Project description:Visual interpretation of cervical biopsies is subjective and variable, generally showing fair to moderate inter-reader agreement in distinguishing high from low grade cervical intraepithelial neoplasia (CIN). We investigated the performance of two objective p16 quantitative tests in comparison with visual assessment: (i) p16-mRNA assay and (ii) digital analysis of sections stained for p16 protein. The primary analysis considered 232 high-risk human papilloma virus positive (HPV+) samples from diagnostic cervical specimens. A p16 RT-qPCR (p16-mRNA assay) was run on mRNA extracted from formalin-fixed paraffin-embedded sections. Two p16 immunohistochemistry (IHC) readings, a visual read by a histopathologist (Visual IHC) and a digital read of a high-resolution scan (Digital IHC), were done on adjacent sections. The worst reviewed CIN grade (agreed by at least two histopathologists) from up to two biopsies and a loop excision was taken, with CIN2/3 as the primary endpoint. Visual IHC attained a specificity of 70% (95%CI 61-77) for 85% (95%CI 77-91%) sensitivity. The four-point Visual IHC staining area under the curve (AUC) was 0.77 (95%CI 0.71-0.82), compared with 0.71 (95%CI 0.64-0.77) for p16-mRNA and 0.67 (95%CI 0.60-0.74) for Digital IHC. Spearman rank-order correlations were: visual to p16-mRNA 0.41, visual to digital 0.49 and p16-mRNA to digital: 0.22. The addition of p16-mRNA assay to visual reading of p16 IHC improved the AUC from 0.77 to 0.84 (p = 0.0049). p16-mRNA testing may be complementary to visual IHC p16 staining for a more accurate diagnosis of CIN, or perhaps a substitute in locations with a lack of skilled pathologists.
Project description:Senecavirus A (SVA) is an oncolytic RNA virus, and it is the ideal oncolytic virus that can be genetically engineered for editing. However, there has not been much exploration into creating SVA viruses that carry antitumor genes to increase their oncolytic potential. The construction of SVA viruses carrying antitumor genes that enhance oncolytic potential has not been fully explored. In this study, a recombinant SVA-CH-01-2015 virus (p15A-SVA-clone) expressing the human p16INK4A protein, also known as cell cycle-dependent protein kinase inhibitor 2A (CDKN2A), was successfully rescued and characterized. The recombinant virus, called SVA-p16, exhibited similar viral replication kinetics to the parent virus, was genetically stable, and demonstrated enhanced antitumor effects in Ishikawa cells. Additionally, another recombinant SVA virus carrying a reporter gene (iLOV), SVA-iLOV, was constructed and identified using the same construction method as an auxiliary validation. Collectively, this study successfully created a new recombinant virus, SVA-p16, that showed increased antitumor effects and could serve as a model for further exploring the antitumor potential of SVA as an oncolytic virus.
Project description:Exposure of murine and human tissues to ionizing radiation (IR) induces the expression of p16INK4a, a tumor suppressor gene and senescence/aging biomarker. Increased p16INK4a expression is often delayed several weeks post exposure to IR. In this context, it remains unclear if it occurs to suppress aberrant cellular growth of potentially transformed cells or is simply a result of IR-induced loss of tissue homeostasis. To address this question, we used a conditional p16INK4a null mouse model and determined the impact of p16INK4a inactivation long-term post exposure to IR. We found that, in vitro, bone marrow stromal cells exposed to IR enter DNA replication following p16INK4a inactivation. However, these cells did not resume growth; instead, they mostly underwent cell cycle arrest in G2. Similarly, delayed inactivation of p16INK4a in mice several weeks post exposure to IR resulted in increased BrdU incorporation and cancer incidence. In fact, we found that the onset of tumorigenesis was similar whether p16INK4a was inactivated before or after exposure to IR. Overall, our results suggest that IR-induced p16INK4a dependent growth arrest is reversible in mice and that sustained p16INK4a expression is necessary to protect against tumorigenesis.
