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The Functional Role of Prostate Cancer Metastasis-related Micro-RNAs

The mortality of patients with hormone-resistant prostate cancer can be ascribed to a large degree to metastasis to distant organs, predominantly to the bones. In this review, we discuss the contribution of micro-RNAs (miRs) to the metastatic process of prostate cancer. The criteria for selection of miRs for this review were the availability of preclinical in vivo metastasis-related data in conjunction with prognostic clinical data. Depending on their function in the metastatic process, the corresponding miRs are up- or down-regulated in prostate cancer tissues when compared to matching normal tissues. Up-regulated miRs preferentially target suppressors of cytokine signaling or tumor suppressor-related genes and metastasis-inhibitory transcription factors. Down-regulated miRs promote epithelial–mesenchymal transition or mesenchymal–epithelial transition and diverse pro-metastatic signaling pathways. Some of the discussed miRs exert their function by simultaneously targeting epigenetic pathways as well as cell-cycle-related, anti-apoptotic and signaling-promoting targets. Finally, we discuss potential therapeutic options for the treatment of prostate cancer-related metastases by substitution or inhibition of miRs.

validated by in vitro and in vivo preclinical experiments and exclude those which are in earlier stages of preclinical development.

Process of PC Metastasis
PC metastasis can be dissected into several functional steps (8)(9)(10)(11)(12). The metastatic process starts with cancer cells at the tumor edge undergoing epithelial-mesenchymal transition (EMT) associated with acquiring invasive properties, intravasation as single cells or multicellular aggregates, activation of survival programs, protection against attack by immune cells and finally extravasation and colonization of distant organs with a high preference for bone (12). Stromalderived factor 1/C-X-C receptor 4 (SDF1/CXCR4) interactions are a prerequisite for bone tropism of PC cells and for access to metastatic niches in the bone marrow (13). The disseminated tumor cells undergo bi-directional interactions with bone-forming cells (osteoblasts), bonedegrading cells (osteoclasts) and other cells in the tumor microenvironment. The metastatic niche is populated by androgen-independent and chemotherapy-resistant cancer stem cells (CSCs), which are supported by mesenchymal cells with respect to growth and survival, while CSCs contribute to recruitment of cancer-associated fibroblasts (14). Furthermore, angiogenesis is essential for the outgrowth of bone metastases. Osteoblastic, osteoclastic and mixed lesions have been identified as PC-related metastases due to the imbalance of osteoblast-mediated bone formation and osteoclast-mediated bone resorption (9,10). Very often, bone metastases display both osteoblastic and osteolytic elements.
An essential component of bone destruction and osteolytic metastasis is the axis formed by the receptor activator of nuclear factor-ĸB (NFĸB)/receptor activator of NFĸB (RANK)/RANK ligand (RANKL)/osteoprotegerin OPG) and members of the tumor necrosis factor (TNF)/tumor necrosis factor receptor (TNFR) superfamily (9,11).Tumor cellsecreted parathyroid hormone, parathyroid hormone-related protein, interleukin1 (IL-1), IL-6 and RANKL mediate activation of osteoclasts and thus facilitate bone destruction (9,11). RANK/RANKL interactions result in the activation of NFĸB signaling and stimulate final effectors of osteolysis such as carboanhydrase II, H+ ATPase and cathepsin K. A vicious cycle is initiated during which osteolysis factors such as transforming growth factor β (TGFβ), insulin-like growth factor 1 (IGF1) and Ca 2+ are released, which promote tumor cell proliferation and production of parathyroid hormonerelated protein. Osteoblastic metastases are stimulated by factors, which promote osteoblast activity such as fibroblast growth factor (FGF), RANKL, platelet-derived growth factor, IGF1, and endothelin 1 (9,11). TGFβ is activated from latent TGFβ; IGF1 can be released from inhibitory IGF binding proteins; and the osteolytic factor parathyroid hormone-related protein can be inactivated by proteases such as urokinase (9). Bone metastasis is associated with skeleton-related events such as intractable pain, bone fractions, spinal cord suppression, neurological deficits and paralysis (15).

