p18INK4C Expression Research Articles

Bone morphogenetic protein 4 rescues the bone regenerative potential of old muscle-derived stem cells via regulation of cell cycle inhibitors

BackgroundBone morphogenetic protein 4 (BMP4) promotes the osteogenic differentiation and the bone regenerative potential of muscle-derived stem cells (MDSCs). BMP4 also promotes the self-renewal of both embryonic and somatic stem cells; however, BMP4 signaling activity significantly decreases with age. Cyclin-dependent kinase inhibitors P16INK4A (P16) and P18INK4C (P18) induce early G1-phase cell cycle blockade by targeting cyclin-dependent kinase 4/6. It is still unclear if BMP4 affects the bone regenerative potential of old MDSCs through regulation of P16 and P18 expression.MethodsYoung and old MDSCs were isolated from 3 week (young) and 2-year-old (old) mice. In vitro cell proliferation and multipotent differentiation were performed for young and old MDSCs both before and after BMP4/GFP transduction. Cell cycle genes were analyzed using Q-PCR. The bone regenerative potential of young and old MDSCs transduced with BMP4/GFP were compared using Micro-CT and histological analysis. The bone regenerative potential of young and old MDSCs was also compared between single and double transduction (higher BMP4 levels expression). The cell proliferation, mitochondrial function and osteogenic differentiation was also compared in vitro between cells that have been transduced with BMP4GFP (single and double transduction). The correlation of bone regeneration capacity of young and old MDSCs with P16 and P18 expression was further evaluated at 10 days after cell transplantation using histology and western blot analysis.ResultsOld murine MDSCs (MDSCs) exhibit reduced proliferation and multi-lineage differentiation potential with or without BMP4 stimulation, when compared to young murine MDSCs. Old MDSCs express significantly higher P16 and lower P18, with more cells in the G0/1 phase and fewer cells in the G2/M phase, compared to young MDSCs. Old MDSCs retrovirally transduced to express BMP4 regenerated less bone in a critical size skull defect in CD-1 nude mice when compared to young retrovirally transduced MDSCs expressing similar BMP4 levels and contribute less to the new regenerated new bone. Importantly, both young and old MDSCs can regenerate more bone when BMP4 expression levels are increased by double-transduction with the retroviral-BMP4/GFP. However, the bone regeneration enhancement with elevated BMP4 was more profound in old MDSCs (400% at 2 weeks) compared to young MDSCs (200%). Accordingly, P18 is upregulated while P16 is downregulated after BMP4 transduction. Double transduction did not further increase cell proliferation nor mitochondrial function but did significantly increase Osx expression in both young and old MDSCs. Old MDSCs had even significant higher Osx levels as compared to young MDSCs following double transduction, while a similar Alp expression was observed between young and old MDSCs after double transduction. In addition, at 10 days after cell transplantation, old MDSCs having undergone double transduction regenerated bone more rapidly as showed by Alcian blue and Von Kossa staining. Western blot assays demonstrated that old MDSCs after retro-BMP4/GFP double transduction have significantly lower P18 expression levels when compared to young BMP4-transduced MDSCs. In addition, P18 expression was slightly increased in old MDSCs after double transduction when compared to single transduction. P16 expression was not detectable for both young and two old BMP4/GFP transduced MDSCs groups.ConclusionsIn summary, BMP4 can offset the adverse effect of aging on the osteogenic differentiation and the bone regenerative potential of old MDSCs via up-regulation of P18 and down-regulation P16 expression.

MCL1 binding to the reverse BH3 motif of P18INK4C couples cell survival to cell proliferation

Commitment to cell cycle entry and cellular duplication is a tightly coordinated and regulated process. Once initiated, a series of multiple checkpoints ensure both accurate genomic replication and chromosomal separation. In the event of unsuccessful cell division, parallel pathways exist that induce the cell to undergo programmed cell death, or apoptosis. At the center of such stress-induced, intrinsic apoptotic regulation lies the BCL2 family of pro- and anti-apoptotic regulatory proteins. In a proliferative state the balance of pro- and anti-apoptotic signaling proteins would be expected to favor an excess population of anti-apoptotic members. While the anti-apoptotic BCL2 family member, MCL1, has been identified to oversee mitotic progression, direct communication between the BCL2 family and cell proliferation has not been observed. In this study, we demonstrate a direct protein–protein interaction between MCL1 and the G1/S checkpoint protein, P18INK4C. This interaction is mediated by a reverse BH3 (rBH3) motif located in P18INK4C’s C-terminal ankyrin repeat. MCL1 is further shown to decrease P18INK4C expression and thereby regulate cell cycle entry in a retinoblastoma (RB1)-dependent manner. Our findings establish a mechanism for translation independent and direct communication between the BCL2 family regulation of apoptosis and CDK4/6-RB regulation of early G1/S transition during cellular division/growth.

