ARF regulates the stability of p16 protein via REGγ-dependent proteasome degradation.

UNLABELLED: The cell-cycle regulatory gene INK4A-ARF (CDKN2A) has two alternative transcripts that produce entirely different proteins, namely p14(ARF) and p16, which have complementary functions as regulators of p53 and pRB tumor suppressor pathways, respectively. The unusual organization of INK4A-ARF has long led to speculation of a need for coordinated regulation of p14(ARF) and p16. We now show that p14(ARF) (ARF) regulates the stability of p16 protein in human cancer cell lines, as well as in mouse embryonic fibroblasts (MEFs). In particular, ARF promotes rapid degradation of p16 protein, which is mediated by the proteasome and, more specifically, by interaction of ARF with one of its subunits, REGγ. Furthermore, this ARF-dependent destabilization of p16 can be abrogated by knockdown of REGγ or by pharmacologic blockade of its nuclear export. Thus, our findings have uncovered a novel crosstalk of 2 key tumor suppressors mediated by a REGγ-dependent mechanism. The ability of ARF to control p16 stability may influence cell-cycle function. IMPLICATIONS: The ability of ARF to control p16 stability may influence cell cycle function. Visual Overview: http://mcr.aacrjournals.org/content/current.

Oncomir miR-125b suppresses p14(ARF) to modulate p53-dependent and p53-independent apoptosis in prostate cancer.

MicroRNAs are a class of naturally occurring small non-coding RNAs that target protein-coding mRNAs at the post-transcriptional level and regulate complex patterns of gene expression. Our previous studies demonstrated that in human prostate cancer the miRNA miR-125b is highly expressed, leading to a negative regulation of some tumor suppressor genes. In this study, we further extend our studies by showing that miR-125b represses the protein product of the ink4a/ARF locus, p14(ARF), in two prostate cancer cell lines, LNCaP (wild type-p53) and 22Rv1 (both wild type and mutant p53), as well as in the PC-346C prostate cancer xenograft model that lentivirally overexpressed miR-125b. Our results highlight that miR-125b modulates the p53 network by hindering the down-regulation of Mdm2, thereby affecting p53 and its target genes p21 and Puma to a degree sufficient to inhibit apoptosis. Conversely, treatment of prostate cancer cells with an inhibitor of miR-125b (anti-miR-125b) resulted in increased expression of p14(ARF), decreased level of Mdm2, and induction of apoptosis. In addition, overexpression of miR-125b in p53-deficient PC3 cells induced down-regulation of p14(ARF), which leads to increased cell proliferation through a p53-independent manner. Thus, we conclude that miR-125b acts as an oncogene which regulates p14(ARF)/Mdm2 signaling, stimulating proliferation of prostate cancer cells through a p53-dependent or p53-independent function. This reinforces our belief that miR-125b has potential as a therapeutic target for the management of patients with metastatic prostate cancer.

p53 binds to and is required for the repression of Arf tumor suppressor by HDAC and polycomb.

The expression of tumor suppressor Arf is tightly repressed during normal cell growth at a young age and is activated by oncogenic insults, and during aging, results in p53 activation and cell-cycle arrest to prevent hyperproliferation. The mechanisms of both transcriptional repression and activation of Arf are not understood. We show that p53 binds to and represses Arf expression and that this repression requires the function of both histone deacetylases (HDAC) and polycomb group (PcG) proteins. Inactivation of p53 leads to increased Arf transcription in both mouse embryonic fibroblasts (MEF) cultured in vitro and in tissues and organs of p53 null mice. Activation of endogenous p53 enhances Arf repression, and reintroduction of p53 back into p53 null MEFs restores Arf repression. Both DNA binding and transactivation activities of p53 are required for Arf repression. We show that p53 is required for both HDAC and PcG to repress Arf expression. Bindings of both HDAC and PcG to Arf are disrupted by inactivation of p53 and can be restored in p53 null MEFs by the reintroduction of wild-type, but not mutant, p53. These results indicate that p53 recruits both HDAC and PcG to Arf locus to repress its expression, and this repression constitutes a second feedback loop in p53 regulation.

Loss of p14(ARF) confers resistance to heat shock- and oxidative stress-mediated cell death by upregulating ß-catenin.

