Senescent cancer-associated fibroblasts in pancreatic adenocarcinoma restrict CD8+ T cell activation and limit responsiveness to immunotherapy in mice.

Senescent cells within tumors and their stroma exert complex pro- and anti-tumorigenic functions. However, the identities and traits of these cells, and the potential for improving cancer therapy through their targeting, remain poorly characterized. Here, we identify a senescent subset within previously-defined cancer-associated fibroblasts (CAFs) in pancreatic ductal adenocarcinomas (PDAC) and in premalignant lesions in mice and humans. Senescent CAFs isolated from mouse and humans expressed elevated levels of immune-regulatory genes. Depletion of senescent CAFs, either genetically or using the Bcl-2 inhibitor ABT-199 (venetoclax), increased the proportion of activated CD8+ T cells in mouse pancreatic carcinomas, whereas induction of CAF senescence had the opposite effect. Combining ABT-199 with an immune checkpoint therapy regimen significantly reduced mouse tumor burden. These results indicate that senescent CAFs in PDAC stroma limit the numbers of activated cytotoxic CD8+ T cells, and suggest that their targeted elimination through senolytic treatment may enhance immunotherapy.

Senescence of human pancreatic beta cells enhances functional maturation through chromatin reorganization and promotes interferon responsiveness.

Senescent cells can influence the function of tissues in which they reside, and their propensity for disease. A portion of adult human pancreatic beta cells express the senescence marker p16, yet it is unclear whether they are in a senescent state, and how this affects insulin secretion. We analyzed single-cell transcriptome datasets of adult human beta cells, and found that p16-positive cells express senescence gene signatures, as well as elevated levels of beta-cell maturation genes, consistent with enhanced functionality. Senescent human beta-like cells in culture undergo chromatin reorganization that leads to activation of enhancers regulating functional maturation genes and acquisition of glucose-stimulated insulin secretion capacity. Strikingly, Interferon-stimulated genes are elevated in senescent human beta cells, but genes encoding senescence-associated secretory phenotype (SASP) cytokines are not. Senescent beta cells in culture and in human tissue show elevated levels of cytoplasmic DNA, contributing to their increased interferon responsiveness. Human beta-cell senescence thus involves chromatin-driven upregulation of a functional-maturation program, and increased responsiveness of interferon-stimulated genes, changes that could increase both insulin secretion and immune reactivity.

Regulation of Cellular Heterogeneity and Rates of Symmetric and Asymmetric Divisions in Triple-Negative Breast Cancer.

Differentiation events contribute to phenotypic cellular heterogeneity within tumors and influence disease progression and response to therapy. Here, we dissect mechanisms controlling intratumoral heterogeneity within triple-negative basal-like breast cancers. Tumor cells expressing the cytokeratin K14 possess a differentiation state that is associated with that of normal luminal progenitors, and K14-negative cells are in a state closer to that of mature luminal cells. We show that cells can transition between these states through asymmetric divisions, which produce one K14+ and one K14- daughter cell, and that these asymmetric divisions contribute to the generation of cellular heterogeneity. We identified several regulators that control the proportion of K14+ cells in the population. EZH2 and Notch increase the numbers of K14+ cells and their rates of symmetric divisions, and FOXA1 has an opposing effect. Our findings demonstrate that asymmetric divisions generate differentiation transitions and heterogeneity, and identify pathways that control breast cancer cellular composition.

Programming asynchronous replication in stem cells.

Many regions of the genome replicate asynchronously and are expressed monoallelically. It is thought that asynchronous replication may be involved in choosing one allele over the other, but little is known about how these patterns are established during development. We show that, unlike somatic cells, which replicate in a clonal manner, embryonic and adult stem cells are programmed to undergo switching, such that daughter cells with an early-replicating paternal allele are derived from mother cells that have a late-replicating paternal allele. Furthermore, using ground-state embryonic stem (ES) cells, we demonstrate that in the initial transition to asynchronous replication, it is always the paternal allele that is chosen to replicate early, suggesting that primary allelic choice is directed by preset gametic DNA markers. Taken together, these studies help define a basic general strategy for establishing allelic discrimination and generating allelic diversity throughout the organism.

