Investigations in almost every solid malignancy suggest that a thorough understanding of the molecular defects of tumor histological subtypes is critical for the development of effective targeted therapy. The identification of distinct molecular alterations characterizing acral lentiginous melanoma, mucosal melanoma, and uveal melanoma has prompted the initiation of phase II trials evaluating the efficacy of the c-KIT inhibitor Imatinib as part of a subtype-specific melanoma treatment strategy (Eton et al., 2004
; Wyman et al., 2006
). Acral lentiginous melanoma, mucosal melanoma, and uveal melanoma, however, comprises only a small percentage of melanomas diagnosed in the United States (<5%). On the contrary, superficial spreading and nodular melanoma histological subtypes, which represent 70 and 20% of newly diagnosed cases, respectively, are not currently taken into account in decision making, and their molecular classification has not been defined yet.
Although previous studies have mainly focused on the identification of microRNAs associated with melanoma progression (Mueller et al., 2009
; Caramuta et al., 2010
; Philippidou et al., 2010
), in this study we have identified the 134-microRNA signature that distinguishes SSM and NM primary melanoma subtypes. The subset of microRNAs that we found selectively downregulated in SSM compared with both nevi and NM was further studied in order to formally prove that SSM and NM are two different biological entities that do not always arise one from the other. By using an initial screening in “RGP/SSM-like” and “VGP/NM-like” melanoma cell lines, and then SSM and NM primary melanoma specimens, we were in fact able to identify seven microRNAs whose genomic loci
are selectively lost in SSM. We argue that, although the compensatory amplification of the remaining allele can in theory take place, it is unlikely that a genomic locus
that is partially lost at a certain point of tumor progression is regained at later stages. Therefore, we consider the microRNA loci
that we found selectively lost in SSM as a proof of principle that SSM and NM are not sequential phases of melanoma progression.
SSM is the most common histopathological subtype of melanoma and its incidence has risen in the past decades in spite of increased surveillance and earlier detection (Linos et al., 2009
). Although SSM tends to be less aggressive than NM, it does have the ability to metastasize and be fatal. Furthermore, owing to the higher prevalence rate of SSM compared with NM, SSM accounts for a significant portion of melanoma-related deaths (Gimotty et al., 2007
; Shaikh et al., 2011
). Therefore, it is of pivotal importance to identify the alterations that can specifically drive SSM pathogenesis and that can potentially be exploited to develop tailored therapeutic approaches.
Our microarray analysis on the 82 primary melanoma samples composing the training cohort and the 97 primary melanoma samples composing the validation cohort indicates that let-7g, miR-15a, miR-16, miR-138, miR-181a, and miR-191 are consistently expressed at lower levels in SSM than in NM ( and ), possibly because of genomic deletion ( and ). Several pieces of evidence suggest that the diminished expression of these six microRNAs in SSM may contribute to the onset of this melanoma subtype, as described below.
First, these microRNAs have been shown to have an oncosuppressive role in other cancers (see Supplementary Table S4
online for references). In general, they are expressed at a lower level in tumors compared with non-tumoral tissues, and oppose tumor progression by decreasing the abundance of multiple oncogenic targets. Interestingly, some of these targets, such as WNT3A, are known to have an oncogenic role in melanoma as well, although their possible differential contribution to the pathogenesis of SSM and NM has not been considered before.
The canonical WNT1/WNT3A-β-catenin pathway is crucial in the early phases of melanoma. β-Catenin causes the malignant transformation of melanocytes, because it directly represses p16/CDKN2A transcription and, consequently, provides melanocytes with an escape from senescence (O’Connell and Weeraratna, 2009
). The loss of p16 is known to be a more common event in SSM than in NM, and the mechanisms reported thus far (genomic deletion, promoter methylation, and mutation) fail to explain all the cases where the loss occurs (Poetsch et al., 2003
). As amplification or activating mutations in WNT3A have not been detected, we postulate that the loss of miR-15a
might be an SSM-specific mechanism by which WNT3A is upregulated, WNT signaling increases, and p16 transcription is blunted, a mechanism that has not been previously reported. It is also noteworthy that another target of miR-15a/miR-16, the proto-oncogene c-MYB, is upregulated in SSM compared with NM according to two previously published data sets (Jaeger et al., 2007
; Scatolini et al., 2010
). As c-MYB has been recently shown to cooperate with the WNT signaling pathway in colorectal cancer tumorigenesis (Ciznadija et al., 2009
), it is tempting to speculate that the downregulation/deletion of miR-15a/16 might result in two, cooperative oncogenic hits.
