Reuven Agami

Stable suppression of tumorigenicity by virus-mediated RNA interference

Most human tumors harbor multiple genetic alterations, including dominant mutant oncogenes. It is often not clear which of these oncogenes are continuously required and which, when inactivated, may inhibit tumorigenesis. Recently, we developed a vector that mediates suppression of gene expression through RNA interference. Here, we use a retroviral version of this vector to specifically and stably inhibit expression of only the oncogenic K-RAS(V12) allele in human tumor cells. Loss of expression of K-RAS(V12) leads to loss of anchorage-independent growth and tumorigenicity. These results indicate that viral delivery of small interfering RNAs can be used for tumor-specific gene therapy to reverse the oncogenic phenotype of cancer cells.

A Genetic Screen Implicates miRNA-372 and miRNA-373 As Oncogenes in Testicular Germ Cell Tumors

Endogenous small RNAs (miRNAs) regulate gene expression by mechanisms conserved across metazoans. While the number of verified human miRNAs is still expanding, only few have been functionally annotated. To perform genetic screens for novel functions of miRNAs, we developed a library of vectors expressing the majority of cloned human miRNAs and created corresponding DNA barcode arrays. In a screen for miRNAs that cooperate with oncogenes in cellular transformation, we identified miR-372 and miR-373, each permitting proliferation and tumorigenesis of primary human cells that harbor both oncogenic RAS and active wild-type p53. These miRNAs neutralize p53-mediated CDK inhibition, possibly through direct inhibition of the expression of the tumorsuppressor LATS2. We provide evidence that these miRNAs are potential novel oncogenes participating in the development of human testicular germ cell tumors by numbing the p53 pathway, thus allowing tumorigenic growth in the presence of wild-type p53.

A large-scale RNAi screen in human cells identifies new components of the p53 pathway

RNA interference (RNAi) is a powerful new tool with which to perform loss-of-function genetic screens in lower organisms and can greatly facilitate the identification of components of cellular signalling pathways. In mammalian cells, such screens have been hampered by a lack of suitable tools that can be used on a large scale. We and others have recently developed expression vectors to direct the synthesis of short hairpin RNAs (shRNAs) that act as short interfering RNA (siRNA)-like molecules to stably suppress gene expression. Here we report the construction of a set of retroviral vectors encoding 23,742 distinct shRNAs, which target 7,914 different human genes for suppression. We use this RNAi library in human cells to identify one known and five new modulators of p53-dependent proliferation arrest. Suppression of these genes confers resistance to both p53-dependent and p19ARF-dependent proliferation arrest, and abolishes a DNA-damage-induced G1 cell-cycle arrest. Furthermore, we describe siRNA bar-code screens to rapidly identify individual siRNA vectors associated with a specific phenotype. These new tools will greatly facilitate large-scale loss-of-function genetic screens in mammalian cells.

The microRNAs miR-373 and miR-520c promote tumour invasion and metastasis

MicroRNAs (miRNAs) are single-stranded, noncoding RNAs that are important in many biological processes. Although the oncogenic and tumour-suppressive functions of several miRNAs have been characterized, the role of miRNAs in mediating tumour metastasis was addressed only recently and still remains largely unexplored. To identify potential metastasis-promoting miRNAs, we set up a genetic screen using a non-metastatic, human breast tumour cell line that was transduced with a miRNA-expression library and subjected to a trans-well migration assay. We found that human miR-373 and miR-520c stimulated cancer cell migration and invasion in vitro and in vivo, and that certain cancer cell lines depend on endogenous miR-373 activity to migrate efficiently. Mechanistically, the migration phenotype of miR-373 and miR-520c can be explained by suppression of CD44. We found significant upregulation of miR-373 in clinical breast cancer metastasis samples that correlated inversely with CD44 expression. Taken together, our findings indicate that miRNAs are involved in tumour migration and invasion, and implicate miR-373 and miR-520c as metastasis-promoting miRNAs.

Regulation of the p27Kip1 tumor suppressor by miR‐221 and miR‐222 promotes cancer cell proliferation

MicroRNAs (miRNAs) are potent post‐transcriptional regulators of protein coding genes. Patterns of misexpression of miRNAs in cancer suggest key functions of miRNAs in tumorigenesis. However, current bioinformatics tools do not entirely support the identification and characterization of the mode of action of such miRNAs. Here, we used a novel functional genetic approach and identified miR‐221 and miR‐222 (miR‐221&222) as potent regulators of p27Kip1, a cell cycle inhibitor and tumor suppressor. Using miRNA inhibitors, we demonstrate that certain cancer cell lines require high activity of miR‐221&222 to maintain low p27Kip1 levels and continuous proliferation. Interestingly, high levels of miR‐221&222 appear in glioblastomas and correlate with low levels of p27Kip1 protein. Thus, deregulated expression of miR‐221&222 promotes cancerous growth by inhibiting the expression of p27Kip1.

Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector

We have designed a doxycycline‐regulated form of the H1 promoter of RNA polymerase III that allows the inducible knockdown of gene expression by small interfering RNAs (siRNAs). As a proof‐of‐principle, we have targeted β‐catenin in colorectal cancer (CRC) cells. T‐cell factor (TCF) target‐gene expression is induced by accumulated β‐catenin, and is the main transforming event in these cells. We have shown previously that the disruption of β‐catenin/TCF4 activity in CRC cells by the overexpression of dominant‐negative TCF induces rapid G1 arrest and differentiation. Stable integration of our inducible siRNA vector allowed the rapid production of siRNAs on doxycycline induction, followed by specific downregulation of β‐catenin. In these CRC cells, TCF reporter‐gene activity was inhibited, and G1 arrest and differentiation occurred. The inhibition of two other genes using this vector system shows that it should be useful for the inducible knockdown of gene expression.

MicroRNA regulation by RNA-binding proteins and its implications for cancer

Non-protein-coding transcripts have been conserved throughout evolution, indicating that crucial functions exist for these RNAs. For example, microRNAs (miRNAs) have been found to modulate most cellular processes. The protein classes of RNA-binding proteins include essential regulators of miRNA biogenesis, turnover and activity. RNA–RNA and protein–RNA interactions are essential for post-transcriptional regulation in normal development and may be deregulated in disease. In reviewing emerging concepts of the interplay between miRNAs and RNA-binding proteins, we highlight the implications of these complex layers of regulation in cancer initiation and progression.

The PTEN-regulating microRNA miR-26a is amplified in high-grade glioma and facilitates gliomagenesis in vivo

Activated oncogenic signaling is central to the development of nearly all forms of cancer, including the most common class of primary brain tumor, glioma. Research over the last two decades has revealed the particular importance of the Akt pathway, and its molecular antagonist PTEN (phosphatase and tensin homolog), in the process of gliomagenesis. Recent studies have also demonstrated that microRNAs (miRNAs) may be responsible for the modulation of cancer-implicated genes in tumors. Here we report the identification miR-26a as a direct regulator of PTEN expression. We also show that miR-26a is frequently amplified at the DNA level in human glioma, most often in association with monoallelic PTEN loss. Finally, we demonstrate that miR-26a-mediated PTEN repression in a murine glioma model both enhances de novo tumor formation and precludes loss of heterozygosity and the PTEN locus. Our results document a new epigenetic mechanism for PTEN regulation in glioma and further highlight dysregulation of Akt signaling as crucial to the development of these tumors.

Distinct Initiation and Maintenance Mechanisms Cooperate to Induce G1 Cell Cycle Arrest in Response to DNA Damage

DNA damage causes stabilization of p53, leading to G1 arrest through induction of p21cip1. As this process requires transcription, several hours are needed to exert this response. We show that DNA damage causes an immediate and p53-independent G1 arrest, caused by rapid proteolysis of cyclin D1. Degradation is mediated through a previously unrecognized destruction box in cyclin D1 and leads to a release of p21cip1 from CDK4 to inhibit CDK2. Interference with cyclin D1 degradation prevents initiation of G1 arrest and renders cells more susceptible to DNA damage, indicating that cyclin D1 degradation is an essential component of the cellular response to genotoxic stress. Thus, induction of G1 arrest in response to DNA damage is minimally a two step process: a fast p53-independent initiation of G1 arrest mediated by cyclin D1 proteolysis and a slower maintenance of arrest resulting from increased p53 stability.

Alternative cleavage and polyadenylation: extent, regulation and function

The 3′ end of most protein-coding genes and long non-coding RNAs is cleaved and polyadenylated. Recent discoveries have revealed that a large proportion of these genes contains more than one polyadenylation site. Therefore, alternative polyadenylation (APA) is a widespread phenomenon, generating mRNAs with alternative 3′ ends. APA contributes to the complexity of the transcriptome by generating isoforms that differ either in their coding sequence or in their 3′ untranslated regions (UTRs), thereby potentially regulating the function, stability, localization and translation efficiency of target RNAs. Here, we review our current understanding of the polyadenylation process and the latest progress in the identification of APA events, mechanisms that regulate poly(A) site selection, and biological processes and diseases resulting from APA.