The distinct effects of P18 overexpression on different stages of hematopoiesis involve TGF-ß and NF-κB signaling.

Deficiency of P18 can significantly improve the self-renewal potential of hematopoietic stem cells (HSC) and the success of long-term engraftment. However, the effects of P18 overexpression, which is involved in the inhibitory effects of RUNX1b at the early stage of hematopoiesis, have not been examined in detail. In this study, we established inducible P18/hESC lines and monitored the effects of P18 overexpression on hematopoietic differentiation. Induction of P18 from day 0 (D0) dramatically decreased production of CD34highCD43- cells and derivative populations, but not that of CD34lowCD43- cells, changed the cell cycle status and apoptosis of KDR+ cells and downregulated the key hematopoietic genes at D4, which might cause the severe blockage of hematopoietic differentiation at the early stage. By contrast, induction of P18 from D10 dramatically increased production of classic hematopoietic populations and changed the cell cycle status and apoptosis of CD45+ cells at D14. These effects can be counteracted by inhibition of TGF-ß or NF-κB signaling respectively. This is the first evidence that P18 promotes hematopoiesis, a rare property among cyclin-dependent kinase inhibitors (CKIs).

Host Tumor Suppressor p18INK4c Functions as a Potent Cell-Intrinsic Inhibitor of Murine Gammaherpesvirus 68 Reactivation and Pathogenesis.

Gammaherpesviruses are common viruses associated with lifelong infection and increased disease risk. Reactivation from latency aids the virus in maintaining infection throughout the life of the host and is responsible for a wide array of disease outcomes. Previously, we demonstrated that the virus-encoded cyclin (v-cyclin) of murine gammaherpesvirus 68 (γHV68) is essential for optimal reactivation from latency in normal mice but not in mice lacking the host tumor suppressor p18INK4c (p18). Whether p18 plays a cell-intrinsic or -extrinsic role in constraining reactivation remains unclear. Here, we generated recombinant viruses in which we replaced the viral cyclin with the cellular p18INK4c gene (p18KI) for targeted expression of p18, specifically within infected cells. We find that the p18KI virus is similar to the cyclin-deficient virus (cycKO) in lytic infection, establishment of latency, and infected cell reservoirs. While the cycKO virus is capable of reactivation in p18-deficient mice, expression of p18 from the p18KI virus results in a profound reactivation defect. These data demonstrate that p18 limits reactivation within latently infected cells, functioning in a cell-intrinsic manner. Further, the p18KI virus showed greater attenuation of virus-induced lethal pneumonia than the cycKO virus, indicating that p18 could further restrict γHV68 pathogenesis even in p18-sufficient mice. These studies demonstrate that host p18 imposes the requirement for the viral cyclin to reactivate from latency by functioning in latently infected cells and that p18 expression is associated with decreased disease, thereby identifying p18 as a compelling host target to limit chronic gammaherpesvirus pathogenesis.IMPORTANCE Gammaherpesviruses are ubiquitous viruses associated with multiple malignancies. The propensity to cycle between latency and reactivation results in an infection that is never cleared and often difficult to treat. Understanding the balance between latency and reactivation is integral to treating gammaherpesvirus infection and associated disease outcomes. This work characterizes the role of a novel inhibitor of reactivation, host p18INK4c, thereby bringing more clarity to a complex process with significant outcomes for infected individuals.

Abnormal p53 and p16 staining patterns distinguish uterine leiomyosarcoma from inflammatory myofibroblastic tumour.

