Behavior Of Prostate Cancer Cells Research Articles

MicroRNA-425-5p promotes the development of prostate cancer via targeting forkhead box J3.

MicroRNAs (miRNAs) have critical roles in the progression of prostate cancer (PCa) and have the potential to be used as prognosis biomarkers. In this study, we aimed to investigate the role of miR-425-5p in the progression of PCa. miR-425-5p expression in PCa tumor tissues and cell lines was measured by Quantitative Real-time PCR (RT-qPCR). Effects of miR-425-5p expression on PCa cell proliferation, colony formation, cell migration, and cell invasion were measured. We found miR-425-5p expression was elevated in both PCa tissues and cell lines. Importantly, we found overexpression of miR-425-5p promoted proliferation, colony formation, migration and invasion of PCa cell lines in vitro. Forkhead box J3 (FOXJ3) was validated as a downstream target of miR-425-5p. Finally, we found the stimulation effects of miR-425-5p on PCa cell behaviors were fulfilled through directly regulating the expression of FOXJ3, which validated FOXJ3 as a functional target of miR-425-5p in PCa. Taken together, our results demonstrated miR-425-5p may contribute the malignancy progression of PCa in a mechanism involving FOXJ3, implicating miR-425-5p may be developed as therapeutic target for PCa in the future.

ASPM promotes prostate cancer stemness and progression by augmenting Wnt-Dvl-3-β-catenin signaling.

Recurrent and hormone-refractory prostate cancer (PCA) exhibits aggressive behaviors while current therapeutic approaches show little effect of prolonging the survival of patients with PCA. Thus, a deeper understanding of the patho-molecular mechanisms underlying the disease progression in PCA is crucial to identify novel diagnostic and/or therapeutic targets to improve the outcome of patients. Recent evidence suggests that activation of Wnt signaling in cancer stem cells (CSCs) contributes to cancer progression in malignant tumors. Here, we report that a novel Wnt co-activator ASPM (abnormal spindle-like microcephaly associated) maintains the prostate CSC subpopulation by augmenting the Wnt-β-catenin signaling in PCA. ASPM expression is incrementally upregulated in primary and metastatic PCA, implicating its potential role in PCA progression. Consistently, downregulation of ASPM expression pronouncedly attenuated the proliferation, colony formation, and the invasive behavior of PCA cells, and dramatically reduced the number of ALDH+ CSCs and inhibited cancer stemness and tumorigenicity. Mechanistically, ASPM interacts with disheveled-3 (Dvl-3), a cardinal upstream regulator of canonical Wnt signaling, and inhibits its proteasome-dependent degradation, thereby increasing its protein stability and enabling the Wnt-induced β-catenin transcriptional activity in PCA cells. In keeping with the role of ASPM as a CSC-regulator, ASPM co-localizes with ALDH in PCA tissues and its expression exhibits high intra-tumoral heterogeneity. The proportion of high-ASPM-expressing cells in the tumor inversely correlates with the relapse-free survival of PCA patients. Collectively, our data points to ASPM as a novel oncoprotein and an essential regulator of Wnt signaling and cancer stemness in PCA, which has important clinical and therapeutic significance.

SSeCKS/Akap12 suppresses metastatic melanoma lung colonization by attenuating Src-mediated pre-metastatic niche crosstalk.

SSeCKS/Gravin/AKAP12 (SSeCKS) controls metastasis-associated PKC and Src signaling through direct scaffolding activity. SSeCKS is downregulated in the metastases of many human cancer types, and its forced re-expression suppresses the metastatic behavior of prostate cancer cells. SSeCKS is also downregulated in breast and prostate cancer stroma, and SSeCKS-null mice (KO) are metastasis-prone, suggesting a role in suppressing formation of the pre-metastatic niche. Here, we show that lung colonization and metastasis formation by B16F10 and SM1WT1[BrafV600E] mouse melanoma cells is 9-fold higher in syngeneic KO compared to WT hosts, although there is no difference in orthotopic tumor volumes. Although melanoma cells adhered equally to KO or WT lung fibroblasts (LF), co-injection of melanoma cells with KO (vs. WT) LF increased lung macrometastasis formation in WT hosts, marked by increased melanoma colonization at foci of leaky vasculature. Increased melanoma adhesion on KO lung endothelial cells (LEC) was facilitated by increased E-Selectin levels and by increased STAT3-regulated secretion of senescence-associated factors from KO-LF, such as Vegf. Finally, the ability of SSeCKS to attenuate IFNα-induced Stat3 activation in KO-LF required its Src-scaffolding domain. Taken together, these data suggest that SSeCKS normally suppresses metastatic colonization in the lung by attenuating the expression of Selectin adhesion proteins, which can be controlled autonomously by local endothelial cells or enhanced by senescence factors secreted by neighboring fibroblasts in a SSeCKS-regulated, Src/Stat3-dependent manner.

