P16 Ink4a Research Articles

NKAP Regulates Senescence and Cell Death Pathways in Hematopoietic Progenitors.

NKAP is a multi-functional nuclear protein that has been shown to be essential for hematopoiesis. Deletion of NKAP in hematopoietic stem cells (HSCs) was previously found to result in rapid lethality and hematopoietic failure. NKAP deficient cells also exhibited diminished proliferation and increased expression of the cyclin dependent kinase inhibitors (CDKIs) p19 Ink4d and p21 Cip1. To determine how dysregulation of CDKI expression contributes to the effects of NKAP deficiency, NKAP was deleted in mice also deficient in p19 Ink4d or p21 Cip1 using poly-IC treatment to induce Mx1-cre. Hematopoietic failure and lethality were not prevented by deficiency in either CDKI when NKAP was deleted. Inducible deletion of NKAP in cultured hematopoietic progenitors ex vivo resulted in a senescent phenotype and altered expression of numerous cell cycle regulators including the CDKI p16 INK4a. Interestingly, while combined deficiency in p16 INK4a and p21 Cip1 did not reverse the effect of NKAP deficiency on hematopoiesis in vivo, it did shift the consequence of NKAP deficiency from senescence to apoptosis in ex vivo cultures. These results suggest that NKAP may limit cellular stress that can trigger cell cycle withdrawal or cell death, a role critical for the maintenance of a viable pool of hematopoietic progenitors.

Grading immunohistochemical markers p16INK4a and HPV E4 identifies productive and transforming lesions caused by low- and high-risk HPV within high-grade anal squamous intraepithelial lesions.

SummaryObjectivesBecause current guidelines recognise high‐grade anal squamous intraepithelial lesions (HSILs) and low‐grade SILs (LSILs), and recommend treatment of all HSILs although not all progress to cancer, this study aims to distinguish transforming and productive HSILs by grading immunohistochemical (IHC) biomarkers p16INK 4a (p16) and E4 in low‐risk human papillomavirus (lrHPV) and high‐risk (hr)HPV‐associated SILs as a potential basis for more selective treatment.MethodsImmunostaining for p16 and HPV E4 was performed and graded in 183 biopsies from 108 HIV‐positive men who have sex with men. The causative HPV genotype of the worst lesion was identified using the HPV SPF10‐PCR‐DEIA‐LiPA25 version 1 system, with laser capture microdissection for multiple infections. The worst lesions were scored for p16 (0–4) to identify activity of the hrHPV E7 gene, and panHPV E4 (0–2) to mark HPV production and life cycle completion.ResultsThere were 37 normal biopsies, 60 LSILs and 86 HSILs, with 85% of LSILs caused by lrHPV and 93% of HSILs by hrHPV. No normal biopsy showed E4, but 43% of LSILs and 37% of HSILs were E4 positive. No differences in E4 positivity rates were found between lrHPV and hrHPV lesions. Most of the lesions caused by lrHPV (90%) showed very extensive patchy p16 staining; p16 grade in HSILs was variable, with frequency of productive HPV infection dropping with increasing p16 grade.ConclusionsCombined p16/E4 IHC identifies productive and nonproductive HSILs associated with hrHPV within the group of HSILs defined by the Lower Anogenital Squamous Terminology recommendations. This opens the possibility of investigating selective treatment of advanced transforming HSILs caused by hrHPV, and a ‘wait and see’ policy for productive HSILs. What's already known about this topic? For preventing anal cancer in high‐risk populations, all patients with high‐grade squamous intraepithelial lesions (HSILs) are treated, even though this group of lesions is heterogeneous, the histology is variable and regression is frequent. What does this study add? By adding human papillomavirus (HPV) E4 immunohistochemistry to p16 INK4a (p16), and grading expression of both markers, different biomarker expression patterns that reflect the heterogeneity of HSILs can be identified.Moreover, p16/E4 staining can separate high‐risk HPV‐associated HSILs into productive and more advanced transforming lesions, providing a potential basis for selective treatment.

Clinical Utility of Biomarkers in Diagnosis and Management of High Grade Cervical Intraepithelial Neoplasia (HSIL)

