Yang Sook Chun

Bortezomib inhibits tumor adaptation to hypoxia by stimulating the FIH-mediated repression of hypoxia-inducible factor-1

Bortezomib (PS-341), a proteasome inhibitor, has been examined clinically for the treatment of multiple myeloma and several solid tumors. Bortezomib directly induces tumor cell death and has also been reported to inhibit tumor adaptation to hypoxia by functionally inhibiting hypoxia-inducible factor-1alpha (HIF-1alpha). However, the mechanism underlying HIF-1 inhibition by bortezomib remains obscure. In the present study, we demonstrated that bortezomib attenuated the hypoxic induction of erythropoietin and vascular endothelial growth factor at subnanomolar concentrations in multiple myeloma and liver cancer cell lines, regardless of cytotoxic concentrations of bortezomib. Bortezomib repressed HIF-1alpha activity by inhibiting the recruitment of p300 coactivator. Specifically, bortezomib targeted HIF-1alpha C-terminal transactivation domain (CAD) but not the CAD lacking Asn803, which is a hydroxylation site by the factor inhibiting HIF-1 (FIH). Accordingly, this effect of bortezomib on CAD was augmented by FIH expression and abolished by FIH knock-down. Furthermore, bortezomib stimulated the interaction between CAD and FIH under hypoxic conditions, and FIH inhibition reversed the suppressions of erythropoietin and vascular endothelial growth factor by bortezomib. We propose that the mechanism underlying the inhibitory effects of bortezomib on tumor angiogenesis and hypoxic adaptation involves the repression of HIF-1alpha transcriptional activity by reinforcing the FIH-mediated inhibition of p300 recruitment.

Bone density at interradicular sites: Implications for orthodontic mini-implant placement

OBJECTIVESnImplant stability is primarily related to local bone density; Few studies have evaluated interradicular bone density related to mini-implant placement for orthodontic anchorage. Therefore, this study evaluated bone density differences between interradicular sites.nnnSETTING AND SAMPLE POPULATIONnComputed tomographic (CT) images were obtained from 14 males and 14 females (mean age 27 years, range 23-35 years). Bone density in Hounsfield units (HU) was measured at 13 interradicular sites and four bone levels.nnnRESULTSnBone densities in most areas were higher than 850 HU. Statistically significant differences in bone density were detected at different levels and sites. Bone densities in both maxilla and mandible significantly increased from the alveolar crest toward basal bone in posterior areas, while the opposite was observed in anterior areas. There were statistically significant differences in bone densities between the maxilla and mandible in posterior areas. Bone densities progressively increased from anterior to posterior areas in the mandible.nnnCONCLUSIONnThe results suggest that mini-implants for orthodontic anchorage may be effective when placed in most areas with equivalent bone density up to 6 mm apical to the alveolar crest. Site selection should be adjusted according to bone density assessment.

Comparison of cortical bone thickness and root proximity at maxillary and mandibular interradicular sites for orthodontic mini‐implant placement

OBJECTIVESnTo compare maxillary and mandibular cortical bone thickness and rootic proximity for optimal mini-implant placement.nnnSETTING AND SAMPLE POPULATIONnCT images from 14 men and 14 women were used to evaluate buccal interradicular cortical bone thickness and root proximity from mesial of the central incisor to the 2nd molar. Cortical bone thickness was measured at 0 degrees , 15 degrees , 30 degrees , and 45 degrees angles relative to the root surface using three-dimensional images.nnnRESULTSnFor the cortical bone thickness, there was no statistically significant difference between the maxilla and the mandible in the anterior area; however, there was a significant difference in the posterior area. Cortical bone in the maxilla, mesial and distal to canine interradicular sites, was thickest while thickness in the mandible exhibited a gradual anterior to posterior increase. Cortical bone thickness in the maxilla increased as both level and angle increased, while the cortical bone thickness in the mandible was greatest at 4 mm from the alveolar crest. Root proximity mesial and distal to 2nd premolar interradicular sites was greatest.nnnCONCLUSIONnBased on our results, cortical bone thickness depends on the interradicular site rather than sex or individual differences.

