PPARγ Modulation of Cytokine-Stimulated MUC16 (CA125) Expression in Breast and Ovarian Cancer-Derived Cells.

CA125 is serum tumor marker consisting of an epitope carried by a portion of the extremely large (>3 MDa), heavily glycosylated cell surface transmembrane mucin, MUC16. In malignancies, membrane bound mucins lose their polarized distribution, become aberrantly over-expressed and protect tumor cells from the actions of chemotherapeutic agents as well as the immune system. Previously, we described stimulation of MUC16 expression by the proinflammatory cytokines, tumor necrosis factor α (TNFα) and interferon γ (IFNγ), in breast and ovarian cancer cells and tissues. Herein, we show that PPARγ modulates cytokine-stimulated MUC16 in a complex manner: at low concentrations (<10 µM) rosiglitazone further potentiates cytokine-driven MUC16 expression while at high concentrations (>20 µM) rosiglitazone antagonizes cytokine stimulation. Rosiglitazone actions were fully reversible by the PPARγ antagonist, GW9662. Furthermore, siRNA-mediated PPARγ knockdown also prevented a large portion of high dose rosiglitazone suppression of MUC16 expression indicating that rosiglitazone inhibition is largely PPARγ-dependent. Cytokines greatly (>75%) suppressed PPARγ expression. Conversely, PPARγ activation by rosiglitazone at either low or high concentrations greatly (>75%) suppressed NFκB/p65 expression. NFκB/p65 expression was largely preserved in the presence of cytokines at low, but not high, rosiglitazone concentrations accounting for the different concentration dependent effects on MUC16 expression. Collectively, these studies demonstrate that PPARγ is an important modulator of MUC16 expression. The ability to deliver high doses of PPARγ agonists to MUC16-expressing tumors offers an avenue to reduce expression of this protective glycoprotein and increase tumor sensitivity to killing by chemotherapeutic drugs and the immune system. J. Cell. Biochem. 118: 163-171, 2017. © 2016 Wiley Periodicals, Inc.

Transmembrane Mucin Expression and Function in Embryo Implantation and Placentation.

Transmembrane mucins (TMs) are extremely large, complex glycoproteins that line the apical surfaces of simple epithelia including those of the female reproductive tract. TMs provide a physical barrier consistent with their role as part of the innate immune system. This barrier function must be overcome in the context of embryo implantation to permit blastocyst attachment. Three major TMs have been identified in uterine epithelia of multiple species: MUC1, MUC4, and MUC16. MUC1 has been found in all species studied to date, whereas expression of MUC4 and MUC16 have been less well studied and may be species specific. The strategies for removing mucins to permit embryo attachment also vary in a species-specific way and include both hormonal suppression of TM gene expression and membrane clearance via cell surface proteases. Studies emerging from the cancer literature indicate that TMs can modulate a surprisingly wide variety of signal transduction processes. Furthermore, various cell surface proteins have been identified that bind either the oligosaccharide or protein motifs of TMs suggesting that these molecules may support cell attachment in some contexts, including trophoblast interactions with cells of the immune system. The intimate association of TMs at sites of embryo-maternal interaction and the varied functions these complex molecules can play make them key players in embryo implantation and placentation processes.

p62/SQSTM1 is required for cell survival of apoptosis-resistant bone metastatic prostate cancer cell lines.