Inflammation-Induced Expression and Secretion of MicroRNA 122 Leads to Reduced Blood Levels of Kidney-Derived Erythropoietin and Anemia.

BACKGROUND & AIMS: Anemia is associated commonly with acute and chronic inflammation, but the mechanisms of their interaction are not clear. We investigated whether microRNA 122 (MIR122), which is generated in the liver and is secreted into the blood, is involved in the development of anemia associated with inflammation. METHODS: We characterized the primary transcript of the human liver-specific MIR122 using Northern blot, quantitative real-time polymerase chain reaction, and 3' and 5' rapid amplification of cDNA ends analyses. We studied regulation of MIR122 in human hepatocellular carcinoma cell lines (Huh7 and HepG2) as well as in C57BL/6 and mice with disruption of the tumor necrosis factor (Tnf) gene. Liver tissues were collected and analyzed by bioluminescence imaging or immunofluorescence. Inflammation in mice was induced by lipopolysaccharide (LPS) or by cerulein injections. Mice were given 4 successive injections of LPS, leading to inflammation-induced anemia. Steatohepatitis was induced with a choline-deficient, high-fat diet. Hemolytic anemia was stimulated by phenylhydrazine injection. MIR122 was inhibited in mice by tail-vein injection of an oligonucleotide antagonist of MIR122. MicroRNA and messenger RNA levels were determined by quantitative real-time polymerase chain reaction. RESULTS: The primary transcript of MIR122 spanned 5 kb, comprising 3 exons; the third encodes MIR122. Within the MIR122 promoter region we identified a nuclear factor-κB binding site and showed that RELA (NF-κB p65 subunit), as well as activators of NF-κB (TNF and LPS), increased promoter activity of MIR122. Administration of LPS to mice induced secretion of MIR122 into blood, which required TNF. Secreted MIR122 reached the kidney and reduced expression of erythropoietin (Epo), which we identified as a MIR122 target gene. Injection of mice with an oligonucleotide antagonist of MIR122 increased blood levels of EPO, reticulocytes, and hemoglobin. We found an inverse relationship between blood levels of MIR122 and EPO in mice with acute pancreatitis or steatohepatitis, and also in patients with acute inflammation. CONCLUSION: In mice, we found that LPS-induced inflammation increases blood levels of MIR122, which reduces expression of Epo in the kidney; this is a mechanism of inflammation-induced anemia. Strategies to block MIR122 in patients with inflammation could reduce the development or progression of anemia.

Directed elimination of senescent cells by inhibition of BCL-W and BCL-XL.

Senescent cells, formed in response to physiological and oncogenic stresses, facilitate protection from tumourigenesis and aid in tissue repair. However, accumulation of such cells in tissues contributes to age-related pathologies. Resistance of senescent cells to apoptotic stimuli may contribute to their accumulation, yet the molecular mechanisms allowing their prolonged viability are poorly characterized. Here we show that senescent cells upregulate the anti-apoptotic proteins BCL-W and BCL-XL. Joint inhibition of BCL-W and BCL-XL by siRNAs or the small-molecule ABT-737 specifically induces apoptosis in senescent cells. Notably, treatment of mice with ABT-737 efficiently eliminates senescent cells induced by DNA damage in the lungs as well as senescent cells formed in the epidermis by activation of p53 through transgenic p14(ARF). Elimination of senescent cells from the epidermis leads to an increase in hair-follicle stem cell proliferation. The finding that senescent cells can be eliminated pharmacologically paves the way to new strategies for the treatment of age-related pathologies.

p16(Ink4a)-induced senescence of pancreatic beta cells enhances insulin secretion.

