ARF Promoter Research Articles

Abstract 5202: Functional analysis of a chimera of p16 and ARF: All is not lost.

Abstract Adults in the US are now 28 times more likely to develop melanoma than 75 years ago. Among other genetic abnormalities, mutations in the INK4a locus on chromosome 9p21 are prominent in melanoma and can be found in nearly 50% of familial cases. Two independent tumor suppressors which are transcribed from the INK4a locus, p16 (CDKN2A) and ARF (p14ARF), share common exons 2 and 3 but are translated in unique reading frames. p16 causes cell cycle and growth arrest in the G1 phase by binding cyclin-dependent kinases 4 and 6 (CDK4/6), thereby displacing Cyclin D1 and inhibiting the enzymes. ARF, meanwhile, causes cell cycle and growth arrest in the G1 and G2/M phases by binding MDM2, in turn stabilizing p53. Mutations in melanoma often affect one or both genes, suggesting p16 and ARF may play equally important roles in tumor suppression. We report a 2 bp deletion, 1 bp insertion (CC to T) in exon 2 of p16/ARF found in a melanoma cell line that alters the reading frames of both genes and creates unique proteins. One transcript initiates from the ARF promoter and codes for the first 94 amino acids of ARF before switching to the reading frame of p16 to code for another 75 residues. We named the resulting protein chimera ARF (chARF). The other transcript initiates from the p16 promoter, codes for the first 80 amino acids of p16, then switches to a non-p16/non-ARF reading frame to code for an additional 63 residues. We called this protein p16-Alternate Carboxyl Terminus (p16-ACT). Based on sequence, we hypothesized that chARF could function both as ARF and p16 since it contains the MDM2 binding domain of ARF as well as most of the CDK4/6 binding domain of p16. Meanwhile, we hypothesized that p16-ACT would have no function since it is missing most of the CDK4/6 binding region of p16. To assess the function of these proteins, we overexpressed both mutant and wild-type proteins in the p16/ARF-inactivated tumor cell line U2OS. In CDK4 immunoprecipitation assays, chARF bound CDK4, but unlike wild-type p16, did not displace Cyclin D1. p16-ACT neither bound CDK4 nor displaced Cyclin D1. chARF expression strongly activated the p53/p21 pathway and arrested U2OS cells in the G1 and G2/M phases of the cell cycle in a manner identical to wild-type ARF. p16-ACT, however, had no noticeable effect on cell cycle distribution. Finally, we looked at cell proliferation and found that chARF expression suppressed monolayer cell growth and colony formation in U2OS cells by 75-85%, similar to wild-type ARF. p16-ACT expression had a small suppressive effect on colony formation, but no effect on monolayer growth. We conclude that chARF functions as wild-type ARF but neither chARF nor p16-ACT function as wild-type p16. Citation Format: Richard T. Williams, Lisa M. Barnhill, Huan Hsien Kuo, Wen Der Lin, Alice LS Yu, Mitchell B. Diccianni. Functional analysis of a chimera of p16 and ARF: All is not lost. [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 5202. doi:10.1158/1538-7445.AM2013-5202

Abstract 779: ARF activation is responsible for the tumor-suppressing function of p53 in basal cell carcinomas (BCCs) in Ptch1+/- mice.

Abstract Basal cell carcinomas (BCCs) are the most common cancer among people of European ancestry. Aberrant activation of hedgehog (HH) signaling plays a pivotal role in BCC carcinogenesis. Besides mutations in genes encoding HH pathway components, mutations in the p53 gene frequently are found in human BCCs. Consistently, our previous studies have found that the loss of p53 can dramatically accelerate BCC formation in ionizing radiation (IR)-treated Ptch1+/- mice. It remains elusive how p53 is activated in BCC development. DNA damage response (DDR) and oncogenic induced stress (OIS) are two essentially independent pathways leading to p53 activation although limited evidence suggests crosstalk between these pathways. In response to OIS, ARF is activated and leads to p53 activation, functioning as a tumor suppressor, in many cancers. However, the loss of ARF, unlike loss of p53, failed to affect the development of medulloblastoma in Ptch1+/- mice (Wetmore C., et al, 2001). In the present study, we investigated whether ARF affects BCC development in the same mice. To address this, we bred ARFGFP/GFP mice, in which ARF function is disabled and GFP is expressed under the control of the endogenous ARF promoter, with our Ptch1+/- mice to generate Ptch1+/- ARFGFP/GFP (designated as ARF mice) for comparison with Ptch1+/- K14CreER2 p53fl/fl (PF) mice. Following our standard protocol of tamoxifen injection at age 7.5 weeks to delete p53 specifically in K14-expressing keratinocytes (for PF mice) and treating both ARF and PF mice with ionizing radiation at age 8 weeks, we compared BCC development between the above 2 lines. We find that at age 5 months, ARF mice formed numbers of microscopic BCCs comparable to those in PF mice though the latter exhibited greater mouse-to-mouse variation. More importantly, both mouse lines developed initial visible BCCs at age 5 to 7 months, which is dramatically earlier than the age 9 to 18 months for the Ptch1+/- mice (ARF+/+ and p53+/+). In addition, ARFGFP/GFP mice responded to IR-induced DNA damage in a pattern essentially similar to ARF+/+ mice, indicating an intact DDR pathway as reported in the literature. In conclusion, our data demonstrate that ARF is involved in BCC carcinogenesis as a tumor suppressor, probably playing the major, perhaps the exclusive, role in activating p53 in this process. Citation Format: Grace Y. Wang, Carla Wood, Joy Wang, Lynn Wang, Eileen Libove, Ervin Epstein. ARF activation is responsible for the tumor-suppressing function of p53 in basal cell carcinomas (BCCs) in Ptch1+/- mice. [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 779. doi:10.1158/1538-7445.AM2013-779

