Knockdown of RREB1 inhibits cell proliferation via enhanced p16 expression in gastric cancer.

Gastric cancer (GC) is the most common gastrointestinal malignancy worldwide. However, the molecular mechanisms of the progression of GC are not fully understood. Ras-responsive element binding protein 1 (RREB1) is an oncogene in many types of cancer that is involved in various biological processes, such as DNA damage repair, cell growth and proliferation, cell differentiation, fat development, and fasting glucose balance. In this study, we demonstrate the role of RREB1 in gastric cancer. First, by immunohistochemistry staining (IHC) and bioinformatics analysis, we demonstrated the expression of RREB1 in gastric cancer and paired normal gastric tissues. Then, we established RREB1 overexpression and knockdown cell lines via lentiviral transfection and detected cell proliferation by using MTT, colony-forming, cell cycle and apoptosis assays in vitro. We demonstrated the effect of RREB1 on cell proliferation in vivo by using a subcutaneous xenograft tumor model in nude mice. Finally, by using Western blotting and IHC, we demonstrated the possible mechanism by which RREB1 affects cell proliferation. The IHC and bioinformatics analyses demonstrated that RREB1 was highly expressed in gastric cancer and showed that RREB1-expressing patients had a larger tumor size and more lymphovascular invasion than RREB1-negative patients. Knockdown of RREB1 inhibited cell proliferation in vivo and in vitro. Knockdown of RREB1 enhanced p16 expression in vivo and in vitro, and p16 expression was negatively related to RREB1 in gastric cancer tissue. RREB1 was highly expressed in gastric cancer, and knockdown of RREB1 inhibited cell proliferation via enhanced p16 expression.

Role of P16 Expression in the Prognosis of Patients With Laryngeal Cancer: A Single Retrospective Analysis.

BACKGROUND: A possible oncogenic role of human papillomavirus (HPV) in head and neck cancers (mainly oropharynx tumors) has been suggested. This significant association has been considered true for oropharynx tumors; however, the association between HPV infection and laryngeal carcinomas is yet to be established. The aim of this study was to evaluate the relationship between p16 expression and long-term overall, disease-free, and disease-specific survival (OS, DF, and DSS, respectively) in patients surgically treated for laryngeal carcinoma. MATERIALS AND METHODS: Seventy-four previously untreated laryngeal carcinoma patients who underwent surgical treatment were considered for this retrospective study. The tissue specimens were processed for immunohistochemical p16 protein (surrogate HPV marker) detection. RESULTS: Survival analysis of the p16 expression of the primary tumor showed that the 5-year OS rates were 90% and 29.7% for the p16-positive and negative groups, respectively (P = .003). The 5-year DFS and DSS also differed between both groups (P < .001), whereas the 5-year DSS seemed to be related to tumor/lymph node classification and p16 expression. However, only p16 expression was identified as an independent prognostic factor associated with OS and DSS. CONCLUSIONS: Surgically treated p16-positive laryngeal cancer patients may represent a subset of patients with a better prognosis than their p16-negative counterparts.

Characterisation of anal intraepithelial neoplasia and anal cancer in HIV-positive men by immunohistochemical markers p16, Ki-67, HPV-E4 and DNA methylation markers.

Human papillomavirus (HPV)-induced anal intraepithelial neoplasia (AIN, graded 1-3) is highly prevalent in HIV-positive (HIV+) men who have sex with men (MSM), but only a minority of lesions progresses to cancer. Our study aimed to characterise comprehensively anal tissue samples from a cross-sectional series (n = 104) of HIV+ MSM and longitudinal series (n = 40) of AIN2/3 progressing to cancer using different biomarkers. The cross-sectional series consisted of 8 normal, 26 AIN1, 45 AIN2, 15 AIN3 and 10 anal squamous cell carcinoma. Tissue sections were immunohistochemically (IHC) stained for p16 (viral transformation marker), Ki-67 (cellular proliferation marker) and HPV-E4 (viral production marker). We evaluated the expression of IHC markers and compared it with DNA methylation, a marker for malignant transformation. E4 positivity decreased, whereas p16 and Ki-67 scores and methylation marker positivity increased (P values < .001) with increasing severity of anal lesions. Within AIN2, a heterogeneous biomarker pattern was observed concerning E4, p16 and methylation status, reflecting the biological heterogeneity of these lesions. In the longitudinal series, all AIN2/3 and carcinomas showed high p16 and Ki-67 expression, strong methylation positivity and occasional E4 positivity. We earlier showed that high methylation levels are associated with progression to cancer. The observed E4 expression in some AIN2/3 during the course of progression to cancer and absence of E4 in a considerable number of AIN1 lesions make the potential clinical significance of E4 expression difficult to interpret. Our data show that IHC biomarkers can help to characterise AIN; however, their prognostic value for cancer risk stratification, next to objective methylation analysis, appears to be limited.

