p16INK4a Expression Research Articles

SMARCB1 Loss in Poorly Differentiated Chordomas Drives Tumor Progression

Poorly differentiated (PD) chordoma, a rare, aggressive tumor originating from notochordal tissue, shows loss of SMARCB1 expression, a core component of the Switch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes. To determine the impact of SMARCB1 re-expression on cell growth and gene expression, two SMARCB1-negative PD chordoma cell lines with an inducible SMARCB1 expression system were generated. After 72 hours of induction of SMARCB1, both SMARCB1-negative PD chordoma cell lines continued to proliferate. This result contrasted with those observed with SMARCB1-negative rhabdoid cell lines in which SMARCB1 re-expression caused the rapid inhibition of growth. We found that the lack of growth inhibition may arise from the loss of CDKN2A (p16INK4A) expression in PD chordoma cell lines. RNA-sequencing of cell lines after SMARCB1 re-expression showed a down-regulation for rRNA and RNA processing as well as metabolic processing and increased expression of genes involved in cell adhesion, cell migration, and development. Taken together, these data establish that SMARCB1 re-expression in PD chordomas alters the repertoire of SWI/SNF complexes, perhaps restoring those associated with cellular differentiation. These novel findings support a model in which SMARCB1 inactivation blocks the conversion of growth-promoting SWI/SNF complexes to differentiation-inducing ones, and they implicate SMARCB1 loss as a late event in tumorigenic progression. Importantly, the absence of growth inhibition after SMARCB1 restoration creates a unique opportunity to identify therapeutic vulnerabilities.

Abstract B021: Breast cancer survivors and expression of p16INK4a, DNA damage, and the SASP prior to and following treatment

Abstract Purpose: To examine whether breast cancer therapy accelerates aging via increasing expression of cellular senescence (p16INK4a), the DNA damage response (DDR), or the proinflammatory senescence associated secretory phenotype (SASP), we measured gene expression of these pathways in a cohort of women recruited following a diagnosis of breast cancer (Stage 0-III). Methods: The RISE study is a Los Angeles-based longitudinal, observational study of biological and behavioral processes following surgery, radiation (RT) and/or chemotherapy (CT); and more persistent changes in these processes in the months after adjuvant treatment completion and initiation of endocrine therapy (6, 12, 18 month post-treatment follow-ups). Of the 270 women enrolled in the study, gene expression data was available for 184 women (aged average of 55.5 years, SD 11.2). Peripheral blood mononuclear cells (PBMC) gene expression was determined using RNA sequencing on quality-verified RNA. We determined transcript abundance for cellular senescence marker p16INK4a, genes that express proteins of the SASP, and genes responsive to DNA damage. Longitudinal data for p16INK4a, DDR and SASP were modeled using mixed models, including a quadratic term for time and its interaction with four treatment groups (1: Surgery alone [no RT or CT], RT alone, CT alone, and CT+RT), and a zero-inflated two-part model with p16INK4a. Covariates included age, BMI, race, initial surgery type, receipt of interim surgery, and endocrine therapy. Results: Analyses testing whether there was a change in p16INK4a over time by treatment group revealed that all groups experienced increases in p16INK4a expression over time. At baseline 53% of women had no detectable p16INK4a transcripts in their PBMC samples, and this proportion changed over time across all groups indicating significant increases in women expressing the marker for cellular senescence, p<0.0001. Women receiving CT or CT+RT had a 40% and 36% increase in percentage expressing p16INK4a (26% increase among those with surgery alone and 31% for RT alone) by 18 months post treatment compared to their pretreatment values. Analyses found a significant initial increase in DDR over time in all groups (p's<0.002) followed by a deceleration in all three groups except CT+RT, which continued an upward trajectory. There were also significant increases in SASP across all groups (p’s<0.05), with a significant quadratic effect in the RT group only (p<0.001). Conclusions: These results indicate that there was activation of genes associated with acceleration of biological aging across all groups after breast cancer diagnosis. Survivors had increases in p16INK4a, DNA damage response genes, and SASP from pre to 18 months post treatment for breast cancer. (NCI R01 CA237535) Citation Format: Judith E. Carroll, Catherine M. Crespi, Laura Petersen, Julie E. Bower. Breast cancer survivors and expression of p16INK4a, DNA damage, and the SASP prior to and following treatment [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B021.

SATB2, coordinated with CUX1, regulates IL-1β-induced senescence-like phenotype in endothelial cells by fine-tuning the atherosclerosis-associated p16INK4a expression.

