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plasma osmolality with mannitol may further deteriorate rather than improve outcomes.

This antihemostatic effect of PK is remark-able, as recent studies have showed that the inhibition or absence of the PK-activating FXIIa profoundly limits pathological thrombus forma-tion and protects mice from ischemic stroke10,14. Notably, this protection is not achieved at the expense of increased ICH, indicating that the contact activation system—FXIIa—is not required to prevent intracranial bleeding, even under inflammatory conditions10,12.

The combination of these two observations suggests that inhibition of the contact activa-tion system, for instance by a specific FXIIa inhibitor14 or a PK inhibitor4, may not only prevent thrombotic events in the setting of ischemic stroke, but also at the same time lower the risk of ICH in hyperglycemic animals. Further studies will be required to test this intriguing hypothesis in animals and, if con-firmed, also in people with ICH.

COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.

1. Qureshi, A.I., Mendelow, A.D. & Hanley, D.F. Lancet 373, 1632–1644 (2009).

2. Broderick, J. et al. Circulation 116, e391–e413 (2007).3. Stead, L.G. et al. Neurocrit. Care 13, 67–74 (2010). 4. Liu, J. et al. Nat Med. 17, 206–210 (2011).5. Furie, B. & Furie, B.C. N. Engl. J. Med. 359, 938–949

(2008). 6. Kannemeier, C. et al. Proc. Natl. Acad. Sci. USA 104,

6388–6393 (2007). 7. Müller, F. et al. Cell 139, 1143–1156 (2009). 8. Gailani, D. & Renné, T. J. Thromb. Haemost. 5,

1106–1112 (2007). 9. Renné, T. et al. J. Exp. Med. 202, 271–281 (2005). 10. Kleinschnitz, C. et al. J. Exp. Med. 203, 513–518

(2006). 11. Nieswandt, B. & Watson, S.P. Blood 102, 449–461

(2003). 12. Gray, C.S. et al. Lancet Neurol. 6, 397–406 (2007). 13. Wakai, A., Roberts, I. & Schierhout, G. Cochrane

Database Syst. Rev. 1, CD001049 (2007).14. Hagedorn, I. et al. Circulation 121, 1510–1517

(2010).

platelet adhesion and activation at sites of vascular injury in mice11.

PK bound collagen and blocked the GPVI binding sites on the collagen macromolecules in vitro4. As a consequence, PK inhibited collagen-induced platelet activation in vitro, and, remarkably, this effect was greatly enhanced not only in the presence of high glucose concentra-tions but also in the presence of hyperosmolar mannitol or salt. In mice, inhibition or absence of GPVI markedly augmented hematoma expan-sion in nondiabetic animals, suggesting that collagen-induced platelet activation is crucial to confining intracerebral hematoma formation. Liu et al.4 also showed that local administration of a specific platelet GPVI activator reduced PK-induced hematoma expansion in diabetic rats, confirming that platelet activation through this receptor is sufficient to limit intracerebral bleeding in experimental ICH.

Taken together, the findings by Liu et al.4 provide a crucial link between ICH outcome—cerebral hematoma expansion—and hypergly-cemia in the whole-blood model of ICH. As lowering glucose in plasma did not improve outcomes in people with ICH and hypergly-cemia12, the timing of glucose normalization may be essential. How hyperosmolality pro-motes PK interaction with collagen and thus inhibition of collagen-induced platelet activa-tion awaits further clarification.

Nevertheless, the findings reported by Liu et al.4 point to hyperosmolality as a major risk factor for poor outcomes in ICH. In line with this, the use of mannitol for the treat-ment of raised intracranial pressure in acute traumatic brain injury is controversial13. Both ICH and traumatic brain injury often cause life- threatening mass effects requiring anti-edematous treatments. As a note of caution, it is conceivable that this iatrogenic increase in

(both rats and mice) in the whole-blood model. In this model, the animal’s own blood or donor blood injected directly into the brain compresses the surrounding brain tissue. Autologous blood or exogenous PK, but not prekallikrein, directly administered to the brain aggravated cerebral hemorrhage in the context of hyperglycemia in a streptozotozin-induced diabetes rat model as well as in diabetic mice. Conversely, hematoma expansion was blocked in diabetic rats by a PK inhibitor and in mice with streptozotozin-induced diabetes that were genetically engineered to lack PK.

Hematoma expansion after intracerebral PK injection was also greater in acutely hypergly-cemic nondiabetic rats and in rats intravenously treated with a 20% (wt/vol) solution of the hyperosmotic agent mannitol, indicating that plasma hyperosmolality rather than the diabetes per se may facilitate hematoma expansion when plasma glucose is elevated4.

