Search

Background: The senescence marker p16INK4a, which constitutes part of the genome 9p21.3 cardiovascular disease (CVD) risk allele, is believed to play a role in foam cells formation. This study aims to unravel the role of p16INK4a in mediating macrophage foam cells formation, cellular senescence, and autophagy lysosomal functions. Methods: The mammalian expression plasmid pCMV-p16INK4a was used to induce p16INK4a overexpression in THP-1 macrophages. Next, wild-type and p16INK4a-overexpressed macrophages were incubated with oxidized LDL to induce foam cells formation. Lipids accumulation was evaluated using Oil-red-O staining and cholesterol efflux assay, as well as expression of scavenger receptors CD36 and LOX-1. Cellular senescence in macrophage foam cells were determined through analysis of senescence-associated β-galactosidase activity and other SASP factors expression. Meanwhile, autophagy induction was assessed through detection of autophagosome formation and LC3B/p62 markers expression. Results: The findings showed that p16INK4a enhanced foam cells formation with increased scavenger receptors CD36 and LOX-1 expression and reduced cholesterol efflux in THP-1 macrophages. Besides, β-galactosidase activity was enhanced, and SASP factors such as IL-1α, TNF-α, and MMP9 were up-regulated. In addition, p16INK4a is also shown to induce autophagy, as well as increasing autophagy markers LC3B and p62 expression. Conclusions: This study provides insights on p16INK4a in mediating macrophages foam cells formation, cellular senescence, and foam cells formation. [ABSTRACT FROM AUTHOR]

The evidence of the correlation between cellular senescence and aging has increased in research with animal models. These models have been intentionally generated to target and regulate cellular senescent cells with the promoter activity of p16Ink4a or p19Arf, genes that are highly expressed in aging cells. However, the senolytic efficiency in various organs and cells from these models represents unexpected variation and diversity in some cases. We have generated a novel knock‐in model, p16tdT‐hDTR mice, which possess tdTomato and human diphtheria toxin receptor (hDTR) downstream of Cdkn2a, an endogenous p16Ink4a gene. We successfully demonstrated that p16‐derived tdTomato and hDTR expressions are observed in these mouse embryo fibroblasts and following treatment with diphtheria toxin (DT) eliminates those cells. Furthermore, we demonstrated the efficacy of eliminating p16‐positive cells in vivo, and also observed a tendency to decrease their cutaneous SA‐β‐gal activity after subcutaneous DT injection into p16tdT‐hDTR mice. In particular, comprehensive gene expression analysis in skin revealed that upregulated genes related to lipid metabolisms with aging exhibited remarkable expressions under the senolysis. These results clearly unveiled p16‐positive senescent cells contribute to age‐related changes in skin. [ABSTRACT FROM AUTHOR]

Fragmentation/loss of the structural protein elastin represents the precipitating event translating to aortic expansion and subsequent aneurysm formation. The present study tested the hypothesis that greater protein expression of tissue inhibitor of matrix metalloproteinase‐1 (TIMP‐1) and neointimal growth secondary to a reduction of medial elastin content represent sex‐dependent events limiting aortic vessel expansion in females. TIMP‐1 protein levels were higher in the ascending aorta of female versus male patients diagnosed with a bicuspid aortic valve (BAV). The latter paradigm was recapitulated in the aorta of adult male and female rats complemented by greater TIMP‐2 expression in females. CaCl2 (0.5 M) treatment of the infrarenal aorta of adult male and female rats increased the in situ vessel diameter and expansion was significantly smaller in females despite a comparable reduction of medial elastin content. The preferential appearance of a neointimal region of the CaCl2‐treated infrarenal aorta of female rats may explain in part the smaller in situ expansion and neointimal growth correlated positively with the % change of the in situ diameter. Neointimal formation was secondary to a significant increase in the density of medial/neointimal vascular smooth muscle cells (VSMCs) that re‐entered the G2‐M phase whereas VSMC cell cycle re‐entry was attenuated in the CaCl2‐treated infrarenal aorta of male rats. Thus, greater TIMP‐1 expression in the aorta of female BAV patients may prevent excessive elastin fragmentation and preferential neointimal growth following CaCl2‐treatment of the infrarenal aorta of female rats represents a sex‐dependent biological event limiting vessel expansion secondary to a significant loss of the structural protein. [ABSTRACT FROM AUTHOR]

Aging progresses through the interaction of metabolic processes, including changes in the immune and endocrine systems. Glucocorticoids (GCs), which are regulated by the hypothalamic–pituitary–adrenal (HPA) axis, play an important role in regulating metabolism and immune responses. However, the age‐related changes in the secretion mechanisms of GCs remain elusive. Here, we found that corticosterone (CORT) secretion follows a circadian rhythm in young mice, whereas it oversecreted throughout the day in aged mice >18 months old, resulting in the disappearance of diurnal variation. Furthermore, senescent cells progressively accumulated in the zF of the adrenal gland as mice aged beyond 18 months. This accumulation was accompanied by an increase in the number of Ad4BP/SF1 (SF1), a key transcription factor, strongly expressing cells (SF1‐high positive: HP). Removal of senescent cells with senolytics, dasatinib, and quercetin resulted in the reduction of the number of SF1‐HP cells and recovery of CORT diurnal oscillation in 24‐month‐old mice. Similarly, administration of a neutralizing antibody against IL1β, which was found to be strongly expressed in the adrenocortical cells of the zF, resulted in a marked decrease in SF1‐HP cells and restoration of the CORT circadian rhythm. Our findings suggest that the disappearance of CORT diurnal oscillation is a characteristic of aging individuals and is caused by the secretion of IL1β, one of the SASPs, from senescent cells that accumulate in the zF of the adrenal cortex. These findings provide a novel insight into aging. Age‐related hypersecretory GCs could be a potential therapeutic target for aging‐related diseases. [ABSTRACT FROM AUTHOR]

Simple Summary: The skin is the outermost barrier of the human body and consists of different layers and cell types. Several environmental and genetic factors can induce skin aging and age-related diseases. One of the main problems in skin aging is that senescent cells are accumulated and secrete factors, which can induce senescence in other tissues. Many researchers are trying to identify treatment modalities (known as senotherapies) to eliminate the senescent cells and reverse the aging process for chronic age-related diseases. The aim of this study is to address the mechanisms that induce senescence and the molecules with potential HAFi effects that are currently investigated for skin aging. Further studies should be conducted to elucidate all the effects of current senotherapies on the skin and other organs. Current data suggest that ongoing research projects in the field may lead to the discovery of new effective anti-senescence strategies in the skin. The skin is the layer of tissue that covers the largest part of the body in vertebrates, and its main function is to act as a protective barrier against external environmental factors, such as microorganisms, ultraviolet light and mechanical damage. Due to its important function, investigating the factors that lead to skin aging and age-related diseases, as well as understanding the biology of this process, is of high importance. Indeed, it has been reported that several external and internal stressors contribute to skin aging, similar to the aging of other tissues. Moreover, during aging, senescent cells accumulate in the skin and express senescence-associated factors, which act in a paracrine manner on neighboring healthy cells and tissues. In this review, we will present the factors that lead to skin aging and cellular senescence, as well as ways to study senescence in vitro and in vivo. We will further discuss the adverse effects of the accumulation of chronic senescent cells and therapeutic agents and tools to selectively target and eliminate them. [ABSTRACT FROM AUTHOR]

