Akiyo Morinibu

The Akt/mTOR Pathway Assures the Synthesis of HIF-1α Protein in a Glucose- and Reoxygenation-dependent Manner in Irradiated Tumors

Transcriptional activity of HIF-1 (hypoxia-inducible factor-1) has been reported to be up-regulated in solid tumors after ionizing radiation; however, the molecular mechanism underlying the response remains to be elucidated. In the present study, we performed a series of molecular imaging experiments using a HIF-1-dependent reporter gene, 5HREp-ODD-luc, and found an essential role of the Akt/mTOR pathway. Hypoxic tumor cells distant from blood vessels were dramatically reoxygenated at 24 h postirradiation, and HIF-1 activity increased as HIF-1α accumulated in the reoxygenated regions. The accumulation was inhibited with a nonmetabolizable glucose analog, 2-deoxy-d-glucose, through the suppression of radiation-induced phosphorylation of Akt in the reoxygenated regions. Akt knockdown and an mTOR inhibitor revealed the importance of the Akt/mTOR pathway in the postirradiation accumulation of HIF-1α. In vitro experiments confirmed that an increase in glucose availability induced Akt phosphorylation under reoxygenated conditions and consequently up-regulated HIF-1α translation. Moreover, both the accelerated translation and the previously reported reactive oxygen species-mediated stabilization of HIF-1α protein were essential to the activation of HIF-1. All of these results indicate that Akt/mTOR-dependent translation of HIF-1α plays a critical role in the postirradiation up-regulation of intratumoral HIF-1 activity in response to radiation-induced alterations of glucose and oxygen availability in a solid tumor.

Cancer cells that survive radiation therapy acquire HIF-1 activity and translocate towards tumour blood vessels

Tumour recurrence frequently occurs after radiotherapy, but the characteristics, intratumoural localization and post-irradiation behaviour of radioresistant cancer cells remain largely unknown. Here we develop a sophisticated strategy to track the post-irradiation fate of the cells, which exist in perinecrotic regions at the time of radiation. Although the perinecrotic tumour cells are originally hypoxia-inducible factor 1 (HIF-1)-negative, they acquire HIF-1 activity after surviving radiation, which triggers their translocation towards tumour blood vessels. HIF-1 inhibitors suppress the translocation and decrease the incidence of post-irradiation tumour recurrence. For the first time, our data unveil the HIF-1-dependent cellular dynamics during post-irradiation tumour recurrence and provide a rational basis for targeting HIF-1 after radiation therapy.

UCHL1 provides diagnostic and antimetastatic strategies due to its deubiquitinating effect on HIF-1α

Hypoxia-inducible factor 1 (HIF-1) plays a role in tumour metastases; however, the genes that activate HIF-1 and subsequently promote metastases have yet to be identified. Here we show that Ubiquitin C-terminal hydrolase-L1 (UCHL1) abrogates the von Hippel–Lindau-mediated ubiquitination of HIF-1α, the regulatory subunit of HIF-1, and consequently promotes metastasis. The aberrant overexpression of UCHL1 facilitates distant tumour metastases in a HIF-1-dependent manner in murine models of pulmonary metastasis. Meanwhile, blockade of the UCHL1–HIF-1 axis suppresses the formation of metastatic tumours. The expression levels of UCHL1 correlate with those of HIF-1α and are strongly associated with the poor prognosis of breast and lung cancer patients. These results indicate that UCHL1 promotes metastases as a deubiquitinating enzyme for HIF-1α, which justifies exploiting it as a prognostic marker and therapeutic target of cancers.

Treatment regimen determines whether an HIF-1 inhibitor enhances or inhibits the effect of radiation therapy

Hypoxia-inducible factor-1 (HIF-1) has been reported to promote tumour radioresistance; therefore, it is recognised as an excellent target during radiation therapy. However, the inhibition of HIF-1 in unsuitable timing can suppress rather than enhance the effect of radiation therapy because its anti-angiogenic effect increases the radioresistant hypoxic fraction. In this study, we imaged changes of HIF-1 activity after treatment with radiation and/or an HIF-1 inhibitor, YC-1, and optimised their combination. Hypoxic tumour cells were reoxygenated 6 h postirradiation, leading to von Hippel-Lindau (VHL)-dependent proteolysis of HIF-1α and a resultant decrease in HIF-1 activity. The activity then increased as HIF-1α accumulated in the reoxygenated regions 24 h postirradiation. Meanwhile, YC-1 temporarily but significantly suppressed HIF-1 activity, leading to a decrease in microvessel density and an increase in tumour hypoxia. On treatment with YC-1 and then radiation, the YC-1-mediated increase in tumour hypoxia suppressed the effect of radiation therapy, whereas on treatment in the reverse order, YC-1 suppressed the postirradiation upregulation of HIF-1 activity and consequently delayed tumour growth. These results indicate that treatment regimen determines whether an HIF-1 inhibitor enhances or inhibits the therapeutic effect of radiation, and the suppression of the postirradiation upregulation of HIF-1 activity is important for the best therapeutic benefit.

