CeLL CyCLe news & views CeLL CyCLe news & views Cell Cycle 11:8, 1485-1489; April 15, 2012; © 2012 Landes Bioscience Cell Cycle News & Views Length matters: C-terminal tails regulate Mdm2–MdmX complexes Comment on: Dolezelova P, et al. Cell Cycle 2012; 11:953–62; PMID:22333590; http://dx.doi.org/10.4161/cc.11.5.19445 Masha V. Poyurovsky; Kadmon Research Institute; New York, NY USA; Email: [email protected]; http://dx.doi.org/10.4161/cc.20047 Mechanisms controlling the p53 regulatory a p53-dependent embryonic lethality. These Dolezelova et al. present unexpected network remain the focus of numerous inves- data implicate the RinG domain of MdmX as experimental evidence for the heterocomplex tigations in hopes of identifying more robust the sole region of importance in the ability of being structurally and functionally distinct cancer therapies. Both Mdm2 and MdmX are MdmX to regulate p53 and, by extension, the from the Mdm2 homodimer, while providing found overexpressed in tumors with wild-type Mdm2-MdmX complex (and not the Mdm2 a mechanism for the observed in vivo func- p53 and represent a key molecular device homodimer), as the principle negative regula- tional differences between the complexes. modulating p53 function. Thus, examining tor of p53 activity during development. Although the work casts slight doubt on the the interplay between these three proteins The growing body of evidence describ- complete accuracy of the existing structures, becomes highly relevant in the search for new ing the presence of MdmX in the complex it nicely aligns with the above-mentioned pharmacological interventions in oncology. as crucial for target selectivity as well as the results, showing the singular importance of Mdm2 is a RinG-type e3 ubiquitin ligase processivity of the holoezyme somewhat flies the MdmX RinG domain in the activity of the capable of forming homo-oligomers and in the face of the existing structural data. Two holoenzyme. in light of these results, addi- hetero-oligomerization with MdmX via the published structures of the Mdm2 homodimer tional structural studies that will take in to © 2012 Landes Bioscience. extreme C termini of their RinG domains. since and Mdm2/MdmX heterodimer indicate virtu- account reported differences between the its discovery 15 years ago, MdmX has been ally no difference in the complexes.7,8 in the complexes will undoubtedly be informative assigned many roles in the regulation of p53, absence of structural differences, how then and contribute to our understanding of the either on its own or in concert with MdDm2. oar e nsucho sigtnif icdant idisfferetncres iinb funuctiotn ebio.chemistry of RinG-type ubiquitin ligases while clearly an essential negative regulator or accomplished? and the mechanisms regulating p53 in cells. p53 in development, its lack of intrinsic ubiqui- A hypothesis unifying structural and func- tin ligase activity has made the mechanism of tional data is brought forth by a very intriguing References p53 regulation more elusive than in the case study from the Uldrijan group, which sys- 1. Linares LK, et al. Proc natl Acad sci UsA 2003; 100:12009-14; PMiD:14507994; http://dx.doi. of Mdm2. The capacity of MdmX to stimulate tematically looks at the differences between org/10.1073/pnas.2030930100. Mdm2-mediated p53 ubiquitination was first complex formation and activity of Mdm2 and 2. Okamoto K, et al. FeBs Lett 2009; 583:2710-4; PMiD:19619542; http://dx.doi.org/10.1016/j.febs- reported in 2003.1 subsequent biochemical MdmX.9 Phylogenetic analysis showed that the let.2009.07.021. comparisons of the activity of Mdm2–MdmX last cystein of the RinG domain is followed by 3. Kawai H, et al. Cancer Res 2007; 67:6026-30; complexes showed that not only does the exactly 13 amino acids in all Mdm orthologs PMiD:17616658; http://dx.doi.org/10.1158/0008- 5472.CAn-07-1313. presence of MdmX in the complex alter the of vertebrate origin. Based on this, the authors 4. wang X, et al. J Biol Chem 2011; 286:23725-34; substrate specificity of the holo-enzyme, it hypothesized that not only the sequence of PMiD:21572037; http://dx.doi.org/10.1074/jbc. also allows for poly-ubiquitin chain forma- the C-terminal tails, but also their exact length M110.213868. 