Dataset Information

Functional roles of Src and Fgr in ovarian carcinoma.

ABSTRACT:

Purpose

Src is an attractive target because it is overexpressed in a number of malignancies, including ovarian cancer. However, the effect of Src silencing on other Src family kinases (SFKs) is not known. We hypothesized that other SFK members could compensate for the lack of Src activity.

Experimental design

Cell viability after either Src or Fgr silencing was examined in ovarian cancer cell lines by MTT assay. Expression of SFKs after Src silencing in ovarian cancer cells was examined by real-time reverse transcriptase (RT)-PCR. Therapeutic effect of in vivo Src and/or Fgr silencing was examined using siRNA incorporated into chitosan nanoparticles (siRNA/CH-NP). Microvessel density, cell proliferation, and apoptosis markers were determined by immunohistochemical staining in ovarian tumor tissues.

Results

Src silencing enhanced cytotoxicity of docetaxel in both SKOV3ip1 and HeyA8 cells. In addition, Src silencing using siRNA/CH-NP in combination with docetaxel resulted in significant inhibition of tumor growth compared with control siRNA/CH-NP (81.8% reduction in SKOV3ip1, P = 0.017; 84.3% reduction in HeyA8, P < 0.005). These effects were mediated by decreased tumor cell proliferation and angiogenesis, and increased tumor cell apoptosis. Next, we assessed the effects of Src silencing on other SFK members in ovarian cancer cell lines. Src silencing resulted in significantly increased Fgr levels. Dual Src and Fgr silencing in vitro resulted in increased apoptosis that was mediated by increased caspase and AKT activity. In addition, dual silencing of Src and Fgr in vivo using siRNA/CH-NP resulted in the greatest reduction in tumor growth compared with silencing of either Src or Fgr alone in the HeyA8 model (68.8%, P < 0.05).

Conclusions

This study demonstrates that, in addition to Src, Fgr plays a biologically significant role in ovarian cancer growth and might represent an important target.

SUBMITTER: Kim HS

PROVIDER: S-EPMC3077122 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Src family kinases are often overexpressed and constitutively active in cancer cells, where they represent promising targets for therapeutic intervention. In myeloid hematopoietic cells, Hck, Lyn, and Fgr are associated with both chronic and acute forms of myeloid leukemia. All three of these kinases are expressed in acute myeloid leukemia (AML), where high-level expression correlates with poor survival. The pyrrolopyrimidine Src-family kinase inhibitor A-419259 (also known as RK-20449) has been shown to significantly inhibit patient-derived AML bone marrow cell growth in immunocompromised mice. Recent work has identified an N-phenylbenzamide kinase inhibitor, known as TL02-59, which potently inhibited Fgr kinase activity in vitro and reversed orthotopic engraftment of AML cells in the bone marrow and spleen of immunocompromised mice following oral administration. The inhibitory mechanism of this compound with Fgr or other Src-family members has not been reported. Both wild-type Fgr and a tail mutant (i.e., replacement of Tyr527 with Phe) were shown to produce equal numbers of transformed colonies when expressed in rodent fibroblasts, whereas wild-type Hck transforming activity was completely repressed. In both Hck and Fgr, however, the tail tyrosine residue was shown to be phosphorylated, and hydrogen deuterium exchange mass spectrometry (HDX MS) of near-full-length Fgr was consistent with the closed conformation in solution, with the SH3 domain bound to the SH2-kinase linker and the tail bound to SH2. Here we present the first X-ray crystal structures of near-full-length Fgr bound to the ATP site inhibitors A-419259 and TL02-59. Structural changes induced by the inhibitors in the crystalline state were validated in solution using HDX MS, with A-419259 stabilizing the closed conformation and TL02-59 enhancing exposure of the SH3 domain to solvent. These new structures help to explain the constitutive activity of wild-type Fgr in cells as well as its unique sensitivity to TL02-59, which is currently in development for AML and other maladies.