Unraveling Human Tumor Suppressor Pathways

[Cell Cycle 3:5, 616-620; May 2004]; ©2004 Landes Bioscience

Unraveling Human Tumor Suppressor Pathways Spotlight on Human Cell Transformation

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A Tale of the INK4A Locus

Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=859

KEY WORDS

TELOMERASE ACTIVITY

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p14ARF, p16INK4A, p53, p19arf, telomerase, neoplastic transformation, RAS, oncogene, tumor suppressor, RNAi

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Received 03/12/04; Accepted 03/15/04

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*Correspondence to: Reuven Agami; Division of Tumor Biology; The Netherlands Cancer Institute; Plesmanlaan 121; Amsterdam 1066 CX The Netherlands; Tel.: +31.20.512.2079; Fax: +31.20.512.2029; Email: [email protected]

Research on tumor suppressors has for a long time run on two tracks: analysis of the mutations found in human tumor material, and active genetic manipulation in mice. As primary human cells were not easily amenable to genetic alterations, the proof to designate a suspected gene as a tumor suppressor was often by generation of knockout mice and analysis of their phenotypes. In this way, a vast amount of information has been gathered on the actions of major players in carcinogenesis. However, it has recently become apparent that there are major differences in the requirements for oncogenic transformation between human and mouse cells. Among these are the expression of hTERT, SV40 small t, and the response to Ras induced growth arrest by the tumor suppressor pathways involving p53, pRb and the INK4A locus. The potential contribution of these tumor suppressors to the prevention of transformation of human cells can now begin to be unraveled by the recent emergence of novel RNA interference genetic tools.

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Division of Tumor Biology; The Netherlands Cancer Institute; Amsterdam, The Netherlands

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ABSTRACT

P. Mathijs Voorhoeve Reuven Agami*

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Primary human fibroblasts are limited in their lifespan by telomere degeneration which results from the inability to faithfully replicate the end of telomeres during each cell division.1 Mouse (but not human) somatic cells express high levels of a telomerase enzyme complex that repairs the telomere ends and thus protect these cells from telomere attrition which triggers a growth arrest. Most human tumors also contain high levels of telomerase activity, mainly by derepressing the expression of hTERT, the catalytic subunit of the telomerase complex.2 The cloning of hTERT opened the way to systematically investigate the steps which are required to transform primary human cells into cells capable of growing as tumors in vivo. Human oncogenes such as constitutively active RAS (e.g., H-RasV12, referred to as Ras in this review) and viral oncogenes, such as SV40 Large T antigen (LT) which simultaneously inactivates the tumor suppressors p53 and pRb, cooperate in transformation assays of mouse primary cells. Based on this knowledge, Hahn et al. showed that various human primary cells could be transformed by combined expression of hTERT, HRasV12 and the SV40 genomic region that expresses LT.3

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SMALL T EXPRESSION Serendipity revealed that, in addition to the expression of hTERT, RasV12 and SV40 LT, expression of an additional protein is required for transformation of human cells. This protein, SV40 small t, was fortunately also expressed from the SV40 early region used in the initial studies, and its expression was subsequently shown to be required for anchorage independent growth of human but not mouse cells.4 Small t can bind to and compromise the function of a collection of cellular phosphatase complexes which are known under the name of PP2A. A PP2A complex comprises a constant core dimer which consists of a catalytic C and a structural A subunit, complemented by one of more than a dozen of different regulatory B subunits. These B subunits are thought to direct the substrate specificity of PP2A, and small t exerts its transforming activity by displacing several of these subunits from their respective complexes. Depletion of a particular B subunit (B56γ) by antisense or short interfering RNA (siRNA) can replace expression of small t, suggesting that the main transforming effect of small t is not exerted through relocalization of PP2A to new substrates but by prevention of dephosphorylation of PP2A substrates.5 PP2A-B56 has been reported to bind to the tumor suppressor APC and the p53 target gene cyclin G5-8 but it is unclear whether these substrates are functionally related to the actions of small t. However, as inhibition of expression of this one subunit resulted in a 40% reduction of Cell Cycle

