Abstract

Background

   miRNAs are considered important players in oncogenesis, serving either
   as oncomiRs or suppressormiRs. Although the accumulation of somatic
   alterations is an intrinsic aspect of cancer development and many
   important cancer-driving mutations have been identified in
   protein-coding genes, the area of functional somatic mutations in miRNA
   genes is heavily understudied.

Methods

   Here, based on the analysis of large genomic datasets, mostly the
   whole-exome sequencing of over 10,000 cancer/normal sample pairs
   deposited within the TCGA repository, we undertook an analysis of
   somatic mutations in miRNA genes.

Findings

   We identified and characterized over 10,000 somatic mutations and
   showed that some of the miRNA genes are overmutated in Pan-Cancer
   and/or specific cancers. Nonrandom occurrence of the identified
   mutations was confirmed by a strong association of overmutated miRNA
   genes with KEGG pathways, most of which were related to specific cancer
   types or cancer-related processes. Additionally, we showed that
   mutations in some of the overmutated genes correlate with miRNA
   expression, cancer staging, and patient survival.

Interpretation

   Our study is the first comprehensive Pan-Cancer study of cancer somatic
   mutations in miRNA genes. It may help to understand the consequences of
   mutations in miRNA genes and the identification of miRNA functional
   mutations. The results may also be the first step (form the basis and
   provide the resources) in the development of computational and/or
   statistical approaches/tools dedicated to the identification of
   cancer-driver miRNA genes.

Funding

   This work was supported by research grants from the Polish National
   Science Centre 2016/22/A/NZ2/00184 and 2015/17/N/NZ3/03629.

   Keywords: miRNA, Somatic mutations, Pan-Cancer, TCGA, Non-coding
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Research in Context Section.

Evidence before this study

   Cancer genome analyses supported by data generated in large projects
   such as The Cancer Genome Atlas (TCGA) have led to the identification
   of hundreds of cancer-driving genes and thousands of cancer-driving
   mutations in protein-coding regions, which encompass barely 2% of the
   genome. Some of these genes/mutations, serve as important biomarkers
   for cancer-targeted therapies. However, very little (close to nothing)
   is known about genetic alterations in regions that encode non-coding
   RNAs, including miRNAs that are considered important players in
   oncogenesis, serving either as oncomiRs or suppressormiRs.

Added value of this study

   With the use of sequencing datasets generated within the large
   cancer-genome projects, mostly TCGA, we identified and characterized
   >10,000 miRNA gene mutations in 33 types of cancers. Among the
   identified variants were mutations in well-known oncomiRs and miRNA
   suppressors, including let-7, miR-21, and miR-205. We identified and
   characterized dozens of significantly overmutated miRNA genes and
   hotspot nucleotide positions that are recurrently mutated in particular
   cancer types or the overall Pan-Cancer dataset and showed that some of
   these mutations affect miRNA expression, cancer staging, and patient
   survival as well as occur more frequently in miRNA genes playing role
   in cancer.

Implication of all the available evidence

   This is the first comprehensive analysis of somatic mutations in miRNA
   genes in cancer. It may serve as the first step in the identification
   of driver mutations in miRNA genes and may help in understanding the
   role of particular miRNAs in cancer. After further functional
   validation, some of the mutations may be utilized as cancer biomarkers.

   Alt-text: Unlabelled box

1. Introduction

   Cancer encompasses a broad spectrum of heterogeneous diseases whose
   development (i.e., initiation, promotion, and progression) is
   associated with the accumulation of numerous genetic alterations in the
   cancer genome, which is the hallmark of all cancers. Although most of
   these alterations are neutral, some of the randomly occurring mutations
   are functional, providing a growth advantage to a neoplastic cell
   [35][1]. As a result of clonal selection of the fastest dividing cells,
   functional (driver) mutations often recur in genes playing an important
   role in cancer development (driver genes) and therefore may serve as
   indicators of such genes. Numerous large cancer genome sequencing
   studies (mostly whole-exome sequencing, WES) have been performed, and
   hundreds of cancer-driving genes and thousands of cancer-driving
   mutations have been detected. Some of these genes/mutations, e.g.,
   EGFR, BRAF, and JAK2, serve as important biomarkers for cancer-targeted
   therapies. As the overwhelming majority of the cancer genome studies
   have focused on protein-coding genes, the most identified cancer-driver
   mutations are in protein-coding sequences, which encompass barely 2% of
   the genome. The spectacular exception are TERT promoter mutations,
   which occur most frequently in melanoma, brain, and bladder cancers but
   have also been identified in other cancers [36][2], [37][3], [38][4].

