Senescence-like Cell Research Articles

RagC GTPase regulates mTOR to promote chemoresistance in senescence-like HepG2 cells.

Radiotherapy and chemotherapy can arrest cancer cells in a senescence-like state, which can lead to therapy resistance and cancer relapse. mTOR is hyperactivated in senescent cells but the mechanisms remain unclear. In this study, we examine the roles of several mTOR-regulated GTPases in senescence-like liver cancer cells and the mechanisms in drug resistance. We show that although RagC, Rheb, Rab1A, Rab5 and Arf1 GTPases were required for optimal mTOR activation in proliferating HepG2 cells, only RagC and Rheb are required in the senescence-like counterparts. Consistently, the drug resistance of the senescence-like HepG2 can be reduced by knocking down RagC and Rheb but not the other GTPases. Autophagic and lysosomal activity were increased in senescence-like cells; pharmacological inhibition of autophagy-lysosome decreased mTOR activity and preferentially sensitized senescence-like HepG2 cells to chemotherapy drugs including trametinib, cisplatin, and doxorubicin. In liver cancer patients, expression of RagC and Rheb but not other GTPases examined was associated with unfavorable prognosis. Our study therefore has defined a key role of Rag-Rheb GTPase in mediating mTOR activation and drug resistance in senescence-like HepG2 cells, which could have important implications in developing second-line treatments for liver cancer patients.

HMGA2 drives the IGFBP1/AKT pathway to counteract the increase in P27KIP1 protein levels in mtDNA/RNA-less cancer cells.

Recent comprehensive analyses of mtDNA and orthogonal RNA-sequencing data revealed that in numerous human cancers, mtDNA copy numbers and mtRNA amounts are significantly reduced, followed by low respiratory gene expression. Under such conditions (called mt-Low), cells encounter severe cell proliferation defects; therefore, they must acquire countermeasures against this fatal disadvantage during malignant transformation. This study elucidated a countermeasure against the mt-Low condition-induced antiproliferative effects in hepatocellular carcinoma (HCC) cells. The mechanism relied on the architectural transcriptional regulator HMGA2, which was preferably expressed in HCC cells of the mt-Low type in vitro and in vivo. Detailed in vitro analyses suggest that HMGA2 regulates insulin-like growth factor binding protein 1 (IGFBP1) expression, leading to AKT activation, which then phosphorylates the cyclin-dependent kinase inhibitor (CKI), P27KIP1, and facilitates its ubiquitin-mediated degradation. Accordingly, intervention in the HMGA2 function by RNAi resulted in an increase in P27KIP1 levels and an induction of senescence-like cell proliferation inhibition in mt-Low-type HCC cells. Conclusively, the HMGA2/IGFBP1/AKT axis has emerged as a countermeasure against P27KIP1 CKI upregulation under mt-Low conditions, thereby circumventing cell proliferation inhibition and supporting the tumorigenic state. Notably, similar to in vitro cell lines, HMGA2 was likely to regulate IGFBP1 expression in HCC in vivo, thereby contributing to poor patient prognosis. Considering the significant number of cases under mt-Low or the threat of CKI upregulation cancer-wide, the axis is noteworthy as a vulnerability of cancer cells or target for tumor-agnostic therapy inducing irreversible cell proliferation inhibition via CKI upregulation in a large population with cancer.

Proteomic characterisation of triple negative breast cancer cells following CDK4/6 inhibition

When used in combination with hormone treatment, Palbociclib prolongs progression-free survival of patients with hormone receptor positive breast cancer. Mechanistically, Palbociclib inhibits CDK4/6 activity but the basis for differing sensitivity of cancer to Palbociclib is poorly understood. A common observation in a subset of Triple Negative Breast Cancers (TNBCs) is that prolonged CDK4/6 inhibition can engage a senescence-like state where cells exit the cell cycle, whilst, remaining metabolically active. To better understand the senescence-like cell state which arises after Palbociclib treatment we used mass spectrometry to quantify the proteome, phosphoproteome, and secretome of Palbociclib-treated MDA-MB-231 TNBC cells. We observed altered levels of cell cycle regulators, immune response, and key senescence markers upon Palbociclib treatment. These datasets provide a starting point for the derivation of biomarkers which could inform the future use CDK4/6 inhibitors in TNBC subtypes and guide the development of potential combination therapies.

