Lineage Factors and Differentiation States in Lung Cancer Progression

REVIEW Lineage factors and differentiation states in lung cancer progression

WKC Cheung1 and DX Nguyen1,2

Lung cancer encompasses a heterogeneous group of malignancies. Here we discuss how the remarkable diversity of major lung cancer subtypes is manifested in their transforming cell of origin, oncogenic dependencies, phenotypic plasticity, metastatic competence and response to therapy. More speciﬁcally, we review the increasing evidence that links this biological heterogeneity to the deregulation of cell lineage-speciﬁc pathways and the transcription factors that ultimately control them. As determinants of pulmonary epithelial differentiation, these poorly characterized transcriptional networks may underlie the etiology and biological progression of distinct lung cancers, while providing insight into innovative therapeutic strategies.

Oncogene (2015) 34, 5771–5780; doi:10.1038/onc.2015.85; published online 30 March 2015

INTRODUCTION intermediates during this process.5 In the post-natal lungs, AT2 cells 4,6 Pathologists have long been familiar with the existence of various also acquire progenitor-like functions to generate AT1 cells. lung cancer histological subtypes and the challenges in accurately Following severe injury and inflammation, distal epithelial regen- 7,8 classifying them. Thoracic cancer can be divided into the following eration can also occur from putative lineage-negative stem cells. two major histotypes: small cell lung cancer (SCLC) and non-small Having compiled a working catalog of pulmonary epithelial cell lung cancer (NSCLC). Most lung cancer patients are diagnosed stem/progenitor lineages, recent studies have attempted to with NSCLC, which can be further subclassified into lung determine which of these cells are targets for transformation in adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC) various lung cancers by using genetically engineered mouse and large cell carcinoma. Updated histological subclasses of LUAD models (GEMMs).9 Several GEMMs exploit cell type-specific have been proposed for diagnosis and treatment.1 As revealed by promoters, such as surfactant protein C (SPC) in AT2 cells, club recent genome-wide studies, individual lung tumors also possess cell 10-kDa protein (CC10) in club cells and calcitonin gene-related highly diverse genomes, which further underscore the biological peptide in pulmonary neuroendocrine (NE) cells, to express complexity of these diseases. Untangling the relationship between mutations commonly seen in lung cancer patients in a lineage- lung tumor histotype, phenotype and molecular heterogeneity specific manner.10 In sum, both the oncogenic mutation and represents a critical barrier in improving the clinical outcome epithelial cell type in which it is expressed can influence which of thoracic malignancies, which collectively account for most lung cancer subtypes will form in mice. This topic has been cancer-related deaths to date.2 extensively reviewed elsewhere,11–14 and recent updates are discussed here (Figure 1). LUNG CANCER: PLEIOTROPIC ORIGINS AND DRIVERS Current views on the cellular origins of different lung cancers are Small cell lung cancer shaped by our understanding of normal pulmonary development Because SCLC arises in the central airways and expresses NE and homeostasis. The lung epithelium arises from the ventral side markers, it has long been postulated that this lung cancer type of the anterior foregut endoderm, where primary lung buds are originates from pulmonary NE cells. These progenitors are rare 15 formed.3 Following extensive branching of the proximal conducting and usually cluster as NE bodies in the bronchioles. Most human airways including the trachea, bronchi and bronchioles, cells at the SCLCs harbor inactivating mutations in the prototypical tumor 16 distal branch tips differentiate into alveolar type 1 (AT1) and 2 suppressors TP53 and the retinoblastoma 1 (RB1) genes. By using (AT2) cells, which constitute the gas-exchanging alveoli. In the cell type-specific conditional knockouts of these two tumor developing and adult lungs, multiple regional epithelial cell types suppressors,17 independent groups have confirmed that pulmonary – can serve as pools of progenitor cells. In the trachea and main NE cells are the predominant cells of origin of SCLC.18 20 bronchi, basal cells give rise to secretory and ciliated cells of the Interestingly, inactivation of Tp53 and Rb1 in a subset of AT2 luminal layer, whereas in bronchiolar epithelium, club cells cells, but not in club cells, may also lead to SCLC at reduced (formerly known as Clara cells) can self-renew and generate efficiency.18 The progression of Tp53/Rb1 null SCLC can be ciliated cells. In the distal airway, AT1 and AT2 cells arise directly accelerated by the additional loss of Pten, which has recently from a bipotent progenitor during embryogenesis.4 A recent been proposed as a frequent, but late genetic event during SCLC single-cell transcriptome analysis identified several maturation tumorigenesis.20,21