Genome-wide association studies (GWAS) have validated a strong association of atherosclerosis with the CDKN2A/B locus, a locus harboring three tumor suppressor genes: p14ARF , p15INK4b , and p16INK4a . Post-GWAS functional analysis reveals that CUX is a transcriptional activator of p16INK4a via its specific binding to a functional SNP (fSNP) rs1537371 on the atherosclerosis-associated CDKN2A/B locus, regulating endothelial senescence. In this work, we characterize SATB2, another transcription factor that specifically binds to rs1537371. We demonstrate that even though both CUX1 and SATB2 are the homeodomain transcription factors, unlike CUX1, SATB2 is a transcriptional suppressor of p16INK4a and overexpression of SATB2 competes with CUX1 for its binding to rs1537371, which inhibits p16INK4a and p16INK4a -dependent cellular senescence in human endothelial cells (ECs). Surprisingly, we discovered that SATB2 expression is transcriptionally repressed by CUX1. Therefore, upregulation of CUX1 inhibits SATB2 expression, which enhances the binding of CUX1 to rs1537371 and subsequently fine-tunes p16INK4a expression. Remarkably, we also demonstrate that IL-1β, a senescence-associated secretory phenotype (SASP) gene itself and a biomarker for atherosclerosis, induces cellular senescence also by upregulating CUX1 and/or downregulating SATB2 in human ECs. A model is proposed to reconcile our findings showing how both primary and secondary senescence are activated via the atherosclerosis-associated p16INK4a expression.

Immunohistochemical p16 expression in the prognosis of patients with sinonasal squamous cell carcinoma.

In sinonasal squamous cell carcinoma (SNSCC), the prognostic relevance of p16INK4a (p16) expression has been reported rarely. This study aims to examine the immunohistochemical expression of p16 and investigate the possibility of p16 as a prognostic factor for SNSCC. The medical records of 173 individuals with SNSCC between 2010 and 2022 were retrospectively reviewed. The researchers examined patients' demographics, p16 status, staging, tumor histological subtypes, treatment details, recurrence, metastasis, and survival outcomes. p16 was found in 22.0% (38/173) of SNSCC patients, and there was no difference between inverted papilloma-SNSCC (19.6%) and de novo SNSCC (23.0%). p16 status did not correlate with all the cases' age, gender, clinical stage, or therapy features. p16-positive patients had a considerably superior 5-year overall survival (OS) rate (80.7% vs. 57.5%, p=0.039) and a slight tendency in progression-free survival (PFS) rate (68.1% vs. 52.0%, p=0.15), except in stage T4b cases. In maxillary sinus lesions, p16-positive SNSCC had a better 5-year OS (87.4% vs. 49.2%, p=0.03) rate and PFS (79.1% vs. 40.7%, p=0.01) rate than p16-negative SNSCC. Among patients without skull base involvement (82.9% vs. 57.7%, p=0.037) or orbital invasion (86.9% vs. 57.3%, p=0.02), p16-positive SNSCC confers benefits in OS rates more than p16-negative SNSCC. Immunohistochemical p16 expression may be a predictive predictor in individuals with maxillary sinus SCC, non-T4b stage, without skull base involvement, and without orbital invasion.

P16INK4a and γ-H2AX as Biomarkers of Senescence in Skin Tissue: A Cadaveric Cross-sectional Study at a Tertiary Care Centre in Punjab, India

Introduction: Baseline expression of p16INK4a characterises cellular senescence. Similarly, the measurement of γ-GammaH2AX (γ-H2AX) foci levels in cells provides a reliable method for quantification of Deoxyribonucleic Acid (DNA) damage. Aim: To explore the role of p16INK4a and γ-H2AX as biomarkers of senescence in skin tissue. Materials and Methods: This was a cross-sectional study conducted in the Department of Anatomy, PGIMER, Chandigarh, Punjab, India from June 2022 to January 2023. Skin tissue was obtained from 30 cadavers, aged 20-90 years, from the anterior abdominal wall. The time duration of sample collection after death varied from six hours to 12 hours. Samples were divided into two groups: Group-I <30 years and Group-II >70 years, with n=15 in each group. The relative change in the expression pattern of p16INK4a and γH2AX markers, as well as the microstructure of the skin (thickness of epidermis and dermis, distribution of collagen I/II/III fibres, architecture of sebaceous glands), were statistically analysed using an unpaired t-test. Results: Intense staining was observed with p16INK4a in Group-II, showing positivity in 60.75% of the cells, while Group-I depicted a weak staining pattern (15%). On immunostaining with γ-H2AX, only Group-II cells showed intense positivity (45%). Significant differences were observed in the epidermal and dermal thickness: Group-I (165.5267±37.73 μm; 2394.6±874.13 μm); Group-II (54.6±22.79 μm; 566.67±242.23 μm), and collagen Type-II/III fibres were found predominantly in aging skin tissue. Conclusion: The present study provides comprehensive data regarding age-associated changes between p16INK4a, γ-H2AX, and skin microstructure, which result in decreased repair and regenerative capacity of skin tissue and various age-related skin diseases.

P-MSC-derived extracellular vesicles facilitate diabetic wound healing via miR-145-5p/ CDKN1A-mediated functional improvements of high glucose-induced senescent fibroblasts.

