Prostate Cancer Metastasis Suppressor Research Articles

CircUBE3A(2,3,4,5) promotes adenylate-uridylate-rich binding factor 1 nuclear translocation to suppress prostate cancer metastasis

Metastatic progression is the primary cause of mortality in prostate cancer (PCa) patients. Although circular RNAs (circRNAs) have been implicated in cancer progression and metastasis, our current understanding of their role in PCa metastasis remains limited. In this study, we identified that circUBE3A(2,3,4,5), which originated from exons 2, 3, 4 and 5 of the human ubiquitin-protein ligase E3A (UBE3A) gene, was specifically downregulated in PCa tissues and correlated with the Gleason score, bone metastasis, and D'Amico risk classification. Through the in vitro and in vivo experiments, we demonstrated that overexpression of circUBE3A(2,3,4,5) inhibited PCa cell migration, invasion, metastasis, and proliferation. Mechanistically, circUBE3A(2,3,4,5) was found to bind to adenylate-uridylate-rich binding factor 1 (AUF1), promoting the translocation of AUF1 into the nucleus. This led to decreased AUF1 in the cytoplasm, resulting in methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) mRNA instability and a subsequent reduction at the protein level. The downregulation of MTHFD2 further inhibited vimentin expression, thereby suppressing PCa cell epithelial-mesenchymal transition. Additionally, two pairs of the short-inverted repeats (TSIRs) in flanking introns were identified to synergistically facilitate the generation of circUBE3A(2,3,4,5) and other circRNAs. In summary, TSIRs-induced circUBE3A(2,3,4,5) acts as a suppressor of PCa metastasis by enhancing AUF1 nuclear translocation, reducing MTHFD2, and subsequently inhibiting vimentin expression. This study characterizes circUBE3A(2,3,4,5) as a functional circRNA and proposes it as a highly promising target for preventing PCa metastasis.

PTEN-regulated PI3K-p110 and AKT isoform plasticity controls metastatic prostate cancer progression

PTEN loss, one of the most frequent mutations in prostate cancer (PC), is presumed to drive disease progression through AKT activation. However, two transgenic PC models with Akt activation plus Rb loss exhibited different metastatic development: Pten/RbPE:−/− mice produced systemic metastatic adenocarcinomas with high AKT2 activation, whereas RbPE:−/− mice deficient for the Src-scaffolding protein, Akap12, induced high-grade prostatic intraepithelial neoplasias and indolent lymph node dissemination, correlating with upregulated phosphotyrosyl PI3K-p85α. Using PC cells isogenic for PTEN, we show that PTEN-deficiency correlated with dependence on both p110β and AKT2 for in vitro and in vivo parameters of metastatic growth or motility, and with downregulation of SMAD4, a known PC metastasis suppressor. In contrast, PTEN expression, which dampened these oncogenic behaviors, correlated with greater dependence on p110α plus AKT1. Our data suggest that metastatic PC aggressiveness is controlled by specific PI3K/AKT isoform combinations influenced by divergent Src activation or PTEN-loss pathways.

Abstract 3523: Arginyltransferase1 as a novel suppressor of prostate cancer metastasis

Abstract Arginylation is a post-translational modification wholly mediated by Arginyltransferase 1 (Ate1). Our studies have shown that Ate1 and arginylation are upregulated during stress conditions. In addition, it was recently shown that Ate1-deficient fibroblasts demonstrate cancer-like characteristics including genomic instability and mutagenesis. While Ate1’s role in cancer is poorly understood, our preliminary studies suggest that Ate1 is an important regulator of metastasis and cancer progression in prostate cancer models. In this study, our data suggests that Ate1 is essential for normal cell stress response in conditions including cellular oxidant, apoptosis inducing drugs, and radiation. Further, we show that a loss of Ate1 in LnCaP prostate cancer cells increases anchorage-independent growth in the soft-agar assay, and that a loss of Ate1 in PC-3 prostate cancer cells increases matrigel invasion in the Boyden chamber assay. To text how Ate1 affects metastasis in vivo, PC-3 cells with Ate1 or control shRNA were orthotopically implanted into the prostates of immunocompromised mice to observe metastasis. While very little metastasis was observed in PC-3 control cells, PC-3 cells with reduced Ate1 achieved much higher levels of metastasis. In conclusion, our data suggests that the loss of Ate1 in prostate cancer promotes cancer progression phenotypes and cell survival. In future studies, we will examine if Ate1 levels in human prostate cancers in prostate cancer prognosis. Citation Format: Michael Birnbaum, Akhilesh Kumar, William Morgan, Fangliang Zhang. Arginyltransferase1 as a novel suppressor of prostate cancer metastasis [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 3523. doi:10.1158/1538-7445.AM2017-3523

