We analyzed the small RNA transcriptome from 5‐month‐old, 24‐month‐old, and 36‐month‐old mouse liver and found 56 miRNAs that changed their expression profile with age. Among these is a cluster of 18 miRNAs that are upregulated between 50‐ and 1,000‐fold at 24 and 36 months of age. This cluster is located in a 60‐kb region of the X‐chromosome that is devoid of other coding sequences and is part of a lamin‐associated domain. Potential targets of the miRNAs in the cluster suggest they may regulate several pathways altered in aging, including the PI3K‐Akt pathway. Total transcriptome analyses indicate that expression of several potential genes in the PI3K‐Akt pathway that may be targeted by the mir‐465 family (mmu‐mir‐465a, mmu‐mir‐465b, and mmu‐mir‐465c) is downregulated with age. Transfection of the liver cell line AML12 with mir‐465 family members leads to a reduction of three of these potential targets at the mRNA level: a 40% reduction of the growth hormone receptor (GHR), and a 25% reduction in Kitl and PPP2R3C. Further investigation of the GHR 3′UTR revealed that the mir‐465 family directly targets the GHR mRNA. Cells transfected with mir‐465 showed a reduction of JAK2 and STAT5 phosphorylation upon growth hormone (GH) stimulation, resulting in a reduction in insulin‐like growth factor 1 (IGF‐1) and IGF‐1‐binding protein 3 expression. With age, GH signaling falls and there is a reduction in circulating IGF‐1. Our data suggest that an increase in expression of the mir‐465 family with age may contribute to the reduction in the GH signaling.
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.
Objectives Cellular senescence is a stress response involving permanent replicative arrest. It is accompanied by a complex senescence‐associated secretory phenotype (SASP), which is characterized by the presence of pro‐inflammatory cytokines and chemokines, growth factors, and tissue‐remodeling metalloproteinases. Little is known regarding the role of senescence and the SASP in the context of arrhythmias post myocardial infarction (MI). Here, we characterized the arrhythmogenic cardiac consequences and molecular mechanisms underlying tissue remodeling changes in impaired or accelerated cardiac myofibroblast (CMF) senescence and the pro‐inflammatory SASP during post‐MI wound healing process in genetically engineered mice. Material‐Methods Impaired or accelerated CMF‐specific senescence was achieved by using cre‐lox mouse models to delete p53 or MDM2 genes. MI surgery via direct ligation of the left anterior descending artery (LAD) was performed in 4‐month‐old wild‐type (WT), Pstn‐Cre(+/‐) p53(fl/fl), Pstn‐MerCreMer(+/‐) MDM2(fl/fl), and respective littermate control (LMC) mice of both sexes. Changes in cardiac structure and function post‐MI were assessed by echocardiography. Senescence‐associated β‐galactosidase (SA‐βgal) staining was applied to detect and quantify senescent cells in three zones: infarct zone (IZ), infarct border zone (IBZ), and remote zone (RZ). Immunofluorescence staining identified the types of senescent cells. Masson’s trichrome staining was used for fibrosis assessment. RT‐qPCR was performed to quantitate the expression of known senescence genes and canonical SASP factors. Results Pstn‐Cre(+/‐) p53(fl/fl) mice displayed significantly decreased CMF senescence two weeks post‐MI in the scar compared to littermate control p53(fl/fl) and WT mice. We noticed a similar trend in the IBZ, but this did not reach statistical significance. Pstn‐MCM(+/‐) MDM2(fl/fl) mice showed significantly increased CMF senescence in the IZ compared littermate control MDM2(fl/fl) mice. We observed a similar trend in the IBZ, but this did not reach statistical significance. In all mice, we observed essentially no senescence in the RZ. Our data did not show any significant difference in fibrosis between any genotype in any zone. We did not observe any overall trend in differential expression in senescence and SASP genes between p53(fl/fl) and Pstn‐Cre(+/‐) p53(fl/fl) mice in any zone. There was no evidence of any sex‐specific differences in aforementioned results. Conclusions A significant increase and persistence of CMF senescence likely contributes to pathological tissue remodeling during the post‐MI wound healing process. This is likely due to SASP‐induced pro‐arrhythmogenic chronic inflammation or via direct cell‐cell interactions between senescent CMFs and myocytes.
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