Metastatic Prostate Cancer Cells Research Articles

The role of KiSS1 gene on the growth and migration of prostate cancer and the underlying molecular mechanisms

Metastatic prostate cancers have a high mortality rate. KiSS1 was originally identified as a metastasis suppressor gene in metastatic melanoma and breast cancer, but its role in prostate cancer has been contradictory. This study was therefore undertaken to investigate the effects of KiSS1 overexpression on the growth and migration of human metastatic prostate cancer cells. We first tested the effect of KiSS1 overexpression on the growth and migration of DU145 human metastatic prostate cancer cells in vitro. DU145 cells were infected with the culture medium of 293T cells, which produce lentivirus particles containing KiSS1. A 2.5-fold increase in proliferation of KiSS1-overexpressing cancer cells was observed, and these cells formed tumor spheroids about 3 times larger than the vector control group. qPCR and immunoblotting revealed the association between increased cell growth and regulation of the PI3K/Akt and cell cycle genes, and also that increases in β-catenin and CD133 contribute to tumor aggregation. KiSS1 overexpression resulted in upregulation of the β-arrestin1/2 and Raf-MEK-ERK-NF-κB pathways via KiSS1R. Moreover, the migration and invasion of KiSS1-overexpressing cells were determined to be faster than the control group, along with 1.6-fold increased metastatic colonization of the KiSS1-overexpressing cancer cells. These were associated to the regulation of EMT gene expressions, such as E-cadherin and N-cadherin, and the upregulation of MMP9. In a xenograft mouse model inoculated with DU145 cells infected GFP or KiSS1 via a lentiviral vector, KiSS1 statistically significantly increased the tumor growth, with upregulation of PCNA and Ki-67 in the tumor tissues. In addition, KiSS1 increased the angiogenic capacity by upregulating VEGF-A and CD31, both in vitro and in vivo. Taken together, our results indicate that KiSS1 not only induces prostate cancer proliferation, but also promotes metastasis by increasing the migration, invasion, and angiogenesis of malignant cells.

Prostate cancer cells of increasing metastatic potential exhibit diverse contractile forces, cell stiffness, and motility in a microenvironment stiffness-dependent manner.

During metastasis, all cancer types must migrate through crowded multicellular environments. Simultaneously, cancers appear to change their biophysical properties. Indeed, cell softening and increased contractility are emerging as seemingly ubiquitous biomarkers of metastatic progression which may facilitate metastasis. Cell stiffness and contractility are also influenced by the microenvironment. Stiffer matrices resembling the tumor microenvironment cause metastatic cells to contract more strongly, further promoting contractile tumorigenic phenotypes. Prostate cancer (PCa), however, appears to deviate from these common cancer biophysics trends; aggressive metastatic PCa cells appear stiffer, rather than softer, to their lowly metastatic PCa counterparts. Although metastatic PCa cells have been reported to be more contractile than healthy cells, how cell contractility changes with increasing PCa metastatic potential has remained unknown. Here, we characterize the biophysical changes of PCa cells of various metastatic potential as a function of microenvironment stiffness. Using a panel of progressively increasing metastatic potential cell lines (22RV1, LNCaP, DU145, and PC3), we quantified their contractility using traction force microscopy (TFM), and measured their cortical stiffness using optical magnetic twisting cytometry (OMTC) and their motility using time-lapse microscopy. We found that PCa contractility, cell stiffness, and motility do not universally scale with metastatic potential. Rather, PCa cells of various metastatic efficiencies exhibit unique biophysical responses that are differentially influenced by substrate stiffness. Despite this biophysical diversity, this work concludes that mechanical microenvironment is a key determinant in the biophysical response of PCa with variable metastatic potentials. The mechanics-oriented focus and methodology of the study is unique and complementary to conventional biochemical and genetic strategies typically used to understand this disease, and thus may usher in new perspectives and approaches.

Site-Specific Intact N-Linked Glycopeptide Characterization of Prostate-Specific Membrane Antigen from Metastatic Prostate Cancer Cells.

