Anindya Dutta

p21 in cancer: intricate networks and multiple activities

One of the main engines that drives cellular transformation is the loss of proper control of the mammalian cell cycle. The cyclin-dependent kinase inhibitor p21 (also known as p21WAF1/Cip1) promotes cell cycle arrest in response to many stimuli. It is well positioned to function as both a sensor and an effector of multiple anti-proliferative signals. This Review focuses on recent advances in our understanding of the regulation of p21 and its biological functions with emphasis on its p53-independent tumour suppressor activities and paradoxical tumour-promoting activities, and their implications in cancer.

Muscle-specific microRNA miR-206 promotes muscle differentiation

Three muscle-specific microRNAs, miR-206, -1, and -133, are induced during differentiation of C2C12 myoblasts in vitro. Transfection of miR-206 promotes differentiation despite the presence of serum, whereas inhibition of the microRNA by antisense oligonucleotide inhibits cell cycle withdrawal and differentiation, which are normally induced by serum deprivation. Among the many mRNAs that are down-regulated by miR-206, the p180 subunit of DNA polymerase α and three other genes are shown to be direct targets. Down-regulation of the polymerase inhibits DNA synthesis, an important component of the differentiation program. The direct targets are decreased by mRNA cleavage that is dependent on predicted microRNA target sites. Unlike small interfering RNA–directed cleavage, however, the 5′ ends of the cleavage fragments are distributed and not confined to the target sites, suggesting involvement of exonucleases in the degradation process. In addition, inhibitors of myogenic transcription factors, Id1-3 and MyoR, are decreased upon miR-206 introduction, suggesting the presence of additional mechanisms by which microRNAs enforce the differentiation program.

MicroRNAs in Cancer

Within the past few years, studies on microRNA (miRNA) and cancer have burst onto the scene. Profiling of the miRNome (global miRNA expression levels) has become prevalent, and abundant miRNome data are currently available for various cancers. The pattern of miRNA expression can be correlated with cancer type, stage, and other clinical variables, so miRNA profiling can be used as a tool for cancer diagnosis and prognosis. miRNA expression analyses also suggest oncogenic (or tumor-suppressive) roles of miRNAs. miRNAs play roles in almost all aspects of cancer biology, such as proliferation, apoptosis, invasion/metastasis, and angiogenesis. Given that many miRNAs are deregulated in cancers but have not yet been further studied, it is expected that more miRNAs will emerge as players in the etiology and progression of cancer. Here we also discuss miRNAs as a tool for cancer therapy.

Small RNAs with Imperfect Match to Endogenous mRNA Repress Translation IMPLICATIONS FOR OFF-TARGET ACTIVITY OF SMALL INHIBITORY RNA IN MAMMALIAN CELLS

A 21-base pair RNA duplex that perfectly matches an endogenous target mRNA selectively degrades the mRNA and suppresses gene expression in mammalian tissue culture cells. A single base mismatch with the target is believed to protect the mRNA from degradation, making this type of interference highly specific to the targeted gene. A short RNA with mismatches to a target sequence present in multiple copies in the 3′-untranslated region of an exogenously expressed gene can, however, silence it by translational repression. Here we report that a mismatched RNA, targeted to a single site in the coding sequence of an endogenous gene, can efficiently silence gene expression by repressing translation. The antisense strand of such a mismatched RNA requires a 5′-phosphate but not a 3′-hydroxyl group. G·U wobble base pairing is tolerated as a match for both RNA degradation and translation repression. Together, these findings suggest that a small inhibitory RNA duplex can suppress expression of off-target cellular proteins by RNA degradation or translation repression. Proper design of experimental small inhibitory RNAs or a search for targets of endogenous micro-RNAs must therefore take into account that these short RNAs can affect expression of cellular genes with as many as 3–4 base mismatches and additional G·U mismatches.

Depletion of Human Micro-RNA miR-125b Reveals That It Is Critical for the Proliferation of Differentiated Cells but Not for the Down-regulation of Putative Targets during Differentiation

Micro-RNAs are small non-coding RNAs that regulate target gene expression post-transcriptionally through base pairing with the target messenger RNA. Functional characterization of micro-RNAs awaits robust experimental methods to knock-down a micro-RNA as well as to assay its function in vivo. In addition to the recently developed method to sequester micro-RNA with 2′-O-methyl antisense oligonucleotide, we report that small interfering RNA against the loop region of a micro-RNA precursor can be used to deplete the micro-RNA. The depletion of miR-125b by this method had a profound effect on the proliferation of adult differentiated cancer cells, and this proliferation defect was rescued by co-transfected mature micro-RNA. This technique has unique advantages over the 2′-O-methyl antisense oligonucleotide and can be used to determine micro-RNA function, assay micro-RNAs in vivo, and identify the contribution of a predicted micro-RNA precursor to the pool of mature micro-RNA in a given cell. miR-125b and let-7 micro-RNAs are induced, whereas their putative targets, lin-28 and lin-41, are decreased during in vitro differentiation of Tera-2 or embryonic stem cells. Experimental increase or decrease of micro-RNA concentrations did not, however, affect the levels of the targets, a finding that is explained by the fact that the down-regulation of the targets appears to be mostly at the transcriptional level in these in vitro differentiation systems. Collectively these results reveal the importance of micro-RNA depletion strategies for directly determining micro-RNA function in vivo.

