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RAD51 is the central protein that catalyzes DNA repair via homologous recombination, a process that ensures genomic stability. RAD51 protein is commonly expressed at high levels in cancer cells relative to their noncancerous precursors. High levels of RAD51 expression can lead to the formation of genotoxic RAD51 protein complexes on undamaged chromatin. We developed a therapeutic approach that exploits this potentially toxic feature of malignancy, using compounds that stimulate the DNA-binding activity of RAD51 to promote cancer cell death. A panel of immortalized cell lines was challenged with the RAD51-stimulatory compound RS-1. Resistance to RS-1 tended to occur in cells with higher levels of RAD54L and RAD54B, which are Swi2/Snf2-related translocases known to dissociate RAD51 filaments from dsDNA. In PC3 prostate cancer cells, RS-1-induced lethality was accompanied by the formation of microscopically visible RAD51 nuclear protein foci occurring in the absence of any DNA-damaging treatment. Treatment with RS-1 promoted significant antitumor responses in a mouse model, providing proof-of-principle for this novel therapeutic strategy., (©2014 American Association for Cancer Research.)

Peroxisome proliferator-activated receptor (PPAR) represents a ligand-dependent nuclear receptor family that regulates multiple metabolic processes associated with fatty acid beta-oxidation, glucose utilization, and cholesterol transport. These and other receptor-mediated actions pertain to their role in hypolipidemic and antidiabetic therapies and as potential targets for cancer chemopreventive agents. The present study evaluated the chemopreventive activity of two highly potent and selective PPARgamma and PPARdelta agonists in a progestin- and carcinogen-induced mouse mammary tumorigenesis model. Animals treated with the PPARgamma agonist GW7845 exhibited a moderate delay in tumor formation. In contrast, animals treated with the PPARdelta agonist GW501516 showed accelerated tumor formation. Significantly, tumors from GW7845-treated mice were predominantly ductal adenocarcinomas, whereas tumors from GW501516-treated animals were adenosquamous and squamous cell carcinomas. Gene expression analysis of tumors arising from GW7845- and GW501516-treated mice identified expression profiles that were distinct from each other and from untreated control tumors of the same histopathology. Only tumors from mice treated with the PPARgamma agonist expressed estrogen receptor-alpha in luminal transit cells, suggesting increased ductal progenitor cell expansion. Tumors from mice treated with the PPARdelta agonist exhibited increased PPARdelta levels and activated 3-phosphoinositide-dependent protein kinase-1 (PDK1), which co-associated, suggesting a link between the known oncogenic activity of PDK1 in mammary epithelium and PPARdelta activation. These results indicate that PPARdelta and PPARgamma agonists produce diverse, yet profound effects on mammary tumorigenesis that give rise to distinctive histopathologic patterns of tumor differentiation and tumor development.

Activation of the PI3k/Akt pathway controls key cellular processes and contributes to tumorigenesis in vivo, but investigation of the PI3k/Akt pathway has been limited by the lack of specific inhibitors directed against Akt. To develop Akt inhibitors, we used molecular modeling of the pleckstrin homology (PH) domain of Akt to guide synthesis of structurally modified phosphatidylinositol ether lipid analogues (PIAs). Here, we characterize the biochemical and cellular effects of PIAs. Of 24 compounds tested, five PIAs with modifications at two sites on the inositol ring inhibited Akt with IC(50)s < 5 micro M. Molecular modeling identified putative interactions of PIAs with the phosphoinositide-binding site in the PH domain of Akt, and growth factor-induced translocation of Akt to the plasma membrane was inhibited by PIA administration. Inhibition of Akt occurred rapidly and was maintained for hours. PIAs decreased phosphorylation of many downstream targets of Akt without affecting upstream kinases, such as PI3k or phosphoinositide-dependent kinase-1, or members of other kinase pathways such as extracellular signal-regulated kinase. Importantly, PIAs increased apoptosis 20-30-fold in cancer cell lines with high levels of endogenous Akt activity but only 4-5-fold in cancer cell lines with low levels of Akt activity. These studies identify PIAs as effective Akt inhibitors, and provide proof of principle for targeting the PH domain of Akt.

Protein kinase C (PKC) has been implicated in enhancing cellular sensitivity to cis-diamminedichloroplatinum(II) (CP). We have synthesized a series of novel analogues of lyngbyatoxin A (7-linalylindolactam V), a natural tumor promoter and a potent activator of PKC, and investigated the effects of these synthetic compounds on PKC activity and the antiproliferative activity of CP. Lyngbyatoxin A was as effective as phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate, in enhancing the sensitivity of HeLa cells to CP. A 24-h pretreatment of HeLa cells with 1 to 100 nM lyngbyatoxin A caused an approximately 9-fold sensitization to CP. All analogues of lyngbyatoxin A that retained the lactam ring portion of the molecule but contained different hydrophobic substituents at C-7 including indolactam V (ILV), tert-butyl-ILV, or n-hexyl-ILV increased cellular sensitivity to CP in a concentration-dependent manner. Maximum cellular sensitization to CP (9-fold) was seen with 10 nM n-hexyl or tert-butyl compounds, and ILV devoid of any C-7 substitution required higher concentrations (1 microM) for equivalent sensitization. The ability of lyngbyatoxin A analogues to sensitize cells to CP correlated directly with their ability to activate PKC in vitro. Synthetic analogues that lacked the lactam ring structure neither activated PKC nor sensitized cells to CP. The C-9 epi analogue of n-hexyl-ILV was less effective than the corresponding natural stereoisomer in activating PKC as well as sensitizing cells to CP. Exposure of HeLa cells to 100 nM lyngbyatoxin A for 24 h caused a substantial decrease in cellular PKC activity to 20% of the untreated control value, but a similar treatment of cells with n-hexyl- or tert-butyl-ILV led to only a 25% reduction in PKC activity. Concentrations of ILV (e.g. 1 microM) that sensitized HeLa cells to CP caused no down-regulation of PKC. Thus, on the basis of results with these novel lyngbyatoxin A analogues, we conclude that activation but not down-regulation of PKC is necessary for sensitization of HeLa cells to CP.