Expression of p27Kip1 and p18Ink4c in human multiple endocrine neoplasia type 1-related pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumors are a major manifestation of multiple endocrine neoplasia type 1 (MEN1). This tumor syndrome is caused by germline mutations in MEN1, encoding menin. Insight into pathogenesis of these tumors might lead to new biomarkers and therapeutic targets for these patients. Several lines of evidence point towards a role for p27Kip1 and p18Ink4c in MEN1-related tumor development in animal models for MEN1, but their contribution to human MEN1-related pancreatic neuroendocrine tumor development is not known. In this study, we characterized protein expression of p27Kip1 and p18Ink4c in human MEN1-related PanNETs by immunohistochemistry. From the nationwide DutchMEN1 Study Group database including>90% of the Dutch MEN1 population, MEN1-patients, who underwent pancreatic surgery, were selected. A tissue micro-array was constructed with available paraffin tissue blocks, and PanNETs from 61 MEN1 patients were eligible for analysis. Expression of p27Kip1 was high in 57 (93%) PanNETs and 67% of the tumors showed low expression of p18Ink4c (67.3%). No association was found between expression of either p27Kip1 or p18Ink4c and clinic-pathological characteristics. These findings indicate that loss of p18Ink4c, but not p27Kip1, is a common event in the development of MEN1-related PanNETs. Restoration of p18Ink4c function through CDK4/6 inhibitors could be a therapeutic option for MEN1-related PanNETs.

Down-regulation of ribosomal protein S15A inhibits proliferation of human glioblastoma cells in vivo and in vitro via AKT pathway

Background: Ribosomal protein s15a (RPS15A), a highly conserved cytoplasmic protein, promotes mRNA/ribosome interaction in translation. Recent evidence showed that RPS15A would be essential for tumor cell growth.Objective: The aim of this study was to investigate whether RPS15A has prognostic relevance in human glioblastoma.Material and methods: Levels of RPS15A expression were measured in glioblastoma and normal brain (NB) tissues samples. Stably decreased expression of RPS15A was established by the pLVTHM-GFP lentiviral RNAi expression system. Molecular mechanisms and the effect of RPS15A on cell growth and migration were investigated using MTT assay, wound healing assay, transwell migration assay, Flow cytometry analysis, western blot assay, immunofluorescence double labeling and tumorigenesis in nude mice.Results: Here we report that RPS15A is specifically overexpressed in human glioblastoma tumor tissues, and RPS15A knockdown inhibits proliferation and migration of glioblastoma cells in vitro. Lentivirus, transduced with shRPS15A, lead to depression of phosphorylated AKT, cell cycle arrest in G0/G1 phase in U87 cells, and also reduce expression of cell cycle regulation protein CDK4 while enhancing expression of p18INK4C. Furthermore, the growth of glioblastoma cell-transplanted tumors in nude mice is inhibited by transduction with Lv-shRPS15A.Conclusion: Our findings indicate that RPS15A promotes cell proliferation and migration in glioblastoma for the first time. RPS15A might play a distinct role in glioblastoma and serve as a potential target for therapy. Background: Ribosomal protein s15a (RPS15A), a highly conserved cytoplasmic protein, promotes mRNA/ribosome interaction in translation. Recent evidence showed that RPS15A would be essential for tumor cell growth. Objective: The aim of this study was to investigate whether RPS15A has prognostic relevance in human glioblastoma. Material and methods: Levels of RPS15A expression were measured in glioblastoma and normal brain (NB) tissues samples. Stably decreased expression of RPS15A was established by the pLVTHM-GFP lentiviral RNAi expression system. Molecular mechanisms and the effect of RPS15A on cell growth and migration were investigated using MTT assay, wound healing assay, transwell migration assay, Flow cytometry analysis, western blot assay, immunofluorescence double labeling and tumorigenesis in nude mice. Results: Here we report that RPS15A is specifically overexpressed in human glioblastoma tumor tissues, and RPS15A knockdown inhibits proliferation and migration of glioblastoma cells in vitro. Lentivirus, transduced with shRPS15A, lead to depression of phosphorylated AKT, cell cycle arrest in G0/G1 phase in U87 cells, and also reduce expression of cell cycle regulation protein CDK4 while enhancing expression of p18INK4C. Furthermore, the growth of glioblastoma cell-transplanted tumors in nude mice is inhibited by transduction with Lv-shRPS15A. Conclusion: Our findings indicate that RPS15A promotes cell proliferation and migration in glioblastoma for the first time. RPS15A might play a distinct role in glioblastoma and serve as a potential target for therapy.