Persistent hyperglycaemia in diabetes causes functional abnormalities of human dermal fibroblasts (HDFs), partially leading to delayed skin wound healing. Extracellular vesicles (EVs) containing multiple pro-healing microRNAs (miRNAs) have been shown to exert therapeutic effects on diabetic wound healing. The present study aimed to observe the effects of EVs derived from placental mesenchymal stem cells (P-MSC-EVs) on diabetic wound healing and high glucose (HG)-induced senescent fibroblasts and to explore the underlying mechanisms. P-MSC-EVs were isolated by differential ultracentrifugation and locally injected into the full-thickness skin wounds of diabetic mice, to observe the beneficial effects on wound healing in vivo by measuring wound closure rates and histological analysis. Next, a series of assays were conducted to evaluate the effects of low (2.28 x 1010 particles/ml) and high (4.56 x 1010 particles/ml) concentrations of P-MSC-EVs on the senescence, proliferation, migration, and apoptosis of HG-induced senescent HDFs in vitro. Then, miRNA microarrays and real-time quantitative PCR (RT-qPCR) were carried out to detect the differentially expressed miRNAs in HDFs after EVs treatment. Specific RNA inhibitors, miRNA mimics, and small interfering RNA (siRNA) were used to evaluate the role of a candidate miRNA and its target genes in P-MSC-EV-induced improvements in the function of HG-induced senescent HDFs. Local injection of P-MSC-EVs into diabetic wounds accelerated wound closure and reduced scar widths, with better-organized collagen deposition and decreased p16INK4a expression. In vitro, P-MSC-EVs enhanced the antisenescence, proliferation, migration, and antiapoptotic abilities of HG-induced senescent fibroblasts in a dose-dependent manner. MiR-145-5p was found to be highly enriched in P-MSC-EVs. MiR-145-5p inhibitors effectively attenuated the P-MSC-EV-induced functional improvements of senescent fibroblasts. MiR-145-5p mimics simulated the effects of P-MSC-EVs on functional improvements of fibroblasts by suppressing the expression of cyclin-dependent kinase inhibitor 1A and activating the extracellular signal regulated kinase (Erk)/protein kinase B (Akt) signaling pathway. Furthermore, local application of miR-145-5p agomir mimicked the effects of P-MSC-EVs on wound healing. These results suggest that P-MSC-EVs accelerate diabetic wound healing by improving the function of senescent fibroblasts through the transfer of miR-145-5p, which targets cyclin-dependent kinase inhibitor 1A to activate the Erk/Akt signaling pathway. P-MSC-EVs are promising therapeutic candidates for diabetic wound treatment.

Changes in hormones, Leukocyte Telomere Length (LTL), and p16INK4a expression in SM-exposed individuals in favor of the cellular senescence

Sulfur mustard (SM) is a chemical warfare agent with well-known severe toxic effects and may cause long-term debilitating injuries. We aimed to evaluate aging and longevity in Iranian SM-exposed survivors using some endocrine and molecular biomarkers for the first time. Dehydroepiandrosterone (DHEA), prolactin (PRL), cortisol, testosterone, and luteinizing hormone (LH) were measured in 289 male SM-veterans and 66 age-matched males using the ELISA method. Leukocyte Telomere Length (LTL) measurement and p16INK4a expression were measured in the peripheral blood leukocytes of 55 males who were exposed to SM. We found a significantly lower serum DHEAS level and higher serum PRL level in SM-exposed groups (without any related to the severity of lung injuries) compared to healthy controls, but no significant difference in serum levels of cortisol, testosterone, and LH. The molar ratio of DHEAS/cortisol was significantly higher in controls compared to the SM-exposed individuals especially those with severe lung damage. Some biological parameters of allostatic load score such as DHEAS and DHEAS/cortisol ratio significantly decreased long-term after the SM exposure. Additionally, we found that LTL was shorter in SM-exposed veterans rather than unexposed controls while p16INK4a gene expression significantly increased in these groups. It seems that DHEAS, DHEAS/cortisol ratio, LTL, and p16INK4a gene expression have changed significantly in favor of cellular senescence in SM-exposed patients. Therefore, it seems that SM exposure increases biological age compared to chronological age in SM-exposed survivors.

CIRCULATING BIOMARKERS OF CELL SENESCENCE IN A RANDOMIZED TRIAL OF CALORIC RESTRICTION WEIGHT LOSS

Abstract There are no circulating markers exclusive to cell senescence for clinical trials in middle-aged/older adults. In this study we evaluated T-cell expression of tumor suppressor protein p16INK4a and plasma senescence associated secretory proteins (SASP)-factors in the context of an 18-week randomized trial of caloric restriction (CR) compared to a weight stable Control. The analysis includes 55 middle-aged/older adults (75%Women; 33%African American; 57.6±5.8 years) with obesity and prediabetes. We measured mRNA expression of select senescence and apoptosis gene transcripts (p16INK4a, p21, BCL2L, BAK1) in peripheral blood CD3+ T-cells (quantitative-RT-PCR) and a panel of 27 plasma SASP proteins (Luminex/multiplex; ELISA). Weight loss in those randomized to CR was -10.8±0.9 kg compared to +0.5±0.9 kg in Control. Using mixed models, T-cell expression of senescence-biomarkers p16INK4a, p21, BCL-2L, and apoptosis-marker BAK1 were not different between CR and control at 18 weeks (time × treatment adjusted for age, race, and sex, all p>0.05). We explored associations between biomarkers with weight loss; moderate spearman correlations (rs=0.28 to 0.64) were observed between weight loss and change in plasma SASP-factors (Activin A, I-CAM, MMP1, PAI-1, TNF-RI, TNF-RII, VEGF, uPAR, p< 0.05 all), but not T-cell biomarker expression. We evaluated associations between biomarkers and found change in T-cell p16INK4a was associated with change in plasma SASP-factors, Fas (rs=0.51), osteopontin (rs=0.34), myeloperoxidase (rs=0.39), and MMP2 (rs=0.40) (CR+Control combined). Among middle-aged/older persons with obesity undergoing 18-weeks CR, changes in T cell p16INK4a expression were correlated with reductions in key SASP factors, and weight loss was associated with lower pro-inflammatory SASP profile.