The prostate metastasis suppressor gene NDRG1 differentially regulates cell motility and invasion.

Experimental and clinical evidence suggests that N‐myc downregulated gene 1 (NDRG1) functions as a suppressor of prostate cancer metastasis. Elucidating pathways that drive survival and invasiveness of NDRG1‐deficient prostate cancer cells can help in designing therapeutics to target metastatic prostate cancer cells. However, the molecular mechanisms that lead NDRG1‐deficient prostate cancer cells to increased invasiveness remain largely unknown. In this study, we demonstrate that NDRG1‐deficient prostate tumors have decreased integrin expression and reduced cell adhesion and motility. Our data indicate that loss of NDRG1 differentially affects Rho GTPases. Specifically, there is a downregulation of active RhoA and Rac1 GTPases with a concomitant upregulation of active Cdc42 in NDRG1‐deficient cells. Live cell imaging using a fluorescent sensor that binds to polymerized actin revealed that NDRG1‐deficient cells have restricted actin dynamics, thereby affecting cell migration. These cellular and molecular characteristics are in sharp contrast to what is expected after loss of a metastasis suppressor. We further demonstrate that NDRG1‐deficient cells have increased resistance to anoikis and increased invasiveness which is independent of its elevated Cdc42 activity. Furthermore, NDRG1 regulates expression and glycosylation of EMMPRIN, a master regulator of matrix metalloproteases. NDRG1 deficiency leads to an increase in EMMPRIN expression with a concomitant increase in matrix metalloproteases and thus invadopodial activity. Using a three‐dimensional invasion assay and an in vivo metastasis assay for human prostate xenografts, we demonstrate that NDRG1‐deficient prostate cancer cells exhibit a collective invasion phenotype and are highly invasive. Thus, our findings provide novel insights suggesting that loss of NDRG1 leads to a decrease in actin‐mediated cellular motility but an increase in cellular invasion, resulting in increased tumor dissemination which positively impacts metastatic outcome.

FOXO3a modulates WNT/β-catenin signaling and suppresses epithelial-to-mesenchymal transition in prostate cancer cells

Emerging evidence has revealed a negative correlation between Forkhead box-O (FOXO) expression and prostate cancer grade and spread, indicating its role as a suppressor of prostate cancer metastasis. However, there is still incomplete understanding about the role of FOXO transcription factors in prostate cancer progression. In this investigation, we demonstrate that FOXO3a significantly inhibits the expression β-catenin in prostate cancer cells. The mechanism of inhibiting β-catenin expression involves the FOXO3a-mediated transactivated microRNA-34b/c, which consequently suppressed β-catenin mRNA expression by targeting the untranslated regions (UTRs) of β-catenin. Additionally, FOXO3a can directly bind to β-catenin, and competes with TCF for interaction with β-catenin, thereby inhibiting β-catenin/TCF transcriptional activity and reducing the expression of β-catenin target genes. Furthermore, prostate cancer cells expressing FOXO3a shRNAs display mesenchymal characteristics, including enhanced cell migration and differential regulation of the EMT markers, whereas knockdown of β-catenin results in reversal of shFOXO3a-mediated EMT phenotypic changes. Collectively, these observations demonstrated that FOXO3a inhibits malignant phenotypes that are dependent on β-catenin-dependent modulation of EMT-related genes, and provided fresh insight into the mechanisms by which a FOXO3a-miR-34b/c axis restrains canonical β-catenin signaling cascades in prostate cancer cell.