Liu et al.4 showed that the enzymatic activity of PK is not required for its hematoma-promoting effect, thereby implying that the intrinsic pathway of coagulation does not participate in this disease state. They also excluded PK-produced bradyki-nin or the central protease of the fibrinolytic sys-tem, plasmin, as crucial mediators in this process as neither of them reproduced the hematoma-expanding effect of PK in diabetic rats4.

Instead, Liu et al.4 proposed that the hyperglycemia-dependent antihemostatic effect of PK is due to the specific inhibition of platelet activation at sites of tissue trauma (Fig. 1). Vascular injury results in the expo-sure of subendothelial collagens that are potent triggers of platelet activation and plug forma-tion. Glycoprotein VI (GPVI) is the activating collagen receptor on platelets, and its absence or inhibition abrogates collagen-induced plate-let activation in vitro and markedly reduces

with stem cell properties drives tumor growth, invasion and metastasis1.

By virtue of their relative resistance to conven-tional therapies, including radiation, chemother-apy and hormone therapies2, these cells may be responsible for treatment resistance and recur-rence. If this is the case, it will be necessary to develop strategies to effectively target the cancer

The rapid progress in understanding the molecular underpinnings of common malig-nancies has not been matched by equally

impressive progress in cancer therapeutics. As a result, most common human malig-nancies, including prostate cancer, are not curable with current treatment modalities once they spread beyond the primary sites. Accumulating evidence suggests that many human malignancies follow a ‘stem cell’ model where a subpopulation of tumor cells

Max S. Wicha is at the University of Michigan

Comprehensive Cancer Center, Ann Arbor,

Michigan, USA.

e-mail: mwicha@umich.edu

Stemming a tumor with a little miRMax S Wicha

A microRNA decreases the expression of the adhesion molecule CD44 in prostate cancer stem cells (CSCs), blocking tumor growth and metastasis in mice (pages 211–215). Systemic delivery of this negative regulator may open new avenues for targeting CSCs to halt cancer.

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successful targeting of all CSCs. Although a reduction in tumor growth and increased survival of mice with orthotopic prostate tumors treated systemically with miR34a was shown by Liu et al.6, the efficient targeting of miRNAs to all CSCs represents a formidable technical challenge.

CD44 is expressed in CSCs of various can-cers including those of the breast, prostate, ovary, colon, and head and neck, suggesting the potential use of miR34a in the treatment of these tumors. Nevertheless, the complex-ity of regulatory pathways in CSCs, as well as the heterogeneity of these cell populations, suggests that it may also be necessary to com-bine multiple CSC-targeting agents to elimi-nate this cell population.

As normal and malignant stem cells may use similar regulatory pathways, the potential detrimental effects of CSC-targeting agents on normal stem cells remains a concern. No toxicity in mice receiving systemic miR34a was shown in the study; however, it will be necessary to carefully monitor the effects of CSC-targeting agents on normal stem cells as these agents move into clinical trials. Indeed, several early stage clinical trials designed to target CSCs in leukemia, myeloma and breast cancer have been initiated. These studies will indicate whether CSCs can be safely and successfully targeted and whether targeting CSCs will result in improved therapeutic outcome.

CSCs. Furthermore, the study by Liu et al.6 supports the feasibility of directly targeting CD44-positive CSCs through systemic deliv-ery of this miRNA.

In light of these results, CD44 may not merely be a marker of CSCs, but instead it may serve a crucial functional role. Studies have suggested that the v6 isoform—CD44 adhe-sion proteins are a family of molecules gen-erated by alternative splicing—may be the most important in facilitating metastasis of CSCs9. It has previously been shown that monoclo-nal antibodies against CD44 can reduce CSC numbers in colon cancer10, breast cancer11 and leukemias12.

The potential advantage of using miRNAs, as compared to antibody-based approaches, is that, in addition to targeting CD44, miRNAs may simultaneously target other molecules that regulate CSCs. Liu et al.6 showed that miR34a also reduced the expression of other mol-ecules involved in stem cell pathways including cyclin D1, cyclin- dependent kinase-4 and cyclin- dependent kinase-6 and cMyc and NOTCH. Despite this theoretical advantage, the superi-ority of miRNAs compared to antibody-based CSC therapeutics remains to be proven.

The use of miRNAs as therapeutic agents poses a number of technical challenges, such as how to achieve efficient systemic delivery. Because, in theory, each CSC has the potential to generate a tumor, preven-tion of tumor recurrence would require the

stem cell population to improve the outcome of those individuals with advanced tumors.

Prostate cancer is the most common malig-nancy in men, with over 217,000 cases and 32,000 deaths per year in the US. Despite the initial hormone responsiveness of advanced prostate cancer, resistance to these therapies inevitably develops, highlighting the need for new therapeutic approaches. The exis-tence of CSCs has been recently described in murine3 and human prostate cancers4, where these cells have been found to drive cancer growth and metastasis. CSCs from a number of human cancers, including prostate cancer, have been characterized as expressing CD44, which is an adhesion molecule regulated by the Wnt pathway5.

