A Translational Approach to Cancer Research, Education and Training.

The demand for biomedical researchers and health science professionals has increased over the past several decades. This need is particularly acute in the fields of cancer research and oncology in which technological advances have fueled an unprecedented pace of laboratory discoveries and their applications in novel diagnostic and therapeutic strategies. Internships that expose undergraduate students to cancer research and patient care serve an important function in meeting this need by educating trainees about careers in this field and inspiring them to pursue these professional paths. Moreover, the translational impetus of cancer research incorporates research, regulatory, business, and clinical components, providing students with even more cancer-focused career options. With the goal of providing hands-on experiences in cancer research and oncology to undergraduate students who comprise the next generation of cancer physician-scientists and will fill this demand in our professional workforce, the Nathan Schnaper Intern Program in Translational Cancer Research (NSIP) has grown from a small laboratory-based local summer internship to a competitive national program. In this study, we evaluate three new modules of the NSIP research, education, and clinical components that have been implemented in the first 2 years of National Cancer Institute Cancer Research Education Grants Program funding. The impact of these modules on intern satisfaction, learning, and near-term career trajectory is assessed to identify the most effective approaches and key measures of program outcomes.

The Roles of RNase-L in Antimicrobial Immunity and the Cytoskeleton-Associated Innate Response.

The interferon (IFN)-regulated endoribonuclease RNase-L is involved in multiple aspects of the antimicrobial innate immune response. It is the terminal component of an RNA cleavage pathway in which dsRNA induces the production of RNase-L-activating 2-5A by the 2'-5'-oligoadenylate synthetase. The active nuclease then cleaves ssRNAs, both cellular and viral, leading to downregulation of their expression and the generation of small RNAs capable of activating retinoic acid-inducible gene-I (RIG-I)-like receptors or the nucleotide-binding oligomerization domain-like receptor 3 (NLRP3) inflammasome. This leads to IFNß expression and IL-1ß activation respectively, in addition to broader effects on immune cell function. RNase-L is also one of a growing number of innate immune components that interact with the cell cytoskeleton. It can bind to several cytoskeletal proteins, including filamin A, an actin-binding protein that collaborates with RNase-L to maintain the cellular barrier to viral entry. This antiviral activity is independent of catalytic function, a unique mechanism for RNase-L. We also describe here the interaction of RNase-L with the E3 ubiquitin ligase and scaffolding protein, ligand of nump protein X (LNX), a regulator of tight junction proteins. In order to better understand the significance and context of these novel binding partners in the antimicrobial response, other innate immune protein interactions with the cytoskeleton are also discussed.

RNase L interacts with Filamin A to regulate actin dynamics and barrier function for viral entry.

UNLABELLED: The actin cytoskeleton and its network of associated proteins constitute a physical barrier that viruses must circumvent to gain entry into cells for productive infection. The mechanisms by which the physical signals of infection are sensed by the host to activate an innate immune response are not well understood. The antiviral endoribonuclease RNase L is ubiquitously expressed in a latent form and activated upon binding 2-5A, a unique oligoadenylate produced during viral infections. We provide evidence that RNase L in its inactive form interacts with the actin-binding protein Filamin A to modulate the actin cytoskeleton and inhibit virus entry. Cells lacking either RNase L or Filamin A displayed increased virus entry which was exacerbated in cells lacking both proteins. RNase L deletion mutants that reduced Filamin A interaction displayed a compromised ability to restrict virus entry, supporting the idea of an important role for the RNase L-Filamin A complex in barrier function. Remarkably, both the wild type and a catalytically inactive RNase L mutant were competent to reduce virus entry when transfected into RNase L-deficient cells, indicating that this novel function of RNase L is independent of its enzymatic activity. Virus infection and RNase L activation disrupt its association with Filamin A and release RNase L to mediate its canonical nuclease-dependent antiviral activities. The dual functions of RNase L as a constitutive component of the actin cytoskeleton and as an induced mediator of antiviral signaling and effector functions provide insights into its mechanisms of antiviral activity and opportunities for the development of novel antiviral agents. IMPORTANCE: Cells constantly face and sample pathogens on their outer surface. The actin cytoskeleton and interacting proteins associate with the cell membrane and constitute a barrier to infection. Disruption of the actin cytoskeleton allows viruses to enter the cell and induces innate immune responses to clear infections. The molecular mechanisms that link virus-induced physical perturbations to host defense pathways remain unclear. Our studies identified a novel interaction between the antiviral endoribonuclease RNase L and the actin-binding protein Filamin A that enhances host defense by preventing viral entry into naive cells. This role for RNase L is independent of its enzymatic function. Virus infection alters actin dynamics, disrupts the RNase L-Filamin A complex, and releases RNase L to mediate antiviral signaling and effector functions via its established nucleolytic activities. These dual roles for RNase L provide an efficient strategy to protect cells from infection and rapidly respond upon pathogen exposure.