   On the other hand, a growing body of evidence indicates that miRNAs, a
   class of short (~21 nt long) single-stranded non-coding RNAs, play an
   important role in cancer, and it was shown that particular miRNAs can
   either drive (oncomiRs, often upregulated in cancer) or suppress
   (suppressormiRs, often downregulated in cancer) oncogenesis. It was
   also proposed that miRNAs have great potential as cancer biomarkers
   and/or targets of cancer therapies [39][5], [40][6], [41][7], [42][8].

   Among the most intensively studied miRNAs whose function in cancer is
   best documented are the let-7 family, miR-17-92 cluster (oncomiR-1),
   miR-21, and miR-205 (reviewed in [43][9]). Although the global level of
   miRNA is generally downregulated in cancer, many miRNAs are
   consistently either upregulated or downregulated in particular cancer
   types or specific cancer conditions. It was also shown that miRNA genes
   frequently show copy number alterations (either amplification or
   deletion) in cancer [[44]10,[45]11]. The cancer-related processes that
   are regulated by miRNAs include cell proliferation,
   epithelial-mesenchymal transformation (EMT), migration, angiogenesis,
   inflammation, apoptosis, and response to cancer treatment (reviewed in
   [46][12], [47][13], [48][14], [49][15]).

   Despite the great interest in the role of miRNA in cancer, very little
   (close to nothing) is known about somatic mutations in miRNA genes
   (defined here as sequences coding for the most crucial part of miRNA
   precursors) occurring in cancer. Considering subsequent steps of miRNA
   biogenesis and the mechanism of miRNA posttranscriptional gene
   regulation, mutations may be expected to affect different attributes of
   miRNA genes. In addition to the most obvious consequences of mutations
   in seed sequences that affect the pivotal function of miRNAs, i.e., the
   ability to recognize and downregulate their specific targets, mutations
   in any part of the miRNA precursor may affect the effectiveness or
   precision of miRNA biogenesis by altering/destabilizing the hairpin
   structure of the miRNA precursor, by altering DROSHA or DICER1 cleavage
   sites, or by altering protein-interacting or other regulatory
   sequences/structure motifs [50][16], [51][17], [52][18], [53][19].
   Additionally, mutations destabilizing one of the miRNA duplex ends may
   alter 5p/3p miRNA preference. Despite the scarcity of identified miRNA
   gene mutations, the individual examples of SNPs, germline or somatic
   mutations provide proof, at least for some of the scenarios listed
   above. Examples include (i) the mutation in the seed sequence of
   miR-204-5p, affecting target recognition, that causes inherited retinal
   dystrophy [54][20]; (ii) the mutation in the passenger strand of
   hsa-miR-96 that destabilizes the structure of the miRNA precursor,
   affects its processing, and decreases the miR-96-5p level, eventually
   resulting in the same phenotypic effect as mutations in the seed
   sequence of the guide strand, i.e., nonsyndromic inherited hearing loss
   [[55]21,[56]22]; (iii) the G>C substitution (SNP rs138166791) in the
   penultimate position of the 3p passenger strand of hsa-miR-890 that
   significantly lowers the cleavage efficiency by DROSHA and consequently
   decreases the levels of both mature miR-890-5p and passenger miR-890-3p
   [57][23]; (iv) the G>C substitution (SNP rs2910164) located in the 3p
   passenger strand of hsa-miR-146a that is associated with an increased
   risk of papillary thyroid carcinoma, where it was shown that the C
   allele of the SNP alters the structure of the precursor, decreases
   expression of the mature miRNA and activates the passenger strand,
   which becomes the second mature miRNA modulating many genes involved in
   the regulation of apoptosis [58][24]; (v) interesting example is a
   mutation in the seed sequence of miR-184-3p, causing familial
   keratoconus, whose effect is not the disruption of miR-184-3p target
   recognition but the inability to mask overlapping targets for miR-205
   in INPPL1 and ITGB4 [59][25]; (vi) mutations in hsa-miR-30c-1 and
   hsa-miR-17 that affect the precursor structure and thereby increase the
   levels of mature miRNAs, downregulating BRCA1 in familial breast cancer
   cases without BRCA1/2 mutations [60][26]; and finally, (vii) two
   different somatic mutations in the seed sequence of miR-142-3p, found
   in acute myeloid leukemia (AML) samples, that were shown to decrease
   miR-142-5p and reverse the miR-5p/3p ratio (in favor of miR-3p)
   [61][27] (more details and references on mutations in hsa-miR-142 in