Inhibited Prostate Cancer Cell Invasion Research Articles

Potent Inhibitor of Human Trypsins from the Aeruginosin Family of Natural Products.

Serine proteases regulate many physiological processes and play a key role in a variety of cancers. Aeruginosins are a family of natural products produced by cyanobacteria that exhibit pronounced structural diversity and potent serine protease inhibition. Here, we sequenced the complete genome of Nodularia sphaerocarpa UHCC 0038 and identified the 43.7 kb suomilide biosynthetic gene cluster. Bioinformatic analysis demonstrated that suomilide belongs to the aeruginosin family of natural products. We identified 103 complete aeruginosin biosynthetic gene clusters from 12 cyanobacterial genera and showed that they encode an unexpected chemical diversity. Surprisingly, purified suomilide inhibited human trypsin-2 and -3, with IC50 values of 4.7 and 11.5 nM, respectively, while trypsin-1 was inhibited with an IC50 of 104 nM. Molecular dynamics simulations suggested that suomilide has a long residence time when bound to trypsins. This was confirmed experimentally for trypsin-1 and -3 (residence times of 1.5 and 57 min, respectively). Suomilide also inhibited the invasion of aggressive and metastatic PC-3M prostate cancer cells without affecting cell proliferation. The potent inhibition of trypsin-3, together with a long residence time and the ability to inhibit prostate cancer cell invasion, makes suomilide an attractive drug lead for targeting cancers that overexpress trypsin-3. These results substantially broaden the genetic and chemical diversity of the aeruginosin family and suggest that aeruginosins may be a source of selective inhibitors of human serine proteases.

M6 A modification of lncRNA PCAT6 promotes bone metastasis in prostate cancer through IGF2BP2-mediated IGF1R mRNA stabilization.

BackgroundBone metastasis is the leading cause of tumor‐related death in prostate cancer (PCa) patients. Long noncoding RNAs (lncRNAs) have been well documented to be involved in the progression of multiple cancers. Nevertheless, the role of lncRNAs in PCa bone metastasis remains largely unclear.MethodsThe expression of prostate cancer‐associated transcripts was analyzed in published datasets and further verified in clinical samples and cell lines by RT‐qPCR and in situ hybridization assays. Colony formation assay, MTT assay, cell cycle analysis, EdU assay, Transwell migration and invasion assays, wound healing assay, and in vivo experiments were carried out to investigate the function of prostate cancer‐associated transcript 6 (PCAT6) in bone metastasis and tumor growth of PCa. Bioinformatic analysis, RNA pull‐down, and RIP assays were conducted to identify the proteins binding to PCAT6 and the potential targets of PCAT6. The therapeutic potential of targeting PCAT6 by antisense oligonucleotides (ASO) was further explored in vivo.Results PCAT6 was upregulated in PCa tissues with bone metastasis and increased PCAT6 expression predicted poor prognosis in PCa patients. Functional experiments found that PCAT6 knockdown significantly inhibited PCa cell invasion, migration, and proliferation in vitro, as well as bone metastasis and tumor growth in vivo. Mechanistically, METTL3‐mediated m6A modification contributed to PCAT6 upregulation in an IGF2BP2‐dependent manner. Furthermore, PCAT6 upregulated IGF1R expression by enhancing IGF1R mRNA stability through the PCAT6/IGF2BP2/IGF1R RNA‐protein three‐dimensional complex. Importantly, PCAT6 inhibition by ASO in vivo showed therapeutic potential against bone metastasis in PCa. Finally, the clinical correlation of METTL3, IGF2BP2, IGF1R, and PCAT6 was further demonstrated in PCa tissues and cells.ConclusionsOur study uncovers a novel molecular mechanism by which the m6A‐induced PCAT6/IGF2BP2/IGF1R axis promotes PCa bone metastasis and tumor growth, suggesting that PCAT6 may serve as a promising prognostic marker and therapeutic target against bone‐metastatic PCa.

LncRNA KCNQ1OT1 Promotes Proliferation, Invasion and Metastasis of Prostate Cancer by Regulating miR-211-5p/CHI3L1 Pathway.

