The INK4a/ARF locus in murine tumorigenesis

Manuel Serrano

Department of Immunology and Oncology, Centro Nacional de Biotecnología, Campus de Cantoblanco, Madrid E-28049, Spain Email: mserrano{at}cnb.uam.es

Abstract

The INK4a/ARF locus is regarded as one of the most important anti-tumoral defenses that mammalian organisms possess. The characterization of its two gene products, p16INK4a and p19ARF, has provided a great insight on the functioning of the tumor suppressors Rb and p53, respectively. Present evidence indicates that the INK4a/ARF locus is transcriptionally activated by oncogenic stresses, resulting in cell-cycle arrest or apoptosis. Here, I review the evidence accumulated on the involvement of the INK4a/ARF locus in murine tumorigenesis. Also, I summarize the phenotype of the different transgenic mouse models based on the inactivation of the INK4a/ARF locus.

Introduction

The p16INK4a protein was initially cloned as a protein associated with the cyclin D-dependent kinase CDK4, and it was shown to be an efficient inhibitor of the CDK4 kinase activity (1). Several proteins closely related to p16INK4a have subsequently been identified that together form the INK4 family: p16INK4a, p15INK4b, p18INK4c and p19INK4d (2). The four INK4 proteins have similar biochemical activities, all of them binding and inhibiting CDK4 and its related kinase CDK6. The relevant feature that characterizes each INK4 protein appears to be its specific transcriptional regulation. In particular, the expression of p16INK4a is activated by oncogenic stresses (see below), while the expression of p15INK4b is upregulated by the negative growth factor transforming growth factor ß (3).

One of the most surprising discoveries of recent years has been the realization that the INK4a locus contains an overlapping gene named ARF and encoding p19ARF (also called p14ARF when referring specifically to the human version) (47). The INK4a and ARF genes have their own separate promoters, each of which produces a different transcript: the INK4a transcript is formed by exons 1{alpha}, 2 and 3; and the ARF transcript is formed by exons 1ß, 2 and 3 (Figure 1Go). Although exons 2 and 3 are common to INK4a and ARF transcripts, they are read in different frames and, consequently, the corresponding proteins do not share amino acid sequence homology. The p19ARF protein has turned out to be a negative regulator of the p53-destabilizing oncogene MDM2 (8). The structure of the INK4a/ARF locus is conserved among humans, mice and rats (47,9), but an equivalent to p19ARF is absent in the loci encoding the other three members of the INK4 family. It is also important to mention that the INK4b gene is located immediately upstream of the INK4a/ARF locus (Figure 1Go).



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Fig. 1. Structure of the INK4a/ARF and INK4b loci. This structure is conserved in human, mouse and rat.

 
The p16INK4a/Rb pathway
Due to their ability to inhibit the CDK4–6/cycD kinases, INK4 proteins contribute to maintain the Rb protein and its related homologs, p107 and p130, in their unphosphorylated, growth-suppressive state. Among all the possible pathways that relate these proteins, the one formed by p16INK4a/CDK4/cycD1/Rb (Figure 2Go) and, to a lesser extent, the one formed by p15INK4b/CDK4/cycD1/Rb seem of special relevance for tumorigenesis. Importantly, the p16INK4a/Rb pathway behaves as a single mutagenic target in many human tumors (2). For example, the inactivation of p16INK4a or Rb are, in general, mutually exclusive alterations. All together, the p16INK4a/Rb pathway appears to be deregulated in the large majority of human tumors either by loss of p16INK4a or Rb, or by overexpression of CDK4 or cyclin D1 (Figure 2Go).



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Fig. 2. Tumor suppressor pathways controlled by the products of the INK4a/ARF locus. These pathways are activated by the presence of oncogenic stresses. Multiple crosstalks are known to exist between the p16INK4a/Rb and p19ARF/p53 pathways (see text).