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UNRAVELING HUMAN TUMOR SUPPRESSOR PATHWAYS

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Expression of oncogenic RAS induces a stress response in both mouse and human primary fibroblasts that results in growth arrest and a flat cell phenotype. As mutant forms of RAS are found in a large fraction of human tumors, considerable interest exists in this anti-oncogenic response and in the tumor suppressors that are critical for its function. This protective response depends in mouse cells on stabilization of p53 as a direct result of inhibition of mdm2 activity which normally targets p53 for degradation. Mdm2 is inhibited by p19arf, a protein that shares its second exon (in an alternative reading frame) with p16ink4a which is an activator of the retinoblastoma pathway. p19arf and p16ink4a are encoded by the ink4a locus in a quite unique genomic configuration of two genes that activate two different tumor suppressors, p53 and pRb respectively.11 The fact that this locus is often found silenced or mutated in tumors12 is therefore not entirely surprising, as both the p53 and the Rb pathways are found to be directly or indirectly compromised in the vast majority of tumors. However, the data derived from mouse knockout studies and detailed analysis of the INK4A locus in human tumors regarding the relative contribution of p19arf and p16ink4a to tumor suppression is conflicting. In mice, p19arf is clearly the major tumor suppressor, with only minor consequences of deletion of p16ink4a for tumor induction.13,14 In contrast, analysis of more subtle disruptions of the INK4A locus reveal point mutations or small deletions which specifically affect p16INK4A but leave p14ARF (the human homologue of mouse p19arf) functional, indicating that in human tumors p16INK4A is most likely the major target.15 Also studies in cultured cells show major differences between man and mouse in the contribution of the two ink4a encoded proteins. p19arf is transcriptionally upregulated after expression of Ras, and p53-/- or p19arf-/- MEFs are refractory to the growth arrest induced by Ras. Furthermore, there is little evidence for selection against loss of p16ink4a,16 despite the fact that overexpression of Ras induces p16ink4a expression.17 Also, MEFs that still express p19arf but lack p16INK4A expression are still responsive to Ras induced arrest18 p16ink4a does seem to play a role in later progression of tumorigenesis, as lymphomas from mice that lack p16ink4a are resistant to chemotherapy, whereas lymphomas expressing p16ink4a exhibited a senescent phenotype.19 The dominant role of p19arf in MEFs in response to Ras expression is not reflected in human fibroblasts. p14ARF transcription is not induced by RAS,20,21 and the subsequent stabilization of p53 is also not a general rule in human fibroblasts.21,22 This does not exclude however the possibility that p14ARF still plays a critical role in protecting human cells from RAS transformation like it does in mouse cells. p16INK4A is induced by RAS in human cells as it is in mouse cells. p16INK4A is also gradually induced when human cells are cultured for prolonged periods, eventually leading to cell cycle arrest and so-called senescence.23 Therefore RAS induced growth arrest is often called premature senescence, although it is unclear whether these two forms of growth arrest are executed by the same players.

Investigation of the pathways involved in the response to RAS expression in human cells has until recently depended on the expression of dominant viral or mutant oncogenes, the time consuming in vitro generation of knockout cells, or the use of cells from patients that carry mutations affecting p16INK4A but not p14ARF function. That the conclusions from these studies seem to conflict is not surprising given the diverse nature of the cells and methods used. Several studies suggest that in human fibroblasts not only the p53 pathway but in addition also the pRb pathway needs to be inhibited to allow growth in the presence of oncogenic Ras. Expression of dominant negative p53 alone was not enough to protect IMR90 fibroblasts from RAS induced growth arrest.17 Both the inactivation of pRb and p53 were required for full transformation of BJ fibroblasts expressing hTERT, oncogenic RAS and small t, as was shown by using (mutants of ) viral oncogenes that inactivate pRb and p53 independently.4 LF1 fibroblasts whose p53 genes had been disrupted by homologous recombination showed a premature senescence phenotype in response to RAS expression20 and failed to grow anchorage independently unless p16INK4A function was inhibited.24 Although convincing, these data stand in contrast with studies that seem to stress the dominance of the p16INK4A response in protecting human cells from RAS induced transformation. Two different fibroblasts strains have been isolated from patients that carry either a homozygous deletion in exon 2 of p16INK4A, or two point mutations that disable p16INK4A function. By all available methods the p14ARF protein derived from these cells seems to function at least as well as wild type.21,25 These “natural p16INK4A knockout” cells express wild type p53, but are surprisingly insensitive to RAS induced growth arrest. Although p53 is still stabilized and activated in response to DNA damage, it does not seem to be involved in the arrest of these cells in response to RAS induced stress. Moreover, when transformed with oncogenic Ras, these p16INK4A null cells can also grow anchorage independently and form tumors in nude mice without the requirement for small t expression. However, the authors themselves mentioned that these cells need to be grown for a relatively long latency period before tumors arise, suggesting that an additional genetic change is required. It would therefore be interesting to see if the tumors that arise have activated PI3-Kinase compared to cells that are freshly transformed, explaining the apparent lack of requirement for small t in this system.10 Alternatively, expression of small t should considerably decrease the lag period before tumors are formed.

