Establishment and evaluation of module-based immune-associated gene signature to predict overall survival in patients of colon adenocarcinoma.

BACKGROUND: Patients with colon adenocarcinoma (COAD) exhibit significant heterogeneity in overall survival. The current tumor-node-metastasis staging system is insufficient to provide a precise prediction for prognosis. Identification and evaluation of new risk models by using big cancer data may provide a good way to identify prognosis-related signature. METHODS: We integrated different datasets and applied bioinformatic and statistical methods to construct a robust immune-associated risk model for COAD prognosis. Furthermore, a nomogram was constructed based on the gene signature and clinicopathological features to improve risk stratification and quantify risk assessment for individual patients. RESULTS: The immune-associated risk model discriminated high-risk patients in our investigated and validated cohorts. Survival analyses demonstrated that our gene signature served as an independent risk factor for overall survival and the nomogram exhibited high accuracy. Functional analysis interpreted the correlation between our risk model and its role in prognosis by classifying groups with different immune activities. Remarkably, patients in the low-risk group showed higher immune activity, while those in the high-risk group displayed a lower immune activity. CONCLUSIONS: Our study provides a novel tool that may contribute to the optimization of risk stratification for survival and personalized management of COAD.

Placental glycosylation senses the anti-angiogenic milieu induced by human sFLT1 during pregnancy.

Abnormal placental angiogenesis during gestation resulting from high levels of anti-angiogenic factors, soluble fms-like tyrosine kinase-1 (sFLT1) and soluble endoglin, has been implicated in the progression of preeclampsia (PE). This heterogeneous syndrome (defined by hypertension with or without proteinuria after 20 weeks of pregnancy) remains a major global health burden with long-term consequences for both mothers and child. Previously, we showed that in vivo systemic human (hsFLT1) overexpression led to reduced placental efficiency and PE-like syndrome in mice. Galectins (gal-1, -3 and -9) are critical determinants of vascular adaptation to pregnancy and dysregulation of the galectin-glycan circuits is associated with the development of this life-threatening disease. In this study, we assessed the galectin-glycan networks at the maternal-fetal interface associated with the hsFLT1-induced PE in mice. We observed an increase on the maternal gal-1 expression in the decidua and junctional zone layers of the placenta derived from hs FLT1high pregnancies. In contrast, placental gal-3 and gal-9 expression were not sensitive to the hsFLT1 overexpression. In addition, O- and N-linked glycan expression, poly-LacNAc sequences and terminal sialylation were down-regulated in hsFLT1 high placentas. Thus, the gal-1-glycan axis appear to play an important role counteracting the anti-angiogenic status caused by sFLT1, becoming critical for vascular adaptation at the maternal-fetal interface.

IFNγ-Stat1 axis drives aging-associated loss of intestinal tissue homeostasis and regeneration.

The influence of aging on intestinal stem cells and their niche can explain underlying causes for perturbation in their function observed during aging. Molecular mechanisms for such a decrease in the functionality of intestinal stem cells during aging remain largely undetermined. Using transcriptome-wide approaches, our study demonstrates that aging intestinal stem cells strongly upregulate antigen presenting pathway genes and over-express secretory lineage marker genes resulting in lineage skewed differentiation into the secretory lineage and strong upregulation of MHC class II antigens in the aged intestinal epithelium. Mechanistically, we identified an increase in proinflammatory cells in the lamina propria as the main source of elevated interferon gamma (IFNγ) in the aged intestine, that leads to the induction of Stat1 activity in intestinal stem cells thus priming the aberrant differentiation and elevated antigen presentation in epithelial cells. Of note, systemic inhibition of IFNγ-signaling completely reverses these aging phenotypes and reinstalls regenerative capacity of the aged intestinal epithelium.

Single-cell atlas of the aging mouse colon.

We performed massive single-cell sequencing in the aging mouse colonic epithelium and immune cells. We identified novel compartment-specific markers as well as dramatic aging-associated changes in cell composition and signaling pathways, including a shift from absorptive to secretory epithelial cells, depletion of naive lymphocytes, and induction of eIF2 signaling. Colon cancer is one of the leading causes of death within the western world, incidence of which increases with age. The colonic epithelium is a rapidly renewing tissue, tasked with water and nutrient absorption, as well as hosting intestinal microbes. The colonic submucosa is populated with immune cells interacting with and regulating the epithelial cells. However, it is unknown whether compartment-specific changes occur during aging and what impact this would cause. We show that both epithelial and immune cells differ significantly between colonic compartments and experience significant age-related changes in mice. We found a shift in the absorptive-secretory cell balance, possibly linked to age-associated intestinal disturbances, such as malabsorption. We demonstrate marked changes in aging immune cells: population shifts and interactions with epithelial cells, linking cytokines (Ifn-γ, Il1B) with the aging of colonic epithelium. Our results provide new insights into the normal and age-associated states of the colon.

