Inhibition of SUV39H1 reduces tumor angiogenesis via Notch1 in oral squamous cell carcinoma.

Targeting tumor angiogenesis is an important approach in advanced tumor therapy. Here we investigated the effect of the suppressor of variegation 3-9 homolog 1 (SUV39H1) on tumor angiogenesis in oral squamous cell carcinoma (OSCC). The GEPIA database was used to analyze the expression of SUV39H1 in various cancer tissues. The expression of SUV39H1 in OSCC was detected by immunohistochemistry, and the correlation between SUV39H1 and Notch1 and microvascular density (MVD) was analyzed. The effect of SUV39H1 inhibition on OSCC was investigated in vivo by chaetocin treatment. The migration and tube formation of vascular endothelial cells by conditioned culture-medium of different treatments of oral squamous cell cells were measured. The transcriptional level of SUV39H1 is elevated in various cancer tissues. The transcription level of SUV39H1 in head and neck squamous cell carcinoma was significantly higher than that in control. Immunohistochemistry result showed increased SUV39H1 expression in OSCC, which was significantly correlated with T staging. The expression of SUV39H1 was significantly correlated with Notch1 and CD31. In vivo experiment chaetocin treatment significantly inhibit the growth of tumor, and reduce SUV39H1, Notch1, CD31 expression. The decreased expression of SUV39H1 in OSCC cells lead to the decreased expression of Notch1 and VEGF proteins, as well as the decreased migration and tube formation ability of vascular endothelial cells. Inhibition of Notch1 further enhance this effect. Our results suggest inhibition of SUV39H1 may affect angiogenesis by regulating Notch1 expression. This study provides a foundation for SUV39H1 as a potential therapeutic target for OSCC.

Transforming growth factor-ß signals promote progression of squamous cell carcinoma by inducing epithelial-mesenchymal transition and angiogenesis.

At present, the molecular mechanisms driving the progression and metastasis of oral squamous cell carcinoma (OSCC) remain largely uncharacterized. The activation of transforming growth factor-ß (TGF-ß) signaling in the tumor microenvironment has been observed in various types of cancer and has been implicated their progression by enhancing the migration and invasion of epithelial cancer cells. However, its specific roles in the oral cancer progression remain unexplored. In this study, we examined the effects of TGF-ß signaling on the murine squamous cell carcinoma, SCCVII cells in vitro and in vivo. The incubation of SCCVII cells with TGF-ß induced the activation of TGF-ß signals and epithelial-mesenchymal transition (EMT). Notably, the motility of SCCVII cells was increased upon the activation of the TGF-ß signaling. RNA sequencing revealed upregulation of genes related to EMT and angiogenesis. Consistent with these in vitro results, the inhibition of TGF-ß signals in SCCVII cell-derived primary tumors resulted in suppressed angiogenesis. Furthermore, we identified six candidate factors (ANKRD1, CCBE1, FSTL3, uPA, TSP-1 and integrin ß3), whose expression was induced by TGF-ß in SCCVII cells, and associated with poor prognosis for patients with head and neck squamous cell carcinoma. These results highlight the role of TGF-ß signals in the progression of OSCC via multiple mechanisms, including EMT and angiogenesis, and suggest novel therapeutic targets for the treatment of OSCC.

Semaphorin 4A restricts tumor progression by inhibiting angiogenesis of oral squamous cell carcinoma cells.

OBJECTIVE: To investigate the effects of Semaphorin 4A (Sema4A) on the angiogenesis, migration and invasion of oral squamous cell carcinoma (OSCC) cells. METHODS: Sema4A expression in OSCC patients was detected by Immunohistochemistry, and its relationship with clinicopathological features and prognosis of patients was analyzed. The mRNA and protein expression of Sema4A in primary human oral keratinocytes (HOKs) and OSCC cells (SCC-25, HSC-3, CAL-27) were determined by Western blotting and qRT-PCR. After HOKs, HSC-3 cells and SCC-25 cells transfected with Control/Sema4A CRISPR activation plasmid, the migration and invasion abilities were detected by Wound healing and Transwell invasion. Tube formation assay was also performed on endothelial cells and the contents of VEGF and bFGF were quantified using qRT-PCR and ELISA. RESULTS: Cytoplasmic Sema4A expression was related to T classification, clinical stage and nodal metastasis of OSCC patients. Patients with low cytoplasmic Sema4A expression showed the higher microvessel density (MVD) and the poorer prognosis in OSCC. Compared with HOK, OSCC cells (SCC-25, HSC-3, CAL-27) declined apparently in Sema4A expression, which was much more significant in metastatic HSC-3 and SCC-25 cells. After HOKs, HSC-3 cells and SCC-25 cells transfected with Sema4A over-expression plasmid, the invasion and migration abilities were decreased. Besides, overexpression of Sema4A could significantly inhibit the tube formation of HUVEC induced by OSCC cells with reductions of angiogenic factors (VEGF and bFGF). CONCLUSION: Over-expression of Sema4A could restrict tumor progression through inhibiting the angiogenesis, invasion and migration of OSCC cells.

