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Aim: To evaluate the biological function of titanium implants coated with cell-derived mineralized extracellular matrix, which mimics a bony microenvironment. Materials & methods: A biomimetic titanium implant was fabricated primarily by modifying the titanium surface with TiO2 nanotubes or sand-blasted, acid-etched topography, then was coated with mineralized extracellular matrix constructed by culturing bone marrow mesenchymal stromal cells. The osteogenic ability of biomimetic titanium surface in vitro and in vivo were evaluated. Results: In vitro and in vivo studies revealed that the biomimetic titanium implant enhanced and accelerated osteogenesis of bone marrow stromal cells by increasing cell proliferation and calcium deposition. Conclusion: By combining surface topography modification with biological coating, the results provided a valuable method to produce biomimetic titanium implants with excellent osteogenic ability.

Background The gasdermin E gene (GSDME, also known as DFNA5) is mutated in familial aging-related hearing loss. Recent studies have also revealed that the expression of DFNA5 is suppressed in many cancer types; however, little is known about the function of DFNA5 in head and neck squamous cell carcinoma (HNSCC). Accordingly, the aim of the present study was to evaluate the expression of DFNA5 and explore its prognostic value in HNSCC. Result We used a set of bioinformatics tools, including Oncomine, TIMER, TISIDB, cBioPortal, and GEPIA, to analyze the expression of DFNA5 in patients with HNSCC from public databases. Kaplan-Meier plotter was used to evaluate the potential prognostic significance of DFNA5. DFNA5 mRNA levels were significantly higher in HNSCC tissues than in normal tissues, and high DFNA5 expression was correlated with worse survival. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that DFNA5 expression has a strong positive correlation with cell adhesion and the integrin signaling pathway, whereas its expression was negatively correlated with the levels of infiltrating B cells (cor = − 0.223, P = 8.57e-07) and CD8 T cells (cor = − 0.223, P = 2.99e-07). Conclusion This study demonstrates that DFNA5 expression has prognostic value for HNSCC patients. Moreover, these results suggest that regulation of lymphocyte infiltration is the mechanism underlying the function of DFNA5 in HNSCC.

The gasdermin E gene (GSDME, also known as DFNA5) is mutated in familial aging-related hearing loss. Recent studies have also revealed that the expression of DFNA5 is suppressed in many cancer types; however, little is known about the function of DFNA5 in head and neck squamous cell carcinoma (HNSCC). Accordingly, the aim of the present study was to evaluate the expression of DFNA5 and explore its prognostic value in HNSCC. We used a set of bioinformatics tools, including Oncomine, TIMER, TISIDB, cBioPortal, and GEPIA, to analyze the expression of DFNA5 in patients with HNSCC from public databases. Kaplan-Meier plotter was used to evaluate the potential prognostic significance of DFNA5. DFNA5 mRNA levels were significantly higher in HNSCC tissues than in normal tissues, and high DFNA5 expression was correlated with worse survival. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that DFNA5 expression has a strong positive correlation with cell adhesion and the integrin signaling pathway, whereas its expression was negatively correlated with the levels of infiltrating B cells (cor = –0.223, P = 8.57e-07) and CD8 T cells (cor = –0.223, P = 2.99e-07). Overall, this study demonstrates that DFNA5 expression has prognostic value for HNSCC patients. Moreover, these results suggest that regulation of lymphocyte infiltration is the mechanism underlying the function of DFNA5 in HNSCC.

Background Few related surveys about reasons for tooth extraction was shown in mainland China. Our aim was to investigate the primary reasons and main pattern for extractions among patients presenting at a hospital of stomatology in Guangzhou, China. Methods This descriptive cross-sectional study was conducted over a period of 3 months, 2000 patients who underwent tooth extraction were recorded and analyzed by age, gender, the tooth type and the extraction reasons. Chi-square test was used to examine the differences in the extraction reasons between different genders, age groups and tooth types. Results A total of 3106 teeth were removed from 2000 patients, with an age ranging from 6 to 91. This study revealed details of extraction reasons and suggested that dental caries (25.42%) was the main reasons for tooth extraction and pointed out preventive extraction of third molar (19.89%) had displaced periodontal diseases (12.89%) as the dominant reason for extractions. Overall, third molar was the most frequently extracted tooth (1567). Significant statistical differences were found among different age groups, genders and tooth types for tooth extraction reasons (p༜0.05). For example, caries was the main reason for tooth extraction in 30–59 years old patients, while 20–29 years old patients asked for preventive extraction of third molar teeth mostly. Posterior teeth were mostly removed because of caries and periodontal disease was the leading cause for anterior teeth extraction. Conclusions We found the primary reasons and the new patterns for tooth extraction among patients in Guangzhou. It was proposed for the first time that the dominant reason for extractions was preventive extraction of third molar. Our finding could help to put forward some predictors and adjust the preventive strategies in oral disorders.