Radiation-Induced Senescence in p16+/LUC Mouse Lung Compared to Bone Marrow Multilineage Hematopoietic Progenitor Cells.

We defined the time course of ionizing radiation-induced senescence in lung compared to bone marrow of p16+/LUC mice in which the senescence-induced biomarker (p16) is linked to a luciferase reporter gene. Periodic in situ imaging revealed increased luciferase activity in the lungs of 20 Gy thoracic irradiated, but not 8 Gy total-body irradiated (TBI) mice beginning at day 75 and increasing to day 170. In serial sections of explanted lungs, senescent cells appeared in the same areas as did fibrosis in the 20 Gy thoracic irradiated, but not the 8 Gy TBI group. Lungs from 8 Gy TBI mice at one year did show increased RNA levels for p16, p21, p19 and TGF-ß. Individual senescent cells in 20 Gy irradiated mouse lung included those with epithelial, endothelial, fibroblast and hematopoietic cell biomarkers. Rare senescent cells in the lungs of 8 Gy TBI mice at one year were of endothelial phenotype. Long-term bone marrow cultures (LTBMCs) were established at either day 60 or one year after 8 Gy TBI. In freshly removed marrow at both times after irradiation, there were increased senescent cells. In LTBMCs, there were increased senescent cells in both weekly harvested single cells and in colonies of multilineage hematopoietic progenitor cells producing CFU-GEMM (colony forming unit-granulocyte, erythrocyte, monocyte/macrophage, mega-karyocyte) that were formed in secondary cultures when these single cells were plated in semisolid media. LTBMCs from TBI mice produced fewer CFU-GEMM; however, the relative percentage of senescent cell-containing colonies was increased as measured by both p16-luciferase and ß-galactosidase. Therefore, 20 Gy thoracic radiation, as well as 8 Gy TBI, induces senescent cells in the lungs. With bone marrow, 8 Gy TBI induced senescence in both hematopoietic cells and in colony-forming progenitors. The p16+/LUC mouse strain provides a valuable system in which to compare the kinetics of radiation-induced senescence between organs in vivo, and to evaluate the potential role of senescent cells in irradiation pulmonary fibrosis.

The long non-coding RNA MIR31HG regulates the senescence associated secretory phenotype.

Oncogene-induced senescence provides a barrier against malignant transformation. However, it can also promote cancer through the secretion of a plethora of factors released by senescent cells, called the senescence associated secretory phenotype (SASP). We have previously shown that in proliferating cells, nuclear lncRNA MIR31HG inhibits p16/CDKN2A expression through interaction with polycomb repressor complexes and that during BRAF-induced senescence, MIR31HG is overexpressed and translocates to the cytoplasm. Here, we show that MIR31HG regulates the expression and secretion of a subset of SASP components during BRAF-induced senescence. The SASP secreted from senescent cells depleted for MIR31HG fails to induce paracrine invasion without affecting the growth inhibitory effect. Mechanistically, MIR31HG interacts with YBX1 facilitating its phosphorylation at serine 102 (p-YBX1S102) by the kinase RSK. p-YBX1S102 induces IL1A translation which activates the transcription of the other SASP mRNAs. Our results suggest a dual role for MIR31HG in senescence depending on its localization and points to the lncRNA as a potential therapeutic target in the treatment of senescence-related pathologies.

Patterns of SATB2 and p16 reactivity aid in the distinction of atypical polypoid adenomyoma from myoinvasive endometrioid carcinoma and benign adenomyomatous polyp on endometrial sampling.