Micro RNAs
miRs are small noncoding RNAs with a length between 18 and 25 nucleotides. They are transcribed in the nucleus as primary pre-miRs that are capped, spliced and polyadenylated. Thirty percent of miRs are processed from introns of protein-coding genes, the others are encoded by dedicated loci (16)(17)(18). Primary pre-miRs are cleaved by a complex called the microprocessor to 60-to 70-nucleotide hair-pin looped pre-miRs, which are exported to the cytoplasm by exportin 5 and subsequently are processed by multi-protein complex DICER to produce mature miRs. One strand of the mature miR (guide strand) is loaded into the miR-induced silencing complex to target mRNA by sequence complementarity. This interaction results in gene suppression by targeted mRNA degradation or translational repression in processing bodies (18). More than 1,000 miR genes have been identified and a single miR can target hundreds to thousands of mRNAs, while a single gene can be targeted by multiple miRs (19). Therefore, miRs can function as regulators of complex signaling networks.
The importance of miRs in cancer was demonstrated by correlating the deletion of miR15/miR16 with chronic lymphocytic leukemia and its induction in a mouse model by deletion of miR15/miR16 (20). Furthermore, miRs are involved in pro-oncogenic and tumor-suppressive pathways in a context-dependant manner (21). The crucial role of miRs in metastasis has been demonstrated for breast cancer (22,23), ovarian cancer (24), colorectal cancer (25), and hepatocellular carcinoma (26). Here, we focus on the role of specific miRs involved in the metastatic process of PC.

Prostate Cancer Cell Lines and In Vivo Models
In this review, we describe PC metastasis related miRs which have been validated in at least one in vivo model. We excluded miRs with metastasis-related prognostic impact, but lacking in vivo validation in a PC-related metastasis model.
Several subtypes of PC such as adenocarcinoma, transitional cell, squamous cell and small cell carcinoma (neuroendocrine) have been described (27). The most abundant subtype is prostate adenocarcinoma (≥90%) and consequently most PC cell lines used for in vitro and in vivo studies correspond to this phenotype. The most commonly used PC cell lines are DU-145, PC3, LNCaP, C42B, LAPC4, VCaP and 22Rv as well as BPH1, PIN, RWE-1,2 as CANCER GENOMICS & PROTEOMICS 16: 1-19 (2019) immortalized prostate cells. The generation and functional characterization of these cell lines is described in detail in (28). For functional in vivo metastasis-related studies, several standard in vivo models are used (28)(29)(30). Hematogenous metastasis-mimicking models are based on either the injection of PC cells into the tail vein of mice and monitoring the development of lung metastases, or on intracardiac tumor cell inoculation and following-up of their metastasis to distant organs. Spontaneous metastasis can be assessed by subcutaneous implantation or by orthotopic prostatic implantation. Injection of PC cells into the bones mimics the growth of PC cells in the bone microenvironment, but is not a model of metastasis per se (28,29). Vossicle models are based on implantation of vertebrae from transgenic mice together with PC cells into athymic mice to study the effects of genetic alterations in the tumor microenvironment on tumor development and metastasis (28,29). The transgenic adenocarcinoma of the mouse prostate (TRAMP) model was developed as the first in vivo transgenic model with spontaneous PC metastasis (30,31). In this model, SV40 large T-antigen is expressed in prostate cells under the control of the rat probasin promoter. Metastasis to the lymph nodes, the lungs and occasionally to the kidney, adrenal glands and bones has been observed in this model. (miR-9, miR-194, miR-210). miR-9 is up-regulated in 75% of patients with PC in comparison to corresponding normal tissues (33) and Figure 1. miR-9 promoted the growth and invasion of the PC cell line M12. Subcutaneously injected M12 cells expressing a miR-9 sponge showed reduced tumor growth. Intraprostatic injection of miR-9 sponge expressing M12 cells revealed no metastatic sites in contrast to the control cell line (33). Cadherin 1 (CDH1) and SOCS5 were identified as direct targets of miR-9 (33). Cleavage of CDH1 leads to accumulation of β-catenin in the nucleus, driving transcription of survival factors such as c-MYC and cyclin D1 (34). c-MYC induces additional miR-9. The second miR-9 target, SOCS5, prevents phosphorylation of Janus kinase and signal transducer and activator of transcription 3 (STAT3) (35) (Figure 2A).

