ZBTB2 links p53 deficiency to HIF-1-mediated hypoxia signaling to promote cancer aggressiveness.

Aberrant activation of the hypoxia-inducible transcription factor HIF-1 and dysfunction of the tumor suppressor p53 have been reported to induce malignant phenotypes and therapy resistance of cancers. However, their mechanistic and functional relationship remains largely unknown. Here, we reveal a mechanism by which p53 deficiency triggers the activation of HIF-1-dependent hypoxia signaling and identify zinc finger and BTB domain-containing protein 2 (ZBTB2) as an important mediator. ZBTB2 forms homodimers via its N-terminus region and increases the transactivation activity of HIF-1 only when functional p53 is absent. The ZBTB2 homodimer facilitates invasion, distant metastasis, and growth of p53-deficient, but not p53-proficient, cancers. The intratumoral expression levels of ZBTB2 are associated with poor prognosis in lung cancer patients. ZBTB2 N-terminus-mimetic polypeptides competitively inhibit ZBTB2 homodimerization and significantly suppress the ZBTB2-HIF-1 axis, leading to antitumor effects. Our data reveal an important link between aberrant activation of hypoxia signaling and loss of a tumor suppressor and provide a rationale for targeting a key mediator, ZBTB2, to suppress cancer aggressiveness.

Increased 14C-acetate accumulation in IDH-mutated human glioblastoma: implications for detecting IDH-mutated glioblastoma with 11C-acetate PET imaging.

PURPOSE: Recently, the potential value of isocitrate dehydrogenase (IDH) mutation as a prognostic marker in glioblastomas has been established. Glioblastomas are classified by their IDH mutation status under the 2016 WHO classification system. However, noninvasive diagnostic methods for the mutation status in glioblastoma patients have not been established so far. The purpose of this study was to evaluate the difference of acetate metabolism between in glioblastomas with wild-type IDH and in those with IDH mutation by comparing the uptake of 14C-acetate using genetically engineered glioblastoma cell lines in vitro and in vivo. METHODS: We established glioblastoma cells (U251) expressing IDH1 R132H and examined the cell uptake of [1-14C]acetate. Biodistribution studies and an autoradiographic study for U251 cell tumor-bearing mice (BALB/c-nu/nu) with or without the IDH1 mutation were performed 1 h after [1-14C]acetate administration. RESULTS: Significantly higher uptake of [1-14C]acetate was observed in U251/IDH1 R132H cells than in U251/IDH1 wild-type cells both in vitro (10.11 ± 0.94 vs. 4.26 ± 0.95%dose/mg, p = 0.0047) and in vivo (0.97 ± 0.14 vs. 0.66 ± 0.05%ID/g; p = 0.0037). Tumor-to-muscle ratios were also significantly higher in U251/IDH1 R132H tumors (3.36 ± 0.41 vs. 1.88 ± 0.59, p = 0.0030). The autoradiographic study shows the entirely higher radioactivity of the U251/IDH1 R132H tumor tissue section than that of the U251/IDH1 Wild-type tumor. CONCLUSIONS: In vitro and in vivo studies demonstrated that the uptake of radiolabeled acetate was significantly higher in IDH-mutated cells than in IDH-wild-type cells.

HIF-1 maintains a functional relationship between pancreatic cancer cells and stromal fibroblasts by upregulating expression and secretion of Sonic hedgehog.

Hypoxic and stroma-rich microenvironments, characteristic features of pancreatic cancers, are strongly associated with a poor prognosis. However, whether and how hypoxia increases stromal compartments remain largely unknown. Here, we investigated the potential importance of a master regulator of the cellular adaptive response to hypoxia, hypoxia-inducible factor-1 (HIF-1), in the formation of stroma-rich microenvironments of pancreatic tumors. We found that pancreatic cancer cells secreted more Sonic hedgehog protein (SHH) under hypoxia by upregulating its expression and efficiency of secretion in a HIF-1-dependent manner. Recombinant SHH, which was confirmed to activate the hedgehog signaling pathway, accelerated the growth of fibroblasts in a dose-dependent manner. The SHH protein secreted from pancreatic cancer cells under hypoxic conditions promoted the growth of fibroblasts by stimulating their Sonic hedgehog signaling pathway. These results suggest that the increased secretion of SHH by HIF-1 is potentially responsible for the formation of detrimental and stroma-rich microenvironments in pancreatic cancers, therefore providing a rational basis to target it in cancer therapy.