A short peptide reverses the aggressive phenotype of prostate cancer cells

Previous studies have demonstrated that fibroblast growth factor 8b (FGF8b) is up-regulated in a large proportion of prostate cancer patients, and plays a key role in the aggressive progress of prostate cancer. Herein, we investigated the effects of a short peptide derived from the gN helix domain of FGF8b on the metastatic behaviors of prostate cancer cells. The results demonstrated that the synthetic peptide might reverse the effects of FGF8b on cell proliferation, migration and invasion by suppressing the activation of MAPK and Akt signaling cascades, and reducing the expressions of the metastasis-related proteins, resulting in suppression of the aggressive phenotype of the prostate cancer cells. Collectively, these results underline the therapeutic potential of the FGF8b mimic peptide in advanced prostate cancer.

Chronic hypoxia-induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin-B1.

Advanced solid tumors are exposed to hypoxic conditions over longer periods of time as they grow. Tumor hypoxia is a major factor that induces malignant progression, but most previous studies on tumor hypoxia were performed under short‐term hypoxia for up to 72 hours and few studies have focused on tumor response to chronic hypoxic conditions. Here we show a molecular mechanism by which chronic hypoxia promotes invasive behavior in prostate cancer cells. We found that an epithelial‐mesenchymal transition (EMT)‐driving transcription factor, slug, is specifically upregulated under chronic hypoxia and promotes tumor cell migration and invasion. Unexpectedly, processes associated with EMT, such as loss of E‐cadherin, are not observed under chronic hypoxia. Instead, expression of ephrin‐B1, a ligand of Eph‐related receptor tyrosine kinases, is markedly induced by slug through E‐box motifs and promotes cell migration and invasion. Furthermore, slug and ephrin‐B1 are highly coexpressed in chronic hypoxic cells of human prostate adenocarcinoma tissues after androgen deprivation, which is known to cause tumor hypoxia. Taken together, these results indicate that chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating the expression of ephrin‐B1. In addition, ephrin‐B1 may be a novel therapeutic target in combination with androgen deprivation therapy for aggressive prostate cancer.

Abstract B086: Monocytes-produced Chitinase-3-like 1 is a driver of metastatic behavior in advanced prostate cancer patients

Abstract Purpose: The identification, functional status, distribution, and interactions of immune cells in PCa patients have yet to be fully characterized. Understanding these details could provide key insights as to how promising current and developing immunotherapies might be directed toward PCa. Recruited myeloid cells promote cancer initiation, malignant progression, metastasis, and resistance to therapy in the tumor niche. In the present study, we tested the hypothesis that circulating blood monocytes from advanced prostate cancer (PCa) patients exhibit a tumor-promoting phenotype and directly influence the tumor microenvironment in response to tumor-derived signals. Experimental Design: Monocytes from metastatic (PCa-M) and nonmetastatic (PCa-N) patient blood samples were isolated, cultured, and conditioned media (CM) was obtained. To evaluate the role of monocytes in metastatic behavior of PCa cells, in vitro invasion (via Matrigel) and motility assays (Incucyte Live Cell Analysis System) were performed using monocyte-CM. To identify the candidate PCa-M monocyte-secreted protein(s) that might be implicated in tumor progression, we analyzed the CM using Proteome Profiler Analysis (R&D). Data were validated using ELISA and qPCR. Results: Herein, we report that CM from circulating monocytes in patients with PCa/mCRPC (metastatic castration-resistant PCa) increased motility and invasion of epithelial PCa cells. Proteome Profiler Analysis revealed that monocyte-derived CM from metastatic PCa/mCRPC patients presented high levels of YKL-40 (Chitinase-3-like 1; CHI3L1) when compared with PCa-N patients and healthy controls (HC), classified as individuals with no known history of cancer. The only described receptor for YKL-40, interleukin-13 receptor α2 (IL-13Rα2), was significantly upregulated in ARCaPm cells; accordingly, we observed that the activation of IL-13Rα2 from PCa-M CM increased the invasiveness of ARCaPm. In addition, siRNA directed against this receptor in ARCaPm cells caused a significant reduction in the invasiveness of these cells in the presence of CM from PCa-M patients. Conclusions: We show that circulating monocytes from metastatic PCa/mCRPC patients exert a tumor-promoting role via the secretion of YKL-40, and YKL-40 demands further exploration as a possible therapeutic target in advanced PCa. Citation Format: Karen A. Cavassani, Rebecca Meza, David Habiel, Jie-Fu Chen, Alexander Montes, Manisha Tripathi, Gislaine Martins, Timothy R. Crother, Cory M. Hogaboam, Sungyong You, Neil Bhowmick, Edwin Posadas. Monocytes-produced Chitinase-3-like 1 is a driver of metastatic behavior in advanced prostate cancer patients [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B086.