AIMS: Uterine myxoid leiomyosarcoma may show relatively bland histological appearances, despite its aggressive behaviour. Distinguishing uterine leiomyosarcoma from the more indolent inflammatory myofibroblastic tumour (IMT), which is amenable to targeted therapies, can be challenging. A significant subset of leiomyosarcomas harbour TP53 and/or CDKN2A genomic alterations. Here, we examined the diagnostic value of p53 and p16 immunohistochemistry in the distinction of uterine conventional and myxoid leiomyosarcoma from IMT, in correlation with targeted sequencing of TP53 and CDKN2A. METHODS AND RESULTS: We performed p53 and p16 immunohistochemistry in 49 tumours, including 23 uterine leiomyosarcomas (12 myxoid, 11 conventional) and 26 IMT (12 uterine, 14 extrauterine). TP53 and CDKN2A coding regions were sequenced in 20 cases (four myxoid, 11 conventional uterine leiomyosarcomas; four uterine, one extrauterine IMT). Abnormal p53 staining patterns (strong/diffuse or null) were observed in six of 12 (50%) myxoid and six of 11 (55%) conventional leiomyosarcomas but none of the IMT (P < 0.0001), correlating with TP53 mutation/deletion (P = 0.0001). P16 loss was detected in five of 10 (50%) myxoid and two of 11 (18%) conventional leiomyosarcomas, but none of the IMT (P = 0.0005), correlating with CDKN2A deletion (P = 0.014). Strong/diffuse p16 staining in six of 21 (29%) leiomyosarcomas and three of 26 (12%) IMT did not correlate with CDKN2A alterations. CONCLUSIONS: Abnormal p53 staining and p16 loss are observed frequently in uterine leiomyosarcomas, with 100% specificity and 70% sensitivity against IMT, and correlating with genomic alterations. Conversely, IMT shows normal p53 and p16 staining, highlighting the use of these markers in the differential diagnosis of uterine mesenchymal neoplasms.

Novel epigenetic changes in CDKN2A are associated with progression of cervical intraepithelial neoplasia.

OBJECTIVE: To conduct a comprehensive mapping of the genomic DNA methylation in CDKN2A, which codes for the p16(INK4A) and p14(ARF) proteins, and 14 of the most promising DNA methylation marker candidates previously reported to be associated with progression of low-grade cervical intraepithelial neoplasia (CIN1) to cervical cancer. METHODS: We analyzed DNA methylation in 68 HIV-seropositive and negative women with incident CIN1, CIN2, CIN3 and invasive cervical cancer, assaying 120 CpG dinucleotide sites spanning APC, CDH1, CDH13, CDKN2A, CDKN2B, DAPK1, FHIT, GSTP1, HIC1, MGMT, MLH1, RARB, RASSF1, TERT and TIMP3 using the Illumina Infinium array. Validation was performed using high resolution mapping of the target genes with HELP-tagging for 286 CpGs, followed by fine mapping of candidate genes with targeted bisulfite sequencing. We assessed for statistical differences in DNA methylation levels for each CpG loci assayed using univariate and multivariate methods correcting for multiple comparisons. RESULTS: In our discovery sample set, we identified dose dependent differences in DNA methylation with grade of disease in CDKN2A, APC, MGMT, MLH1 and HIC1, whereas single CpG locus differences between CIN2/3 and cancer groups were seen for CDH13, DAPK1 and TERT. Only those CpGs in the gene body of CDKN2A showed a monotonic increase in methylation between persistent CIN1, CIN2, CIN3 and cancers. CONCLUSION: Our data suggests a novel link between early cervical disease progression and DNA methylation in a region downstream of the CDKN2A transcription start site that may lead to increased p16(INK4A)/p14(ARF) expression prior to development of malignant disease.

Functional Studies on Primary Tubular Epithelial Cells Indicate a Tumor Suppressor Role of SETD2 in Clear Cell Renal Cell Carcinoma.