BACKGROUND: Bone marrow stromal cell (BMSC) paracrine factor(s) can induce apoptosis in bone metastatic prostate cancer (PCa) cell lines. However, the PCa cells that escape BMSC-induced apoptosis can upregulate cytoprotective autophagy. METHODS: C4-2, C4-2B, MDA PCa 2a, MDA PCa 2b, VCaP, PC3, or DU145 PCa cell lines were grown in BMSC conditioned medium and analyzed for mRNA and/or protein accumulation of p62 (also known as sequestome-1/SQSTM1), Microtubule-associated protein 1 light chain 3B (LC3B), or lysosomal-associated membrane protein 1 (LAMP1) using quantitative polymerase chain reaction (QPCR), Western blot, or immunofluorescence. Small interfering RNA (siRNA) was used to determine if p62 is necessary PCa cell survival. RESULTS: BMSC paracrine signaling upregulated p62 mRNA and protein in a subset of the PCa cell lines. The PCa cell lines that were insensitive to BMSC-induced apoptosis and autophagy induction had elevated basal p62 mRNA and protein. In the BMSC-insensitive PCa cell lines, siRNA knockdown of p62 was cytotoxic and immunostaining showed peri-nuclear clustering of autolysosomes. However, in the BMSC-sensitive PCa cell lines, p62 siRNA knockdown was not appreciably cytotoxic and did not affect autolysosome subcellular localization. CONCLUSIONS: A pattern emerges wherein the BMSC-sensitive PCa cell lines are known to be osteoblastic and express the androgen receptor, while the BMSC-insensitive PCa cell lines are characteristically osteolytic and do not express the androgen receptor. Furthermore, BMSC-insensitive PCa may have evolved a dependency on p62 for cell survival that could be exploited to target and kill these apoptosis-resistant PCa cells in the bone.

Mechanisms of resistance to oncogenic KRAS inhibition in pancreatic cancer.

KRAS inhibitors demonstrate clinical efficacy in pancreatic ductal adenocarcinoma (PDAC); however, resistance is common. Among patients with KRASG12C-mutant PDAC treated with adagrasib or sotorasib, mutations in PIK3CA and KRAS, and amplifications of KRASG12C, MYC, MET, EGFR, and CDK6 emerged at acquired resistance. In PDAC cell lines and organoid models treated with the KRASG12D inhibitor MRTX1133, epithelial-to-mesenchymal transition and PI3K-AKT-mTOR signaling associate with resistance to therapy. MRTX1133 treatment of the KrasLSL-G12D/+;Trp53LSL-R172H/+;p48-Cre (KPC) mouse model yielded deep tumor regressions, but drug resistance ultimately emerged, accompanied by amplifications of Kras, Yap1, Myc, and Cdk6/Abcb1a/b, and co-evolution of drug-resistant transcriptional programs. Moreover, in KPC and PDX models, mesenchymal and basal-like cell states displayed increased response to KRAS inhibition compared to the classical state. Combination treatment with KRASG12D inhibition and chemotherapy significantly improved tumor control in PDAC mouse models. Collectively, these data elucidate co-evolving resistance mechanisms to KRAS inhibition and support multiple combination therapy strategies.

Effects of Physical Activity and Nutrition Education on the Gut Microbiota in Overweight and Obese Children.

Childhood obesity continues to represent a growing challenge, and it has been associated with gut microbiota dysbiosis. This study examines the gut microbiota composition in overweight and obese school children and assesses whether a 12-week multidisciplinary intervention can induce changes in the gut microbiota. The intervention, which combined recreational football and nutritional education, was implemented among 15 school children, aged 7-10 years, with a Body Mass Index ≥ 85th percentile. The children were assigned into two groups: Football Group (n = 9) and Nutrition and Football Group (n = 6). Faecal samples were collected at the beginning and end of the program and analysed by sequencing the 16S rRNA gene. Over the intervention, a significant decrease was found collectively for Bifidobacterium genera (p = 0.011) and for Roseburia genera in the Football Group (p = 0.021). The relative abundance of Roseburia (p = 0.002) and Roseburia faecis (p = 0.009) was negatively correlated with moderate to vigorous physical activity (MVPA), while Prevotella copri was positively correlated with MVPA (p = 0.010) and with the daily intake of protein (p = 0.008). Our findings suggest that a multidisciplinary intervention was capable of inducing limited but significant positive changes in the gut microbiota composition in overweight and obese school children.

Effects of "Football and Nutrition for Health" program on body composition, physical fitness, eating behaviours, nutritional knowledge, and psychological status among 7 to 10 years school children.

