Pulmonary Perspective Lung Cancer and Lung Stem Cells Strange Bedfellows? Adam Giangreco1, Karen R. Groot2, and Sam M. Janes2 1
Keratinocyte Laboratory, Cancer Research UK, London, United Kingdom; and 2Centre of Respiratory Research, University College London, London, United Kingdom
Lung cancer is a significant disease with survival rates remaining poor despite numerous therapeutic advances during the last 30 years. Understanding lung cancer pathogenesis through murine modeling may improve future human therapies, and new data indicate that mutations within different endogenous stem cells situated throughout airways can drive cancer formation. Airway stem cells maintain prototumorigenic characteristics, including high proliferative capacity, multipotent differentiation, and a long lifespan relative to other cells. These cells localize to proximal airway submucosal glands/intercartilagenous rings, neuroepithelial bodies, and terminal bronchioles/bronchoalveolar duct junctions. Recent studies suggest that endogenous stem cell signaling and differentiation pathways are maintained within distinct cancer types, and that destabilization of this signaling machinery may initiate regionspecific lung cancers. A better understanding of this relationship among stem cell regulation, cellular mutation, and lung cancer oncogenesis is critical for developing the next wave of lung cancer therapies. Keywords: lung cancer; stem cells; pathogenesis; signaling; progenitors
Lung cancer kills more people than any other cancer. The majority of patients present too late for curative surgery and the 5-year survival rate remains relatively poor despite active medical therapy and significant therapeutic advances. It is estimated that more people in the West die of lung cancer than prostate, breast, colon, and cervical cancer combined (1). Sadly, this devastating disease is also the most preventable cancer, with cigarette smoking causing 90% of lung cancers worldwide (2). With the increased social vilification and awareness of dangers of smoking in Europe and the United States, it was hoped that the worldwide incidence of lung cancer would decline. Unfortunately, this has not yet occurred, and an increase in lung cancer incidence in several developing countries has recently been fuelled by a new, predominately young, smoking population. Indeed, China expects one-third of its male population to die of tobacco-related disease (3). Although cancer prevention through public awareness and lifestyle choice remains a primary strategy in the fight against lung cancer, clinicians and scientists together must develop a better appreciation of the underlying cellular and molecular events driving this disease to design more effective therapies. In contrast to the slow improvement in lung cancer prevention and treatment, pulmonary stem cell biology (driven by mouse
(Received in original form July 19, 2006; accepted in final form December 7, 2006 ) Correspondence and requests for reprints should be addressed to Sam M. Janes, M.D., Ph.D., Centre of Respiratory Research, Rayne Building, University College London, 5 University Street, London, WC1E 6JJ UK. E-mail:
[email protected] Am J Respir Crit Care Med Vol 175. pp 547–553, 2007 Originally Published in Press as DOI: 10.1164/rccm.200607-984PP on December 7, 2006 Internet address: www.atsjournals.org
models) is rapidly revealing progenitor cell populations throughout lungs (4, 5). Multipotent, long-lived cells (stem cells) have been identified throughout airways and give rise to both transiently amplifying (TA) and terminally differentiated (TD) daughters (see Table 1). These cells, like stem cells in other organs, are critically important for local tissue maintenance and repair after injury (6, 7). Despite an established tissue-maintenance role, recent mouse data also support a stem cell–mediated origin for leukemia (8–13). It appears leukemias arise either from transformation of hematopoietic stem cells or via mutation in partially committed cells, resulting in selective expression of genes and enhancing their self-renewal potential (10). Hence, stem cells might accurately be considered prototumorigenic, lacking only those genetic mutations that may induce aberrant, cancerous growth and tissue invasiveness. Thus, stem cells and solid tumors may not be such strange bedfellows after all. This Pulmonary Perspective highlights evidence that supports roles for lung stem cells as likely originating cells for specific lung cancers. Recently, several studies have also demonstrated that some tumors contain their own subpopulations of drugresistant, cancerous stem cells (see Table 1) (11–17). Despite being distinct entities, normal tissue and cancerous stem cells appear to share common properties, including enhanced pollutant/drug resistance, robust differentiation, and increased mitotic capacity relative to neighboring cells (14). Whether these similarities are indicative of a common originating population remains unexplored. In the interest of space, we will not discuss the phenomenon of cancer stem cells within lung tumors. Rather, the current Perspective will highlight links between normal lung stem cell function and these stems cells’ involvement during lung cancer oncogenesis.
