Cell Biochem Biophys (2014) 69:389–398 DOI 10.1007/s12013-014-9844-4
REVIEW PAPER
Lung Cancer Stem Cells and Implications for Future Therapeutics Jing Wang • Ze-hong Li • James White Lin-bo Zhang
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Published online: 20 February 2014 Ó Springer Science+Business Media New York 2014
Abstract Lung cancer is the most dreaded of all cancers because of the higher mortality rates associated with it worldwide. The various subtypes of lung cancer respond differently to a particular treatment regime, which makes the therapeutic interventions all the more complicated. The concept of cancer stem cells (CSCs) is based primarily on the clinical and experimental observations that indicate the existence of a subpopulation of cells with the capacity to self-renew and differentiate as well as show increased resistance to radiation and chemotherapy. They are considered as the factors responsible for the cases of tumor relapse. The CSCs may have significant role in the development of lung tumorigenesis based on the identification of the CSCs which respond during injury. The properties of multi-potency and self-renewal are shared in common by the lung CSCs with the normal pluripotent stem cells which can be isolated using the similar markers. This review deals with the origin and characteristics of the lung cancer stem cells. The role of different markers used to isolate lung CSCs like CD44, ALDH (aldehyde dehydrogenase), CD133 and ABCG2 (ATP binding cassette sub family G member 2) have been discussed in detail. Analysis of the developmental signaling pathways such as Wnt/b-catenin, Notch, hedgehog in the regulation and maintenance of the lung CSCs have been done. Finally, before targeting the J. Wang (&) Z. Li L. Zhang (&) School of Life Sciences, Jilin Agricultural University, Changchun 130118, China e-mail: [email protected] L. Zhang e-mail: [email protected] J. White School of Medicine, University of Maryland, Baltimore, MD 20201, USA
lung CSC biomarkers for potential therapeutics, challenges faced in lung cancer stem cell research need to be taken into account. With the accepted notion that the CSCs are to blame for cancer relapse and drug resistance, targeting them can be an important aspect of lung cancer therapy in the future. Keywords Lung cancer Cancer stem cells (CSCs) CD44 ALDH (aldehyde dehydrogenase) CD133 ABCG2 (ATP binding cassette sub family G member 2)
Introduction The diagnosis of lung cancer usually takes place at a very advanced stage because of poor prognosis and as a result it becomes the leading death causing cancer throughout the world [1, 2]. In 2011, *221,000 new lung cancer cases have been diagnosed with 157,000 deaths while that in Europe the mortality rate is 148,000/year [3–5]. This signifies the disease burden associated with lung cancer. The two main categories of lung cancer include small cell lung cancer (SCLC) which is observed in only 15–20 % of the lung cancer cases; and NSCLC (non-small cell lung cancer), the more common of the two and which accounts for *80–85 % of the cases [1, 3, 5, 6]. Based on the histology, NSCLC is again subtyped into squamous cell carcinoma (accounting for 30 % of the cases) and non-squamous carcinoma (observed in 70 % of NSCLCs). The nonsquamous cell carcinomas consist of mostly adenocarcinomas as well as large cell carcinomas. and at times. some lesser differentiated variants are also observed [1, 7, 8]. There are ever-increasing evidences that support the role of cancer stem cells (CSCs) in driving the development and progression of a plethora of cancers including lung cancers.
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It is also suggested that the poor prognosis and unsuccessful clinical outcomes associated with lung cancers are rendered by the CSCs [9, 10]. Therefore, it becomes important to study the various stem cell markers that are enriched in lung cancers and can be used to isolate the cells with stem-like characteristics. It is also necessary to understand the various signaling pathways that are involved in the regulation as well as the maintenance of the lung CSCs. The critical pathways involved in such activities present themselves as promising targets for lung cancer treatment. The CSCs which result in the generation of cancer cells of heterogenous population consist of a unique, small number of undifferentiated cells with the capacity of multipotent progenitor generation and formation of tumors. This subpopulation of cancer cells when introduced into transgenic animal models mimics the original tumorigenic potential in vivo. Under in vitro conditions, the cells could give rise to spheres [11–13]. The CSCs make the tumors become more aggressive in nature because of their resistance to chemotherapy and radiation, and hence result in poor prognosis [9]. In addition, the epithelial to mesenchymal transition (EMT) identified in connection with the CSCs drives the cancer cells toward metastasis and migration, making them more dangerous component of tumor progression. The CSCs have their major impact in the cancer recurrence. The current treatment modalities can affect and treat most of the cancer cell population. However, in the small subpopulation of treatment-resistant cancer cells, the CSCs serve as the reservoir for the cancer cells and replenish the population which gives rise to tumor relapse [10, 14]. Same is the case with the lung cancer where *20 % of NSCLCs can be operated at present. However, the recurrence rate among those operated is quite high at 30–50 % [15]. The overall survival rate is as low as 7–20 % observed in a 5-year period when radical chemoradiotherapy is applied in the advanced cases of NSCLC [16, 17]. Significant responses have been observed in the adenocarcinoma patients [where the tumors show mutations for analplastic lymphoma kinase (ALK) or epidermal growth factor receptor (EGFR)]. However, within 2–3 years, almost all the patients succumb to recurrence of drug resistance [18, 19]. Similar responses are encountered in SCLC cases where platinum-based chemotherapy induces high response among the patients; however, such responses are short-lived with drug-resistant relapse. The survival rate among such patients is very poor (at *5 %) [20, 21]. Such ability of the lung cancers to recur, despite showing complete response and tumor regression at one point, suggests the presence of a cell population with tremendous regenerative and self-renewal capacities, which is only comparable with the embryonic stem cells. Therefore, the targeting of lung CSCs presents the potential of delaying or inhibiting tumor relapse. Although the
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significance of CSCs in the development and progression of lung cancers is quite evident, a precise review that details about it is, however, not available except for some specific information and articles related to the recent studies in the field. This review will try to focus on the different aspects of lung cancer with respect to the CSCs in the progression and development of tumorigenesis. A better understanding of the different markers used to identify and characterize the lung CSCs as well as the signaling mechanisms will enable the development of more effective therapeutics in the treatment of lung cancer and prevention of relapse.
Method The review has been developed using the published literature in the field of lung cancer stem cells. We have searched the Pubmed, the database for articles on life sciences and biomedical research. The search included only the articles from January 1, 2000 to October 1, 2013 to have the most updated information in the field. The keywords used to search the articles included lung cancer, stem cell, lung cancer stem cell, CSCs, markers for CSCs, signaling pathways in CSCs, cancer initiating cells (CICs). Some of the relevant journals related to the field of stem cell research like stem cell, CSCs were searched separately to find any additional information that might have been missed. Only those peer-reviewed relevant articles were used for the current review and have been aptly referenced. Although we put no restriction according to language, only the English articles were found relevant to our purpose.
Lung Cancer Stem Cells The CSCs are also referred to as the CICs since these cells when transplanted in animal models can reproduce the phenotypic features of the tumors of their primary origin [22]. Several studies have linked the CSCs with enhanced proliferation and invasiveness, metastasis and migration, stem cell marker expression, resistance to chemo or radiotherapy, resistance to apoptosis and hypoxia, and an overall increased tumorigenic potential [23–27]. Notwithstanding the increase in the number of studies in this field in recent times, the observations and findings from such studies have to be interpreted and exploited with caution, since there are sufficient controversies regarding the selection criteria of the CSCs like the absence of proper validation of the studies or well-defined markers [28]. It was demonstrated in a study in 1997 that a small subpopulation of cells from the acute myeloid leukemia (AML) could develop AML in NOD/SCID mice giving rise to one
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of the first ever possibilities of CSC existence [11]. It was not before 6 years from thence that the isolation of CSCs took place from solid tumor in 2003 (Clarke’s group). In this study with breast cancer, the researchers found that the CD44?/CD24 low-isolated cell population could only reproduce the phenotypic and morphologic characteristics of the primary cancer through tumor formation in immunodeficient mice [29]. There exists substantial overlap between the regulation of the stemness characteristics of the CSCs and ESCs (embryonic stem cells) such as selfrenewal, stemness, pluripotency, proliferation, and differentiation which are controlled by similar signaling pathways such as the Wnt, Notch, and Hedgehog (Hh) [30]. These are the same pathways involved in the development of lung and in adults, due to acquisitioned aberration results in lung oncogenesis by dysregulating the balance between differentiation and self-renewal [31]. The critical decision to make during the stem cell division is whether to differentiate or to self-renew. Some of the important transcription factors associated with the maintenance of stem cells and tumorigenesis promotion are Nanog, Oct4, and Sox2, which induce self-renewal and prevent differentiation [32]. The CSCs from non-small cell lung carcinoma are usually identified by the use of the markers like the surface marker CD133 or an enhanced effluxing capacity of the Hoechst dye (that binds to DNA) and gives rise to the SP (side-population) [33–38]. When comparing with the NSCLC, very few studies with the stem cells from the SCLC have been performed till date. Nevertheless, in one of the studies, it was shown that the SP from SCLC had increased tumorigenic potential in vivo [37]. In yet another study, it was observed that CD87 could be used to isolate SCLC stem-like cells successfully, which possessed the capacity to form spheres and showed resistance against multiple drugs in vitro [39]. The presence of cell surface markers on the CSCs has been routinely exploited to isolate the subpopulations of the cells from the pool of cancer cells with stemness properties such as enhanced colony and sphere-forming capacities, increased rate of invasion and proliferation, increased resistance to therapy, and overall increase in tumorigenicity [40–49]. The use of cell-surface markers (although a number of them are available) to isolate cancer cells with stem-like characteristics is very challenging. As a matter of fact there exists significant variation in their level of expression and types available in tumor cells. This variation is even existent among surface markers of same tumor origin from different cell lines or patients. Moreover, often the isolated cell population is heterogenous in nature and/or the expression of the markers is not sufficient that causes a compromise in the study of the lung CSCs [50]. In a recent report, it was shown that both the CD133? and CD133-enriched cell populations from the lung cancer cells contained equal percentage of
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lung CSCs. This suggests that CD133 may not be sufficient alone as a marker for the isolation of lung CSCs [36, 51]. Others have found similar results with prostate and colon CSCs [52, 53]. Accordingly, the cells with stem-like characteristics isolated using CD133 as a marker has produced controversial results. In an interesting finding, the ALDH? SP cells demonstrated the unique stem-like characteristics of quiescence, dormancy, and slow division. Nonetheless, with such conflicting results available regarding the various markers used to identify, isolate, and characterize the lung cancer stem cells, a thorough analysis of the various lung CSC-associated markers is essential to understand their potential fully before putting them into application.
