We used CDK4/hTERT–immortalized normal human bronchial epithelial cells (HBEC) from several individuals to study lung cancer pathogenesis by introducing combinations of common lung cancer oncogenic changes (p53, KRAS, and MYC) and followed the stepwise transformation of HBECs to full malignancy. This model showed that: (i) the combination of five genetic alterations (CDK4, hTERT, sh-p53, KRASV12, and c-MYC) is sufficient for full tumorigenic conversion of HBECs; (ii) genetically identical clones of transformed HBECs exhibit pronounced differences in tumor growth, histology, and differentiation; (iii) HBECs from different individuals vary in their sensitivity to transformation by these oncogenic manipulations; (iv) high levels of KRASV12 are required for full malignant transformation of HBECs, however, prior loss of p53 function is required to prevent oncogene-induced senescence; (v) overexpression of c-MYC greatly enhances malignancy but only in the context of sh-p53+KRASV12; (vi) growth of parental HBECs in serum-containing medium induces differentiation, whereas growth of oncogenically manipulated HBECs in serum increases in vivo tumorigenicity, decreases tumor latency, produces more undifferentiated tumors, and induces epithelial-to-mesenchymal transition (EMT); (vii) oncogenic transformation of HBECs leads to increased sensitivity to standard chemotherapy doublets; (viii) an mRNA signature derived by comparing tumorigenic versus nontumorigenic clones was predictive of outcome in patients with lung cancer. Collectively, our findings show that this HBEC model system can be used to study the effect of oncogenic mutations, their expression levels, and serum-derived environmental effects in malignant transformation, while also providing clinically translatable applications such as development of prognostic signatures and drug response phenotypes.

Visual Overview:http://mcr.aacrjournals.org/content/11/6/638/F1.large.jpg.

Mol Cancer Res; 11(6); 638–50. ©2013 AACR.

This article is featured in Highlights of This Issue, p. 555

Human lung cancer develops as a multistep process, usually after prolonged smoke-related tobacco exposure resulting in specific proto-oncogene and tumor suppressor gene alterations in lung epithelial cells (1). Genome-wide analyses have identified multiple genetic and epigenetic alterations in lung tumors (2–5). To translate these findings to the clinic, however, it is essential to identify the best targets for early detection and therapeutic intervention by determining which alterations represent “driver” and which represent “passenger” changes.

Functional tests are a critical step in determining “driver” status and are most commonly conducted by genetically or pharmacologically “correcting” the defect in a lung cancer line and monitoring its effect. Another approach is to introduce putative oncogenic changes into normal lung epithelial cells and ascertain their contribution to malignancy. To undertake the latter, we previously developed an in vitro model of immortalized human bronchial epithelial cells (HBEC) establishing cell lines from more than 30 different individuals (6). HBECs were immortalized by overexpressing Cdk4 and human telomerase reverse transcriptase (hTERT) to emulate 2 of the earliest events and almost universal events in lung cancer pathogenesis: abrogation of the p16/Rb cell-cycle checkpoint pathway and bypass of replicative senescence. Importantly, this was the first report of immortalizing lung epithelial cells in the absence of viral oncoproteins, as used previously by other groups (7, 8). Viral oncoproteins such as SV40 early region (containing large T and small t antigens) are known to cause malignant transformation through inactivation of retinoblastoma (Rb) and/or p53, as well as inhibit the tumor suppressor gene PP2A phosphatase (9, 10). They also have other less characterized interactions with cellular processes, thereby contributing to cancer pathogenesis in unknown ways. Thus, the critical genetic manipulations that lead to malignant transformation of epithelial cells immortalized with viral oncoproteins are not completely “defined.” Moreover, they are likely more oncogenically progressed than nonviral models as shown in mammary epithelial cell systems where full transformation with SV40 early region required 3 manipulations (hTERT, SV40 early region, and RAS; ref. 8) as compared with 6 manipulations (hTERT, dominant-negative p53, CDK4, c-MYC, and cyclin D1) in the absence of SV40 early region (11).

While HBECs used in our study are immortalized, a phenotype they share with cancer cells, they do not display other cancer cell phenotypes such as disruption of the p53 pathway, extensive copy number changes, lack of contact-inhibition, anchorage-independent growth, and the ability to form tumors in immunodeficient mice (6, 12). Furthermore, we have shown that immortalized HBECs retain the ability to differentiate into structures found in the normal bronchial epithelium (13, 14). These features make HBECs a physiologically appropriate in vitro model for studying the transformation process of bronchial epithelial cells to lung cancer.

Transformation of lung epithelial cells to full malignancy using defined genetic manipulations has only been described in 2 studies (8, 15). One study using viral oncoproteins-transformed tracheobronchial and small airway epithelial cells with hTERT, SV40 early region, and oncogenic HRAS or KRAS (8). The second study showed fully transformed human small airway epithelial cells (HSAEC) occurred with CDK4, hTERT, mutant p53, mutant KRAS, and either c-MYC, PIK3CA, cyclin D1, or LKB1 manipulations (15). In our bronchial epithelial cell model, we have previously shown the combination of CDK4, hTERT, and p53 knockdown with mutant EGFR, or moderate levels of mutant KRASV12 progressed cells partially but not completely to malignancy, as evidenced by the failure to form tumors in immunodeficient mice (12). As lung cancer develops in both the central bronchus and peripheral small airways, development of an in vitro model of malignant transformation in bronchial epithelial cells is essential.

In the present study, we aimed to confirm the genetic tractability of HBECs and fully transform cells to malignancy using oncogenic manipulations commonly found in lung tumors. Loss of p53 function and oncogenic KRAS are 2 well-known genetic alterations in lung cancer occurring in approximately 50% and 30% of non–small cell lung carcinoma (NSCLC), respectively (16, 17). Aberrant expression of c-MYC, through amplification or overexpression, is found in approximately 20% of NSCLCs (1). While it is known that protein levels of oncogenic RAS can influence its oncogenic ability (18), expression of oncogenic KRASV12 can also lead to premature senescence of normal human epithelial cells (19). The prevalence of KRAS alterations in NSCLC indicates however that malignant transformation requires the cell to adapt to this oncogenic stress, perhaps assisted through preceding oncogenic transformations (20). Here, we present one of the first reports of full malignant transformation of lung epithelial cells with defined genetic manipulations. Furthermore, we characterize the effect of oncogenic stress and environmental effects such as growth factors upon tumorigenic transformation in HBECs, illustrate divergent clonal heterogeneity, and determine the capability of this in vitro model for developing and testing lung cancer therapeutics.

Cells and culture conditions

HBEC3 (HBEC3KT), HBEC4 (HBEC4KT), and HBEC17 (HBEC17KT) immortalized normal HBEC lines were established by introducing mouse Cdk4 and hTERT into normal HBECs (6). HBECs were cultured with keratinocyte serum-free medium (KSFM; Life Technologies Inc.) media containing 50 μg/mL of bovine pituitary extract (BPE; Life Technologies Inc.) and 5 ng/mL of EGF (Life Technologies Inc.). Partially transformed HBECs (soft agar clones) were also cultured with RPMI-1640 (Life Technologies Inc.) media supplemented with 10% FBS (R10). Lung cancer cell lines were established by our laboratory and maintained in RPMI-1640 (Life Technologies Inc.) with 5% FBS (21, 22). All cell lines were DNA-fingerprinted (PowerPlex 1.2 Kit; Promega) and Mycoplasma-free (e-Myco Kit; Boca Scientific).

