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is deficient? Komatsu et al. note that overex-pression of p62 alone in hepatic cells does not cause cell lethality, hinting that the cause of pathology may not be strictly cell-autonomous in an organ setting. A closely related study published recently by White and colleagues6 provides some explanations to this puzzle. Aiming at explaining the tumour-suppressor role of autophagy in a hepatic cancer model, this team overexpressed p62 in mice hetero-zygous for the autophagy regulator Beclin 1. In this genetic background, where autophagy is partially inhibited, spontaneous tumours develop, in which TNF--induced canoni-cal signalling through the pro-inflammatory transcription factor NF-B is suppressed, apparently because of p62 accumulation. They

observed an inverse relationship between p62 accumulation and NF-B signalling activity in spontaneously developing tumours of Beclin 1+/ mice. Although not conclusive at this point, these findings may provide a plausible expla-nation for non-cell-autonomous hypertrophy and carcinogenic effects of autophagy, through cytokine action7,8. A possible confounding issue in both studies is the existence of a second p62-like protein, neighbour of BRCA1 gene 1 (NBR1), which could act redundantly with p62 in some cell types9.

With the multiple p62-interacting proteins that are now being identified (refs 1 and 4), the race is on to find new autophagic cargoes that are involved in diseases associated with autophagy deficiency. Some of these might

turn out to be relevant drug targets and, given the involvement of p62 in an increas-ing number of cellular processes that range from kinase regulation to degradation and transcriptional regulation, it is perhaps not so surprising that this protein behaves as a hero in some situations and a culprit in others.

CoMpeting FinAnCiAl inteRestsThe authors declare no competing financial interests.

1. Lamark, T., Kirkin, V., Dikic, I. & Johansen, T. Cell Cycle 8, 19861990 (2009).

2. Komatsu, M. et al. Cell 131, 11491163 (2007).3. Nezis, I. P. et al. J. Cell Biol. 180, 10651071 (2008).4. Komatsu, M. et al. Nature Cell Biol. 12, 213223

(2010).5. Komatsu, M. et al. Nature 441, 880884 (2006).6. Mathew, R. et al. Cell 137, 10621075 (2009).7. Chang, L. et al. Cell 124, 601613 (2006).8. Luedde, T. et al. Cancer Cell 11, 119132 (2007).9. Kirkin, V. et al. Mol. Cell 33, 505516 (2009).

Mycmodulated miR9 makes more metastasesYeesim Khew-goodall and gregory J. goodall

The microRna miR9 is induced by Myc in breast cancer cells where it targets the major epithelial adherens junction protein, ecadherin. This primes the cancer cells for epithelialmesenchymal transition (eMT) and also stimulates angiogenesis in tumours.

We are becoming more and more aware of the almost ubiquitous involvement of microRNAs in shaping cellular properties. Several microRNAs influence steps involved in metastasis, including EMT, the name given to the conversion of adher-ent, non-motile epithelial cells to mesenchymal cells, with their repertoire of migratory and invasive capacities. On page 247 of this issue1, Ma et al. present evidence that upregulation of miR-9 in cancers may present a double whammy, not only priming cancer cells for EMT and inva-sion, but also promoting angiogenesis, thus help-ing the tumour to grow, and providing a nearby escape route for migrating cancer cells.

Prompted by earlier indications that miR-9 is increased in some breast cancer cell lines and tumours, Ma et al. checked the target predictions for miR-9 and noted that E-cadherin has a highly conserved predicted target site for this microRNA. This finding was of interest because E-cadherin, which forms

the adherens junctions between neighbouring epithelial cells, is essential for maintaining epi-thelial cell morphology and function. Without it, epithelial cells acquire migratory and inva-sive capabilities. Sure enough, when they introduced miR-9 into breast epithelial cells, E-cadherin levels were reduced and the cells gained enhanced motility and invasiveness in vitro, an effect that was largely reversed by co-transfection with an E-cadherin gene lack-ing the miR-9 target region. Experiments with luciferase reporters containing the E-cadherin 3UTR confirmed that the single target site for miR-9 confers regulation by this microRNA.

When breast cancer cells with enforced miR-9 expression were implanted into mice to form breast tumours, the mesenchymal marker vimen-tin was expressed by cancer cells at the tumour edge, suggesting that these cells were undergoing EMT under the combined influence of miR-9 and inducing signals from the tumour environment. This is interesting because, although it is well established that some cancer cells can undergo EMT in vitro, there are few clear indications of the change occurring in vivo.