AIMS: Atypical polypoid adenomyoma (APAM) is an uncommon uterine lesion composed of complex endometrioid glands with frequent squamous morular metaplasia and fibromuscular stroma. On endometrial curettage, biopsy or polypectomy specimens, the admixture of endometrioid glands and smooth muscle raises the differential diagnosis of myoinvasive endometrioid carcinoma. Reproductive-age APAM patients may opt for fertility preservation, whereas myoinvasive carcinoma is treated surgically. One previous study reported an incidental finding that the stroma of APAM, in contrast to that of other polypoid lesions, was SATB2-positive. APAM has also been reported to show increased stromal p16 staining. We aimed to assess whether SATB2 and p16 are useful stains for the distinction of APAM from myoinvasive carcinoma and benign adenomyomatous polyps. METHODS AND RESULTS: Cases of 'atypical polypoid adenomyoma' (n = 32), 'adenomyomatous polyp' (n = 39) and 'myoinvasive endometrioid carcinoma' (n = 30) were identified. Morphological features were assessed, along with the intensity and extent of SATB2 and p16 staining in the stromal component of each lesion. SATB2 expression was seen in the stromal components of 30 of 32 (94%) APAMs, versus none of 39 (0%) benign adenomyomatous polyps and five of 30 (17%) myoinvasive endometrioid carcinomas. Stromal p16 expression was seen in 31 of 31 (100%) APAMs, versus 20 of 39 (51%) benign adenomyomatous polyps and 12 of 30 (40%) myoinvasive endometrioid carcinomas. CONCLUSIONS: Patchy to diffuse SATB2 and block-type p16 staining of fibromuscular stroma separating atypical endometrioid glands is more consistent with APAM than with myoinvasive endometrioid carcinoma. These stains are potentially useful adjuncts to careful morphological evaluation of endometrial biopsies/curettings.

IL-6/STAT3-mediated autophagy participates in the development of age-related glomerulosclerosis.

The standard of age-related glomerulosclerosis is unclear. Both signal transducer and activator of transcription 3 (STAT3) and autophagy are involved in age-related kidney disease. Therefore, we aimed to explore the standard, as well as the potential mechanism(s). A total of 44 patients who underwent radical nephrectomy were enrolled. Pearson analysis was performed to investigate the parameters with ages. The patients were divided into the young- and aged-kidney groups. Kidney morphological changes were evaluated by histology staining, senescence was evaluated by senescence-associated-ß-galactosidase (SA-ß-gal) staining, and autophagosome was measured by transmission electron microscopy. Moreover, Western blot and/or immunohistochemistry were accomplished to assess the expression of p16, STAT3, and glycoprotein130 (GP130) and autophagy-related proteins. Furthermore, human glomerular mesangial cells were administrated with tocilizumab (TCZ) and/or IL-6, and then the above indexes were tested again. Sclerotic glomerular density and glomerular sclerosis rate were significantly higher in individuals more than 40 years old, and they were strongly correlated with ages. Moreover, the expression of p16, STAT3, GP130, and p62 was significantly increased, while LC3II and autophagosome were statistically decreased in the aged-kidney. Glomeruli were hardly to stain with SA-ß-gal. For the in vitro experiments, we observed that IL-6 significantly increased p16, STAT3, GP130, and p62, induced higher SA-ß-gal staining, while downregulated LC3II and autophagosome. Furthermore, TCZ statistically reversed the effects of IL-6 on the above expression of proteins. Glomerular sclerosis rate might be one standard for natural renal aging, and IL-6/STAT3-mediated autophagy may participate in the development of age-related glomerulosclerosis.

A Study of Prognostic Factors in Young Patients With Non-HPV Oral Cancer in Central Europe.

The etiological factors of squamous cell carcinomas of the head and neck have been well known for a long time. It is also well known that the incidence of oral cancer diagnosed in younger patients is on the rise. Due to the young age of these patients, the increase in the number of these cases and the fact that many of them neither smoke nor drink alcohol it has been suggested that other factors might be at play in the carcinogenesis of oral cancer. Thus, along the classic etiological factors of smoking and alcohol abuse certain molecular marker anomalies and the human papilloma virus (HPV) have emerged as potential factors. The aim of the present study is to verify the potential prognostic factors and to map the differences in biomarker expression between the young and the old patient groups. In the present study the immunohistochemical profile of samples obtained from oral squamous cell carcinomas was studied and compared with various clinico-pathological parameters. In 88 samples the expressions of p16, p53, Ki67, EGFR were studied with a tissue microarray technique under standard reaction conditions as well as the detection and typing of HPV infection with the Full Spectrum HPV DNA method. The biomarker expression profile of young patients with oral squamous cell carcinoma was compared to that of older patients (above 50). A significant difference was found between the immunohistochemical profile of the young and old patient groups in p16, Ki67 expression. The overall survival and progression free survival were influenced by p16 expression in young age.