SOCS-targeting miRs
Serum levels of miR-194 are higher in men with CRPC than those in men with localized disease (36). miR-194 is induced by transcription factor GATA binding protein 2 (37,38) and PC3 cells ectopically expressing miR-194 showed significantly enhanced cell invasion in the chorionic allantoic membrane assay and exhibited spontaneous metastasis to visceral organs such as kidney, lung, liver and spleen, whereas control PC3 cells remained contained in the prostate (36). SOCS2, a  ubiquitin ligase, was identified as a direct target of miR-194 (36). Inhibition of SOCS2 induced expression of prometastatic genes such as Fms-like tyrosine kinase 3 (FLT3) and JAK1 through activation of extracellular regulated kinase (ERK)-and STAT3 signaling (39,40) (Figure 2A).
Targeting of intercellular adhesion molecule-1 (ICAM1) by miR-296-3p. Clinical tissue microarrays have revealed frequent up-regulation of miR-296-3p in PC patients and concomitant down-regulation of ICAM1 (67). Knock-down of miR-296-3p in M12 PC cells reduced pulmonary metastasis in a model of experimental metastasis in nude mice (22). miR-296-3p inhibits ICAM1 by directly targeting its 3'UTR and is pivotal for the metastasis of PC9 cells to the lungs, probably by enhancing survival of circulating tumor cells by mediating resistance to natural killer (NK) cells (67). The described in vivo effects were abolished by a natural killer cell-eliminating anti-sialo GM1 antibody (67). ICAM1 has been identified as a co-stimulator of natural killer cell-mediated cytotoxicity (68,69).
AR as a target of miR-301a. miR-301a is a predictor of PC metastasis, which is induced in tumor cells by adipocytes (70). It was shown that PC recruits more adipocytes than normal prostate tissues and in vitro enhanced pre-adipocyte recruitment increases PC cell invasion (70). Overexpression of miR-301a enhanced lung metastasis of LNCaP and PC3 cells after subcutaneous implantation into nude mice (70).     Orthotopically engrafted PC CWR22Rv-1 cells gave rise to increased metastatic foci when co-implanted with immortalized pre-adipocytes. Mechanistically, the observed effects were due to the inhibition of AR by miR-301 in tumor cells (70). AR has been identified as a negative regulator of TGFβ/SMAD/MMP9 signaling (71)(72)(73)(74)(75).
miRs Inhibiting PC Metastasis miR-34a and miR-383 target CD44. It has been shown that PC stem cells express CD44 and confer tumorigenic properties on these cells (76,77). CD44 has been identified as a direct target of miR-34a and miR-383 in PC cells (78,79) (Figure 4). In addition, miR-373, miR-520c and miR-708 were shown to target CD44 and modulate in vitro-invasion of PC cells (80,81). miR-34a inhibited clonal and clonogenic properties of PC cells (78). Tail vein injection of miR-34a into nude mice with orthotopic LAPC9 tumors inhibited lung metastasis and extended the survival of the animals without affecting tumor growth (78). Ectopic expression of miR-383 suppressed the tumorigenicity of DU145, PC3 and LNCaP PC cell lines, while silencing of miR-383 induced tumorigenicity of normal prostate epithelial cells (78). miR-383 inhibited the capacity of CD44 + PC cells to initiate tumors, and its ectopic expression in PC3-3M cells tagged with luciferase suppressed growth of xenografts in vivo (78). Inoculation of these cells expressing miR-383 ectopically into the left ventricles of nude mice reduced metastasis (78). Thus, CD44 is a crucial mediator of metastasis (82). Low expression of miR-383 is associated with poor survival outcome in patients with PC and an inverse correlation of expression of miR-383 in PC tissues and lymph node metastasis has been found (78).
miR-573 targets FGF receptor R1 and attenuates EMT. miR-573 is down-regulated in PC and its expression levels inversely correlate with survival (83). miR-573 is induced by transcription factor GATA-binding protein 3, a key factor in preventing PC progression through antagonizing AKT signaling (84). miR-573 suppressed migration and invasion of VCaP and PC3 PC cell lines (83). VCaP transfected with miR-573 gave rise to significantly fewer lung metastases compared to control cells after implantation into the dorsal flank of immuno-deficient mice (83). FGFR1 was identified as a direct target of miR-573 (83). FGFR1 is a mediator of EMT and an activator of downstream signaling pathways such as AKT, SMAD2, SMAD3 and p38 mitogen-activated protein kinase (85) ( Figure  5). An inverse correlation between expression levels of miR-573 and FGFR1 has been found in prostate tumors (83).

