Background: Breast cancers can be subgrouped into ER-positive and ER-negative breast cancers according to their estrogen receptor status. Few effective targeted treatments are available for the more aggressive ER-negative breast cancers, 80% of which carry p53 mutation. Our lab conducted a human kinome study which identified 52 kinases highly expressed in ER-negative breast tumors. One highly expressed kinase, DAPK-1, was selected for further study. Death associated protein kinase-1 (DAPK-1) transduces death signaling through P53-dependent or -independent pathways. In this study, we determined a novel role of DAPK-1 in tumorigenesis of ER-negative, p53 mutant breast cancers.
Methods: To perform expression and survival analysis of DAPK-1 in breast tumors, we used data from VandeVijver, Desmedt and Ivshina datasets. Inducible DAPK-1 knockdown cell lines were constructed through pTRIPZ lentiviral system. Anchorage-independent growth was performed in soft agar and colonies were enumerated after 15-30 days. Xenograft experiments were performed in nude mice. Proteomic analysis was used to determine downstream signaling.
Results: Gene expression analysis showed that high DAPK-1 expression correlates significantly with the ER-negative subgroup of breast cancer. Using an inducible knockdown system, we found that depletion of DAPK-1 strongly suppressed in vitro growth and in vivo tumorigenesis of ER-negative, P53-mutant cells. Through a siRNA knockdown study in an expanded panel of breast cancer cell lines, we established an association between p53 mutation status and sensitivity of the cells to DAPK-1 depletion. ER-negative breast cancer cell lines with P53 mutations are very sensitive to DAPK-1 knockdown, while ER-positive breast cancer cell lines with wild-type P53 are resistant to DAPK-1 knockdown. By depleting P53 in P53 wild-type cells, we then showed that P53 wild-type breast cancer cells become partially dependent on DAPK-1 upon P53 dysfunction. Through proteomic analysis, we determined that DAPK-1 regulates cell growth through phosphorylating TSC2 at Ser939 and interfering with TSC1/TSC2 complex, which is a well-known repressor of mTOR pathway. Our survival analysis confirmed that high DAPK-1 expression in breast tumors is associated with worse clinical outcome, especially in patients that carry P53 mutations.
Conclusion: P53-mutant breast cancer cells are sensitive to DAPK-1 inhibition in vitro and in vivo. P53 dysfunction causes breast cancer cells to become dependent on DAPK-1 signaling for their growth. High expression of DAPK-1 in P53-mutant breast tumor activates the mTOR pathway through relieving the TSC1/TSC2-mediated repressor of mTOR signaling. Our results demonstrate a dual role of DAPK-1 in regulating breast cancer proliferation and apoptosis depending on the P53 status. This study is supported by a PROMISE grant (KG081694) from Susan G. Komen for the Cure Foundation.
Citation Format: Jing Zhao, Yun Zhang, Jamal L. Hill, Zack Hartman, Abhijit Mazumdar, Gordon B. Mills, Powel H. Brown. DAPK-1, a death signaling transducer, is critical for the tumorigenicity of p53 mutant breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2329. doi:10.1158/1538-7445.AM2013-2329