MicroRNAs (miRNAs) regulate gene expression and at the same time mediate tumorigenesis. miR-373-3p has diverse effects in tumors, but its role in tongue squamous cell carcinoma (TSCC) remains unknown. The purpose of this study is to determine the function of miR-373-3p in the progression of TSCC. Our results brought to light that miR-373-3p is markedly upregulated in clinical TSCC tissues compared with paired adjacent normal tissues and has significant correlation with a more aggressive TSCC phenotype in patients. Gain-of-function and loss-of-function studies revealed that ectopic miR-373-3p overexpression promoted the metastasis of TSCC cells. Notably, Wnt/β-catenin signaling was hyperactivated in TSCC cells overexpressing miR-373-3p, and this pathway was responsible for the epithelial-mesenchymal transition (EMT) induced by miR-373-3p. Furthermore, miR-373-3p directly targeted and suppressed Dickkopf-1 (DKK1), a negative regulator of the Wnt/β-catenin signaling cascade. These results demonstrate that, by directly targeting DKK1, miR-373-3p constitutively activated Wnt/β-catenin signaling, thus promoting the EMT-induced metastasis of TSCC. Taken together, our findings reveal a new regulatory mechanism for miR-373-3p and suggest that miR-373-3p might be a potential target in TSCC therapy.

Chemoresistance is often associated with other clinical characteristics such as enhanced migratory/invasive potential. However, the correlation and underlying molecular mechanisms remain unclear. The aim of this study was to elucidate the function of the miR-222-ABCG2 pathway in the correlation between cisplatin (DDP) resistance and enhanced cell migration/invasion in tongue squamous cell carcinoma (TSCC). Using TSCC cell lines and primary cultures from TSCC cases, we first confirmed the correlation among DDP resistance (measured by IC50 values and ABCG2/ERCC1 expression), migratory/invasive potential (assessed by migration/invasion assays) and miR-222 expression. In TSCC cells, siRNA-mediated ABCG2 knockdown led to enhanced DDP responsiveness and reduced migratory/invasive potential, whereas ABCG2 overexpression induced DDP resistance and enhanced cell migration/invasion. Luciferase assays revealed that ABCG2 is a direct target of miR-222. In addition to reducing cell migration/invasion, functional analyses in TSCC cells indicated that miR-222 can reduce expression of the ABCG2 gene and enhance DDP responsiveness. However, co-transfection with ABCG2 cDNA restored both DDP resistance and migration/invasion. Moreover, miR-222 mimics and ABCG2 siRNA inhibited tumor growth and lung metastasis in vivo. Thus, our results verified that DDP resistance is correlated with enhanced migratory/invasive potential in TSCC. ABCG2 is a direct target of miR-222,and deregulation of the miR-222-ABCG2 regulatory module in TSCC contributes to both DDP resistance and enhanced migratory/invasive potential.

Summary Objectives The role of autophagy is still a controversy in cancer development. In our previous study, we confirmed that decrease of autophagy activity promotes malignant progression of tongue squamous cell carcinoma (TSCC). However, the role of autophagy-related protein, Beclin1, has not well been documented in TSCC. In this study, we aim to elucidate the role of beclin1 in TSCC progression and investigate its potential mechanisms. Materials and methods TSCC cell lines, SCC9 and SCC15 were used to generate the stable cells with transfection lentivirus BECN1 and sh-BECN1. Then, Beclin1 expression was detected with qPCR and western blot. Moreover, the expressions of autophagy-related proteins and tumor metastasis associated proteins were examined by western blot and ELISA. For functional analysis, MTT assay were performed to evaluate the proliferation activity and transwell assay was used to assess the migration and invasion ability. Finally, TSCC xenograft models were established to confirm the effect of Beclin1 on TSCC in vivo. Results The results showed that BECN1 and sh-BECN1 virus transfection significantly increased or decreased the mRNA and protein expression of Beclin1 in the transfected TSCC cells. Meanwhile, we also observed that Beclin1 could enhance the expression levels of LC3-II, ATG4 and ATG5. Then, we revealed that overexpression of Beclin1 inhibited proliferation, migration and invasion while knockdown of Beclin1 promoted proliferation, migration and invasion in TSCC cells. Furthermore, we demonstrated that vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 and -9 were involved in Beclin1-mediated inhibition of migration and invasion. More importantly, our data also confirmed that Beclin1 inhibited TSCC xenograft growth in vivo. Conclusion Taken together, the results indicate that autophagy regulating gene, Beclin1, may contribute to the malignant phenotypes of TSCC cells and can be a potential target for oral cancer gene therapy.

