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phase 0 trial is widely evaluated, we expect that pharmacodynamic-driven early therapeutic studies, as was the case for pharmacokinetic monitoring nearly 20 years ago21, will become a routine part of future early-phase oncological drug development.
Robert Kinders, Larry Rubinstein, Ralph E. Parchment, Anthony J. Murgo, Jerry Collins, Oxana Pickeral, Jennifer Low, Sherry Yang, Joseph E. Tomaszewski and James H. Doroshow are at the Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland, USA. Shivaani Kummar, Seth M. Steinberg, Martin Gutierrez, Lee Helman, Robert Wiltrout and James H. Doroshow are at the Center for Cancer Research, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland USA. Correspondence to J.H.D. e-mail doroshoj@mail.nih.gov doi:10.1038/nrc2066
1. US Food and Drug Administration. Guidance for Industry, Investigators, and Reviewers. US Food and Drug Administration [online], http://www.fda.gov/cder/ guidance/7086fnl.pdf (2006). Kola, I., Landis, J. Can the pharmaceutical industry reduce attrition rates? Nature Rev. Drug Discov. 3, 711715 (2004). Johnson, J. I. et al. Relationships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials. Br. J. Cancer 84, 14241431 (2001). Olson, H., Betton, G., Robinson D. et al. Concordance of the toxicity of pharmaceuticals in humans and in animals. Regul. Toxicol. Pharmacol. 32, 5667 (2000). Tomaszewski, J. E., Smith, A. C., Covey, J. M., Donohue, S. J., Rhie, J. K. & Schweikart, K. M. in Anti-Cancer Drug Design (ed. Baguley, B. C.) Chpt 17, 301328 (San Diego, Academic Press, 2001). Tomaszewski, J. E., Doroshow, J. H. in Molecular Targets in Oncology (ed. Antman, K.) (Humana Press, Totowa, USA, in the press). Fox, E., Curt, G. A. & Balis, F. M. Clinical trial design for target-based therapy. Oncologist. 7, 401409 (2002). Millar, A. W., Lynch, K. P. Rethinking clinical trials for cytostatic drugs. Nature Rev. Cancer. 3, 540545 (2003). Rothenberg, M. L., Carbone, D. P. & Johnson, D. H. Improving the evaluation of new cancer treatments: challenges and opportunities. Nature Rev. Cancer. 3, 303309 (2003). Kummar, S., Gutierrez, M. E., Doroshow, J. H. & Murgo, A. J. Drug development in oncology: classical cytotoxics and molecularly targeted agents. Br. J. Clin. Pharmacol. 62, 1526 (2006). Workman, P. et al. Minimally invasive pharmacokinetic and pharmacodynamic technologies in hypothesistesting clinical trials of innovative therapies. J. Natl Cancer Inst. 98, 580598 (2006). Parulekar, W. R., Eisenhauer, E. A. Phase I trial design for solid tumor studies of targeted, non-cytotoxic agents: theory and practice. J. Natl. Cancer Inst. 96, 990997 (2004). Bartlett, J. M. Pharmacodiagnostic testing in breast cancer: focus on HER2 and trastuzumab therapy. Am. J. Pharmacogenomics 5, 303315 (2005). Lehmann, F., Lacombe, D., Therasse, P., Eggermont A. M. M. Integration of translational research in the european organization for research and treatment of cancer research (EORTC) clinical trial cooperative group mechanisms. J. Transl. Med. 1, 2 (2003). Hidalgo, M., and Eckhardt, S. G. Matrix metalloproteinase inhibitors: how can we optimize their development? Ann. Oncol. 12, 285287 (2001). Moore, M. J. et al. Comparison of gemcitabine versus the matrix metalloproteinase inhibitor BAY 129566 in patients with advanced or metastatic adenocarcinoma of the pancreas: a phase III trial of the National Cancer Institute of Canada Clinical Trials Group. J. Clin. Oncol. 21, 32963302 (2003). Van Den Bossche, B., Van de Wiele, C. Receptor imaging in oncology by means of nuclear medicine: current status. J. Clin. Oncol. 22, 35933607 (2004). 18. Sun, H. et al. Imaging the pharmacokinetics of [F-18]FAU in patients with tumors: PET studies. Cancer Chemother. Pharmacol. 57, 343348 (2006). 19. Liu, G. et al. Dynamic contrast-enhanced magnetic resonance imaging as a pharmacodynamic measure of response after acute dosing of AG-013736, an oral angiogenesis inhibitor, in patients with advanced solid tumors: results from a phase I study. J. Clin. Oncol. 23, 54645473 (2005). 20. Collins, J. M. Imaging and other biomarkers in early clinical studies: one step at a time or re-engineering drug development? J. Clin. Oncol. 23, 54175419 (2005). 21. Collins, J. M., Grieshaber, C. K. & Chabner, B. A. Pharmacologically guided phase I clinical trials based upon preclinical drug development. J. Natl. Cancer Inst. 82, 13211326 (1990). mention of trade names, commercial products or organizations imply endorsement by the US Government. This research was supported by the Division of Cancer Treatment and Diagnosis and the Center for Cancer Research of the National Cancer Institute.

Competing interests statement

The authors declare no competing financial interests.

FURTHER INFORMATION
National Cancer Institute: http://www.cancer.gov Division of Cancer Treatment and Diagnosis: http://www. cancer.gov/dctd Center for Cancer Research, National Cancer Institute: http://ccr.cancer.gov Cancer Therapy and Evaluation Program, National Cancer Institute Guidelines for Correlative Studies in Clinical Trials: http://ctep.info.nih.gov/guidelines/index.html Office of Biorepositories and Biospecimen Research, National Cancer Institute: http://biospecimens.cancer.gov Steps for pharmacodynamic assay development: http://dtp.nci.nih.gov/docs/phase0/PharmacoDynamicAssay Develoment.html Access to this links box is available online.