RUVBL2 is a novel repressor of ARF transcription

ARF is the second most commonly inactivated tumor suppressor behind p53. It has been implicated in the control of cell proliferation, cell senescence, and tumor suppression. However, the detailed mechanism underlying the transcriptional control of ARF remains largely unknown. Here we report RUVBL2 as a novel transcriptional repressor of ARF. Ectopic expression of RUVBL2 decreases the levels of ARF, whereas knockdown of RUVBL2 results in a marked increase in ARF levels. In addition, RUVBL2 down-regulates the levels of p53 in an ARF-dependent manner. Mechanistically, RUVBL2 binds to the distal region of ARF promoter, thus leading to the repression of ARF transcription. These results suggest an important role of RUVBL2 in the regulation of ARF-p53 pathway.

Direct promoter induction of p19Arf by Pit-1 explains the dependence receptor RET/Pit-1/p53-induced apoptosis in the pituitary somatotroph cells

Somatotrophs produce growth hormone (GH) and are the most abundant secretory cells of the pituitary. Somatotrophs express the transcription factor Pit-1 and the dependence receptor RET, its co-receptor GFRa1 and ligand GDNF. Pit-1 is a transcription factor essential for somatotroph proliferation and differentiation and for GH expression. GDNF represses excess Pit-1 expression preventing excess GH. In the absence of GDNF, RET behaves as a dependence receptor, becomes intracellularly processed and induces strong Pit-1 expression leading to p53 accumulation and apoptosis. How accumulation of Pit-1 leads to p53 expression is unknown. We have unveiled the relationship of Pit-1 with the p19Arf gene. There is a parallel correlation of RET processing, Pit-1 increase and ARF protein and mRNA expression. Interfering the pathway with RET, Pit-1 or p19Arf siRNA blocked apoptosis. We have found a Pit-1 DNA-binding element within the ARF promoter. Pit-1 directly regulates the CDKN2A locus and binds to the p19Arft promoter inducing p19Arf gene expression. The Pit-1-binding element is conserved in rodents and humans. RET/Pit-1 induces p19Arf/p53 and apoptosis not only in a somatotroph cell line but also in primary cultures of pituitary somatotrophs, where ARF siRNA interference also blocks p53 and apoptosis. Analyses of the somatotrophs in whole pituitaries supported the above findings. Thus Pit-1, a differentiation factor, activates the oncogene-induced apoptosis (OIA) pathway as oncogenes exerting a tight control in somatotrophs to prevent the disease due to excess of GH (insulin-resistance, metabolic disease, acromegaly).

Functional interactions between Lmo2, the Arf tumor suppressor, and Notch1 in murine T-cell malignancies

AbstractLMO2 is a target of chromosomal translocations in T-cell tumors and was activated by retroviral vector insertions in T-cell tumors from X-SCID patients in gene therapy trials. To better understand the cooperating genetic events in LMO2-associated T-cell acute lymphoblastic leukemia (T-ALL), we investigated the roles of Arf tumor suppressor loss and Notch activation in murine models of transplantation. Lmo2 overexpression enhanced the expansion of primitive DN2 thymocytes, eventually facilitating the stochastic induction of clonal CD4+/CD8+ malignancies. Inactivation of the Arf tumor suppressor further increased the self-renewal capacity of the primitive, preleukemic thymocyte pool and accelerated the development of aggressive, Lmo2-induced T-cell lympholeukemias. Notch mutations were frequently detected in these Lmo2-induced tumors. The Arf promoter was not directly engaged by Lmo2 or mutant Notch, and use of a mouse model in which activation of a mutant Notch allele depends on previous engagement of the Arf promoter revealed that Notch activation could occur as a subsequent event in T-cell tumorigenesis. Therefore, Lmo2 cooperates with Arf loss to enhance self-renewal in primitive thymocytes. Notch mutation and Arf inactivation appear to independently cooperate in no requisite order with Lmo2 overexpression in inducing T-ALL, and all 3 events remained insufficient to guarantee immediate tumor development.