Markers Used in the Identification of Lung CSCs Although a number of CSC markers are being reported in recent times in the available literatures, very few of them are eventually validated. However, the identification of the different CSCs with significant variation from those of the other cancer cells has been possible because of the elaborate studies performed within this field. The CSCs are known to express a number of different markers at the same time and the use of a single marker may not be sufficient to isolate a pure subpopulation of stem-like cells from the pool of cancer cells [36]. Therefore, there is a definite need to study the available CSC markers in relation to lung cancer which might as well improve prognosis and overall therapeutic intervention in this cancer type. In this section, the present knowledge regarding the lung CSC markers-CD44 and CD24, ABCG2, CD133, and ALDH has been highlighted. CD44 and CD24 Almost all types of tumor cells express the hyaluronic acid receptor CD44 which is, therefore, one of the most frequently studied surface markers [54]. The marker has been found in association with the regulatory proteins for growth, proliferation, cell adhesion, motility, and migration [55]. In addition, it has been found to be involved in cell migration and takes part in the signaling cascades involved in tumor initiation and progression. The CD44 gene due to alternate splicing expresses various isoforms that are associated with the different subtypes of cancer. Hence, it is an ideal surface marker candidate for the isolation of CSCs from carcinomas of different origins [56]. Another stem cell surface marker CD24 is an antigen that is heat stable and expressed in a multitude of cancers. It is an important marker for the diagnosis as well as prognosis of tumorigenesis since it plays significant roles in cell to
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matrix as well as cell to cell interactions thereby is involved in cell adhesion and migration [57]. CD24 causes enhanced proliferation of the tumor cells and induces them to get adhered to collagen, laminin and fibronectin. Therefore, a CD24 high cell population can be linked to increased progression of tumorigenesis and metastasis. CD24 and CD44 in combination have been used for the isolation of solid tumor CSCs. Since, they are expressed in only a few types of cancers their usage have been limited [58]. The lung cancer CD44? cells have shown enhanced ability to initiate tumors both in vitro and in vivo. Besides, the EMT markers like Vimentin, Snail1 and Cadherin2 were highly upregulated in the CD44? cells and hence it might be involved in EMT and thereby in the maintenance of stemness [59]. ATP-Binding Cassette Sub-Family G Member 2 (ABCG2) Another important marker used in the identification of lung CSCs is the ATP binding cassette reporter that causes decreased drug concentration within the cells by pumping the chemotherapeutic drugs out of the cell [60]. A poor prognosis in cancers has been associated with ABCG2 as it is highly upregulated in the small stem-like cell subpopulation [61]. There are studies to suggest that the expression of ABCG2 is not limited to CSCs of a few cancer types and is even overexpressed in lung CSCs. ABCG2 has been implicated in the increase in hematopoietic stem cell survival in the hypoxic condition as well as in the protection of stem cells [62]. It also protects the stem cells from oxidative stress and contributes to the capacity of proliferation of the progenitor cells both in vivo and in vitro [63]. The stem cells isolated via Hoechst dye efflux method and FACS from the lung cancer cells such as HTB-58, H460, H2170, H23, A549 and H441 exhibited a stem cell SP with increased drug resistance and ABCG2 overexpression [64]. In a recent study, it was found that low molecular weight heparin (LMWH) could reduce the expression of ABCG2 and hence decreased the SP from lung cells which had lesser clonogenic ability in vitro. In addition, cisplatin and LMWH together showed the potential in inducing apoptosis and overcoming drug resistance [42]. Cd133 The most commonly used among the CSC markers is CD133 which is a surface glycoprotein with two extracellular glycosylated loops and five transmembrane stretches. In a study involving 10 NSCLC patients and lung cancer cell lines, the CD133? and CD133- cells from the samples of those patients and five lung cancer cell lines
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(H1299, A549, C299, CCL5 and CCL1) were analyzed [35]. The CD133? cells were demonstrated to possess higher tumor initiation, self-renewal and drug-resistance abilities. In addition, the cells showed increased level of Oct4 (a common embryonic stem cell transcription factor) expression [35, 65]. There are studies that indicate for the drug-induced enrichment of the CD133? population from the lung cancer cells. In one such study, it was observed that the exposure of the A549 cells to cisplatin at cytotoxic concentration caused the increase of the CD133? cell population by eightfolds [34]. The results were reproduced in vivo when cisplatin was used to treat different mice xenograft models. Such findings further confirm the idea that the elimination of the CD133-enriched CICs is not possible through chemotherapy. However, CD133 is not universally expressed in the different subtypes of lung cancer cells. In a related study, it was demonstrated that the CD133? stem-like characteristics was present in the SCLC cell line H446 but not in the NSCLC cells like H157, H292, Calu-1, A549 and Hee6 [40]. On similar lines, the NSCLC specimens showed enhanced CD133- expression that contributed significantly to neovascularization [66]. But CD133 presented near negligible prognostic value in early stage resected NSCLC [67]. Aldehyde Dehydrogenase (ALDH) Another marker linked to stemness properties is ALDH which is perhaps the most important of all lung CSC markers. This enzyme is involved in the oxidation of acetaldehyde as well as in the control of stem cell differentiation [68]. It has been established as a specific marker in lung adenocarcinomas [46]. The ALDH1A1? lung cancer cells exhibited resistance against the EGFR, gefitinib but not the ALDH1A1- cells [69]. Thus, ALDH has been very useful in providing important insights into tumor initiation and is of high prognostic value [70]. Others have observed a correlationship between poor prognosis in lung cancer patients and ALDH1A1 expression [47]. A majority of the NSCLC from different lung cancer tumor samples and cell lines display the presence of increased activity of ALDH (mainly ALDH1A1) in a cell subpopulation. In addition, the ALDH expressing lung cancer cells are proven to be more clonogenic with the increased tumorigenic and self-renewing potential. The Notch pathway has been found to be intricately associated in the maintenance of the lung cancer cells that are positive for ALDH, and accordingly, the Notch-signaling suppression yielded much reduced ALDH? cell population with decreased proliferation and clonogenic capacity of the tumor cells [47]. Therefore, ALDH can be successfully utilized in the selection of NSCLC subpopulation with stem-like properties that possess greater capacity of being tumorigenic.
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Pathways Involved in Lung Cancer Stem Cells The signaling pathways that are involved in embryogenesis are also highly implicated in oncogenesis. Some of the important pathways involved in the maintenance of lung cancer stem cells include Wnt/b-catenin, Notch, and Hh. These pathways are linked with the maintenance of tissue homeostasis and normal stem cell renewal [71]. Any deregulation of such signaling drives the activity of the CSCs in multiple cancer types that even include lung cancer [72]. Hedgehog (Hh) Signaling The Hh signaling is an important component of embryogenesis that regulates proliferation, differentiation and migration of the embryonic stem cells but its presence is near negligible in adult tissues [73]. The activation of the pathway takes place through smoothened, a transmembrane domain containing protein and further activation of transcription factors like GLI1, GLI2 and GLI3 which are involved in the regulation of the downstream genes [74]. The Hh-Gli signaling has been involved in the maintenance of a number of different cancers including breast, melanoma and lung cancers [75–79]. The Hh signaling is among one of the first pathways demonstrated in SCLC. The tumorigenicity in SCLC was lost upon the application of cyclopamine (a smoothened antagonist) to inhibit Hh signaling [80]. These findings indicate that aberrant Hh signaling could give rise to SCLC. According to a similar study involving genetically engineered mice, it was demonstrated that SCLC initiation is contributed significantly by Hh signaling. The use of smoothened antagonists to inhibit the Hh pathway shows good promise in the treatment of lung cancer with potential to prevent cases of tumor relapse [81]. Notch Pathway The highly conserved mammalian embryogenesis pathway Notch is frequently involved in the determination of cellular fate. The asymmetric division-mediated viability of the stem cells is maintained through cell-to-cell interaction that leads to Notch signaling activation [82]. The normal lung development, as well as the determination of the distal and proximal epithelial cellular fates, is controlled by the Notch signaling as demonstrated through knockout mice models [83]. Studies with knockout mice models for Hes 1 (hairy and enhancer of split 1), an important downstream target in the Notch pathway, established the contribution of the Notch signaling in the maintenance of the normal lung stem cells [84]. Accumulation of the stem cells for distal airways with delayed differentiation took place upon
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constitutive Notch signaling activation [85]. The NSCLC cell lines depicted much greater transcript levels of Notch that confirmed the role of the pathway in the tumorigenesis of the lung [86, 87]. The antiapoptotic signals are inhibited by Notch through Survivin induction, while it promotes pro-apoptotic signals via Bim [86, 88]. The g-secretase (required for the cleavage and activation of Notch receptors) inhibition results in the Notch pathway blockade that induces apoptosis and inhibits growth both under in vivo and in vitro conditions in NSCLC [88]. It has also been established that the ras-driven cancers are maintained by a dysregulated Notch pathway [89]. The sphere-forming abilities of the ALDH ? NSCLC cells are largely dependent on the active Notch signaling [47]. There are reports that suggest the probable presence of mutations in the Notch1 receptors from NSCLC [90]. Notwithstanding the role of Notch signaling in the progression of lung tumorigenesis, the involvement of the pathway in the CSC phenotype maintenance is not yet clearly evident [91–93]. However, the Notch pathway might be more complex than it is being understood. In the neuroendocrine-mediated malignancy in SCLC, the Notch receptor expression is very rarely found. Nonetheless, the Notch signaling appears to be an attractive target for antilung cancer therapeutic interventions. It is not only due to its role in stem cell maintenance and tumorigenesis but also for its potential involvement in immune function and tumor angiogenesis. Accordingly, the development of specific inhibitors of Notch signaling pathway members is currently under investigation [94, 95]. The Wnt/b-Catenin Pathway The Wnt/b-catenin pathway is highly implicated in a variety of cellular processes like differentiation, proliferation, motility, and apoptosis [96]. The canonical Wnt signaling is the best understood among the Wnt pathways wherein the Wnts get attached to the cell-surface receptors that phosphorylate the Dvl (disheveled family proteins). The kinases Casein kinase1 (CK1) and glycogen synthase kinase 3b (GSK3b) are, in turn, activated by Dvl which facilitates b-catenin degradation. Inhibition of Dvl therefore results in the b-catenin accumulation in the cytoplasm. The pool of b-catenin that enters the nucleus binds to the transcription factor/lymphoid enhancer-binding factor 1 (TCF/LEF) family of transcription factors, which leads to the expression of the specific genes [97]. The ligands Wnt1 and Wnt2 are highly upregulated in a variety of NSCLC tumor specimens as well as cell lines [98]. In a recent study, Oct 4 (the critical player in stem cell differentiation and self-renewal) regulation was demonstrated after the lung cancer cells A549 were treated with cisplatin. The treatment also resulted in higher b-catenin expression. On
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treating the lung cancer cells with LiCl (inhibitor of GSK3b), stabilization of b-catenin took place. It resulted in a dramatic increase in migration, proliferation, drug-resistance, and clonogenic properties. A blockade of the Wnt/b– catenin pathway caused a decrease in such CSC properties [99].
Targeting the Lung CSCs for Therapy It is an established fact that lung CSCs play a significant role in tumor initiation and progression. Accordingly, lung cancer therapy targeted at the CICs has been developed. In recent times, it was found that the CSC-mediated drug resistance in lung cancer could be overcome through the use of gefitinib and trifluoperazine that target the CD44 and CD133 markers [100]. In a study with lung cancer cells lines (H1299, H460. and A549) that are negative and positive for ALDH, it was demonstrated that the telomere length of the lung CSCs are longer than the differentiated lung cells [101]. It was also shown that the response of the cells to MST312 (an inhibitor for telomerase) was dependent on the length of the telomere they possessed (the longer the telomere, the more sensitive were the cells to the MST312 treatment). Such a response was accompanied by the reduction in ALDH? cells post treatment of the drug. The involvement of the Notch pathway in the maintenance of lung CSCs has been established when a much-reduced ALDH? lung cell population was recovered upon targeting the Notch signaling [90]. Similarly, the inhibition of the Wnt/b-catenin signaling, another pathway highly implicated in lung cancer through the knock down of the Wnt ligands (Wnt1 and Wnt2) or by the use of monoclonal antibodies against them results in the increased apoptosis of the NSCLC cells [102]. The Notch3-signaling inhibition through the use of MRK-003(inhibitor for c-secretase) yielded highly promising results. Such treatment resulted in enhanced apoptosis and reduced proliferation of the lung cancer cells as observed through in vitro and in vivo studies [88]. Others have found the Hh pathway inhibitor, GDC0449, to produce effective results in the growth inhibition of SCLC and adenocarcinoma cells. In addition, it stimulated the cisplatin [103]. The Skp, Cullin, F-box containing complex (SCF complex) is produced by the CSCs which can induce the proliferation of the CSCs via c-Kit binding. The anticancer drug imatinib partly exerts its activity by interfering with the SCF-mediated c-Kit activation thereby negatively influencing the growth of the CSCs. However, imatinib activity seems to be futile in most of the NSCLC cell growth because of lack of c-Kit expression [104]. The majority of these studies point toward the effectiveness of targeting the lung CSCs in controlling different subtypes of lung cancer.
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Problems Encountered in the CSC Model Approach The concept of CSC model in tumor initiation and progression has been gaining momentum in recent times. However, the specific characteristics that define the bona fide CSCs across multiple cancer types are still under debate. The proponents of the non-CSC model argue that the current model is a result of data misinterpretation since the phenotypes of the potential CSC population has been largely inconsistent [105, 106]. The typical and strict assay that has been adapted to test the CSCs is the ability of the putative cancer stem-like cells to form tumor through serial dilution (cell population as low as 1–1,000 should be capable of regenerating the original tumor). The fundamental belief for such assay is that tumor is formed through the clonal expansion of the rare population of CSCs. The in vivo assay has been done typically through the injection of a small population of the isolated CSCs into the NOD/ SCID mice and checking for tumorigenic growth. The clonogenic potential of the nonsorted mouse lymphoma and melanoma cells were confirmed when it was found that only ten such cells were able to regenerate the disease in the recipient mice [107, 108]. Although the non-stem-like cells from melanoma and lymphoma can generate tumors in vivo, the potential of the CSCs to develop tumors in other cancer types is a high and real possibility. The factors like stromal cytokines, tumor angiogenesis, and vasculature, hypoxia, and the overall tumor microenvironment can cause significant perturbations on the CSC presence [26, 109–111]. The differentiated tumor cells can be reprogrammed to CSCs through EMT [10, 112]. Therefore, the future of the therapeutics targeted at the CSCs is highly dependent on the better understanding of the factors that contribute to the stem cell properties of drug resistance, dormancy, and quiescence, tumor initiation and progression, tumor heterogeneity, migration, and metastasis.