5. Huang L, et al. Proc natl Acad sci UsA 2011; tion on p53 (modification required for nuclear are of central importance to the function of 108:12001-6; PMiD:21730163; http://dx.doi. exclusion and degradation of p53).2-4 the complexes. subsequent investigation org/10.103/pnas.1102309108. in vitro observations describing the of the ability of Mdm2 and MdmX proteins, 6. Pant v, et al. Proc natl Acad sci UsA 2011; 108:11995- 2000; PMiD:21730132; http://dx.doi.org/10.1073/ importance of the MdmX RinG domain in which have been extended at the C terminus pnas.1102241108. regulation of p53 turnover have now gained by 5, 14 or 18 amino acids, was designed 7. Kostic M, et al. J Mol Biol 2006; 363:433-50; in vivo experimental support from the two to test the importance of the length of the PMiD:16965791; http://dx.doi.org/10.1016/j. jmb.2006.08.027. knock-in animal models.5,6 Consistent with the C-terminal extensions. To the researchers sur- 8. Linke K, et al. Cell Death Differ 2008; 15:841-8; notion that MdmX is an essential component prise, when examined based on their ability PMiD:18219319; http://dx.doi.org/10.1038/ sj.cdd.4402309. of p53 polyubiquitination/proteasomal deg- to hetero-oligomerize and ubiquitinate p53, 9. Dolezelova P, et al. Cell Cycle 2012; 11:953-62. radation pathway, mice expressing either a Mdm2 proteins behaved differently depend- PMiD:22333590; http://dx.doi.org/10.4161/ point mutant in the MdmX RinG domain or a ing on whether the oligomeric partner was cc.11.5.19445. RinG domain deletion mutant succumbed to Mdm2 or MdmX. www.landesbioscience.com Cell Cycle 1485 pRb or its cousins: Who controls the family business? Comment on: Bazarov AV, et al. Cell Cycle 2012; 11:1008–13; PMID:22333593; http://dx.doi.org/10.4161/cc.11.5.19492 Koji Itahana1 and Goberdhan P. Dimri2,*; 1Duke-NUS Graduate Medical School; Singapore; 2George Washington University Medical Center; Washington, DC USA; *Email: [email protected]; http://dx.doi.org/10.4161/cc.20048 More than 90% of human cancers are of epithe- that knockdown of each of the three pRb of human fibroblasts,4 and explain why pRb, lial origin. Cellular senescence of human mam- family proteins individually did not abrogate but not p107 or p130, is frequently mutated in mary epithelial cells (HMeCs) is an important senescence mediated by ectopically expressed cancer. interestingly, although abrogation of barrier that protects cells from immortalization; p16 in the breast cancer cell lines MDA-MB-231 pRb is critical for HMeCs escaping senescence, the first step in breast cancer development.1 and MCF7. However, the senescence induced simultaneous depletion of pRb together with Although induction of tumor suppressor p16 by ectopic p16 was abrogated if they intro- either p107, p130 or both accelerates bypass of is not evident in some types of normal human duced e7, which inactivates all three pRb fam- senescence. This suggests that p107 and p130 fibroblasts undergoing senescence,2 in cul- ily proteins. Their data suggest that two of help pRb to trigger/maintain HMeC senes- tured HMeCs, senescence occurs by a robust pRb family proteins can compensate for the cence in culture and possibly in vivo. Although p16 induction, and cells that acquire silencing loss of each pRb family protein to induce each pRb family protein preferentially binds to of p16Ink4a locus eventually proliferate and p16-mediated senescence in these cancer different members of the e2F family,5 the con- undergo senescence again by telomere short- cells. The remaining question is whether all tribution of each e2F family protein in escap- ening in a p53-dependent manner.1 Therefore, three pRb family members play an additive ing p16-mediated senescence remains unclear. p16 induction is a critical barrier to immortalize role, and whether the inactivation of at least