Abstract 2958: Cyclin D1 regulates the ARF and INK4a promoters

Abstract Cyclin D1 is a member of cyclin protein family, which drives the cell cycle transition from G1 to S phase. The cyclin D1 protein is overexpressed in ∼50% of human breast cancers and is associated with short survival of patients when the gene is amplified. Cyclin D1 forms holoenzymes with cyclin-dependent protein kinase (Cdk) 4 and 6, and the Cyclin D-Cdk4/6 complex phosphorylates and inactivates the pRb retinoblastoma tumor suppressor, releasing E2F transcriptional factors from Rb inhibition. The pRb phosphorylation is blocked by p16INK4a, encoded by the CDKN2A locus, which inhibits the kinase activity of Cyclin D-Cdk4/6 complex. The CDKN2A locus also encodes another tumor suppressor protein named p14ARF (p19Arf in mouse), which stabilizes p53 by inhibiting MDM2-dependent degradation. Previous studies from our laboratory showed that the cyclin D1 collaborated with the Dmp1 (cyclin D binding myb-like protein 1) tumor suppressor to activate the Arf promoter. To further demonstrate the effect of cyclin D1 on p19Arf and p16Ink4a promoter activity, NIH 3T3 cells and MEF cells were transfected a construct expressing the luciferase reporter gene linked with the Arf promoter together with vector or cyclin D1. Using luciferase reporter assay, we found that the Arf promoter was activated upon cyclin D1 overexpression in both cell lines, and the activation was abrogated when cells were co-transfected with E2F-DB, which displaces endogenous E2Fs and acts in a dominant-negative manner. Consistent with reporter assays, the Arf mRNA was increased in MEFs overexpressing cyclin D1, compared to those infected with vector only. By performing chromatin immunoprecipitation in tumors of mammary gland from MMTV-neu transgenic mice that overexpress cyclin D1, we found that cyclin D1 can interact with the Arf promoter although it cannot directly bind to the Arf promoter region in electrophoretic mobility shift assay using recombinant cyclin D1 protein. To identify the effect of cyclin D1 on p16Ink4a promoter, we performed luciferase reporter assay in NIH 3T3 cells by co-transfecting Ink4a promoter together with vector or cyclin D1. We found that the Ink4a promoter activity was increased upon cyclin D1 overexpression, and the activation was not abrogated when cells were transfected with cyclin D1-K114E that does not bind to CDK4. Taken together, our data suggest that overexpression of cyclin D1 activates both p19Arf and p16Ink4a promoters to prevent neoplastic growth of incipient cancer cells, and the activation is mediated by indirect binding of cyclin D1 to the promoter regions. We are currently mapping the cyclin D1-responsive elements on the Arf and Ink4a promoters to identify transcription factors responsible for Arf/Ink4a induction by cyclin D1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2958. doi:10.1158/1538-7445.AM2011-2958

Abstract 1140: C/ebp beta represses Arf induction by tgf-beta2

Abstract Arf is a bona fide tumor suppressor that regulates cell proliferation through p53-dependent and -independent mechanisms. Recent studies show that Arf also has physiological functions in addition to tumor repression. Our group reported that the Arf promoter is strictly controlled during development and Tgf-beta2 is required for Arf promoter activation at several sites in the developing mouse. In a cell culture-based model, Arf induction by Tgf-beta depends on Smads 2/3 and p38 MAPK. But the transcription factors influenced by these signaling pathways are not yet clear. Using a candidate-gene approach, we have identified C/ebp beta as a negative Arf regulator, modified by Tgf-beta 2. The ability of Tgf-beta2 to increase p19Arf expression in wild-type mouse embryo fibroblasts (MEFs) and Arf promoter activity in Arf lacZ/lacZ MEFs correlated with repression of C/ebp beta. Pre-incubation of MEFs with SB431542, a Tgf-beta type I receptor inhibitor, blocked C/ebp beta repression and Arf induction. Retrovirus-mediated ectopic C/ebp beta blocked Arf induction by Tgf-beta2 in Arf lacZ/lacZ and wild-type MEFs. Similarly, RNAi-driven knock-down of C/ebp beta augmented Arf promoter activation by Tgf-beta2. Chromatin immunoprecipitation revealed that C/ebp beta binding to the Arf promoter after Arf promoter is initially activated by Tgf-beta2, and the binding dropped at 48 hours, when Arf expression was significantly induced. In summary, our results indicate that C/ebp beta is modified by and contributes to Arf regulation by Tgf-beta2. In contrast, the p38 MAPK inhibitor SB203580 did not influence C/ebp beta levels but still interfered with Arf induction. We are currently exploring the importance of C/ebp beta in Arf regulation by other signals and in other cell types, and investigating its importance in vivo in the developing mouse. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1140. doi:10.1158/1538-7445.AM2011-1140

Abstract 3076: MEKK1 is a novel modulator of the Arf-p53 pathway via Dmp1 phosphorylation