Conclusions The CSC reservoir controlled via distinct signaling pathways and their identifying markers contribute immensely to the better understanding of chemotherapy resistance, tumor initiation, and recurrence of the different lung cancer subtypes. The lung cancer which is the leading cause of cancer-related deaths worldwide makes the victims highly susceptible because of the drug resistance and tumor relapse. Therefore, the studies relating the CSCs with lung cancer initiation and progression have been gaining momentum. A number of studies have come up in the recent times, which suggest the roles of CSCs both in SCLC and NSCLC lung cancer subtypes both in vitro and in vivo. The CSC markers seem to be expressed
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differentially in the various types of cancers. The variation in expression of the markers is even existent in the lung cancer subtypes making them all the more difficult to be targeted. A precise knowledge about the CSCs that are involved with the specific cancer types is therefore essential for their identification, isolation, and characterization. This can certainly extend the available knowledge regarding the development of lung cancer therapy based on the CSCs. It is also essential to understand the different signaling mechanisms that are involved in the maintenance and self-renewal of lung cancer stem-like cells. Different pathways like Notch or Wnt/b-catenin or Hh are found to be deregulated in specific cancer cases, and hence their roles need to be dissected so that they can be targeted to design better therapeutic modalities. Notwithstanding the controversies regarding the use of CSCs in the lung tumor development, they provide significant promise in the generation of therapies against the drug-resistant lung cancers that can also solve the problem of tumor relapse.
References 1. Herbst, R. S., Heymach, J. V., & Lippman, S. M. (2008). Lung cancer. New England Journal of Medicine, 359, 1367–1380. 2. Martel, C., Ferlay, J., Franceschi, S., et al. (2012). Global burden of cancers attributable to infections in 2008: a review and synthetic analysis. The Lancet Oncology, 13, 607–615. 3. American Cancer Society. (2011). Cancer facts & figures 2011. Atlanta: American Cancer Society. 4. Brodowicz, T., Ciuleanu, T., Crawford, J., et al. (2012). Third CECOG consensus on the systemic treatment of non-small-cell lung cancer. Annals of Oncology, 23, 1223–1229. 5. D’Addario, G., & Felip, E. (2009). ESMO Guidelines Working Group. Non-small-cell lung cancer: ESMO clinical recommendations for diagnosis, treatment and follow-up. Annals of Oncology, 20, 68–70. 6. D’Addario, G., Fru¨h, M., Reck, M., et al. (2010). Metastatic non-small-cell lung cancer: ESMO clinical practice guidelines for diagnosis, treatment and follow-up. Annals of Oncology, 21, 116–119. 7. Goldstraw, P., Ball, D., Jett, J. R., et al. (2011). Non-small-cell lung cancer. Lancet, 378, 1727–1740. 8. Travis, W. D., Travis, L. B., & Devesa, S. S. (1995). Lung cancer. Cancer, 75, 191–202. 9. Alison, M. R., Lin, W. R., Lim, S. M., et al. (2012). Cancer stem cells: in the line of fire. Cancer Treatment Reviews, 38, 589–598. 10. Mani, S. A., Guo, W., Liao, M. J., et al. (2008). The epithelial mesenchymal transition generates cells with properties of stem cells. Cell, 133, 704–715. 11. Bonnet, D., & Dick, J. E. (1997). Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nature Medicine, 3, 730–737. 12. Korkaya, H., & Wicha, M. S. (2007). Selective targeting of cancer stem cells: a new concept in cancer therapeutics. BioDrugs, 21, 299–310. 13. Perona, R., Lopez-Ayllon, B. D., de Castro Carpeno, J., et al. (2011). A role for cancer stem cells in drug resistance and
395
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31. 32.
metastasis in non-small-cell lung cancer. Clinical and Translational Oncology, 13, 289–293. Li, J., & Zhou, B. P. (2011). Activation of beta-catenin and Akt pathways by twist are critical for the maintenance of EMT associated cancer stem cell-like characters. BMC Cancer, 11, 49. Kelsey, C. R., Marks, L. B., Hollis, D., et al. (2009). Local recurrence after surgery for early stage lung cancer: An 11-year experience with 975 patients. Cancer, 115, 5218–5227. Wisnivesky, J. P., Yankelevitz, D., & Henschke, C. I. (2005). Stage of lung cancer in relation to its size: Part 2. Evidence. Chest, 127, 1136–1139. Howlader, N., Noon, A.M., Krapcho, M., et al. (2012). SEER Cancer Statistics Review, 1975–2009 (Vintage 2009 Populations). Bethesda, MD: National Cancer Institute. http://seer.can cer.gov/csr/1975_2009_pops09/, based on November 2011 SEER data submission, posted to the SEER website, April 2012. Pao, W., Miller, V. A., Politi, K. A., et al. (2005). Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Medicine, 2, e73. Katayama, R., Shaw, A. T., Khan, T. M., et al. (2012). Mechanisms of acquired crizotinib resistance in ALK-rearranged lung cancers. Science Translational Medicine, 4, 120ra17. Janne, P. A., Freidlin, B., Saxman, S., et al. (2002). Twentyfive years of clinical research for patients with limited-stage small cell lung carcinoma in North America. Cancer, 95, 1528–1538. Murray, N., Coy, P., Pater, J. L., et al. (1993). Importance of timing for thoracic irradiation in the combined modality treatment of limited-stage small-cell lung cancer. The National Cancer Institute of Canada Clinical Trials Group. Journal of Clinical Oncology, 11, 336–344. Lapidot, T., Sirard, C., Vormoor, J., et al. (1994). A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature, 367, 645–648. Rivera, C., Rivera, S., Loriot, Y., et al. (2011). Lung cancer stem cell: New insights on experimental models and preclinical data. Journal of Oncology, 2011, 549181. O’Flaherty, J. D., Barr, M., Fennell, D., et al. (2012). The cancer stem-cell hypothesis: Its emerging role in lung cancer biology and its relevance for future therapy. Journal of Thoracic Oncology, 7, 1880–1890. Eyler, C. E., & Rich, J. N. (2008). Survival of the fittest: Cancer stem cells in therapeutic resistance and angiogenesis. Journal of Clinical Oncology, 26, 2839–2845. Visvader, J. E., & Lindeman, G. J. (2008). Cancer stem cells in solid tumours: Accumulating evidence and unresolved questions. Nature Reviews Cancer, 8, 755–768. Visvader, J. E., & Lindeman, G. J. (2012). Cancer stem cells: Current status and evolving complexities. Cell Stem Cell, 10, 717–728. Welte, Y., Adjaye, J., Lehrach, H. R., et al. (2010). Cancer stem cells in solid tumors: Elusive or illusive? Cell Communication and Signaling, 8, 6. Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., et al. (2003). Prospective identification of tumorigenic breast cancer cells. Proceedings of the National Academy of Sciences of the United States of America, 100, 3983–3988. Garvalov, B. K., & Acker, T. (2011). Cancer stem cells: A new framework for the design of tumor therapies. Journal of Molecular Medicine (Berlin), 89, 95–107. Lundin, A., & Driscoll, B. (2013). Lung cancer stem cells: Progress and prospects. Cancer Letters, 338, 89–93. He, S., Nakada, D., & Morrison, S. J. (2009). Mechanisms of stem cell self-renewal. Annual Review of Cell and Developmental Biology, 25, 377–406.
123
396 33. Eramo, A., Lotti, F., Sette, G., et al. (2008). Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death and Differentiation, 15, 504–514. 34. Bertolini, G., Roz, L., Perego, P., et al. (2009). Highly tumorigenic lung cancer CD133? cells display stem-like features and are spared by cisplatin treatment. Proceedings of the National Academy of Sciences of the United States of America, 106, 16281–16286. 35. Chen, Y. C., Hsu, H. S., Chen, Y. W., et al. (2008). Oct-4 expression maintained cancer stem-like properties in lung cancer-derived CD133-positive cells. PLoS One, 3, e2637. 36. Meng, X., Li, M., Wang, X., et al. (2009). Both CD133? and CD133- subpopulations of A549 and H446 cells contain cancer-initiating cells. Cancer Science, 100, 1040–1046. 37. Salcido, C. D., Larochelle, A., Taylor, B. J., et al. (2010). Molecular characterisation of side population cells with cancer stem cell-like characteristics in small-cell lung cancer. British Journal of Cancer, 102, 1636–1644. 38. Ho, M. M., Ng, A. V., Lam, S., et al. (2007). Side population in human lung cancer cell lines and tumors is enriched with stemlike cancer cells. Cancer Research, 67, 4827–4833. 39. Gutova, M., Najbauer, J., Gevorgyan, A., et al. (2007). Identification of uPAR-positive chemoresistant cells in small cell lung cancer. PLoS One, 2, e243. 40. Cui, F., Wang, J., Chen, D., et al. (2011). CD133 is a temporary marker of cancer stem cells in small cell lung cancer, but not in non-small cell lung cancer. Oncology Reports, 25, 701–708. 41. Akunuru, S., James Zhai, Q., & Zheng, Y. (2012). Non-small cell lung cancer stem/progenitor cells are enriched in multiple distinct phenotypic subpopulations and exhibit plasticity. Cell Death and Disease, 3, e352. 42. Niu, Q., Wang, W., Li, Y., et al. (2012). Low molecular weight heparin ablates lung cancer cisplatin-resistance by inducing proteasome-mediated ABCG2 protein degradation. PLoS One, 7, e41035. 43. Shi, Y., Fu, X., Hua, Y., et al. (2012). The side population in human lung cancer cell line NCI-H460 is enriched in stem-like cancer cells. PLoS One, 7, e33358. 44. Wang, F., Mi, Y. J., Chen, X. G., et al. (2012). Axitinib targeted cancer stemlike cells to enhance efficacy of chemotherapeutic drugs via inhibiting the drug transport function of ABCG2. Molecular Medicine, 18, 887–898. 45. Nakatsugawa, M., Takahashi, A., Hirohashi, Y., et al. (2011). SOX2 is overexpressed in stemlike cells of human lung adenocarcinoma and augments the tumorigenicity. Laboratory Investigation, 91, 1796–1804. 46. Liang, D., & Shi, Y. (2012). Aldehyde dehydrogenase-1 is a specific marker for stem cells in human lung adenocarcinoma. Medical Oncology, 29, 633–639. 47. Sullivan, J. P., Spinola, M., Dodge, M., et al. (2010). Aldehyde dehydrogenase activity selects for lung adenocarcinoma stem cells dependent on notch signaling. Cancer Research, 70, 9937–9948. 48. Kubo, T., Takigawa, N., Osawa, M., et al. (2013). Subpopulation of small-cell lung cancer cells expressing CD133 and CD87 show resistance to chemotherapy. Cancer Science, 104, 78–84. 49. Hegedus, C., Truta-Feles, K., Antalffy, G., et al. (2012). Interaction of the EGFR inhibitors gefitinib, vandetanib, pelitinib and neratinib with the ABCG2 multidrug transporter: implications for the emergence and reversal of cancer drug resistance. Biochemical Pharmacology, 84, 260–267. 50. Sung, J. M., Cho, H. J., Yi, H., et al. (2008). Characterization of a stem cell population in lung cancer A549 cells. Biochemical and Biophysical Research Communications, 371, 163–167. 51. Meng, X., Wang, X., & Wang, Y. (2009). More than 45 % of A549 and H446 cells are cancer initiating cells: evidence from
123
Cell Biochem Biophys (2014) 69:389–398
52.
53.
54.
55.
