Nicole Meisner-Kober

Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties

UNLABELLED Extracellular vesicles (EVs) are natural nanoparticles that mediate intercellular transfer of RNA and proteins and are of great medical interest; serving as novel biomarkers and potential therapeutic agents. However, there is little consensus on the most appropriate method to isolate high-yield and high-purity EVs from various biological fluids. Here, we describe a systematic comparison between two protocols for EV purification: ultrafiltration with subsequent liquid chromatography (UF-LC) and differential ultracentrifugation (UC). A significantly higher EV yield resulted from UF-LC as compared to UC, without affecting vesicle protein composition. Importantly, we provide novel evidence that, in contrast to UC-purified EVs, the biophysical properties of UF-LC-purified EVs are preserved, leading to a different in vivo biodistribution, with less accumulation in lungs. Finally, we show that UF-LC is scalable and adaptable for EV isolation from complex media types such as stem cell media, which is of huge significance for future clinical applications involving EVs. FROM THE CLINICAL EDITOR Recent evidence suggests extracellular vesicles (EVs) as another route of cellular communication. These EVs may be utilized for future therapeutics. In this article, the authors compared ultrafiltration with size-exclusion liquid chromatography (UF-LC) and ultra-centrifugation (UC) for EV recovery.

The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing

Despite progress in mechanistic understanding of the RNA interference (RNAi) pathways, the subcellular sites of RNA silencing remain under debate. Here we show that loading of lipid‐transfected siRNAs and endogenous microRNAs (miRNA) into RISC (RNA‐induced silencing complexes), encounter of the target mRNA, and Ago2‐mediated mRNA slicing in mammalian cells are nucleated at the rough endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found in most subcellular compartments, the miRNA‐ and siRNA‐loaded Ago2 populations co‐sediment almost exclusively with the rER membranes, together with the RISC loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein activator of the interferon‐induced protein kinase (PACT). Fractionation and membrane co‐immune precipitations further confirm that siRNA‐loaded Ago2 physically associates with the cytosolic side of the rER membrane. Additionally, RLC‐associated double‐stranded siRNA, diagnostic of RISC loading, and RISC‐mediated mRNA cleavage products exclusively co‐sediment with rER. Finally, we identify TRBP and PACT as key factors anchoring RISC to ER membranes in an RNA‐independent manner. Together, our findings demonstrate that the outer rER membrane is a central nucleation site of siRNA‐mediated RNA silencing.

The mRNA Stability Factor HuR Inhibits MicroRNA-16 Targeting of COX-2

Commonly observed in colorectal cancer is the elevated expression of the prostaglandin (PG) synthase COX-2. In normal intestinal epithelium, the COX-2 mRNA is targeted for rapid decay through the 3′-untranslated region (3′-UTR) adenylate- and uridylate (AU)-rich element (ARE), whereas in tumors ARE-mediated decay is compromised. Here we show that the COX-2 ARE can mediate degradation through microRNA (miRNA)-mediated regulation. We identified miR-16 to bind the COX-2 3′-UTR and inhibit COX-2 expression by promoting rapid mRNA decay. In colorectal cancer cells and tumors, miR-16 levels were decreased approximately twofold and miR-16 expression in cancer cells attenuated COX-2 expression and PG synthesis. The COX-2 ARE is also bound by the RNA-binding protein HuR. In colorectal cancer tumors, HuR is overexpressed and localized within the cytoplasm, where it promotes ARE-mRNA stabilization. Under conditions of HuR overexpression, miR-16 was unable to promote rapid mRNA decay through the COX-2 ARE. Ribonucleoprotein immunoprecipitation of HuR showed direct association with miR-16 that was reversed when cytoplasmic trafficking of HuR was inhibited. Furthermore, this interaction between HuR and miR-16 promoted the downregulation of miR-16. These new results identify miR-16 as a central posttranscriptional regulator of COX-2 and show the ability of elevated levels of HuR to antagonize miR-16 function. Along with insight into altered ARE-mediated mRNA decay observed in colorectal cancer, these findings provide a new explanation for tumor-derived loss of miR-16. Mol Cancer Res; 10(1); 167–80. ©2011 AACR.

Exosomes surf on filopodia to enter cells at endocytic hot spots, traffic within endosomes, and are targeted to the ER.