Abstract 3799: Heparan sulfate modifiers and signaling between tumor-associated macrophages and cancer-associated fibroblasts in a biomimetic hydrogel model of bone-metastatic prostate cancer

Abstract The establishment of a new tumor niche for most carcinomas is orchestrated primarily by the bidirectional engagement of cancer cells and fibroblasts. Cancer cells reprogram and activate fibroblasts into a wound healing-like state and, in turn, these cancer-associated fibroblasts (CAFs) react by remodeling the extracellular matrix (ECM) and secreting chemokines and growth factors. Macrophages are rapidly recruited to the tumor and represent the primary immune cell in the microenvironment. Tumor-associated macrophages (TAMs) enter a wound healing-like loop, displaying the full spectrum of macrophage phenotypes and behaviors as the disease progresses. Among these activities is the ability to modulate growth factor bioavailability. Deposition of the heparan sulfate (HS) proteoglycan perlecan/HSPG2 in the ECM and secretion of HS modulators are increased as result of the cancer-CAF-TAM trialogue and play critical roles in the availability and signaling of growth factors, cytokines, and chemokines. In this work, we developed a novel, biomimetic 3D hydrogel system composed of hyaluronic acid and collagen I (1:1) to study prostate cancer bone metastases. Our goal was to explore the molecular signaling between TAMs and CAFs to learn how this interaction affects the expression of the enzymes that modulate HS as a growth factor depot. Essential modulators of HS include heparanase (HPSE) and sulfatases 1 and 2 (SULF1, 2), all of which selectively release growth factors to influence signaling in the tumor microenvironment. We found that alternatively activated macrophages (M2) and TAMs (M2-like) increase levels of HPSE and SULFs in CAFs. We next examined the impact of TAM-CAF co-signaling on prostate cancer viability, growth, and dependence on HS modulators. Our data show that as a consequence of the TAM-CAF interaction, the elevated levels of HS-modifying enzymes are sufficient to trigger substantial changes in prostate cancer cell behavior, including viability, proliferation, and response to growth factors. We currently are assessing if these outcomes are seen in a series of patient-derived prostate xenograft lines representing different prostate cancer subtypes. We conclude that our 3D hydrogel model is a physiologically relevant in vitro system to discover fundamental aspects of HS signaling in the tumor microenvironment and thus uncover new ways to reduce the bioavailability of growth factors in the prostate cancer microenvironment and interfere with cancer progression. (Supported by NIH P01 and CAPES.) Citation Format: Fabio Henrique Brasil da Costa, Nora M. Navone, Mary C. Farach-Carson, Daniel D. Carson. Heparan sulfate modifiers and signaling between tumor-associated macrophages and cancer-associated fibroblasts in a biomimetic hydrogel model of bone-metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3799.

Abstract 4260: Dynamic modeling of macrophage plasticity in bone metastatic prostate cancer

Abstract Tumor-associated macrophages, particularly those with an anti-inflammatory phenotype, have long been implicated in the progression of primary solid malignancies, including prostate cancer. Metastatic prostate cancer typically manifests in the bone where it induces painful osteogenic lesions that are incurable. Bone is naturally rich in myeloid-derived macrophages whose temporal polarization into pro- (M1) and anti-inflammatory (M2) phenotypes is critical for regulating the bone repair program mediated by bone-resorbing osteoclasts and bone-building osteoblasts. However, the dynamics of macrophage polarization in the context of bone metastatic prostate cancer are underexplored and difficult to address with traditional biologic approaches. To address the role of macrophage polarization in the context of bone metastatic prostate cancer, we first investigated the macrophage polarization temporal dynamics in normal bones and analyzed macrophage plasticity in vivo subsequent to intratibial injury. Bone marrows were isolated at several time points and profiled by flow cytometry for pro- and anti-inflammatory monocyte macrophage content. Contralateral tibias were analyzed for bone volume, osteoblast and osteoclast numbers. We then designed a mathematical model describing the different cell population dynamics in the bones. Generation of the model required testing a number of assumptions regarding macrophage polarization behavior. For example, it is unknown whether M1 resolves at the initial stages of bone injury repair through death or by repolarizing into M2. For each aspect, competing mechanistic assumptions were proposed and simulated by sets of ODEs. The best-fitting assumptions for each aspect were integrated into a single comprehensive ODE model to fully describe the dynamics of the bone resident cell populations during bone remodeling. This experimentally validated model is now being used to address how bone resident cells respond to metastatic prostate cancer cells. In conclusion, we have generated an ODE model that describes macrophage polarization over time during bone repair and how pro- and anti-inflammatory macrophages interact with bone stromal cells. The mathematical model predictions are in agreement with our biologic experiments in vivo and will allow us to interrogate how macrophage polarization impacts the behavior of metastatic prostate cancer cells in the bone microenvironment. Citation Format: Etienne A. Baratchart, Chen Hao Lo, Conor Lynch, David Basanta. Dynamic modeling of macrophage plasticity in bone metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4260.