Objective of the study: The increasing inter- and intra-observer variability in the diagnosis of CIN by histopathology has led to the advent of different biomarkers. The aim of this study is to evaluate Ki-67 and p16 INK4a marker in differentiating CIN lesions and the clinical utility of it in the management of high grade squamous intraepithelial lesions (HSIL).
 Materials and methods: This cross-sectional study has been carried out among the biopsy proved cases of CIN2 and CIN3 in which P16 and Ki-67 immunostain was performed in the Colposcopy clinic, Bangabandhu Sheikh Mujib Medical University (BSMMU) and Department of Pathology, BSMMU respectively from 14-3-17 to 30-05-18. Thirty-two cases were incorporated in the study by purposive sampling. Data was analyzed using SPSS version 17.
 Results: Among 32 cases of HSIL, 25 were diagnosed as CIN 2 (78.13 %) and 7 cases as CIN 3 (21.87 %) by histopathology. All the cases were subjected to immunohistochemistry with P16 and Ki 67. Regarding the status of P16, it was found positive only in 32% cases of CIN 2 but in 100% cases of CIN 3. Similarly, Ki67 was found positive in 36 % of CIN2 cases but in all cases of CIN 3 lesions. Thus immunostain confirmed one third histopathologically diagnosed CIN 2 cases. On the other hand, all cases of CIN 3 were confirmed as HSIL with the immunostain. All the cases of CIN 2 were reviewed for consensus opinion. Only 8 (32%) cases of CIN 2 were both immunostain positive , the review diagnosis by consensus opinion was also CIN 2 in those cases. One case (4%) of CIN 2 was P16 negative but Ki 67 positive, which ultimately revealed as immature squamous metaplasia. Sixteen cases of CIN 2 were negative for both P16 and Ki 67. Among these, majority cases (52%) , consensus opinion revealed as CIN 1 and 12% cases as immature squamous metaplasia. Therefore, an HSIL diagnosed by histopathology could be false positive in a significant number of cases and overtreatment of a false positive CIN 2 lesion can affect future reproductive status, also have some psychological consequences and economic burden in the health sector.
 Conclusion: By using the biomarkers, overtreatment and undertreatment of CIN can be avoided. All cases of CIN 2 should be carefully dealt with either review of the slides by consensus opinion or by doing immunohistochemistry if facility is available.
 Bangladesh J Obstet Gynaecol, 2019; Vol. 34(2): 79-86

230 Psoriatic epidermis is associated with upregulation of CDK2 and inhibition of CDK4 activity

Cyclin-dependent kinases (CDKs) are involved in regulation of cell cycle and passage through the G1 restriction point relies on CDK4/6 and their association with cyclin D1 (CycD1) whereas entry into S phase requires CDK2 and its association with cyclin E (CycE). The levels of cyclin partners, their phosphorylation status, and the abundance of inhibitors of CDK (CDKI) regulate CDK activity. CKIs may be implicated in signaling pathways that are involved in psoriasis pathogenesis and regulation of G1 and S phases by CDKs is relevant for inflammatory skin diseases that are associated with proliferative cell disorders. To monitor CDK2 and CDK4 activity in human epidermis we used CDKACT fluorescent biosensors that undergo fluorescence enhancement upon phosphorylation by CDKs. By using confocal and Western blot analysis, we evaluated the expression of CDK4/2, Cyc D1/E and CDKI such as p16 INK4A(p16), p21Cip1(p21) and p27Kip1(p27). A cohort of 24 patients was asked to participate in the study. Punch biopsies were taken from a chronic plaque and from non-lesional skin of the same patient. CDK4 and its CycD1 partner were overexpressed in psoriatic epidermis. Epidermal QPCR analysis showed that the increased CDK4 expression was not due to enhanced transcription. Compared to normal skin there was no increase in CDK4/CyclinD1 activity. CDK2 and CycE were overexpressed in psoriatic epidermis and this overexpression was correlated with an increase in CDK2-CycE activity. Increased CDK2 expression was not related to enhanced transcription. There was an increased expression of p16 and p21 whereas p27 was decreased in psoriatic epidermis. We have have shown that epidermal CDK2 activity was greatly increased in psoriatic epidermis while CDK4 activity was completely inhibited. These alterations are not associated with changes in CDK transcription and instead involve post-translational control that may be mediated by a dysregulation of CDKI expresion in psoriatic epidermis.

Psoriatic epidermis is associated with upregulation of CDK2 and inhibition of CDK4 activity.

