Journal of Thoracic Oncology:
State of the Art: Concise Review
Lung Cancer Stem Cell: Fancy Conceptual Model of Tumor Biology or Cornerstone of a Forthcoming Therapeutic Breakthrough?
Sourisseau, Tony PhD*; Hassan, Khaled A. MD†; Wistuba, Ignacio MD‡; Penault-Llorca, Frédérique MD, PhD§; Adam, Julien MD*; Deutsch, Eric MD, PhD‖; Soria, Jean-Charles MD, PhD*
*Inserm Unit 981, DHU TORINO, Gustave Roussy and University, Paris Sud, France; †Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan; ‡Department of Translational Molecular Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas; §Département de Pathologie et de Biopathologie, Centre Jean Perrin, Clermont-Ferrand, France; and EA 4766, ERTICa, Université d’Auvergne, Clermont-Ferrand, France; and ‖Inserm Unit 1030, DHU TORINO, Gustave Roussy and University Paris Sud, France.
Disclosure: The authors declare no conflict of interest.
Address for correspondence: Tony Sourisseau, Inserm Unit 981, Gustave Roussy Cancer Campus, 114, Rue Edouard-Vaillant, Batiment de Medecine Moleculaire, 1er Etage, Piece 124, Villejuif 94805, France. E-mail: firstname.lastname@example.org
Cancer research has received a fresh impetus from the concept of cancer stem cell (CSC) which postulates the existence of a tumor cell population uniquely endowed with self-renewal capacity and therapy resistance. Despite recent progresses including targeted therapy, lung cancer treatment remains a challenge owing largely to disease recurrence. Providing a conceptual model of tumor resistance and disease relapse, the lung CSC has received extensive attention, leading to a flourishing literature and several ongoing clinical trials. In this study, we will discuss the data suggesting the existence of CSC in lung tumors and the potential clinical utility of CSCs as prognostic markers or cellular targets of new therapeutic strategies. We will also touch on the new fundamental developments of the CSC concept that ought to be considered if the integration of the CSC concept into clinical practice is to be successful and impact on lung cancer treatment.
Lung cancer is the leading cause of cancer-related death in western countries. Available therapeutic options combine surgery, chemo- and radiotherapy, and targeted therapy depending on the stage and histological subtype. Even though an initial response to these treatments is commonly observed, the long-term benefit to patients is compromised by disease relapse driven by therapy-resistant tumor cells. Therefore, the efficient killing of resistant cells is a major endeavor for outcome improvement.
Many fields in cancer research have received a fresh impetus brought about a decade ago with the cancer stem cell (CSC) concept (Fig. 1). Originating from transplantation experiments of hematological tumor cells into immunodeficient mice, the CSC concept initially proposed that human acute myeloid leukemia comprises functionally unequivalent cells organized hierarchically with a leukemia CSC positioned at the apex.1 The growth of cancerous tissue thus shows striking similarities to the corresponding normal hematopoietic tissue, and the defining properties of a leukemia CSC, just as the normal tissue stem cells, include self-renewal and the ability to generate progeny committing to differentiation.
Consequently, the tumor tissue is heterogeneous and comprises phenotypically and functionally distinct cell populations. Sustaining long-term growth in serial transplantation assays, the CSCs are considered to be the tumor-initiating cells (also sometimes called the tumor-seeding cells). Perhaps even more importantly, the CSCs also display resistance to drug and are thought to give rise to therapy-resistant tumor regrowth after treatment, hence their alternative name of tumor-reinitiating cells.
The CSC concept has since been extrapolated to solid tumors, and the existence of CSC in lung tumors has recently been reported. Properties of the CSCs toward anticancer agents have been extensively examined in lung cancer and an abundant literature has ensued. Nevertheless, the available data ought to be considered cautiously before translation into a clinical impact can be reasonably envisaged. In this article, we consider the experimental data supporting the relevance of the CSC concept to lung cancer, and the arguments for its integration into the clinical practice.
STEM AND PROGENITOR CELLS IN THE LUNG
Whether the different cell types of the lung epithelia arise from a single multipotent lung stem cell is still a debated question even though studies mostly carried out on mouse lung rather point to several region-specific, oligopotent progenitors ensuring the lung epithelium homeostasis. Several candidate cell types have been proposed to fulfill this function.