miR-143 and miR-145 induce mesenchymal-epithelial transition (MET).
Down-regulation of miR-143 and miR-145 is associated with tumor progression and metastasis (86). miR-143 and miR-145 were shown to induce transdifferentiation of mesenchymal cells towards epithelial cells (86). As shown with miR-transfected PC3 cells, both miR-143 and miR-145 inhibited growth of tibial injected cells and reduce the size of skeletal lesions (1). miR-143 can interfere with extracellular signal-regulated kinase (ERK) signaling, which is involved in the MET pathway (87,88), and miR-145 has been shown to target MMP11, and a disintegrin and metalloproteinase17 (ADAM17) (89) (Figure 5). miR-143 is down-regulated in prostate tumors when compared with matching normal prostate tissues (Figure 1). miR-200b attenuates EMT. miR-200 was up-regulated by AR and reversed EMT in PC3 cells (90). miR-200 induced an increase in expression of differentiation markers of prostate epithelium such as cytokeratins CK8 and CK18, suppressed mesenchymal features such as zinc finger and homeobox transcription factor1 (ZEB1) and reduced invasion of PC3 cells in vitro (90). Transfection of PC3 cells with miR-200b suppressed growth and angiogenesis of PC3 xenografts (90). Orthotopic injection of these cells dramatically reduced spontaneous metastasis as assessed by monitoring fluoresence. miR-200b also inhibited platelet-derived growth factor-D in PC3 cells (91) (Figure 5). From a mechanistic point of view, the direct targets of miR-200b need to be identified in further detail.
miR-130b targets MMP2. miR-130b is down-regulated in clinical PC specimens (92). Stable expression of miR-130b in M12 PC cells suppressed migration and invasion in wound-healing assays without affecting proliferation. Intracardiac injection of luciferase-tagged PC3 cells reduced tissue metastasis, as detected by bioluminescence. MMP2 was identified as a direct target of miR-130b (92) and acts as a promoter of metastasis through degradation of the extracellular matrix (93) ( Figure 5).

miR-449a targets prostate leucine zipper gene (PrLZ).
Downregulation of miR-449 is related to PC clinical stage and distant metastasis (94). miR-449a suppressed proliferation, promoted apoptosis and restricted invasion and tube formation by LNCaP and PC3 cells (94). miR-449 suppressed tumor formation of subcutaneously implanted transfected PC3 cells and reduced the number of animals with tumor metastases (94). PrLZ has been identified as a direct target of miR-449. PrLZ is a member of the tumor protein D52 (TPD52) family, which is specifically expressed in prostate tumors and is associated with progression of PC (95)(96)(97). CSCs and progenitors (99). miR-25 overexpression reduced integrin αv and integrin α6 in human PC cell lines and PC stem cells (98) (Figure 6). Integrins αv and α6 have been identified as pro-invasive integrins (100,101). In PC stem cells, miR-25 affected morphology and reduced migration, switching them to a less invasive phenotype due to cytoskeletal reorganization (98). The impact of miR-25 on metastasis was monitored by injecting fluorescently labeled miR-25 expressing PC3 cells into the circulatory system of embryonic zebrafish (98,102). It was found that miR-25 disrupted extravasation and colonization of distant sites in vivo (98).