Abstract Dmp1 (cyclin D-interacting myb-like protein 1; Dmtf1) is a transcription factor isolated in yeast two-hybrid screen as a cyclin D2 binding partner. As a transcription factor, Dmp1 binds to nonameric DNA consensus sequences CCCG(G/T)ATG(T/C) to act as an activator or a repressor. Extensive molecular and genetic evidence links tumor suppressor function of Dmp1 to regulation of the ARF-p53 pathway. In fact, Dmp1 directly binds to p19Arf (p14ARF in humans) promoter and induces its expression. Increase in ARF level leads to protection of p53 from Mdm2-mediated degradation and subsequent increase in p53 target genes involved in cell cycle arrest and apoptosis. Oncogenic Ras and Her2/neu induce Dmp1 expression leading to Arf-, p53-dependent cell cycle arrest. Human DMP1 locus is hemizygously deleted in ∼50% of breast carcinomas, which is mutually exclusive of ARF and p53 loss. Based on the amino acid sequence, predicted molecular weight of Dmp1 is 85kDa. However, Dmp1 migrates to ∼120-130kDa on a SDS-PAGE gel, suggesting post-translational modifications. Although signal transduction pathways regulating Dmp1 transcripton have been studied, the role of Dmp1 protein modification is unknown. Treatment of Dmp1 protein with calf intestine phosphatase and running a 2D SDS-PAGE gel, we have shown that one mode of post-translational modification is via phosphorylation. The presence of phosphates on several Serines and/or Threonines was confirmed with mass spectrometry. Using an Arf luciferase reporter assay in 3T3 cells, we found that MEKK1 (MEK kinase 1) synergizes with Dmp1 on the Arf promoter activity, with the synergy attenuated when several of the putative phosphorylation sites on Dmp1 were mutated. The effect observed was Dmp1-dependent since MEKK1 was unable to activate the Arf promoter in Dmp1-null cells. Full length (inactive) MEKK1 was unable to synergize with Dmp1 on the Arf promoter activity suggesting necessity of the kinase activity. The ability of MEKK1 to directly phosphorylate Dmp1 was confirmed in in vitro kinase assay using purified MEKK1 and Flag-Dmp1 proteins and Arf reporter assays where known kinases downstream of MEKK1(MEK1, MEK4/7, JNK1/2) were inhibited or knocked down. MEKK1 and Dmp1 co-immunoprecipitated when expressed in 3T3 cells. To study the effect of MEKK1 on endogenous Dmp1 and Arf, we either expressed MEKK1 or treated cells with MEKK1-activating drugs, cisplatin and etoposide. In this setting, endogenous p14ARF was induced and the banding pattern of Dmp1 protein was shifted in a SDS-PAGE gel indicating protein modification. Here we show that Dmp1 can be directly phosphorylated by MEKK1 to modulate Dmp1 transcriptional activity on the Arf promoter. Therefore, MEKK1 may activate the Arf-p53 pathway via direct Dmp1 protein phosphorylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3076. doi:10.1158/1538-7445.AM2011-3076

Critical Roles of DMP1 in Human Epidermal Growth Factor Receptor 2/neu-Arf-p53 Signaling and Breast Cancer Development

Human epidermal growth factor receptor 2 (HER2) overexpression stimulates cell growth in p53-mutated cells while it inhibits cell proliferation in those with wild-type p53, but the molecular mechanism is unknown. The Dmp1 promoter was activated by HER2/neu through the phosphatidylinositol-3'-kinase-Akt-NF-κB pathway, which in turn stimulated Arf transcription. Binding of p65 and p52 subunits of NF-κB was shown to the Dmp1 promoter and that of Dmp1 to the Arf promoter on HER2/neu overexpression. Both Dmp1 and p53 were induced in premalignant lesions from mouse mammary tumor virus-neu mice, and mammary tumorigenesis was significantly accelerated in both Dmp1+/- and Dmp1-/- mice. Selective deletion of Dmp1 and/or overexpression of Tbx2/Pokemon was found in >50% of wild-type HER2/neu carcinomas, although the involvement of Arf, Mdm2, or p53 was rare. Tumors from Dmp1+/-, Dmp1-/-, and wild-type neu mice with hemizygous Dmp1 deletion showed significant downregulation of Arf and p21Cip1/WAF1, showing p53 inactivity and more aggressive phenotypes than tumors without Dmp1 deletion. Notably, endogenous hDMP1 mRNA decreased when HER2 was depleted in human breast cancer cells. Our study shows the pivotal roles of Dmp1 in HER2/neu-p53 signaling and breast carcinogenesis.

Tgfβ Signaling Directly Induces Arf Promoter Remodeling by a Mechanism Involving Smads 2/3 and p38 MAPK

We have investigated how the Arf gene product, p19(Arf), is activated by Tgfβ during mouse embryo development to better understand how this important tumor suppressor is controlled. Taking advantage of new mouse models, we provide genetic evidence that Arf lies downstream of Tgfβ signaling in cells arising from the Wnt1-expressing neural crest and that the anti-proliferative effects of Tgfβ depend on Arf in vivo. Tgfβ1, -2, and -3 (but not BMP-2, another member of the Tgfβ superfamily) induce p19(Arf) expression in wild type mouse embryo fibroblasts (MEFs), and they enhance Arf promoter activity in Arf(lacZ/lacZ) MEFs. Application of chemical inhibitors of Smad-dependent and -independent pathways show that SB431542, a Tgfβ type I receptor (TβrI) inhibitor, and SB203580, a p38 MAPK inhibitor, impede Tgfβ2 induction of Arf. Genetic studies confirm the findings; transient knockdown of Smad2, Smad3, or p38 MAPK blunt Tgfβ2 effects, as does Cre recombinase treatment of Tgfbr2(fl/fl) MEFs to delete Tgfβ receptor II. Chromatin immunoprecipitation reveals that Tgfβ rapidly induces Smads 2/3 binding and histone H3 acetylation at genomic DNA proximal to Arf exon 1β. This is followed by increased RNA polymerase II binding and progressively increased Arf primary and mature transcripts from 24 through 72 h, indicating that increased transcription contributes to p19(Arf) increase. Last, Arf induction by oncogenic Ras depends on p38 MAPK but is independent of TβrI activation of Smad 2. These findings add to our understanding of how developmental and tumorigenic signals control Arf expression in vivo and in cultured MEFs.

