Dual Role of the Alternative Reading Frame ARF Protein in Cancer.

The CDKN2a/ARF locus expresses two partially overlapping transcripts that encode two distinct proteins, namely p14ARF (p19Arf in mouse) and p16INK4a, which present no sequence identity. Initial data obtained in mice showed that both proteins are potent tumor suppressors. In line with a tumor-suppressive role, ARF-deficient mice develop lymphomas, sarcomas, and adenocarcinomas, with a median survival rate of one year of age. In humans, the importance of ARF inactivation in cancer is less clear whereas a more obvious role has been documented for p16INK4a. Indeed, many alterations in human tumors result in the elimination of the entire locus, while the majority of point mutations affect p16INK4a. Nevertheless, specific mutations of p14ARF have been described in different types of human cancers such as colorectal and gastric carcinomas, melanoma and glioblastoma. The activity of the tumor suppressor ARF has been shown to rely on both p53-dependent and independent functions. However, novel data collected in the last years has challenged the traditional and established role of this protein as a tumor suppressor. In particular, tumors retaining ARF expression evolve to metastatic and invasive phenotypes and in humans are associated with a poor prognosis. In this review, the recent evidence and the molecular mechanisms of a novel role played by ARF will be presented and discussed, both in pathological and physiological contexts.

Regulatory Network of ARF in Cancer Development.

ARF is a tumor suppressor protein that has a pivotal role in the prevention of cancer development through regulating cell proliferation, senescence, and apoptosis. As a factor that induces senescence, the role of ARF as a tumor suppressor is closely linked to the p53-MDM2 axis, which is a key process that restrains tumor formation. Thus, many cancer cells either lack a functional ARF or p53, which enables them to evade cell oncogenic stress-mediated cycle arrest, senescence, or apoptosis. In particular, the ARF gene is a frequent target of genetic and epigenetic alterations including promoter hyper-methylation or gene deletion. However, as many cancer cells still express ARF, pathways that negatively modulate transcriptional or post-translational regulation of ARF could be potentially important means for cancer cells to induce cellular proliferation. These recent findings of regulators affecting ARF protein stability along with its low levels in numerous human cancers indicate the significance of an ARF post-translational mechanism in cancers. Novel findings of regulators stimulating or suppressing ARF function would provide new therapeutic targets to manage cancer- and senescence-related diseases. In this review, we present the current knowledge on the regulation and alterations of ARF expression in human cancers, and indicate the importance of regulators of ARF as a prognostic marker and in potential therapeutic strategies.

P14ARF deficiency and its correlation with overexpression of p53/MDM2 in sporadic vestibular schwannomas.

Recent studies have shed considerable light into schwannomas. To date, only merlin has been identified as a hallmark or pathogenesis of both sporadic and NF2-related schwannomas. Here, we show, by immunoblot and immunohistochemical analyses of 58 sporadic vestibular schwannomas, that upregulation of p53 was observed in 90 % of tumors examined. No p53 mutations were found in 12 % tumors analyzed. Expression of p14ARF was negative in 95 % of tumors, while overexpression of MDM2 was found in all specimens. Aberrant DNA hypermethylation of the p14ARF promoter was observed in three of seven tumors examined (43 %), associated with remarkably decreased mRNA levels. The very high degree of concordance in the aberrations of the p14ARF/MDM2/p53 pathway in this tumor may be considered to be a new player in the pathogenesis of sporadic vestibular schwannomas. Moreover, expression of p21 (waf1) was negative in all tumors, suggesting that the aberration of this pathway is associated with greater attenuation of p21-mediated signals that are critical for growth inhibition. These data are in agreement with the model in RT-4 rat schwannoma cells: i.e., overexpression of ARF was associated with accumulation of p21 expression both at protein and mRNA levels. ShRNA knock-down of p53 expression attenuated p21-mediated increase in cellular arrest in the G1-phase, suggesting that p14ARF regulates p21 protein levels through a p53-dependent pathway. Thus, this study reveals a high degree of concordance in the aberrations of the p14ARF/MDM2/p53 pathway with the development of sporadic vestibular schwannomas.

Abnormal MDMX degradation in tumor cells due to ARF deficiency.

