Valproic acid inhibits the invasion of PC3 prostate cancer cells by upregulating the metastasis suppressor protein NDRG1

Valproic acid (VPA) is a clinically available histone deacetylase inhibitor with promising anticancer attributes. Recent studies have demonstrated the anticancer effects of VPA on prostate cancer cells. However, little is known about the differential effects of VPA between metastatic and non-metastatic prostate cancer cells and the relationship between the expression of metastasis suppressor proteins and VPA. In the present study, we demonstrate that inhibition of cell viability and invasion by VPA was more effective in the metastatic prostate cancer cell line PC3 than in the tumorigenic but non-metastatic prostate cell line, RWPE2. Further, we identified that the metastasis suppressor NDRG1 is upregulated in PC3 by VPA treatment. In contrast, NDRG1 was not increased in RWPE2 cells. Also, the suppressed invasion of PC3 cells by VPA treatment was relieved by NDRG1 knockdown. Taken together, we suggest that the anticancer effect of VPA on prostate cancer cells is, in part, mediated through upregulation of NDRG1. We also conclude that VPA has differential effects on the metastasis suppressor gene and invasion ability between non-metastatic and metastatic prostate cancer cells.     

miR-145靶向调控DAB2对前列腺癌PC3细胞迁移和侵袭能力的影响

已有研究表明, miR-145在多种肿瘤中低表达, 并与细胞增殖和转移相关。文章通过生物信息学分析并结合体外实验鉴定, 发现DAB2(Disabled homolog 2)为miR-145在肿瘤转移过程中累及的新靶点。DAB2一直被认为是一个重要的抑癌基因, 在多种肿瘤标本中表达低下。然而, 研究发现, 在具高侵袭能力的前列腺癌细胞株PC3中DAB2基因却呈较高水平表达。另外, 外源表达miR-145能显著下调 DAB2表达水平, 并抑制PC3细胞的迁移和侵袭能力, 且这种miR-145诱导的PC3细胞功能缺陷能被DAB2过表达修复。上述结果表明, miR-145能通过靶向调控DAB2而影响高侵袭前列腺癌细胞的迁移和侵袭能力。     

Does toll-like receptor-3 (TLR-3) have any role in Indian AMD phenotype?

In our previous study, the mouse double minute 2 (MDM2) was identified as one of the leading genes that promote the metastasis of pancreatic cancer (PC). However, the mechanism by which MDM2 promotes metastasis of PC is not understood. In this study, we show that down-regulation of MDM2 through lentivirus-mediated RNA interference could also suppress in vitro proliferation and in vivo tumor growth, and led to an obvious inhibition of both in vitro invasion and in vivo live metastases of SW1990HM cells which had an over-expression of MDM2 and a higher metastatic potential. Moreover, we also show that the down-regulation of MDM2 induced a significant decrease in MMP9, Ki-67 and increase in P53, E-Cadherin expression, and results in an altered expression of genes involved in metastasis, apoptosis, and cell proliferation. Our results suggest that MDM2 plays an important role in metastasis as well as tumor growth of PC. MDM2 could be a hopeful target for the control of PC.     

Loss of mir-146a function in hormone-refractory prostate cancer

The pattern of microRNA (miRNA) expression is associated with the degree of tumor cell differentiation in human prostate cancer. MiRNAs bind complementarily to either oncogenes or tumor suppressor genes, which are consequently silenced, resulting in alterations of tumorigenecity. We have detected eight down-regulated and three up-regulated known miRNAs in androgen-independent human prostate cancer cells compared to those in androgen-dependent cells, using miRNA microarray analyses. These identified miRNAs showed the same expression patterns in hormone-refractory prostate carcinomas (HRPC) compared to androgen-sensitive noncancerous prostate epithelium as determined by fluorescent in situ hybridization assays in human prostate cancer tissue arrays. One of the eight down-regulated miRNAs, mir-146a, was selected and constitutively expressed to examine its effects on suppression of prostate cancer transformation from androgen-dependent to -independent cells as determined by in vitro tumorigenecity assays. Transfection of mir-146a, which perpetually express the miRNA, suppressed >82% of the expression of the targeted protein-coding gene, ROCK1, in androgen-independent PC3 cells, consequently markedly reducing cell proliferation, invasion, and metastasis to human bone marrow endothelial cell monolayers. Given that ROCK1 is one of the key kinases for the activation of hyaluronan (HA)-mediated HRPC transformation in vivo and in PC3 cells, mir-146a may function as a tumor-suppressor gene in modulating HA/ROCK1-mediated tumorigenecity in androgen-dependent prostate cancer.     

