Effect of Antigen Retrieval on Genomic DNA From Immunodissected Samples.

Immunohistochemical (IHC) staining is an established technique for visualizing proteins in tissue sections for research studies and clinical applications. IHC is increasingly used as a targeting strategy for procurement of labeled cells via tissue microdissection, including immunodissection, computer-aided laser dissection (CALD), expression microdissection (xMD), and other techniques. The initial antigen retrieval (AR) process increases epitope availability and improves staining characteristics; however, the procedure can damage DNA. To better understand the effects of AR on DNA quality and quantity in immunodissected samples, both clinical specimens (KRAS gene mutation positive cases) and model system samples (lung cancer patient-derived xenograft tissue) were subjected to commonly employed AR methods (heat induced epitope retrieval [HIER], protease digestion) and the effects on DNA were assessed by Qubit, fragment analysis, quantitative PCR, digital droplet PCR (ddPCR), library preparation, and targeted sequencing. The data showed that HIER resulted in optimal IHC staining characteristics, but induced significant damage to DNA, producing extensive fragmentation and decreased overall yields. However, neither of the AR methods combined with IHC prevented ddPCR amplification of small amplicons and gene mutations were successfully identified from immunodissected clinical samples. The results indicate for the first time that DNA recovered from immunostained slides after standard AR and IHC processing can be successfully employed for genomic mutation analysis via ddPCR and next-generation sequencing (NGS) short-read methods.

Microdissection Methods Utilizing Single-Cell Subtype Analysis and the Impact on Precision Medicine.

Improving the utilization of tumor tissue from diagnostic biopsies is an unmet medical need. This is especially relevant today in the rapidly evolving precision oncology field where tumor genotyping is often essential for the indication of many advanced and targeted therapies. National Comprehensive Cancer Network (NCCN) guidelines now mandate molecular testing for clinically actionable targets in certain malignancies. Utilizing advanced stage lung cancer as an example, an improved genotyping approach for solid tumors is possible. The strategy involves optimization of the microdissection process and analysis of a large number of identical target cells from formalin-fixed paraffin-embedded (FFPE) specimens sharing similar characteristics, in other words, single-cell subtype analysis. The shared characteristics can include immunostaining status, cell phenotype, and/or spatial location within a histological section. Synergy between microdissection and droplet digital PCR (ddPCR) enhances the molecular analysis. We demonstrate here a methodology that illustrates genotyping of a solid tumor from a small tissue biopsy sample in a time- and cost-efficient manner, using immunostain targeting as an example.

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.

Tristetraprolin: roles in cancer and senescence.

Cancer and senescence are both complex transformative processes that dramatically alter many features of cell physiology and their interactions with surrounding tissues. Developing the wide range of cellular features characteristic of these conditions requires profound alterations in global gene expression patterns, which can be achieved by suppressing, activating, or uncoupling cellular gene regulatory pathways. Many genes associated with the initiation and development of tumors are regulated at the level of mRNA decay, frequently through the activity of AU-rich mRNA-destabilizing elements (AREs) located in their 3'-untranslated regions. As such, cellular factors that recognize and control the decay of ARE-containing mRNAs can influence tumorigenic or senescent phenotypes mediated by products of these transcripts. In this review, we discuss evidence showing how suppressed expression and/or activity of the ARE-binding protein tristetraprolin (TTP) can contribute to these processes. Next, we outline current findings linking TTP suppression to exacerbation of individual tumorigenic phenotypes, and the roles of specific TTP substrate mRNAs in mediating these effects. Finally, we survey potential mechanisms that cells may employ to suppress TTP expression in cancer, and propose potential diagnostic and therapeutic strategies that may exploit the relationship between TTP expression and tumor progression or senescence.

Coordinated expression of tristetraprolin post-transcriptionally attenuates mitogenic induction of the oncogenic Ser/Thr kinase Pim-1.

The serine/threonine kinase Pim-1 directs selected signaling events that promote cell growth and survival and is overexpressed in diverse human cancers. Pim-1 expression is tightly controlled through multiple mechanisms, including regulation of mRNA turnover. In several cultured cell models, mitogenic stimulation rapidly induced and stabilized PIM1 mRNA, however, vigorous destabilization 4-6 hours later helped restore basal expression levels. Acceleration of PIM1 mRNA turnover coincided with accumulation of tristetraprolin (TTP), an mRNA-destabilizing protein that targets transcripts containing AU-rich elements. TTP binds PIM1 mRNA in cells, and suppresses its expression by accelerating mRNA decay. Reporter mRNA decay assays localized the TTP-regulated mRNA decay element to a discrete AU-rich sequence in the distal 3'-untranslated region that binds TTP. These data suggest that coordinated stimulation of TTP and PIM1 expression limits the magnitude and duration of PIM1 mRNA accumulation by accelerating its degradation as TTP protein levels increase. Consistent with this model, PIM1 and TTP mRNA levels were well correlated across selected human tissue panels, and PIM1 mRNA was induced to significantly higher levels in mitogen-stimulated fibroblasts from TTP-deficient mice. Together, these data support a model whereby induction of TTP mediates a negative feedback circuit to limit expression of selected mitogen-activated genes.