A second consequence of miR-9 expression in experimental breast tumours was enhancement

of angiogenesis. Ma et al. describe a downstream pathway that, at least partly, accounts for this effect. The intracellular domain of E-cadherin anchors a complex that connects the actin cytoskeleton to the adherens junctions (Fig. 1). This complex includes -catenin, which, when liberated, potentially becomes available to act as a transcription factor in partnership with TCF/LEF proteins, subject to the status of the GSK3 pathway, which controls the stability of the free -catenin2. This pathway has been implicated in VEGF induction following disruption of E-cadherin in a mouse lung cancer model3. Ma et al. observed -catenin in the nucleus of cells transfected with miR-9, and found that a reporter gene for -catenin-mediated tran-scription was turned on in response to miR-9. Overexpression of miR-9 led to increased levels of VEGF mRNA in cells that express E-cadherin, but not in cells devoid of E-cadherin, or in cells in which E-cadherin downregulation was pre-vented by enforced expression, supporting a pathway in which the reduction in E-cadherin levels by miR-9 allows liberation of -catenin, which then activates the VEGF gene (Fig. 1).

What is the consequence of this miR-9 path-way for cancer? For one thing, the enhanced

Yeesim Khew-Goodall and Gregory J Goodall are in the Centre for Cancer Biology, SA Pathology, Adelaide, Australia.e-mail: greg.goodall@health.sa.gov.auPublished online 21 February 2010; DOI:10.1038/ncb0310-209

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vascularization seems to allow the tumour cells to proliferate more rapidly than they otherwise would; although the miR-9-expressing cells grew more slowly in vitro than their control counterparts, the tumours they formed grew faster and the cells had a higher proliferation rate, as indicated by the expression of the Ki67 proliferation marker. A second important effect of the microRNA was enhancement of metas-tasis. The SUM149 human breast cancer cells are not normally metastatic, but with enforced miR-9 expression they gave rise to numer-ous micrometastases in the lungs. To test the involvement of miR-9 in a different model, Ma et al. turned to 4T1 mouse mammary tumour cells, which naturally express high levels of miR-9 and are highly metastatic. As the cells were from mouse tumours, this experiment could be performed in mice that had a normal immune system, rather than in immunocom-promised mice, which are required to test the properties of human tumours. To block the activity of miR-9 in the 4T1 cells, Ma et al. made the cells express a microRNA spongea transcript with multimerized,near-perfect binding sites for the microRNA that would effectively compete with endogenous targets for binding of miR-9, but retain a mismatch in the centre of the binding region to avoid acti-vating siRNA-type cleavage of the sponge tran-script. Introduction of the sponge-expressing 4T1 cells into the mammary fat pad gave rise to primary tumours that grew at a rate similar to the control cells, but the number of metastases

was reduced by half, nicely complementing the enhancement of metastases that occurred when miR-9 was introduced into SUM149 cells.

What drives the increased miR-9 expression that is typical of breast cancers? Several lines of evidence have pointed to Myc as the cul-prit. In particular, miR-9 expression is greatly increased in Myc-induced mouse mammary tumours4. Ma et al. verified that Myc induces miR-9 in breast cancer cells. Chip-on-chip analysis showed a direct interaction of Myc and n-Myc with the miR-9-3 gene in neu-roblastoma cells, suggesting that activation of miR-9 expression in breast cancers that over-express Myc may occur through activation of the miR-9 gene by Myc. As Myc is a target of -catenin5, it will be interesting to test whether a feed-forward loop amplifies Myc and miR-9 expression in some breast cancers.

Does the pathway influence breast cancer metastasis in humans? The answer seems to be yes: miR-9 levels were elevated in primary tumours from breast cancer patients with metastases, compared with metastasis-free patients. It is interesting to note that miR-9 is upregulated in primary hepatocellular car-cinomas with metastasis6, so miR-9 may promote metastasis in a variety of human cancers. Neuroblastomas, however, do not express E-cadherin, so the pathway involv-ing E-cadherin deduced here in breast cancer cells does not apply. It will be interesting to see the identification of miR-9 targets in the neuroblastoma context, and whether they are

neuronal-specific, or could also contribute to the miR-9 effects in breast cancer.