Viral vector construction and viral transduction

Stable p53 knockdown and moderate expression of KRASV12 was achieved as described previously (12). Expression of high KRASV12 levels used a lentiviral vector, pLenti6-KRASV12, as described previously (23). Lentiviral vectors expressing KRASWT, KRASC12, and KRASD12 were constructed from pLenti6-KRASV12 as described previously (24). A c-MYC-expressing retroviral vector (designated pMSCV-MYC) was constructed by ligating a BamHI/SalI–digested c-MYC insert from pCTV3K (ref. 25; a gift from Dr. J. Michael Ruppert, University of Alabama, Tuscaloosa, AL) into BglII/XhoI–digested pMSCV-hyg (Clontech). Lentivirus and retrovirus-containing medium were produced as described previously (12). Transduced cells were selected with zeocin (12.5 μg/mL), blasticidin (2 μg/mL), or hygromycin (20 μg/mL) for 7 to 14 days. The presence of mutant KRASV12 in stable cell lines was confirmed using a reverse transcription PCR (RT-PCR)/RFLP assay, as described previously (12).

Immunoblotting and southern blotting

Preparation of total cell lysates and Western blotting were carried out as described previously (26). Primary antibodies are listed in Supplementary Table S1 and were detected with horseradish peroxidase (HRP)–conjugated anti-rabbit or anti-mouse secondary antibodies (1:2,000 dilution; Thermo Fisher Scientific). Actin or HSP90 protein levels were used as loading controls. Southern blotting for HBEC clones derived from large soft agar colonies was carried out as described previously (27) using Phototope-Star Detection Kit (New England Biolabs). DNA was probed with the blasticidin-resistance gene amplified from pLenti6-KRASV12 vector using 5′-ATGGCCAAGCCTTTGTCTCAAG-3′ and 5′-TTAGCCCTCCCACACATAACC-3′ primers.

Biochemical and in vitro transformation assays

Senescent cells were stained with Senescence β-Galactosidase Staining Kit (Cell Signaling) and blue-stained senescent cells were counted under a microscope (×20 total magnification). Percentage of positively stained cells was averaged across 6 fields. Cell-cycle analysis was conducted on subconfluent populations of cells harvested 48 hours after seeding, unless otherwise stated, and cell-cycle analysis was conducted as described previously (28). Cell proliferation assays were conducted by seeding 5,000 cells in 6-well plates and counting cells every 4 days. Cells were expanded when subconfluent, as necessary. Anchorage-dependent colony formation assays were conducted as previously described (12); 200 to 600 viable cells were seeded in triplicate in 100-mm plates and cultured for 2 weeks before staining colonies with methylene blue. Acute KRASV12 toxicity assays were conducted by transducing cells with KRASV12 or LacZ lentivirus and selecting for 3 days with blasticidin before seeding anchorage-dependent colony formation assays. Anchorage-independent (soft agar) growth assays were conducted as previously described (12) seeding 1,000 viable cells in 12-well plates. MTS assays to measure drug response to standard platinum-based doublets [paclitaxel–carboplatin (2/3 wt/wt), gemcitabine–cisplatin (25/2 wt/wt), and pemetrexed–cisplatin (20/3 wt/wt)] were conducted as previously described (29). Cells were treated for 96 hours with 4-fold dilutions from a maximum dose of 1,000/3,501 nmol/L (paclitaxel/carboplatin), 1,000/298 μmol/L (pemetrexed–cisplatin), or 2,000/140 nmol/L (gemcitabine/cisplatin). Each experiment was carried out in quadruplicate with 8 replicates per experiment. Fitting of data to dose–response inhibition curves, calculation of ED50 values, and comparisons based on one-way ANOVA with Dunnett posttest were conducted using GraphPad Prism version 5.00 for Windows (GraphPad Software).

In vivo tumorigenicity assays and histologic analysis

In vivo tumorigenicity was evaluated by injection of cells in female 5- to 6-week-old nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Each mouse was given a subcutaneous injection on its flank of 3 to 5 × 106 viable cells in 0.2 mL of PBS. Mice were monitored every 2 to 3 days for tumor formation for up to 6 months. All animal care was in accord with institutional guidelines and approved Institutional Animal Care and Use Committee (IACUC) protocols. Formalin-fixed, paraffin-embedded (FFPE) xenograft tumor tissue was sectioned and stained with hematoxylin and eosin (H&E), Alcian Blue–PAS, PAS, and mucicarmine for histologic analysis. Immunohistochemical staining for p63 (BioCare; clone BC4A4), Napsin A, CK5, and CK7 was carried out commercially by ProPath. Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) staining was conducted using DeadEnd Fluorometric TUNEL System (Promega).

Statistical testing and microarray analysis

For comparison of colony formation and senescent cells between different genetically manipulated cell strains, we used a two-tailed Student t test and P < 0.05 was considered significant. mRNA microarray profiling was conducted with Illumina HumanHT-12 v4 Expression BeadChips (Illumina Inc.) following the manufacturer's guidelines and analyzed with in-house Visual Basic software MATRIX V1.483. Functional analysis of differentially expressed genes was conducted using Ingenuity Pathway Analysis (IPA; Ingenuity Systems, Inc.). The data discussed in this publication have been made available in the National Center for Biotechnology Information's Gene Expression Omnibus (GEO) public repository (http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE40828. The predictive ability of the soft agar gene signature was tested using 2 independent mRNA microarray lung tumor datasets; 209 primary lung adenocarcinomas and squamous cell carcinomas (SCC; SPORE dataset) GSE41271, and 442 primary lung adenocarcinomas [National Cancer Institute (NCI) Director's Challenge Consortium dataset; ref. 30). A detailed description of these 2 datasets and the prediction analysis is given in the Supplementary Methods.

Oncogenic transformation of HBECs correlates with level of exogenous KRASV12 expression

NSCLC cell lines harboring a mutation in KRAS express wide-ranging endogenous levels of the protein (Fig. 1A). Thus, to create HBECs that express comparable levels of oncogenic KRAS protein, we used 2 different expression vectors: a retroviral vector, which resulted in modest levels of expression, and a lentiviral vector that resulted in high levels of expression (Fig. 1A). Compared with HBEC3 expressing moderate levels (retroviral-mediated) of KRASV12 protein, HBEC3 expressing high levels of KRASV12 (lentiviral-mediated) exhibited a significant increase in soft agar colonies in the background of both wild-type p53 (HBEC3) and p53 knockdown (HBEC3p53; Fig. 1B). To confirm the contribution of the level of KRASV12 expression toward HBEC transformation, we examined 7 clonal populations of HBEC3p53,KRAS (sh-p53 and lentiviral-delivered KRASV12; Fig. 1C). Clones with high levels of KRAS expression had increased anchorage-dependent (liquid) colony-forming ability (Fig. 1D and Supplementary Fig. S1A).

Figure 1.

High exogenous levels of KRASV12, comparable with endogenous levels found in mutant KRAS NSCLC cell lines, increases transformation of HBECs and induces senescence, which is largely bypassed with p53 knockdown. A, immunoblot for KRAS protein expression in HBEC3 cells infected with KRASV12 using either a moderately expressing retroviral (pBabe-hyg-KRASV12) or high-expressing lentiviral (pL6-KRASV12) vector. Actin was used as loading control. B, anchorage-independent (soft agar) colony formation in HBEC3 with high (lentiviral) or moderate (retroviral) levels of KRASV12 in the background of both wild-type p53 and p53 knockdown (sh-p53; t test). C, immunoblot of HBEC3p53,KRAS soft agar clones confirming p53 and KRASV12 manipulations. The presence (+) or absence (−) of each manipulation is shown. D, anchorage-dependent (liquid) colony formation ability of HBEC3p53,KRAS soft agar clones. E, quantification of SA-β-gal staining found KRASV12-induced senescence in HBEC3 cells was significantly lower in cells with p53 knockdown compared with p53 wild-type (t test). F, anchorage-dependent colony formation assay to compare acute KRASV12-induced toxicity in HBEC3 and HBEC4 with wild-type p53 or p53 knockdown. G, immunoblot of HBEC3 cell lysates harvested 7 days after infection with KRASV12 or LacZ lentivirus. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Full-length blots are presented in Supplementary Fig. S8.

