SUMO and Cancer Caretakers and Gatekeepers

As seen from the previous account, there has been significant progress in our understanding of the role of sumoylation in a variety of biological processes.

In this part of the review we attempt to relate some of these findings to oncogenic and tumour-suppressor mechanisms, at the heart of which lies genomic and organismal integrity [16].

Tumourigenesis is strictly limited to complex organisms with renewable tissues that contain dividing or proliferation competent cells. Cancer can be simplistically defined as a case of aberrant cell hyperproliferation that develops in post-natal tissues and causes organismal disorder that, in most cases, leads to the death of the organism. Complex organisms have evolved specific tumour-suppressor mechanisms to curb uncontrolled cell proliferation. The molecules involved in these mechanisms can be broadly divided into caretakers and gatekeepers [57].

Caretaker tumour suppressors act predominantly on the genome, in principle, by preventing, sensing and repairing DNA damage. Gatekeeper tumour suppressors, on the other hand, act on cells by regulating and implementing apoptosis or cellular senescence. Apoptosis kills and ultimately eliminates cancerous cells, while cellular senescence irreversibly arrests cell growth and thus immobilizes cancerous cells (for further details see, for example, recent reviews on apoptosis and cellular senescence as tumour-suppressor mechanisms [16,20,22,67]). To date, several caretaker proteins have been validated as SUMO substrates, the RecQ-like DNA-dependent helicases Bloom (BLM) and Werner (WRN) [24, 56, 125], topoisomerases I (Topo-I) [69] and -II (Topo-II) [68], PARP, Ku80, TRAX and XRCC1 [34].

The Bloom gene is mutated in a rare hereditary disorder, Bloom syndrome (BS). BS cells are hypermutable, showing numerous chromatid gaps and breaks and many sister chromatid exchanges (SCEs) [25]. The BLM he-licase is a nuclear protein that is differentially regulated during the cell cycle and is distributed throughout the nucleoplasm as well as concentrated in PML nuclear bodies [10, 32]. One of the salient feature of the BLM helicase is its colocalization with proteins involved in DNA damage repair after treatment of cells with DNA damaging agents [122]. Within minutes after DNA damage, BLM starts to appear in foci harbouring yH2AX, RAD50 complex, RAD51, FANCD2 and BRCA1 [21, 29, 88]. Elegant experiments by Eladad et al. [24] provided evidence that sumoylation of BLM plays an essential role for intra-nuclear trafficking and the authors established SUMO modification as a negative regulator of BLM's function in the maintenance of genomic stability. Moreover, cells lacking functional PML show an increased number of sister-chromatid exchanges, most likely due to mislocalization of the BLM protein [129] and it is therefore tempting to speculate that PML nuclear bodies (NBs) act as a storage and modification site for sumoylated BLM.

Defects in WRN, another SUMO substrate [56,125], have also been linked to a hereditary disorder, Werner's syndrome (WS). WS shares several features with BS, most notably a high incidence of cancer. In addition, WS cells, like BS cells, are hypermutable. However, there are marked differences. WS individuals are asymptomatic before puberty, but thereafter develop a panoply of age-related disorders, including cardiovascular disease, cataracts, and osteoporosis. At the biochemical level WRN distinguishes itself from other RecQ-like helicases by possessing, apart from its helicase activity, also an N-terminal 3'-5' exonuclease activity. Several studies have linked WRN function to various DNA metabolic processes as for example replication, restoration of stalled replication forks, rDNA transaction mechanisms, homologous recombination and telomere maintenance [8, 83]. WRN is a nuclear protein that is located predominantly in the nucleolus in interphase cells. Upon DNA damage, however, it delocalizes into discrete DNA damage induced foci in the nucleoplasm [36, 70]. Its redistribution appears to be at least in part driven by p14ARF induced sumoylation [125] supporting the notion that WRN plays a crucial role in the cellular response to DNA damage in that its activity is modulated by DNA damage-induced post-translational modifications and possibly WRN-interacting proteins. Moreover, these findings imply that there is an intimate relationship between rDNA metabolism and home-ostasis of the cell, which is in part communicated by the modification status of WRN.

