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. 2009 Nov 15;69(22):8733-41.
doi: 10.1158/0008-5472.CAN-09-1359. Epub 2009 Nov 3.

Retinoblastoma family proteins have distinct functions in pulmonary epithelial cells in vivo critical for suppressing cell growth and tumorigenesis

David S Simpson  1 , Nicole A Mason-Richie, Caitlin A Gettler, Kathryn A Wikenheiser-Brokamp
Affiliations

Retinoblastoma family proteins have distinct functions in pulmonary epithelial cells in vivo critical for suppressing cell growth and tumorigenesis

David S Simpson et al. Cancer Res. .

Abstract

Lung cancer is the leading cause of cancer deaths, accounting for more deaths than breast, colon, and prostate cancer combined. The retinoblastoma (Rb)/p16 tumor suppressive pathway is deregulated in most cancers. Loss of p16 occurs more frequently than Rb loss, suggesting that p16 suppresses cancer by regulating Rb as well as the related proteins p107 and p130. However, direct evidence demonstrating that p130 or p107 cooperate with Rb to suppress epithelial cancers associated with p16 loss is currently lacking. Moreover, the roles of p130 and p107 in lung cancer are not clear. In the present studies, Rb ablation was targeted to the lung epithelium in wild-type, p107, or p130 null mice to determine unique and overlapping Rb family functions critical in tumor suppression. Rb ablation during development resulted in marked epithelial abnormalities despite p107 upregulation. In contrast, p130 and p107 were not required during development but had distinct functions in the Rb-deficient epithelium: p107 was required to suppress proliferation, whereas a novel proapoptotic function was identified for p130. Adult Rb-ablated lungs lacked the epithelial phenotype seen at birth and showed compensatory p107 upregulation and p16 induction in epithelial cell lineages that share phenotypic characteristics with human non-small cell lung cancers (NSCLC) that frequently show p16 loss. Importantly, Rb/p107-deficient, but not Rb/p130-deficient, lungs developed tumors resembling NSCLC. Taken together, these studies identify distinct Rb family functions critical in controlling epithelial cell growth, and provide direct evidence that p107 cooperates with Rb to protect against a common adult cancer.

