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Tumor angiogenesis is essential for tumor progression. The inhibition of tumor angiogenesis is a promising therapy for tumors. Bovine lactoferrin (bLF) has been reported as an anti-tumor agent. However, bLF effects on tumor angiogenesis are not well demonstrated. This study evaluated the inhibitory effects of bLF on tumor angiogenesis in vivo and in vitro. Herein, tumor endothelial cells (TECs) and normal endothelial cells (NECs) were used. Proliferation, migration, tube formation assays, RT-PCR, flow cytometry, Western blotting, siRNA experiments and immunoprecipitation were conducted to clarify the mechanisms of bLF-induced effects. CD-31 immunoexpression was examined in tumor tissues of oral squamous cell carcinoma mouse models with or without Liposomal bLF (LbLF)-administration. We confirmed that bLF inhibited proliferation/migration/tube formation and increased apoptosis in TECs but not NECs. TNF receptor-associated factor 6 (TRAF6), p-p65, hypoxia inducible factor-α (HIF-1α) and vascular endothelial growth factor (VEGF) were highly expressed in TECs. In TECs, bLF markedly downregulated VEGF-A, VEGF receptor (VEGFR) and HIF-1α via the inhibition of p-p65 through binding with TRAF6. Since NECs slightly expressed p-p65, bLF-TRAF-6 binding could not induce detectable changes. Moreover, orally administrated LbLF decreased CD31-positive microvascular density only in TECs. Hence, bLF specifically suppressed tumor angiogenesis through p-p65 inhibition by binding to TRAF6 and suppressing HIF-1α activation followed by VEGF/VEGFR down-regulation. Collectively, bLF can be an anti-angiogenic agent for tumors.

Oral squamous cell carcinoma (OSCC) impairs functionality and sensuousness resulting in poor quality of life. Biomarkers can predict disease trajectory and lead to effective treatments. Transcriptomics have identified genes that are upregulated in tumor endothelial cells (TECs) compared with normal endothelial cells (NECs). Among them, chemokine receptor 7 (CXCR7) is highly expressed in TECs of several cancers and involved in angiogenesis of TECs. However, levels of CXCR7 in OSCC blood vessels have not been fully investigated. In this study, we analyzed the correlation between CXCR7 expression in TECs and clinicopathological factors in OSCC. Immunohistochemistry for CXCR7 and CD34 was performed on 59 OSCC tissue specimens resected between 1996 and 2008 at Hokkaido University Hospital. CXCR7 expression in blood vessels was evaluated by the ratio of CXCR7+/CD34+ blood vessels. CXCR7 expression was 42% and 19% in tumor and non-tumor parts, respectively, suggesting that CXCR7 expression is higher in TECs than in NECs. CXCR7 expression in TECs correlated with advanced T-stage and cancer stage. Overall survival and disease-free survival rates were higher in low-expressing CXCR7 patients than in high-expressing. These results suggest that CXCR7 expression in blood vessels may be a useful diagnostic and prognostic marker for OSCC patients., (© 2021 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

AU-rich elements (AREs) are RNA elements that enhance the rapid decay of mRNAs, including those of genes required for cell growth and proliferation. HuR, a member of the embryonic lethal abnormal vision (ELAV) family of RNA-binding proteins, is involved in the stabilization of ARE-mRNA. The level of HuR in the cytoplasm is up-regulated in most cancer cells, resulting in the stabilization of ARE-mRNA. We developed the adenoviruses AdARET and AdAREF, which include the ARE of TNF- α and c-fos genes in the 3'-untranslated regions of the E1A gene, respectively. The expression of the E1A protein was higher in cancer cells than in normal cells, and virus production and cytolytic activities were also higher in many types of cancer cells. The inhibition of ARE-mRNA stabilization resulted in a reduction in viral replication, demonstrating that the stabilization system was required for production of the virus. The growth of human tumors that formed in nude mice was inhibited by an intratumoral injection of AdARET and AdAREF. These results indicate that these viruses have potential as oncolytic adenoviruses in the vast majority of cancers in which ARE-mRNA is stabilized., Competing Interests: The authors declare no conflicts of interest.

AU-rich elements (AREs) are RNA elements that enhance the rapid decay of mRNA. The fate of ARE-mRNA is controlled by ARE-binding proteins. HuR, a member of the embryonic lethal abnormal vision (ELAV) family of RNA-binding proteins, is involved in the export and stabilization of ARE-mRNA. In the vast majority of cancer cells, HuR constitutively relocates to the cytoplasm, resulting in the stabilization of ARE-mRNA. Previously, we described that the adenovirus gene product, E4orf6, which is necessary for virus replication, participates in ARE-mRNA export and stabilization. In the present study, we showed the oncolytic potential of E4orf6-deleted adenovirus dl355, which is expected to be replicated selectively in cancer cells. Virus production and cytolytic activity of dl355 were higher in cancer cells than in normal cells. HuR-depletion downregulated dl355 replication, demonstrating that ARE-mRNA stabilization is required for the production of this virus. Tumor growth was inhibited in nude mice by an intratumoral injection of dl355. Furthermore, dl355 had a stronger oncolytic effect than E1B55k-deleted adenovirus. These results indicate that dl355 has potential as an oncolytic adenovirus for a large number of cancers where ARE-mRNA is stabilized.

Special AT-rich sequence-binding protein 2 (SATB2)-associated syndrome (SAS) is characterized by alterations of SATB2. Its clinical features include intellectual disability and craniofacial abnormalities, such as cleft palate, dysmorphic features, and dental abnormalities. Here, we describe three previously undiagnosed, unrelated patients with SAS who exhibited dental abnormalities, including multiple odontomas. Although isolated odontomas are common, multiple odontomas are rare. Individuals in families 1 and 3 underwent whole-exome sequencing. Patient 2 and parents underwent targeted amplicon sequencing. On the basis of the hg19/GRCh37 reference and the RefSeq mRNA NM_001172517, respective heterozygous mutations were found and validated in Patients 1, 2, and 3: a splice-site mutation (chr2:g.200137396C > T, c.1741-1G > A), a nonsense mutation (chr2:g.200213750G > A, c.847C > T, p.R283*), and a frame-shift mutations (chr2:g.200188589_200188590del, c.1478_1479del, p.Q493Rfs*19). All mutations occurred de novo. The mutations in Patients 1 and 3 were novel; the mutation in Patient 2 has been described previously. Tooth mesenchymal cells derived from Patient 2 showed diminished SATB2 expression. Multiple odontomas were evident in the patients in this report; however, this has not been recognized previously as a SAS-associated phenotype. We propose that multiple odontomas be considered as an occasional manifestation of SAS., (© 2018 Wiley Periodicals, Inc.)