Small extracellular vesicle-bound vascular endothelial growth factor secreted by carcinoma-associated fibroblasts promotes angiogenesis in a bevacizumab-resistant manner.

The blood vessel growth inhibitor bevacizumab targets vascular endothelial growth factor (VEGF), a crucial regulator of angiogenesis. Recently, small extracellular vesicles (sEVs) have been demonstrated to be important vehicles in the transport of growth factors to target cells. In this study, we isolated primary carcinoma-associated fibroblasts (CAFs) from four human oral squamous cell carcinoma (OSCC) specimens. Compared with other non-extracellular vesicle components, CAF-derived sEVs were found to be the main regulators of angiogenesis. The ability of CAF sEVs to activate VEGF receptor 2 (VEGFR2) signaling in human umbilical vein endothelial cells (HUVEC) was dependent on the association between sEVs and VEGF. In addition, sEV-bound VEGF secreted by CAFs further activated VEGFR2 signaling in HUVEC in a bevacizumab-resistant manner. VEGF was found to interact with heparan sulfate proteoglycans on the CAF sEV surface and could be released by heparinase I/III. The bioactivity of the dissociated VEGF was retained in vitro and in vivo and could be neutralized by bevacizumab. These findings suggest that the combined use of heparinase and bevacizumab might inhibit angiogenesis in patients with high levels of sEV-bound VEGF.

Ki67, CD105, and α-SMA Expression Supports Biological Distinctness of Oral Squamous Cell Carcinoma Arising in the Background of Oral Submucous Fibrosis.

BACKGROUND: The clinicopathological distinctness of oral squamous cell carcinoma arising in the background of oral submucous fibrosis (OSCC-OSF) is well known; however, the molecular distinctness of this unique OSCC-OSF has not been investigated to date. With this in mind, we compared the expression of Ki67, CD105, and α-SMA between OSCC-OSF and oral squamous cell carcinoma (OSCC). METHODS: Formalin-fixed paraffin-embedded tissues of 105 OSCC-OSF and 112 OSCC cases were subjected to immunohistochemistry for evaluation of Ki67, CD105, and α-SMA expression. RESULTS: Ki67 (labeling index) LI, MVD and α-SMA expression were significantly higher in OSCC compared to OSCC-OSF. Ki67 LI and MVD was significantly higher in OSCC compared to OSCC-OSF in parameters such as well-differentiated, early TNM stage, non-metastatic, and more than 3-year survival. α-SMA expression was significantly higher in OSCC compared to OSCC-OSF in parameters such as moderate differentiation, metastatic lesions, and survival less than 3 years. Ki67 LI, MVD and α-SMA showed significant positive correlation with each other in OSCC and OSCC-OSF. CONCLUSION: Proliferation, neoangiogenesis and myofibroblast differentiation were significantly higher in the OSCC group compared to the OSCC-OSF group. This suggests the biological distinctness of OSCC-OSF, which could help the future development of targeted therapies.

Activin A triggers angiogenesis via regulation of VEGFA and its overexpression is associated with poor prognosis of oral squamous cell carcinoma.