1Department of Pathology, Pathology and Cancer Center, Yale University School of Medicine, New Haven, CT, USA and 2Yale Cancer Center, Yale University School of Medicine, New Haven, CT, USA. Correspondence: Dr DX Nguyen, Department of Pathology, Pathology and Cancer Center, Yale University School of Medicine, PO Box 208023, New Haven, CT 06520-8023, USA. E-mail: [email protected] Received 18 December 2014; revised 13 February 2015; accepted 16 February 2015; published online 30 March 2015 Transcription control of lineage states in lung cancer WKC Cheung and DX Nguyen 5772

Figure 1. Known and predicted inﬂuence of the cell of origin vs oncogenic mutation in the formation of different lung cancer subtypes. BADJ, bronchoalveolar duct junction; CGRP, calcitonin gene-related peptide; PNEC, pulmonary neuroendocrine cell.

Lung squamous cell carcinoma bronchioles can also occur if the Tp53 tumor suppressor gene is + 30 LUSC expresses basal cell markers (including KRT5, p63 and SOX2) concomitantly deleted in CC10 cells. Some of these effects may and frequently occurs in the proximal airway. Therefore, it has depend on distinct oncogenic activities of variant Kras mutations. been proposed that LUSC arises from basal progenitors. Until Inflammatory responses may also enhance the overall transforming 27 recently, the lack of LUSC GEMM has precluded the identification effects of associated genotypes/cell phenotypes. By contrast, + of its cellular origin, and several attempts have been made to mutant Egfr expression in CC10 cells can give rise to LUAD that 31 model this malignancy. Although rarely mutated in human LUSC, rarely metastasizes. Moreover, the cell surface antigenic profile of tumor-initiating cells varies when comparing LUADs driven by inactivation of the tumor suppressor Stk11 (also known as Lkb1)in 32 Kras-mutant mice can generate LUSC, but also creates a wide Egfr and Kras mutations. Altogether, the experimental evidence spectrum of NSCLCs including mixed adenosquamous carcinoma to date implicates a combination of unique genetic and and large cell carcinoma.22 In addition, IKKα kinase inactivation in environmental contexts within the distal airway that is required mice generates spontaneous LUSC exclusively, albeit at a relatively for LUAD initiation. The function of other driver mutations in LUAD low efficiency.23 The comprehensive catalog of genomic altera- might be equally variable across regional epithelial cell lineages, tions in LUSC identified recurrent PTEN mutations.24 Concomitant contributing even further to the heterogeneous nature of this inactivation of Lkb1 and Pten leads to LUSC in GEMM with 100% NSCLC subtype. penetrance.25 Notably, the resulting tumors resemble many histological and transcriptional features of the human LUSC basal SWITCHING PATHS TO PROGRESS subtype. Given this result, it is expected that the cellular origin(s) fi of LUSC will soon be identified using conditional knockouts of Following tumor initiation from speci c cell types, lung cancers Lkb1 and Pten in GEMM. can seemingly adopt various aberrant differentiation states. In solid epithelial tumors, one of the most extensively studied manifestations of this phenomenon is epithelial-to-mesenchymal Lung adenocarcinoma transition (EMT). EMT is a developmental process that is not only Among different lung cancer subtypes, the cellular origins of linked to tumor cell invasion but also to oncogenesis and tumor LUAD are well studied owing to the availability of multiple initiation.33 In human lung cancers, clinical evidence for EMT is GEMMs. Several of these rely on the spatial and temporal seen in a fraction of LUADs that have acquired resistance to induction of an oncogenic Kras-mutant allele. In humans, KRAS tyrosine kinase inhibitors.34–36 Independent of EMT, lung cancer is most frequently mutated in tissues of endodermal origin, histopathological variations may correlate with cell lineage states including the lung epithelium.26 Independent groups have shown that are unique or selective for the airways. For instance, at the that Kras-driven murine LUAD preferentially arises from AT2 cells, genomic level, resected human NSCLCs can be distinguished by consistent with its predominant site of diagnosis at the distal gene expression profiles associated with different stages of airway and the immunohistochemical staining of alveolar markers pulmonary development.37 Despite arising from the alveolar – in early stage patient tumors.27 30 In contrast, Kras activation epithelium, LUADs can convert to SCLCs, the latter being a NE driven by the CC10 promoter, presumably in club cells, cancer more typically located in the central lungs.38 LUAD bronchioalveolar stem cells and a small percentage of AT2 cells, conversion to an ‘SCLC-like’ phenotype is associated with drug leads to hyperplasia at the bronchoalveolar duct junction, but not resistance.35,39–41 Furthermore, a significant proportion of high- frank LUAD.28 In principle, tumor progression in the terminal grade LUADs histologically resembles LUSCs, which express

