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IFIX, a newly discovered member of the interferon-inducible HIN-200 family, has been identified as a tumor suppressor in breast cancer; however, the involvement of IFIX in oral cancer are poorly understood. Here, we demonstrate a relationship between the level of IFIX expression and the invasive or migratory abilities of oral squamous cell carcinoma. Higher IFIX expression significantly correlated with clinicopathological parameters such as the histopathological grade of clinical samples. In vitro, IFIX overexpression suppressed the invasiveness of human tongue squamous cell carcinoma CAL-27 cells, and this inhibitory effect was mediated by stabilization of the cytoskeleton through various cytokeratins along with downregulation of paxillin, an intracellular adaptor protein that promotes tumor invasion. This inhibitory effect does not appear to affect the transformation of cancer stem-like cells in this cell culture model. Altogether, these data provide novel insights into the tumor-suppressive function of IFIX, namely, stabilization of the cancer cell cytoskeleton.

BACKGROUND: Resistance to PD-1 blocking agents is not uncommon, limiting their wide clinical success. Certain tumor-infiltrating immune cells (e.g., TILs/CTLs) have emerged as biomarkers of response, and absence of such immune cells contributes to resistance. OBJECTIVE: We deconvoluted the dynamic immune microenvironment in a mouse model of oral carcinogenesis for augmenting the resistance to PD-1 blocking agents by combination. METHODS: Bioinformatics methods and routine biological experiments were adopted such as morphological analysis and ELISA in the 4NQO-treated mice model. RESULTS: Our findings revealed that dysplastic tongue tissues from 4NQO-treated mice were characterized by an immunosuppressive tumor microenvironment. Tongue tissues from mice treated with 4NQO for 12 weeks had higher levels of Th2 cells and Tregs compared to tissues taken from control mice or mice treated with 4NQO for 28 weeks; these results suggested a potential therapeutic benefit of anti-PD-1 in the oral cancer. The IL-17 pathway was significantly upregulated during progression from normal mucosa to hyperplasia and tumor formation in mice. Inhibition of IL-17α combined with PD-1 blockade delayed the development of 4NQO-induced precancerous and cancerous lesions and prolonged the survival of 4NQO-treated mice. CONCLUSIONS: Our data suggested a strong rationale of IL-17α blockade as a potential approach to augment the tumor-eliminating effects of anti-PD-1 therapy.

PIWIL2 is a human Argonaute protein, which is guided by small RNAs to its targets, plays a role in germ cell maintenance and has been proposed to be expressed in precancerous stem cells and tumor stem cells. However, the significance of PIWIL2 expression in oral cancer and precancerous lesions has not been investigated. In this study, we analyzed the expression of the stem cell protein PIWIL2 in oral squamous-cell carcinoma (OSCC) and in premalignant oral leukoplakia (OL) with predominant expression in malignant and premalignant tissues. In the evaluated patients, we found that PIWIL2 was associated significantly with OSCC prognosis and OL. Furthermore, PIWIL2 was found to be expressed in tumor epithelial cells and macrophages in the tumor microenvironment, which are not derived from enlarged lymph nodes. Cytological experiments confirmed that the human squamous cell carcinoma cell line SCC-25, can promote the PIWIL2 and Nanog level in THP-1 cells, which are extensively used to study the modulation of monocytes and macrophages. Our findings showed that PIWIL2 can predict effectively OSCC prognosis and OL with a high risk of OSCC development and substantiate the deduction that cancer stem(-like) cells in oral cancer have the ability to reconstitute the heterogeneity of the bulk tumor and contribute to poor outcome and immunosuppression.