MDMX is a heterodimeric partner of MDM2 and a critical regulator of p53. The MDMX level is generally elevated in tumors with wild-type p53 and contributes to p53 inactivation. MDMX degradation is controlled in part by MDM2-mediated ubiquitination. Here, we show that MDMX turnover is highly responsive to changes in MDM2 level in non-transformed cells, but not in tumor cells. We found that loss of alternate reading frame (ARF) expression, which occurs in most tumors with wild-type p53, significantly reduces MDMX sensitivity to MDM2. Restoration of ARF expression in tumor cells enables MDM2 to degrade MDMX in a dose-dependent manner. ARF binds to MDM2 and stimulates a second-site interaction between the central region of MDM2 and MDMX, and thus increases MDMX-MDM2 binding and MDMX ubiquitination. These results reveal an important abnormality in the p53-regulatory pathway as a consequence of ARF deficiency. Loss of ARF during tumor development not only prevents p53 stabilization by proliferative stress but also causes accumulation of MDMX that compromises p53 activity. This phenomenon may reduce the clinical efficacy of MDM2-specific inhibitors by preventing MDMX downregulation.

Losses of both products of the Cdkn2a/Arf locus contribute to asbestos-induced mesothelioma development and cooperate to accelerate tumorigenesis.

The CDKN2A/ARF locus encompasses overlapping tumor suppressor genes p16(INK4A) and p14(ARF), which are frequently co-deleted in human malignant mesothelioma (MM). The importance of p16(INK4A) loss in human cancer is well established, but the relative significance of p14(ARF) loss has been debated. The tumor predisposition of mice singly deficient for either Ink4a or Arf, due to targeting of exons 1α or 1ß, respectively, supports the idea that both play significant and nonredundant roles in suppressing spontaneous tumors. To further test this notion, we exposed Ink4a(+/-) and Arf(+/-) mice to asbestos, the major cause of MM. Asbestos-treated Ink4a(+/-) and Arf(+/-) mice showed increased incidence and shorter latency of MM relative to wild-type littermates. MMs from Ink4a(+/-) mice exhibited biallelic inactivation of Ink4a, loss of Arf or p53 expression and frequent loss of p15(Ink4b). In contrast, MMs from Arf(+/-) mice exhibited loss of Arf expression, but did not require loss of Ink4a or Ink4b. Mice doubly deficient for Ink4a and Arf, due to deletion of Cdkn2a/Arf exon 2, showed accelerated asbestos-induced MM formation relative to mice deficient for Ink4a or Arf alone, and MMs exhibited biallelic loss of both tumor suppressor genes. The tumor suppressor function of Arf in MM was p53-independent, since MMs with loss of Arf retained functional p53. Collectively, these in vivo data indicate that both CDKN2A/ARF gene products suppress asbestos carcinogenicity. Furthermore, while inactivation of Arf appears to be crucial for MM pathogenesis, the inactivation of both p16(Ink4a) and p19(Arf) cooperate to accelerate asbestos-induced tumorigenesis.

Brain tumor stem cells.

Primary malignant brain cancer, one of the most deadly diseases, has a high rate of recurrence after treatment. Studies in the past several years have led to the hypothesis that the root of the recurrence may be brain tumor stem cells (BTSCs), stem-like subpopulation of cells that are responsible for propagating the tumor. Current treatments combining surgery and chemoradiotherapy could not eliminate BTSCs because these cells are highly infiltrative and possess several properties that can reduce the damages caused by radiation or anti-cancer drugs. BTSCs are similar to NSCs in molecular marker expression and multi-lineage differentiation potential. Genetic analyses of Drosophila CNS neoplasia, mouse glioma models, and human glioma tissues have revealed a link between increased NSC self-renewal and brain tumorigenesis. Furthermore, data from various rodent models of malignant brain tumors have provided compelling evidence that multipotent NSCs and lineage-restricted neural progenitor cells (NPCs) could be the cell origin of brain tumors. Thus, the first event of brain tumorigenesis might be the occurrence of oncogenic mutations in the stem cell self-renewal pathway in an NSC or NPC. These mutations convert the NSC or NPC to a BTSC, which then initiates and sustains the growth of the tumor. The self-renewal of BTSCs is controlled by several evolutionarily conserved signaling pathways and requires an intact vascular niche. Targeting these pathways and the vascular niche could be a principle in novel brain tumor therapies aimed to eliminate BTSCs.