BackgroundBone metastasis after failure of castration therapy is the main reason of death in patients with prostate cancer (PCa). Therefore, full awareness of the metastasis mechanism of PCa and discovery of new therapeutic targets are necessary. Studies showed that lncRNA was involved in the development of cancer. However, its potential role and molecular mechanism in PCa metastasis are still unclear. YKL-40 is an 18 glycosyl hydrolase family protein encoded by CHI3L1, which is involved in the invasion and metastasis of various tumors. A previous study of the authors found that YKL-40 was related to the invasion and metastasis of PCa cells. However, the cause of its abnormal expression in PCa remains unclear. The present study explored the role of lncRNA KCNQ1OT1/miR-211-5p/CHI3L1 regulatory axis in the proliferation, invasion, and metastasis of PCa.MethodsRT-PCR and Western blot were used to measure the expression profiles of KCNQ1OT1 and YKL. CCK-8 and Transwell assays were used to examine their effects on cell proliferation and migration. Double luciferase reporter assay was used to verify the interactions between miR-211-5p and CHI3L1 3′-UTR.ResultsKCNQ1OT1 expression was upregulated in PCa tissues and cells. Downregulating this expression inhibited PCa cell invasion, proliferation, and metastasis. KCNQ1OT1 bound miR-211-5p competitively, and miR-211-5p targeted CHI3L1 3′-UTR. miR-211-5p expression was downregulated, whereas CHI3L1 (YKL-40) expression was upregulated. miR-211-5p levels were negatively correlated with KCNQ1OT1 expression and CHI3L1 mRNA. The decrease in YKL-40 expression in PCa cells induced by the downregulation of KCNQ1OT1 expression could be offset by miR-211-5p inhibitor transfection.ConclusionThis study showed that lncRNA KCNQ1OT1, as a ceRNA, upregulated CHI3L1 and promoted PCa progression through competitive binding to miR-211-5p.

The FTO m6A demethylase inhibits the invasion and migration of prostate cancer cells by regulating total m6A levels

AimsN6-Methyladenosine (m6A) is the most frequent posttranscriptional modification and plays important roles in tumorigenesis and metastasis. The roles of fat mass and obesity-associated (FTO) in metabolic diseases have been widely explored. However, the molecular mechanisms and physiological functions of FTO in prostate cancer remain largely unknown. This study aimed to explore the exact functions of FTO in the progression of prostate cancer metastasis. Main methodsDot blot and m6A RNA methylation quantification assays were performed to determine m6A levels. The protein and mRNA expression levels were detected using immunoblot (IB) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses. Cell invasion and migration abilities were measured using transwell and wound healing assays. Bioinformatics was used to measure the expression level of FTO and possible correlation between FTO levels and advanced tumor stage. Immunofluorescence (IF) was performed to measure the cellular localization of FTO. Key findingsFTO was downregulated in prostate cancer tissues and cell lines, and the m6A content was increased. Importantly, patients with lower FTO expression had advanced tumor stage and higher Gleason scores. Gain- and loss-of-function assays revealed that FTO inhibits prostate cancer cell invasion and migration in vitro. Moreover, we confirmed that FTO can decrease the total m6A level. SignificanceThe present study revealed that the FTO m6A demethylase inhibits prostate cancer cell invasion and migration by regulating total m6A levels.

StarD13 differentially regulates migration and invasion in prostate cancer cells.

Prostate cancer is the second most commonly diagnosed cancer in men and one of the main leading causes of cancer deaths among men worldwide. Rapid uncontrolled growth and the ability to metastasize to other sites are key hallmarks in cancer development and progression. The Rho family of GTPases and its activators the GTPase-activating proteins (GAPs) are required for regulating cancer cell proliferation and migration. StarD13 is a GAP for Rho GTPases, specifically for RhoA and Cdc42. We have previously shown that StarD13 acts as a tumor suppressor in astrocytoma as well as breast and colorectal cancer. In this study, we performed a functional comparative analysis of StarD13 targets/and or interacting molecules to understand the general role that StarD13 plays in cancers. Our data highlight the importance of StarD13 in modulating several hallmarks of cancer. Findings from database mining and immunohistochemistry revealed that StarD13 is underexpressed in prostate cancers, in addition knocking down Stard13 increased cancer cell proliferation, consistent with its role as a tumor suppressor. Stard13 depletion, however, led to an increase in cell adhesion, which inhibited 2D cell migration. Most interestingly, StarD13 depletion increases invasion and matrix degradation, at least in part, through its regulation of Cdc42. Altogether, the data presented suggest that StarD13 acts as a tumor suppressor inhibiting prostate cancer cell invasion.