Objective: A few studies indicate that human papillomavirus (HPV) induces aberrant expression of microRNAs (miRNAs) and correlate this with p16INK4a in oral dysplasia (OD) and oral squamous cell carcinoma (OSCC). Therefore, this study aimed to evaluate the expression of miRNA-21, miRNA-22, and miRNA-224 by q-PCR and the p16INK4a by immunohistochemical (IHC) as markers for HPV-positive OSCC and OD in comparison to controls as miRNA expression can be altered by the HPV oncogenes and hence can be used as a biomarker for HPV positive cases. Material and Methods: Fifty-two specimens were collected from archived paraffin blocks for patients aged between 19 and 88 (31 males and 21 females) from various oral sites. They were examined by IHC using p16INK4a, by RT-PCR for the detection of HPV (6, 11, 16, 18, 31, 33, 35, 42, 43, 44, 45, 52, 53, 56), and by q-PCR for the expression of miRNA-21, miRNA-22, and miRNA-224 in positive specimens. Results: Out of the 15 OD, three were positive by both techniques. Meanwhile, 17 out of all OSCC specimens showed intense nuclear and cytoplasmic staining by p16INK4a, and only 16 were also positive by RT-PCR. However, all control specimens were negative. MiRNA-21, miRNA-22, and miRNA-224 were overexpressed in 3 specimens of OD and 16 of OSCC. Conclusion: MiRNA-21, miRNA-22, and miRNA-224, besides p16INK4a, could be used as indicators for HPV-associated OD and OSCC as their expression is attributed to the HPV oncoprotein. Further studies using follow-up data should be done to correlate it with miRNA overexpression. [ABSTRACT FROM AUTHOR]

Copyright of Journal of China Medical University is the property of Journal of China Medical University Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

AbstractSulfur mustard (SM) exposure has delayed harmful effects, including premature biological aging. This study aimed to evaluate the expression of aging markers (i.e., ANRIL, P16INK4a, TBX2, and TERRA) and assess their correlation with the severity of SM exposure in the long term. The study was conducted on two volunteer groups. 1) SM-exposed group, exposed to SM once in 1987 during the war; divided into three subgroups based on the injury severity, asymptomatic (without any clinical signs), mild, and severe; 2) Non-exposed group. In the SM-exposed group, ANRIL transcript was decreased, especially in subgroups of mild and severe. TBX2 transcript was also decreased in the total SM-exposed group. This decrease was more significant in the mild and severe subgroups than in asymptomatic ones. P16INK4a transcript was increased in the SM-exposed group, especially in the asymptomatic subgroup. The increase in TERRA transcript was also significant in all subgroups. There was a positive correlation between the TERRA transcript and the severity of injury, while this correlation was negative for the ANRIL. It is concluded that the delayed toxicity of SM may be associated with dysregulation of aging markers leading to premature cellular aging. These markers’ alterations differed according to the severity of SM injury. [ABSTRACT FROM AUTHOR]

Anterior cruciate ligament (ACL) injuries pose a significant challenge due to their limited healing potential, often resulting in premature arthritis. The factors and mechanisms contributing to this inadequate healing process remain elusive. During the acute phase of injury, ACL tissues express elevated periostin levels that decline over time. The functional significance of periostin in ligament biology remains understudied. In this study, we investigated the functional and mechanistic implications of periostin deficiency in ACL biology, utilizing ligament fibroblasts derived from patients and a murine model of ACL rupture. Our investigations unveiled that periostin knockdown compromised fibroblast growth characteristics, hindered the egress of progenitor cells from explants, and arrested cell‐cycle progression, resulting in the accumulation of cells in the G0/G1 phase and moderate apoptosis. Concurrently, a significant reduction in the expression of cell‐cycle and matrix‐related genes was observed. Moreover, periostin deficiency triggered apoptosis through STAT3Y705/p38MAPK signaling and induced cellular senescence through increased production of reactive oxygen species (ROS). Mechanistically, inhibition of ROS production mitigated cell senescence in these cells. Notably, in vivo data revealed that ACL in Postn−/− mice exhibited a higher tearing frequency than wild‐type mice under equivalent loading conditions. Furthermore, injured ACL with silenced periostin expression, achieved through nanoparticle‐siRNA complex delivery, displayed an elevated propensity for apoptosis and senescence compared to intact ACL in C57BL/6 mice. Together, our findings underscore the pivotal role of periostin in ACL health, injury, and potential for healing. [ABSTRACT FROM AUTHOR]

Drug delivery systems (DDSs) enable the controlled release of drugs in the body. DDSs have attracted increasing attention for the treatment of various disorders, including cancer, inflammatory diseases, and age-related diseases. With recent advancements in our understanding of the molecular mechanisms of aging, new target molecules and drug delivery carriers for age-related diseases have been reported. In this review, we will summarize the recent research on DDSs for age-related diseases and identify DDS strategies in the treatment of age-related diseases. [ABSTRACT FROM AUTHOR]

Background: The association between p16INK4a and p21, a marker of cellular senescence, and the Immunoscore, an immunological prognostic indicator, in rectal cancer patients undergoing curative surgery were investigated. Methods: A total of 82 patients who underwent curative surgery for rectal cancer were evaluated. The resected specimens were analyzed for p16INK4a, p21, CD3 and CD8 expression by immunohistochemistry. Immunoscore was calculated on the basis of CD3 and CD8 expressions. The clinicopathological characteristics and long-term outcomes were evaluated. Results: Among the 82 patients, 24 (29.3%) were p16INK4a-positive and 11 (13.4%) were p21-positive. The patients were classified into the following five Immunoscore groups (IS0–5). IS0, IS1 and IS2 were classified as the low Immunoscore group (45 patients, 54.9%) and IS3 and IS4 as the high Immunoscore group (37 patients, 45.1%). There was no significant difference in age, sex, body mass index, American Society of Anesthesiologists physical status, depth of invasion of the tumor, lymph node metastasis and histological classification of the tumor with p16INK4a or p21 expression or Immunoscore. p16INK4a-positive expression and low Immunoscore each showed a tendency to indicate poor prognosis of disease-free survival (DFS). Patients with the combination of p16INK4a and p21 positivity and with p16INK4a positivity and low Immunoscore showed significantly poor prognosis of DFS. Patients with p21 positive positivity and low Immunoscore tended to have worse DFS. Conclusions: p16INK4a, p21 and Immunoscore may be prognostic indicators of rectal cancer. The combination of them may provide more accurate prognostic prediction than either factor alone. [ABSTRACT FROM AUTHOR]

Objective: A few studies indicate that human papillomavirus (HPV) induces aberrant expression of microRNAs (miRNAs) and correlate this with p16INK4a in oral dysplasia (OD) and oral squamous cell carcinoma (OSCC). Therefore, this study aimed to evaluate the expression of miRNA-21, miRNA-22, and miRNA-224 by q-PCR and the p16INK4a by immunohistochemical (IHC) as markers for HPV-positive OSCC and OD in comparison to controls as miRNA expression can be altered by the HPV oncogenes and hence can be used as a biomarker for HPV positive cases. Material and Methods: Fifty-two specimens were collected from archived paraffin blocks for patients aged between 19 and 88 (31 males and 21 females) from various oral sites. They were examined by IHC using p16INK4a, by RT-PCR for the detection of HPV (6, 11, 16, 18, 31, 33, 35, 42, 43, 44, 45, 52, 53, 56), and by q-PCR for the expression of miRNA-21, miRNA-22, and miRNA-224 in positive specimens. Results: Out of the 15 OD, three were positive by both techniques. Meanwhile, 17 out of all OSCC specimens showed intense nuclear and cytoplasmic staining by p16INK4a, and only 16 were also positive by RT-PCR. However, all control specimens were negative. MiRNA-21, miRNA-22, and miRNA-224 were overexpressed in 3 specimens of OD and 16 of OSCC. Conclusion: MiRNA-21, miRNA-22, and miRNA-224, besides p16INK4a, could be used as indicators for HPV-associated OD and OSCC as their expression is attributed to the HPV oncoprotein. Further studies using follow-up data should be done to correlate it with miRNA overexpression.

