Plasma extracellular vesicle messenger RNA profiling identifies prognostic EV signature for non-invasive risk stratification for survival prediction of patients with pancreatic ductal adenocarcinoma.

BACKGROUND: The prognosis of pancreatic ductal adenocarcinoma (PDAC) is one of the most dismal of all cancers and the median survival of PDAC patients is only 6-8 months after diagnosis. While decades of research effort have been focused on early diagnosis and understanding of molecular mechanisms, few clinically useful markers have been universally applied. To improve the treatment and management of PDAC, it is equally relevant to identify prognostic factors for optimal therapeutic decision-making and patient survival. Compelling evidence have suggested the potential use of extracellular vesicles (EVs) as non-invasive biomarkers for PDAC. The aim of this study was thus to identify non-invasive plasma-based EV biomarkers for the prediction of PDAC patient survival after surgery. METHODS: Plasma EVs were isolated from a total of 258 PDAC patients divided into three independent cohorts (discovery, training and validation). RNA sequencing was first employed to identify differentially-expressed EV mRNA candidates from the discovery cohort (n = 65) by DESeq2 tool. The candidates were tested in a training cohort (n = 91) by digital droplet polymerase chain reaction (ddPCR). Cox regression models and Kaplan-Meier analyses were used to build an EV signature which was subsequently validated on a multicenter cohort (n = 83) by ddPCR. RESULTS: Transcriptomic profiling of plasma EVs revealed differentially-expressed mRNAs between long-term and short-term PDAC survivors, which led to 10 of the top-ranked candidate EV mRNAs being tested on an independent training cohort with ddPCR. The results of ddPCR enabled an establishment of a novel prognostic EV mRNA signature consisting of PPP1R12A, SCN7A and SGCD for risk stratification of PDAC patients. Based on the EV mRNA signature, PDAC patients with high risk displayed reduced overall survival (OS) rates compared to those with low risk in the training cohort (p = 0.014), which was successfully validated on another independent cohort (p = 0.024). Interestingly, the combination of our signature and tumour stage yielded a superior prognostic performance (p = 0.008) over the signature (p = 0.022) or tumour stage (p = 0.016) alone. It is noteworthy that the EV mRNA signature was demonstrated to be an independent unfavourable predictor for PDAC prognosis. CONCLUSION: This study provides a novel and non-invasive prognostic EV mRNA signature for risk stratification and survival prediction of PDAC patients.

miR-132-3p and KLF7 as novel regulators of aortic stiffening-associated EndMT in type 2 diabetes mellitus.

BACKGROUND: The prevalence of diabetes mellitus has risen considerably and currently affects more than 422 million people worldwide. Cardiovascular diseases including myocardial infarction and heart failure represent the major cause of death in type 2 diabetes (T2D). Diabetes patients exhibit accelerated aortic stiffening which is an independent predictor of cardiovascular disease and mortality. We recently showed that aortic stiffness precedes hypertension in a mouse model of diabetes (db/db mice), making aortic stiffness an early contributor to cardiovascular disease development. Elucidating how aortic stiffening develops is a pressing need in order to halt the pathophysiological process at an early time point. METHODS: To assess EndMT occurrence, we performed co-immunofluorescence staining of an endothelial marker (CD31) with mesenchymal markers (α-SMA/S100A4) in aortic sections from db/db mice. Moreover, we performed qRT-PCR to analyze mRNA expression of EndMT transcription factors in aortic sections of db/db mice and diabetic patients. To identify the underlying mechanism by which EndMT contributes to aortic stiffening, we used aortas from db/db mice and diabetic patients in combination with high glucose-treated human umbilical vein endothelial cells (HUVECs) as an in vitro model of diabetes-associated EndMT. RESULTS: We demonstrate robust CD31/α-SMA and CD31/S100A4 co-localization in aortic sections of db/db mice which was almost absent in control mice. Moreover, we demonstrate a significant upregulation of EndMT transcription factors in aortic sections of db/db mice and diabetic patients. As underlying regulator, we identified miR-132-3p as the most significantly downregulated miR in the micronome of db/db mice and high glucose-treated HUVECs. Indeed, miR-132-3p was also significantly downregulated in aortic tissue from diabetic patients. We identified Kruppel-like factor 7 (KLF7) as a target of miR-132-3p and show a significant upregulation of KLF7 in aortic sections of db/db mice and diabetic patients as well as in high glucose-treated HUVECs. We further demonstrate that miR-132-3p overexpression and KLF7 downregulation ameliorates EndMT in high glucose-treated HUVECs. CONCLUSIONS: We demonstrate for the first time that EndMT contributes to aortic stiffening in T2D. We identified miR-132-3p and KLF7 as novel EndMT regulators in this context. Altogether, this gives us new insights in the development of aortic stiffening in T2D.

