Salutary effects of moderate but not high intensity aerobic exercise training on the frequency of peripheral T-cells associated with immunosenescence in older women at high risk of breast cancer: a randomized controlled trial.

BACKGROUND: Immunosenescence is described as age-associated changes within the immune system that are responsible for decreased immunity and increased cancer risk. Physically active individuals have fewer 'senescent' and more naïve T-cells compared to their sedentary counterparts, but it is not known if exercise training can rejuvenate 'older looking' T-cell profiles. We determined the effects of 12-weeks supervised exercise training on the frequency of T-cell subtypes in peripheral blood and their relationships with circulating levels of the muscle-derived cytokines (i.e. 'myokines') IL-6, IL-7, IL-15 and osteonectin in older women at high risk of breast cancer. The intervention involved 3 sessions/week of either high intensity interval exercise (HIIT) or moderate intensity continuous exercise (MICT) and were compared to an untrained control (UC) group. RESULTS: HIIT decreased total granulocytes, CD4+ T-cells, CD4+ naïve T-cells, CD4+ recent thymic emigrants (RTE) and the CD4:CD8 ratio after training, whereas MICT increased total lymphocytes and CD8 effector memory (EM) T-cells. The change in total T-cells, CD4+ naïve T-cells, CD4+ central memory (CM) T-cells and CD4+ RTE was elevated after MICT compared to HIIT. Changes in [Formula: see text] after training, regardless of exercise prescription, was inversely related to the change in highly differentiated CD8+ EMRA T-cells and positively related to changes in ß2-adrenergic receptor (ß2-AR) expression on CM CD4+ and CM CD8+ T-cells. Plasma myokine levels did not change significantly among the groups after training, but individual changes in IL-7 were positively related to changes in the number of ß2-AR expressing CD4 naïve T cells in both exercise groups but not controls. Further, CD4 T-cells and CD4 naive T-cells were negatively related to changes in IL-6 and osteonectin after HIIT but not MICT, whereas CD8 EMRA T-cells were inversely related to changes in IL-15 after MICT but not HIIT. CONCLUSIONS: Aerobic exercise training alters the frequency of peripheral T-cells associated with immunosenescence in middle aged/older women at high risk of breast cancer, with HIIT (pro-senescent) and MICT (anti-senescent) evoking divergent effects. Identifying the underlying mechanisms and establishing whether exercise-induced changes in peripheral T-cell numbers can alter the risk of developing breast cancer warrants investigation.

The impact of high-intensity interval exercise training on NK-cell function and circulating myokines for breast cancer prevention among women at high risk for breast cancer.

PURPOSE: Preclinical evidence suggests that natural killer cell (NK-cell) function and myokines facilitate the protective effects of exercise for breast cancer prevention. Since higher-intensity exercise acutely promotes greater mobilization and larger changes in NK-cell cytotoxicity than lower-intensity, high-intensity interval training (HIIT) might offer increased immune protection compared to moderate-intensity continuous-training (MICT). This study compared a 12-week HIIT program to a 12-week MICT program and usual care on changes in resting NK-cell function and circulating myokines among women at high risk for breast cancer. METHODS: Thirty-three women were randomized to HIIT, MICT, or usual care, for a supervised exercise intervention. Blood was collected at baseline and end-of-study. The cytotoxic activity of CD3-/CD56+ NK-cells against the K562 target cell line in vitro was determined by flow cytometry. Circulating myokines (IL-15, IL-6, irisin, OSM, osteonectin, IL-7) were assessed with luminex multiplex assays and ELISA. One-way ANOVA and paired sample t-tests assessed between- and within-group differences, respectively. Pearson correlation coefficients determined relationships between baseline fitness and change variables. RESULTS: Significant differences were not observed between groups for change in NK-cell function or circulating myokines (p > 0.05). Significant correlations were only observed for baseline peak aerobic capacity (ml/kg/min) and change in NK-cell-specific lysis (r = - 0.43, p = 0.02) and hemacytotoxicity for the total sample (r = - 0.46, p = 0.01). CONCLUSION: Our findings suggest that exercise intensity may not significantly impact change in resting NK-cell function and circulating myokines among women at high risk for breast cancer. Structured exercise training may have a larger impact on NK-cell function in those with lower levels of cardiorespiratory fitness. CLINICAL TRIAL REGISTRATION: NCT02923401; Registered on October 4, 2016.