Upregulation of CRMP4, a new prostate cancer metastasis suppressor gene, inhibits tumor growth in a nude mouse intratibial injection model

Prostate cancer, the most commonly diagnosed male cancer in North America, has a high incidence of bone metastasis. Our previous study showed collapsin response mediator protein 4 (CRMP4) gene inhibited prostate cancer migration and invasion. In this study, we investigated whether overexpression of CRMP4 gene in prostate cancer cells inhibit tumor bone metastasis. The stable prostate cancer cells overexpressing the CRMP4 gene were constructed using lentivirus infection. Prostate cancer bone metastasis nude mouse model was built though orthotopic prostate implantation, intracardiac injection and intratibial injection with CRMP4 overexpress and control cancer cells. Small animal PET/CT scanning results showed no difference of bone metastatic capacity in orthotopic and intracardiac injection models between CRMP4 overexpression and control group, while CRMP4 overexpression inhibited tumor growth in the intratibial injection model. Moreover, our invitro study showed CRMP4 overexpression downregulates the Neuropilin1 (NRP1) expression and upregulate the Noggin expression. Immunohistochemical staining of the hind limbs of intratibial injection model was confirmed with cytological experiments. Taken together, our research indicated CRMP4 inhibits prostate cancer cells growth in the nude mouse bone microenvironment and this effect may relate with regulation of NRP1 and Noggin expression.

LncRNA H19/miR-675 axis represses prostate cancer metastasis by targeting TGFBI.

Prostate cancer is a leading cause of cancer-related mortality in men worldwide and there is a lack of effective treatment options for advanced (metastatic) prostate cancer. Currently, limited knowledge is available concerning the role of long non-coding RNAs in prostate cancer metastasis. In this study, we found that long non-coding RNA H19 (H19) and H19-derived microRNA-675 (miR-675) were significantly downregulated in the metastatic prostate cancer cell line M12 compared with the non-metastatic prostate epithelial cell line P69. Upregulation of H19 in P69 and PC3 cells significantly increased the level of miR-675 and repressed cell migration; however, ectopic expression of H19 in M12 cells could not increase the level of miR-675 and therefore had no effect on cell migration. Furthermore, we found that the expression level of either H19 or miR-675 in P69 cells was negatively associated with the expression of transforming growth factor β induced protein (TGFBI), an extracellular matrix protein involved in cancer metastasis. Dual luciferase reporter assays showed that miR-675 directly bound with 3'UTR of TGFBI mRNA to repress its translation. Taken together, we show for the first time that the H19-miR-675 axis acts as a suppressor of prostate cancer metastasis, which may have possible diagnostic and therapeutic potential for advanced prostate cancer.

Interaction of KAI1 on tumor cells with DARC on vascular endothelium leads to metastasis suppression

CD82, also known as KAI1, was recently identified as a prostate cancer metastasis suppressor gene on human chromosome 11p1.2 (ref. 1). The product of CD82 is KAI1, a 40- to 75-kDa tetraspanin cell-surface protein also known as the leukocyte cell-surface marker CD82 (refs. 1,2). Downregulation of KAI1 has been found to be clinically associated with metastatic progression in a variety of cancers, whereas overexpression of CD82 specifically suppresses tumor metastasis in various animal models. To define the mechanism of action of KAI1, we used a yeast two-hybrid screen and identified an endothelial cell-surface protein, DARC (also known as gp-Fy), as an interacting partner of KAI1. Our results indicate that the cancer cells expressing KAI1 attach to vascular endothelial cells through direct interaction between KAI1 and DARC, and that this interaction leads to inhibition of tumor cell proliferation and induction of senescence by modulating the expression of TBX2 and p21. Furthermore, the metastasis-suppression activity of KAI1 was significantly compromised in DARC knockout mice, whereas KAI1 completely abrogated pulmonary metastasis in wild-type and heterozygous littermates. These results provide direct evidence that DARC is essential for the function of CD82 as a suppressor of metastasis.

