Role of adipocyte-derived extracellular vesicles during the progression of liver inflammation to hepatocellular carcinoma.

Extracellular vesicles are membrane-bound cargos that vary in size and are stably transported through various bodily fluids. Extracellular vesicles communicate information between the cells and organs. Extracellular vesicles from the diseased cells alter cellular responses of the recipient cells contributing to disease progression. In obesity, adipocytes become hypertrophic and the extracellular vesicles from these dysfunctional adipocytes showed altered cargo contents instigating pathophysiological response leading to chronic liver diseases. In this review, the role of adipocyte-derived extracellular vesicles on the progression of liver inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma are extensively discussed. Newer approaches are crucial to take advantage of extracellular vesicles and their content as biomarkers to diagnose initial liver inflammation before reaching to an irreversible liver failure stage.

Potential nanocarrier-mediated miRNA-based therapy approaches for multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune neuroinflammatory disorder attributed to neurodegeneration and demyelination, resulting in neurological impairment. miRNA has a significant role in biological processes in MS. In this review, we focus on the feasibility of delivering miRNAs through nanoformulations for managing MS. We provide a brief discussion of miRNA synthesis and evidence for miRNA dysregulation in MS. We also highlight formulation strategies and resulting technologies for the effective delivery of miRNAs through nanocarrier systems for achieving high therapeutic benefits.

"Janus-Faced" α-Synuclein: Role in Parkinson's Disease.

Parkinson's disease (PD) is a pathological condition characterized by the aggregation and the resultant presence of intraneuronal inclusions termed Lewy bodies (LBs) and Lewy neurites which are mainly composed of fibrillar α-synuclein (α-syn) protein. Pathogenic aggregation of α-syn is identified as the major cause of LBs deposition. Several mutations in α-syn showing varied aggregation kinetics in comparison to the wild type (WT) α-syn are reported in PD (A30P, E46K, H 50Q, G51D, A53E, and A53T). Also, the cell-to-cell spread of pathological α-syn plays a significant role in PD development. Interestingly, it has also been suggested that the pathology of PD may begin in the gastrointestinal tract and spread via the vagus nerve (VN) to brain proposing the gut-brain axis of α-syn pathology in PD. Despite multiple efforts, the behavior and functions of this protein in normal and pathological states (specifically in PD) is far from understood. Furthermore, the etiological factors responsible for triggering aggregation of this protein remain elusive. This review is an attempt to collate and present latest information on α-syn in relation to its structure, biochemistry and biophysics of aggregation in PD. Current advances in therapeutic efforts toward clearing the pathogenic α-syn via autophagy/lysosomal flux are also reviewed and reported.

Mitochondrial DNA modification by CRISPR/Cas system: Challenges and future direction.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR associated endonuclease), a hotshot genome editing tool which is originally known to be the form of prokaryotic adaptive immune system against viral infections has gained all the attention of scientific community as a promising genome editing platform. This review encompasses a brief description of mitochondrial disease conditions associated with the alteration in mitochondrial genome (mtDNA) and highlights the key role of the CRISPR/Cas system pertaining to its working mechanism and its involvement in gene-based therapeutics in treating the foresaid mitochondrial diseases. Here, we also extend the perception related to the detailed mechanism of CRISPR/Cas system in mtDNA modification.

The chromatin modifier MORC2 affects glucose metabolism by regulating the expression of lactate dehydrogenase A through a feed forward loop with c-Myc.

Microrchidia family CW-type zinc finger 2 (MORC2) is a recently identified chromatin modifier with an emerging role in cancer metastasis. However, its role in glucose metabolism, a hallmark of malignancy, remains to be explored. We found that MORC2 is a glucose-inducible gene and a target of c-Myc. Our meta-analysis revealed that MORC2 expression is positively correlated with the expression of enzymes involved in glucose metabolism in breast cancer patients. Furthermore, overexpression of MORC2 in MCF-7 and BT-549 cells augmented the expression and activity of a key glucose metabolism enzyme, lactate dehydrogenase A (LDHA). Conversely, selective knockdown of MORC2 by siRNA markedly decreased LDHA expression and activity and in turn reduced cancer cell migration. Collectively, these findings provide evidence that MORC2, a glucose-inducible gene, modulates the migration of breast cancer cells through the MORC2-c-Myc-LDHA axis.

Centchroman prevents metastatic colonization of breast cancer cells and disrupts angiogenesis via inhibition of RAC1/PAK1/ß-catenin signaling axis.