The cyclin-dependent kinases (CDKs) CDK2 and CDK4 are involved in regulation of cell-cycle progression, and psoriasis is characterized by hyperproliferation of basal epidermal cells. CDK inhibitory proteins (CKIs) such as p16INK 4A (p16) bind CDK4/6 kinases and prevent their interaction with D-type cyclins. CKIs such as p21Cip1 (p21) and p27Kip1 (p27) associate with CDK-cyclin complexes and prevent their activation. To gain insight into the molecular implication of CDK2 and CDK4 kinases in psoriasis, we sought to characterize expression of these kinases and associated cyclins, as well as of CKIs, and addressed the status of CDK2 and CDK4 activity in human psoriatic epidermis. A cohort of 24 patients with psoriasis participated in the study. Biopsies were removed from a chronic plaque and from nonlesional skin. CDK2, CDK4, cyclin D1, cyclin E and CKI protein expression was assessed by immunoblotting, immunohistochemistry and immunofluorescence. CDK4 and CDK2 mRNA expression was determined by real-time polymerase chain reaction. Specific kinase activities of CDK2 and CDK4 were evaluated using fluorescent peptide biosensors. CDK2-cyclin E expression and activity were significantly increased in psoriatic epidermis compared with uninvolved adjacent skin. In contrast, CDK4-cyclin D1 activity was inhibited, although its expression was increased in psoriatic epidermis and its transcription slightly inhibited. p27 expression was reduced, while p16 and p21 expression was induced in psoriatic epidermis. Epidermal CDK2 activity is increased in psoriatic epidermis while CDK4 activity is completely inhibited. These alterations are not associated with changes in CDK transcription and instead involve post-translational control mediated by decreased expression of p27 and p16 overexpression, respectively. What's already known about this topic? Cyclin-dependent kinases (CDKs) are involved in cell-cycle progression. The levels of cyclin partners and CDK inhibitors regulate their activity. Psoriasis is a chronic T-cell-driven inflammatory skin disease characterized by hyperproliferation of basal epidermal cells. What does this study add? Thanks to fluorescent peptide biosensors, this study demonstrates that epidermal CDK2 activity is increased in psoriatic epidermis while CDK4 activity is completely inhibited. These alterations involve post-translational control mediated by decreased expression of p27, and p16 overexpression, respectively. What is the translational message? CDK2 and CDK4 are involved in regulation of cell-cycle progression, and psoriasis is characterized by hyperproliferation of basal epidermal cells. Epidermal CDK2 activity is increased in psoriatic epidermis while CDK4 activity is completely inhibited. These alterations are not associated with changes in CDK transcription and instead involve post-translational control mediated by decreased expression of p27 and p16 overexpression, respectively. Pharmacological modulation of CDK2 and CDK4 may constitute a promising therapeutic strategy.

Anti-aging Effect of Free Curcumin and Niosome Entrapping Curcumin in H2O2-induced Aging in Human Fibroblast Cell Lines

Aging is not a disease but it a changes that come with time, aging may make an individual susceptible to disease. Exposure to smoke, UV radiation, chemicals or other exogenous agents over time can damage cells, as can produce DNA replication errors or oxidative stress. The use of antioxidant curcumin can produce intracellular defenses in addition to low expensive and no side effect. In the present study, hydrogen peroxide (H2O2) was used to produce oxidative stress in Human Fibroblast cell lines (HFB4) and free curcumin and niosome entrapping curcumin treatments are used for aged HFB4 cell lines. Using Flowcytometry analysis, p 16 INK4a and p53 BP1 genes levels were investigated as aging biomarkers. Result show that H2O2 treated cell lines are highly elevate p16 INK4a and p53 BP1 genes. While HFB4 cells treated with free curcumin and niosome entrapping curcumin were significantly protected against aged process.

Knockout of Canopy 2 activates p16INK4a pathway to impair cardiac repair

BackgroundCardiac repair depends on angiogenesis and cell proliferation. Previously we identified Canopy 2 (CNPY2) as a secreted angiogenic growth factor which promotes neovascularization. We investigated the role of CNPY2 in cardiac repair following myocardial infarction (MI) and the possible mediators involved using Cnpy2 knockout (KO) mice and human cardiac tissue. Methods and resultsCardiac tissue from patients with end-stage heart failure had significantly lower endogenous CNPY2 expression compared to samples from control patients. CNPY2 expression in mouse hearts significantly decreased following MI. Significantly less leukocyte and endothelial cell proliferation was found in Cnpy2 KO than wild-type (WT) mice post MI which contributed to impaired angiogenesis, tissue repair, and decreased cardiac function (fractional shortening: WT: 21.1 ± 2.1% vs. KO: 16.4 ± 1.6%, p < .01 at day 28 post MI). RT-qPCR revealed significantly increased p16INK4a expression in Cnpy2 KO mouse hearts (WT: 1.0 ± 0.04 vs. KO: 2.33 ± 0.11 [relative expression of p16 INK4a], p < .01) which was confirmed by immunostaining (WT: 8.47 ± 1.22 vs. KO: 12.9 ± 1.22 [% total cells], p < .05) for the p16INK4a protein. Expression of cell cycle-related proteins, cyclin D1, cyclin-dependent kinases 4 and 6, and phosphorylated retinoblastoma protein (pRb) was significantly decreased in Cnpy2 KO mouse hearts. The up-regulation of the p16INK4a/cyclin D1/Rb pathway by knockout of Cnpy2 was accompanied by attenuation of PDK1/Akt phosphorylation. MI exacerbated the detrimental effects of p16INK4a on tissue repair in Cnpy2 KO mice. Overexpression of CNPY2 in the cardiac tissue of transgenic mice reversed the inhibition of cell proliferation through suppression of the p16INK4a pathway. ConclusionsCardiac injury and progressive heart failure were associated with decreased CNPY2 levels in both humans and mice. Knockout of Cnpy2 resulted in up-regulation of p16INK4a which impaired cardiac function and tissue repair. These data suggest that CNPY2 is an important regulator of p16INK4a and promotes cell proliferation and tissue repair through inhibition of the p16INK4a pathway. CNPY2 treatment may offer a new approach to restore cardiac function after an MI.