An In Silico Analysis Reveals an EMT-Associated Gene Signature for Predicting Recurrence of Early-Stage Lung Adenocarcinoma.

Background: The potential micrometastasis tends to cause recurrence of lung adenocarcinoma (LUAD) after surgical resection and consequently leads to an increase in the mortality risk. Compelling evidence has suggested the underlying mechanisms of tumor metastasis could involve the activation of an epithelial-mesenchymal transition (EMT) program. Hence, the objective of this study was to develop an EMT-associated gene signature for predicting the recurrence of early-stage LUAD. Methods: The mRNA expression data of patients with early-stage LUAD were downloaded from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) available databases. Gene Set Variation Analysis (GSVA) was first performed to provide an assessment of EMT phenotype, whereas Weighted Gene Co-expression Network Analysis (WGCNA) was constructed to determine EMT-associated key modules and genes. Based on the genes, a novel EMT-associated signature for predicting the recurrence of early-stage LUAD was identified using a least absolute shrinkage and selection operator (LASSO) algorithm and a stepwise Cox proportional hazards regression model. Kaplan-Meier survival analysis, receiver operating characteristic (ROC) curves and Cox regression analyses were used to estimate the performance of the identified gene signature. Results: GSVA revealed diverse EMT states in the early-stage LUAD. Further correlation analyses showed that the EMT states presented high correlations with several hallmarks of cancers, tumor purity, tumor microenvironment cells, and immune checkpoint genes. More importantly, Kaplan-Meier survival analyses indicated that patients with high EMT scores had worse recurrence-free survival (RFS) and overall survival (OS) than those with low EMT scores. A novel 5-gene signature (AGL, ECM1, ENPP1, SNX7, and TSPAN12) was established based on the EMT-associated genes from WGCNA and this signature successfully predicted that the high-risk patients had a higher recurrence rate compared with the low-risk patients. In further analyses, the signature represented robust prognostic values in 2 independent validation cohorts (GEO and TCGA datasets) and a combined GEO cohort as evaluated by Kaplan-Meier survival (P-value < .0001) and ROC analysis (AUC = 0.781). Moreover, the signature was corroborated to be independent of clinical factors by univariate and multivariate Cox regression analyses. Interestingly, the combination of the signature-based recurrence risk and tumor-node-metastasis (TNM) stage showed a superior predictive ability on the recurrence of patients with early-stage LUAD. Conclusion: Our study suggests that patients with early-stage LUAD exhibit diverse EMT states that play a vital role in tumor recurrence. The novel and promising EMT-associated 5-gene signature identified and validated in this study may be applied to predict the recurrence of early-stage LUAD, facilitating risk stratification, recurrence monitoring, and individualized management for the patients after surgical resection.

Extravesicular TIMP-1 is a non-invasive independent prognostic marker and potential therapeutic target in colorectal liver metastases.

Molecular reprogramming of stromal microarchitecture by tumour-derived extracellular vesicles (EVs) is proposed to favour pre-metastatic niche formation. We elucidated the role of extravesicular tissue inhibitor of matrix metalloproteinase-1 (TIMP1EV) in pro-invasive extracellular matrix (ECM) remodelling of the liver microenvironment to aid tumour progression in colorectal cancer (CRC). Immunohistochemistry analysis revealed a high expression of stromal TIMP1 in the invasion front that was associated with poor progression-free survival in patients with colorectal liver metastases. Molecular analysis identified TIMP1EV enrichment in CRC-EVs as a major factor in the induction of TIMP1 upregulation in recipient fibroblasts. Mechanistically, we proved that EV-mediated TIMP1 upregulation in recipient fibroblasts induced ECM remodelling. This effect was recapitulated by human serum-derived EVs providing strong evidence that CRC release active EVs into the blood circulation of patients for the horizontal transfer of malignant traits to recipient cells. Moreover, EV-associated TIMP1 binds to HSP90AA, a heat-shock protein, and the inhibition of HSP90AA on human-derived serum EVs attenuates TIMP1EV-mediated ECM remodelling, rendering EV-associated TIMP1 a potential therapeutic target. Eventually, in accordance with REMARK guidelines, we demonstrated in three independent cohorts that EV-bound TIMP1 is a robust circulating biomarker for a non-invasive, preoperative risk stratification in patients with colorectal liver metastases.

