Chemotherapy-induced Senescence Research Articles

Sprouty1 is a broad mediator of cellular senescence

Genes of the Sprouty family (Spry1-4) restrain signaling by certain receptor tyrosine kinases. Consequently, these genes participate in several developmental processes and function as tumor suppressors in adult life. Despite these important roles, the biology of this family of genes still remains obscure. Here we show that Sprouty proteins are general mediators of cellular senescence. Induction of cellular senescence by several triggers in vitro correlates with upregulation of Sprouty protein levels. More importantly, overexpression of Sprouty genes is sufficient to cause premature cellular senescence, via a conserved N-terminal tyrosine (Tyrosine 53 of Sprouty1). Accordingly, fibroblasts from knockin animals lacking that tyrosine escape replicative senescence. In vivo, heterozygous knockin mice display delayed induction of cellular senescence during cutaneous wound healing and upon chemotherapy-induced cellular senescence. Unlike other functions of this family of genes, induction of cellular senescence appears to be independent of activation of the ERK1/2 pathway. Instead, we show that Sprouty proteins induce cellular senescence upstream of the p38 pathway in these in vitro and in vivo paradigms.

Chemotherapy-induced adipocyte senescence triggers bone loss through RANKL-mediated osteoclastogenesis

Chemotherapy-induced adipocyte senescence triggers bone loss through RANKL-mediated osteoclastogenesis

Abstract 2956: Chemotherapy-induced adipocyte senescence triggers bone loss through osteoclast activation

Abstract Despite breakthroughs in cancer treatment, chemotherapy-induced osteo-toxicity is a major problem that compromises the quality of life and overall survival of cancer patients regardless of cancer type. Recently we demonstrated that chemotherapy-induced senescence drives bone loss by both limiting mineralization of new bone and increasing bone resorption. However, the underlying mechanism of action remains elusive. Therefore, it is critical that we understand the mechanisms that drive these toxicities and develop approaches to mitigate their severity. To establish whether senescent bone resident cells or systemic responses to chemotherapy drove therapy-induced bone loss, we used a vertebral body transplant (vossicle) model. Using this approach, we found that the specific elimination of senescent cells in L4 and L5 donor vossicles (INK-ATTAC) implanted into wildtype mice, protects from chemotherapy-induced bone loss within the vossicles but not the femur of the recipient mice. This demonstrated that chemotherapy-induced senescence in resident bone cells is responsible for bone loss. To determine which bone resident cell(s) underwent senescence in response to chemotherapy and how their gene expression was impacted, we used the p16-CreERT2-tdTomato mouse model and found that chemotherapy triggers senescence in bone marrow adipocytes. Subsequently, we observed that postnatal fat ablation in adipoqCre-inducible DTR transgenic mice (iDTRADQ) prevented chemotherapy-induced bone loss, indicating senescent adipocytes trigger bone loss. Furthermore, we observed chemotherapy-induced bone loss is attributed to RANKL-mediated high osteoclasts activity. Collectively, our data demonstrate that chemotherapy causes senescence in marrow adipocytes which in turn triggers bone loss via RANKL-mediated osteoclasts activation and bone loss can be protected by eliminating senescent cells. Citation Format: Ganesh Kumar Raut, Taylor Malachowski, Taylor Holt, Renata Oliveira, Xianmin Luo, Douglas Faget, Qihao Ren, Sheila Stewart. Chemotherapy-induced adipocyte senescence triggers bone loss through osteoclast activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2956.

Temporal regulation of gene expression and pathways in chemotherapy-induced senescence in HeLa cervical cancer cell line

Cellular senescence is the state of permanent growth arrest. Chemotherapeutic drugs induce senescence, known as therapy-induced senescence. Although there are studies deciphering processes in senescence, more studies providing detailed information on therapy-induced senescence at the transcriptome level are needed. In order to understand temporal molecular changes of doxorubicin treatment in the course of senescence formation, two data sets from HeLa cells at 16 h and 72 h doxorubicin treatment were analyzed. GO BP enrichment, KEGG pathways and hub genes specific to or shared between 16 h and 72 h doxorubicin treated HeLa cells were identified. Genes functioning in p53 signaling were upregulated only in 16 h, while genes functioning in extracellular matrix organization were upregulated only in 72 h doxorubicin treated HeLa cells. Wound healing genes were gradually upregulated from 16 h to 72 h doxorubicin treatment and metabolic pathways were downregulated at both. ncRNA processing and ribosome biogenesis GO BP terms were enriched in upregulated genes at 16 h, while these terms were enriched in downregulated genes at 72 h senescent HeLa cells. According to our results, genes functioning in p53 signaling may be involved in the induction of senescence, but may not be required to maintain senescence in HeLa cells.