Transcriptional downregulation of miR-127-3p by CTCF promotes prostate cancer bone metastasis by targeting PSMB5.

Prostate cancer (PCa) is one of the most common cancers in males and particularly tends to metastasize to bone. Currently, metastatic bone disease is incurable, and new therapies need to be developed. Our study aims to determine the role of miR-127-3p in PCa metastasis to bone. The results demonstrate that miR-127-3p is markedly reduced in bone metastasis-positive PCa tissues relative to that in bone metastasis-negative PCa tissues. Furthermore, overexpressing miR-127-3p inhibits PCa cell invasion and migration invitro by targeting the proteasome β-subunit PSMB5. Moreover, CCCTC-binding factor (CTCF) transcriptionally inhibits miR-127-3p by interacting with the miR-127-3p promoter. Collectively, this study uncovers a novel mechanism of the CTCF/miR-127-3p/PSMB5 axis in promoting PCa bone metastasis, indicating that miR-127-3p could function as a promising therapeutic target against bone metastasis.

MicroRNA‐140 inhibit prostate cancer cell invasion and migration by targeting YES proto‐oncogene 1

Prostate cancer (PCa), which is an aggressive malignancy of the male genitourinary system. In the present study, the effects of microRNA-140 (miR-140) on PCa were determined. We transfected miR-140 mimics or negative control into PCa cells, and we used MTT, wound healing, and Transwell assays for determining the capacities of miR-140 in cell proliferation, migration, and invasion, respectively. We also confirmed the relationship between miR-140 and YES proto-oncogene 1 (YES1) using Luciferase reporter assay. The results showed that miR-140 was downregulated in PCa cells and tissues, and overexpression of miR-140 could significantly suppress their capacities of proliferation, migration, and invasion. Moreover, YES1 was shown to be a direct target of miR-140. Moreover, miR-140 expression is negatively correlated with YES1 levels. miR-140 exhibits significant tumor-suppressive effects in PCa by inhibiting YES1. The study indicated that miR-140 and YES1 could be the potential targets for PCa therapy.

Upregulation of SPOCK2 inhibits the invasion and migration of prostate cancer cells by regulating the MT1-MMP/MMP2 pathway.

BackgroundIt is known that secreted protein acidic and cysteine rich (osteonectin), cwcv and kazal-like domains proteoglycan 2 (SPOCK2) plays a significant role in the development and progression of several human cancers; however, the role of SPOCK2 in prostate cancer (PCa) remains unclear. This study aimed to find the role and mechanism of SPOCK2 in the development and progression of PCa.MethodsThe messenger ribonucleic acid (mRNA) expression of SPOCK2 in PCa tissue was detected by real-time polymerase chain reaction (PCR). Upregulation of the SPOCK2 gene was achieved using the DU145 and LNCaP cells by transfecting the cells with SPOCK2 recombinant fragment. Cell invasion and migration ability were detected by transwell chamber and wound healing assay. The expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) and matrix metalloproteinase 2 (MMP2) in the cells was detected by Western Blot and zymography gel assay.ResultsThe mRNA level of SPOCK2 was significantly lower in the PCa tissue compared to benign prostate hyperplasia. Upregulation of SPOCK2 inhibited cell invasion and migration in DU145 and LNCaP cells, inhibited the expression of MT1-MMP and MMP2 and, inhibited activation of MMP2 in DU145 and LNCaP cells.ConclusionSPOCK2 is associated with the progression of PCa. Upregulation of SPOCK2 can inhibit PCa cell invasion and metastasis by decreasing MT1-MMP and MMP2 gene expression and decreasing MMP2 protein activation.