Hydroxyurea Induces Bone Marrow Mesenchymal Stromal Cells Senescence and Modifies Cell Functionality In Vitro.

Hydroxyurea (HU) is an antineoplastic agent that functions as an antimetabolite compound by inhibiting the ribonucleotide reductase. HU acts mainly as a cytostatic drug that through DNA replication stress may trigger a premature senescence-like cell phenotype, though its influence on bone marrow-derived mesenchymal stem/stromal cell (BMMSC) functions has not elucidated yet. Our results indicate that HU inhibits the growth of human BMMSC alongside senescence-like changes in both morphology and replicative potential, provokes cell cycle arrest at the S phase without affecting cellular viability and induces the expression of senescence-associated β-galactosidase and p16INK4. Moreover, HU-induced senescent BMMSC, although they did not change MSC markers expression, exhibited reduced capacity osteogenic and adipogenic differentiation. Conversely, HU treatment increased immunoregulatory functions of BMMSC compared with untreated cells and determined by T-cell proliferation. Interestingly, HU did not influence the capacity of BMMSC to induce monocytic myeloid-derived suppressor cells. Thus, these results suggest that HU improves the BMMSC functions on the T-cell inhibition and preserves their interaction with myeloid cell compartment. Mechanistically, BMMSC under HU treatment displayed a downregulation of mTOR and p38 MAPK signaling that may explain the reduced cell differentiation and increased immunomodulation activities. Together, the results obtained in this investigation suggest that HU by inducing senescence-like phenotype of BMMSC influences their cellular differentiation and immunoregulatory functions.

PRPF19 regulates p53-dependent cellular senescence by modulating alternative splicing of MDM4 mRNA

Alteration of RNA splicing is a hallmark of cellular senescence, which is associated with age-related disease and cancer development. However, the roles of splicing factors in cellular senescence are not fully understood. In this study, we identified the splicing factor PRPF19 as a critical regulator of cellular senescence in normal human diploid fibroblasts. PRPF19 was downregulated during replicative senescence, and PRPF19 knockdown prematurely induced senescence-like cell cycle arrest through the p53–p21 pathway. RNA-sequencing analysis revealed that PRPF19 knockdown caused a switch of the MDM4 splicing isoform from stable full-length MDM4-FL to unstable MDM4-S lacking exon 6. We also found that PRPF19 regulates MDM4 splicing by promoting the physical interaction of other splicing factors, PRPF3 and PRPF8, which are key components of the core spliceosome, U4/U6.U5 tri-snRNP. Given that MDM4 is a major negative regulator of p53, our findings imply that PRPF19 downregulation inhibits MDM4-mediated p53 inactivation, resulting in induction of cellular senescence. Thus, PRPF19 plays an important role in the induction of p53-dependent cellular senescence.

Chemotherapy Induces Senescence-Like Resilient Cells Capable of Initiating AML Recurrence.

Patients with acute myeloid leukemia (AML) frequently relapse after chemotherapy, yet the mechanism by which AML reemerges is not fully understood. Herein, we show that primary AML cells enter a senescence-like phenotype following chemotherapy in vitro and in vivo. This is accompanied by induction of senescence/inflammatory and embryonic diapause transcriptional programs, with downregulation of MYC and leukemia stem cell genes. Single-cell RNA sequencing suggested depletion of leukemia stem cells in vitro and in vivo, and enrichment for subpopulations with distinct senescence-like cells. This senescence effect was transient and conferred superior colony-forming and engraftment potential. Entry into this senescence-like phenotype was dependent on ATR, and persistence of AML cells was severely impaired by ATR inhibitors. Altogether, we propose that AML relapse is facilitated by a senescence-like resilience phenotype that occurs regardless of their stem cell status. Upon recovery, these post-senescence AML cells give rise to relapsed AMLs with increased stem cell potential. SIGNIFICANCE: Despite entering complete remission after chemotherapy, relapse occurs in many patients with AML. Thus, there is an urgent need to understand the relapse mechanism in AML and the development of targeted treatments to improve outcome. Here, we identified a senescence-like resilience phenotype through which AML cells can survive and repopulate leukemia.This article is highlighted in the In This Issue feature, p. 1307.

