Characterization of methionine dependence in melanoma cells.

Dietary methionine restriction is associated with a reduction in tumor growth in preclinical studies and an increase in lifespan in animal models. The mechanism by which methionine restriction inhibits tumor growth while sparing normal cells is incompletely understood. We do know that normal cells can utilize methionine or homocysteine interchangeably (methionine independence) while most cancer cells are strictly dependent on methionine availability. Here, we compared a typical methionine dependent and a rare methionine independent melanoma cell line. We show that replacing methionine, a methyl donor, with its precursor homocysteine generally induced hypomethylation in gene promoters. This decrease was similar in methionine dependent and methionine independent cells. There was only a low level of pathway enrichment, suggesting that the hypomethylation is generalized rather than gene specific. Whole proteome and transcriptome were also analyzed. This analysis revealed that contrarily to the effect on methylation, the replacement of methionine with homocysteine had a much greater effect on the transcriptome and proteome of methionine dependent cells than methionine independent cells. Interestingly, methionine adenosyltransferase 2A (MAT2A), responsible for the synthesis of S-adenosylmethionine from methionine, was equally strongly upregulated in both cell lines. This suggests that the absence of methionine is equally detected but triggers different outcomes in methionine dependent versus independent cells. Our analysis reveals the importance of cell cycle control, DNA damage repair, translation, nutrient sensing, oxidative stress and immune functions in the cellular response to methionine stress in melanoma.

Exosomal MicroRNA and Protein Profiles of Hepatitis B Virus-Related Hepatocellular Carcinoma Cells.

Infection with hepatitis B virus (HBV) is a main risk factor for hepatocellular carcinoma (HCC). Extracellular vesicles, such as exosomes, play an important role in tumor development and metastasis, including regulation of HBV-related HCC. In this study, we have characterized exosome microRNA and proteins released in vitro from hepatitis B virus (HBV)-related HCC cell lines SNU-423 and SNU-182 and immortalized normal hepatocyte cell lines (THLE2 and THLE3) using microRNA sequencing and mass spectrometry. Bioinformatics, including functional enrichment and network analysis, combined with survival analysis using data related to HCC in The Cancer Genome Atlas (TCGA) database, were applied to examine the prognostic significance of the results. More than 40 microRNAs and 200 proteins were significantly dysregulated (p < 0.05) in the exosomes released from HCC cells in comparison with the normal liver cells. The functional analysis of the differentially expressed exosomal miRNAs (i.e., mir-483, mir-133a, mir-34a, mir-155, mir-183, mir-182), their predicted targets, and exosomal differentially expressed proteins (i.e., POSTN, STAM, EXOC8, SNX9, COL1A2, IDH1, FN1) showed correlation with pathways associated with HBV, virus activity and invasion, exosome formation and adhesion, and exogenous protein binding. The results from this study may help in our understanding of the role of HBV infection in the development of HCC and in the development of new targets for treatment or non-invasive predictive biomarkers of HCC.

Targeting mitochondria in the aged cerebral vasculature with SS-31, a proteomic study of brain microvessels.

Cognitive impairment and dementias during aging such as Alzheimer's disease are linked to functional decline and structural alterations of the brain microvasculature. Although mechanisms leading to microvascular changes during aging are not clear, loss of mitochondria, and reduced efficiency of remaining mitochondria appear to play a major role. Pharmacological agents, such as SS-31, which target mitochondria have been shown to be effective during aging and diseases; however, the benefit to mitochondrial- and non-mitochondrial proteins in the brain microvasculature has not been examined. We tested whether attenuation of aging-associated changes in the brain microvascular proteome via targeting mitochondria represents a therapeutic option for the aging brain. We used aged male (> 18 months) C57Bl6/J mice treated with a mitochondria-targeted tetrapeptide, SS-31, or vehicle saline. Cerebral blood flow (CBF) was determined using laser speckle imaging during a 2-week treatment period. Then, isolated cortical microvessels (MVs) composed of end arterioles, capillaries, and venules were used for Orbitrap Eclipse Tribrid mass spectrometry. CBF was similar among the groups, whereas bioinformatic analysis revealed substantial differences in protein abundance of cortical MVs between SS-31 and vehicle. We identified 6267 proteins, of which 12% were mitochondria-associated. Of this 12%, 107 were significantly differentially expressed and were associated with oxidative phosphorylation, metabolism, the antioxidant defense system, or mitochondrial dynamics. Administration of SS-31 affected many non-mitochondrial proteins. Our findings suggest that mitochondria in the microvasculature represent a therapeutic target in the aging brain, and widespread changes in the proteome may underlie the rejuvenating actions of SS-31 in aging.