Human antigen R (HuR) is a RNA-binding protein, which binds to the AU-rich element (ARE) in the 3'-untranslated region (3'-UTR) of certain mRNA and is involved in the export and stabilization of ARE-mRNA. HuR constitutively relocates to the cytoplasm in many cancer cells, however the mechanism of intracellular HuR trafficking is poorly understood. To address this question, we examined the functional role of the cytoskeleton in HuR relocalization. We tested the effect of actin depolymerizing macrolide latrunculin A or myosin II ATPase activity inhibitor blebbistatin for HuR relocalization induced by the vasoactive hormone Angiotensin II in cancer and control normal cells. Western blot and confocal imaging data revealed that both inhibitors attenuated the cytoplasmic HuR in normal cells but no such alteration was observed in cancer cells. Concomitant with changes in intracellular HuR localization, both inhibitors markedly decreased the accumulation and half-lives of HuR target ARE-mRNAs in normal cells, whereas no change was observed in cancer cells. Furthermore, co-immunoprecipitation experiments with HuR proteins revealed clear physical interaction with ß-actin only in normal cells. The current study is the first to verify that cancer cells can implicate a microfilament independent HuR transport. We hypothesized that when cytoskeleton structure is impaired, cancer cells can acquire an alternative HuR trafficking strategy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

HuR is an RNA-binding protein of the embryonic lethal abnormal vision (ELAV) family, which binds to the AU-rich element (ARE) in the 3'-untranslated region (UTR) of certain mRNAs and is involved in the nucleo-cytoplasmic export and stabilization of ARE-mRNAs. The cytoplasmic relocalization of ARE-mRNAs with several proteins such as HuR and pp32 increases in cells transformed by the adenovirus oncogene product E4orf6. Additionally, these ARE-mRNAs were stabilized and acquired the potential to transform cells. Although, the relocalization of HuR and the stabilization of ARE-mRNAs are crucial for cell transformation, evidence regarding the relationship of HuR and ARE-mRNAs with adenovirus replication is lacking. In this report, we demonstrate that adenovirus infection induces the relocation of HuR to the cytoplasm of host cells. Analysis using the luciferase-ARE fusion gene and the tetracycline (tet)-off system revealed that the process of stabilizing ARE-mRNAs is activated in adenovirus-infected cells. Heat shock treatment or knockdown-mediated depletion of HuR reduced adenovirus production. Furthermore, expression of ARE-including viral IVa2 mRNA, decreased in HuR-depleted infected cells. These results indicate that HuR plays an important role in adenovirus replication, at least in part, by up-regulating IVa2 mRNA expression and that ARE-mRNA stabilization is required for both transformation and virus replication., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Tumor blood vessels play an important role in tumor progression and metastasis. We previously reported that tumor endothelial cells (TEC) exhibit several altered phenotypes compared with normal endothelial cells (NEC). For example, TEC have chromosomal abnormalities and are resistant to several anticancer drugs. Furthermore, TEC contain stem cell-like populations with high aldehyde dehydrogenase (ALDH) activity (ALDH high TEC). ALDH high TEC have proangiogenic properties compared with ALDH low TEC. However, the association between ALDH high TEC and drug resistance remains unclear. In the present study, we found that ALDH mRNA expression and activity were higher in both human and mouse TEC than in NEC. Human NEC:human microvascular endothelial cells (HMVEC) were treated with tumor-conditioned medium (tumor CM). The ALDH high population increased along with upregulation of stem-related genes such as multidrug resistance 1, CD90, ALP, and Oct-4. Tumor CM also induced sphere-forming ability in HMVEC. Platelet-derived growth factor (PDGF)-A in tumor CM was shown to induce ALDH expression in HMVEC. Finally, ALDH high TEC were resistant to fluorouracil (5-FU) in vitro and in vivo. ALDH high TEC showed a higher grade of aneuploidy compared with that in ALDH low TEC. These results suggested that tumor-secreting factor increases ALDH high TEC populations that are resistant to 5-FU. Therefore, ALDH high TEC in tumor blood vessels might be an important target to overcome or prevent drug resistance., (© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Objective: Hypoxia is a common feature and prognostic factor in cancer. 18 F-fluoromisonidazole (FMISO) positron emission tomography (PET) can detect tumor hypoxia noninvasively. The aim of this study was to assess the correlations between FMISO-PET and 18 F-fluorodexyglucose (FDG)-PET parameters with cell proliferation and hypoxia in patients with oral squamous cell carcinoma (OSCC)., Study Design: Twenty-three preoperative patients with OSCC were included. The tumor/muscle ratio (TMR) of FMISO-PET, the maximum standardized uptake values (SUV max ) of FDG-PET, metabolic tumor volume, and total lesion glycolysis were measured. Ki-67 and hypoxia-inducible factor-1α (HIF-1α) expression was immunohistochemically evaluated., Results: FMISO TMR (P = .003) and FDG SUV max (P = .04) were significantly higher in patients with high expression of Ki-67 compared with those with low expression of Ki-67. FMISO TMR (P = .006) and FDG SUV max (P = .01) were also significantly higher in patients with HIF-1α expression than in those without HIF-1α expression. Metabolic tumor volume was not significantly related to either Ki-67 or HIF-1α expression. Multivariate analysis showed that FMISO TMR was independently predictive of Ki-67 (P = .002; odds ratio 31.1) and HIF-1α (P = .049; odds ratio 10.5) expression., Conclusions: FMISO-PET showed significant relationships with Ki-67 and HIF-1α expression, which are key features of cell proliferation and hypoxia in OSCC., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Tumor progression is dependent on tumor angiogenesis. We previously reported that the phenotype of tumor endothelial cells (TECs) is distinct from normal endothelial cells (NECs). Herein, we conducted a pathway analysis using a public TEC microarray database and identified several putative TEC-specific miRNAs. We found that miR-145 expression was upregulated in TECs and that miR-145 enhanced cell adhesion and anoikis resistance and upregulated Bcl-2 and Bcl-xl via ERK1/2 in human microvascular endothelial cells. These findings suggested that miR-145 is involved in the acquisition of the TEC phenotype, partially. Therefore, miR-145 and its target genes may be molecular targets for anti-angiogenic therapy., (© The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Reactive oxygen species (ROS) are unstable molecules that activate oxidative stress. Because of the insufficient blood flow in tumors, the tumor microenvironment is often exposed to hypoxic condition and nutrient deprivation, which induces ROS accumulation. We isolated tumor endothelial cells (TECs) and found that they have various abnormalities, although the underlying mechanisms are not fully understood. Here we showed that ROS were accumulated in tumor blood vessels and ROS enhanced TEC migration with upregulation of several angiogenesis related gene expressions. It was also demonstrated that these genes were upregulated by regulation of Nuclear factor erythroid 2-related factor 2 (NRF2). Among these genes, we focused on Biglycan, a small leucine-rich proteoglycan. Inhibition of Toll-like receptors 2 and 4, known BIGLYCAN (BGN) receptors, cancelled the TEC motility stimulated by ROS. ROS inhibited NRF2 expression in TECs but not in NECs, and NRF2 inhibited phosphorylation of SMAD2/3, which activates transcription of BGN. These results indicated that ROS-induced BGN caused the pro-angiogenic phenotype in TECs via NRF2 dysregulation.

Aim: We evaluated the prognostic value of vasohibin-1 (VASH1) expression in head and neck squamous cell carcinoma., Materials and Methods: Immunohistochemistry for VASH1 and cluster of differentiation 34 (CD34) was performed on 61 head and neck squamous cell carcinoma specimens. The association between VASH1 expression in the tumour and clinical outcomes was analyzed statistically., Results: VASH1 staining in normal tissue adjacent to cancerous tissue was negative, whereas it was positive in tumour blood vessels and AE1/AE3 and Ki67-positive tumour cells. Therefore, we examined the association between VASH1 expression in the tumour and clinical outcomes. Patients with high VASH1 expression in tumour had significantly shorter disease-free survival and more frequently had lymph node recurrence than those with low VASH1 expression., Conclusion: These results suggest that VASH1 expression is associated with tumour progression and may be useful as a prognostic marker of head and neck squamous cell carcinoma., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Oral leukoplakia (OL) is a clinically diagnosed preneoplastic lesion of the oral cavity with an increased oral cancer risk. However, the risk of malignant transformation is still difficult to assess. The objective of the present study was to examine the expression patterns of aldehyde dehydrogenase 1 (ALDH1) and podoplanin in OL, and to determine their roles in predicting oral cancer development. In the present study, the expression patterns of ALDH1 and podoplanin were determined in samples from 79 patients with OL. The association between protein expression and clinicopathological parameters, including oral cancer-free survival, was analyzed during a mean follow-up period of 3.4 years. Expression of ALDH1 and podoplanin was observed in 61 and 67% patients, respectively. Kaplan-Meier analysis demonstrated that the expression of the proteins was correlated with the risk of progression to oral cancer. Multivariate analysis revealed that expression of ALDH1 and podoplanin was associated with 3.02- and 2.62-fold increased risk of malignant transformation, respectively. The malignant transformation risk of OL was considerably higher in cases with expression of both proteins. Point-prevalence analysis revealed that 66% of patients with co-expression of ALDH1 and podoplanin developed oral cancer. Taken together, our data indicate that ALDH1 and podoplanin expression patterns in OL are associated with oral cancer development, suggesting that ALDH1 and podoplanin may be useful biomarkers to identify OL patients with a substantially high oral cancer risk.

