Loss of the SWI/SNF ATPase subunits BRM and BRG1 drives lung cancer development

Abstract

Inactivation of Brg1 and Brm accelerated lung tumor development, shortened tumor latency, and caused a loss of differentiation. Tumors with Brg1 and/or Brm loss recapitulated the evolution of human lung cancer as observed by the development of local tumor invasion as well as distal tumor metastasis, thereby making this model useful in lung cancer studies. Brg1 loss contributed to metastasis in part by driving E-cadherin loss and Vimentin up-regulation. By changing more than 6% of the murine genome with the down-regulation of tumor suppressors, DNA repair, differentiation and cell adhesion genes, and the concomitant up-regulation of oncogenes, angiogenesis, metastasis and antiapoptosis genes, caused by the dual loss of Brg1/Brm further accelerated tumor development. Additionally, this Brg1/Brm-driven change in gene expression resulted in a nearly two-fold increase in tumorigenicity in Brg1/Brm knockout mice compared with wild type mice. Most importantly, Brg1/Brm-driven lung cancer development histologically and clinically reflects human lung cancer development thereby making this GEMM model potentially useful.

Keywords: lung cancer; smarca2; smarca4; swi/snf; tumor.

Figure 1

A. shows the four different mouse genotypes (wild type, Brg1-knockout, Brm-null and DKO) and the means by which they were generated. B. illustrates the process by which the Brg1-knockout mice were generated; Brg1 exons 16 and 17 were flanked by LoxP sites [45]. Upon tetracycline administration, the Cre enzyme was expressed, which then cleaved the LoxP sites, resulting in a deletion of these two exons. C. shows the experimental design of the Brg1 and Brm knockout model, which results in tumor development. Ethyl carbamate and tetracycline administration occurred at 8 and 12 weeks, respectively. Adenoma development occurred at 12 weeks followed by progression and the development of malignant adenocarcinomas beginning at 22 weeks. No further tumors were found in these mice at 60 weeks and thus the experiment was ended.

Figure 2

A. illustrates Pcna expression by IHC in adenomas from WT (Brg1+/+Brm+/+) (a), Brg1 knockout (Brg1−/−Brm+/+) (b), Brm-null (Brg1+/+Brm−/−) (c) and double KO (Brg1−/−Brm−/−) (d) mice. B. shows the relative Pcna expression in lung adenocarcinomas from DKO (d)>Brg1-KO (b), Brm-null (c) and > WT (a) mice. Magnification bar (right lower corner) = 40 μm. According to C. very few cells express Ki-67 in the nucleus (arrows) in adenomas from a WT mouse (a), Brg1 KO mouse (b), Brm-null mouse (c), and a DKO mouse (d). D. shows that, compared with adenomas, a higher percentage of cells express Ki-67 protein (arrows) in adenocarcinomas from a WT mouse (a), a Brg1 KO mouse (b), a Brm-null mouse (c) and a DKO mouse (d). Microscope bar = 20 μM.

Figure 3

A. shows local progression of a malignant adenocarcinoma into a blood vessel (a), a cluster of tumor cells invading a bronchiolar airway (b), the presence of tumor cells in the stroma (large tumor cells, arrow) alongside spindle-shaped fibroblasts (arrowheads) (c) and tumor cells surrounding and invading a nerve (d). B and C. contain representative images of Brm and Brg1 expression in tumors, respectively, as detected by IHC in tumors from the four genotypes: WT (a), Brg1-KO (b), Brm-null (c) and DKO (d); arrows indicate Brg1-positive cells within the Brg1-KO and DKO tumors.

Figure 4

A. shows the various differentiation states (well, moderate and poor) as illustrated by H&E staining. The well-differentiated tumor shows well-formed papillary and glandular structures and mild cytological atypia (a). The moderately-differentiated tumor is composed of irregular glandular structures, moderate cytological atypia and occasional mitotic figures (b). The poorly-differentiated tumor shows a predominantly solid growth pattern, marked cytological atypia and increased mitotic figures (c). Low (d) and high (e) magnification of a high-grade tumor with prominent nucleoli and mitotic figures. Magnification bar = 20 μM in a-c; 10 μM in e. B. illustrates a tumor with sarcomatoid morphology, the most poorly differentiated variant (a), a cluster of signet ring cells (arrows) (b), mucinous tumor cells with cytoplasmic vacuoles (black arrows) (c), and a pale staining area of fibrosis with tumor cells (black arrows) (d).

Figure 5

A. shows a sheet of lymphocytes (arrowhead) and a cluster of macrophages (arrow) (a), as well as several multinucleated giant cells (arrows) have infiltrated these tumors; arrowhead, lymphocytes (b). Also illustrated is the studding (tip of white triangles) of the ribcage after gross dissection of a DKO mouse with the lungs in place and after the lungs were removed (c). One of the tumors noted in the MRI was removed and examined and was revealed to be ∼2 cm in length and contain a clear fluid indicative of a pleural effusion (black arrow) (d). MRI images of the larger adenocarcinomas of the lungs, the ribcage mets (denoted by white triangle tips), as well as and pleural effusions (white lines) are shown in (e). H&E stain of malignant lung adenocarcinoma cells that have penetrated through the chest cavity into the intercostal muscles of the ribcage in a BRG−/− mouse at low mag 10x (f) and high mag 40x (g). B. shows strong E-cadherin expression in a lung adenocarcinoma derived from a WT mouse (a), but much weaker staining in tumors from a Brg1-KO mouse (b), a Brm-null mouse (c), and a DKO mouse, where E-cadherin expression is almost completely absent (d). There is minimal to no expression of E-Cadherin in a metastatic rib met (e), which was derived from the mouse in (b).

Figure 6

A. shows a low (a) and high (b) magnified image of a kidney met from a Brg1-KO mouse; (c) and (d) show a liver met from the same mouse; a low (e) and high (f) mag image of a kidney met from a DKO mouse that features a dense sheet of tumor cells; a colon met from a Brg1-KO mouse displays spindle-shaped sarcomatoid tumor cells surrounding the normal glandular structures (g-h). B. shows the results of qPCR of multiple tumors for E-cadherin and the fold-change of E-cadherin expression in several Brm-null, Brg1-KO and DKO tumors relative to WT tumors (a). A colon met (b), a kidney met (c) and a liver met (d) from several BRG-deficient mice; the arrow in (c) denotes normal kidney. Magnification bar = 20 μm.

Figure 7

A. illustrates the rate of tumor development of lung adenocarcinomas from each of the four genotypes over the 60-week experimental period. B. shows the comparisons of various genotypes along with the hazard ratios (HR) and 95% confidence intervals (CI). The p-values obtained from a Cox proportional hazard model are also given.