Musashi Exerts Control of Gonadotrope Target mRNA Translation During the Mouse Estrous Cycle.

The anterior pituitary controls key biological processes, including growth, metabolism, reproduction, and stress responses through distinct cell types that each secrete specific hormones. The anterior pituitary cells show a remarkable level of cell type plasticity that mediates the shifts in hormone-producing cell populations that are required to meet organismal needs. The molecular mechanisms underlying pituitary cell plasticity are not well understood. Recent work has implicated the pituitary stem cell populations and specifically, the mRNA binding proteins of the Musashi family in control of pituitary cell type identity. In this study we have identified the target mRNAs that mediate Musashi function in the adult mouse pituitary and demonstrate the requirement for Musashi function in vivo. Using Musashi RNA immunoprecipitation, we identify a cohort of 1184 mRNAs that show specific Musashi binding. Identified Musashi targets include the Gnrhr mRNA, which encodes the gonadotropin-releasing hormone receptor (GnRHR), and the Fshb mRNA, encoding follicle-stimulating hormone (FSH). Reporter assays reveal that Musashi functions to exert repression of translation of the Fshb mRNA, in addition to the previously observed repression of the Gnrhr mRNA. Importantly, mice engineered to lack Musashi in gonadotropes demonstrate a failure to repress translation of the endogenous Gnrhr and Fshb mRNAs during the estrous cycle and display a significant heterogeneity in litter sizes. The range of identified target mRNAs suggests that, in addition to these key gonadotrope proteins, Musashi may exert broad regulatory control over the pituitary proteome in a cell type-specific manner.

Characterization of methionine dependence in melanoma cells.

Dietary methionine restriction is associated with a reduction in tumor growth in preclinical studies and an increase in lifespan in animal models. The mechanism by which methionine restriction inhibits tumor growth while sparing normal cells is incompletely understood. We do know that normal cells can utilize methionine or homocysteine interchangeably (methionine independence) while most cancer cells are strictly dependent on methionine availability. Here, we compared a typical methionine dependent and a rare methionine independent melanoma cell line. We show that replacing methionine, a methyl donor, with its precursor homocysteine generally induced hypomethylation in gene promoters. This decrease was similar in methionine dependent and methionine independent cells. There was only a low level of pathway enrichment, suggesting that the hypomethylation is generalized rather than gene specific. Whole proteome and transcriptome were also analyzed. This analysis revealed that contrarily to the effect on methylation, the replacement of methionine with homocysteine had a much greater effect on the transcriptome and proteome of methionine dependent cells than methionine independent cells. Interestingly, methionine adenosyltransferase 2A (MAT2A), responsible for the synthesis of s-adenosylmethionine from methionine, was equally strongly upregulated in both cell lines. This suggests that the absence of methionine is equally detected but triggers different outcomes in methionine dependent versus independent cells. Our analysis reveals the importance of cell cycle control, DNA damage repair, translation, nutrient sensing, oxidative stress and immune functions in the cellular response to methionine stress in melanoma.

Maternal undernutrition results in transcript changes in male offspring that may promote resistance to high fat diet induced weight gain.

Maternal nutrition during embryonic development and lactation influences multiple aspects of offspring health. Using mice, this study investigates the effects of maternal caloric restriction (CR) during mid-gestation and lactation on offspring neonatal development and on adult metabolic function when challenged by a high fat diet (HFD). The CR maternal model produced male and female offspring that were significantly smaller, in terms of weight and length, and females had delayed puberty. Adult offspring born to CR dams had a sexually dimorphic response to the high fat diet. Compared to offspring of maternal control dams, adult female, but not male, CR offspring gained more weight in response to high fat diet at 10 weeks. In adipose tissue of male HFD offspring, maternal undernutrition resulted in blunted expression of genes associated with weight gain and increased expression of genes that protect against weight gain. Regardless of maternal nutrition status, HFD male offspring showed increased expression of genes associated with progression toward nonalcoholic fatty liver disease (NAFLD). Furthermore, we observed significant, sexually dimorphic differences in serum TSH. These data reveal tissue- and sex-specific changes in gene and hormone regulation following mild maternal undernutrition, which may offer protection against diet induced weight gain in adult male offspring.

Repurposing live attenuated trivalent MMR vaccine as cost-effective cancer immunotherapy.

