Author Correction: EZH2 inhibition sensitizes BRG1 and EGFR mutant lung tumours to TopoII inhibitors.

We wish to correct two mutations in Supplementary Table 4 of this Letter. The NCI-H460 cell line was annotated as being mutant for TP53. NCI-H460 has been verified to be TP53 wild type by several sources1. The NCI-H2009 cell line was annotated as being mutant for PIK3CA. As annotated by COSMIC (ref. 24 of the original Letter) and CCLE (ref. 25 of the original Letter), the NCI-H2009 cell line has a mutation in PIK3C3, rather than PIK3CA. The cell line is wild type for PIK3CA. The Supplementary Information of this Amendment contains the corrected Supplementary Table 4. These errors do not affect our conclusions. The original Letter has not been corrected.

EZH2 inhibition sensitizes BRG1 and EGFR mutant lung tumours to TopoII inhibitors.

Non-small-cell lung cancer is the leading cause of cancer-related death worldwide. Chemotherapies such as the topoisomerase II (TopoII) inhibitor etoposide effectively reduce disease in a minority of patients with this cancer; therefore, alternative drug targets, including epigenetic enzymes, are under consideration for therapeutic intervention. A promising potential epigenetic target is the methyltransferase EZH2, which in the context of the polycomb repressive complex 2 (PRC2) is well known to tri-methylate histone H3 at lysine 27 (H3K27me3) and elicit gene silencing. Here we demonstrate that EZH2 inhibition has differential effects on the TopoII inhibitor response of non-small-cell lung cancers in vitro and in vivo. EGFR and BRG1 mutations are genetic biomarkers that predict enhanced sensitivity to TopoII inhibitor in response to EZH2 inhibition. BRG1 loss-of-function mutant tumours respond to EZH2 inhibition with increased S phase, anaphase bridging, apoptosis and TopoII inhibitor sensitivity. Conversely, EGFR and BRG1 wild-type tumours upregulate BRG1 in response to EZH2 inhibition and ultimately become more resistant to TopoII inhibitor. EGFR gain-of-function mutant tumours are also sensitive to dual EZH2 inhibition and TopoII inhibitor, because of genetic antagonism between EGFR and BRG1. These findings suggest an opportunity for precision medicine in the genetically complex disease of non-small-cell lung cancer.

Non-small-cell lung cancers: a heterogeneous set of diseases.

Non-small-cell lung cancers (NSCLCs), the most common lung cancers, are known to have diverse pathological features. During the past decade, in-depth analyses of lung cancer genomes and signalling pathways have further defined NSCLCs as a group of distinct diseases with genetic and cellular heterogeneity. Consequently, an impressive list of potential therapeutic targets was unveiled, drastically altering the clinical evaluation and treatment of patients. Many targeted therapies have been developed with compelling clinical proofs of concept; however, treatment responses are typically short-lived. Further studies of the tumour microenvironment have uncovered new possible avenues to control this deadly disease, including immunotherapy.

Neurotrophin receptor TrkB promotes lung adenocarcinoma metastasis.

Lung cancer is notorious for its ability to metastasize, but the pathways regulating lung cancer metastasis are largely unknown. An in vitro system designed to discover factors critical for lung cancer cell migration identified brain-derived neurotrophic factor, which stimulates cell migration through activation of tropomyosin-related kinase B (TrkB; also called NTRK2). Knockdown of TrkB in human lung cancer cell lines significantly decreased their migratory and metastatic ability in vitro and in vivo. In an autochthonous lung adenocarcinoma model driven by activated oncogenic Kras and p53 loss, TrkB deficiency significantly reduced metastasis. Hypoxia-inducible factor-1 directly regulated TrkB expression, and, in turn, TrkB activated Akt signaling in metastatic lung cancer cells. Finally, TrkB expression was correlated with metastasis in patient samples, and TrkB was detected more often in tumors that did not have Kras or epidermal growth factor receptor mutations. These studies demonstrate that TrkB is an important therapeutic target in metastatic lung adenocarcinoma.

Loss of Lkb1 and Pten leads to lung squamous cell carcinoma with elevated PD-L1 expression.

Lung squamous cell carcinoma (SCC) is a deadly disease for which current treatments are inadequate. We demonstrate that biallelic inactivation of Lkb1 and Pten in the mouse lung leads to SCC that recapitulates the histology, gene expression, and microenvironment found in human disease. Lkb1;Pten null (LP) tumors expressed the squamous markers KRT5, p63 and SOX2, and transcriptionally resembled the basal subtype of human SCC. In contrast to mouse adenocarcinomas, the LP tumors contained immune populations enriched for tumor-associated neutrophils. SCA1(+)NGFR(+) fractions were enriched for tumor-propagating cells (TPCs) that could serially transplant the disease in orthotopic assays. TPCs in the LP model and NGFR(+) cells in human SCCs highly expressed Pd-ligand-1 (PD-L1), suggesting a mechanism of immune escape for TPCs.