Suppression of Metastatic Colonization by the Context-Dependent Activation of the c-Jun NH2-Terminal Kinase Kinases JNKK1/MKK4 and MKK7

Advances in clinical, translational, and basic studies of metastasis have identified molecular changes associated with specific facets of the metastatic process. Studies of metastasis suppressor gene function are providing a critical mechanistic link between signaling cascades and biological outcomes. We have previously identified c-Jun NH2-terminal kinase (JNK) kinase 1/mitogen-activated protein kinase (MAPK) kinase 4 (JNKK1/MKK4) as a prostate cancer metastasis suppressor gene. The JNKK1/MKK4 protein is a dual-specificity kinase that has been shown to phosphorylate and activate the JNK and p38 MAPKs in response to a variety of extracellular stimuli. In this current study, we show that the kinase activity of JNKK1/MKK4 is required for suppression of overt metastases and is sufficient to prolong animal survival in the AT6.1 model of spontaneous metastasis. Ectopic expression of the JNK-specific kinase MKK7 suppresses the formation of overt metastases, whereas the p38-specific kinase MKK6 has no effect. In vivo studies show that both JNKK1/MKK4 and MKK7 suppress the formation of overt metastases by inhibiting the ability of disseminated cells to colonize the lung (secondary site). Finally, we show that JNKK1/MKK4 and MKK7 from disseminated tumor cells are active in the lung but not in the primary tumor, providing a biochemical explanation for why their expression specifically suppressed metastasis while exerting no effect on the primary tumor. Taken together, these studies contribute to a mechanistic understanding of the context-dependent function of metastasis regulatory proteins.

The biology of a prostate cancer metastasis suppressor protein: Raf kinase inhibitor protein

Raf kinase inhibitor protein (RKIP) was originally identified as a protein that bound membrane phospholipids and was named phosphatidylethanolamine binding protein-2 (PEBP-2). RKIP was than identified as a protein that bound Raf and blocked its ability to phosphorylate MEK, thus earning its new name of RKIP. Subsequent to identification of its role in the Raf:MEK pathway, RKIP has been demonstrated to regulate several other signaling pathways including G-protein signaling and NF-kappaB signaling. Its involvement in several signaling pathways has engendered RKIP to contribute to several physiological processes including membrane biosynthesis, spermatogenesis, neural development, and apoptosis. RKIP is expressed in many tissues including brain, lung, and liver and thus, dysregulation of RKIP expression or function has potential to contribute to pathophysiology in these tissues. Loss of RKIP expression in prostate cancer cells confers a metastatic phenotype on them. Additionally, restoration of RKIP expression in a metastatic prostate cancer cell line does not effect primary tumor growth, but it does inhibit prostate cancer metastasis. These parameters identify RKIP as a metastasis suppressor gene. In this review, the biology and pathophysiology of RKIP is described.

CD82, and CD63 in thyroid cancer.