Acknowledgements
This project has been funded in whole or in part with federal funds from the US National Cancer Institute, National Institutes of Health. The content of this publication does not necessarily reflect the views or policies of the US Department of Health and Human Services, nor does any

TUMOUR MICROENVIRONMENT OPINION

2.

3.

4.

The tumour microenvironment as a target for chemoprevention

Adriana Albini and Michael B. Sporn

5.

6.

7. 8.

9.

10.

Abstract | New data indicate that primary dysfunction in the tumour microenvironment, in addition to epithelial dysfunction, can be crucial for carcinogenesis. These recent findings make a compelling case for targeting the microenvironment for cancer chemoprevention. We review new insights into the pathophysiology of the microenvironment and new approaches to control it with chemopreventive agents. The microenvironment of a cancer is an integral part of its anatomy and physiology, and functionally, one cannot totally dissociate this microenvironment from what have traditionally been called cancer cells. Finally, we make suggestions for more effective clinical implementation of this knowledge in preventive strategies.
The continuing pandemic of cancer deaths requires a reassessment of our basic assumptions about the nature of cancer and how to control it. Directly bearing on this, there has been an explosion of new information about the tumour microenvironment, which is a complex system of many cell types, including endothelial cells and their precursors, pericytes, smooth-muscle cells, fibroblasts of various phenotypes, myofibroblasts, neutrophils and other granulocytes (eosinophils and basophils), mast cells, T, B and natural killer lymphocytes, and antigenpresenting cells such as macrophages and dendritic cells. All these cells can participate in tumour progression. If the process of carcinogenesis and its end result, invasive and metastatic cancer, are viewed as the maladaptive response of an entire tissue or organ to both genetic and epigenetic stress13, then knowledge and control of the immediate microenvironment within a developing tumour become as important as the corresponding knowledge and control of the dysfunctional epithelial cells within that tumour. New data from studies on the tumour microenvironment suggest that we might need to revise the very definition of the term carcinoma (currently defined in classical terms as a malignancy derived from epithelial cells), and that to control cancer in the future, we need to regard carcinogenesis and carcinomas as phenomena that occur in tissues, not in individual cancer cells. From this perspective, the microenvironment becomes an integral, essential part of the cancer.

11.

12.

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a Acute inflammation
Stimulus (injury, infection)

b Carcinogenesis
Epithelia

Mast cells

Basal lamina Stroma ECM

Neutrophils Mutagenesis Proliferation Inflammation Macrophages

Effector immune cells

Hyperplasia Dysplasia Further mutagenesis Inflammation

there might be molecular lesions in cells of the microenvironment and in epithelial cells themselves. Although there are many excellent recent reviews on the tumour microenvironment, and it has recently been proposed as a target for therapy4,5, the application of chemoprevention to control the tumour microenvironment during the early stages of carcinogenesis has so far not received concerted attention. This Perspective is not intended to be a comprehensive review; rather, it attempts to indicate new directions and to highlight new priorities for future research. Although the microenvironment is also involved in the genesis of leukaemias and sarcomas, this Perspective deals only with carcinomas (the most common forms of malignancy), which originate in epithelia and their associated stroma.
New insights The concept that the microenvironment of a developing tumour is a crucial regulator of carcinogenesis was originally proposed by Paget in his famous seed and soil hypothesis. Recent data indicate that carcinogenesis and tumour angiogenesis result not only from the interaction of cancer cells with endothelial cells of various origin (vascular or lymphatic), but that surrounding normal stromal and inflammatory cells and tissue also have a crucial role in directing the formation of the blood vessels that nourish a developing tumour. This newer version of an old hypothesis now enables us to consider that stromal cells and their associated matrix, as well as cells of the immune system, have important roles in tumour angiogenesis and carcinogenesis69. There is a discrete order of events in physiologically acute inflammation and repair10 (FIG. 1a). However, these events become chaotically disorganized during chronic unresolved inflammation and carcinogenesis (FIG. 1b). This chaotic local microenvironment has led to the suggestion that tumours are wounds that do not heal11. The constant disruption of homeostasis by proliferating epithelial cells produces a chronic inflammatory reaction, which is an abortive attempt to re-establish homeostasis through tissue remodelling12. However, the classic players in acute inflammation (granulocytes, macrophages, endothelial cells and fibroblasts) that ordinarily lead to the resolution of a wound through an orderly series of events, instead react paradoxically to the presence of dysfunctional epithelial cells by promoting their survival and replication12. This process includes inflammatory angiogenesis.