In this issue of Nature Medicine, Liu et al.6 show how a microRNA, miR34a, degrades CD44, resulting in impaired tumor growth and decreased metastases in mouse models of prostate cancer. The increased survival of mice treated with a systemically delivered miR34a suggests a new strategy to target prostate CSCs, thereby blocking tumor growth and metastatic disease.

miRNAs, which contain 18–24 nucleotides, regulate gene expression by binding the 3′ untranslated region (UTR) of target mRNAs with complementary sequences, reducing mRNA stability and translation7. Previous studies have shown that miRNAs can regulate normal and malignant stem cells8 by simulta-neously regulating the expression of hundreds of genes. These miRNAs may therefore regu-late multiple pathways involved in stem cell fate decisions, including self-renewal, prolifera-tion and differentiation—pathways frequently altered in cancer.

Using miRNA expression profiling of human prostate cancer cells, Liu et al.6 found miR34a—a p53 target—to be highly regulated during prostate cancer cell differentiation. Prostate CSCs identified by their expression of CD44 expressed low levels of miR34a com-pared to CD44-negative cells. More important, miR34a was shown to decrease CD44 expres-sion by binding the 3′ UTR of CD44 mRNA.

Enforced expression of miR34a by gene transfection into prostate CSCs downregu-lated their expression of CD44, resulting in decreased clonogenicity in vitro and tumor growth in vivo. To demonstrate that systemi-cally delivered miR34A inhibited tumor growth and metastasis in prostate cancer xenografts, the authors used a liposome-based delivery system that resulted in decreased lung metas-tasis and extended animal survival6 (Fig. 1).

These studies show that miR34a, through its ability to modulate CD44 expression, represents a negative regulator of prostate

Liposomal nanovectordelivery system

CD44

CD44

Wnt

miR34amiR34a

CD44+ CSC

Metastasis to lung

TCF-Lef

Differentiated cancer cells

p53

Prostate cancer

CSC

Invasion

Blood vessels

β-cat

CD44 mRNA

Figure 1 miR34a inhibits prostate CSCs, which drive the growth and metastatic potential of prostate cancers. These CSCs express CD44, a cell surface adhesive protein regulated by the Wnt pathway. The p53-regulated microRNA miR34a inhibits CD44 expression by binding the 3′ UTR of CD44 mRNA. Liu et al.6 now show that systemic delivery of miR34a using a liposomal nanovector delivery system can inhibit growth and metastasis of prostate cancer xenografts in a mouse model, significantly prolonging survival. β-cat, β-catenin; TCF-Lef, T-cell factor/lymphocyte enhancer factor.

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9. Pacheco-Rodriguez, G. et al. Cancer Res. 67, 10573–10581 (2007).

10. Todaro, M. et al. Gastroenterology 138, 2151–2162 (2010).

11. Marangoni, E. et al. Br. J. Cancer 100, 918–922 (2009).

12. Jin, L. et al. Nat. Med. 12, 1167–1174 (2006).

3. Lukacs, R.U. et al. Cell Stem Cell 7, 682–693 (2010).4. Wang, X. et al. Nature 461, 495–500 (2009).5. Tu, L.C. et al. Curr. Stem Cell Res. Ther. 4, 147–153

(2009).6. Liu, C. et al. Nat. Med. 17, 211–215 (2011).7. Grimson, A. et al. Mol. Cell 27, 91–105 (2007).8. Zimmerman, A.L. & Wu, S. Cancer Lett. 300, 10–19

(2010).

COMPETING FINANCIAL INTERESTSThe author declares competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/naturemedicine/.

1. Wicha, M.S., Liu, S. & Dontu, G. Cancer Res. 66, 1883–1890 (2006).

2. Li, X. et al. J. Natl. Cancer Inst. 100, 672–679 (2008).

an anti-inflammatory phenotype in Nlrp3−/− mice. Interferon-γ from activated T cells can also promote inflammation in adipose tissue of obese mice. The authors showed that, compared with adipose tissue from HFD-fed mice, Nlrp3−/− adipose tissue contained fewer activated T cells and had diminished produc-tion of interferon-γ5.

