TGF-β and αvβ6 integrin act in a common pathway to suppress pancreatic cancer progression

Abstract

The TGF-β pathway is under active consideration as a cancer drug target based on its capacity to promote cancer cell invasion and to create a protumorigenic microenvironment. However, the clinical application of TGF-β inhibitors remains uncertain as genetic studies show a tumor suppressor function of TGF-β in pancreatic cancer and other epithelial malignancies. Here, we used genetically engineered mouse models to investigate the therapeutic impact of global TGF-β inhibition in pancreatic cancer in relation to tumor stage, genetic profile, and concurrent chemotherapy. We found that αvβ6 integrin acted as a key upstream activator of TGF-β in evolving pancreatic cancers. In addition, TGF-β or αvβ6 blockade increased tumor cell proliferation and accelerated both early and later disease stages. These effects were dependent on the presence of Smad4, a central mediator of TGF-β signaling. Therefore, our findings indicate that αvβ6 and TGF-β act in a common tumor suppressor pathway whose pharmacologic inactivation promotes pancreatic cancer progression.

Fig 1. αvβ6 activates TGFβ in the pancreatic epithelium

A) IHC staining for phospho-Smad2 (top row, 400X) and integrin β6 (bottom row, 200X), during multi-stage progression of the Kras-p53^Lox/+ PDAC model. Duct (D), acinar (A), islet (I), metaplastic acini (M), and PanIN (P) cells indicated. P-Smad2+ neoplastic and stromal cells designated by yellow and red arrowheads, respectively. B) IHC for integrinβ6 in normal human pancreas, and in multistage PDAC progression. C) Left: phospho-Smad2 IHC in PanIN and PDAC from Kras-p53^Lox/+ mice treated with αvβ6 blocking antibodies or control. Right: Phospho-Smad2+ nuclei quantified by automated analysis.

Fig 2. TGFβ or αvβ6 blockade accelerates PDAC initiation and progression in GEM models

A, B) Evaluation of the impact of anti-αvβ6 and anti-Tgf-β antibodies on early disease in Kras-p53^Lox/+ mice. A) Schematic indicating the course of PDAC progression in control animals (top), as well as the treatment interval (red line). Mice were euthanized for analysis at 12 weeks. B) Left: Representative histological images to quantify the proportion diseased pancreas (top) and Ki67 staining to evaluate proliferation of PanIN epithelium (bottom). Right panel: Quantification of % of pancreatic area occupied by PanIN and PDAC lesions, treatment groups have significantly increased disease burden compared with controls (*p<0.05). Quantification of Ki67 staining; treatment groups have increased epithelial proliferation compared with controls (**p< 0.005; ***p< 0.001). C) Impact of anti-αvβ6 and anti-Tgf-β antibodies on tumor progression in Kras-p53^Lox/+ mice. Schematic: treatment was initiated at later stages of disease and continued until clinical signs of illness. Lower Panels: Kaplan-Meier analysis. Left: Survival is shortened in the anti-αvβ6 (mean 6.6 weeks; p=0.03) or anti-Tgfβ (mean 5.6 weeks; p=0.007) cohorts compared with isotype control treated animals (mean 8.9 weeks). Survival of untreated animals is shown for comparison (grey line); *p< 0.05. Right: Anti-Tgfβ and anti-αvβ6 antibody treatments reduce survival of gemcitabine (Gem) treated mice.

Fig 3. αvβ6 functions through Smad4 tumor suppressor in the pancreas

A) The relationship between Smad4 and β6 expression was evaluated in Smad4 wildtype and mutant PDAC models. % β6- neoplastic cells in individual tumors (graph on left) and representative IHC images (right panels) are shown. All Smad4 null tumors retain β6 expression (left), whereas subsets of Smad4 wild type tumors either lose (middle) or retain (right) Smad4 expression (*p<0.05). Insets show higher magnification. B) Impact of anti-αvβ6 treatment on PDAC progression in the Kras-Smad4^Lox/Lox; Ink4a/Arf^Lox/+ model. Left: Schematic showing experimental design. Right: Kaplan-Meier analysis showing that αvβ6 blockade does not affect survival (n.s.= not significant).