Constitutively active Akt1 cooperates with KRas(G12D) to accelerate in vivo pancreatic tumor onset and progression

Abstract

Background and aims: Pancreatic adenocarcinoma is a deadly disease characterized by metastatic progression and resistance to conventional therapeutics. Mutation of KRAS is the most frequent early event in pancreatic tumor progression. AKT isoforms are frequently activated in pancreatic cancer, and reports have implicated hyperactivation of AKT1, as well as AKT2, in pancreatic tumor formation. The objective here is to delineate the role of AKT in facilitating in vivo pancreatic tumor progression in the context of KRAS mutation and predisposition to pancreatic cancer.

Methods: Mice with Akt1 and KRas mutant alleles expressed using the pancreas Pdx promoter were mated to characterize the incidence and frequency of histologic and genetic alterations known to occur commonly in human pancreatic ductal adenocarcinoma.

Results: Active Akt1 (Akt1(Myr), containing a myristoylation sequence) cooperated with active mutant KRas(G12D) to accelerate pancreatic carcinoma onset and progression and increase phosphorylation of downstream effectors in the Akt pathway. Mucin and smooth muscle actin expression was found in and around pancreatic intraepithelial neoplasms (PanINs), and accelerated time to metastasis was found in Akt1(Myr)/KRas(G12D) mice.

Conclusions: In contrast to prior reports of pancreatic KRas mutant mice mated with mice deficient for various tumor suppressor genes, which resulted in aggressive disease within a few months of age, Akt1(Myr)/KRas(G12D) mice enabled the study of PanINs and spontaneous pancreatic transformation more characteristic of human pancreatic progression in elderly individuals. The Akt1(Myr)/KRas(G12D) model holds promise for delineating the tumor biology and biomarkers critical for understanding their cooperation in cancer oncogenesis and future targeting in therapeutic strategies.

Figure 1

Tumor latency in Akt1^Myr/KRas^G12Dversus KRas^G12D mice. Akt1^Myr/KRas^G12D mice (broken line) developed pancreatic tumors (PDACs) at a faster rate than KRas^G12D mice (solid line). Curves were significantly different with a P value < .0001 by log rank (Mantel-Cox) or Gehan-Breslow-Wilcoxon tests (GraphPad Prism 5).

Figure 2

Activation of the Akt/mTor/S6K pathway in pancreatic tumor progression. The panels show representative early ductal pancreatic lesions, similar to human low-grade PanINs, with strong activation (brown DAB stain) for phospho-Akt, phospho-mTor, and phospho-p70S6 kinase in PanINs of (A) Akt1^Myr/KRas^G12D and (B) KRas^G12D mice (40 × objective). (C) Immunohistochemical staining of primary PDAC and metastatic specimens from a ~ 43-week-old Akt1^Myr/KRas^G12D mouse for phospho-Akt, phospho-mTor, and phospho-p70S6 kinase and cytokeratin 17/19; a set of panels corresponding to PDAC metastasis to liver (10 × objective and a scale bar corresponding to 200 μm, with boxed-in close ups from the 40 × objective and a scale bar of 50 μm). In the metastasis panels, L = liver and T = tumor. Images were acquired using a Leica DM 2000 microscope with a digital DFC 295 camera.

Figure 3

Pancreatic histologic alterations in Akt1^Myr/KRas^G12D and KRas^G12D mice. The panels from (A) Akt1^Myr/KRas^G12D and (B) KRas^G12D mice show staining of representative pancreatic tissues. Sections showed staining for H&E, Alcian Blue staining of ducts for detection of mucin (dark blue), Muc-4 (brown color) in areas of ducts, trichrome stain of red acinar cells, and green-blue collagen-rich fibrotic areas of the PDAC tumor and α-SMA marker (brown color) in areas of acinar cells near fibrotic regions. Boxed-in highlighted areas (10 × objective, scale bar of 200 μm) were magnified for a focal view with the 40 × objective (scale bar of 50 μm).

Figure 4

Phospho-Akt and tumor suppressors in mouse and human pancreatic tumor cells. (A) (Left) Representative Western blots from each of three KRas^G12D (mouse numbers 190, 148, and 117) and three Akt1^Myr/KRas^G12D (mouse numbers 505, 9C, and 533) tumor cell cultures analyzed for expression of total Akt, phospho-Akt (Ser473), and tumor suppressor genes p53, p16Ink4a, and p19Arf. Actin is a loading control. (Right) Representative human pancreatic tumor cell lines run adjacent to mouse tumor cells showing relative amount of total Akt, phospho-Akt (Ser473), and actin. (B) Genomic DNA PCR showing retention or loss of Tp53, p16Ink4a, or p19Arf.