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Salivary gland scintigraphy was performed on 52 patients who were suspected of having Sjögren's syndrome (SS), and the results were compared with clinicopathologic features of the salivary and lacrimal glands. The time-activity curves which were obtained from computer-assisted analysis of 99mTc-pertechnetate (99mTc) scintigraphy were classified into four types (normal, median, flat and sloped types). The stimulated parotid flow rate decreased and the incidence of SS-related sialographic and histopathologic findings increased significantly as the scintigraphic abnormality advanced. In addition, the lacrimal gland function decreased and the proportion of patients diagnosed as having keratoconjunctivitis sicca (KCS) increased significantly as the scintigraphic abnormality advanced. These results indicate that the results of scintigraphy are related not only to the clinicopathologic features of the salivary glands but also to the lacrimal gland function in SS. [ABSTRACT FROM AUTHOR]

Unilateral maxillary or mandibular molars were extracted from twenty-four, 25-day-old male syrian golden hamsters; 25 days after extraction, 20 received MNU intragastrically twice a week for 3 months. All animals were killed 1 month later. High degrees and incidence of invasion of gingival SCC were observed in dentulous jaws without functional occlusion, followed by dentulous jaws with functional occlusion and edentulous jaws. Invasion of carcinomas with extensive bone destruction was observed commonly in the gingiva of dentulous maxillary jaws without functional occlusion, but was scarcely observed in the gingiva of edentulous jaws. By analogy, disuse atrophy of the periodontium due to loss of functional occlusion may result in early deeper invasion, while loss of peridontal ligament due to tooth extraction and disappearance of so-called inner gingival epithelium may result in less invasive carcinomas. [ABSTRACT FROM AUTHOR]

src homology 2 (SH2)-containing protein-tyrosine phosphatase SHP2 is known to transduce positive signals from activated receptor protein-tyrosine kinases such as platelet-derived growth factor receptor (PDGFR) beta and insulin receptor. Here, we demonstrate the physiological expression of SHP2 in rats. In northern and western blot analyses, SHP2 expressions were recognized in all tissues, but their expression levels varied significantly among tissues: it is lowest in the liver and kidney. Immunohistochemical staining and in situ hybridization showed SHP2 was expressed ubiquitously but predominantly in vascular smooth muscle cells (SMC). During the development of granulations. SHP2 was expressed predominantly in vascular SMC and also highly expressed in capillary cells. The functional associations of SHP2 with PDGFR beta, which transduces major growth signals in vascular SMC, identify a crucial function of SHP2 in blood vessels in consert with PDGFR beta. [ABSTRACT FROM AUTHOR]

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.

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.)