Prostate Cancer Cell Tumor Growth Research Articles

Androgen deprivation induces neuroendocrine phenotypes in prostate cancer cells through CREB1/EZH2-mediated downregulation of REST.

Although effective initially, prolonged androgen deprivation therapy (ADT) promotes neuroendocrine differentiation (NED) and prostate cancer (PCa) progression. It is incompletely understood how ADT transcriptionally induces NE genes in PCa cells. CREB1 and REST are known to positively and negatively regulate neuronal gene expression in the brain, respectively. No direct link between these two master neuronal regulators has been elucidated in the NED of PCa. We show that REST mRNA is downregulated in NEPC cell and mouse models, as well as in patient samples. Phenotypically, REST overexpression increases ADT sensitivity, represses NE genes, inhibits colony formation in culture, and xenograft tumor growth of PCa cells. As expected, ADT downregulates REST in PCa cells in culture and in mouse xenografts. Interestingly, CREB1 signaling represses REST expression. In studying the largely unclear mechanism underlying transcriptional repression of REST by ADT, we found that REST is a direct target of EZH2 epigenetic repression. Finally, genetic rescue experiments demonstrated that ADT induces NED through EZH2's repression of REST, which is enhanced by ADT-activated CREB1 signaling. In summary, our study has revealed a key pathway underlying NE gene upregulation by ADT, as well as established novel relationships between CREB1 and REST, and between EZH2 and REST, which may also have implications in other cancer types and in neurobiology.

MAGI2 antisense RNA 3 (MAGI2-AS3) knockdown contributes to prostate cancer by downregulating proline-rich membrane anchor 1 (PRIMA1) via miR-142-3p

IntroductionLong non-coding RNA Membrane-Associated Guanylate Kinase Inverted 2 Antisense RNA 3 (MAGI2-AS3) has been identified as a predictive characteristic for prostate cancer (PC). However, the underlying mechanism of MAGI2-AS3 in PC is yet unclear.Material and methodsThe gene expression was detected via reverse transcription quantitative PCR. Cell viability, apoptosis and invasion were detected via cell counting kit-8, caspase-3 activity, flow cytometry, and transwell assays. Tumor xenograft experiment was performed to measure the tumor growth in vivo. Luciferase and radioimmunoprecipitation assays were utilized to determine the association between microRNA (miR)-142-3p and MAGI2-AS3 or proline-rich membrane anchor 1 (PRIMA1).ResultsMAGI2-AS3 and PRIMA1 were established to be downregulated in PC, whereas miR-142-3p exhibited the upregulation in PC. In vitro and in vivo loss-of-function assays demonstrated that MAGI2-AS3 silencing increased the viability and invasiveness, enhanced the tumor growth of PC cells, while reducing apoptosis. PRIMA1 silencing in PC cells showed the similar effect as MAGI2-AS3 silencing. Moreover, the miR-142-3p inhibitor reversed the impacts of the downregulation of MAGI2-AS3 or PRIMA1 on the malignant behavior of PC cells.ConclusionsMAGI2-AS3 knockdown enhanced the malignant behavior of PC cells by targeting miR-142-3p to suppress PRIMA1 expression. Our findings reveal that MAGI2-AS3 can be a promising therapeutic target for the treatment of PC.

Caffeic acid phenethyl ester suppresses EGFR/FAK/Akt signaling, migration, and tumor growth of prostate cancer cells