Tumor-propagating cells and Yap/Taz activity contribute to lung tumor progression and metastasis.

Metastasis is the leading cause of morbidity for lung cancer patients. Here we demonstrate that murine tumor propagating cells (TPCs) with the markers Sca1 and CD24 are enriched for metastatic potential in orthotopic transplantation assays. CD24 knockdown decreased the metastatic potential of lung cancer cell lines resembling TPCs. In lung cancer patient data sets, metastatic spread and patient survival could be stratified with a murine lung TPC gene signature. The TPC signature was enriched for genes in the Hippo signaling pathway. Knockdown of the Hippo mediators Yap1 or Taz decreased in vitro cellular migration and transplantation of metastatic disease. Furthermore, constitutively active Yap was sufficient to drive lung tumor progression in vivo. These results demonstrate functional roles for two different pathways, CD24-dependent and Yap/Taz-dependent pathways, in lung tumor propagation and metastasis. This study demonstrates the utility of TPCs for identifying molecules contributing to metastatic lung cancer, potentially enabling the therapeutic targeting of this devastating disease.

Lung stem and progenitor cells in tissue homeostasis and disease.

The mammalian lung is a complex organ containing numerous putative stem/progenitor cell populations that contribute to region-specific tissue homeostasis and repair. In this review, we discuss recent advances in identifying and studying these cell populations in the context of lung homeostasis and disease. Genetically engineered mice now allow for lineage tracing of several lung stem and progenitor cell populations in vivo during different types of lung injury repair. Using specific sets of cell surface markers, these cells can also be isolated from murine and human lung and tested in 3D culture systems and in vivo transplant assays. The pathology of devastating lung diseases, including lung cancers, is likely in part due to dysregulation and dysfunction of lung stem cells. More precise characterization of stem cells with identification of new, unique markers; improvement in isolation and transplant techniques; and further development of functional assays will ultimately lead to new therapies for a host of human lung diseases. In particular, lung cancer biology may be greatly informed by findings in normal lung stem cell biology as evidence suggests that lung cancer is a disease that begins in, and may be driven by, neoplastic lung stem cells.

The bed and the bugs: interactions between the tumor microenvironment and cancer stem cells.

Tumors have been increasingly recognized as organs with a complexity that approaches, and may even exceed, that of healthy tissues. When viewed from this perspective, the biology of a tumor can be understood only by studying tumor cell heterogeneity and the microenvironment that is constructed during the course of tumorigenesis and malignant progression. Recent work has revealed the existence of cancer stem cells, the "bugs", with the capacity for self-renewal and tumor propagation. In addition, it is now recognized that the tumor microenvironment, the "bed", plays a critical role in supporting cancer stem cells and also may promote neoplasia and malignant progression. The interdependence of the cell-intrinsic features of cancer, including the cancer stem cell "bugs" and the tumor microenvironment "bed", is only beginning to be understood. In this review, we highlight the rapidly evolving concepts about the interactions between tumor stem cells and their microenvironment, the insights gained from studying their normal tissue counterparts, and the questions and controversies surrounding this area of research, with an emphasis on breast and lung cancer. Finally, we address evidence supporting the notion that eliminating the bed as well as the bugs should lead to more effective and personalized cancer treatments that improve patient outcome.

Lung stem cell self-renewal relies on BMI1-dependent control of expression at imprinted loci.

BMI1 is required for the self-renewal of stem cells in many tissues including the lung epithelial stem cells, Bronchioalveolar Stem Cells (BASCs). Imprinted genes, which exhibit expression from only the maternally or paternally inherited allele, are known to regulate developmental processes, but what their role is in adult cells remains a fundamental question. Many imprinted genes were derepressed in Bmi1 knockout mice, and knockdown of Cdkn1c (p57) and other imprinted genes partially rescued the self-renewal defect of Bmi1 mutant lung cells. Expression of p57 and other imprinted genes was required for lung cell self-renewal in culture and correlated with repair of lung epithelial cell injury in vivo. Our data suggest that BMI1-dependent regulation of expressed alleles at imprinted loci, distinct from imprinting per se, is required for control of lung stem cells. We anticipate that the regulation and function of imprinted genes is crucial for self-renewal in diverse adult tissue-specific stem cells.

Stochastic state transitions give rise to phenotypic equilibrium in populations of cancer cells.