Chemotherapy-Induced Cellular Senescence Promotes Stemness of B-Cell Non-Hodgkin's Lymphoma Via CCL21/CCR7/NF-Κb Signaling Activation

A bs tract: Resistance to existing therapies is currently the main reason for treatment failure in refractory/relapsed B-NHL patients, and treatment-induced cellular senescence (TIS) is one of the important mechanisms of tumor drug resistance. In our previous studies, we found that the anti-apoptotic and tumorigenic ability of chemotherapy-induced senescent B-NHL cells was significantly enhanced, and the survival period of tumor-bearing mice was significantly shortened, but the mechanism is still unclear. In this study, we identified the C-C chemokine receptor 7 (CCR7) by single-cell RNA-sequencing, which showed significant differential expression in senescent B-NHL cell population and non-senescent cell population. CCL21/CCR7 dependent B-NHL-TIS initiates downstream transcription of NF-κB, thereby enhancing phenotypic and functional stemness features, including the up-regulation of stemness markers, CD34, CD44, LGR5, CD150, and stronger colony forming ability and cell sphere forming ability. The analysis results of clinical patients showed that the expression of CCR7 in the peripheral blood of B-NHL patients was significantly higher than that of healthy normal people and leukemia patients. The expression of CCR7 in peripheral blood of B-NHL patients before treatment was significantly lower than that of relapsed patients, and The expression in abnormal B lymphocyte population is significantly higher than that in normal T and B lymphocyte population, high expression of CCR7 is associated with recurrence and poor prognosis in B-NHL patients. Targeting CCR7/NF-κB signaling has positive significance in inhibiting the acquisition and maintenance of stemness in aging B-NHL cells and preventing tumor drug resistance and recurrence. K ey words: Stress-induced senescence; CCR7; CCL21; senescence related stemness; B-cell non-Hodgkin's lymphoma

Co-targeting BCL-XL and BCL-2 by PROTAC 753B eliminates leukemia cells and enhances efficacy of chemotherapy by targeting senescent cells.

BCL-XL and BCL-2 are key anti-apoptotic proteins and validated cancer targets. 753B is a novel BCL-XL/BCL-2 proteolysis targeting chimera (PROTAC) that targets both BCL-XL and BCL-2 to the von Hippel-Lindau (VHL) E3 ligase, leading to BCLX L/BCL-2 ubiquitination and degradation selectively in cells expressing VHL. Because platelets lack VHL expression, 753B spares on-target platelet toxicity caused by the first-generation dual BCL-XL/BCL-2 inhibitor navitoclax (ABT-263). Here, we report pre-clinical single-agent activity of 753B against different leukemia subsets. 753B effectively reduced cell viability and induced dose-dependent degradation of BCL-XL and BCL-2 in a subset of hematopoietic cell lines, acute myeloid leukemia (AML) primary samples, and in vivo patient-derived xenograft AML models. We further demonstrated the senolytic activity of 753B, which enhanced the efficacy of chemotherapy by targeting chemotherapy-induced cellular senescence. These results provide a pre-clinical rationale for the utility of 753B in AML therapy, and suggest that 753B could produce an added therapeutic benefit by overcoming cellular senescence-induced chemoresistance when combined with chemotherapy.

Chemotherapy-Induced Senescence Reprogramming Promotes Nasopharyngeal Carcinoma Metastasis by circRNA-Mediated PKR Activation.

Senescence is associated with tumor metastasis and chemotherapy resistance, yet the mechanisms remain elusive. Here, it is identified that nasopharyngeal carcinoma (NPC) patients who developed distant metastasis are characterized by senescence phenotypes, in which circWDR37 is a key regulator. CircWDR37 deficiency limits cisplatin or gemcitabine-induced senescent NPC cells from proliferation, migration, and invasion. Mechanistically, circWDR37 binds to and dimerizes double-stranded RNA-activated protein kinase R (PKR) to initiate PKR autophosphorylation and activation. Independent of its kinase activity, phosphorylated PKR induces I-kappaB kinase beta (IKKβ) phosphorylation, binds to and releases RELA from NF-κB inhibitor alpha (IκBα) to trigger nuclear factor kappa B (NF-κB) activation, thereby stimulating cyclin D1 (CCND1)and senescence-associated secretory phenotype component gene transcription in a circWDR37-dependent manner. Low circWDR37 levels correlate with chemotherapy response and favorable survival in NPC patients treated with gemcitabine or cisplatin induction chemotherapy. This study uncovers a new mechanism of circWDR37 activated PKR in senescence-driven metastasis and provides appealing therapeutic targets in NPC.