Abstract 1097: Arf induction by Tgf-beta2 is mediated by Smad-2/3 dependent enhancement of Arf transcription

Abstract The Arf tumor suppressor gene product, p19Arf, is induced in response to certain oncogenic signals to block cancer development and progression. How the oncogenic cues are coupled to Arf promoter activation remains poorly defined, though. We took advantage of the fact that the Arf promoter is strictly controlled during development to investigate the normal regulatory mechanisms. We have recently published that Tgf-beta2 is required for Arf promoter activation at several sites in the developing mouse. To define mechanisms for the regulation, we developed a cell culture-based assay. We now show that Tgf-beta2 (5 ng/ml) induced p19Arf expression in WT mouse embryo fibroblasts (MEFs), enhanced Arf promoter activity in Arf lacZ/lacZ MEFs, and maintained a G1 cell cycle arrest in an Arf-dependent manner. The Arf promoter was also increased by Tgf-beta1, which functionally overlaps with Tgf-beta2, but not by Pdgf-B, a potent mitogen, nor BMP-2, another Tgf-beta superfamily member. To elucidate which pathway mediated the Tgf-beta2 effects, we used a panel of chemical inhibitors of Smad-dependent and -independent pathways. Among them, SB431542, a Tgf-beta type I receptor inhibitor, blocked Smad2 phosphorylation and impeded Tgf-beta2 induction of Arf. Genetic studies confirmed this because RNAi-driven knock-down of Tgf-beta receptor I, Smad2 or Smad3 impaired ArflacZ induction by Tgf-beta2. Further, Tgf-beta2 failed to induce p19Arf in Cre-treated Tgfbr2 flox/flox MEFs, in which Tgf-beta receptor II was deleted. Interestingly, the p38 MAPK inhibitor SB203580 partially impeded Arf induction, showing cross-talk with Smad signals. Chromatin immunoprecipitation revealed that Tgf-beta2 induced the binding of Smad2/3 and RNA Polymerase II to genomic DNA proximal to Arf exon 1 beta. Increased promoter binding preceded the progressively induced Arf mRNA and primary Arf transcripts at 24 and 48 hours, indicating that the induction was due to increased transcription. In summary, our results indicate that Tgf-beta2 induces the Arf promoter via a direct Smad-dependent signaling pathway. This study provides the first evidence that Arf expression is coupled to cell extrinsic signals. We are currently exploring this pathway in other type of cells and testing whether Arf induction by oncogenes also depends on Tgf-beta signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1097.

Abstract SY29-03: Regulation of cellular self-renewal by the ARF

Abstract The Ink4-Arf locus encodes three tumor suppressor proteins, two of which (p16Ink4a and p15Ink4b) inhibit the activities of cyclin D-dependent kinases (Cdk4 and Cdk6) and a third (p19Arf) that inhibits the Mdm2 E3 ligase to activate p53. Arf induction in response to aberrantly increased and persistent signaling thresholds mediated by activated oncogenes triggers a p53-dependent transcriptional program that can lead to cell cycle arrest or apoptosis, thereby eliminating incipient tumor cells. Arf inactivation compromises these p53-dependent protective effects and synergizes with oncogene activation to drive tumor progression. The Ink4-Arf locus is epigenetically silenced in self-renewing stem cells by Polycomb Group (PcG) complexes but is remodeled as stem cells differentiate to yield progeny that have lost their unlimited self-renewal potential. Thus, although it is generally not engaged in most normal tissues, the Ink4-Arf locus is “poised” to respond to oncogenic stress signals in non-stem cells and can thereby act as a differentiation stage-specific tumor suppressor. Deletion of Ink4-Arf is a frequent event in many forms of cancer, helping to reconfer the ability of tumor cells to self-renew indefinitely. The fact that INK4A-ARF deletions and p53 mutations occur in a mutually exclusive manner in many human tumors provides the strongest argument that disruption of the ARF-HDM2-p53 “pathway” is a hallmark of cancer in humans as well as in mice. We have produced several strains of mice in which functional Arf coding sequences in the mouse genome have been replaced by reporter genes. These include cassettes encoding green fluorescent protein (GFP) or Cre recombinase that are driven by the cellular Arf promoter. These experiments revealed that, apart from monitoring latent oncogenic signals in living animals, the Arf promoter is transiently engaged in several normal cell types. For example, Arf is expressed in the male germ cell lineage in mitotically amplifying spermatogonia but not in the primary spermatocytes that derive from them. Notably, in this setting, Arf expression is compatible with cell division and does not trigger apoptosis or cellular senescence. However, inactivation of Arf alters the homeostatic relationship between spermatogonia and spermatocytes and leads to a progressive depletion of male germ stem cells and to precocious infertility as animals age. Surprisingly, although the Ink4a gene is co-expressed with Arf in spermatogonia, its inactivation results in increased sperm production, thereby offsetting the effects of Arf loss. In short, despite their intimate linkage and ability to be epigenetically silenced en bloc, Ink4a and Arf inactivation lead to opposing phenotypes in this setting. We suspect that the ability of transient Arf and Ink4a expression to “gate” stem cell self-renewal may reflect a primordial function of this locus distinct from its role in tumor suppression. This may potentially explain why this gene cluster is evolutionarily conserved in mammals, despite its strong propensity to undergo deletion in cancer cells. Citation Format: Charles J. Sherr, Adam Gromley, Michelle L. Churchman, Emmanuel J. Volanakis. Regulation of cellular self-renewal by the ARF [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY29-03