The p14(ARF) is a key tumor suppressor induced mainly by oncogenic stimuli. Although p14(ARF) does not seem to respond to DNA damage, there are very few data regarding its role in other forms of stress, such as heat shock (HS) and oxidative stress (OS). Here, we report that suppression of p14(ARF) increased resistance to cell death when cells were treated with H(2) O(2) or subjected to HS. In this setting, protection from cell death was mediated by elevated levels and activity of ß-catenin, as downregulation of ß-catenin alleviated the protective role of p14(ARF) silencing. Moreover, Hsp70 was shown to regulate ß-catenin protein levels by interacting with p14(ARF) , suggesting that Hsp70, p14(ARF) and ß-catenin form a regulatory network. This novel pathway triggers cell death signals when cells are exposed to HS and OS.

Downregulation of DeltaNp63alpha in keratinocytes by p14ARF-mediated SUMO-conjugation and degradation.

The tumor suppressor p14(ARF) inhibits cell growth in response to oncogenic stress in a p53-dependent and independent manner. However, new physiologic roles for ARF activation have been proposed. We have previously demonstrated that ARF interacts with p63, influencing its transcriptional activity. p63 is a member of the p53 family involved in skin and limb development, as well as in the homeostasis of mature epidermis. Here, we show that, in human keratinocytes, as well as in tumor-derived cell lines, ARF targets DeltaNp63alpha, the most abundantly expressed p63 isoform, to proteasomal degradation by stimulating its sumoylation. Interestingly, we have observed an increase of ARF expression in differentiating keratinocytes, that is concomitant to the already described upregulation of SUMO2/3. Remarkably, we found that DeltaNp63alpha is preferentially sumoylated by SUMO2, instead of SUMO1, and p14(ARF) increases the efficiency of this process.

Induction of p14ARF by E2F1 contributes to 8-chloro-adenosine-induced apoptosis in human lung cancer H1299 cells.

BACKGROUND: 8-Chloro-adenosine (8-Cl-Ado) inhibits tumor cell proliferation by inducing cell-cycle arrest and apoptosis. We speculate that upregulation of p14ARF by E2F1 might contribute to 8-Cl-Ado-induced late apoptosis. METHODS: Hoechst staining, cell proliferation and TUNEL assays, real-time quantitative PCR, Western blotting, chromatin immunoprecipitation and RNA interference were employed in investigating the role of induction of p14ARF by E2F1 in 8-Cl-Ado-induced apoptosis in human lung cancer H1299 cells. RESULTS: Exposure of H1299 to 8-Cl-Ado led to apoptosis after long exposure (48 h), revealed by the appearance of nucleus fragmentation and apoptotic bodies and the activation of procaspase-3 pathway. Western blotting and RT-PCR showed that the upregulation of p14ARF was in parallel with E2F1 expression during exposure. Furthermore, induction of p14ARF was attributed to increased E2F1 expression, evidenced by E2F1 transfection and chromatin immunoprecipitation/real-time quantitative PCR. Knockdown of p14ARF expression in H1299 decreased TUNEL-positive cell numbers and relatively increased survival cell numbers during 8-Cl-Ado exposure, indicating insensitivity of p14ARF-knocked down cells to 8-Cl-Ado. CONCLUSIONS: Induction of p14ARF by E2F1 contributes to 8-Cl-Ado-induced late apoptosis.

TBX3 is overexpressed in breast cancer and represses p14 ARF by interacting with histone deacetylases.

TBX3 is a transcription factor of the T-box gene family. Mutations in the TBX3 gene can cause hypoplastic or absent mammary glands. Previous studies have shown that TBX3 might be associated with breast cancer. Here, we show that TBX3 is overexpressed in malignant cells of primary breast cancer tissues by immunohistochemistry. TBX3 interacts with histone deacetylases (HDAC) 1, 2, 3, and 5. TBX3 interacts with HDAC1, 2, and 3 via two distinct binding sites. However, deletion of the repression domain (amino acids 566-624) of TBX3 completely abolishes its interaction with HDAC5. Endogenous TBX3 and HDACs interaction and colocalization are found in a breast cancer cell line by coimmunoprecipitation and immunofluorescence, respectively. The functional significance of the interaction between TBX3 and HDAC is also tested in a p14(ARF)-luciferase reporter system. Results indicate that TBX3 represses expression of p14(ARF) tumor suppressor and that a HDAC inhibitor is able to reverse the TBX3 repressive function in a dosage-dependent manner. This study suggests that TBX3 may function by recruiting HDACs to the T-box binding site in the promoter region. TBX3 repression to its targets is dependent on HDAC activity. TBX3 may serve as a biomarker for breast cancer and have significant applications in both breast cancer diagnosis and treatment.

An endothelial cell genetic screen identifies the GTPase Rem2 as a suppressor of p19ARF expression that promotes endothelial cell proliferation and angiogenesis.