A multipotent lung stem cell was first proposed by Kim et al.2 This cell expresses both Clara cell and pneumocyte type II specific proteins, namely secretoglobin 1a1 (scgb1a1, also called CC10 or Clara cell secretory protein [CCSP]) and surfactant protein C (SP-C) (prosurfactant apoprotein C), respectively. This candidate lung stem cell can give rise to three epithelial cell types, Clara cells and type I and II pneumocytes, localizes at the bronchoalveolar duct junction and was hence named bronchoalveolar stem cells. By using lineage tracing, Rock et al.3 revealed role of the Clara cells in lung airway epithelia homeostasis as transit amplifiers derived from basal cells and able to self-renew and differentiate into ciliated cells. The alveolar epithelium, however, was found to have a distinct cellular origin.4 A specific progenitor for the alveolar epithelium has indeed been reported recently.5 In another elegant study also using lineage tracing, Chen et al.6 too generated data strongly suggesting the existence of several conducting airway epithelial progenitors distributed along the proximal-to-distal axis. More extensive tissue regeneration triggered, for example, by experimental lung injury may, however, rely on bona fide stem cell activity.7 The few reports suggesting the existence of a lung stem cell population able to differentiate into all the different lung epithelial cells are currently still awaiting experimental confirmation.8,9
The cellular target of oncogenic transformation in the lung has also been the topic of a number of reports. Adenocarcinomas, for instance, were first suggested to originate from the bronchoalveolar stem cells before an alveolar origin was recently demonstrated in mouse lung cancer models.2,10,11 Neuroendocrine cells, and to a lesser extent alveolar type II pneumocytes, have been proposed as the cellular origin of small-cell lung cancer.12
MARKERS OF LUNG CSCS
Advances in the understanding of normal and CSCs rely on specific markers. The combined expression of several lung epithelial cell–type markers such as CCSP or SP-C may enable the characterization of putative tissue stem cells, but has not been reported to identify lung CSCs so far. The transmembrane glycoprotein prominin 1, also known as CD133, has no known function but was identified as a hematopoietic stem cell marker.13 Its expression has since been reported in other tissues and CSCs. After CD133 was shown to label a population of lung tumor cells endowed with CSC properties,14 many reports have followed suit and used CD133 as a CSC marker.15–19
ALDH1 is a member of the detoxifying aldehyde dehydrogenase family. High expression of ALDH1 is observed in tissue stem cell (SC) as well as CSC in lung tumors.20–25 The stromal cell–derived factor-1 (SDF-1, also known as CXCL12) membrane receptor CXCR4 is an another commonly used marker for lung CSC.26,27 A number of other membrane markers, previously demonstrated to label CSC in other cancers, have been used to identify lung CSC, as for example CD44, CD166, interleukin-6R, or urokinase-type plasminigen activator receptor (uPAR).28–31 Interestingly, stem cell–related pathways such as Notch activity have been associated with lung CSCs.25,32
When assessed across 10 human non–small-cell lung cancer (NSCLC) cell lines, the expression of the afore-mentioned markers varied greatly among cell lines and did not correlate to each other.28 Besides, CXRC4-sorted lung CSC did not consistently coexpress other previously reported CSC markers.27 Therefore, there is no reliable single lung CSC marker reported as yet. Nevertheless, other well-established strategies such as the exclusion of the DNA-binding dye Hoechst 33342 can be used to identify and isolate stem cells. A Hoechst-negative lung CSC population, also called side population, has been identified and isolated by flow cytometry from lung cancer cell lines and tumor samples.33,34 However, the side population isolated either from lung cancer–derived cell lines or from resected tumor samples did not coexpress the previously described CSC markers.27,34 The preclinical data generated based on these CSC markers, while prompting cautious interpretation, have nonetheless contributed to the current understanding of therapeutics resistance.
PROPERTIES OF LUNG CSC TOWARD ANTICANCER TREATMENTS
Resistance to Chemotherapy
Cytotoxic chemotherapy is the main first-line treatment for advanced/metastatic lung cancers, often achieve an initial therapeutic response but provides limited long-term benefit to patient because of the recurrence of the disease. The topic of resistance of lung CSCs to chemotherapy is therefore an outstanding issue for the improvement of lung cancer treatment. The experimental exploration of CSC properties toward chemotherapy has relied on three main experimental strategies (Fig. 2 and Table 1).
