2002 Nature Publishing Group402 | JUNE 2002 | VOLUME 2 www.nature.com/reviews/cancer
H I G H L I G H T S
As well as deleting or mutatingtumour-suppressor genes, cancercells can turn off their expressionby epigenetic means. It has been a
challenge, however, to determineexactly which genes are transcrip-tionally silenced in a particulartumour. A new microarray-basedscreen can identify genes that aresuppressed by hypermethylationand histone deacetylase (HDAC)activity, revealing 74 genes that aredownregulated by this process incolorectal cancer cells.
Epigenetic gene silencinginvolves increased methylation ofDNA sequences known as CpGislands, which usually lie in genepromoter regions, and also theactivity of HDACs, which controlchromosome condensation. Butbecause the sites that are regulatedby these mechanisms are often out-side the coding regions of genes, itis laborious to search for nearbygenes and determine whether theirexpression patterns are altered.Sometimes, it is a challenge to evenidentify hypermethylated sites, assome of these do not contain CpGislands, and there is no establishedmethod to identify genes that areregulated by HDAC activity.
Hiromu Suzuki et al. have over-come these obstacles by usingmicroarrays to compare the expres-sion patterns of untreated cells withthose treated with 5-aza-2 deoxycy-tidine (DAC), which blocks DNAmethylation, and/or trichostatin A(TSA), which inhibits HDAC activ-ity. A screen of over 10,000 genes ineight different colorectal cancer celllines revealed 74 genes that wereupregulated by treatment with DACand/or TSA. Of these genes, 51required treatment with both drugsfor suppression to be relieved,whereas 23 upregulated expressionafter treatment with TSA alone. Somost genes seem to require a com-bination of hypermethylation andHDAC activity to be silenced.
Many of these genes werealready known to be involved intumorigenesis, such as SEZ6L, agene of unknown function that ismutated in lung and breast cancer,and TIMP2, an inhibitor of thematrix metalloproteinase family ofgenes that are required for cellinvasion. One unexpected finding
There have been many attempts to directthe cytolytic activity of adenovirusestowards tumours. The tricky part,however, is manipulating them to killcancer cells but not normal cells. Arecently engineered adenovirus, ONYX-411, only lyses cells that lack a functionalform of the tumour suppressorretinoblastoma (RB) making it a saferand more effective oncolytic virus.
Oncolytic adenoviruses caused muchexcitement when they were first describedin 1996. ONYX-015 the first oncolyticvirus shown to preferentially kill cells inwhich p53 is inactivated reducedtumour growth in mice and is, at present,in clinical trials for the treatment of headand neck cancer. One drawback of thisagent, however, is that it must be directlyinjected into the patients tumour, becauseit has been shown to have toxic effectswhen administered intravenously. Thislimits its use to patients who have largeaccessible tumours.
Leisa Johnson et al. have improved on theoncolytic virus concept to create a versionthat can be applied systemically. The newvirus, ONYX-411, was engineered topropagate only in cells with defects in the RBpathway which includes almost all cancercells. So, how does ONYX-411 work? Theauthors placed the adenoviral E1A and E4genes which are required for viralpropagation under control of the humancellular E2F1 promoter. This promoter isactivated by E2F and is selectively active incancer cells that contain high levels of E2Fowing to loss of RB activity. The E1A gene ofONYX-411 also contains a point mutationthat prevents its interaction with RB,although it can still mediate S-phase entry.This mutant form of E1A therefore cannotdisrupt RB activity in normal cells, and onlyreplicates in cells that lack functional RB.
The authors tested ONYX-411 and foundthat it killed many different types of cancercells, but not normal cells, in vitro. But can itbe safely administered intravenously? A
detailed analysis in mice revealed thatONYX-411 caused less systemic toxicity thanall other adenoviral vectors it was comparedwith, including ONYX-015. But mostimportantly, intravenously administeredONYX-411 prolonged survival of micebearing human cervical cancer xenografts,curing 6070% of mice without damage tonormal tissues.
Although the mechanisms responsiblefor the lower systemic toxicity associatedwith ONYX-411 are not clearlyunderstood, the multiple systems forensuring that the virus replicates only inRB-deficient cells are likely to beresponsible. ONYX-411 might therefore beuseful for treating metastatic tumours andother cancers that require systemicadministration of therapy.
References and linksORIGINAL RESEARCH PAPER Johnson, L. et al. Selectivelyreplicating adenoviruses targeting deregulated E2F activity arepotent, systemic antitumor agents. Cancer Cell 1, 325337(2002)FURTHER READING Dubensky, T. W. Engineering tumor cell-selective replicating adenoviruses: a step in the right directiontoward systemic therapy for metastatic disease? Cancer Cell1, 307310 (2002) | McCormick, F. Cancer gene therapy:fringe or cutting edge? Nature Rev. Cancer 1, 130141 (2001)WEB SITEFrank McCormicks lab:http://cc.ucsf.edu/people/mccormick_frank.html
T H E R A P E U T I C S
Breaking the silence
E P I G E N E T I C S
2002 Nature Publishing Group
H I G H L I G H T S
NATURE REVIEWS | CANCER VOLUME 2 | JUNE 2002 | 403
The oncogene c-Myc controls the fine linebetween life and death, as it can induce both cellproliferation and apoptosis. But whether c-Myc-induced cell death can actually restrain tumourgrowth has remained undetermined. StellaPelengaris et al. have now shown that c-Myc-induced apoptosis can indeed prevent tumourformation, and that switching off apoptosis allowsthe tumorigenic capability of c-Myc to proceedunchecked.