Background This study aimed to evaluate the relationship between survivin expression and melanoma after 5-aminolevulinic acid (5-ALA)-mediated sonodynamic therapy. Methods Immunohistochemistry was used to detect survivin protein expression in human melanoma clinical samples. Subsequently, the effects of 5-ALA-mediated sonodynamic therapy were determined by measuring the volume of melanoma xenografts and the bodyweights of melanoma-bearing nude mice. The MTT assay was used to detect the viability of melanoma B16-F10 cells under the action of 5-ALA-mediated sonodynamic therapy, and Western blotting and PCR were used to detect survivin expression in melanoma cells and in the melanoma-xenograft model. Results Survivin expression was significantly upregulated in human melanoma tissues compared with that of non-melanoma tissues. In the in vivo case, 5-ALA-mediated sonodynamic therapy significantly delayed tumor growth, prolonged the survival of mice, and inhibited the expression of survivin. In the in vitro case, 5-ALA-mediated sonodynamic therapy inhibited B16-F10 cell proliferation and decreased survivin expression at both protein and mRNA levels. Conclusion Our results suggest that 5-ALA-mediated sonodynamic therapy inhibited B16-F10 cell proliferation and melanoma-xenograft growth and prolonged survival of melanoma-bearing nude mice, which might be through downregulation of survivin expression.

In the present study, the presence of extracellular matrix components, including collagenous and elastic fibers, and the expression of their key regulating enzymes, were investigated in different stages of hamster tongue carcinoma development. Immunohistochemical and computer-assisted morphological analyses were performed to quantify the staining intensity and area (integral optical density) of matrix metalloproteinase (MMP)-1 and -2, tissue inhibitors of metalloproteinase (TIMP)-1 and -2, and the extent of elastic and collagenous fibers in histological sections. MMP-1, MMP-2, TIMP-1 and TIMP-2 expression levels gradually increased with tongue cancer progression, and were associated with disease pathology staging (r=0.705, 0.633, 0.759 and 0.751, respectively). By contrast, elastic fiber levels gradually decreased with cancer progression and were negatively correlated with disease staging (r=-0.881). The levels of collagenous fibers gradually increased with cancer progression and showed a positive correlation (r=0.619). In summary, the study demonstrated that MMP1/2 and TIMP1/2 expression levels, and collagenous and elastic fiber levels were significantly correlated with disease progression in a hamster model of tongue cancer.

Background Neuropilin-2 (NRP2), a receptor for vascular endothelial growth factor C (VEGF-C) and semaphorin 3 F (SEMA3F), is a possible regulator of tumor progression and angiogenesis. However, little evidence of a correlation between NRP2 expression and lymphangiogenesis has been reported. SEMA3F might suppress lymphangiogenesis by competing with VEGF-C for binding to NRP2. Methods We evaluated lymphatic vessel density (LVD), lymphatic vessel invasion (LVI), the expression levels of VEGF-C, SEMA3F, and NRP2 by immunohistochemistry in 80 cases of oral squamous cell carcinomas (OSCCs). Results In these tumors, the expression of NRP2 was positively associated with T stage classification, lymph node metastasis, LVD, LVI, and the expression of VEGF-C. In contrast, low expression of SEMA3F was significantly related to poor differentiation and higher incidence of lymph node metastasis. Patients expressing high levels of VEGF-C or NRP2, or low levels of SEMA3F had a higher risk of recurrence and shorter overall survival. Multivariate analysis showed that VEGF-C and NRP2 were independent prognostic markers for overall survival. Conclusions Results indicate SEMA3F is an inhibitor of tumor progression and provide evidence for an association between NRP2 and VEGF-C, lymphangiogenesis, lymph node metastasis. This suggests the prognostic significance and potential therapeutic value of the VEGF-C/SEMA3F/NRP2 axis for OSCCs. J. Surg. Oncol. 2015 111:382–388. © 2014 Wiley Periodicals, Inc.