Therapeutic potential of FLANC, a novel primate-specific long non-coding RNA in colorectal cancer.

OBJECTIVE: To investigate the function of a novel primate-specific long non-coding RNA (lncRNA), named FLANC, based on its genomic location (co-localised with a pyknon motif), and to characterise its potential as a biomarker and therapeutic target. DESIGN: FLANC expression was analysed in 349 tumours from four cohorts and correlated to clinical data. In a series of multiple in vitro and in vivo models and molecular analyses, we characterised the fundamental biological roles of this lncRNA. We further explored the therapeutic potential of targeting FLANC in a mouse model of colorectal cancer (CRC) metastases. RESULTS: FLANC, a primate-specific lncRNA feebly expressed in normal colon cells, was significantly upregulated in cancer cells compared with normal colon samples in two independent cohorts. High levels of FLANC were associated with poor survival in two additional independent CRC patient cohorts. Both in vitro and in vivo experiments demonstrated that the modulation of FLANC expression influenced cellular growth, apoptosis, migration, angiogenesis and metastases formation ability of CRC cells. In vivo pharmacological targeting of FLANC by administration of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine nanoparticles loaded with a specific small interfering RNA, induced significant decrease in metastases, without evident tissue toxicity or pro-inflammatory effects. Mechanistically, FLANC upregulated and prolonged the half-life of phosphorylated STAT3, inducing the overexpression of VEGFA, a key regulator of angiogenesis. CONCLUSIONS: Based on our findings, we discovered, FLANC as a novel primate-specific lncRNA that is highly upregulated in CRC cells and regulates metastases formation. Targeting primate-specific transcripts such as FLANC may represent a novel and low toxic therapeutic strategy for the treatment of patients.

Interplay between epigenetic abnormalities and deregulated expression of microRNAs in cancer.

Epigenetic abnormalities and aberrant expression of non-coding RNAs are two emerging features of cancer cells, both of which are responsible for deregulated gene expression. In this review, we describe the interplay between the two. Specific themes include epigenetic silencing of tumor suppressor miRNAs, epigenetic activation of oncogenic miRNAs, epigenetic aberrations caused by miRNAs, and naturally occurring compounds which modulate miRNA expression through epigenetic mechanisms.

Aurora B kinase phosphorylates and instigates degradation of p53.

Aurora B is a mitotic checkpoint kinase that plays a pivotal role in the cell cycle, ensuring correct chromosome segregation and normal progression through mitosis. Aurora B is overexpressed in many types of human cancers, which has made it an attractive target for cancer therapies. Tumor suppressor p53 is a genome guardian and important negative regulator of the cell cycle. Whether Aurora B and p53 are coordinately regulated during the cell cycle is not known. We report that Aurora B directly interacts with p53 at different subcellular localizations and during different phases of the cell cycle (for instance, at the nucleus in interphase and the centromeres in prometaphase of mitosis). We show that Aurora B phosphorylates p53 at S183, T211, and S215 to accelerate the degradation of p53 through the polyubiquitination-proteasome pathway, thus functionally suppressing the expression of p53 target genes involved in cell cycle inhibition and apoptosis (e.g., p21 and PUMA). Pharmacologic inhibition of Aurora B in cancer cells with WT p53 increased p53 protein level and expression of p53 target genes to inhibit tumor growth. Together, these results define a mechanism of p53 inactivation during the cell cycle and imply that oncogenic hyperactivation or overexpression of Aurora B may compromise the tumor suppressor function of p53. We have elucidated the antineoplastic mechanism for Aurora B kinase inhibitors in cancer cells with WT p53.

Anti-miR-93-5p therapy prolongs sepsis survival by restoring the peripheral immune response.

