PCa cells. Our results further reveal that overexpression of REST contributes to miR-133b downexpression via transcriptional repression in PCa tissues. Importantly, silencing miR-133b enhances invasion and migration abilities in vitro and bone metastasis ability in vivo in REST-silenced PCa cells. The clinical correlation of miR-133b with TGFBRI, TGFBRII, REST and TGF-β signaling activity is verified in PCa tissues. Therefore, our results uncover a novel mechanism of miR-133b downexpression that REST transcriptionally inhibits miR-133b expression in PCa cells, and meanwhile support the notion that administration of miR-133b may serve as a rational regimen in the treatment of PCa bone metastasis.

Downregulation of miR-141-3p promotes bone metastasis via activating NF-\u03baB signaling in prostate cancer

BackgroundClinically, prostate cancer (PCa) exhibits a high avidity to metastasize to bone. miR-141-3p is an extensively studied miRNA in cancers and downregulation of miR-141-3p has been widely reported to be involved in the progression and metastasis of several human cancer types. However, the clinical significance and biological roles of miR-141-3p in bone metastasis of PCa are still unclear.MethodsmiR-141-3p expression was examined in 89 non-bone metastatic and 52 bone metastatic PCa tissues by real-time PCR. Statistical analysis was performed to investigate the clinical correlation between miR-141-3p expression levels and clinicopathological characteristics in PCa patients. The biological roles of miR-141-3p in bone metastasis of PCa were evaluated both in vitro and a mouse intracardial model in vivo. Bioinformatics analysis, Western blot, luciferase reporter and miRNA immunoprecipitation assays were performed to explore and examine the relationship between miR-141-3p and its potential targets. Clinical correlation of miR-141-3p with its targets was examined in clinical PCa tissues.ResultsmiR-141-3p expression is reduced in bone metastatic PCa tissues compared with non-bone metastatic PCa tissues. Low expression of miR-141-3p positively correlates with serum PSA levels, Gleason grade and bone metastasis status in PCa patients. Furthermore, upregulating miR-141-3p suppresses the EMT, invasion and migration of PCa cells in vitro. Conversely, silencing miR-141-3p yields an opposite effect. Importantly, upregulating miR-141-3p dramatically reduces bone metastasis of PC-3 cells in vivo. Our results further show that miR-141-3p inhibits the activation of NF-κB signaling via directly targeting tumor necrosis factor receptor-associated factor 5(TRAF5) and 6 (TRAF6), which further suppresses invasion, migration and bone metastasis of PCa cells. The clinical negative correlation of miR-141-3p expression with TRAF5, TRAF6 and NF-κB signaling activity is demonstrated in PCa tissues.ConclusionOur findings unravel a novel mechanism underlying the bone metastasis of PCa, suggesting that miR-141-3p mimics might represent a potential therapeutic avenue for the treatment of PCa bone metastasis.

Oncogenic miR-210-3p promotes prostate cancer cell EMT and bone metastasis via NF-\u03baB signaling pathway

BackgroundThe primary issue arising from prostate cancer (PCa) is its high prevalence to metastasize to bone, which severely affects the quality of life and survival time of PCa patients. miR-210-3p is a well-documented oncogenic miRNA implicated in various aspects of cancer development, progression and metastasis. However, the clinical significance and biological roles of miR-210-3p in PCa bone metastasis remain obscure.MethodsmiR-210-3p expression was evaluated by real-time PCR in 68 bone metastatic and 81 non-bone metastatic PCa tissues. The biological roles of miR-210-3p in the bone metastasis of PCa were investigated both in vitro by EMT and Transwell assays, and in vivo using a mouse model of left cardiac ventricle inoculation. Bioinformatics analysis, real-time PCR, western blot and luciferase reporter analysis were applied to discern and examine the relationship between miR-210-3p and its potential targets. RT-PCR was performed to identify the underlying mechanism of miR-210-3p overexpression in bone metastasis of PCa. Clinical correlation of miR-210-3p with its targets was examined in human PCa and metastatic bone tissues.ResultsmiR-210-3p expression is elevated in bone metastatic PCa tissues compared with non-bone metastatic PCa tissues. Overexpression of miR-210-3p positively correlates with serum PSA levels, Gleason grade and bone metastasis status in PCa patients. Upregulating miR-210-3p enhances, while silencing miR-210-3p represses the EMT, invasion and migration of PCa cells in vitro. Importantly, silencing miR-210-3p significantly inhibits bone metastasis of PC-3 cells in vivo. Our results further demonstrate that miR-210-3p maintains the sustained activation of NF-κB signaling via targeting negative regulators of NF-κB signaling (TNF-α Induced Protein 3 Interacting Protein 1) TNIP1 and (Suppressor Of Cytokine Signaling 1) SOCS1, resulting in EMT, invasion, migration and bone metastasis of PCa cells. Moreover, our results further indicate that recurrent gains (amplification) contribute to miR-210-3p overexpression in a small number of PCa patients. The clinical correlation of miR-210-3p with SOCS1, TNIP1 and NF-κB signaling activity is verified in PCa tissues.ConclusionOur findings unravel a novel mechanism for constitutive activation of NF-κB signaling pathway in the bone metastasis of PCa, supporting a functional and clinical significance of epigenetic events in bone metastasis of PCa.