Introduction: Noncommunicable diseases and obesity are between the major health threat due to consumption of unhealthy foods and limited time spent on physical activities, a situation of particular concern among children. Since children spend most of their time at school, this study intends to investigate the effect of a school intervention program, which combines recreational football and nutrition education, on body composition, physical fitness, physical activity, blood pressure and heart rate, eating behaviours, nutritional knowledge, and psychological status in elementary school children. Methods: A total of 67 children, between 7 and 10 years old, were allocated into three groups: the Football Group (FG) which held 2 weekly sessions of 60 min of recreational football, the Nutrition and Football Group (NFG) which held 2 sessions per week of 60 min of recreational football plus 60 min of nutritional education and the Control Group (CG) which maintained its usual curriculum. The intervention lasted 12 weeks. All measurements were collected before and after the intervention. Results: Intervention groups significantly (p < 0.05) improved BMI Z-score, rest heart rate, horizontal jump and shuttle test, physical activity level, and psychosocial health. The NFG group significantly decreased (p < 0.05) waist-to-height ratio and blood pressure, and significantly increased (p < 0.05) nutritional knowledge, fruit, and fish consumption. While FG significantly decreased (p < 0.05) the percentage of fat mass and significantly increased (p < 0.05) muscle mass and performance in the 20 m sprint. Discussion: The results have shown to improve nutritional status, explosive strength, aerobic and neuromuscular fitness, as well as increase the level of physical activity. The nutritional education sessions contributed to increase nutritional knowledge and to improve the consumption of healthy food groups in a ludic-educational way. The "Football and Nutrition for Health" program was able to induce short-term improvements in several health markers, highlighting the role of the school curriculum in children's health.

Exploring Gut Microbiota and the Influence of Physical Activity Interventions on Overweight and Obese Children and Adolescents: A Systematic Review.

The recognition that the gut microbiota of obese children differs from lean children has grown, and some studies suggest that physical activity positively influences the gut microbiota. This systematic review explores the changes in the gut microbiota composition of obese and non-obese children and adolescents and provides an understanding of the effects of physical activity interventions in modulating their microbiota. The PRISMA protocol was used across PubMed, Scopus, and Web of Science. Overall, twenty-four research papers were included in accordance with the chosen inclusion and exclusion criteria, eighteen studies compared the gut microbiota of obese and normal-weight children and adolescents, and six studies explored the effect of physical activity interventions on the gut microbiota. The analysis indicated that obese gut microbiota is reduced in Bacteroidetes, Bifidobacterium and alpha diversity but enriched in Proteobacteria and Lactobacillus. Interventions with physical activity seem to improve the alpha diversity and beneficial bacteria linked to body weight loss in children and adolescents. The gut microbiota of obese children exhibited a remarkably individual variation. More interventions are needed to clearly and accurately explore the relationships between child obesity, gut microbiota, and physical activity and to develop approaches to decrease the incidence of paediatric obesity.

Spatial Mapping and Immunomodulatory Role of the OX40/OX40L Pathway in Human Non-Small Cell Lung Cancer.