56. 57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
cloning and tumorigenic analyses. Oncology Reports, 21, 995–1000. Zhou, J., Wang, H., Cannon, V., et al. (2011). Side population rather than CD133(?) cells distinguishes enriched tumorigenicity in hTERT-immortalized primary prostate cancer cells. Molecular Cancer, 10, 112. Shmelkov, S. V., Butler, J. M., Hooper, A. T., et al. (2008). CD133 expression is not restricted to stem cells, and both CD133? and CD133- metastatic colon cancer cells initiate tumors. Journal of Clinical Investigation, 118, 2111–2120. Jaggupilli, A., & Elkord, E. (2012). Significance of CD44 and CD24 as cancer stem cell markers: An enduring ambiguity. Clinical and Developmental Immunology, 2012, 708036. Du, L., Wang, H., He, L., et al. (2008). CD44 is of functional importance for colorectal cancer stem cells. Clinical Cancer Research, 14, 6751–6760. Bapat, S. A. (2010). Human ovarian cancer stem cells. Reproduction, 140, 33–41. Lee, H. J., Choe, G., Jheon, S., et al. (2010). CD24, a novel cancer biomarker, predicting disease-free survival of non-small cell lung carcinomas: A retrospective study of prognostic factor analysis from the viewpoint of forthcoming (seventh) new TNM classification. Journal of Thoracic Oncology, 5, 649–657. Slomiany, M. G., Dai, L., Tolliver, L. B., et al. (2009). Inhibition of functional hyaluronan-CD44 interactions in CD133positive primary human ovarian carcinoma cells by small hyaluronan oligosaccharides. Clinical Cancer Research, 15, 7593–7601. Leung, E. L., Fiscus, R. R., Tung, J. W., et al. (2010). Non-small cell lung cancer cells expressing CD44 are enriched for stem cell-like properties. PLoS One, 5, e14062. An, Y., & Ongkeko, W. M. (2009). ABCG2: The key to chemoresistance in cancer stem cells? Expert Opinion on Drug Metabolism and Toxicology, 5, 1529–1542. Ross, D. D., & Nakanishi, T. (2010). Impact of breast cancer resistance protein on cancer treatment outcomes. Methods in Molecular Biology, 596, 251–290. Krishnamurthy, P., Ross, D. D., Nakanishi, T., et al. (2004). The stem cell marker Bcrp/ABCG2 enhances hypoxic cell survival through interactions with heme. Journal of Biological Chemistry, 279, 24218–24225. Ahmed, F., Arseni, N., Glimm, H., et al. (2008). Constitutive expression of the ATP-binding cassette transporter ABCG2 enhances the growth potential of early human hematopoietic progenitors. Stem Cells, 26, 810–818. Levina, V., Marrangoni, A. M., & De Marco, R. (2008). Drugselected human lung cancer stem cells: Cytokine network, tumorigenic and metastatic properties. PLoS One, 3, e3077. Rosner, M. H., Vigano, M. A., Ozato, K., et al. (1990). A POUdomain transcription factor in early stem cells and germ cells of the mammalian embryo. Nature, 345, 686–692. Hilbe, W., Dirnhofer, S., Oberwasserlechner, F., et al. (2004). CD133 positive endothelial progenitor cells contribute to the tumour vasculature in non-small cell lung cancer. Journal of Clinical Pathology, 57, 965–969. Herpel, E., Jensen, K., Muley, T., et al. (2011). The cancer stem cell antigens CD133, BCRP1/ABCG2 and CD117/c-KIT are not associated with prognosis in resected early-stage non-small cell lung cancer. Anticancer Research, 31, 4491–4500. Russo, J. E., & Hilton, J. (1998). Characterization of cytosolic aldehyde dehydrogenase from cyclophosphamide resistant L1210 cells. Cancer Research, 48, 2963–2968. Huang, C. P., Tsai, M. F., Chang, T. H., et al. (2013). ALDH positive lung cancer stem cells confer resistance to epidermal growth factor receptor tyrosine kinase inhibitors. Cancer Letters, 328, 144–151.
Cell Biochem Biophys (2014) 69:389–398 70. Lu, Y., Thomson, J. M., Wong, H. Y., et al. (2007). Transgenic over-expression of the microRNA miR-17-92 cluster promotes proliferation and inhibits differentiation of lung epithelial progenitor cells. Development Biology, 310, 442–453. 71. Karamboulas, C., & Ailles, L. (2013). Developmental signaling pathways in cancer stem cells of solid tumors. Biochimica et Biophysica Acta, 1830, 2481–2495. 72. Takebe, N., & Ivy, S. P. (2010). Controversies in cancer stem cells: targeting embryonic signaling pathways. Clinical Cancer Research, 16, 3106–3112. 73. Varjosalo, M., & Taipale, J. (2008). Hedgehog: Functions and mechanisms. Genes and Development, 22, 2454–2472. 74. Sasaki, H., Nishizaki, Y., Hui, C., et al. (1999). Regulation of GLI2 and GLI3 activities by an amino-terminal repression domain: Implication of GLI2 and GLI3 as primary mediators of Shh signaling. Development, 126, 3915–3924. 75. Clement, V., Sanchez, P., de Tribolet, N., et al. (2007). HEDGEHOG-GLI1 signaling regulates human glioma growth, cancer stem cell selfrenewal, and tumorigenicity. Current Biology, 17, 165–172. 76. Peacock, C. D., Wang, Q., Gesell, G. S., et al. (2007). Hedgehog signaling maintains a tumor stem cell compartment in multiple myeloma. Proceedings of the National Academy of Sciences of the United States of America, 104, 4048–4053. 77. Stecca, B., Mas, C., Clement, V., et al. (2007). Melanomas require HEDGEHOG-GLI signaling regulated by interactions between GLI1 and the RAS-MEK/AKT pathways. Proceedings of the National Academy of Sciences of the United States of America, 104, 5895–5900. 78. Mukherjee, S., Frolova, N., Sadlonova, A., et al. (2006). Hedgehog signaling and response to cyclopamine differ in epithelial and stromal cells in benign breast and breast cancer. Cancer Biology and Therapy, 5, 674–683. 79. Yuan, Z., Goetz, J. A., Singh, S., et al. (2007). Frequent requirement of hedgehog signaling in non-small cell lung carcinoma. Oncogene, 26, 1046–1055. 80. Watkins, D. N., Berman, D. M., Burkholder, S. G., et al. (2003). Hedgehog signalling within airway epithelial progenitors and in small-cell lung cancer. Nature, 422, 313–317. 81. Park, K. S., Martelotto, L. G., Peifer, M., et al. (2011). A crucial requirement for Hedgehog signaling in small cell lung cancer. Nature Medicine, 17, 1504–1508. 82. Collins, B. J., Kleeberger, W., & Ball, D. W. (2004). Notch in lung development and lung cancer. Seminars in Cancer Biology, 14, 357–364. 83. Richter, S., McWhirter, E., Chen, E. X., et al. (2012). A phase I study of R04929097, an oral gamma secretase inhibitor, in combination with gemcitabine, in patients with advanced solid tumors (PHL-078/CTEP 8575). Journal of Clinical Oncology, 30, 3082. 84. Ito, T., Udaka, N., Yazawa, T., et al. (2000). Basic helix-loophelix transcription factors regulate the neuroendocrine differentiation of fetal mouse pulmonary epithelium. Development, 127, 3913–3921. 85. Dang, T. P., Eichenberger, S., Gonzalez, A., et al. (2003). Constitutive activation of Notch3 inhibits terminal epithelial differentiation in lungs of transgenic mice. Oncogene, 22, 1988–1997. 86. Chen, H., Thiagalingam, A., Chopra, H., et al. (1997). Conservation of the Drosophila lateral inhibition pathway in human lung cancer: A hairy-related protein (HES-1) directly represses achaete-scute homolog-1 expression. Proceedings of the National Academy of Sciences of the United States of America, 94, 5355–5360. 87. Osanyingbemi-Obidi, J., Dobromilskaya, I., Illei, P. B., et al. (2011). Notch signaling contributes to lung cancer clonogenic
397
88.
89.
90.
91.
92.
93.
94. 95. 96. 97.
98.
99.
100.
101.
102.
103.
104.
105. 106. 107. 108.
capacity in vitro but may be circumvented in tumorigenesis in vivo. Molecular Cancer Research, 9, 1746–1754. Konishi, J., Kawaguchi, K. S., Vo, H., et al. (2007). Gammasecretase inhibitor prevents Notch3 activation and reduces proliferation in human lung cancers. Cancer Research, 67, 8051–8057. Weijzen, S., Rizzo, P., Braid, M., et al. (2002). Activation of Notch-1 signaling maintains the neoplastic phenotype in human Ras-transformed cells. Nature Medicine, 8, 979–986. Westhoff, B., Colaluca, I. N., D’Ario, G., et al. (2009). Alterations of the Notch pathway in lung cancer. Proceedings of the National Academy of Sciences of the United States of America, 106, 22293–22298. Nicolas, M., Wolfer, A., Raj, K., et al. (2003). Notch1 functions as a tumor suppressor in mouse skin. Nature Genetics, 33, 416–421. Klinakis, A., Lobry, C., Abdel-Wahab, O., et al. (2011). A novel tumour suppressor function for the Notch pathway in myeloid leukaemia. Nature, 473, 230–233. Liu, Z., Turkoz, A., Jackson, E. N., et al. (2011). Notch1 loss of heterozygosity causes vascular tumors and lethal hemorrhage in mice. Journal of Clinical Investigation, 121, 800–808. Phng, L. K., & Gerhardt, H. (2009). Angiogenesis: A team effort coordinated by Notch. Developmental Cell, 16, 196–208. Radtke, F., Fasnacht, N., & Macdonald, H. R. (2010). Notch signaling in the immune system. Immunity, 32, 14–27. Willert, K., & Jones, K. A. (2006). Wnt signaling: Is the party in the nucleus? Genes and Development, 20, 1394–1404. He, B., Barg, R. N., You, L., et al. (2005). Wnt signaling in stem cells and non-small-cell lung cancer. Clinical Lung Cancer, 7, 54–60. Uematsu, K., He, B., You, L., et al. (2003). Activation of the Wnt pathway in non small cell lung cancer: Evidence of dishevelled overexpression. Oncogene, 22, 7218–7221. Teng, Y., Wang, X., Wang, Y., et al. (2010). Wnt/beta-catenin signaling regulates cancer stem cells in lung cancer A549 cells. Biochemical and Biophysical Research Communications, 392, 373–379. Yeh, C. T., Wu, A. T., Chang, P. M., et al. (2012). Trifluoperazine, an antipsychotic agent, inhibits cancer stem cell growth and overcomes drug resistance of lung cancer. American Journal of Respiratory and Critical Care Medicine, 186, 1180–1188. Serrano, D., Bleau, A. M., Fernandez-Garcia, I., et al. (2011). Inhibition of telomerase activity preferentially targets aldehyde dehydrogenase-positive cancer stem-like cells in lung cancer. Molecular Cancer, 10, 96. He, B., You, L., Uematsu, K., et al. (2004). A monoclonal antibody against Wnt-1 induces apoptosis in human cancer cells. Neoplasia, 6, 7–14. Tian, F., Mysliwietz, J., Ellwart, J., et al. (2012). Effects of the Hedgehog pathway inhibitor GDC-0449 on lung cancer cell lines are mediated by side populations. Clinical and Experimental Medicine, 12, 25–30. Levina, V., Marrangoni, A., Wang, T., et al. (2010). Elimination of human lung cancer stem cells through targeting of the stem cell factor-c-kit autocrine signaling loop. Cancer Research, 70, 338–346. Hill, R. P. (2006). Identifying cancer stem cells in solid tumors: Case not proven. Cancer Research, 66, 1891–1895. Kern, S. E., & Shibata, D. (2007). The fuzzy math of solid tumor stem cells: A perspective. Cancer Research, 67, 8985–8988. Kelly, P. N., Dakic, A., Adams, J. M., et al. (2007). Tumor growth need not be driven by rare cancer stem cells. Science, 317, 337. Quintana, E., Shackleton, M., Sabel, M. S., et al. (2008). Efficient tumour formation by single human melanoma cells. Nature, 456, 593–598.
123
398 109. Li, Z., Bao, S., Wu, Q., et al. (2009). Hypoxia-inducible factors regulate tumorigenic capacity of glioma stem cells. Cancer Cell, 15, 501–513. 110. Heddleston, J. M., Li, Z., McLendon, R. E., et al. (2009). The hypoxic microenvironment maintains glioblastoma stem cells and promotes reprogramming towards a cancer stem cell phenotype. Cell Cycle, 8, 3274–3284.
123
Cell Biochem Biophys (2014) 69:389–398 111. Li, L., & Neaves, W. B. (2006). Normal stem cells and cancer stem cells: The niche matters. Cancer Research, 66, 4553–4557. 112. Gupta, P. B., Chaffer, C. L., & Weinberg, R. A. (2009). Cancer stem cells: Mirage or reality? Nature Medicine, 15, 1010–1012.