Sepsis remains a leading cause of death for humans and currently has no pathogenesis-specific therapy. Hampered progress is partly due to a lack of insight into deep mechanistic processes. In the past decade, deciphering the functions of small noncoding miRNAs in sepsis pathogenesis became a dynamic research topic. To screen for new miRNA targets for sepsis therapeutics, we used samples for miRNA array analysis of PBMCs from patients with sepsis and control individuals, blood samples from 2 cohorts of patients with sepsis, and multiple animal models: mouse cecum ligation puncture-induced (CLP-induced) sepsis, mouse viral miRNA challenge, and baboon Gram+ and Gram- sepsis models. miR-93-5p met the criteria for a therapeutic target, as it was overexpressed in baboons that died early after induction of sepsis, was downregulated in patients who survived after sepsis, and correlated with negative clinical prognosticators for sepsis. Therapeutically, inhibition of miR-93-5p prolonged the overall survival of mice with CLP-induced sepsis, with a stronger effect in older mice. Mechanistically, anti-miR-93-5p therapy reduced inflammatory monocytes and increased circulating effector memory T cells, especially the CD4+ subset. AGO2 IP in miR-93-KO T cells identified important regulatory receptors, such as CD28, as direct miR-93-5p target genes. In conclusion, miR-93-5p is a potential therapeutic target in sepsis through the regulation of both innate and adaptive immunity, with possibly a greater benefit for elderly patients than for young patients.

Evaluation of cytokine expression in BEAS cells exposed to fine particulate matter (PM2.5) from specialized indoor environments.

Fine particles were collected in three indoor environments and an outdoor reference site. Samples were acid and aqueous extracted for metal analyses and cytokine expression study using a BEAS-2B line. Results revealed that the average PM(2.5) concentration indoors was 5.8 µg/m(3) while outside, it was 9.4 µg/m(3). The airborne metal concentrations in indoor air ranged from 0.01 ng/m(3) (Cd) to 620 ng/m(3) (Al). All metals analyzed were higher indoors when compared to outdoor (I/O ratio) indicating a contribution from the workplace. Some metals were more efficiently extracted (e.g., Ni, V, As) in the aqueous phase than others (e.g., Fe and Al). Toxicological assays showed that the aqueous extracts at 20% induced IL-6 and subsequently inhibited it at a higher concentration (50%); both IL-8 and MCP-1 were inhibited at 20 and 50%. As, Ni and V concentrations seem to be the most important metals associated with the cytokine induction/inhibition response probably due to the higher bioavailability.

Inhibition of G Protein-Coupled Receptor Kinase 2 Promotes Unbiased Downregulation of IGF1 Receptor and Restrains Malignant Cell Growth.

The ability of a receptor to preferentially activate only a subset of available downstream signal cascades is termed biased signaling. Although comprehensively recognized for the G protein-coupled receptors (GPCR), this process is scarcely explored downstream of receptor tyrosine kinases (RTK), including the cancer-relevant insulin-like growth factor-1 receptor (IGF1R). Successful IGF1R targeting requires receptor downregulation, yet therapy-mediated removal from the cell surface activates cancer-protective ß-arrestin-biased signaling (ß-arr-BS). As these overlapping processes are initiated by the ß-arr/IGF1R interaction and controlled by GPCR-kinases (GRK), we explored GRKs as potential anticancer therapeutic targets to disconnect IGF1R downregulation and ß-arr-BS. Transgenic modulation demonstrated that GRK2 inhibition or GRK6 overexpression enhanced degradation of IGF1R, but both scenarios sustained IGF1-induced ß-arr-BS. Pharmacologic inhibition of GRK2 by the clinically approved antidepressant, serotonin reuptake inhibitor paroxetine (PX), recapitulated the effects of GRK2 silencing with dose- and time-dependent IGF1R downregulation without associated ß-arr-BS. In vivo, PX treatment caused substantial downregulation of IGF1R, suppressing the growth of Ewing's sarcoma xenografts. Functional studies reveal that PX exploits the antagonism between ß-arrestin isoforms; in low ligand conditions, PX favored ß-arrestin1/Mdm2-mediated ubiquitination/degradation of IGF1R, a scenario usually exclusive to ligand abundancy, making PX more effective than antibody-mediated IGF1R downregulation. This study provides the rationale, molecular mechanism, and validation of a clinically feasible concept for "system bias" targeting of the IGF1R to uncouple downregulation from signaling. Demonstrating system bias as an effective anticancer approach, our study reveals a novel strategy for the rational design or repurposing of therapeutics to selectively cross-target the IGF1R or other RTK. SIGNIFICANCE: This work provides insight into the molecular and biological roles of biased signaling downstream RTK and provides a novel "system bias" strategy to increase the efficacy of anti-IGF1R-targeted therapy in cancer.