Abstract 2563: Upregulation of miR-155 is associated with cisplatin resistance in the head and neck cancer UMSCC-10B/15S cells

Abstract Introduction: Cisplatin combination chemotherapy is the cornerstone of treatment for many cancers including head and neck cancer. These complexes bind to the DNA and form adducts such that they ultimately trigger apoptosis. Although initial platinum responsiveness is usually high, the majority of cancer patients will eventually relapse with cisplatin-resistant disease. Thus, chemotherapy resistance is a major clinical obstacle in the successful treatment of head and neck cancer. This study aims to investigate the role of microRNAs (miRNAs) in cisplatin resistance and to better understand the underlying mechanism of cisplatin resistance in the head and neck cancer UMSCC-10B/15S cells. Methods: To determine whether aberrant miRNA expression is associated with cisplatin resistance in human head and neck cancer cells, we profiled miRNA expression in the cisplatin resistant UMSCC-10b/15S and the parental line UMSCC-10B cells by real-time PCR. Luciferase reporter assays and western blot were used to confirm targets for miR-155. We also suppressed miR-155 by anti-sense locked nucleic acid (LNA) oligo to further determine the role of miR-155 in cisplatin resistance. Results: We showed that several miRNAs were altered in UMSCC-10B/15S and among them, miR-155 was the most significantly upregulated miRNA. This was also confirmed by in situ hybridization. Furthermore, ectopic expression of miR-155 was able to increase the resistance of UMSCC-10B cells to cisplatin, which is likely in part through targeting DMTF1, a tumor suppressor that directly binds to the Arf promoter to activate its expression, thereby inducing p53-dependent cell cycle arrest and apoptosis. To determine whether miR-155 directly targets DMTF1, we cloned 1.2 kb 3′-untranslated region (UTR) of DMTF1 into a luciferase reporter and demonstrated that miR-155 was able to suppress luciferase activity by over 70%. We also showed that the endogenous DMFT1 protein was reduced by over 50% in the miR-155 highly expressed resistant cells compared to the parental sensitive cells. Experiments are under way to determine whether ectopic expression of DMTF1 or its suppression by RNAi impacts cisplatin resistance in these cells. Conclusion: These findings suggest that deregulation of miRNAs is in resistant head and neck cancer cells and miR-155 may play a causal role in cisplatin resistance through targeting the DMTF1 gene. As a result, suppression of miR-155 may provide a strategy to overcome the resistance in head and neck cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2563.

Abstract 1096: Arf-independent activation of p53 by the Dmp1 tumor suppressor

Abstract Cyclin D binding myb-like protein 1 (Dmp1; Dmtf1) is a tumor suppressor that is deleted in ∼40 % of human non-small cell lung carcinomas. The Dmp1 promoter receives signals from oncogenic Ras and the protein shows its activity as a tumor suppressor by directly binding and transactivating the Arf promoter, and thereby inducing Arf-, p53-dependent cell cycle arrest. Both Eµ-Myc and K-rasLA-induced tumor development was accelerated in Dmp1+/− and Dmp1−/− mice, suggesting that Dmp1 is haplo-insufficient for tumor suppression. In Eµ-Myc lymphomas, the combined frequencies of p53 mutation and Arf deletion in the Dmp1+/− or Dmp1−/− mice were significantly lower (∼10 %) than that in Dmp1+/+ littermates (∼50 %), indicating that Dmp1 is a physiological regulator of the Arf-p53 pathway in vivo. We recently found that the frequency of p53 mutation (∼40 %) was significantly decreased in both Dmp1+/− and Dmp1−/− backgrounds (< 10 %) in the K-rasLA lung cancer model where the Ink4a/Arf involvement is very rare. Moreover, our recent data show that Dmp1 physically interacts with p53 to neutralize the activity of Hdm2. To demonstrate the Arf-independent function of Dmp1 on p53 activation in vivo, we injected doxorubicin into wild-type, Arf-null, and Dmp1-null mice, harvested the thymus at 2 hr intervals, and studied the expression of the p53 targets, p21cip1 and bbc3, by real-time PCR and immunohistochemistry. Thymus was chosen as a target tissue since Dmp1 is highly expressed in the medulla in our histochemical studies. Western blotting analyses with DO-1 and phosphoserine-specific antibodies verified p53 activation by doxorubicin. Cleaved caspase 3 was detectable in the thymic medulla of wild-type mice 6 hrs after tail injection of doxorubicin, indicating the induction of apoptotic cell death by the genotoxic drug. Cleaved caspase 3 staining was significantly decreased in the thymus from Arf−/− mice as compared to wild-type mice, but the staining was even lower in Dmp1−/− mice. Consistent with these findings, the p21cip1 mRNA induction (∼23-fold increase in wild-type mice) was more significantly compromised in Dmp1−/− mice (∼7-fold) than in Arf−/− mice (∼15-fold). Bbc was undetectable in Dmp1−/− thymus even 4-6 hrs after injection of doxorubicin while it was significantly induced (∼3-fold) in both wild-type and Arf−/− mice. These mRNA data were confirmed by immunohistochemical staining of thymic tissues with p21cip1 and bbc3-specific antibodies. Together, our data demonstrate the Arf-independent activation of p53 by Dmp1 in response to genotoxic stress in vivo. We are currently conducting the same series of experiments in the mouse lung. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1096.