AIMS: We have previously reported that Centchroman (CC), an oral contraceptive drug, inhibits breast cancer progression and metastasis. In this study, we investigated whether CC inhibits local invasion of tumor cells and/or their metastatic colonization with detailed underlying mechanisms. MAIN METHODS: The effect of CC on the experimental metastasis and spontaneous metastasis was demonstrated by using tail-vein and orthotopic 4T1-syngeneic mouse tumor models, respectively. The anti-angiogenic potential of CC was evaluated using well established in vitro and in vivo models. The role of RAC1/PAK1/ß-catenin signaling axis in the metastasis was investigated and validated using siRNA-mediated knockdown of PAK1 as well as by pharmacological PAK1-inhibitor. KEY FINDINGS: The oral administration of CC significantly suppressed the formation of metastatic lung nodules in the 4T1-syngeneic orthotopic as well as experimental metastatic models. More importantly, CC treatment suppressed the tube formation and migration capacities of human umbilical vein endothelial cells (HUVEC) and inhibited pre-existing vasculature as well as the formation of neovasculature. The suppression of migration and invasion capacities of metastatic breast cancer cells upon CC treatment was associated with the inhibition of small GTPases (Rac1 and Cdc42) concomitant with the downregulation of PAK1 and downstream ß-catenin signaling. In addition, CC upregulated the expression of miR-145, which is known to target PAK1. SIGNIFICANCE: This study warrants the repurposing of CC as a potential therapeutic agent against metastatic breast cancer.

Author Correction: Cancer Testis Antigen Promotes Triple Negative Breast Cancer Metastasis and is Traceable in the Circulating Extracellular Vesicles.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cancer Testis Antigen Promotes Triple Negative Breast Cancer Metastasis and is Traceable in the Circulating Extracellular Vesicles.

Triple negative breast cancer (TNBC) has poor survival, exhibits rapid metastases, lacks targeted therapies and reliable prognostic markers. Here, we examined metastasis promoting role of cancer testis antigen SPANXB1 in TNBC and its utility as a therapeutic target and prognostic biomarker. Expression pattern of SPANXB1 was determined using matched primary cancer, lymph node metastatic tissues and circulating small extracellular vesicles (sEVs). cDNA microarray analysis of TNBC cells stably integrated with a metastasis suppressor SH3GL2 identified SPANXB1 as a potential target gene. TNBC cells overexpressing SH3GL2 exhibited decreased levels of both SPANXB1 mRNA and protein. Silencing of SPANXB1 reduced migration, invasion and reactive oxygen species production of TNBC cells. SPANXB1 depletion augmented SH3GL2 expression and decreased RAC-1, FAK, A-Actinin and Vinculin expression. Phenotypic and molecular changes were reversed upon SPANXB1 re-expression. SPANXB1 overexpressing breast cancer cells with an enhanced SPANXB1:SH3GL2 ratio achieved pulmonary metastasis within 5 weeks, whereas controls cells failed to do so. Altered expression of SPANXB1 was detected in the sEVs of SPANXB1 transduced cells. Exclusive expression of SPANXB1 was traceable in circulating sEVs, which was associated with TNBC progression. SPANXB1 represents a novel and ideal therapeutic target for blocking TNBC metastases due to its unique expression pattern and may function as an EV based prognostic marker to improve TNBC survival. Uniquely restricted expression of SPANXB1 in TNBCs, makes it an ideal candidate for targeted therapeutics and prognostication.

Microbiome Diversity in Sputum of Nontuberculous Mycobacteria Infected Women with a History of Breast Cancer.

