MicroRNAs as emerging key players in the etiology and progression of prostate cancer Clinical implications

MicroRNAs (miRNAs o miRs) are short non-coding RNAs (18-24 nucleotides) regarded as a novel class of regulatory molecules that suppress gene expression at the post-transcriptional level. miRNA genes are, in general, regulated and transcribed in the same manner as a protein-coding gene. They are transcribed by the RNA polymerase II into long primary transcripts (pri-miRNAs) that can contain the precursors of one to several clustered miRNAs. These primary transcripts are then cleaved by endonucleases (Drosha) to produce the pre-miRNAs which consist of ~70-nucleotide hairpin structures. The pre-miRNAs are further processed in the cytoplasm by the Dicer complex into the mature miRNAs which are incorporated into the RNA-induced silencing complex (RISC) that execute the regulatory activity through the binding to the 3' untranslated region (3'UTR) of target mRNAs having complementary sequences. The formation of the mRNA/miRNA duplexes, lead to mRNA degradation, inhibition of translation, or a combination of both.

At present, there are more than 1,600 human miRNAs entries in the miRBase release 19 [77]. Each of these molecules may regulate the expression of hundreds of genes within one cell, and one particular target may be regulated by several miRNAs via different binding sites, creating an extremely complex regulatory network for gene expression. Indeed, it has been estimated that about 60% of the protein-coding genes are targets of miRNAs [78]. In recent years, rapidly growing evidence has established the significance of miRNAs in different physiological processes such as development and differentiation, cell cycle, metabolism, he-mostasis and apoptosis [79].

On the contrary, an altered expression of these regulators play an important role in diseases, including carcinogenesis [80]. Quantitative alterations, either genetic or epigenetic, may modify the expression levels of miRNAs, and are associated with tumor development and progression in various tumors. More than half of the deregulated miRNAs map at, or near to, cancer-associated loci prone to deletions, amplifications and translocations [81]. Qualita tive changes can also arise when there are mutations that disrupt or create miRNA recognition sites. Therefore, miRNAs may contribute to carcinogenesis acting as oncogenes, called oncomirs, if they promote tumor growth when they are over-expressed. They may also act as tumor suppressors when they stimulate cancer development and progression when they are down-regulated. As a general rule, oncomirs target tumor-suppressor gene mRNAs (e.g. miR-21 regulates PTEN), and tumor-suppressor miRNAs target proto-oncogene mRNAs (e.g. let-7 regulates KRAS).

miRNAs, as well as mRNAs, display tissue-specific expression profiles and, therefore, they may have different roles in cells from different origins. An example of this disparity is miR-125b which can have a tumor suppressor activity in ovarian and breast cancers but act as an oncomir in prostate cancer, thyroid cancer, neuroblastoma and glioblastoma [82]. The study of the global miRNA expression levels (miRNAome) has been rising in the past years and abundant miRNAome data are currently available for several cancers. The miRNA expression patterns in different types of tissues have been reported to be more predictive of tumor origin and differentiation status than mRNA profiles because, unlike mRNA expression, a modest number of miRNAs (~200 in total) might be sufficient to classify human cancers [83]. In prostate cancer, the expression of several miRNAs and their target mRNAs are altered and involved in development, invasion and metastasis. Nevertheless, the data on miRNA expression in prostatic tumors are still conflicting and, at present, a conclusive miR-NA profile cannot be recognized. In this section we describe miRNAs that have been studied in the context of prostate cancer and summarize their possible application in disease diagnosis and prognosis.

2.3.1. miRNAs associated to prostate cancer

The expression of miR-21 is up-regulated in many types of cancers, including prostate cancer, glioblastoma, lymphoma, pancreatic cancer, and lung cancer, among others [84, 85]. miR-21 can act as an oncomir that contributes to prostate tumor growth, resistance to apop-tosis, invasiveness and metastasis. Its regulatory activity probably involves the down-regulation of the tumor-suppressor gene PTEN (commonly lost or down-regulated) programmed cell death 4 (PDCD4), tropomyosin 1 alpha (TPM1), and myristoylated ala-nine-rich proteinase kinase C substrate (MARCKS), among other genes. miR-21 was found to be over-expressed in androgen-independent prostate cancer cell lines but its expression is low in androgen-dependent prostate cancer cells; therefore, it may be responsible, at least in part, for the development of castrate-resistant tumors. AR can bind to miR-21 promoter resulting in an androgen-dependent transcriptional regulation of miR-21; consequently andro-gen-dependent miR-21 expression may contribute to prostate cancer pathogenesis. In support of these findings, an in vivo study showed that miR-21 is over-expressed in human prostate tumor samples compared to the matching normal tissue, and tumor growth was accelerated in xenograph models when miR-21 expression was elevated [86].