Regulation of cell proliferation by a novel POK family protein HKR3

POK family proteins have been shown to play roles in cell proliferation, development, oncogenesis, and immunity. HKR3 is a ubiquitous transcription factor consisted of 688 amino acids and contains N‐terminus BTB/POZ‐domain and eleven Krüppel‐like zinc fingers. We investigated whether the HKR3 has a regulatory effect on cell cycle progression by regulating gene expression of p53 pathway (ARF‐HDM2‐p53‐p21CIP1). The HKR3 is a potent activator of the pathway and activates transcription of ARF, p53, and p21CIP1 genes. In particular, HKR3 potently activated transcription of tumor suppressor gene ARF. The activation of ARF by HKR3 involved the acetylation of histone H3 and H4 around the ARF promoter through interaction with transcription co‐activator p300. HKR3 induced cell cycle arrest and inhibited cell proliferation by decreasing the number of cells in S phase. Also, we found that mRNA expression of HKR3 is greatly increased when cells were treated with a DNA damage agent, etoposide. Our investigation suggests that HKR3 is one of the major regulators of the p53 pathway by acting at the level of transcription and may be a potential a tumor suppressor.

Developmentally Restricted Protection From Notch1-Induced T Cell Acute Lymphoblastic Leukemia by the Arf Tumor Suppressor.

Abstract 143At presentation, 50% of T cell acute lymphoblastic leukemia (T ALL) cases harbor activating mutations of the NOTCH1 transmembrane receptor, virtually all of which have also sustained deletions of the CDKN2A (INK4A-ARF) locus. Although the concordance of these genetic anomalies has been well documented, its basis remains unclear. The CDKN2A gene cluster encodes two structurally and functionally distinct tumor suppressors, p16INK4A and p14ARF (p19Arf in the mouse), the activation of which modulates the activities of the retinoblastoma protein and p53, respectively, to induce cell cycle arrest and/or apoptosis in response to oncogenic stress. A novel approach for modeling T ALL in the mouse now reveals that the ability of Arf to suppress Notch1-induced tumors is conferred at a specific stage of T cell development. Bone marrow cells or thymocytes transduced with a vector encoding the constitutively active intracellular fragment of NOTCH1 (ICN1) together with green fluorescent protein (GFP) were cultured ex vivo under conditions that support T lymphocyte differentiation and proliferation. These cultures were quickly dominated by early T cell precursors that expressed Thy-1 but not B cell or myeloid markers, and which produced a rapidly fatal CD4+/CD8+ (”double-positive”) T ALL when transferred into healthy, non-irradiated syngeneic mice. A phenotypically identical disease resulted upon infusion of the transduced cells into athymic nude mice, demonstrating that the deregulated ICN1 signal is sufficient to drive T cell development to the CD4+/CD8+ stage without any requirement for input from the thymic microenvironment. In recipients of bone marrow-derived ICN1+, Arf+/+ progenitors, T ALLs arose at high frequency, retained the Arf locus, but universally failed to express p19Arf. In turn, T ALLs initiated with bone marrow-derived ICN1+, Arf-/- donor cells exhibited only a modest acceleration of disease progression. In marked contrast, retention of the intact Arf locus in thymocyte-derived ICN1+ donor cells significantly prolonged disease latency and decreased tumor penetrance, indicating that Arf tumor suppression can be activated in thymic progenitors, but not in their less mature bone marrow precursors. Polycomb complexes epigenetically silence the Ink4a-Arf locus in primitive hematopoietic progenitors and early T cell precursors, preventing p19Arf expression and licensing a robust proliferative response to ICN1 signals. However, the locus is subsequently remodeled before T cells reach the double-positive stage, and its activation enables the Arf-p53 axis to cull T cells exposed to aberrant ICN1-induced oncogenic signals. In order to directly demonstrate that ICN1 overexpression in thymocyte-derived progenitors can induce the Arf locus, thymocytes from a homozygous Arf-GFP “knock-in” mouse were transduced with a vector encoding ICN1 and cherry-fluorescent protein (CFP), cultured short-term, and infused into healthy recipient mice. Notably, the Arf-GFP allele, while functionally null, expresses GFP in lieu of p19Arf when the cellular Arf promoter is induced. Healthy syngeneic recipients of ICN1/CFP, ArfGfp/Gfp donor cells developed CFP-marked T ALLs that co-expressed GFP, providing direct evidence that Arf is induced during thymus-derived leukemogenesis in vivo. Notably, Arf-induction not only provides p53-dependent elimination of incipient tumor cells but also generates a selective pressure for the emergence and survival of rare clones that have sustained Arf deletions. We reason that deletion of INK4A-ARF observed in human NOTCH1-induced CD4+/CD8+ T ALL strongly argues for the obligate induction of the locus at an earlier stage of T cell maturation. Our mouse model recapitulates this requirement, and should provide a useful platform to further elucidate disease mechanisms and potential therapies in T ALLs arising in nonirradiated mice that retain hematopoietic and immune functions. Disclosures:No relevant conflicts of interest to declare.