REVIEW PAPER
Lung Cancer Stem Cells and Implications for Future Therapeutics Jing Wang • Ze-hong Li • James White Lin-bo Zhang
•
Published online: 20 February 2014 Ó Springer Science+Business Media New York 2014
Abstract Lung cancer is the most dreaded of all cancers because of the higher mortality rates associated with it worldwide. The various subtypes of lung cancer respond differently to a particular treatment regime, which makes the therapeutic interventions all the more complicated. The concept of cancer stem cells (CSCs) is based primarily on the clinical and experimental observations that indicate the existence of a subpopulation of cells with the capacity to self-renew and differentiate as well as show increased resistance to radiation and chemotherapy. They are considered as the factors responsible for the cases of tumor relapse. The CSCs may have significant role in the development of lung tumorigenesis based on the identification of the CSCs which respond during injury. The properties of multi-potency and self-renewal are shared in common by the lung CSCs with the normal pluripotent stem cells which can be isolated using the similar markers. This review deals with the origin and characteristics of the lung cancer stem cells. The role of different markers used to isolate lung CSCs like CD44, ALDH (aldehyde dehydrogenase), CD133 and ABCG2 (ATP binding cassette sub family G member 2) have been discussed in detail. Analysis of the developmental signaling pathways such as Wnt/b-catenin, Notch, hedgehog in the regulation and maintenance of the lung CSCs have been done. Finally, before targeting the J. Wang (&) Z. Li L. Zhang (&) School of Life Sciences, Jilin Agricultural University, Changchun 130118, China e-mail: [email protected] L. Zhang e-mail: [email protected] J. White School of Medicine, University of Maryland, Baltimore, MD 20201, USA
lung CSC biomarkers for potential therapeutics, challenges faced in lung cancer stem cell research need to be taken into account. With the accepted notion that the CSCs are to blame for cancer relapse and drug resistance, targeting them can be an important aspect of lung cancer therapy in the future. Keywords Lung cancer Cancer stem cells (CSCs) CD44 ALDH (aldehyde dehydrogenase) CD133 ABCG2 (ATP binding cassette sub family G member 2)
Introduction The diagnosis of lung cancer usually takes place at a very advanced stage because of poor prognosis and as a result it becomes the leading death causing cancer throughout the world [1, 2]. In 2011, *221,000 new lung cancer cases have been diagnosed with 157,000 deaths while that in Europe the mortality rate is 148,000/year [3–5]. This signifies the disease burden associated with lung cancer. The two main categories of lung cancer include small cell lung cancer (SCLC) which is observed in only 15–20 % of the lung cancer cases; and NSCLC (non-small cell lung cancer), the more common of the two and which accounts for *80–85 % of the cases [1, 3, 5, 6]. Based on the histology, NSCLC is again subtyped into squamous cell carcinoma (accounting for 30 % of the cases) and non-squamous carcinoma (observed in 70 % of NSCLCs). The nonsquamous cell carcinomas consist of mostly adenocarcinomas as well as large cell carcinomas. and at times. some lesser differentiated variants are also observed [1, 7, 8]. There are ever-increasing evidences that support the role of cancer stem cells (CSCs) in driving the development and progression of a plethora of cancers including lung cancers.
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It is also suggested that the poor prognosis and unsuccessful clinical outcomes associated with lung cancers are rendered by the CSCs [9, 10]. Therefore, it becomes important to study the various stem cell markers that are enriched in lung cancers and can be used to isolate the cells with stem-like characteristics. It is also necessary to understand the various signaling pathways that are involved in the regulation as well as the maintenance of the lung CSCs. The critical pathways involved in such activities present themselves as promising targets for lung cancer treatment. The CSCs which result in the generation of cancer cells of heterogenous population consist of a unique, small number of undifferentiated cells with the capacity of multipotent progenitor generation and formation of tumors. This subpopulation of cancer cells when introduced into transgenic animal models mimics the original tumorigenic potential in vivo. Under in vitro conditions, the cells could give rise to spheres [11–13]. The CSCs make the tumors become more aggressive in nature because of their resistance to chemotherapy and radiation, and hence result in poor prognosis [9]. In addition, the epithelial to mesenchymal transition (EMT) identified in connection with the CSCs drives the cancer cells toward metastasis and migration, making them more dangerous component of tumor progression. The CSCs have their major impact in the cancer recurrence. The current treatment modalities can affect and treat most of the cancer cell population. However, in the small subpopulation of treatment-resistant cancer cells, the CSCs serve as the reservoir for the cancer cells and replenish the population which gives rise to tumor relapse [10, 14]. Same is the case with the lung cancer where *20 % of NSCLCs can be operated at present. However, the recurrence rate among those operated is quite high at 30–50 % [15]. The overall survival rate is as low as 7–20 % observed in a 5-year period when radical chemoradiotherapy is applied in the advanced cases of NSCLC [16, 17]. Significant responses have been observed in the adenocarcinoma patients [where the tumors show mutations for analplastic lymphoma kinase (ALK) or epidermal growth factor receptor (EGFR)]. However, within 2–3 years, almost all the patients succumb to recurrence of drug resistance [18, 19]. Similar responses are encountered in SCLC cases where platinum-based chemotherapy induces high response among the patients; however, such responses are short-lived with drug-resistant relapse. The survival rate among such patients is very poor (at *5 %) [20, 21]. Such ability of the lung cancers to recur, despite showing complete response and tumor regression at one point, suggests the presence of a cell population with tremendous regenerative and self-renewal capacities, which is only comparable with the embryonic stem cells. Therefore, the targeting of lung CSCs presents the potential of delaying or inhibiting tumor relapse. Although the
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significance of CSCs in the development and progression of lung cancers is quite evident, a precise review that details about it is, however, not available except for some specific information and articles related to the recent studies in the field. This review will try to focus on the different aspects of lung cancer with respect to the CSCs in the progression and development of tumorigenesis. A better understanding of the different markers used to identify and characterize the lung CSCs as well as the signaling mechanisms will enable the development of more effective therapeutics in the treatment of lung cancer and prevention of relapse.
Method The review has been developed using the published literature in the field of lung cancer stem cells. We have searched the Pubmed, the database for articles on life sciences and biomedical research. The search included only the articles from January 1, 2000 to October 1, 2013 to have the most updated information in the field. The keywords used to search the articles included lung cancer, stem cell, lung cancer stem cell, CSCs, markers for CSCs, signaling pathways in CSCs, cancer initiating cells (CICs). Some of the relevant journals related to the field of stem cell research like stem cell, CSCs were searched separately to find any additional information that might have been missed. Only those peer-reviewed relevant articles were used for the current review and have been aptly referenced. Although we put no restriction according to language, only the English articles were found relevant to our purpose.
Lung Cancer Stem Cells The CSCs are also referred to as the CICs since these cells when transplanted in animal models can reproduce the phenotypic features of the tumors of their primary origin [22]. Several studies have linked the CSCs with enhanced proliferation and invasiveness, metastasis and migration, stem cell marker expression, resistance to chemo or radiotherapy, resistance to apoptosis and hypoxia, and an overall increased tumorigenic potential [23–27]. Notwithstanding the increase in the number of studies in this field in recent times, the observations and findings from such studies have to be interpreted and exploited with caution, since there are sufficient controversies regarding the selection criteria of the CSCs like the absence of proper validation of the studies or well-defined markers [28]. It was demonstrated in a study in 1997 that a small subpopulation of cells from the acute myeloid leukemia (AML) could develop AML in NOD/SCID mice giving rise to one
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of the first ever possibilities of CSC existence [11]. It was not before 6 years from thence that the isolation of CSCs took place from solid tumor in 2003 (Clarke’s group). In this study with breast cancer, the researchers found that the CD44?/CD24 low-isolated cell population could only reproduce the phenotypic and morphologic characteristics of the primary cancer through tumor formation in immunodeficient mice [29]. There exists substantial overlap between the regulation of the stemness characteristics of the CSCs and ESCs (embryonic stem cells) such as selfrenewal, stemness, pluripotency, proliferation, and differentiation which are controlled by similar signaling pathways such as the Wnt, Notch, and Hedgehog (Hh) [30]. These are the same pathways involved in the development of lung and in adults, due to acquisitioned aberration results in lung oncogenesis by dysregulating the balance between differentiation and self-renewal [31]. The critical decision to make during the stem cell division is whether to differentiate or to self-renew. Some of the important transcription factors associated with the maintenance of stem cells and tumorigenesis promotion are Nanog, Oct4, and Sox2, which induce self-renewal and prevent differentiation [32]. The CSCs from non-small cell lung carcinoma are usually identified by the use of the markers like the surface marker CD133 or an enhanced effluxing capacity of the Hoechst dye (that binds to DNA) and gives rise to the SP (side-population) [33–38]. When comparing with the NSCLC, very few studies with the stem cells from the SCLC have been performed till date. Nevertheless, in one of the studies, it was shown that the SP from SCLC had increased tumorigenic potential in vivo [37]. In yet another study, it was observed that CD87 could be used to isolate SCLC stem-like cells successfully, which possessed the capacity to form spheres and showed resistance against multiple drugs in vitro [39]. The presence of cell surface markers on the CSCs has been routinely exploited to isolate the subpopulations of the cells from the pool of cancer cells with stemness properties such as enhanced colony and sphere-forming capacities, increased rate of invasion and proliferation, increased resistance to therapy, and overall increase in tumorigenicity [40–49]. The use of cell-surface markers (although a number of them are available) to isolate cancer cells with stem-like characteristics is very challenging. As a matter of fact there exists significant variation in their level of expression and types available in tumor cells. This variation is even existent among surface markers of same tumor origin from different cell lines or patients. Moreover, often the isolated cell population is heterogenous in nature and/or the expression of the markers is not sufficient that causes a compromise in the study of the lung CSCs [50]. In a recent report, it was shown that both the CD133? and CD133-enriched cell populations from the lung cancer cells contained equal percentage of
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lung CSCs. This suggests that CD133 may not be sufficient alone as a marker for the isolation of lung CSCs [36, 51]. Others have found similar results with prostate and colon CSCs [52, 53]. Accordingly, the cells with stem-like characteristics isolated using CD133 as a marker has produced controversial results. In an interesting finding, the ALDH? SP cells demonstrated the unique stem-like characteristics of quiescence, dormancy, and slow division. Nonetheless, with such conflicting results available regarding the various markers used to identify, isolate, and characterize the lung cancer stem cells, a thorough analysis of the various lung CSC-associated markers is essential to understand their potential fully before putting them into application.