ATRX protein loss and deregulation of PI3K/AKT pathway is frequent in pilocytic astrocytoma with anaplastic features.

Pilocytic astrocytoma (PA) with anaplastic features (PAAF) is a rare entity associated with decreased survival. It is characterized by hypercellularity, atypia, brisk mitotic activity, variable necrosis, and association with a classic PA component or anaplastic transformation in a recurrent tumor with a previously-documented classic PA. We present 5 PAAF cases with clinical, radiological, pathological, and molecular correlation. We interrogated ATRX, IDH, TP53, PTEN, EGFR, BRAF, 6q23, p16(Ink4a) by sequencing, FISH, and immunohistochemistry. Four tumors were located in the cerebellum, and 1 was supratentorial. All showed ATRX protein loss by immunohistochemistry, loss of heterozygosity for PTEN, and had no IDH/TP53/BRAF mutations, nor EGFR amplification. Two of 5 tumors showed BRAF duplication by pyrosequencing. All showed loss of PTEN nuclear expression in subsets of tumor cells, which was associated with variable cytoplasmic positivity for pS6. There was a relative correlation between loss of PTEN expression and pS6 cytoplasmic expression. p53 was expressed in ~ 50% of tumor cells in all tumors. P16 was variably lost in all cases. One tumor showed MYB/6q23 deletion. We confirm ATRX protein loss suggestive of ATRX alteration as well as dysregulation of the PI3K/AKT pathway and, less often, of the MAPK/ERK pathway in PAAF. .

Length doesn't matter-telomere damage triggers cellular senescence in the ageing heart.

Telomere shortening induces cellular senescence in proliferative cells. Yet, it is presently unclear how it is triggered in post‐mitotic cells such as cardiac myocytes. A new study by Anderson et al (2019) reports that during ageing of the heart, cellular senescence develops independently of telomere length, but is evoked by DNA damage, which preferentially accumulates at the telomere. Removal of senescent cells using senolytic drugs ameliorated cardiac hypertrophy and fibrosis and may inform novel approaches to improve the conditions for the ageing heart.

Cells exhibiting strong p16 INK4a promoter activation in vivo display features of senescence

Cells exhibiting strong p16 INK4a promoter activation in vivo display features of senescence

Enhanced Selective Inhibition of KRAS Mutant Hematopoietic Progenitor Cell Expansion By MEK and Bcl-2 Inhibition

Amidst the complex genetic landscape of cancer, the mutagenic and oncogenic effects caused by RAS mutations alone can be overlooked particularly in the context of hematopoietic malignancies. Therefore identifying drugs that could modulate aberrant RAS-MAPK signaling is clinically important. Here we demonstrate the utilization of an iPS cell-based system that could be adapted for compound screening to identify inhibitors with selective inhibition activity against the expansion of KRAS-mutant hematopoietic progenitor cells (HPCs).RAS-associated autoimmune lymphoproliferative disorder (RALD) is a leukemia-like disease caused by a single acquired RAS mutation. In this study, BM CD34+ cells from RALD patients were reprogrammed into iPS cells. KRAS-mutant cells could be obtained without necessitating the use of gene editing techniques. Non-mutant cells were used as isogenic controls thus permitting the assessment of unwanted toxicity against healthy cells. Constitutive activation of the RAS-MAPK pathway was confirmed by western blotting in mutant iPS cells. As expected, whole exome sequencing (WES) showed that mutant KRAS (G13C) was the only mutation present in these cells. Therefore, the inherent characteristics of mutant cell expansion can be attributed to this mutation only.Previously we reported that oncogenic KRAS enforces retention of self-renewal in human iPS cells (Kubara et al. Stem Cell Reports. 2018) due to constitutive activation of the KRAS-MAPK signaling pathway. Notably mutant iPS cells retained pluripotency marker expression even in the absence of bFGF, but not isogenic control cells. When we tested compounds with different mechanisms of action, direct MEK inhibition was the most effective in triggering a loss of pluripotency marker expression in mutant iPS cells in the absence of bFGF. This method permits rapid determination of compounds that could modulate aberrant KRAS-MAPK signaling. However, further verification is required to determine whether inhibitory effects on mutant HPC expansion could also be observed.Next, differentiated multi-potent (Lin-34+43+) HPCs from iPS cells were expanded in “standard” ex vivo culture conditions using SCF, TPO Flt3L and IL-3. This favored the expansion of control cells. We also found “selective” culture conditions that could selectively expand mutant HPCs. RNA-seq analysis on pre-expanded and expanded HPCs revealed the unique molecular signature of expanding mutant cells, meaning genes that showed upregulation in both “standard” and “selective” culture conditions. There was upregulation of CCND1 and CDK4 in mutant HPCs, which facilitates progression to the S phase of the cell cycle. This was also accompanied by upregulation of p16INK4a and p21Cip1, which is characteristic of senescent cells. Indeed, it could be shown that expanded mutant HPCs acquired senescence features such as increased p16 INK4a and Bcl-xL expression at the protein level and increased SA-b-gal activity compared with control cells. Gene correction of KRAS (G13C) using CRISPR/Cas9 reversed these phenotypes. Therefore CCND1/CDK4 and Bcl-2 may be potential inhibitory targets.We developed a high-throughput screening method whereby differentiated HPCs were sorted directly into 96-well plates and following expansion, cell numbers were quantified by luminescence detection of ATP. First we screened drugs for selective activity (IC50 value for mutant cells lower than control cells). Consistent with the iPS cell pluripotency assay, we identified Trametinib (MEK), as having selective effects against mutant HPCs in addition to Palbociclib (CCND1/CDK4) and Navitoclax (Bcl-2). When used in combination, the selective inhibitory effect was enhanced without compromising the viability of control cells.To test these drugs in an in vivo setting, highly purified transduced (KRAS or control) mouse HSCs (Lin-CD34-KSL) were transplanted in a competitive repopulation assay. Recipient mice were given Trametinib and Navitoclax. In the KRAS group, donor cells contributed to a lower level of chimerism. We also developed the use of droplet digital PCR (ddPCR) to monitor the percentage of mutant cells within bulk BM CD34+ cells (mutant and isogenic control) when cultured in the presence of inhibitors. Taken together, our study offers novel insights into mutant KRAS-driven HPC expansion and demonstrate an efficient platform for drug screening. DisclosuresKubara:Eisai Co., Ltd.: Employment. Yamazaki:Eisai Co., Ltd.: Employment.

