Differential development of large-cell neuroendocrine or small-cell lung carcinoma upon inactivation of 4 tumor suppressor genes

Abstract

High-grade neuroendocrine lung malignancies (large-cell neuroendocrine cell carcinoma, LCNEC, and small-cell lung carcinoma, SCLC) are among the most deadly lung cancer conditions with no optimal clinical management. The biological relationships between SCLC and LCNEC are still largely unknown and a current matter of debate as growing molecular data reveal high heterogeneity with potential therapeutic consequences. Here we describe murine models of high-grade neuroendocrine lung carcinomas generated by the loss of 4 tumor suppressors. In an Rbl1-null background, deletion of Rb1, Pten, and Trp53 floxed alleles after Ad-CMVcre infection in a wide variety of lung epithelial cells produces LCNEC. Meanwhile, inactivation of these genes using Ad-K5cre in basal cells leads to the development of SCLC, thus differentially influencing the lung cancer type developed. So far, a defined model of LCNEC has not been reported. Molecular and transcriptomic analyses of both models revealed strong similarities to their human counterparts. In addition, a ⁶⁸Ga-DOTATOC-based molecular-imaging method provides a tool for detection and monitoring the progression of the cancer. These data offer insight into the biology of SCLC and LCNEC, providing a useful framework for development of compounds and preclinical investigations in accurate immunocompetent models.

Keywords: LCNEC; SCLC; cell of origin; tumor suppressor.

Fig. 1.

Genetic deletion of 4 tumor suppressors in mouse lung epithelial cells results in LCNEC development. Basal cell-type restricted genetic deletion renders SCLC. (A) Schematic of mouse models experiment. Rb1^F/F;Rbl1^−/−;Trp53^F/F;Pten^F/F (QKO) mice were infected with Ad5-CMVcre (CMV-QKO) or Ad5-K5cre (K5-QKO). (B) Kaplan-Meier percent tumor-free survival curve of Rb1^F/F;Rbl1^−/−;Trp53^F/F;Pten^F/F mice infected with Ad5-CMVcre virus (green line) or Ad5-K5Cre virus (orange line). x axis: latency (weeks after adenovirus injection). Total number of mice analyzed for CMV-QKO, n = 67; K5-QKO, n = 67. (C) The pie charts represent the histopathology spectrum of tumors that arose from CMV-QKO or K5-QKO mice. Total number of tumors for CMV-QKO, n = 886; K5-QKO, n = 537. (D) Hematoxylin–eosin (H&E) representative images and immunohistochemical analyses of the quoted proteins in CMV-QKO LCNEC and human LCNEC (1st and 2nd panels) and K5-QKO SCLC and human SCLC (3rd and 4th panels). (Scale bars, 50 μm.) CHRA, Chromogranin A; CGRP, Calcitonin Gene Related Protein; TTF-1, Thyroid Transcription Factor-1; LCNEC, large-cell neuroendocrine carcinoma; SCLC, small-cell neuroendocrine carcinoma.

Fig. 2.

Characterization of LCNEC and SCLC mouse tumors and metastasis. (A) Immunohistochemical analysis of LCNEC in CMV-QKO (Top) and SCLC in K5-QKO (Bottom) mice. Representative images of immunohistochemical staining for the quoted proteins. (Scale bars, 50 μm.) (B) Differential tumor growth localization in CMV-QKO and K5-QKO lungs. Representative images of H&E staining performed on lungs isolated from CMV-QKO mice (Left) with LCNEC growing in lung parenchyma and K5-QKO mice and (Middle) with SCLC growing lining bronchiole, respectively. P, parenchyma; B, bronchiole; T, tumor. Quantification of the comparison of parenchymal versus central tumor growth localization in Ad5-CMVcre and Ad5-K5cre-infected QKO mice (Right). Data represent mean ± SEM. ***P value = 0.0001 and ****P value ≤ 0.0001, determined by Mann–Whitney U test. (Scale bars, 1 mm.) (C) Development of metastases in K5-QKO mice. SCLC primary tumor in the lung (Top Left) of a K5-QKO mouse showing metastases in liver (Top Middle) and lymph node (Top Right). Immunohistochemical detection of the quoted neuroendcrine markers CGRP, Calcitonin Gene Related Protein (Bottom Left); CHRA, Chromoganin A (Bottom Middle); and SYN, synaptophysin in a liver metastasis of a K5-QKO mouse (Bottom Right). (Scale bars, 50 μm.)

Fig. 3.

Gene expression analyses of SCLC tumors arising from K5-QKO mice (moSCLC) and LCNEC tumors arising from CMV-QKO mice (moLCNEC). (A) PCA plot showing the distribution of the samples along PC1 and PC2. (B) Genes significantly (FDR ≤ 0.01) up-regulated or down-regulated more than 2-fold in moSCLC or in moLCNEC compared to lung. Numbers indicate Affymetrix probe set identifiers. FDR, false discovery rate; FC, fold change. (C) Venn diagram showing the overlap between the up-regulated genes in moSCLC versus moLung and moLCNEC versus moLung. Hypergeometric test was used to assess the statistical significance of the overlap. The rectangular boxes contain the main signaling pathways enriched in the indicated groups (gene ontology biological processes, category GEOTERM_BP_DIRECT). P values in brackets. The table shows some of the most relevant up-regulated genes in each group. Expression FC in brackets. In the case of common tumor genes: FC in moSCLC/FC in moLCNEC. (D) Heatmap representing the expression values of the LCNEC/SCLC classifier genes differentially expressed (t test, P < 0.05) in moLCNEC versus moSCLC (normalized gene/rows, unsupervised hierarchical clustering, Pearson correlation, average linking). Red and blue indicate high and low gene expression (−1 to 1 log2 gene expression value), respectively. Color clusters at the right side of the heatmap mark the genes specifically up-regulated in human LCNEC/SCLC clustering class (I-IV).

Fig. 4.

Comparison of positron emission tomography (PET) imaging with ¹⁸F-fluorodeoxyglucose (FDG) (Top) and ⁶⁸Ga-(tetraxetan-d-Phe1,Tyr3)-octreotide (DOTATOC) (Bottom). (A) Combined micro PET/CT images of a representative mouse K5-QKO. No increased ¹⁸F-FDG utilization is observed in micro-PET in an animal with spontaneous SCLC, whereas a higher avidity for ⁶⁸Ga-DOTATOC is detected. (B) Gross tumor appearance of dissected lung (arrow). (C) H&E of the SCLC at necropsy. (D) Detection of SSTR2 by immunohistochemistry staining. (Scale bars, 50 μm.) w.p.i., weeks postinfection.