Death-associated protein kinase 1 promotes growth of p53-mutant cancers
Jing Zhao, … , Gordon B. Mills, Powel H. Brown
Jing Zhao, … , Gordon B. Mills, Powel H. Brown
Published June 15, 2015
Citation Information: J Clin Invest. 2015;125(7):2707-2720. https://doi.org/10.1172/JCI70805.
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Research Article Oncology

Death-associated protein kinase 1 promotes growth of p53-mutant cancers

Abstract

Estrogen receptor–negative (ER-negative) breast cancers are extremely aggressive and associated with poor prognosis. In particular, effective treatment strategies are limited for patients diagnosed with triple receptor–negative breast cancer (TNBC), which also carries the worst prognosis of all forms of breast cancer; therefore, extensive studies have focused on the identification of molecularly targeted therapies for this tumor subtype. Here, we sought to identify molecular targets that are capable of suppressing tumorigenesis in TNBCs. Specifically, we found that death-associated protein kinase 1 (DAPK1) is essential for growth of p53-mutant cancers, which account for over 80% of TNBCs. Depletion or inhibition of DAPK1 suppressed growth of p53-mutant but not p53-WT breast cancer cells. Moreover, DAPK1 inhibition limited growth of other p53-mutant cancers, including pancreatic and ovarian cancers. DAPK1 mediated the disruption of the TSC1/TSC2 complex, resulting in activation of the mTOR pathway. Our studies demonstrated that high DAPK1 expression causes increased cancer cell growth and enhanced signaling through the mTOR/S6K pathway; evaluation of multiple breast cancer patient data sets revealed that high DAPK1 expression associates with worse outcomes in individuals with p53-mutant cancers. Together, our data support targeting DAPK1 as a potential therapeutic strategy for p53-mutant cancers.

Authors

Jing Zhao, Dekuang Zhao, Graham M. Poage, Abhijit Mazumdar, Yun Zhang, Jamal L. Hill, Zachary C. Hartman, Michelle I. Savage, Gordon B. Mills, Powel H. Brown

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Figure 6

RPPA analysis identifies DAPK1 as a modulator of mTOR pathway.

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RPPA analysis identifies DAPK1 as a modulator of mTOR pathway. RPPA anal...
RPPA analysis was performed using triplicate samples of MCF7, HCC1143, MDAMB 231, and MDAMB 468 cells with inducible DAPK1 knockdown capacity. (A) Heat maps of protein level (left panel) and protein level change (middle panel) in cells before (–Doxy) and after (+Doxy) DAPK1 knockdown. (B) Level of phospho-S6 at Ser235/236 and Ser240/244 and of phospho-S6K at T389 before and after DAPK1 knockdown in HCC1143 cells. (C) Expression of phospho-S6 at Ser235/236 and Ser240/244 and of phospho-S6K at T389 before and after DAPK1 knockdown in MCF7 cells. (D) Growth and levels of phospho-S6 at Ser235/236 in HCC1143 ctrl and DAPK1 overexpression cell lines. Western blot analysis was done 3 times. A representative blot and quantification of phospho-S6/S6 ratio are shown. (E) Co-IP of TSC1/TSC2 complex using TSC1 antibody in HCC1143 before and after DAPK1 knockdown. This experiment was done 3 times. A representative IP Western blot and quantification of TSC2/TSC1 ratio are shown. (F) Co-IP of TSC1/TSC2 complex using TSC1 antibody in HCC1143 before and after DAPK1 overexpression. This experiment was done 3 times. A representative IP Western blot is shown, and quantification of the TSC2/TSC1 ratio is shown. (G) Phosphorylation of TSC2 by DAPK1 in in vitro kinase assay. (H) Level of phospho-TSC2 at Ser939 in HCC1143 control and HCC1143 DAPK1 overexpression cell lines. All lysates were collected 4 days after treatment. Western blot was performed 3 times. A representative blot and quantification of phospho-TSC2/TSC2 are shown. (I) Fold induction of apoptosis in HCC1143 (p53-mutant) and MCF7 (p53-WT) cells treated with 50 ng/ml TNF-α for 48 hours, measured by FACS analysis of annexin V/PI staining. Cell lines were either shEmpty vector control or shDAPK1-induced, as indicated. Each data point represents 3 technical replicates, with results reported as average ± SEM. *P < 0.01; **P < 0.001, 2-tailed Student’s t test.