The study by Ma et al. clearly establishes that miR-9 has a role in regulating VEGF production by targeting E-cadherin and thereby releasing -catenin to activate VEGF transcription in the nucleus. However, as the authors point out, additional miR-9 targets must be involved in this pathway leading to VEGF production, because knocking down E-cadherin by siRNA did not mimic the miR-9 effect and neither did introduc-tion of a constitutively active -catenin pro-tein. In addition, restoration of E-cadherin expression in miR-9-expressing cells did not completely block the effect of miR-9 on migration and invasion, again opening the possibility that other miR-9 targets may be important. In this regard it would be interest-ing to consider -catenin, another predicted target of miR-9 that resides with -catenin in the cytoskeleton-anchoring complex on the E-cadherin cytoplasmic tail. Reduced -catenin is a prognostic indicator of poor survival in many cancers7. Furthermore, the interaction of -catenin with -catenin can inhibit -catenin from acting as a transcrip-tion factor7. It is tempting to speculate, there-fore, that miR-9 enhances its effect on this pathway by simultaneously targeting both E-cadherin and -catenin.

MiR-9 has now been added to a growing list of microRNAs that influence cancer metas-tasis. miR-373, miR-520c8, miR-10b9 and

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Figure 1 miR-9 downregulates E-cadherin to disrupt adherens junctions and allow -catenin-mediated transcription. In normal epithelial cells, there is little free -catenin. It is tethered to the cytoplasmic tail of E-cadherin on one end and to -catenin on the other, which then links it to the actin cytoskeleton. miR-9 expression downregulates E-cadherin and releases -catenin from the complex. Normally, free -catenin is targeted for degradation, but in cells where the degradative pathway is downregulated, free -catenin can accumulate and enter the nucleus, bind to TCF/LEF and act as a transcription factor to activate transcription of target genes such as VEGF. This results in increased metastasis and tumour angiogenesis.

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miR-21 (ref. 10) have all been identified as enhancers of invasion and metastasis, whereas miR-335, miR-126 (ref. 11), miR-31 (ref. 12) and miR-200 family13 have been found to repress invasion and metastasis. This raises the question of how many microRNAs will eventually be found that affect metastasis and why so many microRNAs are able to have such effects? The answer to the latter question may lie in the nature of the change that epithelial-derived cancer cells must undergo to become invasive. A prevailing concept is that this involves recapitulation of EMT, the transition that occurs at various stages in development to allow tissue remodelling in the growing embryo. The extensive changes in cell struc-ture and function that occur during EMT

require many changes in gene expression, so it would not be surprising if multiple micro-RNAs and transcription factors were enlisted in effecting and coordinating these changes. In addition, EMT takes place at various stages and in various tissues during development, giving scope for tissue- or stage-specific microRNAs to be involved. Very few studies to date have addressed the roles of microRNAs in developmental EMTs, so much remains to be learned in this regard.

In the case of miR-9, its major defined role so far had been as a regulator of neural devel-opment14. However, conservation across many species of the miR-9 site in the E-cadherin gene (and perhaps in the -catenin gene) suggests that targeting of these genes may be a normal

function for miR-9 at some stage of develop-ment, which remains to be discovered.

CoMpeting FinAnCiAl inteRestsThe authors declare no competing financial interests.

1. Ma, L. et al. Nature Cell Biol. 12, 247256 (2010).2. Jeanes, A., Gottardi, C. J. & Yap, A. S. Oncogene 27,

69206929 (2008).3. Ceteci, F. et al. Cancer Cell 12, 145159 (2007).4. Sun, Y. et al. Breast Cancer Res. Treat. 118, 185196

(2009).5. He, T. C. et al. Science 281, 15091512 (1998).6. Hao-Xiang, T. et al. Med. Oncol. (2009).7. Benjamin, J. M. & Nelson, W. J. Semin. Cancer Biol.

18, 5364 (2008).8. Huang, Q. et al. Nature Cell Biol. 10, 202210 (2008).9. Ma, L., Teruya-Feldstein, J. & Weinberg, R. A. Nature

449, 682688 (2007).10. Zhu, S. et al. Cell Res. 18, 350359 (2008).11. Tavazoie, S. F. et al. Nature 451, 147152 (2008).12. Valastyan, S. et al. Cell 137, 10321046 (2009).13. Gibbons, D. L. et al. Genes Dev. 23, 21402151

(2009).14. Leucht, C. et al. Nature Neurosci. 11, 641648

(2008).

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