RNase L attenuates mitogen-stimulated gene expression via transcriptional and post-transcriptional mechanisms to limit the proliferative response.

The cellular response to mitogens is tightly regulated via transcriptional and post-transcriptional mechanisms to rapidly induce genes that promote proliferation and efficiently attenuate their expression to prevent malignant growth. RNase L is an endoribonuclease that mediates diverse antiproliferative activities, and tristetraprolin (TTP) is a mitogen-induced RNA-binding protein that directs the decay of proliferation-stimulatory mRNAs. In light of their roles as endogenous proliferative constraints, we examined the mechanisms and functional interactions of RNase L and TTP to attenuate a mitogenic response. Mitogen stimulation of RNase L-deficient cells significantly increased TTP transcription and the induction of other mitogen-induced mRNAs. This regulation corresponded with elevated expression of serum-response factor (SRF), a master regulator of mitogen-induced transcription. RNase L destabilized the SRF transcript and formed a complex with SRF mRNA in cells providing a mechanism by which RNase L down-regulates SRF-induced genes. TTP and RNase L proteins interacted in cells suggesting that RNase L is directed to cleave TTP-bound RNAs as a mechanism of substrate specificity. Consistent with their concerted function in RNA turnover, the absence of either RNase L or TTP stabilized SRF mRNA, and a subset of established TTP targets was also regulated by RNase L. RNase L deficiency enhanced mitogen-induced proliferation demonstrating its functional role in limiting the mitogenic response. Our findings support a model of feedback regulation in which RNase L and TTP target SRF mRNA and SRF-induced transcripts. Accordingly, meta-analysis revealed an enrichment of RNase L and TTP targets among SRF-regulated genes suggesting that the RNase L/TTP axis represents a viable target to inhibit SRF-driven proliferation in neoplastic diseases.

RNase-L control of cellular mRNAs: roles in biologic functions and mechanisms of substrate targeting.

RNase-L is a mediator of type 1 interferon-induced antiviral activity that has diverse and critical cellular roles, including the regulation of cell proliferation, differentiation, senescence and apoptosis, tumorigenesis, and the control of the innate immune response. Although RNase-L was originally shown to mediate the endonucleolytic cleavage of both viral and ribosomal RNAs in response to infection, more recent evidence indicates that RNase-L also functions in the regulation of cellular mRNAs as an important mechanism by which it exerts its diverse biological functions. Despite this growing body of work, many questions remain regarding the roles of mRNAs as RNase-L substrates. This review will survey known and putative mRNA substrates of RNase-L, propose mechanisms by which it may selectively cleave these transcripts, and postulate future clinical applications.

RNase-L deficiency exacerbates experimental colitis and colitis-associated cancer.