Zinc promotes prostate cancer cell chemosensitivity to paclitaxel by inhibiting epithelial-mesenchymal transition and inducing apoptosis.

Paclitaxel (PTX) is a first-line chemotherapeutic drug for the treatment of prostate cancer. However, most patients develop resistance and metastasis, and thus new therapeutic approaches are urgently required. Recent studies have identified widespread anti-tumor effects of zinc (Zn) in various tumor cell lines, especially prostate cancer cells. In this study, we examined the effects of Zn as an adjuvant to PTX in prostate cancer cells. PC3 and DU145 cells were treated with different concentrations of Zn and/or PTX. MTT assay was used to detect cell viability. Real-time cell analysis (RTCA) and microscopy were used to observe morphological changes in cells. Western blotting was used to detect the expression of epithelial-mesenchymal transition (EMT)-related proteins. qPCR (reverse transcription-polymerase chain reaction) was used to examine changes in TWIST1 mRNA levels. Cell invasion and migration were detected by scratch and transwell assays. shRNA against TWIST1 was used to knockdown TWIST1. Colony formation assay was used to detect cell proliferation, while Annexin V and propidium iodide (PI) staining was used to detect cell apoptosis. Zn and PTX increased proliferation inhibition in a dose- and time-dependent manner in prostate cancer cells, while Zn increased prostate cancer cell chemosensitivity to PTX. Combined Zn and PTX inhibited prostate cancer cell invasion and migration by downregulating the expression of TWIST1. Furthermore, knockdown of TWIST1 increased the sensitivity of prostate cancer cells to PTX. In addition, Zn and PTX reduced cell proliferation and induced apoptosis in prostate cancer cells. Our results demonstrated that Zn and PTX combined therapy inhibits EMT by reducing the expression of TWIST1, which reduces the invasion and migration of prostate cancer cells. SiTWIST1 increased the sensitivity of prostate cancer cells to PTX. In addition, with prolonged treatment, Zn and PTX inhibited proliferation and led to prostate cancer cell apoptosis. Therefore, Zn may be a potential adjuvant of PTX in treating prostate cancer and combined treatment may offer a promising therapeutic strategy for prostate cancer.

Expression of forkhead transcription factor O4 in prostate cancer and its effect on prostate cancer cell invasion

To examine the expression of forkhead transcription factor O4 (FOXO4) in prostate cancer and to explore its effect on prostate cancer cell invasion. Methods: Immunohistochemistry was used to detect the expression of FOXO4 in prostate hyperplasia tissues and prostate cancer tissues. Western blot was used to detect the expression of FOXO4 in prostate hyperplasia cell line BPH-1 and prostate cancer cell lines: PC-3 and DU145. PC-3 cells with high relative expression of FOXO4 were transfected with FOXO4 siRNA and scramble siRNA; DU145 cells with low expression of FOXO4 were transfected with FOXO4 plasmid and blank vector. Matrigel Transwell assay was used to detect the invasive ability of transfected cells. The expression of endothelial-mesenchymal transition (EMT)-related proteins E-cadherin, N-cadherin, and vimentin in the transfected cells was detected by Western blot. Results: The expression of FOXO4 in prostate cancer cells and tissues was significantly lower than that in the prostate hyperplasia cells and tissues (both P<0.05). In the prostate cancer tissues, the expression of FOXO4 in cancer tissues with prostate cancer specific antigen (PSA) value <4 was significantly higher than that in the tissues with 4≤PSA≤10 and PSA>10 (all P<0.05). The expression of FOXO4 in cancer tissues with Gleason score <8 was significantly higher than that in the cancer tissues with Gleason ≥8 (P<0.05). The expression of FOXO4 in clinical stage T1-T2 prostate cancer tissues was higher than that in the clinical stage T3-T4 prostate cancer tissues (P<0.05). The expression of FOXO4 in prostate cancer tissues without lymph node metastasis was significantly higher than that in the prostate cancer tissues with lymph node metastasis (P<0.05). Down-regulation of FOXO4 in PC-3 cells could significantly promote the EMT and invasion, with the decreased expression of E-cadherin and the increased expression of N-cadherin and vimentin (all P<0.05); Up-regulation of FOXO4 in DU145 cells could inhibit the EMT and invasion of cells, with the increased expression of E-cadherin and the decreased expression of N-cadherin and vimentin (all P<0.05). Conclusion: FOXO4 is involved in prostate cancer progression, and it can inhibit prostate cancer cell invasion by regulating EMT of prostate cancer cells.