Cancer cells within individual tumors often exist in distinct phenotypic states that differ in functional attributes. While cancer cell populations typically display distinctive equilibria in the proportion of cells in various states, the mechanisms by which this occurs are poorly understood. Here, we study the dynamics of phenotypic proportions in human breast cancer cell lines. We show that subpopulations of cells purified for a given phenotypic state return towards equilibrium proportions over time. These observations can be explained by a Markov model in which cells transition stochastically between states. A prediction of this model is that, given certain conditions, any subpopulation of cells will return to equilibrium phenotypic proportions over time. A second prediction is that breast cancer stem-like cells arise de novo from non-stem-like cells. These findings contribute to our understanding of cancer heterogeneity and reveal how stochasticity in single-cell behaviors promotes phenotypic equilibrium in populations of cancer cells.

Estrogen expands breast cancer stem-like cells through paracrine FGF/Tbx3 signaling.

Many tumors contain heterogeneous populations of cells, only some of which exhibit increased tumorigenicity and resistance to anticancer therapies. Evidence suggests that these aggressive cancer cells, often termed "cancer stem cells" or "cancer stem-like cells" (CSCs), rely upon developmental signaling pathways that are important for survival and expansion of normal stem cells. Here we report that, in analogy to embryonic mammary epithelial biology, estrogen signaling expands the pool of functional breast CSCs through a paracrine FGF/FGFR/Tbx3 signaling pathway. Estrogen or FGF9 pretreatment induced CSC properties of breast cancer cell lines and freshly isolated breast cancer cells, whereas cotreatment of cells with tamoxifen or a small molecule inhibitor of FGFR signaling was sufficient to prevent the estrogen-induced expansion of CSCs. Furthermore, reduction of FGFR or Tbx3 gene expression was able to abrogate tumorsphere formation, whereas ectopic Tbx3 expression increased tumor seeding potential by 100-fold. These findings demonstrate that breast CSCs are stimulated by estrogen through a signaling pathway that similarly controls normal mammary epithelial stem cell biology.

Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy.

INTRODUCTION: The phenotypic and functional differences between cells that initiate human breast tumors (cancer stem cells) and those that comprise the tumor bulk are difficult to study using only primary tumor tissue. We embarked on this study hypothesizing that breast cancer cell lines would contain analogous hierarchical differentiation programs to those found in primary breast tumors. METHODS: Eight human breast cell lines (human mammary epithelial cells, and MCF10A, MCF7, SUM149, SUM159, SUM1315 and MDA.MB.231 cells) were analyzed using flow cytometry for CD44, CD24, and epithelial-specific antigen (ESA) expression. Limiting dilution orthotopic injections were used to evaluate tumor initiation, while serial colony-forming unit, reconstitution and tumorsphere assays were performed to assess self-renewal and differentiation. Pulse-chase bromodeoxyuridine (5-bromo-2-deoxyuridine [BrdU]) labeling was used to examine cell cycle and label-retention of cancer stem cells. Cells were treated with paclitaxel and 5-fluorouracil to test selective resistance to chemotherapy, and gene expression profile after chemotherapy were examined. RESULTS: The percentage of CD44+/CD24- cells within cell lines does not correlate with tumorigenicity, but as few as 100 cells can form tumors when sorted for CD44+/CD24-/low/ESA+. Furthermore, CD44+/CD24-/ESA+ cells can self-renew, reconstitute the parental cell line, retain BrdU label, and preferentially survive chemotherapy. CONCLUSION: These data validate the use of cancer cell lines as models for the development and testing of novel therapeutics aimed at eradicating cancer stem cells.

Inactivation of the Snf5 tumor suppressor stimulates cell cycle progression and cooperates with p53 loss in oncogenic transformation.

Snf5 (Ini1/Baf47/Smarcb1), a core member of the Swi/Snf chromatin remodeling complex, is a potent tumor suppressor whose mechanism of action is largely unknown. Biallelic loss of Snf5 leads to the onset of aggressive cancers in both humans and mice. We have developed an innovative and widely applicable analytical technique for cross-species validation of cancer models and show that the gene expression profiles of our Snf5 murine models closely resemble those of human Snf5-deficient rhabdoid tumors. We exploit this system to produce what we believe to be the first report documenting the effects on gene expression of inactivating a Swi/Snf subunit in normal mammalian cells and to identify the transcriptional pathways regulated by Snf5. We demonstrate that the tumor suppressor activity of Snf5 depends on its regulation of cell cycle progression; Snf5 inactivation leads to aberrant up-regulation of E2F targets and increased levels of p53 that are accompanied by apoptosis, polyploidy, and growth arrest. Further, conditional mouse models demonstrate that inactivation of p16Ink4a or Rb (retinoblastoma) does not accelerate tumor formation in Snf5 conditional mice, whereas mutation of p53 leads to a dramatic acceleration of tumor formation.