BACKGROUND: The endoribonuclease RNase-L is a type-I interferon (IFN)-regulated component of the innate immune response that functions in antiviral, antibacterial, and antiproliferative activities. RNase-L produces RNA agonists of RIG-I-like receptors, sensors of cytosolic pathogen-associated RNAs that induce cytokines including IFN-ß. IFN-ß and RIG-I-like receptors signaling mediate protective responses against experimental colitis and colitis-associated cancer and contribute to gastrointestinal homeostasis. Therefore, we investigated a role for RNase-L in murine colitis and colitis-associated cancer and its association with RIG-I-like receptors signaling in response to bacterial RNA. METHODS: Colitis was induced in wild type-deficient and RNase-L-deficient mice (RNase-L⁻/⁻) by administration of dextran sulfate sodium (DSS). Colitis-associated cancer was induced by DSS and azoxymethane (AOM). Histological analysis and immunohistochemistry were performed on colon tissue to analyze immune cell infiltration and tissue damage after induction of colitis. Expression of cytokines was measured by quantitative real-time-PCR and ELISA. RESULTS: DSS-treated RNase-L⁻/⁻ mice exhibited a significantly higher clinical score, delayed leukocyte infiltration, reduced expression of IFN-ß, tumor necrosis factor α, interleukin-1ß, and interleukin-18 at early times post-DSS exposure, and increased mortality as compared with wild-type mice. DSS/AOM-treated RNase-L⁻/⁻ mice displayed an increased tumor burden. Bacterial RNA triggered IFN-ß production in an RNase-L-dependent manner and provided a potential mechanism by which RNase-L contributes to the gastrointestinal immune response to microbiota and protects against experimental colitis and colitis-associated cancer. CONCLUSIONS: RNase-L promotes the innate immune response to intestinal damage and ameliorates murine colitis and colitis-associated cancer. The RNase-L-dependent production of IFN-ß stimulated by bacterial RNA may be a mechanism to protect against gastrointestinal inflammatory disease.

Regulation of human RNase-L by the miR-29 family reveals a novel oncogenic role in chronic myelogenous leukemia.

The endoribonuclease RNase-L is the terminal component of an interferon-regulated RNA decay pathway known as the 2'-5'-oligoadenylate (2-5A) system, whose established functions include antimicrobial and tumor suppressive activities. RNase-L activity requires binding of the small molecule 2-5A, leading to RNase-L dimerization and cleavage of single-stranded RNA. RNase-L expression is controlled post-transcriptionally by its 3'-untranslated region (3' UTR), which exerts a strong negative effect on RNase-L levels. MicroRNAs (miRNAs) are a class of small noncoding RNAs that repress expression of target genes by binding to regions of complementarity often in the 3' UTR. The miR-29 family acts as a tumor suppressor in several cancers, including acute and chronic myelogenous leukemia (CML), and has many oncogenic targets. We report that the miR-29 family represses RNase-L protein expression across several cell types. Using a luciferase reporter, we showed that miR-29 acts via 4 target sites within the RNASEL 3' UTR. Mutation of all sites is required for abrogation of miR-29 repression. In light of the reported tumor suppressive role of miR-29 in K562 CML cells and miR-29 repression of RNase-L in these cells, we generated K562 cells with stable RNase-L knockdown and demonstrated that loss of RNase-L inhibits proliferation in vitro as well as tumor growth in a xenograft model. Our findings identify a previously unknown miRNA regulator of RNase-L expression and support a novel oncogenic role for RNase-L in CML and potentially other hematopoietic malignancies.

Pathologic effects of RNase-L dysregulation in immunity and proliferative control.