Epidermal growth factor receptor (EGFR) is upregulated in prostate cancer (PCa). However, suppression of EGFR did not improve the patient outcome, possibly due to the activation of PI3K/Akt signaling in PCa. Compounds able to suppress both PI3K/Akt and EGFR signaling may be effective for treating advanced PCa. We examined if caffeic acid phenethyl ester (CAPE) simultaneously suppresses the EGFR and Akt signaling, migration and tumor growth in PCa cells. Wound healing assay, transwell migration assay and xenograft mice model were used to determine the effects of CAPE on migration and proliferation of PCa cells. Western blot, immunoprecipitation, and immunohistochemistry staining were performed to determine the effects of CAPE on EGFR and Akt signaling. CAPE treatment decreased the gene expression of HRAS, RAF1, AKT2, GSK3A, and EGF and the protein expression of phospho-EGFR (Y845, Y1069, Y1148, Y1173), phospho-FAK, Akt, and ERK1/2 in PCa cells. CAPE treatment inhibited the EGF-induced migration of PCa cells. Combined treatment of CAPE with EGFR inhibitor gefitinib showed additive inhibition on migration and proliferation of PCa cells. Injection of CAPE (15mg/kg/3 days) for 14 days suppressed the tumor growth of prostate xenografts in nude mice as well as suppressed the levels of Ki67, phospho-EGFR Y845, MMP-9, phospho-Akt S473, phospho-Akt T308, Ras, and Raf-1 in prostate xenografts. Our study suggested that CAPE can simultaneously suppress the EGFR and Akt signaling in PCa cells and is a potential therapeutic agent for advanced PCa.

Demethyltransferase FTO alpha-ketoglutarate dependent dioxygenase (FTO) regulates the proliferation, migration, invasion and tumor growth of prostate cancer by modulating the expression of melanocortin 4 receptor (MC4R)

ABSTRACT N6-Methyladenosine (m6A) is the most abundant modifications in human messenger RNAs (mRNAs). This study aimed at investigating the function and mechanism of demethyltransferase fat mass and obesity-associated protein (FTO) in prostate cancer(PCa). The expression level of FTO in PCa was detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Besides, the impacts of FTO on the proliferation, migration and invasion of PCa cells were also detected by cell counting kit-8 (CCK-8), 5-ethynyl-2ʹ-deoxyuridine (EdU) and transwell assays. Furthermore, we also explored the potential mechanism of FTO in PCa. The results showed that FTO expression was decreased in PCa, and the low expression of FTO showed an obvious relevance to the clinical characteristics. Downregulation of FTO facilitated the proliferation, migration, invasion and tumor growth of PCa cells. Besides, MC4R displayed a remarkably high expression in PCa tissues, whose expression and m6A level were regulated by FTO. Meanwhile, the in vitro experiments revealed that highly expressed FTO partially reversed the facilitating effect of highly expressed MC4R on the malignant phenotype of PCa cells. Overall, FTO was downregulated in PCa and its expression level showed a relevance to the prognosis of PCa patients. Additionally, FTO could regulate the proliferation, migration and invasion of PCa via regulating the expression level of MC4R.

Knockdown of sterol O-acyltransferase 1 (SOAT1) suppresses SCD1-mediated lipogenesis and cancer procession in prostate cancer

Prostate cancer (PCa) is one of the most fatal malignant tumors that occurs in the prostate epithelium, especially in older men, the mortality of which ranks sixth among all cancer-related deaths. It has been urgently needed to elucidate the pathogenesis of PCa and provide promising therapeutic targets for PCa treatment. The Sterol O-acyltransferase 1 (SOAT1), cholesterol metabolism enzyme, was widely expressed in various cancer tissues, resulting in cancer progression. SOAT1 has been demonstrated to be highly expressed in prostate cancer tissues, whereas the underlying mechanism has not been elucidated. Herein, we found the expression of SOAT1 was elevated in human PCa tissues, which demonstrated SOAT1 level was correlated with lymph node metastasis (p = 0.006), clinical stage (p = 0.032), grading (p = 0.036), and Gleason score (p = 0.030) of PCa patients. In addition, we revealed that SOAT1 promoted proliferation and liposynthesis of PCa cells by targeting Stearoyl-CoA Desaturase 1 (SCD1). Our data further confirmed that SCD1 overexpression reversed the proliferation and liposynthesis defects caused by SOAT1 depletion in PCa cells, however, SOAT1 depletion inhibited tumor growth of PCa cells in mice. We further found SOAT1 contributed to the progression of PCa via SREBF1 pathway. Taken together, our data revealed the mechanism underlying SOAT1 promoting PCa progression in vitro and in vivo.

