SOX2 Is the Determining Oncogenic Switch in Promoting Lung Squamous Cell Carcinoma from Different Cells of Origin

Abstract

Lung squamous cell carcinoma (LSCC) is a devastating malignancy with no effective treatments, due to its complex genomic profile. Therefore, preclinical models mimicking its salient features are urgently needed. Here we describe mouse models bearing various combinations of genetic lesions predominantly found in human LSCC. We show that SOX2 but not FGFR1 overexpression in tracheobronchial basal cells combined with Cdkn2ab and Pten loss results in LSCC closely resembling the human counterpart. Interestingly, Sox2;Pten;Cdkn2ab mice develop LSCC with a more peripheral location when Club or Alveolar type 2 (AT2) cells are targeted. Our model highlights the essential role of SOX2 in commanding the squamous cell fate from different cells of origin and represents an invaluable tool for developing better intervention strategies.

Graphical abstract

Figure 1

In Vivo Characterization of Basal Cell-Specific Adenoviral Vectors Using mT/mG Reporter Mice GFP IHC staining of longitudinal sections of tracheas (A) and lungs (B) isolated from mT/mG reporter mice 3 weeks after intratracheal injection of Ad5-CMV-Cre, Ad5-K14-Cre, and Ad5-K5-Cre following pretreatment with corn oil or naphthalene as indicated. Scale bars, 100 μm (A) and 200 μm (B). Scale bars in insets, 50 μm (B). See also Figure S1.

Figure 2

Generation of Mouse Models that Recapitulate the Genomic Complexity of Human LSCC (A) Genetic alterations of CDKN2A, CDKN2B, PTEN, FGFR1, and SOX2 in the 178 human LSCC specimens in the TCGA dataset. Exon skipping and DNA methylation for the CDKN2A locus are excluded. (B) Schematic representation of the experimental design used for all cohorts of mice: PC, Fgfr1PC, and Sox2PC. Mice are treated with naphthalene on day 0 and intratracheally injected with Ad5-K14-Cre or Ad5-K5-Cre on day 3. (C) Representative H&E staining on various lung lesions of PC mice. Scale bars, 100 μm. See also Table S1.

Figure 3

Combined Overexpression of FGFR1 Partially Drives Squamous Differentiation within Heterogeneous Neoplastic Lesions (A) Schematic representation of the Col1a1 locus targeted with LSL-Fgfr1^K656E transgene before Cre infection. Fgfr1PC mice were infected with basal specific adenoviruses. (B and C) H&E staining of lung sections showing squamous differentiation with nest of cells with evident intercellular bridges (arrow in B) in circular arrangement (C). (D–G) IHC analysis of lung sections possessing tumors with squamous differentiation, for K5 (D), GFP (E), FGFR1 (F), and p63 (G). Scale bars, 10 μm (B) and 50 μm (C–G). See also Figure S2 and Table S1.

Figure 4

SOX2 Promotes the Switch from Heterogeneous Neoplastic Lesions to Typical LSCC (A) Schematic representation of the Col1a1 locus targeted with LSL-Sox2 transgene before Cre infection. Sox2PC mice were generated and infected with basal specific adenoviruses. (B and C) H&E staining on mouse lung sections collected 7–9 months after injection. The tumor showed prominent cornification (arrow in B), keratin pearls, and inflammatory infiltration (arrows in C). (D) H&E staining of well-differentiated human LSCC. (E) IHC staining on human and mouse lung sections with the indicated histopathological markers. (F) IHC staining for GFP, K14, and K5 on mouse lung sections. (G and H) Real-time RT-PCR of Sox2 endogenous (Sox2 end) and exogenous (SOX2 exo) levels performed on tumor tissues isolated from the indicated mouse tumor samples (G) and MEFs untreated (UNT) or treated with tamoxifen (TAM) (H). Data represent means ± SD. Samples are normalized by using actin RNA level. Scale bars, 200 μm (B) and 50 μm (C–F). See also Figures S2 and S3; Tables S1 and S2.

Figure 5

Gene Expression Profile Analysis (A) Venn diagram showing the commonly DE genes between mouse and human LSCC found by comparing DE genes between Sox2PC versus Kras;p53±Eed and human LSCC versus LADC. (B and C) GSEA for genes upregulated in Sox2PC (B) and hLSCC (C) compared with Kras;p53±Eed and hLADC, respectively. (D) Hierarchical clustering of upregulated genes in both mouse and human LSCC, which are enriched for a squamous differentiation signature. (E and F) Significant GO categories for molecular functions (E) and cellular components (F). (G) Real-time RT-PCR of the indicated genes performed on tumor tissues isolated from the indicated mouse tumor samples. Data represent means ± SD. Samples are normalized by using actin RNA level. See also Figure S4 and Table S3.

Figure 6

Tumor Microenvironment in the Three Cohorts of Mice and Comparative Analysis of Human and Mouse LSCC (A) IHC analysis performed on adult lung tissues of Sox2PC, PC, and Fgfr1PC mice, using the indicated antibodies against markers of inflammatory cells. (B) IHC analysis performed on human and mouse LSCC for the indicated markers of immune cells. Scale bars, 200 μm (A) and 50 μm (B). Scale bars in insets, 50 μm (A). See also Table S4.

Figure 7

Club and AT2 Cells Are Transformed to Give Rise to LSCC in Sox2PC Mice (A) Scan images of GFP staining performed on lungs isolated from Sox2PC mice injected with the indicated adenoviruses. (B) Quantification of the comparison of early lesions versus carcinoma in Ad5-SPC-Cre- and Ad5-CC10-Cre-injected mice (Ad-SPC-Cre, p < 0.0001; Ad5-CC10-Cre, p < 0.05). Data represent means ± SD. (C–F) Dual IF staining with the indicated markers, performed on lung tissues isolated from Sox2PC mice injected with the indicated adenoviruses, representative of single/small clusters of targeted cells (C), early lesions (D), intermediate lesions (E), and advanced LSCC (F). Scale bars, 20 μm (C, D) and 50 μm (E, F). See also Figure S5 and Table S5.

Figure 8

FFGR1 and SOX2 Are Both Tumor Drivers but Only SOX2 Imposes an LSCC Phenotype (A) Lung carcinoma-free survival curve of mice with the indicated genotypes. (B) Schematic representation of tumors arising in mice with different genetic lesions, activated in distinct lung cell types: LSCC arises from PC mice following overexpression of SOX2 but not FGFR1. Sox2PC mice develop LSCC also from AT2 and Club cells. (C) LSCC arises in different locations according to the targeted cell population. Scale bars, 50 μm. (D) Hierarchical clustering of transcriptional profiling of the indicated Basal-, AT2-, and Club-derived LSCC samples. One hundred genes with the largest variability across samples were used. Columns are for samples and rows are for genes.