The composition of N-linked glycans that are conjugated to the prostate-specific membrane antigen (PSMA) and their functional significance in prostate cancer progression have not been fully characterized. PSMA was isolated from two metastatic prostate cancer cell lines, LNCaP and MDAPCa2b, which have different tissue tropism and localization. Isolated PSMA was trypsin-digested, and intact glycopeptides were subjected to LC-HCD-EThcD-MS/MS analysis on a Tribrid Orbitrap Fusion Lumos mass spectrometer. Differential qualitative and quantitative analysis of site-specific N-glycopeptides was performed using Byonic and Byologic software. Comparative quantitative analysis demonstrates that multiple glycopeptides at asparagine residues 51, 76, 121, 195, 336, 459, 476, and 638 were in significantly different abundance in the two cell lines (p < 0.05). Biochemical analysis using endoglycosidase treatment and lectin capture confirm the MS and site occupancy data. The data demonstrate the effectiveness of the strategy for comprehensive analysis of PSMA glycopeptides. This approach will form the basis of ongoing experiments to identify site-specific glycan changes in PSMA isolated from disease-stratified clinical samples to uncover targets that may be associated with disease progression and metastatic phenotypes.

Antiproliferative Effect and Autophagy Inhibition of Dihydropyrimidinone‐Cinnamic Acid Hybrids

AbstractHere we report the design, synthesis and evaluation of dihydropyrimidinones (DHPMs)‐cinnamic acids hybrids as potential anti‐cancer compounds. The synthetic route was successfully performed and the products obtained in good to excellent yields. These compounds were tested against LNCaP and PC‐3 metastatic prostate cancer cells and RWPE‐1 prostate cells. The results indicate that hybridization was effective, since the hybrids were much more cytotoxic than DHPMs and cinnamic acids evaluated separately. Compound 3 a was the most potent hybrid against PC‐3 cells (IC50 of 15.7 μM) and LNCaP cells (IC50 of 11.5 μM) and was further optimized by bioisosterism and molecular simplification strategies leading to three new analogues (5, 7 and 8). Compounds 3 a, 7 and 8 showed an antiproliferative effect against PC‐3 cells without inducing cell death nor cell cycle arrest. The mechanism of action of compound 8 was further investigated. The results indicated that compound 8 inhibits autophagic flux, and this effect could be linked to the antiproliferative activity. Moreover, in silico prediction of some molecular properties related to pharmacokinetics showed that all the hybrids meet the criteria for a drug prototype to have a good absorption and permeation for oral administration. These results highlight the potential use of DHPMs‐cinnamic acids hybrids as antiproliferative agents for further evaluation of the antitumor activity in vivo.

Enzyme Responsive Rigid-Rod Aromatics Target "Undruggable" Phosphatases to Kill Cancer Cells in a Mimetic Bone Microenvironment.

Bone metastasis remains a challenge in cancer treatment. Here we show enzymatic responsive rigid-rod aromatics acting as the substrates of "undruggable" phosphatases to kill cancer cells in a mimetic bone microenvironment. By phosphorylation and conjugating nitrobenzoxadiazole (NBD) to hydroxybiphenylcarboxylate (BP), we obtained pBP-NBD (1P) as a substrate of both acid and alkaline phosphatases. 1P effectively kills both metastatic castration-resistant prostate cancer cells (mCRPCs) and osteoblast mimic cells in their coculture. 1P enters Saos2 almost instantly to target the endoplasmic reticulum (ER) of the cells. Co-culturing with Saos2 cells boosts the cellular uptake of 1P by mCRPCs. Cryo-EM reveals the nanotube structures of both 1P (2.4 Å resolution, pH 5.6) and 1 (2.2 Å resolution, pH 7.4). The helical packing of both nanotubes is identical, held together by strong pi-stacking interactions. Besides reporting the atomistic structure of nanotubes formed by the assembly of rigid-rod aromatics, this work expands the pool of molecules for designing EISA substrates that selectively target TME.