It has been described that tumor progression has many similarities to inflammation and wound healing in terms of the signaling processes involved. Among biological responses, angiogenesis, which is necessary for tumor progression and metastasis, is a common hallmark; therefore, tumor blood vessels have been considered as important therapeutic targets in anticancer therapy. We focused on pentraxin 3 (PTX3), which is a marker of cancer-related inflammation, but we found no reports on its expression and function in tumor blood vessels. Here we showed that PTX3 is expressed in mouse and human tumor blood vessels based on immunohistochemical analysis. We found that PTX3 is upregulated in primary mouse and human tumor endothelial cells compared to normal endothelial cells. We also showed that PTX3 plays an important role in the proliferation of the tumor endothelial cells. These results suggest that PTX3 is an important target for antiangiogenic therapy., (© 2016 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

The risk of malignant transformation in oral preneoplastic lesions (OPLs) is challenging to assess. The objective of the present study was to determine the expression of ELAV like RNA binding protein 1 (HuR) and podoplanin in OPLs, and to evaluate the use of each protein as biomarkers for the risk assessment of malignant transformations. Immunohistochemistry for HuR and podoplanin was performed on the tissues of 51 patients with OPL, including cases of low grade dysplasia (LGD) and high grade dysplasia (HGD). The association between the protein expression patterns and clinicopathological parameters, including oral cancer free survival (OCFS) time, was analyzed during the follow-up period. HuR and podoplanin expression was observed in 28 (55%) and 36 (71%) of 51 patients, respectively. Kaplan-Meier analysis showed that the expression of HuR and podoplanin was associated with the risk of progression to oral cancer (P<0.05). Multivariate analysis revealed that HuR and podoplanin expression was associated with a 2.93-fold (95% confidence interval (CI), 0.98-10.34; P=0.055) and 2.06-fold (95% CI, 0.55-8.01; P=0.283) increase in risk of malignant transformation, respectively. The risk of OPL malignant transformation was considerably increased with the coexpression of HuR and podoplanin compared with the histological grading (95% CI, 1.64-23.59; P=0.005). The results of the present study demonstrated that the expression of HuR and podoplanin associates with malignant transformation and suggests that the proteins may be used as biomarkers to identify OPL patients with an increased risk of cancer development.

Tumour blood vessels are gateways for distant metastasis. Recent studies have revealed that tumour endothelial cells (TECs) demonstrate distinct phenotypes from their normal counterparts. We have demonstrated that features of TECs are different depending on tumour malignancy, suggesting that TECs communicate with surrounding tumour cells. However, the contribution of TECs to metastasis has not been elucidated. Here, we show that TECs actively promote tumour metastasis through a bidirectional interaction between tumour cells and TECs. Co-implantation of TECs isolated from highly metastatic tumours accelerated lung metastases of low metastatic tumours. Biglycan, a small leucine-rich repeat proteoglycan secreted from TECs, activated tumour cell migration via nuclear factor-κB and extracellular signal-regulated kinase 1/2. Biglycan expression was upregulated by DNA demethylation in TECs. Collectively, our results demonstrate that TECs are altered in their microenvironment and, in turn, instigate tumour cells to metastasize, which is a novel mechanism for tumour metastasis.

Cellular interactions with the extracellular matrix play critical roles in tumor progression. We previously reported that receptor activator of NF-κB ligand (RANKL) specifically facilitates head and neck squamous cell carcinoma (HNSCC) progression in vivo. Here, we report a novel role for RANKL in the regulation of cell adhesion. Among the major type I collagen receptors, integrin α2 was significantly upregulated in RANKL-expressing cells, and its knockdown suppressed cell adhesion. The mRNA abundance of integrin α2 positively correlated with that of RANKL in human HNSCC tissues. We also revealed that RANK-NF-κB signaling mediated integrin α2 expression in an autocrine/paracrine manner. Interestingly, the amount of active integrin β1 on the cell surface was increased in RANKL-expressing cells through the upregulation of integrin α2 and endocytosis. Moreover, the RANK-integrin α2 pathway contributed to RANKL-dependent enhanced survival in a collagen gel and inhibited apoptosis in a xenograft model, demonstrating an important role for RANKL-mediated cell adhesion in three-dimensional environments.

We describe two patients with anti-BP180-type mucous membrane pemphigoid (MMP), who were correctly diagnosed and treated in early stages through the cooperation of dentists and dermatologists. Patient 1 was a 74-year-old woman who visited our dental department due to blisters over the oral mucosa and eruptions on the skin. She had also experienced bleeding of the gingiva and palate mucosa. Biopsy specimens from the oral mucosa revealed detachment of epithelial basement membrane and subepithelial lamina propria with slight chronic inflammation. Direct immunofluorescence (DIF) revealed linear IgG and IgA deposits along the basement membrane zone (BMZ). Indirect immunofluorescence (IIF) using 1 M-NaCl split normal human skin showed binding of IgG and IgA on the epidermal side. On immunoblot analysis, IgG and IgA autoantibodies reacted with the C-terminal protein of BP180. These findings indicated a diagnosis of anti-BP180-type MMP. Patient 2 was a 59-year-old woman who was referred to our dental department with a history of blisters and large erosions on the gingiva. Biopsy specimens from the oral mucosa revealed partial junctional separation at the level of the basement membrane. DIF showed linear depositions of IgG and C3 along the BMZ. IIF, using 1 M-NaCl split normal human skin, revealed circulating anti-BMZ-IgG antibodies bound to the epidermal side. These findings indicated a diagnosis of anti-BP180-type MMP. Both patients were treated successfully with systemic or topical steroids and oral health care. In conclusion, appropriate clinical examination and cooperation among medical specialists are important for the early diagnosis and treatment of patients with recurrent and chronic stomatitis and for their good prognosis.

We reported that tumor endothelial cells (TECs) differ from normal endothelial cells (NECs) in many aspects, such as gene expression profiles. Although CXCR7 is reportedly highly expressed in blood vessels of several tumors, its function in TECs is still unknown. To investigate this role, we isolated TECs from mouse tumor A375SM xenografts, and compared them with NECs from normal mouse dermis. After confirming CXCR7 upregulation in TECs, we analyzed its function using CXCR7 siRNA and CXCR7 inhibitor; CCX771. CXCR7 siRNA and CCX771 inhibited migration, tube formation and resistance to serum starvation in TECs but not in NECs. ERK1/2 phosphorylation was inhibited by CXCR7 knockdown in TECs. These results suggest that CXCR7 promotes angiogenesis in TECs via ERK1/2 phosphorylation. Using ELISA, we also detected CXCL12, a ligand of CXCR7, in conditioned medium from TECs, but not from NECs. CXCL12 neutralizing antibody significantly inhibited TEC random motility. VEGF stimulation upregulated CXCR7 expression in NECs, implying that VEGF mediates CXCR7 expression in endothelial cells. A CXCR7 inhibitor, CCX771 also inhibited tumor growth, lung metastasis and tumor angiogenesis in vivo. Taken together, the CXCL12-CXCR7 autocrine loop affects TEC proangiogenic properties, and could be the basis for an antiangiogenic therapy that specifically targets tumor blood vessels rather than normal vessels., (© 2015 UICC.)

