The type 1 insulin-like growth factor receptor (lGF-1 R) plays a significant role in tumor growth and spread, and IGF-1 R inhibitors and antibodies are now undergoing clinical testing. However, factors that regulate sensitivity to IGF-1 R inhibition remain unclear. The aim of this project is to identify proteins whose depletion regulates sensitivity to IGF-1 R inhibition, in order to design effective combination treatments to benefit patients. An IGF-1 R kinase inhibitor, AZ12253801 (provided by AstraZeneca) was able to block IGF-induced phosphorylation of IGF-1 R in DU145 prostate cancer and MCF-7 breast cancer cells, inhibited downstream signalling in DU145 cells, and also inhibited proliferation and cell survival of both cell lines. AZ12253801 was used in an unbiased siRNA screen in both cell lines, using two s'iRNA libraries (779 kinase-related Kinome and 230 DNA repair-associated siRNAs). Eight Kinome and five DNA repair-associated hits have been identified after primary and second round screens, and further validated. The strongest hit was dishevelled homolog 3 (DVL3), a member of the WNT signalling pathway, which is highly expressed in both cell lines. DVL3 silencing caused reduction in active l3-catenin and inactivated the mTOR pathway, consistent with previous studies, and did not affect IGF-1 Rand AKT activity. However, DVL3 silencing led to activation of MEK1/2-ERK1/2 in serum-starved cells and sensitized this pathway to IGF-1 stimulation, with translocation of ERK1/2 into the nucleus and increased expression of ERK1/2 target genes. A DVL PDZ domain inhibitor (DVLi) showed similar effects on active l3-catenin, mTOR signalling and ERK1/2 signalling activity. The administration of DVLi increased sensitivity to AZ12253801 in cell lines with detectable ERK1/2 activation, but not prostate cancer cells in which ERK signalling was suppressed and AKT was activated in the context of loss of functional PTEN. Furthermore, DVL3 regulated activation of ERKs by influencing signaling downstream of the IGF-1 R and upstream of RAS, and DVL3 was found in a complex with the adaptor proteins GRB2 and DAB2. GRB2 knockdown was capable of abolishing ERK1/2 activation induced by DVLi, further implicating involvement of GRB2, and DAB2 silencing sensitized to IGF-1 R inhibition, mimicking effects of DVL3 depletion. Taken together, DVL3 silencing or inhibition enhances sensitivity to IGF-1 R inhibition by negatively regulating the ERK1/2 signaling pathway. These investigations shed new light on the factors that regulate IGF signaling, and provide a rational basis for design of clinical trials of IGF-1 R inhibitors.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:565965 |
| Date | January 2012 |
| Creators | Gao, Shan |
| Contributors | Macaulay, Valentin |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
Page generated in 0.0017 seconds