DNA methyltransferase 3A promotes cell proliferation by silencing CDK inhibitor p18INK4C in gastric carcinogenesis

Little is known about the roles of DNA methyltransferase 3A (DNMT3A) in gastric carcinogenesis. Here, we reported that the exogenous expression of DNMT3A promoted gastric cancer (GC) cell proliferation by accelerating the G1/S transition. Subsequently, p18INK4C was identified as a downstream target of DNMT3A. The elevated expression of DNMT3A suppressed p18INK4C at least at the transcriptional level. Depletion of p18INK4C expression in GC cells induced cell cycle progression, whereas its re-expression alleviated the effect of DNMT3A overexpression on G1/S transition. Furthermore, we found that DNMT3A modulated p18INK4C by directly binding to and silencing the p18INK4C gene via promoter hypermethylation. In clinical GC tissue specimens analyzed, the level of methylation of p18INK4C detected in tumor tissues was significantly higher than that in paired non-tumor tissues. Moreover, elevated level of DNMT3A expression was associated with the differentiation of GC tissues and was negatively correlated with the p18INK4C expression level. Taken together, our results found that DNMT3A contributes to the dysregulation of the cell cycle by repressing p18INK4C in a DNA methylation-dependent manner, suggesting that DNMT3A-p18INK4C axis involved in GC. These findings provide new insights into gastric carcinogenesis and a potential therapeutic target for GC that may be further investigated in the future.

PPARγ1 phosphorylation enhances proliferation and drug resistance in human fibrosarcoma cells

Post-translational regulation plays a critical role in the control of cell growth and proliferation. The phosphorylation of peroxisome proliferator-activated receptor γ (PPARγ) is the most important post-translational modification. The function of PPARγ phosphorylation has been studied extensively in the past. However, the relationship between phosphorylated PPARγ1 and tumors remains unclear. Here we investigated the role of PPARγ1 phosphorylation in human fibrosarcoma HT1080 cell line. Using the nonphosphorylation (Ser84 to alanine, S84A) and phosphorylation (Ser84 to aspartic acid, S84D) mutant of PPARγ1, the results suggested that phosphorylation attenuated PPARγ1 transcriptional activity. Meanwhile, we demonstrated that phosphorylated PPARγ1 promoted HT1080 cell proliferation and this effect was dependent on the regulation of cell cycle arrest. The mRNA levels of cyclin-dependent kinase inhibitor (CKI) p21Waf1/Cip1 and p27Kip1 descended in PPARγ1S84D stable HT1080 cell, whereas the expression of p18INK4C was not changed. Moreover, compared to the PPARγ1S84A, PPARγ1S84D up-regulated the expression levels of cyclin D1 and cyclin A. Finally, PPARγ1 phosphorylation reduced sensitivity to agonist rosiglitazone and increased resistance to anticancer drug 5-fluorouracil (5-FU) in HT1080 cell. Our findings establish PPARγ1 phosphorylation as a critical event in human fibrosarcoma growth. These findings raise the possibility that chemical compounds that prevent the phosphorylation of PPARγ1 could act as anticancer drugs.

Abstract 576: Transforming growth factor-beta (TGF-β) inhibits growth of human myeloid leukemia cells and downregulates the expression of CDK inhibitor, p18INK4C.