Project description:The tumor suppressor protein p16INK4a (p16) is a well-established hallmark of aging that induces cellular senescence in response to stress. Previous studies have focused primarily on p16 regulation at the transcriptional level; comparatively little is known about the protein's intracellular localization and degradation. The autophagy-lysosomal pathway has been implicated in the subcellular trafficking and turnover of various stress-response proteins and has also been shown to attenuate age-related pathologies, but it is unclear whether p16 is involved in this pathway. Here, we investigate the role of autophagy, vesicular trafficking, and lysosomal degradation on p16 expression and localization in human epithelial cells. Time-lapse fluorescence microscopy using an endogenous p16-mCherry reporter revealed that serum starvation, etoposide, and hydrogen peroxide stimulate autophagy and drive p16 recruitment to acidic cytoplasmic vesicles within 4 hr. Blocking lysosomal proteases with leupeptin and ammonium chloride resulted in the accumulation of p16 within lysosomes and increased total p16 levels suggesting that p16 is degraded by this pathway. Furthermore, autophagy blockers chloroquine and bafilomycin A1 caused p16 aggregation within stalled vesicles containing autophagosome marker LC3. Increase of p16 within these vesicles coincided with the accumulation of LC3-II. Knockdown of autophagosome chaperone p62 attenuated the formation of p16 aggregates in lysosomes, suggesting that p16 is targeted to these vesicles by p62. Taken together, these results implicate the autophagy pathway as a novel regulator of p16 degradation and localization, which could play a role in the etiology of cancer and age-related diseases.
Project description:The expression of markers of cellular senescence increases exponentially in multiple tissues with aging. Age-related physiological changes may contribute to adverse outcomes in cancer survivors. To investigate the impact of high dose chemotherapy and stem cell transplantation on senescence markers in vivo, we collected blood and clinical data from a cohort of 63 patients undergoing hematopoietic cell transplantation. The expression of p16INK4a, a well-established senescence marker, was determined in T-cells before and 6months after transplant. RNA sequencing was performed on paired samples from 8 patients pre- and post-cancer therapy. In patients undergoing allogeneic transplant, higher pre-transplant p16INK4a expression was associated with a greater number of prior cycles of chemotherapy received (p=0.003), prior autologous transplantation (p=0.01) and prior exposure to alkylating agents (p=0.01). Transplantation was associated with a marked increase in p16INK4a expression 6months following transplantation. Patients receiving autologous transplant experienced a larger increase in p16INK4a expression (3.1-fold increase, p=0.002) than allogeneic transplant recipients (1.9-fold increase, p=0.0004). RNA sequencing of T-cells pre- and post- autologous transplant or cytotoxic chemotherapy demonstrated increased expression of transcripts associated with cellular senescence and physiological aging. Cytotoxic chemotherapy, especially alkylating agents, and stem cell transplantation strongly accelerate expression of a biomarker of molecular aging in T-cells.
Project description:p16INK4a (CDKN2A) is a central tumor suppressor, which induces cell-cycle arrest and senescence. Cells expressing p16INK4a accumulate in aging tissues and appear in premalignant lesions, yet their physiologic effects are poorly understood. We found that prolonged expression of transgenic p16INK4a in the mouse epidermis induces hyperplasia and dysplasia, involving high proliferation rates of keratinocytes not expressing the transgene. Continuous p16INK4a expression increases the number of epidermal papillomas formed after carcinogen treatment. Wnt-pathway ligands and targets are activated upon prolonged p16INK4a expression, and Wnt inhibition suppresses p16INK4a-induced hyperplasia. Senolytic treatment reduces p16INK4a-expressing cell numbers, and inhibits Wnt activation and hyperplasia. In human actinic keratosis, a precursor of squamous cell carcinoma, p16INK4a-expressing cells are found adjacent to dividing cells, consistent with paracrine interaction. These findings reveal that chronic p16INK4a expression is sufficient to induce hyperplasia through Wnt-mediated paracrine stimulation, and suggest that this tumor suppressor can promote early premalignant epidermal lesion formation.
Project description:Cathepsin L (CTSL) has been implicated in aging and age-related diseases, such as cardiovascular diseases, specifically atherosclerosis. However, the underlying mechanism(s) is not well documented. Recently, we demonstrated a role of CUT-like homeobox 1 (CUX1) in regulating the p16INK4a-dependent cellular senescence in human endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) via its binding to an atherosclerosis-associated functional SNP (fSNP) rs1537371 on the CDKN2A/B locus. In this study, to determine if CTSL, which was reported to proteolytically activate CUX1, regulates cellular senescence via CUX1, we measured the expression of CTSL, together with CUX1 and p16INK4a, in human ECs and VSMCs undergoing senescence. We discovered that CUX1 is not a substrate that is cleaved by CTSL. Instead, CTSL is an upstream regulator that activates CUX1 transcription indirectly in a process that requires the proteolytic activity of CTSL. Our findings suggest that there is a transcription factor in between CTSL and CUX1, and cleavage of this factor by CTSL can activate CUX1 transcription, inducing endothelial senescence. Thus, our findings provide new insights into the signal transduction pathway that leads to atherosclerosis-associated cellular senescence.