SOCS6 Functions as a Tumor Suppressor by Inducing Apoptosis and Inhibiting Angiogenesis in Human Prostate Cancer.

Our previous studies revealed that the downregulation of Suppressor of cytokine signaling 6 (SOCS6) was correlated with malignant progression of human prostate cancer (PCa). In the current study, we aimed to investigate the tumor suppressive roles of SOCS6 and the underlying mechanisms in PCa. SOCS6 expression in PCa and non-cancerous prostate tissues was compared by immunohistochemistry. Statistical associations of SOCS6 expression with various clinicopathological features and patients prognosis were evaluated. In addition, we investigated SOCS6's functions by overexpressing it in vitro (cell apoptosis, migration and invasion assays) and in vivo (tumor formation, angiogenesis and apoptosis). Moreover, SOCS6-regulated genes were identified by nextgeneration RNA-sequencing analysis, followed by pathway enrichment analysis and in vitro experimental validation. SOCS6 downregulation was significantly associated with advanced clinical stage (P=0.029) and positive lymph node metastasis (P=0.013) in PCa patients. We also identified SOCS6 as an independent prognostic factor for disease-free survival in PCa patients (P=0.045). Moreover, overexpression of SOCS6 inhibited PCa cell invasion, migration, tumor xenografts growth and angiogenesis, but induced PCa cell apoptosis (P values <0.05). Mechanically, we revealed that SOCS6 expression may induce cell apoptosis coincident with down-regulation of Bcl2 and Hspa1a, and may suppress tumor angiogenesis with downregulation of F7, Fak3 and Frzb. These findings suggest that the reduced expression of SOCS6 may be predictive of unfavorable prognosis in PCa. Thus, SOCS6 may serve as a tumor suppressor and a novel therapeutic target for this cancer.

An In Vivo Screen Identifies PYGO2 as a Driver for Metastatic Prostate Cancer.

Advanced prostate cancer displays conspicuous chromosomal instability and rampant copy number aberrations, yet the identity of functional drivers resident in many amplicons remain elusive. Here, we implemented a functional genomics approach to identify new oncogenes involved in prostate cancer progression. Through integrated analyses of focal amplicons in large prostate cancer genomic and transcriptomic datasets as well as genes upregulated in metastasis, 276 putative oncogenes were enlisted into an in vivo gain-of-function tumorigenesis screen. Among the top positive hits, we conducted an in-depth functional analysis on Pygopus family PHD finger 2 (PYGO2), located in the amplicon at 1q21.3. PYGO2 overexpression enhances primary tumor growth and local invasion to draining lymph nodes. Conversely, PYGO2 depletion inhibits prostate cancer cell invasion in vitro and progression of primary tumor and metastasis in vivo In clinical samples, PYGO2 upregulation associated with higher Gleason score and metastasis to lymph nodes and bone. Silencing PYGO2 expression in patient-derived xenograft models impairs tumor progression. Finally, PYGO2 is necessary to enhance the transcriptional activation in response to ligand-induced Wnt/β-catenin signaling. Together, our results indicate that PYGO2 functions as a driver oncogene in the 1q21.3 amplicon and may serve as a potential prognostic biomarker and therapeutic target for metastatic prostate cancer.Significance: Amplification/overexpression of PYGO2 may serve as a biomarker for prostate cancer progression and metastasis. Cancer Res; 78(14); 3823-33. ©2018 AACR.