The endoribonuclease RNase-L is the terminal component of an RNA cleavage pathway that mediates antiviral, antiproliferative and immunomodulatory activities. Inactivation or dysregulation of RNase-L is associated with a compromised immune response and increased risk of cancer, accordingly its activity is tightly controlled and requires an allosteric activator, 2',5'-linked oligoadenylates, for enzymatic activity. The biological activities of RNase-L are a result of direct and indirect effects of RNA cleavage and microarray analyses have revealed that RNase-L impacts the gene expression program at multiple levels. The identification of RNase-L-regulated RNAs has provided insights into potential mechanisms by which it exerts antiproliferative, proapoptotic, senescence-inducing and innate immune activities. RNase-L protein interactors have been identified that serve regulatory functions and are implicated as alternate mechanisms of its biologic functions. Thus, while the molecular details are understood for only a subset of RNase-L activities, its regulation by small molecules and critical roles in host defense and as a candidate tumor suppressor make it a promising therapeutic target.

A central role for RNA in the induction and biological activities of type 1 interferons.

In mammals the type 1 interferon (IFN) system functions as the primary innate antiviral defense and more broadly as a stress response and regulator of diverse homeostatic mechanisms. RNA plays a central role in the induction of IFN and in its biologic activities. Cellular toll-like receptors (TLR), RIG-I-like receptors (RLR), and nucleotide organization domain-like receptors (NLR) sense pathogen- and danger-associated RNAs as nonself based on structural features and subcellular location that distinguish them from ubiquitous host RNAs. Detection of nonself RNAs activates signaling pathways to induce IFN transcription and secretion. In turn, IFN binds cell surface receptors to initiate signaling that results in the induction of IFN-stimulated genes (ISGs) that mediate its biologic activities. RNA also plays a critical role in this effector phase of the IFN system, serving as an activator of enzyme activity for protein kinase RNA-dependent (PKR) and oligoadenylate synthetase (OAS), and as a substrate for 2('), 5(') -linked oligoadenylate dependant-endoribonuclease (RNase-L). In contrast to the transcriptional response induced by RNA receptors, these key ISGs mediate their activities primarily through post transcriptional mechanisms to regulate the translation and stability of host and microbial RNAs. Together RNA-sensing and RNA-effector molecules comprise a network of coordinately regulated proteins with integrated feedback and feed-forward loops that tightly regulate the cellular response to RNA. This stringent regulation is essential to prevent deleterious effects of uncontrolled IFN expression and effector activation. In light of this extensive crosstalk, targeting key mediators of the cellular response to RNA represents a viable strategy for therapeutic modulation of immune function and treatment of diseases in which this response is dysregulated (e.g., cancer).

An essential role for the antiviral endoribonuclease, RNase-L, in antibacterial immunity.

Type I IFNs were discovered as the primary antiviral cytokines and are now known to serve critical functions in host defense against bacterial pathogens. Accordingly, established mediators of IFN antiviral activity may mediate previously unrecognized antibacterial functions. RNase-L is the terminal component of an RNA decay pathway that is an important mediator of IFN-induced antiviral activity. Here, we identify a role for RNase-L in the host antibacterial response. RNase-L(-/-) mice exhibited a dramatic increase in mortality after challenge with Bacillus anthracis and Escherichia coli; this increased susceptibility was due to a compromised immune response resulting in increased bacterial load. Investigation of the mechanisms of RNase-L antibacterial activity indicated that RNase-L is required for the optimal induction of proinflammatory cytokines that play essential roles in host defense from bacterial pathogens. RNase-L also regulated the expression of the endolysosomal protease, cathepsin-E, and endosome-associated activities, that function to eliminate internalized bacteria and may contribute to RNase-L antimicrobial action. Our results reveal a unique role for RNase-L in the antibacterial response that is mediated through multiple mechanisms. As a regulator of fundamental components of the innate immune response, RNase-L represents a viable therapeutic target to augment host defense against diverse microbial pathogens.

Post-transcriptional regulation of RNase-L expression is mediated by the 3'-untranslated region of its mRNA.

