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Soft Tissue Sarcoma Cells Are Highly Sensitive to AKT Blockade: A Role for p53-Independent Up-regulation of GADD45

Departments of ¹ Surgical Oncology, ² Systems Biology, ³ Cancer Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas; and ⁴ Department of Radiation Oncology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania

Requests for reprints: Dina Lev, Department of Cancer Biology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 1104, Houston, TX 77030. Phone: 713-792-1637; Fax: 713-563-1185; E-mail: dlev{at}mdanderson.org.

Key Words: soft tissue sarcoma • AKT • GADD45 • G₂ arrest • p53 • apoptosis

The AKT signaling pathway is activated in soft tissue sarcoma (STS). However, AKT blockade has not yet been studied as a potential targeted therapeutic approach. Here, we examined the in vitro and in vivo effects of AKT inhibition in STS cells. Western blot analysis was used to evaluate the expression of AKT pathway components and the effect of AKT stimulation and inhibition on their phosphorylation. Cell culture assays were used to assess the effect of AKT blockade (using a phosphatidylinositol 3-kinase inhibitor and a specific AKT inhibitor) on STS cell growth, cell cycle, and apoptosis. Oligoarrays were used to determine gene expression changes in response to AKT inhibition. Reverse transcription–PCR was used for array validation. Specific small inhibitory RNA was used to knockdown GADD45. Human STS xenografts in nude mice were used for in vivo studies, and immunohistochemistry was used to assess the effect of treatment on GADD45 expression, proliferation, and apoptosis. Multiple STS cell lines expressed activated AKT. AKT inhibition decreased STS downstream target phosphorylation and growth in vitro; G₂ cell cycle arrest and apoptosis were also observed. AKT inhibition induced GADD45 mRNA and protein expression in all STS cells treated independent of p53 mutational status. GADD45 knockdown attenuated the G₂ arrest induced by AKT inhibition. In vivo, AKT inhibition led to decreased STS xenograft growth. AKT plays a critical role in survival and proliferation of STS cells. Modulation of AKT kinase activity may provide a novel molecularly based strategy for STS-targeted therapies. [Cancer Res 2008;68(8):2895–903]