miR-221 and miR-222 are two highly homologous oncomirs that are frequently over-expressed in different cancers. In primary prostate carcinomas and cell lines, these two miR-NAs inversely correlate with the expression of the tumor suppressor gene p27, which is a well-established marker of poor prognosis in prostate cancer and other types of tumors [87]. In vitro and in vivo experiments link these two miRNAs to prostate cancer development and progression. Furthermore, miR-221 and miR-222 contribute to the growth and maintenance of castration-resistant prostate cancer (CRPC) through mechanisms that comprise the AR signaling.

Another oncomir, miR-125b, was reported to be over-expressed in androgen-independent prostate cancer lines and was also implicated in the hormone independent growth. The mRNA of the pro-apoptotic protein Bak1, which was found down-regulated in CRPC, is a target of miR-125b. However, this miRNA was also suggested to act as a tumor suppressor in a different context because it was found to be down-regulated in CRPC and in breast cancer where it silences the expression of HER-2/new [88]. Interestingly, it was also reported that HER-2/new is over-expressed in the progressing prostate tumors [89]. Therefore, the relevance of miR-125b in prostate cancer progression needs further investigation to assess its role in prostate carcinogenesis.

miR-101-1 and miR-101-2 map in two locus (1p31.3 and 9p24.2, respectively) that are commonly deleted in localized and metastatic prostate cancer. In addition, the loss of miR-101-1 or -2 is associated with the over-expression of EZH2, a histone methyltransferase enzyme that is a direct target of miR-101. The up-regulation of this miRNA reduced the proliferation and the invasive potential of the DU145 cell line. COX-2 is another target of miR-101, linking the miRNAs portray to chronic inflammation and tumor development via the COX-2/pros-taglandins pathway [90]. In vitro studies have shown that there is an inverse correlation between miR-101 and COX-2 in different prostate-derived cell lines, and the over-expression of miR-101 reduces the proliferation rate of the COX-2-associated benign prostatic hyperplasia cell line [91]. Similarly, experimental models by inoculation of cells into BALB/c athymic nude mice demonstrated that the miR-101 over-expressing clone showed a slower tumor growth. Furthermore, the treatment of the tumorigenic BPH1 cell line (BPHCAFTD) with exogenous miR-101 resulted in an inhibition of prostate cancer growth in vitro and in vivo [91]. Similarly, the over-expression of miR-128a reduced invasion capability of the androgen independent prostate tumor cell line, DU145, and was found to be progressively decreased in tissues from benign prostatic hyperplasia, to localized prostate cancer and to distant metastasis [92].

Another tumor suppressor miRNA that was reported to play a role in prostate cancer progression to CRPC is miR-146. This miRNA is down-regulated in androgen-independent cell lines and CRPC tissues compared to androgen-dependent cell lines and non-tumor epithelial tissues [93]. The mechanism of action of miR-146 consists of the inhibition of the expression of ROCK1 (Rho-activated protein kinase 1), which is a member of the hyaluronan/ CD168 pathway involved in prostate cancer invasion and metastasis.

PKCe (protein kinase C epsilon) and ZEB2 (zinc finger E-box binding homeobox 2) are two proteins involve in the migration and invasion capabilities of prostate cancer cells and their expression is regulated, at least in part, by miR-205. This miRNA was reported to be down-regulated in prostate cancer cell lines and carcinomas compared to the non-tumorigenic cell line RWPE-1 and normal prostate tissues, respectively. miR-205 also induces genes involved in cell-cell junctions and down-regulates genes associated with prostate cancer progression such as IL6, caveolin-1, EZH2, ERBB3, E2F1 and E2F5.

This list is just a small part of all miRNA alterations found in prostate cancer (For a more complete list of miRNAs in prostate cancer, the reader may refer to the review written by Coppola et al. [85] and Pang et al. [94]), but other players cannot be discarded.