EGR1 Haploinsufficiency Confers a Fitness Advantage to Hematopoietic Stem Cells Following Chemotherapy

Therapy-related myeloid neoplasms (t-MNs) share many clinical and molecular characteristics with AML de novo in the elderly. One common factor is that they arise in the setting of chronic inflammation, likely because of advanced age or chemotherapy-induced senescence. Here, we examined the effect of haploinsufficient loss of the del(5q) tumor suppressor gene, EGR1, commonly deleted in high-risk MNs. In mice, under the exogenous stress of either serial transplant or successive doses of the alkylating agent N-ethyl-nitrosourea (ENU), Egr1-haploinsufficient hematopoietic stem cells (HSCs) exhibit a clonal advantage. Complete loss of EGR1 function is incompatible with transformation; mutations of EGR1 are rare and are not observed in the remaining allele in del(5q) patients, and complete knockout of Egr1 in mice leads to HSC exhaustion. Using chromatin immunoprecipitation sequencing (ChIP-seq), we identified EGR1 binding sites in human CD34+ cord blood-derived stem and progenitor cells (HSPCs) and found that EGR1 binds genes critical for stem cell differentiation, inflammatory signaling, and the DNA damage response. Notably, in the chromosome 5 sequences frequently deleted in patients, there is a significant enrichment of innate and inflammatory genes, which may confer a fitness advantage in an inflammatory environment. Short hairpin RNA (shRNA)-mediated silencing of EGR1 biases HSPCs toward a self-renewal transcriptional signature. In the absence of EGR1, HSPCs are characterized by upregulated MYC-driven proliferative signals, downregulated CDKN1A (p21), disrupted DNA damage response, and downregulated inflammation-adaptations anticipated to confer a relative fitness advantage for stem cells especially in an environment of chronic inflammation.

A Novel ALDH1A1 Inhibitor Blocks Platinum-Induced Senescence and Stemness in Ovarian Cancer.

Simple SummaryOvarian cancer is the deadliest amongst the gynecologic malignancies. Most ovarian cancer patients initially respond to chemotherapy but will eventually relapse and become chemoresistant. A specialized subpopulation of cells within the tumor known as cancer stem cells are known to contribute to recurrence and chemoresistance. Ovarian cancer stem cells have a high expression of ALDH1A1, and patients with a high level of ALDH1A1 in their tumor have worse survival. Thus, specifically targeting ALDH1A1 could be an effective strategy to inhibit cancer stemness and disease relapse. We describe the discovery of a novel ALDH1A1 inhibitor called 974 and show that targeting ALDH1A1 with 974 decreases the population of ovarian cancer stem cells. Furthermore, inhibiting ALDH1A1 suppresses chemotherapy-induced senescence and stemness. Collectively, our data demonstrate that targeting ALDH1A1 in cancer stem cells could be an effective strategy to overcome chemotherapy resistance in ovarian cancer.Ovarian cancer is a deadly disease attributed to late-stage detection as well as recurrence and the development of chemoresistance. Ovarian cancer stem cells (OCSCs) are hypothesized to be largely responsible for the emergence of chemoresistant tumors. Although chemotherapy may initially succeed at decreasing the size and number of tumors, it leaves behind residual malignant OCSCs. In this study, we demonstrate that aldehyde dehydrogenase 1A1 (ALDH1A1) is essential for the survival of OCSCs. We identified a first-in-class ALDH1A1 inhibitor, compound 974, and used 974 as a tool to decipher the mechanism of stemness regulation by ALDH1A1. The treatment of OCSCs with 974 significantly inhibited ALDH activity, the expression of stemness genes, and spheroid and colony formation. An in vivo limiting dilution assay demonstrated that 974 significantly inhibited CSC frequency. A transcriptomic sequencing of cells treated with 974 revealed a significant downregulation of genes related to stemness and chemoresistance as well as senescence and the senescence-associated secretory phenotype (SASP). We confirmed that 974 inhibited the senescence and stemness induced by platinum-based chemotherapy in functional assays. Overall, these data establish that ALDH1A1 is essential for OCSC survival and that ALDH1A1 inhibition suppresses chemotherapy-induced senescence and stemness. Targeting ALDH1A1 using small-molecule inhibitors in combination with chemotherapy therefore presents a promising strategy to prevent ovarian cancer recurrence and has the potential for clinical translation.

