Cancers appear when changes in the genetic information of a cell, also called mutations, allow it to multiply uncontrollably. The disease we know as “lung cancer” kills more people than any other cancer, but this term actually refers to different types of tumors that appear because of various mutations that happen in different kinds of lung cells.

To complicate matters further, as lung cancer cells become more aggressive, they can stop appearing and behaving like the type of lung cell they came from. Yet, knowing the exact origin of the cancer is key, since it determines which treatment will work best to stop the disease in its tracks.

Despite these differences, many lung cancer cells contain mutations that over-activate two molecular cascades called the MAP kinase and the PI3’-kinase pathways. Under normal conditions, these signaling pathways relay external messages to the inside of the cell, where they help cells multiply. Two separate mutations can respectively over-stimulate either the MAP kinase or the PI3’-kinase pathway, but it was unclear how these could work together to start and maintain aggressive lung tumors. Another unanswered question was how these cancer cells lose the characteristics of the healthy cells they came from.

To address these issues, van Veen et al. genetically engineered mice that carry a mutation which activates the MAP kinase pathway. The lung cells with this genetic change also made a red fluorescent protein that marked cancer cells, so that these could be separated from the rest of the lung and analyzed.

This revealed that cells with only the MAP kinase mutation turned into small and benign tumors that began in lung cells, known as “type 2” cells. The PI3’-kinase mutation alone could not even start a tumor. However, together the mutations made tumors much more aggressive. Cells that carried both mutations also stopped producing proteins normally made by type 2 cells, therefore causing the cells to lose their original identity.

The mice created by van Veen et al. could help to understand how lung cancers develop in these animals and also in human lung cancer patients. Ultimately, this information could be used to design new cancer treatments, especially since both the MAP kinase and PI3’-kinase pathways contain many proteins that can be targeted with drugs.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death, with lung adenocarcinoma (LUAD) being the most common NSCLC subtype (Siegel et al., 2016). Due to the morbidity and mortality associated with LUAD, there is an urgent need to better characterize how key genetic drivers contribute to the pathogenesis of this disease. To that end, since the original discovery of KRAS mutations in human lung cancer cells (Capon et al., 1983), it has emerged that ~75% of LUADs display mutational activation of key components of receptor tyrosine kinase (RTK) signaling that, in turn, promote activation of RAS and its key downstream effectors: the RAF→MEK→ERK→MAP kinase (MAPK) and the PI3’-lipid pathways (Cancer Genome Atlas Research Network, 2014; Heist and Engelman, 2012). Moreover, mutational activation of RTKs or downstream signaling proteins (e.g. EGFR/ERBB1, ALK, ROS1, NTRK, BRAF) serve as predictive biomarkers for the clinical deployment of FDA-approved inhibitors of these oncoprotein kinases for the treatment of genetically-defined subsets of lung cancer (Drilon et al., 2018; Hyman et al., 2015; Rosell et al., 2012; Scagliotti et al., 2010; Shaw et al., 2013; Shaw et al., 2014).

Mutational activation of BRAF occurs in ~8% of LUAD, with the most common single mutation (BRAF^T1799A) encoding the BRAF^V600E oncoprotein kinase (Cancer Genome Atlas Research Network, 2014). To model BRAF^V600E driven cancers, we previously described Braf^CA mice carrying a CRE-activated allele of Braf that expresses normal BRAF prior to CRE-mediated recombination, after which BRAF^V637E (orthologous to human BRAF^V600E and for simplicity henceforth referred to as BRAF^V600E), is expressed from the endogenous chromosomal locus (Dankort et al., 2007). This mouse has proven useful in modeling many cancer types in which BRAF^V600E is implicated as a driver oncoprotein (Charles et al., 2011; Dankort et al., 2009; Sakamoto et al., 2017; Trejo et al., 2013; Wang et al., 2012). Taken together, these studies indicate that BRAF mutation serves as a foundational initiating event for tumorigenesis in many target tissues. However, the progression of benign tumors initiated by BRAF^V600E expression into malignant cancer invariably requires additional events such as silencing of tumor suppressors (e.g. INK4A-ARF, TP53, PTEN, CDX2) or activation of cooperating oncogenes (PIK3CA, CTNNB1, c-MYC) (Charles et al., 2014; Dankort et al., 2007; Dankort et al., 2009; Huillard et al., 2012; Juan et al., 2014; Sakamoto et al., 2017; Trejo et al., 2013; Tsao et al., 2004; Yu et al., 2009).