miR-1 targets v-src sarcoma viral oncogene homolog (SRC).
miR-1 is reduced in PC compared with benign tissue and its expression further decreases with metastatic progression (103)(104)(105). miR-1 was induced by AR and inhibits in vitro proliferation and in vivo growth of RASB1 cells, a cell line derived from experimental bone metastases of DU145 PC cells expressing mutated RAS V12G37 (103,106). SRC was identified as a direct target of miR-1 (103) (Figure 6). Bone and brain metastases of RASB1 cells transfected with miR-1 were evaluated after intracardiac inoculation. Developing metastases were delayed in time from 3 weeks to 10 weeks, diminished in size and mainly found in the brain. Expression of SRC in miR-1-expressing RASB1 cells restored metastatic ability. Furthermore, it was shown that EGFR promotes PC bone metastases by down-regulating miR-1 and activating the transcription factor TWIST1 (107). In this context, translocation of EGFR into nuclei and increased nuclear EGFR as well as reduced miR-1 expression correlate with worse clinical outcome (107).

miR-16 targets BCL2 and cell-cycle-related genes. miR-16
was identified in a functional screen for miRs which inhibit proliferation of 22Rv1 PC cells (108). Expression of miR-16 was low in the majority of PC clinical samples in comparison to matching normal tissues. miR-16 can induce apoptosis of PC cells by targeting BCL2 by inhibiting the translation of its mRNA (20) (Figure 6). Transfection of PC with miR-16 down-regulated the expression of several cell cycle-related genes such as cyclin D3 as well as cyclindependent kinases 1 and 3 (108). In vivo bone metastasis of luciferase-tagged PC3 cells ectopically expressing miR-16 cells was inhibited after intracardiac injection. A miR-16 mimetic complexed to atelocollagen administered i.v. into mice inhibited metastasis into the thorax, jaws and legs, suggesting that systemic delivery of miR-16 could be a novel strategy to inhibit prostate tumor growth in the bones (108).
miR-27b and miR101 target nuclear receptor subfamily 2 group F member 2 (NR2F2) (previously COUP-TFII). miRs-27b and miR-101 directly target NR2F2, a master regulator of the metastatic gene regulatory network and member of the orphan nuclear receptor family, by modulating the expression of transcription factors ZEB1 and ZEB2 (115, 116) ( Figure  6). Loss of miR-101 correlated with increase in the NR2F2forkhead box M1 (FOXM1)-centromer protein F precursor (CENPF) signaling activity in clinical data sets (115). FOXM1 is a transcription factor which is overexpressed in PC and contributes to proliferation, angiogenesis and metastasis (117). CENPF is a structural component of the kinetochore and a known target of FOXM1 (118). Both FOXM1 and CENPF levels are increased in metastatic PC (48). In vivo, lymphatic metastasis was promoted in LNCaP cells in which miR-101 and miR-27b were inhibited after injection into mouse prostate (115). Clinical data revealed that loss of function of miR-101 and miR-27b leads to overexpression of NR2F2, FOXM1 and CENPF, and enhancement of metastasis and drug resistance (115).

miRs Targeting Bone-related Targets
miR-466 targets Runt-related transcription factor 2 (RUNX2). miR-466 was underexpressed in PC compared to normal tissues and inhibited invasion and migration of PC3 and DU145 PC cells (119). In vivo, PC3 cells constitutively expressing miR-145 exhibited reduced growth of the primary tumor after orthotopic implantation into the posterior prostatic lobe of athymic mice (119). Intracardiac injection of these cells suppressed spontaneous metastases compared to controls. miR-466 directly targets bone-related transcription factor RUNX2, which is involved in expression of several genes related to tumor growth and metastasis, such as osteopontin, osteocalcin, angiopoietins 1 and 4, MMP11, vimentin, focal adhesion kinase (FAK) and AKT (120,121) (Figure 4).