Angiogenesis

Fibroblasts and fibrosis

Angiogenesis Uncontrolled growth Progression

Tissue remodelling

Figure 1 | Correlations and contrasts between acute inflammation and carcinogenesis. a | The sequence of events in acute inflammation and tissue repair (see REF. 9 for a more detailed description). The process of inflammation initiates a series of catabolic and anabolic processes that occur in a defined order, first eliminating foreign pathogens and then remodelling tissue, thereby establishing homeostasis. Shown in this figure are: first, the activation of resident cells (mast cells, resident macrophages and dendritic cells) and rapid entry of granulocytes in response to injury; second, further recruitment of macrophages; third, infiltration of effector immune cells (lymphocytes) to enable specific immune responses; fourth, the recruitment and activation of mesenchymal cells such as endothelial cells and fibroblasts to form new blood vessels and a collagenous matrix; and fifth, tissue remodelling. In its initial stages, inflammation is an aggressive state that can destroy both exogenous pathogens and host tissues; this is followed by a switch to a state that promotes cell survival and tissue regeneration. b | Carcinogenesis as the chaotic disorganization of inflammation and repair. In contrast to the orderly series of events shown in part a, during chronic unresolved inflammation and carcinogenesis these events are chaotically disorganized and homeostasis is not achieved. During carcinogenesis, both epithelial and stromal elements might initially undergo alterations that promote epithelial cell proliferation and mutation. This alteration in tissue homeostasis can in turn lead to an inflammatory response, which then further promotes tumour growth through the activation of the surrounding stroma, especially neovascularization. Continued hyperplasia and dysplasia eventually lead to an invasive neoplastic state. The process shown here has been described with the metaphor that tumours are wounds that do not heal11. Both epithelial cells and cells of the microenvironment are targets for chemopreventive agents at all the steps shown. Chemopreventive agents might be anti-mutagenic, anti-proliferative, anti-inflammatory or anti-angiogenic, therefore restoring tissue homeostasis that has been disrupted during carcinogenesis. Early genetic or epigenetic changes in epithelia or stroma are shown in yellow. ECM, extracellular matrix.

Therefore, it is necessary to consider the microenvironment of a cancer and its associated abnormal epithelium as a functional whole. In addition to existing preventive efforts directed at dysfunctional epithelium, we propose that it is now essential to develop new chemopreventive measures

targeted to the tumour microenvironment to control the process of carcinogenesis and prevent cancer. The implementation of this new approach, in addition to the accepted approach of targeting dysfunctional epithelial cells, becomes increasingly important as new data, summarized below, indicate that

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Continuing angiogenesis driven by myeloid inflammatory cells is well recognized as an important component of chronic inflammatory disorders such as rheumatoid arthritis13; recent data indicate that it can also have a key role in the progression of cancer12,14,15. Epithelial cells can become dysfunctional if their microenvironment is severely perturbed. This control of epithelia by their stroma is a default condition in normal tissues, as adult organs do not change composition, size or shape by uncontrolled remodelling. Therefore, rather than being a passive reaction to the cancer cell, the microenvironment might be a primary active factor in determining whether dysfunctional epithelial cells will continue to grow and invade in a particular local milieu or, alternatively, merely become an indolent micro-hyperplasia or even be eliminated. Recent observations suggest that paracrine control in a developing tumour depends not so much on the intrinsic identity of its cellular components, but on the phenotypic expression of specific factors that either promote or suppress carcinogenesis. For example, the phenotype of tumourassociated macrophages (termed M2) might be quite different compared with normal macrophages (termed M1)16, and additional subsets based on microenvironmental cues have been proposed17. M2 macrophages might also directly influence tumour cells themselves and promote aggressiveness; for example, macrophages might have a key role in freeing prostate cancer cells from androgen dependence18, a crucial step in malignant progression. Neutrophils are another important cell in the microenvironment. They can promote tumour destruction, but might also have an opposite effect and increase the growth of tumour cells12,19. Several chemokines that act on the CXCR2 receptor (also known as the interleukin 8 receptor, ) are known to be angiogenic factors produced within tumours, and this seems to involve the neutrophil-dependent release of vascular endothelial growth factor A (VEGFA)20,21. A cascade of events that includes neutrophil recruitment followed by VEGF and matrix metalloproteinase 9 (MMP9) release is induced by these chemokines, which subsequently leads to endothelial cell invasion and vessel formation19. Experimentally, events such as Ras transformation have been shown to enhance this cascade15. Furthermore, fibroblasts in a tumour can also promote tumorigenesis22. These include, among others, tumour fibroblasts such as carcinoma-associated fibroblasts
Table 1 | Molecular targets and chemopreventive agents in the microenvironment

Molecular targets
Oestrogen receptors Akt and NFB NRF2KEAP1 COX2 COX1/2 Histone deacetylases TGF pathway HIF1 STATs VEGF

Chemopreventive agents
Tamoxifen; raloxifene; arzoxifene Curcumin; N-acetyl cysteine; silibinin; xanthohumol; deguelin; EGCG; resveratrol Sulphoraphane; oltipraz Rofecoxib; celecoxib; EGCG Aspirin and other NSAIDs Sulphoraphane CDDO-Imidazolide EGCG; resveratrol; apigenin; sulphoraphane CDDO-Imidazolide Sulphoraphane; EGCG; fenretinide

This is not intended as a comprehensive list, but only to show specific targets in the microenvironment, and indicate specific agents that interact with these targets, as discussed in the text. CDDO, 2-cyano-3,12dioxooleana-1,9-dien-28-oic acid; COX2, cyclooxygenase 2; EGCG, epigallocatechin-3-gallate; HIF1, hypoxiainducible factor 1; KEAP1, kelch-like ECH-associated protein 1; NFB, nuclear factor B; NSAIDs, non-steroidal anti-inflammatory drugs; STATs, signal transducers and activators of transcription; TGF, transforming growth factor-; VEGF, vascular endothelial growth factor.