‘MUSCADINE GRAPE SKIN EXTRACT INHIBITS ANDROGEN‐INDEPENDENT PROSTATE CANCER CELL GROWTH, INDUCING CELL‐CYCLE ARREST, AND DECREASING MIGRATION BY TARGETING HEAT SHOCK PROTEIN 40

Previously we demonstrated that muscadine grape skin extract (MSKE), a natural product, significantly inhibited androgen‐responsive prostate cancer cell growth by inducing apoptosis through the targeting of survival pathways. However, the therapeutic effect of MSKE on more aggressive androgen‐independent prostate cancer remains unknown. This study examined the effects of MSKE treatment in metastatic prostate cancer using complementary PC‐3 cells and xenograft model. MSKE significantly inhibited PC‐3 human prostate cancer cell tumor growth in vitro and in vivo. The growth‐inhibitory effect of MSKE appeared to be through the induction of cell‐cycle arrest. This induction was accompanied by a reduction in the protein expression of Hsp40 and cell‐cycle regulation proteins, cyclin D1 and NF‐kBp65. In addition, MSKE induced p21 expression independent of wild‐type p53 induced protein expression. Moreover, we demonstrate that MSKE significantly inhibited cell migration in PC‐3 prostate cancer cells. Overall, these results demonstrate that MSKE inhibits prostate tumor growth and migration, and induces cell‐cycle arrest by targeting Hsp40 and proteins involved in cell‐cycle regulation and proliferation. This suggests that MSKE may also be explored either as a neo‐adjuvant or therapeutic for castration resistant prostate cancer.

Muscadine grape skin extract inhibits prostate cancer cells by inducing cell-cycle arrest, and decreasing migration through heat shock protein 40

Previously we demonstrated that muscadine grape skin extract (MSKE), a natural product, significantly inhibited androgen-responsive prostate cancer cell growth by inducing apoptosis through the targeting of survival pathways. However, the therapeutic effect of MSKE on more aggressive androgen-independent prostate cancer remains unknown. This study examined the effects of MSKE treatment in metastatic prostate cancer using complementary PC-3 cells and xenograft model. MSKE significantly inhibited PC-3 human prostate cancer cell tumor growth in vitro and in vivo. The growth-inhibitory effect of MSKE appeared to be through the induction of cell-cycle arrest. This induction was accompanied by a reduction in the protein expression of Hsp40 and cell-cycle regulation proteins, cyclin D1 and NF-kBp65. In addition, MSKE induced p21 expression independent of wild-type p53 induced protein expression. Moreover, we demonstrate that MSKE significantly inhibited cell migration in PC-3 prostate cancer cells. Overall, these results demonstrate that MSKE inhibits prostate tumor growth and migration, and induces cell-cycle arrest by targeting Hsp40 and proteins involved in cell-cycle regulation and proliferation. This suggests that MSKE may also be explored either as a neo-adjuvant or therapeutic for castration resistant prostate cancer.

MUSCADINE GRAPE SKIN EXTRACT INHIBITS ANDROGEN‐INDEPENDENT PROSTATE CANCER CELL GROWTH, INDUCING CELL‐CYCLE ARREST, AND DECREASING MIGRATION BY TARGETING HEAT SHOCK PROTEIN 40

Previously we demonstrated that muscadine grape skin extract (MSKE), a natural product, significantly inhibited prostate cancer cell growth by inducing apoptosis through the targeting of phosphatidylinositol 3‐kinase‐Akt and mitogen‐activated protein kinase survival pathways in androgen‐responsive transformed human prostate cancer epithelial cell lines. However, the preventive effect of MSKE on more aggressive androgen‐independent prostate cancer remains unknown. This study examined the effects of MSKE treatment using complementary PC‐3 prostate cancer cell culture and xenograft models. MSKE significantly inhibited PC‐3 human prostate cancer cell tumor growth in vitro and in vivo. The growth‐inhibitory effect of MSKE appeared to be through the induction of cell‐cycle arrest. This induction was accompanied by a reduction in the protein expression of Hsp40 protein and cell‐cycle regulation proteins, cyclin D1 and NF‐kBp65. In addition, MSKE induced p21 expression independent of wild‐type p53 induced protein expression. Moreover, we demonstrated that MSKE significantly inhibited cell migration in PC‐3 prostate cancer cells which was complimented by siRNA‐HSP40 knock‐down that also inhibited cell migration and growth. Overall, these results demonstrate that MSKE inhibits prostate tumor growth and migration, and induces cell‐cycle arrest by targeting Hsp40 and proteins involved in cell‐cycle regulation and proliferation. This suggests that MSKE may also be explored as a novel therapeutic in castration resistant prostate cancer.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In a randomized trial in prostate cancer patients, dietary protein restriction modifies markers of leptin and insulin signaling in plasma extracellular vesicles.