Colorectal cancer (CRC) is one of the most prevalent cancer types worldwide, exhibiting significant variance in incidence rates across different ethnicities and geographical regions. Notably, there is a rising incidence of CRC among younger adults, particularly evident in advanced stages, with a more pronounced trend observed in developing nations. Epigenetic alterations potentially play a role in the early onset of CRC and could elucidate interpopulation disparities. This study aimed to examine DNA methylation levels in the tumor suppressor genes MLH1 and p16INK4a, comparing Nepalese and Swedish patients with CRC.

Simple Summary: Ten-eleven-translocation (TET) 2 is implicated in human cancers such as lung, breast, skin, and kidney cancers, T-cell lymphomas, and leukemia. It is unclear whether TET2 is involved in the re-expression of the p16 tumor suppressor, which was partially silenced by CDKN2A gene methylation. The effect of DNA methyltransferase (DNMT) 1 gene status on TET2 expression following DNMT inhibitor treatment remains unknown in cancer. We found that deleting the DNMT1 gene made cancer cells prone to increased TET2 expression and re-expression of p16 after drug treatment. TET2 is involved in the demethylation of the CDKN2A gene and activation of p16 expression, and concomitant resistance to DNMT inhibitors when DNMT1 is obliterated. The long-sought data for the first time revealed a link between TET2 expression and the activation of p16 expression. The findings should have an impact on reactivating tumor suppressors and cancer treatment. Background: Ten-eleven-translocation (TET) 2 is a member of the TET family of proteins (TET1-3). DNMT1 gene deletion confers resistance to DNA methyltransferase (DNMT) inhibitors in colorectal, breast, and ovarian cancer cells. Currently, the effect of DNMT1 gene status on TET2 phenotype following DNMT inhibitor treatment is unclear in human malignancies. Methods: Human colorectal carcinoma HCT116 cells (DNMT+/+) and their isogenic DNMT1 knockout (DNMT1–/–) counterpart were treated with DNMT inhibitors. Expression of TET2 and tumor suppressor (p16ink4A and p15ink4B) proteins were examined by Western blot. Apoptosis and CDKN2A promoter demethylation following drug treatment were detected by Annexin-V apoptosis assay and methylation-specific PCR. Results: TET2 expression was robustly increased in DNMT1−/− cells by 0.5 µM and 5 µM decitabine and azacitidine treatment. Augmentation of TET2 expression was accompanied by re-expression of p16ink4A and p15ink4B proteins and CDKN2A promoter demethylation. TET2 upregulation and tumor suppressor re-expression were associated with resistance conferred by DNMT1 deletion. Treatment with 5-aza-4′-thio-2′-deoxycytidine at a low 0.5 µM dose only upregulated TET2 and reduced CDKN2A promoter methylation, and re-expression of p16ink4A in DNMT1−/− cells. DNMT inhibitors showed minimal effects on TET2 upregulation and re-expression of tumor suppressor proteins in cells with intact DNMT1. Conclusions: DNMT1 gene deletion made cancer cells prone to TET2 upregulation and activation of tumor suppressor expression upon DNMT inhibitor challenge. TET2 augmentation is concomitant with resistance to DNMT inhibitors in a DNMT1-deleted state. [ABSTRACT FROM AUTHOR]

Abstract Age-dependent accumulation of amyloid plaques in patients with sporadic Alzheimer’s disease (AD) is associated with reduced amyloid clearance. Older microglia have a reduced ability to phagocytose amyloid, so phagocytosis of amyloid plaques by microglia could be regulated to prevent amyloid accumulation. Furthermore, considering the aging-related disruption of cell cycle machinery in old microglia, we hypothesize that regulating their cell cycle could rejuvenate them and enhance their ability to promote more efficient amyloid clearance. First, we used gene ontology analysis of microglia from young and old mice to identify differential expression of cyclin-dependent kinase inhibitor 2A (p16ink4a), a cell cycle factor related to aging. We found that p16ink4a expression was increased in microglia near amyloid plaques in brain tissue from patients with AD and 5XFAD mice, a model of AD. In BV2 microglia, small interfering RNA (siRNA)-mediated p16ink4a downregulation transformed microglia with enhanced amyloid phagocytic capacity through regulated the cell cycle and increased cell proliferation. To regulate microglial phagocytosis by gene transduction, we used poly (D,L-lactic-co-glycolic acid) (PLGA) nanoparticles, which predominantly target microglia, to deliver the siRNA and to control microglial reactivity. Nanoparticle-based delivery of p16ink4a siRNA reduced amyloid plaque formation and the number of aged microglia surrounding the plaque and reversed learning deterioration and spatial memory deficits. We propose that downregulation of p16ink4a in microglia is a promising strategy for the treatment of Alzheimer’s disease.

Introduction: Carcinoma cervix is the most common cancer and the invasive stage of cervical cancer is preceded by pre-invasive phases and all the phases can be picked up and diagnosed by Pap smear. p16INK4 is a tumour suppressor protein which serves as a surrogate marker for the oncogenic activities of Human Papilloma virus and its overexpression is well established in cervical intraepithelial neoplasia (CIN) and invasive cancers. Aim: To evaluate the pattern of cervical cytology at a tertiary care hospital and to correlate the findings with histopathological diagnosis and to determine the importance of p16INK4a as a diagnostic marker of HPV by IHC staining method. Materials and Methods: This is a two year retrospective study done on Pap smears reported between April 2021 to May 2023 in the Department of Pathology, Gayatri Vidya Parishad Institute of Healthcare and Medical Technology, Visakhapatnam. Frequency and percentage statistics was used to present the results. Results: A total of 844 Pap smears were included in the study, out of which 52 were cytologically abnormal. Biopsy was done on 42 cases, out of which 33 cases showed concordance which included18 LSIL, 8 HSIL, 5 squamous cell carcinoma and 2 cases of adenocarcinoma, among them p16INK4a was found to be positive in 6 cases of LSIL, 5 cases of HSIL and 4 cases of SCC and 2 cases of adenocarcinoma. Conclusion: Pap smear is a sensitive and cost effective screening modality for premalignant and malignant lesions of cervix and expression of p16 as IHC marker has the potential to accredit the lesions with an increased risk of progression to high-grade lesions. [ABSTRACT FROM AUTHOR]

Background and Objectives: Human papillomavirus (HPV) was previously investigated in lung cancer with wide inter-geographic discrepancies. p16INK4a has been used as a surrogate for detecting high-risk HPV (HR-HPV) in some cancer types. This study assessed the evidence of HPV in non-small-cell lung cancer (NSCLC) among Jordanian patients, investigated the expression of p16INK4a, and evaluated its prognostic value and association with HPV status. Materials and Methods: The archived samples of 100 patients were used. HPV DNA detection was performed by real-time polymerase chain reaction (RT-PCR). p16INK4a expression was assessed by immunohistochemistry (IHC). The Eighth American Joint Committee on Cancer protocol (AJCC) of head and neck cancer criteria were applied to evaluate p16INK4a positivity considering a moderate/strong nuclear/cytoplasmic expression intensity with a distribution in ≥75% of cells as positive. Results: HPV DNA was detected in 5% of NSCLC cases. Three positive cases showed HR-HPV subtypes (16, 18, 52), and two cases showed the probable HR-HPV 26 subtype. p16INK4a expression was positive in 20 (20%) NSCLC cases. None of the HPV-positive tumors were positive for p16INK4a expression. A statistically significant association was identified between p16INK4a expression and the pathological stage (p = 0.029) but not with other variables. No survival impact of p16INK4a expression was detected in NSCLC cases as a group; however, it showed a statistically significant association with overall survival (OS) in squamous cell carcinoma (SqCC) cases (p = 0.033). Conclusions: This is the first study to assess HPV and p16INK4a expression in a Jordanian population. HPV positivity is rare in NSCLC among a Jordanian subpopulation. P16 INK4a reliability as a surrogate marker for HPV infection in lung cancer must be revisited. [ABSTRACT FROM AUTHOR]