A circadian clock gene, PER2, activates HIF-1 as an effector molecule for recruitment of HIF-1α to promoter regions of its downstream genes.

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor functioning in cellular adaptive responses to hypoxia. Recent studies have suggested that HIF-1 activity is upregulated by one of the important circadian clock genes, period circadian clock 2 (PER2); however, its underlying mechanism remains unclear. Here, we show that PER2 functions as an effector protein for the recruitment of HIF-1α to its cognate enhancer sequence, the hypoxia-response element (HRE). We found that the forced expression of PER2 enhanced HIF-1 activity without influencing expression levels of the regulatory subunit of HIF-1, HIF-1α, at either mRNA or protein levels. A series of coimmunoprecipitation-based experiments revealed that PER2 interacted with HIF-1α and facilitated the recruitment of HIF-1α to HRE derived from vascular endothelial growth factor (VEGF) promoter. The PER2-mediated activation of HIF-1 was observed only when the asparagine residue at position 803 of HIF-1α (HIF-1α N803) was kept unhydroxylated by hypoxic stimulation, by introducing an N803A point mutation, or by an inhibitor of N803-dioxygenase, deferoxamine. However, the extent of PER-2-HIF-1α interaction was equivalent regardless of the N803 hydroxylation status. Taken together, these results suggest that, with the help of an unknown sensor molecule for the N803 hydroxylation status, PER2 functions as an effector molecule for the recruitment of HIF-1 to promoter regions of its downstream genes. Our findings reveal a novel regulatory step in the activation of HIF-1, which can be targeted to develop therapeutic strategies against HIF-1-related diseases, such as cancers.

UCHL1-HIF-1 axis-mediated antioxidant property of cancer cells as a therapeutic target for radiosensitization.

Hypoxia-inducible factor 1 (HIF-1) has been recognized as an important mediator of the reprogramming of carbohydrate metabolic pathways from oxidative phosphorylation to accelerated glycolysis. Although this reprogramming has been associated with the antioxidant and radioresistant properties of cancer cells, gene networks triggering the HIF-1-mediated reprogramming and molecular mechanisms linking the reprogramming with radioresistance remain to be determined. Here, we show that Ubiquitin C-terminal hydrolase-L1 (UCHL1), which we previously identified as a novel HIF-1 activator, increased the radioresistance of cancer cells by producing an antioxidant, reduced glutathione (GSH), through HIF-1-mediated metabolic reprogramming. A luciferase assay to monitor HIF-1 activity demonstrated that the overexpression of UCHL1, but not its deubiquitination activity-deficient mutant (UCHL1 C90S), upregulated HIF-1 activity by stabilizing the regulatory subunit of HIF-1 (HIF-1α) in a murine breast cancer cell line, EMT6. UCHL1 overexpression induced the reprogramming of carbohydrate metabolism and increased NADPH levels in a pentose phosphate pathway (PPP)-dependent manner. The UCHL1-mediated reprogramming elevated intracellular GSH levels, and consequently induced a radioresistant phenotype in a HIF-1-dependent manner. The pharmacological inhibition of PPP canceled the UCHL1-mediated radioresistance. These results collectively suggest that cancer cells acquire antioxidant and radioresistant phenotypes through UCHL1-HIF-1-mediated metabolic reprogramming including the activation of PPP and provide a rational basis for targeting this gene network for radiosensitization.

pH-Responsive near-infrared fluorescent cyanine dyes for molecular imaging based on pH sensing.

Indocyanine green (ICG) derivatives having nucleophilic substituents were synthesized as pH-responsive near-infrared dyes. pH-responsive dyes 1-C with closed-ring structures smoothly internalized and converted to emissive open-ring structures 1-O in response to relatively low pHs in acidic intracellular compartments of HeLa cells.

LY6E: a conductor of malignant tumor growth through modulation of the PTEN/PI3K/Akt/HIF-1 axis.