Therefore, it will be interesting to see whether HMeCs in culture. p16 inhibits kinase activity two members of the pRb family is required to the critical role of pRb, and a supportive role of © 2012 Landes Bioscience. of Cdk4/6-cyclinD complexes, which inactivate overcome p16-induced senescence in breast p130 and p107, in p16-mediated HMeC senes- three pRb family proteins: pRb, p107 and p130. cancer cells. On the other hand, they showed cence depend on how each pRb family protein However, the relative contribution of these that abrogation of pRb, but not of p107 and/or interacts with an e2F family protein. three pRb family proteins to HMeC senesceDnce op1 3n0, atotenutate s dseneiscsencte irn HiMbeCsu, sugt- e.Bazarov et al. also showed that even aggres- is not well understood. gesting a non-redundant critical role of pRb in sive p53-negative breast cancer cells undergo in a recent issue of Cell Cycle, Bazarov et al. HMeC senescence. These data are consistent cellular senescence upon ectopic p16 expres- examined the role of each pRb family protein with a recent report demonstrating that pRb sion. These results are quite encouraging from in p16-mediated senescence in breast cancer has a non-redundant role in repressing DnA an epigenetic therapy point of view. silencing cell lines and in HMeCs (Fig. 1).3 They showed replication during H-ras-induced senescence of p16 often occurs in breast cancer cells via promoter methylation. During DnA replica- tion, cells require new p16 promoter methyla- tion to keep p16 silenced. The observations of Bazarov et al. suggest that we may be able to stop the growth of even aggressive p53-nega- tive breast cancers in patients by inducing p16 expression in cancer cells using DnA methyla- tion inhibitors. Back to the question of running family business: “it appears that pRb is still the boss, but in some cases, it may get a helping hand from his cousins- p107 and p130.” References 1. stampfer MR, et al. Cancer Lett 2003; 194:199-208; PMiD:12757978; http://dx.doi.org/10.1016/s0304- 3835(02)00707-3. 2. itahana K, et al. Mol Cell Biol 2003; 23:389-401; Figure 1. Contribution of pRb family proteins to p16-mediated senescence in breast cancer cells PMiD:12482990; http://dx.doi.org/10.1128/ MCB.23.1.389-401.2003. and HMeCs. Knockdown of each of the three pRb family proteins in breast cancer cells does not 3. Bazarov Av, et al. Cell Cycle 2012; 11:1008-13; abrogate ectopic p16-induced senescence, suggesting that either two of pRb family proteins can PMiD:22333593; http://dx.doi.org/10.4161/ compensate for the loss of each pRb family proteins or all three of pRb family proteins play an cc.11.5.19492. additive role in p16-mediated senescence in breast cancer cells. On the other hand, knockdown of 4. Chicas A, et al. Cancer Cell 2010; 17:376-87; pRb, but not of p107 or p130, abrogates HMeC senescence, suggesting a non-redundant critical role PMiD:20385362; http://dx.doi.org/10.1016/j. for pRb in senescence of HMeCs. However, the knockdown of either p107 or p130, in conjunction ccr.2010.01.023. with pRb depletion, abrogates HMeC senescence more efficiently than pRb knockdown alone. This 5. Classon M, et al. nat Rev Cancer 2002; 2:910-7; PMiD:12459729; http://dx.doi.org/10.1038/nrc950. suggests a supporting role for p107 and p130 in maintaining HMeC senescence. 1486 Cell Cycle volume 11 issue 8 Milking the stroma in triple-negative breast cancer Comment on: Witkiewicz AK, et al. Cell Cycle 2012; 11: 1108–1117; PMID:22313602; http://dx.doi.org/10.4161/cc.11.6.19530 Alastair M. Thompson1,* and Timothy J. Newman2; 1Dundee Cancer Centre; 2College of Life Sciences; University of Dundee; Dundee, UK; *Email: [email protected]; http://dx.doi.org/10.4161/cc.20049 investment in the post-genomic molecular in the stromal infrastructure of the breast. This challenges the cancer treatment paradigm. dissection of breast cancer has resulted in an supports the concept of epithelial malignant The long-established antidiabetes biguanide emphasis on prognostic and predictive