Posttranscriptional Regulation of PTEN Dosage by Noncoding RNAs

Science Signaling  02 Nov 2010:
Vol. 3, Issue 146, pp. pe39
DOI: 10.1126/scisignal.3146pe39


The classic “two-hit” model of tumor-suppressor inactivation, originally established by mathematical modeling of cancer incidence, implies that tumorigenesis requires complete loss of function of tumor-suppressor genes. Although this is true in some tumor types, the exact nature of tumor-suppressor deregulation varies depending on tissue type, stage of cancer development, nature of coexisting molecular lesions, and environmental factors. Emerging evidence has indicated the functional importance of PTEN (phosphatase and tensin homolog) dosage during tumor development. Among the key regulators of PTEN dosage are a number of noncoding RNAs, including microRNAs (miRNAs) and pseudogenes, which regulate PTEN abundance at the posttranscriptional level. Various studies have revealed the essential roles of these PTEN-targeting noncoding RNAs during tumor development, thus providing a paradigm to explore the molecular mechanisms underlying the dosage-dependent effects of key oncogenes and tumor suppressors.

In addition to complete loss of function, alterations of tumor suppressor genes in cancer can also cause partial loss of function, gain of function, or dominant negative phenotypes, which are all important for tumor initiation and progression in a context-dependent manner (1). In particular, there is growing interest in the various monoallelic inactivating tumor-suppressor alterations that occur as an early event of tumorigenesis, which precede the ultimate biallelic inactivation of the same tumor suppressor in late-stage cancers (2). These findings implicate dosage-dependent effects on tumor suppression and suggest that the molecular basis for gene dosage regulation plays a key role in cancer development.

One of the best-studied dosage-dependent tumor suppressors is phosphatase and tensin homolog (PTEN), the essential lipid phosphatase repressor of the phosphoinositidyl-3 kinase (PI3K)–AKT pathway, which promotes proliferation and can contribute to tumorigenesis (3, 4). In various human cancers, such as prostate, breast, colon, and lung, monoallelic PTEN mutations or deletions occur frequently at an early stage of tumorigenesis, but the second PTEN allele remains active (2). It is in advanced and metastatic cancers that biallelic PTEN inactivation becomes prevalent (2). Furthermore, in mouse models in which endogenous Pten expression is genetically altered, Pten inactivation has dosage-dependent effects on tumor progression, latency, and invasiveness (57). For example, heterozygous Pten loss in the mouse model for prostate cancer leads to prostate epithelial hyperplasia and low-grade lesions with incomplete penetrance (7). Additional reductions in Pten dosage causes massive prostate hyperplasia with complete penetrance, accelerating tumor progression (7). Ultimately, complete inactivation of Pten gives rise to invasive and aggressive malignancies (7). Surprisingly, even a subtle reduction in Pten dosage, such as a hypomorphic allele with 80% of wild-type activity, increases tumor formation in mice with cell type–dependent penetrance (5). The complete loss of PTEN triggers cellular senescence, which protects against tumor initiation or progression (8). Therefore, it is tempting to speculate that partial loss of PTEN is likely to be advantageous for tumorigenesis during the initial stage, whereas complete loss of PTEN promotes rapid tumor growth and metastasis after senenscence mechanisms are impaired in advanced tumors.

Although reduced PTEN activity in cancer is often associated with genetic mutations or chromosomal alterations, PTEN expression and its activity are subject to other regulatory mechanisms. Transcriptional repression, epigenetic silencing, posttranscriptional gene regulation, posttranslational modification, and aberrant PTEN localization can all contribute to reduced PTEN activity, thus affecting the progression and the invasiveness of the resulted malignancies (2). If small changes in PTEN dosage cause phenotypical consequences in humans as they do in mouse genetic models, it is likely that genetic or chromosomal alterations affecting these gene regulatory pathways could alter clinical outcome by modulating PTEN abundance. This is consistent with the finding that, in some sporadic tumors with monoallelic PTEN mutations, PTEN abundance can be decreased or lost without detectable mutations or deletions of the second allele (9).

