Senescence delay and repression of p16INK4a by Lsh via recruitment of histone deacetylases in human diploid fibroblasts

Lymphoid specific helicase (Lsh) belongs to the family of SNF2/helicases. Disruption of Lsh leads to developmental growth retardation and premature aging in mice. However, the specific effect of Lsh on human cellular senescence remains unknown. Herein, we report that Lsh overexpression delays cell senescence by silencing p16^INK4a in human fibroblasts. The patterns of p16^INK4a and Lsh expression during cell senescence present the inverse correlation. We also find that Lsh requires histone deacetylase (HDAC) activity to repress p16^INK4a and treatment with trichostatin A (TSA) is sufficient to block the repressor effect of Lsh. Moreover, overexpression of Lsh is correlated with deacetylation of histone H3 at the p16 promoter, and TSA treatment in Lsh-expressing cells reverses the acetylation status of histones. Additionally, we demonstrate an interaction between Lsh, histone deacetylase 1 (HDAC1) and HDAC2 in vivo. Furthermore, we demonstrate that Lsh interacts in vivo with the p16 promoter and recruits HDAC1. Our data suggest that Lsh represses endogenous p16^INK4a expression by recruiting HDAC to establish a repressive chromatin structure at the p16^INK4a promoter, which in turn delays cell senescence.     

Restored immune cell functions upon clearance of senescence in the irradiated splenic environment

Some studies show eliminating senescent cells rejuvenate aged mice and attenuate deleterious effects of chemotherapy. Nevertheless, it remains unclear whether senescence affects immune cell function. We provide evidence that exposure of mice to ionizing radiation (IR) promotes the senescent‐associated secretory phenotype (SASP) and expression of p16^INK4a in splenic cell populations. We observe splenic T cells exhibit a reduced proliferative response when cultured with allogenic cells in vitro and following viral infection in vivo. Using p16‐3MR mice that allow elimination of p16^INK4a‐positive cells with exposure to ganciclovir, we show that impaired T‐cell proliferation is partially reversed, mechanistically dependent on p16^INK4a expression and the SASP. Moreover, we found macrophages isolated from irradiated spleens to have a reduced phagocytosis activity in vitro, a defect also restored by the elimination of p16^INK4a expression. Our results provide molecular insight on how senescence‐inducing IR promotes loss of immune cell fitness, which suggest senolytic drugs may improve immune cell function in aged and patients undergoing cancer treatment.     

Geroconversion of aged muscle stem cells under regenerative pressure

Regeneration of skeletal muscle relies on a population of quiescent stem cells (satellite cells) and is impaired in very old (geriatric) individuals undergoing sarcopenia. Stem cell function is essential for organismal homeostasis, providing a renewable source of cells to repair damaged tissues. In adult organisms, age-dependent loss-of-function of tissue-specific stem cells is causally related with a decline in regenerative potential. Although environmental manipulations have shown good promise in the reversal of these conditions, recently we demonstrated that muscle stem cell aging is, in fact, a progressive process that results in persistent and irreversible changes in stem cell intrinsic properties. Global gene expression analyses uncovered an induction of p16^INK4a in satellite cells of physiologically aged geriatric and progeric mice that inhibits satellite cell-dependent muscle regeneration. Aged satellite cells lose the repression of the INK4a locus, which switches stem cell reversible quiescence into a pre-senescent state; upon regenerative or proliferative pressure, these cells undergo accelerated senescence (geroconversion), through Rb-mediated repression of E2F target genes. p16^INK4a silencing rejuvenated satellite cells, restoring regeneration in geriatric and progeric muscles. Thus, p16^INK4a/Rb-driven stem cell senescence is causally implicated in the intrinsic defective regeneration of sarcopenic muscle. Here we discuss on how cellular senescence may be a common mechanism of stem cell aging at the organism level and show that induction of p16^INK4a in young muscle stem cells through deletion of the Polycomb complex protein Bmi1 recapitulates the geriatric phenotype.     

p16INK4A Positively Regulates p21WAF1 Expression by suppressing AUF1-dependent mRNA decay

Background

p16^INK4a and p21^WAF1 are two independent cyclin-dependent kinase inhibitors encoded by the CDKN2A and CDKN1A genes, respectively. p16^INK4a and p21^WAF1 are similarly involved in various anti-cancer processes, including the regulation of the critical G1 to S phase transition of the cell cycle, senescence and apoptosis. Therefore, we sought to elucidate the molecular mechanisms underlying the link between these two important tumor suppressor proteins.

