N E W S A N D V I E W S

NATURE MEDICINE VOLUME 12 | NUMBER 8 | AUGUST 2006 887

How might the downregulation of mutant SOD1 expression in microglia account for such a profound slowing of disease progres-sion? The underlying mechanism remains unclear. Does mutant SOD1 interfere with cellular metabolism and prevent full activa-tion of microglial cells? This might inhibit the expression or release of neuroprotective fac-tors required by stressed motor neurons in the neighborhood. Conversely, the intracellular accumulation (or extracellular secretion) of mutant human SOD1 might induce a height-ened state of microglial activation, leading to higher levels of neuronal damage associated with increased expression of inflammatory mediators.

Boillee et al. suggest that targeting therapy to microglial cells may be an effective strategy for human ALS—but the key question is: what

type of therapy? Do we need to inhibit activa-tion of microglial inflammatory functions? This might prevent damage to motor neurons by secreted free radical species, glutamate, TNF-α or NO, but might also be harmful, by inhibiting the upregulation of neurotrophic factors and the increased uptake of excito-toxic glutamate by astrocytes that occurs after microglial activation.

Furthermore, the clearance of apoptotic neurons, an important function of micro-glial cells11, might also be compromised and this could exacerbate disease progres-sion. The role of microglia in motor neuron injury in the more common subtypes of ALS, not caused by SOD1 mutations, also needs to be further examined.

These questions must be addressed before a new route to ALS therapy can be mapped

out based on manipulating microglia to minimize neurotoxicity and maximize ‘good neighborhood’ effects on motor neurons.

1. Boillee, S. et al. Science 312, 1389–1392 (2006).

2. Haverkamp, L.J., Appel, V. & Appel, S.H. Brain 118 (Pt 3), 707–719 (1995).

3. Rosen, D.R. et al. Nature 362, 59–62 (1993).4. Wilms, H. et al. J. Neuroimmunol. 144, 139–142

(2003).5. Troost, D., Van den Oord, J.J. & Vianney de Jong,

J.M. Neuropathol. Appl. Neurobiol. 16, 401–410 (1990).

6. Sargsyan, S.A., Monk, P.N. & Shaw, P.J. Glia 51, 241–253 (2005).

7. McGeer, E.G. & McGeer, P.L. BioDrugs 19, 31–37 (2005).

8. Wang, J. et al. Neurobiol. Dis. 20, 943–952 (2005).

9. Coull, J.A. et al. Nature 438, 1017–1021 (2005).10. Zhao, W. et al. J. Neuropathol. Exp. Neurol. 63,

964–977 (2004).11. Stolzing, A. & Grune, T. FASEB J. 18, 743–745

(2004).

Stopping cancer before it colonizesCarrie W Rinker-Schaeffer & Jonathan A Hickson

Controlling the growth of cancer cells at metastatic sites is one goal of cancer drug development. Studies of metastasis suppressor function bring this long-sought goal closer (pages 933–938).

Carrie W. Rinker-Schaeffer is in the Departments of

Surgery, Medicine and Obstetrics and Gynecology,

and Jonathan A. Hickson is in theDepartment

of Obstetrics and Gynecology, The University of

Chicago, Chicago, Illinois 60637, USA.

E-mail: crinkers@midway.uchicago.edu

Primary tumor formation and metastasis are distinct processes. For instance, can-cer cells can express metastasis suppressors that block cell proliferation at secondary or metastatic sites yet permit cell proliferation at primary sites1. The cell membrane pro-tein KAI1 (kang ai, Chinese for ‘anticancer’) was identified as a metastasis suppressor a decade ago, but determining exactly how it functions has been a challenge.

In this issue, Bandyopadhyay et al.2 report that KAI1 binds the Duffy blood group glyco-protein (DARC, also known as gp-Fy), a pro-tein expressed on the vascular endothelium. This binding inhibits cancer cell proliferation at distant sites and ultimately induces expres-sion of markers of cellular senescence (Fig. 1). The findings identify a previously unknown function for KAI1—and an unanticipated bio-logical outcome resulting from restoration of its expression in metastatic cells. Such expres-sion may enable control of cancer cell growth at distant sites.