Bioactive Oligopeptides from Ginseng (Panax ginseng Meyer) Suppress Oxidative Stress-Induced Senescence in Fibroblasts via NAD+/SIRT1/PGC-1α Signaling Pathway.

The physicochemical properties and multiple bioactive effects of ginseng oligopeptides (GOPs), plant-derived small molecule bioactive peptides, suggest a positive influence on health span and longevity. Given this, cellular senescence is the initiating factor and key mechanism of aging in the organism, and thus the current study sought to explore the effects of GOPs on H2O2-induced cellular senescence and its potential mechanisms. Senescence was induced in mouse embryonic fibroblasts NIH/3T3 by 4 h of exposure to 200 µM H2O2 and confirmed using CCK-8 assay and Western blot analyses of p16INK4A and p21Waf1/Cip1 after 24 h of growth medium administration with or without GOPs supplementation (25, 50, and 100 µg/mL). We found that GOPs delayed oxidative stress-induced NIH/3T3 senescence by inhibiting the G1 phase arrest, increasing DNA synthesis in the S phase, decreasing the relative protein expression of p16INK4A and p21Waf1/Cip1, promoting cell viability, protecting DNA, and enhancing telomerase (TE) activity. Further investigation revealed that the increase in antioxidative capacity and anti-inflammation capacity might form the basis for the retarding of the senescence effects of GOPs. Furthermore, GOPs supplementation significantly improved mitochondrial function and mitochondrial biogenesis via the NAD+/SIRT1/PGC-1𝛼 pathway. These findings indicate that GOPs may have a positive effect on health span and lifespan extension via combating cellular senescence, oxidative stress, and inflammation, as well as modulating longevity regulating pathway NAD+/SIRT1/PGC-1𝛼.

Abnormal expression of cortical cell cycle regulators underlying anxiety and depressive-like behavior in mice exposed to chronic stress.

The cell cycle is a critical mechanism for proper cellular growth, development and viability. The p16INK4a and p21Waf1/Cip1 are important regulators of the cell cycle progression in response to internal and external stimuli (e.g., stress). Accumulating evidence indicates that the prefrontal cortex (PFC) is particularly vulnerable to stress, where stress induces, among others, molecular and morphological alterations, reflecting behavioral changes. Here, we investigated if the p16INK4a and p21Waf1/Cip1 expression are associated with behavioral outcomes. Prefrontal cortex mRNA and protein levels of p16INK4A and p21Waf1/Cip1 of mice (six independent groups of C57BL/6J, eight mice/group, 50% female) exposed from 0 to 35 days of chronic restraint stress (CRS) were quantified by qPCR and Western Blot, respectively. Correlation analyses were used to investigate the associations between cyclin-dependent kinase inhibitors (CKIs) expression and anxiety- and depression-like behaviors. Our results showed that the PFC activated the cell cycle regulation pathways mediated by both CKIs p16INK4A and p21Waf1/Cip1 in mice exposed to CRS, with overall decreased mRNA expression and increased protein expression. Moreover, correlation analysis revealed that mRNA and protein levels are statistically significant correlated with anxiety and depressive-like behavior showing a greater effect in males than females. Our present study extends the existing literature providing evidence that PFC cells respond to chronic stress exposure by overexpressing CKIs. Furthermore, our findings indicated that abnormal expression of p16INK4A and p21Waf1/Cip1 may significantly contribute to non-adaptive behavioral responses.

Cellular senescence marker p16INK4a and NFKB1 gene polymorphisms in lower gastro-intestinal acute graft versus host disease

