Julia Resch

Expression of hypoxia-inducible factor 1α in thyroid carcinomas

Hypoxia-inducible factor 1α (HIF-1α) is upregulated by hypoxia and oncogenic signalling in many solid tumours. Its regulation and function in thyroid carcinomas are unknown. We evaluated the regulation of HIF-1α and target gene expression in primary thyroid carcinomas and thyroid carcinoma cell lines (BcPAP, WRO, FTC-133 and 8505c). HIF-1α was not detectable in normal tissue but was expressed in thyroid carcinomas. Dedifferentiated anaplastic tumours (ATCs) exhibited high levels of nuclear HIF-1α staining. The HIF-1 target glucose transporter 1 was expressed to a similar level in all tumour types, whereas carbonic anhydrase-9 was significantly elevated in ATCs. In vitro studies revealed a functionally active HIF-1α pathway in thyroid cells with transcriptional activation observed after graded hypoxia (1% O2, anoxia) or treatment with a hypoxia mimetic cobalt chloride. High basal and hypoxia-induced expression of HIF-1α in FTC-133 cells that harbour a phosphatase and tensin homologue (PTEN) mutation was reduced by introduction of wild-type PTEN. Similarly, pharmacological inhibition of the phosphoinositide 3-kinase (PI3K) pathway using LY294002 inhibited HIF-1α and HIF-1α targets in all cell lines, including those with B-RAF mutations (BcPAP and 8505c). In contrast, the effects of inhibition of the RAF/MEK/extracellular signal-regulated kinase pathway were restricted by environmental condition and B-RAF mutation status. HIF-1 is functionally expressed in thyroid carcinomas and is regulated not only by hypoxia but also via growth factor signalling pathways and, in particular, the PI3K pathway. Given the strong association of HIF-1α with an aggressive disease phenotype and therapeutic resistance, this pathway may be an attractive target for improved therapy in thyroid carcinomas.

GDC-0941 inhibits metastatic characteristics of thyroid carcinomas by targeting both the phosphoinositide-3 kinase (PI3K) and hypoxia-inducible factor-1α (HIF-1α) pathways.

CONTEXT 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. OBJECTIVE 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. DESIGN 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. RESULTS 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 (P < 0.05-0.0001). Antiproliferative effects of GDC-0941 were more pronounced in PTEN mutant compared with PTEN-restored cells (P < 0.05). Hypoxia increased migration in papillary cells and cell spreading/migration in FTC cells (P < 0.01). GDC-0941 reduced spreading and migration in all O(2) conditions, whereas dnHIF had an impact only on hypoxia-induced migration (P < 0.001). 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 (P < 0.05). CONCLUSIONS 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.

Hypoxia-inducible factor in thyroid carcinoma.

Intratumoural hypoxia (low oxygen tension) is associated with aggressive disease and poor prognosis. Hypoxia-inducible factor-1 is a transcription factor activated by hypoxia that regulates the expression of genes that promote tumour cell survival, progression, metastasis, and resistance to chemo/radiotherapy. In addition to hypoxia, HIF-1 can be activated by growth factor-signalling pathways such as the mitogen-activated protein kinases- (MAPK-) and phosphatidylinositol-3-OH kinases- (PI3K-) signalling cascades. Mutations in these pathways are common in thyroid carcinoma and lead to enhanced HIF-1 expression and activity. Here, we summarise current data that highlights the potential role of both hypoxia and MAPK/PI3K-induced HIF-1 signalling in thyroid carcinoma progression, metastatic characteristics, and the potential role of HIF-1 in thyroid carcinoma response to radiotherapy. Direct or indirect targeting of HIF-1 using an MAPK or PI3K inhibitor in combination with radiotherapy may be a new potential therapeutic target to improve the therapeutic response of thyroid carcinoma to radiotherapy and reduce metastatic burden.