SummaryObesity, metabolic syndrome, and hyperleptinemia are associated with aging and age‐associated diseases including prostate cancer. One experimental approach to inhibit tumor growth is to reduce dietary protein intake and hence levels of circulating amino acids. Dietary protein restriction (PR) increases insulin sensitivity and suppresses prostate cancer cell tumor growth in animal models, providing a rationale for clinical trials. We sought to demonstrate that biomarkers derived from plasma extracellular vesicles (EVs) reflect systemic leptin and insulin signaling and respond to dietary interventions. We studied plasma samples from men with prostate cancer awaiting prostatectomy who participated in a randomized trial of one month of PR or control diet. We found increased levels of leptin receptor in the PR group in total plasma EVs and in a subpopulation of plasma EVs expressing the neuronal marker L1CAM. Protein restriction also shifted the phosphorylation status of the insulin receptor signal transducer protein IRS1 in L1CAM+ EVs in a manner suggestive of improved insulin sensitivity. Dietary PR modifies indicators of leptin and insulin signaling in circulating EVs. These findings are consistent with improved insulin and leptin sensitivity in response to PR and open a new window for following physiologic responses to dietary interventions in humans.

MP66-10 CHARACTERIZATION OF SUSCEPTIBILITY LOCI IN HEREDITARY PROSTATE CANCER FAMILIES USING COPY NUMBER VARIATION AND LINKAGE ANALYSIS

INTRODUCTION AND OBJECTIVES: The intratumoral androgen synthesis is one of the mechanisms by which androgen receptor (AR) is aberrantly re-activated in castration-resistant prostate cancer (CRPC) after androgen ablation. However, pathways controlling steroidogenic enzyme expression and de novo androgen synthesis in prostate cancer (PCa) cells are largely unknown. In this study, we explored the potential roles of DAB2IP/AKR1C3 signaling in androgen synthesis and castration-resistant progression of PCa. METHODS: Tumor growth of two PCa cells (androgenindependent C4-2 and androgen-dependent LAPC-4) with or without DAB2IP expression was compared under castrated condition by MTT assay and subcutaneous xenograft model. Expression levels of steroidogenic enzymes were measured by real-time RT-PCR. Regulation of AKR1C3 expression by DAB2IP, subsequent alteration of AR activity and prostate-specific antigen (PSA) re-expression were examined by luciferase reporter assays or western blotting. Intracrine levels of androgens were measured by EIA in PCa cells, xenografts or castrated DAB2IP-/mice prostate tissues. The correlation between DAB2IP and AKR1C3 expression in PCa xenografts, DAB2IP-/mice prostate and human PCa tissues were examined by immunohistochemical (IHC) staining or western blotting. RESULTS: DAB2IP loss could potentiate the castration-resistant tumor growth of PCa cells in vitro and in vivo, and reprogram the expression profiles of steroidogenic enzymes, such as AKR1C3, CYP17A1, 17BHSD2, 17BHSD3, AKR1C1/2 and UGT2B17. Furthermore, DAB2IP coulddramatically inhibit thepromoter activity ofAKR1C3andsuppress its expression. Consistently, the levels of total testosteronewere significantly higher in DAB2IP-deficient PCa cells, xenografts or DAB2IP-/mice prostate tissues, in which more AR phosphorylation or nuclear staining and higher PSA expression were observed. Moreover, knockdown of AKR1C3 using shRNA could abolish all of these phenomena. In addition, DAB2IP expression inversely correlated with AKR1C3 in PCa xenografts, DAB2IP-/mice prostate and human PCa tissues. CONCLUSIONS: Loss of DAB2IP in PCa cells enhances AKR1C3 expression and de novo androgen synthesis, which may contribute to the development of CPRC.