BackgroundLower gastrointestinal (GI) graft versus host disease (GVHD) represents a severe complication in allogeneic hematopoietic stem cell transplant (HSCT) recipients with high rates of transplant-related mortality. Deregulated innate immunity reactions are the features of its pathogenesis. Cellular senescence has been considered a program of the innate immunity. We focused on lower GI GVHD from the perspective of cellular senescence. ObjectiveWe analyzed the impact of p16INK4a expression, a hallmark of cellular senescence, in intestinal biopsies of patients with lower GI GVHD symptoms and NFKB1 gene polymorphisms (rs3774937 C/T and rs3774959 A/G) on HSCT outcome. Study designFifty-two single-center patients who presented with symptoms of lower GI GVHD were analyzed in a retrospective manner. Two SNPs located in the NFKB1 gene regions (rs3774937 C/T and rs3774959 A/G) were genotyped from the peripheral blood samples collected before the start of the conditioning. All patients underwent proctosigmoidoscopy with biopsy of the mucosa. The expression of p16INK4a was analyzed in normal intestinal crypts and stroma. ResultsFifty-two patients (50% male) received HSCT for hematological diseases (acute leukemias in 67%) and developed lower GI symptoms. Patients with p16INK4a expression in the intestinal stroma were in lower risk of developing histological grade 3–4 aGVHD (RR 0.18 [95% CI 0.05–0.65]; p = 0.009). The multivariate linear regression confirmed the independent effect of p16INK4a expression on time of the lower GI aGVHD symptoms onset (Coef. 38.9 [95% CI 12.7–65.1]; p = 0.005). The NFKB1 rs3774937 CC and TT/TC genotype were present in 40 and 80% of patients with p16INK4a expression, respectively (p = 0.04). The rs3774959 AA and GG/AG genotype were present among 43 and 82% of patients with p16INK4a expression, respectively (p = 0.02). Expression of p16INK4a was associated with no clinical variable but NFKB1 genotype. ConclusionsOur results address possible new mechanisms that may lead to better understanding of HSCT-related immune complications. Cellular senescence may bring novel approaches in GVHD diagnostics and therapy.

P16INK4a in the Gut Mucosa Associates with NFKB1 Gene Polymorphisms and Decreased Serum Levels of Inflammation-Associated Proteins in Patients with Gastrointestinal Acute Graft Versus Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation

Cellular senescence is characterized by irreversible cell cycle arrest and pro-inflammatory secretion, mostly regulated by NF-kB. Expression of p16INK4a - a cell cycle inhibitor, is considered a hallmark of cellular senescence. Based on overlapping features observed in inflammatory signaling of both GVHD and cellular senescence, we have postulated a hypothesis whether genetic variants of the NFKB1 gene and expression of p16INK4a in intestinal mucosa of the recipients have impact on transplant-related inflammatory complications. Here we present 52 consecutive patients with symptoms of lower gastrointestinal GVHD after allogeneic stem cell transplantation. The patients were genotyped for GVHD-prone polymorphisms of the NFKB1 gene, rs3774937 C/T and rs3774959 A/G. All patients underwent diagnostic proctosigmoidoscopy with gut mucosa biopsy. Immunohistochemical detection of p16INK4a expression was evaluated in normal intestinal crypts and stroma. On day +14 after graft infusion serum levels of inflammation - associated proteins were analyzed. Increased p16INK4a expression in intestinal crypts was independently associated with late manifestation of lower gastrointestinal symptoms. Patients with and without p16INK4a expression in intestinal crypts developed lower gastrointestinal symptoms in median of 43 days (range,12-140) and 24 days (range,13-80), respectively. The effect was observed in the whole study cohort (HR 2.3 [95%CI 0.99-5.47];p=0.006) as well as in a subgroup of patients who fulfilled the clinical diagnostic criteria of GVHD only (HR 3.1 [95%CI 0.95-10.01];p=0.002). Increased risk of histological grade 3-4 aGVHD was observed in patients without stromal p16INK4a expression (HR 3.45 [95%CI 1.26-9.42];p=0.01). The impact was confirmed by means of multivariate Cox regression (p=0.009). No clinical variable had effect on p16INK4a expression but NFKB1 genotype. The GVHD-prone genotypes rs3774937 CC and rs3774959 AA were significantly less frequent in patients with increased p16INK4a expression (p=0.04 and p=0.02, respectively). In 13 patients, measurement of serum levels of 92 inflammation-associated proteins from the Proseek Multiplex lnflammation - I kit on day +14 after graft infusion was performed. Patients with increased expression of p16INK4a in intestinal crypts at the time of gut biopsy had significantly lower serum levels of proinflammatory proteins (IL-17A, IL-18, IL-4, CXCL10 and TNFRSF9) on day +14. Using the STRING web engine we performed the enrichment analysis with statistically significant confirmation of interactions among these proteins suggesting various T-cell cytokine production or Type -2 immune response from the gene ontology analysis. Cellular senescence is considered a program of innate immunity. In GVHD, innate immunity becomes seriously decontrolled. Cellular senescence may thus bring novel approaches towards GVHD diagnostics and therapy. Supported by Ministry of Health, Czech Republic - conceptual development of research organization (FNOl, 00098892) Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

TMIC-14. DIFFUSE P16INK4A EXPRESSION IS ASSOCIATED WITH TUMOR CELL SENESCENCE AND SENESCENCE-ASSOCIATED SECRETARY PHENOTYPE RELATED TO IMMUNE MICROENVIRONMENT IN GLIOBLASTOMAS