MiR-148a Attenuates Paclitaxel Resistance of Hormone-refractory,Drug-resistant Prostate Cancer PC3 Cells by Regulating MSK1 Expression

MicroRNAs are involved in cancer pathogenesis and act as tumor suppressors or oncogenes. It has been recently reported that miR-148a expression is down-regulated in several types of cancer. The functional roles and target genes of miR-148a in prostate cancer, however, remain unknown. In this report, we showed that miR-148a expression levels were lower in PC3 and DU145 hormone-refractory prostate cancer cells in comparison to PrEC normal human prostate epithelial cells and LNCaP hormone-sensitive prostate cancer cells. Transfection with miR-148a precursor inhibited cell growth, and cell migration and invasion, and increased the sensitivity to anti-cancer drug paclitaxel in PC3 cells. Computer-aided algorithms predicted mitogen- and stress-activated protein kinase, MSK1, as a potential target of miR-148a. Indeed, miR-148a overexpression decreased expression of MSK1. Using luciferase reporter assays, we identified MSK1 as a direct target of miR-148a. Suppression of MSK1 expression by siRNA, however, showed little or no effects on malignant phenotypes of PC3 cells. In PC3PR cells, a paclitaxel-resistant cell line established from PC3 cells, miR-148a inhibited cell growth, and cell migration and invasion, and also attenuated the resistance to paclitaxel. MiR-148a reduced MSK1 expression by directly targeting its 3′-UTR in PC3PR cells. Furthermore, MSK1 knockdown reduced paclitaxel-resistance of PC3PR cells, indicating that miR-148a attenuates paclitaxel-resistance of hormone-refractory, drug-resistant PC3PR cells in part by regulating MSK1 expression. Our findings suggest that miR-148a plays multiple roles as a tumor suppressor and can be a promising therapeutic target for hormone-refractory prostate cancer especially for drug-resistant prostate cancer.     

Cystatin C Is Downregulated in Prostate Cancer and Modulates Invasion of Prostate Cancer Cells via MAPK/Erk and Androgen Receptor Pathways

Cystatin C is believed to prevent tumor progression by inhibiting the activities of a family of lysosomal cysteine proteases. However, little is known about the precise mechanism of cystatin C function in prostate cancer. In the present study, we examined the expression of cystatin C and its association with matrix metalloproteinases 2 (MMP2) and androgen receptor (AR) in a tissue microarray comparing benign and malignant specimens from 448 patients who underwent radical prostatectomy for localized prostate cancer. Cystatin C expression was significantly lower in cancer specimens than in benign tissues (p<0.001) and there was a statistically significant inverse correlation between expression of cystatin C and MMP2 (r_s ² = −0.056, p = 0.05). There was a clear trend that patients with decreased level of cystatin C had lower overall survival. Targeted inhibition of cystatin C using specific siRNA resulted in an increased invasiveness of PC3 cells, whereas induction of cystatin C overexpression greatly reduced invasion rate of PC3 in vitro. The effect of cystatin C on modulating the PC3 cell invasion was provoked by Erk2 inhibitor that specifically inhibited MAPK/Erk2 activity. This suggests that cystatin C may mediate tumor cell invasion by modulating the activity of MAPK/Erk cascades. Consistent with our immunohistochemical findings that patients with low expression of cystatin C and high expression of androgen receptor (AR) tend to have worse overall survival than patients with high expression of cystatin C and high AR expression, induced overexpression of AR in PC3 cells expressing cystatin C siRNA greatly enhanced the invasiveness of PC3 cells. This suggests that there may be a crosstalk between cystatin C and AR-mediated pathways. Our study uncovers a novel role for cystatin C and its associated cellular pathways in prostate cancer invasion and metastasis.     

Forced expression of MMP9 rescues the loss of angiogenesis and abrogates metastasis of pancreatic tumors triggered by the absence of host SPARC

Pancreatic adenocarcinoma is characterized by desmoplasia, local invasion, and metastasis. These features are regulated in part by MMP9 and SPARC. To explore the interaction of SPARC and MMP9 in cancer, we first established orthotopic pancreatic tumors in SPARC-null and wild-type mice with the murine pancreatic adenocarcinoma cell line, PAN02. MMP9 expression was higher in tumors from wild-type compared to SPARC-null mice. Coincident with lower MMP9 expression, tumors grown in SPARC-null mice were significantly larger, had decreased ECM deposition and reduced microvessel density compared to wild-type controls. In addition, metastasis was enhanced in the absence of host SPARC. Therefore, we next analyzed the orthotopic tumor growth of PAN02 cells transduced with MMP9 or a control empty vector. Forced expression of MMP9 by the PAN02 cells resulted in larger tumors in both wild-type and SPARC-null animals compared to empty vector controls and further diminished ECM deposition. Importantly, forced expression of MMP9 within the tumor reversed the decrease in angiogenesis and abrogated the metastatic potential displayed by control tumors grown in SPARC-null mice. Finally, contrary to the in vivo results, MMP9 increased cell migration in vitro, which was blocked by the addition of SPARC. These results suggest that SPARC and MMP9 interact to regulate many stages of tumor progression including ECM deposition, angiogenesis and metastasis.     