Fluorescence-Activated Cell Sorting/Magnetic Beads Sorting of Lung CSC
This experimental approach consists in sorting a cell population based on the expression of the previously discussed CSC markers. In most reports, the stemness of the sorted population is confirmed in vitro, for example, by the sphere-formation assay described below or by using the standard procedure subcutaneous transplantation assay as an in vivo read out of tumorigenicity. The sensitivity of the sorted cells to chemotherapeutic agents can then be evaluated and compared with the bulk, parental population or to the non–stem-cell population in in vitro assays (Fig. 2).
By using this approach, we have shown that lung cancer cell line–derived CD133-positive subpopulations were able to form spheres and displayed increased resistance to cisplatin and other chemotherapeutic agents.14,35
To model human cancer more faithfully, xenograft models of human tumors have been established in which the tumorigenic potential of the CD133-expressing lung CSCs was examined.16 Of note, no correlation was found between the expression of CD133 in the original tumor and the ability to form a xenograft, suggesting that histological assessment of CD133 expression may not directly relate to the tumor-seeding potential. Nevertheless, combining in vitro and in vivo assays, CSC properties of the CD133-expressing population could indeed be demonstrated. Furthermore, in cisplatin-treated xenografts, CD133-positive tumor cells were enriched in a fraction of the samples. However, sorting CD133-expressing CSCs derived from human lung cancer cell lines, Meng et al.36 did not observe resistance of the putative CSC population to chemotherapy when assessed by in vivo tumorigenicity assays.
The two alternative markers CXCR4 and CD44 have also been shown to label cisplatin-resistant lung CSC.27,28 In line with the reports suggesting an increased resistance of lung CSC to chemotherapy, the side population of a panel of human lung cancer cell lines was also shown to be resistant to the chemotherapeutic agents cisplatin, etoposide, gemcitabine, daunorubicin, doxorubicin, vinorelbine, and docetaxel in in vitro cell-proliferation assays.34
Sphere-Forming Cell Population
Another commonly used approach to study CSCs relies on their unique ability to grow in suspension as spheres, sometimes also called spheroids or spheroid bodies. This is usually achieved when cells are cultured in specific conditions including low adherence substrate and the tightly controlled growth factor composition of culture media.37,38 As mentioned previously, this assay can not only be used to confirm stemness of a prospectively isolated population but can also be a mean to enrich a population of CSC cells in vitro (Fig. 2). One obvious advantage of this approach is that CSC markers are not required.
Cancer cell line–derived or primary tumor-derived sphere-forming cells were shown to be more tumorigenic than the corresponding bulk population in transplantation experiments and are also more resistant to cisplatin and irradiation. Two reports have identified a specific DNA damage response (DDR) of the sphere-forming cells as the mechanistic basis of resistance to DNA damage-generating treatment, even though a context-specific regulation of the DDR machinery was observed.39,40
Exploring the pathways required for the maintenance of chemotherapy-resistant lung CSCs, Levina et al.41 identified the stem cell factor-c-kit axis activity as a major determinant of stemness and therapy resistance in NSCLC cell lines. Disrupting this signaling pathway prevents both the formation of CSC sphere and the emergence of resistant clones when tested in vitro.
Selection of Therapy-Resistant Cell Population
Finally, a third common approach to study the ability of stem cells to resist therapeutic agents relies on the selection of a resistant population (Fig. 2).
A number of articles report on the in vitro selection of a population of lung cancer–derived cell lines based on their ability to survive chemotherapeutic treatments such as 5-fluorouracil and methotrexate,30 cisplatin,18,42,43 doxorubicin, or etoposide42 (Table 1). Several in vitro assays were performed to assess whether the drug-resistant cells did display CSC properties, such as the expression of membrane (CD133, CD44, interleukin-6R) and functional (ALDH, ABCG2) markers. Stem cell–associated transcriptional programs were also expressed in resistant cells. This selected population also formed spheres in vitro and tumors in transplantation assays, providing functional evidence of their CSC activity. One study, however, reports no enrichment of stem cells after drug selection raising questions about the reproducibility of this experimental strategy.18
To address the resistance of lung CSC to therapeutic agents in vivo, Bertolini et al.16 analyzed expression of CSC markers in residual human tumor xenograft after cisplatin treatment. The residual, resistant tumor cells were enriched for CD133 expression in a subset of the samples tested. However in a conflicting study, Hegde et al.44 used xenograft and a genetically modified mouse model of lung cancer relapse after chemotherapy. Upon treatment, no consistent enrichment in expression of CSC markers was observed in the residual disease. Instead, the proportion of CD133-, CD44-, or CD117-expressing cells varied across the models. The tumor-reinitiating capacity of the residual, chemotherapy-resistant cancer cells and the expression of the CSC markers were not correlated when tested in subcutaneous or orthotopic transplantation experiments. Showing that the cells responsible for relapse do not have all CSC features, this report suggests that tumor-initiating capacities and tumor regrowth after treatment may rely on distinct cell population or be a dynamic trait.