Emerging evidence highlights the importance of posttranscriptional silencing in gene regulation and reveals its essential roles in diverse developmental, physiological, and pathological processes. A key player in this gene regulation is a family of small noncoding RNAs known as microRNAs (miRNAs). First identified in Caenorhabditis elegans as regulators of larval developmental timing (10), miRNAs are now recognized as a large family of noncoding RNAs found in nearly all metazoans. Despite a high degree of functional divergence, most animal miRNAs share a common molecular structure, biogenesis machinery, and effector pathway. The mature miRNA is incorporated into the RNA-induced silencing complex (RISC), which recognizes specific mRNA targets by imperfect sequence complementarity and subsequently mediates their posttranscriptional gene silencing by a combined mechanism of mRNA degradation and translational repression (11). The small size of miRNAs and the imperfect base-pairing with their targets together give miRNAs the capacity to regulate many target mRNAs (12). Therefore, miRNAs often act as global regulators for gene expression, and a single mRNA can be subjected to regulation by multiple miRNAs. The collective action of multiple miRNAs on a particular mRNA may result in a continuum of gene dosage in a cell type– and context-dependent manner. Such dosage-dependent modulation by miRNAs during tumor development could have a considerable effect on the biological outcome.

The connection between miRNAs and cancer was first implied by their frequent genomic alteration and dysregulated abundance in various human tumors (13, 14). Genetic and epigenetic alterations of global miRNA biogenesis machinery also exhibit oncogenic effects (15). Given the importance of PTEN dosage during tumor development, it is not surprising that multiple miRNAs have been identified to modulate PTEN abundance at the posttranscriptional level in the context of malignant transformation. These PTEN-targeting miRNAs include those derived from miRNA precursors containing a single hairpin structure, such as miR-21 (16, 17), miR-22 (18), miR-214 (19), and miR-205 (20), as well as those with a polycistronic structure, such as mir-17-92 (2123), mir-106b-25 (18), mir-367-302b (18), and mir-221-222 (16) (Fig. 1). Many polycistronic PTEN-targeting miRNAs are particularly interesting. Unlike protein-coding genes in which one transcript gives rise to one protein product, a single precursor transcript from a polycistronic miRNA gene yields multiple mature miRNAs (Fig. 1). This gene structure gives miRNA clusters a unique regulatory ability, because specific components of the same miRNA polycistron often have synergistic effects on the same target mRNA. For example, miR-19a and miR-19b, two highly homologous components of mir-17-92, both target PTEN through the same binding sites in the 3′ untranslated region (3′UTR) (22). In mir-106b-25, two nonhomologous components, miR-25 and miR-93, repress PTEN through separate target sites (18). In both cases, although each individual miRNA component moderately decreases PTEN abundance, cooperative effects among different components can achieve greater reduction. Complex modes of posttranscriptional regulation of PTEN abundance by miRNAs present a redundant yet powerful mechanism to generate a range of PTEN dosages. Fine-tuned PTEN abundance translates to precise PI3K-Akt signaling level, which, in turn, determines the exact biological readout of PTEN physiological functions in a cell type– and context-dependent manner.

Fig. 1

Posttranscriptional regulation of PTEN by miRNAs and pseudogenes. miRNAs derived from precursors with either a single-hairpin structure or a polycistronic structure can recognize conserved target sites in both the PTEN 3′UTR and PTENP1 through imperfect base-pairing. PTEN repression can be achieved through miRNA targeting, whereas PTENP1 acts as a decoy for the same miRNA species to derepress PTEN expression.

Credit: Y. Hammond/Science Signaling

Besides directly targeting PTEN, some PTEN-targeting miRNAs act to repress additional components of the same pathway, further increasing the activity of the PI3K-Akt signaling. miR-19 miRNAs, for example, repress both PTEN and the protein phosphatase PP2A, suggesting that similar sequence motifs may exist in the 3′UTRs of functionally related genes to coordinate their posttranscriptional regulation (21, 22, 24). Given the polycistronic structure of several PTEN-targeting miRNA genes, it is conceivable that multiple mature miRNAs encoded by the same miRNA precursor could target different components of the same signaling pathway, thus quantitatively modulating the dosage of the ultimate PI3K-Akt signaling readout.