Activated T cell exosomes promote tumor invasion via Fas signaling pathway

Activated T cells release bioactive Fas ligand (FasL) in exosomes, which subsequently induce self-apoptosis of T cells. However, their potential effects on cell apoptosis in tumors are still unknown. In this study, we purified exosomes expressing FasL from activated CD8(+) T cell from OT-I mice and found that activated T cell exosomes had little effect on apoptosis and proliferation of tumor cells but promoted the invasion of B16 and 3LL cancer cells in vitro via the Fas/FasL pathway. Activated T cell exosomes increased the amount of cellular FLICE inhibitory proteins and subsequently activated the ERK and NF-κB pathways, which subsequently increased MMP9 expression in the B16 murine melanoma cells. In a tumor-invasive model in vivo, we observed that the activated T cell exosomes promoted the migration of B16 tumor cells to lung. Interestingly, pretreatment with FasL mAb significantly reduced the migration of B16 tumor cells to lung. Furthermore, CD8 and FasL double-positive exosomes from tumor mice, but not normal mice, also increased the expression of MMP9 and promoted the invasive ability of B16 murine melanoma and 3LL lung cancer cells. In conclusion, our results indicate that activated T cell exosomes promote melanoma and lung cancer cell metastasis by increasing the expression of MMP9 via Fas signaling, revealing a new mechanism of tumor immune escape.     

p53 regulates the expression of the tumor suppressor gene maspin

Maspin has been shown to inhibit tumor cell invasion and metastasis in breast tumor cells. Maspin expression was detected in normal breast and prostate epithelial cells, whereas tumor cells exhibited reduced or no expression. However, the regulatory mechanism of maspin expression remains unknown. We report here a rapid and robust induction of maspin expression in prostate cancer cells (LNCaP, DU145, and PC3) and breast tumor cells (MCF7) following wild type p53 expression from an adenovirus p53 expression vector (AdWTp53). p53 activates the maspin promoter by binding directly to the p53 consensus-binding site present in the maspin promoter. DNA-damaging agents and cytotoxic drugs induced endogenous maspin expression in cells containing the wild type p53. Maspin expression was refractory to the DNA-damaging agents in cells containing mutant p53. These results, combined with recent studies of the tumor metastasis suppressor gene KAI1 and plasminogen activator inhibitor 1 (PAI1), define a new category of molecular targets of p53 that have the potential to negatively regulate tumor invasion and/or metastasis.     

Finasteride Inhibits Human Prostate Cancer Cell Invasion through MMP2 and MMP9 Downregulation

Introduction

The use of the 5-alpha reductase inhibitors (5-ARIs) finasteride and dutasteride for prostate cancer prevention is still under debate. The FDA recently concluded that the increased prevalence of high-grade tumors among 5-ARI-treated patients must not be neglected, and they decided to disallow the use of 5-ARIs for prostate cancer prevention. This study was conducted to verify the effects of finasteride on prostate cell migration and invasion and the related enzymes/proteins in normal human and tumoral prostatic cell lines.

Materials and Methods

RWPE-1, LNCaP, PC3 and DU145 cells were cultivated to 60% confluence and exposed for different periods to either 10 µM or 50 µM finasteride that was diluted in culture medium. The conditioned media were collected and concentrated, and MMP2 and MMP9 activities and TIMP-1 and TIMP-2 protein expression were determined. Cell viability, migration and invasion were analyzed, and the remaining cell extracts were submitted to androgen receptor (AR) detection by western blotting techniques. Experiments were carried out in triplicate.

Results

Cell viability was not significantly affected by finasteride exposure. Finasteride significantly downregulated MMP2 and MMP9 activities in RWPE-1 and PC3 cells and MMP2 in DU145 cells. TIMP-2 expression in RWPE-1 cells was upregulated after exposure. The cell invasion of all four tested cell lines was inhibited by exposure to 50 µM of finasteride, and migration inhibition only occurred for RWPE-1 and LNCaP cells. AR was expressed by LNCaP, RWPE-1 and PC3 cells.

Conclusions

Although the debate on the higher incidence of high-grade prostate cancer among 5-ARI-treated patients remains, our findings indicate that finasteride may attenuate tumor aggressiveness and invasion, which could vary depending on the androgen responsiveness of a patient’s prostate cells.     