Expression of p16INK4a and human papillomavirus 16 with associated risk factors in cervical premalignant and malignant lesions.

Introduction:Human papilloma virus (HPV) which is causative factor for cervical cancer may interact with p16 leading to malignant transformation of cervical epithelial cells. The present study was conducted to assess the immunoexpression of p16 INK4a in premalignant and malignant lesions of cervix and to correlate it with HPV 16 expression. It was also intended to study the various risk factors which may be associated with cervical cancer in this north Himalayan region of India.Material and Methods:The study included 50 cases of premalignant and malignant cervical lesions and 50 controls diagnosed on histopathology over a period of one year. All the relevant clinical details were noted and both cases and controls were subjected to HPV 16 and p16 INK4a immunohistochemical staining.Results:67% of subjects (including cases and controls) and 94% of the cases were positive for HPV 16 expression. p16 INK4a expression was negative in all the controls, positive in 96% of invasive cancer, 66.6% in HSIL and 37.5% in LSIL.Conclusion:Cervical cancer is associated with low socio economic status, illiteracy, smoking, early age of marriage and conception in north Himalayan region of India. HPV 16 infection is positive in both cases and controls indicating high prevalence of HPV 16 in this region. Neoplastic transformation by HPV is identified by over expression of p16 INK4a in premalignant and malignant cases. The immunopositivity of p16 INK4a increases with the severity of cervical lesions and thus may play an important role in stratification of premalignant and malignant lesions.

Kallistatin attenuates endothelial senescence by modulating Let-7g-mediated miR-34a-SIRT1-eNOS pathway.

Kallistatin, a plasma protein, protects against vascular and organ injury. This study is aimed to investigate the role and mechanism of kallistatin in endothelial senescence. Kallistatin inhibited H2O2‐induced senescence in human endothelial cells, as indicated by reduced senescence‐associated‐β‐galactosidase activity, p16INK 4a and plasminogen activator inhibitor‐1 expression, and elevated telomerase activity. Kallistatin blocked H2O2‐induced superoxide formation, NADPH oxidase levels and VCAM‐1, ICAM‐1, IL‐6 and miR‐34a synthesis. Kallistatin reversed H2O2‐mediated inhibition of endothelial nitric oxide synthase (eNOS), SIRT1, catalase and superoxide dismutase (SOD)‐2 expression, and kallistatin alone stimulated the synthesis of these antioxidant enzymes. Moreover, kallistatin's anti‐senescence and anti‐oxidant effects were attributed to SIRT1‐mediated eNOS pathway. Kallistatin, via interaction with tyrosine kinase, up‐regulated Let‐7g, whereas Let‐7g inhibitor abolished kallistatin's effects on miR‐34a and SIRT1/eNOS synthesis, leading to inhibition of senescence, oxidative stress and inflammation. Furthermore, lung endothelial cells isolated from endothelium‐specific kallistatin knockout mice displayed marked reduction in mouse kallistatin levels. Kallistatin deficiency in mouse endothelial cells exacerbated senescence, oxidative stress and inflammation compared to wild‐type mouse endothelial cells, and H2O2 treatment further magnified these effects. Kallistatin deficiency caused marked reduction in Let‐7g, SIRT1, eNOS, catalase and SOD‐1 mRNA levels, and elevated miR‐34a synthesis in mouse endothelial cells. These findings indicate that endogenous kallistatin through novel mechanisms protects against endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐SIRT1‐eNOS pathway.