It has long been known that oncolytic viruses wield their therapeutic capability by priming an inflammatory state within the tumor and activating the tumor immune microenvironment, resulting in a multifaceted antitumor immune response. Vaccine-derived viruses, such as measles and mumps, have demonstrated promising potential for treating human cancer in animal models and clinical trials. However, the extensive cost of manufacturing current oncolytic viral products makes them far out of reach for most patients. Here by analyzing the impact of intratumoral (IT) administrations of the trivalent live attenuated measles, mumps, and rubella viruses (MMR) vaccine, we unveil the cellular and molecular basis of MMR-induced anti-cancer activity. Strikingly, we found that IT delivery of low doses of MMR correlates with tumor control and improved survival in murine hepatocellular cancer and colorectal cancer models via increased tumor infiltration of CD8+ granzyme B+ T-cells and decreased macrophages. Moreover, our data indicate that MMR activates key cellular effectors of the host's innate and adaptive antitumor immunity, culminating in an immunologically coordinated cancer cell death. These findings warrant further work on the potential for MMR to be repurposed as safe and cost-effective cancer immunotherapy to impact cancer patients globally.

Circulating Exosomal microRNAs as Predictive Biomarkers of Neoadjuvant Chemotherapy Response in Breast Cancer.

BACKGROUND: Neoadjuvant chemotherapy (NACT) is an increasingly used approach for treatment of breast cancer. The pathological complete response (pCR) is considered a good predictor of disease-specific survival. This study investigated whether circulating exosomal microRNAs could predict pCR in breast cancer patients treated with NACT. METHOD: Plasma samples of 20 breast cancer patients treated with NACT were collected prior to and after the first cycle. RNA sequencing was used to determine microRNA profiling. The Cancer Genome Atlas (TCGA) was used to explore the expression patterns and survivability of the candidate miRNAs, and their potential targets based on the expression levels and copy number variation (CNV) data. RESULTS: Three miRNAs before that NACT (miR-30b, miR-328 and miR-423) predicted pCR in all of the analyzed samples. Upregulation of miR-127 correlated with pCR in triple-negative breast cancer (TNBC). After the first NACT dose, pCR was predicted by exo-miR-141, while miR-34a, exo-miR182, and exo-miR-183 predicted non-pCR. A significant correlation between the candidate miRNAs and the overall survival, subtype, and metastasis in breast cancer, suggesting their potential role as predictive biomarkers of pCR. CONCLUSIONS: If the miRNAs identified in this study are validated in a large cohort of patients, they might serve as predictive non-invasive liquid biopsy biomarkers for monitoring pCR to NACT in breast cancer.

Raman Spectroscopy and Machine Learning Reveals Early Tumor Microenvironmental Changes Induced by Immunotherapy.

Cancer immunotherapy provides durable clinical benefit in only a small fraction of patients, and identifying these patients is difficult due to a lack of reliable biomarkers for prediction and evaluation of treatment response. Here, we demonstrate the first application of label-free Raman spectroscopy for elucidating biomolecular changes induced by anti-CTLA4 and anti-PD-L1 immune checkpoint inhibitors (ICI) in the tumor microenvironment (TME) of colorectal tumor xenografts. Multivariate curve resolution-alternating least squares (MCR-ALS) decomposition of Raman spectral datasets revealed early changes in lipid, nucleic acid, and collagen content following therapy. Support vector machine classifiers and random forests analysis provided excellent prediction accuracies for response to both ICIs and delineated spectral markers specific to each therapy, consistent with their differential mechanisms of action. Corroborated by proteomics analysis, our observation of biomolecular changes in the TME should catalyze detailed investigations for translating such markers and label-free Raman spectroscopy for clinical monitoring of immunotherapy response in cancer patients. SIGNIFICANCE: This study provides first-in-class evidence that optical spectroscopy allows sensitive detection of early changes in the biomolecular composition of tumors that predict response to immunotherapy with immune checkpoint inhibitors.

Multi-omics data integration reveals correlated regulatory features of triple negative breast cancer.