mark- changes consequent with ecological and evo- drugs offer a low-toxicity opportunity to dis- ers, signatures derived to stratify the disease lutionary opportunity.4 rupt the reverse warburg effect. Metformin and the drive to generate targeted therapies. The “parasitic” character of tumor cells may target the cancer mitochondria3 and However, there remain significant challenges feeding off stromal cells highlights the need phenformin induce stromal sclerosis, at least in to individualize therapeutic targeting and to seriously consider both ecological and bio- a breast cancer xenograft model,8 in addition improve the prognosis for the thousands of physical concepts.5 we need to think beyond to in vivo AMPK pathway and insulin-mediated women who die each year from the heteroge- “intraspecific” competition among clonal sub- systemic effects of metformin in women with neous range of breast cancers. This is particu- populations in the tumor and to consider breast cancer.9 larly true for poor prognosis “triple-negative” tumor and stromal cells as distinct popula- The reverse warburg effect challenges our breast cancers (TnBC), most prevalent in tions in a cancer ecosystem, with a range of therapeutic focus on breast cancer epithelium. young and African American women, lacking “interspecific” competitive, exploitative and stromal MCT4 expression with caveolin-1 loss the established therapeutic targets of estro- opportunistic interactions. Furthermore, the identifies poor prognostic TnBC patients and gen receptor, progesterone receptor or HeR2. reverse warburg effect relies on the inefficient emphasizes the roles of the tumor microenvi- © 2012 Landes Bioscience. Research has largely focused on the epi- diffusion of nutrients from stromal cells to ronment and ecological interactions between thelial component of breast cancer rather than tumor cells in a complex three-dimensional distinct populations of cells. The challenges the tumor microenvironment, now recognized space. The extracellular space is brought to now revolve around therapeutic manipula- as a key hallmark of cancer.1 in vitro, aniDmal oth e nforeogroutnd , dand ipshysitcalr pirobpertiues otf etio.n of the stroma/epithelial interaction and models and observations on clinical material2 molecular transport in this space may have the extracellular space, and testing these con- are now moving to consider physiological as much impact on tumor growth as intricate cepts in pre-invasive and metastatic settings mechanisms by which stromal cells may influ- cellular processes. where stromal changes may provide tissue ence breast epithelial and carcinoma cells. The importance of the spatial arena is niches of evolutionary opportunity for malig- witkiewicz et al.3 build on published evi- also apparent when contrasting the reverse nant cells. dence from the Lisanti group that cancer cells warburg effect with angiogenesis. in the for- secrete hydrogen peroxide, initiating oxidative mer, tumor cells are exploiting their local envi- References stress and aerobic glycolysis in tumor stroma, ronment, which will presumably be of limited 1. Hanrahan D, et al. Cell 2011; 144:5646-74 2. Place Ae, et al. Breast Cancer Res 2011; 13:227; http// with L-lactate secretion from cancer-associ- yield, whereas angiogenesis taps the nutrients breast-cancer-research.com/content/13/6/227; ated fibroblasts fueling oxidative mitochon- of the entire organism— an effectively infinite PMiD:22078026; http://dx.doi.org/10.1186/ bcr2912. drial metabolism in epithelial cancer cells: the reservoir for a growing tumor. in the reverse 3. witkiewicz AK, et al. Cell Cycle 2012; 11:1108-17; “reverse warburg effect.” warburg effect, a balance of ecological and PMiD:22313602; http://dx.doi.org/10.4161/ They demonstrate stromal monocarboxyl- biophysical factors underpins the sustainabil- cc.11.6.19530. 4. Gatenby RA, et al. nat Rev Cancer 2011; 11:237- ate transporter 4 (MCT4), detected by immu- ity of this mode of cancer nutrition. 