Thrombospondin-1 mediates oncogenic Ras–induced senescence in premalignant lung tumors
Kwan-Hyuck Baek, … , Gerard I. Evan, Sandra Ryeom
Kwan-Hyuck Baek, … , Gerard I. Evan, Sandra Ryeom
Published September 9, 2013
Citation Information: J Clin Invest. 2013;123(10):4375-4389. https://doi.org/10.1172/JCI67465.
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Research Article Oncology

Thrombospondin-1 mediates oncogenic Ras–induced senescence in premalignant lung tumors

Abstract

Progression of premalignant lesions is restrained by oncogene-induced senescence. Oncogenic Ras triggers senescence in many organs, including the lung, which exhibits high levels of the angiogenesis inhibitor thrombospondin-1 (TSP-1). The contribution of TSP-1 upregulation to the modulation of tumorigenesis in the lung is unclear. Using a mouse model of lung cancer, we have shown that TSP-1 plays a critical and cell-autonomous role in suppressing Kras-induced lung tumorigenesis independent of its antiangiogenic function. Overall survival was decreased in a Kras-driven mouse model of lung cancer on a Tsp-1–/– background. We found that oncogenic Kras–induced TSP-1 upregulation in a p53-dependent manner. TSP-1 functioned in a positive feedback loop to stabilize p53 by interacting directly with activated ERK. TSP-1 tethering of ERK in the cytoplasm promoted a level of MAPK signaling that was sufficient to sustain p53 expression and a senescence response. Our data identify TSP-1 as a p53 target that contributes to maintaining Ras-induced senescence in the lung.

Authors

Kwan-Hyuck Baek, Dongha Bhang, Alexander Zaslavsky, Liang-Chuan Wang, Anil Vachani, Carla F. Kim, Steven M. Albelda, Gerard I. Evan, Sandra Ryeom

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Figure 6

TSP-1 coprecipitates with pERK, and its loss alters pERK subcellular localization.

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TSP-1 coprecipitates with pERK, and its loss alters pERK subcellular loc...
(A) Endogenous TSP-1 coprecipitated with endogenous pERK after HrasV12 expression. Top: WT lung fibroblasts were infected with HrasV12 retrovirus, harvested 10 days after infection, and subjected to IP with isotype-matched IgG and TSP-1 or pERK antibodies. IP samples were then probed with antibodies for TSP-1 and pERK. Bottom: WT lung fibroblasts with HrasV12 retrovirus were also subjected to IP with TSP-1, ERK, pERK, and MEK-1/2 antibodies probed for MEK-1/2, ERK, and pERK. (B) Interaction of TSP-1 with pERK in vivo was assessed by Duolink immunostaining for TSP-1 and pERK in KrasG12D×Tsp-1+/+ and KrasG12D×Tsp-1–/– lung tumors 21 weeks after Ad-Cre infection. (C) Subcellular localization of pERK, determined by immunofluorescence staining for pERK in KrasG12D×Tsp-1+/+ and KrasG12D×Tsp-1–/– lung tumors 21 weeks after Ad-Cre infection. (D) Subcellular localization of pERK, assessed by cell fractionation followed by immunoblotting for pERK, in HrasV12-infected Tsp-1+/+ and Tsp-1–/– lung fibroblasts. Lamin B1 and α-tubulin served as markers for nuclear (N) and cytosolic (C) compartments, respectively. Nuclear relative to cytosolic gel density was quantified by densitometry. (E) pp90RSK and pELK-1, assessed by immunoblotting, in Tsp-1+/+ and Tsp-1–/– lung fibroblasts after retroviral HrasV12 infection. β-actin served as loading control. Gel density of phospho- relative to total substrates was quantified by densitometry. (F) TSP-1 coprecipitated with pERK, even in the presence of neutralizing TSP-1 antibody in the culture media. WT primary lung fibroblasts were infected with HrasV12 retrovirus and treated with 10 μg/ml neutralizing TSP-1 antibody (nAb) or an isotype-matched control (IgG) for 10 days, followed by IP with anti–TSP-1 and immunoblot for TSP-1 and pERK. Scale bars: 100 μm (B); 50 μm (C); 25 μm (C, insets).