Memory of Divisional History Directs the Continuous Process of Primitive Hematopoietic Lineage Commitment.

SummaryHematopoietic stem cells (HSCs) exist in a dormant state and progressively lose regenerative potency as they undergo successive divisions. Why this functional decline occurs and how this information is encoded is unclear. To better understand how this information is stored, we performed RNA sequencing on HSC populations differing only in their divisional history. Comparative analysis revealed that genes upregulated with divisions are enriched for lineage genes and regulated by cell-cycle-associated transcription factors, suggesting that proliferation itself drives lineage priming. Downregulated genes are, however, associated with an HSC signature and targeted by the Polycomb Repressive Complex 2 (PRC2). The PRC2 catalytic subunits Ezh1 and Ezh2 promote and suppress the HSC state, respectively, and successive divisions cause a switch from Ezh1 to Ezh2 dominance. We propose that cell divisions drive lineage priming and Ezh2 accumulation, which represses HSC signature genes to consolidate information on divisional history into memory.

DNMT3B Oncogenic Activity in Human Intestinal Cancer Is Not Linked to CIMP or BRAFV600E Mutation.

SummaryApproximately 10% of human colorectal cancer (CRC) are associated with activated BRAFV600E mutation, typically in absence of APC mutation and often associated with a CpG island methylator (CIMP) phenotype. To protect from cancer, normal intestinal epithelial cells respond to oncogenic BRAFV600E by activation of intrinsic p53 and p16-dependent tumor suppressor mechanisms, such as cellular senescence. Conversely, CIMP is thought to contribute to bypass of these tumor suppressor mechanisms, e.g. via epigenetic silencing of tumor suppressor genes, such as p16. It has been repeatedly proposed that DNMT3B is responsible for BRAFV600E-induced CIMP in human CRC. Here we set out to test this by in silico, in vitro, and in vivo approaches. We conclude that although both BRAFV600E and DNMT3B harbor oncogenic potential in vitro and in vivo and show some evidence of cooperation in tumor promotion, they do not frequently cooperate to promote CIMP and human intestinal cancer.

Hydroxyurea-induced senescent peripheral blood mesenchymal stromal cells inhibit bystander cell proliferation of JAK2V617F-positive human erythroleukemia cells.

Hydroxyurea (HU) is a nonalkylating antineoplastic agent used in the treatment of hematological malignancies. HU is a DNA replication stress inducer, and as such, it may induce a premature senescence-like cell phenotype; however, its repercussion on bystander cell proliferation has not been revealed so far. Our results indicate that HU strongly inhibited peripheral blood mesenchymal stromal cells (PBMSC) proliferation by cell cycle arrest in S phase, and that, consequently, PBMSC acquire senescence-related phenotypical changes. HU-treated PBMSC display increased senescence-associated β-galactosidase levels and p16INK4 expression, as well as DNA damage response and genotoxic effects, evidenced by expression of γH2A.X and micronuclei. Moreover, HU-induced PBMSC senescence is mediated by increased reactive oxygen species (ROS) levels, as demonstrated by the inhibition of senescence markers in the presence of ROS scavenger N-acetylcysteine and NADPH oxidase inhibitor Apocynin. To determine the HU-induced bystander effect, we used the JAK2V617F-positive human erythroleukemia 92.1.7 (HEL) cells. Co-culture with HU-induced senescent PBMSC (HU-S-PBMSC) strongly inhibited bystander HEL cell proliferation, and this effect is mediated by both ROS and transforming growth factor (TGF)-β expression. Besides induction of premature senescence, HU educates PBMSC toward an inhibitory phenotype of HEL cell proliferation. Finally, our study contributes to the understanding of the role of HU-induced PBMSC senescence as a potential adjuvant in hematological malignancy therapies.

Triphosphate Tunnel Metalloenzyme Function in Senescence Highlights a Biological Diversification of This Protein Superfamily.