Resistance to Targeted Therapy
Endothelial Growth Factor Receptor
Similar to chemotherapy, the therapeutic efficacy of targeted agents such as endothelial growth factor receptor (EGFR) inhibitors is hampered by the emergence of resistance. Well-described, genetically driven mechanisms of resistance to EGFR tyrosine kinase inhibitor (TKI) involve either desensitizing mutations within the EGFR receptor itself or genetic alteration of other genes along the pathway. Nevertheless, these alterations explain only a fraction of the resistance observed in the clinic and other mechanisms based on the activity of lung CSCs have been proposed.
By selecting an EGFR-TKI–resistant lung cell line population, CSC traits were shown to be acquired by the resistant cells.45 The CXCR4/SDF-1α axis was shown to be involved in the generation of stem cell–like resistant cells.26 In another report, an insulin-like growth factor-1–mediated transient chromatin modification was the basis of a spontaneously arising phenotypic heterogeneity within the population that allowed the generation of resistant cells.46 Of note, the drug-tolerant cells were enriched in the stem cell markers CD133 and CD24. The enrichment was then lost as these persistent cells started to proliferate again, confirming the transient nature of the epigenetically induced CSC phenotype and the dynamic nature of heterogeneity.
Resistance to Ionizing Radiation
Normal tissue and CSCs are uniquely equipped to survive genotoxic stresses such as ionizing radiation.47,48 The rationale to examine the response to radiation in lung CSC is obvious as resistance to radiotherapy is often observed in the clinic. Several of the previously mentioned reports assessing the resistance of CSC to chemo- and targeted therapy have also examined the resistance of lung CSC to radiation. Lundholm et al.,39 for example, studied sphere-forming CSC-like cells and found that when compared with their more differentiated counterpart, these cells display an increased radioresistance. Jung et al.26 also assessed the radiosentitivity of CXCR4-positive, gefitinib-resistant cells and observed a moderate resistance to ionizing radiation. In an interesting preclinical study, the targeting of ALDH-positive CSCs by the telomerase inhibitor MTS312 combined to radiation resulted in a cumulative effect in vitro, suggesting that the radiation-resistant population is an ALDH-positive CSC population.24 Finally, focusing specifically on the CSC resistance to ionizing radiation, Mihatsch et al.49 selected in vitro a radioresistant lung cancer cell line subpopulation. When compared with the parental cell line, the radioselected cells neither displayed a stem cell–related gene expression program nor were they enriched for CD133 expression. However, an increased activity of ALDH1 could be observed in the resistant cells.
PROGNOSTIC AND PREDICTIVE SIGNIFICANCE OF CSC IN LUNG CANCER
The presence of CSC has been correlated with poor survival in a number of solid tumors including glioblastoma, colon cancer, or pancreatic cancer.50–52 An increase in CD133 level was also reported in cisplatin-relapsed NSCLC.35 Even though CD133 expression is correlated to the expression of genes involved in the repair of chemotherapy-induced DNA damage such as thymidylate synthase and O6-methylguanine-DNA methyltransferase, its expression was not correlated to survival.53 A series of other reports also concluded by the absence of prognostic value for CD133 expression.19,25,54
The prognostic value of CD44 in NSCLC was also examined and led to conflicting results.28,55 On the contrary, reports converge toward a prognostic role of the CSC quantification when based on functional CSC markers. ALDH activity in NSCLC correlates with poor survival.21,25 The ATP-binding cassette-transporter breast cancer resistance protein 1 (BRCP1) was also associated with poor prognosis and a poor response to cisplatin-based chemotherapy, pointing to a potential predictive value of BRCP1 expression that was however not confirmed in early-stage NSCLC.54,56,57
In an interesting study, NSCLC CSCs were sorted based on the expression of the CSC membrane marker CD166.29 Transcriptomic analysis of the tumor-initiating CSCs identified glycine decarboxylase (GLDC) as an enzyme specifically expressed in tumor-initiating CSC. Importantly, GLDC-expressing CSCs represented only a subset of the CD166-positive cells, and although CD166 did not have a prognostic significance in NSCLC, high expression of GLDC correlated with poor survival. This study corroborates the potential superiority of functional markers in identifying clinically relevant lung CSC populations.