Abrogation of DNA vector-based RNAi during apoptosis in mammalian cells due to caspase-mediated cleavage and inactivation of Dicer-1.

RNA interference (RNAi) is used as a reverse-genetic tool to examine functions of a gene in different cellular processes including apoptosis. As key cellular proteins are inactivated during apoptosis, and as RNAi requires cooperation of many cellular proteins, we examined whether DNA vector-based RNAi would continue to function during apoptosis. The short hairpin RNA transcribed from the DNA vector is processed by Dicer-1 to form small interfering RNA that is incorporated in the RNA-induced silencing complex (RISC) to guide a sequence-specific silencing of the target mRNA. We report here that DNA vector-based RNAi of three different genes, namely poly(ADP-ribose) polymerase-1, p14(ARF) and lamin A/C are abrogated during apoptosis. The failure of DNA vector-based RNAi was not at the level of Ago-2 or RISC-mediated step of RNAi but due to catalytic inactivation of Dicer-1 on specific cleavage at the STTD(1476) and CGVD(1538) sites within its RNase IIIa domain. Using multiple approaches, caspase-3 was identified as the major caspase responsible for the cleavage and inactivation of Dicer-1. As Dicer-1 is also the common endonuclease required for formation of microRNA (miRNA) in mammalian cells, we observed decreased levels of mature forms of miR-16, miR-21 and let-7a. Our results suggest a role for apoptotic cleavage and inactivation of Dicer-1 in controlling apoptotic events through altered availability of miRNA.

ARF functions as a melanoma tumor suppressor by inducing p53-independent senescence.

Inactivation of the p53 pathway represents the most common molecular defect of human cancer. But in the setting of melanoma, a highly aggressive and invariably fatal malignancy in its advanced disseminated form, mutation/deletion of p53 is relatively rare, whereas its positive regulator ARF is often lost. Here, we show that genetic deficiency in Arf but not p53 facilitates rapid development of melanoma in a genetically engineered mouse model. This difference is accounted for, at least in part, by the unanticipated observation that, unlike fibroblasts, senescence control in melanocytes is strongly regulated by Arf and not p53. Moreover, oncogenic NRAS collaborates with deficiency in Arf, but not p53, to fully transform melanocytes. Our data demonstrate that ARF and p53, although linked in a common pathway, suppress tumorigenesis through distinct, lineage-dependent mechanisms and suggest that ARF helps restrict melanoma progression by executing the oncogene-induced senescence program in benign nevi. Thus, therapeutics designed to restore wild-type p53 function may be insufficient to counter melanoma and other malignancies in which ARF holds p53-independent tumor suppressor activity.

Frequent loss of chromosome 9, homozygous CDKN2A/p14(ARF)/CDKN2B deletion and low TSC1 mRNA expression in pleomorphic xanthoastrocytomas.

The molecular pathogenesis of pleomorphic xanthoastrocytoma (PXA), a rare astrocytic brain tumor with a relatively favorable prognosis, is still poorly understood. We characterized 50 PXAs by comparative genomic hybridization (CGH) and found the most common imbalance to be loss on chromosome 9 in 50% of tumors. Other recurrent losses affected chromosomes 17 (10%), 8, 18, 22 (4% each). Recurrent gains were identified on chromosomes X (16%), 7, 9q, 20 (8% each), 4, 5, 19 (4% each). Two tumors demonstrated amplifications mapping to 2p23-p25, 4p15, 12q13, 12q21, 21q21 and 21q22. Analysis of 10 PXAs with available high molecular weight DNA by high-resolution array-based CGH indicated homozygous 9p21.3 deletions involving the CDKN2A/p14(ARF)/CDKN2B loci in six tumors (60%). Interphase fluorescence in situ hybridization to tissue sections confirmed the presence of tumor cells with homozygous 9p21.3 deletions. Mutational analysis of candidate genes on 9q, PTCH and TSC1, revealed no mutations in PXAs with 9q loss and no evidence of TSC1 promoter methylation. However, PXAs consistently showed low TSC1 transcript levels. Taken together, our study identifies loss of chromosome 9 as the most common chromosomal imbalance in PXAs and suggests important roles for homozygous CDKN2A/p14(ARF)/CDKN2B deletion as well as low TSC1 mRNA expression in these tumors.