Heusermann et al. use a single-vesicle dye-tracing analysis in live cells showing that exosomes enter cells as intact vesicles, primarily at filopodia-active regions, and sort into endocytic vesicle circuits that are targeted to scan the ER before being directed to lysosomes.

The mRNA-binding protein HuR promotes hypoxia-induced chemoresistance through posttranscriptional regulation of the proto-oncogene PIM1 in pancreatic cancer cells.

Previously, it has been shown that pancreatic ductal adenocarcinoma (PDA) tumors exhibit high levels of hypoxia, characterized by low oxygen pressure (pO2) and decreased O2 intracellular perfusion. Chronic hypoxia is strongly associated with resistance to cytotoxic chemotherapy and chemoradiation in an understudied phenomenon known as hypoxia-induced chemoresistance. The hypoxia-inducible, pro-oncogenic, serine–threonine kinase PIM1 (Proviral Integration site for Moloney murine leukemia virus 1) has emerged as a key regulator of hypoxia-induced chemoresistance in PDA and other cancers. Although its role in therapeutic resistance has been described previously, the molecular mechanism behind PIM1 overexpression in PDA is unknown. Here, we demonstrate that cis-acting AU-rich elements (ARE) present within a 38-base pair region of the PIM1 mRNA 3′-untranslated region mediate a regulatory interaction with the mRNA stability factor HuR (Hu antigen R) in the context of tumor hypoxia. Predominantly expressed in the nucleus in PDA cells, HuR translocates to the cytoplasm in response to hypoxic stress and stabilizes the PIM1 mRNA transcript, resulting in PIM1 protein overexpression. A reverse-phase protein array revealed that HuR-mediated regulation of PIM1 protects cells from hypoxic stress through phosphorylation and inactivation of the apoptotic effector BAD and activation of MEK1/2. Importantly, pharmacological inhibition of HuR by MS-444 inhibits HuR homodimerization and its cytoplasmic translocation, abrogates hypoxia-induced PIM1 overexpression and markedly enhances PDA cell sensitivity to oxaliplatin and 5-fluorouracil under physiologic low oxygen conditions. Taken together, these results support the notion that HuR has prosurvival properties in PDA cells by enabling them with growth advantages in stressful tumor microenvironment niches. Accordingly, these studies provide evidence that therapeutic disruption of HuR’s regulation of PIM1 may be a key strategy in breaking an elusive chemotherapeutic resistance mechanism acquired by PDA cells that reside in hypoxic PDA microenvironments.

mRNA stability alterations mediated by HuR are necessary to sustain the fast growth of glioma cells

Regulation of mRNA decay is an important mechanism controlling gene expression. Steady state levels of mRNAs can be markedly altered by changes in the decay rate. The control of mRNA stability depends on sequences in the transcript itself and on RNA-binding proteins that dynamically bind to these sequences. A well characterized sequence motif, which has been shown to be present in many short-lived mRNAs, is the de-stabilizing adenylate/uridylate-rich element (ARE) located at the 3′ untranslated region (3′UTR) of mRNAs. HuR is an RNA-binding protein, which binds to AREs and in doing so, increases the half-life and steady state levels of the corresponding mRNA. Using tissue microarray technology, we found that HuR is over-expressed in human gliomas. We also found that there is a change in HuR localization from being solely in the nucleus to being expressed at high levels in the cytosol. Moreover, a positive correlation was found between total HuR levels, cytosolic localization and tumor grade. We also studied the decay rate of several HuR target mRNAs and found that these mRNAs have a slower rate of decay in glioma cell lines than in astrocytes. Finally, we have been able to decrease both the stability and steady state level of these transcripts in glioma cells using an RNA decoy. More importantly, the decoy transfected cells and cells exposed to a HuR inhibitor have reduced cell growth. In addition, pharmacological inhibition of HuR also resulted in glioma cell growth inhibition. In conclusion, our data suggest that post-transcriptional control abnormalities mediated by HuR are necessary to sustain the rapid growth of this devastating type of cancer.