KLF9, a transcription factor induced in flutamide-caused cell apoptosis, inhibits AKT activation and suppresses tumor growth of prostate cancer cells.

Kruppel-like factors (KLFs) are involved in various biological processes; emerging studies have indicated that KLF9 plays a critical role in regulating tumorigenesis. The role of KLF9 in prostate cancer (PCa), however, has not yet been investigated. The expression of KLF members, AKT- and apoptosis-related proteins were analyzed by Western blot or qRT-PCR. Tet-On inducible KLF9 expression was established for the evaluation of the effects of KLF9 on cell proliferation, apoptosis, and xenograft tumor growth in nude mice. Cell cycle and apoptosis were determined by flow cytometry. KLF9 was induced in a time-dependent manner in flutamide-caused apoptosis, and knockdown of KLF9 significantly decreased flutamide-induced growth inhibition and apoptosis in LNCaP cells. The levels of KLF9 were relatively lower in PCa cell lines, particularly in androgen-independent cell lines compared with those in nontumorous prostate epithelial cell lines. Overexpression of KLF9 dramatically suppressed cell proliferation and caused cell cycle arrest in the G2/M phase and cell apoptosis in the androgen-independent cell lines, PC3 and DU145. Intriguingly, KLF9 expression severely suppressed the activation of AKT and its downstream targets. AKT reactivation partially rescued the KLF9-mediated inhibitory effects on the proliferation of PCa cells. More importantly, we found that KLF9 overexpression efficiently inhibited the xenograft tumor growth of PCa cells. These data collectively showing that KLF9 substantially inhibits AKT activation and abrogates tumor growth of PCa cells, suggest the potential of either genetic or pharmacological activation of KLF9 in the therapeutic treatment of castration-resistant PCa.

Abstract 5600: Destruction of prostate cancer cell xenografts by FSH-Lytic peptide conjugates.

Abstract Background: In previous studies (Hansel, et al., Mol. and Cell. Endocrinol. 269:26-33, 2007),we showed that conjugates of membrane destroying lytic peptides with either LHRH or with a 15-amino acid segment of the β chain of human chorionic gonadotropin (hCG) target and destroy human prostate, breast and ovarian cancer cells in tumor bearing nude mice. Recently (Radu et al., N. England J. Med. 363:1621-1630, 2012) reported that endothelial cells in blood vessels supplying cancers express follicle-stimulating hormone (FSH) receptors. Objectives:The objectives of this study were to synthesize a bioconjugate of a lyticpeptide (Phor18) to each of three segments of the β chain of FSH that are known to bind to the FSH receptor, and test these conjugates (FSH90-95-Phor18, FSH81-95-Phor18 and FSH33-53-Phor18) for their ability to target and lyse prostate cancer cells in vitro and in vivo. Results: In in vivo experiments, administration of FSH90-95-Phor18 and FSH81-95-Phor18 significantly (p < 0.05) inhibited prostate cancer cell (PC-3) growth, as measured by tumor volumes and tumor weights at necropsy. FSH33-53-Phor18 also caused a significant reduction in tumor volume at necropsy at a dose of 1 mg/kg, but the reduction in tumor weight was not statistically significant (p > 0.05). The average tumor volume was maintained at significantly lower levels in the mice treated with FSH90-95-Phor18 (p=0.027), FSH81-95-Phor18 (p=0.029) when compared with vehicle and free peptide treatment groups during the study (p < 0.05). Immunohistochemical analyses for FSHR were performed on the tumor samples. FSHR-positive endothelial cells were found in many vessels supplying the tumors of control mice, but few FSHR-positive endothelial cells were present in tumors of mice treated with FSH90-95-Phor18, FSH81-95-Phor 18 or FSH33-53-Phor 18. FSH81-95-Phor 18 was the most effective of the three conjugates tested in destroying the FSHR-bearing endothelial cells and in inhibiting tumor growth of the PC-3 xenografts in nude mice. Conclusion: These data show that Phor18 conjugates of FSH β chain segments that bind to FSHR expressed by the endothelial cells of the blood vessels supplying the tumors are capable of inhibiting prostate cancer cell tumor growth by inhibiting angiogenesis. These FSH β chain-lytic peptide conjugates may be useful in treating prostate and other FSHR expressing cancers. Citation Format: Sita Aggarwal, Ted Gauthier, Hector Alila, Carola Leuschner, Namrata Karki, Rajasree Solipuram, William Hansel. Destruction of prostate cancer cell xenografts by FSH-Lytic peptide conjugates. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5600. doi:10.1158/1538-7445.AM2013-5600