Figure 1.

High exogenous levels of KRASV12, comparable with endogenous levels found in mutant KRAS NSCLC cell lines, increases transformation of HBECs and induces senescence, which is largely bypassed with p53 knockdown. A, immunoblot for KRAS protein expression in HBEC3 cells infected with KRASV12 using either a moderately expressing retroviral (pBabe-hyg-KRASV12) or high-expressing lentiviral (pL6-KRASV12) vector. Actin was used as loading control. B, anchorage-independent (soft agar) colony formation in HBEC3 with high (lentiviral) or moderate (retroviral) levels of KRASV12 in the background of both wild-type p53 and p53 knockdown (sh-p53; t test). C, immunoblot of HBEC3p53,KRAS soft agar clones confirming p53 and KRASV12 manipulations. The presence (+) or absence (−) of each manipulation is shown. D, anchorage-dependent (liquid) colony formation ability of HBEC3p53,KRAS soft agar clones. E, quantification of SA-β-gal staining found KRASV12-induced senescence in HBEC3 cells was significantly lower in cells with p53 knockdown compared with p53 wild-type (t test). F, anchorage-dependent colony formation assay to compare acute KRASV12-induced toxicity in HBEC3 and HBEC4 with wild-type p53 or p53 knockdown. G, immunoblot of HBEC3 cell lysates harvested 7 days after infection with KRASV12 or LacZ lentivirus. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Full-length blots are presented in Supplementary Fig. S8.

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High levels of mutant KRASV12 induce senescence in HBECs, in a p53-dependent manner

Lentiviral-mediated introduction of high levels of KRASV12 caused a large subset of HBEC3 cells to display significant morphologic changes, including flattened, enlarged shape, and a vacuole-rich cytoplasm, suggestive of oncogene-induced senescence. This effect was not observed following exogenous expression of moderate levels of KRASV12 (Supplementary Fig. S1B). Senescence-associated β-galactosidase (SA-β-gal) staining confirmed the morphologic changes corresponded with senescence (Supplementary Fig. S1C). Significantly less RAS-induced senescence was observed in HBEC3 with stable p53 knockdown (HBEC3p53) compared with HBEC3 with wild-type p53 (Fig. 1E and Supplementary Fig. S1C). An acute, senescence-associated p53-mediated KRASV12 toxicity was shown in anchorage-dependent colony formation assays seeded 4 days after transduction with KRASV12 lentivirus. Significant toxicity was observed in HBECs with wild-type p53 (HBEC3 and another HBEC line, HBEC4) following transduction with KRASV12 lentivirus compared with control LacZ lentivirus but not in HBECs with p53 knockdown (Fig. 1F and Supplementary Fig. S1D and S1E), suggesting induced senescence was p53-dependent. p53-mediated mitigation of oncogenic KRAS stress was also observed with other codon 12 KRAS mutants (KRASC12 and D12; Supplementary Fig. S1F and S1G), whereas overexpression of wild-type KRAS in HBEC3 did not induce a senescence phenotype (Supplementary Fig. S1G). Immunoblotting for cleaved caspase-3 confirmed KRASV12-induced toxicity was not apoptosis-associated (Fig. 1G), whereas cell-cycle analysis found no increased accumulation of cells in G1-phase in HBEC3p53 cells infected with KRASV12 as compared with the control vector (Supplementary Fig. S1H).

These results show that oncogenic KRAS mediates a potent cellular stress response in CDK4/hTERT–immortalized HBECs, whereby the cells resist oncogenic transformation by engaging cellular senescence. Loss of p53 function however impedes this cellular senescence response, indicating that in these cells, p53 signaling is a primary mediator of RAS-induced senescence. Furthermore, as high levels of oncogenic KRAS expression are required for malignant transformation, it shows that loss of p53 function is a critical co-oncogenic step in the malignant transformation of the large majority of HBECs.

The combination of p53 knockdown and KRASV12 in HBECs significantly increases in vitro transformation, which is further augmented with c-MYC overexpression

The single introduction of p53 knockdown, mutant KRASV12 or c-MYC overexpression resulted in quantitatively modest but significant increases in soft agar colony number (Fig. 2A–C). The combination of p53 knockdown and KRASV12 (HBEC3p53,KRAS) resulted in a significant increase in transformation not observed in other dual combinations [p53 knockdown+c-MYC (HBEC3p53,MYC) or KRASV12+c-MYC (HBEC3KRAS,MYC)], whereas introduction of all 3 manipulations (HBEC3p53,KRAS,MYC) resulted in the most transformed phenotype (Fig. 2B). Higher expression of c-MYC was achieved if overexpressed in HBEC3p53 (p53 knockdown), HBEC3KRAS (KRASV12), or HBEC3p53,KRAS (p53 knockdown and KRASV12) cells (Fig. 2A), suggesting partial transformation of immortalized HBEC3 with either p53 knockdown or addition of mutant KRASV12 is required for the cells to tolerate high levels of c-MYC.

Figure 2.

Stepwise in vitro transformation of HBEC3 with sh-p53, KRASV12, and c-MYC. A, immunoblot of isogenic derivatives of HBEC3 with sh-p53 or overexpression of KRASV12 or c-MYC, alone or in combination. The presence (+) or absence (−) of each manipulation is shown. B, transformation as defined by anchorage-independent growth in soft agar assays for HBEC3 with each manipulation alone or in combination. C, representative photographs of soft agar assays showing the formation of large, macroscopic (>1 mm) colonies in HBEC3p53,KRAS and HBEC3p53,KRAS,MYC (×4 magnification). Full-length blots are presented in Supplementary Fig. S8.

Figure 2.

Stepwise in vitro transformation of HBEC3 with sh-p53, KRASV12, and c-MYC. A, immunoblot of isogenic derivatives of HBEC3 with sh-p53 or overexpression of KRASV12 or c-MYC, alone or in combination. The presence (+) or absence (−) of each manipulation is shown. B, transformation as defined by anchorage-independent growth in soft agar assays for HBEC3 with each manipulation alone or in combination. C, representative photographs of soft agar assays showing the formation of large, macroscopic (>1 mm) colonies in HBEC3p53,KRAS and HBEC3p53,KRAS,MYC (×4 magnification). Full-length blots are presented in Supplementary Fig. S8.