In mouse models of lung carcinogenesis, there are key similarities between the early stages of tumorigenesis observed in response to expression of either the KRAS^G12D or BRAF^V600E oncoproteins (Dankort et al., 2007; Trejo et al., 2012). While tumors initiated by BRAF^V600E remain as benign adenomas with certain features of senescence (Dankort et al., 2007; Jackson et al., 2001), a proportion of KRAS^G12D initiated lung tumors progress to frank adenocarcinomas within six months, most likely due to the ability of KRAS^G12D to activate the PI3’-kinase signaling pathway (Castellano et al., 2013; Murillo et al., 2018; Rodriguez-Viciana et al., 1994; Vivanco and Sawyers, 2002; Yuan and Cantley, 2008). Consistent with this hypothesis, co-expression of BRAF^V600E and PIK3CA^H1047R, a mutationally-activated form of PI3’-kinase-α (PI3Kα), in AT2 pneumocytes leads to rapid growth of lung tumors, many of which display progression to frank malignancy bearing various hallmarks of the cognate human disease (Kinross et al., 2012; Trejo et al., 2013). Thus, these genetically manipulated mice provide a unique opportunity to genetically and biochemically separate and analyze the effects of activation of these two critical downstream arms of RTK→RAS signaling individually or in combination.

Whereas the original Braf^CA mouse allowed insights into cancer initiation, progression and therapy, there remain many questions that this mouse is inadequately configured to address. For example, it is not trivial to identify and isolate pure populations of tumor cells without significant stromal contamination, particularly in contexts when tumor cells are rare, such as in the earliest stages of tumorigenesis, or in the context of minimal residual disease following pathway-targeted inhibition of BRAF^V600E signaling (Dankort et al., 2007). To address these issues we and others have used mice carrying CRE-activated alleles that express fluorescent proteins, such as the mT-mG allele in which the activity of CRE recombinase silences the expression of tdTomato and elicits expression of EGFP (Muzumdar et al., 2007). However, this approach is confounded by the observation that not all cells expressing the desired oncoprotein also express EGFP and vice versa.

In order to unequivocally identify BRAF^V600E expressing cells we have generated Braf^CAT mice carrying a new CRE-activated Braf allele. Like the original Braf^CA allele, Braf^CAT encodes normal BRAF prior to CRE-mediated recombination, after which the recombined allele expresses a bicistronic Braf^T1910A-P2A-tdTomato mRNA encoding both BRAF^V600E and the red fluorophore tdTomato. Moreover, here we report the use of Braf^CAT mice to explore the cooperation of oncogenic BRAF^V600E and PI3Kα^H1047R in lung carcinogenesis in greater mechanistic detail. In brief, BRAF^V600E-driven lung tumors maintain expression of markers of AT2 identity, including the known regulators of AT2 identity, NKX2-1, FOXA1, and FOXA2 (Bruno et al., 1995; Camolotto et al., 2018; DeFelice et al., 2003; Hamvas et al., 2013; Lazzaro et al., 1991; Minoo et al., 1995; Snyder et al., 2013; Stahlman et al., 1996; Winslow et al., 2011; Yuan et al., 2000). By contrast, co-expression of BRAF^V600E and PI3Kα^H1047R leads to development of lung tumors that show variable and widespread loss of expression of markers of AT2 pneumocyte terminal differentiation including the well-characterized surfactant proteins, SFTPA, SFTPB, and SFTPC. Notably reduced expression of AT2 markers begins early in tumor development and occurs despite sustained NKX2-1, FOXA1, and FOXA2 expression in tumor cells. Hence, these data shed light on the mechanisms by which pathways that cooperate in lung tumorigenesis also cooperate to influence the differentiation state of tumor cells. Indeed, these findings bear similarity to observations in human lung adenocarcinomas in which poorly differentiated and metastatic cancers often show loss of expression of functional markers of lung identity despite maintaining expression of NKX2-1 (Yatabe et al., 2002). Consequently, our results may shed light on our understanding of human lung cancer progression and how normal lung epithelial cells may lose their differentiation status following activation of cooperating oncogenic pathways.

