Dysregulation and functional roles of miR-183-96-182 cluster in cancer cell proliferation, invasion and metastasis

Previous studies have reported aberrant expression of the miR-183-96-182 cluster in a variety of tumors, which indicates its' diagnostic or prognostic value. However, a key characteristic of the miR-183-96-182 cluster is its varied expression levels, and pleomorphic functional roles in different tumors or under different conditions. In most tumor types, the cluster is highly expressed and promotes tumorigenesis, cancer progression and metastasis; yet tumor suppressive effects have also been reported in some tumors. In the present study, we discuss the upstream regulators and the downstream target genes of miR-183-96-182 cluster, and highlight the dysregulation and functional roles of this cluster in various tumor cells. Newer insights summarized in this review will help readers understand the different facets of the miR-183-96-182 cluster in cancer development and progression.

IntroductIon microRNA (miRNA) is a small non-coding RNA molecule (containing about 22 nucleotides) that silences cognate target genes via base-pairing with complementary sequences within 3'UTRs (sometimes 5'UTRs or coding regions) in corresponding mRNAs, resulting in inhibition of translation or mRNA degradation [1]. miRNA is involved in various biological processes, including cell proliferation, apoptosis, metabolism and differentiation. miR-183-96-182 cluster is a highly conserved miRNA cluster [2]. Members of this cluster are located within a 5-kb region on human chromosome 7q32.2 [3], transcribed in the same direction from telomere to centromere, and have similar biological functions in some of the closely related signaling pathways.
While investigating its regulatory effect on the downstream target genes, the miR-183-96-182 cluster was Review discovered as being a regulator of tumor development, the nervous system and the immune system [2,[28][29][30][31][32][33][34]. Recent studies have documented localization of some tumor-related genes, such as CDK6, BRAF, and c-MET at the upstream/downstream domain of miR-183-  cluster [23,35,36], suggesting that these genes might be regulated to process similar functions of tumor-related molecules. Furthermore, over-expression of miR-183-96-182 cluster has been described in most malignant tumors including, hepatocarcinoma [37][38][39] The available evidence, thus, suggests much variability in the role played by the miR-183-96-182 cluster in tumorigenesis, tumor progression and metastasis. In this review, we profile the dysregulation and functional roles of the miR-183-96-182 cluster during tumorigenesis in various tumor cells, and its prognostic relevance in clinical settings. The outline of this paper is provided in Appendix 1-1.

mir-183-96-182 cluster inhibits cancer cell proliferation
Interestingly, in certain cancers, over-expression of miR-183-96-182 cluster had an inhibitory effect on cell proliferation, a finding which is not consistent with the earlier reports related to most cancer types. The miR-96 target gene, ATG7, is a key factor in the autophagy pathway, which protects the cancer cells against stress responses such as hypoxia or starvation [87]. Highexpression of miR-96 is thought to inhibit autophagy through directly targeting ATG7, and subsequently inhibit the survival of cancer cells under hypoxic conditions [23]. In addition, miR-96 is known to down-regulate RAD51 (a DNA repair protein) and REV1 (a DNA polymerase) to promote cellular sensitivity to cisplatin, which binds to and cause crosslinking of DNA to ultimately trigger apoptosis [36]. Similar results were also found for miR-182 in acute myelogenous leukemia [21]. Thus, the over-expression of miR-96/miR-182 appears to dramatically promote drug sensitization in cancer cells [36]. miR-96 was also shown to inhibit cell proliferation of ALK-expressing cancer cells via suppressing ALK expression, as well as those ALKtargeted genes, including AKT, STAT3, JNK and IGF-1 [88].
Notably, the inhibitory effect of miR-96 on pancreatic cancer cell proliferation has been clearly elucidated in the past few years [20, 51, 89

contradictory results
However the functions of miR-183-96-182 cluster in lung and gastric cancer are yet to be confirmed (Table  1). In non-small cell lung cancer, miR-96 was shown to promote cell proliferation by targeting FOXO3 and RECK mRNA (A549, SK-MES-1, H1299 and SPC-A-1 cell lines) [61,84], while according to a study by Vishwamitra et al., miR-96 inhibits cell proliferation by targeting ALK (H2228 cell line) [88]. This reported discrepancy in results may be attributable to the inclusion of different cell types for analysis or involvement of different signaling pathways. Two studies on gastric carcinoma simultaneously reported contradictory results with respect to the function of miR-183 during cell proliferation in SGC-7901 cells [78,103]

miR-182
Oncogene Colon cancer (tissue) FBXW7 Associated with prognosis (Survival) [145]. www.impactjournals.com/oncotarget findings suggest that the regulation of cell proliferation by miR-183-96-182 cluster is a complicated synergic process, and the different functions of this cluster may be due to that the target genes might be expressed at different levels, contain mutations, or compete with other molecules.
Other possible reasons for the contradictory results are summarized in Appendix 1-3.