Impact of diabetes on promoting the growth of breast cancer.

BACKGROUND: Type II diabetes mellitus (DM2) is a significant risk factor for cancers, including breast cancer. However, a proper diabetic breast cancer mouse model is not well-established for treatment strategy design. Additionally, the precise diabetic signaling pathways that regulate cancer growth remain unresolved. In the present study, we established a suitable mouse model and demonstrated the pathogenic role of diabetes on breast cancer progression. METHODS: We successfully generated a transgenic mouse model of human epidermal growth factor receptor 2 positive (Her2+ or ERBB2) breast cancer with DM2 by crossing leptin receptor mutant (Leprdb/+ ) mice with MMTV-ErbB2/neu) mice. The mouse models were administrated with antidiabetic drugs to assess the impacts of controlling DM2 in affecting tumor growth. Magnetic resonance spectroscopic imaging was employed to analyze the tumor metabolism. RESULTS: Treatment with metformin/rosiglitazone in MMTV-ErbB2/Leprdb/db mouse model reduced serum insulin levels, prolonged overall survival, decreased cumulative tumor incidence, and inhibited tumor progression. Anti-insulin resistance medications also inhibited glycolytic metabolism in tumors in vivo as indicated by the reduced metabolic flux of hyperpolarized 13 C pyruvate-to-lactate reaction. The tumor cells from MMTV-ErbB2/Leprdb/db transgenic mice treated with metformin had reprogrammed metabolism by reducing levels of both oxygen consumption and lactate production. Metformin decreased the expression of Myc and pyruvate kinase isozyme 2 (PKM2), leading to metabolism reprogramming. Moreover, metformin attenuated the mTOR/AKT signaling pathway and altered adipokine profiles. CONCLUSIONS: MMTV-ErbB2/Leprdb/db mouse model was able to recapitulate diabetic HER2+ human breast cancer. Additionally, our results defined the signaling pathways deregulated in HER2+ breast cancer under diabetic condition, which can be intervened by anti-insulin resistance therapy.

Use of human bronchial epithelial cells (BEAS-2B) to study immunological markers resulting from exposure to PM(2.5) organic extract from Puerto Rico.

Fine particulate air pollutants, mainly their organic fraction, have been demonstrated to be associated with cardiovascular and respiratory health problems. Puerto Rico has been reported to have the highest prevalence of pulmonary diseases (e.g., asthma) in the United States. The aim of this study was to assess, for the first time, the immunological response of human bronchial epithelial cells (BEAS-2B) to organic extracts isolated from airborne particulate matter (PM(2.5)) in Puerto Rico. Organic extracts from PM(2.5) collected throughout an 8-month period (2000-2001) were pooled (composite) in order to perform chemical analysis and biological activity testing. BEAS-2B cells were exposed to PM(2.5) organic extract to assess cytotoxicity, levels of cytokines and relative gene expression of MHC-II, hPXR and CYP3A5. Our findings show that organic PM(2.5) consist of toxic as well as bioactive components that can regulate the secretion of cytokines in BEAS-2B, which could modulate inflammatory response in the lung. Trace element analyses confirmed the presence of metals in organic extracts highlighting the relative high abundance of Cu and Zn in polar organic extracts. Polar organic extracts exhibited dose-dependant toxicity and were found to significantly induce the release of interleukin 6 (IL-6), IL-1beta and IL-7 while significantly inhibiting the secretion of IL-8, G-CSF and MCP-1. Moreover, MHC-II transcriptional activity was up-regulated after 24 h of exposure, whereas PXR and CYP3A5 were down-regulated. This research provides a new insight into the effects of PM(2.5) organic fractions on specific effectors and their possible role in the development of respiratory inflammatory diseases in Puerto Rico.

Diabetes mellitus type 2 drives metabolic reprogramming to promote pancreatic cancer growth.