A p53-Dependent Checkpoint Pathway Prevents Rereplication

Eukaryotic cells control the initiation of DNA replication so that origins that have fired once in S phase do not fire a second time within the same cell cycle. Failure to exert this control leads to genetic instability. Here we investigate how rereplication is prevented in normal mammalian cells and how these mechanisms might be overcome during tumor progression. Overexpression of the replication initiation factors Cdt1 and Cdc6 along with cyclin A-cdk2 promotes rereplication in human cancer cells with inactive p53 but not in cells with functional p53. A subset of origins distributed throughout the genome refire within 2-4 hr of the first cycle of replication. Induction of rereplication activates p53 through the ATM/ATR/Chk2 DNA damage checkpoint pathways. p53 inhibits rereplication through the induction of the cdk2 inhibitor p21. Therefore, a p53-dependent checkpoint pathway is activated to suppress rereplication and promote genetic stability.

Replication from oriP of Epstein-Barr virus requires human ORC and is inhibited by geminin

A hypomorphic mutation made in the ORC2 gene of a human cancer cell line through homologous recombination decreased Orc2 protein levels by 90%. The G1 phase of the cell cycle was prolonged, but there was no effect on the utilization of either the c-Myc or beta-globin cellular origins of replication. Cells carrying this mutation failed to support the replication of a plasmid bearing the oriP replicator of Epstein Barr virus (EBV), and this defect was rescued by reintroduction of Orc2. Orc2 specifically associates with oriP in cells, most likely through its interaction with EBNA1. Geminin, an inhibitor of the mammalian replication initiation complex, inhibits replication from oriP. Therefore, ORC and the human replication initiation apparatus is required for replication from a viral origin of replication.

PCNA-dependent regulation of p21 ubiquitylation and degradation via the CRL4Cdt2 ubiquitin ligase complex

The DNA polymerase delta processivity factor Proliferating Cell Nuclear Antigen (PCNA) promotes the DNA damage-induced degradation of the replication initiation factor Cdt1 via the CRL4(Cdt2) E3 ubiquitin ligase complex. Here we demonstrate that PCNA promotes the ubiquitylation and degradation of the CDK inhibitor p21 in cells irradiated with low dose of ultraviolet (UV) by a similar mechanism. Human cells that are depleted of Cul4, DDB1 (damage-specific DNA-binding protein-1), or the DCAF Cdt2, are deficient in the UV-induced ubiquitylation and degradation of p21. Depletion of mammalian cells of PCNA by siRNA, or mutations in p21 that abrogate PCNA binding, prevent UV-induced p21 ubiquitylation and degradation, indicating that physical binding with PCNA is necessary for the efficient ubiquitylation of p21 via the CRL4(Cdt2) ubiquitin ligase. Cdt2 functions as the substrate recruiting factor for p21 to the rest of the CRL4 ubiquitin ligase complex. The CRL4(Cdt2) E3 ubiquitin ligase ubiquitylates p21 both in vivo and in vitro, and its activity is dependent on the interaction of p21 with PCNA. Finally, we show that the CRL4(Cdt2) and the SCF(Skp2) ubiquitin ligases are redundant with each other in promoting the degradation of p21 during an unperturbed S phase of the cell cycle.

Right Place, Right Time, and Only Once: Replication Initiation in Metazoans

DNA replication is tightly regulated at the initiation step by both the cell cycle machinery and checkpoint pathways. Here, we discuss recent advances in understanding how replication is initiated in metazoans at the correct chromosome positions, at the appropriate time, and only once per cell cycle.

miR-206 and -486 Induce Myoblast Differentiation by Downregulating Pax7

ABSTRACT The Pax7 transcription factor is required for muscle satellite cell biogenesis and specification of the myogenic precursor lineage. Pax7 is expressed in proliferating myoblasts but is rapidly downregulated during differentiation. Here we report that miR-206 and -486 are induced during myoblast differentiation and downregulate Pax7 by directly targeting its 3′ untranslated region (UTR). Expression of either of these microRNAs in myoblasts accelerates differentiation, whereas inhibition of these microRNAs causes persistence of Pax7 protein and delays differentiation. A microRNA-resistant form of Pax7 is sufficient to inhibit differentiation. Since both these microRNAs are induced by MyoD and since Pax7 promotes the expression of Id2, an inhibitor of MyoD, our results revealed a bistable switch that exists either in a Pax7-driven myoblast state or a MyoD-driven myotube state.