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Combination of sh-p53+KRASV12 or sh-p53+KRASV12+c-MYC oncogenically transforms HBEC3 in vivo

To test the tumor-forming ability of HBEC3p53,KRAS and HBEC3p53,KRAS,MYC, cells grown in defined KSFM media were injected subcutaneously into NOD/SCID mice. We had previously found HBEC3p53 with moderate levels of oncogenic KRASV12 (using a retroviral expression vector) failed to form tumors when injected into immunodeficient mice (12). In contrast, transforming with higher levels of KRASV12 resulted in tumor growth in 5 of 24 (21%) injections (Table 1). Despite the significant increase in in vitro anchorage-independent growth observed in HBEC3p53,KRAS,MYC compared with HBEC3p53,KRAS (Fig. 2B), HBEC3p53,KRAS,MYC was only slightly more tumorigenic in vivo with tumors in 3 of 10 (30%) injections (Table 1). Histopathologic analysis showed that the oncogenically manipulated HBEC populations formed SCCs, adenocarcinomas, adenosquamous carcinomas, and poorly differentiated carcinomas with typical morphologic features of each histology (Table 1 and Fig. 3). Squamous and adenocarcinoma differentiation was confirmed by p63 and mucicarmine/Alcian Blue–PAS positivity, respectively, and pathology was verified by an independent lung cancer pathology expert. Adenocarcinomas were found to be strongly cytokeratin 5–positive, whereas negative for Napsin A and the squamous cell marker, cytokeratin 7 (Supplementary Fig. S2A). In all cases, adenocarcinoma differentiation was TITF1(NKX2-1)–negative (data not shown), most likely a reflection of the HBECs being derived from central airway cells. The fact that glandular cells are negative for both TITF-1 and Napsin A is not unexpected as the vast majority of adenocarcinomas (and glandular component in adenosquamous carcinomas) in whole tissue sections are either positive for both or negative for both. The development of different tumor histologies from the same HBEC-manipulated population suggests either clonal selection or the cells respond to differentiation signals in vivo.

Figure 3.

Representative FFPE sections of subcutaneous xenografts derived from HBEC3p53,KRAS and HBEC3p53,KRAS,MYC. HBEC3p53,KRAS and HBEC3p53,KRAS,MYC formed subcutaneous tumor reflective of naturally arising lung carcinomas with adenosquamous differentiation (top), adenocarcinoma (middle left), and squamous differentiation (bottom left), as well as undifferentiated large cell carcinomas, some of which also exhibited a giant cell component (middle and bottom on right). Squamous and adenocarcinoma differentiation was confirmed with p63 and mucicarmine and/or Alcian Blue PAS staining, respectively. The example of adenosquamous cell carcinoma (top) clearly shows dual differentiation of peripheral squamous/basal-like cells (p63+/mucin−) and central glandular cells (p63−/mucin+). Muc, mucicarmine; AB-PAS, Alcian Blue PAS. Original magnification of images at ×10 except adenosquamous H&E and P63 (×20); large cell carcinoma with giant cell component H&E (×20); and large cell carcinoma H&E (×40).

Figure 3.

Representative FFPE sections of subcutaneous xenografts derived from HBEC3p53,KRAS and HBEC3p53,KRAS,MYC. HBEC3p53,KRAS and HBEC3p53,KRAS,MYC formed subcutaneous tumor reflective of naturally arising lung carcinomas with adenosquamous differentiation (top), adenocarcinoma (middle left), and squamous differentiation (bottom left), as well as undifferentiated large cell carcinomas, some of which also exhibited a giant cell component (middle and bottom on right). Squamous and adenocarcinoma differentiation was confirmed with p63 and mucicarmine and/or Alcian Blue PAS staining, respectively. The example of adenosquamous cell carcinoma (top) clearly shows dual differentiation of peripheral squamous/basal-like cells (p63+/mucin−) and central glandular cells (p63−/mucin+). Muc, mucicarmine; AB-PAS, Alcian Blue PAS. Original magnification of images at ×10 except adenosquamous H&E and P63 (×20); large cell carcinoma with giant cell component H&E (×20); and large cell carcinoma H&E (×40).

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Table 1.

In vivo tumorigenicity of manipulated HBECs

Cell lineMediumaTumor formation ratebLatency, dcHistology
HBEC3 p53,KRAS 
 Population KSFM 5/24 (21%) 152 SCC (2) Adenosquamous (2)Poorly differentiated carcinoma (1) 
 Population R10 7/17 (41%) 128 Large cell/giant cell carcinoma (3)Adenocarcinoma (2)SCC (1)Adenosquamous (1) 
 Clone 1 R10 8/8 (100%) 38 Large cell/giant cell carcinoma (7)SCC (1) 
 Clone 5 R10 7/8 (88%) 57 Large cell/giant cell carcinoma (5)Papillary adenocarcinoma (1)Poorly differentiated carcinoma (1) 
 Clone 11 R10 6/8 (75%) 117 Large cell/giant cell carcinoma (3)Poorly differentiated carcinoma (2)Adenocarcinoma (1) 
 Clone 6 R10 0/6 (0%) — — 
 Clone 7 R10 0/7 (0%) — — 
 Clone 8 R10 0/8 (0%) — — 
 Clone 9 R10 0/9 (0%) — — 
HBEC3 p53,KRAS,MYC 
 Population KSFM 3/10 (30%) 115 Adenosquamous (2)SCC (1) 
 Population R10 12/12 (100%) 27 Large cell/giant cell carcinoma (5)Poorly differentiated carcinoma (4)SCC (2) 
 Clone 1 R10 9/9 (100%) 119 Large cell/giant cell carcinoma (7)Adenocarcinoma (2) 
 Clone 2 R10 3/8 (38%) 90 SCC (2) Large cell/giant cell carcinoma (1) 
 Clone 7 R10 10/10 (100%) 37 Large cell/giant cell carcinoma (7)SCC (3) 
 Clone 8 R10 1/6 (17%) 78 Large cell/giant cell carcinoma (1) 
HBEC17 p53,KRAS 
 Population KSFM 0/9 (0%) — — 
 Population R10 0/8 (0%) — — 
HBEC17 p53,KRAS,MYC 
 Population KSFM 0/10 (0%) — — 
 Population R10 3/8 (38%) n.d. SCC (2)Sarcomatoid carcinoma features (1) 
Cell lineMediumaTumor formation ratebLatency, dcHistology
HBEC3 p53,KRAS 
 Population KSFM 5/24 (21%) 152 SCC (2) Adenosquamous (2)Poorly differentiated carcinoma (1) 
 Population R10 7/17 (41%) 128 Large cell/giant cell carcinoma (3)Adenocarcinoma (2)SCC (1)Adenosquamous (1) 
 Clone 1 R10 8/8 (100%) 38 Large cell/giant cell carcinoma (7)SCC (1) 
 Clone 5 R10 7/8 (88%) 57 Large cell/giant cell carcinoma (5)Papillary adenocarcinoma (1)Poorly differentiated carcinoma (1) 
 Clone 11 R10 6/8 (75%) 117 Large cell/giant cell carcinoma (3)Poorly differentiated carcinoma (2)Adenocarcinoma (1) 
 Clone 6 R10 0/6 (0%) — — 
 Clone 7 R10 0/7 (0%) — — 
 Clone 8 R10 0/8 (0%) — — 
 Clone 9 R10 0/9 (0%) — — 
HBEC3 p53,KRAS,MYC 
 Population KSFM 3/10 (30%) 115 Adenosquamous (2)SCC (1) 
 Population R10 12/12 (100%) 27 Large cell/giant cell carcinoma (5)Poorly differentiated carcinoma (4)SCC (2) 
 Clone 1 R10 9/9 (100%) 119 Large cell/giant cell carcinoma (7)Adenocarcinoma (2) 
 Clone 2 R10 3/8 (38%) 90 SCC (2) Large cell/giant cell carcinoma (1) 
 Clone 7 R10 10/10 (100%) 37 Large cell/giant cell carcinoma (7)SCC (3) 
 Clone 8 R10 1/6 (17%) 78 Large cell/giant cell carcinoma (1) 
HBEC17 p53,KRAS 
 Population KSFM 0/9 (0%) — — 
 Population R10 0/8 (0%) — — 
HBEC17 p53,KRAS,MYC 
 Population KSFM 0/10 (0%) — — 
 Population R10 3/8 (38%) n.d. SCC (2)Sarcomatoid carcinoma features (1) 

aR10, RPMI-1640 supplemented with 10% FBS.

bNumber of subcutaneous tumors/number of injections (%).

cMedian time (days) for subcutaneous xenografts to reach 250 mm3; n.d., not determined.

