Cancer is a complex genetic disease involving structural and expression abnormalities of both coding and non-coding genes. The search for cancer causing genes has identified two major classes namely the oncogenes and tumor suppressor genes (TSGs). Activation or over expression of a proto-oncogene, such as cMyc,¹ or inactivation of a tumor suppressor, such as TP53,²,³ causes tumorigenicity. Oncogene activation in tumors is straightforward, while the inactivation of TSGs is a complex process attained by different means including mutations, deletions, down regulation by microRNAs, epigenetic silencing etc. Although complete loss of TSG is common in tumors, recent studies indicate that a partial compromise in TSG function termed ‘haploinsufficiency’ contributes to the development and progression of many cancers.⁴ The condition where one functional allele of a gene is lost by mutation or deletion, and the remaining normal allele is insufficient to execute its original physiological function, is called haploinsufficiency. This phenomenon is extensively applicable to TSGs⁵ as they offer a benefit for cancer cells in regards to proliferation,⁶ survival,⁷ and metastasis.⁸

In principle, haploinsufficient TSGs are impaired by a 50% reduction in expression or activity. However, in vivo studies demonstrate that even a subtle 20% reduction of PTEN protein level—termed as ‘quasi-insufficiency’— could contribute to the development of cancer.⁹ Another example is that TP53, when targeted by short hairpin RNAs (shRNAs), is shown to elicit distinct phenotypes ranging from hyperplasia to malignancy in mouse models depending on the reduction in its protein level.¹⁰ This proves that some TSGs, like PTEN, are exquisitely sensitive to dose, while some, like TP53, are intermediately sensitive. Based on such observations, Berger et al has proposed a continuum model that accounts for subtle dosage effects of tumor suppressors including their regulation by microRNAs.¹¹ The dosage and function of haploinsufficient genes are critical, and understanding the impact of haploinsufficiency is important for assessment of interindividual genetic variation, as well as the molecular basis of haploinsufficiency disorders.

Haploinsufficiency of multiple genes cooperate to promote tumorigenesis, a phenomenon called ‘compound haploinsufficiency’. The 5q deletion syndrome (5q-) is a paradigm of compound haploinsufficiency and demonstrates the importance of combinatorial interactions to elicit specific phenotypes.¹² Experimental evidence has shown that co-suppression of linked 8p TSGs promotes tumor formation more potently than any individual gene.¹³ All the available evidence indicates that the functioning of a cell depends on the appropriate expression levels of proteins and, more importantly, that cell signalling pathways involve complex interactions between many proteins. Not only genes, but also microRNAs (miRNAs), a class of small noncoding RNAs, are shown to be haploinsufficient and to cause developmental abnormalities in humans.¹⁴ In addition, genes involved in miRNA biosynthesis pathway, such as DICER1, TARBP2 and XPO5, are identified as haploinsufficient tumor suppressors.¹⁵–¹⁷

miRNAs act at the post-transcriptional level of gene regulation through RNA-induced silencing complex (RISC) mediated translational inhibition or mRNA cleavage. The miRNA target recognition is mediated through the sequence complementarity between the 2–8 nt at the 5′ end of miRNA (seed sequence) and the 3′-UTR of target mRNA.¹⁸ Based on the degree of complementarity, the mRNA is either guided for inhibition of translation or degradation resulting in the decrease of protein encoded by the target messenger.¹⁹ This has extended the dimensions of haploinsufficiency as miRNAs can lead to the production of insufficient amount of proteins. Bioinformatics prediction and experimental analysis suggested that miRNAs can regulate approximately half of the mammalian genes, with a significant number of important oncogenes and tumor suppressor genes involved.²⁰,²¹ A recent genome-wide association study has suggested that a gene with more than two miRNA target sites will have higher variability of expression than a gene which is not regulated by a miRNA. The variability is further increased by SNPs in the miRNA target sites.²² Polymorphisms in the miRNA regulatory pathway are a novel class of functional polymorphisms present in the human genome. The initial demonstration that miRNA binding site variations can result in a phenotype was provided by Abelson et al who identified a mutation in the miR-189 binding site of SLITRK1 and its association with Tourette’s syndrome.²³ A pioneering study conducted by Carlo Croce’s group showed that a germline mutation in pri-miR-16-1 resulted in low levels of miR-16-1 expression in familial chronic lymphocytic leukemia,²⁴,²⁵ providing evidence that sequence variation in miRNA genes may affect function and result in cancer susceptibility. Pre- and pri-miRNA SNPs in miR-124-1, miR-146a, miR-196-a2, miR-218, miR-219-1, miR-26a-1, miR-27a, miR-423, miR-492, and miR-499 have already been shown to increase/decrease cancer risk in various populations.²⁶

