Inactivation of TIF1gamma cooperates with Kras to induce cystic tumors of the pancreas

Abstract

Inactivation of the Transforming Growth Factor Beta (TGFbeta) tumor suppressor pathway contributes to the progression of Pancreatic Ductal AdenoCarcinoma (PDAC) since it is inactivated in virtually all cases of this malignancy. Genetic lesions inactivating this pathway contribute to pancreatic tumor progression in mouse models. Transcriptional Intermediary Factor 1 gamma (TIF1gamma) has recently been proposed to be involved in TGFbeta signaling, functioning as either a positive or negative regulator of the pathway. Here, we addressed the role of TIF1gamma in pancreatic carcinogenesis. Using conditional Tif1gamma knockout mice (Tif1gamma(lox/lox)), we selectively abrogated Tif1gamma expression in the pancreas of Pdx1-Cre;Tif1gamma(lox/lox) mice. We also generated Pdx1-Cre;LSL-Kras(G12D);Tif1gamma(lox/lox) mice to address the effect of Tif1gamma loss-of-function in precancerous lesions induced by oncogenic Kras(G12D). Finally, we analyzed TIF1gamma expression in human pancreatic tumors. In our mouse model, we showed that Tif1gamma was dispensable for normal pancreatic development but cooperated with Kras activation to induce pancreatic tumors reminiscent of human Intraductal Papillary Mucinous Neoplasms (IPMNs). Interestingly, these cystic lesions resemble those observed in Pdx1-Cre;LSL-Kras(G12D);Smad4(lox/lox) mice described by others. However, distinctive characteristics, such as the systematic presence of endocrine pseudo-islets within the papillary projections, suggest that SMAD4 and TIF1gamma don't have strictly redundant functions. Finally, we report that TIF1gamma expression is markedly down-regulated in human pancreatic tumors by quantitative RT-PCR and immunohistochemistry supporting the relevance of these findings to human malignancy. This study suggests that TIF1gamma is critical for tumor suppression in the pancreas, brings new insight into the genetics of pancreatic cancer, and constitutes a promising model to decipher the respective roles of SMAD4 and TIF1gamma in the multifaceted functions of TGFbeta in carcinogenesis and development.

Figure 1. Homozygous deletion of Tif1γ cooperates with activated Kras^G12D mutation to induce cystic tumors of the pancreas.

Magnetic Resonance Imaging (MRI) (A–C). Positron Emission Tomography (PET) (D–F). Gross anatomy of the pancreas after dissection (G–I). Hematoxylin Phloxine Saffron (HPS) staining of pancreas sections (J–L). Acinar tissue (stars), scattered endocrine islets (arrow) and exocrine ducts (arrowhead). When notified, pancreas is circled in red dashed lines. Pdx1-Cre;LSL-Kras^G12D;Tif1γ^lox/lox used in this experiment was 154 days old and representative of the 4 mice we analyzed at the age of 18–27 weeks.

Figure 2. Inactivation of Tif1γ accelerates activated Kras^G12D–mediated pancreatic neoplasia.

Sections of wild-type (A–D), Pdx1-Cre;LSL-Kras^G12D (E–H) and Pdx1-Cre;LSL-Kras^G12D;Tif1γ^lox/lox (I–L) pancreas from mice at different ages were stained with HPS. Number of mice: 3 weeks, n = 3; 6–13 weeks, n = 5; 18–27 weeks, n = 4. Inset pictures: Higher magnification.

Figure 3. Endocrine and exocrine compartments destruction in the pancreas of Pdx1-Cre;LSL-Kras^G12D;Tif1γ^lox/lox mice.

Wild-type (A–C′) and Pdx1-Cre; LSL-Kras^G12D; Tif1γ^lox/lox (D–L′) pancreas sections were stained with HPS (A,D,G,J), immuno-revealed either with an anti-chymotrypsin antibody (B,E,H,K) or an anti-insulin antibody (C,F,I,L and C′,F′,I′,L′ that represent the boxed region in C,F,I,L at a higher magnification). For Pdx1-Cre;LSL-Kras^G12D;Tif1γ^lox/lox mice, analysis were performed at different ages. Number of mice: 3 weeks, n = 3; 6–13 weeks, n = 5; 18–27 weeks, n = 4. Boxes: Higher magnification. The pictures show one representative mouse for each group.

Figure 4. Immunophenotyping of pancreas cysts observed in Pdx1-Cre;LSL-Kras^G12D;Tif1γ^lox/lox mice.

Wild-type (A–D) and Pdx1-Cre;Tif1γ^lox/lox;LSL-Kras^G12D (E–H) pancreas sections were stained to detect glandular cells (CK19), mucus-secreting cells (Alcian Blue), Tif1γ and Smad4. The pictures show one representative mouse at the age of 22 weeks.

Figure 5. TIF1γ expression in human pancreatic neoplasia.

The expression of TIF1γ from 20 human PDAC and 16 peritumoral tissues was determined by quantitative RT–PCR (A). The average expression (±sd) is plotted for each group (B). The significance (P value) for the difference between peritumoral and tumoral groups, determined by a Student's T test, is shown above the graphs. The expression of TIF1γ protein was evaluated by immunohistochemistry in different pancreatic lesions: PDAC (n patients = 20) (peritumoral (C) and tumoral tissues (D, E). IPMN (n patients = 10) of high grade dysplasia (F), high-grade PanINs (n patients = 8) (G) and MCN (n patients = 15) (H). Inset pictures: higher magnification.