Project description:To evaluate the possible involvement of epigenetic modulation by HPV16-p16INK4a in oral potentially malignant disorder (OPMD). We generated DNA-methylation profiles, according to p16INK4a expression and HPV16 genotype (positive or negative), of OPMD samples and p16INK4a-HPV16 negative samples (used as control), using reduced-representation bisulphite sequencing (RRBS-Seq- Illumina) technology. Twelve samples, four for each group, as follows: 1) p16INK4a+ HPV16+; 2) p16INK4a+ HPV16-; 3) p16INK4a- HPV16-, were analysed in triplicate for DNA-methylation profiles. Fifty-four per cent of DMRs were hypermethylated and 46% were hypomethylated. An increase in methylation of loci in OPMD was independent of the presence of HPV. The hypermethylated genes in HPV+ samples were associated with signalling pathways such as NICD traffics to nucleus, signalling by NOTCH1 (p = 0.008), Interferon-gamma (p = 0.008) and Interleukin-6 signalling (p = 0.027). The hypomethylated genes in HPV infection were associated with TRAF3-dependent IRF activation pathway (p = 0.002), RIG-I/MDA5 mediated induction of IFN-alpha/beta pathways (p = 0.005), TRAF6 mediated IRF7 activation (p = 0.009), TRIF-mediated TLR3/TLR4 signalling (p = 0.011) and MyD88-independent cascade release of apoptotic factors (p = 0.011). Protein association analysis of DMRs in OPMD revealed 19 genes involved in the cell cycle regulation, immune system, and focal adhesion. Aberrantly methylated loci in OPMD were observed in p16INK4a positive samples which suggests that a shift in global methylation status may be important for cancer progression. The results suggest that HPV infection in OPMD induces modulation of genes related to the immune system and regulation of the cellular cycle.
Project description:T-cell dysfunction arising upon repeated antigen exposure prevents effective immunity and immunotherapy. Using various clinically and physiologically relevant systems, we show that a prominent feature of PD-1-expressing exhausted T cells is the development of cellular senescence features both in vivo and ex vivo. This is associated with p16INK4a expression and an impaired cell cycle G1 to S-phase transition in repeatedly stimulated T cells. We show that these T cells accumulate DNA damage and activate the p38MAPK signaling pathway, which preferentially leads to p16INK4a upregulation. However, in highly dysfunctional T cells, p38MAPK inhibition does not restore functionality despite attenuating senescence features. In contrast, p16INK4a targeting can improve T-cell functionality in exhausted CAR T cells. Collectively, this work provides insights into the development of T-cell dysfunction and identifies T-cell senescence as a potential target in immunotherapy.
Project description:Oncogene induced senescence is a tumor suppressing defense mechanism, in which the cell cycle-dependent protein kinase (CDK) inhibitor p16INK4A (encoded by the CDKN2A gene) plays a key role. We previously reported that a transcriptional co-activator chromodomain helicase DNA binding protein 7 (CHD7) mediates oncogenic ras-induced senescence by inducing transcription of the p16INK4A gene. In the current study, we identified myeloid zinc finger 1 (MZF1) as the transcriptional factor that recruits CHD7 to the p16INK4A promoter, where it mediates oncogenic ras-induced p16INK4A transcription and senescence through CHD7, in primary human cells from multiple origins. Moreover, the expression of MZF1 is induced by oncogenic ras in senescent cells through the c-Jun and Ets1 transcriptional factors upon their activation by the Ras-Raf-1-MEK-ERK signaling pathway. In non-small cell lung cancer (NSCLC) and pancreatic adenocarcinoma (PAAD) where activating ras mutations occur frequently, reduced MZF1 expression is observed in tumors, as compared to corresponding normal tissues, and correlates with poor patient survival. Analysis of single cell RNA-sequencing data from PAAD patients revealed that among the tumor cells with normal RB expression levels, those with reduced levels of MZF1 are more likely to express lower p16INK4A levels. These findings have identified novel signaling components in the pathway that mediates induction of the p16INK4A tumor suppressor and the senescence response, and suggested that MZF1 is a potential tumor suppressor in at least some cancer types, the loss of which contributes to the inactivation of the p16INK4A/RB pathway and disruption of senescence in tumor cells with intact RB.