Exploration of interindividual differences in HBEC transformation

To compare interindividual differences in malignant transformation using the same combination of oncogenic changes, we tested HBEC17, derived from a different individual than HBEC3. We observed a similar transformed phenotype in vitro (Supplementary Fig. S2B) however, in contrast to HBEC3, HBEC17 was significantly more resistant to full in vivo transformation (Table 1). The difference in tumor formation rate between HBECs derived from different donors suggests the existence of interindividual differences in susceptibility to specific oncogene-induced malignant transformation.

Growth in serum-containing medium enhances full tumorigenic transformation of HBECs

In addition to viral oncoproteins, many previous studies that succeeded in transforming normal cells to malignancy also used serum-containing medium instead of defined serum-free medium (31–33). We have previously shown that CDK4/hTERT–immortalized HBECs with no additional oncogenic manipulations require EGF, a supplement present in KSFM medium, for cell growth, but oncogenic transformation with p53 knockdown and KRASV12 allows the cells to become EGF-independent (12). In the present study, we show while HBEC3 cannot tolerate growth in RPMI-1640 supplemented with serum (FBS), media commonly used for growth of cancer cell lines, HBEC3p53,KRAS, is adaptable and will proliferate (Supplementary Fig. S3A). Serum-supplemented media has been shown to induce differentiation of epithelial cells in culture (34). Therefore, to further delineate the differences between HBEC3 compared with HBEC3p53,KRAS and HBEC3p53,KRAS,MYC following growth in serum-supplemented media, cells grown in either serum-free KSFM or serum-supplemented (5% FBS) KSFM for 96 hours were then analyzed for expression of a panel of lung differentiation and cancer stem cell (CSC) markers by quantitative RT-PCR (qRT-PCR). All 3 cell lines expressed high levels of basal markers (KRT5 and KRT14), low levels of central airway epithelial cell markers (MUC1 and TUBB4), with undetectable expression of peripheral airway markers (CC10 and SPC) in line with cells derived from bronchial epithelial cells (Supplementary Fig. S3B). Aldehyde dehydrogenase (ALDH) activity has been shown to be a marker of CSCs in the lung (28). We therefore measured the expression of 2 ALDH isozymes, ALDH1A1 and ALDH1A3, to find all the 3 cell lines that expressed ALDH1A3 with much lower expression of ALDH1A1. Growth in serum-containing medium resulted in all 3 cell lines exhibiting significant decrease in the expression of basal markers (KRT5 and KRT14), most notably in HBEC3 (Supplementary Fig. S3C). Expression of central airway markers, particularly MUC1, increased more than 10-fold in HBEC3 when cells were grown in serum-supplemented medium, whereas HBEC3p53,KRAS and HBEC3p53,KRAS,MYC showed little if any increase in these markers. Expression of CSC markers ALDH1A1 and ALDH1A3 increased in HBEC3 and HBEC3p53,KRAS but not in HBEC3p53,KRAS,MYC. Morphologically, growth in serum-supplemented medium resulted in HBEC3 cells developing a flattened morphology representative of a differentiated state (Supplementary Fig. S3D), whereas this was largely absent in HBEC3p53,KRAS cells and completely absent in HBEC3p53,KRAS,MYC. Furthermore, HBEC3p53,KRAS,MYC and to a lesser extent HBEC3p53,KRAS developed an elongated mesenchymal morphology after short-term growth in serum (Supplementary Fig. S3D). Together, these data show that serum-supplemented medium induces differentiation in parental HBEC3 cells; however, oncogenic transformation enables the cells to resist serum-induced differentiation and instead undergo epithelial-to-mesenchymal transition (EMT).

We reasoned that partially transformed HBECs that have adapted to growth in serum-supplemented RPMI-1640 may differ in tumorigenicity compared with cells grown in KSFM. Therefore, to compare the effect of genetic and environmental manipulations, HBEC3p53,KRAS and HBEC3p53,KRAS,MYC were grown in either defined KSFM (serum-free) medium or RPMI medium supplemented with 10% FBS (R10) for at least 3 weeks, then injected subcutaneously in NOD/SCID mice (Table 1). Growth in serum-containing medium markedly increased in vivo tumorigenicity, decreased tumor latency, and tumors in general were more undifferentiated (e.g., large cell and giant cell carcinomas), particularly in HBEC3p53,KRAS,MYC (Table 1 and Fig. 3). This effect is similar to what is observed in patients where poorly differentiated lung tumors are generally associated with aggressive tumor growth. To determine if the rate of apoptosis differed between poor and well-differentiated xenograft tumors, FFPE sections were analyzed with a TUNEL assay. There was no significant difference in the rate of apoptosis in relation to differentiation although a moderate well-differentiated SCC and giant cell carcinoma showed the greatest amount of TUNEL staining (Supplementary Fig. S3E). Overall, the increase in tumorigenicity of oncogenically progressed HBECs after growth in serum shows the influential role of exogenous serum-derived factors in the malignant progression of lung cancer.

Malignant transformation of HBECs is enhanced by epithelial-to-mesenchymal transition, induced by either c-MYC or growth in serum

In HBEC3p53,KRAS cells, overexpression of c-MYC (in defined KSFM medium) or growth in serum-containing RPMI-1640 medium led to increased oncogenic transformation, as shown by soft agar colony formation and in vivo tumor growth. Both of these manipulations also led to HBEC3p53,KRAS cells exhibiting a more mesenchymal-like morphology (Fig. 4A). Loss of E-cadherin (epithelial marker) and gain of vimentin and N-cadherin (mesenchymal markers) confirmed an EMT (Fig. 4B). c-MYC- or serum-induced EMT was also seen in HBEC17 cells, derived from another individual, with p53 and KRASV12 manipulations (HBEC17p53,KRAS; Fig. 4B). Whole-genome mRNA profiling of HBEC3p53,KRAS and HBEC3p53,KRAS,MYC cells grown in KSFM or R10 confirmed a significant over-representation of EMT-related genes (P = 1.04 × 10−16 and P = 1.38 × 10−09 for HBEC3p53,KRAS and HBEC3p53,KRAS,MYC, respectively; Fig. 4C), with upregulation of EMT-promoting genes following growth in serum-containing media.

Figure 4.

c-MYC overexpression or growth in serum-containing media induces EMT in HBEC3p53,KRAS. A, phase contrast photomicrographs showing the morphologic effect observed in HBEC3p53,KRAS (left) following overexpression of c-MYC (middle) or switching from defined serum-free media to serum-containing media (right; ×20 magnification). B, immunoblot for EMT markers in oncogenically manipulated HBEC3 and HBEC17 grown in either KSFM (serum-free) or serum-containing (R10) media. The presence (+) or absence (−) of serum is shown. C, EMT-related genes altered 4-fold or greater in pairwise analysis of HBEC3p53,KRAS and HBEC3p53,KRAS,cMYC comparing cells grown in serum or defined medium (KSFM). Values log2 transformed. *, P < 0.05; **, P < 0.01 (t test). Full-length blots are presented in Supplementary Fig. S8.