Other miR-related Targets
miR-23b and miR-27b target Huntington-interacting protein 1related protein (HIP1R). miR-23b and miR-27b are encoded by the miR cluster 23b/27b and are down-regulated in CRPC (122). Ectopic expression of miR-23b, -27b in CRPC cell lines ALV31 and PC3 ML reduced mobility and invasion, increased E-cadherin and reduced expression of Ras-related C3 botulinum toxin substrate 1 (RAC1), even though the proliferation rate was not altered (122,123). In vivo, orthotopic xenografts of aggressive PC cells transduced with miR-23b and miR-27b were not affected with respect to growth of the primary tumor, however, distant metastases were significantly reduced (123). miR-23b and miR-27b down-regulated HIP1R, a component of clathrin-coated pits and vesicles that may link the endocytic pathway to the actin cytoskeleton (123, 124) ( Figure 4). HIP1R binds 3-phosphoinosites and may promote survival by stabilizing receptor tyrosine kinases following ligand-induced endocytosis (124).
miR-188-5p targets lysosome transmembrane protein 4 (LAPTM4B). Down-regulation of miR-188-5p is associated with metastasis and poor prognosis in PC (125). miR-188-5p inhibited proliferation, invasion and migration of PC3 and LNCaP PC cell lines in vitro (125). Ectopic expression of miR-188-5p inhibited tumor growth and metastasis of PC3 cells implanted into the dorsal flank of nude mice (125). LAPTM4B has been identified as a direct target of miR-188-5p in PC3 and LNCaP cells (125) (Figure 4). LAPTM4B is located in late endosomes and lysosomes and can promote proliferation, invasion and inhibit apoptosis and is able to initiate autophagy (126). LAPTM4B is associated with poor prognosis and metastasis in several types of tumors (127)(128)(129)(130).
miR-195 targets breast cancer overexpressed gene 1 (KIAA0100, previously BCOX1). PC patients with low miR-195 expression were found to have a poorer prognosis (131). Restoring miR-195 expression in PC3 and LNCaP cells inhibited proliferation, migration and invasion (131). miR-195 down-regulates BCOX1 (Figure 4, which is associated with the recurrence and progression of triplenegative breast cancer (132,133). BCOX1 is significantly overexpressed in patients with PC and lymph node metastasis and is associated with PC progression (131). miR-195 reduced growth and metastasis of PC3 and LNCaP cells implanted into the dorsal flanks of immunodeficient mice (131). (134) and targets RNF31, also known as HOIL-1 interacting protein (135,136) (Figure 4). RNF31 catalyzes the formation of linear peptide bonds between the amino terminus of methionine and the carboxy-terminal glycine (137). RNF31 promotes degradation of p53 (138) and facilitates NFĸB signaling (139). RNF31 knockdown in PC3 cells attenuated metastasis of these cells implanted subcutaneously into the dorsal flanks of nude mice (134). RNF31-positive staining is associated with PC progression and unfavorable outcome (134). miR-708 targets karyopherin. miR-708 targets karyopherin (KPNA4) (Figure 4). The expression levels of karyopherin correlate with PC progression. miR-708 inhibited migration of PC3 cells. KPNA4 knockdown attenuated primary tumor invasion of PC3 xenografts as well as bone metastasis (140). Central mediators of karyopherin function are TNFα and TNFβ, which enhance PC cell migration, stimulate osteoclasts and bone resorption (141,142) and can alter macrophage polarization towards the M2 subtype in the tumor microenvironment (143).