Finally, another mean to assess the abundance of CSC within tumors could be the search for embryonic stem cell expression signature, as Hassan et al.58 suggested a correlation between embryonic stem cell expression program and markers of poor prognosis and worse overall survival in lung adenocarcinomas.
CSC, A POTENTIAL CONTRIBUTOR TO IMPROVEMENT OF LUNG CANCER TREATMENT?
Targeted Eradication of CSC
Several preclinical studies have identified pathways involved in the stemness maintenance of CSC that can potentially be pharmacologically disrupted and lead to CSC depletion by enforcing their differentiation (Fig. 3). The Notch pathway, for instance, is required for the regulation of self-renewal in many biological systems and its inhibition by a γ-secretase inhibitor leads to the specific elimination of CSC in a preclinical study.25 The efficacy of a γ-secretase inhibitor, RO4929097, as a single agent in NSCLC is currently being evaluated clinically (NCT01193868).
The Wnt pathway is another key regulator of self-renewal. The disruption of its activity using a monoclonal antibody antagonist of Wnt-1 and -2 has shown promising antitumor effects in lung cancer, even though these effects were not shown to be underpinned by CSCs.59 Inhibitors of the canonical Wnt pathway β-catenin transcriptional activity have also been developed and one of them has entered phase I clinical trial (NCT01302405).
Finally, the Hedgehog pathway is another important contributor to the regulation of stemness. There are some preclinical indication that interfering with hedgehog signaling can have antitumor effects in the lung through alteration of the CSC function.60 Clinical studies have yet to be undertaken to investigate the effect of Hedgehog pathway inhibition as a single agent in lung cancer. However, there are currently four clinical trials evaluating the inhibition of the Hedgehog pathway in combination with chemotherapy.
As hedgehog pathway disruption hits CSC in the lung,60 and that the CSC population has been shown to resist chemotherapy, there is a clear rational to combine both therapies in order to increase treatment efficacy (Fig. 3). Four clinical trials are currently evaluating the combination of Hedgehog pathway inhibitors to chemotherapy (NCT00887159, NCT01579929, NCT01722292, NCT00927875).
In a preclinical study, inhibiting the Notch pathway could prevent the emergence of CD133-positive, therapy-resistant lung CSC.35 Whether these findings could be confirmed in a clinical setting is currently under investigation in several clinical trials, one of them testing the combination of Notch pathway disruption using monoclonal antibody to Notch ligand DLL4 (delta like 4) and cytotoxic chemotherapy (NCT01189968). In another clinical study, the targeted therapeutic agent Erlotinib is combined to a γ-secretase inhibitor (NCT01193881). Whether this combination prevents the systematically observed emergence of EGFR-TKI–resistant cancer cells is of outstanding clinical and basic interest.
Like Notch inhibition, disruption of the SDF1-CXCR4 can interfere with lung CSC maintenance. Thus inhibition of STAT3, an effector of the CXCR4 signaling pathway, in this context by WP1066 or siSTAT3 can prevent the emergence of gefitinib-resistant NSCLC cell.26 The relevance of these findings remains to be evaluated clinically.
Finally, in a preclinical study, c-kit signaling was found to be involved in maintenance of lung CSC.41 In these experimental conditions, the inhibition of the SCF-c-kit axis by imatinib interfered with CSC self-renewal and lead to a better response to cisplatin. There is therefore also a strong rational of combining chemotherapy to c-kit pharmacological inhibition which is currently being tested in a phase II clinical trial (NCT00156286).