Angiogenesis requires an increase in endothelial cell proliferation to support an increase in mass of blood vessels. We designed an in vitro endothelial cell model to functionally screen for genes that regulate endothelial cell proliferation. A gain of function screen for genes that bypass p53 endothelial cell arrest identified Rem2, a Ras-like GTPase. We show that ectopic Rem2 suppresses p14(ARF) (human) or p19(ARF) (mouse) expression that leads to increased endothelial cell proliferation. Conversely, loss of ectopic Rem2 by RNA interference restores p19(ARF) expression in endothelial cells. We further show that Rem2-interacting 14-3-3 proteins are involved in the cell localization of Rem2, regulation of p19(ARF) expression, and endothelial cell proliferation. Finally, we demonstrate using the RIP1 tag2 mouse model of pancreatic disease that Rem2 is up-regulated in endothelial cells of stage IV disease. The data unravel a possible molecular mechanism for Rem2-induced angiogenesis and suggests Rem2 as a potential novel target for treating pathological angiogenesis.

An inducible mouse model of melanoma expressing a defined tumor antigen.

Cancer immunotherapy based on vaccination with defined tumor antigens has not yet shown strong clinical efficacy, despite promising results in preclinical models. This discrepancy might result from the fact that available preclinical models rely on transplantable tumors, which do not recapitulate the long-term host-tumor interplay that occurs in patients during progressive tumor development and results in tumor tolerance. To create a faithful preclinical model for cancer immunotherapy, we generated a transgenic mouse strain developing autologous melanomas expressing a defined tumor antigen recognized by T cells. We chose the antigen encoded by P1A, a well-characterized murine cancer germ line gene. To transform melanocytes, we aimed at simultaneously activating the Ras pathway and inactivating tumor suppressor Ink4a/Arf, thereby reproducing two genetic events frequently observed in human melanoma. The melanomas are induced by s.c. injection of 4-OH-tamoxifen (OHT). By activating a CreER recombinase expressed from a melanocyte-specific promoter, this treatment induces the loss of the conditional Ink4a/Arf gene in melanocytes. Because the CreER gene itself is also flanked by loxP sites, the activation of CreER also induces the deletion of its own coding sequence and thereby allows melanocyte-specific expression of genes H-ras and P1A, which are located downstream on the same transgene. All melanomas induced in those mice with OHT show activation of the Ras pathway and deletion of gene Ink4a/Arf. In addition, these melanomas express P1A and are recognized by P1A-specific T lymphocytes. This model will allow to characterize the interactions between the immune system and naturally occurring tumors and thereby to optimize immunotherapy approaches targeting a defined tumor antigen.

Myeloid leukemia-associated nucleophosmin mutants perturb p53-dependent and independent activities of the Arf tumor suppressor protein.

Nucleophosmin (NPM or B23) plays key roles in ribosome biogenesis, centrosome duplication, and maintenance of genomic integrity. Mutations affecting the carboxylterminal domain of NPM occur in a significant percentage of adult patients with acute myeloid leukemia (AML), and these alterations create an additional nuclear export signal that relocalizes much of the protein from its normal nucleolar stores to the cytoplasm. When induced by oncogenic stress, the Arf tumor suppressor protein accumulates within the nucleolus, where it is physically associated with, and stabilized by, NPM. Ectopic overexpression of an NPM cytoplasmic mutant (NPMc) relocalized p19Arf and the endogenous NPM protein to the cytoplasm. NPMc-dependent export of p19Arf from the nucleus inhibited its functional interaction with the p53 negative regulator, Mdm2, and blunted Arf-induced activation of the p53 transcriptional program. Cytoplasmic NPM relocalization also attenuated Arf-induced sumoylation of Mdm2 and NPM and prevented wild type NPM from inhibiting p19Arf protein turnover. However, despite the ability of NPMc to interfere with these p53-dependent and independent activities of Arf, NPMc exhibited anti-proliferative activity in Arf-null NIH-3T3 cells. Overexpression of wild type NPM, but not NPMc, overcame premature senescence of Atm-null cells, a phenotype that can be rescued by inactivation of Arf or p53. Therefore, perturbation of Arf function appears to be insufficient to explain the oncogenic effects of the NPMc mutation. We favor the idea that NPMc also contributes to AML by dominantly perturbing other functions of the wild type NPM protein.

ARF impedes NPM/B23 shuttling in an Mdm2-sensitive tumor suppressor pathway.