TS-1 enhances the effect of radiotherapy by suppressing radiation-induced hypoxia-inducible factor-1 activation and inducing endothelial cell apoptosis.

The therapeutic effect of concurrent chemoradiotherapy with TS‐1 has been confirmed in various solid tumors; however, the detailed mechanism of action has not yet been fully elucidated. In the present study, we identified hypoxia‐inducible factor‐1 (HIF‐1) as one of the targets of TS‐1 in chemoradiotherapy. In growth delay assays using a tumor xenograft of non‐small‐cell lung carcinoma, H441, TS‐1 treatment enhanced the therapeutic effect of single γ‐ray radiotherapy (14 Gy) and significantly delayed tumor growth by 1.58‐fold compared to radiotherapy alone (P < 0.01). An optical in vivo imaging experiment using a HIF‐1‐dependent 5HRE‐luc reporter gene revealed that TS‐1 treatment suppressed radiation‐induced activation of HIF‐1 in the tumor xenografts. The suppression led to apoptosis of endothelial cells resulting in both a significant decrease in microvessel density (P < 0.05; vs radiation therapy alone) and a significant increase in apoptosis of tumor cells (P < 0.01; vs radiation therapy alone) in tumor xenografts. All of these results indicate that TS‐1 enhances radiation‐induced apoptosis of endothelial cells by suppressing HIF‐1 activity, resulting in an increase in radiosensitivity of the tumor cells. Our findings strengthen the importance of both HIF‐1 and its downstream gene, such as vascular endothelial cell growth factor, as therapeutic targets to enhance the effect of radiotherapy. (Cancer Sci 2008; 99: 2327–2335)

HIF-1-mediated metabolic reprogramming reduces ROS levels and facilitates the metastatic colonization of cancers in lungs

Hypoxia-inducible factor 1 (HIF-1) has been associated with distant tumor metastasis; however, its function in multiple metastatic processes has not yet been fully elucidated. In the present study, we demonstrated that cancer cells transiently upregulated HIF-1 activity during their metastatic colonization after extravasation in the lungs in hypoxia-independent and reactive oxygen species (ROS)-dependent manners. Transient activation induced the expression of lactate dehydrogenase A and phosphorylation of the E1α subunit of pyruvate dehydrogenase, which indicated the reprogramming of glucose metabolic pathways from mitochondrial oxidative phosphorylation to anaerobic glycolysis and lactic acid fermentation. The administration of the HIF-1 inhibitor, YC-1, inhibited this reprogramming, increased intratumoral ROS levels, and eventually suppressed the formation of metastatic lung tumors. These results indicate that HIF-1-mediated metabolic reprogramming is responsible for the survival of metastatic cancers during their colonization in lungs by reducing cytotoxic ROS levels; therefore, its blockade by HIF-1-inhibitors is a rational strategy to prevent tumor metastasis.

A novel strategy to tag matrix metalloproteinases-positive cells for in vivo imaging of invasive and metastatic activity of tumor cells.