Targeting Resistance Pathways
Preventing Drug Efflux
The expression of efflux transporters is a hallmark of stem cells and is exploited experimentally to isolate the side population. There are a number of conflicting reports on the role of the efflux transporters in the resistance of tumor cells to chemotherapy. Decreased uptake of drugs is commonly reported, and changes in transporters expression often correlates to resistance.61 When tested in genetically modified mouse model of lung cancers, however, this mechanism did not play a major role in resistance and whether interfering with transport can increase chemotherapy efficacy could not be confirmed in clinical trial as yet (NCT00042315).62
Modulating the DDR to Avoid Resistance to DNA Damage
Among the mechanisms that have been proposed for the increased resistance of CSC to radiotherapy and DNA-damaging chemotherapeutic agents, the increased ability to repair DNA damage stands as a good candidate. The PI3K/Akt signaling is an important regulator of the DNA double-strand break repair pathways, particularly the nonhomologous end joining pathway, through its DNA-dependent protein kinase (DNA-PK) regulatory activity.63 On the basis of the observation that a CSC-like radioresistant population also displayed an increased DNA double-strand break repair capacity, Mihatsch et al.49 found that inhibition of PI3K could sensitize this resistant population to ionizing radiation. These results provide a mechanistic rational to future clinical trials.
Finally, the checkpoint protein CHK1 was identified as a mediator of the DNA damage checkpoint specifically activated in drug-resistant lung CSCs.40 As a result, the combination of a CHK1 inhibitor with the chemotherapeutic agents cisplatin and gemcitabine seems effective at preventing tumor growth in vivo. A phase II clinical trial testing the combination of the CHK1 inhibitor LY2603618 to chemotherapy is currently testing these preclinical findings (NCT01139775).
Transient epigenetic modulation is the basis of a recently identified mechanism of resistance of a lung cancer cell line to EGFR-TKI.46 Interfering with the corresponding epigenetic machinery or with the upstream signaling that involves insulin-like growth factor-1 can prevent the emergence of resistant cells. Whether this mechanism also takes place in vivo and can have a therapeutic effect remains to be explored.
Targeting Telomerase Activity
Finally, NSCLC cell line–derived CSCs that display high ALDH activity also have longer telomeres and as a result were preferentially targeted by the telomerase inhibitor MTS312 in vitro and in xenograft models.24 To our knowledge, no clinical trial has confirmed these findings yet.
The role of lung CSCs in tumor development and disease relapse has still to be firmly demonstrated. On the basis of an incomplete basic understanding of the lung CSC biology, conflicting results have been generated on candidate markers as well as on their potential clinical impact, be it as a mean to improve prognosis, or as a cellular target for novel therapeutics. Of note, the architectural complexity of the lung and the possible existence of multiple region-specific tissue stem cells may make the characterization of lung CSC markers a particularly challenging endeavor.
One of the main impediments to the exploration of lung CSC properties probably lies in the experimental approaches most often combining cell lines and in vitro assays. First, the use of cell lines does not recapitulate the various sources of tumor heterogeneity. Indeed, many tumors are now widely recognized to be composed of multiple cancer cell clones (genetic-based heterogeneity), within which a phenotypical, non–genetic-based heterogeneity brings an additional level of complexity.46,64–66 Second, CSC markers and functional tests such as the sphere-forming assays have been validated in other systems and are assumed to be applicable to lung CSC. Perhaps, the use of primary tumor material in experimental settings modeling more closely the natural tumor microenvironment—such as the orthotopic xenograft or the embryonic lung organoid assay—is mandatory to gain the basic insight into the identity and property of lung CSC that could enable the validation of more convenient experimental models (Fig. 4). Indeed, in the absence of the tumor microenvironment, the behavior of the CSC in these experimental settings may have limited relevance to the clinical setting, especially regarding their ability to sustain tumor growth and to resist therapeutic agents. Finally, among the most recent fundamental developments in the field of CSC research, recent reports point toward the dynamic nature of the CSCs phenotype, enabling differentiated cells to acquire CSC phenotype.67,68 This property of CSC should be considered for experimental strategy as well as therapeutic design.
In conclusion, the CSC hypothesis has generated much excitement in the field of lung cancer basic and clinical research. The concept bears unquestionable interest to understand the mechanisms of tumorigenesis and resistance to therapy. It is probably still early times to predict how the basic knowledge generated will impact clinical practice, but considering the recent advances in the field, we believe that a positive impact on lung cancer treatment can be reasonably hoped for.
Dr. Sourisseau is supported by a Roche postdoctoral fellowship. The authors thank Cedric Verjat for his help in the illustrations design.
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