Schwann cell-derived exosomes: Janus-faced mediators of regeneration and disease.

The phenotypic plasticity of Schwann cells (SCs) has contributed to the regenerative potential of the peripheral nervous system (PNS), but also pathological processes. This double-sided effect has led to an increasing attention to the role of extracellular vesicles (EVs) or exosomes in SCs to examine the intercellular communication between SCs and their surroundings. Here, we first describe the current knowledge of SC and EV biology, which forms the basis for the updates on advances in SC-derived exosomes research. We seek to explore in-depth the exosome-mediated molecular mechanisms involved in the regulation of SCs and their microenvironment. This review concludes with potential applications of SC-derived exosomes as delivery vehicles for therapeutics and biomarkers. The goal of this review is to emphasize the crucial role of SC-derived exosomes in the functional integration of the PNS, highlighting an emerging area in which there is much to explore and re-explore.

Proteomic Analyses of Fibroblast- and Serum-Derived Exosomes Identify QSOX1 as a Marker for Non-invasive Detection of Colorectal Cancer.

The treatment of colorectal cancer (CRC) has improved during the last decades, but methods for crucial early diagnosis are yet to be developed. The influence of the tumour microenvironment on liquid biopsies for early cancer diagnostics are gaining growing interest, especially with emphasis on exosomes (EXO), a subgroup of extracellular vesicles (EVs). In this study, we established paired cancer-associated (CAFs) and normal fibroblasts (NF) from 13 CRC patients and investigated activation status-related protein abundance in derived EXOs. Immunohistochemical staining of matched patient tissue was performed and an independent test cohort of CRC patient plasma-derived EXOs was assessed by ELISA. A total of 11 differentially abundant EV proteins were identified between NFs and CAFs. In plasma EXOs, the CAF-EXO enriched protein EDIL3 was elevated, while the NF-EXO enriched protein QSOX1 was diminished compared to whole plasma. Both markers were significantly reduced in patient-matched CRC tissue compared to healthy colon tissue. In an independent test cohort, a significantly reduced protein abundance of QSOX1 was observed in plasma EXOs from CRC patients compared to controls and diagnostic ROC curve analysis revealed an AUC of 0.904. In conclusion, EXO-associated QSOX1 is a promising novel marker for early diagnosis and non-invasive risk stratification in CRC.

Combined use of irinotecan with histone deacetylase inhibitor belinostat could cause severe toxicity by inhibiting SN-38 glucuronidation via UGT1A1.

SN-38, the active metabolite of irinotecan, and histone deacetylase inhibitors (HDACis) such as belinostat, vorinostat and panobinostat, have all been shown to be deactivated by glucuronidation via UGTs. Since they all compete for UGTs for deactivation, we aimed to investigate the inhibitory effect of various HDACis on the glucuronidation of SN-38. This inhibitory effect was determined by measuring the formation rate of SN-38 glucuronide after SN-38 incubation with human recombinant UGT1A isoforms (1A1, 1A6, 1A7 and 1A9) and pooled human liver microsomes (HLM, wild type, UGT1A1*1*28 and UGT1A1*28*28 allelic variants), with and without HDACis. The data showed that belinostat at 100 and 200 µmol/L inhibited SN-38 glucuronidation via UGT1A1 in a dose-dependent manner, causing significant decrease in Vmax and CLint (p < 0.05) from 12.60 to 1.95 pmol/min/mg and 21.59 to 4.20 µL/min/mg protein respectively. Similarly, in HLMs, Vmax dropped from 41.13 to 10.54, 24.96 to 3.77 and 6.23 to 3.30 pmol/min/mg, and CLint reduced from 81.25 to 26.11, 29.22 to 6.10 and 5.40 to 1.34 µL/min/mg protein for the respective wild type, heterozygous and homozygous variants. Interestingly, belinostat at 200 µmol/L that is roughly equivalent to the average Cmax, 183 µmol/L of belinostat at a dose of 1,400 mg/m2 given intravenously once per day on days 1 to 5 every 3 weeks, was able to inhibit both heterozygous and homozygous variants to same extents (~64%). This highlights the potential clinical significance, as a large proportion of patients could be at risk of developing severe toxicity if irinotecan is co-administered with belinostat.

UGT1A1 Mediated Drug Interactions and its Clinical Relevance.