The blockage of downstream P2Y2 receptor signaling inhibits the prostate cancer cell adhesion to endothelial cells

AimsProstate cancer is the second most frequently malignancy in men worldwide. Most deaths are caused by metastasis, and tumor cell dissemination involves the interaction with endothelial cells. However, the endothelial cell signaling involved in such interaction is not entirely understood. The tumor microenvironment contains extracellular ATP, an endogenous agonist of the purinergic P2Y2 receptor (P2Y2R). P2Y2R signaling changes endothelial cell phenotype, which may be relevant to cancer pathophysiology. Therefore, we hypothesized that P2Y2R activation could favor the metastatic prostate cancer cells adhesion to endothelial cells. Main methodsFor adhesion assays, confluent endothelial cells EA.hy926 were treated with P2Y2R agonists before adding and imaging stained DU-145 cells. Alternatively, fluorescent probes and antibodies were used to determine intracellular endothelial Ca2+, nitric oxide (NO), and flow cytometry assays. Key findingsEndothelial P2Y2R activation with ATP, UTP, or the selective agonist 2-thio-UTP increased DU-145 cell adhesion to EA.hy926 cells. This effect required endothelial cell Ca2+ mobilization and relied on the endothelial expression of VCAM-1 and ICAM-1. Conversely, inhibiting this proadhesive endothelial phenotype could impair DU-145 cell adhesion. To evaluate this, we chose atorvastatin based on its notable improvement of endothelial cell dysfunction. Atorvastatin blocked UTP-induced DU-145 cell adhesion to endothelial cell monolayer in a NO-dependent manner, unveiling a P2Y2R and NO signaling crosstalk. SignificanceEndothelial P2Y2R signaling contributes to the adhesion of metastatic prostate cancer cells suggesting that the downstream signaling blockade by statins could be a putative mechanism to reduce prostate cancer metastasis.

The effects of boric acid and disodium pentaborate dechydrate in metastatic prostate cancer cells

Boron and their derived molecules have prevention or treatment potential against prostate cancer. In this study, we aim to investigate the effects of Boric acid (BA) and Disodium Pentaborate Dechydrate (DPD) in metastatic prostate cancer cells such as DU-145 which is brain metastatic prostate cancer, and PC3 which is bone metastatic prostate cancer. Metastatic human prostate cancer cell lines, PC-3 and DU-145, were used to show whether inhibition effects of BA and DPD on prostate cancer cells in this study. BA and DPD were applied for 24 hours to the cells. Cell viability determination was performed using WST-1 assay. Apoptotic cell death was evaluated with Annexin-V/PI flow cytometric analysis and caspase-3 expression immunohistochemically. A wound healing assay was also used to measure cancer cell migration after exposure to BA and DPD. Applying BA and DPD made inhibition of cell proliferation in both BA (1 mM) and DPD (7 mM) at 24 h. The results of Annexin-V/PI showed that DPD induced higher levels of apoptosis than BA in both prostate cancer cells. Caspase-3 expressions were also higher than BA with DPD in both metastatic prostate cancer cells. We evaluated cell migration using a wound healing assay and the result showed that cell migration was inhibited with BA and DPD in both cells. Both BA and DPD inhibited the cell viability of metastatic prostate cancer cells. Apoptotic cell death with applying DPP had a higher rate than BA treatment. Moreover, BA and DPD inhibited cell migration in both cells when we compared them with control. This study’s results showed that BA and DPD of boron derivates significantly induced cells to apoptosis and the migration was inhibited by the derived form of boron in metastatic prostate cancer cells.

Synergistic Benefit of Adoptive T Cells in Combination With Chemoradiotherapy Against Metastatic Prostate Cancer Cells.

Prostate cancer (PC) is one of the major diseases that affects male health and ranks as the second most frequent cancer in men worldwide. Although most newly-diagnosed PCs are well-differentiated tumors with a high cure probability, there are some patients with aggressive malignancies that show potential for recurrence and metastasis. Cytotoxic T lymphocytes are a specific immune effector cell population that mediates immune responses against cancer. In the present study, the cytotoxicity of peripheral blood mononuclear cells (PBMCs)-derived γδ T cells and cytokine-induced killer (CIK) cells in combination with chemoradiotherapy against PC cells was evaluated using Alamar blue cell viability and cell membrane permeability assays. Advanced PC-3 cells, which were more resistant to docetaxel (Doc), also showed higher viability following pretreatment with radiation. The cell proliferation inhibition was significantly increased upon additional γδ T or CIK treatment. Furthermore, the proportion of apoptotic cells was significantly (p<0.05) increased in the Doc-γδ T cell co-treatment group as compared with the Doc or γδ T cell treated alone group. γδ T cell therapy may provide additional benefit compared to traditional chemoradiotherapy for PC treatment.