SET domain-containing 2 (SETD2) is responsible for the trimethylation of histone H3 lysine36 (H3K36me3) and is one of the genes most frequently mutated in clear cell renal cell carcinoma (ccRCC). It is located at 3p21, one copy of which is lost in the majority of ccRCC tumors, suggesting that SETD2 might function as a tumor suppressor gene. However, the manner in which loss of SETD2 contributes to ccRCC development has not been studied in renal primary tubular epithelial cells (PTECs). Therefore, we studied the consequences of SETD2 knockdown through lentiviral shRNA in human PTECs. Consistent with its known function, SETD2 knockdown (SETD-KD) led to loss of H3K36me3 in PTECs. In contrast to SETD2 wild-type PTECs, which have a limited proliferation capacity; the SETD2-KD PTECs continued to proliferate. The expression profiles of SETD2-KD PTECs showed a large overlap with the expression profile of early-passage, proliferating PTECs, whereas nonproliferating PTECs showed a significantly different expression profile. Gene set enrichment analysis revealed a significant enrichment of E2F targets in SETD2-KD and proliferating PTECs as compared with nonproliferating PTECs and in proliferating PTEC compared with SETD2-KD. The SETD2-KD PTECs maintained low expression of CDKN2A and high expression of E2F1, whereas their levels changed with continuing passages in untreated PTECs. In contrast to the nonproliferating PTECs, SETD2-KD PTECs showed no ß-galactosidase staining, confirming the protection against senescence. Our results indicate that SETD2 inactivation enables PTECs to bypass the senescence barrier, facilitating a malignant transformation toward ccRCC.

Immunohistochemical evaluation of p16 expression in cutaneous histiocytic, fibrohistiocytic and undifferentiated lesions.

BACKGROUND: Expression of p16 is frequently evaluated in melanocytic lesions. Expression of p16 in cutaneous histiocytic, fibrohistiocytic and undifferentiated lesions has not been well characterized. METHODS: We evaluated p16 expression in a cohort of histiocytic (reticulohistiocytoma, Langerhans cell histiocytosis, xanthogranuloma, Rosai Dorfman disease and xanthoma), fibrohistiocytic (dermatofibroma, epithelioid fibrous histiocytoma and dermatofibrosarcoma protuberans) and undifferentiated (atypical fibroxanthoma and pleomorphic undifferentiated sarcoma) lesions. A group of melanocytic lesions (Spitz nevus, ordinary nevus, spitzoid melanoma and non-spitzoid melanoma) were also evaluated as reference. Each case was scored by the proportion of p16-positive cells and by staining intensity. RESULTS: Immunoreactivity for p16 was found in almost all histiocytic (28/30, 93%) and fibrohistiocytic (22/24, 92%) lesions. About half of the undifferentiated lesions also exhibited p16 staining (9/17, 53%). Most of the melanocytic cases examined in this study expressed p16. A wide range of staining intensity and proportion of p16-positive cells was observed in most groups. CONCLUSION: Expression of p16 is common, albeit variable in proportion and intensity, amongst a wide variety of cutaneous histiocytic, fibrohistiocytic and undifferentiated lesions. Further studies are required to determine if p16 expression is useful in distinguishing benign from malignant neoplasms of these types.

Genetic variation at a Yin-Yang 1 response site regulates the transcription of cyclin-dependent kinase inhibitor p18INK4C transcript in lupus-prone mice.

We have previously shown that a novel -74 C-to-T mutation in the promoter of the cyclin-dependent kinase inhibitor p18(Ink4c) (p18) gene was associated with a reduced p18 expression in B cells from mice carrying the Sle2c1 lupus susceptibility locus. To determine the function of the -74 C/T single nucleotide polymorphism, we have characterized the proximal promoter of the mouse p18 gene. Functional analysis of the 5' flanking region by sequential deletions revealed crucial elements between -300 and +1, confirming the in silico prediction that the -74 T allele created a novel Yin-Yang 1 (YY-1) binding site adjacent to an existing one common to both alleles. Moreover, we found that YY-1, E2F1, and Sp-1 can synergistically enhance the activity of the p18 promoter. Mutational inactivation revealed that YY-1 binding regulates the p18 activity in an allele-dependent fashion. EMSAs with splenic B cell extracts directly demonstrated that YY-1 binds to the p18 promoter with differences between the C and the T alleles. We also determined in vivo by chromatin immunoprecipitation that the T allele resulted in increased YY-1 and decreased Nrf-2 binding to the p18 promoter as compared with the C allele in B cells. Thus, YY-1 is a direct regulator of p18 gene expression in an allele-dependent fashion that is consistent with the lupus-associated T allele, inducing a lower p18 transcriptional activity by increasing YY-1 binding. These results establish the p18 -74 C/T mutation as the leading causal variant for the B1a cell expansion that characterizes the NZB and NZM2410 lupus-prone strains.