Poor prognosis associated with the dysregulated expression of activin A in a number of malignancies has been related to with numerous aspects of tumorigenesis, including angiogenesis. The present study investigated the prognostic significance of activin A immunoexpression in blood vessels and cancer cells in a number of oral squamous cell carcinoma (OSCC) cases and applied in vitro strategies to determine the impact of activin A on angiogenesis. In a cohort of 95 patients with OSCC, immunoexpression of activin A in both blood vessels and tumor cells was quantified and the association with clinicopathological parameters and survival was analyzed. Effects of activin A on the tube formation, proliferation and migration of human umbilical vein endothelial cells (HUVECs) were evaluated in gain­of­function (treatment with recombinant activin A) or loss­of­function [treatment with activin A­antagonist follistatin or by stable transfection with short hairpin RNA (shRNA) targeting activin A] conditions. Conditioned medium from an OSCC cell line with shRNA­mediated depletion of activin A was also tested. The profile of pro­ and anti­angiogenic factors regulated by activin A was assessed with a human angiogenesis quantitative PCR (qPCR) array. Vascular endothelial growth factor A (VEGFA) and its major isoforms were evaluated by reverse transcription­qPCR and ELISA. Activin A expression in blood vessels demonstrated an independent prognostic value in the multivariate analysis with a hazard ratio of 2.47 [95% confidence interval (CI), 1.30­4.71; P=0.006) for disease­specific survival and 2.09 (95% CI, 1.07­4.08l: P=0.03) for disease­free survival. Activin A significantly increased tubular formation of HUVECs concomitantly with an increase in proliferation. This effect was validated by reduced proliferation and tubular formation of HUVECs following inhibition of activin A by follistatin or shRNA, as well as by treatment of HUVECs with conditioned medium from activin A­depleted OSCC cells. Activin A­knockdown increased the migration of HUVECs. In addition, activin A stimulated the phosphorylation of SMAD2/3 and the expression and production of total VEGFA, significantly enhancing the expression of its pro­angiogenic isoform 121. The present findings suggest that activin A is a predictor of the prognosis of patients with OSCC, and provide evidence that activin A, in an autocrine and paracrine manner, may contribute to OSCC angiogenesis through differential expression of the isoform 121 of VEGFA.

Mast Cells and Blood Vessels Profile in Oral Carcinogenesis: An Immunohistochemistry Study.

BACKGROUND: The objectives of the present study were to evaluate angiogenesis and mast cell density in oral epithelial dysplasia and oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: This was an observational, retrospective and quantitative study. The samples consisted of 60 tissue specimens from patients with squamous cell carcinoma, epithelial dysplasia and controls (n=20/group). Immunohistochemistry was performed using an anti-tryptase antibody to mast cells and anti-CD31 and anti-CD34 for blood vessels and we count the number of mast cells and determine the percentage of CD31 and CD34 antibody staining (vascular density). RESULTS: The mast cells had lower density in OSCC compared to control and dysplasia (p = 0.009). In angiogenesis, the expression of CD31 showed a higher percentage of blood vessels in OSCC (p < 0.001), however, CD34 showed no difference between groups (p=0.092). The CD31 antibody presented as a high immunostaining in oral mucosa than CD34. CONCLUSIONS: The increased vascularity in squamous cell carcinoma suggests that angiogenesis begins when malignant transformation starts that seems to be inversely associated with the number of mast cells.

Pseudomyogenic Hemangioendothelioma: A Rare Vascular Tumor of the Oral Cavity.

Pseudomyogenic hemangioendothelioma is a vascular neoplasm that presents a borderline biological behavior, intermediate between entirely benign hemangiomas and highly malignant angiosarcomas. Up to date, only 1 case of this entity has been reported in the oral cavity.

Prospective feasibility analysis of salvage surgery in recurrent oral cancer in terms of quality of life.

OBJECTIVES: The goals of the present study were to prospectively analyze salvage surgery with microvascular reconstruction in recurrent squamous cell carcinoma of the oral cavity (OSCC) in terms of oncological outcome and quality of life. PATIENTS AND METHODS: From 2012 to 2015, 28 patients underwent salvage surgery due to recurrent OSCC or second primary OSCC without the option of curative re-irradiation. Endpoints were disease-specific survival and progression-free survival after 12 months. The survival was estimated by using the Kaplan-Meier blotting. Quality of life data (European Organization for Research and Treatment of Cancer - EORTC: QLQ-C30 and QLQ-H&N35) was assessed at baseline and subsequently every 3 months up to one year. RESULTS: Estimated 1-year-survival was 68.4% and progression-free survival was 38.5%. Overall quality of life was significantly reduced three months after salvage surgery [baseline (mean 64.15) versus time 1 (mean 53.04); p = 0.002]. However, the patients experienced a recovery within the first year [baseline (mean 64.15) versus time 4 (mean 70.33); p = 0.176]. Furthermore, the sensation of pain is significantly reduced after salvage surgery [baseline (mean 47.53) versus time 2 (mean 31.25); p = 0.036]. Microvascular reconstruction success rate was 93.1%. CONCLUSION: Salvage surgery is a curative treatment option in recurrent and intensively pretreated OSCC. Microvascular reconstruction is feasible with acceptable morbidity and high success rates. Quality of life can be preserved. Further studies combining checkpoint inhibition with salvage surgery are justified.

