Identification and Validation of Oncologic miRNA Biomarkers for Luminal A-like Breast Cancer

Introduction

Breast cancer is a common disease with distinct tumor subtypes phenotypically characterized by ER and HER2/neu receptor status. MiRNAs play regulatory roles in tumor initiation and progression, and altered miRNA expression has been demonstrated in a variety of cancer states presenting the potential for exploitation as cancer biomarkers. Blood provides an excellent medium for biomarker discovery. This study investigated systemic miRNAs differentially expressed in Luminal A-like (ER+PR+HER2/neu-) breast cancer and their effectiveness as oncologic biomarkers in the clinical setting.

Methods

Blood samples were prospectively collected from patients with Luminal A-like breast cancer (n = 54) and controls (n = 56). RNA was extracted, reverse transcribed and subjected to microarray analysis (n = 10 Luminal A-like; n = 10 Control). Differentially expressed miRNAs were identified by artificial neural network (ANN) data-mining algorithms. Expression of specific miRNAs was validated by RQ-PCR (n = 44 Luminal A; n = 46 Control) and potential relationships between circulating miRNA levels and clinicopathological features of breast cancer were investigated.

Results

Microarray analysis identified 76 differentially expressed miRNAs. ANN revealed 10 miRNAs for further analysis (miR-19b, miR-29a, miR-93, miR-181a, miR-182, miR-223, miR-301a, miR-423-5p, miR-486-5 and miR-652). The biomarker potential of 4 miRNAs (miR-29a, miR-181a, miR-223 and miR-652) was confirmed by RQ-PCR, with significantly reduced expression in blood of women with Luminal A-like breast tumors compared to healthy controls (p = 0.001, 0.004, 0.009 and 0.004 respectively). Binary logistic regression confirmed that combination of 3 of these miRNAs (miR-29a, miR-181a and miR-652) could reliably differentiate between cancers and controls with an AUC of 0.80.

Conclusion

This study provides insight into the underlying molecular portrait of Luminal A-like breast cancer subtype. From an initial 76 miRNAs, 4 were validated with altered expression in the blood of women with Luminal A-like breast cancer. The expression profiles of these 3 miRNAs, in combination with mammography, has potential to facilitate accurate subtype-specific breast tumor detection.     

Arrestin-dependent Angiotensin AT1 Receptor Signaling Regulates Akt and mTor-mediated Protein Synthesis

Control of protein synthesis is critical to both cell growth and proliferation. The mammalian target of rapamycin (mTOR) integrates upstream growth, proliferation, and survival signals, including those transmitted via ERK1/2 and Akt, to regulate the rate of protein translation. The angiotensin AT₁ receptor has been shown to activate both ERK1/2 and Akt in arrestin-based signalsomes. Here, we examine the role of arrestin-dependent regulation of ERK1/2 and Akt in the stimulation of mTOR-dependent protein translation by the AT₁ receptor using HEK293 and primary vascular smooth muscle cell models. Nascent protein synthesis stimulated by both the canonical AT₁ receptor agonist angiotensin II (AngII), and the arrestin pathway-selective agonist [Sar¹-Ile⁴-Ile⁸]AngII (SII), is blocked by shRNA silencing of βarrestin1/2 or pharmacological inhibition of Akt, ERK1/2, or mTORC1. In HEK293 cells, SII activates a discrete arrestin-bound pool of Akt and promotes Akt-dependent phosphorylation of mTOR and its downstream effector p70/p85 ribosomal S6 kinase (p70/85S6K). In parallel, SII-activated ERK1/2 helps promote mTOR and p70/85S6K phosphorylation, and is required for phosphorylation of the known ERK1/2 substrate p90 ribosomal S6 kinase (p90RSK). Thus, arrestins coordinate AT₁ receptor regulation of ERK1/2 and Akt activity and stimulate protein translation via both Akt-mTOR-p70/85S6K and ERK1/2-p90RSK pathways. These results suggest that in vivo, arrestin pathway-selective AT₁ receptor agonists may promote cell growth or hypertrophy through arrestin-mediated mechanisms despite their antagonism of G protein signaling.     