Markers Used in the Identification of Lung CSCs Although a number of CSC markers are being reported in recent times in the available literatures, very few of them are eventually validated. However, the identification of the different CSCs with significant variation from those of the other cancer cells has been possible because of the elaborate studies performed within this field. The CSCs are known to express a number of different markers at the same time and the use of a single marker may not be sufficient to isolate a pure subpopulation of stem-like cells from the pool of cancer cells [36]. Therefore, there is a definite need to study the available CSC markers in relation to lung cancer which might as well improve prognosis and overall therapeutic intervention in this cancer type. In this section, the present knowledge regarding the lung CSC markers-CD44 and CD24, ABCG2, CD133, and ALDH has been highlighted. CD44 and CD24 Almost all types of tumor cells express the hyaluronic acid receptor CD44 which is, therefore, one of the most frequently studied surface markers [54]. The marker has been found in association with the regulatory proteins for growth, proliferation, cell adhesion, motility, and migration [55]. In addition, it has been found to be involved in cell migration and takes part in the signaling cascades involved in tumor initiation and progression. The CD44 gene due to alternate splicing expresses various isoforms that are associated with the different subtypes of cancer. Hence, it is an ideal surface marker candidate for the isolation of CSCs from carcinomas of different origins [56]. Another stem cell surface marker CD24 is an antigen that is heat stable and expressed in a multitude of cancers. It is an important marker for the diagnosis as well as prognosis of tumorigenesis since it plays significant roles in cell to
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matrix as well as cell to cell interactions thereby is involved in cell adhesion and migration [57]. CD24 causes enhanced proliferation of the tumor cells and induces them to get adhered to collagen, laminin and fibronectin. Therefore, a CD24 high cell population can be linked to increased progression of tumorigenesis and metastasis. CD24 and CD44 in combination have been used for the isolation of solid tumor CSCs. Since, they are expressed in only a few types of cancers their usage have been limited [58]. The lung cancer CD44? cells have shown enhanced ability to initiate tumors both in vitro and in vivo. Besides, the EMT markers like Vimentin, Snail1 and Cadherin2 were highly upregulated in the CD44? cells and hence it might be involved in EMT and thereby in the maintenance of stemness [59]. ATP-Binding Cassette Sub-Family G Member 2 (ABCG2) Another important marker used in the identification of lung CSCs is the ATP binding cassette reporter that causes decreased drug concentration within the cells by pumping the chemotherapeutic drugs out of the cell [60]. A poor prognosis in cancers has been associated with ABCG2 as it is highly upregulated in the small stem-like cell subpopulation [61]. There are studies to suggest that the expression of ABCG2 is not limited to CSCs of a few cancer types and is even overexpressed in lung CSCs. ABCG2 has been implicated in the increase in hematopoietic stem cell survival in the hypoxic condition as well as in the protection of stem cells [62]. It also protects the stem cells from oxidative stress and contributes to the capacity of proliferation of the progenitor cells both in vivo and in vitro [63]. The stem cells isolated via Hoechst dye efflux method and FACS from the lung cancer cells such as HTB-58, H460, H2170, H23, A549 and H441 exhibited a stem cell SP with increased drug resistance and ABCG2 overexpression [64]. In a recent study, it was found that low molecular weight heparin (LMWH) could reduce the expression of ABCG2 and hence decreased the SP from lung cells which had lesser clonogenic ability in vitro. In addition, cisplatin and LMWH together showed the potential in inducing apoptosis and overcoming drug resistance [42]. Cd133 The most commonly used among the CSC markers is CD133 which is a surface glycoprotein with two extracellular glycosylated loops and five transmembrane stretches. In a study involving 10 NSCLC patients and lung cancer cell lines, the CD133? and CD133- cells from the samples of those patients and five lung cancer cell lines
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(H1299, A549, C299, CCL5 and CCL1) were analyzed [35]. The CD133? cells were demonstrated to possess higher tumor initiation, self-renewal and drug-resistance abilities. In addition, the cells showed increased level of Oct4 (a common embryonic stem cell transcription factor) expression [35, 65]. There are studies that indicate for the drug-induced enrichment of the CD133? population from the lung cancer cells. In one such study, it was observed that the exposure of the A549 cells to cisplatin at cytotoxic concentration caused the increase of the CD133? cell population by eightfolds [34]. The results were reproduced in vivo when cisplatin was used to treat different mice xenograft models. Such findings further confirm the idea that the elimination of the CD133-enriched CICs is not possible through chemotherapy. However, CD133 is not universally expressed in the different subtypes of lung cancer cells. In a related study, it was demonstrated that the CD133? stem-like characteristics was present in the SCLC cell line H446 but not in the NSCLC cells like H157, H292, Calu-1, A549 and Hee6 [40]. On similar lines, the NSCLC specimens showed enhanced CD133- expression that contributed significantly to neovascularization [66]. But CD133 presented near negligible prognostic value in early stage resected NSCLC [67]. Aldehyde Dehydrogenase (ALDH) Another marker linked to stemness properties is ALDH which is perhaps the most important of all lung CSC markers. This enzyme is involved in the oxidation of acetaldehyde as well as in the control of stem cell differentiation [68]. It has been established as a specific marker in lung adenocarcinomas [46]. The ALDH1A1? lung cancer cells exhibited resistance against the EGFR, gefitinib but not the ALDH1A1- cells [69]. Thus, ALDH has been very useful in providing important insights into tumor initiation and is of high prognostic value [70]. Others have observed a correlationship between poor prognosis in lung cancer patients and ALDH1A1 expression [47]. A majority of the NSCLC from different lung cancer tumor samples and cell lines display the presence of increased activity of ALDH (mainly ALDH1A1) in a cell subpopulation. In addition, the ALDH expressing lung cancer cells are proven to be more clonogenic with the increased tumorigenic and self-renewing potential. The Notch pathway has been found to be intricately associated in the maintenance of the lung cancer cells that are positive for ALDH, and accordingly, the Notch-signaling suppression yielded much reduced ALDH? cell population with decreased proliferation and clonogenic capacity of the tumor cells [47]. Therefore, ALDH can be successfully utilized in the selection of NSCLC subpopulation with stem-like properties that possess greater capacity of being tumorigenic.
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Pathways Involved in Lung Cancer Stem Cells The signaling pathways that are involved in embryogenesis are also highly implicated in oncogenesis. Some of the important pathways involved in the maintenance of lung cancer stem cells include Wnt/b-catenin, Notch, and Hh. These pathways are linked with the maintenance of tissue homeostasis and normal stem cell renewal [71]. Any deregulation of such signaling drives the activity of the CSCs in multiple cancer types that even include lung cancer [72]. Hedgehog (Hh) Signaling The Hh signaling is an important component of embryogenesis that regulates proliferation, differentiation and migration of the embryonic stem cells but its presence is near negligible in adult tissues [73]. The activation of the pathway takes place through smoothened, a transmembrane domain containing protein and further activation of transcription factors like GLI1, GLI2 and GLI3 which are involved in the regulation of the downstream genes [74]. The Hh-Gli signaling has been involved in the maintenance of a number of different cancers including breast, melanoma and lung cancers [75–79]. The Hh signaling is among one of the first pathways demonstrated in SCLC. The tumorigenicity in SCLC was lost upon the application of cyclopamine (a smoothened antagonist) to inhibit Hh signaling [80]. These findings indicate that aberrant Hh signaling could give rise to SCLC. According to a similar study involving genetically engineered mice, it was demonstrated that SCLC initiation is contributed significantly by Hh signaling. The use of smoothened antagonists to inhibit the Hh pathway shows good promise in the treatment of lung cancer with potential to prevent cases of tumor relapse [81]. Notch Pathway The highly conserved mammalian embryogenesis pathway Notch is frequently involved in the determination of cellular fate. The asymmetric division-mediated viability of the stem cells is maintained through cell-to-cell interaction that leads to Notch signaling activation [82]. The normal lung development, as well as the determination of the distal and proximal epithelial cellular fates, is controlled by the Notch signaling as demonstrated through knockout mice models [83]. Studies with knockout mice models for Hes 1 (hairy and enhancer of split 1), an important downstream target in the Notch pathway, established the contribution of the Notch signaling in the maintenance of the normal lung stem cells [84]. Accumulation of the stem cells for distal airways with delayed differentiation took place upon
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constitutive Notch signaling activation [85]. The NSCLC cell lines depicted much greater transcript levels of Notch that confirmed the role of the pathway in the tumorigenesis of the lung [86, 87]. The antiapoptotic signals are inhibited by Notch through Survivin induction, while it promotes pro-apoptotic signals via Bim [86, 88]. The g-secretase (required for the cleavage and activation of Notch receptors) inhibition results in the Notch pathway blockade that induces apoptosis and inhibits growth both under in vivo and in vitro conditions in NSCLC [88]. It has also been established that the ras-driven cancers are maintained by a dysregulated Notch pathway [89]. The sphere-forming abilities of the ALDH ? NSCLC cells are largely dependent on the active Notch signaling [47]. There are reports that suggest the probable presence of mutations in the Notch1 receptors from NSCLC [90]. Notwithstanding the role of Notch signaling in the progression of lung tumorigenesis, the involvement of the pathway in the CSC phenotype maintenance is not yet clearly evident [91–93]. However, the Notch pathway might be more complex than it is being understood. In the neuroendocrine-mediated malignancy in SCLC, the Notch receptor expression is very rarely found. Nonetheless, the Notch signaling appears to be an attractive target for antilung cancer therapeutic interventions. It is not only due to its role in stem cell maintenance and tumorigenesis but also for its potential involvement in immune function and tumor angiogenesis. Accordingly, the development of specific inhibitors of Notch signaling pathway members is currently under investigation [94, 95]. The Wnt/b-Catenin Pathway The Wnt/b-catenin pathway is highly implicated in a variety of cellular processes like differentiation, proliferation, motility, and apoptosis [96]. The canonical Wnt signaling is the best understood among the Wnt pathways wherein the Wnts get attached to the cell-surface receptors that phosphorylate the Dvl (disheveled family proteins). The kinases Casein kinase1 (CK1) and glycogen synthase kinase 3b (GSK3b) are, in turn, activated by Dvl which facilitates b-catenin degradation. Inhibition of Dvl therefore results in the b-catenin accumulation in the cytoplasm. The pool of b-catenin that enters the nucleus binds to the transcription factor/lymphoid enhancer-binding factor 1 (TCF/LEF) family of transcription factors, which leads to the expression of the specific genes [97]. The ligands Wnt1 and Wnt2 are highly upregulated in a variety of NSCLC tumor specimens as well as cell lines [98]. In a recent study, Oct 4 (the critical player in stem cell differentiation and self-renewal) regulation was demonstrated after the lung cancer cells A549 were treated with cisplatin. The treatment also resulted in higher b-catenin expression. On
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treating the lung cancer cells with LiCl (inhibitor of GSK3b), stabilization of b-catenin took place. It resulted in a dramatic increase in migration, proliferation, drug-resistance, and clonogenic properties. A blockade of the Wnt/b– catenin pathway caused a decrease in such CSC properties [99].
Targeting the Lung CSCs for Therapy It is an established fact that lung CSCs play a significant role in tumor initiation and progression. Accordingly, lung cancer therapy targeted at the CICs has been developed. In recent times, it was found that the CSC-mediated drug resistance in lung cancer could be overcome through the use of gefitinib and trifluoperazine that target the CD44 and CD133 markers [100]. In a study with lung cancer cells lines (H1299, H460. and A549) that are negative and positive for ALDH, it was demonstrated that the telomere length of the lung CSCs are longer than the differentiated lung cells [101]. It was also shown that the response of the cells to MST312 (an inhibitor for telomerase) was dependent on the length of the telomere they possessed (the longer the telomere, the more sensitive were the cells to the MST312 treatment). Such a response was accompanied by the reduction in ALDH? cells post treatment of the drug. The involvement of the Notch pathway in the maintenance of lung CSCs has been established when a much-reduced ALDH? lung cell population was recovered upon targeting the Notch signaling [90]. Similarly, the inhibition of the Wnt/b-catenin signaling, another pathway highly implicated in lung cancer through the knock down of the Wnt ligands (Wnt1 and Wnt2) or by the use of monoclonal antibodies against them results in the increased apoptosis of the NSCLC cells [102]. The Notch3-signaling inhibition through the use of MRK-003(inhibitor for c-secretase) yielded highly promising results. Such treatment resulted in enhanced apoptosis and reduced proliferation of the lung cancer cells as observed through in vitro and in vivo studies [88]. Others have found the Hh pathway inhibitor, GDC0449, to produce effective results in the growth inhibition of SCLC and adenocarcinoma cells. In addition, it stimulated the cisplatin [103]. The Skp, Cullin, F-box containing complex (SCF complex) is produced by the CSCs which can induce the proliferation of the CSCs via c-Kit binding. The anticancer drug imatinib partly exerts its activity by interfering with the SCF-mediated c-Kit activation thereby negatively influencing the growth of the CSCs. However, imatinib activity seems to be futile in most of the NSCLC cell growth because of lack of c-Kit expression [104]. The majority of these studies point toward the effectiveness of targeting the lung CSCs in controlling different subtypes of lung cancer.
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Problems Encountered in the CSC Model Approach The concept of CSC model in tumor initiation and progression has been gaining momentum in recent times. However, the specific characteristics that define the bona fide CSCs across multiple cancer types are still under debate. The proponents of the non-CSC model argue that the current model is a result of data misinterpretation since the phenotypes of the potential CSC population has been largely inconsistent [105, 106]. The typical and strict assay that has been adapted to test the CSCs is the ability of the putative cancer stem-like cells to form tumor through serial dilution (cell population as low as 1–1,000 should be capable of regenerating the original tumor). The fundamental belief for such assay is that tumor is formed through the clonal expansion of the rare population of CSCs. The in vivo assay has been done typically through the injection of a small population of the isolated CSCs into the NOD/ SCID mice and checking for tumorigenic growth. The clonogenic potential of the nonsorted mouse lymphoma and melanoma cells were confirmed when it was found that only ten such cells were able to regenerate the disease in the recipient mice [107, 108]. Although the non-stem-like cells from melanoma and lymphoma can generate tumors in vivo, the potential of the CSCs to develop tumors in other cancer types is a high and real possibility. The factors like stromal cytokines, tumor angiogenesis, and vasculature, hypoxia, and the overall tumor microenvironment can cause significant perturbations on the CSC presence [26, 109–111]. The differentiated tumor cells can be reprogrammed to CSCs through EMT [10, 112]. Therefore, the future of the therapeutics targeted at the CSCs is highly dependent on the better understanding of the factors that contribute to the stem cell properties of drug resistance, dormancy, and quiescence, tumor initiation and progression, tumor heterogeneity, migration, and metastasis.