Oncogene (2015) 5771 – 5780 © 2015 Macmillan Publishers Limited Transcription control of lineage states in lung cancer WKC Cheung and DX Nguyen 5773 markers of proximal airway basal cells. Mixed adenosquamous lungs, Rb is required for NE cell fate, while the specification of tumors retain epithelial markers, yet are more invasive and have a other airway cell types can occur in the absence of Rb due to worse prognosis.42–44 Within the LUAD subtype specifically, a compensation from other pocket proteins.62 This selective gene module of normal alveolar differentiation stratifies tumors requirement for RB1 during mammalian lung development is with distinct grades, biological properties and clinical outcome.45 consistent with the NE origins of SCLC and the potent SCLC The transcriptomic portrait of lung epithelial specification is phenotype generated via Rb and p53 mutations in GEMMs. ultimately reflected in the nomenclature adopted by The Cancer 24,46 Genome Atlas to subclassify human LUADs and LUSCs. This Basal factors in LUSC remarkable molecular heterogeneity seen between and within fl In LUSCs, Sry-related HMG box 2 (SOX2) is one of the most human lung cancer subtypes may re ect the relative abundance frequently amplified and overexpressed genes.24,63–65 SOX2 of various progenitor cell lineages in a tumor mass. Instead of fi encodes for a TF best known for its ability to reprogram being xed over time, tumor histopathology can also deviate due embryonic stem cells. During normal development, however, to the capacity of a tumor-initiating cell population to dediffer- SOX2 is essential for basal cell commitment in the proximal entiate or transdifferentiate relative to its original lineage. 66 fi airways. As an oncogene, SOX2 is required for the survival of Signi cantly, molecular aberrations or extracellular signals can LUSC cells, but its aberrant expression alone is not sufficient for directly drive this phenotypic plasticity. transformation.65 A combination of genetic alterations, such as the In GEMMs, lineage-tracing experiments have demonstrated that loss of Lkb1,67 might be required for Sox2-driven LUSC in mice. Kras-transformed AT2 cells self-renew without additional changes fi 4 SOX2 is usually co-ampli ed with the basal lineage TF p63 at in cell fate, at least up until the formation of adenomas. By 63 + − chromosome 3q26. Their interaction and colocalization at target contrast, Kras-mutant CC10 hyperplasias can switch to a CC10 / 68 + gene loci support oncogenesis instead of pluripotency. Within SPC -like phenotype to develop into LUAD in the terminal the same amplicon as SOX2 is PRKCI, which encodes for protein 27 fl bronchioles. This may be induced by local in ammation or kinase Cι that phosphorylates SOX2, leading to the activation of oncogenic RAS-mediated AT2 differentiation, a lineage state that Sonic hedgehog (Shh) signaling and LUSC self-renewal.69 is more conducive to LUAD progression. Phenotypic conversion of In contrast to its role in promoting LUSCs, high levels of Sox2 in AT2-derived LUADs is seen when mutating the tumor suppressor CC10+ cells restrict the formation of LUAD in the bronchioles by Stk11 in conjunction with Kras, which generates tumors that suppressing Notch activation.70 transdifferentiate into LUSCs.22,47 LUSC metaplasia from LUADs is regulated by conserved developmental signals including the Hippo/Yap48 and the transforming growth factor/SMAD Alveolar factors in LUAD pathways.49 Another developmental program required for respira- In distal epithelial cells, a different SOX family member, SOX9, tion is the canonical WNT/β-catenin pathway.50–52 Mutations in marks the branch tips of early lungs and functions downstream of components of this pathway such as APC, GSK3B and CTNNB1 are receptor tyrosine kinase signaling to suppress premature alveolar 53 differentiation.71,72 SOX9 is overexpressed in human LUAD and its detected in 15% of human LUADs. Multiple studies report 