Knockout of the caspase 8-associated protein 2 gene improves recombinant protein expression in HEK293 cells through up-regulation of the cyclin-dependent kinase inhibitor 2A gene.

Cell lines used in bioproduction are routinely engineered to improve their production efficiency. Numerous strategies, such as random mutagenesis, RNA interference screens, and transcriptome analyses have been employed to identify effective engineering targets. A genome-wide small interfering RNA screen previously identified the CASP8AP2 gene as a potential engineering target for improved expression of recombinant protein in the HEK293 cell line. Here, we validate the CASP8AP2 gene as an engineering target in HEK293 cells by knocking it out using CRISPR/Cas9 genome editing and assessing the effect of its knockout on recombinant protein expression, cell growth, cell viability, and overall gene expression. HEK293 cells lacking CASP8AP2 showed a seven-fold increase in specific expression of recombinant luciferase and a 2.5-fold increase in specific expression of recombinant SEAP, without significantly affecting cell growth and viability. Transcriptome analysis revealed that the deregulation of the cell cycle, specifically the upregulation of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene, contributed to the improvement in recombinant protein expression in CASP8AP2 deficient cells. The results validate the CASP8AP2 gene is a viable engineering target for improved recombinant protein expression in the HEK293 cell line.

Tonsillar p16-Positive Follicular Dendritic Cell Sarcoma Mimicking HPV-Related Oropharyngeal Squamous Cell Carcinoma: A Case Report and Review of Reported Cases.

Follicular dendritic cell sarcoma (FDCS) is a rare entity which can share morphologic features with non-keratinizing squamous cell carcinoma. Recent reports suggest that up to half of FDCSs show immunohistochemical positivity for p16 (Zhang et al., in Hum Pathol 66:40-47, 2017), a stain that is conventionally used in the risk stratification of oropharyngeal squamous cell carcinoma (OPSCC). Herein, we report a case of p16-positive FDCS with clinical and histomorphologic overlap with human papilloma virus (HPV)-related OPSCC.

Immortalization of human primary prostate epithelial cells via CRISPR inactivation of the CDKN2A locus and expression of telomerase.

BACKGROUND: Immortalization of primary prostate epithelial cells (PrEC) with just hTERT expression is particularly inefficient in the absence of DNA tumor viral proteins or p16INK4A knockdown. MATERIALS AND METHODS: Here, we describe the establishment of immortalized normal prostate epithelial cell line models using CRISPR technology to inactivate the CDKN2A locus concomitantly with ectopic expression of an hTERT transgene. RESULTS: Using this approach, we have obtained immortal cell clones that exhibit fundamental characteristics of normal cells, including diploid genomes, near normal karyotypes, normal p53 and pRB cell responses, the ability to form non-invasive spheroids, and a non-transformed phenotype. Based on marker expression, these clones are of basal cell origin. CONCLUSIONS: Use of this approach resulted in the immortalization of independent clones of PrEC that retained normal characteristics, were stable, and non-transformed. Thus, this approach could be used for the immortalization of normal primary prostate cells. This technique could also be useful for establishing cell lines from prostate tumor tissues of different tumor grades and/or from patients of diverse ethnicities to generate cell line models that facilitate the study of the molecular basis of disease disparity.

Trop2 inhibition of P16 expression and the cell cycle promotes intracellular calcium release in OSCC.

Trop2 is an intracellular calcium signal transducer and a prognostic biomarker in many cancers. P16 is a cell cycle gene that negatively regulates cell proliferation and division in most human cancers. Oral squamous cell carcinoma (OSCC) is a common malignant tumor subgroup of head and neck squamous cell carcinoma worldwide. Both Ca2+-dependent and cell cycle signaling pathways play vital roles in OSCC, although the molecular mechanisms remain to be elucidated. We aimed to examine the function of Trop2 and P16 in regulating intracellular calcium ions and the cell cycle in OSCC cell lines. Furtherly, the mRNA and protein expression levels of Trop2 and P16 in OSCC tissue samples were assessed, and their function was evaluated as potential clinical prognostic biomarkers. Trop2 promoted intracellular calcium ion release in OSCC and induced S phase of the cell cycle. Moreover, Trop2-mediated Ca2+ inhibited P16 expression through the AMP-activated protein kinase pathway in OSCC. Interestingly, P16 overexpression could not reverse these phenomena in vitro. We also demonstrated that human OSCC tissues showed high Trop2 mRNA and protein expression, and Trop2+/P16- expression is an independent prognostic marker for OSCC patients. Our data suggest that Trop2+/P16- may be a valuable prognostic marker for OSCC and that Trop2 inhibits P16 expression and induces S phase by promoting intracellular calcium release in OSCC.