Analysis of the expression and association of retinoblastoma binding protein 6 with the JNK signaling pathway in prostate cancers

This study aims to investigate the expression of retinoblastoma binding protein 6 (RBBP6) in prostate cancer (PCa) and its association with the c‐Jun N‐terminal kinase (JNK) pathway. Immunohistochemistry was used to detect RBBP6 and JNK1/2 expression in PCa and benign prostatic hyperplasia tissues. RBBP6 expression in PCa cells (LNCap, PC3, and DU145) and noncancerous prostate epithelial cells (RWPE‐1) was determined by quantitative real‐time polymerase chain reaction and western blot analysis. PC3 and DU145 cells were transfected with RBBP6 small interfering RNAs (siRNAs) to examine the biological characteristics. Anisomycin (a JNK activator) with/without RBBP6 siRNA was used to treat PC3 cells for further investigating the ramification of the RBBP6‐mediated JNK pathway in PCa. PCa tissues and cells showed higher RBBP6 and JNK1/2 expression. RBBP6 was positively correlated with JNK1/2 in PCa tissues. Besides, RBBP6 expression was correlated to clinical tumor stage, lymph node metastasis, Gleason grade, preoperative prostate‐specific antigen level, as well as prognosis of PCa. RBBP6 siRNA reduced cell proliferation, arrested cells at G2/M, and promoted cell apoptosis, and suppressed JNK pathway. In addition, migration and invasion decreased after the RBBP6 siRNA transfection with downregulated matrix metallopeptidase‐2 (MMP‐2) and MMP‐9. Anisomycin promoted the proliferation, invasion, and migration of PC3 cells and inhibited PC3 cell apoptosis, which could be reversed by RBBP6 siRNA. RBBP6 expression was upregulated in PCa tissues and positively correlated with expression level of JNK1/2. With inhibition of RBBP6 expression, the proliferation, invasion, and migration of PCa cells decreased dramatically, while PC3 cell apoptosis increased appreciably, accompanied by the suppression of the JNK pathway.     

Mesenchymal stem cell-derived CCL-9 and CCL-5 promote mammary tumor cell invasion and the activation of matrix metalloproteinases

Stromal chemokine gradients within the breast tissue microenvironment play a critical role in breast cancer cell invasion, a prerequisite to metastasis. To elucidate which chemokines and mechanisms are involved in mammary cell migration we determined whether mesenchymal D1 stem cells secreted specific chemokines that differentially promoted the invasion of mammary tumor cells in vitro. Results indicate that mesenchymal D1 cells produced concentrations of CCL5 and CCL9 4- to 5-fold higher than the concentrations secreted by 4T1 tumor cells (P < 0.01). Moreover, 4T1 tumor cell invasion toward D1 mesenchymal stem cell conditioned media (D1CM), CCL5 alone, CCL9 alone or a combination CCL5 and CCL9 was observed. The invasion of 4T1 cells toward D1 mesenchymal stem CM was dose-dependently suppressed by pre-incubation with the CCR1/CCR5 antagonist met-CCL5 (P < 0.01). Furthermore, the invasion of 4T1 cells toward these chemokines was prevented by incubation with the broad-spectrum MMP inhibitor GM6001. Additionally, the addition of specific MMP9/MMP13 and MMP14 inhibitors prevented the MMP activities of supernatants collected from 4T1 cells incubated with D1CM, CCL5 or CCL9. Taken together these data highlight the role of CCL5 and CCL9 produced by mesenchymal stem cells in mammary tumor cell invasion.     

Parathyroid hormone-related protein upregulates integrin expression via an intracrine pathway in PC-3 prostate cancer cells

Parathyroid hormone-related protein (PTHrP) is expressed by human prostatic tissue and prostate cancer cell lines, and enhances prostate tumor cell growth both in vivo and in vitro. PTHrP expression also plays a role in the development of bone metastasis, which is a frequent complication in patients with prostate carcinoma. Tumor cell adhesion to extracellular matrix (ECM) components is mediated via integrin subunits, and plays a major role in the invasion and metastasis of tumor cells. We previously showed that PTHrP overexpression increases adhesion of the human prostate cancer cell line PC-3 to the ECM molecules collagen type I, fibronectin, and laminin. Increased adhesion is accompanied by upregulation in the expression of alpha1, alpha5, alpha6, and beta4 integrin subunits. We used the same cell line to study the mechanism via which PTHrP upregulates integrin expression. Clonal PC-3 cells were established overexpressing wild-type PTHrP or PTHrP mutated in the nuclear localization sequence (NLS). Mutation of the NLS negated the effects of PTHrP on alpha1, alpha5, alpha6, and beta4 integrin expression, indicating that these effects are mediated via an intracrine pathway requiring nuclear localization. Expression of the alpha2, alpha3, alphav, and beta1 integrin subunits were comparable in wild-type and NLS-mutated PTHrP transfectants. These findings indicate that PTHrP may play a role in prostate tumor invasion and metastasis by upregulating the expression of specific integrin subunits via an intracrine pathway.