73,74 frequent overexpression of various WNT pathway components expression correlates with poor patient survival. Induction of and epigenetic silencing of WNT pathway antagonists in human Sox9 expression has been observed in murine LUADs harboring 54 fi activating mutations in Kras and β-catenin, and this overexpres- LUADs. In GEMM, lung-speci c expression of a constitutively 56,57 active mutant β-catenin is not sufficient to initiate LUAD, but sion is associated with high-grade tumors. Moreover, SOX9 is preferentially upregulated in human KRAS mutant adenomas and may increase the pool of progenitor cells available for 75 transformation.55,56 Deletion of the WNT antagonist APC coop- is also induced by Notch activation. 57 NK2 homeobox 1 (NKX2-1), also known as thyroid TF-1, is erates with Kras to induce dedifferentiated invasive LUADs. 76 Moreover, transcriptional activation of the WNT/TCF effector LEF1 expressed in AT2 cells and a subset of bronchiolar cells. NKX2-1 58,59 is essential for lung morphogenesis and alveolar cell is a marker and mediator of LUAD metastasis to the brain. The 77 mechanism by which WNT/TCF promotes LUAD may involve both differentiation. Thyroid TF-1 protein is a biomarker of thymic the expansion of the cell of origin and epigenetic reprograming of cancers and LUADs. About 15% of LUADs harbor NKX2-1 amplification78–80 that correlates with poor outcome81,82 and is transformed cells into a primitive state that has increased 80,83 metastatic potential. required for tumor cell viability. NKX2-1 can support pro- tumorigenic signaling downstream of mutant epidermal growth factor receptor (EGFR)84 and is required for EGFR-mediated LINEAGE TRANSCRIPTION FACTORS IN CONTEXT transformation in vivo.85 Paradoxically, NKX2-1 expression also correlates with better outcome, as its expression is frequently Underlying the diverse pathways, molecular perturbations and fi differentiation states in lung cancers are transcription factors (TFs) suppressed in high-grade LUADs with wild-type/non-ampli ed NKX2-1.81,82,86,87 Importantly, in murine LUAD models, wild-type and cofactors whose expression and activity can be restricted for 85,88 certain cell lineages.3,60 As will be discussed below, the genes Nkx2-1 inhibits LUAD progression driven by Kras. This tumor- suppressive effect is closely linked to epithelial differentiation encoding several such lineage TFs can be directly mutated, fi 85 activated or silenced in particular lung cancer subtypes, suggesting states, as Nkx2-1 haploinsuf ciency promotes mucinous LUAD and activation of enteric lineage markers that are normally that they are context-dependent drivers of tumor progression and 88 phenotypic heterogeneity. repressed in alveolar cells. Repression of Nkx2-1 is also required for stochastic dissemination and metastasis by tumors with Kras and p53 mutations.86 NE factors in SCLC The contradictory role of certain lineage TFs likely depends on Inactivating mutations in RB1 are ubiquitous to all SCLCs.16 The the epigenetic context in which they function. One determinant of extended pRb family of pocket proteins interacts with a myriad of such contexts is the relative abundance and accessibility of DNA binding TFs and cofactors required for cell cycle progression, different cofactors in a given progenitor cell. The levels and self-renewal, apoptosis, senescence and cellular differentiation.61 dependencies of cofactors can be initially dictated by cell lineage Although components of the pRB pathway are frequently states (for example, tumor cell of origin or stage) and further inactivated across many cancers, the strong selection for dysregulated by somatic alterations (for example, mutation, gene mutations in RB1 exclusively within SCLC suggests that pRb itself amplification and DNA methylation). NKX2-1, for instance, can is a lineage-specific tumor suppressor. Remarkably, in the murine interact with multiple DNA binding transcriptional repressors or