Detection of Human papillomavirus and the role of p16INK4a in colorectal carcinomas.

INTRODUCTION: Human papillomavirus (HPV) infection is associated with the development of anogenital and head and neck cancers. In recent years a potential role of HPV in colorectal cancer (CRC) has been suggested. OBJECTIVE: To investigate the presence of HPV in colorectal carcinomas and to study the role of p16INK4a as a marker of transcriptionally active HPV infection. In addition, to investigate the correlation between these findings and the CRC prognostic factors. METHODS: Case control study with 92 cases of colorectal cancers, 75 controls of normal tissue adjacent to the tumor, and 30 controls of precursor lesions, including polyps and colorectal adenomas. Paraffinized samples were used, HPV detection and genotyping were performed by PCR and reverse hybridization by using the INNO LIPA kit, with SPF10 plus primers. The expression of the p16INK4a protein was investigated using immunohistochemistry. Data analysis was performed using descriptive, univariate statistics and survival curves were calculated by using the Kaplan Meier and log-rank method. RESULTS: HPV was detected in 13% of the cases and the most prevalent genotype was HPV 16. HPV DNA was not detected in either control groups. The high expression of p16INK4a was observed in 30% of the cases, but it was not associated to the presence of HPV. The overall survival was 53.3% and was influenced by prognostic factors such as later stage, lymph node and distant metastasis. CONCLUSIONS: Based on these results, HPV is unlikely to be involved in colorectal carcinogenesis and p16INK4a expression is not a relevant marker of transcriptionally active HPV infection in CRC.

High frequency of p16 and SOX10 coexpression but not androgen receptor expression in triple-negative breast cancers.

Triple-negative breast cancers (TNBCs) represent approximately 12-17% of all breast cancers and have distinctively aggressive clinical courses. Because routine biomarkers for breast cancer do not apply for TNBCs, it is essential to find novel prognostic markers and potential targets for therapeutic agents. p16 and SOX10 are emerging biomarkers with relatively unexplored expressions in TNBCs. We present an analysis of the expression of p16 and SOX10 in combination with that of androgen receptor (AR) and cytokeratin (CK) 5/6 in TNBCs. In addition, we used tissue microarrays (TMAs) to compare frequencies of p16 and SOX10 between TNBCs and non-TNBCs. Fifty-six TNBC samples with clinical data were stained immunohistochemically with p16, SOX10, AR, and CK5/6. Fifty-four cases (96.4%) were invasive ductal carcinoma, not otherwise specified, and 46 cases (82.1%) were Nottingham histologic grade 3. The majority of TNBC cases were positive for p16 (n = 44; 78.6%) and SOX10 (n = 48; 85.7%). AR was positive in 15 cases (26.8%). CK5/6 was positive in 24 cases (42.9%), which were classified as basal-like breast cancer (BLBC) subtype. The frequencies of p16 and SOX10 expression in BLBC and non-BLBC subtypes did not reveal significant statistical difference in a separate analysis. Using archived TNBC and non-TNBC TMAs, we observed that 56% of TNBC cases were positive for p16 compared with 16% of non-TNBC cases (p-value <0.0001). SOX10 was positive in 80% of TNBC cases compared with 35% of non-TNBC cases (p-value <0.0001). A significant correlation was observed between p16 and SOX10 coexpression in TNBC cases (n = 56/80, p = 0.02) but not in non-TNBC cases (n = 23/348; p = 0.626). In conclusion, p16 and SOX10 are frequently expressed in TNBC, regardless of CK5/6 expression. Furthermore, p16 and SOX10 are often coexpressed in TNBCs compared with non-TNBCs.

Prevalence and Prognostic Value of HPV among Tunisian Patients with Laryngeal Cancer and Relationship between DNA HPV and p16, IGF-1R, Survivin, p53 Expressions.