Abstract 5208: Monocyte-produced Chitinase-3-like 1 is a driver of metastatic behavior in prostate cancer patients

Abstract Purpose: The identification, functional status, distribution, and interactions of immune cells in prostate cancer (PCa) patients have yet to be fully characterized. Understanding these details could provide key insights as to how promising current and developing immunotherapies might be directed toward PCa. Recruited myeloid cells are known to promote cancer initiation, malignant progression, metastasis, and resistance to therapy in the tumor niche. In the present study, we tested the hypothesis that circulating blood monocytes from advanced PCa patients exhibit a protumor phenotype and directly influence the tumor microenvironment in response to tumor-derived signals. Experimental Design: Monocytes from metastatic (PCa-M) and nonmetastatic (PCa-N) patient blood samples were isolated, cultured, and conditioned media (CM) was obtained. To evaluate the role of monocytes in metastatic behaviors of PCa cells, in vitro invasion (via Matrigel) and motility assays (Incucyte® Live Cell Analysis System) were performed using monocyte-CM. To identify the candidate PCa-M monocyte-secreted protein(s) that might be implicated in tumor progression, we analyzed the CM using Proteome Profiler Analysis (R&D). Data were validated using ELISA and qPCR. Results: Herein, we report that CM from circulating monocytes in patients with PCa/mCRPC (metastatic castration-resistant PCa) increased motility and invasion of epithelial PCa cells in vitro. Proteome Profiler Analysis revealed that monocyte-derived CM from metastatic PCa/mCRPC patients presented high levels of Chitinase-3-like 1 (CHI3L1, YKL-40) when compared with PCa-N patients and healthy-control individuals (HC). The only described receptor for CHI3L1, interleukin-13 receptor α2 (IL-13Rα2), was significantly upregulated in the human metastatic prostate cancer cell line, ARCaPM. Accordingly, we observed that the activation of IL-13Rα2 from PCa-M CM increased the invasiveness of ARCaPM cells, and siRNA directed against this receptor significantly reduced invasiveness of these cells in the presence of CM from PCa-M patients. Conclusions: Thus, we show that circulating monocytes from metastatic PCa/mCRPC patients exert a tumor-promoting role via the secretion of CHI3L1, and CHI3L1 demands further exploration as a possible therapeutic target in advanced PCa. Citation Format: Karen A. Cavassani, Rebecca J. Meza, David M. Habiel, Jie-Fu Chen, Alexander Montes, Manisha Tripathi, Gislaine A. Martins, Timothy R. Crother, Sungyong You, Cory M. Hogaboam, Neil Bhowmick, Edwin M. Posadas. Monocyte-produced Chitinase-3-like 1 is a driver of metastatic behavior in prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5208.

Using high throughput microtissue culture to study the difference in prostate cancer cell behavior and drug response in 2D and 3D co-cultures

BackgroundThere is increasing appreciation that non-cancer cells within the tumour microenvironment influence cancer progression and anti-cancer drug efficacy. For metastatic prostate cancer (PCa), the bone marrow microenvironment influences metastasis, drug response, and possibly drug resistance.MethodsUsing a novel microwell platform, the Microwell-mesh, we manufactured hundreds of 3D co-culture microtissues formed from PCa cells and bone marrow stromal cells. We used luciferase-expressing C42B PCa cells to enable quantification of the number of PCa cells in complex microtissue co-cultures. This strategy enabled us to quantify specific PCa cell growth and death in response to drug treatment, in different co-culture conditions. In parallel, we used Transwell migration assays to characterize PCa cell migration towards different 2D and 3D stromal cell populations.ResultsOur results reveal that PCa cell migration varied depending on the relative aggressiveness of the PCa cell lines, the stromal cell composition, and stromal cell 2D or 3D geometry. We found that C42B cell sensitivity to Docetaxel varied depending on culture geometry, and the presence or absence of different stromal cell populations. By contrast, the C42B cell response to Abiraterone Acetate was dependent on geometry, but not on the presence or absence of stromal cells.ConclusionIn summary, stromal cell composition and geometry influences PCa cell migration, growth and drug response. The Microwell-mesh and microtissues are powerful tools to study these complex 3D interactions.