PROTOTUMORIGENIC STEM CELLS Characteristics that define stem cells include their capacity for self-renewal, production of daughter cells, and extensive proliferative capacity (18, 19). In this sense, stem cells could be considered ideal tumor initiation candidates, because dysregulation of this robust proliferative capacity through mutation may rapidly cause dysplastic, tumorlike growth. Drug-dependent, regulable transgenic models that reversibly mimic single oncogenic mutations (resulting in suppression of tumor suppressor genes or activation of oncogenes) provide crucial evidence that continued signaling is required to maintain tumor phenotypes (20–22). However, it appears that simply expanding stem cells without additional genetic mutation may not produce fully invasive tumors (20). Thus, stem cells appear to be prototumorigenic, and must first receive at least one permanent genetic mutation to destabilize their growth prior to cancer initiation (23). In rapidly dividing tissues such as the blood, gut, and skin, stem cells persist throughout an individual’s life and can easily acquire numerous oncogenic mutations. In contrast, committed
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AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 175 2007 TABLE 1. DEFINITION OF TERMS Name Progenitor cell Stem cell
TA cell TD cell Cancer cell Cancer stem cell
Definition
Examples
Any proliferation-competent cell capable of giving rise to at least one offspring Long-lived, self-renewing progenitor that exhibits extensive proliferative capacity and typically multipotent differentiation Short-lived progenitor cell with limited proliferative capacity and restricted differentiation potential Fully differentiated cell with no identified proliferative capacity Mutated, tumor-derived cell with dysregulated proliferation and differentiation Cancer cell exhibiting enhanced tumor-regenerative potential and/or pollutant resistance
PNEC BASC, variant CCSP-expressing cell (vCE)
Clara cell, type II pneumocyte Type I pneumocyte, ciliated cell
Definition of abbreviations: BASC ⫽ bronchioalveolar stem cell; CCSP ⫽ Clara cell secretory protein; PNEC ⫽ pulmonary neuroendocrine cell; TA ⫽ transiently amplifying; TD ⫽ terminally differentiated.
daughter progenitor cells (TA cells) and fully differentiated cells appear to have too limited a lifespan to accumulate this damage. Indeed, targeted oncogenic mutagenesis within these committed daughters has repeatedly failed to yield appropriate tumor models (24–26). It is therefore unsurprising that, within rapidly dividing tissues, most evidence supports a stem cell–mediated origin for cancers (10, 27). Alternatively, in tissues where cell turnover is slow (e.g., lung) daughter cells, including ciliated and Clara cells, exhibit significantly longer tissue transit times. In these organs, stem cells could theoretically accumulate only some oncogenic mutations, with their long-lived daughter cells receiving additional transforming mutations (10). Whether this actually occurs in lung and other slow-turnover organs remains largely unstudied. An additional stem cell characteristic that allows for their prototumorigenic classification is an inherent resistance to potentially toxic compounds (28). In murine lung, several distinct properties confer pollutant resistance among airway progenitors. Bronchiolar progenitor cells localized within neuroepithelial body (NEB) microenvironments express reduced levels of cytochrome p450 xenobiotic metabolizing enzymes (29). This reduces intracellular bioactivation of lipophilic compounds to toxic metabolites, allowing stem cells to survive several types of airway injury. These same cells also exhibit efficient drug efflux or a “side population” phenotype via the overexpression of ABCGtype transporters (30, 31). ABCG2 (breast cancer resistance protein 1) is an ATP binding cassette (ABC) transporter with affinity for numerous cytotoxic molecules, including mitoxantrone (32). ABC transporter–dependent “side” population (SP) cells have also been observed within other murine lung preparations, and most likely represent subsets of lung endothelial or fibroblast-like cells (31, 33). However, it remains untested in vivo whether any of these isolated SP cells truly exhibit bona fide lung stem cell function (33). ABCG-dependent drug efflux has also been observed within lung and other tissue tumor cell lines in vitro (12, 14, 34). SP cells within these tumor tissues appear to have the same pollution-resistance phenotype as endogenous stem cells and have cancer stem cell characteristics, including enhanced clonogenicity and secondary tumor formation capacity. The ABCG transporter proteins that define SP cells also pump various chemotherapy agents from these cells, protecting them from many current therapeutic drug strategies (14).