Matrix metalloproteinases (MMPs) are endopeptidases responsible for degrading the extracellular matrix (ECM) and remodeling tissue in both physiological and pathological processes. MMP2 and membrane-type 1 MMP (MT1-MMP) have been associated with tumor invasion, metastasis and angiogenesis; therefore, a molecular imaging strategy assessing their activity may help to predict the malignancy of tumors. Here, we established a novel method of specifically tagging the surface of MMP2- and MT1-MMP-positive cells, and applied it to the development of an optical imaging probe. We constructed a protein-based probe composed of a glutathione-S-transferase (GST)-tag (Inhibitory [I]-domain), a polypeptide as a specific substrate for both MMP2 and MT1-MMP (Cleaved [C]-domain), a transmembrane domain of the epidermal growth factor receptor (Transmembrane [TM]-domain), and DsRed2 (Fluorescent [F]-domain). In vitro experiments clearly demonstrated that, after the probe was cleaved at the C-domain by the MMPs, the resultant TM-F-domain was inserted into the cellular membrane. Optical imaging experiments in vivo demonstrated that the probe was cleaved and specifically remained in tumor xenografts in a MMP-dependent manner. These results indicate that the release of the I-C-domain through the proteolytic cleavage of the C-domain by MMP2 and MT1-MMP triggers the tagging of cellular membranes with the TM-F-domain. The present feasibility study opens the door to the development of a novel imaging probe for tumor malignancy using positron emission tomography as well as an optical imaging device.

Aberrant IDH3α expression promotes malignant tumor growth by inducing HIF-1-mediated metabolic reprogramming and angiogenesis

Cancer cells gain a growth advantage through the so-called Warburg effect by shifting glucose metabolism from oxidative phosphorylation to aerobic glycolysis. Hypoxia-inducible factor 1 (HIF-1) has been suggested to function in metabolic reprogramming; however, the underlying mechanism has not been fully elucidated. We found that the aberrant expression of wild-type isocitrate dehydrogenase 3α (IDH3α), a subunit of the IDH3 heterotetramer, decreased α-ketoglutarate levels and increased the stability and transactivation activity of HIF-1α in cancer cells. The silencing of IDH3α significantly delayed tumor growth by suppressing the HIF-1-mediated Warburg effect and angiogenesis. IDH3α expression was associated with the poor postoperative overall survival of lung and breast cancer patients. These results justify the exploitation of IDH3 as a novel target for the diagnosis and treatment of cancers.

Involvement of decreased hypoxia-inducible factor 1 activity and resultant G1–S cell cycle transition in radioresistance of perinecrotic tumor cells

Cancer patients often suffer from local tumor recurrence after radiation therapy. Some intracellular and extracellular factors, such as activity of hypoxia-inducible factor 1 (HIF-1), cell cycle status and oxygen availability, have been suggested to affect DNA damage responses and eventual radioresistant characteristics of cancer cells. But when, where, and how these factors affect one another and induce cellular radioresistance is largely unknown. Here, we analyzed mechanistic and spatio-temporal relationships among them in highly heterogeneous tumor microenvironments. Experiments in vitro demonstrated that a decrease in the glucose concentration reduced the transcriptional activity of HIF-1 and expression of a downstream gene for the cell cycle regulator p27Kip1 even under hypoxic conditions. Then, the proportion of cells in the radioresistant S phase increased, whereas that in the radiosensitive G1 phase decreased, significantly. Immunohistochemical analyses showed that cancer cells in perinecrotic hypoxic regions, which should be under low-glucose conditions, expressed little HIF-1α, and therefore, were mainly in S phase and less damaged by radiation treatment. Continuous administration of glucagon, which increases the blood glucose concentration and so improves glucose availability in perinecrotic hypoxic regions, induced HIF-1α expression and increased radiation-induced DNA damage. Taken all together, these results indicate that cancer cells in perinecrotic regions, which would be under low-glucose and hypoxic conditions, obtain radioresistance by decreasing the level of both HIF-1 activity and p27Kip1 expression, and adjusting their cell cycle to the radioresistant S phase.

Diameter of tumor blood vessels is a good parameter to estimate HIF-1-active regions in solid tumors

As the transcriptional activity of hypoxia-inducible factor 1 (HIF-1) is associated with resistance of tumor cells to current antitumor therapies, the spatiotemporal dynamics of HIF-1-active regions has been of great interest as a therapeutic target. In the present study, we established a unique cancer cell line, which changes color HIF-1-dependently, and monitored it during tumor progression. In imaging experiments, HIF-1-active cells appeared over the tumor xenograft, but dramatically decreased in number as blood vessels developed around the tumor. The remaining HIF-1-active cells at the center of the xenograft also disappeared after neovascularization. Thereafter, tumor growth was accelerated and HIF-1-active cells reappeared in different regions. The distance between HIF-1-active cells and the nearest vessels correlated to the diameter of the vessel (r=0.801). These results provide a basic knowledge of how to estimate the spatiotemporal dynamics of HIF-1-active cells using information about the image-guided architecture of tumor blood vessels.