High C-X-C motif chemokine5 expression is associated with malignant phenotypes of prostate cancer cells via autocrine and paracrine pathways.

The present study aimed to examine the effects and mechanisms of exogenous C-X-C motif chemokine5(CXCL5) and lentiviral CXCL5 overexpression on the regulation of malignant behaviors of prostate cancer cells invitro and in a nude mouse xenograft model. The expression levels of CXCL5 and a number of tumor-related genes were assessed by using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), western blotting, ELISA, or immunohistochemistry in normal and cancerous prostate cells and tissues. Cell proliferation, colony formation, and Transwell assays were performed to determine the effects of exogenous, autocrine, and paracrine CXCL5 on prostate cancer cell proliferative and migratory capacity. The results indicated that CXCL5 expression was upregulated in PC‑3 and DU145 prostate cancer cells, in WPMY‑1 normal prostate stromal cells, and in RWPE‑1 prostate epithelial cells, as well as in prostate cancer tissue specimens. Exogenous CXCL5 exposure resulted in increase in prostate cancer cell proliferation, colony formation, and migration. In cells transfected with a CXCL5 overexpression vector, in cells cultured in conditioned medium from CXCL5-overexpressing WPMY cells, and in cells co-cultured with CXCL5‑OE WPMY cells prostate cancer cell malignant phenotypes were induced in an autocrine/paracrine fashion invitro; similar results were observed in nude mouse xenografts. CXCL5 overexpression also regulated expression of tumor-related genes, including BAX, N-Myc downstream-regulated gene3, extracellular signal-regulated kinase1/2, C-X-C chemokine receptor type 2, interleukin18, Bcl‑2, and caspase‑3. These data demonstrated that CXCL5 expression was upregulated in prostate cancer tissues and that exogenous CXCL5 protein exposure or CXCL5 overexpression promoted malignant phenotypes of prostate cancer cells invitro and invivo.

Micro-RNA-186-5p inhibition attenuates proliferation, anchorage independent growth and invasion in metastatic prostate cancer cells

BackgroundDysregulation of microRNA (miRNA) expression is associated with hallmarks of aggressive tumor phenotypes, e.g., enhanced cell growth, proliferation, invasion, and anchorage independent growth in prostate cancer (PCa).MethodsSerum-based miRNA profiling involved 15 men diagnosed with non-metastatic (stage I, III) and metastatic (stage IV) PCa and five age-matched disease-free men using miRNA arrays with select targets confirmed by quantitative real-time PCR (qRT-PCR). The effect of miR-186-5p inhibition or ectopic expression on cellular behavior of PCa cells (i.e., PC-3, MDA-PCa-2b, and LNCaP) involved the use bromodeoxyuridine (BrdU) incorporation, invasion, and colony formation assays. Assessment of the impact of miR-186-5p inhibition or overexpression on selected targets entailed microarray analysis, qRT-PCR, and/or western blots. Statistical evaluation used the modified t-test and ANOVA analysis.ResultsMiR-186-5p was upregulated in serum from PCa patients and metastatic PCa cell lines (i.e., PC-3, MDA-PCa-2b, LNCaP) compared to serum from disease-free individuals or a normal prostate epithelial cell line (RWPE1), respectively. Inhibition of miR-186-5p reduced cell proliferation, invasion, and anchorage-independent growth of PC-3 and/or MDA-PCa-2b PCa cells. AKAP12, a tumor suppressor target of miR-186-5p, was upregulated in PC-3 and MDA-PCa-2b cells transfected with a miR-186-5p inhibitor. Conversely, ectopic miR-186-5p expression in HEK 293 T cells decreased AKAP12 expression by 30%. Both pAKT and β-catenin levels were down-regulated in miR-186-5p inhibited PCa cells.ConclusionsOur findings suggest miR-186-5p plays an oncogenic role in PCa. Inhibition of miR-186-5p reduced PCa cell proliferation and invasion as well as increased AKAP12 expression. Future studies should explore whether miR-186-5p may serve as a candidate prognostic indicator and a therapeutic target for the treatment of aggressive prostate cancer.

HepaCAM inhibits the malignant behavior of castration-resistant prostate cancer cells by downregulating Notch signaling and PF-3084014 (a γ-secretase inhibitor) partly reverses the resistance of refractory prostate cancer to docetaxel and enzalutamide in vitro.