PURPOSE: To evaluate the tissue distribution and clinical significance of OX40 and OX40L in human non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Using multiplexed quantitative immunofluorescence, we conducted simultaneous and localized measurements of OX40 and OX40L proteins, major T-cell subsets, and conventional type 1 dendritic cells (cDC1) in 614 primary NSCLCs from three independent cohorts represented in tissue microarrays. We also measured OX40L protein in samples from a phase I clinical trial of intratumor administration of a lipid nanoparticle encapsulated mRNA encoding OX40L (mRNA-2416) in human solid tumors. Finally, we studied the OX40 pathway in 212 uterine/ovarian serous carcinomas. RESULTS: OX40 protein was expressed in approximately 90% of NSCLCs, and OX40L was detected in approximately 10% of cases. Increased expression of OX40 was associated with higher CD4+ and CD8+ T lymphocytes, as well as cDC1s. Elevated expression of OX40L was consistently associated with increased CD4+ tumor-infiltrating lymphocytes and longer overall survival. No association was found between OX40 or OX40L levels and oncogenic driver mutations in EGFR and KRAS in lung adenocarcinomas. Delivering OX40L mRNA using intratumor mRNA-2416 injection mediated increased local OX40L protein levels that was most prominent in a patient with ovarian serous carcinoma. Detectable OX40L protein levels were observed in 15% of primary uterine/ovarian serous malignancies and associated with longer survival. CONCLUSIONS: The OX40 pathway is expressed in a fraction of NSCLCs and is associated with a favorable immune contexture. Although OX40L is uncommonly expressed in NSCLC and serous malignancies, it is associated with better prognosis and can be introduced using exogenous mRNA.

Discovery of putative tumor suppressors from CRISPR screens reveals rewired lipid metabolism in acute myeloid leukemia cells.

CRISPR knockout fitness screens in cancer cell lines reveal many genes whose loss of function causes cell death or loss of fitness or, more rarely, the opposite phenotype of faster proliferation. Here we demonstrate a systematic approach to identify these proliferation suppressors, which are highly enriched for tumor suppressor genes, and define a network of 145 such genes in 22 modules. One module contains several elements of the glycerolipid biosynthesis pathway and operates exclusively in a subset of acute myeloid leukemia cell lines. The proliferation suppressor activity of genes involved in the synthesis of saturated fatty acids, coupled with a more severe loss of fitness phenotype for genes in the desaturation pathway, suggests that these cells operate at the limit of their carrying capacity for saturated fatty acids, which we confirm biochemically. Overexpression of this module is associated with a survival advantage in juvenile leukemias, suggesting a clinically relevant subtype.

Differential changes in TGF-ß/BMP signaling pathway in the right ventricular myocardium of newborns with hypoplastic left heart syndrome.

BACKGROUND: Hypoplastic left heart syndrome (HLHS) is characterized by underdevelopment of the left ventricle (LV) and increased biomechanical stress on the right ventricle (RV) from single ventricle physiology. Despite the clinical significance, the signaling pathways active during RV remodeling and disease progression are not known. To address this, we examined differential changes in expression of genes associated with transforming growth factor-beta (TGF-beta)/bone morphogenetic protein (BMP) signaling in RV tissue isolated from HLHS patients relative to RV and LV tissue from control subjects. METHODS AND RESULTS: Quantitative real-time polymerase chain reaction was used to detect changes in expression of 84 genes involved in TGF-beta/BMP-mediated cardiac development, cell growth, and differentiation in RV tissue collected from 6 neonates with HLHS undergoing stage 1 Norwood procedure (age, 1-7 days; mean, 4 days) and RV and LV tissue obtained from 5 infants with noncardiac pathology (age range, 1-135 days: mean, 85 days) that served as controls. Analysis of gene expression profiles between control-LV and control-RV revealed significant depression of TGF-beta/BMP signaling in RV compared with LV. Of the 84 genes analyzed, 38 were differentially expressed between HLHS-RV and control-RV, whereas only 22 compared with control-LV. Significant changes were observed in: tissue remodeling genes including Activin receptor type IIA (ACVR2A) (+2.13) and Activin receptor-like kinase 1 (ACVRL1) (+2.22); and cell survival, growth, and differentiation genes including CDC25A (+2.18), p21 (-3.64), p15 (+2.15), BMP5 (+4.58), BMP3 (+2.16), GDF3 (+8.59), NODAL (+2.32), and BMP binding endothelial regulator (BMPER) (+4.58). The most significant changes common to HLHS-RV versus control-RV and control-LV sample groups is observed for Anti müllerian hormone receptor 2 (AMHR2) (+18.79 control-RV, +3.38 control-LV), and the BMP antagonist Inhibin alpha (INHA) (+11.47 control-RV, +5.73 control-LV). CONCLUSIONS: Although this descriptive study does not allow cause-effect inferences, our results suggest changes in cardiac development pathways and upregulation of genes associated with cell growth and differentiation in the neonatal RV of children with HLHS. These molecular profiles are more closely related to those observed in the normal LV rather than normal RV at similar maturational age. This work provides the basis for future mechanistic studies to elucidate the molecular mechanisms regulating RV remodeling in HLHS.