BACKGROUND/AIMS: The nontuberculous mycobacterial lung disease (NTM), caused by Mycobacterium avium complex (MAC) is an increasing health problem in the USA and worldwide. The NTM disease is prevalent in Caucasian women with a current diagnosis or history of breast cancer (BCa), posing a significant challenge towards treatment. We hypothesize that NTM affected women with considerable therapeutic resistance may harbor pathogenic microbes other than nontuberculous mycobacterium, aiding in disease progression and therapeutic resistance. METHODS: We assessed microbiome diversity in sputa from healthy women, women with nontuberculous mycobacterial lung disease (NTM) and women with both nontuberculous mycobacterial lung disease and breast cancer (NTM-BCa). First, we collected sputa and isolated DNA from sputa of these healthy women and women with NTM and NTM-BCa. We also isolated DNA from sera derived extracellular vesicles from women with NTM-BCa. To identify diverse pathogenic microbes in various groups of subjects, we then performed 16S rDNA sequencing. Data analysis was performed utilizing the analytical pipelines at the Center for Metagenomic and Microbiome Research (CMMR), Baylor College of Medicine. RESULTS: A large community of resident microbes, including bacteria, virus, Archeas and Fungi live in the human body are being increasingly recognized as the key components of human health and disease. We identified a diverse microbiome community in the sputa and the extracellular vesicles dominated by Streptococcus, Haemophillus, Veillonella, Neisseria, Prevotella, Fusobacterium, Bacteroides, Allistipes, Faecalibacterium and Staphylococcus in women with nontuberculous mycobacterial lung disease as well as women with both nontuberculous mycobacterial lung disease and breast cancer. Some of these genera, including Fusobacterium, Bacteroides, and Allistipes have estrobolome activity and associated with breast and other neoplasms. CONCLUSION: This work confirms the presence of a distinct pathogenic microbiome other than nontuberculous mycobacteria in the sputa and the circulating extracellular vesicles of these patients. This information could be useful for better therapeutic design to treat the NTM patients.

Sputum Detection of Predisposing Genetic Mutations in Women with Pulmonary Nontuberculous Mycobacterial Disease.

Nontuberculous mycobacterial lung disease (NTM), including Mycobacterium avium complex (MAC), is a growing health problem in North America and worldwide. Little is known about the molecular alterations occurring in the tissue microenvironment during NTM pathogenesis. Utilizing next generation sequencing, we sequenced sputum and matched lymphocyte DNA in 15 MAC patients for a panel of 19 genes known to harbor cancer susceptibility associated mutations. Thirteen of 15 NTM subjects had a diagnosis of breast cancer (BCa) before or after NTM infection. Thirty three percent (4/12) of these NTM-BCa cases exhibited at least 3 somatic mutations in sputa compared to matched lymphocytes. Twenty four somatic mutations were detected with at least one mutation in ATM, ERBB2, BARD1, BRCA1, BRCA2, AR, TP53, PALB2, CASP8, BRIP1, NBN and TGFB1 genes. All four NTM-BCa patients harboring somatic mutations also exhibited 15 germ line BRCA1 and BRCA2 mutations. The two NTM subjects without BCa exhibited twenty somatic mutations spanning BRCA1, BRCA1, BARD1, BRIP1, CHEK2, ERBB2, TP53, ATM, PALB2, TGFB1 and 3 germ line mutations in BRCA1 and BRCA2 genes. A single copy loss of STK11 and AR gene was noted in NTM-BCa subjects. Periodic screening of sputa may aid to develop risk assessment biomarkers for neoplastic diseases in NTM patients.

Morin impedes Yap nuclear translocation and fosters apoptosis through suppression of Wnt/ß-catenin and NF-κB signaling in Mst1 overexpressed HepG2 cells.

Recent clinical and experimental evidences strongly acclaim Yes-associated protein (Yap), a key oncogenic driver in liver carcinogenesis, as a therapeutic target. Of the known multiple schemes to inhibit Yap activity, activation of Mammalian Sterile 20-like Kinase 1 (Mst1), an upstream regulator of Yap, appears to be a promising one. In this study, we hypothesize that morin, a bioflavonoid, mediates its anti-cancer effect through the activation of Mst1/hippo signaling in liver cancer cells. To test this hypothesis, both full length Mst1 (F-Mst1) and kinase active N-terminal Mst1 (N-Mst1)-overexpressed HepG2 cells were used. Exposure of F-Mst1 overexpressed HepG2 cells to morin activated Mst1 by caspase-3 cleavage and thereby inhibited Yap nuclear translocation and fostered apoptosis. Morin suppressed NF-κB p65 and Wnt/ß-catenin signaling through Mst1 activation via cleavage and phosphorylation, leading to cell death. Annexin-V/PI staining further confirmed the induction of apoptosis in morin treated F-Mst1 overexpressed cells. The present study shows that morin targets cell survival molecules such as NF-κB p65 and ß-catenin through activation of hippo signaling. Therefore, morin could be considered as a potential anti-cancer agent against liver cancer.

Genetic Mutation and Exosome Signature of Human Papilloma Virus Associated Oropharyngeal Cancer.