Castration-resistant prostate cancer (CRPC) continues to be a major challenge in the treatment of prostate cancer (PCa). The expression of hepatocyte cell adhesion molecule (HepaCAM), a novel tumor suppressor, is frequently downregulated or lost in PCa. Overactivated Notch signaling is involved in the development and progression of PCa, including CRPC. In this study, we found that the activities of Notch signaling were elevated, while HepaCAM expression was decreased in CRPC tissues compared with matched primary prostate cancer (PPC) tissues. In addition, HepaCAM negativity was found to be associated with a worse progression-free survival (PFS). Furthermore, the overexpression of HepaCAM induced by transfection with a HepaCAM overexpression vector (Ad-HepaCAM) exerted antitumor effects by decreasing the proliferation, and suppressing the invasion and migration of bicalutamide-resistant (Bica-R) cells and enzalutamide-resistant (Enza-R) cells. Importantly, we found that the antitumor effects of HepaCAM on the resistant cells were associated with the downregulation of Notch signaling. Moreover, we revealed that PF-3084014 (a γ-secretase inhibitor) re-sensitized Enza-R cells to enzalutamide, and sequential dual-resistant (E+D-R) cells to docetaxel. Additionally, the findings of this study demonstrated that the use of PF-3084014 alone exerted potent antitumor effect on the resistant cells in vitro. On the whole, this study indicates that HepaCAM potentially represents a therapeutic target and PF-3084014 may prove to a promising agent for use in the treatment of refractory PCa.

PRL‑3 increases the aggressive phenotype of prostate cancer cells in�vitro and its expression correlates with high-grade prostate tumors in patients

The increased expression of phosphatase of regenerating liver-3 (PRL-3) has been shown to be associated with the aggressive and metastatic phenotype of different solid tumors. However, it is not known whether PRL-3 plays a similar role in the progression of prostate cancer (PCa). In this study, immunoblot analysis of androgen receptor (AR)-positive PCa lines (LNCaP and LNCaP-SF) revealed the constitutive cytoplasmic expression of PRL-3, and stimulation with R1881 (AR agonist) rapidly increased the nuclear translocation of PRL-3. The AR-negative cell lines exhibited negligible PRL-3 expression, and the ectopic overexpression of PRL-3 increased both the proliferative and invasive potential of PC3 and DU145 cells. In addition, we measured PRL-3 protein expression in human prostate tumor sections. A high-density prostate tumor microarray (TMA) was immunostained to assess whether PRL-3 expression and its subcellular localization (cytoplasmic and nuclear levels) is associated with the Gleason score (GS), Gleason grade (GG) and tumor stage (T-stage). Digital image analysis (DIA) revealed that PRL-3 expression was significantly higher in the malignant cores, as compared to the non-malignant areas. Increases in both total and nuclear PRL-3 levels were also associated with a higher GS and GG. Metastatic tumors (T4-stage) had lower cytoplasmic, but higher nuclear PRL-3 levels. Furthermore, the nuclear/cytoplasmic ratio for PRL-3 in the tumors graded as GS7 could effectively distinguish between indolent (3+4) and aggressive (4+3) disease. Thus, our experiments using PCa lines suggested that PRL-3 is an AR-regulated gene and its androgen-induced nuclear localization may increase the aggressive behavior of PCa cells. Furthermore, the digital analysis of immunostained tumor sections suggested that PRL-3 may be an effective biomarker of high-grade PCa, and its nuclear/cytoplasmic ratio may be used to distinguish between indolent vs. aggressive tumors.

