Since some hits are involved in intricate signaling pathways, there could be other drug targetable molecules within the same pathway, which could impact paclitaxel sensitivity. For these example, a top hit in our screen, RPS6KB1, is downstream of mTOR and PI3K, two prominent signaling pathways in breast cancer with known direct inhibitors, rapamycin and LY294002, and that have been shown to sensitize cells to paclitaxel. Three gene targets from our list were of particular interest. These genes encode proteins to which agents have been developed and thus we could test the com pounds in combination with paclitaxel for biological effect. The first was PPM1D, a member of the PP2C fam ily of serine theronine protein phosphatases, and a known negative regulator of cell stress response pathways including those regulated by p53, CHEK1, and p38 MAP kinase.

PPM1D is amplified and overexpressed in breast cancers and inhibition of its activity, through use of small molecules such as CCT007093, Inhibitors,Modulators,Libraries inhibits the growth of tumor cell lines that overexpress PPM1D. The second gene target of interest was SP1, a constitutively expressed transcription factor Inhibitors,Modulators,Libraries that regulates basal promoter activity of many housekeeping genes. SP1 binding activity has been shown to be higher in human breast carcinomas than in normal tissues and may play a role in tumorigenesis by regulating the expres sion of genes involved in angiogenesis, cell growth, and apoptosis resistance. Mithramycin A binds to dsDNA and inhibits SP1 binding sites thus inhibiting SP1 transcriptional activity.

Finally, TGFB1 is a ligand that regulates a signaling pathway that Inhibitors,Modulators,Libraries becomes Inhibitors,Modulators,Libraries deregulated in many types of malignancies including breast cancer. TGFB1 can act in a paracrine manner to promote tumor growth and can activate PI3K AKT, a signaling program associated with drug resistance. Thus, the ligand TGFB1 and its receptors TGFB receptor type I and II have been pursued as anti cancer targets. LY2109761 is a small mol ecule inhibitor of TGFBR I and II and has been shown to inhibit tumor cell migration, invasion, as well as sup pressing metastasis in vivo. Pharmacological agents enhance paclitaxel cell growth inhibition of breast cancer cells To observe potential enhanced activity of drug combina tions, IC50 concentrations of CCT007093 or mithramy cin were combined with a IC50 concentration Inhibitors,Modulators,Libraries of paclitaxel. These combinations resulted in increased growth inhibition of three breast cancer cell lines tested, MDA MB 231, MDA MB 468, and MCF 7 relative to single agent treatment. CCT007093 alone had little effect on MDA MB 231 or MDA MB 468 cell growth but significantly decreased proliferation in combination with paclitaxel, 47% sellckchem and 55% inhibition, respectively.