Abstract The cell cycle progression controlled by cyclin-dependent kinases (CDKs) is counterbalanced by CDK inhibitors (CKIs). The CKIs have two families: CIP/KIP and INK. The CIP/KIP members are composed of p21WAF1/CIP1, p27KIP1 and p57KIP2, which bind to and inhibit both cyclin D-CDK4/6 kinases and cyclin-E/A-CDK2. The INK members consist of p15INK4B, p16INK4A, p18INK4C and p19INK4D. The members in this family only bind to and inhibit G1CDKs (CDK4 and CDK6). We have previously reported that p27KIP1 plays both a positive and a negative role in human myeloid leukemia cells. In this study, we investigated whether p18INK4C plays any role in TGF-β-mediated cell cycle control of human myeloid leukemia cells. TGF-β significantly inhibited proliferation of MV4-11 and TF-1 cells, with upregulation of p27KIP1and downregulation of multiple CDKs and cyclins. Surprisingly, TGF-β significantly inhibited the expression of p18INK4C, a traditional G1 CDK inhibitor. The inhibitory effect of TGFβ on p18INK4C was time dependent. The time course showed that early inhibition of p18INK4C occurred at 3h after initiating TGFβ treatment in the cells with a maximal inhibition of 70% being observed at 72h incubation with TGF-β. The control cells treated with the solvent for TGFβ did not show any significant changes in the levels of p18INK4C. As a sample loading control, the cells treated with or without TGF-β expressed about the same levels of actin.TGFβ-induced p18INK4C inhibition is also dose dependent with a maximal inhibition being detected by Western blot when 30ng/ml of TGFβ was added to the culture. Low concentrations (5ng/ml or less) did not markedly affect the expression of p18INK4C. The association of p18INK4C with CDK4 was also decreased in response to TGF-β treatment detected by immunoprecipitation. Our data also showed that the two cell lines tested barely expresses p15INK4B, p16INK4A, p19INK4D, and p21WAF1/CIP1 and these inhibitors do not appear to play an important role, either positively or negatively, in the cell cycle control of the leukemia cells. Our data suggest that p18INK4C may be required for cell cycle progression in human myeloid leukemia cells tested. Whether such a positive role of p18INK4C is caused by a possible gene mutation(s) and thereby contributes to leoplasia of the leukemia cells is currently under investigation (supported by NIH-NIGMS MBRS RISE: R25 GM059244, Barry University). . Citation Format: Talia Guardia, Reshma Badaloo, Valentina Serrano, Alejandra Toro, Xiaotang Hu. Transforming growth factor-beta (TGF-β) inhibits growth of human myeloid leukemia cells and downregulates the expression of CDK inhibitor, p18INK4C. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 576. doi:10.1158/1538-7445.AM2013-576

The function of hematopoietic stem cells is altered by both genetic and inflammatory factors in lupus mice

AbstractHematopoietic stem cells (HSCs) are protected in a metabolically dormant state within the bone marrow stem cell niche. Inflammation has been shown to disrupt HSC dormancy and cause multiple functional changes. Here, we investigated whether HSC functions were altered in systemic lupus erythematosus (SLE)-prone mice and whether this contributed to clinical manifestations of SLE. We found that HSCs were significantly expanded in lupus mice. The increase in HSC cellularity was caused by both genetic lupus risk factors and inflammatory cytokines in lupus mice. In addition, the inflammatory conditions of lupus led to HSC mobilization and lineage-biased hematopoiesis. Strikingly, these functionally altered HSCs possessed robust self-renewal capacity and exhibited repopulating advantages over wild-type HSCs. A single-nucleotide polymorphism in the cdkn2c gene encoding p18INK4c within a SLE susceptibility locus was found to account for reduced p18INK4c expression and the increase in HSC self-renewal capacity in lupus mice. Lupus HSCs with enhanced self-renewal capacity and resistance to stress may compete out transplanted healthy HSCs, thereby leading to relapses after HSC transplantation.

Glucocorticoid alternative effects on proliferating and differentiated mammary epithelium are associated to opposite regulation of cell‐cycle inhibitor expression

Glucocorticoids influence post-natal mammary gland development by sequentially controlling cell proliferation, differentiation, and apoptosis. In the mammary gland, it has been demonstrated that glucocorticoid treatment inhibits epithelial apoptosis in post-lactating glands. In this study, our first goal was to identify new glucocorticoid target genes that could be involved in generating this effect. Expression profiling, by microarray analysis, revealed that expression of several cell-cycle control genes was altered by dexamethasone (DEX) treatment after lactation. Importantly, it was determined that not only the exogenous synthetic hormone, but also the endogenous glucocorticoids regulated the expression of these genes. Particularly, we found that the expression of cell cycle inhibitors p21CIP1, p18INK4c, and Atm was differentially regulated by glucocorticoids through the successive stages of mammary gland development. In undifferentiated cells, DEX treatment induced their expression and reduced cell proliferation, while in differentiated cells this hormone repressed expression of those cell cycle inhibitors and promoted survival. Therefore, differentiation status determined the effect of glucocorticoids on mammary cell fate. Particularly, we have determined that p21CIP1 inhibition would mediate the activity of these hormones in differentiated mammary cells because over-expression of this protein blocked DEX-induced apoptosis protection. Together, our data suggest that the multiple roles played by glucocorticoids in mammary gland development and function might be at least partially due to the alternative roles that these hormones play on the expression of cell cycle regulators.