Lymphocyte antigen 6 complex, locus E (LY6E) has been implicated in the malignant progression of various types of cancers; however, the underlying mechanism remains unclear. Here, we identified LY6E as an activator of HIF-1 and revealed their mechanistic and functional links in malignant tumor growth. The aberrant overexpression of LY6E increased HIF-1α gene expression principally at the transcription level. This, in turn, led to the expression of the pro-angiogenic factors, VEGFA and PDGFB, through decreases in the expression levels of PTEN mRNA and subsequent activation of the PI3K/Akt pathway. The LY6E-HIF-1 axis functioned to increase tumor blood vessel density and promoted tumor growth in immunodeficient mice. LY6E expression levels were significantly higher in human breast cancers than in normal breast tissues, and were strongly associated with the poor prognoses of various cancer patients. Our results characterized LY6E as a novel conductor of tumor growth through its modulation of the PTEN/PI3K/Akt/HIF-1 axis and demonstrated the validity of targeting this pathway for cancer therapy.

PLK1 blockade enhances therapeutic effects of radiation by inducing cell cycle arrest at the mitotic phase.

The cytotoxicity of ionizing radiation depends on the cell cycle phase; therefore, its pharmacological manipulation, especially the induction of cell cycle arrest at the radiosensitive mitotic-phase (M-phase), has been attempted for effective radiation therapy. Polo-like kinase 1 (PLK1) is a serine/threonine kinase that functions in mitotic progression, and is now recognized as a potential target for radiosensitization. We herein investigated whether PLK1 blockade enhanced the cytotoxic effects of radiation by modulating cell cycle phases of cancer cells using the novel small molecule inhibitor of PLK1, TAK-960. The TAK-960 treatment exhibited radiosensitizing effects in vitro, especially when it increased the proportion of M-phase cells. TAK-960 did not sensitize cancer cells to radiation when an insufficient amount of time was provided to induce mitotic arrest. The overexpression of a PLK1 mutant, PLK1-R136G&T210D, which was confirmed to cancel the TAK-960-mediated increase in the proportion of mitotic cells, abrogated the radiosensitizing effects of TAK-960. A tumor growth delay assay also demonstrated that the radiosensitizing effects of TAK-960 depended on an increase in the proportion of M-phase cells. These results provide a rational basis for targeting PLK1 for radiosensitization when considering the therapeutic time window for M-phase arrest as the best timing for radiation treatments.

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.

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.

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.

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.

The Akt/mTOR pathway assures the synthesis of HIF-1alpha 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-1alpha 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-1alpha. In vitro experiments confirmed that an increase in glucose availability induced Akt phosphorylation under reoxygenated conditions and consequently up-regulated HIF-1alpha translation. Moreover, both the accelerated translation and the previously reported reactive oxygen species-mediated stabilization of HIF-1alpha protein were essential to the activation of HIF-1. All of these results indicate that Akt/mTOR-dependent translation of HIF-1alpha 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.

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 gamma-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.

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.

The combination of hypoxia-response enhancers and an oxygen-dependent proteolytic motif enables real-time imaging of absolute HIF-1 activity in tumor xenografts.

The transcriptional activity of hypoxia-inducible factor-1 (HIF-1) is associated with tumor malignancies; therefore, it is important to comprehend its dynamism in solid tumors. However, a molecular imaging strategy to accurately access it remains to be developed. We constructed here a novel HIF-1-dependent reporter gene, 5HREp-ODD-luc, in which 5 copies of the hypoxia-response element (5HRE) enhance expression of the oxygen-dependent degradation (ODD) domain and luciferase (luc) fusion under hypoxia. Because the ODD domain caused the oxygen-dependent degradation of the ODD-Luc protein, the novel reporter gene showed little leak of luminescence under normoxia. Such a property caused an increase of the hypoxia-responsiveness up to about 4.7 x 10(4) -fold. Moreover, the ODD domain caused rapid degradation of the ODD-Luc protein under normoxia, the luminescence reflected the dynamism of HIF-1 activity in real-time. The superiority of the novel reporter gene will surely accelerate analysis of the intratumoral HIF-1 activity during tumor progression and cancer treatments.