Cellular senescence is thought to contribute to age-associated deterioration of tissue physiology. The senescence effector p16(Ink4a) is expressed in pancreatic beta cells during aging and limits their proliferative potential; however, its effects on beta cell function are poorly characterized. We found that beta cell-specific activation of p16(Ink4a) in transgenic mice enhances glucose-stimulated insulin secretion (GSIS). In mice with diabetes, this leads to improved glucose homeostasis, providing an unexpected functional benefit. Expression of p16(Ink4a) in beta cells induces hallmarks of senescence--including cell enlargement, and greater glucose uptake and mitochondrial activity--which promote increased insulin secretion. GSIS increases during the normal aging of mice and is driven by elevated p16(Ink4a) activity. We found that islets from human adults contain p16(Ink4a)-expressing senescent beta cells and that senescence induced by p16(Ink4a) in a human beta cell line increases insulin secretion in a manner dependent, in part, on the activity of the mechanistic target of rapamycin (mTOR) and the peroxisome proliferator-activated receptor (PPAR)-γ proteins. Our findings reveal a novel role for p16(Ink4a) and cellular senescence in promoting insulin secretion by beta cells and in regulating normal functional tissue maturation with age.

Evolving views of breast cancer stem cells and their differentiation States.

Cellular heterogeneity is a prominent characteristic of breast cancers, and accumulating evidence indicates that variability in the differentiation state of tumor cells contributes to this phenomenon. Breast cancers are among the tumor types in which the existence of cancer stem cells has been widely supported, and specific markers, including CD44/CD24 and ALDH1, have been consistently used to identify such cells. Recent studies have revealed the potential for dynamic bidirectional transitions of breast cancer cells between differentiated and stem-like phenotypes. In addition, the potential importance of intermediate states along differentiation axes, including the epithelial-to-mesenchymal transition and the basal-luminal differentiation axis, has emerged. These findings provide a view of breast cancer stem cells that departs from the traditional unidirectional hierarchical model, as well as new insights into the mechanisms by which differentiation events contribute to breast cancer biology. Herein we discuss these advances.

Transduction of fetal mice with a feline lentiviral vector induces liver tumors which exhibit an E2F activation signature.

Lentiviral vectors are widely used in basic research and clinical applications for gene transfer and long-term expression; however, safety issues have not yet been completely resolved. In this study, we characterized hepatocarcinomas that developed in mice 1 year after in utero administration of a feline-derived lentiviral vector. Mapped viral integration sites differed among tumors and did not coincide with the regions of chromosomal aberrations. Furthermore, gene expression profiling revealed that no known cancer-associated genes were deregulated in the vicinity of viral integrations. Nevertheless, five of the six tumors exhibited highly significant upregulation of E2F target genes, of which a majority are associated with oncogenesis, DNA damage response, and chromosomal instability. We further show in vivo and in vitro that E2F activation occurs early on following transduction of both fetal mice and cultured human hepatocytes. On the basis of the similarities in E2F target gene expression patterns among tumors and the lack of evidence implicating insertional mutagenesis, we propose that transduction of fetal mice with a feline lentiviral vector induces E2F-mediated major cellular processes that drive hepatocytes toward uncontrolled proliferation culminating in tumorigenesis.

Gene transfer to chicks using lentiviral vectors administered via the embryonic chorioallantoic membrane.

The lack of affordable techniques for gene transfer in birds has inhibited the advancement of molecular studies in avian species. Here we demonstrate a new approach for introducing genes into chicken somatic tissues by administration of a lentiviral vector, derived from the feline immunodeficiency virus (FIV), into the chorioallantoic membrane (CAM) of chick embryos on embryonic day 11. The FIV-derived vectors carried yellow fluorescent protein (YFP) or recombinant alpha-melanocyte-stimulating hormone (α-MSH) genes, driven by the cytomegalovirus (CMV) promoter. Transgene expression, detected in chicks 2 days after hatch by quantitative real-time PCR, was mostly observed in the liver and spleen. Lower expression levels were also detected in the brain, kidney, heart and breast muscle. Immunofluorescence and flow cytometry analyses confirmed transgene expression in chick tissues at the protein level, demonstrating a transduction efficiency of ∼0.46% of liver cells. Integration of the viral vector into the chicken genome was demonstrated using genomic repetitive (CR1)-PCR amplification. Viability and stability of the transduced cells was confirmed using terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) assay, immunostaining with anti-proliferating cell nuclear antigen (anti-PCNA), and detection of transgene expression 51 days post transduction. Our approach led to only 9% drop in hatching efficiency compared to non-injected embryos, and all of the hatched chicks expressed the transgenes. We suggest that the transduction efficiency of FIV vectors combined with the accessibility of the CAM vasculature as a delivery route comprise a new powerful and practical approach for gene delivery into somatic tissues of chickens. Most relevant is the efficient transduction of the liver, which specializes in the production and secretion of proteins, thereby providing an optimal target for prolonged study of secreted hormones and peptides.