Conclusions The CSC reservoir controlled via distinct signaling pathways and their identifying markers contribute immensely to the better understanding of chemotherapy resistance, tumor initiation, and recurrence of the different lung cancer subtypes. The lung cancer which is the leading cause of cancer-related deaths worldwide makes the victims highly susceptible because of the drug resistance and tumor relapse. Therefore, the studies relating the CSCs with lung cancer initiation and progression have been gaining momentum. A number of studies have come up in the recent times, which suggest the roles of CSCs both in SCLC and NSCLC lung cancer subtypes both in vitro and in vivo. The CSC markers seem to be expressed
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differentially in the various types of cancers. The variation in expression of the markers is even existent in the lung cancer subtypes making them all the more difficult to be targeted. A precise knowledge about the CSCs that are involved with the specific cancer types is therefore essential for their identification, isolation, and characterization. This can certainly extend the available knowledge regarding the development of lung cancer therapy based on the CSCs. It is also essential to understand the different signaling mechanisms that are involved in the maintenance and self-renewal of lung cancer stem-like cells. Different pathways like Notch or Wnt/b-catenin or Hh are found to be deregulated in specific cancer cases, and hence their roles need to be dissected so that they can be targeted to design better therapeutic modalities. Notwithstanding the controversies regarding the use of CSCs in the lung tumor development, they provide significant promise in the generation of therapies against the drug-resistant lung cancers that can also solve the problem of tumor relapse.
References 1. Herbst, R. S., Heymach, J. V., & Lippman, S. M. (2008). Lung cancer. New England Journal of Medicine, 359, 1367–1380. 2. Martel, C., Ferlay, J., Franceschi, S., et al. (2012). Global burden of cancers attributable to infections in 2008: a review and synthetic analysis. The Lancet Oncology, 13, 607–615. 3. American Cancer Society. (2011). Cancer facts & figures 2011. Atlanta: American Cancer Society. 4. Brodowicz, T., Ciuleanu, T., Crawford, J., et al. (2012). Third CECOG consensus on the systemic treatment of non-small-cell lung cancer. Annals of Oncology, 23, 1223–1229. 5. D’Addario, G., & Felip, E. (2009). ESMO Guidelines Working Group. Non-small-cell lung cancer: ESMO clinical recommendations for diagnosis, treatment and follow-up. Annals of Oncology, 20, 68–70. 6. D’Addario, G., Fru¨h, M., Reck, M., et al. (2010). Metastatic non-small-cell lung cancer: ESMO clinical practice guidelines for diagnosis, treatment and follow-up. Annals of Oncology, 21, 116–119. 7. Goldstraw, P., Ball, D., Jett, J. R., et al. (2011). Non-small-cell lung cancer. Lancet, 378, 1727–1740. 8. Travis, W. D., Travis, L. B., & Devesa, S. S. (1995). Lung cancer. Cancer, 75, 191–202. 9. Alison, M. R., Lin, W. R., Lim, S. M., et al. (2012). Cancer stem cells: in the line of fire. Cancer Treatment Reviews, 38, 589–598. 10. Mani, S. A., Guo, W., Liao, M. J., et al. (2008). The epithelial mesenchymal transition generates cells with properties of stem cells. Cell, 133, 704–715. 11. Bonnet, D., & Dick, J. E. (1997). Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nature Medicine, 3, 730–737. 12. Korkaya, H., & Wicha, M. S. (2007). Selective targeting of cancer stem cells: a new concept in cancer therapeutics. BioDrugs, 21, 299–310. 13. Perona, R., Lopez-Ayllon, B. D., de Castro Carpeno, J., et al. (2011). A role for cancer stem cells in drug resistance and
395
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31. 32.
metastasis in non-small-cell lung cancer. Clinical and Translational Oncology, 13, 289–293. Li, J., & Zhou, B. P. (2011). Activation of beta-catenin and Akt pathways by twist are critical for the maintenance of EMT associated cancer stem cell-like characters. BMC Cancer, 11, 49. Kelsey, C. R., Marks, L. B., Hollis, D., et al. (2009). Local recurrence after surgery for early stage lung cancer: An 11-year experience with 975 patients. Cancer, 115, 5218–5227. Wisnivesky, J. P., Yankelevitz, D., & Henschke, C. I. (2005). Stage of lung cancer in relation to its size: Part 2. Evidence. Chest, 127, 1136–1139. Howlader, N., Noon, A.M., Krapcho, M., et al. (2012). SEER Cancer Statistics Review, 1975–2009 (Vintage 2009 Populations). Bethesda, MD: National Cancer Institute. http://seer.can cer.gov/csr/1975_2009_pops09/, based on November 2011 SEER data submission, posted to the SEER website, April 2012. Pao, W., Miller, V. A., Politi, K. A., et al. (2005). Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Medicine, 2, e73. Katayama, R., Shaw, A. T., Khan, T. M., et al. (2012). Mechanisms of acquired crizotinib resistance in ALK-rearranged lung cancers. Science Translational Medicine, 4, 120ra17. Janne, P. A., Freidlin, B., Saxman, S., et al. (2002). Twentyfive years of clinical research for patients with limited-stage small cell lung carcinoma in North America. Cancer, 95, 1528–1538. Murray, N., Coy, P., Pater, J. L., et al. (1993). Importance of timing for thoracic irradiation in the combined modality treatment of limited-stage small-cell lung cancer. The National Cancer Institute of Canada Clinical Trials Group. Journal of Clinical Oncology, 11, 336–344. Lapidot, T., Sirard, C., Vormoor, J., et al. (1994). A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature, 367, 645–648. Rivera, C., Rivera, S., Loriot, Y., et al. (2011). Lung cancer stem cell: New insights on experimental models and preclinical data. Journal of Oncology, 2011, 549181. O’Flaherty, J. D., Barr, M., Fennell, D., et al. (2012). The cancer stem-cell hypothesis: Its emerging role in lung cancer biology and its relevance for future therapy. Journal of Thoracic Oncology, 7, 1880–1890. Eyler, C. E., & Rich, J. N. (2008). Survival of the fittest: Cancer stem cells in therapeutic resistance and angiogenesis. Journal of Clinical Oncology, 26, 2839–2845. Visvader, J. E., & Lindeman, G. J. (2008). Cancer stem cells in solid tumours: Accumulating evidence and unresolved questions. Nature Reviews Cancer, 8, 755–768. Visvader, J. E., & Lindeman, G. J. (2012). Cancer stem cells: Current status and evolving complexities. Cell Stem Cell, 10, 717–728. Welte, Y., Adjaye, J., Lehrach, H. R., et al. (2010). Cancer stem cells in solid tumors: Elusive or illusive? Cell Communication and Signaling, 8, 6. Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., et al. (2003). Prospective identification of tumorigenic breast cancer cells. Proceedings of the National Academy of Sciences of the United States of America, 100, 3983–3988. Garvalov, B. K., & Acker, T. (2011). Cancer stem cells: A new framework for the design of tumor therapies. Journal of Molecular Medicine (Berlin), 89, 95–107. Lundin, A., & Driscoll, B. (2013). Lung cancer stem cells: Progress and prospects. Cancer Letters, 338, 89–93. He, S., Nakada, D., & Morrison, S. J. (2009). Mechanisms of stem cell self-renewal. Annual Review of Cell and Developmental Biology, 25, 377–406.
123
396 33. Eramo, A., Lotti, F., Sette, G., et al. (2008). Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death and Differentiation, 15, 504–514. 34. Bertolini, G., Roz, L., Perego, P., et al. (2009). Highly tumorigenic lung cancer CD133? cells display stem-like features and are spared by cisplatin treatment. Proceedings of the National Academy of Sciences of the United States of America, 106, 16281–16286. 35. Chen, Y. C., Hsu, H. S., Chen, Y. W., et al. (2008). Oct-4 expression maintained cancer stem-like properties in lung cancer-derived CD133-positive cells. PLoS One, 3, e2637. 36. Meng, X., Li, M., Wang, X., et al. (2009). Both CD133? and CD133- subpopulations of A549 and H446 cells contain cancer-initiating cells. Cancer Science, 100, 1040–1046. 37. Salcido, C. D., Larochelle, A., Taylor, B. J., et al. (2010). Molecular characterisation of side population cells with cancer stem cell-like characteristics in small-cell lung cancer. British Journal of Cancer, 102, 1636–1644. 38. Ho, M. M., Ng, A. V., Lam, S., et al. (2007). Side population in human lung cancer cell lines and tumors is enriched with stemlike cancer cells. Cancer Research, 67, 4827–4833. 39. Gutova, M., Najbauer, J., Gevorgyan, A., et al. (2007). Identification of uPAR-positive chemoresistant cells in small cell lung cancer. PLoS One, 2, e243. 40. Cui, F., Wang, J., Chen, D., et al. (2011). CD133 is a temporary marker of cancer stem cells in small cell lung cancer, but not in non-small cell lung cancer. Oncology Reports, 25, 701–708. 41. Akunuru, S., James Zhai, Q., & Zheng, Y. (2012). Non-small cell lung cancer stem/progenitor cells are enriched in multiple distinct phenotypic subpopulations and exhibit plasticity. Cell Death and Disease, 3, e352. 42. Niu, Q., Wang, W., Li, Y., et al. (2012). Low molecular weight heparin ablates lung cancer cisplatin-resistance by inducing proteasome-mediated ABCG2 protein degradation. PLoS One, 7, e41035. 43. Shi, Y., Fu, X., Hua, Y., et al. (2012). The side population in human lung cancer cell line NCI-H460 is enriched in stem-like cancer cells. PLoS One, 7, e33358. 44. Wang, F., Mi, Y. J., Chen, X. G., et al. (2012). Axitinib targeted cancer stemlike cells to enhance efficacy of chemotherapeutic drugs via inhibiting the drug transport function of ABCG2. Molecular Medicine, 18, 887–898. 45. Nakatsugawa, M., Takahashi, A., Hirohashi, Y., et al. (2011). SOX2 is overexpressed in stemlike cells of human lung adenocarcinoma and augments the tumorigenicity. Laboratory Investigation, 91, 1796–1804. 46. Liang, D., & Shi, Y. (2012). Aldehyde dehydrogenase-1 is a specific marker for stem cells in human lung adenocarcinoma. Medical Oncology, 29, 633–639. 47. Sullivan, J. P., Spinola, M., Dodge, M., et al. (2010). Aldehyde dehydrogenase activity selects for lung adenocarcinoma stem cells dependent on notch signaling. Cancer Research, 70, 9937–9948. 48. Kubo, T., Takigawa, N., Osawa, M., et al. (2013). Subpopulation of small-cell lung cancer cells expressing CD133 and CD87 show resistance to chemotherapy. Cancer Science, 104, 78–84. 49. Hegedus, C., Truta-Feles, K., Antalffy, G., et al. (2012). Interaction of the EGFR inhibitors gefitinib, vandetanib, pelitinib and neratinib with the ABCG2 multidrug transporter: implications for the emergence and reversal of cancer drug resistance. Biochemical Pharmacology, 84, 260–267. 50. Sung, J. M., Cho, H. J., Yi, H., et al. (2008). Characterization of a stem cell population in lung cancer A549 cells. Biochemical and Biophysical Research Communications, 371, 163–167. 51. Meng, X., Wang, X., & Wang, Y. (2009). More than 45 % of A549 and H446 cells are cancer initiating cells: evidence from
123
Cell Biochem Biophys (2014) 69:389–398
52.
53.
54.
55.