Abstract 2679: Cyanobacterial trypsin-3 inhibitor inhibits prostate cancer cell invasion

Abstract Trypsin-3 is a highly active protease that has recently been identified as a potential therapeutic target for the reduction of tumor growth and metastasis in prostate, breast and pancreatic cancers. Current trypsin inhibitors typically inhibit a broad range of different trypsin-like enzymes. Thus, novel approaches to identify trypsin-3 selective inhibitors are needed. Cyanobacteria produce a vast diversity of natural products with complex chemical structures many of which are potent protease inhibitors. We screened extracts of 140 cyanobacteria strains and discovered numerous strains showing selectivity towards trypsin-2 and -3 inhibition. We isolated a complex glycopeptide from one of these strains, which selectively inhibited human trypsin-2 and -3 with IC50 values of about 100 nM, while trypsin-1 was not inhibited. Importantly, we also found that this peptide inhibited invasion of aggressive and metastatic PC-3M prostate cancer cells though ECM preparation, while it did not affect the proliferation of the cells. Our results suggest that microbial natural products may offer a viable alternative source of potent and selective trypsin-3 inhibitors. Such inhibitors may be suitable, after further development, for targeting the mechanisms associated with the invasion and metastatic dissemination of cancer cells, i.e., for treatment of aggressive cancers. Citation Format: Hannu Koistinen, Matti Wahlsten, Muhammad N. Ahmed, Kaarina Sivonen, Matthias Nees, Ulf-Håkan Stenman, David P. Fewer. Cyanobacterial trypsin-3 inhibitor inhibits prostate cancer cell invasion [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 2679.

Protective effect of stromal Dickkopf-3 in prostate cancer: opposing roles for TGFBI and ECM-1

Aberrant transforming growth factor–β (TGF-β) signaling is a hallmark of the stromal microenvironment in cancer. Dickkopf-3 (Dkk-3), shown to inhibit TGF-β signaling, is downregulated in prostate cancer and upregulated in the stroma in benign prostatic hyperplasia, but the function of stromal Dkk-3 is unclear. Here we show that DKK3 silencing in WPMY-1 prostate stromal cells increases TGF-β signaling activity and that stromal cell-conditioned media inhibit prostate cancer cell invasion in a Dkk-3-dependent manner. DKK3 silencing increased the level of the cell-adhesion regulator TGF-β–induced protein (TGFBI) in stromal and epithelial cell-conditioned media, and recombinant TGFBI increased prostate cancer cell invasion. Reduced expression of Dkk-3 in patient tumors was associated with increased expression of TGFBI. DKK3 silencing reduced the level of extracellular matrix protein-1 (ECM-1) in prostate stromal cell-conditioned media but increased it in epithelial cell-conditioned media, and recombinant ECM-1 inhibited TGFBI-induced prostate cancer cell invasion. Increased ECM1 and DKK3 mRNA expression in prostate tumors was associated with increased relapse-free survival. These observations are consistent with a model in which the loss of Dkk-3 in prostate cancer leads to increased secretion of TGFBI and ECM-1, which have tumor-promoting and tumor-protective roles, respectively. Determining how the balance between the opposing roles of extracellular factors influences prostate carcinogenesis will be key to developing therapies that target the tumor microenvironment.

Extracellular redox state shift: A novel approach to target prostate cancer invasion.

Extracellular superoxide dismutase (ECSOD) and the cysteine/glutamate transporter (Cys)/(xCT) are tumor microenvironment (TME) redox state homeostasis regulators. Altered expression of ECSOD and xCT can lead to imbalance of the TME redox state and likely have a profound effect on cancer invasion. In the present study, we investigated whether ECSOD and xCT could be therapeutic targets for prostate cancer (PCa) invasion. Immunohistochemistry of tumor microarray PCa tissues (N = 165) with high Gleason scores indicated that xCT protein expression is significantly increased while ECSOD protein expression is significantly decreased. Metastatic PCa indicated ECSOD protein expression is significantly decreased in epithelial area whereas xCT protein expression is significantly increased in stromal area. Furthermore, inhibition of extracellular O2•- by overexpression of ECSOD or alteration of the extracellular Cys/CySS ratio by knockdown of xCT protein inhibited PCa cell invasion. Simultaneous overexpression of ECSOD and knockdown xCT inhibited PCa cell invasion more than overexpression of ECSOD or knockdown of xCT alone. In the co-culturing system, simultaneous overexpression of ECSOD and knockdown of xCT in prostate stromal WPMY-1 cells inhibited PCa cell invasiveness more than overexpression of ECSOD alone. The decrease in PCa invasion correlated with increased of extracellular H2O2 levels. Notably, overexpression of catalase in TME reversed the inhibitory effect of ECSOD on cancer cell invasion. Impaired ECSOD activity and an upregulated of xCT protein expression may be clinical features of an aggressive PCa, particularly metastatic cancers and/or those with a high Gleason score. Therefore, shifting the extracellular redox state toward an oxidizing status by targeted modulation of ECSOD and xCT, in both cancer and stromal cells, may provide a greater strategy for potential therapeutic interventions of aggressive PCa.