CO16. The Mitogen Activated Protein Kinase pathway regulates the expression of p18INK4c and p21Waf1/Cip1 cyclin-dependent kinase inhibitors through the activating protein-1 transcription factor

CO16. The Mitogen Activated Protein Kinase pathway regulates the expression of p18INK4c and p21Waf1/Cip1 cyclin-dependent kinase inhibitors through the activating protein-1 transcription factor

Upregulation of E2F1 in cerebellar neuroprogenitor cells and cell cycle arrest during postnatal brain development

In the developing cerebellum, proliferation of granular neuroprogenitor (GNP) cells lasts until the early postnatal stages when terminal maturation of the cerebellar cortex occurs. GNPs are considered cell targets for neoplastic transformation, and disturbances in cerebellar GNP cell proliferation may contribute to the development of pediatric medulloblastoma. At the molecular level, proliferation of GNPs is regulated through an orchestrated action of the SHH, NOTCH, and WNT pathways, but the underlying mechanisms still need to be dissected. Here, we report that expression of the E2F1 transcription factor in rat GNPs is inversely correlated with cell proliferation rate during postnatal development, as opposed to its traditional SHH-dependent induction of cell cycle. Proliferation of GNPs peaked at postnatal day3 (P3), with a subsequent continuing decrease in proliferation rates occurring until P12. Such gradual decline in proliferating neuroprogenitors paralleled the extent of cerebellum maturation confirmed by histological analysis with cresyl violet staining and temporal expression profiling of SHH, NOTCH2, and WNT4 genes. A time course analysis of E2F1 expression in GNPs revealed significantly increased levels at P12, correlating with decreased cell proliferation. Expression of the cell cycle inhibitor p18 ( Ink4c ), a target of E2F1, was also significantly higher at P12. Conversely, increased E2F1 expression did not correlate with either SMAC/DIABLO and BCL2 expression profiles or apoptosis of cerebellar cells. Altogether, these results suggest that E2F1 may also be involved in the inhibition of GNP proliferation during rat postnatal development despite its conventional mitogenic effects.

Upregulation of p18Ink4c expression by oncogenic HPV E6 via p53‐miR‐34a pathway

Binding of p53 to miR-34a promoter activates the expression of tumor-suppressive miR-34a. Oncogenic human papillomavirus (HPV) infection downregulates miR-34a expression through viral E6 degradation of p53. In our report, we found that miR-34a specifically targets p18Ink4c, a CDK4 and CDK6 inhibitor induced by E2F transactivation. HPV18(+) HeLa cells with ectopic miR-34a expression or by E6 siRNA knockdown-induced expression of endogenous miR-34a exhibited a substantial reduction of p18Ink4c in a dose-dependent manner, but had no effect on p16Ink4a, another member of CDK4/6 inhibitor family. In contrast, de novo infection by oncogenic HPVs of human keratinocyte-derived raft tissues increased p18Ink4c expression. Suppression of endogenous miR-34a in cell lines with a miR-34a inhibitor also increased p18Ink4c. We found that miR-34a suppresses the expression of p18Ink4c by binding to a specific seed match in the 5' UTR of p18Ink4c. Further investigation found remarkable increase of p18Ink4c in cervical precancer lesions and cervical cancer. Immunohistochemical staining of cervical tissue arrays showed increased expression of p18Ink4c in 68% of cervical cancer, 8.3% of chronic cervical inflammation and 4.8% of normal cervix. Although p18Ink4c inhibits cell proliferation in general and regulates E2F1 expression in HCT116 cells, it appears not to function as a tumor suppressor in cervical cancer cells lacking an intact G1 checkpoint because of viral E7 degradation of pRB. In summary, our study demonstrates an intimate connection among oncogenic HPV E6, p53, miR-34a and p18Ink4c and identifies p18Ink4c as a possible biomarker for cervical cancer.