Human papilloma virus-16 (HPV-16) associated oropharyngeal cancer (HPVOPC) is increasing alarmingly in the United States. We performed whole genome sequencing of a 44 year old, male HPVOPC subject diagnosed with moderately differentiated tonsillar carcinoma. We identified new somatic mutation in MUC16 (A.k.a. CA-125), MUC12, MUC4, MUC6, MUC2, SIRPA, HLA-DRB1, HLA-A and HLA-B molecules. Increased protein expression of MUC16, SIRPA and decreased expression of HLA-DRB1 was further demonstrated in this HPVOPC subject and an additional set of 15 HPVOPC cases. Copy number gain (3 copies) was also observed for MUC2, MUC4, MUC6 and SIRPA. Enhanced expression of MUC16, SIRPA and HPV-16-E7 protein was detectable in the circulating exosomes of numerous HPVOPC subjects. Treatment of non-tumorigenic mammary epithelial cells with exosomes derived from aggressive HPVOPC cells harboring MUC16, SIRPA and HPV-16-E7 proteins augmented invasion and induced epithelial to mesenchymal transition (EMT) accompanied by an increased expression ratio of the EMT markers Vimentin/E-cadherin. Exosome based screening of key HPVOPC associated molecules could be beneficial for early cancer diagnosis, monitoring and surveillance.

Exosome secretome and mediated signaling in breast cancer patients with nontuberculous mycobacterial disease.

Bronchiectasis Nontuberculous mycobacterium (NTMnb) infection is an emerging health problem in breast cancer (BCa) patients. We measured sera exosome proteome in BCa-NTMnb subjects and controls by Mass Spectroscopy. Extracellular matrix protein 1 (ECM1) was detected exclusively in the circulating exosomes of 82% of the BCa-NTMnb cases. Co-culture of ECM1+ exosomes with normal human mammary epithelial cells induced epithelial to mesenchymal transition accompanied by increased Vimentin/CDH1 expression ratio and Glutamate production. Co-culture of the ECM1+ exosomes with normal human T cells modulated their cytokine production. The ECM1+ exosomes were markedly higher in sera obtained from BCa-NTMnb subjects. Exclusive expression of APN, APOC4 and AZGP1 was evident in the circulating exosomes of these BCa-NTMnb cases, which predicts disease prevalence independent of the body max index in concert with ECM1. Monitoring ECM1, APN, APOC4 and AZGP1 in the circulating exosomes could be beneficial for risk assessment, monitoring and surveillance of BCa-NTMnb.

Mitochondrial Reprogramming Regulates Breast Cancer Progression.

PURPOSE: The goal of this study was to understand the role of altered mitochondrial function in breast cancer progression and determine the potential of the molecular alteration signature in developing exosome-based biomarkers. EXPERIMENTAL DESIGN: This study was designed to characterize the critical components regulating mitochondrial function in breast tumorigenesis. Experiments were conducted to assess the potential of these molecules for exosome-based biomarker development. RESULTS: We observed a remarkable reduction in spontaneous metastases through the interplay in mitochondria by SH3GL2, vesicular endocytosis-associated protein and MFN2, an important regulator of mitochondrial fusion. Following its overexpression in breast cancer cells, SH3GL2 translocated to mitochondria and induced the production of superoxide and release of cytochrome C from mitochondria to the cytoplasm. These molecular changes were accompanied by decreased lung and liver metastases and primary tumor growth. SH3GL2 depletion reversed the above phenotypic and associated molecular changes in nontumorigenic and tumorigenic breast epithelial cells. Loss of SH3GL2 and MFN2 expression was evident in primary human breast cancer tissues and their positive lymph nodes, which was associated with disease progression. SH3GL2 and MFN2 expression was detected in sera exosomes of normal healthy women, but barely detectable in the majority of the women with breast cancer exhibiting SH3GL2 and MFN2 loss in their primary tumors. CONCLUSIONS: This study identified a new mitochondria reprogramming pathway influencing breast cancer progression through SH3GL2 and MFN2. These proteins were frequently lost in breast cancer, which was traceable in the circulating exosomes. Clin Cancer Res; 22(13); 3348-60. ©2016 AACR.

Complex-I Alteration and Enhanced Mitochondrial Fusion Are Associated With Prostate Cancer Progression.

