The therapeutic implications of oxygen deficiency have been fuelling cancer research for over 100 years. However, detailed studies on the impact of hypoxia on the cytotoxic and/or radiosensitising effects of anticancer drugs are lacking. As the adaptation of tumour cells to hypoxia is primarily mediated by stabilisation of HIF-1, we evaluated the role of functional HIF-1 in the response to chemoradiotherapy. Interestingly, several studies have shown that the presence of HIF-1α is a negative prognostic factor for human breast cancer [21–23]. Animal studies of metastatic breast cancer have demonstrated that lack of HIF-1α in malignant cells significantly reduced tumour progression and metastasis . Moreover, high HIF-1α levels were shown to be predictive of response to epirubicin therapy in patients with breast cancer .
In our study, Western blotting showed a consistent upregulation of HIF-1α protein level under hypoxia, whereas the PCR array indicated that HIF-1α was downregulated on mRNA level under hypoxia. Similarly, exposure of HeLa cells to hypoxia (1% O2) or the oxygen mimetic CoCl2 for 2.5 h did not change HIF-1α mRNA levels significantly, while HIF-1α protein levels increased . In patients with colon cancer, high HIF-1 expression was demonstrated using immunohistochemistry, but no significant difference in HIF-1α mRNA expression between tumour groups and control groups was noticed . One possible explanation for such a discrepancy between mRNA and protein levels is that induction of HIF-1α protein expression is not due to enhanced HIF-1α gene transcription or elevated mRNA stability, but instead results from a longer half-life of the protein due to increased HIF-1α translation and decreased HIF-1α proteolysis .
Importantly, due to the above-described transient stabilisation and short half-life of endogenous HIF-1α, HIF targets such as CA9 and the glucose transporter 1 (GLUT-1) have been used to detect hypoxic response in tumour tissues. In breast cancer, abundant expression of CA9 and GLUT-1 was shown to be associated with high-grade cancers and poor prognosis [28, 29]. Moreover, CA9 has also been suggested as a predictive marker for response to doxorubicin treatment and adjuvant endocrine therapy in patients with breast cancer [30, 31]. In addition, several gene and miRNA expression signatures have been described to be associated with poor prognosis in breast carcinoma . In this respect, there is a pressing need for better biomarkers of hypoxia (including gene expression profiles, serum proteins, circulating tumour cells or functional imaging) that could be used non-invasively in patients to enable more rigorous testing of its prognostic/predictive value .
Concerning the cytotoxic effect of gemcitabine, no significant influence of hypoxia was observed in the breast carcinoma cell lines included in the present study. In addition, for the first time, the effect of gemcitabine’s main metabolite dFdU was investigated under reduced oxygen conditions and a similar cytotoxic effect was shown under normoxia and hypoxia. This might be explained by the fact that hypoxia has been shown to have no effect on protein expression of several key enzymes (including dCK and cytidine monophosphate kinase) responsible for metabolism of gemcitabine .
Moreover, we noticed that both gemcitabine and dFdU induced a clear S phase block in normoxic and hypoxic cells, independent on HIF-1 functionality. Also, no reduction of cellular uptake and DNA incorporation of gemcitabine under hypoxia was reported in pancreatic carcinoma and hepatoma-derived cell lines .
Other papers however showed that oxygen deficiency did compromise the cytotoxic effect of gemcitabine, suggesting a cell type dependency of this phenomenon. For example, treatment of transitional cell carcinoma cells with gemcitabine was less effective under hypoxia . For pancreatic cancer cells, several studies reported that hypoxia induced resistance to gemcitabine, by altered signalling through PI3K/Akt/NF-κB pathways and partially through MAPK signalling pathway , by reducing both inhibition of proliferation and induction of apoptosis by gemcitabine , and by decreasing the synthesis of active gemcitabine deoxynucleotides, possibly also through downregulation of dCK . As such, the impact of tumour-associated hypoxia on the cytotoxic effect of gemcitabine is still not completely resolved.
The present report showed no association between radiosensitisation by gemcitabine or dFdU and HIF-1 functionality. Previous work either focused on the relationship between HIF-1 and the cytotoxic effect of gemcitabine or between HIF-1 and radiosensitivity per se.
Firstly, previous observations have been somewhat controversial regarding HIF-1α expression and the sensitivity to gemcitabine. Suppression of HIF-1α using siRNA resulted in an enhanced efficacy of gemcitabine in the treatment of several pancreatic tumour cell lines [40, 41]. Nevertheless, in line with our results, knockdown of HIF-1α has also been reported to have no effect on the sensitivity of pancreatic PANC-1 cells when treated with gemcitabine under hypoxic conditions . In addition, HIF-1α expression levels after platinum/gemcitabine therapy did not correlate with outcome of patients with stage II/III non-small cell lung cancer and HIF-1α expression was not associated with adverse effects or outcome in patients with pancreatic cancer . As such, the therapeutic value of an approach by which gemcitabine is combined with inactivation of HIF-1α signalling by novel strategies remains to be fully elucidated.
Secondly, the effects of HIF-1 blockade on tumour radiosensitivity are complex. Downstream effects of HIF-1 serve to help tumour cells to adapt to hypoxic stress. In doing so, they change the tumour phenotype in ways that might impact radiosensitivity, some positively and some negatively, but the degree and direction of that influence appears to be dependent on the context. For example, inhibition of HIF-1 activation using siRNA clearly increased radiosensitivity of hypoxic fibrosarcoma cells . Other studies however suggested that HIF-1 would not affect radiosensitivity . From the experimental model used in this study, two conclusions can be drawn. Firstly, no significant difference in radiosensitivity was observed for HIF-1 proficient versus deficient cells, with an OER around 1.50 for all three cell lines. A comparable and relatively low OER of 1.86 ± 0.73 has been reported for MDA-MB-231 by Lagadec et al., who suggested that a negative correlation exists between the OER and increasing malignancy of the breast cancer subtype the cell lines were originally derived from . Secondly, none of the radiobiological parameters (ID50, MID, SF2) calculated were significantly influenced by HIF-1 functionality after treatment with gemcitabine or dFdU in combination with radiation.
One important limitation of our in vitro study is the lack of the microenvironment that would surround tumours in vivo. Therefore, further studies using tumour animal models would certainly be warranted. Only in this way, an in-depth understanding and characterisation of hypoxia in breast cancer and other relevant tumour types can be established, ultimately enabling an enhanced prediction of prognosis, optimisation of (gemcitabine and/or radiation) treatment and information on whether and how to target tumour hypoxia.