Abstract 5999: TBX2 promotes prostate cancer bone-metastatic phenotype through exosomal microRNA-375-3p

Abstract Introduction: Bone is the preferred site of metastasis in about 80% of advanced prostate cancer (PCa) patients. The molecular mechanisms that drive bone metastatic PCa are not well understood. Recent reports have increasingly shed light on the role of cancer-cell-derived extracellular vesicles (exosomes) in driving the metastatic cascade including priming the secondary site for metastatic manifestation. We have previously reported that TBX2 promotes multiple facets of the PCa bone metastatic cascade via a cell-intrinsic manner. In this study, we sought to determine if the bone metastasis promoted by TBX2 is driven by microRNAs (miRs) in an exosome-mediated paracrine manner. Methods: Two converse approaches were utilized for the genetic modulation of TBX2: a) TBX2 was blocked in the metastatic PC3 and C4-2B human PCa cells using a dominant negative (DN) approach, and b) TBX2 was over-expressed in the low-TBX2 expressing LNCaP human PCa cells. Exosomes isolated from TBX2-modulated human PCa cells were added to MC3T3 pre-osteoblastic cells or mouse calvaria bone cells to examine the differential effects of TBX2 modulation on osteoblast or osteoclast differentiation. Next, unbiased next generation sequencing (NGS) was performed to identify the differential miR expression in exosomes derived from the TBX2-blocked PC3 cells. Results: Our results demonstrate that compared with control exosomes, exosomes derived from TBX2-blocked PC3 cells slowed the proliferation and migration/wound healing when added to 22Rv1 cells. Further, compared with control exosomes, exosomes derived from TBX2-blocked C4-2B cells resulted in reduced osteoblast differentiation when added to: a) MC3T3 pre-osteoblastic cells, or b) mouse calvaria bone cells. Along similar lines, when compared with the control exosomes, exosomes derived from TBX2-blocked PC3 and C4-2B cells resulted in reduced osteoclast differentiation when added to bone-marrow derived cells. These results were confirmed by q RT-PCR analysis of the osteoblastic/osteolytic bone markers. In order to identify exosomal miRs downstream of TBX2 signaling, NGS analysis of exosomal miRs obtained from TBX2-blocked PC3 human PCa cells identified miR-375-3p as the top-most differentially-expressed (over-expressed miR) when compared with control exosomes. In silico analysis revealed that miR-375-3p could potentially bind and target the 3’UTR of RBPJ, an established driver of PCa bone metastasis. In agreement, q RT-PCR analysis revealed that rescue of miR-375-3p in TBX2 modulated human PCa cells rescued RBPJ expression. Conclusions: Our studies have identified the exosome-mediated TBX2/miR-375-3p/RBPJ signaling axis as a promoter of the bone-metastatic phenotype in human PCa. Citation Format: Girijesh Kumar Patel, Sayanika Dutta, Hamed Khedmatgozar, Manisha Tripathi, Srinivas Nandana. TBX2 promotes prostate cancer bone-metastatic phenotype through exosomal microRNA-375-3p [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5999.

Abstract LB082: Genome-wide CRISPR activation screen identifies PRRX2 as an enzalutamide resistance gene in prostate cancer

Abstract Androgen receptor pathway inhibitors are the mainstay treatment for advanced prostate cancer, but resistance is common. Here, we used a CRISPR activation screen to identify genes that promote enzalutamide resistance in the metastatic castration-sensitive prostate cancer cell line LNCaP. We found that activation of the TGF-β target gene, PRRX2, promoted enzalutamide resistance. PRRX2 expression is highest in double negative prostate cancer (DNPC) patients and a PRRX2-related gene signature identified a subset of DNPC patients with lower overall survival. PRRX2 expressing cells showed alterations in the CDK4/6/Rb/E2F and BCL2 pathways. Accordingly, CDK4/6 and BCL2 inhibitors sensitized PRRX2-expressing enzalutamide-resistant tumors to enzalutamide. High PRRX2 expression/signature in prostate cancer merit investigation as markers of enzalutamide resistance that could be reversed with CDK4/6 and BCL2 inhibitors. Citation Format: Yara R. Rodriguez, Kenji Unno, Mihai Truica, Zachary R. Chalmers, Young A Yoo, Rajita Vatapalli, Vinay Sagar, Jindan Yu, Barbara Lysy, Huiying Han, Sarki A. Abdulkadir. Genome-wide CRISPR activation screen identifies PRRX2 as an enzalutamide resistance gene in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB082.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Dual STAT‑3 and IL‑6R inhibition with stattic and tocilizumab decreases migration, invasion and proliferation of prostate cancer cells by targeting the IL‑6/IL‑6R/STAT‑3 axis