Delivery of mesenchymal stem cells-derived extracellular vesicles with enriched miR-185 inhibits progression of OPMD.

Oral leukoplakia is one of the most common oral potentially malignant disorders (OPMDs) and its malignant transformation to oral cancer is highly associated with chronic inflammation. Extracellular vesicles (EVs) or exosome-delivered microRNAs modulate inflammatory responses and alleviate irritations that predisposes to cancer. We previously reported that microRNA-185 (miR-185) was significantly decreased in the buccal tissue of patients with oral cancer. In this study, we utilized genetically modified mesenchymal stem cells (MSCs) derived EVs with high expression of miR-185 to pasted MSC-EV-miR-185 on buccal lesions in dimethylbenzanthracene (DMBA) induced OPMD model. We found that treatment with MSC-EV-miR-185 remarkably attenuated inflammation severity and significantly decreased the incidence and the number of dysplasia in the OPMD tissue. Immunohistochemistry showed significantly decreased expression of proliferation marker PCNA and angiogenic marker CD31 in the lesion treated with MSC-EV-miR-185. Furthermore, miR-185 specifically targeted Akt genes by promoting activation of the apoptotic pathway, confirmed by the increased levels of activated caspase-3 and 9. In conclusion, genetically modified MSC-derived EVs enriched with miR-185 alleviate inflammatory response, inhibit cell proliferation and angiogenesis, and induce cell apoptosis, suggesting that their potential role as a novel therapeutic option for OPMD.

Red propolis and L-lysine on angiogenesis and tumor growth in a new model of hamster cheek pouch inoculated with Walker 256 tumor cells.

OBJECTIVE: To evaluate the effect of red propolis and L-lysine on angiogenesis and tumor growth in a new model of hamster cheek pouch inoculated with Walker 256 tumor cells. METHODS: The study consisted of two experiments with four groups each (total: 57 hamsters). In the experiment 1, the animals were inoculated with Walker tumor cells, followed by administration of test substances (red propolis 200mg/5mL/kg or L-lysine 150mg/kg) or control substances (gum arabic 5mL/kg or water 5mL/kg) for 10 days. The animals in the experiment 2 received red propolis, L-lysine, gum arabic or water at the same doses, for 33 days prior to inoculation of Walker tumor cells, followed by 10 days of treatment with the same substances. Based on single-plane images, angiogenesis was quantified (mean vascular area), in percentage, and tumor area (mm2) and perimeter (mm). RESULTS: In the experiment 1, compared to animals receiving water, the mean vascular area expressed in percentage was significantly smaller in animal treated with propolis (p<0.05) and L-lysine (p<0.001). CONCLUSION: Both red propolis and L-lysine inhibited tumor angiogenesis in the new hamster cheek pouch model when administered after tumor inoculation.

HMGA2 Contributes to Distant Metastasis and Poor Prognosis by Promoting Angiogenesis in Oral Squamous Cell Carcinoma.

The highly malignant phenotype of oral squamous cell carcinoma (OSCC), including the presence of nodal and distant metastasis, reduces patient survival. High-mobility group A protein 2 (HMGA2) is a non-histone chromatin factor that is involved in advanced malignant phenotypes and poor prognosis in several human cancers. However, its biological role in OSCC remains to be elucidated. The purpose of this study was to determine the clinical significance and role of HMGA2 in the malignant potential of OSCC. We first investigated the expression pattern of HMGA2 and its clinical relevance in 110 OSCC specimens using immunohistochemical staining. In addition, we examined the effects HMGA2 on the regulation of vascular endothelial growth factor (VEGF)-A, VEGF-C, and fibroblast growth factor (FGF)-2, which are related to angiogenesis, in vitro. High expression of HMGA2 was significantly correlated with distant metastasis and poor prognosis. Further, HMGA2 depletion in OSCC cells reduced the expression of angiogenesis genes. In OSCC tissues with high HMGA2 expression, angiogenesis genes were increased and a high proportion of blood vessels was observed. These findings suggest that HMGA2 plays a significant role in the regulation of angiogenesis and might be a potential biomarker to predict distant metastasis and prognosis in OSCC.