Wnt signaling in estrogen-induced lactotroph proliferation

Prolactinomas are the most common type of functioning pituitary adenoma in humans, but the control of lactotroph proliferation remains unclear. Here, using microarray analysis, we show that estrogen treatment increased expression of Wnt4 mRNA in adult Fischer rat pituitary tissue. Dual immunofluorescence analysis revealed that Wnt4 expression was not confined to lactotrophs, but that it was expressed in all anterior pituitary cell types. Estradiol induced proliferation in the somatolactotroph GH3 cell line, in parallel with Wnt4 mRNA and protein induction. A reporter gene assay for TCF- and LEF-dependent transcription revealed that there was no activation of the canonical Wnt pathway in GH3 cells upon stimulation with Wnt-conditioned culture medium or coexpression of constitutively active mutant β-catenin. Expression of β-catenin in both GH3 cells and normal rat anterior pituitary cells was restricted to the cell membrane and was unaltered by treatment with estradiol, with no nuclear β-catenin being detected under any of the conditions tested. We show for the first time that Wnt4 affects non-canonical signaling in the pituitary by inhibiting Ca2+ oscillations in GH3 cells, although the downstream effects are as yet unknown. In summary, Wnt4 is expressed in the adult pituitary gland, and its expression is increased by estrogen exposure, suggesting that its involvement in adult tissue plasticity is likely to involve β-catenin-independent signaling pathways.

Expression of hypoxia-inducible factor 1 in thyroid carcinomas

Hypoxia-inducible factor 1α (HIF-1α) is upregulated by hypoxia and oncogenic signalling in many solid tumours. Its regulation and function in thyroid carcinomas are unknown. We evaluated the regulation of HIF-1α and target gene expression in primary thyroid carcinomas and thyroid carcinoma cell lines (BcPAP, WRO, FTC-133 and 8505c). HIF-1α was not detectable in normal tissue but was expressed in thyroid carcinomas. Dedifferentiated anaplastic tumours (ATCs) exhibited high levels of nuclear HIF-1α staining. The HIF-1 target glucose transporter 1 was expressed to a similar level in all tumour types, whereas carbonic anhydrase-9 was significantly elevated in ATCs. In vitro studies revealed a functionally active HIF-1α pathway in thyroid cells with transcriptional activation observed after graded hypoxia (1% O2, anoxia) or treatment with a hypoxia mimetic cobalt chloride. High basal and hypoxia-induced expression of HIF-1α in FTC-133 cells that harbour a phosphatase and tensin homologue (PTEN) mutation was reduced by introduction of wild-type PTEN. Similarly, pharmacological inhibition of the phosphoinositide 3-kinase (PI3K) pathway using LY294002 inhibited HIF-1α and HIF-1α targets in all cell lines, including those with B-RAF mutations (BcPAP and 8505c). In contrast, the effects of inhibition of the RAF/MEK/extracellular signal-regulated kinase pathway were restricted by environmental condition and B-RAF mutation status. HIF-1 is functionally expressed in thyroid carcinomas and is regulated not only by hypoxia but also via growth factor signalling pathways and, in particular, the PI3K pathway. Given the strong association of HIF-1α with an aggressive disease phenotype and therapeutic resistance, this pathway may be an attractive target for improved therapy in thyroid carcinomas.

Phosphatidylinositide 3-kinase (PI3K) and PI3K-related kinase (PIKK) activity contributes to radioresistance in thyroid carcinomas