Mitochondria (mt) encoded respiratory complex-I (RCI) mutations and their pathogenicity remain largely unknown in prostate cancer (PCa). Little is known about the role of mtDNA loss on mt integrity in PCa. We determined mtDNA mutation in human and mice PCa and assessed the impact of mtDNA depletion on mt integrity. We also examined whether the circulating exosomes from PCa patients are transported to mt and carry mtDNA or mt proteins. We have employed next generation sequencing of the whole mt genome in human and Hi-myc PCa. The impact of mtDNA depletion on mt integrity, presence of mtDNA, and protein in sera exosomes was determined. A co-culture of human PCa cells and the circulating exosomes followed by confocal imaging determined co-localization of exosomes and mt. We observed frequent RCI mutations in human and Hi-myc PCa which disrupted corresponding complex protein expression. Depletion of mtDNA in PCa cells influenced mt integrity, increased expression of MFN1, MFN2, PINK1, and decreased expression of MT-TFA. Increased mt fusion and expression of PINK1 and DNM1L were also evident in the Hi-myc tumors. RCI-mtDNA, MFN2, and IMMT proteins were detected in the circulating exosomes of men with benign prostate hyperplasia (BPH) and progressive PCa. Circulating exosomes and mt co-localized in PCa cells. Our study identified new pathogenic RCI mutations in PCa and defined the impact of mtDNA loss on mt integrity. Presence of mtDNA and mt proteins in the circulating exosomes implicated their usefulness for biomarker development.

MDA-9/Syntenin Control.

MDA-9/Syntenin is a small PDZ domain containing scaffolding protein with diverse array of functions regulating membrane trafficking, cell adhesion, neural, and synaptic development, ubiquitination, and exosome biogenesis. An appreciable number of studies also established a pivotal role of MDA-9/Syntenin in cancer development and progression. In this review, we will discuss the dynamic role of MDA-9/Syntenin in regulating normal and abnormal fate of various cellular processes.

Receptor channel TRPC6 orchestrate the activation of human hepatic stellate cell under hypoxia condition.

Hepatic stellate cells (HSCs), a specialized stromal cytotype have a great impact on the biological behaviors of liver diseases. Despite this fact, the underlying mechanism that regulates HSC still remains poorly understood. The aim of the present study was to understand the role of TRPC6 signaling in regulating the molecular mechanism of HSCs in response to hypoxia. In the present study we showed that under hypoxia condition, the upregulated Hypoxia Inducible Factor 1α (HIF1α) increases NICD activation, which in turn induces the expression of transient receptor potential channel 6 (TRPC6) in HSC line lx-2. TRPC6 causes a sustained elevation of intracellular calcium which is coupled with the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway which activates the synthesis of extracellular matrix proteins. TRPC6 also activates SMAD2/3 dependent TGF-ß signaling in facilitating upregulated expression of αSMA and collagen. As activated HSCs may be a suitable target for HCC therapy and targeting these cells rather than the HCC cells may result in a greater response. Collectively, our studies indicate for the first time the detailed mechanism of activation of HSC through TRPC6 signaling and thus being a promising therapeutic target.

Caveolin-1 promotes gastric cancer progression by up-regulating epithelial to mesenchymal transition by crosstalk of signalling mechanisms under hypoxic condition.

Gastric cancer is the second most fatal common form of cancer. The crosstalk among signalling pathways that results in the acceleration of epithelial to mesenchymal transition (EMT) plays a pivotal role in the molecular mechanism of gastric carcinogenesis. To understand the role of caveolin-1 (Cav-1), the expression pattern was studied in human gastric adenocarcinoma tissues and also in AGS and KATO III cell lines. Here, we show that during hypoxic condition, the increase in the levels of hypoxia-inducible factor-1α (HIF-1α) results in a significant decrease in the expression of caveolin-1 which is regulated by heat shock protein 90 (HSP90). The reduced levels of Cav-1 correlated with the increased epidermal growth factor receptor (EGFR) activation resulting in the significant activation of its downstream target STAT3. Accumulation of pSTAT3 in the nucleus results in the decreased expression of E-cadherin and increased expression of mesenchymal markers (Slug, α-SMA, N-cadherin and vimentin). Crosstalk of EGFR and transforming growth factor ß (TGF-ß) signalling with Wnt signalling enhances cell proliferation, cell survival and upregulates EMT. There was no significant alteration in the expression of epithelial and mesenchymal molecules in both the cell lines studied. Thus, we provide evidence that Cav-1 was modulated by HSP90 and functions as a crucial regulator of EMT in gastric cancer.