Methodology/Principal Findings

We have shown here that the p16^INK4a protein positively controls the expression of p21^WAF1 in both human and mouse cells. p16^INK4a stabilizes the CDKN1A mRNA through negative regulation of the mRNA decay-promoting AUF1 protein. Immunoprecipitation of AUF1-associated RNAs followed by quantitative RT-PCR indicated that endogenous AUF1 binds to the CDKN1A mRNA in a p16^INK4A-dependent manner. Furthermore, while AUF1 down-regulation increased the expression level of the CDKN1A mRNA, the concurrent knockdown of AUF1 and CDKN2A, using specific silencing RNAs, restored the normal expression of the gene. Moreover, we used EGFP reporter fused to the CDKN2A AU-rich element (ARE) to demonstrate that p16^INK4A regulation of the CDKN1A mRNA is AUF1- and ARE-dependent. Furthermore, ectopic expression of p16^INK4A in p16^INK4A-deficient breast epithelial MCF-10A cells significantly increased the level of p21^WAF1, with no effect on cell proliferation. In addition, we have shown direct correlation between p16^INK4a and p21^WAF1 levels in various cancer cell lines.

Conclusion/Significance

These findings show that p16^INK4a stabilizes the CDKN1A mRNA in an AUF1-dependent manner, and further confirm the presence of a direct link between the 2 important cancer-related pathways, pRB/p16^INK4A and p14^ARF/p53/p21^WAF1.     

Co-regulation of senescence-associated genes by oncogenic homeobox proteins and polycomb repressive complexes

Cellular senescence is a stable cell cycle arrest that can be induced by stresses such as telomere shortening, oncogene activation or DNA damage. Senescence is a potent anticancer barrier that needs to be circumvented during tumorigenesis. The cell cycle regulator p16^INK4a is a key effector upregulated during senescence. Polycomb repressive complexes (PRCs) play a crucial role in silencing the INK4/ARF locus, which encodes for p16^INK4a, but the mechanisms by which PRCs are recruited to this locus as well as to other targets remain poorly understood. Recently we discovered the ability of the homeobox proteins HLX1 (H2.0-like homeobox 1) and HOXA9 (Homeobox A9) to bypass senescence. We showed that HLX1 and HOXA9 recruit PRCs to repress INK4a, which constitutes a key mechanism explaining their effects on senescence. Here we provide evidence for the regulation of additional senescence-associated PRC target genes by HLX1 and HOXA9. As both HLX1 and HOXA9 are oncogenes implicated in leukemogenesis, we discuss the implications that the collaboration between Homeobox proteins and PRCs has for senescence and cancer.     

Telomerase induces immortalization of human esophageal keratinocytes without p16INK4a inactivation

Normal human somatic cells have a finite life span and undergo replicative senescence after a limited number of cell divisions. Erosion of telomeric DNA has emerged as a key factor in senescence, which is antagonized during cell immortalization and transformation. To clarify the involvement of telomerase in the immortalization of keratinocytes, catalytic subunit of telomerase (hTERT) expression was restored in normal human esophageal epithelial cells (EPC2). EPC2-hTERT cells overcame senescence and were immortalized without p16INK4a genetic or epigenetic alterations. p16INK4a was expressed at moderate levels and remained functional as evidenced by induction with UV treatment and binding to cyclin-dependent kinase 4 and 6. There were no mutations in the p53 gene, and p53 was functionally intact. Importantly, senescence could be activated in the immortalized EPC2-hTERT cells by overexpression of oncogenic H-ras or p16INK4a. Furthermore, the EPC2-hTERT cells yielded basal cell hyperplasia in an innovative organotypic culture system in contrast to a normal epithelium from parental cells. These comprehensive results indicate that the expression of telomerase induces immortalization of normal human esophageal keratinocytes without inactivation of p16INK4a/pRb pathway or abrogation of the p53 pathway.     

p16INK4a and its regulator miR-24 link senescence and chondrocyte terminal differentiation-associated matrix remodeling in osteoarthritis

Introduction

Recent evidence suggests that tissue accumulation of senescent p16^INK4a-positive cells during the life span would be deleterious for tissue functions and could be the consequence of inherent age-associated disorders. Osteoarthritis (OA) is characterized by the accumulation of chondrocytes expressing p16^INK4a and markers of the senescence-associated secretory phenotype (SASP), including the matrix remodeling metalloproteases MMP1/MMP13 and pro-inflammatory cytokines interleukin-8 (IL-8) and IL-6. Here, we evaluated the role of p16^INK4a in the OA-induced SASP and its regulation by microRNAs (miRs).