Prostate cancer (PCa) is a key public health problem worldwide; at diagnosis, a high percentage of patients exhibit tumor cell invasion of adjacent tissue. STAT-3, IL-6 receptor (R) and IL-6 serum levels are associated with enhanced PCa migratory, invasive, clonogenic and metastatic ability. Inhibiting the STAT-3 pathway at different levels (cytokines, receptors, and kinases) exhibits relative success in cancer. The present study investigated the effect of Stattic (Stt) + Tocilizumab (Tcz) on proliferative, clonogenic, migratory and invasive ability of human metastatic PCa (assessed by colony formation, wound healing and migration assay). RWPE-1 (epithelial prostate immortalized cells), 22Rv1 (Tumor cells), LNCaP (Metastatic cells) and DU-145 (metastatic, castration-resistant prostate cells) cells were used in vitro to evaluate levels of cytokines, chemokines, growth factors (Cytometric Bead Array), STAT-3, phosphorylated STAT-3 (In-Cell Western), IL-6R, vimentin and epithelial (E-) cadherin (Western Blot). The effect of inhibition of STAT-3 (expressed constitutively in DU-145 cells) with Stt and/or Tcz on expression levels of vimentin, VEGF, and E-cadherin, as well as proliferative, clonogenic, migratory and invasive capacity of metastatic PCa cells was assessed. The expression levels of IL-6, C-X-C chemokine ligand 8, VEGF and vimentin, as well as proliferation and migration, were increased in metastatic PCa cells. Treatment with Stt or Tcz decreased vimentin and VEGF and increased E-cadherin expression levels and inhibited proliferative, clonogenic, migratory and invasive capacity of DU-145 cells; addition of IL-6 decreased this inhibitory effect. However, Stt + Tcz maintained inhibition even in the present of high concentrations of IL-6. Stt + Tcz decreased expression of vimentin and VEGF and inhibited the proliferative, clonogenic, migratory and invasive capacity of metastatic PCa cells. To the best of our knowledge, the present study is the first to combine Stt, a STAT-3 inhibitor, with Tcz, an antibody against IL-6R, to target tumor cells.

Protein Kinase A (PKA) as a Master Regulator of the Early Metabolic Reprogramming in Bone Metastatic Prostate Cancer Cells

PURPOSE The arrival of metastatic prostate cancer (PCa) tumor cells to the bone niche requires a metabolic adaptation. We sought to identify metabolic dysregulations fueling PCa metastasis, modulated by bone secreted factors. METHODS By an indirect co-culture system of PCa (PC3) and bone progenitors (MC3T3, pre-osteoblasts, or Raw 264.7, pre-osteoclasts) we assessed the transcriptome of PC3 cells modulated by soluble factors released from bone precursors. We validated the transcriptional profile of metabolic genes in open-access transcriptomic datasets. We performed an Ingenuity Pathway Analysis (IPA) to delineate the regulators of these metabolic genes. Bone secretome was profiled on the conditioned media (CM) by ESI-MS/MS. RESULTS PC3 cells co-cultured with bone progenitors displayed an activation of lipidic categories, including PPAR-signaling and fat absorption/digestion. Principal Component and Unsupervised Clustering analyses using transcriptomic data from human PCa and bone metastatic samples (GSE74685) showed that the metabolic genes deregulated in PC3 accurately clustered samples in primary tumor or bone metastasis. Moreover, four lipid-associated genes, PPARA, VDR, SLC16A1 and GPX1, were associated with a shorter survival time (SU2C-PCF dataset), and were independent risk-predictors of death ( P &lt; .05). An IPA revealed that these genes are regulated by the Protein Kinase A (PKA). Accordingly, PC3 cells treated with the CM of the co-culture presented a decreased ATP content compared to the treatment with the CM of PC3 grown alone, which was restored upon PKA inhibition. Finally, the secretome analysis revealed soluble factors secreted by bone progenitors (Col1a1, Fn1) which could regulate PKA activity. CONCLUSION We identified a novel lipid-associated gene signature important for metastatic PCa, triggered by the dialogue with bone cells. Moreover, PKA could regulate this signature in response to bone-secreted factors reprogramming the metabolic phenotype of metastatic cells, emerging as a potential druggable target for this disease.