Paneth cells drive intestinal stem cell competition and clonality in aging and calorie restriction.

Calorie restriction has been recently shown to increase intestinal stem cell competition and to reduce mutation fixation in young mice. However, the impact of aging on this process is unknown. By employing Confetti reporter mice, here we show that, unexpectedly, old mice have more intestinal stem cell (ISC) competition than young mice. Moreover, differently from what observed in young mice, calorie restriction, when applied at late-life, decreases this process. Importantly, we also observed a strong correlation between the ISC competition and Paneth cell number. In vivo analysis and in vitro organoid experiments indicated that Paneth cells play a major role in driving intestinal stem cell competition and crypt clonality. Taken together, our results provide evidence that increasing the number of Paneth cells can increase the number of competitive ISCs, representing a valuable therapeutic target to delay fixation of mutated intestinal stem cells.

Characterization of Novel α-Mangostin and Paeonol Derivatives With Cancer-Selective Cytotoxicity.

α-Mangostin (aMan) and Paeonol (Pae) have shown anticancer and anti-inflammatory properties. However, these two natural compounds have no clinical value because of their low solubility and low membrane permeability. In this study, we screened chemically synthesized derivatives from these two natural compounds as potential novel chemicals that increase cancer cell cytotoxicity over nontransformed human cells. We found that two derivative compounds, named α-Mangostin-1 (aMan1) and Paeonol-1 (Pae1) more efficiently and more specifically induced cytotoxicity in HCT116, HT29, and SW48 colorectal cancer cell lines than the parental compounds. Both aMan1 and Pae1 arrested HCT116 cells in the G1 phase and HT29 and SW48 cells in the G2-M phase of the cell cycle. Both aMan1 and Pae1 induced apoptosis in human colorectal cancer cells, through a caspase-dependent mechanism. aMan1 and Pae1 induced selective transcriptional responses in colorectal cancer cells involving genes related to metabolic stress and DNA damage response signaling pathways. Finally, experiments on primary colon organoids showed that both derivatives were able to kill cancer-derived organoids without affecting the viability of organoids derived from healthy tissue, where the parental compounds and the currently used chemotherapeutic drug irinotecan failed. In conclusion, our findings expand the knowledge of natural compound derivatives as anticancer agents and open new avenues of research in the derivation of lead compounds aimed at developing novel chemotherapeutic drugs for colorectal cancer treatment that selectively target cancer, but not healthy cells.

Inflammaging is driven by upregulation of innate immune receptors and systemic interferon signaling and is ameliorated by dietary restriction.

Aging is characterized by a chronic low-grade inflammation known as inflammaging in multiple tissues, representing a risk factor for age-related diseases. Dietary restriction (DR) is the best-known non-invasive method to ameliorate aging in many organisms. However, the molecular mechanism and the signaling pathways that drive inflammaging across different tissues and how they are modulated by DR are not yet understood. Here we identify a multi-tissue gene network regulating inflammaging. This network is characterized by chromatin opening and upregulation in the transcription of innate immune system receptors and by activation of interferon signaling through interferon regulatory factors, inflammatory cytokines, and Stat1-mediated transcription. DR ameliorates aging-induced alterations of chromatin accessibility and RNA transcription of the inflammaging gene network while failing to rescue those alterations on the rest of the genome. Our results present a comprehensive understanding of the molecular network regulating inflammation in aging and DR and provide anti-inflammaging therapeutic targets.

The natural compound atraric acid suppresses androgen-regulated neo-angiogenesis of castration-resistant prostate cancer through angiopoietin 2.