CD82 (KAI1) and CD63 (ME491) are highly glycosylated proteins which belong to the transmembrane 4 superfamily (TM4SF). CD82 has been implicated as a possible prostate cancer metastasis suppressor gene, whereas CD63 is involved in the progression of human melanoma cancer. Down-regulation of both CD82 and CD63 expression has been associated with the metastatic potential of several solid tumors. Currently, information is lacking on the role of CD82 and CD63 during thyroid carcinogenesis. The aim of this study was to determine whether the expression of CD82 and CD63 is a useful prognostic indicator in patients with thyroid carcinoma. The expression of CD82 and CD63 was analysed by reverse transcriptase-PCR (RT-PCR) and immunohistochemistry in benign goiter (n=12) and 75 primary thyroid carcinoma tissue specimens (PTC: 33, FTC: 24, UTC: 18) out of which 36 were non-metastasized primary tumors and 39 were metastasized tumors (regional lymph node and/or distant metastases). All of the benign goiter tissues showed CD82 expression. By contrast, a significant decrease in CD82 mRNA and protein levels was detected in carcinoma tissues as compared to benign goiter tissues (p<0.001). A similar down-regulation was observed in metastasized tumor tissues when compared with non-metastasized tumors (all p<0.05). CD82 expression was correlated with pTNM status of differentiated and undifferentiated thyroid tumor and the pathologic stage of differentiated thyroid tumor. In contrast to CD82, CD63 mRNA and protein expression was unchanged in all thyroid carcinomas. Benign goiter tissues showed weak expression of CD63. There were no significant correlation between CD63 mRNA/protein expression and any clinical/pathological parameters. Our results support the hypothesis that down-regulation of CD82 expression may reflect an increased in vivo metastatic potential of thyroid cancer cells. CD82 may serve as a prognostic marker of metastasis in thyroid cancer. Constitutive expression of CD63 may indicate that this factor does not play a direct role in thyroid carcinogenesis.

KAI1, a prostate metastasis suppressor: prediction of solvated structure and interactions with binding partners; integrins, cadherins, and cell-surface receptor proteins.

The solution structure of the transmembrane-4 superfamily protein KAI1, a recently identified prostate cancer metastasis suppressor gene that encodes a 267-amino acid protein, was modeled. The structure of this four-helical transmembrane protein was developed by defining and modeling sections individually. A complete three-dimensional structure for the solvated protein was developed by combining the individually modeled sections. The four-helix transmembrane bundle structure was predicted combining information from several methods including Fourier transform analysis of residue variability for helix orientation. The structure of the KAI1 large extracellular domain was modeled based on the solved crystal structure of the extracellular domain of another tetraspanin superfamily protein member, CD81 (hepatitis C virus envelope E2 glycoprotein receptor). This is a novel protein fold consisting of five alpha helices held together by two disulfide bonds for which the CD81 protein is the first solved representative. Molecular dynamics studies were performed to test stability and to relax the total model KAI1 structure's solution. The resulting KAI1 structural model should be a useful tool for predicting modes of self-association and associations with other TM4SF proteins, integrins, cadherins, and other KAI1 binding partners. Mutations for probing the interactions of KAI1 with antibodies and with other binding partners are suggested. Published 2001 Wiley-Liss, Inc.

Expression of the prostate cancer metastasis suppressor gene KAI1 in primary prostate cancers: a biphasic relationship with tumour grade.

The KAI1 gene, isolated from human chromosome 11p11.2, has been implicated as a prostate cancer metastasis suppressor gene. Recent studies have demonstrated that the expression of KAI1 protein is reduced in metastases of human prostate cancers and is inversely correlated with tumour grade. The objectives of the present work were to determine whether alterations of KAI1 at a genetic level in localized prostate cancers correlate with degrees of differentiation. This paper reports the application of semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Southern analysis to two different regions of the KAI1 gene on 35 microdissected primary prostate cancer specimens and demonstrates a biphasic pattern of KAI1 expression according to histological grade. KAI1 mRNA, relative to the housekeeping gene beta -actin, was elevated in low-grade primary prostate cancer (2.7+/-0.4) compared with non-malignant (hyperplastic) prostatic tisues (0.92+/-0.02, p< 0.05), yet reduced in high-grade primary cancers (0.61+/-0.11, p< 0. 05). These data demonstrate, for the first time, that KAI1 is biphasically expressed in primary prostate cancers and suggest that hyperexpression of KAI1 in low-grade prostate cancer may be associated with restraint of tumour progression, whereas a relative decrease in KAI1 gene expression may accompany more aggressive cancers through loss of such restraint. This differential expression of the metastasis suppressor gene KAI1 in primary prostate cancers may have important prognostic implications for the development of subsequent metastases. Should the level of KAI1 in primary prostate cancer be correlated with patient outcome such information may, in the future, enable more intensive adjuvant therapy to be directed to those patients identified to be at greatest risk of metastasis.