Single nucleotide polymorphisms (SNPs) are single base pair changes in DNA that occur with a frequency of about 1 in 12,500 bp in the genome.²⁷ Several studies have shown that SNPs in microRNA networks moderately increase the risk of cancer incidence.²⁸ In general, sequence variations in pri-miRNAs, premiRNAs, mature miRNAs, and microRNA binding sites potentially affects the processing and/or target selection of miRNAs. As a consequence, aberrant expression of hundreds of genes and pathways greatly affecting miRNA function may occur.²⁹ SNPs in mature miRNAs and miRNA binding sites function analogously to modulate the miRNA-mRNA interaction and create or destroy miRNA binding sites. Supporting this idea, SNPs within the miRNA binding sites of genes have been implicated in susceptibility to various types of cancer.³⁰–³³ Additionally, functional support for individual miR-SNPs implicated in cancer do exist.

In this study we analyzed the role of SNPs that occur at miRNA binding sites and miR-SNPs and their contribution towards haploinsufficiency of tumor suppressor genes.

With the expanding understanding of the roles of miRNAs in various cellular processes and diseases, this is the first report to discuss the role of miRNAs and its contribution towards tumor suppressor gene haploinsufficiency at the protein level via 3′UTR SNPs and miR-SNPs. Further, this could be an alternate means for attaining compound heterozygosity observed in several tumors and the mechanism is in complete agreement with the continuum model of tumor suppression.¹¹ Our data suggests that 26% of the tumorigenic haploinsufficient genes were brought under the control of new miRNAs due to 3′UTR SNPs. The identification of 10 SNPs that drive haploinsufficiency by bringing the polymorphic mRNA under the control of miRNAs, other than the miRNAs which are deleted, needs experimental validation in tumors. This alteration in the miRNA mediated gene regulation may cause predisposition to cancer initiation and progression.

Evidence for co-operative contribution of oncogenic mutations with tumor suppressor haploinsufficiency also exist.⁴⁷ The realization that miR-SNPs play central roles in the aberrant regulation of tumor suppressor genes has provided a new perspective on our understanding of pathophysiologic mechanisms. In addition, networking analysis reveals strong interactions between the haploinsufficient tumor suppressor genes. Any subtle alteration in this network of genes due to SNPs at the 3′UTR and miR-SNPs may contribute to pathogenesis. We suggest that at least a few among these SNPs and their effect on miRNA binding will aid in the diagnosis and/or prognosis of such type of cancers, if experimentally validated. While challenges remain in this regard, the pace of development in this field suggests that new discoveries are forthcoming.

Our approach has currently focused only on analyzing 3′UTRs, although a small subset of miRNAs can target 5′UTRs, coding regions, and gene promoters. miR-SNPs leading to non-specific binding of miRNAs to tumor suppressors may also result in the loss of binding to their original targets. If the target is an oncogene, it will be over expressed and will result in tumorigenesis. This has not been focused on in this study. Since the rules for miRNA binding to its target changes constantly,⁴⁸ the databases need constant updating. The 110 tumor suppressor genes chosen for our study were already proven to be haploinsufficient in several cancers through experimental evidence. This list may likely grow due to continuous identification of haploinsufficient tumor suppressors.

fn1-cin-11-2012-157Author Contributions

AKM conceived and designed the study. MM and GR performed the experiments. AKM, MM, and GR analyzed the data. MM and AKM prepared the manuscript. All authors read and approved the final manuscript.

fn2-cin-11-2012-157Competing Interests

Author(s) disclose no potential conflicts of interest.

fn3-cin-11-2012-157Disclosures and Ethics

As a requirement of publication author(s) have provided to the publisher signed confirmation of compliance with legal and ethical obligations including but not limited to the following: authorship and contributorship, conflicts of interest, privacy and confidentiality and (where applicable) protection of human and animal research subjects. The authors have read and confirmed their agreement with the ICMJE authorship and conflict of interest criteria. The authors have also confirmed that this article is unique and not under consideration or published in any other publication, and that they have permission from rights holders to reproduce any copyrighted material. Any disclosures are made in this section. The external blind peer reviewers report no conflicts of interest.

fn4-cin-11-2012-157Funding Sources

This research was supported in part by a grant from the Department of Biotechnology (DBT), New Delhi (Grant Number BT/PR10023/AGR/36/27/2007) to AKM. MM is a recipient of research fellowship from Council of Scientific and Industrial Research (CSIR), New Delhi. We also thank DST-FIST and UGC-SAP for the infrastructure provided by grants to the department.