MicroRNA-26a/b directly regulate La-related protein 1 and inhibit cancer cell invasion in prostate cancer.

Our past studies of microRNA (miRNA) expression signatures of cancers including prostate cancer (PCa) revealed that microRNA-26a and microRNA-26b (miR-26a and miR-26b) were significantly downregulated in cancer tissues. In the present study, we found that restoration of miR-26a or miR-26b significantly inhibited PCa cell invasion. Gene expression data and insilico analysis showed that the gene encoding La-related protein 1 (LARP1) was a putative candidate of miR-26a and miR-26b regulation. Moreover, luciferase reporter assays revealed that LARP1 was a direct target of both miR-26a and miR-26b. Overexpression of LARP1 was observed in PCa clinical specimens and knockdown of LARP1 inhibited cancer cell migration. Therefore, LARP1 acted as an oncogene in PCa cells. Moreover, 'ribosome', 'RNA transport' and 'mTOR signaling pathway' were identified as LARP1-regulated pathways. Our present data suggested that loss of tumor-suppressive miR-26a and miR-26b enhanced cancer cell invasion in PCa through direct regulation of oncogenic LARP1. Elucidation of the molecular networks regulated by tumor-suppressive miRNAs will provide insights into the molecular mechanisms of PCa oncogenesis and metastasis.

Dysregulated microRNA-224/apelin axis associated with aggressive progression and poor prognosis in patients with prostate cancer.

Our previous study revealed that microRNA (miR)-224 down-regulation could promote tumor progression of prostate cancer (PCa) and might be associated with poor biochemical recurrence-free survival of patients with this malignancy. However, the underlying mechanisms of miR-224 have not been fully elucidated. In the current study, apelin (APLN) was identified as a target gene of miR-224. Forced expression of miR-224 inhibited PCa cell invasion and migration by suppressing the expression of APLN. In addition, the down-regulation of miR-224 was negatively correlated with the up-regulation of APLN mRNA in PCa tissues. Moreover, miR-224 down-regulation was significantly associated with advanced clinical stage (P = .027) and metastasis (P = .001), whereas APLN up-regulation more frequently occurred in PCa tissues with advanced pathologic stage (P = .003), metastasis (P < .001), and prostate-specific antigen failure (P = .001). Furthermore, patients with PCa in the miR-224-low/APLN-high group more frequently had shorter biochemical recurrence-free survival than those in groups with other expression patterns of the 2 molecules. Taken together, our data strongly confirmed for the first time that the dysregulated miR-224/APLN axis may be associated with tumorigenesis and aggressive progression of PCa. More importantly, miR-224 down-regulation and APLN up-regulation may synergistically predict biochemical recurrence-free survival in patients with PCa.

Antitumor Effects of Saffron-Derived Carotenoids in Prostate Cancer Cell Models

Crocus sativus L. extracts (saffron) are rich in carotenoids. Preclinical studies have shown that dietary intake of carotenoids has antitumor effects suggesting their potential preventive and/or therapeutic roles. We have recently reported that saffron (SE) and crocin (CR) exhibit anticancer activity by promoting cell cycle arrest in prostate cancer (PCa) cells. It has also been demonstrated that crocetin esters are produced after SE gastrointestinal digestion by CR hydrolysis. The aim of the present report was to investigate if SE, crocetin (CCT), and CR affected in vivo tumor growth of two aggressive PCa cell lines (PC3 and 22rv1) which were xenografted in male nude mice treated by oral gavage with SE, CR, and CCT. We demonstrated that the antitumor effects of CCT were higher when compared to CR and SE and treatments reverted the epithelial-mesenchymal transdifferentiation (EMT) as attested by the significant reduction of N-cadherin and beta-catenin expression and the increased expression of E-cadherin. Additionally, SE, CR, and CCT inhibited PCa cell invasion and migration through the downmodulation of metalloproteinase and urokinase expression/activity suggesting that these agents may affect metastatic processes. Our findings suggest that CR and CCT may be dietary phytochemicals with potential antitumor effects in biologically aggressive PCa cells.