8; PMiD:21548400; http://dx.doi.org/10.1038/ nohistochemistry, as a functional marker of A two-compartment model coupling oxi- nrc3036. stromal hypoxia, oxidative stress, aerobic gly- dative epithelial cells with glycolytic fibroblasts 5. Moore nM, et al. Phys Biol 2011; 8:010302; PMiD:21368346; http://dx.doi.org/10.1088/1478- colysis and L-lactate efflux. High stromal MCT4 reflects increased expression of hypoxia-asso- 3975/8/1/010302. expression (but, critically, not epithelial MCT4) ciated genes as a component part of prog- 6. Finak G, et al. nat Med 2008; 14:518-27; was associated with poor prognosis in TnBC nostic stromal signatures.6 Further evidence of PMiD:18438415; http://dx.doi.org/10.1038/nm1764 7. Coates PJ, et al. Cancer Res 2010; 70:1-8; http:// patients. Combined high stromal MCT4 and stromal/epithelial interaction comes from evi- dx.doi.org/10.1158/0008-5472.CAn-10-1118. loss of stromal caveolin-1 identify particularly dence that the effects of radiation on normal 8. Appleyard MvCL, et al. Br J Cancer 2012; 106:1117- poor prognostic TnBC. breast epithelium in vivo is at least partially 22; PMiD:22361631; http://dx.doi.org/10.1038/ bjc.2012.56. Thus, development of cancer may not lie dependent on the stromal context.7 9. Hadad s, et al. Breast Cancer Res Treatment 2011; solely in genetic or epigenetic epithelial Manipulation of the tumor microenviron- 128:783-94. changes, but with acquired functional changes ment to promote an anticancer phenotype www.landesbioscience.com Cell Cycle 1487 Stress, specificity and the NEDD8 proteome Comment on: Leidecker O, et al. Cell Cycle 2012; 1142–50; PMID: 22370482; http://dx.doi.org/10.4161/cc.11.6.19559 David P. Lane; p53 Laboratory; A*STAR; Singapore; Email: [email protected]; http://dx.doi.org/10.4161/cc.20073 in an exciting and surprising paper in a recent been uncovered, with initial studies identify- improved mass spectrometry methods devel- issue of Cell Cycle, Leidecker et al. show that ing p53 and Mdm2 as substrates for ned- oped in this paper using Lys-C to digest ned- the balance between protein modification by dylation, and Mdm2 as a e3 ligase for both dylated proteins allow one to distinguish ubiquitin or the ubiquitin like protein neDD8 neDD8 and ubiquitin.6 Proteomic approaches neDD8 modification from ubiquitination. This is dramatically altered by cellular stress. in have now identified many more substrates, helps to further refine our knowledge of this a variety of conditions that reduce the con- notable among them being the ribosomal fascinating system, but, meanwhile, protein centration of free ubiquitin, a very dramatic proteins involved in signaling to p53.7,8 in the neddylation may provide a new biomarker for increase in protein modification by ned- current study, the authors found that a high cellular stress. Many critical issues remain to dylation is revealed. importantly, this process level of neDD8-conjugated proteins were rap- be resolved: are there proteins with ubiqui- is shown to arise as neDD8 is activated under idly induced by proteasome inhibition with tin/neDD8 binding domains that specifically these conditions by the ubiquitin-activating MG132, but that this reaction was not inhib- recognize the ubiquitin neDD8 hybrid chains enzyme Ube1 and not by the typical neDD8 ited by MLn4924, even while the same com- that result from these stress signals? which specific ei enzyme, nAe. This results in many pound was blocking cullin neddylation. This e2s and e3s are responsible for stress-induced proteins in stressed cells being modified by meant that another e1 had to be in play for neddylation? should Ube1 inhibitors be devel- mixed ubiquitin neDD8 chains, which is highly the neddylation of these new substrates, and oped to complement the nAe inhibitor in relevant in the development of novel can- knockdown of Ube1 (which was known to be cancer treatments, or would they prove too © 2012 Landes Bioscience. cer therapeutics, as the nAe specific inhibitor able to activate neDD8 in vitro)9 showed that toxic? The next few years promise to reveal