OBJECTIVES: Tobacco and alcohol are the main etiological factors common to laryngeal cancers. However, the Human Papilloma Virus (HPV) constitutes an alternative risk factor according to several studies. In Tunisia, despite the annual increasing incidence of laryngeal squamous cell carcinoma (LSCC), the prevalence and prognostic significance of HPV have never been explored.In this study, we sought to highlight HPV DNA in 70 biopsies of laryngeal cancer, and to analyze the status of HPV infection in association with p53, p16, survivin, and IGF-1R expressions. METHODS: HPV high risk (HPV HR) DNA was detected in tumors by in situ hybridization. However, the expression of p53, p16, survivin and IGF-1R were stained by immunohistochemistry test. The correlations of HPV status with clinicopathological parameters, overall survival, disease-free survival and proteins expressions were statistically evaluated. RESULTS: HPV HR DNA was detected in 39 out of 70 (55.71%) laryngeal tumors. HPV+ patients have a better overall survival (P = .081) and long disease-free-survival (P = .016) with a low rate of recurrence (P = .006) than HPV- patients. No significant correlations were found between HPV HR status and clinicopathological parameters (all P > .005). Moreover, HPV+ tumors were not associated with expression of p53, p16 and survivin. However, HPV HR status correlates with weak to moderate IGF-1R expression (P = .043). CONCLUSION: The substantial detection of HPV HR in LSCC tumors suggest that this virus plays an important part in laryngeal cancer in Tunisia. It is a good prognostic factor. In addition, HPV infection could act to block the pathway of IGF-1R expression.

Amplification of MDM2 and Loss of p16 Expression: Do They Have a Role in Malignant Transformation of Ovarian Brenner Tumor?

OBJECTIVES: To review the significance of MDM2 and cyclin D1 expression and loss of p16 expression in malignant and borderline Brenner tumors (BTs) of the ovary. METHODS: We describe 2 new cases of ovarian BT, 1 malignant and 1 borderline. We studied MDM2, p16, and cyclin D1 expression by immunohistochemistry in the benign, borderline, and malignant components of these 2 cases and in 5 additional cases of benign BT. We also reviewed and summarized the literature on the clinical, immunohistochemical and molecular characteristics of borderline and malignant BTs (BdBTs and MBTs). RESULTS: Nuclear expression of MDM2 was seen only in the MBT. Loss of p16 expression was seen in both BdBT and MBT. Cyclin D1 expression was in proportion to the degree of malignancy. Amplification of MDM2, loss of CDKN2A (p16-encoding gene), and amplification of CCND1 (cyclin D1-encoding gene) were confirmed by commercial next-generation sequencing in the case of MBT. CONCLUSIONS: We are the first to report immunohistochemical expression of MDM2 in an MBT. Amplification of MDM2 and loss of p16 expression may have a role in malignant transformation of BT.

Loss of ARF/INK4A Promotes Liver Progenitor Cell Transformation Toward Tumorigenicity Supporting Their Role in Hepatocarcinogenesis.

Liver progenitor cells (LPCs) contribute to liver regeneration during chronic damage and are implicated as cells of origin for liver cancers including hepatocellular carcinoma (HCC). The CDKN2A locus, which encodes the tumor suppressors alternate reading frame protein (ARF) and INK4A, was identified as one of the most frequently altered genes in HCC. This study demonstrates that inactivation of CDKN2A enhances tumorigenic transformation of LPCs. The level of ARF and INK4A expression was determined in a panel of transformed and nontransformed wild-type LPC lines. Moreover, the transforming potential of LPCs with inactivated CDKN2A was shown to be enhanced in LPCs derived from Arf-/- and CDKN2Afl/fl mice and in wild-type LPCs following CRISPR-Cas9 suppression of CDKN2A. ARF and INK4A abundance is consistently reduced or ablated following LPC transformation. Arf-/- and CDKN2A-/- LPCs displayed hallmarks of transformation such as anchorage-independent and more rapid growth than control LPC lines with unaltered CDKN2A. Transformation was not immediate, suggesting that the loss of CDKN2A alone is insufficient. Further analysis revealed decreased p21 expression as well as reduced epithelial markers and increased mesenchymal markers, indicative of epithelial-to-mesenchymal transition, following inactivation of the CDKN2A gene were required for tumorigenic transformation. Loss of ARF and INK4A enhances the propensity of LPCs to undergo a tumorigenic transformation. As LPCs represent a cancer stem cell candidate, identifying CDKN2A as a driver of LPC transformation highlights ARF and INK4A as viable prognostic markers and therapeutic targets for HCC.