The triphosphate tunnel metalloenzyme (TTM) superfamily comprises a group of enzymes that hydrolyze organophosphate substrates. They exist in all domains of life, yet the biological role of most family members is unclear. Arabidopsis (Arabidopsis thaliana) encodes three TTM genes. We have previously reported that AtTTM2 displays pyrophosphatase activity and is involved in pathogen resistance. Here, we report the biochemical activity and biological function of AtTTM1 and diversification of the biological roles between AtTTM1 and 2 Biochemical analyses revealed that AtTTM1 displays pyrophosphatase activity similar to AtTTM2, making them the only TTMs characterized so far to act on a diphosphate substrate. However, knockout mutant analysis showed that AtTTM1 is not involved in pathogen resistance but rather in leaf senescence. AtTTM1 is transcriptionally up-regulated during leaf senescence, and knockout mutants of AtTTM1 exhibit delayed dark-induced and natural senescence. The double mutant of AtTTM1 and AtTTM2 did not show synergistic effects, further indicating the diversification of their biological function. However, promoter swap analyses revealed that they functionally can complement each other, and confocal microscopy revealed that both proteins are tail-anchored proteins that localize to the mitochondrial outer membrane. Additionally, transient overexpression of either gene in Nicotiana benthamiana induced senescence-like cell death upon dark treatment. Taken together, we show that two TTMs display the same biochemical properties but distinct biological functions that are governed by their transcriptional regulation. Moreover, this work reveals a possible connection of immunity-related programmed cell death and senescence through novel mitochondrial tail-anchored proteins.

MiR-137 Modulates a Tumor Suppressor Network-Inducing Senescence in Pancreatic Cancer Cells.

Activating K-Ras mutations occurs frequently in pancreatic cancers and is implicated in their development. Cancer-initiating events, such as oncogenic Ras activation, lead to the induction of cellular senescence, a tumor suppressor response. During senescence, the decreased levels of KDM4A lysine demethylase contribute to p53 activation, however, the mechanism by which KDM4A is downregulated is unknown. We show that miR-137 targets KDM4A mRNA during Ras-induced senescence and activates both p53 and retinoblastoma (pRb) tumor suppressor pathways. Restoring the KDM4A expression contributed to bypass of miR-137-induced senescence and inhibition of endogenous miR-137 with an miRNA sponge-compromised Ras-induced senescence. miR-137 levels are significantly reduced in human pancreatic tumors, consistent with previous studies revealing a defective senescence response in this cancer type. Restoration of miR-137 expression inhibited proliferation and promoted senescence of pancreatic cancer cells. These results suggest that modulating levels of miR-137 may be important for triggering tumor suppressor networks in pancreatic cancer.

Mechanism of heat stress-induced cellular senescence elucidates the exclusive vulnerability of early S-phase cells to mild genotoxic stress.

Heat stress is one of the best-studied cellular stress factors; however, little is known about its delayed effects. Here, we demonstrate that heat stress induces p21-dependent cellular senescence-like cell cycle arrest. Notably, only early S-phase cells undergo such an arrest in response to heat stress. The encounter of DNA replication forks with topoisomerase I-generated single-stranded DNA breaks resulted in the generation of persistent double-stranded DNA breaks was found to be a primary cause of heat stress-induced cellular senescence in these cells. This investigation of heat stress-induced cellular senescence elucidates the mechanisms underlying the exclusive sensitivity of early S-phase cells to ultra-low doses of agents that induce single-stranded DNA breaks.

Ezh1 Is Required for Hematopoietic Stem Cell Maintenance and Prevents Senescence-like Cell Cycle Arrest

Polycomb group (PcG) proteins are key epigenetic regulators of hematopietic stem cell (HSC) fate. The PcG members Ezh2 and Ezh1 are important determinants of embryonic stem cell identity, and the transcript levels of these histone methyltransferases are inversely correlated during development. However, the role of Ezh1 in somatic stem cells is largely unknown. Here we show that Ezh1 maintains repopulating HSCs in a slow-cycling, undifferentiated state, protecting them from senescence. Ezh1 ablation induces significant loss of adult HSCs, with concomitant impairment of their self-renewal capacity due to a potent senescence response. Epigenomic and gene expression changes induced by Ezh1 deletion in senesced HSCs demonstrated that Ezh1-mediated PRC2 activity catalyzes monomethylation and dimethylation of H3K27. Deletion of Cdkn2a on the Ezh1 null background rescued HSC proliferation and survival. Our results suggest that Ezh1 is an important histone methyltransferase for HSC maintenance.