MLn49242does not block this new pathway it was, indeed, responsible. exploring further critical insights into the crosstalk between the despite its promising anticancer activity. stress signals showed that this increased ned- different Ubl pathways. initial comparative studies on the ubiDqui- ody lantion orespton sed wasi insductedr byi hbeat ushoctk e. tin and ubiquitin-like (Ubl) protein pathways and by elevated levels of reactive oxygen References have established that each pathway has sepa- species (ROs). since all of these stress path- 1. Leidecker O, et al. Cell Cycle 2012; 11:1142-50; PMiD:22370482; http://dx.doi.org/10.4161/ rate and specific enzymes both for activating ways reduce free ubiquitin levels, the authors cc.11.6.19559. the Ubl and for removing it.3 in the case of asked if nAe-independent neddylation could 2. soucy TA, et al. nature 2009; 458:732-6; PMiD:19360080; http://dx.doi.org/10.1038/ neDD8, the e1 is nAe; the e2s are Ubc12 and be triggered simply by reducing free ubiquitin nature07884. Ube2F, and the e3s include the Rbx1 and Rbx2 levels. The clearly positive results of this study 3. Kerscher O, et al. Annu Rev Cell Dev Biol 2006; 22:159- RinG finger proteins as well as members of suggested that competition with ubiquitin 80; PMiD:16753028; http://dx.doi.org/10.1146/ annurev.cellbio.22.010605.093503. the DCn family of proteins. The first studies for Ube1 may normally limit Ube1 activation 4. watson iR, et al. Cancer Cell 2011; 19:168-76; of the neDD8 system suggested that there of neDD8 and the neddylation of non-cullin PMiD:21316600; http://dx.doi.org/10.1016/j. were very few substrates for this modifica- substrates (Fig. 1). ccr.2011.01.002. 5. Duda DM, et al. Curr Opin struct Biol 2011; 21:257- tion, with most emphasis placed on the cullin in stress conditions then, when free ubiqui- 64; PMiD:21288713; http://dx.doi.org/10.1016/j. proteins. The cullins are components of the tin levels fall, Ube1 acts as a sensor of this state sbi.2011.01.003. cullin-RinG ligases (CRLs) that are responsible and neddylation increases. why would this 6. Xirodimas DP, et al. Cell 2004; 118:83-97; PMiD:15242646; http://dx.doi.org/10.1016/j. for the ubiquitylation of many critical sub- be useful? The speculation is that the modi- cell.2004.06.016. strates, for example, oncoproteins such as fication of substrate proteins by neDD8 may 7. Xirodimas DP, et al. eMBO Rep 2008; 9:280- cyclin e and c-myc. The cullins are modified help the cell to cope with stress signals, for 6; PMiD:18274552; http://dx.doi.org/10.1038/ embor.2008.10. by neddylation, which increases the e3 activ- example, by promoting cell survival through 8. sundqvist A, et al. eMBO Rep 2009; 10:1132- ity of the CRLs, probably through structural inhibition of the degradation of very labile 9; PMiD:19713960; http://dx.doi.org/10.1038/ embor.2009.178. alterations that free the Ring domain of the e3 pro-survival proteins, such as Mcl-1. After the 9. whitby FG, et al. .J Biol Chem 1998; 273:34983- and/or by blocking the binding of inhibitory stress signal abates, the many effective de- 91; PMiD:9857030; http://dx.doi.org/10.1074/ proteins such as CAnD 1.4,5 Recently, many ubiquitinating and de-neddylating enzymes jbc.273.52.34983. new substrates and e3 ligases for neDD8 have can come into play to restore homeostasis. www.landesbioscience.com Cell Cycle 1488 © 2012 Landes Bioscience. Do not distribute. Figure 1. nedd8 pathway and stress. (A) in unstressed cells, two parallel and non-overlapping pathways are in play. nedd8 activation is through the action of nAe, while ubiquitin is activated by Ube1. substrate selectivity of the e2 and e3 results in many proteins being ubiquitinated, but few are nedd8-modified, notably, the cullins. (B) Low free ubiquitin levels in stress conditions results in nedd8 being activated by the ubiquitin Ube1 as well as nAe1. This, in turn, results in a large increase in the variety of protein substrates that are neDD8-modified, in addition to the cullins. www.landesbioscience.com Cell Cycle 1489