Abstract 5297: ONC201 suppresses cancer cell growth in a reconstructed tumor microenvironment that includes chemotherapy-induced senescent fibroblasts

Abstract Cellular senescence and its associated secretory phenotype (SASP) can promote cancer progression in the tumor microenvironment (TME). The TME includes tumor cells, stromal cells, immune cells, endothelial cells, and extracellular matrix. Senescent cancer-associated fibroblasts (CAF) may contribute to tumor growth and therapy resistance. Targeting senescent CAF by means of removal, modulation of the SASP, or through cellular reprogramming might provide therapeutic avenues for treating cancer. We investigated the impact of chemotherapy-induced fibroblast senescence in the TME on tumor growth and response to cancer therapy. Expression of cytokines in chemo-induced senescent fibroblasts and cancer cells was assessed by bulk and single cell cytokine profiling. As expected, there were alterations in SASP factors with increased pro-tumorigenic immune factors and decreased anti-tumor cytokines during IMR90 etoposide-induced fibroblast senescence. We co-cultured luciferase-labeled HT29 cancer cells with senescent IMR90 and found that the senescent fibroblasts promoted HT29 cell growth in culture and accelerated xenograft tumor formation in mice. We next inhibited cellular senescence and its SASP with the senolytic drug ABT263 or the senostatic/senomorphic drug lamivudine (3TC). Both ABT263 and 3TC significantly reduced bioluminescence of HT29-Luc cells co-cultured with senescent IMR90 compared to non-treated IMR90 cells. Therapy-induced senescence confers 5-Fluorouracil (5-FU) resistance in colorectal cancer. We found that 5-FU treatment significantly reduced colony formation of HT29 cells in the presence of proliferating or senescent IMR90 cells, with a lesser reduction in the presence of the senescent IMR90 cells. This suggests that a microenvironment that includes senescent cells promotes tumor cell resistance to 5-FU. We hypothesize that SASP factors might confer cancer cell resistance to 5-FU treatment. Cytokine profiling showed that TRAIL expression is reduced in senescent cells. Treatment with the TRAIL-inducer ONC201 reduced colony formation and cell viability of HT29 cells co-cultured with senescent IMR90 fibroblasts. Single-cell cytokine profiling showed subpopulations of cancer cells with increased polyfunctionality strength index (PSI, secretion of more than 2 dominant types of cytokines per cell in a population). Combined treatment with ABT263 and ONC201 synergically reduced viability HT29 cells co-cultured with senescent IMR90, and this correlated with reduced PSI. Our results indicate that TME targeting by increasing antitumor cytokines in conjunction with senolytic therapies can inhibit tumor growth. We are continuing to unravel the cytokine landscape of chemotherapy-induced cell senescence to gain insights into therapeutic strategies targeting chemotherapy-induced TME-senescence and drug resistance. Citation Format: Shengliang Zhang, Kelsey E. Huntington, Bianca Kun, Lanlan Zhou, Jill Kreiling, John M. Sedivy, Wafik S. El-Deiry. ONC201 suppresses cancer cell growth in a reconstructed tumor microenvironment that includes chemotherapy-induced senescent fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5297.

Abstract 897: Targeting ALDH1A1 and stemness regulatory networks in ovarian cancer