CITED2 mediates the paradoxical responses of HIF-1α to proteasome inhibition

Hypoxia-inducible factor-1α (HIF-1α) is destabilized via the ubiquitin–proteasome system. Thus HIF-1α expression is robustly upregulated by proteasome inhibition, but paradoxically its activity is reduced. In the present study, we investigated the mechanism underlying the paradoxical response of HIF-1α to proteasome inhibition. In both Hep3B and HEK293 cells, a proteasome inhibitor MG132 noticeably attenuated hypoxic induction of erythropoietin and VEGF mRNAs. MG132 inactivated HIF-1α C-terminal transactivation domain (CAD), independently of factor inhibiting HIF-1 (FIH) and inhibited p300 recruitment by HIF-1α. We next tested the possibility that CITED2 is involved in the HIF-1 inactivation. CITED2 was found to be degraded via the ubiquitin–proteasome system and thus was stabilized by proteasome inhibition. Both the activity and the p300 binding of HIF-1α were inhibited by CITED2 expression and recovered by CITED2 siRNA in the presence of MG132. These results suggest that CITED2 is stabilized by proteasome inhibition and inactivates HIF-1 by interfering with the HIF-1α–p300 interaction. This may be an important mode-of-action for proteasome inhibition-based cancer therapy.

PHF2 histone demethylase acts as a tumor suppressor in association with p53 in cancer

Plant homeodomain finger 2 (PHF2) has a role in epigenetic regulation of gene expression by demethylating H3K9-Me2. Several genome-wide studies have demonstrated that the chromosomal region including the PHF2 gene is often deleted in some cancers including colorectal cancer, and this finding encouraged us to investigate the tumor suppressive role of PHF2. As p53 is a critical tumor suppressor in colon cancer, we tested the possibility that PHF2 is an epigenetic regulator of p53. PHF2 was associated with p53, and thereby, promoted p53-driven gene expression in cancer cells under genotoxic stress. PHF2 converted the chromatin that is favorable for transcription by demethylating the repressive H3K9-Me2 mark. In an HCT116 xenograft model, PHF2 was found to be required for the anticancer effects of oxaliplatin and doxorubicin. In PHF2-deficient xenografts, p53 expression was profoundly induced by both drugs, but its downstream product p21 was not, suggesting that p53 cannot be activated in the absence of PHF2. To find clinical evidence about the role of PHF2, we analyzed the expressions of PHF2, p53 and p21 in human colon cancer tissues and adjacent normal tissues from patients. PHF2 was downregulated in cancer tissues and PHF2 correlated with p21 in cancers expressing functional p53. Colon and stomach cancer tissue arrays showed a positive correlation between PHF2 and p21 expressions. Informatics analyses using the Oncomine database also supported our notion that PHF2 is downregulated in colon and stomach cancers. On the basis of these findings, we propose that PHF2 acts as a tumor suppressor in association with p53 in cancer development and ensures p53-mediated cell death in response to chemotherapy.

ITF2 Prevents Activation of the β-Catenin–TCF4 Complex in Colon Cancer Cells and Levels Decrease With Tumor Progression

BACKGROUND & AIMSnImmunoglobulin transcription factor 2 (ITF2) was believed to promote neoplastic transformation via activation of β-catenin. However, ITF2 recently was reported to suppress colon carcinogenesis. We investigated the roles of ITF2 in colorectal cancer cell lines and tumor formation and growth in mice.nnnMETHODSnLevels of ITF2, β-catenin, and c-Myc were measured in 12 human colorectal tumor samples and by immunohistochemistry. ITF2 regulation of β-catenin and T-cell factor (TCF) were analyzed using luciferase reporter, reverse-transcription quantitative polymerase chain reaction, flow cytometry, and immunoblot analyses. Mice were given subcutaneous injections of human colorectal cancer cell lines that stably express ITF2, small hairpin RNAs to reduce levels of ITF2, or control plasmids; xenograft tumor growth was assessed. Human colorectal carcinoma tissue arrays were used to associate levels of ITF2 expression and clinical outcomes.nnnRESULTSnLevels of β-catenin, cMyc, and ITF2 were increased in areas of human colon adenomas and carcinomas, compared with nontumor areas of the same tissues. ITF2 levels were reduced and cMyc levels were increased in areas of carcinoma, compared with adenoma. In human colorectal cancer cell lines, activation of the β-catenin-TCF4 complex and expression of its target genes were regulated negatively by ITF2. ITF2 inhibited formation ofxa0the β-catenin-TCF4 complex by competing with TCF4 for β-catenin binding. Stable transgenic expression of ITF2 in human colorectal cancer cell lines reduced their proliferation and tumorigenic potential in mice, whereas small hairpin RNA knockdown of ITF2 promoted growth of xenograft tumors in mice. In an analysis of colorectal tumor tissue arrays, loss of ITF2 from colorectal tumor tissues was associated with poor outcomes of patients. A gene set enrichment analysis supported the negative correlation between the level of ITF2 and activity of the β-catenin-TCF4 complex.nnnCONCLUSIONSnIn human colorectal cancer cell lines and tissue samples, ITF2 appears to prevent activation of the β-catenin-TCF4 complex and transcription of its gene targets. Loss of ITF2 promotes the ability of colorectal cancer cells to form xenograft tumors, and is associated with tumor progression and shorter survival times of patients.