Age-dependent accumulation of amyloid plaques in patients with sporadic Alzheimer's disease (AD) is associated with reduced amyloid clearance. Older microglia have a reduced ability to phagocytose amyloid, so phagocytosis of amyloid plaques by microglia could be regulated to prevent amyloid accumulation. Furthermore, considering the aging-related disruption of cell cycle machinery in old microglia, we hypothesize that regulating their cell cycle could rejuvenate them and enhance their ability to promote more efficient amyloid clearance. First, we used gene ontology analysis of microglia from young and old mice to identify differential expression of cyclin-dependent kinase inhibitor 2A (p16ink4a), a cell cycle factor related to aging. We found that p16ink4a expression was increased in microglia near amyloid plaques in brain tissue from patients with AD and 5XFAD mice, a model of AD. In BV2 microglia, small interfering RNA (siRNA)-mediated p16ink4a downregulation transformed microglia with enhanced amyloid phagocytic capacity through regulated the cell cycle and increased cell proliferation. To regulate microglial phagocytosis by gene transduction, we used poly (D,L-lactic-co-glycolic acid) (PLGA) nanoparticles, which predominantly target microglia, to deliver the siRNA and to control microglial reactivity. Nanoparticle-based delivery of p16ink4a siRNA reduced amyloid plaque formation and the number of aged microglia surrounding the plaque and reversed learning deterioration and spatial memory deficits. We propose that downregulation of p16ink4a in microglia is a promising strategy for the treatment of Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Purpose: The ocular surface microenvironment changes with aging. However, it remains unclear if cellular senescence influences the ocular surface. We investigated the presence of p16INK4a-expressing senescent cells in healthy human conjunctiva. Study design: Clinical and experimental. Methods: Healthy conjunctival tissue samples were obtained from middle-aged and elderly subjects. RT-qPCR was performed to assess the expression of senescence markers CDKN2A (p16INK4a) and CDKN1A (p21CIP1/WAF1) and immunostaining was performed to examine the expression of the senescence marker p16INK4a, stem cell markers Ki67 and p63, tight-junction marker ZO-1. Results: Our study involved 19 conjunctival tissue samples (10 elderly and 9 middle-aged), mean age [elderly: 75.8 ± 3.7 years (72–81), middle-aged: 52.7 ± 7 years (38–59)], sex (elderly: 3 men, 7 women; middle-aged: 3 men, 6 women). The expression of p16INK4a was significantly increased at the RNA level in the elderly compared to middle-aged (p < 0.05). Positivity rate of p16INK4a was significantly elevated in the elderly (15.0 ± 7.8%) compared to middle-aged (0.2 ± 0.6%) (p < 0.05). Positivity rate of Ki67and p63 was significantly reduced in the elderly (1.7 ± 1.7% and 16.5 ± 9.5%) compared to middle-aged (3.9 ± 1.8% and 24.7 ± 5.7%) (p < 0.05). ZO-1 expression was reduced in tissue samples showing p16INK4a-positivity but retained in tissue samples in which p16INK4a was undetectable. Conclusions: Senescent cells accumulate with age in the conjunctival epithelium, accompanied by a decrease in Ki67, p63 and ZO-1 expressing cells. [ABSTRACT FROM AUTHOR]

Introduction: Tissue expression of P16ink4A is correlated with cervical lesions. In this study we determined the association between serum P16ink4A concentrations and cervical lesions among women attending the cervical cancer clinic at Mbarara Regional Hospital (MRRH) South Western Uganda. Material and Methods: We recruited 90 cervical intraepithelial neoplasia (CIN) cases, 90 cervical cancer (CC) cases before treatment and 90 controls. Clinical and demographic data were recorded. Serum P16ink4A concentrations were measured by quantitative Elisa. Cases were confirmed with cytology and/or histology. Descriptive statistics and logistic regression were done with STATA 17 and P-values of

The skin is the layer of tissue that covers the largest part of the body in vertebrates, and its main function is to act as a protective barrier against external environmental factors, such as microorganisms, ultraviolet light and mechanical damage. Due to its important function, investigating the factors that lead to skin aging and age-related diseases, as well as understanding the biology of this process, is of high importance. Indeed, it has been reported that several external and internal stressors contribute to skin aging, similar to the aging of other tissues. Moreover, during aging, senescent cells accumulate in the skin and express senescence-associated factors, which act in a paracrine manner on neighboring healthy cells and tissues. In this review, we will present the factors that lead to skin aging and cellular senescence, as well as ways to study senescence in vitro and in vivo. We will further discuss the adverse effects of the accumulation of chronic senescent cells and therapeutic agents and tools to selectively target and eliminate them.

Drug delivery systems (DDSs) enable the controlled release of drugs in the body. DDSs have attracted increasing attention for the treatment of various disorders, including cancer, inflammatory diseases, and age-related diseases. With recent advancements in our understanding of the molecular mechanisms of aging, new target molecules and drug delivery carriers for age-related diseases have been reported. In this review, we will summarize the recent research on DDSs for age-related diseases and identify DDS strategies in the treatment of age-related diseases.

Epigenetic alterations of tumor suppressor genes (TSG) involved in the onset and progression of Breast Cancer (BC) may serve as biomarkers for early detection and prediction of disease prognosis. We have herein tried to determine the methylation status of TSG, p16INK4a, in our 50 BC patients and their association with clinicopathological parameters. The methylation status of the p16INK4a gene in fresh tissue samples from 50 patients with BC was assessed by methylation-specific polymerase chain reaction (MS-PCR). The mean age of BC patients was 49.30 ± 9.75 years. Of 50 BC samples tested, 21 (42%) had methylated p16INK4a gene. p16INK4a gene hypermethylation was significantly associated with age ≤ 50 years, premenopausal status and advanced BC stage. Multivariate analysis revealed a strong association between advanced BC stage (Stage III and Stage IV) and p16INK4a hypermethylation (P = 0.008, RR = 5.996, 95% CI = 1.581—22.739). p16INK4a methylation was significantly associated with Triple Negative BC (TNBC) (P = 0.045, OR = 4.181, 95% CI = 1.030–16.981). These findings indicate that p16INK4a hypermethylation frequently occurs in BC. Hypermethylation of p16INK4a in young, premenopausal, TNBC and with advance stage in BC patients suggests its association with aggressive BC. [ABSTRACT FROM AUTHOR]

Contrast-induced acute kidney injury (CI-AKI) is the third leading cause of hospital-acquired acute kidney injury. Cellular senescence is associated with CI-AKI. P16INK4a (p16) is a cell cycle regulator and link to aging and senescence. We found that the expression of p16 was elevated in CI-AKI renal tissues, however its role in CI-AKI remains insufficiently understood. In this study, we used p16 knockout (p16KO) mice and wild-type (WT) littermates to establish CI-AKI mice model to elucidate the impact of p16 on CI-AKI. The results showed that serum creatinine (SCr), blood urea nitrogen (BUN), and serum neutrophil gelatinase-associated lipocalin (NGAL) levels were markedly reduced in p16KO CI-AKI mice. Both immunohistochemistry and western blot analyses confirmed that p16 knockout alleviated renal cell apoptosis. Furthermore, interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) were attenuated by downregulating NLRP3 and NF-κB inflammasomes. Additionally, ROS levels were diminished via activating Nrf2/Keap-1 pathway in p16KO CI-AKI mice. Collectively, our findings suggest that p16 deletion exerts protective effects against apoptosis, inflammation, and oxidative stress in CI-AKI mice model, p16 deletion might be a potential therapeutic strategy for ameliorating CI-AKI.