JMJD2A Promotes Cellular Transformation by Blocking Cellular Senescence through Transcriptional Repression of the Tumor Suppressor CHD5

Senescence is a cellular response preventing tumorigenesis. The Ras oncogene is frequently activated or mutated in human cancers, but Ras activation is insufficient to transform primary cells. In a search for cooperating oncogenes, we identify the lysine demethylase JMJD2A/KDM4A. We show that JMJD2A functions as a negative regulator of Ras-induced senescence and collaborates with oncogenic Ras to promote cellular transformation by negatively regulating the p53 pathway. We find CHD5, a known tumor suppressor regulating p53 activity, as a target of JMJD2A. The expression of JMJD2A inhibits Ras-mediated CHD5 induction leading to a reduced activity of the p53 pathway. In addition, we show that JMJD2A is overexpressed in mouse and human lung cancers. Depletion of JMJD2A in the human lung cancer cell line A549 bearing an activated K-Ras allele triggers senescence. We propose that JMJD2A is an oncogene that represents a target for Ras-expressing tumors.

Beryllium sulfate induces p21 CDKN1A expression and a senescence-like cell cycle arrest in susceptible cancer cell types

In fibroblasts, beryllium salt causes activation of the p53 transcription factor and induction of a senescence-like state. It is not known whether Be(2+) can affect the proliferation of cancer cells, which are generally unsusceptible to senescence. A172 glioblastoma and RKO colon carcinoma cell lines each have wildtype p53, so these cell types have the potential to be responsive to agents that activate p53. In A172 cells, BeSO(4) produced a G(0)/G(1)-phase cell cycle arrest and increased expression of senescence-associated β-galactosidase, an enzymatic marker of senescence. BeSO(4) caused phosphorylation of serine-15 of p53, accumulation of p53 protein, and expression of p21, the cyclin-dependent kinase inhibitor that is prominent during senescence. BeSO(4) inhibited A172 growth with an IC(50) = 4.7 μM in a 6-day proliferation assay. In contrast, BeSO(4) had no effect on RKO cells, even though Be(2+) uptake was similar for the two cell types. This differential responsiveness marks BeSO(4) as a reagent capable of activating a separable branch of the p53 signaling network. A172 and RKO cells are known to exhibit p53-dependent upregulation of p21 in response to DNA damage. The RKO cells produced high levels of p21 when exposed to DNA damaging agents, yet failed to express p21 when treated with BeSO(4). Conversely, BeSO(4) did not cause DNA damage in A172 cells, yet it was a potent inducer of p21 expression. These observations indicate that the growth control pathway affected by BeSO(4) is distinct from the DNA damage response pathway, even though both ultimately converge on p53 and p21.

IGFBP7 Is Not Required for B-RAF-Induced Melanocyte Senescence

Induction of senescence permanently restricts cellular proliferation after oncogenic stimulation thereby acting as a potent barrier to tumor development. The relevant effector proteins may therefore be fundamental to cancer development. A recent study identified IGFBP7 as a secreted factor mediating melanocyte senescence induced by oncogenic B-RAF, which is found commonly in cutaneous nevi. In contrast to the previous report, we demonstrate that B-RAF signaling does not induce IGFBP7 expression, nor the expression of the IGFBP7 targets, BNIP3L, SMARCB1, or PEA15, in human melanocytes or fibroblasts. We also found no correlation between B-RAF mutational status and IGFBP7 protein expression levels in 22 melanoma cell lines, 90 melanomas, and 46 benign nevi. Furthermore, using a lentiviral silencing strategy we show that B-RAF induces senescence in melanocytes and fibroblasts, irrespective of the presence of IGFBP7. Therefore, we conclude that the secreted protein IGFBP7 is dispensable for B-RAF(V600E)-induced senescence in human melanocytes.

