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Phosphoinositide 3-kinase (PI3K) regulates the transcription factor hypoxia-inducible factor-1 (HIF-1) in thyroid carcinoma cells. Both pathways are associated with aggressive phenotype in thyroid carcinomas.Our objective was to assess the effects of the clinical PI3K inhibitor GDC-0941 and genetic inhibition of PI3K and HIF on metastatic behavior of thyroid carcinoma cells in vitro and in vivo.Vascular endothelial growth factor ELISA, HIF activity assays, proliferation studies, and scratch-wound migration and cell spreading assays were performed under various O(2) tensions [normoxia, hypoxia (1 and 0.1% O(2)), and anoxia] with or without GDC-0941 in a panel of four thyroid carcinoma cell lines (BcPAP, WRO, FTC133, and 8505c). Genetic inhibition was achieved by overexpressing phosphatase and tensin homolog (PTEN) into PTEN-null cells and by using a dominant-negative variant of HIF-1α (dnHIF). In vivo, human enhanced green fluorescence protein-expressing follicular thyroid carcinomas (FTC) were treated with GDC-0941 (orally). Spontaneous lung metastasis was confirmed by viewing enhanced green fluorescence protein-positive colonies cultured from lung tissue.GDC-0941 inhibited hypoxia/anoxia-induced HIF-1α and HIF-2α expression and HIF activity in thyroid carcinoma cells. Basal (three of four cell lines) and/or hypoxia-induced (four of four) secreted vascular endothelial growth factor was inhibited by GDC-0941, whereas selective HIF targeting predominantly affected hypoxia/anoxia-mediated secretion (P0.05-0.0001). Antiproliferative effects of GDC-0941 were more pronounced in PTEN mutant compared with PTEN-restored cells (P0.05). Hypoxia increased migration in papillary cells and cell spreading/migration in FTC cells (P0.01). GDC-0941 reduced spreading and migration in all O(2) conditions, whereas dnHIF had an impact only on hypoxia-induced migration (P0.001). In vivo, GDC-0941 reduced expression of HIF-1α, phospho-AKT, GLUT-1, and lactate dehydrogenase A in FTC xenografts. DnHIF expression and GDC-0941 reduced FTC tumor growth and metastatic lung colonization (P0.05).PI3K plays a prominent role in the metastatic behavior of thyroid carcinoma cells irrespective of O(2) tension and appears upstream of HIF activation. GDC-0941 significantly inhibited the metastatic phenotype, supporting the clinical development of PI3K inhibition in thyroid carcinomas.

Background The ataxia telangiectasia mutated and Rad3-related kinase (ATR) has a key role in the signalling of stalled replication forks and DNA damage to cell cycle checkpoints and DNA repair. It has long been recognised as an important target for cancer therapy but inhibitors have proved elusive. As NU6027, originally developed as a CDK2 inhibitor, potentiated cisplatin in a CDK2-independent manner we postulated that it may inhibit ATR. Methods Cellular ATR kinase activity was determined by CHK1 phosphorylation in human fibroblasts with inducible dominant-negative ATR-kinase dead expression and human breast cancer MCF7 cells. Cell cycle effects and chemo- and radiopotentiation by NU6027 were determined in MCF7 cells and the role of mismatch repair and p53 was determined in isogenically matched ovarian cancer A2780 cells. Results NU6027 is a potent inhibitor of cellular ATR activity (IC(50)=6.7 μM) and enhanced hydroxyurea and cisplatin cytotoxicity in an ATR-dependent manner. NU6027 attenuated G2/M arrest following DNA damage, inhibited RAD51 focus formation and increased the cytotoxicity of the major classes of DNA-damaging anticancer cytotoxic therapy but not the antimitotic, paclitaxel. In A2780 cells sensitisation to cisplatin was greatest in cells with functional p53 and mismatch repair (MMR) and sensitisation to temozolomide was greatest in p53 mutant cells with functional MMR. Importantly, NU6027 was synthetically lethal when DNA single-strand break repair is impaired either through poly(ADP-ribose) polymerase (PARP) inhibition or defects in XRCC1. Conclusion NU6027 inhibits ATR, impairing G2/M arrest and homologous recombination thus increasing sensitivity to DNA-damaging agents and PARP inhibitors. It provides proof of concept data for clinical development of ATR inhibitors.

Background: Radiotherapy is used in the treatment of over 50% of cancer patients. Although seen as a positive intervention, there are reports of radiation enhancing the metastatic characteristics of cells. We have previously observed that radiotherapy activates a number of pathways associated with tumour metastases and aggressive phenotype including Src and phosphoinositide 3-kinase (PI3K). Here we wished to relate pathway activation to cellular phenotype and to investigate the impact of pharmacological inhibition on radiation response and metastases. Methods: The effects of radiotherapy alone, and combined with Src (AZD0530) and/or PI3K (GDC-0941) inhibition, on cell adhesion, migration and clonogenic survival were assessed in vitro using well-characterised assays. Initial in vivo studies, using FTC133 xenografts, evaluated the effects of radiation treatment on the Src and PI3K pathways and inhibition thereof using pAKT (Ser473) and pFAK (Tyr861) as biomarkers. The effect of single versus combined pathway inhibition with radiation therapy (5x2Gy fractions) on primary tumour growth and lung metastasis (via ex vivo clonogenic assay) was then evaluated. Results: In vitro, radiation combined with Src and/or PI3K inhibition resulted in a reduction in cell adhesion, cell spreading and cell migration compared to radiation alone, with the combined effects greater than either inhibitor alone. Radiation both in vivo and in vitro enhanced activity of the PI3K/AKT and the Src/FAK axis. In vivo, radiation combined with Src or PI3K inhibition showed little effect on tumour growth but did reduce metastatic lung colonisation, compared to radiation alone, with Src inhibition proving more efficacious. When radiation was combined with inhibition of both pathways a significant reduction in both primary tumour growth and metastatic lung colonisation was observed. Conclusion: Radiation enhances multiple pathways, two predominant ones being PI3K and Src, and by using rationale drug combinations we saw improved local control and reduced metastatic spread. From these data we can suggest that the PI3K and Src pathways are involved in the radiation enhancement in metastatic phenotype and this provides potential for application of these therapies in the clinic. Citation Format: Emily J. Rowling, Brian Telfer, Paul Elvin, Kaye J. Williams. Analyzing the effects of radiotherapy on the metastatic phenotype: a role for combined therapeutic approaches incorporating Src and PI3K targeting. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3957. doi:10.1158/1538-7445.AM2014-3957