Abstract 4344: Cd146 modulates the malignant phenotype in human prostate cancer

Abstract Prostate Cancer (PCa) is the most frequent cancer in males and the second leading cause of death from cancer in men. When PCa progress from androgen-responsive to castration resistance, the formation of incurable metastases, mainly in the bone, is almost inevitable. Therefore, understanding the factors that regulate homing and survival of metastatic cancer cells in the bone is important for the identification of new therapeutic targets. High CD146 expression has been measured in the stroma of lytic and blastic lesions in preclinical models of PCa bone metastasis. The objective of this study is to characterize the role of CD146 in the maintenance of the aggressive and invasive phenotype in human PCa. We used shRNAs to knockdown the expression of CD146 in the lytic PC-3M-Pro4Luc2dTomato and in the blastic C4-2BdTomato PCa cell lines. We validated the knockdown at protein level and tested the effect with functional assays such as migration, proliferation. We used RT-qPCR to test CD146 knockdown on EMT markers. We measured the effect of the knockdown on the maintenance of cancer stem/progenitor-like cells by ALDEFLUOR assay. CD146 knockdown reduced proliferation in PC-3M-Pro4Luc2dTomato PCa cells and resulted in increased E-Cadherin expression. Conversely, no effect on proliferation was measured on C4-2BdTomato cells. It has been described that metastatic human PCa cells target the hematopoietic stem cell (HSC) niche in the bone marrow at the level of an “endosteal/osteoblast” niche and a “vascular/perivascular” niche. We optimized an in vitro model of “osteoblast niche” to study the behavior of prostate cancer cells upon co-culture with osteoblasts and to measure the resulting effects on cancer stem/progenitor-like markers. Our results showed that CD146 is required for the osteoblast-mediated induction of ALDH activity on PCa cells and CD146 knockdown prevented the increase in the size of the ALDHhigh subpopulation in the tumor cells, mediated by human osteoblasts. Additionally, CD146 knockdown in PCa cells co-cultured with osteoblast, reduced the amount of CD146 expressed by osteoblasts compared to non-targeted control. Finally, we showed that CD146 is significantly increased in the highly metastatic ALDHhigh cells and identified a new subset of ALDHhigh / CD146high cells which could be depleted upon CD146 knockdown. In Conclusion, we detected a novel subset of ALDHhigh/CD146high cells and found that CD146 influences the maintenance of an aggressive-mesenchymal phenotype in human PCa. Therefore, CD146 represents a promising molecule to modulate the behavior of aggressive PCa cells. Note: This abstract was not presented at the meeting. Citation Format: Eugenio Zoni, Letizia Astrologo, Janine Melsen, Sofia Karkampouna, Irena Klima, Joël Grosjean, Peter C. Gray, Gabri van der Pluijm, Marco G. Cecchini, Marianna Kruithof-de Julio, George N. Thalmann. Cd146 modulates the malignant phenotype in human prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4344. doi:10.1158/1538-7445.AM2017-4344

Behavior of prostate cancer cells in a nanohydroxyapatite/collagen bone scaffold.

Prostate cancer (PCa) is the second leading cause of death among men in Europe and U.S. The metastatic dissemination pattern of PCa is unique, developing bone metastasis as the only site of progression, consequently with a prognosis very poor. The cancer cells interactions within the surrounding bone environment are critical for tumor growth and progression. Secreted protein, acidic and rich in cysteine (SPARC) is described to be involved in PCa cells migration and invasion into bone. Three-dimensional (3D) in vitro systems that are able to closely resemble the in vivo microenvironment are recently taking importance in cancer research. Original nanohydroxyapatite/collagen scaffolds were designed to resemble bone microenvironment in order to be applied as substitutes in bone defects and as potential biomaterials to mimic skeletal tumors. In fact, these 3D structures were cytocompatible and able to support osteoblast (MC3T3-E1) colonization and to promote bone ingrowth. Additionally, SPARC adsorption onto the scaffolds affected PC3 and LNCaP PCa cell lines behavior. PC3 cells were found to adapt and colonize the scaffolds, differing from LNCaP where cells underwent morphogenic changes and grew as clusters. Furthermore, for the tested SPARC concentration, SPARC plays a role in retaining LNCaP cells at the latter time points while with PC3 cells no significant differences were observed. This characterization study is required to establish a bone model to provide new insights into the poorly understood PCa mechanisms of metastasis to bone and the generation of improved therapies. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2035-2046, 2017.

Comparative Study of Multicellular Tumor Spheroid Formation Methods and Implications for Drug Screening.

Improved in vitro models are needed to better understand cancer progression and bridge the gap between in vitro proof-of-concept studies, in vivo validation, and clinical application. Multicellular tumor spheroids (MCTS) are a popular method for three-dimensional (3D) cell culture, because they capture some aspects of the dimensionality, cell-cell contact, and cell-matrix interactions seen in vivo. Many approaches exist to create MCTS from cell lines, and they have been used to study tumor cell invasion, growth, and how cells respond to drugs in physiologically relevant 3D microenvironments. However, there are several discrepancies in the observations made of cell behaviors when comparing between MCTS formation methods. To resolve these inconsistencies, we created and compared the behavior of breast, prostate, and ovarian cancer cells across three MCTS formation methods: in polyNIPAAM gels, in microwells, or in suspension culture. These methods formed MCTS via proliferation from single cells or passive aggregation, and therefore showed differential reliance on genes important for cell-cell or cell-matrix interactions. We also found that the MCTS formation method dictated drug sensitivity, where MCTS formed over longer periods of time via clonal growth were more resistant to treatment. Toward clinical application, we compared an ovarian cancer cell line MCTS formed in polyNIPAAM with cells from patient-derived malignant ascites. The method that relied on clonal growth (PolyNIPAAM gel) was more time and cost intensive, but yielded MCTS that were uniformly spherical, and exhibited the most reproducible drug responses. Conversely, MCTS methods that relied on aggregation were faster, but yielded MCTS with grapelike, lobular structures. These three MCTS formation methods differed in culture time requirements and complexity, and had distinct drug response profiles, suggesting the choice of MCTS formation method should be carefully chosen based on the application required.