Tumor angiogenesis plays an important role in tumor progression and metastasis. Tumor endothelial cells (TECs) are a therapeutic target of antiangiogenic chemotherapy that was recently developed and is currently being investigated in the clinic with promising results. Low-dose chemotherapy, which is the long-term administration of relatively low doses of chemotherapeutic agents, has been proposed for targeting tumor angiogenesis in various types of cancers. Although the efficacy of low-dose chemotherapy has been confirmed in several clinical models, some studies show insufficient therapeutic effect for malignant cancers. As a possible mechanism of the treatment failure, it has been considered that tumor cells may acquire resistance to this therapy. However, drug resistance by TECs may also be due to another mechanism for resistance of tumor cells to low-dose chemotherapy. We reported elsewhere that TECs were resistant to the anticancer drug paclitaxel, which is a mitotic inhibitor, concomitant with P-glycoprotein up-regulation. Verapamil, a P-glycoprotein inhibitor, abrogated TEC resistance in vitro. Herein, we demonstrated that verapamil coadministration enhanced the effects of low-dose paclitaxel concomitant with inhibiting tumor angiogenesis in a preclinical in vivo mouse melanoma xenograft model. Furthermore, verapamil coadministration reduced lung metastasis. These results suggest that inhibiting P-glycoprotein in TECs may be a novel strategy for low-dose chemotherapy targeting TECs., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Tumor blood vessels play an important role in tumor progression and metastasis. It has been reported that tumor endothelial cells (TECs) exhibit highly angiogenic phenotypes compared with those of normal endothelial cells (NECs). TECs show higher proliferative and migratory abilities than those NECs, together with upregulation of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2). Furthermore, compared with NECs, stem cell markers such as Sca-1, CD90, and multidrug resistance 1 are upregulated in TECs, suggesting that stem-like cells exist in tumor blood vessels. In this study, to reveal the biological role of stem-like TECs, we analyzed expression of the stem cell marker aldehyde dehydrogenase (ALDH) in TECs and characterized ALDHhigh TECs. TECs and NECs were isolated from melanoma-xenografted nude mice and normal dermis, respectively. ALDH mRNA expression and activity were higher in TECs than those in NECs. Next, ALDHhigh/low TECs were isolated by fluorescence-activated cell sorting to compare their characteristics. Compared with ALDHlow TECs, ALDHhigh TECs formed more tubes on Matrigel-coated plates and sustained the tubular networks longer. Furthermore, VEGFR2 expression was higher in ALDHhigh TECs than that in ALDHlow TECs. In addition, ALDH was expressed in the tumor blood vessels of in vivo mouse models of melanoma and oral carcinoma, but not in normal blood vessels. These findings indicate that ALDHhigh TECs exhibit an angiogenic phenotype. Stem-like TECs may have an essential role in tumor angiogenesis.

Recent studies have reported that stromal cells contribute to tumor progression. We previously demonstrated that tumor endothelial cells (TEC) characteristics were different from those of normal endothelial cells (NEC). Furthermore, we performed gene profile analysis in TEC and NEC, revealing that suprabasin (SBSN) was upregulated in TEC compared with NEC. However, its role in TEC is still unknown. Here we showed that SBSN expression was higher in isolated human and mouse TEC than in NEC. SBSN knockdown inhibited the migration and tube formation ability of TEC. We also showed that the AKT pathway was a downstream factor of SBSN. These findings suggest that SBSN is involved in the angiogenic potential of TEC and may be a novel TEC marker., (© 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.)

Purpose: Hypoxia, a prognostic factor in many types of cancer, can be detected by (18)F-fluoromisonidazole (FMISO) positron emission tomography (PET). It is unclear whether hypoxia reflects the response to chemotherapy in patients with oral squamous cell carcinoma (OSCC). The correlations of FMISO-PET and FDG-PET with histological response to preoperative chemotherapy were therefore assessed in patients with OSCC., Methods: This study enrolled 22 patients with OSCC undergoing preoperative chemotherapy. The T-stages were T2 in 6 patients, T3 in 3, and T4a in 13, and the N-stages were N0 in 14 patients, N1 in 3, and N2 in 5. Each patient was evaluated by both FMISO-PET and FDG-PET before surgery, and the maximum standardized uptake value (SUVmax) of FDG- and FMISO-PET and tumor-muscle ratio (TMR) of FMISO-PET were measured. The threshold for the hypoxic volume based on TMR was set at 1.25. The histological response to preoperative chemotherapy was evaluated using operative materials., Results: FMISO-PET and FDG-PET detected uptake by primary OSCCs in 15 (68%) and 21 (95%) patients, respectively, and median SUVmaxs of FMISO- and FDG-PET in the primary site were 2.0 (range, 1.3-3.5) and 16.0 (range, 1.0-32.2), respectively. The median of FMISO TMR was 1.5 (range, 0.99-2.96). There were five cases whose FMISO TMR was less than 1.25. Histological evaluation showed good response to preoperative chemotherapy in 7 patients (32%) and poor response in 15 (68%). Good response was significantly more prevalent in patients with negative than positive FMISO uptake (P < 0.001) and without the hypoxic area evaluated by FMISO-PET TMR (P = 0.04), whereas FDG uptake was not significantly correlated with response to chemotherapy response. Multivariate logistic regression analysis showed that FMISO uptake was an independent significant predictor of response to preoperative chemotherapy (P = 0.03, odds ratio = 0.06, 95% confidence interval = 0.004-0.759)., Conclusions: An advantage of FMISO-PET over FDG-PET for predicting histological response to preoperative chemotherapy in patients with OSCC was observed.

Recent studies have shown that certain types of transformed cells are extruded from an epithelial monolayer. However, it is not known whether and how neighbouring normal cells play an active role in this process. In this study, we demonstrate that filamin A and vimentin accumulate in normal cells specifically at the interface with Src- or RasV12-transformed cells. Knockdown of filamin A or vimentin in normal cells profoundly suppresses apical extrusion of the neighbouring transformed cells. In addition, we show in zebrafish embryos that filamin plays a positive role in the elimination of the transformed cells. Furthermore, the Rho/Rho kinase pathway regulates filamin accumulation and filamin acts upstream of vimentin in the apical extrusion. This is the first report demonstrating that normal epithelial cells recognize and actively eliminate neighbouring transformed cells and that filamin is a key mediator in the interaction between normal and transformed epithelial cells.

Oral verrucous carcinoma (OVC) is a low grade variant of oral squamous cell carcinoma, and oral verrucous hyperplasia (OVH) is a benign lesion without malignant features. However, pathologists are sometimes presented with borderline lesions and are indecisive as to diagnose them as benign or malignant. Thus, these lesions are tentatively termed oral verrucous lesions (OVLs). HuR is an ARE mRNA-binding protein, normally localized in the nucleus but cytoplasmic exportation is frequently observed in cancer cells. The present study aimed to elucidate whether expression of the HuR protein facilitates the diagnosis of true malignant lesions. Clinicopathological features were evaluated, and immunohistochemical analysis for p53, Ki67 and HuR proteins was performed in 48 cases of OVH, OVC and OVL, and the outcomes were correlated using appropriate statistical analysis. The association of these three proteins in relation to malignant transformation was analyzed after a 3-year follow-up of 25 OVL cases. The basal characteristics (age, gender and location) of all cases had no significant association with the types of lesions. Gingiva (39.4%) was the common site for all lesions. Distribution of the examined proteins had a significant association with the lesions. As compared with the OVLs, the number of immunostained-positive cells was significantly higher in the OVCs and lower in the OVH cases. During follow-up, 24% of the OVLs underwent malignant transformation for which high HuR expression and a diffuse staining pattern in the epithelium were observed. Taken together, the high degree of HuR expression with diffuse staining pattern in the epithelium may be an effective diagnostic tool that determines the potential of OVLs for malignant transformation.

pp32 is a tumor suppressor and is one of the associated proteins of the RNA-binding protein HuR. The pp32-HuR complex is exported to the cytoplasm of cells under stress conditions, and HuR is degraded by caspases in the cytoplasm. In the present study, we examined the role of pp32r1, a member of the pp32 family that has oncogenic properties, in the decay of HuR. pp32r1 was found to be abundantly expressed in cancer cells, and overexpression of pp32r1 induced colony formation in soft-agar. pp32r1 was expressed in both the nucleus and cytoplasm, whereas pp32 was predominantly localized in the nucleus. Even with lethal stress such as staurosporine (STS), HuR in the cytoplasm was never downregulated, and caspase-3 activity was inhibited when cells expressed pp32r1. pp32r1 bound to HuR without interacting with pp32. In cancer cells, HuR survived in the cytoplasm of cells overexpressing pp32r1, although HuR was not expressed in the cytoplasm of pp32-expressing cells, similar to lethal stress conditions. Taken together, these results indicate that pp32r1 binds to HuR to avoid the caspase-mediated decay of HuR in the cytoplasm of cells. We suggest that this function contributes to the oncogenic activity of pp32r1.