Bone Progenitors Pull the Strings on the Early Metabolic Rewiring Occurring in Prostate Cancer Cells.

Simple SummaryAlmost 90% of prostate cancer metastases occur in bone. The arrival of tumor cells to the homing organ requires a metabolic adaptation to different nutrients and oxygen availability in the new microenvironment to fulfill sustained growth and proliferation rates. In this study, we characterized the alterations in tumor metabolism occurring in human prostate cancer cells when they interact with bone cells. We hypothesized that elucidating how these cells obtain their energy and “building blocks”, and that identifying key determinants underlying this phenomenon, could be a promising strategy to halt disease progression. Accordingly, we discovered five genes related to the metabolism of lipids that play a critical role in the survival of metastatic patients, and we established a communication axis between tumor and bone cells that includes bone-secreted collagen and the tumor Protein Kinase A, which drives the early metabolic reprogramming of metastatic cells.Metastatic prostate cancer (PCa) cells soiling in the bone require a metabolic adaptation. Here, we identified the metabolic genes fueling the seeding of PCa in the bone niche. Using a transwell co-culture system of PCa (PC3) and bone progenitor cells (MC3T3 or Raw264.7), we assessed the transcriptome of PC3 cells modulated by soluble factors released from bone precursors. In a Principal Component Analysis using transcriptomic data from human PCa samples (GSE74685), the altered metabolic genes found in vitro were able to stratify PCa patients in two defined groups: primary PCa and bone metastasis, confirmed by an unsupervised clustering analysis. Thus, the early transcriptional metabolic profile triggered in the in vitro model has a clinical correlate in human bone metastatic samples. Further, the expression levels of five metabolic genes (VDR, PPARA, SLC16A1, GPX1 and PAPSS2) were independent risk-predictors of death in the SU2C-PCF dataset and a risk score model built using this lipid-associated signature was able to discriminate a subgroup of bone metastatic PCa patients with a 23-fold higher risk of death. This signature was validated in a PDX pre-clinical model when comparing MDA-PCa-183 growing intrafemorally vs. subcutaneously, and appears to be under the regulatory control of the Protein Kinase A (PKA) signaling pathway. Secretome analyses of conditioned media showcased fibronectin and type-1 collagen as critical bone-secreted factors that could regulate tumoral PKA. Overall, we identified a novel lipid gene signature, driving PCa aggressive metastatic disease pointing to PKA as a potential hub to halt progression.

Overexpression of regucalcin blocks the migration, invasion, and bone metastatic activity of human prostate cancer cells: Crosstalk between cancer cells and bone cells.

Prostate cancer is a bone metastatic cancer and is the second leading cause of cancer-related death in men. Prolonged progression-free survival of prostate cancer patients is associated with high regucalcin expression in the tumor tissues. This study investigates the underlying mechanism by which regucalcin prevents bone metastatic activity of prostate cancer cells. Human prostate cancer PC-3 or DU-145 wild-type cells or regucalcin-overexpressing PC-3 or DU-145 cells (transfectants) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Overexpressed regucalcin suppressed the migration and invasion of bone metastatic human prostate cancer cells in vitro, and it reduced the levels of key proteins in metastasis including Ras, Akt, MAPK, RSK-2, mTOR, caveolin-1, and integrin β1. Invasion of prostate cancer cells was promoted by coculturing with preosteoblastic MC3T3-E1 or preosteoclastic RAW264.7 cells. Coculturing with cancer cells and bone cells repressed the growth of preosteoblastic cells and enhanced osteoclastogenesis of preosteoclastic cells, and these alterations were caused by a conditioned medium from cancer cell culture. Disordered differentiation of bone cells was prevented by regucalcin overexpression. Production of tumor necrosis factor-α (TNF-α) in cancer cells was blocked by overexpressed regucalcin. Of note, the effects of conditioned medium on bone cells were prevented by NF-κB inhibitor. TNF-α may be important as a mediator in the crosstalk between cancer cells and bone cells. Overexpression of regucalcin suppressed the migration, invasion, and bone metastatic activity of human prostate cancer cells. This study may provide a new strategy for therapy with the regucalcin gene transfer.

