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The resistance of hypoxic cells to conventional chemotherapy is well documented. Using both adenovirus-mediated gene delivery and small molecules targeting hypoxia-inducible factor-1 (HIF-1), we evaluated the impact of HIF-1 inhibition on the sensitivity of hypoxic tumor cells to etoposide. The genetic therapy exploited a truncated HIF-1␣ protein that acts as a dominant-negative HIF-1␣ (HIF-1␣-no-TAD). Its functionality was validated in six human tumor cell lines using HIF-1 reporter assays. An EGFP-fused protein demonstrated that the dominant-negative HIF-1␣ was nucleus-localized and constitutively expressed irrespective of oxygen tension. The small molecules studied were quinocarmycin monocitrate (KW2152), its analog 7-cyanoquinocarcinol (DX-52-1), and topotecan. DX-52-1 and topotecan have been previously established as HIF-1 inhibitors. HT1080 and HCT116 cells were treated with either AdHIF-1␣-no-TAD or nontoxic concentrations (0.1 M; ϽIC 10 ) of KW2152 and DX-52-1 and exposed to etoposide in air or anoxia (Ͻ0.01% oxygen). Topotecan inhibited HIF-1 activity only at cytotoxic concentrations and was not used in the combination study. Etoposide IC 50 values in anoxia were 3-fold higher than those in air for HT1080 (2.2 Ϯ 0.3 versus 0.7 Ϯ 0.2 M) and HCT116 (9 Ϯ 4 versus 3 Ϯ 2 M) cells. KW2152 and DX-52-1 significantly reduced the anoxic etoposide IC 50 in HT1080 cells, whereas only KW2152 yielded sensitization in HCT116 cells. In contrast, AdHIF-1␣-no-TAD (multiplicity of infection 50) ablated the anoxic resistance in both cell lines (IC 50 values: HT1080, 0.7 Ϯ 0.04 M; HCT116, 3 Ϯ 1 M). HIF-1␣-no-TAD expression inhibited HIF-1-mediated down-regulation of the proapoptotic protein Bid under anoxia. These data support the potential development of HIF-1 targeted approaches in combination with chemotherapy, where hypoxic cell resistance contributes to treatment failure.

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.

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