PTEN loss accelerates KrasG12D-induced pancreatic cancer development

Abstract

KRAS mutations are found in ∼90% of human pancreatic ductal adenocarcinomas (PDAC). However, mice genetically engineered to express Kras(G12D) from its endogenous locus develop PDACs only after a prolonged latency, indicating that other genetic events or pathway alterations are necessary for PDAC progression. The PTEN-controlled phosphatidylinositol 3-kinase (PI3K)/AKT signaling axis is dysregulated in later stages of PDAC. To better elucidate the role of PTEN/PI3K/AKT signaling in Kras(G12D)-induced PDAC development, we crossed Pten conditional knockout mice (Pten(lox/lox)) to mice with conditional activation of Kras(G12D). The resulting compound heterozygous mutant mice showed significantly accelerated development of acinar-to-ductal metaplasia (ADM), malignant pancreatic intraepithelial neoplasia (mPanIN), and PDAC within a year. Moreover, all mice with Kras(G12D) activation and Pten homozygous deletion succumbed to cancer by 3 weeks of age. Our data support a dosage-dependent role for PTEN, and the resulting dysregulation of the PI3K/AKT signaling axis, in both PDAC initiation and progression, and shed additional light on the signaling mechanisms that lead to the development of ADM and subsequent mPanIN and pancreatic cancer.

Figure 1. Mice with Kras^G12D activation and Pten heterozygosity have decreased survival

A Kaplan-Meier analysis reveal a median survival of approximately 17 days in Pdx1-Cre⁺;Kras^G12D/+;Pten^lox/lox mice (n=26), significantly less than wild-type mice (n=16). Pten heterozygosity decreases the survival of mice with an activated Kras^G12D mutation (n=45) which survive a significantly shorter time than mice with only an activated Kras^G12D mutation alone (n=25) (p < 0.001, log-rank test, for each pair-wise combination). Low grade mPanIN lesion in a 3 month old Pdx1-Cre⁺;Kras^G12D/+ mouse (right side, top) and a PDAC in a Pdx1-Cre⁺;Kras^G12D/+;Pten^lox/+ mouse of the same age (200x). B: Histological, immunofluorescence, and immunohistochemical analysis of 3 month old Pdx1-Cre⁺;Kras^G12D/+;Pten^lox/+ (bottom row) and wild type mice (top row). H&E staining of representative histology of WT and Pdx1-Cre⁺;Kras^G12D/+;Pten^lox/+ mice shows PDAC lesions disrupting the normal glandular architecture found in WT mice (200x). Cells which display nuclear atypia, a high nuclear–to- cytoplasmic ratio, and loss of normal cell polarity can be observed in well-differentiated cancers in Pdx1-Cre⁺;Kras^G12D/+;Pten^lox/+ mice. These lesions exhibit invasive behavior as determined by the presence of ductal epithelium (Cytokeratin-19; red; white arrow) which is found invading into the surrounding desmoplastic stromal compartment (identified by smooth muscle actin (SMA) staining, green) (200x). The cells of these invasive cancers demonstrate the presence of lectin Dolichos biflorus agglutinin (DBA) which also marks normal ducts of WT mice (200x). Tumors also showed positive PDX-1 staining (200x; inset 400x), which stained islet cells but not ductal cells in WT mice (200x; inset 400x). Scale bars, 50μm.

Figure 2. Mutant mice with Kras^G12D activation and Pten heterozygosity have an earlier onset of mPanIN and PDAC

A Pdx1-Cre⁺;Kras^G12D/+;Pten^lox/+ mice (black bar) show earlier mPanIN development at 1 month of age and more frequent mPanIN development at 3 months of age compared to Pdx1-Cre⁺;Pten^L/+ (white bar) or Pdx1-Cre⁺;Kras^G12D/+ mice (gray bar). The mPanIN lesions that develop in both Pdx1-Cre⁺;Kras^G12D/+;Pten^lox/+ mice (age 3 months) and Pdx1-Cre⁺;Kras^G12D/+ mice (age 3–6 months) show up-regulation of phosphorylated ERK but not phosphorylated AKT or S6 as demonstrated by IHC on sections containing mPanINs (right columns) (400x). B: Pdx1-Cre⁺;Kras^G12D/+;Pten^lox/+ mice show earlier and more frequent onset of PDAC compared to mice with Kras^G12D activation alone. Neoplastic ducts in PDACs of Pdx1-Cre⁺;Kras^G12D/+;Pten^lox/+ mice (age 6 months) show up-regulation of membrane bound phosphorylated AKT, ERK, and S6 compared to the neoplastic ducts in PDACs of Pdx1-Cre⁺;Kras^G12D mice (age 6 months) which show only up-regulation of phosphorylated ERK (400x). A minimum of 4 non consecutive H&E sections from each mouse were examined for lesion scoring for each genotype. Scale bars, 50μm.