P16INK4a gene promoter methylation as a biomarker for the diagnosis of non-small cell lung cancer: An updated meta-analysis.

BackgroundThis meta‐analysis was conducted to investigate the diagnostic performance of P16 INK4a gene promoter methylation as a biomarker of non‐small cell lung cancer (NSCLC).MethodsTwo reviewers independently searched the Web of Science, PubMed, Cochrane, Embase, China National Knowledge Infrastructure, and Chinese Biomedical Literature databases. Publications relevant to P16 INK4a gene promoter methylation in serum or bronchoalveolar fluid/sputum were screened and included in this meta‐analysis. Pooled diagnostic sensitivity, specificity, and symmetric receiver operating characteristic curve were calculated.ResultsTwenty‐six publications with 1768 lung cancer cases and 1323 controls were included. The pooled sensitivity, specificity, positive and negative likelihood ratios, and diagnostic odds ratio were 0.46 (95% confidence interval [CI] 0.43–0.48), 0.90 (95% CI 0.88–0.91), 6.33 (95% CI 3.89–10.30), 0.57 (95% CI 0.50–0.65) and 10.72 (95% CI 6.94–16.56), respectively, for P16 INK4a gene promoter methylation as a biomarker for the diagnosis of NSCLC. The area under the symmetric receiver operating characteristic curve was 0.75 with a standard error of 0.004. No publication bias was detected via line regression test (t = 0.95; P = 0.35) and Begg's funnel plot.Conclusion P16 INK4a gene promoter methylation detection in serum or bronchoalveolar fluid/sputum may be a potential biomarker for NSCLC diagnosis; however, the sensitivity was relatively low, which is not suitable for NSCLC screening.

Expression of p16INK4a is a biomarker of chondrocyte aging but does not cause osteoarthritis.

SummaryCellular senescence drives a functional decline of numerous tissues with aging by limiting regenerative proliferation and/or by producing pro‐inflammatory molecules known as the senescence‐associated secretory phenotype (SASP). The senescence biomarker p16 INK 4a is a potent inhibitor of the cell cycle but is not essential for SASP production. Thus, it is unclear whether p16 INK 4a identifies senescence in hyporeplicative cells such as articular chondrocytes and whether p16 INK 4a contributes to pathologic characteristics of cartilage aging. To address these questions, we examined the role of p16 INK 4a in murine and human models of chondrocyte aging. We observed that p16 INK 4a mRNA expression was significantly upregulated with chronological aging in murine cartilage (~50‐fold from 4 to 18 months of age) and in primary human chondrocytes from 57 cadaveric donors (r 2 = .27, p < .0001). Human chondrocytes exhibited substantial replicative potential in vitro that depended on the activity of cyclin‐dependent kinases 4 or 6 (CDK4/6), and proliferation was reduced in cells from older donors with increased p16 INK 4a expression. Moreover, increased chondrocyte p16 INK 4a expression correlated with several SASP transcripts. Despite the relationship between p16 INK 4a expression and these features of senescence, somatic inactivation of p16 INK 4a in chondrocytes of adult mice did not mitigate SASP expression and did not alter the rate of osteoarthritis (OA) with physiological aging or after destabilization of the medial meniscus. These results establish that p16 INK 4a expression is a biomarker of dysfunctional chondrocytes, but that the effects of chondrocyte senescence on OA are more likely driven by production of SASP molecules than by loss of chondrocyte replicative function.

MYC Releases Early Reprogrammed Human Cells from Proliferation Pause via Retinoblastoma Protein Inhibition

Here, we report that MYC rescues early human cellsundergoing reprogramming from a proliferation pause induced by OCT3/4, SOX2, and KLF4 (OSK). We identified ESRG as a marker of early reprogramming cells that is expressed as early as day 3 after OSK induction. On day 4, ESRG positive (+) cells converted to a TRA-1-60 (+) intermediate state. These early ESRG (+) or TRA-1-60 (+) cells showed a proliferation pause due to increased p16INK4A and p21 and decreased endogenous MYC caused by OSK. Exogenous MYC did not enhance the appearance of initial reprogramming cells but instead reactivated their proliferation and improved reprogramming efficiency. MYC increased expression of LIN41, which potently suppressed p21 post-transcriptionally. MYC suppressed p16 INK4A. These changes inactivated retinoblastoma protein (RB) and reactivated proliferation. The RB-regulated proliferation pause does not occur in immortalized fibroblasts, leading to high reprogramming efficiency even without exogenous MYC.