(CAFs) that increase tumour growth and angiogenesis through the expression of CXCL1223. CXCL12, a chemokine ligand for the widely distributed chemokine receptor CXCR4, promotes angiogenesis by recruiting marrow-derived precursors that contribute to vessel development. Similar to tumour macrophages, CAFs have a distinct phenotype compared with normal fibroblasts22, which neither produce CXCL12 nor induce angiogenesis, and only marginally contribute to tumour growth23. Another important mesenchymal cell is the myofibroblast, which has different functions in different organs. Myofibroblasts abut on epithelial or glandular cells and produce several growth factors, cytokines, chemokines and extracellular matrix components. They express many receptors and are a source and target of soluble mediators24. Myofibroblasts are activated when tissue integrity is compromised and might actively increase tumour growth and expansion once tissue invasion begins, especially in the colon and liver25. Finally, adipocytes have also been recognized recently as important components of the tumour microenvironment. The explosion of concern about obesity in the United States has heightened awareness about the relationship between fat cells and cancer. Obesity constitutes an independent risk factor for several types of cancer26,27, especially those with a strong inflammatory component. Adipocytes are important producers of various biologically active molecules that influence inflammation and angiogenesis, and adipose tissue is an authentic endocrine and paracrine organ that secretes several

specific cytokines (the adipokines leptin, adiponectin, resistin and visfatin), as well as matrix metalloproteinases that promote the inflammatory process and angiogenesis28. A key question remains: which comes first, the dysfunction of epithelial cells or the dysfunction of their microenvironment29? There is no intrinsic reason to postulate that carcinogenesis necessarily begins with epithelial dysfunction, especially because underlying mesenchymal cells control epithelial differentiation during embryogenesis30,31. Genetic changes in stromal cells adjacent to breast cancer cells have been described in many publications, although these might not always be present (see REF. 32 for further data). Most recently, new studies on clinical material derived from both breast and colon carcinomas indicate that genetic changes occur in the stroma during the earliest stages of human carcinogenesis33,34, and suggest that a genetically unstable stroma might facilitate further genetic instability in the overlying epithelium33,34. Clinical histopathology data, which show lymphoma-specific genetic abnormalities in the microvascular endothelial cells in B-cell lymphomas, support these findings35. Furthermore, animal studies have found cytogenetic abnormalities in the endothelium of lung tumours36, although the primacy of these endothelial lesions is still uncertain. However, given the exposed anatomic location of capillaries in the alveoli of the lung, it would not be surprising, for example, if alveolar capillary endothelial cells in smokers were mutagenized by tobacco carcinogens. Furthermore, recent murine studies, which are discussed more below, have shown that initial genetic lesions

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Hyperplastic and dysplastic foci

Neutrophil ECM
Angioprevention

Macrophage

Fibroblast
Angiogenesis

Apoptotic cell

Apoptosis = proliferation Limited inflammation Slow progression Chronic or subclinical disease

Uncontrolled growth Progression Invasion Metastasis

Figure 2 | Angioprevention as a strategy for chemoprevention. The foci of hyperplastic and dysplastic cells are microscopic, non-invasive lesions. It is known that people might have many such lesions in different organs but still be asymptomatic. These silent lesions will usually not progress until an angiogenic switch occurs, which enables significant neovascularization that results in the progression of the tumour, invasion and metastasis108. Angioprevention seeks to interrupt this sequence of events by blocking the formation of new blood vessels. Many chemopreventive drugs function by various mechanisms to suppress tumour angiogenesis, as discussed in the text. If hyperplastic and dysplastic foci do not become vascularized, then a steady state might be achieved in which programmed cell death (apoptosis) and proliferation are in balance, resulting in lesions that are continually asymptomatic and never life-threatening22. ECM, extracellular matrix.

confined to the stromal compartment of the gastrointestinal tract are sufficient to induce epithelial carcinomas37. A recent and provocative hypothesis suggests that chronic tissue hypoxia within the tumour microenvironment might favour stromal mutagenesis, which subsequently promotes further mutagenesis and genetic instability in the adjacent epithelium38. All of these findings require a re-evaluation of previous notions that the stroma has only a secondary role in the genesis of carcinomas.
Approaches to control As the microenvironment has such a crucial role in carcinogenesis and metastasis, it represents a crucial target not only for cancer therapy but also for preventive strategies. The rationale for chemoprevention is simple and straightforward: it is preferable to fix something in its early stages of dysfunction, before it is beyond repair. The microenvironment of a developing carcinoma is an obvious target for chemoprevention, although in the past, most attention in cancer research has been given to controlling the dysfunctional epithelium. There is already a wealth of information about specific cells and molecules in the tumour microenvironment that are targets for cancer therapy at present4,5.

All of these targets should now be investigated for their use in chemoprevention. The overall topic of chemoprevention has been reviewed at length many times3943, and we will not do this here. Rather, we will briefly summarize recent progress that indicates that the effects of chemopreventive agents on the microenvironment are an important aspect of their preventive action, and that many classes of agents previously shown to have significant chemopreventive actions on epithelia also have similar useful actions on the microenvironment. We will discuss selected examples of important cellular and molecular targets in the microenvironment, and the drugs that interact with these targets. Cellular targets for chemoprevention in the microenvironment. During the earliest stages of carcinogenesis, all of the cells in the microenvironment are targets for chemoprevention, because dysfunctional cytokine networks that promote carcinogenesis are established in these cells. Chemopreventive agents can be directed towards regulating and normalizing the function of macrophages, granulocytes, lymphocytes, endothelial cells and their associated pericytes, as well as fibroblasts. Owing to space limitations only selected examples are cited.