Genetically engineered mouse models have proven to be invaluable tools that complement the significant advances being made in the genetic and biochemical characterization of the initiation, progression and maintenance of human cancers. As genome sequencing efforts catalog clinically actionable mutations and their correlations with the cancer’s observed phenotypes, directly testing how these mutations affect disease initiation, progression, and response to novel therapeutics becomes a high priority. Here we describe a new GEM model that has allowed a deeper dissection of the stages of BRAF^V600E-driven lung cancer, a disease that kills ~4000 patients per year in the U.S.A. (Siegel et al., 2016). To engineer this mouse we employed a strategy in which the previously targeted CRE-activated Braf^CA allele was re-targeted to allow for the expression of both BRAF^V600E and tdTomato from a single bicistronic mRNA following CRE-mediated recombination. Consequently, all BRAF^V600E expressing cells are predicted to be red fluorescent by virtue of co-expression of tdTomato. This is an alternative approach to the more widely employed method of using a fluorophore expressed in trans from a different promoter (e.g. Rosa26) the expression of which is co-activated by CRE recombinase (Livet et al., 2007; Madisen et al., 2010; Muzumdar et al., 2007; Snippert et al., 2010). The approach described here has both advantages and disadvantages compared to the use of a fluorophore expressed in trans approach. The main disadvantage of our approach appears to be the modest fluorescence emission of tdTomato when expressed downstream of a P2A element from the mutationally activated Braf^T1910A mRNA. Many LSL-XFP alleles have been designed for very high levels of expression by the incorporation of enhancers and strong promoters, whereas our model is driven by the endogenous Braf enhancer/promoter sequences. Hence, although the level of tdTomato fluorescence in lung tumors arising in Braf^CAT mice is readily detected by flow cytometry, detection of tdTomato expression by immunohistochemistry in FFPE tissue sections is problematic. However, we have developed protocols that allow for detection of native tdTomato fluorescence in frozen sections (Figure 1D) or by a ligation proximity assay in FFPE sections (Daphne Pringle, unpublished). By contrast, the use of a fluorophore expressed in trans approach inherently relies on the simultaneous activity of CRE at distinct regions of the genome. Thus it is possible for the proto-oncogenic Braf^CA locus to be subject to CRE-mediated recombination without recombination of the reporter, or vice-versa. While this is acceptable for some applications, it adds confounding noise that may be amplified under conditions when tumor cells have been specifically depleted by the application of pathway targeted or immunomodulatory therapy that has negligible effect on normal cells. Our approach avoids this noise entirely and results in a level of fluorescence that is directly correlated with expression of BRAF^V600E on a cell-by-cell basis (van Veen et al., 2016). Importantly, our approach is especially compatible with the emerging technology of single cell RNA sequencing (scRNA-Seq), in which it is desirable to isolate single BRAF^V600E oncoprotein kinase expressing cells for analysis.

Our studies were aided greatly by recent characterizations of the gene expression changes accompanying development of the mouse distal lung epithelium (Treutlein et al., 2014) and KRAS^G12D-mediated oncogenic transformation of AT2 cells (Desai et al., 2014). Using scRNA-Seq, the authors reconstructed the lineage hierarchies of AT1, AT2, club and ciliated cells, as well as provided a new list of markers useful in identifying these major cell types of the distal lung epithelium. We used these marker lists extensively and without bias or selection for most of our analyses. Importantly, we constructed gene sets for the GSEA analyses that showed widespread decreased AT2 marker expression in BRAF^V600E/PIK3CA^H1047R-driven lung tumors. Analysis of the full complement of AT2 markers paints a dramatic picture; nearly every AT2 identity marker is diminished in expression in BRAF^V600E/PIK3CA^H1047R-driven tumors as compared to BRAF^V600E-driven tumors, as early as two weeks p.i. Importantly we noted a similar early and consistent repression of PGC1α expression in BRAF^V600E/PIK3CA^H1047R-driven tumors. Here our data support previous findings that indicate that PGC1α expression is repressed by the PI3K→AKT signaling axis via three insulin response sequences found in the PGC1α promoter shown to bind to the AKT-regulated FOXO1 transcription factor (Daitoku et al., 2003; Kemper et al., 2014). It has also been suggested that BRAF^V600E signaling can suppress PGC1α expression via the MITF transcription factor in melanoma, further demonstrating the complicated interplay between growth factor signaling and oxidative phosphorylation (Haq et al., 2013), though this study did not examine any potential connection of PGC1α to tumor cell differentiation status.

