Wilms tumor 1 (WT1) regulates KRAS-driven oncogenesis and senescence in mouse and human models
Silvestre Vicent, Ron Chen, Leanne C. Sayles, Chenwei Lin, Randal G. Walker, Anna K. Gillespie, Aravind Subramanian, Gregory Hinkle, Xiaoping Yang, Sakina Saif, David E. Root, Vicki Huff, William C. Hahn, E. Alejandro Sweet-Cordero
Silvestre Vicent, Ron Chen, Leanne C. Sayles, Chenwei Lin, Randal G. Walker, Anna K. Gillespie, Aravind Subramanian, Gregory Hinkle, Xiaoping Yang, Sakina Saif, David E. Root, Vicki Huff, William C. Hahn, E. Alejandro Sweet-Cordero
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Research Article

Wilms tumor 1 (WT1) regulates KRAS-driven oncogenesis and senescence in mouse and human models

Abstract

KRAS is one of the most frequently mutated human oncogenes. In some settings, oncogenic KRAS can trigger cellular senescence, whereas in others it produces hyperproliferation. Elucidating the mechanisms regulating these 2 drastically distinct outcomes would help identify novel therapeutic approaches in RAS-driven cancers. Using a combination of functional genomics and mouse genetics, we identified a role for the transcription factor Wilms tumor 1 (WT1) as a critical regulator of senescence and proliferation downstream of oncogenic KRAS signaling. Deletion or suppression of Wt1 led to senescence of mouse primary cells expressing physiological levels of oncogenic Kras but had no effect on wild-type cells, and Wt1 loss decreased tumor burden in a mouse model of Kras-driven lung cancer. In human lung cancer cell lines dependent on oncogenic KRAS, WT1 loss decreased proliferation and induced senescence. Furthermore, WT1 inactivation defined a gene expression signature that was prognostic of survival only in lung cancer patients exhibiting evidence of oncogenic KRAS activation. These findings reveal an unexpected role for WT1 as a key regulator of the genetic network of oncogenic KRAS and provide important insight into the mechanisms that regulate proliferation or senescence in response to oncogenic signals.

Authors

Silvestre Vicent, Ron Chen, Leanne C. Sayles, Chenwei Lin, Randal G. Walker, Anna K. Gillespie, Aravind Subramanian, Gregory Hinkle, Xiaoping Yang, Sakina Saif, David E. Root, Vicki Huff, William C. Hahn, E. Alejandro Sweet-Cordero

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Figure 5

WT1 is a critical regulator of RAS-driven oncogenesis in human lung cancer.

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WT1 is a critical regulator of RAS-driven oncogenesis in human lung canc...
(A) Western blot showing WT1 suppression by 2 independent shRNAs in nuclear lysates of wild-type RAS NSCLC cells, NCI-H1568 (H1568), and mutant RAS NSCLC cells, NCI-H23 (H23). (B) Cell viability analysis of human NSCLC cell lines infected with WT1 shRNAs as measured by an MTT assay at day 5 after selection. RAS wild-type cell lines: NCI-H522, NCI-H1437, NCI-H1568, NCI-H1650, NCI-H1975, and NCI-H2126. RAS mutant cell lines: NCI-H23, NCI-H358, NCI-H441, NCI-H460, NCI-H727, NCI-H1299, NCI-H2009, and NCI-A549. The same WT1 shRNAs were used for all experiments (squares: WT1 shRNA1; circles: WT1 shRNA2). Data points indicate percentage of cell viability (percentage of the ratio of WT1 shRNA–infected cells over GFP shRNA–infected cells). (C) Cell viability of pancreatic cancer cell lines with either wild-type (HPAF-II) or mutant KRAS (ASPC-1) after WT1 knockdown with the same shRNAs used in B. Results were normalized against cell viability of control cells transduced with a GFP shRNA. Error bars show mean ± SD. (D) Annexin V analysis of NSCLC cell lines infected with 2 WT1 shRNAs or a GFP shRNA. Wild-type (NCI-H1568, NCI-H1975, and NCI-H2126) and mutant (NCI-H23, NCI-A549, and NCI-H1299) RAS NSCLC cell lines were used. Data points show difference in annexin V staining in cells infected with WT1 shRNA compared with cells infected with control GFP shRNA. (E) Percentage change of BrdU-positive cells in NSCLC cell lines analyzed in D. Data points represent percentage change of BrdU incorporating cells between cells infected with WT1 shRNAs and control cells carrying a GFP shRNA. (F) Percentage of SA-βgal–positive cells in NSCLC cells used in D. Data points indicate percentage of SA-βgal–positive cells. (G) SA-βgal staining of mutant RAS NSCLC cell lines described in D. Percentage of positive cells is shown in parentheses. Arrows point at senescence cells. Scale bars: 75 μm. All graphs are representative of at least 2 experiments. For all experiments, P values are for a 2-tailed t test.