There is much evidence that hypoxia in the tumor microenvironment enhances tumor progression. In an earlier study, we reported abnormal phenotypes of tumor-associated endothelial cells such as those resistant to chemotherapy and chromosomal instability. Here we investigated the role of hypoxia in the acquisition of chromosomal abnormalities in endothelial cells. Tumor-associated endothelial cells isolated from human tumor xenografts showed chromosomal abnormalities, >30% of which were aneuploidy. Aneuploidy of the tumor-associated endothelial cells was also shown by simultaneous in-situ hybridization for chromosome 17 and by immunohistochemistry with anti-CD31 antibody for endothelial staining. The aneuploid cells were surrounded by a pimonidazole-positive area, indicating hypoxia. Human microvascular endothelial cells expressed hypoxia-inducible factor 1 and vascular endothelial growth factor A in response to either hypoxia or hypoxia-reoxygenation, and in these conditions, they acquired aneuploidy in 7 days. Induction of aneuploidy was inhibited by either inhibition of vascular endothelial growth factor signaling with vascular endothelial growth factor receptor 2 inhibitor or by inhibition of reactive oxygen species by N-acetyl-L-cysteine. These results indicate that hypoxia induces chromosomal abnormalities in endothelial cells through the induction of reactive oxygen species and excess signaling of vascular endothelial growth factor in the tumor microenvironment.

Background: Molecules that are highly expressed in tumour endothelial cells (TECs) may be candidates for specifically targeting TECs. Using DNA microarray analysis, we found that the lysyl oxidase (LOX) gene was upregulated in TECs compared with its expression in normal endothelial cells (NECs). LOX is an enzyme that enhances invasion and metastasis of tumour cells. However, there are no reports on the function of LOX in isolated TECs., Methods: TECs and NECs were isolated to investigate LOX function in TECs. LOX inhibition of in vivo tumour growth was also assessed using β-aminopropionitrile (BAPN)., Results: LOX expression was higher in TECs than in NECs. LOX knockdown inhibited cell migration and tube formation by TECs, which was associated with decreased phosphorylation of focal adhesion kinase (Tyr 397). Immunostaining showed high LOX expression in human tumour vessels in vivo. Tumour angiogenesis and micrometastasis were inhibited by BAPN in an in vivo tumour model., Conclusion: LOX may be a TEC marker and a possible therapeutic target for novel antiangiogenic therapy.

Unlabelled: Hypoxia is a common feature of cancer and a prognostic factor for many types of cancer. (18)F-fluoromisonidazole ((18)F-FMISO) PET can detect tumor hypoxia noninvasively. Hypoxia-inducible factor-1 (HIF-1) is a key player in the transcriptional response to low oxygen tension in many types of cancer. Its activity is mainly dependent on the stability and modification of HIF-1α, which is a composition of HIF-1. However, it is unclear whether (18)F-FMISO PET can identify HIF-1α expression in oral squamous cell carcinoma (OSCC). The present study was performed to elucidate the correlation between (18)F-FMISO PET findings and HIF-1α expression in OSCC., Methods: Twenty-three patients (age range, 42-84 y; 15 men, 8 women) with OSCC were enrolled in this study. The T-stages of cancer were T1 in 1 patient, T2 in 9, T3 in 2, and T4a in 11. The N-stages were N0 in 13 patients, N1 in 5, and N2 in 5. Each patient underwent (18)F-FMISO and (18)F-FDG PET before surgery, and the maximum standardized uptake value (SUV max) of both PET studies was measured. HIF-1α expression in the operation materials was evaluated by immunohistochemical staining. The SUV max of both PET studies and HIF-1α findings were compared statistically., Results: (18)F-FMISO PET detected uptake in the primary site in 14 of the 23 patients (61%). The median SUV max of (18)F-FMISO and (18)F-FDG PET in the primary site was 1.83 (range, 0.8-2.7) and 16.5 (range, 1.0-32.3), respectively. There was a weak significant correlation between (18)F-FMISO and (18)F-FDG PET SUV max (P = 0.02, r = 0.48). HIF-1α expression was clearly detected in 11 of the 23 patients (48%). The (18)F-FMISO PET SUV max was significantly higher in the HIF-1α-positive cases than in the HIF-1α-negative cases (median, 2.1; range, 1.5-2.4, vs. median, 1.6; range, 0.8-2.0, respectively) (P = 0.002). However, there were no significant correlations between (18)F-FDG PET SUV max and HIF-1α expression (median, 21.8; range, 7.7-29.1 vs. 1.0-32.2) (P = 0.06)., Conclusion: (18)F-FMISO uptake in the primary site of OSCC indicates a hypoxic environment with HIF-1α expression.

Parathyroid hormone-related protein (PTHrP) is known to induce bone resorption by activating RANKL as well as PTH. PTHrP plays a central role in humoral hypercalcemia, and its expression has been reported to be closely associated with bone metastasis of breast carcinoma. PTHrP expression in oral squamous carcinoma cell lines was investigated, and PTHrP was expressed in oral squamous cell carcinoma cell lines similar to that in a prostate carcinoma cell line. Mucoepidermoid carcinoma is the most common malignant salivary gland tumor composed of different types of cells including a squamous component. Its clinical behavior is highly variable and ranges from slow-growing and indolent to locally aggressive and highly metastatic. We examined the PTHrP expression in mucoepidermoid carcinoma and assessed the significance of its correlation with clinicopathological features. Immunohistochemical detection of PTHrP was carried out in 21 cases of mucoepidermoid carcinoma in the head and neck region. PTHrP was highly detectable in intermediate and epidermoid cells, and abundant expression of PTHrP in intermediate cells had a significant association with cancer malignancy, including lymph node metastasis and/or tumor recurrence. These results suggest that PTHrP expression can be used as a prognostic factor for mucoepidermoid carcinoma.