The ARF tumor suppressor is widely regarded as an upstream activator of p53-dependent growth arrest and apoptosis. However, recent findings indicate that ARF can also regulate the cell cycle in the absence of p53. In search of p53-independent ARF targets, we isolated nucleophosmin (NPM/B23), a protein we show is required for proliferation, as a novel ARF binding protein. In response to hyperproliferative signals, ARF is upregulated, resulting in the nucleolar retention of NPM and concomitant cell cycle arrest. The Mdm2 oncogene outcompetes NPM/B23 for ARF binding, and introduction of Mdm2 reverses ARF's p53-independent properties: in vitro, NPM is released from ARF-containing protein complexes, and in vivo S phase progression ensues. ARF induction by oncogenes or replicative senescence does not alter NPM/B23 protein levels but rather prevents its nucleocytoplasmic shuttling without inhibiting rRNA processing. By actively sequestering NPM in the nucleolus, ARF utilizes an additional mechanism of tumor suppression, one that is readily antagonized by Mdm2.

Role for PP2A in ARF signaling to p53.

Activation of the ARF-p53 tumor suppressor pathway is one of the cell's major defense mechanisms against cancer induced by oncogenes. The ARF-p53 pathway is dysfunctional in a high proportion of human cancers. The regulation of the ARF-p53 signaling pathway has not yet been well characterized. In this study polyoma virus (Py) is used as a tool to better define the ARF-p53 signaling pathway. Py middle T-antigen (PyMT) induces ARF, which consequently up-regulates p53. We show that Py small T-antigen (PyST) blocks ARF-mediated activation of p53. This inhibition requires the small T-antigen PP2A-interacting domain. Our results reveal a previously unrecognized role of PP2A in the modulation of the ARF-p53 tumor suppressor pathway.

p15INK4b, p14ARF, and p16INK4a inactivation in sporadic and neurofibromatosis type 1-related malignant peripheral nerve sheath tumors.

PURPOSE: Malignant peripheral nerve sheath tumor (MPNST) can arise sporadically or in association with neurofibromatosis type 1. Deletions at the 9p21 locus have been reported in these tumors. To additionally characterize the status of this chromosomal region, in this study we performed a comprehensive, mostly PCR-based molecular analysis of the three tumor suppressor genes p15(INK4b), p14(ARF) and p16(INK4a) located at the 9p21 locus in 26 cryopreserved MPNSTs. EXPERIMENTAL DESIGN: Fourteen neurofibromatosis type 1-related and 12 sporadic cases were investigated for homozygous deletion coupled with fluorescent in situ hybridization, promoter methylation, and mutational analysis, as well as m-RNA expression. RESULTS: The results showed that an inactivation of one or more genes occurred in 77% of MPNSTs and was mainly achieved through homozygous deletion (46%), which, in turn, encompassed all of the three tandemly linked genes in 83% of the deleted cases. Promoter methylation was at a less extent involved in gene silencing (18%), and no mutations were found. Loss of function at DNA level strongly correlated with loss of mRNA expression accounting for 80% of the cases. Because of the close relationship between p14(ARF) and TP53 and between p15(INK4b)/p16(INK4a) and Rb, these results support a model of a coinactivation of TP53 and Rb pathways in 75% of MPNSTs, with functional consequences on cell growth control and apoptosis. CONCLUSIONS: The inactivation of the 9p21 locus is a frequent and peculiar hallmark of MPNST genetic profile leading also to an impaired apoptosis that could be taken into account in treatment planning of these tumors.

Abundant expression of spliced HDM2 in Hodgkin lymphoma cells does not interfere with p14(ARF) and p53 binding.

Recently, comparative genomic hybridization (CGH)- and fluorescence in situ hybridization (FISH)-analyses of native Hodgkin and Reed-Sternberg (H&RS) cells extracted from Hodgkin lymphoma (HL) revealed a recurrent amplification of the HDM2 locus on chromosome 12. HDM2 is known to target, inactivate and to degrade p53. Wild type (wt) p53 protein is detected in high levels in HL. Simultaneously, stabilized wt p53 and spliced hdm2 transcripts have been observed in different tumors. Therefore, we examined the expression and structure of HDM2 in HL cell lines and possible effects on components of the p53 pathway. DNA integrity and induction potential of p53 was verified by DNA sequencing and detection of potential effector proteins (p21(WAF/CIP), HDM2) using immunofluorescence, respectively. All HL cell lines show an overexpression of HDM2 protein. Furthermore, several different spliced hdm2 transcripts (mdm-sv) including five new variants lacking a functional p53 binding site were characterized. If expressed, corresponding proteins were shown to be not restricted to the nucleus. Co-localization of the potential binding partners HDM2/p14(ARF) and HDM2/p53 was found in HL cell lines. We suggest that HDM2-sv have no significant disturbing influence on the interaction of these proteins.