© 2015 Macmillan Publishers Limited Oncogene (2015) 5771 – 5780 Transcription control of lineage states in lung cancer WKC Cheung and DX Nguyen 5774 activators to expand or limit the range of target genes.89 Wild-type The oncogenic function of GATA6 may depend on its amplification levels of NKX2-1 maintain alveolar differentiation and inhibit or other cooperating gene amplifications103 and availability of proliferation by restricting the activity and genomic target loci of interacting TF partners, such as SP1,105 KLF5 and GATA4.104 FOXA1/288 and AP1 factors,85 respectively. In this regulatory Elevated levels of various GATAs correlate with EMT in a murine network, decreases in NKX2-1 causes derepression of genes that LUAD model.108 On the other hand, expression of GATAs is mediate cell proliferation,90 EMT,91 motility92 and resistance to generally silenced in a significant proportion of human anoikis.86 Conversely, NKX2-1 amplification is often accompanied LUADs.46,109 In a subset of high-grade LUADs, GATA6 is co- by overexpression of the lineage TF FOXA1.93 In this latter context, repressed with other alveolar TFs with whom it may cooperate to 45 which includes aberrantly high levels of NKX2-1 and FOXA1, both inhibit LUAD metastasis. Hypermethylation and mutation of 109 TFs can cooperatively access and transactivate de novo genes GATA6 itself are uncommon in LUADs, but, as in other cancers, 110 required for cancer cell survival.93 Thus, genes that would alternative mechanisms may contribute to its downregulation. otherwise not be targeted by physiological levels of a given The homeodomain only protein X (HOPX), reported as a marker 5,6 lineage TF can be co-opted for malignant progression under of AT1 cells, is an atypical homedomain protein that is essential aberrant epigenetic settings (Figure 2). for lung maturation. Although Hopx lacks a DNA binding domain, it acts downstream of Nkx2-1 and Gata6 to modulate alveolar- specific gene expression in a histone-deacetylase-dependent An expanding network of alveologenic tumor suppressors 111 manner. HOPX exhibits tumor inhibitory effects in a wide range In addition to NKX2-1, several other alveolar specifying TFs can of cancers.112–116 In LUADs, HOPX expression correlates tightly limit LUAD progression. Many of these factors genetically and with NKX2-1,93 and it can restrain tumor cell invasion and physically cooperate with one another to regulate the expression metastasis.45 Moreover, ectopic HOPX expression can induce of prototypical alveolar differentiation markers, including surfac- senescence and is preferentially silenced via DNA methylation in tant proteins. This suggests that the transcriptional network that the LUAD subtype, where decreased HOPX levels correlate with controls alveologenesis is broadly linked to tumor suppression in poor clinical outcome.117 Reducing both HOPX and GATA6 in LUAD the LUAD subtype. cells not only reverses alveolar identity but also activates the The GATA family proteins are DNA binding TFs that can prime expression of squamous basal cell markers and pro-invasive target promoters for repression or activation in specific tissues.94 genes.45 Thus, HOPX and its cooperating TFs may function GATA6 is abundantly expressed in the distal lungs where it is similarly to NKX2-1 by controlling latent differentiation programs required for lung branching morphogenesis and alveolar specifi- that limit overall metastatic competence.118 cation, and it restricts the expansion of adult progenitor cells at CCAAT enhancer binding protein-α (C/EBPα) is a family member – the bronchoalveolar duct junction.95 97 Gata6 directly binds to of basic leucine zipper TFs. C/EBPα is required for perinatal AT2 Nkx2-1 and induces the expression of surfactant proteins in AT2 differentiation and surfactant gene expression.119,120 In the adult – – cells.98 100 Similar to NKX2-1, GATA6 has both oncogenic101 105 lungs, loss of C/EBPα impairs epithelial regeneration.121 Although and tumor-suppressive effects106,107 in a variety of cancers. rarely mutated in lung cancer,122 C/EBPα is often suppressed by