Antiviral action of the tumor suppressor ARF.

Oncogenic viruses frequently target the pathways controlled by tumor suppressor genes, suggesting an extra function for these proteins as antiviral factors. The control exerted by the tumor suppressor Arf on cellular proliferation is crucial to restrict tumor development; however, a potential contribution of Arf to prevent viral infectivity has remained unexplored. In the present study, we investigated the consequences of loss or increased expression of Arf on viral infection. Our results reveal that ARF expression is induced by interferon and after viral infection. Furthermore, we show that ARF protects against viral infection in a gene dosage-dependent manner, and that this antiviral action is mediated in part by PKR through a mechanism that involves ARF-induced release of PKR from nucleophosmin complexes. Finally, Arf-null mice were hypersensitive to viral infection compared to wild-type mice. Together, our results reveal a novel and unexpected role for the tumor suppressor ARF in viral infection surveillance.

ROS fusion tyrosine kinase activates a SH2 domain-containing phosphatase-2/phosphatidylinositol 3-kinase/mammalian target of rapamycin signaling axis to form glioblastoma in mice.

Glioblastoma multiforme is the most common and lethal form of primary brain cancer. Diagnosis of this advanced glioma has a poor prognosis due to the ineffectiveness of current therapies. Aberrant expression of receptor tyrosine kinases (RTK) in glioblastoma multiformes is suggestive of their role in initiation and maintenance of these tumors of the central nervous system. In fact, ectopic expression of the orphan RTK ROS is a frequent event in human brain cancers, yet the pathologic significance of this expression remains undetermined. Here, we show that a glioblastoma-associated, ligand-independent rearrangement product of ROS (FIG-ROS) cooperates with loss of the tumor suppressor gene locus Ink4a;Arf to produce glioblastomas in the mouse. We show that this FIG-ROS-mediated tumor formation in vivo parallels the activation of the tyrosine phosphatase SH2 domain-containing phosphatase-2 (SHP-2) and a phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling axis in tumors and tumor-derived cell lines. We have established a fully penetrant preclinical model for adult onset of glioblastoma multiforme in keeping with major genetic events observed in the human disease. These findings provide novel and important insights into the role of ROS and SHP-2 function in solid tumor biology and set the stage for preclinical testing of targeted therapeutic approaches.

Specific contribution of p19(ARF) to nitric oxide-dependent apoptosis.

NO is an important bioactive molecule involved in a variety of physio- and pathological processes, including apoptosis induction. The proapoptotic activity of NO involves the rise in the tumor suppressor p53 and the accumulation and targeting of proapoptotic members of the Bcl-2 family, in particular Bax and the release of cytochrome c from the mitochondria. However, the exact mechanism by which NO induces p53 activation has not been fully elucidated. In this study, we describe that NO induces p19(ARF) through a transcriptional mechanism. This up-regulation of p19(ARF) activates p53, leading to apoptosis. The importance of p19(ARF) on NO-dependent apoptosis was revealed by the finding that various cell types from alternate reading frame-knockout mice exhibit a diminished response to NO-mediated apoptosis when compared with normal mice. Moreover, the biological relevance of alternative reading frame to p53 apoptosis was confirmed in in vivo models of apoptosis. Together, these results demonstrate that NO-dependent apoptosis requires, in part, the activation of p19(ARF).

The PTEN and INK4A/ARF tumor suppressors maintain myelolymphoid homeostasis and cooperate to constrain histiocytic sarcoma development in humans.