Abstract 03: Imaging characteristics of head and neck tumors according to human papillomavirus (HPV) status in Bogotá, Colombia

Abstract Background: HPV-positive head and neck tumors (HNT) correspond to a unique entity given their clinical behavior and molecular characteristics. These tumors can show distinct features on pretreatment imaging, such as well-defined borders and cystic nodal metastases; therefore, the aim of our study was to evaluate the imaging characteristics and determine if there are differences between HPV-positive and HPV-negative HNT tumors in patients studied at our institution. Design: A retrospective pretreatment imaging review from 35 HNT cases recruited under INTERCHANGE- IARC project in Latin America between 11-2014 and 12-2016 was performed. Imaging evaluation included primary lesion and nodal metastases. Initial evaluation by radiologists was blinded for patients' histological findings and HPV status. Radiology was evaluated for: location, tumor size, tumor border morphology, contrast enhancement, nodal metastases, size of metastasis and morphology of nodal metastasis. Radiological findings were correlated with histology and p16 INK4a inmunohistochemistry using clone E6H4 (HPV surrogate marker). Results: Pretreatment images were available for review only in 17 of 35 cases (48,5%). 10 cases had computerized tomography (CT) examination, 1 case magnetic resonance (MR) and 6 cases had 18F-FDG PET-CT. 6/17 cases (35.3%) were excluded due to no visualization of the primary tumor or due to limitation artifacts (amalgam). From the 11 cases available for review, 7 were from the oropharynx (OP), 2 from the oral cavity (OC) and 1 from the larynx (LA). All OP tumors were positive for p16 and all the rest tumors were p16 negative. Tumor size ranged from 7 to 51 mm in greatest dimension. Well-defined borders were present in 4/7 p16 positive tumors vs 2/4 p16 negative tumors. Tumor enhancement with contrast was present and able to evaluate only in 5 out of 11 tumors (3 p16 + and 2 p16 -). All the OP p16 positive tumors presented with nodal metastasis greater than 10 mm in dimension. 6/7 had nodal cystic change and these p16 positive cases were more likely to have large cystic nodal metastases with a cystic component larger than 50% of the nodal size (6/7) than p16/HPV negative tumors (1 out of 4 cases had smaller nodal metastasis with cystic component &amp;lt;50%). Conclusion: Large nodal metastasis (&amp;gt;10 mm) with cystic component larger than 50% of the node size is an important finding seen in p16 HNT. One should consider these findings when examining neck images in patients with unknown primary in order to exclude malignancy given that usually cystic nodes could be misdiagnosed as benign. Our nondiagnostic imaging rate pretreatment was 35%; in order to improve the use of diagnostic tools we recommend the use of MR especially in patients with amalgams and/or tumor located at the oral cavity and oropharynx. Further studies are granted with a larger sample size in order to validate and confirm imaging characteristics in p16/HPV positive HNT. Citation Format: Nicolás Useche, Oscar Torres, Maria M. Rojas, Mauricio Palau-Lázaro, Marcela Mejía-Arango, Ana Margarita Baldión, José A. Hakim, Johanna Campos, Alberto Escallon, Aylen Vanessa Ospina, Sandra Perdomo, Paula A. Rodríguez-Urrego. Imaging characteristics of head and neck tumors according to human papillomavirus (HPV) status in Bogotá, Colombia [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 03.

P1.02-035 Human Papillomavirus Infection in Lung Squamous Cell Carcinoma and Correlation to p16 INK4a Expression from an Argentine Population

Lung cancer is the leading cause of cancer death worldwide. In 1979, Syrjänen suggested a role for human papillomavirus (HPV) infection in bronchial carcinoma. Many studies have found HPV on lung carcinoma, predominantly in squamous cell carcinoma (SCC). There seems to be a geographical factor determining prevalence rates. In Latin America, only 2 studies, altogether including 51 cases of lung SCC, examined this association. However, data from Argentina is lacking. The aim of this study is to assess the incidence of HPV infection in lung SCC of Argentinean population, and to correlate with p16INK4a expression from an Argentine population. The study was approved by CEMIC’s Ethics Committee. Informed consent was obtained. Materials consisted of formalin-fixed paraffin-embedded (FFPE) tissue from 29 surgically excised and 11 transbronchial biopsies of primary L-SCC evaluated between 2006-2016. HPV Genotyping: On 50μm-thick slides, tumor was microdissected and DNA was extracted (columns method). Wide-spectrum HPV DNA (L1-ORF) was amplified by PCR. Positive specimens were genotyped by PCR for types 16 and 18. Immunohistochemistry: All p16 staining’s were performed on VENTANA BenchMark GX using antibody CINtec® p16. Staining patterns were interpreted on a binary way (positive or negative). Only cases with diffusely intense cytoplasmic and/or nucleic staining on tumor cell (TC) were considered positive. Cases in which the normal bronchial epithelium resulted p16 positive but TC were negative, were also registered. HPV was isolated in 10/40 cases (25%). The details of HPV infection and the clinicopathological data is depicted on Table 1. We detected an HPV infection rate of 25%. HPV18 was the common genotype. On 7 cases, normal bronchial epithelium was both p16 and HPV positive, suggesting that adjacent tumor tissue may be HPV infected. p16 should not be used as a surrogate marker for HPV infection, since it is only positive on 60% of cases.