As exemplified by PTEN, the prominent biological effects of dosage-dependent tumor-suppressor activity illustrate the functional importance of miRNAs in tumorigenesis. Both specific miRNAs and components of the miRNA biogenesis machinery undergo genetic and epigenetic alterations in human cancers. Deregulation of several PTEN-targeting miRNAs, in particular, is prevalent in various human cancers. Enforced expression of these miRNAs promotes malignant transformation by enhancing PI3K-Akt signaling in both cultured cells and animal models. Alteration of the PTEN 3′UTR is also likely to affect miRNA regulation and PTEN dosage and activity through an altered posttranscriptional gene regulation. No PTEN 3′UTR mutations have been identified in human cancer so far. However, there are several examples where defective posttranscriptional gene regulation caused by alterations in the 3′UTR lead to aberrant phenotypes. For example, altered polyadenylation sites in oncogenes often give rise to shortened mRNA isoforms that exhibit increased stability and translation, at least in part because of the loss of miRNA-mediated gene repression (25). In addition, sequence polymorphism in the 3′UTR can alter the efficiency and specificity of miRNA targeting (26). Ongoing cancer genome sequencing projects are likely to reveal functionally important 3′UTR alterations in well-characterized oncogenes and tumor suppressors, which may result in defective miRNA regulation, thus promoting tumor initiation, progression, or metatasis.

Yet another unexpected mechanism for the regulation of PTEN dosage has been revealed in which a PTEN pseudogene acts as a miRNA decoy to modulate PTEN abundance during tumorigenesis (9). The protein-centric view of gene function considers pseudogenes to be nonfunctional variants of known genes because they have lost their protein-coding ability through genetic mutations. This viewpoint has been challenged in recent years, as examples of active transcription of pseudogenes have emerged and the gene regulatory role of pseudogenes was recognized (27). A PTEN pseudogene, PTENP1, shares extensive sequence homology with PTEN mRNA, particularly in the open reading frame region and within the first third of its 3′UTR, which are enriched for known miRNA target sites (9). Because both PTEN and PTENP1 can be regulated by the same set of miRNAs, PTENP1 transcripts may sequester PTEN-targeting miRNAs, indirectly derepressing PTEN expression and enhancing its tumor suppressor activity (9). Consistent with this hypothesis, chromosomal deletion of PTENP1 has been identified in colon and breast cancer samples with decreased PTEN abundance (9). As exciting as this finding is, questions still remain. For example, how does PTENP1 act as a powerful and efficient decoy for PTEN-targeting miRNAs despite its lower abundance compared with that of endogenous PTEN mRNA? Do PTEN-targeting miRNAs regulate PTEN and PTENP1 through the same mechanisms? Last, are there additional noncoding RNAs that function as decoys for PTEN-targeting miRNAs? Answers to these questions will help us fully elucidate the molecular mechanisms underlying pseudogene-mediated posttranscriptional regulation of PTEN.

PTEN is not the only gene that exhibits dosage-dependent tumor suppressor activities at different stages of tumorigenesis. Similar scenarios have been described for a number of other tumor suppressors as well. For example, the role of p53 in mediating DNA repair and autophagic response makes partial p53 activity preferable for tumor cell survival at an early stage of cancer development. It is only at a later stage of tumorigenesis that additional mutations obviate the benefit of partial p53 activity (28) and result in complete p53 inactivation. The importance of gene dosage for oncogene function is emerging as well. For example, high c-Myc signaling can trigger widespread apoptosis, which could prevent tumorigenesis, whereas low-grade c-Myc overexpression is advantageous in early cancer development because it promotes proliferation without triggering apoptosis (29). As a result, robust c-Myc signaling is only prevalent in late-stage cancers that have impaired apoptotic responses (29). Given the pathological importance of gene dosage in tumor development, it is tempting to speculate that defective posttranscriptional gene regulation, combined with genetic mutations, chromosomal alterations, and epigenetic modifications, could generate a range of aberrant tumor suppressor or oncogene dosages that are advantageous for specific stages of malignant transformation. Identifying the crucial players that regulate gene dosage in the oncogene and tumor-suppressor network will provide new insights into the molecular basis underlying tumor development.


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