Abstract High grade serous ovarian cancer (HGSOC) is the leading cause of death from gynecologic malignancies. Ovarian cancer stem cells (OCSCs) are hypothesized to contribute to the emergence of chemoresistant tumors. Aldehyde dehydrogenase 1A1 (ALDH1A1) is an intracellular enzyme that oxidizes toxic aldehydes to carboxylic acids and plays a role in controlling cell differentiation pathways. We have previously used ALDH1A1 activity as a functional marker to identify OCSCs and demonstrated that OCSCs persist after chemotherapy and drive recurrent, drug resistant disease. However, the mechanism by which ALDH1A1 maintains stemness phenotype remains poorly understood. To examine the effect of ALDH1A1 on cellular survival signals in OCSCs, we generated a novel ALDH1A1-specific small molecule inhibitor named compound 974. Treatment of HGSOC cell lines with compound 974 reduced ALDH enzyme activity (p<0.01) and inhibited stem-like properties including spheroid formation (p<0.01) and clonogenic survival (p<0.05). Compound 974 treatment inhibited (p<0.01) stemness genes Prom1, Bmi1, Oct4 and Nanog. ALDH1A1 inhibition also reduced (p<0.05) cisplatin IC50 and synergized (p<0.05) with cisplatin treatment. To further examine the effect of compound 974 to inhibit ALDH1A1 and consequently tumor initiation, mice were injected with 106, 105 and 104 OVCAR3 cells treated in vitro with compound 974 (5µM for 48h). In a parallel study, mice were injected with 106, 105 and 104 of ALDH1A1 knockdown or scrambled control cells. In both cases, tumor initiation was significantly delayed compared to the control, and extreme limiting dilution analysis further revealed that compound 974 reduced (p<0.05) CSC frequency compared to control. To better understand the mechanism by which ALDH1A1 maintains OC stemness, OVCAR3 cells treated with vehicle or compound 974 were analyzed by RNA-sequencing and bioinformatics. Transcriptomic analysis revealed that compound 974 significantly (FDR < 0.05, fold change > 2) downregulated expression of markers strongly associated with CSC phenotypes (NF-κB, CD44, Klf4, Sox9, Fzd7) and chemoresistance (ABCB1). Ingenuity Pathway Analysis for downstream regulators of differentially expressed genes revealed that the senescence pathway was significantly altered. Analysis by qRT-PCR revealed that compound 974 inhibited (p<0.01) chemotherapy-induced expression of senescence marker p21 and senescence associated secretory phenotype (SASP) genes including IL6, IL8, CXCL1 and CXCL3. Compound 974 also inhibited(p<0.05) the chemotherapy-induced beta-galactosidase activity. Collectively, our findings further support the functional role for ALDH1A1 in OCSC and reveal a novel role for the enzyme in chemotherapy-induced senescence and disease relapse. Citation Format: Vaishnavi Muralikrishnan, Fang Fang, Tyler C. Given, Ram Podicheti, Douglas B. Rusch, Thomas D. Hurley, Kenneth P. Nephew. Targeting ALDH1A1 and stemness regulatory networks in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 897.

Abstract 1297: Chemotherapy-induced senescence activates robust, parallel programs of immune checkpoint expression that can be targeted with immunotherapy

Abstract We and others have previously shown that the breast cancers most difficult to eradicate with chemotherapy are TP53 wild-type, and these are among the most lethal breast cancers. For instance, chemotherapy-treated TNBC patients with TP53 wild-type tumors have a median overall survival of 45 months, vs 263 months for TP53 mutant tumors. Treatments fail to eradicate these cancers for two reasons: the tumor cells 1) avoid intrinsic cell death, and 2) escape immune clearance. We have shown that p53 drives a program of senescence that arrests the cell cycle to prevent intrinsic modes of cell death such as mitotic catastrophe, apoptosis and nutrient deprivation. However, it is not currently understood how breast cancer cells that enter senescence to survive chemotherapy escape immune clearance. Here, we show tumor cells from mice and human patients that survive chemotherapy activate complex programs of immune modulation. Surprisingly, the surviving, senescent tumor cells are highly enriched for many antigen presentation genes and positive regulators of T cell activation, suggesting they have switched from immunologically “cold” to “hot”. Unfortunately, these senescent tumors concurrently upregulate redundant expression of many T cell inhibitory checkpoint genes, including CD80 and PD-L1. scRNA-seq and cell imaging revealed that CD80 and PD-L1 each mark unique populations of cells in the treated tumor, typified by p53 signaling or interferon signaling, respectively. In p53 wild-type, syngeneic, orthotopic mouse mammary tumor models that recapitulate human breast cancer response to chemotherapy, treatment of tumors with chemotherapy followed by targeting of the PD-L1 and/or CD80 axes improved response, including complete eradication in some instances. Unfortunately, however, even combination strategies failed to elicit a cure in the majority of cases. Our findings reveal the formidable challenge of eliminating residual disease populated by senescent cells that express multiple redundant immune inhibitory pathways and suggest rational strategies are needed based on the specific checkpoint pathways expressed in residual disease. Citation Format: Ashkan Shahbandi, Fang-Yen Chiu, Nathan A. Ungerleider, Ashlyn Y. Anderson, Heather L. Machado, Zachary F. Pursell, Raegan Kvadas, Sonia G. Rao, James G. Jackson. Chemotherapy-induced senescence activates robust, parallel programs of immune checkpoint expression that can be targeted with immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1297.