Prediction of Simulated 1,000 m Kayak Ergometer Performance in Young Athletes.

This study aimed to develop a predictive explanatory model for the 1,000-m time-trial (TT) performance in young national-level kayakers, from biomechanical and physiological parameters assessed in a maximal graded exercise test (GXT). Twelve young male flat-water kayakers (age 16.1 ± 1.1 years) participated in the study. The design consisted of 2 exercise protocols, separated by 48 h, on a kayak ergometer. The first protocol consisted of a GXT starting at 8 km.h-1 with increments in speed of 1 km.h-1 at each 2-min interval until exhaustion. The second protocol comprised the 1,000-m TT. Results: In the GXT, they reached an absolute V∙O2max of 3.5 ± 0.7 (L.min-1), a maximum aerobic power (MAP) of 138.5 ± 24.5 watts (W) and a maximum aerobic speed (MAS) of 12.8 ± 0.5 km/h. The TT had a mean duration of 292.3 ± 15 s, a power output of 132.6 ± 22.0 W and a V∙O2max of 3.5 ± 0.6 (L.min-1). The regression model [TT (s) = 413.378-0.433 × (MAP)-0.554 × (stroke rate at MAP)] presented an R2 = 84.5%. Conclusion: It was found that V∙O2max , stroke distance and stroke rate during the GXT were not different from the corresponding variables ( V∙O2peak , stroke distance and stroke rate) observed during the TT. The MAP and the corresponding stroke rate were strong predicting factors of 1,000 m TT performance. In conclusion, the TT can be useful for quantifying biomechanical parameters (stroke distance and stroke rate) and to monitor training induced changes in the cardiorespiratory fitness ( V∙O2max ).

Heart Rate Variability and Stress Recovery Responses during a Training Camp in Elite Young Canoe Sprint Athletes.

Training camps are typical in elite Canoeing preparation, during which, the care to assure adaptation to avoid undesired fatigue is not always present. This study aimed identifying a specific sex response in perceived training loads, recovery and stress balance, and cardiac autonomic responses. Twenty-one elite athletes (11 males and 10 females) of the Portuguese Canoeing National team participated in the investigation. The daily HRV (lnRMSSD) was monitored. The (RESTQ-52) questionnaire was used to access the recovery and stress state. The 10-day training camp was composed of two consecutive 5-day periods (P1 and P2). Data analyses were performed using confidence limits, effect size, and magnitude-based inference. In the females, Session rating of perceived exertion (sRPE), lnRMSSD, and its coefficient of variation did not change between P1 and P2. However, in males, lnRMSSD showed a small reduction from P1 to P2. Also, sRPE was higher in males over the training period, with a possibly small difference at P2. Regarding RESTQ-52, total stress most likely increased with large and very large differences in males and moderate differences in females during the training period. Male canoeists undertook higher perceived training loads than females, with a consequent higher level of total perceived stress and lnRMSSD during a 10-day training camp.

A 3D in vitro model of patient-derived prostate cancer xenograft for controlled interrogation of in vivo tumor-stromal interactions.