Upregulation of p18Ink4c expression by HPV E6 via p53-miR-34a pathway

Binding of p53 to miR-34a promoter activates the expression of tumor suppressive miR-34a. Our previous study demonstrated that oncogenic HPV infection downregulates miR-34a expression through degradation of p53 mediated by E6. In searching for miR-34a targets, we found that miR-34a mainly downregulates p18Ink4c, a CDK4/6 inhibitor induced by E2F transactivation. HPV18+ HeLa cells with ectopic miR-34a expression or by E6 siRNA knockdown-induced expression of miR-34a had a substantially reduced expression of p18Ink4c in a dose-dependent manner, but had no effect on p16Ink4a, another member of CDK4/6 inhibitor family. Further investigation showed an increased p18Ink4c level in cervical cancer tissues by western blotting as compared to normal cervix. By immunostaining of tissue arrays, an increased expression of p18Ink4c was found in 68% of cervical cancer tissues, but only in 6.6% of normal cervical tissues. Moreover, specific inhibition of miR-34a expression in CaSki cells promoted the expression of p18Ink4c. Raft cultures with HPV16 or HPV18 infections also showed an increased p18Ink4c expression. In a reporter assay, we demonstrated that disruption of the miR-34a seed match from p18Ink4c promoted luciferase activity in the presence of ectopic miR-34a. As a cell cycle regulatory protein in normal cells, the increased p18Ink4c expression by E6-induced p53 destabilization and miR-34a reduction in cervical cancer would lead to suppress cell cycling and proliferation. However, viral E7 expressed in cervical cancer cells inactivates pRB and disassociates E2F from pRB, resulting in disruption of normal G1 checkpoint. Thus, an increased p18Ink4c in the cervical cancer cells would not affect the cell growth. To test this hypothesis, we knocked down p18Ink4c expression by siRNA in both HPV18-positive HeLa cells and HPV-negative HCT116 cells, a colon cancer cell line that contains an intact G1 checkpoint. As we predicted, knocking down p18Ink4c expression promoted the growth of HCT116 cells but had no effect on HeLa cells. Altogether, our data indicate that p18Ink4c is a miR-34a target and its increased expression in cervical cancer tissues can be used as a new biomarker for cervical cancer diagnosis and prognosis.

Abstract B26: Lack of cyclin D1 kinase activity leads to reduced levels of ErbB2-induced senescence and activation of the Wnt signaling pathway in the mammary gland

Abstract Proper orchestration of the Oncogene-induced Senescence (OIS) checkpoint relies upon the activation of the ARF-p53 and/or the p16 INK4a-pRB tumor suppressor pathways. Overexpression of cyclin D1, as observed in a large proportion of human breast cancers, is believed to confer proliferative advantage through pRb inactivation. The aim of the current study is to assess the role of cyclin D1-associated kinase activity during ErbB2-induced senescence in the mammary epithelium using a kinase-deficient cyclin D1 knock-in mouse (cyclin D1KE/KE) as a model. The effect of cyclin D1WT/WT or cyclin D1KE/KE on MMTV-ErbB2-induced senescence was analyzed in mammary glands of double-transgenic animals. Surprisingly, we observed that cyclin D1KE/KE/MMTV-ErbB2 mammary glands fail to upregulate markers of cellular senescence when compared to preneoplastic cyclin D1WT/WT/ MMTV-ErbB2 mammary glands. These findings correlated with high levels of proliferation and increased cellularity per duct section despite the fact that the cyclin D1KE/KE/MMTV-ErbB2 mammary epithelial cells do not progress to tumor formation. Moreover, this ectopic proliferation was not accompanied by phosphorylation at Ser780 of pRb, suggesting that other D-cyclins were unable to compensate for the lack of cyclin D1-associated kinase activity. Interestingly, failure to implement the senescence program in mutant mammary epithelium was accompanied by an activation of the Wnt signaling pathway that led to stabilization of beta-catenin in mutant mammary epithelial cells, an observation that others have found to correlate with senescence bypass in MEFs. In addition, induction of senescence in cyclin D1WT/WT/ MMTV-ErbB2 mammary glands did not lead to the accumulation of the gene products encoded by the INK4a/ARF locus. Instead, p18 INK4c expression was increased in D1WT/WT/ MMTV-ErbB2 mammary glands above the basal levels observed in cyclin D1WT/WT mammary tissue that did not express MMTV-ErbB2. A comparable induction was not observed in cyclin D1KE/KE/ MMTV-ErbB2 mammary glands. Taken together, our results suggest a dual role for cyclin D1-associated kinase activity in ErbB2-induced senescence and tumor formation. Cyclin D1 kinase activity is necessary for the full transformation of ErbB2-expressing cells, but it may also play an active role in the cascade that leads ErbB2-overexpressing cells to senesce such that cells that are kinase-deficient can still proliferate due to Wnt signaling activation. Citation Information: Cancer Res 2009;69(23 Suppl):B26.