 
The p19ARF/p53 pathway
The tumor suppressor p53 is inactivated in ~50% of human cancers. The protein levels of p53 are determined primarily by ubiquitination and subsequent proteolytic degradation. The main ubiquitin ligase for p53 is the oncogene MDM2 (also known as HDM2 when referring specifically to the human gene) (10). Concurring with the role of MDM2 as a repressor of p53, tumors overexpressing MDM2 do not accumulate detectable p53 levels and do not carry mutations in the p53 gene (11). Protein p19ARF associates with MDM2 and sequesters it into the nucleoli (12,13). Theses two functions, binding to MDM2 and nucleolar localization, are essential for p19ARF-mediated stabilization of p53 (12,13). The p19ARF/p53 pathway is also thought to be deregulated in the large majority of human tumors either by loss of p19ARF or p53, or by overexpression of MDM2 (Figure 2Go).

The above-described pathways can be simultaneously deregulated by homozygous deletion of the entire INK4/ARF locus (see below), or they can be deregulated by a combination of mutagenic events, such as amplification of cyclin D1 and loss of p53. Recently, a novel mechanism of deregulation has been identified in relation to the Bmi1 oncogene. Bmi1 belongs to the Polycomb family of chromatin silencers, and when overexpressed is able to repress simultaneously the transcriptional activity of the INK4a and ARF promoters (14).

It is important to emphasize the existence of multiple levels of crosstalk between the p16INK4a/Rb and p19ARF/p53 pathways. For example, p53 transcriptionally activates the expression of p21Cip1 which, in turn, inhibits the CDK2/A–E kinases preventing the full phosphorylation of Rb (15). Also, MDM2 can interfere with the growth-suppressive activity of Rb (16). These and other additional levels of regulation complicate the interpretation of the effects that particular alterations of these pathways may have on cell proliferation and apoptosis.

The INK4a/ARF locus as a sensor of oncogenic stress

The normal levels of expression of p16INK4a and p19ARF are extremely low in most tissues. In mice, p16INK4a and p19ARF expression is only detected in some adult tissues by RT–PCR (17,18). It appears, therefore, that these two tumor suppressors are not continuously restraining proliferation under normal conditions, but rather `awaiting' to be activated in response to the appropriate signals. These signals have begun to be identified as oncogenic stresses (Figure 2Go). In particular, the presence of oncogenic Ras in a normal cell can activate the expression of both p16INK4a and p19ARF resulting in cell-cycle arrest (1923). Also, other oncogenes, such as Myc, E2F1 and the adenoviral oncoprotein E1a, activate the expression of p19ARF (but not of p16INK4a) (2426). When expressed in normal cells, Myc, E2F1 and E1a, elicit a strong apoptotic response mediated primarily through activation of p53. It is now well established that the activation of p53 by Myc and E1a is mediated by the upregularion of p19ARF (24,25). In summary, the INK4a/ARF locus is activated in response to oncogenic stresses, thus preventing the propagation of cells carrying activated oncogenes (8,27).

Inactivation of the INK4a/ARF locus in mouse carcinogenesis

The inactivation of the INK4a/ARF locus has been thoroughly studied in human tumors. Essentially three modes of inactivation have been reported: (i) Homozygous deletion. This is a common mechanism of inactivation which generally involves the entire INK4a/ARF locus and very often the neighbor INK4b gene. It affects up to 14% of all human tumors analyzed (data obtained from ref. 2). (ii) Intragenic mutation. This type of inactivation mechanism is rather infrequent in the INK4a/ARF locus. Most point mutations occur in the second exon common for p16INK4a and p19ARF, usually affecting the amino acid sequence of both proteins. Nevertheless, a fraction of mutations occur in exon 1{alpha}, thus affecting exclusively the p16INK4a coding region. No mutations have been reported yet in exon 1ß. All together, point mutations in the INK4a/ARF coding regions have been detected in 5% of all human tumors analyzed (data obtained from ref. 2). (iii) Promoter silencing by methylation. A significant fraction of human tumors show aberrant methylation of the INK4a promoter which results in a complete inactivation of its transcriptional activity. Silencing of the INK4a promoter by methylation has been found in 19% of all human tumors analyzed (data obtained from ref. 2). Aberrant methylation of the ARF promoter has been recently reported in human colorectal cancers independently of INK4a promoter methylation (28).