Abstract 137: Abrogating therapy induced senescence driven bone loss to improve quality of life

Abstract Despite the central role chemotherapy plays in prolonging survival, the toxicities associated with its use can negatively impact quality of life and in some cases, be so severe that patients forego further life preserving treatments. Therefore, it is critical that we understand the mechanisms that drive these toxicities and develop approaches to mitigate their severity. Recently we demonstrated that chemotherapy-induced senescence drives bone loss by both limiting mineralization of new bone and increasing bone resorption. To establish whether senescent bone resident cells or systemic responses to chemotherapy drove therapy-induced bone loss, we used a vossicle model in which neonatal vertebral bones (L4 and L5) were transplanted from 4-day old wildtype or INKATTAC pups (allow to selectively kill senescent cells) into wildtype or INKATTAC adult mice. Using this approach, we found that the elimination of senescent cells in donor vossicles protects from chemotherapy-induced bone loss, indicating that senescent bone resident cells are responsible for therapy-induced bone loss. To understand the mechanism(s) that contributed to therapy-induced bone loss, we used scRNA-seq to determine which bone resident cells underwent senescence in response to chemotherapy and how their gene expression was impacted. Using this approach, we found that adipocytes, fibroblast, chondrocytes and osteoblasts underwent senescence as evidenced by expression of p16, p21 and loss of Mki67 and displayed evidence of a senescence associated secretory phenotype (SASP), which we postulate contributes to therapy-induced bone loss. Collectively, our data indicated that chemotherapy causes bone loss via senescence and can be protected by eliminating senescent cells. Citation Format: Ganesh Kumar Raut, Zhangting Yao, Tom Cole, Xainmin Luo, Qihao Ren, Sheila A. Stewart. Abrogating therapy induced senescence driven bone loss to improve quality of life [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 137.

Chemotherapy-induced senescence promotes osteoclastogenesis and bone loss by triggering inflammatory responses in vivo

Chemotherapy-induced senescence promotes osteoclastogenesis and bone loss by triggering inflammatory responses in vivo

Blockade of integrin signaling reduces chemotherapy-induced premature senescence in collagen cultured bladder cancer cells.

BackgroundDiminished sensitivity towards chemotherapy remains the major impediment to the clinical treatment of bladder cancer. However, the critical elements in control of chemotherapy resistance remain obscure.MethodsWe adopted improved collagen gels and performed cytotoxicity analysis of doxorubicin (DOX) and mitomycin C (MMC) of bladder cancer cells in a 3D culture system. We then detected the expression of multidrug resistant gene ABCB1, dormancy-associated functional protein chicken ovalbumin upstream-transcription factor 1 (COUPTF1), cell proliferation marker Ki-67, and cellular senescence marker senescence-associated β-galactosidase (SA-β-Gal) in these cells. We further tested the effects of integrin blockade or protein kinase B (AKT) inhibitor on the senescent state of bladder cancer. Also, we examined the tumor growth and survival time of bladder cancer mouse models given the combination treatment of chemotherapeutic agents and integrin α2β1 ligand peptide TFA (TFA).ResultsCollagen gels played a repressive role in bladder cancer cell apoptosis induced by DOX and MMC. In mechanism, collagen activated the integrin β1/AKT cascade to drive bladder cancer cells into a premature senescence state via the p21/p53 pathway, thus attenuating chemotherapy-induced apoptosis. In addition, TFA had the ability to mediate the switch from senescence to apoptosis of bladder cancer cells in xenograft mice. Meanwhile, TFA combined with chemotherapeutic drugs produced a substantial suppression of tumor growth as well as an extension of survival time in vivo.ConclusionsBased on our finding that integrin β1/AKT acted primarily to impart premature senescence to bladder cancer cells cultured in collagen gel, we suggest that integrin β1 might be a feasible target for bladder cancer eradication.

Mechanisms of Senescence-Related NKG2D Ligands Release and Immune Escape Induced by Chemotherapy in Neuroblastoma Cells.