Background Autophagy is a catabolic process involving the degradation of cells' own unnecessary, injured, or aged proteins and recycling of degraded products to maintain hemostasis. Recently, studies indicated that autophagy plays a crucial role in cancer development. However, the role of autophagy in tongue squamous cell carcinoma (TSCC) has not been well documented. This study aims to assess the expression of autophagy-related protein and investigate its effect on TSCC. Materials and methods Archival 50 TSCC samples were enrolled. Immunohistochemistry were performed to examine the expression of Beclin1 and LC3. Statistical analyses were carried out to assess the associations among clinicopathologic parameters. In vitro, cells were treated with rapamycin or 3-MA. Then, qPCR, western blot and immunofluorescence were performed to detect the expression of Beclin1 and LC3. Transmission electron microscopy was utilized to identify autophagsomes. For functional analysis, cell proliferation and cell cycle were evaluated with MTT assay and flow cytometer, respectively. At last, cell migration and invasion potentials were assessed by wound healing assay and transwell assay. Results We confirmed that down-regulation of Beclin1 and LC3 is a frequent event in TSCC. Then, we demonstrated that decreased expression of Beclin1 was associated with T stage, clinical stage and differentiation. Furthermore, we showed that activation of autophagy by rapamycin suppressed proliferation, migration and invasion while inhibition of autophagy by 3-MA promoted proliferation, migration and invasion in TSCC cells. Conclusion Taken together, these data suggest that autophagy plays a pivotal role in the progression of TSCC.

Oral squamous cell carcinoma (OSCC) is a common and lethal malignancy. Thus, improvement in current knowledge of molecular changes associated with OSCC is urgently needed to explore novel avenues of diagnostics and treatment of this disease. While aberrant expression of long non‑coding RNAs (lncRNAs) has been functionally associated with certain types of cancer, including lung, breast and prostate carcinomas, their expression pattern and biological relevance in OSCC is currently unknown. In the present study, the relative abundance of a collection of lncRNAs in tissue or saliva samples from OSCC patients was investigated. It was shown that subsets of lncRNAs are expressed across non‑tumor, tumor and metastatic tissue samples. Some detected lncRNAs were shown to be aberrantly expressed in cases of oral cancer and metastasis. Moreover, whole saliva contained a detectable amount of some lncRNAs, which appeared to be potential markers for OSCC. These findings suggest that the detection of lncRNAs in saliva may be used as a noninvasive and rapid diagnostic tool for the diagnosis of oral cancer.

MicroRNAs (miRNAs) regulate gene expression and at the same time mediate tumorigenesis. miR-373-3p has diverse effects in tumors, but its role in tongue squamous cell carcinoma (TSCC) remains unknown. The purpose of this study is to determine the function of miR-373-3p in the progression of TSCC. Our results brought to light that miR-373-3p is markedly upregulated in clinical TSCC tissues compared with paired adjacent normal tissues and has significant correlation with a more aggressive TSCC phenotype in patients. Gain-of-function and loss-of-function studies revealed that ectopic miR-373-3p overexpression promoted the metastasis of TSCC cells. Notably, Wnt/β-catenin signaling was hyperactivated in TSCC cells overexpressing miR-373-3p, and this pathway was responsible for the epithelial-mesenchymal transition (EMT) induced by miR-373-3p. Furthermore, miR-373-3p directly targeted and suppressed Dickkopf-1 (DKK1), a negative regulator of the Wnt/β-catenin signaling cascade. These results demonstrate that, by directly targeting DKK1, miR-373-3p constitutively activated Wnt/β-catenin signaling, thus promoting the EMT-induced metastasis of TSCC. Taken together, our findings reveal a new regulatory mechanism for miR-373-3p and suggest that miR-373-3p might be a potential target in TSCC therapy.