Lack of Correlation between IGFBP7 Expression and BRAF Mutational Status in Melanoma

Lack of Correlation between IGFBP7 Expression and BRAF Mutational Status in Melanoma

Inhibition of c-Jun N-terminal kinase enhances temozolomide-induced cytotoxicity in human glioma cells

Previous studies have revealed that p38, a member of the family of stress-activated protein kinases (SAPKs), cooperates with the Chk1-pathway to bring about temozolomide (TMZ)-induced G2 arrest, and that the inhibition of either pathway alone is sufficient to sensitize U87MG glioma cells to TMZ-induced cytotoxicity. c-Jun N-terminal kinase (JNK), another SAPK, has been reported to have several roles of cell survival, oncogenesis, growth, differentiation and cell death. To elucidate the functions of JNK in glioma cells treated with TMZ, we analyzed alterations in JNK and the effect of modification of JNK in U87MG human glioma cells treated with TMZ. We found that JNK was phosphorylated 1-2 days after TMZ treatment and that pretreatment (for 24 h) and post-treatment (for 72 h) with a JNK inhibitor SP600125 at a concentration of 200 nM or higher remarkably reduced clonogenicity in the TMZ-treated cells. The phosphorylation of the JNK target protein c-Jun, but not of ATF-2, was inhibited by this concentration of SP600125. Therefore JNK was proved to have a role of survival in glioma cells treated with TMZ, and c-Jun-related responses were suggested to be more important in the JNK-mediated survival of glioma cells with DNA damage. SP600125 amplified the percentage of senescence-like cells and of mitotic catastrophe cells in TMZ-treated U87MG and U87MG-E6 cells, respectively, suggesting that the enhancement of TMZ-induced cytotoxicity by a JNK inhibitor in glioma cells is induced (at least in part) by the potentiation of cell death pathways induced by TMZ alone. Further investigation based on the present data may provide a viable approach for enhancing TMZ-induced cytotoxicity in human gliomas.

Oncogene-Induced Senescence Relayed by an Interleukin-Dependent Inflammatory Network

Oncogene-induced cellular senescence (OIS) is emerging as a potent cancer-protective response to oncogenic events, serving to eliminate early neoplastic cells from the proliferative pool. Using combined genetic and bioinformatic analysis, we find that OIS is linked specifically to the activation of an inflammatory transcriptome. Induced genes included the pleiotropic cytokine interleukin-6 (IL-6), which upon secretion by senescent cells acted mitogenically in a paracrine fashion. Unexpectedly, IL-6 was also required for the execution of OIS, but in a cell-autonomous mode. Its depletion caused the inflammatory network to collapse and abolished senescence entry and maintenance. Furthermore, we demonstrate that the transcription factor C/EBPbeta cooperates with IL-6 to amplify the activation of the inflammatory network, including IL-8. In human colon adenomas, IL-8 specifically colocalized with arrested, p16(INK4A)-positive epithelium. We propose a model in which the context-dependent cytostatic and promitogenic functions of specific interleukins contribute to connect senescence with an inflammatory phenotype and cancer.

Downregulation of caveolin-1 affects bleomycin-induced growth arrest and cellular senescence in A549 cells

Bleomycin is an anti-cancer drug that induces both apoptosis and senescence, two processes thought to involve caveolin-1. Here we investigate the role of caveolin-1 in bleomycin-induced senescence. We show that bleomycin-treated A549 cells exhibit: senescence-like cell morphology; a senescence-associated increase in SA-β-galactosidase activity; cell cycle arrest; and upregulation of p53 and p21. As predicted, we find that caveolin-1 amount increases in response to bleomycin-treatment and that modulation of caveolin-1 affects p21 and p53 levels, cell cycling, and senescence (SA-β-galactosidase activity). Interestingly, senescence-associated cell cycle arrest via p53 and p21 and SA-β-galactosidase activity is reduced in young A549 cells when short hairpin RNA specific for caveolin-1 was applied before bleomycin-treatment. Our results support the hypothesis that downregulation of caveolin-1 expression affects bleomycin-induced cell cycle arrest and subsequent cellular senescence that is driven by p53 and p21.