Pregnancy-upregulated nonubiquitous calmodulin kinase induces ligand-independent EGFR degradation

We describe here an important function of the novel calmodulin kinase I isoform, pregnancy-upregulated nonubiquitous calmodulin kinase (Pnck). Pnck (also known as CaM kinase Iβ2) was previously shown to be differentially overexpressed in a subset of human primary breast cancers, compared with benign mammary epithelial tissue. In addition, during late pregnancy, Pnck mRNA was shown to be strongly upregulated in epithelial cells of the mouse mammary gland exhibiting decreased proliferation and terminal differentiation. Pnck mRNA is also significantly upregulated in confluent and serum-starved cells, compared with actively growing proliferating cells (Gardner HP, Seung HI, Reynolds C, Chodosh LA. Cancer Res 60: 5571–5577, 2000). Despite these suggestive data, the true physiological role(s) of, or the signaling mechanism(s) regulated by Pnck, remain unknown. We now report that epidermal growth factor receptor (EGFR) levels are significantly downregulated in a ligand-independent manner in human embryonic kidney-293 (HEK-293) cells overexpressing Pnck. MAP kinase activation was strongly inhibited by EGFR downregulation in the Pnck-overexpressing cells. The EGFR downregulation was not the result of reduced transcription of the EGFR gene but from protea-lysosomal degradation of EGFR protein. Knockdown of endogenous Pnck mRNA levels by small interfering RNA transfection in human breast cancer cells resulted in upregulation of unliganded EGFR, consistent with the effects observed in the overexpression model of Pnck-mediated ligand-independent EGFR downregulation. Pnck thus emerges as a new component of the poorly understood mechanism of ligand-independent EGFR degradation, and it may represent an attractive therapeutic target in EGFR-regulated oncogenesis

Characterization of RNA from Exosomes and Other Extracellular Vesicles Isolated by a Novel Spin Column-Based Method

<div><p>Exosomes and other extracellular vesicles (commonly referred to as EVs) have generated a lot of attention for their potential applications in both diagnostics and therapeutics. The contents of these vesicles are the subject of intense research, and the relatively recent discovery of RNA inside EVs has raised interest in the biological function of these RNAs as well as their potential as biomarkers for cancer and other diseases. Traditional ultracentrifugation-based protocols to isolate EVs are labor-intensive and subject to significant variability. Various attempts to develop methods with robust, reproducible performance have not yet been completely successful. Here, we report the development and characterization of a spin column-based method for the isolation of total RNA from EVs in serum and plasma. This method isolates highly pure RNA of equal or higher quantity compared to ultracentrifugation, with high specificity for vesicular over non-vesicular RNA. The spin columns have a capacity to handle up to 4 mL sample volume, enabling detection of low-abundance transcripts in serum and plasma. We conclude that the method is an improvement over traditional methods in providing a faster, more standardized way to achieve reliable high quality RNA preparations from EVs in biofluids such as serum and plasma. The first kit utilizing this new method has recently been made available by Qiagen as “<i>exoRNeasy Serum/Plasma Maxi Kit</i>”.</p></div

RNA extraction from high volumes of plasma and serum using several available commercial kits.

<p>EV RNA from 4 mL of plasma or serum was isolated using membrane affinity (spin column), ultracentrifugation (Ultra) and three commercially available methods based on filtration (Kit B) or polymer-based precipitation (Kit S, Kit I) according to the manufacturers recommendations. The method marked with a star had no procedure for processing of high volumes available. The plot depicts raw C_T values of 5 different individuals using an RT-qPCR assay against the GAPDH mRNA. Only column-based purification and ultracentrifugation efficiently recover RNA from high sample volumes.</p

Quantification of RNA yield with increasing plasma volumes.

<p>EV RNA from increasing plasma volumes was extracted with membrane affinity columns and analyzed using RT-qPCR against two mRNAs (BRAF, HPRT1) and a miRNA known to be present in vesicles (let-7a). Shown are raw C_T values with rows as individual extractions and colored diamonds as replicate qPCRs. The signal increase of 1 CT with each doubling of input amount into extraction demonstrates a linear efficiency of EV extraction up to volumes of 4 mL plasma.</p

Recovery of mRNAs and miRNAs from plasma.

<p>EV RNA from 0.2 mL of pre-filtered plasma was isolated using a membrane affinity column and RNA from the flow-through of the spin column was extracted using direct lysis with the miRNeasy Serum/Plasma kit. Shown are raw C_T values from RT-qPCRs with rows as replicate isolations and colored diamonds as replicate qPCRs. Comparing the two fractions shows that the membrane affinity columns capture almost all mRNA and vesicle-specific miRNAs in plasma.</p

Size distribution of total RNA from cancer patient plasma isolated by membrane affinity columns and ultracentrifugation.

<p>Bioanalyzer sizing of vesicle-derived RNA purified by two methods. Total EV RNA from 2 mL plasma of a melanoma patient was isolated using membrane affinity columns and compared total EV RNA from ultracentrifugation, the current gold standard of EV isolation. Both methods purify RNA of similar size and yield.</p

Scanning electron microscopy and western blot analysis of intact vesicles isolated by membrane affinity capture and ultracentrifugation.

<p>(<b>A</b>) Scanning electron microscopy (SEM; 20.000 x magnification) of a solubilized pellet from ultracentrifugation of pre-filtered (0.8 μm) plasma compared to a non-lysed eluate from the membrane affinity spin column. Both preparations contain vesicle-shaped structures with an expected size range from 50–200 nm (white arrows; scale bar 200 nm) indicating that intact vesicles are eluted from the spin column membrane. (<b>B</b>) Exosomes were isolated from four milliliters of normal human plasma using either the membrane affinity column (lane 2) or ultracentrifugation (UC) method (lane 3). Exosomes were concentrated, washed, then lysed, and exosome protein lysates were processed as described in Materials and Methods. The signal for TSG101 runs close to the predicted molecular weight of 43 kDa, the specificity of the TSG101 antibody was confirmed by positive control HeLa cell lysates and further verified by the absence of the 46 kDa band when probed with secondary antibody only. The blot shown is a representative of at least three separate experiments, indicating that the exosome-enriched protein TSG101 is present in vesicles eluted from the membrane affinity column.</p

Workflow for isolating RNA from extracellular vesicles using membrane affinity columns.

<p>EV RNA is isolated from whole blood by separating the plasma or serum, pre-filtering the sample to exclude cell-contamination, and loading on the membrane affinity column followed by a brief wash. The bound vesicles are lysed and eluted with QIAzol; the RNA extracted by addition of chloroform, precipitated by ethanol and further purified using an RNeasy column.</p

Relative quantification of column-bound RNA after treatment with RNase and/or detergent.

<p>Vesicles from 4 mL of pre-filtered plasma were bound to a membrane affinity column and washed. The column was treated for 30 minutes with either RNase A, the detergent Triton X-100, both, or reaction buffer only (mock-treatment). Subsequently, the RNA was isolated and analyzed using RT-qPCR against two mRNAs (GAPDH, HPRT1) and two miRNAs (miR-16, let-7a). The bar plots represent the relative amount of nucleic acids in the sample, compared to mock treatment alone, with columns as mean and whiskers as SD of two replicate isolations each. Assuming a perfect amplification efficiency, the % of PCR signal from mock is calculated as (2^(C_T control–C_T sample))*100 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136133#sec004" target="_blank">methods</a>). Only when a detergent is used to destabilize the lipid-bilayers, the RNase is able to digest the RNA (leftmost columns), indicating that the procedure isolates membrane-protected RNA, a general property of EVs.</p