Introduction. High-risk human papilloma virus (Hpv), especially genotype 16, causes oropharyngeal squamous cell carcinoma (OSCC). It is detected in about 70 % of tumors developing from lymphoid tissue of the tonsils or the base of the tongue. Due to the increased number of Hpv-positive OSCC, Hpv status is considered a marker of OSCC clinical outcome. Easy testing, low cost, reliability, and high sensitivity of immunohistochemical analysis for p16INk4a allowed to widely use this method for Hpv status determination.Aim. To determine the association between programmed death-ligand 1 (pD-L1) and p53 expression and presence of indirect Hpv marker – p16INk4a – in patients with OSCC.Materials and methods. The study included 76 patients with OSCC т1–4N0–3m0 who received treatment at the Republican Specialized Scientific and practical medical Center of Oncology and Radiology (n = 37) and its Tashkent branch (n = 39) between 2015 and 2020. for all selected patients, retrospective immunohistochemical analysis for the presence of p16INk4a, pD-L1 and р53 in tumor samples fixed with formalin in paraffin blocks was performed. In our work, immunohistochemical examination for p16INk4a was the only relevant tool for Hpv status determination. To reinforce its prognostic significance, we used additional molecular markers pD-L1 and p53 which play an important role in carcinogenic transformation and OSCC progression.Results. The results of immunohistochemical analysis showed that p16INk4a overexpression was accompanied by positive pD-L1 reaction in 46 % (6/13) of cases; there were no cases of positive expression of mutant type p53. wild type p53 was identified in only 1 (3 %) case in combination with p16INk4a overexpression.Conclusion. The developed panel consisting of 3 molecular markers (p16INk4a, pD-L1 and р53) may open new horizons in accurate prognosis, risk stratification and understanding of OSCC molecular signature. This, in turn, will help clinicians in selection of individual therapy strategies for treatment de-escalation and outcome optimization.

Abstract Background The role of human parvovirus B19 (B19V) infection in malignant and benign lesions such as head and neck squamous cell carcinomas (HNSCCs) and oral mucocele lesions has not been established. Herein, we examined, for the first time, the presence of B19V in HNSCCs from Iranian subjects. Methods One hundred and eight HNSCC specimens were analyzed for the presence of B19V using nested polymerase chain reaction (nPCR) and TaqMan quantitative PCR assays. Immunohistochemistry procedures were performed to evaluate the expression of B19V VP1/VP2 proteins, p16INK4a, and NF-κB in tumor tissues and their adjacent non-tumor tissues. In addition, 40 oral mucocele, 30 oral buccal mucosa swabs, and 30 nasopharyngeal swabs obtained from healthy adults were analyzed as controls. Results B19V DNA was detected in 36.1% of HNSCCs. Further, 23.3% of HNSCC specimens showed immunoreactivity against B19V VP1/VP2 proteins. There was a significant difference in the frequency of B19V DNA-positive cases between the patient and control groups (p

Background: Ten-eleven-translocation (TET) 2 is a member of the TET family of proteins (TET1-3). DNMT1 gene deletion confers resistance to DNA methyltransferase (DNMT) inhibitors in colorectal, breast, and ovarian cancer cells. Currently, the effect of DNMT1 gene status on TET2 phenotype following DNMT inhibitor treatment is unclear in human malignancies. Methods: Human colorectal carcinoma HCT116 cells (DNMT+/+) and their isogenic DNMT1 knockout (DNMT1–/–) counterpart were treated with DNMT inhibitors. Expression of TET2 and tumor suppressor (p16ink4A and p15ink4B) proteins were examined by Western blot. Apoptosis and CDKN2A promoter demethylation following drug treatment were detected by Annexin-V apoptosis assay and methylation-specific PCR. Results: TET2 expression was robustly increased in DNMT1−/− cells by 0.5 µM and 5 µM decitabine and azacitidine treatment. Augmentation of TET2 expression was accompanied by re-expression of p16ink4A and p15ink4B proteins and CDKN2A promoter demethylation. TET2 upregulation and tumor suppressor re-expression were associated with resistance conferred by DNMT1 deletion. Treatment with 5-aza-4′-thio-2′-deoxycytidine at a low 0.5 µM dose only upregulated TET2 and reduced CDKN2A promoter methylation, and re-expression of p16ink4A in DNMT1−/− cells. DNMT inhibitors showed minimal effects on TET2 upregulation and re-expression of tumor suppressor proteins in cells with intact DNMT1. Conclusions: DNMT1 gene deletion made cancer cells prone to TET2 upregulation and activation of tumor suppressor expression upon DNMT inhibitor challenge. TET2 augmentation is concomitant with resistance to DNMT inhibitors in a DNMT1-deleted state.

Chondrocyte senescence is an important mechanism underlying osteoarthritis in the senile population and is characterized by reduced expressions of the extracellular matrix proteins. The involvement of glycolysis and the tricarboxylic acid cycle in the development of osteoarthritis is inclusive. The present study aims to investigate the role of the glycolytic enzyme M2 isoform of pyruvate kinase (PKM2) in chondrocytes in senescence and inflammation. Primary chondrocytes are isolated from the knee joints of neonatal mice. Small interfering RNAs (siRNAs) against PKM2 are transfected using lipofectamine. RNA sequencing is conducted in primary chondrocytes with the PKM2 gene deleted. Cell apoptosis, autophagy, reactive oxygen species measurement, and senescent conditions are examined. The glycolytic rate in cells is measured by Seahorse examination. Interleukin 1-β (IL-1β) increases the protein expressions of matrix metallopeptidases (MMP)13 and PKM2 and reduces the protein expression of collagen type II (COL2A1) in primary chondrocytes. Silencing of PKM2 alters the protein expressions of MMP13, PKM2, and COL2A1 in the same pattern in quiescent and stimulated chondrocytes. RNA sequencing analysis reveals that PKM2 silencing reduces senescent biomarker p16INK4a expression. Compared with low-passage chondrocytes, high-passage chondrocytes exhibit increased expression of p16 INK4a and reduced expression of COL2A1. Silencing of PKM2 reduces SA-β-Gal signals and increases COL2A1 expression in high-passage chondrocytes. Seahorse assay reveals that PKM2 deletion favors the tricarboxylic acid cycle in mitochondria in low- but not in high-passage chondrocytes. In summary, the glycolytic enzyme PMK2 modulates chondrocyte senescence but does not participate in the regulation of inflammation.

Cellular senescence is one of the important mechanisms of skin aging. In a recent study, we have shown that in patients with dermatoporosis, an extreme senescence condition of the skin, cells positive for p16Ink4a, a biomarker of senescence, were significantly increased in the epidermis. Senescent cells can develop a senescence-associated secretory phenotype (SASP) comprising pro-inflammatory cytokines, chemokines, and other soluble factors, leading to chronic inflammation and tissue dysfunction. These senescent cells and SASP pathways represent therapeutic targets for the development of senotherapeutics either by inducing selective cell death of senescent cells called senolytics, or suppressing markers of the SASP, called senomorphics. In this study where we conducted a retrospective immunohistochemical analysis of p16Ink4a expression in the skin samples of dermatoporosis patients included in a previous clinical study, we describe the senotherapeutic effect of retinaldehyde (RAL) and intermediate-size hyaluronate fragments (HAFi). Topical application of RAL and HAFi significantly reduced the number of p16Ink4a-positive cells in the epidermis and dermis in dermatoporosis patients which also showed a significant clinical improvement.