Background Oral squamous cell carcinoma (OSCC) invades surrounding tissues by upregulating matrix metalloproteinases (MMPs) -2 and −9, which causes over-expression of the Hedgehog signaling proteins Shh and Gli-1 and degradation of the extracellular matrix, thereby creating a “highway” for tumor invasion. We explored the potential of low intensity ultrasound (LIUS) and doxorubicin (DOX) to inhibit the formation of this “highway”. Methods MTT assays were used to examine OSCC cell viability after exposure to LIUS and DOX. The cell morphological changes and ultrastructure were detected by scanning electron microscopy and transmission electron microscopy. Endogenous autophagy-associated proteins were analyzed by immunofluorescent staining and western blotting. Cell migration and invasion abilities were evaluated by Transwell assays. Collagen fiber changes were evaluated by Masson’s trichrome staining. Invasion-associated proteins were analyzed by immunohistochemistry and western blotting. Results LIUS of 1 W/cm2 increased the in vitro DOX uptake into OSCC by nearly 3-fold in three different cell lines and induced transient autophagic vacuoles on the cell surface. The combination of LIUS and 0.2 μg/ml DOX inhibited tumor cell viability and invasion, promoted tumor stromal collagen deposition, and prolonged the survival of mice. This combination also down-regulated MMP-2, MMP-9, Shh and Gli-1 in tumor xenografts. Collagen fiber expression was negatively correlated with the expression of these proteins in human OSCC samples. Conclusions Our findings suggest that effective low dosages of DOX in combination with LIUS can inhibit cell proliferation, migration and invasion, which might be through MMP-2/9 production mediated by the Hedgehog signaling pathway. Electronic supplementary material The online version of this article (10.1186/s13046-017-0633-y) contains supplementary material, which is available to authorized users.

Backgroud Malignant melanoma is a very refractory skin tumor due to its high metastasis, poor prognosis, and insensitivity to chemotherapy. Sonodynamic therapy has recently evolved as a potential method to treat cancers. In this study, 5-aminolevulinic acid-mediated sonodynamic therapy (ALA-SDT) was used to treat malignant melanoma in vivo . Objective To investigate whether ALA-SDT induces anti-tumor effects in malignant melanoma and to see if miRNAs are involved in this process. Methods Tumor transplantation experiments in BALB/c nude mice were used to assess anti-tumor effects after ALA-SDT treatment. Cell apoptosis was evaluated by TUNEL assays and cell proliferation was measured using immunohistochemisty with anti-PCNA antibody. Microarray analysis was performed to measure miRNAs expressions. Endogenous miR-34a and its upstream and downstream genes were assayed by real-time PCR. Western blottings were used to determine these protein expressions. Intracellular ROS levels were detected by measuring the fluorescence intensity of DCF. Results Tumor transplantation experiments revealed that ALA-SDT could inhibit mouse melanoma cell proliferation and tumor growth. Compared with the control group, TUNEL assays revealed that apoptosis was increased and proliferation was inhibited in the SDT group. Real-time PCR analysis showed 14-fold increase of miR-34a expression in the SDT group compared to the control group. In addition, ALA-SDT significantly increased intracellular ROS levels in vitro , which were almost inhibited by the ROS scavenger NAC. Also, the mRNA, total protein, and acetylation levels of p53 were increased, whereas some downstream anti-apoptotic or pro-proliferative factors of miR-34a such as BCL2, CCND1, CDK6, and SIRT1 were decreased in the SDT group compared with the control, ALA alone, and ultrasound alone groups. When miR-34a was inhibited in vitro , the protein expressions of BCL2, CCND1, CDK6, and SIRT1 recovered. By targeting SIRT1, which inhibits p53 acetylation, miR-34a promoted the transcriptional activity of p53, and finally led to increased expression of miR-34a itself. Therefore, the p53, miR-34a, and SIRT1 constituted a positive feedback loop. Conclusion ALA-SDT showed synergistic anti-tumor effects in malignant melanoma by constituting a positive feedback loop of p53-miR-34a-Sirt1 axis.

The aim of this study was to investigate sonic hedgehog (SHH) signaling pathway components (Shh and Gli-1), E-cadherin, and MMP-9 expression in human oral squamous cell carcinoma (OSCC) and to evaluate their role in prognosis. Expression of Shh, Gli-1, and MMP-9 was significantly upregulated in 74 OSCC samples compared with non-cancerous tissue samples (Shh IOD: 162.44 ± 29.35 and 608.82 ± 170.99; Gli-1 IOD: 203.50 ± 71.57 and 831.11 ± 242.352; MMP-9 IOD: 196.69 ± 64.48 and 721.64 ± 197.99 in non-cancerous and tumor tissues, respectively, P

