Senolytic elimination of Cox2-expressing senescent cells inhibits the growth of premalignant pancreatic lesions.

OBJECTIVE: Cellular senescence limits tumourigenesis by blocking the proliferation of premalignant cells. Additionally, however, senescent cells can exert paracrine effects influencing tumour growth. Senescent cells are present in premalignant pancreatic intraepithelial neoplasia (PanIN) lesions, yet their effects on the disease are poorly characterised. It is currently unknown whether senolytic drugs, aimed at eliminating senescent cells from lesions, could be beneficial in blocking tumour development. DESIGN: To uncover the functions of senescent cells and their potential contribution to early pancreatic tumourigenesis, we isolated and characterised senescent cells from PanINs formed in a Kras-driven mouse model, and tested the consequences of their targeted elimination through senolytic treatment. RESULTS: We found that senescent PanIN cells exert a tumour-promoting effect through expression of a proinflammatory signature that includes high Cox2 levels. Senolytic treatment with the Bcl2-family inhibitor ABT-737 eliminated Cox2-expressing senescent cells, and an intermittent short-duration treatment course dramatically reduced PanIN development and progression to pancreatic ductal adenocarcinoma. CONCLUSIONS: These findings reveal that senescent PanIN cells support tumour growth and progression, and provide a first indication that elimination of senescent cells may be effective as preventive therapy for the progression of precancerous lesions.

Stimulation of epidermal hyperplasia and tumorigenesis by resident p16INK4a-expressing cells.

p16INK4a (CDKN2A) is a central tumor-suppressor and activator of senescence. We recently found that prolonged expression of p16INK4a in epidermal cells induces hyperplasia and dysplasia through Wnt-mediated stimulation of neighboring keratinocytes. The study suggests a pro-tumorigenic function of p16INK4a in early epidermal lesions, which could potentially be targeted by senolytic therapy.

Chronic expression of p16INK4a in the epidermis induces Wnt-mediated hyperplasia and promotes tumor initiation.

p16INK4a (CDKN2A) is a central tumor suppressor, which induces cell-cycle arrest and senescence. Cells expressing p16INK4a accumulate in aging tissues and appear in premalignant lesions, yet their physiologic effects are poorly understood. We found that prolonged expression of transgenic p16INK4a in the mouse epidermis induces hyperplasia and dysplasia, involving high proliferation rates of keratinocytes not expressing the transgene. Continuous p16INK4a expression increases the number of epidermal papillomas formed after carcinogen treatment. Wnt-pathway ligands and targets are activated upon prolonged p16INK4a expression, and Wnt inhibition suppresses p16INK4a-induced hyperplasia. Senolytic treatment reduces p16INK4a-expressing cell numbers, and inhibits Wnt activation and hyperplasia. In human actinic keratosis, a precursor of squamous cell carcinoma, p16INK4a-expressing cells are found adjacent to dividing cells, consistent with paracrine interaction. These findings reveal that chronic p16INK4a expression is sufficient to induce hyperplasia through Wnt-mediated paracrine stimulation, and suggest that this tumor suppressor can promote early premalignant epidermal lesion formation.

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.

Dynamics of senescent cell formation and retention revealed by p14ARF induction in the epidermis.

Cellular senescence, a state of cell-cycle arrest accompanied by dramatic morphologic and metabolic changes, is a central means by which cells respond to physiologic stress and oncogene activity. Senescence is thought to play important roles in aging and in tumor suppression, yet the dynamics by which senescent cells are formed, their effects on tissue function and their eventual fate are poorly understood. To study cellular senescence within an adult tissue, we developed transgenic mice inducibly expressing p14(ARF) (human ortholog of murine p19(ARF)), a central activator of senescence. Induction of p14(ARF) in the epidermis rapidly led to widespread apoptosis and cell-cycle arrest, a stage that was transient, and was followed by p53-dependent cellular senescence. The endogenous Cdkn2a products p19(ARF) and p16(Ink4a) were activated by the transgenic p14(ARF) through p53, revealing a senescence-promoting feed-forward loop. Commitment of cells to senescence required continued p14(ARF) expression, indicating that entry into this state depends on a persistent signal. However, once formed, senescent cells were retained in the epidermis, often for weeks after transgene silencing, indicating an absence of an efficient rapidly acting mechanism for their removal. Stem cells in the hair follicle bulge were largely protected from apoptosis upon p14(ARF) induction, but irreversibly lost their ability to proliferate and initiate follicle growth. Interestingly, induction of epidermal hyperplasia prevented the appearance of senescent cells upon p14(ARF) induction. Our findings provide basic insights into the dynamics of cellular senescence, a central tumor- suppressive mechanism, and reveal the potential for prolonged retention of senescent cells within tissues.

Suppression of adipose lipolysis by long-chain fatty acid analogs.

Agonist-induced lipolysis of adipose fat is robustly inhibited by insulin or by feedback inhibition by the long-chain fatty acids (LCFA) produced during lipolysis. However, the mode of action of LCFA in suppressing adipose lipolysis is not clear. ß,ß'-Tetramethyl hexadecanedioic acid (Mßß/ EDICA16) is a synthetic LCFA that is neither esterified into lipids nor ß-oxidized, and therefore, it was exploited for suppressing agonist-induced lipolysis in analogy to natural LCFA. Mßß is shown here to suppress isoproterenol-induced lipolysis in the rat in vivo as well as in 3T3-L1 adipocytes. Inhibition of isoproterenol-induced lipolysis is due to decrease in isoproterenol-induced cAMP with concomitant inhibition of the phosphorylation of hormone-sensitive lipase and perilipin by protein kinase A. Suppression of cellular cAMP levels is accounted for by inhibition of the adenylate cyclase due to suppression of Raf1 expression by Mßß-activated AMPK. Suppression of Raf1 is further complemented by induction of components of the unfolded-protein-response by Mßß. Our findings imply genuine inhibition of agonist-induced adipose lipolysis by LCFA, independent of their ß-oxidation or reesterification. Mßß suppression of agonist-induced lipolysis and cellular cAMP levels independent of the insulin transduction pathway may indicate that synthetic LCFA could serve as insulin mimetics in the lipolysis context under conditions of insulin resistance.

Gating of the mitochondrial permeability transition pore by thyroid hormone.

The calorigenic-thermogenic activity of thyroid hormone (T3) has long been ascribed to uncoupling of mitochondrial oxidative phosphorylation. However, the mode of action of T3 in promoting mitochondrial proton leak is still unresolved. Mitochondrial uncoupling by T3 is reported here to be transduced in vivo in rats and in cultured Jurkat cells by gating of the mitochondrial permeability transition pore (PTP). T3-induced PTP gating is shown here to be abrogated in inositol 1,4,5-trisphosphate (IP(3)) receptor 1 (IP(3)R1)(-/-) cells, indicating that the endoplasmic reticulum IP(3)R1 may serve as upstream target for the mitochondrial activity of T3. IP(3)R1 gating by T3 is due to its increased expression and truncation into channel-only peptides, resulting in IP(3)-independent Ca(2+) efflux. Increased cytosolic Ca(2+) results in activation of protein phosphatase 2B, dephosphorylation and depletion of mitochondrial Bcl2 (S70), and increase in mitochondrial free Bax leading to low-conductance PTP gating. The T3 transduction pathway integrates genomic and nongenomic activities of T3 in regulating mitochondrial energetics and may offer novel targets for thyromimetics designed to modulate energy expenditure.