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THE GROWTH ARREST INDUCED BY ONCOGENIC RAS

DECONSTRUCTING THE RESPONSE TO RAS IN HUMAN CELLS

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total cellular PP2A activity,5 it cannot be excluded that this subunit is required for the activity of several PP2A complexes containing distinct B subunits and thereby indirectly affecting multiple PP2A substrates. This alternative explanation is consistent with the observation that small t displaces more B subunits than just the B56γ. The search for the relevant PP2A substrates will be greatly helped by the discovery that expression of small t can also be functionally replaced by expression of constitutive active variants of either PI3-Kinase or two of its downstream effectors, Rac1/cdc42 and Akt1.9,10

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KNOCKING DOWN … Another level of complexity is added by our data, where we use stable siRNA expressing retroviruses to knock down expression of putative tumor suppressors in polyclonal isogenic strains of telomerase expressing human fibroblasts.22 We show that knockdown of p53 together with either p16INK4A or pRb in combination with small t and RAS expression gave rise to fully transformed cells. However, the RAS induced growth arrest was solely dependent on expression of p53. In contrast to the studies described above, cells with an intact Rb pathway continued to grow in the presence of Ras, although they failed to give rise to cells that could grow anchorage independently, even in de presence of small t expression. We speculate that this contradiction to previously observed data can be explained by taking into account the relatively young BJ fibroblasts which do not yet express high levels of p16INK4A, and the rapid method that we used. Indeed, expression of viral oncogenes or lengthy selections to obtain

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Figure 1. A model for the ARF-mdm2-p53 pathway in mouse and human fibroblasts. (1) During normal growth, p53 activity is kept in check by mdm2, which in its turn is restrained by ARF. (2) After DNA damage, p53 becomes refractory to mdm2 activity, and p53 levels and transcriptional activity increase dramatically. (3) Loss of ARF results in enhanced mdm2 activity (bold arrow), and p53 levels drop. (4) Oncogenic stress such as RasV12 expression results in activation of p53 in mouse and human cells. In mouse, p19arf and mdm2 are both activated by Ras, resulting in a net decrease of mdm2 activity. in human cells, p14ARF expression is not up regulated, and p53 levels do not increase significantly. In both cases p53 transcriptional activity is also activated by various modifications which are indirectly stimulated by Ras (green arrows). (5) Oncogenic stress in cells that have lost the ARF gene has different outcomes in mouse and human cells. In mouse cells, mdm2 activity is stimulated by Ras but not inhibited by ARF anymore, resulting in degradation of p53. In human cells, ras protects p53 from elevated hdm2 activity caused by loss of ARF (purple blocking arrow). Protection of p53 could in principle be through modification of p53, Hdm2, or through modulation of their interaction. This protection is either absent in mouse cells, or to weak to counteract the elevated mdm2 levels (question mark).