Clinical and prognosis value of the CIMP status combined with MLH1 or p16 INK4a methylation in colorectal cancer.

Aberrant DNA methylation of CpG islands occurred frequently in CRC and associated with transcriptional silencing of key genes. In this study, the CIMP combined with MLH1 or p16 INK4a methylation status was determined in CRC patients and correlated with clinicopathological parameters and overall survival. Our data showed that CIMP+ CRCs were identified in 32.9% of cases and that CACNAG1 is the most frequently methylated promoter. When we combined the CIMP with the MLH1 or the p16 INK4a methylation status, we found that CIMP-/MLH1-U (37.8%) and CIMP-/p16 INK4a -U (35.4%) tumors were the most frequent among the four subtypes. Statistical analysis showed that tumor location, lymphovascular invasion, TNM stage, and MSI differed among the group of patients. Kaplan-Meier analyses revealed differences in overall survival according to the CIMP combined with MLH1 or p16 INK4a methylation status. In a multivariate analysis, CIMP/MLH1 and CIMP/p16 INK4a methylation statuses were predictive of prognosis, and the OS was longer for patients with tumors CIMP-/MLH1-M, as well as CIMP-/p16 INK4a -M. Furthermore, DNMT1 is significantly overexpressed in tumors than in normal tissues as well as in CIMP+ than CIMP- tumors. Our results suggest that tumor classification based on the CIMP status combined with MLH1 or p16 INK4a methylation is useful to predict prognosis in CRC patients.

Abstract 2337: miR-6883 and family miRNAs induce G1-arrest in colon cancer cells by targeting CDK4/6

Abstract Dysregulation of the CDK4/6-Rb pathway is a hallmark of various tumor types. Cancer cells subvert different cell cycle checkpoints to continue unchecked growth and proliferation. Some common mechanisms of overcoming cell cycle checkpoints involve loss of tumor-suppressor proteins such as p16 INK4A or Retinoblastoma (Rb). Alternately, cancer cells can also increase expression of oncogenes like cyclin D1, other CDKs by gene amplification or gene translocation. Thus, targeting different oncogenes in the cell cycle pathway, particularly CDKs has been considered an important therapeutic strategy. Events of overexpression of cell cycle oncogenes and suppression of tumor suppressors are mutually exclusive and tumor-type specific. In colorectal cancer (CRC), it has been shown that there is overexpression of CDK4/6 . We conducted TCGA analysis of 50 matched tumor and normal CRC tumors, and validated that CDK4 and CDK6 are significantly overexpressed in patient tumor samples by RNA-seq (p= 1.55 e-09 and p= 4.439 e-05). Thus, ongoing clinical trials for CRC patients with advanced disease are currently evaluating the efficacy of CDK4/6 inhibitors as single agents and in combination with chemotherapy (J Clin Oncol 33, 2015 ,suppl 3; abstr 626; Zhang 2016). We evaluated the efficacy of miRNAs targeting CDK4/6 as novel therapeutics in CRC. We performed in silico analysis using TargetScan to identify miRNAs that can target the 3’UTR regions of CDK4/6. We have identified a novel family of miRNAs (based on seed sequence similarity) that can potently target CDK4/6 mRNA and thereby reduce both the RNA and protein levels of CDK4/6 in cancer cells. Of the four miRNAs in the family, we chose to characterize miR-6883-5p and miR-149* in a panel of CRC, melanoma and pancreatic cancer cell lines. Our preliminary TCGA analysis in 11 matched T/N samples shows miR-149* expression is significantly lost (p= 0.0049) in CRC samples and this further positively correlates with the stage of the tumor. Ongoing in vitro studies using miRNA mimics in CRC cell lines indicate that both miR-6883-5p and miR-149* have anti-proliferative effects as assayed by CellTiterGlo (CTG) and MTT. Further, both miRNAs induce G1-arrest as a consequence of downregulation of CDK4 and CDK6 in all cell lines and we also observe induction of apoptosis in a subset of CRC cell lines. Knockdown of CDK4 and CDK6 mimics the phenotypes observed with restoring expression of both miR-6883 and miR-149* in CRC cell lines. Combination of miR-6883 with FDA-approved drugs Irinotecan and 5-FU showed strong synergy and led to apoptosis as assayed by CTG and PARP cleavage. Our ongoing work is further looking into the mechanisms of synergy and identifying additional target genes in CRC cell lines. Thus, our novel miRNA based strategy to target CDK4 has potential to translate as both a single agent/combinatorial therapy and to identify biomarkers of response, which is critical for understanding the clinical results seen with CDK inhibitors. Citation Format: Amriti R. Lulla, Margret B. Einarson, Yan Zhou, Michael Slifker, David T. Dicker, Wafik S. El-Deiry. miR-6883 and family miRNAs induce G1-arrest in colon cancer cells by targeting CDK4/6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2337. doi:10.1158/1538-7445.AM2017-2337