The status of the INK4a/ARF locus in murine tumors has been analyzed in a number of studies (summarized in Table IGo). Several interesting points can be made from the available data. In first place, the incidence of alterations in the murine INK4a/ARF locus appears rather low in primary solid tumors compared to the high incidence of alterations found in tumor cell lines. A similar situation has been encountered studying human tumors and derived cell lines. Two possible scenarios can be invoked to explain this situation. One possibility is that alterations in the INK4a/ARF locus are as frequent in primary tumors as in tumor cell lines, but their detection in primary tumors is often obscured by the presence of non-tumoral cells and/or by the different clonal populations that may form a tumor. In this regard, immunohistochemical analysis of p16INK4a in mouse lung tumors has shown the presence of focal areas within the tumor that lacked p16INK4a staining (29). Those tumor cells that have inactivated the INK4a/ARF locus may have a proliferative advantage with respect to the other tumoral cells and after in vitro expansion they may eventually dominate the culture. According to an alternative scenario, the inactivation of the INK4a/ARF locus is not present at all within the primary tumor, but occurs post-explantation during in vitro culture. However, a number of observations contradict this scenario and indicate that the establishment of tumor cell lines in vitro does not entail the inactivation of INK4a/ARF. For example, murine lung cancer cell lines of non-metastatic origin have a lower incidence of INK4a/ARF homozygous deletions compared with metastatic ones (30). Also, skin carcinoma cell lines of a differentiated phenotype have a very low (or zero) incidence of deletions, whereas similar cell lines of poorly differentiated phenotype show a high incidence of INK4a/ARF deletions (31; Table IGo). It appears that inactivation of the INK4a/ARF locus is generally a late event during tumorigenesis.


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Table I. INK4a/ARF alterations in mouse tumors
 
It is interesting to mention that a number of studies have found a significant proportion of INK4a/ARF intragenic mutations in lung and liver tumors induced with 3-methylcholanthrene (7 and 19%, respectively; Table IGo) (3234), but not in similar tumors induced with other carcinogens (29,30,35). This is a remarkable observation considering the low incidence of intragenic mutations in the INK4a/ARF locus in human tumors.

A separate discussion must be made of murine lymphomas and leukemias. Regarding T-cell lymphomas, it appears that the loss of p15INK4b expression by promoter methylation is more relevant than the inactivation of the INK4a/ARF locus. This is particularly prominent in T-cell lymphomas induced by {gamma} or neutron radiation (88 and 42% of INK4b promoter methylation, respectively) (36). These observations are in line with similar findings highlighting the pre-eminence of INK4b inactivation in specific human hematological malignancies (3739). In any case, one study has analyzed the status of p16INK4a, p15INK4b, CDK4, cyclin D1 and Rb in the same set of lymphomas, reaching the conclusion that 75% of the radiation-induced T-cell lymphomas have alterations in one or several components of this pathway (40). Regarding B-cell lymphomas developed by transgenic mice expressing the Myc oncogene, there is strong evidence indicating that p19ARF is the key tumor suppressor inactivated by the deletion of the INK4a/ARF locus (4143).

Finally, it is interesting to mention that those tumors that retain a functional INK4a/ARF locus, usually manifest a dramatic upregulation of its expression. In particular, it has been observed that p16INK4a is significantly accumulated in skin carcinomas (31,44,45), lung tumors (35) and bladder carcinomas (46). The upregulation of p16INK4a can be explained by the continued presence of an oncogenic stress, such as activated Ras (see above), in combination with other alterations, such as overexpression of cyclins D, E or A (45), that make these tumor cells insensitive to the anti-proliferative effects of p16INK4a.

