Abstract B22: Dynamics of cancer cell survival dependency on Bcl-2 family members

Abstract

Apoptosis is regulated through the interactions of the Bcl-2 family of proteins. Overexpression of anti-apoptotic Bcl-2 family members plays a key role in promoting survival for cells undergoing various insults that would normally trigger cell death. Consequently, these proteins are believed to serve as contributing factors to the processes of tumor initiation, tumor progression and the development of resistance to therapeutics. ABT-263, an antagonist of Bcl-2, Bcl-xL, and Bcl-w, is currently being tested in clinical trials. Some cancer cells express high amounts of Mcl-1, another Bcl-2 family anti-apoptotic protein that shares pro-survival function with Bcl-2 but is not targeted by ABT-263. Preclinical data indicates Mcl-1 overexpressing cells are not sensitive to ABT-263. To understand this mechanism of Mcl-1 dependence, A2058 melanoma cells were infected with a lentivirus encoding inducible shRNA against Mcl-1. Upon induction of MCL1 knockdown, massive cell death occurred. Interestingly, a small subset of the cell population managed to survive. Subsequent western blot analysis confirmed a sustained depletion of Mcl-1. Further study indicated A2058 cells deficient in Mcl-1 survived by upregulating expression of Bcl-2, a process we term “switching dependency”. In addition, these cells became sensitized to ABT-263 treatment. An A2058 xenograft model indicates that combined knockdown of both Mcl-1 and Bcl-2 significantly inhibited tumor growth when compared to tumors treated with ABT-263 lacking either protein individually. Real-time kinetic studies reveal a rapid sensitization of A2058 cells to ABT-263 upon sh-RNA induction. To understand whether de novo protein synthesis of Bcl-2 plays a mechanistic role in dependency switching, A2058 sh-Mcl-1 cells were treated with the protein synthesis inhibitor cycloheximide. Kinetic analysis indicates that de novo protein synthesis is not required for dependency switching. Real-time quantitative PCR was then employed to determine if a relationship exists between the decrease in Mcl-1 mRNA, and subsequent sensitivity to ABT-263, is a consequence of a change in Bcl-2 mRNA levels. Surprisingly, the results indicate as the loss of Mcl-1 message occurs, there is no concurrent change in Bcl-2 mRNA. Taken together these data indicate that dependency switching, and therefore survival, occurs without de novo Bcl-2 protein synthesis or alterations in protein turnover. Furthermore, dependency switching is a possible mechanism cancer cells employ to develop resistance toward various anti-cancer therapies with novel implications for both the concept of oncogene addiction and development of targeted cancer therapeutics. Therefore there is an immediate need for elucidation of this novel cancer resistance mechanism. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B22.