Figure 4.

c-MYC overexpression or growth in serum-containing media induces EMT in HBEC3p53,KRAS. A, phase contrast photomicrographs showing the morphologic effect observed in HBEC3p53,KRAS (left) following overexpression of c-MYC (middle) or switching from defined serum-free media to serum-containing media (right; ×20 magnification). B, immunoblot for EMT markers in oncogenically manipulated HBEC3 and HBEC17 grown in either KSFM (serum-free) or serum-containing (R10) media. The presence (+) or absence (−) of serum is shown. C, EMT-related genes altered 4-fold or greater in pairwise analysis of HBEC3p53,KRAS and HBEC3p53,KRAS,cMYC comparing cells grown in serum or defined medium (KSFM). Values log2 transformed. *, P < 0.05; **, P < 0.01 (t test). Full-length blots are presented in Supplementary Fig. S8.

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Isogenic soft agar clones of oncogenically manipulated HBEC3 represent independent genetic events with distinct in vivo growth, tumor histology, and differentiation

The genetic combinations of shp53+KRASV12 or shp53+KRASV12+c-MYC in HBEC3 led to the formation of a small subset (0.5%–1.5% of all soft agar colonies) of very large, macroscopically visible (>1 mm) colonies when grown in soft agar, which was not observed with any single manipulation (shp53, KRASV12, or c-MYC) nor dual combination of sh-p53 or KRASV12 with c-MYC (Fig. 2C). Seven HBEC3p53,KRAS clones and 4 HBEC3p53,KRAS,MYC clones were isolated from these large colonies and repeat soft agar assays confirmed that the large colony phenotype was maintained (Fig. 5A). Southern blotting showed the clones represent independent transformation events as indicated by discrete digestion patterns (Supplementary Fig. S4A) and immunoblotting confirmed HBEC3p53,KRAS and HBEC3p53,KRAS,MYC clones maintained their exogenously introduced oncogenic manipulations (Figs. 1C and 5B, respectively; summarized in Supplementary Table S2). While immortalized but nontransformed HBECs preferentially grow in serum-free conditions, they require serum for anchorage-independent growth (Supplementary Fig. S4B). Remarkably, following isolation from soft agar (in KSFM+20% FBS) 9 of 11 clones were serum growth factor–dependent as they could no longer tolerate growth in serum-free KSFM medium (Supplementary Table S2). Henceforth, all clones were grown in RPMI-1640 medium containing 10% FBS (R10).

Figure 5.

Isolation of large soft agar clones from HBEC3p53,KRAS and HBEC3p53,KRAS,MYC identifies tumorigenic and nontumorigenic clones and genome-wide mRNA expression profiling of HBEC3p53,KRAS soft agar clones identifies a clinically applicable signature of prognosis. A, uncloned, parental populations of HBEC3p53,KRAS and HBEC3p53,KRAS,MYC form very large (>1-mm diameter) soft agar colonies (arrowhead). These large colonies were isolated, expanded, and retested for soft agar colony formation where they maintained the ability to form large soft agar colonies (representative soft agar pictures of 2 clones; ×4 magnification). B, immunoblot of HBEC3p53,KRAS,MYC soft agar clones confirming p53, KRASV12, and c-MYC manipulations. The presence (+) or absence (−) of each manipulation is shown. C, anchorage-independent soft agar colony formation ability of HBEC3p53,KRAS soft agar clones. D, unsupervised hierarchical clustering of whole-genome mRNA expression profiles of HBEC3p53,KRAS soft agar clones harvested at 2 time points [denoted “(1)” and “(2)”] spanning a 3-week interval. E, a supervised analysis comparing HBEC3p53,KRAS tumorigenic (clone 1, 5, and 11) with HBEC3p53,KRAS nontumorigenic (clone 6, 7, 8, and 9) clones identified 203 probes, representing 171 unique genes, significantly differentially expressed (SAM, FDR = 5%). Samples (represented horizontally: red, tumorigenic clones; green, nontumorigenic clones) and probes (represented vertically) were clustered using centered Pearson clustering. F, Kaplan–Meier log-rank analysis of overall survival in patients with lung cancer predicted to have good (black) or poor (red) outcome using the 171 probe signature HBEC3p53,KRAS soft agar signature. A supervised principal component analysis was used to train the model in one dataset (Consortium) and test in a second dataset (SPORE; top) then the datasets were reversed to test for model robustness (bottom). Full-length blots are presented in Supplementary Fig. S8.

Figure 5.

Isolation of large soft agar clones from HBEC3p53,KRAS and HBEC3p53,KRAS,MYC identifies tumorigenic and nontumorigenic clones and genome-wide mRNA expression profiling of HBEC3p53,KRAS soft agar clones identifies a clinically applicable signature of prognosis. A, uncloned, parental populations of HBEC3p53,KRAS and HBEC3p53,KRAS,MYC form very large (>1-mm diameter) soft agar colonies (arrowhead). These large colonies were isolated, expanded, and retested for soft agar colony formation where they maintained the ability to form large soft agar colonies (representative soft agar pictures of 2 clones; ×4 magnification). B, immunoblot of HBEC3p53,KRAS,MYC soft agar clones confirming p53, KRASV12, and c-MYC manipulations. The presence (+) or absence (−) of each manipulation is shown. C, anchorage-independent soft agar colony formation ability of HBEC3p53,KRAS soft agar clones. D, unsupervised hierarchical clustering of whole-genome mRNA expression profiles of HBEC3p53,KRAS soft agar clones harvested at 2 time points [denoted “(1)” and “(2)”] spanning a 3-week interval. E, a supervised analysis comparing HBEC3p53,KRAS tumorigenic (clone 1, 5, and 11) with HBEC3p53,KRAS nontumorigenic (clone 6, 7, 8, and 9) clones identified 203 probes, representing 171 unique genes, significantly differentially expressed (SAM, FDR = 5%). Samples (represented horizontally: red, tumorigenic clones; green, nontumorigenic clones) and probes (represented vertically) were clustered using centered Pearson clustering. F, Kaplan–Meier log-rank analysis of overall survival in patients with lung cancer predicted to have good (black) or poor (red) outcome using the 171 probe signature HBEC3p53,KRAS soft agar signature. A supervised principal component analysis was used to train the model in one dataset (Consortium) and test in a second dataset (SPORE; top) then the datasets were reversed to test for model robustness (bottom). Full-length blots are presented in Supplementary Fig. S8.

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Injection of the large, soft agar clones into NOD/SCID mice found all 4 HBEC3p53,KRAS,MYC clones were tumorigenic, whereas only 3 of 7 HBEC3p53,KRAS clones formed tumors (Table 1). Thus, even with 4 oncogenic changes and biologic selection there is dramatic clonal heterogeneity. This also showed that the tumorigenicity of the clones reflected the tumorigenicity of the parental population (when grown in R10) where approximately 40% of mice injected with HBEC3p53,KRAS grew tumors compared with 100% of mice injected with HBEC3p53,KRAS,MYC (Table 1). Growth rate and tumor histology differed between tumorigenic clones. While subcutaneous tumors were largely undifferentiated (e.g., large cell and giant cell carcinomas), some xenograft tumors exhibited squamous and adenocarcinoma differentiation (Table 1). This differentiation was clone specific, as no clone differentiated into multiple histologies.