INK4a/ARF alleles and cancer susceptibility in mice

Many observations indicate that cancer susceptibility is under complex multigenic influences. For some particular types of tumors it has been possible to map susceptibility loci with a high resolution in the mouse genome. One of the susceptibility loci for chemically induced plasmacytomas (a B-lymphocyte malignancy) has been proposed to correspond to the INK4a/ARF locus (47). Similarly, a susceptibility locus for chemically induced lung tumors has also been mapped to the INK4a/ARF locus (48). Interestingly, sequencing of the INK4a/ARF coding regions of `susceptible' and `resistant' mouse strains has shown that a variant allele is specifically present in the `susceptible' strains (such as Balb/c). The variant allele contains two mis-sense codons with respect to the canonical mouse sequence: one in exon 1{alpha} and the other in exon 2. Biochemical analysis of the corresponding p16INK4a and p19ARF `variant' proteins has shown that the `variant' p16INK4a protein is indeed impaired in its CDK4–6/cycD inhibitory function (47,49). No defects were found in the `variant' p19ARF protein. These observations indicate that the INK4a/ARF locus is a tumor susceptibility locus in mice.

Mouse tumor models based on targeted inactivation of the INK4a/ARF locus

Engineered alterations of the INK4a/ARF locus have served to test the function of p16INK4a and p19ARF in tumor suppression, and to generate sophisticated tissue-specific tumor models (Table IIGo). Two mutations have been introduced so far into the INK4a/ARF locus: elimination of exons 2 and 3 (INK4a/ARF{Delta}ex2,3) (50), and elimination of exon 1ß (ARF{Delta}) (51). In the first case, deletion of exons 2 and 3 completely eliminates the function of p16INK4a. However, the situation is not that clear regarding the status of p19ARF because INK4a/ARF{Delta}ex2,3 cells express at low levels a transcript containing exon 1ß and encoding an active p19ARF mutant protein (C.Pantoja, I.Palmero and M.Serrano, manuscript in preparation). This raises the possibility that INK4a/ARF{Delta}ex2,3 mice could be partially functional for p19ARF. In any case, both mutant mice, INK4a/ARF{Delta}ex2,3 and ARF{Delta}, have strikingly similar phenotypes (50–52; Table IIGo), which suggest that p19ARF is more relevant than p16INK4a for tumor suppression in mice. Notwithstanding, this issue will not be unambiguously answered until the generation of new mutations that specifically inactivate p16INK4a without affecting p19ARF.


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Table II. Mouse tumor models based on the targeted inactivation of the INK4a/ARF locus
 
Several mouse tumor models have been generated by combination of the above-mentioned mutations with transgenic oncogenes expressed in specific cell types. Importantly, these mice reproduce the oncogenic cooperation previously demonstrated in in vitro cell culture systems. For example, INK4a/ARF{Delta}ex2,3 mice have a dramatically increased susceptibility to the oncogenic effects of Ras expressed in melanocytes (53,54), or to the EGF receptor expressed in glia precursors (55) (Table IIGo). Also, heterozygous INK4a/ARF{Delta}ex2,3 or ARF{Delta} mice in combination with a Myc-encoding transgene expressed in B-lymphocytes develop acute lymphomas with a very short latency (4143) (Table IIGo).

It is interesting to mention that INK4a/ARF haploinsufficiency may have strong effects on tumorigenesis (43,55). Remarkably, gliomas were produced equally efficiently in homozygous and heterozygous INK4a/ARF{Delta}ex2,3 mice expressing an activated EGF receptor in glia precursors (55). Since this is not a general effect in other tumor types, such as melanomas (53), it appears that different cell types have different sensitivities to changes in the genetic dose of INK4a/ARF.

Concluding remarks

The role of the INK4a/ARF locus in tumor suppression is now relatively well understood. The manipulation of the INK4a/ARF locus in the mouse has already yielded interesting and sophisticated tumor model systems. New manipulations of the INK4a/ARF locus will certainly provide a more clear picture, and will serve to generate additional tumor model systems.

Acknowledgments

The author would like to express his appreciation to David Beach (Cold Spring Harbor Laboratory) in whose laboratory the author made most of his contributions to this field. The laboratory of M.S. is supported by the Spanish Research Council, the Spanish Ministry of Education, the Regional Government of Madrid, the Human Frontier Science Program, and a core grant to the Department of Immunology and Oncology from the consortium between Pharmacia & Upjohn and the Spanish Research Council.

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Received January 31, 2000; accepted February 1, 2000.