Background: The role of human parvovirus B19 (B19V) infection in malignant and benign lesions such as head and neck squamous cell carcinomas (HNSCCs) and oral mucocele lesions has not been established. Herein, we examined, for the first time, the presence of B19V in HNSCCs from Iranian subjects. Methods: One hundred and eight HNSCC specimens were analyzed for the presence of B19V using nested polymerase chain reaction (nPCR) and TaqMan quantitative PCR assays. Immunohistochemistry procedures were performed to evaluate the expression of B19V VP1/VP2 proteins, p16INK4a, and NF-κB in tumor tissues and their adjacent non-tumor tissues. In addition, 40 oral mucocele, 30 oral buccal mucosa swabs, and 30 nasopharyngeal swabs obtained from healthy adults were analyzed as controls. Results: B19V DNA was detected in 36.1% of HNSCCs. Further, 23.3% of HNSCC specimens showed immunoreactivity against B19V VP1/VP2 proteins. There was a significant difference in the frequency of B19V DNA-positive cases between the patient and control groups (p < 0.0001). Moreover, comparing tumoral tissues and their adjacent non-tumor tissues in terms of immunoreactivity against B19V structural proteins, a significant association was found between tumor tissues and B19V infection (p < 0.0001). Finally, investigating the simultaneous presence of B19V and high-risk human papillomaviruses (HPV) DNA, we found a significant association between these two viral infections in HNSCCs (p = 0.031). Conclusions: To sum up, B19V was frequently present in HNSCC tissues of Iranian patients but mostly absent in the adjacent non-tumor tissues as well as oral mucocele lesions, buccal, and nasopharyngeal swabs of healthy subjects. HPV possibly contributes to B19V persistence in HNSCC tissues. Additional research is required to investigate potential etiological or cofactor roles of B19V in the development of HNSCCs. [ABSTRACT FROM AUTHOR]

Glaucoma is a leading cause of irreversible blindness, with advanced age being the single most significant risk factor. However, the mechanisms underlying the relationship between aging and glaucoma remain unclear. Genome‐wide association studies (GWAS) have successfully identified genetic variants strongly associated with increased glaucoma risk. Understanding how these variants function in pathogenesis is crucial for translating genetic associations into molecular mechanisms and, ultimately, clinical applications. The chromosome 9p21.3 locus is among the most replicated glaucoma risk loci discovered by GWAS. Nonetheless, the absence of protein‐coding genes in the locus makes interpreting the disease association challenging, leaving the causal variant and molecular mechanism elusive. In this study, we report the identification of a functional glaucoma risk variant, rs6475604. By employing computational and experimental methods, we demonstrated that rs6475604 resides in a repressive regulatory element. Risk allele of rs6475604 disrupts the binding of YY1, a transcription factor known to repress the expression of a neighboring gene in 9p21.3, p16INK4A, which plays a crucial role in cellular senescence and aging. These findings suggest that the glaucoma disease variant contributes to accelerated senescence, providing a molecular link between glaucoma risk and an essential cellular mechanism for human aging. [ABSTRACT FROM AUTHOR]

Glaucoma is a leading cause of irreversible blindness, with advanced age being the single most significant risk factor. However, the mechanisms underlying the relationship between aging and glaucoma remain unclear. Genome‐wide association studies (GWAS) have successfully identified genetic variants strongly associated with increased glaucoma risk. Understanding how these variants function in pathogenesis is crucial for translating genetic associations into molecular mechanisms and, ultimately, clinical applications. The chromosome 9p21.3 locus is among the most replicated glaucoma risk loci discovered by GWAS. Nonetheless, the absence of protein‐coding genes in the locus makes interpreting the disease association challenging, leaving the causal variant and molecular mechanism elusive. In this study, we report the identification of a functional glaucoma risk variant, rs6475604. By employing computational and experimental methods, we demonstrated that rs6475604 resides in a repressive regulatory element. Risk allele of rs6475604 disrupts the binding of YY1, a transcription factor known to repress the expression of a neighboring gene in 9p21.3, p16INK4A, which plays a crucial role in cellular senescence and aging. These findings suggest that the glaucoma disease variant contributes to accelerated senescence, providing a molecular link between glaucoma risk and an essential cellular mechanism for human aging. [ABSTRACT FROM AUTHOR]

Background and Objectives: Human papillomavirus (HPV) was previously investigated in lung cancer with wide inter-geographic discrepancies. p16INK4a has been used as a surrogate for detecting high-risk HPV (HR-HPV) in some cancer types. This study assessed the evidence of HPV in non-small-cell lung cancer (NSCLC) among Jordanian patients, investigated the expression of p16INK4a, and evaluated its prognostic value and association with HPV status. Materials and Methods: The archived samples of 100 patients were used. HPV DNA detection was performed by real-time polymerase chain reaction (RT-PCR). p16INK4a expression was assessed by immunohistochemistry (IHC). The Eighth American Joint Committee on Cancer protocol (AJCC) of head and neck cancer criteria were applied to evaluate p16INK4a positivity considering a moderate/strong nuclear/cytoplasmic expression intensity with a distribution in ≥75% of cells as positive. Results: HPV DNA was detected in 5% of NSCLC cases. Three positive cases showed HR-HPV subtypes (16, 18, 52), and two cases showed the probable HR-HPV 26 subtype. p16INK4a expression was positive in 20 (20%) NSCLC cases. None of the HPV-positive tumors were positive for p16INK4a expression. A statistically significant association was identified between p16INK4a expression and the pathological stage (p = 0.029) but not with other variables. No survival impact of p16INK4a expression was detected in NSCLC cases as a group; however, it showed a statistically significant association with overall survival (OS) in squamous cell carcinoma (SqCC) cases (p = 0.033). Conclusions: This is the first study to assess HPV and p16INK4a expression in a Jordanian population. HPV positivity is rare in NSCLC among a Jordanian subpopulation. P16 INK4a reliability as a surrogate marker for HPV infection in lung cancer must be revisited.

ABSTRACT BACKGROUND: Managing cervical intraepithelial neoplasia grade 2 (CIN2) is challenging, considering the CIN2 regression rate, perinatal risks associated with excisional procedures, and insufficient well-established risk factors to predict progression. OBJECTIVES: To determine the ability of p16INK4a and Ki-67 staining in biopsies diagnosed with CIN2 to identify patients with higher-grade lesions (CIN3 or carcinoma). DESIGN AND SETTING: Cross-sectional study conducted at a referral center for treating uterine cervical lesions. METHODS: In 79 women, we analyzed the correlation of p16INK4a and Ki-67 expression in CIN2 biopsies with the presence of a higher-grade lesions, as determined via histopathology in surgical specimens from treated women or via two colposcopies and two cytological tests during follow-up for untreated women with at least a 6-month interval. The expression of these two biomarkers was verified by at least two independent pathologists and quantified using digital algorithms. RESULTS: Thirteen (16.8%) women with CIN2 biopsy exhibited higher-grade lesions on the surgical excision specimen or during follow-up. p16INK4a expression positively and negatively predicted the presence of higher-grade lesions in 17.19% and 86.67% patients, respectively. Ki-67 expression positively and negatively predicted the presence of higher-grade lesions in 40% and 88.24% patients, respectively. CONCLUSIONS: Negative p16INK4a and Ki67 immunohistochemical staining can assure absence of a higher-grade lesion in more than 85% of patients with CIN2 biopsies and can be used to prevent overtreatment of these patients. Positive IHC staining for p16INK4a and Ki-67 did not predict CIN3 in patients with CIN2 biopsies.