Abstract Cellular senescence (CS) is a state of irreversible cell cycle arrest, and the expression of p16INK4a in cells is one of the reliable markers for CS. However, senescent cells are metabolically active with the senescence-associated secretory phenotype (SASP), which can influence the tissue microenvironment by paracrine signaling to the adjacent tumor, non-tumor, and immune cells. In the present study, we evaluated p16INK4a expression in glioblastoma and investigated its association with CS and SASPs. We analyzed the expression of p16INK4a in 73 glioblastomas by immunostaining. To examine the association of p16INK4a expression and CS, we performed senescence-associated β-galactosidase (SA-β-Gal) staining, a standard marker of CS, using glioblastoma cell lines and fresh frozen tumor tissues. For SASPs analysis, RNA sequencing with Gene Set Enrichment Analysis (GSEA) was performed. Among 73 glioblastomas, twenty-eight cases (38.4%) revealed diffuse strong p16INK4a expression in tumor cells. The glioblastoma with diffuse p16INK4a expression (GMDP) patients were younger (52.4 vs. 59.2 years) and showed prolonged overall survival (median: 25.5 vs. 12.3 months) compared to those harboring negative expression. In vitro analysis, p16INK4a over-expressed glioblastoma cell line showed increased expression of SA-β-Gal which indicates CS. In addition, fresh frozen tissues from GMDP also revealed SA-β-Gal positivity. RNA sequencing analysis revealed that splicing or protein biosynthetic genes were enriched in GMDP, and GSEA showed significant enrichment of SASP genes (false discovery rate < 0.05), especially chemokines associated with monocytes and macrophages. Our data suggest that increased expression of p16INK4a in glioblastoma is associated with tumor cell senescence, and SASPs from senescent tumor cells could be one of the crucial modulators in the tumor immune microenvironment.

TMIC-15. AGE-RELATED MARKERS FOR SENESCENCE INCREASE IN THE OLDER ADULT EXTRATUMORAL MOUSE BRAIN DUE TO GLIOBLASTOMA

Abstract OBJECTIVE The median age of onset for glioblastoma (GBM; IDHwt) is 68-70 years. Age is a strong prognostic factor for GBM patient outcomes such that overall survival in older adults is less than their younger counterparts – even after adjustment for MGMT promoter methylation status. Aging is associated with increased levels of senescence in the brain. Several age-related neurological disorders have been shown to improve with senolytic treatments. Here, we explored the effects and therapeutic neutralization of syngeneic brain tumors on increasing senescence levels in the extratumoral brain (ie. outside of the brain tumor) of young and old mice. METHODS General RNA-sequencing, as well as single-cell (sc) RNA-sequencing was performed on extratumoral tissue from young (8-12 weeks) and older adult (80-90 weeks) C57BL/6 mice with or without GL261 and key markers were validated with RT-PCR. The combined effects of the senolytics, dasatinib and quercetin, with radiation, anti-PD-1 mAb, and IDO enzyme inhibitor treatment, was also investigated. RESULTS General- and sc-RNA sequencing revealed a distinct gene expression profile in the extratumoral brain of older mice with syngeneic GL261 as compared to all other groups. RT-PCR results confirmed that the brain tumor increased gene expression for senescence levels, p53, and NFkB signaling in the older adult extratumoral brain. Expression of the senescence marker p16INK4A was primarily localized to oligodendrocyte progenitor cells in the older adult brain. The combinatorial treatment of senolytics with RT, anti-PD-1 mAb, and IDO enzyme inhibitor led to a synergistic survival benefit in older adult mice with GL261 as compared to the treatment with senolytics, or immunotherapy, alone. CONCLUSIONS The data suggest that the extratumoral brain may be responsible in-part for the poorer outcomes of older adults with GBM and that treatment approaches that target senescent cells may provide clinical benefit.

Associations between exercise capacity, p16INK4a expression and inflammation among adult survivors of childhood cancer.

Over 50% of childhood cancer survivors are exercise intolerant, with maximal aerobic capacities comparable to individuals decades older, suggesting early physiologic ageing. In addition, 36% of survivors are obese. Optimal exercise capacity provides a foundation to support daily function and healthy body habitus and is associated with benefits to cognition, cardiovascular health, and longevity. Cellular senescence and inflammation are key mechanisms that drive age-related disease, quantifiable as biomarkers in peripheral blood. This study aimed to evaluate associations between p16INKa, a biomarker of cellular senescence, and inflammation and exercise capacity among adult survivors of childhood cancer. Eligible survivors were recruited from the St. Jude Lifetime (SJLIFE) Cohort Study. Exercise capacity was assessed by maximal oxygen uptake (VO2, ml/kg/min) obtained via cardiopulmonary exercise testing using a modified Bruce protocol. Body fat (%) was determined from dual energy x-ray absorptiometry (DEXA). Peripheral blood samples were used to evaluate log2 p16INK4a mRNA expression, a biomarker of cellular senescence, and inflammation with high sensitivity C-reactive protein (hs-CRP) levels. Multivariable regression evaluated associations between p16INK4a, hs-CRP, body fat, and exercise capacity. Participants included 185 five-year childhood cancer survivors (mean age 36.6 [range 20.1 - 55.7] years, 44% male, 77% non-Hispanic white, 53% leukemia/lymphoma). Compared to males, females had lower peak VO2 (mean ± SD, 22.5 ± 8.2 vs. 28.8 ± 7.7 ml/kg/min, p<0.01), higher p16INK4a expression (9.6 ± 1.2 vs. 9.2 ± 1.2 fold, p=0.02), and hs-CRP concentration (5.9 ± 8.4 vs. 3.3 ± 3.9 mg/L, p=0.01). Among females (n=103), hs-CRP concentration (β -0.2, 95% CI -0.34 to -0.05, p=0.01) and p16INK4a expression (β-5.32, 95% CI 10.42 to -0.22, p=0.04) were inversely associated and statistically significant with peak exercise capacity, with a significant interaction between p16INK4a expression and body fat (β 0.15, 95% CI 0.02 to 0.28, p=0.03). Among males (n=82), p16INK4a expression (β -1.01, 95% CI -2.14 to 0.12, p=0.08), and body fat (β -0.54, 95% CI -0.70 to -0.38, p<0.01) were inversely associated with peak exercise capacity. Inflammation and p16INK4a expression, a biomarker of cellular senescence, are associated with lower exercise capacity in childhood cancer survivors, suggesting potential targets or outcome measures for interventions designed to prevent or remediate accelerated physiologic ageing in this population.