The molecular mechanisms underlying bone destruction by invading oral cancer are not well understood. Using IHC, we demonstrated that receptor activator of nuclear factor-κB ligand (RANKL)-positive fibroblasts and cancer cells were located at sites of bone invasion in human oral cancers. HSC3 and HO-1-N-1, human oral cancer cell lines, expressed RANKL and stimulated Rankl expression in the UAMS-32 murine osteoblastic cell line. We discriminated the roles of RANKL synthesized by stromal cells and cancer cells in cancer-associated bone resorption by using species-specific RANKL antibodies against murine RANKL and human RANKL, respectively. Osteoclastogenesis induced by the conditioned medium of HSC3 and HO-1-N-1 cells in a co-culture of murine bone marrow cells and UAMS-32 cells was inhibited by the addition of antibodies against either mouse or human RANKL. HSC3-induced bone destruction was greatly inhibited by the administration of anti-mouse RANKL antibody in a xenograft model. HO-1-N-1-induced bone destruction was inhibited by the administration of either anti-mouse or anti-human RANKL antibody. Bone destruction induced by the transplantation of human RANKL-overexpressing cells (HSC3-R2) was greatly inhibited by the injection of anti-human RANKL antibody. The present study revealed that RANKL produced by both stromal and cancer cells is involved in oral cancer-induced osteoclastic bone resorption. These results provide important information for understanding the cellular and molecular basis of cancer-associated bone destruction and the mechanism of action underlying RANKL antibody (denosumab) therapy., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Tumor angiogenesis is necessary for tumor progression and metastasis; therefore, tumor blood vessels are potential therapeutic targets in anticancer therapy. We previously reported that tumor endothelial cells (TECs) exhibit different phenotypes compared with normal endothelial cells (NECs), and microarray analyses of mouse TECs and NECs have shown that several genes are upregulated in TECs compared with NECs. Among these genes, the expression levels of prostaglandin F receptor (PTGFR) mRNA, which encodes the prostaglandin F receptor (FP), were higher in TECs than in NECs. It has been reported that FP and its ligand, prostaglandin F(2α) , are involved in tumor angiogenesis. However, there have been no reports of the expression of PTGFR in the tumor vessels of renal cell carcinoma (RCC). Thus, we isolated human TECs (hTECs) from RCCs. The expression levels of PTGFR mRNA were also upregulated in hTECs. In addition, immunostaining showed that the PTGFR was expressed in human tumor blood vessels in vivo. These findings suggested that PTGFR is a novel TEC marker and that it may be a novel target for antiangiogenic therapy for RCC., (© 2013 The Authors. Pathology International © 2013 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.)

In this study, we explored the differences in the human adenovirus type 5 (Ad5) production efficiencies of various cell types. The rate of virus production was higher in several cell lines, such as HeLa cells, than in Saos-2 cells. The expression level of the coxsackie and adenovirus receptor (CAR) protein, an adenovirus receptor, was very similar among these cell lines. Although no significant difference in the expression of early region 1A (E1A) mRNA was detected, the amount of E1A protein in the Saos-2 cells was markedly lower than that in HeLa cells. Proteasome inhibitor treatment did not rescue the quantity of E1A in the Saos-2 cells, suggesting that their decreased E1A protein expression is not due to protein decay. To examine the different expression of E1A protein, we employed a bioinformatics approach to identify miRNA that target the 3'-untranslated region (3'-UTR) of E1A mRNA and identified miR-214 as a highly promising candidate. In Saos-2 cells, which have abundant levels of endogenous miR-214, the expression of luciferase was dramatically repressed, when the reporter gene was fused with the 3'-UTR of E1A mRNA including an miR-214 binding site. On the other hand, the activity from the same reporter was unchanged in HeLa cells, which display low-level miR-214 expression. Finally, we confirmed that the knockdown of the miR-214 upregulated the productive efficiency of the virus. These findings indicate that cellular miR-214 is capable of inhibiting adenovirus replication by regulating the translation of E1A protein., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Molecules highly expressed in tumor endothelial cells (TEC) are important for specific targeting of these cells. Previously, using DNA microarray analysis, we found that the prostacyclin receptor (IP receptor) gene was upregulated in TEC compared with normal endothelial cells (NEC). Although prostacyclin is implicated in re-endothelialization and angiogenesis, its role remains largely unknown in TEC. Moreover, the effect of the IP receptor on TEC has not been reported. In the present study we investigated the function of the IP receptor in TEC. The TEC were isolated from two types of human tumor xenografts in nude mice, while NEC were isolated from normal counterparts. Prostacyclin secretion levels in TEC were significantly higher than those in NEC, as shown using ELISA. Real-time RT-PCR showed that the IP receptor was upregulated in TEC compared with NEC. Furthermore, migration and tube formation of TEC were suppressed by the IP receptor antagonist RO1138452. Immunohistostaining showed that the IP receptor was specifically expressed in blood vessels of renal cell carcinoma specimens, but not in glomerular vessels of normal renal tissue. These findings suggest that the IP receptor is a TEC-specific marker and might be a useful therapeutic target., (© 2012 Japanese Cancer Association.)

Tumor angiogenesis is necessary for progression and metastasis of solid tumor. Tumor blood vessels are morphologically different from their normal counterparts. In this study, we isolated tumor endothelial cells (TECs) and revealed their abnormalities. We have compared the gene expression profiles of TECs and normal endothelial cells (NECs) by microarray analysis and found that several genes were upregulated in TECs. Expression of the chemokine receptor CXCR7 mRNA was higher in TECs than in NECs. However, information regarding the expression of CXCR7 in the tumor vessels of renal cell carcinoma is limited. CXCR7 and its ligand CXCL12 have been implicated in tumor cell survival. In this study, the expression of CXCR7 in the tumor vessels of renal cell carcinoma (RCC) was investigated. Real-time PCR revealed higher expression level of CXCR7 in cultured TECs than in cultured NECs. Furthermore, similar to mouse TECs, immunostaining revealed strong expression of CXCR7 in vivo in human tumor vessels. These findings suggest that CXCR7 is a novel TEC marker and a target for antiangiogenic therapy for RCC., (© 2012 The Authors. Pathology International © 2012 Japanese Society of Pathology and Blackwell Publishing Asia Pty Ltd.)

Background: We isolated tumour endothelial cells (TECs), demonstrated their abnormalities, compared gene expression profiles of TECs and normal endothelial cells (NECs) by microarray analysis and identified several genes upregulated in TECs. We focused on the gene encoding biglycan, a small leucine-rich repeat proteoglycan. No report is available on biglycan expression or function in TECs., Methods: The NEC and TEC were isolated. We investigated the biglycan expression and function in TECs. Western blotting analysis of biglycan was performed on sera from cancer patients., Results: Biglycan expression levels were higher in TECs than in NECs. Biglycan knockdown inhibited cell migration and caused morphological changes in TECs. Furthermore, immunostaining revealed strong biglycan expression in vivo in human tumour vessels, as in mouse TECs. Biglycan was detected in the sera of cancer patients but was hardly detected in those of healthy volunteers., Conclusion: These findings suggested that biglycan is a novel TEC marker and a target for anti-angiogenic therapy.