Stem cell exosomes inhibit angiogenesis and tumor growth of oral squamous cell carcinoma.

Recently, exosomes secreted by menstrual mesenchymal stem cells have been identified as inhibitory agents of tumor angiogenesis and modulators of the tumor cell secretome in prostate and breast cancer. However, their direct effect on endothelial cells and paracrine mediators have not yet been investigated. Using a carrier-based cell culture system to test the scalability for exosome production, we showed that different types of endothelial cells present specific kinetics for exosomes internalization. Exosome-treatment of endothelial cells increased cytotoxicity and reduced VEGF secretion and angiogenesis in a dose-dependent manner. Using the hamster buccal pouch carcinoma as a preclinical model for human oral squamous cell carcinoma, we demonstrated a significant antitumor effect of intra-tumoral injection of exosomes associated with a loss of tumor vasculature. These results address up-scaling of exosome production, a relevant issue for their clinical application, and also assess menstrual stem cell exosomes as potential anti-angiogenic agents for the treatment of neoplastic conditions.

Vascular density and inflammatory infiltrate in primary oral squamous cell carcinoma and after allogeneic hematopoietic stem cell transplantation.

Hematopoietic stem cell transplantation (HSCT) recipients have been reported to have an increased risk of chronic graft versus host disease (cGVHD) and hematological and solid cancers. Oral manifestations are the first signs of cGVHD observed in the majority of patients, and oropharyngeal cancer is the most frequent secondary malignancy occurred after HSCT. In this study, we have evaluated the inflammatory infiltrate cell content and correlated with the vascular density in patients affected by primary oral squamous cell carcinoma (OSCC) from previous healthy controls and OSCC after cGVHD. Results have demonstrated that patients with OSCC after GVHD show a more consistent inflammatory infiltrate as compared with the OSCC ones. In detail, the inflammatory background composed of CD3-positive T cells, tryptase-positive mast cells, CD31-positive endothelial cells, and CD68-positive macrophages may be more pronounced in the setting of GVHD + OSCC than in the control group. By contrast, CD20-positive B cells and CD1a-positive dendritic cells were more abundant in the latter population. Finally, a positive correlation was found as between vascular density and inflammatory cell infiltration in both GVHD + OSCC and OSCC groups. Overall, these results confirm the role played by immune cells in enhancing tumor progression and angiogenesis and suggest a potential therapeutic strategy involving inhibition of recruitment of immune cells to the tumor microenvironment and blockade of pro-tumoral effects and pro-angiogenic functions.

Red propolis and L-lysine on angiogenesis and tumor growth in a new model of hamster cheek pouch inoculated with Walker 256 tumor cells / Própolis vermelha e L-lisina na angiogênese e no crescimento tumoral em novo modelo de bolsa jugal de hamster inoculada com células de tumor de Walker 256

ABSTRACT Objective: To evaluate the effect of red propolis and L-lysine on angiogenesis and tumor growth in a new model of hamster cheek pouch inoculated with Walker 256 tumor cells. Methods: The study consisted of two experiments with four groups each (total: 57 hamsters). In the experiment 1, the animals were inoculated with Walker tumor cells, followed by administration of test substances (red propolis 200mg/5mL/kg or L-lysine 150mg/kg) or control substances (gum arabic 5mL/kg or water 5mL/kg) for 10 days. The animals in the experiment 2 received red propolis, L-lysine, gum arabic or water at the same doses, for 33 days prior to inoculation of Walker tumor cells, followed by 10 days of treatment with the same substances. Based on single-plane images, angiogenesis was quantified (mean vascular area), in percentage, and tumor area (mm2) and perimeter (mm). Results: In the experiment 1, compared to animals receiving water, the mean vascular area expressed in percentage was significantly smaller in animal treated with propolis (p<0.05) and L-lysine (p<0.001). Conclusion: Both red propolis and L-lysine inhibited tumor angiogenesis in the new hamster cheek pouch model when administered after tumor inoculation.