BACKGROUND: Diabetes mellitus type 2 (DM2) is a modifiable risk factor associated with pancreatic carcinogenesis and tumor progression on the basis of epidemiology studies, but the biological mechanisms are not completely understood. The purpose of this study is to demonstrate direct evidence for the mechanisms mediating these epidemiologic phenomena. Our hypothesis is that DM2 accelerates pancreatic cancer growth and that metformin treatment has a beneficial impact. METHODS: To determine the effect of glucose and insulin in pancreatic cancer proliferation, we used conditioned media to mimic DM2 conditions. Also, we studied the effect of anti-diabetic drugs, particularly metformin and rosiglitazone on pancreatic cancer growth. We established orthotopic/syngeneic (Lepr db/db ) mouse cancer models to evaluate the effect of diabetes on pancreatic tumor growth and aggressiveness. RESULTS: Our results showed that diabetes promotes pancreatic tumor growth. Furthermore, enhanced tumor growth and aggressiveness (e.g. epithelial-mesenchymal transition) can be explained by functional transcriptomic and metabolomic changes in the mice with diabetes, namely via activation of the AKT/mTOR pathway. Metformin treatment suppressed the diabetes-induced AKT/mTOR pathway activation and tumor growth. The metabolic profile determined by mass spectrum showed important changes of metabolites in the pancreatic cancer derived from diabetic mice treated with metformin. CONCLUSIONS: Diabetes mellitus type 2 has critical effects that promote pancreatic cancer progression via transcriptomic and metabolomic changes. Our animal models provide strong evidence for the causal relationship between diabetes and accelerated pancreatic cancers. This study sheds a new insight into the effects of metformin and its potential as part of therapeutic interventions for pancreatic cancer in diabetic patients.

miR-543 regulates the epigenetic landscape of myelofibrosis by targeting TET1 and TET2.

Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by cytopenia and extramedullary hematopoiesis, resulting in splenomegaly. Multiple pathological mechanisms (e.g., circulating cytokines and genetic alterations, such as JAKV617F mutation) have been implicated in the etiology of MF, but the molecular mechanism causing resistance to JAK2V617F inhibitor therapy remains unknown. Among MF patients who were treated with the JAK inhibitor ruxolitinib, we compared noncoding RNA profiles of ruxolitinib therapy responders versus nonresponders and found miR-543 was significantly upregulated in nonresponders. We validated these findings by reverse transcription-quantitative PCR. in this same cohort, in 2 additional independent MF patient cohorts from the United States and Romania, and in a JAK2V617F mouse model of MF. Both in vitro and in vivo models were used to determine the underlying molecular mechanism of miR-543 in MF. Here, we demonstrate that miR-543 targets the dioxygenases ten-eleven translocation 1 (TET1) and 2 (TET2) in patients and in vitro, causing increased levels of global 5-methylcytosine, while decreasing the acetylation of histone 3, STAT3, and tumor protein p53. Mechanistically, we found that activation of STAT3 by JAKs epigenetically controls miR-543 expression via binding the promoter region of miR-543. Furthermore, miR-543 upregulation promotes the expression of genes related to drug metabolism, including CYP3A4, which is involved in ruxolitinib metabolism. Our findings suggest miR-543 as a potentially novel biomarker for the prognosis of MF patients with a high risk of treatment resistance and as a potentially new target for the development of new treatment options.

S-MiRAGE: A Quantitative, Secreted RNA-Based Reporter of Gene Expression and Cell Persistence.

Nondestructive measurements of cell persistence and gene expression are crucial for longitudinal research studies and for prognostic assessment of cell therapies. Here we describe S-MiRAGE, a platform that utilizes small secreted RNA molecules as sensitive and quantitatively accurate reporters of cellular processes. S-MiRAGE allows cellular numbers or gene expression to be measured from culture media or from biofluids. We show that multiple S-MiRAGE reporters can be multiplexed, and demonstrate the utility of S-MiRAGE by monitoring the differentiation status of human embryonic stem cells in vitro and tumor growth in a mouse model in vivo.

Implications of dietary ω-3 and ω-6 polyunsaturated fatty acids in breast cancer.