Anaplastic (ATC) and certain follicular thyroid-carcinomas (FTCs) are radioresistant. The Phosphatidylinositide 3-kinase (PI3K) pathway is commonly hyperactivated in thyroid-carcinomas. PI3K can modify the PI3K-related kinases (PIKKs) in response to radiation: How PIKKs interact with PI3K and contribute to radioresistance in thyroid-carcinomas is unknown. Further uncertainties exist in how these interactions function under the radioresistant hypoxic microenvironment. Under normoxia/anoxia, ATC (8505c) and FTC (FTC-133) cells were irradiated, with PI3K-inhibition (via GDC-0941 and PTEN-reconstitution into PTEN-null FTC-133s) and effects on PIKK-activation, DNA-damage, clonogenic-survival and cell cycle, assessed. FTC-xenografts were treated with 5 × 2 Gy, ± 50 mg/kg GDC-0941 (twice-daily; orally) for 14 days and PIKK-activation and tumour-growth assessed. PIKK-expression was additionally assessed in 12 human papillary thyroid-carcinomas, 13 FTCs and 12 ATCs. GDC-0941 inhibited radiation-induced activation of Ataxia-telangiectasia mutated (ATM), ATM-and Rad3-related (ATR) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Inhibition of ATM and DNA-PKcs was PI3K-dependent, since activation was reduced in PTEN-reconstituted FTC-133s. Inhibition of PIKK-activation was greater under anoxia: Consequently, whilst DNA-damage was increased and prolonged under both normoxia and anoxia, PI3K-inhibition only reduced clonogenic-survival under anoxia. GDC-0941 abrogated radiation-induced cell cycle arrest, an effect most likely linked to the marked inhibition of ATR-activation. Importantly, GDC-0941 inhibited radiation-induced PIKK-activation in FTC-xenografts leading to a significant increase in time taken for tumours to triple in size: 26.5 ± 5 days (radiation-alone) versus 31.5 ± 5 days (dual-treatment). PIKKs were highly expressed across human thyroid-carcinoma classifications, with ATM scoring consistently lower. Interestingly, some loss of ATM and DNA-PKcs was observed. These data provide new insight into the mechanisms of hypoxia-associated radioresistance in thyroid-carcinoma.

Abstract 1483: Metastatic characteristics and radiosensitivity of thyroid carcinoma cells depends on HIF-1 and PI3K signaling in vitro and in vivo

The purpose of this study was to determine the importance of the HIF-1 and PI3K pathways in the development of a) a metastatic phenotype and b) desensitization to radiotherapy, in thyroid carcinomas. In vitro scratch wound migration assays and cell spreading assays were performed under various oxygen tensions in the presence or absence of two PI3K inhibitors PI-103 and GDC-0941 (GDC-0941 is currently in clinical trials). The role of HIF-1α and PI3K on these processes was also assessed via genetic inhibition of HIF-1α and PI3K by use of a dominant negative variant (dnHIF) and by over expressing WT PTEN in PTEN-null FTC133 cells. For in vivo studies, follicular (FTC133) and anaplastic (8505c) thyroid carcinoma cells were manufactured to stably express eGFP and implanted sub-cutaneously. Mice were treated with PI-103 and GDC-0941 by intra-peritoneal injection and oral gavage. Spontaneous metastatic colonization to the lungs was confirmed by viewing eGFP postive colonies cultured from digested lung tissue and further by immuno-blotting for eGFP. For irradiation studies, cells were exposed to 0,2,4,6 GY external beam radiation. Both PI3K and hypoxia increased cell migration, cell adhesion and cell spreading on extra-cellular matrix molecules. These processes are key in the development of a metastatic phenotype. Genetic and pharmacological inhibition of both PI3K and HIF-1α reduced the migratory, adhesive and cell spreading potential of thyroid carcinoma cells under varying oxygen tensions. Additionally, pharmacological and genetic inhibition of PI3K and HIF-1α reduced both PI3K and HIF-1 activity resulting in reduced activation of PI3K target proteins (pAKT, pGSK-3β) and reduced expression of HIF-1 target genes (CA-9, VEGF, LDH-A, GLUT-1). In vivo, PI-103 and GDC-0941 reduced PI3K and HIF-1α activity as well as PI3K and HIF-1 target genes in primary follicular and anaplastic tumors. Importantly, mice bearing dnHIF-FTC133 tumors and those treated with GDC-0941 had reduced number of metastatic colonies in the lungs of follicular thyroid tumor bearing mice. Radiation induced HIF-1α activity and expression. GDC-0941 combined with radiation blocked HIF-1 induction, prolonged DNA double-strand breaks and reduced clonogenic survival suggesting a radio-sensitising effect. These data link PI3K, HIF-1 activation and aggressive disease in thyroid carcinoma and suggest PI3K may be an important therapeutic target. One attractive approach would be combination therapy with external beam radiation. The latter is the first line treatment in patients suffering from anaplastic thyroid carcinoma but is seldom successful. With the known desensitizing effects of HIF-1 and PI3K activity on radiation treatment, a combined approach involving both radiation and a PI3K inhibitor may improve both the therapeutic response within the local tumor and reduce metastatic potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1483. doi:10.1158/1538-7445.AM2011-1483