Cell culture media supplementation of uncommonly used sugars sucrose and tagatose for the targeted shifting of protein glycosylation profiles of recombinant protein therapeutics

Protein glycosylation is an important post‐translational modification toward the structure and function of recombinant therapeutics. The addition of oligosaccharides to recombinant proteins has been shown to greatly influence the overall physiochemical attributes of many proteins. It is for this reason that protein glycosylation is monitored by the developer of a recombinant protein therapeutic, and why protein glycosylation is typically considered a critical quality attribute. In this work, we highlight a systematic study toward the supplementation of sucrose and tagatose into cell culture media for the targeted modulation of protein glycosylation profiles on recombinant proteins. Both sugars were found to affect oligosaccharide maturation resulting in an increase in the percentage of high mannose N‐glycan species, as well as a concomitant reduction in fucosylation. The latter effect was demonstrated to increase antibody‐dependent cell‐mediated cytotoxicity for a recombinant antibody. These aforementioned results were found to be reproducible at different scales, and across different Chinese hamster ovary cell lines. Through the selective supplementation of these described sugars, the targeted modulation of protein glycosylation profiles is demonstrated, as well as yet another tool in the cell culture toolbox for ensuring product comparability. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1419–1431, 2014     

Systems analysis of immune responses to attenuated P. falciparum malaria sporozoite vaccination reveals excessive inflammatory signatures correlating with impaired immunity

Immunization with radiation-attenuated sporozoites (RAS) can confer sterilizing protection against malaria, although the mechanisms behind this protection are incompletely understood. We performed a systems biology analysis of samples from the Immunization by Mosquito with Radiation Attenuated Sporozoites (IMRAS) trial, which comprised P. falciparum RAS-immunized (PfRAS), malaria-naive participants whose protection from malaria infection was subsequently assessed by controlled human malaria infection (CHMI). Blood samples collected after initial PfRAS immunization were analyzed to compare immune responses between protected and non-protected volunteers leveraging integrative analysis of whole blood RNA-seq, high parameter flow cytometry, and single cell CITEseq of PBMCs. This analysis revealed differences in early innate immune responses indicating divergent paths associated with protection. In particular, elevated levels of inflammatory responses early after the initial immunization were detrimental for the development of protective adaptive immunity. Specifically, non-classical monocytes and early type I interferon responses induced within 1 day of PfRAS vaccination correlated with impaired immunity. Non-protected individuals also showed an increase in Th2 polarized T cell responses whereas we observed a trend towards increased Th1 and T-bet+ CD8 T cell responses in protected individuals. Temporal differences in genes associated with natural killer cells suggest an important role in immune regulation by these cells. These findings give insight into the immune responses that confer protection against malaria and may guide further malaria vaccine development.Trial registration: ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01994525","term_id":"NCT01994525"}}NCT01994525.     

Two imide substances from a soil-isolated Streptomyces atratus strain provide effective biocontrol activity against grapevine downy mildew

Grapevine downy mildew (Plasmopara viticola) is a devastating disease of grapevine. In this study, 151 actinomycete isolates were obtained and tested for antagonistic activity against P. viticola. The assay suggested that 28 isolates displayed antagonistic effects to varying degrees. The greatest reduction in disease severity was observed with isolate PY-1, which reduced disease severity by 92.13% in the detached leaf assay, and by 83% in a field assay. It was identified as Streptomyces atratus using the 16S rDNA sequence analysis. To elucidate the antagonistic mechanism of PY-1 against P. viticola, scanning electron microscopy showed that major damage to the pathogens sporangia and sporangiophores was observed after treatment for PY-1. Furthermore, PY-1 showed antagonistic activity against other pathogens, including: Botrytis cinerea, Colletotrichum orbiculare, Fusarium oxysporum, Phytophthora capsici and Phytophthora infestans. Two imide compounds were purified from the fermentation liquid using silica gel chromatography and high-performance liquid chromatography and identified as 5-acetoxycycloheximide and cycloheximide using nuclear magnetic resonance. Both compounds showed significant antagonistic activity against P. viticola, determining a reduction in disease severity by 65% and 84%, respectively. In conclusion, 5-acetoxycycloheximide and cycloheximide were identified for the first time in a new S. atratus strain able to effectively control grapevine downy mildew.     