LOCAL PULMONARY ENVIRONMENTS INFLUENCE TUMOR FORMATION Lung cancer is not simply a single disease but a collection of several phenotypically diverse, regionally distinct neoplasias.
These encompass several major tumor subclasses and, in murine models, roughly follow a proximal-to-distal distribution pattern: moving distally from the trachea, major tumor types include squamous cell carcinomas (SCCs), small cell lung carcinomas (SCLCs), and adenocarcinomas/bronchoalveolar carcinomas. The regional segregation among tumor types using mouse models suggests that only finite numbers of distinct cells and/or pulmonary environments are capable of supporting tumor growth. Although humans also present with these tumor types in similar lung locations, determining the exact location in which human tumors originate remains difficult. Observed phenotypic heterogeneity between distinct tumor types suggests that the tumor’s local pulmonary environment profoundly impacts a cancer cell’s fate (35). Studies by Franklin and colleagues have assessed whether a case of multiple premalignant lesions localized within discrete lung microenvironments could have been derived from a common progenitor (36). They discovered that identical p53 mutations were present within discontinuous lesions throughout similar caliber airways, supporting their hypothesis that multiple simultaneous lung tumors may be clonally derived. These observations support Slaughter’s 1953 field carcinogenesis theory, which suggests that multiple primary cancers are derived from common, clonally derived precursor cells (37). These data support the role of individual stem cells in generating phenotypically similar lung tumors. To overcome the inherent problems related to human studies, scientists have increasingly favored genetically modified mouse cancer models. Mouse models for lung cancer have traditionally involved either global knockout strategies to remove putative tumor suppressors (e.g., p53) or transgenic expression of oncogenes/proto-oncogenes under the regulation of widely expressed, pulmonary-specific promoters (including Clara cell secretory protein [CCSP] or surfactant protein C [SPC]) (35, 38). These modifications generate identical mutations throughout large portions of lungs in a pattern similar to what would be expected after mutation and subsequent expansion of stem or progenitor cells. Within the context of field carcinogenesis, these global changes should therefore be capable of generating cancers throughout the entire lung. This has not turned out to be the case. Rather, it appears that very particular airway regions exhibit tumorigenic properties, and only when associated with specific cellular mutations. Of particular interest, several independent mouse models have recently been developed that introduce Kras mutations, which are common in human cancers throughout the lungs (21, 39–42). Amazingly, in all models tested, mice only produced
Pulmonary Perspective
adenomatous hyperplastic lesions localized to the bronchoalveolar zone despite identical mutations in virtually all airway cells. This strongly suggests that different lung cancers originate not only because of permissive oncogenic mutations but also because an individual cell’s local environment (niche) supports growth under these conditions. In other words, field carcinogenesis theory may be broadly applicable only within airway regions harboring equivalently functioning progenitor cells and may not be valid when comparing diverse pulmonary environments (e.g., tracheal vs. alveolar epithelia). Intriguingly, most originating sites identified using murine models of SCC, SCLC, and adeno-/ bronchoalveolar carcinomas appear to coincide with recently identified airway stem cell niches (4).