Second, the oncosuppressive activity of the identified microRNAs is reinforced by some of the predicted targets that they share. Multiple microRNAs are known to bind to the same mRNA and cooperate in its downregulation (Hobert, 2004
). As we found that let-7g, miR-15a, miR-16, miR-138, miR-181a, and miR-191 were co-downregulated and codeleted, we looked for those genes that are predicted to be targeted by at least two of them and hence may be the most affected from the concomitant decrease in their levels. We found 13 predicted targets that are shared by three micro-RNAs (Supplementary Figure S6
online). Interestingly, most of these targets are expressed at a higher level in SSM than in NM according to two previously published data sets (Jaeger et al., 2007
; Scatolini et al., 2010
) (Supplementary Figure S7
online), a pattern that is consistent with the downregulation/deletion of the selected microRNAs in SSM compared with NM.
Two among the predicted overlapping targets, TNRC6B and CNOT6L, are known to have a role in microRNA-guided posttranscriptional repression (Baillat and Shiekhattar, 2009
; Piao et al., 2010
). CNOT6L is a deadenylase that removes poly(A) tails from mRNAs destabilized by microRNAs (Baillat and Shiekhattar, 2009
; Piao et al., 2010
). These two predicted targets suggest that the deletion/downregulation of the identified microRNAs could impact the microRNA network as a whole.
Many of the remaining predicted genes have a well-established oncogenic role in other cancer types (see Supplementary Table S5
online for references), and might be responsible for the potential oncosuppressive functions of the microRNAs that we have found deleted/downregulated in SSM. Indeed, MSI1, which is a cancer stem cell marker, has been recently shown to be directly targeted by miR-138 (Vo et al., 2011
), validating the prediction obtained using TargetScan. Furthermore, MSI1 is known to sustain the cancer stem cell pool by activating the WNT pathway (Sanchez-Diaz et al., 2008
; Wang et al., 2008
). Therefore, it is possible that the concomitant downregulation of miR-15a/16 and miR-138 could cause the SSM-specific activation of the WNT signaling by multiple mechanisms.
Third, genomic loss has been previously associated with the downregulation of the identified microRNAs, supporting our results. The miR-15a
on 13q14.3 and the let-7g locus
on 3p21.1 are located in minimal deleted regions, as reported in B-CLL and epithelial cancer, respectively (Calin et al., 2004
). In addition, miR-138-2 locus
on 16q13 is deleted in ovarian cancer and melanoma (Zhang et al., 2006
). It is noteworthy that the analysis of a melanoma SNP array recently published by our group (Rose et al., 2011
) indicates that all the loci
corresponding to the identified microRNAs are not part of large deletions. Some of them are indeed located close to other important tumor suppressor genes with a role in melanoma, such as BRCA1-associated protein 1 on 3p21.1 together with let-7g
, and RAS association domain family member 1 on 3p21.3 together with miR-191
(Harbour et al., 2010
; Yi et al., 2011
). Nevertheless, the “focal” nature of the deletion of the identified microRNA loci
suggest that they might be “drivers” and not simply “passengers” in SSM pathogenesis (Akavia et al., 2010
In conclusion, in this study we have identified downregulation and loss in microRNA genes that are specific for the SSM (but not NM) subtype. We propose that these alterations may constitute an SSM-specific “point of entry” for the activation of oncogenic and possibly cancer stem cell–related pathways. Overall, our data lend support to the incorporation of genetic signature into the histopathologic classification of melanoma subtypes.