An important concept in tumor angiogenesis is that tumor endothelial cells (TECs) are genetically normal and homogeneous. However, we previously reported that TECs differ from normal ECs. Whether the characteristics of TECs derived from different tumors differ remains unknown. To elucidate this, in this study, we isolated two types of TECs from high-metastatic (HM) and low-metastatic (LM) tumors and compared their characteristics. HM tumor-derived TECs (HM-TECs) showed higher proliferative activity and invasive activity than LM tumor-derived TECs (LM-TECs). Moreover, the mRNA expression levels of pro-angiogenic genes, such as vascular endothelial growth factor (VEGF) receptors 1 and 2, VEGF, and hypoxia-inducible factor-1α, were higher in HM-TECs than in LM-TECs. The tumor blood vessels themselves and the surrounding area in HM tumors were exposed to hypoxia. Furthermore, HM-TECs showed higher mRNA expression levels of the stemness-related gene stem cell antigen and the mesenchymal marker CD90 compared with LM-TECs. HM-TECs were spheroid, with a smoother surface and higher circularity in the stem cell spheroid assay. HM-TECs differentiated into osteogenic cells, expressing activated alkaline phosphatase in an osteogenic medium at a higher rate than either LM-TECs or normal ECs. Furthermore, HM-TECs contained more aneuploid cells than LM-TECs. These results indicate that TECs from HM tumors have a more pro-angiogenic phenotype than those from LM tumors., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Tumor endothelial cells (TECs) are therapeutic targets in anti-angiogenic therapy. Contrary to the traditional assumption, TECs can be genetically abnormal and might also acquire drug resistance. In this study, mouse TECs and normal ECs were isolated to investigate the drug resistance of TECs and the mechanism by which it is acquired. TECs were more resistant to paclitaxel with the up-regulation of multidrug resistance (MDR) 1 mRNA, which encodes the P-glycoprotein, compared with normal ECs. Normal human microvascular ECs were cultured in tumor-conditioned medium (CM) and became more resistant to paclitaxel through MDR1 mRNA up-regulation and nuclear translocation of Y-box-binding protein 1, which is an MDR1 transcription factor. Vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and Akt were activated in human microvascular ECs by tumor CM. We observed that tumor CM contained a significantly high level of VEGF. A VEGFR kinase inhibitor, Ki8751, and a phosphatidylinositol 3-kinase-Akt inhibitor, LY294002, blocked tumor CM-induced MDR1 up-regulation. MDR1 up-regulation, via the VEGF-VEGFR pathway in the tumor microenvironment, is one of the mechanisms of drug resistance acquired by TECs. We observed that VEGF secreted from tumors up-regulated MDR1 through the activation of VEGFR2 and Akt. This process is a novel mechanism of the acquisition of drug resistance by TECs in the tumor microenvironment., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Cyclooxygenase (COX)-2 is known to play an important role in cancer growth and invasion, and it activates the signaling pathways controlling cell proliferation, migration, apoptosis, and angiogenesis. COX-2 is reported to be expressed in many cancer cells. Several studies have reported successful treatment of cancer cells with COX-2 inhibitors (COX-2is). However, the effect of COX-2 inhibition on the tumor endothelium remains to be elucidated. Our study shows that COX-2 is expressed in the vasculature of surgically resected human tumors. To investigate the effects of COX-2 inhibition on the tumor endothelium in vitro, we isolated tumor endothelial cells (TECs) from human melanoma and oral carcinoma xenografts in mice, in which we confirmed that tumor growth was suppressed by inhibiting angiogenesis with the COX-2is NS398. COX-2 mRNA was upregulated in TECs compared to normal endothelial cells (NECs). Cell migration and proliferation were suppressed by NS398 in TECs but not in NECs. The effects of NS398 in vivo were consistent with the in vitro results. The number of CD133+ /vascular endothelial growth factor receptor-2+ cells in circulation was significantly suppressed by COX-2 inhibition. In addition, the number of progenitor marker-positive cells decreased in the tumor blood vessels after COX-2i treatment, which suggests that the homing of progenitor cells into the tumor was also blocked. We conclude that NS398 specifically targets both TECs and vascular progenitor cells without affecting NECs., (Copyright © 2011 UICC.)

Background: Increasing evidence indicates that tumor endothelial cells (TEC) differ from normal endothelial cells (NEC). Our previous reports also showed that TEC were different from NEC. For example, TEC have chromosomal abnormality and proangiogenic properties such as high motility and proliferative activity. However, the mechanism by which TEC acquire a specific character remains unclear. To investigate this mechanism, we focused on tumor-derived microvesicles (TMV). Recent studies have shown that TMV contain numerous types of bioactive molecules and affect normal stromal cells in the tumor microenvironment. However, most of the functional mechanisms of TMV remain unclear., Methodology/principal Findings: Here we showed that TMV isolated from tumor cells were taken up by NEC through endocytosis. In addition, we found that TMV promoted random motility and tube formation through the activation of the phosphoinositide 3-kinase/Akt pathway in NEC. Moreover, the effects induced by TMV were inhibited by the endocytosis inhibitor dynasore. Our results indicate that TMV could confer proangiogenic properties to NEC partly via endocytosis., Conclusion: We for the first time showed that endocytosis of TMV contributes to tumor angiogenesis. These findings offer new insights into cancer therapies and the crosstalk between tumor and endothelial cells mediated by TMV in the tumor microenvironment.

Tumor blood vessels play an important role in tumor progression and metastasis. Thus, targeting the tumor blood vessels is an important strategy in cancer therapy. Tumor blood vessels generally arise from pre-existing vessels and have been thought to be genetically normal. However, they have been shown to differ from their normal counterparts, e.g. with regard to the morphological changes. We isolated tumor endothelial cells (TEC) from mouse tumor xenografts and showed that they were abnormal. TEC up-regulate many genes, proliferate more rapidly and migrate more than normal endothelial cells (NEC). Furthermore, the TEC in our study were cytogenetically abnormal. We concluded that TEC can acquire cytogenetic abnormalities while in the tumor microenvironment. In order to develop ideal antiangiogenic therapies, understanding the crosstalk between blood vessels and the tumor microenvironment is important. This review considers the current studies on TEC abnormalities and discusses the possible mechanism by which the tumor microenvironment produces abnormal TEC., (© 2011 The Authors. Pathology International © 2011 Japanese Society of Pathology and Blackwell Publishing Asia Pty Ltd.)

Approximately 85% of human malignant tumors express increased levels of telomerase. The marked association of telomerase activity with malignant tissue provides strong evidence that telomerase activity is a significant marker for the diagnosis of cancer. In this study, telomerase activity was examined in 12 benign salivary gland tumors (8 pleomorphic adenomas and 4 adenolymphomas), 24 malignant tumors (15 mucoepidermoid carcinomas, 6 adenoid cystic carcinomas and 3 acinic cell carcinomas) and 6 non-neoplastic salivary glands. The mRNA expression of the human telomerase reverse transcriptase (hTERT) and additional telomerase‑associated proteins (hTEP1, p23, Hsp90 and dyskerin) was also examined. Of the 24 malignant tumors, 15 revealed strong telomerase activity. The non-neoplastic salivary glands appeared to have a negative telomerase expression. Furthermore, telomerase activity was significantly higher in high-grade mucoepidermoid carcinomas compared to low‑grade ones (Student's t-test, p<0.05). A significant correlation was found between telomerase activity and mRNA expression of hTERT in 15 cases, including non-neoplastic salivary glands and tumors (Spearman's rank correlation test, p<0.05). Furthermore, a significant correlation was found between telomerase activity and mRNA expression of EGFR (Spearman's rank correlation test, p<0.001). The results suggest that not only hTERT, but also EGFR play a significant role in the activation of telomerase. In conclusion, the results suggest that telomerase activity and hTERT/EGFR mRNA expression are useful markers for the detection of malignant cells in salivary gland carcinomas. Moreover, our results indicated that telomerase activity determines the degree of malignancy of mucoepidermoid carcinoma.

E4orf6 is one of the oncogene products of adenovirus, and it also has an important role for transportation of cellular and viral messenger RNA (mRNA) during the late phase of virus infection. We previously revealed that E4orf6 controls the fate of AU-rich element (ARE) containing mRNA by perturbing the chromosome maintenance region 1-dependent export mechanism. Here, we show that E4orf6 stabilizes ARE-mRNA through the region required for its oncogenic activity and ubiquitin E3 ligase assembly. Cells that failed to stabilize ARE-mRNA after HuR knockdown were unable to produce colonies in soft agar, even when E4orf6 was expressed. Furthermore, the stabilized ARE-mRNA induced the transformation of rodent immortalized cells. These findings indicate that stabilized ARE-mRNA is necessary, if not all, for the oncogenic activity of E4orf6 and has the potential to transform cells, at least under a certain condition.