Extracellular Regucalcin Suppresses the Growth, Migration, Invasion, and Adhesion of Metastatic Human Prostate Cancer Cells

Introduction: Regucalcin plays a multifunctional role in the regulation of cellular function including metabolism, signaling process, and transcriptional activity in maintaining cell homeostasis. Downregulated expression or activity of regucalcin contributes to the development of malignancies in various types of human cancer. Survival of cancer patients, including metastatic prostate cancer, is prolonged with high expression of regucalcin in the tumor tissues. Methods: We elucidate whether extracellular regucalcin conquers the growth, migration, invasion, and adhesion of metastatic human prostate cancer PC-3 and DU-145 cells. Results: Extracellular regucalcin (0.1, 1, and 10 nM) of physiologic levels (1 nM at human serum) inhibited colony formation and growth of PC-3 and DU-145 cells, while it did not have an effect on cell death. Repressive effects of extracellular regucalcin on the proliferation were not exhibited by the presence of inhibitors of the cell cycle, intracellular signaling process, and transcriptional activity, suggesting that the signals of extracellular regucalcin are transmitted to block cell growth. Furthermore, extracellular regucalcin (0.1, 1, or 10 nM) inhibited migration, invasion, and adhesion of PC-3 and DU-145 cells. Mechanistically, extracellular regucalcin (10 nM) decreased the levels of various signaling proteins including Ras, posphatidylinositol-3 kinase, mitogen-activated protein kinase, mechanistic target of rapamycin, RSK-2, caveolin-1, and integrin β1 in PC-3 cells. Discussion and Conclusion: Thus, extracellular regucalcin may play a suppressive role in growth, migration, invasion, and adhesion, which are involved in the metastatic activity of human prostate cancer cells, via affecting diverse signaling processes. This study may provide a new strategy in preventing metastatic prostate cancer with exogenous regucalcin.

Calcium signaling in prostate cancer cells of increasing malignancy

Calcium signaling controls a large variety of cell functions, including proliferation and apoptosis, and plays a major role in neoplastic transformation. Prostate cancer (PCa) is one of the most common malignancies in men. The transition to castration-resistant prostate cancer (CRPC), a lethal form that is still lacking an effective cure, could be influenced by fine tuning intracellular calcium ([Ca2+]i) homeostasis. This study investigates [Ca2+]i dynamics in metastatic PCa cell lines that mimic the progression of PCa to CRPC: (i) well differentiated LNCaP cells that require androgen for survival, and (ii) poorly differentiated, highly aggressive androgen-insensitive prostate cancer (AIPC) PC3 and DU145 cells. In AIPC cells, ATP induces a fast rise in [Ca2+]i, due to release from intracellular stores and sensitive to phospholipase C inhibitors, while LNCaP cells do not respond to ATP challenge. Moreover, AIPC cells showed a reduced capacity to store Ca2+ in thapsigargin-sensitive stores and limited store-operated calcium entry, with respect to androgen-dependent LNCaP cells. Finally, green tea extract causes [Ca2+]i elevation and inhibits proliferation in PC3 and DU145 cells, but is ineffective in LNCaP cells. The consequences of these differences are discussed and interpreted in this study with reference to previously proposed models for Ca2+ dependence of prostate carcinogenesis.

The marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol suppresses growth, migration and invasion and stimulates death of metastatic human prostate cancer cells: targeting diverse signaling processes.

Prostate cancer is metastatic cancer and is the second leading cause of cancer-related death in men. It is needed to develop more effective treatment for metastatic prostate cancer. The present study investigates whether the novel factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), which was isolated from marine oyster, suppresses the activity of metastatic human prostate cancer PC-3 or DU-145 cells. Culture of DHMBA (1 or 10 µM) suppressed colony formation and growth of PC-3 or DU-145 cells in vitro. Suppressive effects of DHMBA on cell proliferation were not occurred by culturing with intracellular signaling inhibitors. Mechanistically, DHMBA (10 µM) reduced the levels of key proteins linked to promotion of cell growth, including Ras, PI3K, Akt, MAPK, and mTOR in PC-3 cells. Interestingly, DHMBA increased the levels of cancer suppressor p53, p21, Rb, and regucalcin. Moreover, culture of DHMBA simulated the death of PC-3 and DU-145 cells. This effect was implicated to caspase-3 activation in cells. Interestingly, the effects of DHMBA on cell proliferation and death were blocked by culturing with an inhibitor of aryl hydrocarbon receptor linked to transcriptional regulation. Furthermore, culture of DHMBA inhibited production of reactive oxygen species in PC-3 or DU-145 cells. Of note, DHMBA blocked migration and invasion by diminishing their related protein levels, including NF-κB 65, caveolin-1 and integrin β1. The novel marine factor DHMBA was demonstrated to suppress metastatic prostate cancer cells via targeting diverse signaling pathways. This study may provide a new strategy for prostate cancer therapy with DHMBA.