Chemotherapy-induced senescence promotes immunocyte aggregation in the tumor microenvironment by upregulating the surface expression of activating ligands in cancer cells. However, these senescent tumor cells cannot be completely cleared and can induce tumor recurrence. Previous studiesshowed that soluble natural killer (NK) group 2D (NKG2D) ligands impair the recognition of multiple immune cells. In this study, we established an in vitro senescence model using neuroblastoma cells subjected to low-dose Chemotherapeutic drug doxorubicin or the Aurora A inhibitor MLN8237. The results showed that different neuroblastoma cell lines showed increased secretion of the NKG2D ligand MHC class I polypeptide-related sequence A/B (MICA/B) following proteolysis after treatment, with MICA/B subsequently recruited to exosomes to downregulate NKG2D expression in NK cells. Interestingly, disintegrin and metalloproteinase domain-containing 10 (ADAM10) was upregulated in senescent tumor cells, and combined treatment with the ADAM10 inhibitor GI254023X and chemotherapeutic drugs inhibited MICA/B secretion and enhanced recognition and killing by NK cells. Additionally, we found that expression of the long noncoding RNA MALAT1 was significantly increased in senescent neuroblastoma cells, and that MALAT1 served as a sponge for microRNA (miR)-92a-3p to counteract miR-92a-3p-mediated repression of ADAM10 levels. Furthermore, administration of a MALAT1 inhibitor or an miR-92a-3p mimic reduced the MICA/B shedding and enhanced recognition and killing by NK cells. These results confirmed that low-dose chemotherapy induces senescence in neuroblastoma cells, and that senescent tumor cells promote the shedding of the NKG2D ligand MICA/B through the MALAT1/miR-92a/ADAM10 axis, thereby contributing to the formation of a suppressive immune microenvironment and promoting immune escape.

Sphingomyelin nanosystems decorated with TSP-1 derived peptide targeting senescent cells

Senescent cells accumulation can contribute to the development of several age-related diseases, including cancer. Targeting and eliminating senescence cells, would allow the development of new therapeutic approaches for the treatment of different diseases. The 4N1Ks peptide, a 10 amino acid peptide derived from TSP1 protein, combines both features by targeting the CD47 receptor present in the surface of senescent cells and demonstrating senolytic activity, thereby representing a new strategy to take into account. Nonetheless, peptide drugs are known for their biopharmaceutical issues, such as low short half-life and tendency to aggregate, which reduces their bioavailability and limits their therapeutic potential. In order to overcome this problem, herein we propose the use of biodegradable and biocompatible sphingomyelin nanosystems (SNs), decorated with this peptide for the targeting of senescent cells. In order to efficiently associate the 4N1Ks peptide to the nanosystems while exposing it on their surface for an effective targeting of senescent cells, the 4N1Ks peptide was chemically conjugated to a PEGylated hydrophobic chain. The resulting SNs-4N1Ks (SNs-Ks), were extensively characterized for their physicochemical properties, by dynamic light scattering, multiple-angle dynamic light scattering, nanoparticle tracking analysis and atomic force microscopy. The SNs-Ks demonstrated suitable features in terms of size (∼100 nm), association efficiency (87.2 ± 6.9%) and stability in different biorelevant media. Cell toxicity experiments in MCF7 cancer cells indicated an improved cytotoxic effect of SNs-Ks, decreasing cancer cells capacity to form colonies, with respect to free peptide, and an improved hemocompatibility. Lastly, senescence escape preliminary experiments demonstrated the improvement of SNs-Ks senolytic activity of in chemotherapy-induced senescence model of breast cancer cells. Therefore, these results demonstrate for the first time the potential of the combination of SNs with 4N1Ks peptide for the development of innovative senolytic therapies to battle cancer.

Blocking iASPP/Nrf2/M-CSF axis improves anti-cancer effect of chemotherapy-induced senescence by attenuating M2 polarization

The complex interaction between cancer cells and the immune microenvironment is a central regulator of tumor growth and the treatment response. Chemotherapy-induced senescence is accompanied by the senescence-associated secretion phenotype (SASP). However, the mechanisms underlying the regulation of the SASP remain the most poorly understood element of senescence. Here, we show that nuclear erythroid factor 2-like factor 2 (Nrf2), a master antioxidative transcription factor, accumulates upon doxorubicin-induced senescence. This is due to the increased cytoplasmic Inhibitor of Apoptosis Stimulating Protein of P53, iASPP, which binds with Keap1, interrupting Keap1/Nrf2 interaction and promoting Nrf2 stabilization and activation. Activated Nrf2 transactivates a novel target gene of SASP factor, macrophage colony-stimulating factor (M-CSF), which subsequently acts on macrophages and induces polarization from M1 to M2 via a paracrine mechanism. Genetic inhibition of iASPP-Nrf2 suppresses the growth of apoptosis-resistant xenografts, with further analysis revealing that M-CSF/M-CSFR-regulated macrophage polarization is critical for the functional outcomes delineated above. Overall, our data uncover a novel function of iASPP-Nrf2 in skewing the immune microenvironment under treatment-induced senescence. Targeting the iASPP-Nrf2 axis could be a powerful strategy for the implementation of new chemotherapy-based therapeutic opportunities.