von Hippel-Lindau protein adjusts oxygen sensing of the FIH asparaginyl hydroxylase

Hypoxia inevitably develops in rapidly growing tumors and acts as an important microenvironment that forces changes in tumor behavior. Hypoxia-inducible factor 1α (HIF-1α) is activated during hypoxia and promotes the progression of malignancy by stimulating angiogenesis and by augmenting the ability of tumors to survive. In aerobic conditions, HIF-1α is destabilized by the PHD prolyl-hydroxylases that target HIF-1α for proteolysis via the von Hippel-Lindau protein (pVHL) and further inactivated by the FIH asparaginyl-hydroxylase that precludes the recruitment of transcription coactivators. Although HIF-1α degradation is well understood, little is known about how its transcriptional activity increases gradually in response to decreasing oxygen. In particular, it has been questioned how FIH having a high affinity for oxygen regulates the HIF-1α activity in moderate hypoxia. We here found that the HIF-1α-FIH interaction is disrupted in 1-5% oxygen. Both in vitro and in vivo binding analyses revealed that pVHL acts as an adaptor for FIH to bind HIF-1α. Furthermore, because the pVHL-FIH interaction depends on oxygen tension, the FIH-mediated inactivation of HIF-1α can be exquisitely regulated according to the severity of hypoxia. Based on these findings, we propose that pVHL fine-tunes the transcriptional activity of HIF-1α in graded oxygen tensions.

HIF-1α Upregulation due to Depletion of the Free Ubiquitin Pool

Hypoxia-inducible factor 1alpha (HIF-1α), which transactivates a variety of hypoxia-induced genes, is rapidly degraded under nomoxia through the hydroxylation-ubiquitination-proteasome pathway. In this study, we addressed how HIF-1α is stabilized by proteasome inhibitors. The ubiquitin pool was rapidly reduced after proteasome inhibition, followed by the accumulation of non-ubiquitinated HIF-1α. The poly-ubiquitination of HIF-1α was resumed by restoration of free ubiquitin, which suggests that the HIF-1α stabilization under proteasome inhibition is attributed to depletion of the free ubiquitin pool. Ni2+ and Zn2+ also stabilized HIF-1α with depletion of the free ubiquitin pool and these effects of metal ions were attenuated by restoration of free ubiquitin. Ni2+ and Zn2+ may disturb the recycling of free ubiquitin, as MG132 does. Based on these results, the state of the ubiquitin pool seems to be another critical factor determining the cellular level of HIF-1α. Graphical Abstract

The interdental gingiva, a visible guide for placement of mini-implants

OBJECTIVESnTo determine whether the tip of the interdental gingiva can serve as a visible guide for placement of mini-implants.nnnSETTING AND SAMPLE POPULATIONnComputer tomography (CT) images from 15 males and 15 females (mean age 27 years, range: 23-35 years) were used to evaluate the distance from the tip of the interdental gingiva to the alveolar crest from the central incisor to the 1st molar. The distance from a reference point to the tip of interdental gingiva was recorded from study models using a caliper. The distance between the reference point and the alveolar crest was recorded using CT and added to the model recordings thus providing the distance from the tip of interdental gingiva to the alveolar crest for the various interdental sites. Two-way anova and Student-Newman-Keuls test for multiple comparisons were used for the statistical analysis.nnnRESULTSnThere was no significant difference in the distance from the tip of interdental gingiva to the alveolar crest between maxilla and mandible. The distance between the tip of interdental gingiva and the alveolar crest at the central/lateral incisors was the shortest compared with that of other sites. There was also a statistically significant difference between the male and female groups except for the maxillary 2nd premolar/1st molar interradicular site.nnnCONCLUSIONnThe tip of interdental gingiva appears a reasonable visual guide for the placement of mini-implants for orthodontic anchorage.