Expression of p18INK4C is Down-regulated in Human Pituitary Adenomas

Cyclin-dependent kinase inhibitors represented by the INK4 family comprising p16(INK4A), p15(INK4B), p18(INK4C), and p19(INK4D) are regulators of the cell cycle shown to be aberrant in many types of cancer. Mice lacking p18(Ink4c) exhibit a series of phenotypes including the development of widespread organomegaly and pituitary adenomas. The objective of our study is to examine the role of p18(INK4C) in the pathogenesis of human pituitary tumors. The protein and mRNA levels of p18(INK4C) were examined by immunohistochemistry and real-time reverse transcription-polymerase chain reaction, respectively. The methylation status of the p18(INK4C) gene promoter and somatic mutations of the p18(INK4C) gene were also investigated. p18(INK4C) protein expression was lost or significantly reduced in 64% of pituitary adenomas compared with levels in normal pituitary glands. p18(INK4C) mRNA levels were low in all ACTH adenomas and non-functioning (NF)-FSH and in 42%, 70% and 66% of GH, PRL, and subtype 3 adenomas, respectively. p18(INK4C) mRNA levels were significantly associated with p18(INK4C) protein levels. Neither methylated promoters in pituitary adenomas, except in one NF-FSH adenoma, nor somatic mutations of the p18(INK4C) gene in any pituitary adenomas were detected. The down-regulation of p18(INK4C) expression may contribute to the tumorigenesis of pituitary adenomas.

Identification of p18INK4c as a Tumor Suppressor Gene in Glioblastoma Multiforme

Genomic alterations leading to aberrant activation of cyclin/cyclin-dependent kinase (cdk) complexes drive the pathogenesis of many common human tumor types. In the case of glioblastoma multiforme (GBM), these alterations are most commonly due to homozygous deletion of p16(INK4a) and less commonly due to genomic amplifications of individual genes encoding cyclins or cdks. Here, we describe deletion of the p18(INK4c) cdk inhibitor as a novel genetic alteration driving the pathogenesis of GBM. Deletions of p18(INK4c) often occurred in tumors also harboring homozygous deletions of p16(INK4a). Expression of p18(INK4c) was completely absent in 43% of GBM primary tumors studied by immunohistochemistry. Lentiviral reconstitution of p18(INK4c) expression at physiologic levels in p18(INK4c)-deficient but not p18(INK4c)-proficient GBM cells led to senescence-like G(1) cell cycle arrest. These studies identify p18(INK4c) as a GBM tumor suppressor gene, revealing an additional mechanism leading to aberrant activation of cyclin/cdk complexes in this terrible malignancy.

Synergistic Effect of Oncogenic RET and Loss of p18 on Medullary Thyroid Carcinoma Development

Activating mutations in the RET proto-oncogene are associated with both familial and sporadic medullary thyroid carcinoma (MTC) development; however, the genetic mechanisms underlying MTC tumorigenesis remain largely unknown. Recently, we have identified somatic inactivating mutations in the cell cycle inhibitor gene P18 in human MTC, which coincided with activating RET mutations, suggesting a role for loss of P18 in combination with oncogenic RET in the multistep process of MTC development. Therefore, we crossed transgenic mice expressing oncogenic RET (RET2B) with mice lacking p18 (and p27, another cell cycle inhibitor) and monitored MTC development. RET2B;p18(+/-) mice and RET2B;p18(-/-) mice developed MTC with a highly increased incidence compared with their corresponding single mutant littermates. In addition, expression of oncogenic RET causes an earlier age of onset and larger MTCs in p18(-/-);p27(+/-) mice. In a subset of MTCs of RET2B;p18(+/-)(;p27(+/-)) mice, p18(Ink4c) expression was completely lost. This loss of p18(Ink4c) expression correlated with higher proliferation rates as well as with larger MTCs, indicating that loss of p18 in combination with oncogenic RET not only increases the risk for MTC development but also enhances MTC progression. Our data strongly indicate that oncogenic RET and loss of p18 cooperate in the multistep tumorigenesis of MTC.

