In this work, we showed that miR-1, which promotes myoblast differentiation, is markedly and reproducibly underrepresented in primary RMS and in RMS cell lines, relative to nonneoplastic muscle tissue. For its essentially identical paralog, miR-206, which in mature muscle is roughly 2 orders of magnitude lower than miR-1 and can also vary, depending on the relative abundance of slow- versus fast-twitch fibers (35
), the downregulation in tumors relative to normal muscle is less clear cut. However, it is important to note that, following growth factor deprivation, both miRNAs failed to be induced in RMS cell lines.
In the attempt of identifying a molecular lesion responsible for this defect, we searched for mutations in the predicted myogenic differentiation factor 1–binding (MyoD-binding) and myogenin-binding sites, located upstream of the Mir1
). In parallel, we also looked for possible mutations in the precursors that could compromise their maturation. However, we did not find any significant change in the DNA sequence of RMS cell lines compared to normal muscle controls. An alternative possibility was methylation/deacetylation (14
), but treatment with either the demethylating agent 5Aza-2-deoxycytidine or the histone deacetylase inhibitor Trichostatin A did not enhance miR-1/miR-206 expression in RMS cells. The failure to upregulate transcription of the myomiRs may simply be due to the fact that in RMS, MyoD seems to be nonfunctional, despite its ability to associate with coactivators and to bind to DNA (38
Re-adjusting miR-206 expression in RMS cells at a level comparable to that of differentiating satellite cells suppressed many aspects of the transformed phenotype. However, the most striking effect was the induction of myogenic differentiation, which occurred even in the presence of growth factors. Thus, miR-206 was sufficient to force the neoplastic cells into resuming and completing the myogenic program. This occurred without changes in the phosphorylation of p38. Once activated, p38 promotes the sequential activation of muscle regulatory factors and their transcriptional coactivators, including chromatin remodelling enzymes (reviewed in ref. 39
). Sustained activation of p38 has been proposed as the missing factor required for rescuing MyoD activity in RMS cells. In fact, forced expression of the constitutively active upstream kinase MKK6-EE in RD and RH30 cells in culture was sufficient to increase morphological and biochemical differentiation (40
). Since miR-206 induced differentiation without changes in p38 phosphorylation, this miRNA may bypass the need for p38 activation by acting downstream of it or through parallel pathways.
Gene expression analysis via microarray revealed that miR-206 expression in RMS cells caused a major switch toward a muscle-like profile, as indicated by the fact that among the more than 270 genes found to be upregulated, many were muscle-specific, such as titin, muscle creatine kinase, myosin light chain, troponin C, myomesin 2, and tropomyosin 2. Of the more than 450 downregulated genes many were involved in the cell cycle and DNA metabolism and repair. Conversely, the 2 minor subsets of genes, whose level of expression was more similar to normal muscle in the uninduced rather than in the induced RD18 cells, did not show any significant enrichment for specific functional categories. It is possible that a transient inverse modulation of these genes might be necessary for the conversion from proliferating to differentiated cells. The time dependency of the switch indicates that most of the observed effects of miR-206 were indirect, but among the downregulated mRNAs, there were also validated (Pola1 , PTBP1 ) and predicted (CDK2) direct targets of miR-206. This finding is in line with the emerging concept that in some cases a major component of miRNA-mediated repression is mRNA destabilization (5
We were particularly interested in the role of miR-1/miR-206 on another recently validated target, the Met receptor (37
), which is activated by overexpression in many cancers, including RMS (28
). We found that in normal myogenic cells, at the onset of myogenesis, Met is rapidly downregulated by miR-206 at the posttranscriptional level. Thus, lack of posttranscriptional downregulation may underlie Met overexpression in RMS and possibly in other types of cancer. Restoration of Met signalling in miR-206–expressing RMS cells via its constitutively active form (Tpr-Met) counteracted the effects of the miRNA, proving that sustained Met expression is one of the factors through which the lack of miR-1/miR-206 contributes to the pathogenesis of RMS.