Impact of HuR inhibition by the small molecule MS-444 on colorectal cancer cell tumorigenesis

Colorectal cancer (CRC) is the third most common cancer and a leading cause of cancer-related mortality. Observed during CRC tumorigenesis is loss of post-transcriptional regulation of tumor-promoting genes such as COX-2, TNFα and VEGF. Overexpression of the RNA-binding protein HuR (ELAVL1) occurs during colon tumorigenesis and is abnormally present within the cytoplasm, where it post-transcriptionally regulates genes through its interaction with 3′UTR AU-rich elements (AREs). Here, we examine the therapeutic potential of targeting HuR using MS-444, a small molecule HuR inhibitor. Treatment of CRC cells with MS-444 resulted in growth inhibition and increased apoptotic gene expression, while similar treatment doses in non-transformed intestinal cells had no appreciable effects. Mechanistically, MS-444 disrupted HuR cytoplasmic trafficking and released ARE-mRNAs for localization to P-bodies, but did not affect total HuR expression levels. This resulted in MS-444-mediated inhibition of COX-2 and other ARE-mRNA expression levels. Importantly, MS-444 was well tolerated and inhibited xenograft CRC tumor growth through enhanced apoptosis and decreased angiogenesis upon intraperitoneal administration. In vivo treatment of MS-444 inhibited HuR cytoplasmic localization and decreased COX-2 expression in tumors. These findings provide evidence that therapeutic strategies to target HuR in CRC warrant further investigation in an effort to move this approach to the clinic.

HuR Contributes to TRAIL Resistance by Restricting Death Receptor 4 Expression in Pancreatic Cancer Cells

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal cancers, in part, due to resistance to both conventional and targeted therapeutics. TRAIL directly induces apoptosis through engagement of cell surface Death Receptors (DR4 and DR5), and has been explored as a molecular target for cancer treatment. Clinical trials with recombinant TRAIL and DR-targeting agents, however, have failed to show overall positive outcomes. Herein, we identify a novel TRAIL resistance mechanism governed by Hu antigen R (HuR, ELAV1), a stress-response protein abundant and functional in PDA cells. Exogenous HuR overexpression in TRAIL-sensitive PDA cell lines increases TRAIL resistance whereas silencing HuR in TRAIL-resistant PDA cells, by siRNA oligo-transfection, decreases TRAIL resistance. PDA cell exposure to soluble TRAIL induces HuR translocation from the nucleus to the cytoplasm. Furthermore, it is demonstrated that HuR interacts with the 3′-untranslated region (UTR) of DR4 mRNA. Pre-treatment of PDA cells with MS-444 (Novartis), an established small molecule inhibitor of HuR, substantially increased DR4 and DR5 cell surface levels and enhanced TRAIL sensitivity, further validating HuRs role in affecting TRAIL apoptotic resistance. NanoString analyses on the transcriptome of TRAIL-exposed PDA cells identified global HuR-mediated increases in antiapoptotic processes. Taken together, these data extend HuRs role as a key regulator of TRAIL-induced apoptosis. Implications: Discovery of an important new HuR-mediated TRAIL resistance mechanism suggests that tumor-targeted HuR inhibition increases sensitivity to TRAIL-based therapeutics and supports their re-evaluation as an effective treatment for PDA patients. Mol Cancer Res; 14(7); 599–611. ©2016 AACR.

Structural basis of siRNA recognition by TRBP double‐stranded RNA binding domains

The accurate cleavage of pre‐micro(mi)RNAs by Dicer and mi/siRNA guide strand selection are important steps in forming the RNA‐induced silencing complex (RISC). The role of Dicer binding partner TRBP in these processes remains poorly understood. Here, we solved the solution structure of the two N‐terminal dsRNA binding domains (dsRBDs) of TRBP in complex with a functionally asymmetric siRNA using NMR, EPR, and single‐molecule spectroscopy. We find that siRNA recognition by the dsRBDs is not sequence‐specific but rather depends on the RNA shape. The two dsRBDs can swap their binding sites, giving rise to two equally populated, pseudo‐symmetrical complexes, showing that TRBP is not a primary sensor of siRNA asymmetry. Using our structure to model a Dicer‐TRBP‐siRNA ternary complex, we show that TRBPs dsRBDs and Dicers RNase III domains bind a canonical 19 base pair siRNA on opposite sides, supporting a mechanism whereby TRBP influences Dicer‐mediated cleavage accuracy by binding the dsRNA region of the pre‐miRNA during Dicer cleavage.

