Loss of p130 accelerates tumor development in a mouse model for human small-cell lung carcinoma

Abstract

Small-cell lung carcinoma (SCLC) is a neuroendocrine subtype of lung cancer. Although SCLC patients often initially respond to therapy, tumors nearly always recur, resulting in a 5-year survival rate of less than 10%. A mouse model has been developed based on the fact that the RB and p53 tumor suppressor genes are mutated in more than 90% of human SCLCs. Emerging evidence in patients and mouse models suggests that p130, a gene related to RB, may act as a tumor suppressor in SCLC cells. To test this idea, we used conditional mutant mice to delete p130 in combination with Rb and p53 in adult lung epithelial cells. We found that loss of p130 resulted in increased proliferation and significant acceleration of SCLC development in this triple-knockout mouse model. The histopathologic features of the triple-mutant mouse tumors closely resembled that of human SCLC. Genome-wide expression profiling experiments further showed that Rb/p53/p130-mutant mouse tumors were similar to human SCLC. These findings indicate that p130 plays a key tumor suppressor role in SCLC. Rb/p53/p130-mutant mice provide a novel preclinical mouse model to identify novel therapeutic targets against SCLC.

Figure 1. Deletion of p53, Rb, and p130 in the lungs of adult mice leads to the development of lung tumors

A, schematic representation of the conditional mutant alleles for p53, Rb, and p130. B, representative photographs of the lungs from double and triple mutant mice 6 months after Ad-Cre infection. C, PCR analysis for the deletion of the conditional alleles on genomic DNA obtained from lung tumors (Lu), liver metastasis (Li), and the tails of two representative mice (#1 and #2). D, RT-qPCR analysis of p53, Rb, p130, and p107 expression levels in control lungs (CTRL) (n=3) and double (n=6) and triple mutant tumors (n=3).

Figure 2. Loss of p130 results in accelerated lung cancer development in Rb/p53 mutant mice

A, hematoxylin and eosin (H&E) staining of sections from the lungs of double and triple mutant mice at three time points post-Ad-Cre injection – no triple mutant mice were alive at the 9 months time point. Bar: 400 μm. Arrowheads show small lesions. B, quantification of tumor surface area in triple mutant mice 6 months after Ad-Cre injection compared to double mutant mice 9-12 months after Ad-Cre infection. C, quantification of tumor numbers in double and triple mutant mice 3 and 6 months after Ad-Cre injection. D, analysis of proliferation in double (~9 months) and triple (~6 months) mutant tumors of similar size by immunostaining for phospho-histone 3 (PH3). Left: representative immunostaining (PH3, red – DAPI, blue). Right: quantification. ns, not significant. Bar: 50 μm.

Figure 3. Rb/p53/p130 triple mutant tumors have features of neuroendocrine SCLC

A-B, immunostaining analysis of SCLC at different time points (1, 3, 6, and 9 months) for the expression of SYP (neuroendocrine), CCSP (bronchiolar), and SP-C (alveolar) and the incorporation of BrdU (S phase). DAPI stains the DNA in blue. The small lesion in a triple mutant mouse in B is circled by a dotted line. Bar: 50 μm. C, RT-qPCR analysis for the expression of four neuroendocrine genes in micro-dissected double (n=5) and triple (n=3) mutant tumors compared to control lungs (n=4). **, p<0.005. D, immunoblot analysis for SYP in double and triple mutant tumors compared to a control lung. Tubulin serves as a loading control.

Figure 4. Rb/p53/p130 triple mutant mice provide a novel mouse model for human SCLC

A, Expression of selected genes previously found to be overexpressed and/or implicated in human SCLC is graphed as fold increase relative to control lungs. The values were obtained from SAM analysis of gene expression profiles from 3 Rb/p53/p130 mutant tumors (dissected 6 months after Ad-Cre), 10 Rb/p53 mutant tumors 9 months after Ad-Cre), and 5 control lungs. Noticeably, expression levels for most of the well-known genes are similar in both mouse models. The q-value for all the genes listed above is less than 0.05%. B, Expression profiles of genes in Rb/p53/p130 and Rb/p53 were compared to those of orthologous genes in human array datasets of lung cancers using hierarchical clustering. hAdeno: human adenocarcinoma; hSCC: human squamous cell carcinoma; hSCLC: human SCLC; mSCLC: mouse SCLC.