Double-negative feedback loop between ZEB2 and miR-145 regulates epithelial-mesenchymal transition and stem cell properties in prostate cancer cells.

The invasion and metastasis of tumors are triggered by an epithelial to mesenchymal transition (EMT), which is regulated by microRNAs (miRNAs). EMT also promotes malignant tumor progression and the maintenance of the stem cell property, which endows cancer cells with the capabilities of self-renewal and immortalized proliferation. The transcriptional repressor zinc-finger E-box binding homeobox 2 (ZEB2), as an EMT activator, might be an important promoter of metastasis in some tumors. Here, we report that ZEB2 directly represses the transcription of miR-145, which is a strong repressor of EMT. In turn, ZEB2 is also a direct target of miR-145. Further, our findings show that the downregulation of ZEB2 not only represses invasion, migration, EMT, and the stemness of prostate cancer (PCa) cells, but also suppresses the capability of PC-3 cells to invade bone in vivo. Importantly, the expression level of ZEB2 as revealed by immunohistochemical analysis is positively correlated to bone metastasis, the serum free PSA level, the total PSA level, and the Gleason score in PCa patients and is negatively correlated with miR-145 expression in primary PCa specimens. Thus, our findings demonstrate a double-negative feedback loop between ZEB2 and miR-145 and indicate that the ZEB2/miR-145 double-negative feedback loop plays a significant role in the control of EMT and stem cell properties during the bone metastasis of PCa cells. These results suggest that the double-negative feedback loop between ZEB2 and miR-145 contributes to PCa progression and metastasis and might have therapeutic relevance for the bone metastasis of PCa.

Abstract C73: Src-beta catenin-SNAI1 axis is critical for the stemness and bone metastasis of E-cadherin-lacking prostate cancer cells

Abstract Bone metastasis of prostate cancer (PCA) cells is the main cause of high mortality and morbidity in patients. Recent studies have suggested that cancer stem cells not only sustain the growth and heterogeneity of primary tumors but also play a role in metastasis. Therefore, a better molecular understanding of metastatic cancer stem cells could be exploited towards targeting bone metastasis. Here, we analyzed the role of E-cadherin and Src-beta catenin-SNAI1 axis in regulating the stemness and bone metastasis of PCA cells. Human PCA PC3 cells were transfected with shRNA specific for E-cadherin or random shRNA, and selected for stable knock-down of E-cadherin (ShEC-PC3) or respective control cells (Sh-PC3). MTT, clonogenic and xenograft assays were performed to compare the in vitro and in vivo proliferation potential of Sh-PC3 and ShEC-PC3 cells. Stem cell population was characterized by FACS and prostasphere formation. Orthotopic xenograft model and optical imaging tools were employed to study metastasis. Immunoblotting and immunohistochemistry (IHC) methods were used to analyze proliferation, stemness, EMT, and osteomimicry biomarkers. Our results showed that ShEC-PC3 cells possessed mesenchymal phenotype, enhanced invasiveness and clonogenicity as well as higher growth rate both in cell culture and xenografts. Importantly, compared to Sh-PC3 cells, ShEC-PC3 cells formed significantly higher number and bigger sized prostaspheres suggesting an increase in the stem cell population with E-cadherin knock-down. Also, the ShEC-PC3 prostaspheres disintegration in the presence of serum generated bigger mass of attached proliferating cells compared to Sh-PC3. Cell sorting for established PCA stem cell phenotypic markers (CD44highCD24low) confirmed a two-fold increase in stem cell population along with a nine fold higher CD44high/CD44low population in ShEC-PC3 cells compared to Sh-PC3 cells. Immunoblotting and IHC analyses showed that E-cadherin knock-down increased the expression of stemness markers (CD44, Notch1 and Egr-1) as well as EMT and invasivness markers (Vimentin, pSrc-tyr416, beta catenin, NF-kappaB, integrin alpha 5 and beta 3) in both cell culture and xenograft tissues. Next, to define the role of E-cadherin in PCA metastasis, we orthotopically injected Sh-PC3 or ShEC-PC3 cells in the prostate of nude mice and after 4 weeks, imaged the mice using IR-dye (EGF optical probe). Mice injected with ShEC-PC3 cells showed remarkable metastasis to distant organs mainly to the bones, while mice injected with Sh-PC3 cells showed only prostate localized growth. We also observed increased expression of several osteomimicry molecules namely CXCR4, uPA, RANKL and Runx2 in ShEC-PC3 cells in cell culture as well as xenograft tissues. Importantly, we observed a remarkable increase in SNAI1 expression in both cytoplasmic and nuclear fractions from prostaspheres and xenograft tissues of ShEC-PC3 cells. Furthermore, the SNAI1 knock-down by specific siRNA completely inhibited the prostasphere formation, clonogenicity and invasiveness of ShEC-PC3 cells. The SNAI1 inhibition also decreased the level of pSrc-tyr416 and beta catenin in ShEC-PC3 cells suggesting SNAI1 role in regulating the expression of these molecules. Interestingly, inhibition of Src (by dasatinib) or beta catenin (by FH535) also decreased the SNAI1 expression. These results clearly showed that E-cadherin knock-down in PCA cells promotes the formation of Src-beta catenin-SNAI1 axis which then controls the stemness, invasiveness, and metastasis of PCA cells. Therefore, E-cadherin activators and/or Src-beta catenin-SNAI1 repressors could be useful in targeting the PCA stemness and bone metastasis, and lowering the PCA burden. Citation Format: Gagan Deep, Anil K. Jain, Samiha Mateen, Kavitha C. Vijendra, Subhash C. Gangar, Chapla Agarwal, Rajesh Agarwal. Src-beta catenin-SNAI1 axis is critical for the stemness and bone metastasis of E-cadherin-lacking prostate cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C73.