Abstract A05: The mRNA-binding protein HuR promotes hypoxia-induced chemoresistance through post-transcriptional regulation of the serine-threonine kinase PIM1

Pancreatic ductal adenocarcinoma (PDA) is expected to become the 2nd leading cause of cancer-related mortality in the United States by 2020. One of the reasons PDA remains one of the most lethal malignancies is due to drug resistance mechanisms to both conventional and targeted chemotherapeutic regimens. Previously, it has been shown that PDA tumors exhibit high levels of hypoxia, characterized by low oxygen pressure (pO2) and decreased O2 intracellular perfusion. Importantly, chronic hypoxia is strongly associated with resistance to cytotoxic chemotherapy and chemoradiation in a widely understudied phenomenon known as hypoxia-induced chemoresistance. Recently, the hypoxia-inducible, serine-threonine, pro-oncogenic kinase PIM1 has emerged as a key regulator of hypoxia-induced chemoresistance in PDA and other cancers. While its role in therapeutic resistance has been previously described, the molecular mechanism(s) behind PIM1 overexpression in PDA is unknown. Here, we demonstrate that cis-acting AU-rich elements (ARE) present in the PIM1 mRNA 3′UTR mediate a regulatory interaction with the mRNA-stability factor HuR in the context of tumor hypoxia. We have previously demonstrated that HuR, a predominantly nuclear protein, translocates to the cytoplasm upon acute cancer-associated stress and selectively stabilizes key survival-associated transcripts. Here, we demonstrate by immunofluorescence and western blot analyses of fractionated lysates that PDA cells in hypoxic conditions mobilize HuR to the cytoplasm, where it binds and stabilizes the PIM1 transcript. The functional consequence of this interaction is observed with a 5-fold increase in PIM1 protein expression (p≤0.001). PIM1 imparts its oncogenic role by phosphorylating and inactivating key apoptotic proteins (e.g., BAD) and proteins involved in the DNA repair pathway. In this regard, siRNA-mediated knockdown of HuR abrogates hypoxia-induced PIM1 expression and its downstream phosphorylation of the pro-apoptotic protein BAD. As a result, cells undergo growth arrest and succumb to apoptosis. Most importantly, we demonstrate that HuR9s regulation of PIM1 modulates a potent chemoresistance phenotype. HuR-mediated stabilization of PIM1 promoted dramatic resistance to 5-Fluorouracil (5-FU) and Oxaliplatin, two critical components of the promising combination therapy FOLFIRINOX. We employed confocal microscopy to analyze DNA damage foci formation by tracking and quantifying γH2AX foci in response to 5-FU and oxaliplatin. As a result of PIM1 up-regulation in hypoxia, or forced PIM1 overexpression in normoxia, PDA cells exhibit fewer DNA damage foci (p≤0.01) due to PIM19s ability to regulate DNA repair. From a translational standpoint, we demonstrate that disruption of the HuR:PIM1 axis by HuR knockdown results in PDA cell growth inhibition and enhances sensitivity to cytotoxic chemotherapeutic agents (5-FU and oxaliplatin). Similarly, pharmacological inhibition of HuR by MS-444 (Novartis), a small molecule that inhibits HuR homodimerization and its cytoplasmic translocation, abrogates hypoxia-induced PIM1 overexpression and dramatically enhances PDA cell sensitivity to oxaliplatin and 5-FU under physiologic low oxygen conditions. Taken together, these results support the notion that HuR has pro-oncogenic properties in PDA cells by enabling them with selective growth advantages in stressful tumor microenvironment niches. Accordingly, our studies provide evidence that therapeutic disruption of HuR9s regulation of PIM1 may be a key strategy in breaking an elusive chemotherapeutic resistance mechanism acquired by PDA cells that reside in a hypoxic tumor microenvironment. Citation Format: Fernando F. Blanco, Nicole C. Meisner-Kober, Eric Londin, Isidore Rigoutsos, Makarand V. Risbud, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. The mRNA-binding protein HuR promotes hypoxia-induced chemoresistance through post-transcriptional regulation of the serine-threonine kinase PIM1. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A05.