Inhibition of androgen-responsive LNCaP prostate cancer cell tumor xenograft growth by dietary phenethyl isothiocyanate correlates with decreased angiogenesis and inhibition of cell attachment

Phenethyl isothiocyanate (PEITC) is a candidate anticancer compound found in certain cruciferous vegetables. In our tumor cell xenograft model, dietary administration of PEITC (100–150 mg/kg body weight/d) inhibited androgen-responsive LNCaP human prostate cancer cell tumor growth. We found that dietary treatment with PEITC significantly inhibited tumor platelet/endothelial cell adhesion molecule (PECAM-1/CD31) expression, a marker of angiogenesis. By contrast, we did not find the inhibitory effects of PEITC on tumor growth to be associated with alteration of specific markers for apoptosis, cell proliferation or androgen receptor-mediated pathways. Consistent with in vivo results, PEITC exerted little effects on cell proliferation, cell cycle and androgen-dependent pathways. Interestingly, PEITC significantly attenuated LNCaP cell plating efficiency that correlated with inhibition of integrin family proteins integrin β1, α2 and α6 mRNA expression. Thus, PEITC may be a dietary factor that inhibits androgen-responsive prostate tumor growth indirectly by selectively targeting factors involved in the tumor microenvironment.

Posttranslational regulation of membrane type 1-matrix metalloproteinase (MT1-MMP) in mouse PTEN null prostate cancer cells: Enhanced surface expression and differential O-glycosylation of MT1-MMP

Membrane type 1 (MT1)-matrix metalloproteinase (MT1-MMP) is a membrane-tethered MMP that has been shown to play a key role in promoting cancer cell invasion. MT1-MMP is highly expressed in bone metastasis of prostate cancer (PC) patients and promotes intraosseous tumor growth of PC cells in mice. The majority of metastatic prostate cancers harbor loss-of-function mutations or deletions of the tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome ten). However, the role of PTEN inactivation in MT1-MMP expression in PC cells has not been examined. In this study, prostate epithelial cell lines derived from mice that are either heterozygous ( PTEN +/-) or homozygous ( PTEN −/−) for PTEN deletion or harboring a wild-type PTEN ( PTEN +/+) were used to investigate the expression of MT1-MMP. We found that biallelic loss of PTEN is associated with posttranslational regulation of MT1-MMP protein in mouse PC cells. PTEN −/− PC cells display higher levels of MT1-MMP at the cell surface when compared to PTEN +/+ and PTEN +/− cells and consequently exhibited enhanced migratory and collagen-invasive activities. MT1-MMP displayed by PTEN −/− cells is differentially O-glycosylated and exhibits a slow rate of turnover. MT1-MMP expression in PTEN −/− cells is under control of the PI3K/AKT signaling pathway, as determined using pharmacological inhibitors. Interestingly, rapamycin, an mTOR inhibitor, upregulates MT1-MMP expression in PTEN +/+ cells via PI3K activity. Collectively, these data in a mouse prostate cell system uncover for the first time a novel and complex relationship between PTEN loss-mediated PI3K/AKT activation and posttranslational regulation of MT1-MMP, which may play a role in PC progression.