LUNG CANCERS AND ASSOCIATED PROTO-ONCOGENIC STEM CELL NICHES SCC—Major Airway Basal Cells
SCCs are the most common lung cancer in the United Kingdom and are highly associated with tobacco smoking (2, 43, 44). However, there are relatively few mouse models of human SCC compared with other cancers. Murine SCCs generally occur in the proximal airways down to the second or third bifurcation and are rarely observed distally. Existing SCC models have invariably used chemically mediated carcinogenesis, and to date no genetically modified mouse models of human SCC exist. Despite the obvious disadvantages related to chemical carcinogenesis, including uncontrolled cellular targeting and unknown mutations, these experiments do yield hyperplastic lesions and SCCs that resemble the human disease (45–48). Histologically, carcinogentreated airways undergo stepwise changes, eventually developing a full SCC phenotype. These are staged as a primary, generalized basal cell hyperplasia, followed by squamous metaplasia, dysplasia, carcinoma in situ, and, finally, invasive SCC (Figure 1) (49). It is important to note that, in SCC chemical carcinogenesis studies, most tumors lacked mutations common among other lung cancer types despite numerous carcinogen treatments. This suggests that very particular mutations occurring in specific cell populations are necessary to achieve SCC formation. Recent chemical carcinogenesis studies have identified unique genetic quantitative trait loci (QTL) associated with SCC progression and/or susceptibility that are not found in other lung cancers (48). Mutant mouse models of these QTL-associated genes are now needed to determine their relevance toward human SCC. Although mouse models have not identified the specific genes or cells involved in SCC formation, the morphology and gene expression patterns of mouse SCC-like hyperplastic lesions frequently resemble tracheal basal cell progenitors. These proximal airway progenitors are keratin 5/14 positive and are located at submucosal gland duct junctions or intracartilaginous boundaries (50–52) (Figure 1). Keratin 14 (K14)–positive basal cells decrease in frequency distally toward the carina and, like SCCs, are not normally observed beyond the main bronchi. K14 expression may be a hallmark of basal cells with enhanced regenerative potential, and reactivity is expanded after chemically mediated proximal airway injury and repair. Lineage tagging experiments confirm that these cells contain significant regenerative and differentiation potentials both in vitro and in vivo (51, 53). Similar K14 cell expansion occurs in hyperproliferative, preneoplastic lesions in SCC (54). To confirm the direct linkage between these cells and SCC formation, more rigorous approaches, such as lineage tagging plus chemical carcinogenesis, will be needed. Nonetheless, current data do support a direct relationship between proximal airway basal progenitors and cells associated with carcinogenesis in murine models for human SCC.
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SCLC—NEBs
SCLC is a form of lung cancer with a particularly poor prognosis because of a high rate of metastatic dissemination. Human SCLCs predominately localize to midlevel bronchioles and typically express a range of neuroendocrine cell markers, including calcitonin gene–related peptide (CGRP) and other neuropeptides normally expressed within pulmonary neuroendocrine cells (PNECs). On the basis of these observations, PNECs have been proposed as likely originating cells for SCLC (55). Retinoblastoma (Rb) and TP53 gene mutations are frequently associated with human SCLC. The combination of Rb depletion and knockout of the TP53 gene in mice resulted in the formation of multiple, distinct, SCLC-like hyperplastic foci (56). These foci corresponded to PNEC-containing NEB microenvironments (Figure 1). This model, however, failed to generate fully metastatic lung tumors and mimicked only the early stages of SCLC-like disease. More recently, a lung-specific conditional Rb inactivation model alone yielded strikingly similar results, with exclusive PNEC hyperplasia despite Rb gene deletion throughout the airways (57). Finally, pulmonary models deleting both Rb and TP53 in adult mice resulted in progressive epithelial hyperplasia that was restricted to the NEB microenvironment (58, 59). Importantly, these early lesions went on to form metastatic tumors resembling human SCLC. The observation that only PNECs respond, despite this method’s ability to cause widespread mutation throughout lungs, strongly implicated these cells as obligate targets for SCLC tumorigenesis. NEB microenvironments maintain putative stem cell populations in the bronchiole mucosa. They are defined as widely dispersed clusters of both PNECs and variant CCSP-expressing (vCE) cells and are the first regenerative airway sites after Clara cell injury (60). Suicide gene ablation studies have demonstrated that NEB-associated vCE cells are obligatory progenitors after Clara cell depletion (6), and that they divide infrequently in the steady state and are capable of multipotent differentiation (29). Interestingly, a subset of CCSP-expressing cells exhibit antigens specific to both PNECs and CE cells (60). Expansion of this dual-positive population may relate to the existence of mixed small cell and non–small cell lung carcinoma (NSCLC)–like tumors and/or nonsquamous NSCLC observed throughout airways in some transgenic mouse models of human lung cancer (61–63). Whether a comparable population of Clara or vCE cells