Figure 2. Epigenetic and cellular context dictates the effect of lineage TFs in lung cancer. Given a specific cellular origin, lung cancer progression can be driven by repression of a lineage TF and its tumor-suppressive gene targets. Conversely, amplification of a lineage TF may activate oncogenic genes following de novo TF complex formation at novel target promoters. The tumor-suppressive and oncogenic activities may be adopted by the same TF in different epigenetic contexts. These contexts are defined by the overall abundance and availability of TFs and their partnering cofactors. TF thresholds may be dependent on cooperating mutations and different cellular origins (not depicted).

Oncogene (2015) 5771 – 5780 © 2015 Macmillan Publishers Limited Transcription control of lineage states in lung cancer WKC Cheung and DX Nguyen 5775 DNA hypermethylation,123 and its re-expression was shown to to target promoters.154 Myc has also been suggested to broadly inhibit the proliferation and survival of human NSCLC cells.124 amplify gene transcription in cancer cells.155 In resected human In mice, Cebpa is dispensable for spontaneous LUAD initiation, LUADs, MYC overexpression is linked to increased DNA methyla- however, its loss accelerates stress-induced LUAD formation, tion of known tumor suppressor genes.46 Activation of other indicative of a tumor-suppressive role in response to environ- specific C-myc targets in human LUADs correlates with perturba- mental stimuli.125 Further underlying the complexity of respiratory tions in lineage TF networks and poor clinical outcome.45,156,157 TF networks, C/EBPα binds to NKX2-1126 and Cebpa expression is Consistent with these in silico analysis of human tumors, Myc also regulated downstream of Nkx2-1 during development.119 overexpression from SPC+ AT2 progenitors in mice is sufficient to FOXA1 and FOXA2, previously termed hepatocyte nuclear factor induce macrometastases that have undergone a mixed lineage 3α/β, belong to the subfamily of forkhead box TFs. FOXA1/2 have switch.158 Finally, human mutations in the MYC pathway are similar temporal and spatial expression patterns in developing mutually exclusive to SMARCA4 (or BRG1),148,159 which encodes a lungs. With partially overlapping functions, they have critical roles major component of the SWI/SNF chromatin-remodeling in lung morphogenesis and alveolar differentiation.127,128 Both complex160 and is a mediator of SCLC NE differentiation.148 FOXA1/2 are binding partners of NKX2-1 in LUADs,88,129 and they Therefore, despite its multiple functions, the major consequence can activate latent epithelial differentiation genes when NKX2-1 is of MYC activation in lung cancers may be to epigenetically suppressed.88 FOXA2 also cooperates with GATA6 to regulate the reprogram specific lineage states. expression of cell adhesion genes in the developing lung epithelium.130 In both normal epithelial cells and lung cancer LINEAGE PLASTICITY AND METASTASIS: CAUSE OR cells, FOXA2 induces the expression of C/EBPα119 and vice versa.131 CONSEQUENCE? Similar to other alveolar TFs, FOXA2 silencing is observed in LUADs through promoter hypermethylation.132 Finally, overexpression of Epithelial lineage plasticity has been associated with cancer FOXA2 in human NSCLC cells inhibits cell growth, invasion and metastasis. As core mediators of cell fate, lineage TFs may EMT.131,133 therefore regulate genes required for lung cancer cell dissemina- The runt-related TFs (RUNX) are required for the development tion and distant organ colonization. However, considering the of several mammalian tissues and are dysregulated in epithelial diverse functional targets of these TFs, is their capacity to regulate cancers.134 In the murine lungs, Runx3 is expressed by distal metastasis directly connected to the lineage state in a given cell? epithelial cells including AT2 cells, and its targeted loss causes In lung cancer, this remains an open question, but some insight alveolar hyperplasia.135,136 RUNX3 is yet another alveolar gene may be drawn from recent studies. noted to be methylated at a significantly high frequency in lung The overexpression of EMT inducing TFs in epithelial cancer malignancies.137 RUNX3 methylation status is an independent cells is often interpreted as a marker of increased cell invasiveness 161 prognostic factor in NSCLC patients138 and is associated with and metastasis. Overexpression of EMT inducing TFs has been 162 chemoresistance.139 Other