BACKGROUND: The administration of multiple drugs for the treatment of diseases is an integral aspect of modern medicine. Though its purpose is to create the intended therapeutic effect, the unintended consequences of drug interactions can cause severe side effects and subsequent economic losses. Likewise, herbal extracts and supplements with pharmacologically active moieties also have the potential to interact with medications. There are many possible mechanisms on how these moieties could potentially interact, one of which is mediated by modulation of the activity of metabolizing enzymes. One such enzyme of high clinical significance is uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1). Genetic polymorphism of UGT1A1 has been found to affect the plasma concentrations of many drugs, and may even be linked to treatment outcome. OBJECTIVE: This mini-review summarized the potential in vitro and in vivo interactions mediated by UGT1A1. METHOD: Firstly, literature search was conducted using the Web of Knowledge database. No date limitation was applied to the search. Following which, the interactions were stratified into 3 main categories based on its clinical significance. Both herbal and pharmacological drug moieties are covered within the scope of this mini-review. RESULTS: Of 35 UGT1A1 induced drug interactions, likely and unlikely to be clinically significant interactions are 11 and 6 respectively. The rest of them are inconclusive. CONCLUSION: We hope that this secondary literature can broaden and update the perspective of clinicians, pharmacists and academics on the interactions mediated by UGT1A1.

Garcinol: Current status of its anti-oxidative, anti-inflammatory and anti-cancer effects.

Garcinol is the main medicinal component of the dried fruit rind of Garcinia indica (G. indica), which has traditionally been extensively used to treat gastric ailments and skin irritation. In vitro studies of garcinol revealed its potential therapeutic effects, such as its anti-oxidative, anti-inflammatory and anti-cancer properties. Similarly, in vivo studies in animal models also demonstrated the efficacy of garcinol for the treatment of various inflammatory and cancerous conditions. Despite being well tolerated in preclinical studies, the toxicological profile of garcinol remains elusive. More importantly, systematic pharmacokinetics (PK) studies of garcinol to establish an appropriate route of administration and its effective concentration range under physiological conditions have not yet been performed. PK studies play an essential role in translating the preclinical findings of garcinol from cell line models and animal species to humans, thereby facilitating dose selection, the characterization of the therapeutic index, identification of a metabolic pathway, and the determination of garcinol's potency and tolerability. This paper reviews the current studies of garcinol as a potential anti-oxidant, anti-inflammatory and anti-cancer agent and highlights the importance of performing preclinical PK and toxicological studies on garcinol for its development pipeline.

The anti-cancer activities of Vernonia amygdalina extract in human breast cancer cell lines are mediated through caspase-dependent and p53-independent pathways.

Breast cancer is currently the leading cause of cancer-related deaths among women globally. Notably, medicinal plant extracts may be a potential source for treatments of breast cancer. Vernonia amygdalina (VA) is a woody shrub reported to have not only diverse therapeutic effects but also anti-cancer properties. However, current research about the mechanisms of the anti-cancer potential of VA has been limited. This study aimed to investigate the mechanisms of action of VA that underlie its anti-cancer effects in human breast cancer cell lines (MCF-7 and MDA-MB-231 cells). Results from MTT assay revealed that VA inhibits the proliferation of MCF-7 and MDA-MB-231, in a time- and dose-dependent manner. The underlying mechanism of this growth inhibition involved the stimulation of cell-type specific G1/S phase cell cycle arrest in only MCF-7 cells, and not in MDA-MB-231 cells. While the growth arrest was associated with increased levels of p53 and p21, and a concomitant decrease in the levels of cyclin D1 and cyclin E, it was shown that VA causes cell cycle arrest through a p53-independent pathway as tested by the wild type p53 inhibitor, pifithrin-α. Furthermore, this study revealed that VA induces apoptosis in the two cell lines, as indicated by the increase in Annexin V-positive cells and sub-G1 population, and that this VA-induced apoptosis occurred through both extrinsic and intrinsic apoptotic pathways. The apoptosis in MCF-7 cells was also likely to be caspase-dependent and not p53 transcriptional-dependent. Given that approximately 70% of diagnosed breast cancers express ER-α, a crucial finding was that VA inhibits the expression of ER-α and its downstream player, Akt, highlighting the potential clinical significance of VA. Moreover, VA exhibits synergism when combined with doxorubicin, suggesting that it can complement current chemotherapy. Overall, this study demonstrates the potential applications of VA as an anti-cancer drug for breast cancer treatment.