RESUMO Objetivo: Avaliar o efeito da própolis vermelha e da L-lisina na angiogênese e no crescimento tumoral em novo modelo de bolsa jugal de hamster inoculada com células de tumor de Walker 256. Métodos: O estudo consistiu em dois experimentos com quatro grupos cada (total: 57 hamsters). No experimento 1, os animais foram inoculados com células de tumor de Walker, tendo em seguida administradas as substâncias teste (própolis vermelha 200mg/5mL/kg ou L-lisina 150mg/kg) ou controle (goma arábica 5mL/kg ou água 5mL/kg) por 10 dias. Os animais do experimento 2 receberam própolis vermelha, L-lisina, goma arábica ou água nas mesmas doses, por 33 dias antes do inóculo das células de tumor de Walker, seguido por 10 dias de tratamento com as mesmas substâncias. Baseado em imagens em plano único, foram quantificados a angiogênese (área vascular média), em termos percentuais, e a área (mm2) e o perímetro (mm) do tumor. Resultados: Comparada aos animais que receberam água, a área vascular média, expressa em percentagem, foi significativamente menor nos animais tratados com própolis (p<0,05) e com L-lisina (p<0,001). Conclusão: Tanto a própolis vermelha quanto a L-lisina inibiram a angiogênese no novo modelo de bolsa jugal de hamsters, quando administradas após a inoculação do tumor.

Immunohistochemical analysis of neutrophils, interleukin-17, matrix metalloproteinase-9, and neoformed vessels in oral squamous cell carcinoma.

BACKGROUND: Tumor-associated neutrophils (TAN), matrix metalloproteinase-9 (MMP-9), interleukin-17 (IL-17), and angiogenesis have been proposed as prognostic biomarkers of malignant tumors. The purpose of this study was to investigate these inflammatory markers as prognostic factors for oral squamous cell carcinoma (OSCC). METHODS: Specimens of OSCC (n = 30), healthy oral mucosa (negative control, n = 10), oral leukoplakia (n = 10), and apical granuloma with abscess (positive inflammatory controls, n = 10) were immunostained for CD66b (neutrophils), MMP-9, IL-17, and CD105 (neoformed microvessels). Semiquantitative (IL-17) and quantitative (CD66b, IL-17, MMP-9, and CD105) analyses were performed. Clinical information (TNM stage, metastasis, recurrence, and survival) and tumor histological grade were also obtained. RESULTS: Positivity for TAN, MMP-9, IL-17, and CD105 was higher in OSCC than in the negative control (P < 0.05) and oral leukoplakia, but similar to the positive inflammatory control. Coincident high counts of inflammatory markers (CD66b, MMP-9, IL-17, and CD105) were associated with lymph node metastasis of OSCC. Associations between high numbers of neoformed microvessels and advanced clinical stage and a higher degree of malignancy were also demonstrated. CONCLUSIONS: Combined positivity for TAN, MMP-9, IL-17, and CD105 appears to be associated with the metastasis-prone phenotype of OSCC.

Narrow-band imaging pattern classification in oral cavity.

OBJECTIVE: Narrow-band imaging is widely used in the diagnostic work-up of oral lesions. Different oral subsites present three epithelial types (1, 2a and 2b), each with a different structure and function. The aim of this study was to analyse and describe the different vascular patterns seen on narrow-band imaging according to oral epithelial type and histology. MATERIALS AND METHODS: The narrow-band imaging photographs of healthy, dysplastic and neoplastic oral mucosa were retrospectively reviewed and divided according to epithelial type and histology. The different narrow-band imaging patterns were analysed, related to the clinical appearance of the specific area, accurately described and drawn by a professional designer. RESULTS: The photographs of 302 patients were considered. Six patterns were identified: Normal mucosa exhibited different appearance in each type of epithelium; dysplastic mucosa presented the same pattern in type 1 and 2a epithelia, which differed from that of type 2b epithelium; in cancer, mucosal appearance was identical irrespective of epithelial type, due to complete vascular destruction. CONCLUSIONS: The proposed classification could serve as a guide for clinicians approaching narrowband imaging, especially at early stages of the learning curve, to differentiate normal mucosa from malignant lesions and possibly reduce the number of unnecessary biopsies.

