Cellular adaptation to conditions of stress is critical to the survival of all organisms. In mammalian cells, reduced oxygen availability, or hypoxia, triggers a myriad of alterations within molecular pathways aimed at ensuring sustained viability and functionality of vital organs. The master regulator of the hypoxic response is the heterodimeric HIF transcription factor, composed of an oxygen-labile HIFalpha subunit and a constitutively expressed and stable HIFbeta(ARNT) subunit. While experiments in mice have demonstrated the indispensability of HIF1alpha/HIF2alpha, unchecked hyperactivation of HIF is associated with pathological complications, such as the development of clear-cell renal cell carcinoma (ccRCC), the most common form of kidney cancer. In particular, overexpression of HIF2alpha has been intimately linked to ccRCC molecular pathogenesis. Here, we report that HIF2alpha overexpression leads to Akt-mediated hyperactivation of Hdm2, resulting in decreased activity of p53 and increased resistance to apoptosis. Significantly, we show that restoration of p53 activity via inhibition of Hdm2 reverses chemoresistance of otherwise refractory ccRCC cells. We also demonstrate that the picornavirus EMCV (Encephalomyocarditis virus) efficiently destroys ccRCC tumour cells in an NF-kappaB- and HIFalpha-dependent manner both in vitro and in a mouse xenograft model. This work provides pre-clinical evidence for the potential use of EMCV in an oncolytic virus-based approach to the treatment of advanced ccRCC. In addition, using a cell biology-based approach we reveal that inhibition of the DNA methyltransferase DNMT1 leads to enhanced HIF promoter-binding affinity and transactivation activity in a manner that is independent of changes in HIFalpha protein levels. This study uncovers a novel HIF regulatory mechanism in mammalian cells. In summary, the work presented here provides insight into the molecular mechanisms governing HIF activity and the effects of HIF on the molecular pathogenesis of ccRCC. Our findings have the potential to provide new therapeutic avenues for the treatment of kidney cancer.

Cellular adaptation to conditions of stress is critical to the survival of all organisms. In mammalian cells, reduced oxygen availability, or hypoxia, triggers a myriad of alterations within molecular pathways aimed at ensuring sustained viability and functionality of vital organs. The master regulator of the hypoxic response is the heterodimeric HIF transcription factor, composed of an oxygen-labile HIFalpha subunit and a constitutively expressed and stable HIFbeta(ARNT) subunit. While experiments in mice have demonstrated the indispensability of HIF1alpha/HIF2alpha, unchecked hyperactivation of HIF is associated with pathological complications, such as the development of clear-cell renal cell carcinoma (ccRCC), the most common form of kidney cancer. In particular, overexpression of HIF2alpha has been intimately linked to ccRCC molecular pathogenesis. Here, we report that HIF2alpha overexpression leads to Akt-mediated hyperactivation of Hdm2, resulting in decreased activity of p53 and increased resistance to apoptosis. Significantly, we show that restoration of p53 activity via inhibition of Hdm2 reverses chemoresistance of otherwise refractory ccRCC cells. We also demonstrate that the picornavirus EMCV (Encephalomyocarditis virus) efficiently destroys ccRCC tumour cells in an NF-kappaB- and HIFalpha-dependent manner both in vitro and in a mouse xenograft model. This work provides pre-clinical evidence for the potential use of EMCV in an oncolytic virus-based approach to the treatment of advanced ccRCC. In addition, using a cell biology-based approach we reveal that inhibition of the DNA methyltransferase DNMT1 leads to enhanced HIF promoter-binding affinity and transactivation activity in a manner that is independent of changes in HIFalpha protein levels. This study uncovers a novel HIF regulatory mechanism in mammalian cells. In summary, the work presented here provides insight into the molecular mechanisms governing HIF activity and the effects of HIF on the molecular pathogenesis of ccRCC. Our findings have the potential to provide new therapeutic avenues for the treatment of kidney cancer.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2008. / Härtill 3 uppsatser.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19369号 / 医博第4046号 / 新制||医||1012(附属図書館) / 32383 / 新制||医||1012 / 京都大学大学院医学研究科医学専攻 / (主査)教授 宮本 享, 教授 柳田 素子, 教授 松原 和夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Acute Lung Injury, characterised clinically as the Acute Respiratory Distress Syndrome is a catastrophic response to a range of pulmonary and non-pulmonary insults. Despite much work the key mechanisms involved in generating the exaggerated immune response that results in lung injury are not completely understood. Hypoxia-inducible factor-1 has been shown to be a key transcription factor in the myeloid cell response to inflammatory signals. The aims of this thesis were to develop a model of acute lung injury and to study the role of Hif-1 in the generation of lung injury in this model. A model of direct pulmonary injury as a result of intratracheal instillation of endotoxin is described. Using this model the role of myeloid cell Hif-1α was characterised using a myeloid cell specific conditional knockout system. The injury in Hif-1α deficient mice was quantitatively similar to the injury seen in wild type animals over a range of time points. However, the quality of the injury, assessed by a measure of nitric oxide mediated damage was reduced. The in vivo data were supported by in vitro studies using a murine macrophage cell line which showed that manipulation of the cellular oxygen tension in the presence of endotoxin alters the ability of the cell to generate nitric oxide. Furthermore, pharmacological manipulation of cellular Hif-1 levels by Dimethyloxallyl Glycine (DMOG) in the macrophage cell increased the generation of nitric oxide in response to endotoxin by altering the expression of a number of the isoforms of Nitric Oxide Synthase. In a final set of experiments the response to intratracheal endotoxin was modulated in mice by the concurrent administration of DMOG. As expected the qualitative response to endotoxin was similar but the NO mediated damage was enhanced in the animals administered DMOG. Manipulation of Hif-1 may have a role in the therapy of lung injury by altering the characteristics of the response.