Macrophages are crucial partners for tumour cell migration, invasion and metastasis. These contextually responsive cells of the microenvironment can either promote the formation of extracellular matrix or speed up its destruction, and are primary sources of gaseous signalling molecules such as reactive oxygen and nitrogen species that can be mutagenic and carcinogenic, as well as prostaglandins that regulate inflammation. As such, they have become targets for many pharmacological agents that include suppressors of nitric oxide synthase (iNOS) synthesis and function and suppressors of cyclooxygenase 2 (COX2) synthesis and function, in addition to the antagonists of the cytokines they produce. There is a vast amount of literature on this topic4448. Less attention has been given to lymphocytes as targets for chemoprevention. However, recent studies on the role of Smad signalling (the key signal transduction pathway for transforming growth factor- (TGF)) in T cells, and its importance for the suppression of gastrointestinal cancer, now show that the selective loss of SMAD4dependent signalling in stromal T cells leads to spontaneous epithelial cancers throughout the gastrointestinal tract of mice, whereas deletion of the Smad4 gene in the epithelium only does not37. Loss of TGF signalling is a frequent occurrence in early stages of gastrointestinal cancer in humans, and these new studies therefore suggest that increased stromal TGF signalling could be chemopreventive during the early stages of carcinogenesis. New synthetic triterpenoids, such as the oleanolic acid derivatives CDDO (2-cyano3,12-dioxooleana-1,9-dien-28-oic acid) and its imidazolide derivative, have been shown to facilitate Smad signalling and to increase the expression of TGF-dependent genes, such as plasminogen activator inhibitor-1 (PAI1) and the TGF type 2 receptor itself 49. Further studies on the possible use of these agents to prevent cancer in appropriate mouse models of carcinogenesis seem to be warranted. Molecular targets for chemoprevention in the microenvironment and agents that regulate these targets. There are many molecular targets in the microenvironment, and many drugs that are known to interact with these targets (TABLE 1). Transcription factors and their associated regulatory proteins, by virtue of their pleiotropic actions on many genes, are an obvious target for chemoprevention, especially as carcinogenesis typically involves the dysfunction of several genes. Although this is contrary to conventional approaches

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to finding monofunctional magic bullets, it represents a more realistic approach to the actualities of carcinogenesis and the increasing problem of emerging resistance to monofunctional agents42. With respect to the microenvironment, signal transducers and activators of transcription (STATs), nuclear factor B (NFB) and hypoxia-inducible factor 1 (HIF1) are particularly attractive targets, as these three transcription factors are known to be intimately involved in regulating inflammation, wound healing and angiogenesis. STATs are constitutively overexpressed in many cancers; their phosphorylation, which is required for transcriptional activity, is regulated by a set of kinases (JAKs), phosphatases and binding proteins, all of which are targets for drug development50. Synthetic triterpenoids, which have recently been shown to be potent agents for the chemoprevention of adenocarcinoma of the lung in mice (K. Liby, unpublished observations), have also been shown to be highly active in regulating the phosphorylation of STAT3 and STAT5 (REF. 51). Gene-array studies have shown that STAT3 is a potent inducer of many genes involved in wound healing and repair52. Particularly striking is the ability of STAT3 to increase the expression of fibrinogen genes in lung cancer cells; fibrinogen can increase tumour cell growth and metastasis by controlling cell adhesion, invasion, angiogenesis and chemotaxis52,53. Similarly, the transcriptional activity of STAT3 is markedly upregulated in vivo in the mouse lung during chronic inflammation, and the activation of STAT3 is found at the leading edge of human lung cancers, again suggesting a role for STAT3 in stromal invasion52. Therefore, STATs regulate processes that are crucial for carcinogenesis in the microenvironment, and are ideal targets for the development of new chemopreventive agents. The crucial role of NFB and its associated proteins in many aspects of inflammation47,54 indicates that it, too, is an important target for chemoprevention directed at the microenvironment. The NFB family of transcription factors and the various kinases that regulate NFB (such as phosphatidylinositol 3-kinase (PI3K)Akt), besides participating in the regulation of many genes required for cell growth, survival and invasion, are strongly pro-inflammatory up-stream of COX2. Although these effects have been extensively documented in tumour cells, it is now apparent that they are also exerted on components of the microenvironment. Chemopreventive agents, some of which are under investigation in
IGF1 HGF VEGF

IGF1R Endothelial cell Akt mTOR p70S6K NFB IB RAC1 PI3K

MET

VEGFR GRB2

NADPH oxidase

SOS ROS Ras Raf MEK

HIF1

VEGF MET

HIF1 HIF1

MMPs AP1 NFB IL-8 E-selectin

ERK

Figure 3 | Molecular targets for chemoprevention of angiogenesis (angioprevention). Growth factors and their kinase receptors are frequent targets for cancer chemotherapy. These targets exist in the cells of the tumour microenvironment and epithelial cells, and they are targets for chemopreventive agents. The endothelial cell is shown here as an example of this strategy, with possible chemoprevention targets shown in red. Several growth factors, such as insulin-like growth factor 1 (IGF1), hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF), stimulate the proliferation of otherwise quiescent endothelial cells through the activation of their cognate receptors. These cytokines activate paracrine or autocrine loops in the endothelial cells, resulting in neovascularization of the tumour. Several chemopreventive agents have also been shown to target signalling molecules or cascades that are downstream of the original receptors, such as phosphatidylinositol-3 kinase (PI3K), Aktmammalian target of rapamycin (mTOR) and nuclear factor B (NFB), as well as the generation of reactive oxygen species (ROS) and hypoxia-inducible factor 1 (HIF1). The activation of NFB or HIF1, which are transcription factors, leads to the synthesis of pro-inflammatory and angiogenic factors. NFB regulates many inflammatory genes; HIF1 is a master regulator of VEGF and is induced by hypoxic conditions. Other potential targets downstream of the growth-factor receptors are Ras and extracellular signal-regulated kinase (ERK). See REFS 11,12,56,5861,63,68,70,71,80,91,92 for details. AP1, activator protein 1; GRB2, growth factor receptor-bound protein 2; IL8, interleukin 8; MMP, matrix metalloproteinase.

