Satoshi Itasaka

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.

Significance of HIF-1-active cells in angiogenesis and radioresistance

Human solid tumors contain hypoxic regions that have considerably lower oxygen tension than the normal tissues. Hypoxia offers resistance to radiotherapy and anticancer chemotherapy, as well as predispose to increased tumor metastases. Furthermore, hypoxia induces hypoxia-inducible factor-1 (HIF-1), which in turn increases tumor angiogenesis. Thus, eradication of HIF-1-active/hypoxic tumor cells is very important for cancer therapy. We have previously reported that procaspase-3 fused with a von Hippel–Lindau (VHL)-mediated protein destruction motif of alpha subunit of HIF-1 (HIF-1α) containing Pro564, named TAT-ODD-procaspase-3 (TOP3), specifically induced cell death to hypoxic cells in vivo as well as in vitro. We now report that TOP3 also eradicates the radiation-induced HIF-1-active tumor cells. HIF-1 activity in the xenografts of human tumor cells, which express luciferase under the transcriptional control of HIF-1, were monitored and quantified daily with an in vivo bioluminescence photon-counting device. HIF-1 activity in tumors was more rapidly increased by ionizing radiation (IR) compared to untreated tumors. TOP3 efficiently decreased the HIF-1-activity in irradiated tumors as well as unirradiated ones, indicating TOP3 eradicated tumor cells with HIF-1-activity induced by IR as well as hypoxia. Eradication of HIF-1-active/hypoxic cells in the xenografts during irradiation exhibited significant suppression in angiogenesis and strong enhancement in a long-term growth suppression of tumor xenografts. These results further strengthen the argument that HIF-1-active/hypoxic cells play crucial roles in angiogenesis and radioresistance.

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.

Microenvironment and radiation therapy.

Dependency on tumor oxygenation is one of the major features of radiation therapy and this has led many radiation biologists and oncologists to focus on tumor hypoxia. The first approach to overcome tumor hypoxia was to improve tumor oxygenation by increasing oxygen delivery and a subsequent approach was the use of radiosensitizers in combination with radiation therapy. Clinical use of some of these approaches was promising, but they are not widely used due to several limitations. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that is activated by hypoxia and induces the expression of various genes related to the adaptation of cellular metabolism to hypoxia, invasion and metastasis of cancer cells and angiogenesis, and so forth. HIF-1 is a potent target to enhance the therapeutic effects of radiation therapy. Another approach is antiangiogenic therapy. The combination with radiation therapy is promising, but several factors including surrogate markers, timing and duration, and so forth have to be optimized before introducing it into clinics. In this review, we examined how the tumor microenvironment influences the effects of radiation and how we can enhance the antitumor effects of radiation therapy by modifying the tumor microenvironment.

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.

Hypoxia inducible factor‐1 influences sensitivity to paclitaxel of human lung cancer cell lines under normoxic conditions

Paclitaxel (PTX) is an anticancer drug that is effective against a wide range of solid tumors. The effect of PTX on two human lung cancer cell lines, PC14PE6 and NCI‐H441 cells, was examined in an orthotopically transplanted animal model with an in vivo imaging devise. Although PTX effectively suppressed tumor growth and improved survival rate in NCI‐H441, it did not influence these in PC14PE6. In vitro experiments confirmed that PC14PE6 cells are resistant to PTX under normoxic conditions and that both cell lines were resistant to PTX under hypoxic conditions. It was found that the expression level of endogenous hypoxia inducible factor (HIF)‐1α in PC14PE6 is much higher than that in NCI‐H441 cells under normoxic conditions. Furthermore, sensitivity to PTX in these cell lines was reversed when HIF‐1α expression was decreased by siRNA specific to HIF‐1α in PC14PE6 and increased by overexpression of the exogenous HIF‐1α gene in NCI‐H441. These results suggest that HIF‐1 influences the PTX sensitivity of these cells. The authors further examined β‐tubulin, a target molecule of PTX, with western blotting and immunohistochemical analysis in these cells. The expression level of β‐tubulin was comparable in these cells under both normoxic and hypoxic conditions while the distribution of β‐tubulin and cell morphology were changed according to HIF‐1α expression levels, suggesting that HIF‐1 influences the conformation and dynamics of microtubules. These data support the potential development of HIF‐1 targeted approaches in combination with PTX, where drug resistance tends to contribute to treatment failure. (Cancer Sci 2007; 98: 1394–1401)

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.

Delayed Initial Radioactive Iodine Therapy Resulted in Poor Survival in Patients with Metastatic Differentiated Thyroid Carcinoma: A Retrospective Statistical Analysis of 198 Cases

To elucidate the prognostic role of 131I radioactive iodine therapy (RIT), we conducted a retrospective cohort study analyzing the clinical factors that affect survival of postoperative patients with differentiated thyroid carcinoma (DTC). Methods: We included 198 DTC patients with extrathyroidal extension who received total or near-total thyroidectomy and then RIT in our hospital from January 1997 to June 2009: patients with lymph node metastases only (n = 47), lung metastases without bone metastases (n = 105), or bone metastases and other distant metastases (lung, liver, brain, and skin) (n = 46). Hemithyroidectomy or subtotal thyroidectomy had been performed before total or near-total thyroidectomy in 59 patients. Disease-specific survival after initial RIT was statistically evaluated using relevant clinical parameters, including age at initial RIT, pathology, sex, therapeutic history before initial RIT, pre- and posttherapeutic serum thyroglobulin (“prethyroglobulin” and “postthyroglobulin,” respectively) at initial RIT, thyroglobulin under thyroid-stimulating hormone stimulation at initial RIT (“peak thyroglobulin”), grade of uptake at scintigraphy, extent of metastasis, and number of total RITs. Results: During follow-up after initial RIT (average, 5.37 y), 24 patients died from DTC (11 male patients and 13 female). The most common reasons for death were respiratory failure due to lung metastases (n = 11) and uncontrollable brain metastases (n = 6). Univariate analysis showed that disease-specific survival was related to the following factors: extent of metastasis, age at initial RIT (<45 y), prethyroglobulin (<125 ng/mL), peak thyroglobulin (<1,000 ng/mL), and interval from total thyroidectomy to initial RIT (<180 d). A past history of hemithyroidectomy or subtotal thyroidectomy was not related to disease-specific survival. Multivariate analysis showed 3 factors to be independent prognostic factors—grade of 131I uptake at whole-body scintigraphy, extent of metastasis, and interval to RIT (P ≤ 0.001, 0.010, and 0.005, respectively)—and also showed that risk of death in patients with an interval over 180 d was 4.22 times higher than in those with an interval within 180 d. Kaplan–Meier analysis revealed that a shorter interval (180, 365, or 1,000 d) had prognostic value even in the subgroups 45 y or older, with lung metastases, and with bone metastases or more. Conclusion: The present study suggests that delaying initial RIT until more than 180 d after total thyroidectomy may result in poor survival for DTC patients.

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.