RNase-L mediates critical cellular functions including antiviral, pro-apoptotic, and tumor suppressive activities; accordingly, its expression must be tightly regulated. Little is known about the control of RNASEL expression; therefore, we examined the potential regulatory role of a conserved 3'-untranslated region (3'-UTR) in its mRNA. The 3'-UTR mediated a potent decrease in the stability of RNase-L mRNA, and of a chimeric beta-globin-3'-UTR reporter mRNA. AU-rich elements (AREs) are cis-acting regulatory regions that modulate mRNA stability. Eight AREs were identified in the RNase-L 3'-UTR, and deletion analysis identified positive and negative regulatory regions associated with distinct AREs. In particular, AREs 7 and 8 served a strong positive regulatory function. HuR is an ARE-binding protein that stabilizes ARE-containing mRNAs, and a predicted HuR binding site was identified in the region comprising AREs 7 and 8. Co-transfection of HuR and RNase-L enhanced RNase-L expression and mRNA stability in a manner that was dependent on this 3'-UTR region. Immunoprecipitation demonstrated that RNase-L mRNA associates with a HuR containing complex in intact cells. Activation of endogenous HuR by cell stress, or during myoblast differentiation, increased RNase-L expression, suggesting that RNase-L mRNA is a physiologic target for HuR. HuR-dependent regulation of RNase-L enhanced its antiviral activity demonstrating the functional significance of this regulation. These findings identify a novel mechanism of RNase-L regulation mediated by its 3'-UTR.

VSV disrupts the Rae1/mrnp41 mRNA nuclear export pathway.

Interference with nucleocytoplasmic transport is a strategy employed by certain viruses to compromise host cellular function. While it has been shown that the matrix (M) protein of the vesicular stomatitis virus (VSV) inhibits nuclear export of host cell mRNAs, the underlying mechanism has not been fully established. Here we show that VSV M protein binds the mRNA export factor Rae1/mrnp41. A mutant of M protein defective in Rae1 binding is unable to inhibit mRNA nuclear export. We further show that increased expression of Rae1 fully reverts the inhibition of mRNA export induced by M protein or following virus infection. We found that Rae1 is induced by interferon-gamma, a cytokine that plays a critical role in the immune response to viruses, such as VSV. Thus, these results demonstrate that VSV M protein blocks mRNA export by disrupting Rae1 function, which can be reverted by induction of Rae1 expression.

Expression of a functional secreted YopN-TyeA hybrid protein in Yersinia pestis is the result of a +1 translational frameshift event.

YopN is a secreted protein that prior to secretion directly interacts with the cytosolic SycN/YscB chaperone complex and TyeA. This study identifies a secreted YopN-TyeA hybrid protein that is expressed by Yersinia pestis, but not by Yersinia enterocolitica. DNA sequence analysis and site-directed mutagenesis studies demonstrate that the hybrid protein is the result of a +1 translational frameshift event.

Generation of hepatitis C virus-like particles by use of a recombinant vesicular stomatitis virus vector.

Hepatitis C virus (HCV), a major etiologic agent of hepatocellular carcinoma, presently infects approximately 400 million people worldwide, making the development of protective measures against HCV infection a key objective. Here we have generated a recombinant vesicular stomatitis virus (VSV), which expresses the HCV structural proteins, by inserting the contiguous Core, E1, and E2 coding region of HCV into the VSV genome. Recombinant VSV expressing HCV Core, E1, and E2 (VSV-HCV-C/E1/E2) grew to high titers in vitro and efficiently expressed the incorporated HCV gene product, which became fully processed into the individual HCV structural proteins. Biochemical and biophysical analysis indicated that the HCV Core, E1, and E2 proteins assembled to form HCV-like particles (HCV-LPs) possessing properties similar to the ultrastructural properties of HCV virions. Mice immunized with VSV-HCV-C/E1/E2 generated cell-mediated immune responses to all of the HCV structural proteins, and humoral responses, particularly to E2, were also readily evident. Our data collectively indicate that engineered VSVs expressing HCV Core, E1, and E2 and/or HCV-LPs represent useful tools in vaccine and immunotherapeutic strategies designed to address HCV infection.