mir-183-96-182 cluster in tumor invasion and metastasis mir-183-96-182 cluster promotes tumor invasion and metastasis
It has been demonstrated that miR-183-96-182 cluster promotes tumor invasion and metastasis in most cancers, including thyroid, esophagus, gallbladder, ovary, bladder, kidney, liver cancers, melanoma, medulloblastoma, sarcoma, glioma, and myeloid cell tumor (Table 2). miR-183 promotes tumor invasion and metastasis by targeting PDCD4, protein phosphatase 2A (PP2A), EGR1 and PTEN [76,78,104,105]. In addition, TGF-β and Smad can also promote prostate cancer bone metastasis by induction of miR-96 and activation of the mTOR pathway [106]. Moreover, the inhibitory effect on metastasis in hepatoma carcinoma cells, induced by the suppression of miR-96 [107], was reported as being associated with the inhibition of EFNA5 expression by miR-96-targeting [85]. Similar findings have been reported in case of gastric, bladder, and breast cancers [3, 7, 83,108]. With regard to miR-182, Huynh et al. reported significant suppression of invasive growth tendency and metastasis by suppressing miR-182 in vivo [109]. Moreover, similar to the effects of TGF-β on miR-96, TGF-β up-regulates miR-182, which can target CYLD and thus promote the activation of NF-κB in gliomablastoma. Therefore, TGF-β-mediated up-regulation of miR-182 probably results in the persistent activation of NF-κB in gliomblastoma, which subsequently leads to angiogenesis and tumor invasion. Table 2 shows the target genes of miR-183-96-182 cluster which regulate invasion and metastasis in various tumor cells.

mir-183-96-182 cluster inhibits tumor invasion and metastasis
On the contrary, miR-183-96-182 suppresses tumor metastasis in lung, colon, and pancreatic cancers ( Table  2). Transcriptional repressor Zin C finger E-box-binding homeobox 1 (ZEB1) family is a series of transcription factors which contain zinc finger domain. The highly conserved zinc finger structure can bind to E-box domain of the promoter of target genes, such as E-cadherin, the key epithelial marker for epithelial-mesenchymal transition (EMT and MET) [110,111]. A recent study indicated a ZEB1/miR-200 double negative feedback loop in EMT at different stages of tumor development [112]. Notably, miR-183/96 can inhibit EMT via suppressing ZEB1 expression. Besides, ZEB1 can also block the transcription of miR-183-96-182 cluster by binding to its promoter [9]. miR-183-96-182 cluster and ZEB1 exert a double negative feedback loop in p21-/-cells. However, more recently, p21, an inhibitor of cyclindependent kinase through suppressing the expressions of CDK1 and CDK1 proteins [113], can also inhibit EMT progression [114,115]. There is further evidence that p21 can interact with ZEB1 to form a complex and binds to the promoter of miR-183-96-182 cluster, which suppresses the transcription inhibition by ZEB1 and results in the suppression of EMT. The schematic diagram is provided in Appendix 1-4. Similar results were also reported in lung cancer cells by Kundu et al., where they found that FOXF2 correlates with ZEB1 expression, and miR-183-96-182 can suppress FOXF2 to inhibit tumor invasion and metastasis in lung cancers [116].