Breast cancer represents one of the most common forms of cancer in women worldwide, with an increase in the number of newly diagnosed patients in the last decade. The role of fatty acids, particularly of a diet rich in ω-3 and ω-6 polyunsaturated fatty acids (PUFAs), in breast cancer development is not fully understood and remains controversial due to their complex mechanism of action. However, a large number of animal models and cell culture studies have demonstrated that high levels of ω-3 PUFAs have an inhibitory role in the development and progression of breast cancer, compared to ω-6 PUFAs. The present review focused on recent studies regarding the correlation between dietary PUFAs and breast cancer development, and aimed to emphasize the main molecular mechanisms involved in the modification of cell membrane structure and function, modulation of signal transduction pathways, gene expression regulation, and antiangiogenic and antimetastatic effects. Furthermore, the anticancer role of ω-3 PUFAs through the modulation of microRNA expression levels was also reviewed.

A-to-I miR-378a-3p editing can prevent melanoma progression via regulation of PARVA expression.

Previously we have reported that metastatic melanoma cell lines and tumor specimens have reduced expression of ADAR1 and consequently are impaired in their ability to perform A-to-I microRNA (miRNA) editing. The effects of A-to-I miRNAs editing on melanoma growth and metastasis are yet to be determined. Here we report that miR-378a-3p is undergoing A-to-I editing only in the non-metastatic but not in metastatic melanoma cells. The function of the edited form is different from its wild-type counterpart. The edited form of miR-378a-3p preferentially binds to the 3'-UTR of the PARVA oncogene and inhibits its expression, thus preventing the progression of melanoma towards the malignant phenotype. Indeed, edited miR-378a-3p but not its WT form inhibits melanoma metastasis in vivo. These results further emphasize the role of RNA editing in melanoma progression.

OncomiR-10b hijacks the small molecule inhibitor linifanib in human cancers.

The pervasive role of microRNAs (miRNAs) in cancer pathobiology drives the introduction of new drug development approaches such as miRNA inhibition. In order to advance miRNA-therapeutics, meticulous screening strategies addressing specific tumor targets are needed. Small molecule inhibitors represent an attractive goal for these strategies. In this study, we devised a strategy to screen for small molecule inhibitors that specifically inhibit, directly or indirectly, miR-10b (SMIRs) which is overexpressed in metastatic tumors. We found that the multi-tyrosine kinase inhibitor linifanib could significantly inhibit miR-10b and reverse its oncogenic function in breast cancer and liver cancer both in vitro and in vivo. In addition, we showed that the efficacy of linifanib to inhibit tyrosine kinases was reduced by high miR-10b levels. When the level of miR-10b is high, it can "hijack" the linifanib and reduce its kinase inhibitory effects in cancer resulting in reduced anti-tumor efficacy. In conclusion, our study describes an effective strategy to screen for small molecule inhibitors of miRNAs. We further propose that miR-10b expression levels, due to the newly described "hijacking" effect, may be used as a biomarker to select patients for linifanib treatment.

Regulation of hnRNPA1 by microRNAs controls the miR-18a-K-RAS axis in chemotherapy-resistant ovarian cancer.

The regulation of microRNA (miRNA) biogenesis, function and degradation involves a range of mechanisms, including interactions with RNA-binding proteins. The potential contribution of regulatory miRNAs to the expression of these RNA interactor proteins that could control other miRNAs expression is still unclear. Here we demonstrate a regulatory circuit involving oncogenic and tumor-suppressor miRNAs and an RNA-binding protein in a chemotherapy-resistant ovarian cancer model. We identified and characterized miR-15a-5p and miR-25-3p as negative regulators of hnRNPA1 expression, which is required for the processing of miR-18a-3p, an inhibitor of the K-RAS oncogene. The inhibition of miR-25-3p and miR-15a-5p decreased the proliferation, motility, invasiveness and angiogenic potential and increased apoptosis when combined with docetaxel. Alteration of this regulatory circuit causes poor overall survival outcome in ovarian cancer patients. These results highlight miR-15a-5p and miR-25-3p as key regulators of miR-18a-3p expression and its downstream target K-RAS, through direct modulation of hnRNPA1 expression. Our results demonstrate the therapeutic potential of inhibiting miR-25-3p and miR-15a-5p and the use of miR-18a-3p/KRAS ratio as a prominent outcome prognostic factor.