miR-141 targets CD44, methyltransferase EZH2 and several
Rho-GTPases. miR-141 is a member of the miR-200 family and is underexpressed in PC stem/progenitor populations in xenografts and tumors (144). Forced expression of miR-141 in CD44 + and bulk PC cells suppressed tumor regeneration and invasion and enforced an epithelial phenotype with loss of the mesenchymal phenotype (144). miR-141 inhibited invasion in PC3, DU145, VCaP, LAPC4, LNCaP and C4-2 PC cells. Ectopic expression of miR-141 in CD44 high PC3 and DU-145 PC cells and implantation into the dorsal prostate of non-obese diabetic/severe combined immunodeficient mice resulted in smaller primary tumors and fewer lung metastases. Doxycycline-inducible expression of miR-141 in LAPC9 xenografts reduced growth of xenografts and diminished metastasis (144). The mechanism of action of miR-141 is based on inhibition of CSC-related molecules, cytoskeleton-and cell-cycle-related components, resulting in inhibition of tumor growth and metastasis. CSC-related targets such as CD44 and histone methyltransferase EZH2, a component of the polycomb repressive complex 2, both involved in CSC maintenance, invasion and metastasis, are direct targets of miR-141 (144,145). In addition, several invasion-and motility-modulating Rho GTPases, such as cell division cycle 42 (CDC42), rasrelated C3 botulinum toxin substrate 1 (RAC) and actinrelated protein 2/3 complex subunit 5 (ARPC5) have been identified as direct targets of miR-141 (Figure 7). These targets are involved in formation of actin stress fibers and lamellipodia, and cytoskeletal reorganization (146)(147)(148). Furthermore, miR-141 inhibited cell-cycle-related molecules such as CDK4 and CDK6. However, it is not yet clear, whether these are direct targets of miR-141.
miR-141-3p targets TNF receptor-associated factors (TRAF) 5 and 6. miR-141-3p is an inhibitor of bone metastasis of PC cells and is down-regulated in bone-metastatic PC tissues (149). Intracardiac injection of PC3 cells overexpressing miR-141-3p reduced metastasis to the bones in comparison to controls with fewer metastatic nodules and smaller osteolytic areas of metastatic tumors (149). TRAF5 and TRAF6 were identified as direct targets of miR-141-3p (149). TRAF5 and TRAF6 modulate TNF-induced activation of NFĸB signaling through binding to TNF receptor cytoplasmic domains (150) (Figure 7).

miR-203 inhibits EGFR signaling and anti-apoptotic
proteins. miR-203 levels are reduced during progression of PC. Ectopic expression of miR-203 inhibited proliferation, invasion, migration and adhesion of PC cell lines such as DU145, RASB1 and PC3 (151)(152)(153). miR-203 induced MET in PC cells with concomitant expression of E-cadherin (153). RASB1 cells ectopically expressing miR-203 exhibited a significant decrease in brain and bone metastases after intracardiac injection (151). In a second in vivo model, miR-203 was shown to inhibit growth of DU-145 cells after subcutaneous injection (152). Several miR-203-related targets have been identified, depending on the system under investigation. The first system is based on DU145/RASB1 bone metastasis-derived clones whose parental cell line was transformed with RAS G37 (151). In these cell lines, the mechanism of action of miR-203 is based on activation of EGFR signaling as well as inhibition of anti-apoptotic proteins (151). EGFR ligands amphiregulin (AREG), epiregulin (EREG) and transforming growth factor α (TGFα) as well as apoptosis-inhibiting proteins apoptosis inhibitor 5 (API5), cellular inhibitor of apoptosis protein (c-IAP1) (BIRC2) and p53-regulated inhibitor of apoptosis 1 (TRIAP1) have been identified as targets of miR-203, which result in activation of metastasis-promoting SRC and ERK signaling (151). In a second system, RAP1, a member of the RAS family of GTPases which are involved in cell-cell and cell-extracellular matrix interactions (154,155) was identified as a direct target of miR-203 in DU145 cells (152). In a third system using PC3 cells, survivin, ZEB2, RUNX2, distal-less homeobox 5 (DLX5) and SMAD4 were identified as direct targets of miR-203 (156)(157)(158). In PC3 cells, miR-203 reduced expression of osteopontin and osteocalcin genes, which are key osteoblastic genes, indicating that miR-203 may regulate the osteomimetic properties of PC cells (152). In LNCaP cells, miR-203 was identified as a suppressor of LIM and SH3 protein 1 (LASP1), which impaired proliferation and migration of PC cells (159)(160)(161). The mechanism of action of miR-203 is summarized in Figure 7.