Recent findings have focused attention on the molecular consequences of the microenvironment in tumor progression, but events occurring in cancer cells themselves in response to their ambient conditions remain obscure. Here, we identify receptor activator of nuclear factor κB ligand (RANKL) as a microenvironment-specific factor essential for tumorigenesis in vivo, using head and neck squamous cell carcinoma (HNSCC) as a model. In human HNSCC tissues, RANKL is abundantly expressed, and its expression level correlates with the histological grade of differentiation. RANKL levels are significantly higher in poorly differentiated SCCs than in well or moderately differentiated SCCs. In contrast, all HNSCC cell lines tested displayed extremely low RANKL expression; however, RANKL is efficiently up-regulated when these cell lines are inoculated in the head and neck region of mice. RANKL expression is restored in a microenvironment-specific manner, and cannot be observed when the cells are inoculated in the hindlimbs. Forced expression of RANKL compensates for tumor growth in the hindlimb milieu, promotes epithelial mesenchymal transition, and induces tumor angiogenesis, in a manner independent of vascular endothelial growth factor (VEGF). These results implicate RANKL expression causatively in tumor growth and progression in HNSCC in vivo. RANKL may provide a novel functional marker for biological malignancy and a therapeutic target based on the specific nature of the microenvironment., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Tumour stromal cells differ from its normal counterpart. We have shown that tumour endothelial cells (TECs) isolated from tumour tissues are also abnormal. Furthermore, we found that mRNAs of vascular endothelial growth factor-A (VEGF-A) and cyclooxygenase-2 (COX-2) were upregulated in TECs. Vascular endothelial growth factor-A and COX-2 are angiogenic factors and their mRNAs contain an AU-rich element (ARE). AU-rich element-containing mRNAs are reportedly stabilised by Hu antigen R (HuR), which is exported to the cytoplasm., Methods: Normal endothelial cell (NEC) and two types of TECs were isolated. We evaluated the correlation of HuR and accumulation of VEGF-A and COX-2 mRNAs in TECs and effects of HuR on biological phenotypes of TECs., Results: The HuR protein was accumulated in the cytoplasm of TECs, but not in NECs. Vascular endothelial growth factor-A and COX-2 mRNA levels decreased due to HuR knockdown and it was shown that these ARE-mRNA were bound to HuR in TECs. Furthermore, HuR knockdown inhibited cell survival, random motility, tube formation, and Akt phosphorylation in TECs., Conclusion: Hu antigen R is associated with the upregulation of VEGF-A and COX-2 mRNA in TECs, and has an important role in keeping an angiogenic switch on, through activating angiogenic phenotype in tumour endothelium.

Tumor metastasis to lymph nodes occurs through the lymphatic vessels located in the tumor circumference. However, few studies have focused on the phenotypes of lymphatic vessels around these tumors. We investigated the characteristics of the lymph vessels of tongue squamous cell carcinoma (SCC) and compared them to clinicopathological characteristics. A total of 43 patients diagnosed as having tongue SCC consulted Hokkaido University Hospital were examined. The lymphatic vessels were identified by antibody D2-40 and the number and diameter of tumor lymphatic vessels were measured. The proliferative activity of lymphatic endothelial cells was also examined by immunostaining using antibody MIB-1. We then measured the DNA density of lymphatic endothelial cells in normal and tumor tissues. The number of tumor lymphatic vessels significantly increased in highly metastatic cases of tongue SCC, particularly in cases with a large number of micro lymphatic vessels. A significant correlation was found between the metastatic and proliferative activity of tumor lymphatic endothelial cells. Moreover, the DNA density of tumor lymphatic endothelial cells increased compared to normal tissues. These results suggest that the phenotypes of tumor lymphatic endothelial cells are an indicator of lymph node metastasis of tongue SCC.

Patients with an oral squamous cell carcinoma (OSCC) often develop multiple malignant lesions. This report examined whether individual tumours developed in a patient show the same genetic alteration, such as p53 mutations. This case study describes three SCCs and three leukoplakias which developed simultaneously in a single 67-year-old Japanese man. A p53 mutation was detected in two of the three SCCs and one of the three leukoplakias. One SCC had a missense mutation at codon 285 (GAG>AAG, Glu>Lys) and the other a nonsense mutation at codon 336, and the leukoplakia had a missense mutation at codon 273 (CGT>CAT, Arg>His). This case showed that individual oral tumours may have different genetic changes even when they develop in a single patient. Therefore, this report provided strong evidence that in cases of multiple tumours it is necessary to design tailor-made therapies for each individual tumour rather than a single standardised therapy for all multiple tumours.

Adrenomedullin (AM) is a multifunctional 52-amino acid peptide. AM has several effects and acts as a growth factor in several types of cancer cells. Our previous study revealed that an AM antagonist (AMA) suppressed the growth of pancreatic tumors in mice, although its mechanism was not elucidated. In this study, we constructed an AMA expression vector and used it to treat renal cell carcinoma (RCC) in mice. This AMA expression vector significantly reduced tumor growth in mice. In addition, microvessel density was decreased in AMA-treated tumors. To analyze the effect of AMA on tumor angiogenesis in this model, tumor endothelial cells (TECs) were isolated from RCC xenografts. TEC proliferation was stimulated by AM and it was inhibited by AMA significantly. AM induced migration of TECs and it was also blocked by AMA. However, normal ECs (NECs) were not affected by either AM or AMA. These results demonstrate that AMA has inhibitory effects on TECs specifically, not on NEC, thereby inhibiting tumor angiogenesis. Furthermore, we showed that vascular endothelial growth factor-induced mobilization of endothelial progenitor cell (EPC) into circulation was inhibited by AMA. These results suggest that AMA can be considered a good anti-angiogenic reagent that selectively targets TECs and EPC in renal cancer.

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Increasing evidence indicates that tumor endothelial cells (TECs) are more relevant to the study of tumor angiogenesis than normal endothelial cells (NECs) because their morphologies and gene expression are different from NECs. However, it is challenging to isolate and culture large numbers of pure ECs from tumor tissue since the percentage of ECs is only about 1-2% and tumor cells and fibroblasts easily overgrow them. In addition, there has been concern that isolated TECs may lose their special phenotype once they are dissociated from tumor cells. In this study, we have successfully purified murine TECs from four different human tumor xenografts and NECs from murine dermal tissue. Isolated ECs expressed endothelial markers, such as CD31, VE-cadherin (CD144), and endoglin (CD105), for more than 3 months after isolation. TECs maintained tumor endothelial-specific markers, such as tumor endothelial marker 8 (TEM8) and aminopeptidase N (APN), as in tumor blood vessels in vivo. In addition, TECs were more proliferative and motile than NECs. TECs showed a higher response to VEGF and higher expression of VEGF receptors-1 and -2 than NECs did. Stem cell antigen-1 was up-regulated in all four TECs, suggesting that they have a kind of stemness. Cultured TECs maintain distinct biological differences from NECs as in vivo. In conclusion, it was suggested that TECs are relevant material for tumor angiogenesis research., (2010 Elsevier Inc. All rights reserved.)

HuR binds to AU-rich element-containing mRNA to protect them from rapid degradation. Here, we show that knockdown of HuR changes the oncogenic properties of oral cancer cells. Oral squamous cell carcinoma cell lines, HSC-3 and Ca9.22, which express HuR protein and cytoplasmic AU-rich element mRNA more abundantly than normal cells, were subjected to HuR knockdown. In the HuR-knockdown cancer cells, the cytoplasmic expression of c-fos, c-myc, and COX-2 mRNAs was inhibited compared with those in cells that had been transfected with a control small interfering RNA, and the half-lives of these mRNAs were shorter than those of their counterparts in the control cells. HuR-knockdown cells failed to make colonies in soft agar, suggesting that the cells had lost their ability for anchorage-independent cell growth. Additionally, the motile and invasive activities of the cells decreased remarkably by HuR knockdown. Furthermore, the expression of cell cycle-related proteins, such as cyclin A, cyclin B1, cyclin D1, and cyclin-dependent kinase 1, was reduced in HuR-knockdown cancer cells, and HuR bound to cdk1 mRNA to stabilize it. These findings suggest that HuR knockdown changes the features of oral cancer cells, at least in part, by affecting their cell cycle and shows potential as an effective therapeutic approach., ((c) 2010 AACR.)