Neonatal gene therapy of glycogen storage disease type Ia using a feline immunodeficiency virus-based vector.

Glycogen storage disease type Ia (GSD-Ia), also known as von Gierke disease, is caused by a deficiency of glucose-6-phosphatase-alpha (G6Pase), a key enzyme in glucose homeostasis. From birth, affected individuals cannot maintain normal blood glucose levels and suffer from a variety of metabolic disorders, leading to life-threatening complications. Gene therapy has been proposed as a possible option for treatment of this illness. Vectors have been constructed from feline immunodeficiency virus (FIV), a nonprimate lentivirus, because the wild-type virus does not cause disease in humans. Previously, we have shown that these vectors are capable of integrating stably into hepatocyte cell lines and adult murine livers and lead to long-term transgene expression. In the current work, we have assessed the ability to attenuate disease symptoms in a murine model of GSD-Ia. Single administration of FIV vectors containing the human G6Pase gene to G6Pase-alpha(-/-) mice did not change the biochemical and pathological phenotype. However, a double neonatal administration protocol led to normalized blood glucose levels, significantly extended survival, improved body weight, and decreased accumulation of liver glycogen associated with the disease. This approach shows a promising paradigm for treating GSD-Ia patients early in life thereby avoiding long-term consequences.

Prolonged transgene expression in murine salivary glands following non-primate lentiviral vector transduction.

Salivary glands are an accessible organ for gene therapy, enabling expression of recombinant proteins for both exocrine and endocrine secretion. Lentivirus-based vectors have many advantages for gene therapy, including their ability to infect nondividing cells and to stably integrate into the host genome, enabling long-term transgene expression without eliciting an inflammatory immune response. In the present study, murine salivary glands were inoculated with feline immunodeficiency virus (FIV)-based lentiviral vectors expressing various reporter genes. Luciferase expression was observed as early as 24 h posttransduction, peaked at 17-21 days, and remained stable for more than 80 days. Staining with X-gal suggested that mucous acinar cells were effectively transduced. FIV vector transduction with the secreted alkaline phosphatase gene increased serum levels in treated animals for up to 45 days, and the FIV vector harboring the interferon-gamma (IFN-gamma) expression cassette induced an increase in IFN-gamma serum levels as well as in the supernatant of salivary gland explant cultures. These results demonstrate that the transduction of salivary glands with nonprimate lentiviral vectors may provide a novel and highly effective vehicle for long-term endocrine transgene expression.

Prolonged liver-specific transgene expression by a non-primate lentiviral vector.

Liver-directed gene therapy has the potential for treatment of numerous inherited diseases affecting metabolic functions. The aim of this study was to evaluate gene expression in hepatocytes using feline immunodeficiency virus-based lentiviral vectors, which may be potentially safer than those based on human immunodeficiency virus. In vitro studies revealed that gene expression was stable for up to 24 days post-transduction and integration into the host cell genome was suggested by Alu PCR and Southern blot analyses. Systemic in vivo administration of viral particles by the hydrodynamics method resulted in high levels of gene expression exclusively in the liver for over 7 months whereas injection of plasmid DNA by the same method led to transient expression levels. Our studies suggest that feline immunodeficiency-based lentiviral vectors specifically transduce liver cells and may be used as a novel vehicle of gene delivery for treatment of metabolic disease.