Figure 3. Mutant Kras^G12D activation and Pten heterzygozity leads to earlier onset of ADM

A: H&E staining shows the morphology of a zone of transition where diffuse metaplasia of acinar tissue to a ductal phenotype is observed in a 3 month old Pdx1-Cre⁺;Kras^G12D/+;Pten^lox/+ mouse (left column, white arrow) (400x; inset 600x). Immunofluorescence on a serial section shows that these ADMs contain duct-like cells (pan-cytokeratin-labeled; green; inset 60x; white arrow) and acinar cells (amylase+; red) (400x). H&E staining of slides with sections taken from areas adjacent to PDACs from human patients show similar transition zones where ADMs can be observed (right column, red arrow) (400x; inset 600x). Likewise, immunofluorescence on a serial section confirms the presence of structures containing both ductal (pan-cytokeratin-labeled; green; white arrow) and acinar cells (amylase+; red) (400x; inset 600x). B: Quantification of metaplasia and mPanIN presence in early lesion development. A minimum of five mice per genotype were analyzed with 4 non-consecutive sections examined per mouse. Pdx1-Cre⁺;Kras^G12D/+;Pten^lox/+ mice (black bar, n=6) show a higher number of mice with only ADMs compared to Pdx1-Cre⁺;Kras^G12D/+ mice (gray bar, n=5) at 1 month of age. Scale bars, 50μm.

Figure 4. Mutant Kras^G12D activation and Pten heterzygozity leads to aggressive ductal metaplasia

Quantification of proliferation in mPanINs and metaplasias. Left, sections from Pdx1-Cre⁺;Kras^G12D/+ mice (n=4, age 3–6 months) and Pdx1-Cre⁺;Kras^G12D/+;Pten^lox/+ mice (n=4, age 3 months) were stained with an antibody against the proliferation marker Ki67. The percentage of Ki67 positive cells for each type of lesion was determined by averaging the percentage of Ki67 positive cells per field (10 total which each contained only ADMs or only mPanINs). ADMs and mPanIN lesions in Pdx1-Cre⁺;Kras^G12D/+;Pten^lox/+ mice (right column) show a higher percentage of cells positive for Ki67 than similar lesions in Pdx1-Cre⁺;Kras^G12D/+ mice (left column) (400x); right, quantification. *p<.05. Scale bars, 50μm.

Figure 5. Mice with Pten heterozygosity and mutant Kras^G12D activation show an increase in CD44 positive cells

A Immunohistochemistry on Pdx1-Cre⁺;Kras^G12D/+ and Pdx1-Cre⁺;Kras^G12D/+;Pten^lox/+ mice at 3 months of age showed that the cells of metaplasias (top row), but not mPanINs (bottom row), showed expression of CD44. This pattern of staining was also observed in areas adjacent to PDACs from human tumors which contained metaplasias and mPanINs (400x). B: H&E staining shows the morphology of a PDAC (400×) in a 6 month old Pdx1-Cre⁺;Kras^G12D/+;Pten^lox/+ mouse (top row). Immunofluorescence on a serial section confirms the presence of invading ductal epithelium (right panel, Cytokeratin-19; red/SMA staining, green). Immunohistochemistry shows that these invasive tumors show CD44 staining, matching the expression pattern of CD44 seen in human PDAC samples (bottom panel) (400x). Scale bars, 50μm.

Figure 6. Kras^G12D activation and PTEN loss collaborate to promote ADM, mPanIN, and pancreatic cancer development

In the context of an existing activated Kras^G12D mutation, PTEN haploinsufficiency drives ADM and mPanIN development with complete PTEN loss of function promoting PDAC development.