SDF-1α Induced NF-κB Activation Promotes Adhesion and Trans-Endothelial Migration of Prostate Cancer Cells by Enhancing the Expression of the Chemokine Receptor CXCR4.

Although androgen ablative therapy can suppress growth of localized prostate cancer (PC) cells that are androgen receptor positive, skeletal metastases and tumor relapse ultimately dictate PC related morbidity and mortality. Bone metastasis of PC cells results from a complex cascade of events that involves PC cell intravasation into blood or lymphatic vessels, followed by recruitment by bone marrow microvascular endothelial cells and trans-endothelial migration (TEM) in the bone marrow microenvironment. We have tested the hypothesis that enhanced expression of the chemokine receptor, CXCR4 in PC cells plays a crucial role in their adhesion to the endothelium followed by TEM into the bone microenvironment. To test this hypothesis, PC3 (bone metastatic PC cell line) and LnCaP (lymph node metastatic PC cell line) cells were treated with stromal derived factor-1 alpha (SDF-1α, 100 ng/ml) for 6 or 24 hr and RT-PCR amplification of both CXCR4 and GAPDH mRNAs was carried out. Within 6 hr CXCR4 levels of mRNA expression were increased (3–4 fold) in PC3 cells but not in LnCaP cells. By 24 hr the CXCR4 mRNA levels in PC3 cells returned back to normal and remain unchanged in LnCaP cells. The treatment of PC3 cells with SDF-1α (100 ng/ml) for 6 hr showed a significant (p<0.05) increase (200–225 %) in their adhesion to the confluent human umbilical vein endothelial cells (HUVECs) monolayer and increased the TEM of PC3 cells by 80–100 % in trans-well culture chambers. The CXCR4 expression was shown to be a critical determinant in adhesion of PC3 cells to HUVECs since addition of the monoclonal CXCR4 antibody (1 μg/ml) to SDF-1α treated PC3 cells effectively blocked their adhesion and TEM. Furthermore, SDF-1α (100ng/ml for 6 hr) significantly increased NF-kB transactivational activity in PC-3 cells transfected with NF-kB-luciferase reporter plasmid. However, as predicted this activation was not observed in LnCaP cells under these conditions. Suppression of NF-κB activation in SDF-1α (100ng/ml) treated PC3 cells with an adenoviral vector expressing dominant negative mutant I-κB decreased the CXCR4 mRNA expression by 2–3-fold and resulted in inhibition of their adhesion and TEM by 50–80 %. As expected, over-expression of p65 subunit of NF-κB by transient transfection of PC3 cells with pCMV-p65 expression plasmid (3 μg per 5x105 cells) for 48 hr up-regulated the CXCR4 mRNA expression (2–3 fold) and cell surface CXCR4 receptor expression (2–3 fold) as determined by flow cytometric analysis. Introduction of p65 expression plasmid in PC3 cells increased adhesion (50–80 %) to the endothelium and their TEM (80–100 %) in response to SDF-1α (100 ng/ml). In contrast, introduction of pCMV-p50 expression plasmid in PC3 cells did not affect the CXCR4 gene expression. These data clearly implicate that SDF-1α induced CXCR4 expression and activation of NF-κB plays a significant role in adhesion, TEM and ultimately in bone metastasis of PC cells.