PIK3CA mutation is found in ~4% of human LUAD (Campbell et al., 2016) that, although quite rare, still represents a significant patient population due to the high prevalence of lung cancer in our society. Previous studies have demonstrated that the ability of mutationally-activated KRAS^G12D to bind and activate PI3Kα is critical for its tumor promoting activities, as well as tumor maintenance (Castellano et al., 2013; Gupta et al., 2007b; Molina-Arcas et al., 2013; Murillo et al., 2018). Despite this, activation of PI3’-lipid signaling remains rate limiting with respect to tumor initiation and growth in KRAS^G12D-driven lung tumors as evidenced by the strong cooperation between KRAS^G12D and PIK3CA^H1047R in promoting lung tumorigenesis in a GEM model (Green et al., 2015). This cooperation likely reflects the fact that the initial expression of KRAS^G12D is a relatively weak activator of both PI3’-lipid signaling and the RAF→MEK→ERK pathways (Cicchini et al., 2017). Indeed, whereas KRAS^G12D derived lung cancer cells show little phosphorylation of AKT at a key activating amino acid (S473), KRAS^G12D/PIK3CA^H1047R derived cells show strong pS473-AKT phosphorylation (Green et al., 2015). Our results suggest that the lack of PI3’-lipid signaling in KRAS^G12D-driven lung tumors is not only limiting in tumor growth, but in propensity for AT2 pneumocyte de-differentiation. Intriguingly, recent studies have shown that stromally derived IGF-1 promotes a cancer stem cell like phenotype in Kras mutated cell lines (Chen et al., 2014), via the PI3K→AKT signaling axis. It will be interesting to examine in future studies how various tumor genotypes and tumor microenvironments converge upon the initiation and evolution of LUAD de-differentiation.

Lung adenocarcinoma differentiation status, as judged by pathological criteria, remains a critically important prognostic factor in predicting patient survival (Yoshizawa et al., 2011). However, only in recent years have we begun to understand the genetic aberrations that can directly promote loss of differentiation status. The protein most directly demonstrated to influence both lung adenocarcinoma differentiation status and progression is NKX2-1, a homeodomain transcription factor. Indeed, in the Kras^LSL-G12D GEM model of KRAS^G12D-driven lung adenocarcinoma, NKX2-1 expression is diminished or silenced in the most poorly differentiated tumors. Moreover, shRNA-mediated inhibition of NKX2-1 expression is reported to enhance the metastatic potential of KRAS^G12D/TP53^Null-driven lung cancer cells (Winslow et al., 2011). Interestingly, concomitant expression of KRAS^G12D with genetic silencing of NKX2-1 expression has qualitatively different effects, promoting trans-differentiation of tumor cells into a gastric fate resembling de-differentiated mucinous adenocarcinoma (Snyder et al., 2013), an effect that requires the activity of FOXA1/FOXA2 (Camolotto et al., 2018). Intriguingly, while even haploinsufficiency of NKX2-1 promoted the appearance of mucinous adenocarcinoma in the Kras^LSL-G12D GEM model of lung adenocarcinoma, the same was not true in a lung adenocarcinoma model driven by expression of a mutationally-activated form of the EGF receptor (Maeda et al., 2012). In this model, haploinsufficiency of NKX2-1 slowed tumor progression rather than enhancing it. Importantly, loss-of-function mutations or silencing of NKX2-1 is a relatively infrequent event in lung adenocarcinoma. Instead NKX2-1 is often found as the most significantly focally amplified locus (Campbell et al., 2016). Further, in human NSCLC cell lines in which NKX2-1 is amplified, RNAi-mediated inhibition of NKX2-1 expression elicited decreased cell division and apoptosis (Kwei et al., 2008). Hence, the role of NKX2-1 in LUAD progression is thus simultaneously critically important and also complicated. In this case, GEM models provide an ideal system in which to study the contribution of individual mutations to each step of tumor initiation and progression without the complications of mutagen-induced genome hypermutation as is common in KRAS-mutated human lung cancer cells (Chalmers et al., 2017). While a wealth of literature has shown the direct effect of NKX2-1 on AT2 promoters (Bruno et al., 1995), and the importance of NKX2-1 in normal lung development (DeFelice et al., 2003), it has also been shown that transcriptional co-activators are critical for NKX2-1 function (Cassel et al., 2002; Di Palma et al., 2003; Park et al., 2004; Yi et al., 2002). Here we have shown that the binding motif of the nuclear receptor, NR5A2 is highly enriched in the promoters of AT2 specific genes. We also demonstrate that NR5A2, and its known co-factor PGC1α (Yazawa et al., 2010), can potently enhance the activity of NKX2-1 at the promoters for surfactant proteins A and B. Interestingly, while NR5A2 and PGC1α can activate the promoter of SFTPC alone, the added presence of NKX2-1 and FOXA1 does not further co-activate this promoter. It may be the case that PGC1α and NKX2-1 act independently at this promoter, or it may be that there are additional or alternative transcriptional regulators not present in 293 T cells which, when present, allow PGC1α and NKX2-1 to cooperate. In vivo, in BRAF^V600E/PIK3CA^H1047R-driven tumors, we observed an early decrease of PGC1α mRNA expression, and importantly, a correlation between decreased PGC1α and SFTPA on a cell-by-cell basis within tumors. Combined with functional data in GEM models in which PGC1α expression was genetically silenced, these data argue that mutational-activation of PI3’-lipid signaling in BRAF^V600E-driven LUAD leads to diminished PGC1α expression, and that this reduced expression compromises the ability of NKX2-1/FOXA1 to maintain AT2 pneumocyte identity (Figure 7—figure supplement 2A). It is important to note that while genetic silencing of PGC1α recapitulated some aspects of PI3K activation in BRAF^V600E-driven driven lung tumors, there were interesting differences, including an increase in markers of club and ciliated cell identity, and no significant effect on AT1 marker expression. It is therefore highly likely that amongst the pleiotropic effects of PI3’-lipid signaling, inhibition of PGC1α-mediated signaling represents one of many important effector pathways. This novel mechanism of lung identity regulation is made more important by the observation that it can be induced by mutational-activation of PI3Kα in both KRAS^G12D- and BRAF^V600E-driven driven lung adenocarcinomas. Since the best characterized role of PGC1α is in the regulation of mitochondrial biogenesis, we were initially surprised to discover the cooperativity that PGC1α shows in the regulation of AT2 cell identity. However, it has recently emerged that crippling mitochondrial function via loss of the critical pyruvate transporter, MPC1, potently drives cells into a de-differentiated stem cell fate in drosophila and mouse intestinal cells (Schell et al., 2017). These studies, and the data presented here, suggest exciting future studies to examine the role of PGC1α to act as a pleiotropic effector of tumor cell growth and differentiation state downstream of PI3’-lipid signaling.

