The relationship between HIF-1α and autophagy activity in the hypoxic environment of breast cancer

[摘要] ENGLISH ABSTRACT: Introduction:Among the cancers that afflict females world-wide, neoplastic disease of breast tissue is the most frequently diagnosed form and the leading cause of cancer-related death. Conventional treatment entails the use of doxorubicin, an anticancer agent belonging to the anthracycline family of chemotherapeutic drugs. Cancer cells are becoming increasingly resistant to doxorubicin therapy. The existence of hypoxic zones, which is a common feature of solid tumours, has been shown to promote the selection of therapy resistant clones in proliferating cancer cells. By modifying cellular homeostasis, neoplastic cells are capable of tolerating the hypoxic insult and thriving within the hostile microenvironment of the tumour. This adaptation is known as 'the hypoxic response' and is mediated through the action of the transcriptional regulator, HIF-1. Its expression in cancer tissue has been associated with a dismal prognosis as it promotes the degree of malignancy to an advanced stage.Hypothesis & Aims:We hypothesized that the targeting of HIF-1α would circumvent the 'protective' hypoxic response conferred upon breast cancer and improve the cytotoxicity of doxorubicin treatment. In this study, the first aim was to identify the hypoxic conditions at which the MCF-7 breast cancer cell line manifests a doxorubicin-resistant phenotype. This was followed by examination of the molecular pathways contributing to the hypoxic resistance by elucidating the potential relationship with the hypoxic regulator HIF-1α. Once the involvement of HIF-1α was established, the next aim was to evaluate whether the attenuation of HIF-1α would terminate the resistant phenotype and sensitize the neoplastic MCF-7 cells to doxorubicin treatment. Finally, the reproducibility of the in vitro experiment and efficacy of treatments within an animal model was evaluated.2-Methoxyestradiol is a naturally occurring metabolite originating from 17β-estradiol. It has recently been exploited as an anticancer agent due to its anti-proliferative and anti-angiogenic properties. Among its various mechanisms of action, this compound has been shown to inhibit the expression of HIF-1α. It is for this reason that this study employed 2-methoxyestradiol in the adjuvant therapeutic treatment, along with doxorubicin.Methods:The in vitro experimental model employed the use of the breast adenocarcinoma estrogen receptor (ER-positive cell line, MCF-7. These neoplastic cells were propagated under standard culture conditions until reaching ~70-80% confluency, after which treatment commenced. The treatment regime comprised a 12 hour exposure to the doxorubicin (1 μM) chemotherapeutic agent, either alone or in combination with HIF-1α inhibitors, 2-methoxyestradiol (10 μM) or siRNA duplex (400 nM), with parallel incubations under normoxic (21%) and hypoxic (~0.1%) conditions. To serve as a positive control for HIF-1α expression, cells were treated with CoCl2 (100 μM). Molecular techniques employed included the Caspase-Glo® 3/7 Assay, western blotting, and the bioreductive MTT Assay. Mitochondrial integrity was assessed by live cell imaging/fluorescent microscopy. Cellular viability was monitored at all times. The experiment was then translated into a pre-clinical in vivo model where C57BL/6 mice bearing E0771 xenografts (4 week growth) were allocated into the following treatment groups: (1) control (2) doxorubicin (5 mg.kg-1), (3) 2-methoxyestradiol (45 mg.kg-1), and (4) the combination of the two previously mentioned groups. Body weight and the rate of tumour growth were monitored throughout the experiment.Results:Treatment with CoCl2 effectively stabilized HIF-1α under normoxic conditions. 2-Methoxyestradiol was capable of attenuating HIF-1α expression under both normoxia and hypoxia as compared with siRNA transfection, which was only effective under normoxia. HIF-1α stabilization was accompanied by an increase in autophagy along with the morphological transformation of mitochondria from an elongated network to shorter disc-like forms. On the other hand, HIF-1α attenuation caused an induction in the expression of the apoptotic markers, cleaved caspase 3 and cleaved PARP, as well as the restoration of the normoxic morphology. The exposure of MCF-7 cells to 1 μM doxorubicin for 12 hours produced a differential effect in the bioreductive MTT assay between normoxic and hypoxic conditions (42.97 ± 3.095% vs. normoxic dox, p<0.01), while stimulating the apoptotic and autophagic pathways. Compared to the control, a significant expression of phospho-AMPK became evident at 21% O2, while the levels remained stable at ~0.1% O2 after doxorubicin exposure. Furthermore, chemotherapeutic treatment caused the morphology of the mitochondria to appear dot-like. Although the combination of the two drugs removed the differential effect witnessed in the MTT assay, there was no significant change when compared to doxorubicin. Levels of apoptotic cell death decreased under both oxygen conditions. While HIF-1α and autophagy decreased under normoxia, they remained elevated under hypoxia.In the in vivo component of the study, the administration of doxorubicin and 2-methoxyestradiol, alone or in combination, did not affect the rate of tumour growth or induce systematic toxicity in any of the experimental mice. When drugs were administered separately, a decrease in apoptosis along with a concomitant increase in autophagy and p-AMPK expression became noticeable while neither treatment had any significant effect on the expression of HIF-1α. Adjuvant administration, however, was capable of attenuating HIF-1α along with autophagy.Discussion:By inducing (CoCl2) and inhibiting (2-methoxyestradiol; siRNA duplex) HIF-1α, it was established that the autophagic pathway in the in vitro experimental setting of this study was dependent on the expression of HIF-1α. The bioreductive MTT assay measures the metabolic state of a cell, which is an indirect indication of cellular viability. Based on this, hypoxia was shown to confer survival to neoplastic MCF-7 cells based on the differential effect witnessed after doxorubicin treatment.Apart from the induction of apoptosis and its associated mitochondrial fragmentation, the chemotherapeutic drug increased the activation of the metabolic sensor, AMPK, which upregulated autophagy during normoxia. While this autophagic process may assist in the killing mechanism, we speculate that the autophagy upregulated under hypoxia may be responsible for the survival effect and is most likely dependent on HIF-1α.In contrast to eliciting a synergistic cytotoxic effect, the combination of doxorubicin with 2-methoxyestradiol produced an antagonistic effect on cellular viability instead. We propose that under normoxia, the combined treatment may stimulate the MCF-7 neoplastic cells to enter a state of growth arrest, or senescence, since the results indicate that the decrease in HIF-1α-dependent autophagy did not significantly affect cellular viability. Under hypoxia, despite the incorporation of the pharmacological HIF-1α inhibitor (2-methoxyestradiol), the expression levels of HIF-1α remained unaffected. We speculate that this could be the result of a potentiated stabilization of HIF-1α caused by the build-up of ROS and TCA intermediates which may be the outcome of mitochondrial dysfunction inflicted upon adjuvant therapy under hypoxia. Furthermore, it is also likely that the slight mitogenic effect observed within the MTT assay may be caused by the conversion of 2-methoxyestradiol to a chemically-reactive estrogen derivative, possibly by the action of doxorubicin, and the fact that an ER-positive cancer cell line was employed in this study.With regards to the in vivo experimental model, we speculated that the failure of the molecular changes to manipulate the growth of the tumour could have been the result of an ineffective time- and/or dose regime.Conclusion:We therefore reject our hypothesis based on the fact that an antagonistic rather than synergistic effect was witnessed when the tumorigenic MCF-7 cell line was treated with adjuvant therapy. The results warrant the need for extensive testing on the pharmacodynamics of 2-methoxyestradiol, and more informative techniques to compliment the study.

[发布机构] Stellenbosch University