Cellular senescence is one of the important mechanisms of skin aging. In a recent study, we have shown that in patients with dermatoporosis, an extreme senescence condition of the skin, cells positive for p16Ink4a, a biomarker of senescence, were significantly increased in the epidermis. Senescent cells can develop a senescence-associated secretory phenotype (SASP) comprising pro-inflammatory cytokines, chemokines, and other soluble factors, leading to chronic inflammation and tissue dysfunction. These senescent cells and SASP pathways represent therapeutic targets for the development of senotherapeutics either by inducing selective cell death of senescent cells called senolytics, or suppressing markers of the SASP, called senomorphics. In this study where we conducted a retrospective immunohistochemical analysis of p16Ink4a expression in the skin samples of dermatoporosis patients included in a previous clinical study, we describe the senotherapeutic effect of retinaldehyde (RAL) and intermediate-size hyaluronate fragments (HAFi). Topical application of RAL and HAFi significantly reduced the number of p16Ink4a-positive cells in the epidermis and dermis in dermatoporosis patients which also showed a significant clinical improvement. [ABSTRACT FROM AUTHOR]

Simple Summary: The prevalence of oropharyngeal squamous cell carcinomas (OPSCCs) driven by human papillomavirus (HPV) infection has a wide geographical variability. In addition, several authors have reported steadily increasing prevalence rates over the past two decades. It is important to know the epidemiological landscape of these tumors both to estimate the reliability of diagnostic tests and to guide public health choices aimed at primary prevention. In this retrospective study, we observed a significant increase in the prevalence of HPV-driven OPSCC from 12% during 2000–2006 to 50% during 2019–2022. The use of p16INK4a overexpression as a surrogate marker of transforming HPV infection should consider the prevalence rates of HPV-driven OPSCC as this significantly impacts on its positive predictive value. Health policies should actively promote vaccination against HPV in both females and males also for the prevention of OPSCC. Background: The prevalence and incidence of oropharyngeal squamous cell carcinomas (OPSCCs) driven by human papillomavirus (HPV) infection are increasing worldwide, being higher in high-income countries. However, data from Italy are scanty. p16INK4a overexpression is the standard in determining HPV-driven carcinogenesis, but disease prevalence impacts on its positive predictive value. Methods: This is a multicenter retrospective study enrolling 390 consecutive patients aged ≥18 years, diagnosed with pathologically confirmed OPSCC in Northeastern Italy between 2000 and 2022. High-risk HPV-DNA and p16INK4a status were retrieved from medical records or evaluated in formalin-fixed paraffin-embedded specimens. A tumor was defined as HPV-driven when double positive for high-risk HPV-DNA and p16INK4a overexpression. Results: Overall, 125 cases (32%) were HPV-driven, with a significant upward temporal trend from 12% in 2000–2006 to 50% in 2019–2022. The prevalence of HPV-driven cancer of the tonsil and base of the tongue increased up to 59%, whereas it remained below 10% in other subsites. Consequently, the p16INK4a positive predictive value was 89% for the former and 29% for the latter. Conclusions: The prevalence of HPV-driven OPSCC continued to increase, even in the most recent period. When using p16INK4a overexpression as a surrogate marker of transforming HPV infection, each institution should consider the subsite-specific prevalence rates of HPV-driven OPSCC as these significantly impact on its positive predictive value. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Background Squamous cell carcinoma of the external auditory canal (EACSCC) is an uncommon tumor and responsible for no more than 0.2% of all the head and neck malignancies. Although there is remarkable research evidence exhibiting that high-risk human papillomavirus (HPV) accounts for considerable head and neck malignancies, its role in the pathogenesis of EACSCC is yet to be determined. Methods We evaluated 16 patients with EACSCC treated at our department. We employed PCR to assay for high-risk subtypes of HPV. Two pathologists reviewed the histopathological staining via hematoxylin and eosin along with immunohistochemical staining of p16INK4a and Ki‑67. Results Detection of HPV DNA was done via PCR in 3 (18.75%) patients, and 8 (50%) positive (+) cases were determined via p16INK4a immunostaining. Besides, 3 (37.5%) individuals were HPV positive as per p16INK4a PCR results. In addition, all of the p16INK4a-positive specimens were diagnosed as moderately differentiated carcinomas. Conclusions Expression of Ki-67 was related to HPV status. This is the first report implicating high-risk HPV in squamous cell carcinoma of the external auditory canal. However, p16INK4a immunostaining is a suspectable approach for diagnosing HPV for EACSCC. In addition, HPV might enhance an elevated proliferation rate in EACSCC, illustrated via expression of Ki-67. Graphical Abstract

Secondary prevention of cervical cancer in the conditions of opportunistic cervical screening by co-testing in 5618 women over 30 years of age allowed to identify a high-risk group of cervical cancer for further examination using viral load assessment, immunocytochemical determination of the level of cancer proteins p16INK4a and Ki-67, extended video colposcopy and cervical biopsy with histological examination of surgical material. The obtained data demonstrated the expediency of changing the testing strategy by integrating the methods tested in the framework of opportunistic screening into the nationwide population screening program for cervical cancer.

Tingting Liu, Mengjie Chen, Xueqin Li, He Wang Department of Gynecology, Guangxi Medical University Cancer Hospital, Nanning, Guangxi, People’s Republic of ChinaCorrespondence: He Wang, Department of Gynecology, Guangxi Medical University Cancer Hospital, No. 71 Hedi Road, Nanning, Guangxi, People’s Republic of China, Email wanghe10430@126.comPurpose: To study the expression of serum folate and red blood cell (RBC) folate, folate receptor α (FRα), p16INK4a (p16), and Ki-67 at different levels of cervical intraepithelial neoplasia (CIN) and then analyze their role in the progression of CIN and their value as an early warning indicator of CIN progression.Patients and methods: We randomly collected the data of patients at the Department of Gynecology in Guangxi Medical University Affiliated Cancer Hospital from January 2016 to December 2018: Normal controls (149 cases), CIN1 (150 cases), CIN2 (100 cases), and CIN3 (101 cases). [Ethical approval by Ethics Committee of the Second Hospital of Shanxi Medical University. (2013) No. (001– 1)]. The expression of serum folate and RBC folate was detected by the chemiluminescence method, while the expression of FRα, p16, and Ki-67 was detected by Streptavidin-Perosidase (SP) immunohistochemistry.Results: There was no statistically significant difference in serum folate levels between different grades of CIN (P=0.784), but the RBC folate levels were statistically significant (P=0.015), and there was a negative correlation between RBC folate levels and CIN lesion grades (P< 0.05). The FRα, p16, and Ki-67 levels in the CIN group were significantly different from those in the normal control group (P < 0.01), and a positive correlation was found (P < 0.01); FRα positivity (P=0.000), Ki-67 positivity (P=0.000), and low-level RBC folate (P=0.000) were independent risk factors for the progression of CIN; these indicators were combined to establish a random forest (RF) model in which the Ki-67+FRα model was used as the early warning model of CIN progression.Conclusion: RBC folate, FRα, p16, and Ki-67 can be used as valuable clinical test indicators for predicting the progression of CIN; the combined detection model of Ki-67+FRα can be used as an early warning model for predicting the progression of CIN.Keywords: cervical intraepithelial neoplasia, folate, folate receptor α, p16INK4a, Ki-67

Purpose: Despite the beneficial effects of chemotherapy, therapy-induced senescence (TIS) manifests itself as an undesirable byproduct. Preclinical evidence suggests that tumor cells undergoing TIS can re-emerge as more aggressive divergents and contribute to recurrence, and thus, senolytics were proposed as adjuvant treatment to eliminate senescent tumor cells. However, the identification of TIS in clinical samples is essential for the optimal use of senolytics in cancer therapy. In this study, we aimed to detect and quantify TIS using matched breast cancer samples collected pre- and post-exposure to neoadjuvant chemotherapy (NAC). Methods: Detection of TIS was based on the change in gene and protein expression levels of three senescence-associated markers (downregulation of Lamin B1 and Ki-67 and upregulation of p16INK4a). Results: Our analysis revealed that 23 of 72 (31%) of tumors had a shift in the protein expression of the three markers after exposure to NAC suggestive of TIS. Gene expression sets of two independent NAC-treated breast cancer samples showed consistent changes in the expression levels of LMNB1, MKI67 and CDKN2A. Conclusions: Collectively, our study shows a more individualized approach to measure TIS hallmarks in matched breast cancer samples and provides an estimation of the extent of TIS in breast cancer clinically. Results from this work should be complemented with more comprehensive identification approaches of TIS in clinical samples in order to adopt a more careful implementation of senolytics in cancer treatment. [ABSTRACT FROM AUTHOR]

