Sphingosine kinase 1 (SK1) is a signaling enzyme that catalyzes the formation of sphingosine-1-phosphate. Overexpression of SK1 is causally associated with breast cancer progression and resistance to therapy. SK1 inhibitors are currently being investigated as promising breast cancer therapies. Two major transcriptional isoforms, SK143 kDa and SK151 kDa, have been identified; however, the 51 kDa variant is predominant in breast cancer cells. No studies have investigated the protein-protein interactions of the 51 kDa isoform and whether the two SK1 isoforms differ significantly in their interactions. Seeking an understanding of the regulation and role of SK1, we used a triple-labeling stable isotope labeling by amino acids in cell culture-based approach to identify SK1-interacting proteins common and unique to both isoforms. Of approximately 850 quantified proteins in SK1 immunoprecipitates, a high-confidence list of 30 protein interactions with each SK1 isoform was generated via a meta-analysis of multiple experimental replicates. Many of the novel identified SK1 interaction partners such as supervillin, drebrin, and the myristoylated alanine-rich C-kinase substrate-related protein supported and highlighted previously implicated roles of SK1 in breast cancer cell migration, adhesion, and cytoskeletal remodeling. Of these interactions, several were found to be exclusive to the 43 kDa isoform of SK1, including the protein phosphatase 2A, a previously identified SK1-interacting protein. Other proteins such as allograft inflammatory factor 1-like protein, the latent-transforming growth factor β-binding protein, and dipeptidyl peptidase 2 were found to associate exclusively with the 51 kDa isoform of SK1. In this report, we have identified common and isoform-specific SK1-interacting partners that provide insight into the molecular mechanisms that drive SK1-mediated oncogenicity.

A gene-directed enzyme pro-drug therapy (GDEPT) based on purine nucleoside phosphorylase (PNP)

Advanced prostate cancer is invariably lethal once it becomes androgen independent (AI). With the aim of developing a new treatment we have used the human androgen-independent prostate cancer cell line, PC-3, to evaluate the effectiveness of two enzyme-directed prodrug therapy (EPT) systems as a novel means for promoting tumor cell destruction in vivo. We have confined our study to the use of a PSA promoter, in a preliminary attempt to achieve prostate specificity. The two EPT systems used were the HSVTK/GCV and PNP/6MPDR systems. These were chosen for their differential dependence on DNA replication for their mechanism of action. In the present work, either the HSVTK or PNP gene, each controlled by a PSA promoter fragment, was delivered by an E1-, replication-deficient human adenovirus (Ad5) into PC-3 tumors growing subcutaneously in BALB/c nude mice. Tumors were injected with a single dose of recombinant Ad5 and mice were treated intraperitoneally with the appropriate prodrug, twice daily, for 6 days thereafter. The growth of established PC-3 tumors was significantly suppressed and host survival increased with a single course of HSVTK/GCV or PNP/6MPDR treatment. HSVTK/GCV-treated PC-3 tumor growth was 80% less than that of control treatments on day 33, while PNP/6MPDR-treated tumor growth was approximately 75% less than that of control treatments on day 52. Survival data showed that 20% of HSVTK/GCV- or PNP/6MPDR-treated animals lived >45 and >448 days, respectively, longer than control animals. These results demonstrate that both HSVTK/GCV and PNP/6MPDR therapies interrupt the growth of an aggressive human prostate cancer cell line in vivo.