modes of RUNX3 loss of function, such observed in various models of metastatic LUAD. However, this as EZH2-mediated H3K27 trimethylation140 and MDM2-mediated is confounded by the fact that EMT promoting TFs also target ubiquitination,141 have been reported in other cancers, but have genes that are independently required for other cellular functions such as cell survival, proliferation, DNA damage responses and yet to be explored in LUAD. In GEMMs, heterozygous loss of Runx3 163,164 causes pre-neoplastic lung adenoma.135,136 Notably, Kras activa- self-renewal. In some carcinoma models, EMT is neither fi tion only results in non-mucinous LUADs, whereas Runx3 loss suf cient nor required for cell invasion, while disseminating cancer cooperates with mutant Kras to initiate both mucinous and non- cells that undergo the reverse process, mesenchymal-to-epithelial mucinous LUADs.142 This further suggests that deregulation of cell transition, have a greater advantage for tumor re-initiation and fate-determining TFs can drive transition to different lung tumor metastatic colonization. Intriguingly, some studies suggest that subtypes. The molecular connection between RUNX3 and other mesenchymal carcinoma cells may not invade beyond the local tumor, but rather enhance the dissemination of other epithelial prototypical alveolar TFs is not yet known, but its ability to 165,166 abrogate the ARF-p53 pathway by interacting with the bromodo- cell populations with tumor re-initiating capacity. An main protein BRD2 implicates an epigenetic modulatory role.142 analogous phenomenon has been described in GEMMs of SCLCs, where a fraction of malignant cells can transition from their NE origins into non-NE cells that express mesenchymal genes.167 MYC revisited Although these non-NE cells are clonally related to the rest of the The MYC family proto-oncogenes, which encode C-myc (Myc), SCLC, they do not metastasize. On the other hand, their presence N-myc and L-myc, regulate numerous biological processes. in the tumor mass enhances the ability of the NE lineage-positive Although not restricted to pulmonary lineages, the MYC proteins cells to spread. Thus, modulation of lineage states by TFs may are nonetheless potent transcriptional regulators of lung epithelial indirectly influence metastatic potential by controlling intratu- differentiation. N-myc is expressed at high levels in the distal moral clonal dynamics, presumably via paracrine signals (Figure 3). airway epithelium of mice143 and is controlled by Wnt/β-catenin An alternative mechanistic consequence of tumor metaplasia is signaling.52 Loss of N-myc and C-myc can delay lung branching that it may lead to the activation of lineage gene programs that morphogenesis.144,145 C-myc is amplified in human LUAD.46,146 All directly control the metastatic capacity of a given cell. The three MYC members are frequently amplified in SCLC,16,147 but not activation of latent basal epithelial markers, such as cytokeratins in LUSC.24 Moreover, recent genomic studies have identified loss- (K), has been reported in more invasive tumor cells. For example, of-function mutations in MGA and MAX, two genes that encode for cells at the invasive border of luminal breast tumors express the MYC inhibitory proteins.46,148 In human lung cancers, alterations in basal epithelial marker K14 and basal TF p63. These invasive basal MGA, MAX and MYC are mutually exclusive,46,148 indicating a more cells arise from the well-differentiated luminal cells and are pervasive activation of the MYC pathway via mutations than was responsible for collective tumor cell invasion in a manner that is initially appreciated. independent of EMT.168 In liver cancer, the biliary/progenitor In GEMMs, expression of Myc in SPC+ or CC10+ cells induces marker K19 is expressed in a subset of hepatocellular carcinomas LUAD.149,150 In addition, Myc-induced LUAD requires Kras with a high incidence of microvascular invasion and metastasis.169 activating mutation,150 which can be substituted by Notch1 Similarly, LUAD cells may acquire invasion potential by down- overexpression.151 Myc is also required for tumor maintenance in regulating alveolar differentiation genes and upregulating the a variety of Ras pathway-initiated NSCLC models.152,153 MYC can squamous basal cell markers K6A/B.45 Importantly, knockdown of activate or repress specific genes by recruiting different cofactors these cytokeratins in their respective disease models inhibits tumor