clinical trials, such as curcumin55, N-acetyl cysteine56, EGCG (epigallocatechin-3-gallate) from green tea56, silibinin57, xanthohumol58 and deguelin59 all regulate Akt and NFB in endothelial cells. Many of these agents have also been found to be potent inhibitors of angiogenesis, and the repression of angiogenesis seems to be a key mechanism for the inhibition of tumour growth in animal models12. The anti-angiogenic effects of sulphoraphane60 have been linked to the inhibition of NFB61. Endothelial NFB activity correlates with angiogenesis62, whereas its inhibition is associated with the suppression of angiogenesis56,58,63. New drug discovery directed at regulating NFB function is a highly active field of investigation, and will undoubtedly generate newer agents for chemoprevention. Angiogenesis dependent on inflammation seems to be a central force in tumour growth and expansion12, a concept that is reinforced by evidence that the inhibition of inflammation prevents angiogenesis. Akt

and NFB activation are associated with the downstream upregulation of survivin, an apoptosis inhibitor, in tumour cells64; this has been correlated with angiogenic potential. This observation also relates to the inhibition of the AktNFBsurvivin axis in endothelial cells, as previously suggested in chemoprevention studies57. Survivin is upregulated in endothelial cells in response to VEGF65,66, and vaccination against survivin inhibits angiogenesis67. Therefore, targeting a restricted signalling pathway can repress inflammatory angiogenesis, a process we have called angioprevention68 (FIG. 2). It remains to be determined if the NFB pathway can also target other host components: can it switch macrophage phenotypes away from the carcinogenic M2 profile, or can it suppress the aggressive CAF phenotype? Could chronic inhibition of the NFB pathway repress the colonization of distant sites by bone marrow precursor cell clusters and therefore prevent metastasis?

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Similarly, HIF1 a master regulator in the control of tissue homeostasis, crucial in adaptive responses to tissue oxygenation including energy status, glucose and iron metabolism as well as growth factor signalling69,70 is a key target for the prevention and treatment of cancer. Although HIF1 contributes to an invasive, lethal phenotype in the cancer cell71, in the microenvironment, metabolic responses activated by HIF1 are required for the infiltration of myeloid cells at early stages of inflammation72. The HIF1 subunit is currently the target of significant drug development for therapy. Recent studies with many agents indicate that HIF1 is an important target for chemoprevention. For example, the potent chemopreventive agent sulphoraphane controls HIF1 function in endothelial cells by inhibiting expression of HIF1 and HIF1-regulated genes60. Sulphoraphane is also a histone deacetylase inhibitor73. In addition to being promising chemopreventive agents74, histone deacetylase inhibitors also inhibit HIF independent of the direct acetylation of HIF1 by promoting HIF1 degradation75,76. Recent data indicate that other chemopreventive agents, including green-tea extracts and purified EGCG77, resveratrol78 and apigenin79, also target HIF1. HIF1 is therefore at the crossroads of the main signalling pathways involved in oncogenic tissue remodelling, as its activation promotes both angiogenesis and inflammation80. However, a word of caution needs to be introduced here, as in some contexts the inhibition of either HIF1 or the related factor HIF2 can promote tumour growth81,82. These bifunctional actions of HIFs seem to depend on the context of the microenvironment of tumour cells81,82. The transcription factor NRF2, together with its associated inhibitor kelch-like ECH-associated protein 1 (KEAP1), are other factors that are targets for chemopreventive agents. This system is a primary sensor of oxidative or electrophilic stress, both of which have important roles in the microenvironment83. NRF2 upregulates the transcription of an entire battery of phase 2 enzymes that protect both epithelium and stroma from oxidative and electrophilic stress, and serve to exert an inhibitory tone on mutagenic activity and the inflammatory process. Some of these phase 2 enzymes include glutathione transferases, which detoxify mutagenic electrophiles, as well as superoxide dismutases and catalase, which deactivate reactive oxygen species. Heme oxygenase-1, which is strongly anti-inflammatory84, is another important phase 2
Table 2 | Two scorecards for risk assessment for cardiovascular disease or cancer

Established scorecard for cardiovascular risk (European Society of Cardiology) Personal and lifestyle components
Age and sex Smoking

Proposed new scorecard for cancer risk

Age and sex Smoking Exercise Dietary habits Obesity Reproductive history Use of drugs such as NSAIDs

Genetic analysis

Family history

Family history Oncogene and/or tumour-suppressor mutations Metabolic activity polymorphisms DNA adducts DNA oxidative damage score Proteomics score: tissue proteomics; serum proteomics Microbiopsy dysplasia score Hormone status Chronic infection (for example, Helicobacter pylori) Any new biochemical markers that are developed

Mean LDL cholesterol Laboratory measurements Mean HDL cholesterol/LDL:HDL ratio Mean systolic blood pressure Indications of diabetes

The scorecard of the European Society of Cardiology109 uses 5 principal factors: age, sex, smoking status, serum cholesterol and blood pressure, although indirect consideration is also given to diet, exercise and obesity. For the proposed cancer risk scorecard, there will be a very wide range of risk factors, which will unquestionably vary from one form of cancer to another. Some of the risk factors related to personal lifestyle might be difficult to quantify at the present time, although there are clearly good data at present regarding the risk attendant on most of the other factors, such as specific genetic lesions. There is now a crucial need for algorithms to integrate the relative weight that should be attached to each of these individual risks, with the realization that the algorithm might be different for cancers at different organ sites. HDL, high-density lipoprotein; LDL, low-density lipoprotein; NSAIDs, nonsteroidal anti-inflammatory drugs.