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homozygously deleted null mutants will increase the basal expression levels of p16INK4A,20 which upon further induction by RAS may rise above a threshold level that prohibits cell growth. Intriguingly, cessation of p16INK4A or pRb expression was required to allow our cells to grow in soft agar, indicating that under those conditions either p16INK4A levels are increased further and become limiting, or that the threshold for inhibition of cdks by p16INK4A is lowered, for example by reduction of expression of cyclins that regulate pRb phosphorylation.26 The continued requirement of small t expression indicates that its expression is not required to counteract pleiotropic effects of viral oncogene expression, as suggested by some.21

… THE INK4A LOCUS Encouraged by these results we set out to investigate the relative contribution of p14ARF and p16INK4A in protection from transformation. Although p14ARF was undetectable by Westernblot, knockdown of p14ARF resulted in an increased rate of cell growth in vitro of these primary fibroblasts, which was dependent on p53 expression. This shows that the p14ARF protein in these cells activates p53 to restrict growth. As loss of p53 hardly gave any additional growth advantage in cells that lack p14ARF expression, we conclude that almost all p53 618

in these cells is under control of p14ARF. Indeed, p53 protein levels and transcriptional activity, as apparent from p21 levels, were much reduced in p14ARF knockdown cells, consistent with an expected increase in hdm2 activity. Knock down of p16INK4A expression did not result in a growth advantage, consistent with the idea that the p16INK4A levels in these cells were not growth limiting yet. Interestingly, loss of p16INK4A expression did give a remarkable growth advantage in cells which had reduced p53 levels by knockdown of either p14ARF or p53. Conversely, expression of siRNA’s against p14ARF or p53 had a much more potent growth accelerating effect in cells that had reduced p16INK4A protein levels compared to wild type cells. If this in vitro growth acceleration is somehow reflected in the in vivo situation, this would mean that loss of the entire INK4A locus (both p14ARF and p16INK4A) would give cells a dramatic increase in growth speed compared to cells that lose only p16INK4A expression through a more subtle perturbation of the INK4A locus. The observed synergistic effect of loss of p16INK4A and p53 may also sheds some light on the discrepancy between our work, that of others4,17 and the reported lack of involvement of p53 in RAS induced growth arrest of cells carrying naturally occurring mutations in p16INK4A.21,27 As the latter cells have lacked p16INK4A expression

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IF NOT P14ARF, WHAT?

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Whatever the selective benefit of p14ARF loss on growth speed of untransformed cells may be, the expression of oncogenic RAS in primary cells showed that human p14ARF and p53 are not as functionally interchangeable as mouse p19arf and p53 are p14ARF depleted cells still stopped growing in response to RAS expression, and did not give rise to anchorage independent transformed cells when combined with p16INK4A or pRb siRNA vectors. Indeed, expression of p53 was reinstated to wild type levels in p14ARF cells upon expression of Ras. This rise in p53 levels was exclusive to p14ARF depleted cells, as expression of RAS did not elevate p53 levels in control cells, as also others have found.21 The simplest explanation for this observation is that BJ cells normally have negligible Hdm2 activity which only becomes noticeable upon inhibition of p14ARF and which is then somehow inhibited by RAS in an p14ARF independent way. This is however unlikely, as knockdown of Hdm2 in human fibroblasts resulted in an increase of p53 levels comparable to those elicited by gamma irradiation, indicating that Hdm2 is active in these cells (Voorhoeve M, Drost J; unpublished observation). Therefore, RAS expression only protects p53 against the elevated levels of Hdm2 that result from p14ARF depletion, but does not protect p53 from the regular activity of Hdm2 that keeps p53 levels in check. Only upon induction of, for instance, DNA damage does p53 become totally refractory to Hdm2 activity, resulting in high p53 levels and immediate growth arrest or apoptosis (Fig. 1). In this respect it is interesting to note that the RAS induced growth arrest typically takes days to a week to take hold,22 whereas DNA damage induced p53 effects reach their first peak at six hours.29 Further support for the notion that the stabilization of p53 after DNA damage is qualitatively different from its stabilization by endogenous levels of p14ARF comes from the observation that an HPV E6 mutant, which does not degrade p53 (like wild type E6) but still inhibits its activation by p14ARF, is not able to prevent the activation of p53 by actinomycin D.30 Interestingly, different levels of Mdm2 have recently been implicated in either monoubiquitination or polyubiquitination of p53, resulting in respectively redistribution of p53 or proteasome dependent degradation.31 At present it is unclear whether this selective resistance of p53 to mdm2 is mediated through a RAS induced modification of p53 or whether RAS qualitatively affects hdm2 activity. A third possibility is that the activity of hdm2 can be counteracted by a deubiquitinating enzyme, whose activity could be enhanced after RAS expression. Irrespective of the means by which RAS protects p53 from elevated hdm2 levels, this protection does not seem to play a direct role in the p53 dependent growth arrest that is elicited by RAS expression. Although