Protease-activated receptor-2 dependent and independent responses of bone cells to prostate cancer cell secretory products.

Metastatic prostate cancer lesions in the skeleton are frequently characterized by excessive formation of bone. Prostate cancer cells secrete factors, including serine proteases, that are capable of influencing the behavior of surrounding cells. Some of these proteases activate protease-activated receptor-2 (PAR2 ), which is expressed by osteoblasts (bone-forming cells) and precursors of osteoclasts (bone-resorbing cells). The aim of the current study was to investigate a possible role for PAR2 in regulating the behavior of bone cells exposed to metastatic prostate cancer cells. The effect of medium conditioned by the PC3, DU145, and MDA-PCa-2b prostate cancer cell lines was investigated in assays of bone cell function using cells isolated from wildtype and PAR2 -null mice. Osteoclast differentiation was assessed by counting tartrate-resistant acid phosphatase-positive multinucleate cells in bone marrow cultured in osteoclastogenic medium. Osteoblasts were isolated from calvariae of neonatal mice, and BrdU incorporation was used to assess their proliferation. Assays of alkaline phosphatase activity and quantitative PCR analysis of osteoblastic gene expression were used to assess osteoblast differentiation. Responses of osteoblasts to medium conditioned by MDA-PCa-2b cells were analyzed by RNAseq. Conditioned medium (CM) from all three cell lines inhibited osteoclast differentiation independently of PAR2 . Media from PC3 and DU145 cells had no effect on assays of osteoblast function. Medium conditioned by MDA-PCa-2b cells stimulated BrdU incorporation in both wildtype and PAR2 -null osteoblasts but increased alkaline phosphatase activity and Runx2 and Col1a1 expression in wildtype but not PAR2 -null cells. Functional enrichment analysis of RNAseq data identified enrichment of multiple gene ontology terms associated with lysosomal function in both wildtype and PAR2 -null cells in response to MDA-PCa-2b-CM. Analysis of individual genes identified osteogenesis-associated genes that were either upregulated by MDA-PCa-2b-CM selectively in wildtype cells or downregulated selectively in PAR2 -null cells. Factors secreted by prostate cancer cells influence bone cell behavior through both PAR2 -dependent and -independent mechanisms. Both PAR2 -independent suppression of osteoclast differentiation and PAR2 -dependent stimulation of osteogenesis are likely to determine the nature of prostate cancer metastases in bone.

Personalized 3-Gene Panel for Prostate Cancer Target Therapy.

Many years and billions spent for research did not yet produce an effective answer to prostate cancer (PCa). Not only each human, but even each cancer nodule in the same tumor, has unique transcriptome topology. The differences go beyond the expression level to the expression control and networking of individual genes. The unrepeatable heterogeneous transcriptomic organization among men makes the quest for universal biomarkers and “fit-for-all” treatments unrealistic. We present a bioinformatics procedure to identify each patient’s unique triplet of PCa Gene Master Regulators (GMRs) and predict consequences of their experimental manipulation. The procedure is based on the Genomic Fabric Paradigm (GFP), which characterizes each individual gene by the independent expression level, expression variability and expression coordination with each other gene. GFP can identify the GMRs whose controlled alteration would selectively kill the cancer cells with little consequence on the normal tissue. The method was applied to microarray data on surgically removed prostates from two men with metastatic PCas (each with three distinct cancer nodules), and DU145 and LNCaP PCa cell lines. The applications verified that each PCa case is unique and predicted the consequences of the GMRs’ manipulation. The predictions are theoretical and need further experimental validation.