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Figures

Figure 1
Figure 1. Epithelial hyperplasia and cell death seen in Rb ablated lungs at birth is not present in adult lungs
A, SPC-rtTA+/−/tetCre+/− double transgenic mice were bred to RbLoxP/LoxP or RbLoxP/− mice. Doxycycline (ovals) activates the rtTA (arches) expressed specifically in lung epithelium leading to Cre expression and subsequent recombination at floxed Rb alleles. B, PCR analysis on lung DNA from day 1 pups. Rb recombination (RbRec) of floxed Rb alleles (RbLoxP) is only detected in lungs from mice containing both transgenes (SPC-rtTA, Cre). Mice were homozygous for the floxed Rb allele or heterozygous for the floxed and germline knockout (Rb−) alleles. C, Whole mount ß-galactosidase staining on lungs from day 1 mice pups containing the ROSA26 reporter locus demonstrates uniform staining in double transgenic lungs (SPC-rtTA+/Cre+) but not in controls lacking one or both transgenes (C). ß-galactosidase staining is restricted to the epithelium and present in Clara (arrow), ciliated (dashed arrow) and type II cells (arrowhead) in adult lungs. A subset of CGRP positive epithelial cells (brown, boxed area and open arrowhead) stain for ß-galactosidase (arrow in inset designates double positive cell). n ≥ 3 mice for each group. Original magnification: 200x (middle), 1000x (bottom). D, Hematoxylin and eosin stained lung sections show epithelial hypercellularity and apoptotic bodies (arrows) in Rb ablated E18.5 lungs (Pups) but not in Rb proficient lungs (Control) or Rb ablated lungs from 8 month old mice (Adults). Hypercellular neuroendocrine lesions are present in adult Rb ablated lungs (arrowhead). Original magnification: 400x (top row), 1000x (bottom row).
Figure 2
Figure 2. p107, but not p130, is induced in Rb ablated lungs during development
A, Western blot analyses show increased p107, but not p130, in Rb ablated (Rb -) day 1 and adult lungs as compared to Rb proficient littermate controls (Rb C). Lung lysates were evenly loaded as assessed by reprobing for β-actin. n=4-8 animals per group. B, Quantitative real-time RT-PCR shows increased p107, but not p130, in Rb ablated adult lungs (*p=0.0004, n=3 per group). Rb is significantly reduced in Rb deficient lungs (**p=0.023, n=3 per group). Graphs depict mRNA fold change relative to controls ± SE.
Figure 3
Figure 3. p16 is induced in Rb ablated lungs after birth and is expressed in Clara and type II cells
A, Western blot analyses show increased p16 in Rb ablated (Rb -), and 1 and 6-9 month adult lungs as compared to Rb proficient controls (Rb C). p16 is not detected in Rb ablated or control day 1 lungs. Lung lysates from p16−/− mice (p16−/−) and 3T3-L1 cell lysates (p16 C) represent negative and positive controls, respectively. Lung lysates were evenly loaded as assessed by reprobing for β-actin or tubulin. n=4-9 per group. Quantitative real-time RT-PCR shows increased p16 in Rb ablated 1 month (*p=0.0017) and 6-9 month (**p=0.045) adult lungs. p16 was not detected in E18.5 lungs. n=3 per group. Graphs depict mRNA fold change relative to controls ± SE. B, Immunohistochemical analyses show p16 expression in Clara cells within conducting airways (left panel) and type II cells within the parenchyma (right panel) in both Rb ablated and Rb proficient (Control) 9 month adult lungs. No specific staining is present in p16−/− lungs confirming assay specificity. Higher power views (insets) of boxed areas show representative Clara, ciliated and type II cells. n=3 Rb ablated and control lungs. Original magnification: 1000x. C, Primary type II cell isolate cytospins stained with hematoxylin or stained for SPC and DAPI by immunofluorescence. PCR analysis shows Rb recombination (RbRec) of floxed Rb alleles (RbLoxP) in cells isolated from Rb ablated (+) but not Rb proficient control lungs (−). D, Quantitative real-time RT-PCR shows increased p107, but not p130, in Rb ablated type II cells as compared to Rb proficient controls (*p=0.038). Rb is significantly reduced in Rb ablated type II cells (**p=0.0001). p16 is increased in Rb ablated 1 month lungs (Whole Lung) and type II cells (*p≤0.002). n=3-4 per group. Graphs depict mRNA fold change relative to controls ± SE.
Figure 4
Figure 4. Rb family proteins have distinct roles in regulating epithelial proliferation and survival
A, Hematoxylin and eosin stained E18.5 lung sections show epithelial hyperplasia, dysplasia and apoptotic cell death in Rb ablated, Rb ablated/p130−/− and Rb ablated/p107−/− lungs. Lungs from p130−/− and p107−/− are indistinguishable from Rb family proficient lungs (Control). B, Immunohistochemical staining for the proliferation marker, Ki67 (arrow) shows an increase in proliferating epithelial cells in Rb ablated, Rb ablated/p130−/− and Rb ablated/p107−/− lungs as compared to Rb proficient controls (*p<0.001). Ki67 positive cells are also increased in Rb ablated/p107−/− as compared to Rb ablated lungs (**p<0.001). Proliferation in p130−/− and p107−/− lungs is similar to control Rb proficient lungs (p>0.67). C, TUNEL analysis shows an increase in apoptotic epithelial cells (arrow) in Rb ablated, Rb ablated/p130−/− and Rb ablated/p107−/−lungs as compared to Rb proficient controls (*p<0.05). Apoptotic cells are decreased in Rb ablated/p130−/− as compared to Rb ablated lungs (**p<0.05). Apoptosis in p130−/− and p107−/− lungs is similar to control Rb proficient lungs (p>0.05). Graphs depict percent Ki67 or TUNEL positive epithelial cells ± SE. n≥6 mice per group. Original magnification: 1000x.
Figure 5
Figure 5. Rb/p107 loss results in lung tumors with a type II cell phenotype
A, Hematoxylin and eosin stained lung sections (left) and immunohistochemical analysis (right) of lung tumors arising in double transgenic/p107−/− adult mice not treated with doxycycline show a solid adenoma (t, top left) and a papillary adenocarcinoma invading the airway (t, middle and bottom left and right panel) that are positive for SPC but not CCSP or CGRP. Non-neoplastic cells in the surrounding conducting airway show CCSP and CGRP positive cells (arrows) serving as internal controls. Original magnification: 100x (top and middle left), 400x (bottom left and SPC) 1000x (CCSP and CGRP). B, PCR analysis on DNA from non-neoplastic lung (lung) and lung tumors (tumor) obtained from double transgenic/p107−/− chimeric mice (+) and p107−/− control littermates lacking one or both transgenes (−) that are homozygous for the floxed Rb allele (RbLoxP) show enrichment for the Rb recombined allele (RbRec) in tumors as compared to non-neoplastic lung. Rb recombination is not detected in tail DNA or tissue from controls. C, Co-immunofluorescent staining for SPC (red) and Ki67 (green) shows increased type II cell proliferation in Rb ablated and Rb ablated/p107−/− E18.5 lungs as compared to Rb family proficient controls (*p<0.001). Type II cell proliferation was not increased in Rb ablated/p107−/− lungs as compared to Rb ablated lungs (p=0.07). Higher power views of boxed areas (insets) show representative Ki67 negative (Control), Ki67 positive (Rb ablated) and adjacent Ki67 positive and negative (Rb ablated/p107−/−) SPC positive type II cells. Yellow represents autofluorescent red blood cells. Graph depicts percent SPC positive type II cells with nuclear Ki67 staining ± SE. n=3-4 per group. br=bronchioles. Original magnification: 400x. D, Model for Rb family function in the lung epithelium. Rb is required to suppress epithelial proliferation and death during development despite p107 induction. p107 cooperates with Rb to induce cell cycle arrest whereas p130 promotes apoptosis in the developing Rb deficient lung epithelium. In the adult lung, Rb is required to negatively regulate neuroendocrine cells whereas non-neuroendocrine lineages compensate for Rb loss and show induction of p16 and p107. Rb and p107 cooperate to suppress development of lung tumors resembling NSCLC.
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