56. 57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
cloning and tumorigenic analyses. Oncology Reports, 21, 995–1000. Zhou, J., Wang, H., Cannon, V., et al. (2011). Side population rather than CD133(?) cells distinguishes enriched tumorigenicity in hTERT-immortalized primary prostate cancer cells. Molecular Cancer, 10, 112. Shmelkov, S. V., Butler, J. M., Hooper, A. T., et al. (2008). CD133 expression is not restricted to stem cells, and both CD133? and CD133- metastatic colon cancer cells initiate tumors. Journal of Clinical Investigation, 118, 2111–2120. Jaggupilli, A., & Elkord, E. (2012). Significance of CD44 and CD24 as cancer stem cell markers: An enduring ambiguity. Clinical and Developmental Immunology, 2012, 708036. Du, L., Wang, H., He, L., et al. (2008). CD44 is of functional importance for colorectal cancer stem cells. Clinical Cancer Research, 14, 6751–6760. Bapat, S. A. (2010). Human ovarian cancer stem cells. Reproduction, 140, 33–41. Lee, H. J., Choe, G., Jheon, S., et al. (2010). CD24, a novel cancer biomarker, predicting disease-free survival of non-small cell lung carcinomas: A retrospective study of prognostic factor analysis from the viewpoint of forthcoming (seventh) new TNM classification. Journal of Thoracic Oncology, 5, 649–657. Slomiany, M. G., Dai, L., Tolliver, L. B., et al. (2009). Inhibition of functional hyaluronan-CD44 interactions in CD133positive primary human ovarian carcinoma cells by small hyaluronan oligosaccharides. Clinical Cancer Research, 15, 7593–7601. Leung, E. L., Fiscus, R. R., Tung, J. W., et al. (2010). Non-small cell lung cancer cells expressing CD44 are enriched for stem cell-like properties. PLoS One, 5, e14062. An, Y., & Ongkeko, W. M. (2009). ABCG2: The key to chemoresistance in cancer stem cells? Expert Opinion on Drug Metabolism and Toxicology, 5, 1529–1542. Ross, D. D., & Nakanishi, T. (2010). Impact of breast cancer resistance protein on cancer treatment outcomes. Methods in Molecular Biology, 596, 251–290. Krishnamurthy, P., Ross, D. D., Nakanishi, T., et al. (2004). The stem cell marker Bcrp/ABCG2 enhances hypoxic cell survival through interactions with heme. Journal of Biological Chemistry, 279, 24218–24225. Ahmed, F., Arseni, N., Glimm, H., et al. (2008). Constitutive expression of the ATP-binding cassette transporter ABCG2 enhances the growth potential of early human hematopoietic progenitors. Stem Cells, 26, 810–818. Levina, V., Marrangoni, A. M., & De Marco, R. (2008). Drugselected human lung cancer stem cells: Cytokine network, tumorigenic and metastatic properties. PLoS One, 3, e3077. Rosner, M. H., Vigano, M. A., Ozato, K., et al. (1990). A POUdomain transcription factor in early stem cells and germ cells of the mammalian embryo. Nature, 345, 686–692. Hilbe, W., Dirnhofer, S., Oberwasserlechner, F., et al. (2004). CD133 positive endothelial progenitor cells contribute to the tumour vasculature in non-small cell lung cancer. Journal of Clinical Pathology, 57, 965–969. Herpel, E., Jensen, K., Muley, T., et al. (2011). The cancer stem cell antigens CD133, BCRP1/ABCG2 and CD117/c-KIT are not associated with prognosis in resected early-stage non-small cell lung cancer. Anticancer Research, 31, 4491–4500. Russo, J. E., & Hilton, J. (1998). Characterization of cytosolic aldehyde dehydrogenase from cyclophosphamide resistant L1210 cells. Cancer Research, 48, 2963–2968. Huang, C. P., Tsai, M. F., Chang, T. H., et al. (2013). ALDH positive lung cancer stem cells confer resistance to epidermal growth factor receptor tyrosine kinase inhibitors. Cancer Letters, 328, 144–151.
Cell Biochem Biophys (2014) 69:389–398 70. Lu, Y., Thomson, J. M., Wong, H. Y., et al. (2007). Transgenic over-expression of the microRNA miR-17-92 cluster promotes proliferation and inhibits differentiation of lung epithelial progenitor cells. Development Biology, 310, 442–453. 71. Karamboulas, C., & Ailles, L. (2013). Developmental signaling pathways in cancer stem cells of solid tumors. Biochimica et Biophysica Acta, 1830, 2481–2495. 72. Takebe, N., & Ivy, S. P. (2010). Controversies in cancer stem cells: targeting embryonic signaling pathways. Clinical Cancer Research, 16, 3106–3112. 73. Varjosalo, M., & Taipale, J. (2008). Hedgehog: Functions and mechanisms. Genes and Development, 22, 2454–2472. 74. Sasaki, H., Nishizaki, Y., Hui, C., et al. (1999). Regulation of GLI2 and GLI3 activities by an amino-terminal repression domain: Implication of GLI2 and GLI3 as primary mediators of Shh signaling. Development, 126, 3915–3924. 75. Clement, V., Sanchez, P., de Tribolet, N., et al. (2007). HEDGEHOG-GLI1 signaling regulates human glioma growth, cancer stem cell selfrenewal, and tumorigenicity. Current Biology, 17, 165–172. 76. Peacock, C. D., Wang, Q., Gesell, G. S., et al. (2007). Hedgehog signaling maintains a tumor stem cell compartment in multiple myeloma. Proceedings of the National Academy of Sciences of the United States of America, 104, 4048–4053. 77. Stecca, B., Mas, C., Clement, V., et al. (2007). Melanomas require HEDGEHOG-GLI signaling regulated by interactions between GLI1 and the RAS-MEK/AKT pathways. Proceedings of the National Academy of Sciences of the United States of America, 104, 5895–5900. 78. Mukherjee, S., Frolova, N., Sadlonova, A., et al. (2006). Hedgehog signaling and response to cyclopamine differ in epithelial and stromal cells in benign breast and breast cancer. Cancer Biology and Therapy, 5, 674–683. 79. Yuan, Z., Goetz, J. A., Singh, S., et al. (2007). Frequent requirement of hedgehog signaling in non-small cell lung carcinoma. Oncogene, 26, 1046–1055. 80. Watkins, D. N., Berman, D. M., Burkholder, S. G., et al. (2003). Hedgehog signalling within airway epithelial progenitors and in small-cell lung cancer. Nature, 422, 313–317. 81. Park, K. S., Martelotto, L. G., Peifer, M., et al. (2011). A crucial requirement for Hedgehog signaling in small cell lung cancer. Nature Medicine, 17, 1504–1508. 82. Collins, B. J., Kleeberger, W., & Ball, D. W. (2004). Notch in lung development and lung cancer. Seminars in Cancer Biology, 14, 357–364. 83. Richter, S., McWhirter, E., Chen, E. X., et al. (2012). A phase I study of R04929097, an oral gamma secretase inhibitor, in combination with gemcitabine, in patients with advanced solid tumors (PHL-078/CTEP 8575). Journal of Clinical Oncology, 30, 3082. 84. Ito, T., Udaka, N., Yazawa, T., et al. (2000). Basic helix-loophelix transcription factors regulate the neuroendocrine differentiation of fetal mouse pulmonary epithelium. Development, 127, 3913–3921. 85. Dang, T. P., Eichenberger, S., Gonzalez, A., et al. (2003). Constitutive activation of Notch3 inhibits terminal epithelial differentiation in lungs of transgenic mice. Oncogene, 22, 1988–1997. 86. Chen, H., Thiagalingam, A., Chopra, H., et al. (1997). Conservation of the Drosophila lateral inhibition pathway in human lung cancer: A hairy-related protein (HES-1) directly represses achaete-scute homolog-1 expression. Proceedings of the National Academy of Sciences of the United States of America, 94, 5355–5360. 87. Osanyingbemi-Obidi, J., Dobromilskaya, I., Illei, P. B., et al. (2011). Notch signaling contributes to lung cancer clonogenic
397
88.
89.
90.
91.
92.
93.
94. 95. 96. 97.
98.
99.
100.
101.
102.
103.
104.
105. 106. 107. 108.
capacity in vitro but may be circumvented in tumorigenesis in vivo. Molecular Cancer Research, 9, 1746–1754. Konishi, J., Kawaguchi, K. S., Vo, H., et al. (2007). Gammasecretase inhibitor prevents Notch3 activation and reduces proliferation in human lung cancers. Cancer Research, 67, 8051–8057. Weijzen, S., Rizzo, P., Braid, M., et al. (2002). Activation of Notch-1 signaling maintains the neoplastic phenotype in human Ras-transformed cells. Nature Medicine, 8, 979–986. Westhoff, B., Colaluca, I. N., D’Ario, G., et al. (2009). Alterations of the Notch pathway in lung cancer. Proceedings of the National Academy of Sciences of the United States of America, 106, 22293–22298. Nicolas, M., Wolfer, A., Raj, K., et al. (2003). Notch1 functions as a tumor suppressor in mouse skin. Nature Genetics, 33, 416–421. Klinakis, A., Lobry, C., Abdel-Wahab, O., et al. (2011). A novel tumour suppressor function for the Notch pathway in myeloid leukaemia. Nature, 473, 230–233. Liu, Z., Turkoz, A., Jackson, E. N., et al. (2011). Notch1 loss of heterozygosity causes vascular tumors and lethal hemorrhage in mice. Journal of Clinical Investigation, 121, 800–808. Phng, L. K., & Gerhardt, H. (2009). Angiogenesis: A team effort coordinated by Notch. Developmental Cell, 16, 196–208. Radtke, F., Fasnacht, N., & Macdonald, H. R. (2010). Notch signaling in the immune system. Immunity, 32, 14–27. Willert, K., & Jones, K. A. (2006). Wnt signaling: Is the party in the nucleus? Genes and Development, 20, 1394–1404. He, B., Barg, R. N., You, L., et al. (2005). Wnt signaling in stem cells and non-small-cell lung cancer. Clinical Lung Cancer, 7, 54–60. Uematsu, K., He, B., You, L., et al. (2003). Activation of the Wnt pathway in non small cell lung cancer: Evidence of dishevelled overexpression. Oncogene, 22, 7218–7221. Teng, Y., Wang, X., Wang, Y., et al. (2010). Wnt/beta-catenin signaling regulates cancer stem cells in lung cancer A549 cells. Biochemical and Biophysical Research Communications, 392, 373–379. Yeh, C. T., Wu, A. T., Chang, P. M., et al. (2012). Trifluoperazine, an antipsychotic agent, inhibits cancer stem cell growth and overcomes drug resistance of lung cancer. American Journal of Respiratory and Critical Care Medicine, 186, 1180–1188. Serrano, D., Bleau, A. M., Fernandez-Garcia, I., et al. (2011). Inhibition of telomerase activity preferentially targets aldehyde dehydrogenase-positive cancer stem-like cells in lung cancer. Molecular Cancer, 10, 96. He, B., You, L., Uematsu, K., et al. (2004). A monoclonal antibody against Wnt-1 induces apoptosis in human cancer cells. Neoplasia, 6, 7–14. Tian, F., Mysliwietz, J., Ellwart, J., et al. (2012). Effects of the Hedgehog pathway inhibitor GDC-0449 on lung cancer cell lines are mediated by side populations. Clinical and Experimental Medicine, 12, 25–30. Levina, V., Marrangoni, A., Wang, T., et al. (2010). Elimination of human lung cancer stem cells through targeting of the stem cell factor-c-kit autocrine signaling loop. Cancer Research, 70, 338–346. Hill, R. P. (2006). Identifying cancer stem cells in solid tumors: Case not proven. Cancer Research, 66, 1891–1895. Kern, S. E., & Shibata, D. (2007). The fuzzy math of solid tumor stem cells: A perspective. Cancer Research, 67, 8985–8988. Kelly, P. N., Dakic, A., Adams, J. M., et al. (2007). Tumor growth need not be driven by rare cancer stem cells. Science, 317, 337. Quintana, E., Shackleton, M., Sabel, M. S., et al. (2008). Efficient tumour formation by single human melanoma cells. Nature, 456, 593–598.
123
398 109. Li, Z., Bao, S., Wu, Q., et al. (2009). Hypoxia-inducible factors regulate tumorigenic capacity of glioma stem cells. Cancer Cell, 15, 501–513. 110. Heddleston, J. M., Li, Z., McLendon, R. E., et al. (2009). The hypoxic microenvironment maintains glioblastoma stem cells and promotes reprogramming towards a cancer stem cell phenotype. Cell Cycle, 8, 3274–3284.
123
Cell Biochem Biophys (2014) 69:389–398 111. Li, L., & Neaves, W. B. (2006). Normal stem cells and cancer stem cells: The niche matters. Cancer Research, 66, 4553–4557. 112. Gupta, P. B., Chaffer, C. L., & Weinberg, R. A. (2009). Cancer stem cells: Mirage or reality? Nature Medicine, 15, 1010–1012.