In previous work, we have shown that Met silencing via RNAi reduces the oncogenicity of RMS cells in culture and in vivo, mainly by increasing apoptosis (29
). Recently, 2 papers described the suppressive effect of ectopic expression of miR-1 in hepatocellular carcinoma and non–small cell lung cancer (NSCLC) cells, in which Met and miR-1 are also, respectively, overexpressed and underrepresented, relative to the corresponding nonneoplastic tissues (14
). In these works, growth inhibition, apoptosis, and loss of tumorigenic properties were entirely ascribed by the authors to the ability of miR-1 to silence the Met receptor. Met silencing may play a major role also in the inhibition of the malignant features of RMS by miR-1/miR-206. However, in our hands, the effects of the shRNA and of miR-1/miR-206 were not overlapping. In particular, Met silencing via RNAi was more efficient than miR-1/miR-206 in inducing apoptosis (29
), while the miRNA was only mildly apoptotic but promoted myogenic differentiation. Thus, we conclude that in RMS the mere loss of Met leads to massive apoptosis, but, when occurring in the presence of a concomitant differentiative signal, it leads to differentiation.
Based on the ability of miR-1 and miR-206 to act as a differentiating agent in RMS cells in culture, we proceeded to test the therapeutic potential of miR-206 by inducing its expression in tumors derived from ERMS or ARMS cells transplanted into nude mice. Although there was no regression, the tumors stopped growing, and the vast majority of the cells exited from the cell cycle and underwent full myogenic differentiation. The results of this experiment constitute the first in vivo proof of concept that miR-206 may have therapeutic potential in RMS as a differentiative agent.
It should be noted, however, that while continuous doxycycline administration kept the tumor in check for the entire period of observation (over 3 months), in approximately 2 weeks after doxycycline withdrawal, the tumor resumed an aggressive modality of growth (data not shown). This suggests that a minor but relevant fraction of the RMS cells, in spite of the proliferative block, did not express a sufficient amount of miR-206 to achieve terminal differentiation. Thus, as in the case of retinoic acid and acute promyelocytic leukemia (APL), chronic administration of miR-206 would be necessary to ensure a permanent block of tumor growth.
Recent work has shown that transfection of the neural-enriched miR-124 induces morphological changes and expression of neuronal markers in mouse neural and brain tumor-derived stem cells as well as in human glioblastoma multiforme–derived (GBM-derived) stem cells (42
). This and our findings suggest that other tissue-specific miRNAs could promote differentiation of related solid malignancies. miRNAs that participate in the control of chromatin remodelling may also hold such potential. For example miR-29, a ubiquitous miRNA previously shown to suppress tumorigenicity by normalizing aberrant patterns of methylation in NSCLC cells (43
), has recently been proposed as an enhancer of myogenic differentiation and a suppressor of RMS (31
Initially, RNAi-based therapeutics, in spite of the still largely unsolved problems of delivery, raised great expectations based on their ability to specifically target dominant oncogenes to which the cancer cells may be addicted. However, the recent discovery that individual cancers carry many more mutations than previously thought and that patients with the same diagnosis can harbor different sets of mutations (44
) has cast serious doubts that such drugs will be active against most solid tumors. Strategies based on differentiative agents have so far been successfully applied only to hematological cancers, such as APL (47
). Differentiation-based nontoxic treatments would be most desirable also for solid tumors, especially in the case of pediatric cancers (RMS or neuroblastomas) or of deadly brain tumors that are impossible to treat surgically or are resistant to traditional therapies (GBM). Our results were obtained in vivo, using cells of 2 RMS subtypes, harboring remarkably different genetic lesions (27
), including nonfunctional mutations of p53 (48
). Silber’s results (42
) were obtained in cell culture, using murine tumor-derived stem cells and also long-established human GBM cell lines. Based on these considerations, we propose that tissue-specific miRNAs may hold greater therapeutic potential than targeted drugs, since their differentiative power is based on the ability to influence the expression of thousands of genes and thus may not be compromised by a heterogenous genetic landscape.