GDC-0941 Inhibits Metastatic Characteristics of Thyroid Carcinomas by Targeting both the Phosphoinositide-3 Kinase (PI3K) and Hypoxia- Inducible Factor-1a (HIF-1a) 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 an 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 the metastatic behavior of thyroid carcinoma cells in vitro and in vivo.

Design: Vascular endothelial growth factor (VEGF) ELISA, HIF activity assays, proliferation studies, and scratch-wound migration and cell spreading assays were performed under various O2 tensions (normoxia, hypoxia [1% and 0.1% O2], 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 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 (in three of four cell lines) and/or hypoxia-induced (in all four cell lines) secreted VEGF 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 cells 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 under all O2 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 O2 tension, and appears to act upstream of HIF activation. GDC-0941 significantly inhibited the metastatic phenotype, supporting the clinical development of PI3K inhibition in thyroid carcinomas. (J Clin Endocrinol Metab 96: E1934–E1943, 2011)

Thyroid carcinomas are the most common endocrine neoplasm and are frequently associated with activating mutations in the MAPK and/or the phosphoinositide 3-kinase (PI3K) signaling pathways. Although both pathways regulate disease progression, the PI3K pathway appears dominant and is commonly hyperactivated in aggressive follicular thyroid carcinoma (FTC) and anaplastic thyroid carcinoma (ATC).

We have previously shown that PI3K plays an important role in the activity of hypoxia-inducible factor-1 (HIF-1) in thyroid carcinomas. Like PI3K signaling, hypoxia and HIF-1 are associated with aggressive disease, metastasis, and treatment resistance in many cancers. Activation of HIF-1 is normally observed under hypoxic conditions but can be influenced by oncogene expression. We previously found that basal and hypoxic induction of HIF-1 depend on PI3K activity. Furthermore, expression of the oxygen-labile subunit of HIF-1 (HIF-1α) and the downstream target carbonic anhydrase-9 (CA-9) are elevated in clinically aggressive thyroid tumors. It is therefore conceivable that regulation of HIF-1 contributes to the aggressive phenotype associated with PI3K hyperactivity in thyroid carcinomas.

Here, we assessed how inhibition of the PI3K and HIF-1 pathways influenced metastatic characteristics in a range of thyroid carcinoma cells in vitro and in vivo. We used the PI3K inhibitor GDC-0941 (currently in phase 2 clinical trials) to show: 1) PI3K signaling plays a vital role in metastatic progression, irrespective of O2 tension; 2) hypoxia and PI3K act synergistically to induce HIF-1, further enhancing metastatic characteristics; 3) GDC-0941 inhibits PI3K, HIF-1, and downstream targets in vitro and in vivo; and 4) GDC-0941 and genetic inhibition of HIF-1 inhibit tumor growth and early metastatic lung colonization in mice bearing FTC xenografts.

Furthermore, we show for the first time that the HIF family member HIF-2α, which shares significant homology with HIF-1α and is also associated with tumor progression, is induced by hypoxia/anoxia in papillary thyroid carcinoma (PTC), FTC, and ATC cells and is affected by PI3K.

Discussion

Most thyroid tumors are well differentiated and success- fully treated with surgery and radioiodine. However, ATC prompting the clinical evaluation of VEGF inhibitors (20). Furthermore, VEGF is a well-described target of HIF-1 (17). GDC-0941 inhibited both basal and hypoxia/anoxia- induced VEGF levels in BcPAP, FTC133, and 8505c cells, and hypoxic/anoxic VEGF in WRO cells suggesting that secreted VEGF expression is highly dependent on PI3K signaling under varying O2 tensions. Direct inhibition of HIF-1 by dnHIF significantly reduced basal VEGF in the 8505c model only, whereas hypoxia- and/or anoxia-in- duced VEGF was significantly reduced in all cells. This clearly demonstrates that hypoxia/anoxia-induced VEGF media levels are specifically HIF-1 de- pendent but that HIF may be down- stream of PI3K activity. Because GDC- 0941 inhibited both PI3K and HIF-1- induced VEGF, PI3K inhibitors may be therapeutically useful in targeting VEGF signaling.

Phenotypically, we show that GDC- 0941 is not markedly cytotoxic toward thyroid carcinoma cells, although using the FTC133 model, we demonstrated that sensitivity is greater in cells lacking PTEN than in those where PTEN ex- pression is restored.
Our data show that PI3K signaling is key in thyroid carcinoma cell spreading and migration under varying O2 ten- sions, with the level of GDC-0941-medi- ated inhibition under basal conditions mirroring that previously reported in co- lon, breast, and prostate carcinoma cells (7, 12). Importantly, hypoxia and PI3K act synergistically to induce HIF-1α, which further enhances migration in PTC and FTC cells. Both hyperactive PI3K signaling and hypoxia are linked with promoting migration in many can- cers by inducing metastatic genes (VEGF, CA-9, and lysyl oxidase) (22– 25). The basal migratory phenotype of the cell panel mirrors anticipated ag- gressiveness based upon thyroid carci- noma subtype. Whereas hypoxia in- duces migration in BcPAP, FTC133, and WRO cells, intriguingly, the ana- plastic 8505c cells appear to be primed for maximal migration in basal condi- tions, suggesting deregulation of path- ways managing the transition between behavior in aerobic and hypoxic con- ditions in these cells. This observation perhaps links to the fact that aerobic expression of dnHIF has significant consequence in these cells (in terms of secreted VEGF), which is not apparent in the rest of the panel.

To translate the effects observed in vitro to an antimetastatic pheno- type in vivo, we developed a novel model of detection of early spontane- ous metastasis to the lungs from FTC xenografts (14). By using fluorescently labeled cells, we show the existence of lung micrometas- tasis. Furthermore, culture experiments from lung tissue support that these tumor cells are viable and capable of colony formation and enable a relatively rapid quantifi- cation of early metastatic spread. We show that targeting PI3K with GDC-0941 reduces pAKT, HIF-1α, and down- stream target expression. Both HIF-1α and CA-9 expression were reduced dose dependently in FTC xenografts, which perhaps reflects the relative half-lives and/or stability of the proteins (26). Therapeutically, both tumor growth and early tumor spread were significantly reduced by GDC-0941 or by expression of dnHIF in FTC xenografts. Although the lung clonogenic assay may not necessarily relate to the development of clinically evident lung metastases, one could argue that assessing clonogenecity of metastatic cells residing within the lung is more relevant than histological assessment whereby dormant deposits may contribute to overall score. In summary, our data show that metastatic pro- gression is primarily PI3K driven irrespective of O2 ten- sion, but hypoxia promotes metastatic characteristics that are dependent on HIF-1 in thyroid carcinoma. In- hibition of these processes by clinical PI3K inhibitors such as GDC-0941 in xenograft models highlights the potential therapeutic value of these drugs in the treat- ment of thyroid carcinoma and supports their evalua- tion in other endocrine DRB18 diseases dependent on PI3K signaling.