Members of the phosphoinositide 3-kinase (PI3K) family collectively control multiple cellular responses, including proliferation, growth, chemotaxis, and survival. These diverse effects can partly be attributed to the broad range of downstream effectors being regulated by the products of these lipid kinases, the 3 ¶-phosphoinositides. However, an additional layer of complexity is introduced by the existence of multiple PI3K enzyme isoforms. Much has been learned over the last years on the roles of the classes I and III PI3K members in cellular signaling, but little is known about the isoform-specific tasks done by the class II PI3Ks (C2A, B, and ;). In this study, we used quantitative reverse transcription -PCR and RNA interference in mammalian cells to gain further insight into the function of these lesser studied PI3K enzymes. We find that PI3K-C2A, but not PI3K-C2B, has an important role in controlling cell survival and by using a panel of RNA interference reagents, we were able to determine a critical threshold of PI3K-C2A mRNA levels, below which the apoptotic program is switched on, via the intrinsic cell death pathway. In addition, knockdown of PI3K-C2A to levels that by themselves do not induce apoptosis sensitize cells to the anticancer agent Taxol (paclitaxel). Lastly, we report that lowering the levels of PI3K-C2A in a number of cancer cell lines reduces their proliferation and cell viability, arguing that PI3K inhibitors targeting not only the class IA isoform but also class IIA may contribute to an effective anticancer strategy. (Mol Cancer Res 2008;6(4):614 -23)

Bone metastasis occurs frequently in advanced prostate cancer (PCa) patients; however, it is not known why this happens. The epidermal growth factor receptor (EGFR) ligand EGF is available to early stage PCa; whereas, TGF-α is available when PCa metastasizes. Since the microenvironment of metastases has been shown to play a role in the survival of the tumor, we examined whether the ligands had effects on cell survival and proliferation in early and late PCa. We used LNCaP cells as a model of early stage, non-metastatic PCa and the isogenic C4-2B cells as a model of late stage, metastatic PCa. We found that the proliferation factor MAPK and the survival factor AKT were differentially activated in the presence of different ligands. TGF-α induced growth of C4-2B cells and not of the parental LNCaP cells; however, LNCaP cells expressing a constitutively active AKT did proliferate with TGF-α. Therefore, AKT appeared to be the TGF-α-responsive factor for survival of the late stage PCa cells. LNCaP cells exposed to EGF produced more osteoprotegerin (OPG), an inhibitor of bone remodeling, than C4-2B cells with TGF-α, which had increased expression of RANKL, an activator of bone remodeling. In concordance, TGF-α-treated C4-2B conditioned medium was able to differentiate an osteoclast precursor line to a greater extent than EGF-treated C4-2B or TGF-α-treated LNCaP conditioned media. Taken together, these results suggest that the switch in EGFR ligand availability as PCa progresses affects cell survival and contributes to bone remodeling.

RNA interference (RNAi) has emerged as one of the most powerful tools for functionally characterizing large sets of genomic data. Capabilities of RNAi place it at the forefront of high-throughput screens, which are able to span the human genome in search of novel targets. Although RNAi screens have been used to elucidate pathway components and discover potential drug targets in lower organisms, including Caenorhabditis elegans and Drosophila, only recently has the technology been advanced to a state in which large-scale screens can be performed in mammalian cells. In this review, we will evaluate the major advancements in the field of mammalian RNAi, specifically in terms of high-throughput assays. Crucial points of experimental design will be highlighted, as well as suggestions as to how to interpret and follow-up on potential cell death targets. Finally, we assess the prospective applications of high-throughput screens, the data they are capable of generating, and the potential for this technique to further our understanding of human disease.

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