could account for the development of human nonsquamous NSCLCs originating from pseudostratified airways remains unexplored. NEB-associated PNECs exhibit properties of unipotent progenitor cells and maintain a high proliferative capacity. In this sense, they are not true stem cells, but nonetheless, data support their contributory role within some lung cancer models. During repair-mediated NEB hyperplasia, PNEC-derived growth factors are released, which probably facilitates the rapid expansion of both PNEC and vCE populations (64). Chronic injury models reveal extensive PNEC hyperplasia similar to early hyperplastic lesions observed after lung Rb/TP53 mutations (60). If either vCE cells or PNECs develop mutations that enhance their growth independent of injury, this could in turn rapidly produce hyperplastic lesions similar to those observed in mouse models for human SCLC. Evidence that NEB-associated PNECs and SCLCs use identical signaling pathways further strengthens the possible common origins between these two populations. Sonic hedgehog (Shh) reactivity is elevated in NEBs during lung development and after airway Clara cell depletion, whereas the Hedgehog receptor Patched (Ptc) also shows increased expression within PNECs during repair-associated NEB hyperplasia (65, 66). Similarly, SCLC tumors frequently overexpress both Shh receptor and
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Figure 1. Airway stem cell microenvironments and associated human carcinomas. Fluorescent whole mounts of mouse lungs and schematic drawings reveal identified airway stem cell microenvironments. Upper airways likely contain submucosal gland and intercartilagenous stem cell niches (highlighted in red) defined by keratin 14 (K14)–expressing basal cells (red K14 staining) adjacent to ciliated cells expressing acetylated tubulin (green stained) as shown in A and inset, and drawn in C (highlighted in red). In humans, basal cells may generate squamous cell carcinomas via transitional stages, including metaplasia (D ), moderate dysplasia (E ), carcinoma in situ (F ), and, finally, invasive carcinoma (G ). Highlighted in yellow are midlevel, bronchiolar airway–mediated stem cell populations localized within calcitonin gene–related peptide (CGRP)–expressing neuroepithelial bodies (NEBs) adjacent to pulmonary neuroendocrine cells (PNECs). These are shown in B stained with Clara cell secretory protein (CCSP) (red) and CGRP (green), and drawn schematically in C. Within the NEBs of humans, neuroendocrine cells themselves likely generate small cell carcinomas (H ), whereas variant airway CCSP-expressing cells (vCE) may contribute to bronchiolar adenomas (not shown). CCSP-expressing bronchioalveolar stem cells (BASCs) within green-highlighted terminal airways (CCSP-positive cells in red (B ) and BASCs drawn in C function as distal airway stem cells and, in humans, may generate atypical adenomatous hyperplasia (I ), adenocarcinomas (J ), and bronchoalveolar cell carcinomas (K ). Abnormal human lung tissues are indicated by asterisks (D–K ).
ligand and may require continued Shh signaling for continuous growth. These observations led to the hypothesis that PNECderived human SCLC tumors may undergo specific mutations permitting autonomous Shh signaling, thereby circumventing existing control mechanisms that regulate normal NEB-associated proliferation (66). Similar studies regarding the Notch-delta pathway also indicate roles for this signaling pathway in NEB growth and human SCLC progression (67–69). Central Bronchiolar Adenocarcinoma/Bronchoalveolar Cell Carcinoma—Bronchioalveolar Stem Cells
Central bronchiolar adenocarcinomas and peripheral bronchoalveolar cell carcinomas are among the most common lung cancer types in the United States. Although they occur frequently in smokers, they are also the most common lung cancer type in nonsmokers, with a high incidence in Asian women (2). Central bronchiolar adenocarcinomas classically show acinar–glandular differentiation, and murine models suggest that these adenocarcinomas arise from the junction between the terminal bronchiole and the alveolus termed the “bronchoalveolar duct junction” (BADJ) (70) (Figure 1). These adenocarcinomas frequently exhibit mixed airway and alveolar characteristics, including CCSP
and SPC coexpression (70). Although human cancers largely exhibit either airway or alveolar differentiation, mouse models and some human cancers do express both airway and alveolar differentiation characteristics. This has led to hypotheses that either Clara or alveolar type 2 (AT2) cells function as the originating cells for adenocarcinomas (21, 70). Murine adenocarcinoma models represent a disproportionately high number of the existing models for human lung cancer. One reason for this bias is the extensive use of either airway CCSP or alveolar SPC promoters to express mutated proteins in both lung compartments (35). In humans, CCSP and SPC expression is retained within many adenocarcinomas, indicating their possible Clara or AT2 origins. CCSP and SP-C promoter– driven adenocarcinoma models include mutated epidermal growth factor (EGF) receptor (71, 72), active K-ras (Kras G12D) (40, 41), dominant negative transforming growth factor- (73), and large T antigen (74, 75). Several of these and other transgenic mouse models develop mixed-phenotype tumors adjacent to terminal bronchioles most closely resembling human bronchoalveolar carcinoma. These results strongly suggest that originating cells for adeno- and bronchoalveolar carcinoma localize near BADJs.