TRNA biogenesis and specific aminoacyl-tRNA synthetases regulate senescence stability under the control of mTOR.

Oncogenes or chemotherapy treatments trigger the induction of suppressive pathways such as apoptosis or senescence. Senescence was initially defined as a definitive arrest of cell proliferation but recent results have shown that this mechanism is also associated with cancer progression and chemotherapy resistance. Senescence is therefore much more heterogeneous than initially thought. How this response varies is not really understood, it has been proposed that its outcome relies on the secretome of senescent cells and on the maintenance of their epigenetic marks. Using experimental models of senescence escape, we now described that the stability of this proliferative arrest relies on specific tRNAs and aminoacyl-tRNA synthetases. Following chemotherapy treatment, the DNA binding of the type III RNA polymerase was reduced to prevent tRNA transcription and induce a complete cell cycle arrest. By contrast, during senescence escape, specific tRNAs such as tRNA-Leu-CAA and tRNA-Tyr-GTA were up-regulated. Reducing tRNA transcription appears necessary to control the strength of senescence since RNA pol III inhibition through BRF1 depletion maintained senescence and blocked the generation of escaping cells. mTOR inhibition also prevented chemotherapy-induced senescence escape in association with a reduction of tRNA-Leu-CAA and tRNA-Tyr-GTA expression. Further confirming the role of the tRNA-Leu-CAA and tRNA-Tyr-GTA, results showed that their corresponding tRNA ligases, LARS and YARS, were necessary for senescence escape. This effect was specific since the CARS ligase had no effect on persistence. By contrast, the down-regulation of LARS and YARS reduced the emergence of persistent cells and this was associated with the modulation of E2F1 target genes expression. Overall, these findings highlight a new regulation of tRNA biology during senescence and suggest that specific tRNAs and ligases contribute to the strength and heterogeneity of this tumor suppressive pathway.

Anterior gradient protein 2 is a marker of tumor aggressiveness in breast cancer and favors chemotherapy‑induced senescence escape.

Among the different chemotherapies available, genotoxic drugs are widely used. In response to these drugs, particularly doxorubicin, tumor cells can enter into senescence. Chemotherapy-induced senescence (CIS) is a complex response. Long described as a definitive arrest of cell proliferation, the present authors and various groups have shown that this state may not be complete and could allow certain cells to reproliferate. The mechanism could be due to the activation of new signaling pathways. In the laboratory, the proteins involved in these pathways and triggering cell proliferation were studied. The present study determined a new role for anterior gradient protein 2 (AGR2) in vivo in patients and in vitro in a senescence escape model. AGR2's implication in breast cancer patients and proliferation of senescent cells was assessed based on a SWATH-MS proteomic study of patients' samples and RNA interference technology on cell lines. First, AGR2 was identified and it was found that its concentration is higher in the serum of patients with breast cancer and that this high concentration is associated with metastasis occurrence. An inverse correlation between intratumoral AGR2 expression and the senescence marker p16 was also observed. This observation led to the study of the role of AGR2 in the CIS escape model. In this model, it was found that AGR2 is overexpressed in cells during senescence escape and that its loss considerably reduces this phenomenon. Furthermore, it was shown that the extracellular form of AGR2 stimulated the reproliferation of senescent cells. The power of proteomic analysis based on the SWATH-MS approach allowed the present study to highlight the mammalian target of rapamycin (mTOR)/AKT signaling pathway in the senescence escape mechanism mediated by AGR2. Analysis of the two signaling pathways revealed that AGR2 modulated RICTOR and AKT phosphorylation. All these results showed that AGR2 expression in sera and tumors of breast cancer patients is a marker of tumor progression and metastasis occurrence. They also showed that its overexpression regulates CIS escape via activation of the mTOR/AKT signaling pathway.