Histiocytic sarcoma (HS) is a rare malignant proliferation of histiocytes of uncertain molecular pathogenesis. Here, genetic analysis of coincident loss of Pten and Ink4a/Arf tumor suppressors in the mouse revealed a neoplastic phenotype dominated by a premalignant expansion of biphenotypic myelolymphoid cells followed by the development of HS. Pten protein loss occurred only in the histiocytic portion of tumors, suggesting a stepwise genetic inactivation in the generation of HS. Similarly, human HS showed genetic or epigenetic inactivation of PTEN, p16(INK4A), and p14(ARF), supporting the relevance of this genetically engineered mouse model of HS. These genetic and translational observations establish a cooperative role of Pten and Ink4a/Arf in the development of HS and provide mechanistic insights into the pathogenesis of human HS.

Arf gene loss enhances oncogenicity and limits imatinib response in mouse models of Bcr-Abl-induced acute lymphoblastic leukemia.

Mouse bone marrow cells transduced with retroviral vectors encoding either of two oncogenic Bcr-Abl isoforms (p210(Bcr-Abl) and p185(Bcr-Abl)) induce B cell lympholeukemias when transplanted into lethally irradiated mice. If the activity of the Arf tumor suppressor is compromised, these donor cells initiate a much more highly aggressive and rapidly fatal disease. When mouse bone marrow cells expressing Bcr-Abl are placed in short-term cultures selectively designed to support the outgrowth of pre-B cells, only those lacking one or two Arf alleles can initiate lympholeukemias when inoculated into immunocompetent, syngeneic recipient mice. Although the ABL kinase inhibitor imatinib mesylate (Gleevec) provides highly effective treatment for BCR-ABL-positive chronic myelogenous leukemia, it has proven far less efficacious in the treatment of BCR-ABL-positive acute lymphoblastic leukemias (ALLs), many of which sustain deletions of the INK4A-ARF (CDKN2A) tumor suppressor locus. Mice receiving Arf-/- or Arf+/- p210(Bcr-Abl)-positive pre-B cells do not achieve remission when maintained on high doses of oral imatinib therapy and rapidly succumb to lympholeukemia. Although cells expressing the Bcr-Abl kinase can proliferate in the absence of IL-7, they remain responsive to this cytokine, which can reduce their sensitivity to imatinib. Treatment of Arf-/-, p210(Bcr-Abl)-positive pre-B cells with imatinib together with an inhibitor of JAK kinases abrogates this resistance, suggesting that this combination may prove beneficial in the treatment of BCR-ABL-positive acute lymphoblastic leukemia.

Role of INK4a locus in normal eye development and cataract genesis.

The murine INK4a locus encodes the critical tumor suppressor proteins, p16(INK4a) and p19(ARF). Mice lacking both p16(INK4a) and p19(ARF) (INK4a-/-) in their FVB/NJ genetic backgrounds developed cataracts and microophthalmia. Histopathologically, INK4a-/- mice showed defects in the developmental regression of the hyaloid vascular system (HVS), retinal dysplasia, and cataracts with numerous vacuolations, closely resembling human persistent hyperplastic primary vitreous (PHPV). Ocular defects, such as retinal fold and abnormal migration of lens fiber cells, were observed as early as embryonic day (E) 15.5, thereby resulting in the abnormal differentiation of the lens. We also found that ectopic expression of p16(INK4a) resulted in the induction of gammaF-crystallin, suggesting an important role of INK4a locus during mouse eye development, and also providing insights into the potential genetic basis of human cataract genesis.

Both p16(Ink4a) and the p19(Arf)-p53 pathway constrain progression of pancreatic adenocarcinoma in the mouse.