Oncogenic transformation of HBECs increases sensitivity to standard lung cancer chemotherapies

To determine if oncogenic transformation of HBECs altered their sensitivity to standard lung cancer chemotherapies, we tested 3 platinum-based doublets (paclitaxel–carboplatin, gemcitabine–cisplatin, and pemetrexed–cisplatin) currently in use for NSCLC treatment. Oncogenic manipulation of HBEC3 with sh-p53 and KRASV12 resulted in a significant increase in sensitivity to gemcitabine–cisplatin and pemetrexed–cisplatin doublet therapy in vitro (approximately 6- and 10-fold, respectively) but not paclitaxel–carboplatin (Supplementary Table S3 and Supplementary Fig. S5; one-way ANOVA; P < 0.001). Overall, the soft agar clones of HBEC3p53,KRAS showed sensitivities comparable with the parental HBEC3p53,KRAS cell line with clone 5 and 7 showing intermediate sensitivity. Thus, these tumorigenically progressed HBECs could provide a cell context appropriate, isogenic model system for identifying genetic differences regulating sensitivity and resistance to platinum doublet chemotherapy used in the treatment of NSCLCs.

mRNA profiles of tumorigenic versus nontumorigenic HBEC3p53,KRAS soft agar clones predict prognosis and histology in clinical lung tumor specimens

The identification of tumorigenic and nontumorigenic clones of HBEC3p53,KRAS that share the same genetic manipulations (sh-p53 and KRASV12) and biologic selection (large, soft agar colonies) presents a unique cell model to characterize spontaneous transformation events that progress HBECs to full malignancy. Biochemical assays suggest the expression level of KRASV12 is a major contributor toward full transformation (Figs. 1C and D and 5C), whereas other tumorigenic events such as dysregulation of cell cycle did not differ between clones (Supplementary Fig. S6; summarized in Supplementary Table S2). To analyze molecular differences between tumorigenic versus nontumorigenic clones, the clones were profiled with whole-genome mRNA expression microarrays. The mRNA expression profile of each clone remained stable in culture as shown by comparing mRNAs collected at a 3-week interval in unsupervised clustering (Fig. 5D). Tumorigenic and nontumorigenic clones separated into 2 distinct clusters suggesting the clones exhibit a strong expression profile associated with their tumorigenic phenotype, and supervised analysis comparing tumorigenic with nontumorigenic clones found 171 unique genes (203 probes) that were differentially expressed [Significance Analysis of Microarrays (SAM), False Discovery Rate (FDR) = 5%; Supplementary Table S4; Fig. 5E].

We tested the ability of the expression patterns of the 171 genes to predict overall survival and disease-free survival in 2 independent lung tumor cohorts; the SPORE dataset of resected early-stage NSCLCs (adeno- and squamous carcinomas; n = 209) and a second dataset of primary lung adenocarcinoma samples (n = 442; NCI Director's Challenge Consortium dataset). Prediction models were built using supervised principal component analysis and datasets were interchanged as training and testing datasets to test for robustness. Irrespective of dataset, the soft agar clone tumorigenic versus nontumorigenic signature was able to identify patients with significantly worse overall (Fig. 5F) and recurrence-free (Supplementary Fig. S7) survival. The successful application of a gene signature derived from an isogenic in vitro model of HBEC transformation in predicting outcome in clinical lung tumor samples indicates the power of such a system and provides a preclinical model for testing the functional importance of the genes in the signature.

In this study, we sought to characterize the stepwise progression of lung cancer pathogenesis by introducing defined genetic manipulations commonly found in lung cancer into an in vitro HBEC model system (Fig. 6). We found the expression level of mutant KRAS is a critical transformative factor in HBECs, however, inactivation of p53 signaling is required to evade the tumor-suppressive barrier of RAS-induced senescence. We also show that EMT is an important oncogenic step, where c-MYC or growth in serum-containing medium both spontaneously induced an EMT and led to increased tumorigenicity. In HBECs derived from multiple individuals, we show 5 genetic changes (hTERT and Cdk4 to immortalize the cells, followed by p53 knockdown, mutant KRASV12, and c-MYC overexpression) together with serum-induced EMT are able to transform cells to a fully malignant state. Tumor xenografts of transformed HBECs were typical of lung cancer but varied in histology, suggesting histologically distinct lung cancers from the central bronchus may originate from a multipotent stem-like cell. Interestingly, clonal analysis of sh-p53+KRASV12–transformed HBECs found the isogenic cells exhibited distinct phenotypes in terms of in vivo tumorigenicity, xenograft histology, and drug response. The mRNA profile that distinguished tumorigenic from nontumorigenic clones was also able to identify a subset of primary lung tumor patients with significantly poorer survival. Together, this shows the use of the HBEC system as a preclinical model of lung cancer to understand transformation events.

Figure 6.

Model of malignant transformation of in vitro HBECs following stepwise introduction of common lung cancer mutations. The experimental data presented in this article identify the following steps: step 1, CDK4 and hTERT immortalized, HBECs are nontransformed and lack of anchorage-independent growth in soft agar; step 2, in vitro transformation as defined by anchorage-independent growth in soft agar is achieved with the single manipulation of loss of p53, moderate KRASV12 expression or both, whreas expression of high levels of KRASV12 expression leads to in vitro transformation with significant cellular senescence; step 3, partial in vivo transformation with subcutaneous tumor growth in immunocompromised mice in 30% to 80% of injections is observed with the combination of p53 loss and high KRASV12; step 4, an EMT occurs following overexpression of cMYC or growth in serum-containing media; step 5, combination of cMYC overexpression and growth in serum-containing media results in complete oncogenic transformation of HBECs with tumor growth in vivo observed in more than 80% of injections in immunocompromised mice. Clonal selection of partially transformed HBECs identifies tumorigenic and nontumorigenic clones.

Figure 6.

Model of malignant transformation of in vitro HBECs following stepwise introduction of common lung cancer mutations. The experimental data presented in this article identify the following steps: step 1, CDK4 and hTERT immortalized, HBECs are nontransformed and lack of anchorage-independent growth in soft agar; step 2, in vitro transformation as defined by anchorage-independent growth in soft agar is achieved with the single manipulation of loss of p53, moderate KRASV12 expression or both, whreas expression of high levels of KRASV12 expression leads to in vitro transformation with significant cellular senescence; step 3, partial in vivo transformation with subcutaneous tumor growth in immunocompromised mice in 30% to 80% of injections is observed with the combination of p53 loss and high KRASV12; step 4, an EMT occurs following overexpression of cMYC or growth in serum-containing media; step 5, combination of cMYC overexpression and growth in serum-containing media results in complete oncogenic transformation of HBECs with tumor growth in vivo observed in more than 80% of injections in immunocompromised mice. Clonal selection of partially transformed HBECs identifies tumorigenic and nontumorigenic clones.

Close modal

Mutant KRAS lung cancers exhibit marked differences in the expression level of KRAS. We found the expression level of oncogenic KRASV12 was an important factor in tumorigenic transformation of HBECs, suggesting it exerts its oncogenic effect in a dose-dependent manner, similar to HRASV12 in a human mammary epithelial cell (HMEC) transformation model (18). While higher levels of oncogenic KRAS enhanced HBEC transformation, it also triggered oncogene-induced senescence. Oncogene-induced senescence serves as an important tumor-suppressive barrier in response to persistent oncogenic insult by engaging proliferative arrest through p53 or p16/Rb (35). We found that immortalized HBECs, where the p16/Rb pathway is bypassed as a result of overexpression of CDK4, could largely evade KRASV12-induced senescence with knockdown of p53. Loss of p53 function, therefore, is a key step in the malignant transformation of HBECs by allowing the cells to tolerate high levels of oncogenic KRAS, a driver of malignant transformation. Transformation studies in HMECs, which undergo spontaneous methylation-mediated p16 silencing, also report that high levels, but not low levels, of oncogenic RAS engage in senescence machinery (36, 37). However, in HMECs, HRAS-induced senescence is mediated through TGF-β signaling in a p53-independent manner. Thus, the mechanism of RAS-induced senescence differs between in vitro epithelial cell models, most likely a consequence of immortalization methods or the cell of origin.