p53 levels do not increase like they do in mouse cells after RAS expression, p53 target genes such as p21 are induced, and depletion of p53 suffices to absolve the cells from the RAS induced growth arrest. Somehow RAS increases the transcriptional activity of p53 without changing its stability. p53 can be modified at various residues in distinct ways such as phosphorylation, acetylation and ubiquitylation, resulting in changes in its stability and activity as a transcription factor. It is therefore not unlikely that RAS induces one or more specific modifications on p53 to initiate the growth arrest. It is therefore conceivable to assume that genetic lesions that compromise the activation of p53 by RAS would cause evasion from the cellular protection mechanism against oncogenic transformation. For instance, acetylation of p53 by CBP has been linked to PML, a protein which is induced by oncogenic Ras. PML-/- MEFs have a reduced growth arrest in response to oncogenic Ras, suggesting that one of the ways in which RAS activates p53 may be through activation of PML.32 The stabilization of p53 in p14ARF depleted cells upon expression of Ras could be a side effect of an activating modification, or the result of a process that is independent of such a modification (Fig. 1, purple arrow). Because of the dominance of the p19arf pathway in murine cells, additional pathways that are involved in activation of p53 may have received relatively too little attention. These may, in contrast, play a more significant role in tumor suppression in humans, as inhibition of p14ARF expression is not sufficient to render cells functionally defective in p53.

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during their entire in vitro life span, and still express a form of p14ARF that may even be more active than wild type,28 they may thus have been under increased selective pressure to acquire a compensatory attenuation of the p53 pathway. Although the authors try to assess the functionality of p53 by showing that the p53 response to DNA damage is still intact, this does not show that the response of these cells to oncogenic RAS is completely functional. If the p53 pathway is indeed attenuated in these cells, this would predict that reduction of p53 by siRNA would not enhance their resistance to RAS induced growth arrest. An alternative way to test if their protocol is fundamentally different than that used by others would be to inhibit by RNAi the expression of p16INK4A in their control cells and examine whether these become resistant to RAS induced growth arrest.

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p14ARF: SUPPRESSOR OF TUMORIGENESIS?

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Does this bury then the aspirations of p14ARF as a tumor suppressor in humans? Not entirely. First, we cannot exclude that in other cell types p14ARF has a more essential role in activation of p53 by RAS than it has in the fibroblasts used in our studies. This should be readily testable as the siRNA technology can be easily used in another cell type. However, even in fibroblasts loss of p14ARF and p16INK4A simultaneously will give cells an additional growth advantage compared to cells that have lost p16INK4A expression alone. Therefore, loss of p14ARF could increase the pool of cells that are defective in p16INK4A function, an event that clearly is a step down the road to transformation. Furthermore, loss of p14ARF and the subsequent drop in p53 levels may render cells more genetically unstable. Indeed, expression of a p14ARF siRNA vector made BJ fibroblasts more resistant to DNA damage, although to a somewhat lesser extend than direct inhibition of p53 did (van Leeuwen B, Agami R; unpublished data). Lastly, lesions that compromise the RAS induced protection of p53 from elevated levels of Hdm2 could have minor consequences for the activation of p53 in wild type cells, but result in a loss of p53 phenotype in p14ARF null cells. p14ARF could then be regarded as a conditional tumor suppressor, which enhances the effects of other genetic lesions. Cells which have lost the entire INK4A locus could therefore acquire full transformation not only by loss of p53, but also by mutations in a number of p53 activating pathways. Combined with an enhanced growth rate and an increased genomic instability, loss of p14ARF in this setting would indeed stack the odds for enhanced tumor development. The development of a controlled transformation protocol using human primary cells and the possibility to inactivate large numbers of potential tumor suppressors with siRNA libraries should allow the discovery of any additional pathways involved in the protection of cells from oncogenic transformation.

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