Carcinoma Initiation via Rb Tumor Suppressor Inactivation: A Versatile Approach to Epithelial Subtype-Dependent Cancer Initiation in Diverse Tissues

Carcinomas arise in a complex microenvironment consisting of multiple distinct epithelial lineages surrounded by a variety of stromal cell types. Understanding cancer etiologies requires evaluating the relationship among cell types during disease initiation and through progression. Genetically engineered mouse (GEM) models facilitate the prospective examination of early oncogenic events, which is not possible in humans. Since most solid tumors harbor aberrations in the RB network, we developed an inducible GEM approach for the establishment and assessment of carcinoma initiation in a diverse range of epithelial tissues and subtypes upon inactivation of RB-mediated tumor suppression (RB-TS). The system allows independent assessment of epithelial subtypes that express either cytokeratins (K) 18 or 19. By Cre-dependent expression of a protein that dominantly inactivates RB and functionally redundant proteins p107 and p130, neoplasia could be initiated in either K18 or K19 expressing cells of numerous tissues. By design, because only a single pathway aberration was engineered, carcinomas developed stochastically only after long latency. Hence, this system, which allows for directed cell type-specific carcinoma initiation, facilitates further definition of events that can progress neoplasms to aggressive cancers via engineered, carcinogen-induced and/or spontaneous evolution

RB inactivation in keratin 18 positive thymic epithelial cells promotes non-cell autonomous T cell hyperproliferation in genetically engineered mice

<div><p>Thymic epithelial cells (TEC), as part of thymic stroma, provide essential growth factors/cytokines and self-antigens to support T cell development and selection. Deletion of Rb family proteins in adult thymic stroma leads to T cell hyperplasia <i>in vivo</i>. To determine whether deletion of Rb specifically in keratin (K) 18 positive TEC was sufficient for thymocyte hyperplasia, we conditionally inactivated Rb and its family members p107 and p130 in K18+ TEC in genetically engineered mice (<i>TgK18GT</i>_<i>121</i><i>; K18</i> mice). We found that thymocyte hyperproliferation was induced in mice with Rb inactivation in K18+ TEC, while normal T cell development was maintained; suggesting that inactivation of Rb specifically in K18+ TEC was sufficient and responsible for the phenotype. Transplantation of wild type bone marrow cells into mice with Rb inactivation in K18+ TEC resulted in donor T lymphocyte hyperplasia confirming the non-cell autonomous requirement for Rb proteins in K18+ TEC in regulating T cell proliferation. Our data suggests that thymic epithelial cells play an important role in regulating lymphoid proliferation and thymus size.</p></div

Neoplastic lesions in <i>TgK19GT₁₂₁;</i><i>β-actin Cre</i> mice.

<p>Hyperplasia was observed in most tissues (lung and colon: indicated by black arrows). Multifocal ductal hyperplasia was observed in pancreas, as well as mouse prostate intraepithelial neoplasia (mPIN) in prostate, and hydronephrosis as indicated by white arrows in kidney. All samples were stained with H&E.</p

Summary of histopathological findings in <i>TgK19GT₁₂₁; β-actin Cre</i> and <i>TgK18GT₁₂₁; β-actin Cre</i> mice.

*<p>Average age of <i>TgK19GT₁₂₁; β-actin Cre</i> mice analyzed here was 123 days (2–6 months, mainly line 43) and <i>TgK18GT₁₂₁; β-actin Cre</i> mice 130 days (1–6 months, both lines). Three <i>TgK19GT₁₂₁; β-actin Cre</i> mice were euthanized due to kidney hydronephrosis at 2 months of age. All <i>TgK18GT₁₂₁; β-actin Cre</i> mice were euthanized due to thymic masses at 1–6 months of age.</p>†<p>The onset and extent of lesions were different between <i>TgK19GT₁₂₁; β-actin Cre</i> and <i>TgK18GT₁₂₁; β-actin Cre</i> mice (see text for details).</p>#<p>multifocal hyperplasia. m: male; f: female; N.D.: not determined.</p

Proliferation rates in tissues of <i>TgK19GT₁₂₁;</i><i> </i><i>β-actin Cre</i> and <i>TgK18GT₁₂₁;</i><i> </i><i>β-actin Cre</i> mice.