Methods

We used IL-1-beta-treated primary OA chondrocytes cultured in three-dimensional setting or mesenchymal stem cells differentiated into chondrocyte to follow p16^INK4a expression. By transient transfection experiments and the use of knockout mice, we validate p16^INK4a function in chondrocytes and its regulation by one miR identified by means of a genome-wide miR-array analysis.

Results

p16^INK4a is induced upon IL-1-beta treatment and also during in vitro chondrogenesis. In the mouse model, Ink4a locus favors in vivo the proportion of terminally differentiated chondrocytes. When overexpressed in chondrocytes, p16^INK4a is sufficient to induce the production of the two matrix remodeling enzymes, MMP1 and MMP13, thus linking senescence with OA pathogenesis and bone development. We identified miR-24 as a negative regulator of p16^INK4a. Accordingly, p16^INK4a expression increased while miR-24 level was repressed upon IL-1-beta addition, in OA cartilage and during in vitro terminal chondrogenesis.

Conclusions

We disclosed herein a new role of the senescence marker p16^INK4a and its regulation by miR-24 during OA and terminal chondrogenesis.     

Human keratinocytes that express hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics

Normal human cells exhibit a limited replicative life span in culture, eventually arresting growth by a process termed senescence. Progressive telomere shortening appears to trigger senescence in normal human fibroblasts and retinal pigment epithelial cells, as ectopic expression of the telomerase catalytic subunit, hTERT, immortalizes these cell types directly. Telomerase expression alone is insufficient to enable certain other cell types to evade senescence, however. Such cells, including keratinocytes and mammary epithelial cells, appear to require loss of the pRB/p16(INK4a) cell cycle control mechanism in addition to hTERT expression to achieve immortality. To investigate the relationships among telomerase activity, cell cycle control, senescence, and differentiation, we expressed hTERT in two epithelial cell types, keratinocytes and mesothelial cells, and determined the effect on proliferation potential and on the function of cell-type-specific growth control and differentiation systems. Ectopic hTERT expression immortalized normal mesothelial cells and a premalignant, p16(INK4a)-negative keratinocyte line. In contrast, when four keratinocyte strains cultured from normal tissue were transduced to express hTERT, they were incompletely rescued from senescence. After reaching the population doubling limit of their parent cell strains, hTERT(+) keratinocytes entered a slow growth phase of indefinite length, from which rare, rapidly dividing immortal cells emerged. These immortal cell lines frequently had sustained deletions of the CDK2NA/INK4A locus or otherwise were deficient in p16(INK4a) expression. They nevertheless typically retained other keratinocyte growth controls and differentiated normally in culture and in xenografts. Thus, keratinocyte replicative potential is limited by a p16(INK4a)-dependent mechanism, the activation of which can occur independent of telomere length. Abrogation of this mechanism together with telomerase expression immortalizes keratinocytes without affecting other major growth control or differentiation systems.     

Compromising the constitutive p16INK4a expression sensitizes human neuroblastoma cells to Hsp90 inhibition and promotes premature senescence

The Hsp90 chaperone has become the attractive pharmacological target to inhibit tumor cell proliferation. However, tumor cells can evolve with mechanisms to overcome Hsp90 inhibition. Using human neuroblastoma, we have investigated one such limitation. Here, we demonstrate that neuroblastoma cells overcome the interference of tumor suppressor p16^INK4a in cell proliferation, which is due to its latent interaction with CDK4 and CDK6. Cells also displayed impedance to the pharmacological inhibition of cancer chaperone Hsp90 inhibition with respect to induced cytotoxicity. However, the p16^INK4a knockdown has triggered the activation of cyclin-CDK6 axis and enhanced the cell proliferation. These cells are eventually sensitized to Hsp90 inhibition by activating the DNA damage response mediated through p53-p21^WAF-1 axis and G1 cell cycle exit. While both CDK4 and CDK6 have exhibited low affinity to p16^INK4a, CDK6 has exhibited high affinity to Hsp90. Destabilizing the CDK6 interaction with Hsp90 has prolonged G2/M cell cycle arrest fostering to premature cellular senescence. The senescence driven cells exhibited compromised metastatic potential both in vitro as well as in mice xenografts. Our study unravels that cancer cells can be adapted to the constitutive expression of tumor suppressors to overcome therapeutic interventions. Our findings display potential implication of Hsp90 inhibitors to overcome such adaptations.