contradictory results
Investigations of the effects of miR-183-96-182 cluster on tumor invasion and metastasis have sometimes yielded contradictory results in different tumors, and in some cases, even within the same tumor type. miR-183 was found to be down-regulated by Cao Table 2) and hypothesized to inhibit tumor invasion by suppressing the expressions of BMI1 or EZR proteins in gastric cancers [103,120]. Conversely, Hu et al. reported that miR-183 was up-regulated (20 non-tumor tissue and 80 tumor tissue samples) and promotes gastric cancer cell invasion by inhibiting PDCD4 expression [78]. Similar differences in results were also reported in case of prostate cancers. miR-182 was over-expressed in prostate cancer tissue by  [72,126]. In contrast, over-expression of miR-182 was shown to inhibit tumor invasion in PC3 and LNCaP cells by suppressing GNA13 expression [123]. These findings suggest a context-dependent phenotype for the miR-183-96-182 cluster in carcinogenesis which needs to be further investigated to understand the complex interactions, especially in those cancers where contradictory results have been observed, such as prostate, colon, lung, breast, and gastric cancers (Table 2).

mir-183-96-182 cluster in cancer prognosis
Most of cancer cells display high-expression of miR-183 [127,128]. The up-regulation of miR-183 is known to be associated with poor prognosis in breast cancer, colorectal cancer, hepatocellular cancer, and prostate cancer [13, [129][130][131][132][133][134], while predicts a good prognosis in osteosarcoma [135] (Table 3). This finding is consistent with its functions in cell proliferation, invasion and metastasis in these tumors types. Notably, miR-183 might affect the prediction for PSA-dependent diagnosis and prognosis via regulating PSA expression [136]. With respect to the prediction of miR-183-related prognosis, the available evidence from different studies is contradictory in lung cancer. Lin et al. showed the low expression of miR-183 in the peripheral blood which was associated with increased TNM stage in lung cancer patients (13 squamous-cell carcinoma and 17 adenocarcinoma) [137]. While Zhu et al. demonstrated the up-regulation of miR-183 family in lung cancer tissue (36 squamous-cell carcinoma and 34 adenocarcinoma), and that it appeared to confer a poor prognosis [48]. The wide variability in the reported results may be attributable to the differences between blood and tissue or the heterogeneity in lung cancer cells.
The high expression of miR-96 in prostate cancer is well documented [23,41,63,66,67,138]. Larne et al. recently reported a miRNA index quote (miQ) in prostate cancer, which uses four miRNAs (miR-96, 183, 145, and 221) for more accurate diagnosis (area under the curve, AUC = 0.931) and prognosis (AUC = 0.895 for predicting aggressiveness and AUC = 0.827 for metastasis). miQ was verified in an independent Dutch cohort and three external cohorts, and significantly outperformed the prostate-specific antigen [131]. Schaefer et al. demonstrated that highly expressed miR-96 can predict cancer recurrence after radical prostatectomy [138]. Additionally, Haflidadottir et al. found miR-96 expression correlated with WHO grade, and the overall survival time in prostate cancer [67]. In contrast, a recent investigation found no significant correlation between the expression of miR-96 and clinicopathological parameters [139]. Thus, suggesting that more studies are required to understand the prognostic relevance of miR-96. In addition, miR-96 was reported as a potential biomarker for the predicting recurrence after surgical resection of hepatocellular cancer [140], and as prognostic indicator in lung cancer, colorectal cancer and acute myeloid leukemia [32,48,141] (Table 3).

concludIng remArks
Recent studies suggest an important role of miR-183-96-182 cluster in tumorigenesis, cancer progression, tumor invasion and metastasis. Although most of the reports showed that miR-183-96-182 cluster is an oncogene cluster, it also functions as a TSG by inhibiting cell proliferation and metastasis in certain cancer cells. We hypothesize that the different results observed in expression and function of the miR-183-96-182 cluster may result from different underlying tissue types, different expression abundance of miR-183-96-182 or their target genes, differences between cell lines (Table 1-2), differences between cell line and tumor tissue, tissue and blood (Table 3), and differences between detecting methods used. Recent studies have also indicated diagnostic and prognostic relevance of the members of miR-183-96-182 cluster, either independently or collectively. These new data on the functions of miR-183-96-182 cluster in various tumors suggest that further studies will be needed to clarify its functions in the various stages and histological subtypes in different types of tumors, which will significantly improve the accuracy of the prediction for tumor diagnosis or prognosis. As regards the conflicting results in certain tumors, we believe that miR-183-96-182 cluster might play different roles because of tumor heterogeneity, which will be important for the individual diagnosis and prognosis in anti-tumor treatment.

conflIcts of Interest
The authors disclose no potential conflicts of interest.