<p>Proliferation rates of pancreas, mammary gland, ovary, prostate (dorsolateral lobe), and bladder were quantified on tissue sections stained with Ki-67. *Significantly different from WT control (p<0.001); #significantly different between <i>TgK19GT₁₂₁; β-actin Cre and TgK18GT₁₂₁; β-actin Cre</i> (p = 0.007); @ significantly different between <i>TgK18GT₁₂₁; β-actin Cre</i> and WT (p = 0.043).</p

Proliferation in T₁₂₁-expressing <i>TgK18GT₁₂₁;</i><i> </i><i>β-actin Cre</i> tissues.

<p>Proliferation was assessed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080459#pone-0080459-g005" target="_blank">Figure 5</a> in 2 month old mice. Similar proliferation levels were observed in small intestine, colon, stomach and ovarian follicle as that of wildtype mice shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080459#pone.0080459.s008" target="_blank">Figure S8</a>. Black arrows indicate Ki-67 positive cells. Scale bar = 50 µM.</p

Transgene expression by immunostaining in <i>TgK18GT</i>_<i>121</i> (<i>K18</i>) or induced-<i>K18</i> mice.

<p>(A) Transgene cassette consisting of floxed eGFP stop cassette upstream of truncated SV40 large T antigen (first 121 amino acid; T₁₂₁) was inserted into the 1^st exon of K18 gene on a bacterial artificial chromosome (BAC). Transgene eGFP was driven by K18 regulation. Once K18 mice were crossed to a transgenic mice expressing Cre recombinase, T₁₂₁ was expressed directly under K18 regulation. (B) Representative images of K18 IHC staining in cortex (C) and medulla (M) of <i>WT</i> thymus. Inserts are higher magnification of the images. (C) Representative immunofluorescence images of T₁₂₁ (green), K18 (yellow), K5 (red), and DAPI (blue) in cortex (C) and medulla (M) delineated with a white dotted line, in induced-<i>K18;Cre</i> thymus. Middle and right images are higher magnification of areas in white and red boxes of left image, respectively. Right image: * Cell is positive for T₁₂₁, K18, and K5, and # positive for K5 only. (D) Representative images of K18 (red) and eGFP (green) immunostaining in thymic cortex and medulla (data not shown) of uninduced-<i>K18</i> mice (Cre negative). (E) Representative images of K18 (red) and T₁₂₁ (green) immunostaining in thymic cortex and medulla (data not shown) of induced-<i>K18</i> mice (<i>K18;β-actin Cre</i>).</p

Neoplastic lesions in <i>TgK18GT₁₂₁; β-actin Cre</i> mice.

<p>Hyperplasia was observed in some tissues (ovary and prostate: indicated by arrows). All mice were euthanized at 1–7 months of age due to life-threatening thoracic pressure caused by enlarged thymuses. All samples were stained with H&E.</p

Rb-TS inactivation in K18+ TEC led to decreased survival and thymic hyperplasia.

<p>(A) Kaplan-Meier survival curve of <i>K18;β-actin Cre</i> (n = 74), <i>K18;R26CreER</i> (n = 27), and <i>K18;PbCre4</i> (n = 45) mice with median survival of 94, 41, and 231 days, respectively. Uninduced -<i>K18</i> mice (n = 8) did not develop any gross abnormalities. (B) Gross phenotype of thymuses in <i>WT</i> and <i>K18;β-actin Cre</i> mice. (C) Representative images of H.E. stained thymus sections in <i>WT</i>, <i>K18;R26CreER</i> mice. C: cortex; M: medulla. (D) Representative low magnification images of H.E. stained thymuses in <i>WT</i> and <i>K18;β-actin Cre</i> mice. C: cortex; M: medulla.</p