Our data show microenvironmental signals such as those provided by growth media can strongly influence the transformation of HBECs. Switching immortalized, but nontransformed HBECs from defined, serum-free culture medium to serum-containing medium induced inhibition of cell growth with induction of central airway differentiation markers. When immortalized HBEC with additional oncogenic manipulations are switched to serum-containing medium, however, the cells are able to bypass serum-induced differentiation and instead become mesenchymal and more tumorigenic, with a greater frequency of undifferentiated tumors. Overexpression of c-MYC in HBEC3p53,KRAS also induced an EMT and increased tumorigenicity. c-MYC has been shown to induce EMT in TERT-immortalized HMECs (38). In our study, c-MYC induced EMT only in the presence of p53 and KRASV12 alterations and not with c-MYC overexpression alone (data not shown). While c-MYC overexpression or growth in serum-containing media both caused HBEC3p53,KRAS to undergo EMT and increase tumorigenicity, the presence of both manipulations had a synergistic effect resulting in full malignant transformation in HBECs from 2 individuals. This suggests their tumor-promoting effects signal through mutually exclusive pathways.

In the present study, we found interindividual differences in HBEC transformation suggesting HBECs derived from different donors can vary in their susceptibility to specific oncogene-induced malignant transformation. In terms of in vitro anchorage-independent transformation, neither cell line showed anchorage-independent growth following immortalization (CDK4 and hTERT), yet the combination of 5 genes (Cdk4, hTERT, sh-p53, KRASV12, and cMYC) resulted in a colony-forming efficiency of approximately 60% in HBEC3 compared with less than 15% in HBEC17. These differences could potentially stem from multiple factors including germline polymorphisms [such as single-nucleotide polymorphisms (SNP)], somatically acquired mutations derived from either the patient (such as age or environmental exposures) or laboratory practices (such as time in culture), or epigenetic mechanisms all of which may predispose the cells to oncogenic transformation. The patients from whom HBEC3 and HBEC17 were established, differ in respect to known risk factors for lung cancer such as age and smoking history, and it is likely they also differ in respect to unknown germline and/or somatic alterations. Thus, a comprehensive survey of genomic alterations present in HBECs before genetic manipulation (such as by whole-genome sequencing) would provide better indication of the level of existing premalignant susceptibilities, but these experiments are beyond the scope of the current study.

The lung can be divided into central and peripheral compartments (39). SCCs usually arise from the central compartment, whereas adenocarcinomas may arise from either compartment, illustrating the importance of studying oncogenic transformation in both central and peripheral lung epithelial cell models. Many HBEC-derived tumors in our study were p63-positive with squamous differentiation, which likely reflects a stem/basal cell origin. A fewer number of tumors showed distinct dual squamous and adenocarcinoma differentiation or adenocarcinoma differentiation alone. A study using SV40-immortalized tracheobronchial and small airway epithelial cells found both cell types could be fully transformed with oncogenic RAS (8). Another study using nonviral oncoproteins immortalized HSAECs with CDK4, hTERT, and a dominant-negative form of p53 (p53CT) and transformed the cells using low levels of KRASV12 plus c-MYC, PIK3CAH1047R, cyclin D1, or LKB1D194A (15). In HBECs, we have shown p53 mutation is not required for immortalization (6), and moreover, it increases oncogenic transformation (6, 12). This disparity may signify intrinsic differences between centrally and peripherally derived immortalized epithelial cell models. Taken together, however, our study of defined oncogenic transformation of HBECs [both in the present study and previously (12)] and the study in HSAECs by Sasai and colleagues (15) largely correspond with respect oncogenic transformation of bronchial epithelial cells. We previously showed transformation of HBECs with low levels of KRASV12 (with CDK4, hTERT, and p53 knockdown) resulted in a modest increase in anchorage-independent growth but no tumor formation in vivo (12). Sasai and colleagues reported that transformation of HSAECs with low levels of KRASV12 (with CDK4, hTERT, and p53CT) failed to yield anchorage-independent or in vivo growth. The authors were able to fully transform HSAECs using low levels of KRASV12 only with additional genetic alterations. In the present study, we used a different approach by increasing the level of KRASV12 expression to simulate the high amplification-associated expression often observed in lung cancer (40). We found that higher levels of the oncogene resulted in increased in vitro and in vivo transformation compared with lower levels of KRASV12, which could be further increased with a fifth genetic alteration, such as cMYC overexpression. Thus, studies in HBECs and HSAECs show that increasing the number of oncogenic alterations will increase cellular transformation, whereas in the present study, we also show that increasing the level of certain oncogenic alterations can also increase transformation.

In conclusion, using the HBEC system as a progression model of lung cancer, we were able to study early transformative events in bronchial epithelial cells and the mechanisms used to overcome tumor-suppressive barriers. We show HBECs can be transformed to full malignancy by introducing defined genetic manipulations to produce histologically similar lung tumors in xenografts, indicating our in vitro HBEC model retains characteristics of the tissue of origin. Furthermore, we show HBECs can model preneoplastic changes and spontaneous transformation events such as oncogene-induced senescence and EMT and have clinically translatable applications as shown in isogenic clones exhibiting distinct drug response and tumorigenic phenotypes. Thus, genetically defined in vitro models such as HBECs will be an invaluable tool to assess the contribution of specific genes toward lung cancer pathogenesis, pertinent to recent whole-genome sequencing efforts, and to screen for novel therapeutic compounds directed at oncogenically acquired, tumor-specific vulnerabilities.

J.D. Minna has ownership interest (including patents) in NCI and University of Texas Southwestern Medical Center. No potential conflicts of interest were disclosed by the other authors.

Conception and design: M. Sato, J.E. Larsen, A.F. Gazdar, J.W. Shay, J.D. Minna

Development of methodology: M. Sato, J.E. Larsen, R.D. Ramirez, A.F. Gazdar, J.W. Shay

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Sato, J.E. Larsen, W. Lee, M.P. Dalvi, J.M. DiMaio, I.I. Wistuba, A.F. Gazdar

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Sato, J.E. Larsen, H. Sun, D.S. Shames, M.P. Dalvi, H. Tang, B. Gao, Y. Xie, I.I. Wistuba, A.F. Gazdar, J.W. Shay, J.D. Minna

Writing, review, and/or revision of the manuscript: M. Sato, J.E. Larsen, D.S. Shames, R.D. Ramirez, B. Gao, Y. Xie, I.I. Wistuba, A.F. Gazdar, J.W. Shay, J.D. Minna

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J.E. Larsen, A.F. Gazdar

Study supervision: J.E. Larsen, J.W. Shay, J.D. Minna

The authors thank Natasha Rekhtman (Memorial Sloan-Kettering Cancer Center, New York, NY) for her kind assistance in analyzing immunohistochemical sections. The authors also thank the many current and past members of the Minna laboratory for their technical assistance and article comments, particularly Luc Girard, Suzie Hight, and Michael Peyton.

This work was supported by the NCI Lung Cancer Specialized Program of Research Excellence (SPORE; P50CA70907), NASA NSCOR (NNJ05HD36G). J.E. Larsen was supported by NHMRC Biomedical Fellowship (494511) and TSANZ/Allen & Hanburys Respiratory Research Fellowship.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data