enzyme. Many chemopreventive agents that promote NRF2 function and phase 2 activity have been described85,86. Ligands that bind to members of the nuclear receptor superfamily are important chemopreventive agents in clinical medicine, and some of their actions might be mediated by the tumour microenvironment. Thus, the interaction of tamoxifen with the oestrogen receptor is anti-angiogenic by virtue of its suppression of VEGF synthesis87,88. Similarly, bexarotene, a ligand for the retinoid x receptors, has also recently been reported to have anti-angiogenic activity89. Angiogenesis as a key target for chemoprevention. Although we have reviewed many cellular and molecular targets in the microenvironment for chemoprevention, angioprevention seems to be particularly attractive (FIG. 3). Controlling angiogenesis is clearly an important strategy for the treatment of invasive cancers9092. It is now essential to apply this same strategy for prevention. The

observation that angiogenesis is a key target of chemopreventive agents has led us to look for common molecular mechanisms. Some clues come from the findings that nonsteroidal anti-inflammatory drugs (NSAIDs), COX2 inhibitors in particular, are effective chemopreventive agents, both in animals and in people48,93,94. The suppression of COX2 is now a crucial target for the control of tumours associated with chronic inflammation. Indeed, the first report to indicate that COX2 can have a significant role in colon polyp formation presciently noted that COX2 expression in polyps occurred in the stromal cells, rather than in the epithelium of the polyp95. COX2 modulates angiogenesis96 and seems to be upstream of VEGF, but is downstream of inflammatory signals activated by NFB. There are many other molecular targets in the angiogenic cascade. For example, all of the transcription factors discussed above affect angiogenesis, and they will continue to be important targets for chemoprevention. Interestingly, many anti-angiogenic agents

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also have marked anti-inflammatory activity; good examples are EGCG97, angiostatin20 and fenretinide98,99, which has been clinically effective for the prevention of breast cancer in premenopausal women100.
Translation into effective prevention In the United States, about one in three people will eventually develop cancer, and one in four will die from it. Every chronic disease has a latency period, during which pathogenesis proceeds before symptoms and subjective feelings of pain can be detected. The common misconception that people are healthy during this latency period is the greatest single impediment to the effective implementation of chemoprevention42. However, clinical data on chronic drug use for conditions other than cancer, such as the use of statins for cardiovascular disease or NSAIDs for chronic inflammatory diseases, suggest that prevention has enormous potential to reduce cancer incidence. Statins reduce cancer incidence, and it has been suggested that possible mechanisms for this effect involve the suppression of inflammation and angiogenesis in the microenvironment101. Recent studies have now shown that the chronic use of NSAIDs, which is already known to reduce colon cancer48, is also associated with a striking reduction in the risk of breast cancer94. This study found an approximately 50% reduction in breast cancer risk with aspirin use, and relative risk dropped to 0.29 with specific COX2 inhibitors. These data further confirm the importance of the microenvironment as a target for chemoprevention. NSAIDs are the most widely used medications in the world, and the broad use of these drugs has confirmed their effectiveness and relative safety. The wellrecognized gastrointestinal complications of many NSAIDs led to the search for selective COX2 enzyme inhibitors. However, selective COX2 inhibitors have not found clinical acceptance for chemoprevention because of increased cardiovascular risk associated with chronic use, particularly at high doses. For example, recent findings indicate that although celecoxib is an effective agent for the prevention of colorectal adenomas, it cannot be routinely recommended for this indication at present because of potential cardiovascular events102104. In colon cancer prevention studies, celecoxib at 200 mg or 400 mg twice a day showed a 1.3 to nearly 2-fold increased cardiovascular risk. The trend for a dose-related increase in cardiovascular events raises the possibility that lower doses or alternative dose scheduling (such as the use of rest periods, rather than

the constant administration of drug) might be associated with less cardiovascular risk. Close examination of the current use of these drugs will give more exact indications of the risks associated with their use and of potential strategies to circumvent the negative side effects encountered. It is essential to appreciate that everyone has some finite, measurable potential risk of developing cancer. As there are now new methods to quantify that risk, we need to do so, realizing that, for many people, total risk factors might be miniscule. Sceptics maintain that in cancer we do not have adequate simple markers of risk, such as elevated cholesterol or high blood pressure as indicators for cardiovascular disease. This is indeed true, but the problem might be able to be overcome if we adopt a more sophisticated approach and develop a new integrated personal risk index for the development of cancer (TABLE 2). Although none of the items shown in TABLE 2 are themselves guarantors of future clinically manifest disease (in the same way that neither high serum cholesterol nor high blood pressure necessarily lead to a cardiovascular incident), as an ensemble they might be empirically useful to indicate who might be the best candidates for chemoprevention. Cancer risk tools for the evaluation of specific cancers at specific organ sites, such as the Gail model for breast cancer, have been extremely useful in the design and interpretation of clinical chemoprevention studies105107. We now suggest that a risk index that translates across various cancer types might also be useful, although such an index would require a whole new set of parameters and more effective ways of analysing many markers that predispose to cancer risk. Clearly, the risks are different for cancers at different organ sites, but nevertheless, there is a widespread public desire to have some overall assessment of personal cancer risk. What connection do any of the indicators in TABLE 2 have with the microenvironment? As there are now intensive efforts to develop new biomarkers for assessing cancer risk, we suggest that some of the cellular or biochemical measurements now be directed towards evaluating abnormalities in the stromal microenvironment. In the past, almost all of the efforts in evaluating exfoliative cytology have been directed toward epithelial changes. In the future, it should be possible, with microbiopsy techniques, to assess cellular or molecular changes in the microenvironment of a developing carcinoma. Furthermore, as we have discussed above, chemopreventive agents such as the widely used NSAIDs affect the microenvironment and clearly have a