This study explored the possibility that upregulation of Hypoxia Inducible Factor-1 (Hif-1)-responsive genes in Human Umbilical Vein Endothelial Cells (HUVEC) would promote and stabilize HUVEC formation into inchoate vascular beds within artificial collagen gels. This experiment was designed to explore the above possibility by sub-cloning Hif-1[alpha], the related chimeric construct Hif-1[alpha]/VP16, and the marker gene dsRed into retroviral expression vectors, producing retroviral vectors containing these genes, and stably transducing HUVEC using these retroviruses. Transduced HUVEC were to be observed in cell culture as well as after implantation into artificial collagen gels that have previously supported vascular bed formation by HUVEC. Our results show, preliminarily, that HUVEC transduced with Hif-1[alpha]/VP16 go into cell-cycle arrest. Attempts to transduce HUVEC with Hif-1[alpha] failed to achieve high enough transduction efficiency to determine the cells angiogenic potential. This study concluded that more experiments need to be conducted to better characterize the effects of hypoxia-responsive gene upregulation in controlling HUVEC angiogenesis and cell-cycle signaling and that straightforward transduction of HUVEC by Hif-1[alpha]/VP16 is probably not sufficient, in itself, to induce in vitro vascular bed formation.

The development of intratumoral hypoxia is associated with resistance to therapy in many forms of human cancer, and pre-exposure of tumor cells to hypoxia confers multidrug resistance. Research over the last several years has led to considerable advances in the understanding of the cellular response to oxygen deprivation, however the hypoxia-induced mechanisms that contribute to the chemoresistance phenotype are still not well understood. Recent studies have identified hypoxia-inducible factor 1 (HIF-1), a master transcriptional regulator of oxygen homeostasis, as an important mediator of hypoxia-induced chemoresistance in cancer cells. The research described in this thesis confirms these findings and demonstrates HIF-1 is required for hypoxia-induced resistance to doxorubicin and etoposide in human tumor cells. In addition, novel findings revealed that hypoxia-induced drug resistance occurred independently of changes in the apoptotic fraction and was associated with decreased drug-induced senescence. DNA damage measured at the single-cell level revealed that the increase in survival correlated well with a HIF-1-dependent decrease in etoposide-induced DNA strand breaks, providing direct evidence that exposure of tumor cells to hypoxia leads to protection against some forms of drug-induced DNA damage. Characterization of several classical mechanisms of drug resistance upstream of DNA damage identified multiple determinants of cellular resistance to anticancer agents. The relative contributions of each varied depending on the particular drug and cancer cell line studied. Together, the findings presented here support a model in which hypoxia-induced chemoresistance is a multifactorial phenomenon that is regulated, at least in part, through HIF-1-dependent mechanisms. / Thesis (Ph.D, Anatomy & Cell Biology) -- Queen's University, 2008-08-29 12:35:36.219

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20970号 / 医博第4316号 / 新制||医||1026(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小川 修, 教授 戸井 雅和, 教授 万代 昌紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Lic.-avh. (sammanfattning) Stockholm : Karolinska institutet, 2010.