Background & Aims The mammalian target of rapamycin (mTOR) plays a pivotal role in hepatocellular carcinoma (HCC). Previous studies indicated that inhibition of mTORC1 enhanced histone deacetylase inhibitors (HDACis)-mediated anti-tumor activity, accompanied with feedback activation of AKT. Therefore, dual targeting of mTORC1/C2 should be more efficient in suppressing AKT activity and in enhancing the anti-tumor activity of HDACi in HCC. Methods The interactions between mTOR kinase inhibitors (mTORKis) (i.e., Pp242, AZD8055, OSI027) and HDACis (i.e., SAHA, LBH589) were examined in vitro using HCC cell lines and in vivo using patient-derived primary HCC xenografts on SCID mice. Results mTORKis significantly enhanced HDACi-induced apoptosis in HCC cells. The inhibition of both mTORC1/2 not only efficiently blocked mTORC1 signaling, but also abrogated AKT-feedback activation caused by selective mTORC1 inhibition. The co-treatment of mTORKi and HDACi further inhibited AKT signaling and upregulated Bim. Dysfunction of mTORC2 by shRNA significantly lowered the threshold of HDACi-induced cytotoxicity by abrogating AKT activation. Knockdown of AKT1 sensitized Pp242/HDACi-induced apoptosis and ectopic expression of constitutively active AKT1 abrogated the combination-induced cytotoxicity, indicating AKT plays a vital role in the combination-induced effects. Knockdown of Bim prevented Pp242/HDACis-induced cytotoxicity in HCC. Lastly, in vivo studies indicated that the combination of AZD8055 and SAHA almost completely inhibited tumor-growth, without obvious adverse effects, by abrogating AKT and upregulating Bim; while either agent alone shows only 30% inhibition in primary HCC xenografts. Conclusions Our findings suggest that a combining-regimen of mTORKi and HDACi may be an effective therapeutic strategy for HCC.

The management of infantile hemangiomas remains a subject of controversy. The purpose of this study was to investigate the indications and treatment results of intralesional injection of pingyangmycin for treatment of infantile hemangiomas. In a prospective study of 66 patients, the effectiveness of intralesional injection of pingyangmycin was evaluated and documented. The lesions were all located in the oral and maxillofacial regions. The smallest lesion was 1.0 cm × 0.7 cm and the largest was 4.6 cm × 3.8 cm. Amongst the 66 infants who underwent sclerotherapy with pingyangmycin, cure was obtained in 74% (49/66) of patients, marked improvement of the treated lesion occurred in 14% (9/66), improvement occurred in 12% (8/66), and a lack of response was not observed in any patient. All patients were regularly followed up for 1-4 years following pingyangmycin treatment, and they demonstrated the same good results over this time. Intralesional injection of pingyangmycin was a reliable and effective therapeutic choice for infantile oral and maxillofacial hemangiomas, as it shortened the involution time and decreased the influence induced by these potentially countenance-influenced tumours with few complications.

YM155, a small-molecule survivin suppressant, exhibits anti-tumor activities in vitro, in vivo and in clinical trials. However, the mechanism of YM155 action remains unclear. In this study, YM155 was administered to a panel of cell lines and the effects of YM155 on Bcl-2 family members were analyzed. Our results show that YM155 strikingly downregulates Mcl-1 in a broad spectrum of cancer cell lines and that the Mcl-1 modulation occurs at the transcriptional level, independently of survivin modulation or caspase activity. Furthermore, analysis of the contribution of Mcl-1 or survivin downregulation to YM155-induced cell death in vitro showed that knockdown of Mcl-1 sensitizes cells to YM155-induced cytotoxicity. Finally, our data demonstrate that downregulation of Mcl-1 by YM155 synergistically lowers the threshold of Bcl-2 family member inhibitor ABT-263-induced cell death. Our findings reveal a novel mechanism by which survivin-independent Mcl-1 suppression plays a critical role in YM155-mediated anti-tumor activities. YM155 treatment in combination with ABT-263 thus affords a new strategy for cancer treatment.

Background: The secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein that is produced by tumor and/or neighboring stroma. SPARC expression is thought to facilitate the intracellular accumulation of nanoparticle albumin-bound paclitaxel ( nab -paclitaxel, abraxane [ABX]). Gene hypermethylation is a common mechanism for loss of SPARC expression in non-small cell lung cancer (NSCLC). We aim to demonstrate the role of SPARC expression as biomarker for treatment selection using ABX in NSCLC and to evaluate the presence of synergistic antitumor effect when a demethylating agent is combined with ABX. Methods: We analyzed the SPARC messenger RNA expression and SPARC gene methylation status in 13 NSCLC cell lines and 22 minimally passaged patient-derived (PD) NSCLC tumors using real-time (RT) polymerase chain reaction. The effect of ABX on tumor growth was compared with cremophor-solubilized paclitaxel (taxol) in severe combined immunodeficiency mice bearing SPARC-positive PD xenografts. The effect of pretreatment with a demethylating agent, 5-Aza-2′-deoxycytidine (DEC) in SPARC-negative tumors was assessed. Results: SPARC expression was weak to absent in 62% of established NSCLC cell lines and 68% of PD NSCLC tumor xenografts. SPARC expression could be up-regulated/restored by DEC treatment in both SPARC-negative cell lines and PD xenografts in vitro and in vivo. ABX demonstrated better antitumor efficacy than equitoxic dose of taxol in SPARC-expressing xenografts and some SPARC-negative xenografts. At equimolar doses in vitro, there was similar increased cytotoxicity on DEC pretreatment with either ABX or taxol in SPARC-negative cell lines. At equitoxic doses, there was similar additive antitumor activity of DEC with either ABX or taxol in SPARC-negative PD xenografts. Conclusion: Endogenous SPARC status is somewhat uncorrelated with response to ABX in NSCLC. The greater antitumor effect of ABX compared with equitoxic dose of taxol observed in SPARC-expressing NSCLC tumors can also be seen in some SPARC-negative tumors. DEC pretreatment similarly enhanced antitumor activity with either ABX or taxol in SPARC-negative tumors.