Morus rubra Extract Induces G1 Cell Cycle Arrest and Apoptosis in Human Lung and Prostate Cancer Cells

Abstract: Objective: Cancer is one of the most deadly types of disease and evasion from apoptosis and unstoppable cell proliferation are accepted as its distinctive features. Many studies have evaluated the cytotoxic effect of different Morus species but, there is no study about cytotoxic effect of Morus rubra. In this study we aimed to evaluate phenolic composition, antioxidant properties and cytotoxic effect of acidified dimethyl sulfoxide extract of M. rubra (AMRE). Method: Antioxidant properties, phenolic composition and cytotoxic effect of AMRE were determined using spectrophotometric methods, HPLC, and MTT assay, respectively. Then, mechanisms of cytotoxic effect of AMRE on human prostate (PC-3) and lung (A549) cancer cells were examined in regard to cell cycle, apoptosis and mitochondrial membrane potential using flow cytometric methods. Results: Total phenolic content and reducing power values were 11.9 mg gallic acid equivalents and 42.9 mg trolox equivalents per g sample, respectively. Ascorbic and gallic acid were detected in AMRE as major antioxidant compounds. We determined that AMRE increased cell cycle arrest at G1 phase and exhibited apoptotic features via decreasing mitochondrial membrane potential in both prostate and lung cancer cells. Conclusion: These findings demonstrate that M. rubra extract can affect the behavior of human prostate and lung cancer cells in vitro conditions, and this effect now needs to be investigated in vivo. Key words: Apoptosis, Cell Cycle, Cytotoxicity, Lung Neoplasms, Morus rubra, Prostate Neoplasms.

Targeting of NHERF1 through RNA interference inhibits the proliferation and migration of metastatic prostate cancer cells.

The present study aimed to investigate the effects of Na+/H+ exchanger regulatory factor 1 (NHERF1) gene knockdown, using short-hairpin RNA (shRNA), on the malignant behaviors of prostate cancer cells. A pSuper.puro NHERF1 shRNA vector was transfected into PC-3M prostate cancer cells using Lipofectamine 2000. Stable cell lines were obtained and NHERF1 knockdown was verified through western blot analysis. MTT assays were then used to measure PC-3M cell proliferation; in addition, cell migration was assessed using a wound healing assay. Flow cytometry was employed in order to determine the effects of NHERF1 knockdown on apoptosis. Expression levels of apoptotic pathway proteins B cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein were then determined by western blot analysis. The results demonstrated that shRNA knockdown of NHERF1 significantly suppressed the proliferation of PC-3M cells by >50%. In addition, knockdown of NHERF1 significantly inhibited the migration of PC-3M cells. PC-3M cells harboring NHERF1 shRNA exhibited significantly increased apoptosis, with an ~4-fold increase compared with that of the parental PC-3M cells and cells transfected with an empty vector. Furthermore, the results revealed that knockdown of NHERF1 reduced the protein expression of Bcl-2, although the expression of Bax was unaltered. In conclusion, NHERF1 knockdown using shRNA inhibited the proliferation and migration of PC-3M cells and promoted apoptosis, highlighting the role of NHERF1 in prostate cancer progression.

First Evidence that Sika Deer (Cervus nippon) Velvet Antler Extract Suppresses Migration of Human Prostate Cancer Cells.

Deer velvet antler (DVA) is one of the most popular medicines in China. Numerous studies have demonstrated that velvet antler possess biological effects. However, data regarding its anti-migration activity on prostate cancer is scarce. In this study, we investigated the inhibitory effect of top DVA (T-DVA) on the expression of prostate-specific antigen (PSA) and migration-related genes in the human prostate cancer cell, LNCaP. The T-DVA down-regulated the expression of PSA. In addition, the RadiusTM assay revealed that T-DVA inhibited the migration behavior of prostate cancer cells. Furthermore, the expression of matrix metalloproteinase (MMP)-9 and vascular endothelial growth factor (VEGF) was also decreased with T-DVA. On the contrary, T-DVA increased the tissue inhibition of metalloproteinase (TIMP)-1 and (TIMP)-2. Taken together, our findings indicate that the T-DVA possesses anti-migration activity on prostate cancer cells. This is the first study of DVA to report the anti-migration activity on prostate cancer.