Genistein inhibits the stemness properties of prostate cancer cells through targeting Hedgehog–Gli1 pathway

Cancer stem cells (CSCs) are involved in tumorigenesis and progression of prostate cancer (PCa). Conventional anticancer therapeutics failed to eradicate CSCs, which may eventually lead to the disease relapse and metastasis. Therefore, targeting prostate CSCs may be an ideal strategy to cure PCa. Genistein is a major isoflavone constituent of soybeans and soy products, which has been shown to exhibit potent anticancer effect on many cancers. We have previously reported that genistein can inhibit PCa cell invasion by reversing epithelial to mesenchymal transition, suggesting that genistein may be effective against metastatic PCa. In addition, we have recently demonstrated that PCa tumorsphere cells (TCs) possess CSC properties. Here, we found that tumorsphere formation and colony formation of Pca cells were noticeably suppressed in the presence of genistein. Pretreatment of PCa TCs with genistein also suppressed tumorigenicity in vivo. Additionally, genistein treatment inhibited tumor growth of PCa TCs. Further studies showed that genistein treatment not only led to the down-regulation of PCa CSC markers CD44 in vitro and in vivo, but also inhibited Hedgehog–Gli1 pathway, which may contribute to the anti-CSC effect of genistein in PCa TCs. Therefore, our findings demonstrated that genistein may be a dietary phytochemical with potential to target prostate CSCs.

Abstract 485: Hypoxia increases the expression of stem-cell markers and promotes cell invasion in prostate cancer cells

Abstract Oxygen is an essential regulator of cellular metabolism, survival, and proliferation. Cellular responses to oxygen levels are monitored, in part, by the transcriptional activity of the hypoxia inducible factors (HIFs). The existence of hypoxic microenvironment is a common occurrence in many solid tumors including prostate. Previous studies from our laboratories have shown that in human prostate cancer models, cancer stem cells expressing the embryonic stem cell markers SOX2 and OCT3/4 were highly invasive and, importantly, were capable of altering their E-cadherin expression during the process of invasion. In contrast, non-tumorigenic cells which minimally express SOX2 or OCT3/4 were found to be poorly invasive. Furthermore, targeted knockdown of SOX2 or OCT3/4 markedly suppressed the invasion of prostate cancer cells, suggesting an indispensible role of these two stem cell markers in invasiveness. In the present study, we examined the impact of oxygen deprivation on cell invasiveness and stemness in both stem cell enriched and non-stem cell populations in PC3 and DU145 prostate cancer cells. Moreover, the impact of Notch signaling, which has been shown as a critical regulator of prostate cancer development, progression and metastasis, was examined. When cells were exposed to 1% oxygen for 24 h, the invasive capacity of both the stem- and non-stem cell subpopulations increased. Interestingly the non-stem cell subpopulation showed the most striking increase (&amp;gt; 10-fold) in invasion under hypoxic conditions. The expression of several important stem cell factors, including OCT3/4, SOX2, Nanog and c-Myc, also was upregulated under hypoxia in both subpopulations. In addition, the expression of Notch1, Notch2 and their target genes Hey1, Hey2 and HeyL were increased in both subpopulations that had experienced oxygen deprivation (1% O2, 24 h). These findings suggest that although cancer stem cells per se exhibit significantly greater invasive capacity, aberrant microenvironmental conditions such as hypoxia may dramatically enhance the ability of non-stem cells to invade. Consequently, both the stem and non-stem cell subpopulations of tumors may be potential targets for the development of suitable therapeutic interventions designed to inhibit prostate cancer cell invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 485. doi:10.1158/1538-7445.AM2011-485