The metastasis suppressor gene field was launched in 1988 with the publication of an article identifying nm23 (ref. 3). This semi-nal study provided functional evidence for the existence of proteins, and their cognate signaling pathways, that could specifically regulate metastasis. Efforts over the follow-ing two decades have identified additional

metastasis suppressors ranging from kinases such as MAP kinase kinase 4 to GTP-binding proteins such as Rho-GDI2 (ref. 1).

Although many investigators anticipated that metastasis suppressors would block escape of cells from primary tumors, in vivo studies revealed a novel biological effect of these proteins. In many cases, cancer cells

Proliferation inhibited,cells express senescence

marker (β-gal)

DARC-expressingendothelial cells

Proliferatingcancer cells formovert metastases

Primary tumor

Cancer cells

KAI1

Figure 1 Model for KAI1-mediated metastasis suppression. Data from Bandyopadhyay et al.2 indicate that disseminating cancer cells that express KAI1 can interact with DARC molecules on endothelial cells. This binding transmits a signal that inhibits cancer cell proliferation and induces expression of endogenous markers of cellular senescence (β-galactosidase, blue). In contrast, cells that do not express KAI1 can proliferate at favorable distant sites, giving rise to overt metastases.

Kim

Cae

sar

©20

06 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

urem

edic

ine

N E W S A N D V I E W S

888 VOLUME 12 | NUMBER 8 | AUGUST 2006 NATURE MEDICINE

ectopically expressing metastasis suppres-sors disseminate and lodge at secondary sites but are suppressed in their ability to colonize and form overt metastases in tar-get tissues—suggesting that cancer cells expressing metastasis suppressor proteins respond differ entially to site-specific exter-nal signals1. These findings also support contempor aneous studies that identified colonization of metastatic sites as a rate-limiting step for metastasis formation4.

In our working model, disseminated cells expressing metastasis suppressors may undergo cell death, persist as nondividing cells or be impaired in their ability to proliferate at secondary sites1. The specific biological out-come depends on the gene expression profile, the activation status of key signaling pathways and the cumulative inputs of timing, ampli-tude and duration of signaling responses. The integration of these molecular responses in both cancer cells and cells within their micro-environment enables the expression of tumor phenotypes, such as angiogenesis, which are critical for the development of successful metastatic lesions. Because of this complexity, even in the case of a well-characterized meta-stasis suppressor, determining its biochemical mechanism of action remains daunting.

Dong et al. first identified KAI1 by using a combination of microcell-mediated chro-mosomal transfer and positional cloning techniques5. Subsequently, it was shown that KAI1 is identical to CD82, a tetraspanin pro-tein. Tetraspanins are transmembrane pro-teins ubiquitously expressed in multicellular eukaryotes, and are thought to be involved in processes ranging from cell motility to morphology to differentiation6.

Numerous studies show a correlation between downregulation of KAI1 protein expression and poor prognosis in a wide variety of cancers, including prostate, lung, breast, colon, cervical and ovarian7. Understanding how KAI1 exerts its metastasis-suppressive effect has challenged many investigators, in part because tetraspanins lack intrinsic activity and their effects are mediated by protein interactions.

Several proteins have been shown to interact with KAI1, including integrins, PKCα, B-cell receptors, other tetraspan-ins and receptor tyrosine kinases. Several groups have worked on various aspects of KAI1 regulation and function in clini-cal and experimental metastasis, and key insights have been made into the expression of KAI1 protein and its potential signaling inter actions7. But which of these candidate molecules regulates the metastasis-suppres-sive function of KAI1 was unclear.

Starting at first principles, Bandyopadhyay et al. used a yeast two-hybrid system to iden-tify proteins which specifically interact with KAI1. This approach identified the Duffy antigen receptor for chemokines, DARC, as a potential interactor for KAI1. After demon strating the strength and specificity of the interaction between KAI1 and DARC, studies were designed to determine how an interaction between KAI1 and DARC might suppress metastasis.

The authors harnessed the findings from immunohistochemical studies, which showed DARC protein expression on the vascular endothelium of various organs, to develop a model. They proposed that can-cer cells expressing KAI1 attach to vascular endothelial cells at a secondary site directly through KAI1 and DARC interactions, inhibiting metastasis formation. A variety of in vitro and in vivo assays were used to test this hypothesis.