Areas of hypoxia caused by poor perfusion are a common occurrence in breast cancer. Hypoxia-inducible factors-1 and 2 (HIF1/2) drive the cellular response to hypoxia in such areas, resulting in the upregulation of genes which facilitate the survival of cancer cells and promote growth, invasion, metastasis and angiogenesis, generally leading to more aggressive tumour characteristics. Previous research has demonstrated that growth factor signalling, such as the ligand-mediated activation of HER receptors, can promote the action of HIFs in normoxia, and correlation between HER2 expression and HIFα proteins has been demonstrated in clinical samples of breast cancer. Despite this, little research has been conducted on how the growth factor-driven regulation of HIFα subunits might modify the cellular response to hypoxia. In this thesis, the role of HER2 overexpression in HIFα modulation was assessed in breast cancer cell lines and publically available clinical datasets for breast cancer with the aim of further understanding the implications of hypoxia and HIFα expression in the context of HER2-positive breast cancer. The upregulation of HIF1α and HIF2α by hypoxia was observed across breast cancer cell lines, and the role of HER2 in this process was assessed using an isogenic MCF7 cell line model overexpressing HER2. This demonstrated an increased hypoxic upregulation of HIF2α but not HIF1α when HER2 was overexpressed. The increased upregulation was shown to be facilitated by an increase in normoxic HIF2α, which is driven by a higher transcriptional rate of the EPAS1 (HIF2) gene as a direct result of HER2 overexpression. HER2 overexpression also resulted in the increased hypoxic upregulation of known hypoxia response genes in 2D and 3D culture models. This demonstrates a novel mechanism for growth-factor mediated HIFα regulation in the context of HER2 overexpression, with an important role for HIF2α. Microarray analysis of MCF7 and MCF7-HER2 cells was used to compare the global transcriptional response to acute (24 hrs) and chronic (>10 weeks) hypoxia (0.5% O2) and demonstrated a broadly increased upregulation of hypoxic response genes in the HER2 overexpressing cell line when compared to wild-type MCF7. This included an increase in previously described HIF1 and HIF2 target genes. MCF7-HER2 also illustrated an increased expression of hypoxia response genes in normoxia, and an analysis of the genes involved showed the promotion of a number of pathological processes including proliferation, invasion, angiogenesis and epithelial to mesenchymal transition. Large-scale, publically available expression datasets for breast cancer cell lines and clinical patient data were used to investigate the expression of HIF2α and hypoxia response genes in relation to HER2 expression. A set of pathologically important genes which were primed for hypoxia in MCF7-HER2 were also demonstrated to correlate with HER2 across breast cancer cell lines, suggesting that HER2 may more broadly promote a readiness to respond to hypoxia in breast cancer cells. Assessment of HIF2α in clinical samples has shown its increased expression in the HER2-positive subtype, and HIF2α was shown to be associated with worse disease-specific survival in the context of HER2-positive samples only. To investigate whether HIF2α is a potential target in HER2 overexpressing breast cancer, the effect of HIF2α inhibition through siRNA or HIF2-specific chemical inhibitors was assessed in cell lines with high or low HER2 expression, and this demonstrated an increased sensitivity of HER2 overexpressing cell lines to HIF2α inhibition. This work highlighted HER2 as an important modulator of the cellular response to hypoxia in breast cancer, demonstrating a previously overlooked role for HIF2α in this process. HIF2α expression can be directly driven by HER2 and this differs mechanistically from that previously reported for HIF1α. Finally, further work into the potential for HIF2α as a target for anti-cancer therapy is suggested, as an increased sensitivity of HER2-positive cell lines to anti-HIF2α agents was shown, as well as a HER2-specific relationship between HIF2α expression and worse prognosis. More generally, this work has shown an important interplay between growth factor receptor expression and the cellular response to hypoxia, suggesting that HER2 may promote a stronger response to hypoxia in breast cancer, which may contribute to the increased aggressiveness of HER2-positive tumours.