Activating KRAS mutations and p16(Ink4a) inactivation are near universal events in human pancreatic ductal adenocarcinoma (PDAC). In mouse models, Kras(G12D) initiates formation of premalignant pancreatic ductal lesions, and loss of either Ink4a/Arf (p16(Ink4a)/p19(Arf)) or p53 enables their malignant progression. As recent mouse modeling studies have suggested a less prominent role for p16(Ink4a) in constraining malignant progression, we sought to assess the pathological and genomic impact of inactivation of p16(Ink4a), p19(Arf), and/or p53 in the Kras(G12D) model. Rapidly progressive PDAC was observed in the setting of homozygous deletion of either p53 or p16(Ink4a), the latter with intact germ-line p53 and p19(Arf) sequences. Additionally, Kras(G12D) in the context of heterozygosity either for p53 plus p16(Ink4a) or for p16(Ink4a)/p19(Arf) produced PDAC with longer latency and greater propensity for distant metastases relative to mice with homozygous deletion of p53 or p16(Ink4a)/p19(Arf). Tumors from the double-heterozygous cohorts showed frequent p16(Ink4a) inactivation and loss of either p53 or p19(Arf). Different genotypes were associated with specific histopathologic characteristics, most notably a trend toward less differentiated features in the homozygous p16(Ink4a)/p19(Arf) mutant model. High-resolution genomic analysis revealed that the tumor suppressor genotype influenced the specific genomic patterns of these tumors and showed overlap in regional chromosomal alterations between murine and human PDAC. Collectively, our results establish that disruptions of p16(Ink4a) and the p19(ARF)-p53 circuit play critical and cooperative roles in PDAC progression, with specific tumor suppressor genotypes provocatively influencing the tumor biological phenotypes and genomic profiles of the resultant tumors.

Induction of nevi and skin tumors in Ink4a/Arf Xpa knockout mice by neonatal, intermittent, or chronic UVB exposures.

Nevi and melanomas correlate to childhood and intermittent solar UV exposure, xeroderma pigmentosum patients run increased risk, and p16(Ink4a) expression is often lost in malignant progression. To ascertain the effect of these risk factors, pigmented hairless Ink4a/Arf-, Xpa- knockout mice were subjected to various combinations of neonatal [7,12-dimethylbenz(a)anthracene (DMBA) or UVB exposure] and adult treatments (12-O-tetradecanoylphorbol-13-acetate or subacute daily UVB exposure or intermittent overexposure). Nevi occurred earliest, grew largest, and were most numerous in mice exposed to DMBA followed by intermittent UVB overexposure [effect of six minimal edemal doses (MED), 1 x /2 weeks > 4 MED 1 x /wk]. Neonatal UV exposure enhanced nevus induction but lost its effect after 200 days. The Xpa(-/-) mice proved exquisitely sensitive to UV-driven nevus induction, indicating the involvement of pyrimidine dimer DNA lesions, but Xpa(+/+) mice developed many more nevi (>40 per mouse) at high UV dosages not tolerated by Xpa(-/-) mice. Ink4a/Arf(-/-) mice developed most skin tumors faster, but surprisingly developed nevi slower than their heterozygous counterparts especially after neonatal UV exposure. Despite raising >1,600 nevi, only six melanomas arose in our experiments with Ink4a/Arf knockout mice (five of which in Xpa(+/+) mice at high UV dosages). In contrast to human nevi, these nevi lacked hotspot mutations in Braf or Ras genes, possibly explaining the lack of malignant progression in the Ink4a/Arf(-/-) mice. Hence, although our experiments did not effectively emulate human melanoma, they provided clear evidence that intermittent UV overexposure strongly stimulates and the Ink4a/Arf(-/-) genotype may actually impair nevus development.

The tumor suppressor p16Ink4a regulates T lymphocyte survival.

In contrast to other cell cycle inhibitors, the tumor suppressor p16Ink4a is not detectable or expressed at very low levels in embryonic and adult mouse tissues, and therefore it has often been considered as a specialized checkpoint protein that does not participate in the control of normal cell cycle progression. However, Ink4a-/- mice possess increased thymus size and cellularity, thus suggesting the involvement of p16(Ink4a) in the control of thymocyte proliferation. In this study, we found increased numbers of CD8 and CD4 T lymphocytes in thymus and spleen from Ink4a-/- mice. Unexpectedly, this was not related to an increase in T-cell division rates, which were similar in lymphoid organs of Ink4a-/- and wild-type mice. In contrast, T-cell apoptosis rates were significantly decreased in thymus and spleen from Ink4a-/- mice. Moreover, whereas p16Ink4a-deficient and wild-type T cells were equally sensitive to Fas or TCR-mediated apoptosis, the former were clearly more resistant to apoptosis induced by oxidative stress or gamma irradiation. Our results indicate that p16Ink4a function is associated with T-cell apoptosis, and subsequently contributes to the control of T-cell population size in lymphoid organs.