Concordance of tumor infiltrating lymphocytes, PD-L1 and p16 expression in small biopsies, resection and lymph node metastases of oropharyngeal squamous cell carcinoma.

OBJECTIVE: The incidence of oropharyngeal squamous cell carcinoma (OPSCC), especially human papillomavirus (HPV)-associated, is increasing worldwide. Immunotherapy become available for patients with carcinomas in the head and neck region, however without ideal biomarker. Markers like PD-L1 vary in the clone of the antibody used, and the method of evaluation. Adequate and reliable immune cells characterization and evaluation is still not found. Furthermore, studies analyzing representativeness of different tissue samples are scarce. We analyzed small biopsy, lymph node (LN) metastasis and resected OPSCC, in regards of tumor infiltrating lymphocyte (TIL) density, PD-L1 and p16 expression. MATERIAL AND METHODS: Patients with OPSCC diagnosed from 2000 to 2016, with small biopsy, resection specimen and LN metastasis samples were selected. We analyzed TILs on hematoxylin-eosin stain, and PD-L1 and p16 expression in tumor cells. Concordance between different tumor locations was evaluated. RESULTS: 93 patients, with 65 small biopsies, 72 resection specimens, and 70 LN metastases were included. TILs, p16 and PD-L1 demonstrated very high concordance. Additionally, PD-L1 expression in the small biopsies was more representative of the PD-L1 expression in the resection specimens, than the LN samples. CONCLUSION: TILs density can be reliably assessed using hematoxylin-eosin stain with high concordance between the small biopsy, resection specimen and LN metastasis. Evaluation of concordance of p16 expression is very high, nevertheless some cases might be misdiagnosed on a small biopsy or lymph node metastasis. Evaluation of PD-L1 expression is very reliable on the biopsy specimen. Different PD-L1 clones and methods of evaluation still remain to be addressed.

Absence of Nuclear p16 Is a Diagnostic and Independent Prognostic Biomarker in Squamous Cell Carcinoma of the Cervix.

The tumor-suppressor protein p16 is paradoxically overexpressed in cervical cancer (CC). Despite its potential as a biomarker, its clinical value and the reasons for its failure in tumor suppression remain unclear. Our purpose was to determine p16 clinical and biological significance in CC. p16 expression pattern was examined by immunohistochemistry in 78 CC cases (high-grade squamous intraepithelial lesions (HSILs) and squamous cell carcinomas of the cervix -SCCCs). CC cell proliferation and invasion were monitored by real-time cell analysis and Transwell® invasion assay, respectively. Cytoplasmic p16 interactors were identified from immunoprecipitated extracts by liquid chromatography-tandem mass spectrometry, and colocalization was confirmed by double-immunofluorescence. We observed that SCCCs showed significantly more cytoplasmic than nuclear p16 expression than HSILs. Importantly, nuclear p16 absence significantly predicted poor outcome in SCCC patients irrespective of other clinical parameters. Moreover, we demonstrated that cytoplasmic p16 interacted with CDK4 and other unreported proteins, such as BANF1, AKAP8 and AGTRAP, which could sequester p16 to avoid nuclear translocation, and then, impair its anti-tumor function. Our results suggest that the absence of nuclear p16 could be a diagnostic biomarker between HSIL and SCCC, and an independent prognostic biomarker in SCCC; and explain why p16 overexpression fails to stop CC growth.

Methylation of the Suppressor Gene p16INK4a: Mechanism and Consequences.

Tumor suppressor genes in the CDKN2A/B locus (p15INK4b, p16INK4a, and p14ARF) function as biological barriers to transformation and are the most frequently silenced or deleted genes in human cancers. This gene silencing frequently occurs due to DNA methylation of the promoter regions, although the underlying mechanism is currently unknown. We present evidence that methylation of p16INK4a promoter is associated with DNA damage caused by interference between transcription and replication processes. Inhibition of replication or transcription significantly reduces the DNA damage and CpGs methylation of the p16INK4a promoter. We conclude that de novo methylation of the promoter regions is dependent on local DNA damage. DNA methylation reduces the expression of p16INK4a and ultimately removes this barrier to oncogene-induced senescence.