Patient-derived xenograft (PDX) models better represent human cancer than traditional cell lines. However, the complex in vivo environment makes it challenging to employ PDX models to investigate tumor-stromal interactions, such as those that mediate prostate cancer (PCa) bone metastasis. Thus, we engineered a defined three-dimensional (3D) hydrogel system capable of supporting the co-culture of PCa PDX cells and osteoblastic cells to recapitulate the PCa-osteoblast unit within the bone metastatic microenvironment in vitro. Our 3D model not only maintained cell viability but also preserved the typical osteogenic phenotype of PCa PDX cells. Additionally, co-culture cellularity was maintained over that of either cell type cultured alone, suggesting that the PCa-osteoblast cross-talk supports PCa progression in bone, as is hypothesized to occur in patients with prostatic bone metastasis. Strikingly, osteoblastic cells co-cultured with PCa PDX tumoroids organized around the tumoroids, closely mimicking the architecture of PCa metastases in bone. Finally, tumor-stromal signaling mediated by the fibroblast growth factor axis tightly paralleled that in the in vivo counterpart. Together, these findings indicate that this 3D PCa PDX model recapitulates important pathological properties of PCa bone metastasis, and validate the use of this model for controlled and systematic interrogation of complex in vivo tumor-stromal interactions.

Tumor necrosis factor-α and interferon-γ stimulate MUC16 (CA125) expression in breast, endometrial and ovarian cancers through NFκB.

Transmembrane mucins (TMs) are restricted to the apical surface of normal epithelia. In cancer, TMs not only are over-expressed, but also lose polarized distribution. MUC16/CA125 is a high molecular weight TM carrying the CA125 epitope, a well-known molecular marker for human cancers. MUC16 mRNA and protein expression was mildly stimulated by low concentrations of TNFα (2.5 ng/ml) or IFNγ (20 IU/ml) when used alone; however, combined treatment with both cytokines resulted in a moderate (3-fold or less) to large (> 10-fold) stimulation of MUC16 mRNA and protein expression in a variety of cancer cell types indicating that this may be a general response. Human cancer tissue microarray analysis indicated that MUC16 expression directly correlates with TNFα and IFNγ staining intensities in certain cancers. We show that NFκB is an important mediator of cytokine stimulation of MUC16 since siRNA-mediated knockdown of NFκB/p65 greatly reduced cytokine responsiveness. Finally, we demonstrate that the 250 bp proximal promoter region of MUC16 contains an NFκB binding site that accounts for a large portion of the TNFα response. Developing methods to manipulate MUC16 expression could provide new approaches to treating cancers whose growth or metastasis is characterized by elevated levels of TMs, including MUC16.

Nebulette mutations are associated with dilated cardiomyopathy and endocardial fibroelastosis.

OBJECTIVES: Four variants (K60N, Q128R, G202R, and A592E) in the nebulette gene were identified in patients with dilated cardiomyopathy (DCM) and endocardial fibroelastosis. We sought to determine if these mutations are cardiomyopathy causing. BACKGROUND: Nebulette aligns thin filaments and connects them with the myocardial Z-disk, playing a role in mechanosensation. METHODS: We generated transgenic mice with cardiac-restricted overexpression of human wild-type or mutant nebulette. Chimera and transgenic mice were examined at 4, 6, and 12 months of age by echocardiography and cardiac magnetic resonance imaging. The hearts from embryos and adult mice were assessed by histopathologic, immunohistochemical, ultrastructural, and protein analyses. Rat H9C2 cardiomyoblasts with transient expression of nebulette underwent cyclic mechanical strain. RESULTS: We identified lethal cardiac structural abnormalities in mutant embryonic hearts (K60N and Q128R). Founders of the mutant mouse lines developed DCM with severe heart failure. An irregular localization pattern for nebulette and impaired desmin expression were noted in the proband and chimeric Q128R mice. Mutant G202R and A592E mice exhibited left ventricular dilation and impaired function with specific changes in I-band and Z-disk proteins by 6 months of age. The mutations modulated distribution of nebulette in the sarcomere and Z-disk during stretch of H9C2 cells. CONCLUSIONS: Nebulette is a new susceptibility gene for endocardial fibroelastosis and DCM. Different mutations in nebulette trigger specific mechanisms, converging to a common pathological cascade leading to endocardial fibroelastosis and DCM.