Therapeutic Aspects
We have summarized PC-metastasis related miRs based on preclinical validation by in vitro and in vivo systems, as well as clinical correlations. Some of them are up-regulated in PC tissues in comparison to corresponding normal tissues, but the majority are down-regulated in PC tissues. Reconstitution or inhibition of their functions are potential therapeutic options. Therapeutic strategies and current pitfalls are summarized in several excellent reviews (162)(163)(164)(165)(166)(167) and therefore these issues are not discussed in detail in this review.
Options for reconstitution are ectopic expression by expression vectors for the corresponding miRs, doublestranded synthetic RNAs that mimic endogenous miRs (165), or non-specific induction of corresponding miRs with small molecules (165). To inhibit the functionality of miRs, short nucleotides with chemically enhanced stabilities and nuclease resistance are administered. Types of miRinterfering drugs include antisense oligonucleotides and single-strand DNA-like molecules such as locked nucleic acids. In the same way, small double-stranded interfering RNAs (siRNA) and antagomirs, which are RNAs of 23 nucleotides modified with cholesterol at the 3'-end, ultimately lead to RNA silencing by RNA interference. miR sponges, another approach to inhibiting unwanted actions of miRs, contain multiple binding sites for a miR of interest, thus interfering with their functionality (163)(164)(165)(166)(167). Considerable progress has been achieved by optimizing the pharmacokinetic/pharmacodynamic properties of these agents by chemical modification significantly improving the binding affinity, stability and target modulation (164)(165)(166)(167).
Issues which have to be addressed are: hybridizationassociated off-target effects, hepatotoxicity, design of efficient delivery systems, rapid clearance by the reticuloendothelial system, and enhancement of circulation time when injected into the bloodstream (165)(166)(167).
By increasing the molecular weight of RNAs through conjugation to polyethylene glycol a fast renal clearance can be circumvented. The serum half-life can be further prolonged by coupling of the interfering nucleotides to albumins or cholesterol which bind to circulating lipoproteins, thus shielding the nucleotides from renal clearance (168)(169)(170). Apart from this, complexing the nucleotides with nanoparticles also reduces the risk of sideeffects, e.g. immunogenicity and cytokine release syndrome, while the increased molecular weight is expanding the retention time (171).
As a corollary of the many different factors that influence the delivery of nucleotides, such as tissue-specific targeting, cell penetration and endosomal escape, further studiesespecially in in vivo mouse models -should explore and address the current delivery bottleneck to resolve the limitations of the delivery system.
The extent of delivery of these agents to target cells for achieving therapeutic effects is probably dependent on the specific system under consideration and might be affected by induction of immune responses against released tumor antigens, resulting in death of tumor cells which do not express the therapeutic target.
The miRs discussed in this review were validated in different preclinical metastasis-related models such as experimental metastasis (tail vein injection and lung metastasis), spontaneous metastasis after subcutaneous implantation, metastasis after intracardiac or orthotopic intraprostatic injection, or delivery of tumor cells into the bone. Albeit targets have been identified for the described miRs, in many cases, the precise steps of their interaction in the metastatic cascade remain to be identified. The interpretation of the therapeutic relevance of specific miRs is complicated by the fact that in vivo data are restricted to very specific systems and evaluation of in vivo data simultaneously covering several metastasis-related models are not available.
What should be the profile of a PC-related anti-metastatic miR-related therapeutic agent? Besides inhibiting the migration and invasion, one of the requirements should be an ablative effect on circulating and disseminated tumor cells such as inhibition of proliferation and induction of apoptosis.
If the agent under consideration does not match these features, increased cytotoxicty of an agent used in combination studies or reversal of therapy resistance would be an additional useful therapeutic feature. Interference with growth of established PC-related metastases is another important feature for the selection of miR-related agents as therapeutic agents. Unfortunately, anti-metastatic miR-related agents have not yet been evaluated in settings with PCrelated metastatic burden.
Future experiments will show whether the pitfalls, as outlined, can be tackled successfully.