P18 is a tumor suppressor gene involved in human medullary thyroid carcinoma and pheochromocytoma development.

In multiple endocrine neoplasia syndrome Type 2 (MEN2), medullary thyroid carcinoma (MTC) and pheochromocytoma (PC) are associated with hereditary activating germ-line mutations in the RET proto-oncogene. Also in a large percentage of sporadic MTCs and PCs, somatic RET mutations appear to be involved in tumor formation. In one single MEN2 family an extensive variety in disease expression may be observed, indicating that additional genetic events are responsible for progression of the disease towards a more aggressive phenotype. However, these additional mutations in both hereditary and sporadic MTC and PC development are largely unknown. Here, we show for the first time the presence of somatic mutations in the cell cycle regulator P18 in human RET-associated MTCs and PCs. Each of these mutations causes an amino acid substitution in the cyclin dependent kinase-interacting region of P18(INK4C). Since these mutations partly inhibited P18(INK4C) function and reduced its stability, our findings implicate P18 as a tumor suppressor gene involved in human MTC and PC development.

Sodium butyrate-induced upregulation of p18( INK4C ) gene affects K562 cell G (0)/G (1) arrest and differentiation.

Histone deacetylase inhibitor sodium butyrate (NaBu) can induce G(0)/G(1) arrest and erythroid differentiation in K562 cells, but the molecular mechanisms underlying this process are unclear. Here we show that both p18( INK4C ) mRNA and protein levels were upregulated during K562 cell erythroid differentiation induced by NaBu. Moreover, the NaBu activation of p18( INK4C ) was dependent on the integrity of Sp1 clusters in the promoter. NaBu caused hyperacetylation of histones H3 and H4 on endogenous p18( INK4C ) promoter and enhanced binding of transcription factor Sp1 in vivo. Also, overexpression of p18( INK4C ) in K562 cells resulted in G(0)/G(1) arrest and partial erythroid differentiation. Our results suggested that NaBu-mediated p18( INK4C ) regulation played a role in cell cycle arrest and erythroid differentiation in K562 cells.

Menin regulates pancreatic islet growth by promoting histone methylation and expression of genes encoding p27Kip1 and p18INK4c.

Menin, the product of the Men1 gene mutated in familial multiple endocrine neoplasia type 1 (MEN1), regulates transcription in differentiated cells. Menin associates with and modulates the histone methyltransferase activity of a nuclear protein complex to activate gene expression. However, menin-dependent histone methyltransferase activity in endocrine cells has not been demonstrated, and the mechanism of endocrine tumor suppression by menin remains unclear. Here, we show that menin-dependent histone methylation maintains the in vivo expression of cyclin-dependent kinase (CDK) inhibitors to prevent pancreatic islet tumors. In vivo expression of CDK inhibitors, including p27 and p18, and other cell cycle regulators is disrupted in mouse islet tumors lacking menin. Chromatin immunoprecipitation studies reveal that menin directly associates with regions of the p27 and p18 promoters and increases methylation of lysine 4 (Lys-4) in histone H3 associated with these promoters. Moreover, H3 Lys-4 methylation associated with p27 and p18 is reduced in islet tumors from Men1 mutant mice. Thus, H3 Lys-4 methylation is a crucial function of menin in islet tumor suppression. These studies suggest an epigenetic mechanism of tumor suppression: by promoting histone modifications, menin maintains transcription at multiple loci encoding cell cycle regulators essential for endocrine growth control.