Castration-resistant prostate cancer (CRPC) is an aggressive lethal form of prostate cancer (PCa). Atraric acid (AA) not only inhibits the wild-type androgen receptor (AR) but also those AR mutants that confer therapy resistance to other clinically used AR antagonists, indicating a different mode of AR antagonism. AA induces cellular senescence and inhibits CRPC tumour growth in in vivo xenograft mouse model associated with reduced neo-angiogenesis suggesting the repression of intratumoural neo-angiogenesis by AA. In line with this, the secretome of CRPC cells mediates neo-angiogenesis in an androgen-dependent manner, which is counteracted by AA. This was confirmed by two in vitro models using primary human endothelial cells. Transcriptome sequencing revealed upregulated angiogenic pathways by androgen, being however VEGF-independent, and pointing to the pro-angiogenic factor angiopoietin 2 (ANGPT2) as a key driver of neo-angiogenesis induced by androgens and repressed by AA. In agreement with this, AA treatment of native patient-derived PCa tumour samples ex vivo inhibits ANGPT2 expression. Mechanistically, in addition to AA, immune-depletion of ANGPT2 from secretome or blocking ANGPT2-receptors inhibits androgen-induced angiogenesis. Taken together, we reveal a VEGF-independent ANGPT2-mediated angiogenic pathway that is inhibited by AA leading to repression of androgen-regulated neo-angiogenesis.

Expression of the alternative splicing regulator Rbfox2 during placental development is differentially regulated in preeclampsia mouse models.

PROBLEM: Proper placental development is pivotal to ensure healthy pregnancy outcomes. Among the multiple cellular mechanisms involved in the orchestration of this process, little is known on the role of alternative splicing events in the modulation of trophoblast cell biology. Here, we evaluated the expression of the alternative splicing regulator Rbfox2 in the pre- and post-placentation period in mouse pregnancies in both healthy and pathological settings. METHOD OF STUDY: Immunofluorescence analysis of Rbfox2 expression in mouse implantation sites collected during the pre-placentation period (E5-E7) and post-placentation (E13). RESULTS: We identified a progressive increase of Rbfox2 levels throughout the peri-implantation period with a shift from a cytoplasmatic expression on E5-E6 to a predominantly nuclear expression on E7, together with a prominent expression of this factor in both subcellular compartments of the primitive placenta. Our results further showed that in contrast to healthy gestations, Rbfox2 expression decreased in preeclamptic models during the post-placentation period. Finally, we further demonstrated enhanced expression of Rbfox2 proteins in allogeneic pregnancy compared to syngeneic models. CONCLUSIONS: Our findings uncover a novel role for Rbfox2-controlled splicing events in the modulation of trophoblast function, with potential implications for the pathogenesis of preeclampsia and other pregnancy complications originated from defective placentation.

Characterization of an in vitro 3D intestinal organoid model by using massive RNAseq-based transcriptome profiling.

Organoids culture provides unique opportunities to study human diseases and to complement animal models. Several organs and tissues can be in vitro cultured in 3D structures resembling in vivo tissue organization. Organoids culture contains most of the cell types of the original tissue and are maintained by growth factors mimicking the in vivo state. However, the system is yet not fully understood, and specific in vivo features especially those driven by cell-extrinsic factors may be lost in culture. Here we show a comprehensive transcriptome-wide characterization of mouse gut organoids derived from different intestinal compartments and from mice of different gender and age. RNA-seq analysis showed that the in vitro culture strongly influences the global transcriptome of the intestinal epithelial cells (~ 60% of the total variance). Several compartment-, age- and gender-related transcriptome features are lost after culturing indicating that they are driven by niche or systemic factors. However, certain intrinsic transcriptional programs, for example, some compartment-related features and a minority of gender- and aging- related features are maintained in vitro which suggested possibilities for these features to be studied in this system. Moreover, our study provides knowledge about the cell-extrinsic or cell-intrinsic origin of intestinal epithelial transcriptional programs. We anticipated that our characterization of this in vitro system is an important reference for scientists and clinicians using intestinal organoids as a research model.

Establishment of a fluorescent reporter of RNA-polymerase II activity to identify dormant cells.

Dormancy, a reversible quiescent cellular state characterized by greatly reduced metabolic activity, protects from genetic damage, prolongs survival and is crucial for tissue homeostasis and cellular response to injury or transplantation. Dormant cells have been characterized in many tissues, but their identification, isolation and characterization irrespective of tissue of origin remains elusive. Here, we develop a live cell ratiometric fluorescent Optical Stem Cell Activity Reporter (OSCAR) based on the observation that phosphorylation of RNA Polymerase II (RNApII), a hallmark of active mRNA transcription elongation, is largely absent in dormant stem cells from multiple lineages. Using the small intestinal crypt as a model, OSCAR reveals in real time the dynamics of dormancy induction and cellular differentiation in vitro, and allows the identification and isolation of several populations of transcriptionally diverse OSCARhigh and OSCARlow intestinal epithelial cell states in vivo. In particular, this reporter is able to identify a dormant OSCARhigh cell population in the small intestine. OSCAR therefore provides a tool for a better understanding of dormant stem cell biology.