significant effect on the risk of developing some cancers. An absence of symptoms is not a sign of health; everyone is at some finite risk. Ultimately, personal risk should not be regarded in a benefit versus risk evaluation, but rather in a risk versus risk paradigm, which compares the risk of using an interventional agent with the risk of doing nothing. The risk of doing nothing is the 25% risk of eventually dying of cancer. Therefore, we suggest that an index for selection for cancer chemoprevention is feasible if an individual has a score above a threshold value (to be defined) then that individual is a candidate for chemoprevention (just as blood pressure determines whether someone should receive anti-hypertensive medication, or low-density lipoprotein (LDL) cholesterol levels indicate whether someone is a candidate for statins). These markers have been most useful in the control of cardiovascular disease with chemo-preventive drugs. The control of cancer with new chemopreventive agents should be next. At the same time, we must be fully aware of assuring the long-term safety of any drug to be used for chemoprevention. Safety is always a paramount concern in chemoprevention; clearly the risks associated with the use of chemopreventive agents must be driven as close to zero as possible. Although transcription factors are attractive targets for chemoprevention (and have been clinically useful targets), the multifunctional nature of transcription factors also has attendant risks. However, if used judiciously, chemoprevention should ultimately facilitate the preservation of health and improve the quality of life of many people.
Conclusion Chemoprevention directed towards the control of carcinogenesis in its early stages should ultimately provide a higher quality of life for people than waiting to treat end-stage disease. If we are to provide effective and safe chemoprevention, we need to understand the primary role of the tumour microenvironment in determining the fate of putative cancer cells and controlling their incipient malignant behaviour. In a holistic view of carcinogenesis, one might now consider that the microenvironment is an essential, intrinsic part of the tumour itself. The data we have summarized here clearly show a primary role for the microenvironment during carcinogenesis. These data further indicate that the use of chemopreventive agents to control the function and behaviour of cells in the microenvironment will be an important approach to the overall control of cancer.

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Adriana Albini is at the IRCCS Multimedica Science and Technology Park, Viale Fantoli 15/16, Milan, 20138, Italy. Michael B. Sporn is at the Dartmouth Medical School, Department of Pharmacology, Hanover, New Hampshire 03755, USA. Correspondence to M.B.S. and A.A. e-mails: michael.sporn@dartmouth.edu; adriana.albini@multimedica.it doi:10.1038/nrc2067 Published online 12 January 2007
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Acknowledgements
We thank M. Padgett for expert editorial and stylistic assistance in the preparation of this manuscript. K. Liby, F. Tosetti, R. Benelli and D. Noonan have given valuable suggestions and comments. We are especially indebted to C. Leaf for comments about risk versus risk. A.A. is supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC) and Ministero della Salute. M.B.S. is supported by grants from the US National Cancer Institute and the National Foundation for Cancer Research.

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Competing interests statement

The authors declare no competing financial interests.

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DATABASES
The following terms in this article are linked online to: Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=gene COX2 | CXCR2 | CXCR4 | CXCL12 | HIF1 | iNOS | KEAP1 | MMP9 | NFB | NRF2 | PAI1 | PI3K | SMAD4 | STAT3 | STAT5 | TGF | VEGFA Access to this interactive links box is free online.

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A mesothelioma epidemic in Cappadocia: scientific developments and unexpected social outcomes

Michele Carbone, Salih Emri, A. Umran Dogan, Ian Steele, Murat Tuncer, Harvey I. Pass and Y. Izzettin Baris

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Abstract | In Cappadocia, Turkey, an unprecedented mesothelioma epidemic causes 50% of all deaths in three small villages. Initially linked solely to the exposure to a fibrous mineral, erionite, recent studies by scientists from Turkey and the United States have shown that erionite causes mesothelioma mostly in families that are genetically predisposed to mineral fibre carcinogenesis. This manuscript reports, through the eyes of one of the researchers, the resulting scientific advances that have come from these studies and the social improvements that were brought about by both the scientists and members of the Turkish Government.
Mesothelioma is a cancer arising from the mesothelial cells that line the pleural, pericardial and peritoneal surfaces1,2. Although there are rare benign variants of mesothelioma, such as multicystic mesothelioma or mesothelioma of the atrioventricular node, which are not related to asbestos exposure1,2, this article focuses on the relatively more common malignant mesothelioma. In the United States there are approximately 2,500 cases and deaths per year of malignant mesothelioma, which is often related to asbestos exposure (BOX 1). Median survival is approximately 1 year from diagnosis because current therapies have only marginal effects in altering the natural course of the disease1. Although the link between asbestos exposure and mesothelioma was established in 1960, it is still unclear whether all types of asbestos cause mesothelioma36 (BOX 2).
Mechanisms of mineral fibre carcinogenesis The mechanisms of mineral fibre carcinogenesis have been studied prevalently using crocidolite asbestos, and are summarized below. Carcinogenesis as a result of exposure to crocidolite has been linked to its ability to induce the expression of both tumour-necrosis factor- (TNF) and its receptor (TNFR1) in mesothelial cells and in macrophages that phagocytose asbestos7. Indeed, Tnfr1 knockout mice do not develop fibroproliferative lesions after asbestos

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NATURE REVIEWS | CANCER

2007 Nature Publishing Group

VOLUME 7 | FEBRUARY 2007 | 147