Vandanmagsar et al.5 propose a model whereby activation of the Nlrp3 inflammasome in macrophages may be important for patho-logical inflammation during obesity. Consistent with ATMs as the source of the caspase-1– dependent inflammation, they find that ATMs but not adipocytes express Nlrp3 and Asc5. By contrast, Stienstra et al.4 found that Nlpr3 and caspase-1 also have important roles in adi-pocytes. The differences between the results from the two studies might be attributed to the different assays used. Stienstra et al.4 showed that caspase-1 expression increases during adi-pogenesis, caspase-1 is activated in adipocytes and, in the absence of Nlrp3 and caspase-1, adipocytes have enhanced differentiation and increased insulin signaling. Moreover, caspase-1–deficient mice have higher fat oxi-dation rates4. Collectively, these data suggest that caspase-1 is a crucial regulator of adipo-genesis and tissue mass through its effects on differentiation and insulin signaling, as well as inflammation. Recently, Zhou et al.6 suggested that Nlrp3 inflammasome activation in pan-creatic islets may also contribute to disruption of glucose homeostasis. Thus, expression of the Nlrp3 inflammasome in multiple tissues may contribute to increased inflammation and other pathogenic aspects of obesity (Fig. 1a). These observations are also consistent with a proposed model in which metaflammation involves a complex interaction between meta-bolic and immune cells to disrupt metabolic

Chronic, low-grade, metabolic inflammation—‘metaflammation’—is crucial in the pathogen-esis of obesity and associated disorders such as insulin resistance. Obese humans and mice show chronic inflammation, and blocking of various inflammatory pathways improves insulin action and reduces metabolic pathogenesis. Although metaflammation is triggered by metabolic sig-nals, how nutrient excess interfaces with genetic factors to initiate and sustain inflammation in metabolic tissues is not completely understood1. Defining the molecular triggers and the cellu-lar sensors that induce inflammation is thus of theoretical and therapeutic value.

The Nlrp3 inflammasome is a cytosolic protein complex consisting of the regulatory subunit Nlrp3, the adaptor Asc and the effec-tor subunit caspase-1 (refs. 2,3). It is activated by ‘danger signals’ such as components of necrotic cells or damaged tissues, or noxious exogenous substances. Nlrp3 inflammasome activation leads to caspase-1–dependent pro-duction of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. Unlike many other cyto kines, IL-1β and IL-18 are synthe-sized as latent cytosolic precursors that require caspase-1 processing for secretion. Mice lack-ing Nlrp3, Asc or caspase-1 develop attenu-ated inflammation and its related pathology in gout, atherosclerosis, arthritis, silicosis, ischemia and liver toxi city, indicating that the Nlrp3 inflammasome is important in ‘sterile inflammation’ that occurs in the absence of overt infection2,3.

Two recent studies by Stienstra et al.4 and Vandanmagsar et al.5 show that the Nlrp3 inflammasome contributes to metabolic pathogenesis during obesity. Knockout of the Nlrp3 inflammasome significantly protects a mouse model of high-fat diet (HFD)-induced obesity from increased adiposity, insulin resis-tance, glucose intolerance and inflammation. Therefore, manipulation of the Nlrp3 inflam-masome might be a possible strategy for treat-ing obesity and related pathologies.

The Nlrp3 inflammasome was first sug-gested to regulate adiposity and insulin sen-sitivity during obesity by Zhou et al.6, who showed that Nlrp3−/− mice show improved glucose homeostasis when on a HFD6. The new studies of Stienstra et al.4 and Vandanmagsar et al.5 corroborate these findings and substan-tially expand the earlier analysis. First, the expression of Nlrp3 inflammasome subunits in adipose tissue correlates directly with body weight in mouse models and obese individuals with type 2 diabetic mellitus5. Second, a HFD activates the Nlrp3 inflammasome, as shown by increased caspase-1 processing and IL-1β secretion. Moreover, mice lacking caspase-1 or Nlrp3 have increased insulin signaling in adipose tissue, liver and muscle; improved systemic glucose homeostasis; and reduced adipose tissue mass4,5. Finally, Nlrp3−/− mice are protected against the accumulation of lipid deposits in the liver.

The study from Vandanmagsar et al.5 also links the improved metabolic phenotype of Nlrp3−/− mice to attenuated production of inflammatory products. Nlrp3−/− mice have lower serum concentrations of IL-18 and decreased IL-1β expression in their adipose tissue. Adipose tissue macrophages (ATMs) may contribute to obesity-associated inflam-mation and insulin resistance7 and showed

Tiffany Horng and Gökhan S. Hotamisligil are in the

Department of Genetics and Complex Diseases,

Harvard School of Public Health, Boston,

Massachusetts, USA.

e-mail: thorng@hsph.harvard.edu or

ghotamis@hsph.harvard.edu

Linking the inflammasome to obesity-related diseaseTiffany Horng & Gökhan S Hotamisligil

Chronic inflammation is associated with obesity, but the pathways that mediate this phenomenon are not fully characterized. The nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3 (Nlrp3) inflammasome functions as a sensor to detect danger signals and induce downstream inflammatory signaling that contributes to obesity-associated conditions such as insulin resistance (pages 179–188).

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