First, a series of in vitro cell-to-cell bind-ing assays showed that prostate cancer cells expressing KAI1 specifically bound to endo-thelial cells and control cells that expressed DARC. This suggested that the metastasis suppressor function of KAI1 is due, at least in part, to the trapping of tumor cells on the endothelial linings of vessels. Using DARC-knockout mice, the authors isolated syngenic cancer cell lines that ectopically expressed KAI1. These mice, as well as appropriate littermate controls, were inoculated sub-cutaneously and monitored for primary tumor growth rate and formation of overt meta stasis in the lung. The metastasis sup-pressor activity of KAI1 was compromised in the DARC-knockout mice, whereas KAI1 completely abrogated pulmonary metastases in wild-type and heterozygous littermates.

A final series of experiments addressed how KAI1 regulates growth of disseminated tumor cells. In vitro assays showed that the interaction between KAI1 and DARC sup-pressed the growth of cancer cells but did not increase apoptosis. Instead, the authors found that the interaction with DARC led to senescence in KAI1-positive cancer cells, measured as an increase in the percent-age of cancer cells expressing senescence-associated β-galactosidase. An evaluation of senescence-associated genes confirmed this finding.

The importance of the dynamic and recip-rocal interactions between epithelial cells and stromal cells has been well established. However, the interactions between dissemi-nated cancer cells and their microenviron-ment during metastasis have been mostly logically inferred. Our laboratory’s current

working model is that ectopic expression of metastasis suppressor proteins restores, at least in part, normal cellular interactions and proliferation controls—thereby inhibit-ing metastasis formation. Bandyopadhyay et al.2 provide conclusive evidence in support of a functional role for interactions between cancer cells and endothelial cells and iden-tify a potentially novel function for endo-thelial cells in the regulation of early steps of metastasic colonization.

It is not surprising that several ques-tions arise from work such as this. Is the expression of metastasis suppressor pro-teins like KAI1 static, or do levels change over time and during the progression from disseminated cells to occult (microscopic) meta stasis? Is protein expression of DARC stable throughout the vasculature? An obvi-ous first step to address this second question would be to examine the pattern of DARC expression on the vasculature for correla-tions between expression status, disease pro-gression and patient survival.

Future studies should also address whether the induction of senescence mark-ers is unique to the KAI1-DARC interaction or is a conserved mechanism for metastasis suppressors that inhibit metastatic coloni-zation. It is plausible that re-expression of metastasis suppressor proteins restores the ability of cells to respond to various stress stimuli which are associated with induction of senescence markers8,9.

Finally, the authors present some initial data to identify the critical binding regions necessary for the association of DARC and KAI1. Further defining these binding interfaces is critical for the development of small-molecule inhibitors that may mimic or strengthen this interaction. This report illustrates an exceptional opportunity for metastasis prevention: impeding the growth of disseminated cancer cells using therapies that target early steps in metastatic coloni-zation.

1. Rinker-Schaeffer, C.W., O’Keefe, J.P., Welch, D.R. & Theodorescu, D. Clin. Cancer Res. 12, 3882–3889 (2006).

2. Bandyopadhyay, S. et al. Nat. Med. 12, 933–938 (2006).

3. Steeg, P.S. J. Natl. Cancer Inst. 96, E4 (2004).4. Chambers, A.F., Groom, A.C. & MacDonald, I.C. Nat.

Rev. Cancer 2, 563–572 (2002).5. Dong, J.T. et al. Science 268, 884–886 (1995).6. Hemler, M.E. Nat. Rev. Mol. Cell Biol. 6, 801–811

(2005).7. Sridhar, S.C. & Miranti, C.K. Oncogene 25, 2367–

2378 (2006).8. Collado, M. & Serrano, M. Nat. Rev. Cancer 6, 472–

476 (2006).9. Schwarze, S.R., Fu, V.X., Desotelle, J.A., Kenowski,

M.L. & Jarrard, D.F. Neoplasia 7, 816–823 (2005).

©20

06 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

urem

edic

ine