Pulmonary Perspective
Recently, several improved mouse models for human adenocarcinoma have been developed that use a conditionally activated mutant Kras G12D (39, 40, 42, 76). These include a spontaneously mutating “hit and run” model, a doxicycline-inducible transgenic system, and a transgenic promoter-independent adenovirus-dependent recombination system in which intratracheal and intranasal adenovirus expressing Cre recombinase (AdenoCre) triggers Kras G12D expression throughout infected airways (21, 39–42). Each model produces initial lesions resembling injury-dependent terminal bronchiolar hyperplasia plus alveolar bronchiolization, or induces the transformation of alveolar septae into regions exhibiting a strong airway-like morphology. The AdenoCre model is particularly informative because it provides widespread lung-specific genetic recombination and eliminates confounding developmental effects. Strikingly, each improved model exhibits very similar results typified by mixed airway– alveolar lineage, BADJ-associated adenocarcinomas. Together, results of these newer models indicate that, as well as being progenitor cell specific, certain mutations (here, Kras G12D) are additionally favored during adenocarcinoma initiation relative to other lung cancers. Early observations identified multiple foci of epithelial regeneration localized to both airway branch points and terminal bronchioles after naphthalene-mediated Clara cell depletion (77). Although the former turned out to be NEB-associated vCE progenitors as described above (29), terminal bronchiolar regeneration was due to NEB-independent, BADJ-restricted CE stem cells (78). Significantly, these BADJ stem cells (defined as bronchioalveolar stem cells [BASC]) also demonstrate a steady-state mixed airway–alveolar phenotype and are uniquely capable of in vitro expansion (70). Using the AdenoCre adenocarcinoma model described above, Kim and colleagues have now demonstrated selective, dose-dependent BASC expansion in vitro and in vivo after Kras G12D mutation (70). In addition, in vivo BASC activation through injury followed by Kras mutation resulted in much greater increases to both tumor number and area versus Kras mutation alone. These data provide the most compelling evidence to date that normal airway stem cells can directly act as originating cells for lung cancers.
SUMMARY AND FUTURE DIRECTIONS The relationship between stem cells and cancer formation is becoming increasingly scrutinized with respect to lung cancers. Stem cell populations within proximal airways, NEB microenvironments, and BASCs are ideally situated to serve a role as lung cancer progenitors. Several properties unique to stem cells are similar to those found within different lung cancer types. In addition, there is emerging evidence that signaling pathways governing airway stem cell fate are exploited by lung cancers after mutation. Despite these discoveries and recent studies demonstrating a direct relationship between BASCs and bronchoalveolar carcinomas, the relationship between lung stem cells and lung cancer remains underexplored. Additional studies using cellular lineage tagging of putative stem/cancer progenitors and tightly regulated transgenic mouse models for all human lung cancer types are needed. Only once these cells and lineage relationships are more fully understood can we hope to develop more effective, targeted therapies to treat this devastating disease. Conflict of Interest Statement : None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Acknowledgment : The authors gratefully acknowledge support from the histopathology unit at University College London, specifically Vinu Sheshappanavar, Andrew Nicholson, and Mary Falzon for their assistance in providing human lung carcinoma tissue samples and images. They also thank Mark Griffiths, Soline Estrach, Robert Buttery, and Dawn J. Mazzatti for critical reading of the manuscript.
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