Triple negative breast cancer (TNBC) is an aggressive type of breast cancer with very little treatment options. TNBC is very heterogeneous with large alterations in the genomic, transcriptomic, and proteomic landscapes leading to various subtypes with differing responses to therapeutic treatments. We applied a multi-omics data integration method to evaluate the correlation of important regulatory features in TNBC BRCA1 wild-type MDA-MB-231 and TNBC BRCA1 5382insC mutated HCC1937 cells compared with non-tumorigenic epithelial breast MCF10A cells. The data includes DNA methylation, RNAseq, protein, phosphoproteomics, and histone post-translational modification. Data integration methods identified regulatory features from each omics method that had greater than 80% positive correlation within each TNBC subtype. Key regulatory features at each omics level were identified distinguishing the three cell lines and were involved in important cancer related pathways such as TGFß signaling, PI3K/AKT/mTOR, and Wnt/beta-catenin signaling. We observed overexpression of PTEN, which antagonizes the PI3K/AKT/mTOR pathway, and MYC, which downregulates the same pathway in the HCC1937 cells relative to the MDA-MB-231 cells. The PI3K/AKT/mTOR and Wnt/beta-catenin pathways are both downregulated in HCC1937 cells relative to MDA-MB-231 cells, which likely explains the divergent sensitivities of these cell lines to inhibitors of downstream signaling pathways. The DNA methylation and RNAseq data is freely available via GEO GSE171958 and the proteomics data is available via the ProteomeXchange PXD025238.

Multi-omics data integration considerations and study design for biological systems and disease.

With the advancement of next-generation sequencing and mass spectrometry, there is a growing need for the ability to merge biological features in order to study a system as a whole. Features such as the transcriptome, methylome, proteome, histone post-translational modifications and the microbiome all influence the host response to various diseases and cancers. Each of these platforms have technological limitations due to sample preparation steps, amount of material needed for sequencing, and sequencing depth requirements. These features provide a snapshot of one level of regulation in a system. The obvious next step is to integrate this information and learn how genes, proteins, and/or epigenetic factors influence the phenotype of a disease in context of the system. In recent years, there has been a push for the development of data integration methods. Each method specifically integrates a subset of omics data using approaches such as conceptual integration, statistical integration, model-based integration, networks, and pathway data integration. In this review, we discuss considerations of the study design for each data feature, the limitations in gene and protein abundance and their rate of expression, the current data integration methods, and microbiome influences on gene and protein expression. The considerations discussed in this review should be regarded when developing new algorithms for integrating multi-omics data.

Epigenetic Control of Cdkn2a.Arf Protects Tumor-Infiltrating Lymphocytes from Metabolic Exhaustion.

T-cell exhaustion in cancer is linked to poor clinical outcomes, where evidence suggests T-cell metabolic changes precede functional exhaustion. Direct competition between tumor-infiltrating lymphocytes (TIL) and cancer cells for metabolic resources often renders T cells dysfunctional. Environmental stress produces epigenome remodeling events within TIL resulting from loss of the histone methyltransferase EZH2. Here, we report an epigenetic mechanism contributing to the development of metabolic exhaustion in TIL. A multiomics approach revealed a Cdkn2a.Arf-mediated, p53-independent mechanism by which EZH2 inhibition leads to mitochondrial dysfunction and the resultant exhaustion. Reprogramming T cells to express a gain-of-function EZH2 mutant resulted in an enhanced ability of T cells to inhibit tumor growth in vitro and in vivo. Our data suggest that manipulation of T-cell EZH2 within the context of cellular therapies may yield lymphocytes that are able to withstand harsh tumor metabolic environments and collateral pharmacologic insults. SIGNIFICANCE: These findings demonstrate that manipulation of T-cell EZH2 in cellular therapies may yield cellular products able to withstand solid tumor metabolic-deficient environments. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/21/4707/F1.large.jpg.

Sample storage conditions induce post-collection biases in microbiome profiles.

BACKGROUND: Here we investigated the influence of different stabilization and storage strategies on the quality and composition of the fecal microbial community. Namely, same-day isolated murine DNA was compared to samples stored for 1 month in air at ambient temperature, with or without preservative buffers (i.e. EDTA and lysis buffer), different temperatures (i.e. 4 °C, - 20 °C, and - 80 °C), and hypoxic conditions. RESULTS: Only storage in lysis buffer significantly reduced DNA content, yet without integrity loss. Storage in EDTA affected alpha diversity the most, which was also reflected in cluster separation. Distinct changes were also seen in the phyla and bacterial species abundance per storage strategy. Metabolic function analysis showed 22 pathways not significantly affected by storage conditions, whereas the tyrosine metabolism pathway was significantly changed in all strategies except by EDTA. CONCLUSION: Each long-term storage strategy introduced a unique post-collection bias, which is important to take into account when interpreting data.

Draft Genome Sequences of Four Salmonella enterica Strains Isolated from Turkey-Associated Sources.

We report the draft genomes of four Salmonella enterica isolates evaluated for the contribution of plasmids to virulence. Strains SE163A, SE696A, and SE710A carry plasmids demonstrated to facilitate plasmid-associated virulence, while SE819 is less virulent and has been used as a recipient for conjugation experiments to assess plasmid-encoded virulence mechanisms.