Does walking during chemotherapy impact p16INK4a levels in women with early breast cancer.

Increased p16INK4a (p16) expression is directly related to cellular senescence and is a robust biomarker of aging in humans. Prior studies have shown that levels of p16 dramatically increase in breast cancer patients who have received adjuvant chemotherapy. This study investigated whether moderate physical activity during chemotherapy would attenuate the expected rise in p16 expression. Participants were women with Stage I-III breast cancer enrolled in a walking study for the duration of their chemotherapy (NCT02167932, NCT02328313, NCT03761706). Participants were asked to walk at least 30 min or 6200 steps/day following a structured walking program and to wear an activity tracker. p16 mRNA levels were measured in peripheral blood T-cells before chemotherapy initiation and at approximately 6 months after last chemotherapy treatment (mean 200 days, SD 40 days). In total, 141 participants met inclusion criteria and 10% (n=14) averaged > 6200 steps/day. There was no significant association of daily steps with change in p16 levels pre- to post-chemotherapy (Pearson correlation coefficient=0.11, p=0.17). After adjusting for age, stage, anthracycline-based chemotherapy, and baseline p16, the change in log2 p16 for each 1000 steps was estimated to be 0.03 (p=0.35). Most participants were sedentary prior to chemotherapy and achieved modest levels of physical activity during treatment. A self-guided walking program achieved only modest levels of physical activity and was unable to ameliorate chemotherapy-induced change in p16 levels in women undergoing chemotherapy for early-stage breast cancer. More structured and vigorous exercise programs should be tested for a more definitive exploration of their impact on post-chemotherapy p16 levels.

Upregulation of p16INK4A in Peripheral White Blood Cells as a Novel Screening Marker for Colorectal Carcinoma.

Screening of colorectal cancer (CRC) is important for the early detection. CRC is relating to aging and immuno-senescence. One such senescent marker is p16INK4A expression in immune cells. The objective of the study is to investigate the protein expression of p16INK4A in peripheral white blood cells as a screening marker for colorectal cancer. A case-control studies were conducted. Cases were patients with colorectal cancer and controls were matched with cases based on age and sex. Peripheral blood was collected from patients and controls and the protein p16INK4A was measured with immunofluorescent techniques. The p16INK4A levels from cases and controls were evaluated using ROC analysis to be used as a screening marker in CRC patients. Mean fluorescent intensity of p16INK4A of cases and controls were analyzed in CD45+, CD3+ or CD14+ cells. The p16INK4A levels of cases were also correlated with clinical data. Statistically significant increased expression of p16INK4A levels were found in cases compared to controls. p16INK4A in peripheral immune cells had 78% sensitivity and 71% specificity which can possibly be used as a diagnosis tool for colorectal cancer. P16INK4A-positive cell percentage and mean florescent intensity were significantly higher in CD45+ cells, CD3 positive cells and CD14 positive cells. No significant correlation was observed with the clinical data and p16INK4A level of CRC patients. The significant increase of p16 INK4A expression level in peripheral immune cells represents potential for use as a CRC screening marker.

Overexpression of adrenomedullin (ADM) alleviates the senescence of human dental pulp stem cells by regulating the miR-152/CCNA2 pathway

ABSTRACT The limitation of human dental pulp stem cells (DPSCs), which have potential application value in regenerative medicine, is that they are prone to age in vitro. Studies have shown adrenomedullin (ADM) is believed to promote the proliferation of human DPSCs, but whether it can also affect aging remains to be investigated. A lentivirus vector was used to construct human DPSCs overexpressing ADM. Senescence tests were carried out on cells of the 7th and 15th passage. Transcriptome analysis was conducted to analyze microRNA expression regulation changes after human DPSCs overexpressed ADM. H2O2 induced the aging model of human DPSCs, and we examined the mechanism of recovery of aging through transfection experiments with miR-152 mimic, pCDH-CCNA2, and CCNA2 siRNA. Overexpression of ADM significantly upregulated the G2/M phase ratio of human DPSCs in natural passage culture (P = 0.001) and inhibited the expression of p53 (P = 0.014), P21 WAF1 (P = 0.015), and P16 INK4A (P = 0.001). Decreased ROS accumulation was observed in human DPSCs during long-term natural passage (P = 0.022). Transcriptome analysis showed that miR-152 was significantly upregulated during human DPSC senescence (P = 0.001) and could induce cell senescence by directly targeting CCNA2. Transfection with miR-152 mimic significantly reversed the inhibitory effect of ADM overexpression on p53 (P = 0.006), P21 WAF1 (P = 0.012), and P16 INK4A (P = 0.01) proteins in human DPSCs (H2O2-induced). In contrast, pCDH-CCNA2 weakened the effect of the miR-152 mimic, thus promoting cell proliferation and antiaging. ADM-overexpressing human DPSCs promote cell cycle progression and resist cellular senescence through CCNA2 expression promotion by inhibiting miR-152.