Sequencing data have been deposited in GEO under accession code GSE123126. All R scripts written for this study are available at GitHub (https://github.com/jevanveen/vanveen-elife; copy archived at https://github.com/elifesciences-publications/vanveen-elife).

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Author details

1. J Edward van Veen

   1. Huntsman Cancer Institute, University of Utah, Salt Lake City, United States
   2. Department of Dermatology, University of Utah, Salt Lake City, United States
   3. Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, United States
   4. Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, United States

   Present address

   Department of Integrative Biology and Physiology, Jonsson Comprehensive Cancer Institute, University of California, Los Angeles, Los Angeles, United States

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1798-3210

2. Michael Scherzer

   1. Huntsman Cancer Institute, University of Utah, Salt Lake City, United States
   2. Department of Dermatology, University of Utah, Salt Lake City, United States

   Contribution

   Conceptualization, Data curation, Supervision, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

3. Julia Boshuizen

   1. Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, United States
   2. Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, United States

   Present address

   Division of Molecular Genetics, Netherlands Cancer Institute, Amsterdam, The Netherlands

   Contribution

   Conceptualization, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

4. Mollee Chu

   1. Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, United States
   2. Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, United States

   Present address

   California Northstate University College of Medicine, Elk Grove, United States

   Contribution

   Conceptualization, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

5. Annie Liu

   1. Huntsman Cancer Institute, University of Utah, Salt Lake City, United States
   2. Department of Dermatology, University of Utah, Salt Lake City, United States

   Contribution

   Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

6. Allison Landman

   1. Huntsman Cancer Institute, University of Utah, Salt Lake City, United States
   2. Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, United States

   Present address

   The Lancet Oncology, London, United Kingdom

   Contribution

   Conceptualization, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

7. Shon Green

   1. Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, United States
   2. Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, United States

   Present address

   Altius Institute for Biomedical Sciences, Seattle, United States

   Contribution

   Conceptualization, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

8. Christy Trejo

   1. Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, United States
   2. Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, United States

   Present address

   BioSpyder Technologies, Carlsbad, United States

   Contribution

   Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

9. Martin McMahon

   1. Huntsman Cancer Institute, University of Utah, Salt Lake City, United States
   2. Department of Dermatology, University of Utah, Salt Lake City, United States
   3. Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, United States
   4. Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, United States

   Contribution

   Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Writing—review and editing

   For correspondence

   martin.mcmahon@hci.utah.edu

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0003-2812-1042

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