The dysregulation of exosomal microRNAs (miRNAs) plays a crucial role in the development and progression of cancer. This study investigated the role of a newly identified serum exosomal miRNA miR-4256 in gastric cancer (GC) and the underlying mechanisms. The differentially expressed miRNAs were firstly identified in serum exosomes of GC patients and healthy individuals using next-generation sequencing and bioinformatics. Next, the expression of serum exosomal miR-4256 was analyzed in GC cells and GC tissues, and the role of miR-4256 in GC was investigated by in vitro and in vivo experiments. Then, the effect of miR-4256 on its downstream target genes HDAC5/p16INK4a was studied in GC cells, and the underlying mechanisms were evaluated using dual luciferase reporter assay and Chromatin Immunoprecipitation (ChIP). Additionally, the role of the miR-4256/HDAC5/p16INK4a axis in GC was studied using in vitro and in vivo experiments. Finally, the upstream regulators SMAD2/p300 that regulate miR-4256 expression and their role in GC were explored using in vitro experiments. miR-4256 was the most significantly upregulated miRNA and was overexpressed in GC cell lines and GC tissues; in vitro and in vivo results showed that miR-4256 promoted GC growth and progression. Mechanistically, miR-4256 enhanced HDAC5 expression by targeting the promoter of the HDAC5 gene in GC cells, and then restrained the expression of p16INK4a through the epigenetic modulation of HDAC5 at the p16INK4a promoter. Furthermore, miR-4256 overexpression was positively regulated by the SMAD2/p300 complex in GC cells. Our data indicate that miR-4256 functions as an oncogene in GC via the SMAD2/miR-4256/HDAC5/p16INK4a axis, which participates in GC progression and provides novel therapeutic and prognostic biomarkers for GC. [ABSTRACT FROM AUTHOR]

Simple Summary: CDK4/6 inhibitors (CDKis) are a first-line treatment for metastatic hormone-receptor-positive breast cancer, but resistance frequently develops. One potential resistance mechanism could be induction of cellular senescence in non-tumor tissues. The aim of our study was to identify CDKi-induced changes to host tissues that impact metastasis. Using mouse models, we found that pretreatment with palbociclib can increase metastatic seeding of CDKi-resistant mammary cancer cells in lungs and that this can be mitigated by eliminating senescent host cells. We describe palbociclib-induced gene expression changes in lungs that correlate with this effect and reveal altered intra-lung immune populations. Senescent endothelial cells are identifiable within lung metastases of mice pretreated with palbociclib. Palbociclib-treated primary endothelial cell lines become senescent and increase tumor cell migration and monocyte trans-endothelial invasion. These studies describe how CDKi-induced cellular senescence in host tissues could affect metastasis in breast cancer, which remains a key obstacle to achieving long-term survival. Background: CDK4/6 inhibitors (CDKi) have improved disease control in hormone-receptor-positive, HER2-negative metastatic breast cancer, but most patients develop progressive disease. Methods: We asked whether host stromal senescence after CDK4/6 inhibition affects metastatic seeding and growth of CDKi-resistant mammary cancer cells by using the p16-INK-ATTAC mouse model of inducible senolysis. Results: Palbociclib pretreatment of naïve mice increased lung seeding of CDKi-resistant syngeneic mammary cancer cells, and this effect was reversed by depletion of host senescent cells. RNA sequencing analyses of lungs from non-tumor-bearing p16-INK-ATTAC mice identified that palbociclib downregulates immune-related gene sets and gene expression related to leukocyte migration. Concomitant senolysis reversed a portion of these effects, including pathway-level enrichment of TGF-β- and senescence-related signaling. CIBERSORTx analysis revealed that palbociclib alters intra-lung macrophage/monocyte populations. Notably, lung metastases from palbociclib-pretreated mice revealed senescent endothelial cells. Palbociclib-treated endothelial cells exhibit hallmark senescent features in vitro, upregulate genes involved with the senescence-associated secretory phenotype, leukocyte migration, and TGF-β-mediated paracrine senescence and induce tumor cell migration and monocyte trans-endothelial invasion in co-culture. Conclusions: These studies shed light on how stromal senescence induced by palbociclib affects lung metastasis, and they describe palbociclib-induced gene expression changes in the normal lung and endothelial cell models that correlate with changes in the tumor microenvironment in the lung metastatic niche. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the frequency and clinicopathological characteristics of HPV-independent cervical squamous cell carcinoma (CSCC). A total of 3869 patients with CSCC from 2017 to 2021 were searched. p16INK4a immunochemistry (IHC), two HPV-DNA(L1) polymerase chain reactions and HPV mRNA in situ hybridization were performed. Viral copies were detected using the 21 HPV quantitative test. Six cases showed negative results in all four assays (group 1, 0.16%). Twenty-seven cases showed discordant results (group 2), and 3836 cases presented all-positive results (group 3). p16INK4a IHC showed similar sensitivity, specificity, and positive predictive value compared to the other three direct HPV assays. 21 HPV genotyping showed 100% of negative predictive value. HPV copies were extremely lower in Group 2 than in Group 3 (P < 0.01), but were not significantly different from those in Group 1. Older age, advanced FIGO stage (III-IV) and abnormal p53 (p53abn) IHC were independent predictors of HPV-negative status in univariate and multivariate logistic regression. Group 2 had similar proportions of age >60 years and p53abn IHC with Group 1, but had fewer cases with advanced FIGO stage (P < 0.05) and TILs (P < 0.05). Groups 1 and 2 had worse disease-free survival (DFS) and disease-specific survival (DSS) than Group 3 (P < 0.01), while no significant difference was found between these two groups. HPV-negative status was a risk factor for both DFS (P < 0.05) and DSS (P < 0.01) in univariate but not multivariate Cox regression. Joint detection of multiple technologies and evaluation of clinicopathological characteristics discriminate between HPV-independent and low-copy HPV-associated CSCC cases that present similar prognoses. Additional attention should be paid to these low-copy HPV-associated cases in clinical practice. • Joint detection of HPV-testing technologies discriminated between HPV-independent and low-copy HPV-associated CSCC cases. • p16INK4a IHC was an equally useful HPV-positive indicator compared with the other three direct HPV tests. • Older age, advanced FIGO stage (III-IV) and abnormal p53 (p53abn) IHC were the independent risk factors for HPV-negative status. • Low-copy HPV-associated cases deserved attention by showing similarly poor prognosis with the HPV-independent CSCC. [ABSTRACT FROM AUTHOR]

Objectives: The role of Human papillomavirus (HPV) in the oral squamous cell carcinoma (OSCC) has not been completely elucidated. The purpose of the present study was to investigate the prevalence and localization of HPV‐16 virus in OSCC and to correlate HPV‐16 positivity and p16INK4A expression with the clinical and pathological features of OSCC. Methods: The archives of Oral Pathology at the University of Florida, College of Dentistry were accessed for demographic, clinical, histopathological data, and slides of 114 OSCC patients. HPV‐16 positivity of OSCC was evaluated by p16INK4A immunohistochemistry (IHC) and HPV‐16 E6/E7mRNA by in situ hybridization (ISH). Results: Out of 114 consecutive pathological slides of OSCC, 16 samples (14%) showed positivity for p16INK4A by IHC and 14 samples (12%) were positive for HPV‐16 E6/E7mRNA ISH and the Positivity showed a significant correlation with the patients' age, alcohol consumption, and the degree of OSSC differentiation. The hard palate showed the highest positivity of p16INK4A IHC and HPV‐16 mRNA ISH (38%, 36% respectively). Conclusion: HPV‐16 is a significant factor in oral carcinogenesis. We recommend using p16INK4A as a surrogate marker for HPV detection in OSCC, which can be complemented by RNA ISH for the identification of HPV subtypes. [ABSTRACT FROM AUTHOR]