HPV infection and 5mC/5hmC epigenetic markers in penile squamous cell carcinoma: new insights into prognostics

BackgroundPenile cancer is one of the most aggressive male tumors. Although it is preventable, the main etiologic causes are lifestyle behaviors and viral infection, such as human papillomavirus (HPV). Long-term epigenetic changes due to environmental factors change cell fate and promote carcinogenesis, being an important marker of prognosis. We evaluated epidemiological aspects of penile squamous cell carcinoma (SCC) and the prevalence of HPV infection using high-risk HPV (hrHPV) and p16INK4A expression of 224 participants. Global DNA methylation was evaluated through 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC).ResultsThe incidence of HPV was 53.2% for hrHPV and 22.32% for p16INK4a. hrHPV was not related to systemic or lymph node metastasis and locoregional recurrence, nor influenced the survival rate. P16INK4a seems to be a protective factor for death, which does not affect metastasis or tumor recurrence. Lymph node and systemic metastases and locoregional recurrence increase the risk of death. An increased 5mC mark was observed in penile SCC regardless of HPV infection. However, there is a reduction of the 5hmC mark for p16INK4a + (P = 0.024). Increased 5mC/5hmC ratio (> 1) was observed in 94.2% of penile SCC, irrespective of HPV infection. Despite the increase in 5mC, it seems not to affect the survival rate (HR = 1.06; 95% CI 0.33–3.38).ConclusionsP16INK4a seems to be a good prognosis marker for penile SCC and the increase in 5mC, an epigenetic mark of genomic stability, may support tumor progression leading to poor prognosis.

P16 Regulates Transcription Factor Sp1 Function to Affect Outcome in Oropharyngeal Cancer

<h3>Purpose/Objective(s)</h3> Our group has previously demonstrated that p16 activates a ubiquitin mediated pathway to repress homologous recombination via activation of HUWE1, potentially via the transcription factor Sp1. However, the specific mechanism of this interaction and its clinical significance remain to be determined. We performed <i>in vitro</i> analysis of p16 and Sp1 interaction in multiple contexts and performed correlative analysis using the head and neck TCGA clinical data, as well as analysis of HPV+ and HPV- oropharyngeal cancer (OPC) treated with chemoradiation (CRT) to evaluate the combined importance of p16 and Sp1 on outcome. We hypothesized that p16 increases the Sp1-HUWE1 interaction. <h3>Materials/Methods</h3> For clinical outcome, 100 patients with oropharyngeal cancer evenly divided between p16+ and p16- and treated with chemoradiation were evaluated for gene expression using targeted multiplex analysis platform. Additional clinical correlative analyses between HPV, p16, HUWE1 and Sp1 were performed using the TCGA Head and Neck cohort (n=530). UM-SCC-47 p16 CRISPR KO and HN5 cells harboring doxycycline-inducible p16 were used to perform <i>in vitro</i> studies. Immunoprecipitation (IP) was used to confirm the protein-protein interaction between protein p16 and transcription factor Sp1. Chromatin immunoprecipitation (ChIP) assay was used to determine the existence of the Sp1 protein-HUWE1 DNA promoter interaction. <h3>Results</h3> Sp1 (p=7.9e⁻¹⁶) and HUWE1 (p=7.9e⁻⁴) were expressed at significantly higher levels in HPV+ tumors and Sp1 and HUWE1 gene expression were highly correlated (p=5.3e10⁻⁴⁵) in the TCGA Head and Neck cohort. In addition, Sp1 expression was significantly higher in p16 positive OPC tumors in a separate patient cohort (p=3e⁻³). In OPC patents treated uniformly with CRT, the combination of p16 and Sp1 expression were associated with reduced locoregional recurrence following CRT (p=7e⁻⁶), effectively delineating good prognosis p16- patients and poor prognosis p16+ patients. IP of p16 in HN5 cells showed increased interaction with Sp1 after doxycycline-induced p16INK4a expression, and in combination with IR. ChIP assay demonstrated SP1 dependent enrichment in 2 of 5 GC box regions of the human HUWE1 promoter. More importantly, these SP1 protein-HUWE1 promoter interactions were significantly enhanced after p16 overexpression. <h3>Conclusion</h3> We show Sp1 directly interacts with HUWE1 promoter resulting in an increase of HUWE1 expression, and this interaction is mediated by p16. This increase of HUWE1 expression then leads to down-regulation of homologous recombination, sensitizing cells to DNA damage. Clinical data shows the combined expression of p16, Sp1 and HUWE1 are highly correlated and that the combination of p16 and Sp1 may be an effective biomarker in OPC.