Immunohistochemically Detection of Angiogenesis in Oral Pre-Cancerous Lesions Compared with Oral Invasive Carcinomas

Background: Angiogenic activity is an important event in oral carcinogenesis. During transition of normal oral mucosa to different grades of dysplasia and to invasive carcinoma, significant increase of vascularity occurs. Angiogenesis can be determined by immunohistochemical assessment of several endothelial cell markers like Endogelin (CD 105), expressed in activated endothelial cells and associated with neovasculature, and the vascular endothelial growth factor (VEGF). This study was conducted to evaluate angiogenic activity in oral precancerous lesions compared with oral invasive carcinomas by immunohistochemical staining of VEGF and CD 105 proteins. Methods: In the present cross-sectional study, 20 normal, 20 pre-cancerous mucosa and 20 oral invasive carcinoma samples were immunohistochemically stained. Positive cells were counted in each section and micro vessel density (MVD) was determined. The data were statistically analyzed by Mann-Whitney and Kruskal-Wallis tests, with a P-value ≤0.05 considered significant. Results: The mean expression value for VEGF was 24.6 in oral invasive carcinoma, 16.4 in precancerous mucosa and 15.5 in normal mucosa, with no significant differences between the latter two. Endoglin was negative in all normal mucosa samples, but had scores of 7.58 for precancerous mucosa and 19.4 in oral invasive carcinoma specimens. MVD was significantly higher in SCC than in dysplastic mucosa. Conclusion: Oral invasive carcinoma has more angiogenic activity in comparison with pre-cancerous lesions and normal mucosa. Given the high expression of CD105 positive vessels in malignant lesions, we can argue that determination of mean vessel density (MVD) by application of the CD105 marker could be a useful parameter to differentiate cancerous from pre-cancerous lesions.

ZNF750 inhibited the malignant progression of oral squamous cell carcinoma by regulating tumor vascular microenvironment.

OBJECTIVE: Squamous cell carcinoma is often associated with the deletion or mutation of zinc finger protein 750 (ZNF750), its deletion or mutation is associated with squamous epithelial malignant biological characteristics. The present study is to explore the mechanism of ZNF750 to suppress the tumor malignant process by regulation tumor microenvironment. METHODS: To evaluate the changes of tumor microenvironment in oral squamous cells carcinoma cell line CAL-27 cell, the expression of angiogenin, vascular endothelial growth factor (VEGF), prolyl hydroxylase 2 (PHD2), G protein signal regulated protein 5 (RGS5), integrin A5 (ITGA5), integrin B1 (ITGB1) and CD44 were detected by Western-blot. The changes of platelet derived growth factor (PDGFB) and tumor vascular marker CD105 (Endoglin) mRNA were estimated by qPCR. The effect of over-expressed ZNF750 on cell viability and lateral migration capacity was investigated by CCK-8 and cell scratch assay in three oral squamous cells carcinoma. RESULTS: ZNF750 could effectively inhibit the protein or mRNA expression of angiogenin, VEGF, RGS5 and CD105, repressed the cell adhesion molecules ITGA5, ITGB1 and CD44, but up-regulate the protein or mRNA expression of PHD2 and PDGFB. The cell viability and lateral migration ability of three oral squamous cells carcinoma were reduced by over-expression of ZNF750. CONCLUSION: ZNF750 could modulate the tumor vascular microenvironment to inhibit the oral squamous cells carcinoma malignant progression.

PA28γ acts as a dual regulator of IL-6 and CCL2 and contributes to tumor angiogenesis in oral squamous cell carcinoma.

PA28γ promotes tumor development and progression and is suggested to play a role in tumor angiogenesis, but the molecular mechanisms have not been investigated. Here, we found that PA28γ enhanced the ability of OSCC cells to promote the migration, invasion, and tube formation of HUVECs and promoted tumor-induced angiogenesis in xenograft mice models in vivo. Then, a mechanism study revealed that the expression and secretion of IL-6 and CCL2 were dependent on PA28γ expression. Furthermore, blocking IL-6 or CCL2 or the transcription factor NF-κB induced the inhibition of tube formation in HUVECs co-cultured with PA28γ-overexpression OSCC cell supernatants. Moreover, we revealed that p-STAT3 and p-AKT, which are downstream of the IL-6 and CCL2 signaling axis, were downregulated in HUVECs co-cultured with the PA28γ-silenced supernatant and were upregulated with the PA28γ-overexpressing supernatant. In addition, IL-6, CCL2 and PA28γ expressions were correlated in a clinical OSCC cohort. Collectively, our study indicates that PA28γ contributes to tumor angiogenesis by regulating IL-6 and CCL2. PA28γ may be a novel therapeutic target as a dual regulator of IL-6 and CCL2 for treating PA28γ-positive OSCC.