The apparent molecular size of native alpha-crystallin B in non-lenticular tissues

The apparent molecular size of the native alpha-crystallin B in cytosol preparations from rat heart, brain and retina was determined by gel permeation chromatography, detecting the protein by immunochemical assay (ELISA), using an alpha-crystallin specific antiserum. Native alpha-crystallin from cytosol preparations of rat lens cortex was used as a reference. alpha-Crystallin B present in all three cytosol preparations from non-lenticular tissues eluted in a single symmetrical peak, with the same elution volume as alpha-crystallin from lens cortex cytosol preparations, corresponding to an apparent average molecular size of 0.8 x 10(6) Da. No other species could be detected. The results indicate that the alpha-crystallin aggregates characterized by an apparent average molecular mass of 0.8 x 10(6) Da, and considered to be the native, physiological form of the protein in the lens, are indeed not specific to lens tissue. Furthermore, the size of these alpha-crystallin aggregates is independent of their polypeptide composition. Aggregates found in the lens, composed of alpha A and alpha B polypeptides and their respective phosphorylated forms alpha Ap and alpha Bp, are similar in size to those found in heart, brain and retina, containing the alpha B but not the alpha A polypeptide.     

Endothelin stimulates phosphatidic acid formation in cultured rat mesangial cells: role of a protein kinase C-regulated phospholipase D.

We have previously reported that endothelin-1 stimulates phospholipase C-induced hydrolysis of phosphatidylinositol-4,5-bisphosphate. Other signal transduction pathways that hydrolyze alternative phospholipids through phospholipase D may also mediate endothelin-stimulated cellular responses. We initially evaluated endothelin-dependent generation of 32P-phosphatidic acid as an indirect indication of phospholipase D activity in rat mesangial cells. Endothelin (10(-7) M) induced an elevation of phosphatidic acid that was maximal at 15 min and persisted upward of 60 min. Pretreatment with the diacylglycerol-kinase inhibitor, R59022, did not reduce formation of endothelin-stimulated 32P-phosphatidic acid, demonstrating that the sequential actions of phospholipase C/diacylglycerol kinase do not contribute to endothelin-stimulated phosphatidic acid formation. We next conclusively identified a role for phospholipase D in the generation of phosphatidic acid by assessing the formation of 3H-phosphatidylethanol from 3H-alkyl lyso glycerophosphocholine and exogenous ethanol. Endothelin stimulated 3H-alkyl phosphatidylethanol formation in the presence but not the absence of 0.5% ethanol. Also, endothelin induced a concomitant elevation of 3H-alkyl-phosphatidic acid that was significantly reduced when the cells were exposed to exogenous ethanol, reflecting the formation of phosphatidylethanol. In addition, endothelin stimulated the release of 3H-choline and 3H-ethanolamine, demonstrating that additional phospholipids may serve as substrates for phospholipase D. Phorbol esters and synthetic diglycerides mimicked the effects of endothelin to stimulate phospholipase D and inhibitors of protein kinase C significantly reduced endothelin-stimulated phospholipase D. In addition, endothelin did not stimulate phosphatidylethanol formation in protein kinase C down-regulated cells. The calcium ionophore, ionomycin, did not stimulate phospholipase D and mesangial cells pretreated with BAPTA to chelate cytosolic calcium did not show a diminished endothelin-stimulated phospholipase D. Thus these data demonstrate that mesangial cells possess a protein kinase C-regulated phospholipase D activity that can be stimulated with endothelin.     

Lung Epithelial Cells Coordinate Innate Lymphocytes and Immunity against Pulmonary Fungal Infection

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