Oncogenic activation of tyrosine kinases is a common mechanism of carcinogenesis and, given the druggable nature of these enzymes, an attractive target for anticancer therapy. Here, we show that somatic mutations of the fibroblast growth factor receptor 2 (FGFR2) tyrosine kinase gene, FGFR2, are present in 12% of endometrial carcinomas, with additional instances found in lung squamous cell carcinoma and cervical carcinoma. These FGFR2 mutations, many of which are identical to mutations associated with congenital craniofacial developmental disorders, are constitutively activated and oncogenic when ectopically expressed in NIH 3T3 cells. Inhibition of FGFR2 kinase activity in endometrial carcinoma cell lines bearing such FGFR2 mutations inhibits transformation and survival, implicating FGFR2 as a novel therapeutic target in endometrial carcinoma.endometrial cancer ͉ fibroblast growth factor receptor 2 ͉ oncogene ͉ targeted therapy ͉ tyrosine kinase T yrosine kinases play a major role in transduction of proliferative signals and can become oncogenic when deregulated by somatic mutation (1). Somatically altered tyrosine kinases have proven to be tractable therapeutic targets in several tumor types; examples of successfully targeted tyrosine kinases include ABL1 in chronic myeloid leukemia (2), KIT in gastrointestinal stromal tumors (3), ERBB2 in breast cancer (4), and EGFR in non-small-cell lung cancer (5-7). The tyrosine kinase family has not been exhaustively studied in human cancer, and it is likely that additional tyrosine kinase therapeutic targets remain to be discovered.The fibroblast growth factor receptor (FGFR) tyrosine kinase family, which is comprised of four kinases that differentially respond to 18 FGF ligands (8, 9), has long been implicated in cancer. Translocations involving FGFR3, and activating somatic mutations in FGFR3, have been identified in multiple myeloma patients (10, 11), and translocations of FGFR1 have been found in patients with 8p11 myeloproliferative syndrome (12). Isolated cases of a missense mutation of FGFR4 in a lung adenocarcinoma patient and missense mutations of FGFR2 in a lung squamous cell carcinoma patient and gastric cancer patient have also been reported (13,14). In addition to these documented examples of somatic mutation of FGFR family members in cancer, a germ-line polymorphism in the second intron of FGFR2 was found to be associated with breast cancer in genomewide association studies (15,16).FGFR1-FGFR3 are characterized by alternative splicing of the mRNA encoding the third Ig-like repeat in the extracellular ligand-binding domain. This differential splicing determines ligand specificity such that isoforms expressed primarily in epithelial cells (IIIb) preferentially bind FGF ligands expressed by mesenchymal cells, and isoforms expressed primarily in mesenchymal cells (IIIc) preferentially bind FGF ligands expressed by epithelial cells (17)(18)(19). Alteration of this restricted expression pattern can lead to oncogenic transformation (20). Mutations in FGFR2 and FGF...

A defining hallmark of primary and metastatic cancers is the migration and invasion of malignant cells. These invasive properties involve altered dynamics of the cytoskeleton and one of its major structural components β-actin. Here we identify AIM1 (absent in melanoma 1) as an actin-binding protein that suppresses pro-invasive properties in benign prostate epithelium. Depletion of AIM1 in prostate epithelial cells increases cytoskeletal remodeling, intracellular traction forces, cell migration and invasion, and anchorage-independent growth. In addition, decreased AIM1 expression results in increased metastatic dissemination in vivo. AIM1 strongly associates with the actin cytoskeleton in prostate epithelial cells in normal tissues, but not in prostate cancers. In addition to a mislocalization of AIM1 from the actin cytoskeleton in invasive cancers, advanced prostate cancers often harbor AIM1 deletion and reduced expression. These findings implicate AIM1 as a key suppressor of invasive phenotypes that becomes dysregulated in primary and metastatic prostate cancer.

Methylated DNA binding proteins such as Methyl-CpG Binding Domain Protein 2 (MBD2) can transduce DNA methylation alterations into a repressive signal by recruiting transcriptional co-repressor complexes. Interfering with MBD2 could lead to re-activation of tumor suppressor genes and therefore represents an attractive strategy for epigenetic therapy. We developed and compared fluorescence polarization (FP) and time resolved fluorescence resonance energy transfer (TR-FRET)-based high-throughput screening (HTS) assays to identify small molecule inhibitors of the interaction between the methyl binding domain of MBD2 (MBD2-MBD) and methylated DNA. While both assays performed well in 96-well format, the TR-FRET assay (Z’ factor = 0.58) emerged as a superior screening strategy compared to FP (Z’ factor = 0.08) when evaluated in a HTS 384 well plate format. Using TR-FRET we screened the Sigma® LOPAC library for MBD2-MBD inhibitors and identified 4 compounds that also validated in a dose response series. This included two known DNA intercalators (mitoxantrone and idarubicin) amongst two other inhibitory compounds (NF449, and aurintricarboxylic acid). All four compounds also inhibited the binding of SP-1, a transcription factor with a GC-rich binding sequence, to a methylated oligonucleotide demonstrating that the activity was nonspecific. Our results provide proof-of-principle for using TR-FRET-based HTS to identify small molecule inhibitors of MBD2 and other DNA-protein interactions.