A switchable c-Myc in which the gene isfused to the 4-hydroxytamoxifen (4-OHT)-responsive oestrogen receptor (c-MycERTAM), sothat the protein is activated following theintraperitoneal administration of 4-OHT wasspecifically targeted to pancreatic -cells in miceusing the pIns insulin promoter. Induction ofc-Myc initially resulted in cell proliferation, butthis was accompanied by a faster rate of apoptosis,such that the net effect was -cell ablation andhyperglycaemia caused by loss of insulin-producing cells. Interestingly, when 4-OHT waswithdrawn, which switched off c-Myc, pancreaticislets rapidly regenerated and blood glucose levelsreturned to normal.
If apoptosis overpowers cell proliferation,inhibiting apoptosis should allow proliferation toproceed unchecked. Expressing the apoptosisinhibitor Bcl-x
Lunder the control of the rat
insulin promoter (RIP7) allowed this hypothesisto be tested. pIns-c-MycERTAM/RIP-Bcl-x
transgenic mice had normal pancreatic isletformation until 4-OHT was added, at which pointproliferation was induced throughout thepancreatic -cells. As apoptosis was inhibited byBcl-x
L, this resulted in hyperplasia within 7 days.
But can deregulated expression of c-Mycinduce tumour formation, which is thought torequire the cumulative effect of multiplemutations? Pancreatic -cells in pIns-c-MycERTAM/RIP-Bcl-x
Lmice not only
hyperproliferated, but also underwent de-differentiation as seen by the reducedproduction of insulin and extensiveangiogenesis. Expression of the intercellularadhesion molecule E-cadherin was lost as well,which is a prerequisite for loss of cellcellcontacts and invasion. c-Myc therefore seems tobe able to directly induce several of thehallmarks of cancer. Two weeks after inductionof c-Myc expression, pIns-c-MycERTAM/RIP-Bcl-x
mice had developed pancreatic tumours, and by
8 weeks the tumours were large andvascularized, with sites of local invasion in localblood vessels and draining lymph nodes.
So, expression of a single oncogene c-Myc is sufficient to induce several steps ofcarcinogenesis, as long as its innate apoptoticactivity is curtailed; but is it also required tomaintain the tumours once they have formed?Switching off c-Myc, 14 days after its induction,resulted in a reversal of the tumorigenic process:-cells exited the cell cycle, E-cadherin was re-expressed and cells re-established cellcellcontacts, and endothelial cells and -cellsapoptosed. Even mice that had expressed c-Mycfor 8 weeks, with extensive tumours that hadinvaded into lymph nodes, made a full recoveryfollowing c-Myc deactivation.
These results challenge the paradigm thatcarcinogenesis is a multistep process that requiresmany mutations, and indicate that, instead, it canbe driven by deregulated expression of a singlegrowth-deregulating oncogene, providedapoptosis is suppressed. If this is found to be truefor other commonly mutated oncogenes, newcancer therapeutics should aim to inhibit thesefew crucial molecular targets.
References and linksORIGINAL RESEARCH PAPER Pelengaris, S. et al. Suppression ofMyc-induced apoptosis in -cells exposes multiple oncogenicproperties of Myc and triggers carcinogenic progression. Cell 109,321334 (2002) WEB SITESEncyclopedia of Life Sciences: http://www.els.netapoptosis: molecular mechanisms Gerard Evans lab: http://cc.ucsf.edu/evan/
was the hypermethylation of fourmembers of the SFRP gene family,which have not been previouslyassociated with colorectal cancer.The products of these genes inhibitthe WNT/FZD1 signalling pathway,so the SFRP genes might be a newclass of colorectal tumour suppres-sors. The SFRP gene family provides a new set of hypermethy-lation markers that might be usefulin colon cancer detection.
In the future, this screeningapproach will allow for comparisonsof epigenetic modifications betweenother tumour types, and also aid inthe identification of new tumour-suppressor genes.
References and linksORIGINAL RESEARCH PAPER Suzuki, H. et al.A genomic screen for genes upregulated bydemethylation and histone deacetylase inhibitionin human colorectal cancer. Nature Genet. 31, 18(2002)FURTHER READING Marks, P. et al. Histonedeacetylases and cancer: causes and therapies.Nature Rev. Cancer 1, 194202 (2001)WEB SITEStephen Baylins lab:http://www.hopkinsmedicine.org/graduateprograms/cmm/baylin.html
A life or deathsituation
O N C O G E N E S I S