Background This study aims to investigate the expression and significance of the αvβ6 integrin, collagen fibres and matrix metalloproteinases (MMP)-3 in oral squamous cell carcinoma (OSCC) and to analyse the possible regulatory relationships between αvβ6, collagen fibres and MMP-3. Materials and Methods A series of 80 patients (mean age 56.4 years) diagnosed with OSCC were enrolled. Associations between αvβ6, MMP-3, collagen fibre expression levels and clinicopathological parameters were evaluated using the Fisher exact test. Survival analysis was performed with Kaplan–Meier curves. Spearman rank correlation was used to analyse interactions between αvβ6, MMP-3 and collagen fibres. Results αvβ6 and MMP-3 were strongly expressed in human OSCC, especially at the peripheral borders of invasive tumour islands, and collagen fibres were generally disrupted and degraded in the same areas. The expression intensity of αvβ6 was associated with the differentiation state of cells. β6 mRNA was expressed in almost all cancer cells. In carcinomas, αvβ6 and MMP-3 expression were correlated with the distribution of collagen fibres. Conclusions Tumour cells highly expressing αvβ6 have a strong capability for invasion and migration, due to concomitant protease production and the destruction and remodelling of collagen fibres. Increased αvβ6 integrin and MMP-3 expression and collagen fibre changes in human OSCCs are related to unfavourable clinical prognostic factors and decreased survival.

Studies of sonodynamic therapy (SDT) have mainly focused on its direct cytotoxic effect on tumor cells. Its effects on the tumor microenvironment, especially angiogenesis, remain unknown. In this study, we found that SDT significantly inhibited endothelial cell proliferation, migration, invasion, and tube formation. Furthermore, in a tumor xenograft mouse model, SDT was found to remarkably suppress tumor growth, intratumoral vascularity, and expression of vascular endothelial growth factor in tumor cells. An ultrastructural study showed damage and disruption of tumor microvasculature after STD. Our results indicate that SDT inhibits neovascularization in tumor, which is partially responsible for the anti-tumor effect of SDT.

Background Type IV collagen (ColIV) is the most important scaffold for the basement membrane (BM) proteins, and plays an important role in regulating and limiting tumour invasion and metastasis. Methods Here, we observed the changes in morphology and distribution of type IV collagen (ColIV) in the basement membrane (BM) surrounding nests of carcinoma in 48 patients with oral tongue squamous cell (OTSCC). We examined the correlation between the expressions of ColIV, MMP-2 and MMP-9 and the prognosis of OTSCC patients. The intensity and patterns of expression were assessed immunohistochemically using anti-human mouse monoclonal MMP-2, MMP-9 and Col IV antibodies. Statistical analyses were performed to determine the prognostic correlations of ColIV, MMP-2, and MMP-9 levels. Results MMP-2 and MMP-9 expressions in OTSCC were higher than those in normal oral mucosa and dysplastic oral mucosa group(MMP-2 iOD: 66.40 ± 24.20, 134.69 ± 37.08, and 357.79 ± 116.78; MMP-9 iOD: 88.05 ± 23.85, 307.13 ± 93.22, and 791.31 ± 260.52; in normal, dysplastic oral mucosa, and tumour tissues, respectively, P < 0.01); however, ColIV immunoreactivity was lower (ColIV iOD: 406.87 ± 62.95, 247.83 ± 42.30, and 151.92 ± 38.17 in normal, dysplastic oral mucosa, and tumour tissues, respectively, P < 0.01). High tumour and stromal MMP-2 and MMP-9 expression was significantly associated with positive lymph node status. Col IV expression was associated with positive lymph node status (P < 0.05), and have negatively correlated with the expression of MMP-2 and MMP-9. Overall survival was significantly shorter in patients with high tumour and stromal MMP-2 and MMP-9 expression, and tended to be shorter in patients with low ColIV expression. Conclusions Degradation of ColIV was closely related to increased MMP-2 and MMP-9 expression; MMP-9 have more important function than MMP-2 during the cancer development. Monitoring changes in the expression of ColIV, MMP-2, and MMP-9 may be a useful technique for assessing prognoses in OTSCC patients.