Background Studies of primary patient tumor xenografts grown in immunodeficient mice have shown that these tumors histologically and genetically closely resemble the original tumors. These patient xenograft models are becoming widely used for therapeutic efficacy studies. Because many therapies are directed at tumor stromal components and because the tumor microenvironment also is known to influence the response of a tumor to therapy, it is important to understand the nature of the stroma and, in particular, the vascular supply of patient xenografts. Methods Patient tumor xenografts were established by implanting undisrupted pieces of patient tumors in SCID mice. For this study, formalin fixed, paraffin embedded specimens from several types of solid tumors were selected and, using species-specific antibodies which react with formalin fixed antigens, we analyzed the species origin of the stroma and blood vessels that supported tumor growth in these models. Additionally, we investigated the kinetics of the vascularization process in a colon tumor and a mesothelioma xenograft. In mice bearing a head and neck xenograft, a perfusion study was performed to compare the functionality of the human and mouse tumor vessels. Results In patient tumors which successfully engrafted, the human stroma and vessels which were engrafted as part of the original tumor did not survive and were no longer detectable at the time of first passage (15–25 weeks). Uniformly, the stroma and vessels supporting the growth of these tumors were of murine origin. The results of the kinetic studies showed that the loss of the human vessels and vascularization by host vessels occurred more rapidly in a colon tumor (by 3 weeks) than in a mesothelioma (by 9 weeks). Finally, the perfusion studies revealed that while mouse vessels in the periphery of the tumor were perfused, those in the central regions were rarely perfused. No vessels of human origin were detected in this model. Conclusions In the tumors we investigated, we found no evidence that the human stromal cells and vessels contained in the original implant either survived or contributed in any substantive way to the growth of these xenografts.

The mammalian target of rapamycin (mTOR) consists of two distinct complexes, mTORC1 and mTORC2. mTOR plays a pivotal role in Hepatocellular Carcinoma (HCC). Previous studies indicated that inhibition of mTORC1 only moderately enhanced histone deacetylase inhibitors (HDACis)-mediated anti-tumor activity. This limited effect may be attributable to the increase of AKT activation through S6K1-IRS1-PI3K negative feedback loop triggered by selective mTORC1 inhibition. While as AKT is the downstream effector of mTORC2, we hypothesize that dual targeting of mTORC1/C2 would be more efficient in suppressing AKT and in enhancing anti-tumor activity of HDACi. In this study, the interactions between mTOR kinase inhibitors (mTORKis) and HDACis were examined in vitro using established HCC cell lines and in vivo using patient-derived HCC tumor xenografts on SCID mice. Our results demonstrate that inhibition of both mTORC1/2 not only effectively blocked mTORC1 signaling, but also abrogated negative-feedback activation of AKT caused by mTORC1 inhibition. Furthermore, inactivation of mTORC1/C2 by mTORKis substantially enhanced HDACi-induced anti-tumor activity. Either dysfunction of mTORC2 by knockdown of Rictor (a component of mTORC2), or diminution of AKT1 significantly lowered the threshold of HDACi-induced apoptosis. Lastly, in vivo study indicated that the combination of AZD8055 and Vorinostat yielded much greater tumor-growth inhibition compared to either agent alone in primary HCC xenografts without obvious adverse effects. Conclusion: Dual mTORC1/2 inhibition is more efficient than selective mTORC1 inhibition in enhancing anti-tumor activity of HDACi due to abrogation of negative-feedback activation of AKT induced by selective mTORC1 inhibition. Our findings suggest that combining regimen of mTORKi and HDACi may be an effective therapeutic strategy for HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5363. doi:10.1158/1538-7445.AM2011-5363