P19Ink4d and p18Ink4c cyclin‐dependent kinase inhibitors in the male reproductive axis

The loss of the cyclin-dependent kinase inhibitors (CKIs) p18(Ink4c) and p19(Ink4d) leads to male reproductive defects (Franklin et al., 1998. Genes Dev 12: 2899-2911; Zindy et al., 2000. Mol Cell Biol 20: 372-378; Zindy et al., 2001. Mol Cell Biol 21: 3244-3255). In order to assess whether these inhibitors directly or indirectly affect male germ cell differentiation, we examined the expression of p18(Ink4c) and p19(Ink4d) in spermatogenic and supporting cells in the testis and in pituitary gonadotropes. Both p18(Ink4c) and p19(Ink4d) are most abundant in the testis after 18 days of age and are expressed in purified populations of spermatogenic and testicular somatic cells. Different p18(Ink4c) mRNAs are expressed in isolated spermatogenic and Leydig cells. Spermatogenic cells also express a novel p19(Ink4d) transcript that is distinct from the smaller transcript expressed in Sertoli cells, Leydig cells and in other tissues. Immunohistochemistry detected significant levels of p19(Ink4d) in preleptotene spermatocytes, pachytene spermatocytes, condensing spermatids, and Sertoli cells. Immunoprecipitation-Western analysis detected both CKI proteins in isolated pachytene spermatocytes and round spermatids. CDK4/6-CKI complexes were detected in germ cells by co-immunoprecipitation, although the composition differed by cell type. p19(Ink4d) was also identified in FSH+ gonadotrophs, suggesting that this CKI may be independently required in the pituitary. Possible cell autonomous and paracrine mechanisms for the spermatogenic defects in mice lacking p18(Ink4c) or p19(Ink4d) are supported by expression of these CKIs in spermatogenic cells and in somatic cells of the testis and pituitary.

Reconstituted expression of menin in Men1-deficient mouse Leydig tumour cells induces cell cycle arrest and apoptosis

Multiple endocrine neoplasia type 1 (MEN1) is a hereditary syndrome caused by the inactivation of the responsible gene, MEN1. To date, the lack of MEN1-deficient cell lines derived directly from MEN1 tumours has hampered the detailed study of the MEN1 gene. We have established several stable Men1-deficient Leydig cell tumour (LCT) lines derived from a Leydig cell tumour developed in a male heterozygous Men1 mutant mouse. Our data show that these cell lines maintain the basic characteristics of Leydig cells in terms of both androgen synthesis and gene expression. Interestingly, reconstituted menin expression in one of Men1-deficient LCT cell lines resulted in cell growth inhibition, suggesting that the function of cell growth suppression of the menin pathway, apart from menin itself, is essentially preserved in these cells. Furthermore, we show that menin re-expression in these Men1-deficient cells leads to a block in the transition from G0/G1 to S phase of the cell cycle and an increase in apoptosis, accompanied by a marked increase of p18 INK4C and p27 Kip1 expression. The current study therefore highlights the importance of menin expression in cell cycle and cell survival control in endocrine cells, and may provide insights into the mechanisms of tumour suppression by menin in related endocrine tumours.

Mutation of Tumor Suppressor Gene Men1 Acutely Enhances Proliferation of Pancreatic Islet Cells

Multiple endocrine neoplasia type 1 (MEN1), an inherited tumor syndrome affecting endocrine organs including pancreatic islets, results from mutation of the tumor suppressor gene Men1 that encodes protein menin. Although menin is known to be involved in regulating cell proliferation in vitro, it is not clear how menin regulates cell cycle and whether mutation of Men1 acutely promotes pancreatic islet cell proliferation in vivo. Here we show that excision of the floxed Men1 in mouse embryonic fibroblasts (MEF) accelerates G(0)/G(1) to S phase entry. This accelerated S-phase entry is accompanied by increased cyclin-dependent kinase 2 (CDK2) activity as well as decreased expression of CDK inhibitors p18(Ink4c) and p27(Kip1). Moreover, Men1 excision results in decreased expression of p18(Ink4c) and p27(Kip1) in the pancreas. Furthermore, complementation of menin-null cells with wild-type menin represses S-phase entry. To extend the role of menin in repressing cell cycle in cultured cells to in vivo pancreatic islets, we generated a system in which floxed Men1 alleles can be excised in a temporally controllable manner. As early as 7 days following Men1 excision, pancreatic islet cells display increased proliferation, leading to detectable enlargement of pancreatic islets 14 days after Men1 excision. These observations are consistent with the notion that an acute effect of Men1 mutation is accelerated S-phase entry and enhanced cell proliferation in pancreatic islets. Together, these results suggest a molecular mechanism whereby menin suppresses MEN1 tumorigenesis at least partly through repression of G(0)/G(1) to S transition.