A powerful way to discover key genes playing causal roles in oncogenesis is to identify genomic regions that undergo frequent alteration in human cancers. Here, we report high-resolution analyses of somatic copy-number alterations (SCNAs) from 3131 cancer specimens, belonging largely to 26 histological types. We identify 158 regions of focal SCNA that are altered at significant frequency across multiple cancer types, of which 122 cannot be explained by the presence of a known cancer target gene located within these regions. Several gene families are enriched among these regions of focal SCNA, including the BCL2 family of apoptosis regulators and the NF-κB pathway. We show that cancer cells harboring amplifications surrounding the MCL1 and BCL2L1 anti-apoptotic genes depend upon expression of these genes for survival. Finally, we demonstrate that a large majority of SCNAs identified in individual cancer types are present in multiple cancer types.

Raf kinase inhibitor protein (RKIP) is a modulator of cell signaling that functions as an endogenous inhibitor of multiple kinases. We demonstrate here a positive role for RKIP in the regulation of cell locomotion. We discovered that RKIP is the relevant cellular target of locostatin, a cell migration inhibitor. Locostatin abrogates RKIP's ability to bind and inhibit Raf-1 kinase, and it acts by disrupting a protein-protein interaction, an uncommon mode of action for a small molecule. Small interfering RNA-mediated silencing of RKIP expression also reduces cell migration rate. Overexpression of RKIP converts epithelial cells to a highly migratory fibroblast-like phenotype, with dramatic reduction in the sensitivity of cells to locostatin. RKIP is therefore the compound's valid target and a key regulator of cell motility.

No abstract

Raf kinase inhibitor protein (RKIP) regulates a number of cellular processes, including cell migration. Exploring the role of RKIP in cell adhesion, we found that overexpression of RKIP in Madin-Darby canine kidney (MDCK) epithelial cells increases adhesion to the substratum, while decreasing adhesion of the cells to one another. The level of the adherens junction protein E-cadherin declines profoundly, and there is loss of normal localization of the tight junction protein ZO-1, while expression of the cell-substratum adhesion protein beta1 integrin dramatically increases. The cells also display increased adhesion and spreading on multiple substrata, including collagen, gelatin, fibronectin and laminin. In three-dimensional culture, RKIP overexpression leads to marked cell elongation and extension of long membrane protrusions into the surrounding matrix, and the cells do not form hollow cysts. RKIP-overexpressing cells generate considerably more contractile traction force than do control cells. In contrast, RNA interference-based silencing of RKIP expression results in decreased cell-substratum adhesion in both MDCK and MCF7 human breast adenocarcinoma cells. Treatment of MDCK and MCF7 cells with locostatin, a direct inhibitor of RKIP and cell migration, also reduces cell-substratum adhesion. Silencing of RKIP expression in MCF7 cells leads to a reduction in the rate of wound closure in a scratch-wound assay, although not as pronounced as that previously reported for RKIP-knockdown MDCK cells. These results suggest that RKIP has important roles in the regulation of cell adhesion, positively controlling cell-substratum adhesion while negatively controlling cell-cell adhesion, and underscore the complex functions of RKIP in cell physiology.

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.