Topo-I and -II play essential roles during DNA replication, transcription, recombination and mitosis by relaxation of negatively and positively super-coiled DNA [121]. Topo-II exists in two isoforms a and p, the a isoform being the predominant in proliferating cells [110]. Whereas Topo-I transiently cleaves only a single strand of duplex DNA, Topo-II cleaves both DNA strands simultaneously. Topoisomerases are found in multi-protein complexes that include RP-A, BLM and the RAD51/DMC-I complex among others [79,73]. Several studies have demonstrated an increased topoisomerase inhibitor sensitivity of cells defective in DNA damage repair proteins, replication checkpoints, or both. For this reason topoisomerases now constitute major cellular targets for numerous anti-cancer drugs, e.g. Topo-I for camptothecin (CPT) and its analogues or Topo-II for VP-16 [63]. Recent results have implicated sumoylation in the regulation of Topo-I [46,49,78] and -II activity [4,5]. In the case of Topo-I, asimple model in which conjugation or de-conjugation of SUMO alters enzymatic activity cannot explain experimental outcomes. Rather, it appears that transient cycles of Topo-I sumoylation and de-sumoylation at different sites within the protein regulate the dynamic association with other protein complexes, thereby modulating the various Topo-dependent processes. Topo-II sumoylation has been shown to play critical roles in centromeric function and sister chromatid segregation during mitosis (mentioned in Sect. 2.1), as well as for the proteasome-dependent turnover of Topo-IIb [4,5,48].

For other proteins mentioned in the list above, no clear function has been established for their SUMO-modified forms. Given, however, the functional consequences of SUMO modification on other caretaker tumour suppressors, there is every reason to believe that, in these cases, SUMO will also play a critical role in altering their mode of action.

The ultimate tumour-suppressor gatekeepers in mammalian cells are p53 and the retinoblastoma protein pRB. Both proteins are instrumental in implementing an apoptosis and cellular senescence response as a result of aberrant proliferation, and their genes are found frequently mutated or inactivated in human cancer [103].

The p53 tumour suppressor—a transcription factor that establishes programmes for apoptosis, cellular senescence and repair in response to a variety of cellular insults—is subject to SUMO modification [74], particularly as a consequence of DNA damage [59]. Post-translational SUMO modification was shown to be regulated by MDM2 (murine double minute 2) and p14ARF, two pivotal upstream regulators of p53 stability [17]. MDM2 and its sibling MDMX are themselves subject to sumoylation, but the functional importance of their modification remains unclear. One functional consequence of p53 sumoylation appears to be the modulation of p53 transcriptional activity, but this remains a matter of debate [35,80,93,96]. It also remains an open question as to what extent and in what cellular context(s) sumoylation is important for proper p53 function. Several scenarios are possible. For example, a restricted local modification may alter a specific function of a p53 subset or the entire pool of p53 may undergo transient sumoylation, in both cases to control, for example, p53 residence time in specific protein/protein or protein/DNA complexes. Of further interest is under which physiological conditions (apart from DNA damage) p53 becomes sumoylated and whether sumoylated p53 accumulates during apoptosis or cellular senescence, as has been shown for other post-translational modifications [85, 120]. In this context, work from our laboratory [131] has recently shown that the SUMO E3 ligase PIASy induces premature senescence, and further, that sumoylated p53 plays a role in the execution of this senescence programme.

Identified as the first tumour-suppressor protein, pRB has since been shown to be a master regulator for cellular senescence [14]. Its functionality is predominantly controlled by way of post-translational modifications, in particular phosphorylation. Hypophosphorylated pRB corresponds here with its active state, whereas hyperphosphorylation renders pRB inactive. Recently, also sumoylation of pRB has found its way into spotlight. Ledl et al. [60] identified SUMO modification as a negative regulator of pRB activity. It remains to be seen, however, to what extent sumoylated pRB is found under physiological conditions and what role pRB-SUMO plays in these conditions.

An important mediator for p53 and pRB function is PML [11]. The PML gene was initially identified in patients with acute promyelocytic leukaemia (APL) in which it is fused to the retinoic acid receptor a (RARa) gene as a result of the t(15;17) chromosomal translocation [75]. One of the main features of PML is its concentration within discrete subnuclear structures, termed PML NBs, which are disrupted in a retinoic acid-reversible manner in APL cells [75]. PML was also found among the first proteins subject to sumoylation, and PML modification was shown to be required for proper formation of NBs, recruitment of NB-associated proteins [98] and the expression of the full-blown leukaemogenic potential of the PML-RARa fusion protein [130]. Moreover, PML was implicated in telomere maintenance of cancer cells exhibiting the ALT phenotype (alternative way of telomere lengthening via homologous recombination) [91], where it was found to co-localize with telomeres in so-called ALT-associated promyelocytic leukaemia bodies (APBs). Disruption of this interaction was recently demonstrated by over-expression of a permanent PML NB resident SP100 in ALT cells, leading to repression of ALT-mediated lengthening of telomeres in these cells [50]. Given that both PML and SP100 are important SUMO targets, it would be interesting to see whether sumoylation also plays a significant role in APB function.

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