2.3.2. miRNAs as biomarkers for prostate cancer diagnosis and prognosis

Based on the evidence that miRNAs may be deregulated in different pathologies in a tissue-specific manner, multiple studies have investigated the potential use of the miR-NAome as a biomarker. As a consequence, a growing amount of evidence proposes that the miRNAome can be used as a tool to better define pathological signatures and, in turn, to accurately differentiate tumors according to their origin and cellular linage. In addition, miRNAs meet other important requisites that may allow their use as biomarkers for cancer diagnosis and prognosis: 1) miRNAs are remarkably stable molecules in different types of clinical samples, including formalin-fixed paraffin-embedded (FFPE) tissues which is the standard technique used for long-term conservation of biological samples, 2) they can be analyzed by simple methods such as quantitative retro-transcriptase polymer-ase chain reaction (qRT-PCR), and 3) the lack of intricate transcriptional and translational regulation compared to mRNA.

The tumoral expression of miR-1 and miR-133a correlates with tumor progression. Interestingly, the relapse-free survival of patients with prostate cancer can be predicted by the expression of miR-1 in the tumor specimens. Patients with tumors having low miR-1 expression are more likely to have a biochemical relapse than patients with tumors having high miR-1 expression [95].

Besides their intracellular function, miRNAs can also be released by cells and circulate in the blood stream. Consequently, miRNAs can be isolated from serum and plasma; evenmore, they can be isolated from other body fluids such as urine, saliva and semen. The discovery of circulating miRNAs opened up intriguing possibilities to use the circulating miRNAome as one additional biomarker to improve cancer diagnosis, determine tumor staging more accurately and predict prognosis. Some reports demonstrate that miRNA levels in body fluids may change under certain pathological conditions, including prostate cancer [96]. For this reason, within the past years, studies on miRNAs in cancer have burst onto the scene, and evidence that miRNAs may represent new diagnostic and prognostic molecules in human cancers is rapidly accumulating. However miRNA levels as tools for diagnosis and prognosis in prostate cancer are still limited [96].

Although, serum and plasma levels of miR-141 seems to be one of the most promising markers for prostate cancer diagnosis because they are consistently increased in men diagnosed with this carcinoma compared to healthy individuals; the differences are statistical significant only when the comparisons are made between healthy persons and advanced prostate cancer patients [96]. miR-141 is also elevated in prostatic tumor specimens, suggesting that the raise of this molecule in the body fluids is originated by the tumor cells and increases as disease progresses. Serum levels of other miRNAs are also altered in specimens from men with prostate cancer when compared to healthy individuals (e.g. miR-21, miR-200, miR-221, miR-375, and others), but results are inconsistent among reports.

miR-141 was also studied as a predictor factor for prostate cancer classification. One study showed increased levels of serum miR-141 and miR-375 in high-risk patients (Gleason score >8 or N1) compared to low-risk patients (Gleason score 7 or N0) [97]. Another study found that serum miR-21 is increased in patients with CRPC resistant to docetaxel, opening the possibility to use serum miRNAs as markers of therapeutic response as well [98]. Unfortunately, the specificity and sensitivity of miRNAs when used as single markers for prostate cancer diagnosis and prognosis are similar to the specificity and sensitivity of other markers currently used (e.g. PSA).

In summary, miRNAome from serum or plasma samples may not add much information for prostate cancer diagnosis, outcome and response to therapy when used as a single biomark-er. In addition, it is unlikely to achieve the desire level of accuracy for prostate cancer diagnosis or prognosis, because one miRNAs may be altered in many different diseases. Furthermore, one mRNA can be affected by several miRNAs. Therefore, circulating miR-NAome should be considered an additional tool to improve the accuracy of current diagnostic molecules such as PSA, and other diagnostic tests such as the digital rectal exam, echography and others. Similarly, the tumor miRNAome may help to improve the pathological classification of prostate tumors. Up to date the miRNA profile cannot substitute other clinical tools, but can efficiently supplement them.

2.3.3. Targeting miRNAs as therapeutic strategies

The discovery of miRNAs a decade ago and the subsequent study of their role in the patho-genesis of disease, unveiled a new scenario where miRNA modulators could be used in order to restore the homeostasis of an altered cell or tissue. Recently, a novel class of synthetic inhibitory molecules (antagomirs) that compete with target mRNAs for the binding of miR-NAs, allowing mRNA translation, has been introduced as silencers of oncomirs. The antago-mirs uncover the way to miRNA-base therapeutic strategies. As the number of in vivo studies that analyze the use of miRNAs as therapeutic molecules is restricted to a very small number, further investigations are needed. In spite of all the data being generated, the knowledge and understanding of miRNA in prostate cancer is still at the early stage. Once the normal/pathological role of each alteration is deciphered, and the results validated in a vast cohort of patients, the selected miRNAs might be attractive candidates for prostate cancer diagnosis, patients' management and therapeutic strategy.

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