Figure 3. Hypothetical mechanisms by which cell lineage plasticity and heterogeneity promote lung cancer metastasis. (a) Epithelial lineage- positive malignant cells, such as NE cells in SCLC, constitute the majority of distant metastasis, but their dissemination is enhanced via non-cell autonomous signals from mesenchymal transitioned tumor cells in the primary tumor. (b) LUAD cells may disseminate via a classical EMT/ mesenchymal-to-epithelial transition model. (c) Alternatively, LUAD cells may undergo multiple switches in the expression of airway epithelial lineage-speciﬁc markers that promote metastasis via cell autonomous mechanisms.

cell invasiveness, suggesting an unexpected direct role for basal/ kinase and BCL-Xl.182 In contrast, LUADs that have undergone biliary epithelial markers in malignant invasion and metastasis. lineage transition become drug refractory, even when those treatments target their driver oncogenes. At the same time, such lineage transitions may have foreseeable shifts in signaling HARNESSING LINEAGE STATES FOR THERAPY dependencies. For example, the survival of tyrosine kinase The TFs discussed here are but a few examples linking the inhibitor-resistant EGFR mutant tumor cells, which are more molecular control of cell lineage fate to lung cancer progression. mesenchymal like, requires activation of the receptor tyrosine fi Despite the broad biological signi cance of these observations, kinase AXL,34,183 which can be inhibited with small molecules. their clinical and therapeutic impacts remain underexplored. Part of Given the fact that lung cancers can adopt various differentia- the reason for this is the intrinsic technical barriers in pharmaco- tion states, new biological insight may be revealed by compre- logically manipulating pathways or TFs that are not only tissue fi fi fi hensively de ning their underlying transcriptional circuits. These speci c but also likely to have dosage- and stage-speci c effects circuits may in turn modify the oncogenic dependencies of a during tumor progression. Nevertheless, exploiting aberrant tumor given tumor, providing novel therapeutic opportunities. Major differentiation states or lineage TFs for therapy has been shown to challenges remain in directly inducing or reverting these lineage- be clinically beneficial in leukemia and neuroblastoma170 and pre- specific pathways at different stages of disease progression. clinically feasible in melanoma,171,172 glioma,173,174 nasopharyngeal 175 176 Nevertheless, several lung cancer differentiation states, defined carcinoma and rhabdomyosarcoma. Driving the tumors toward more differentiated states may not only delay their growth either transcriptionally or histologically, may already be used as but also potentially increase their sensitivity to commonly used both prognostic and predictive biomarkers. Moving forward, a fi cytotoxic agents. systematic search for ef cacious agents tailored not only to the Several novel compounds have recently been explored to oncogenic drivers of various lung cancer subtypes but also to target the expression or activity of TFs. For example, drugs the cell lineage of their malignant compartment, holds the that inhibit the chromatin-remodeling bromodomain and promise of abating metastatic progression. extraterminal proteins are being actively investigated to target the lineage-dependent activity of TFs in multiple cancers, including LUAD.177,178 Another potential approach is to use covalent inhibitors of cyclin-dependent kinase 7, which preferen- CONFLICT OF INTEREST tially mediates the expression of super-enhancer associated The authors declare no conflict of interest. genes.179,180 Because such genes include oncogenic MYC and NE TFs, this class of transcription targeting drugs may be particularly effective against SCLC.181 Assessing the lineage state of the bulk tumor may have more ACKNOWLEDGEMENTS fi immediate clinical bene ts for anticipating dynamic therapeutic We thank all members of the Nguyen laboratory for their discussions and critical responses. This is relevant in the treatment of KRAS mutant LUADs, reviews of the manuscript. We apologize for omitting some examples and primary which are highly heterogeneous. Notably, a subset of KRAS mutant references due to space constraints. Our research is funded by grants from Uniting LUADs that are well differentiated and retain epithelial markers Against Lung Cancer (to WKCC) and the National Cancer Institute (1R01CA166376, respond specifically to a combination of drugs that inhibit MAP 1R21CA170537 and 1R01CA191489 to DXN).

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