Expression of theArftumor suppressor gene is controlled by Tgfβ2 during development

The Arf tumor suppressor (also known as Cdkn2a) acts as an oncogene sensor induced by ;abnormal' mitogenic signals in incipient cancer cells. It also plays a crucial role in embryonic development: newborn mice lacking Arf are blind due to a pathological process resembling severe persistent hyperplastic primary vitreous (PHPV), a human eye disease. The cell-intrinsic mechanism implied in the oncogene sensor model seems unlikely to explain Arf regulation during embryo development. Instead, transforming growth factor beta2 (Tgfbeta2) might control Arf expression, as we show that mice lacking Tgfbeta2 have primary vitreous hyperplasia similar to Arf(-/-) mice. Consistent with a potential linear pathway, Tgfbeta2 induces Arf transcription and p19(Arf) expression in cultured mouse embryo fibroblasts (MEFs); and Tgfbeta2-dependent cell cycle arrest in MEFs is maintained in an Arf-dependent manner. Using a new model in which Arf expression can be tracked by beta-galactosidase activity in Arf(lacZ/+) mice, we show that Tgfbeta2 is required for Arf transcription in the developing vitreous as well as in the cornea and the umbilical arteries, two previously unrecognized sites of Arf expression. Chemical and genetic strategies show that Arf promoter induction depends on Tgfbeta receptor activation of Smad proteins; the induction correlates with Smad2 phosphorylation in MEFs and Arf-expressing cells in vivo. Chromatin immunoprecipitation shows that Smads bind to genomic DNA proximal to Arf exon 1beta. In summary, Tgfbeta2 and p19(Arf) act in a linear pathway during embryonic development. We present the first evidence that p19(Arf) expression can be coupled to extracellular cues in normal cells and suggest a new mechanism for Arf control in tumor cells.

Transient expression of theArftumor suppressor during male germ cell and eye development inArf-Crereporter mice

The Arf tumor suppressor is expressed transiently during mouse male germ cell and eye development. Its inactivation compromises spermatogenesis as mice age and leads to aberrant postnatal proliferation of cells in the vitreous of the eye, resulting in blindness. In the testis, expression of p19(Arf) is limited to spermatogonia but is extinguished completely in spermatocytes, suggesting that Arf plays a physiologic role in setting the balance between mitotic and meiotic germ cell division. A knock-in allele encoding Cre recombinase regulated by the mouse cellular Arf promoter was used to trace Arf gene induction in vivo. Interbreeding to a reporter strain that expresses Cre-dependent YFP provided proof-of-principle that the Arf-Cre allele was appropriately expressed in the male germ cell lineage. However, Cre expression resulted in male sterility, limiting germ line transmission of the knock-in allele to females. Arf-null mice fail to resorb the hyaloid vasculature within the ocular vitreous where pericyte-like cells that express the PDGF-beta receptor (Pdgfrbeta) proliferate aberrantly and destroy the retina and lens. Interbreeding of Arf-Cre females to males containing "floxed" (FL) Arf alleles yielded Arf(Cre/FL) progeny that exhibited variably penetrant defects in visual acuity ranging to total blindness. Crossing the Arf(Cre/FL) alleles onto a Pdgfrbeta(FL/FL) background normalized all histopathology and restored vision fully.

8‐chloro‐adenosine‐induced E2F1 promotes p14ARF gene activation in H1299 cells through displacing Sp1 from multiple overlapping E2F1/Sp1 sites

The regulation of p14ARF gene by E2F transcription factor, which differs from that of classical E2F targets, has recently been attributed to a variant E2F-response element. However, promoter assays suggest multiple elements present in the p14ARF promoter and argue against the idea that the ARF promoter has a unique ability to distinguish between aberrant and physiological levels of E2F1. Therefore, the functional characterization of the promoter still needs to be done. We demonstrate that at least two overlapping E2F1/Sp1 binding sites are present in the p14ARF promoter, and E2F1 activates the promoter through displacing constitutive Sp1 from the overlapping sites. We found that 8-chloro-adenosine (a metabolite of 8-Cl-cAMP) exposure induced the p14ARF gene in human lung cancer H1299 cells, followed by increased expression of E2F1 and constitutive expression of Sp1. The combination of cotransfection and electrophoretic mobility shift assay (EMSA) indicated that constitutive binding of Sp1 to the overlapping sites contributed to a constitutive expression of the ARF gene in unexposed H1299, whereas displacing Sp1 from the overlapping sites by E2F1 promoted the gene activation after exposure. EMSA and chromatin immunoprecipitation revealed increased association of E2F1 with the overlapping sites in the active promoter in 8-Cl-Ado-exposed cells. Together, these data suggest that the overlapping E2F1/Sp1 site, being present in multiple copies in the p14ARF promoter, may serve as the targets for both E2F1 and Sp1, thereby playing a crucial role in response to some oncogenic signals and stimulators, which activate the ARF gene through inducing E2F in the cell.

The ARFul truth about melanoma susceptibility genes

The ARFul truth about melanoma susceptibility genes