miR-181a shows tumor suppressive effect against oral squamous cell carcinoma cells by downregulating K-ras

MicroRNAs (miRNAs) are epigenetic regulators of gene expression, and their deregulation plays an important role in human cancer, including oral squamous cell carcinoma (OSCC). Recently, we found that miRNA-181a (miR-181a) was upregulated during replicative senescence of normal human oral keratinocytes. Since senescence is considered as a tumor suppressive mechanism, we thus investigated the expression and biological role of miR-181a in OSCC. We found that miR-181a was frequently downregulated in OSCC. Ectopic expression of miR-181a suppressed proliferation and anchorage independent growth ability of OSCC. Moreover, miR-181a dramatically reduces the growth of OSCC on three dimensional organotypic raft culture. We also identified K-ras as a novel target of miR-181a. miR-181a decreased K-ras protein level as well as the luciferase activity of reporter vectors containing the 3′-untranslated region of K-ras gene. Finally, we defined a minimal regulatory region of miR-181a and found a positive correlation between its promoter activity and the level of miR-181a expression. In conclusion, miR-181a may function as an OSCC suppressor by targeting on K-ras oncogene. Thus, miR-181a should be considered for therapeutic application for OSCC.     

Prevalence of regulatory T cells is increased in peripheral blood and tumor microenvironment of patients with pancreas or breast adenocarcinoma

Regulatory T cells (T(reg)) that prevent autoimmune diseases by suppression of self-reactive T cells may also suppress the immune response against cancer. In mice, depletion of T(reg) by Ab therapy leads to more efficient tumor rejection. T(reg)-mediated suppression of antitumor immune responses may partly explain the poor clinical response to vaccine-based immunotherapy for human cancer. In this study, we measured the prevalence of T(reg) that coexpress CD4 and CD25 in the PBLs, tumor-infiltrating lymphocytes, and regional lymph node lymphocytes from 65 patients with either pancreas or breast cancer. In breast cancer patients (n = 35), pancreas cancer patients (n = 30), and normal donors (n = 35), the prevalence of T(reg) were 16.6% (SE 1.22), 13.2% (SE 1.13), and 8.6% (SE 0.71) of the total CD4(+) cells, respectively. The prevalence of T(reg) were significantly higher in breast cancer patients (p < 0.01) and pancreas cancer patients (p < 0.01) when compared with normal donors. In tumor-infiltrating lymphocytes and lymph node lymphocytes, the T(reg) prevalence were 20.2% (SE 3.93) and 20.1% (SE 4.3), respectively. T(reg) constitutively coexpressed CTLA-4 and CD45RO markers, and secreted TGF-beta and IL-10 but did not secrete IFN-gamma. When cocultured with activated CD8(+) cells or CD4(+)25(-) cells, T(reg) potently suppressed their proliferation and secretion of IFN-gamma. We conclude that the prevalence of T(reg) is increased in the peripheral blood as well as in the tumor microenvironment of patients with invasive breast or pancreas cancers. These T(reg) may mitigate the immune response against cancer, and may partly explain the poor immune response against tumor Ags.     

Increased frequency of suppressive regulatory T cells and T cell-mediated antigen loss results in murine melanoma recurrence

Therapeutic treatment of large established tumors using immunotherapy has yielded few promising results. We investigated whether adoptive transfer of tumor-specific CD8(+) T cells, together with tumor-specific CD4(+) T cells, would mediate regression of large established B16BL6-D5 melanomas in lymphopenic Rag1(-/-) recipients devoid of regulatory T cells. The combined adoptive transfer of subtherapeutic doses of both TRP1-specific TCR transgenic Rag1(-/-) CD4(+) T cells and gp100-specific TCR transgenic Rag1(-/-) CD8(+) T cells into lymphopenic recipients, who received vaccination, led to regression of large (100-400 mm(2)) melanomas. The same treatment strategy was ineffective in lymphoreplete wild-type mice. Twenty-five percent of mice (15/59) had tumors recur (15-180 d postregression). Recurrent tumors were depigmented and had decreased expression of gp100, the epitope targeted by the CD8(+) T cells. Mice with recurrent melanoma had increased CD4(+)Foxp3(+) TRP1-specific T cells compared with mice that did not show evidence of disease. Importantly, splenocytes from mice with recurrent tumor were able to suppress the in vivo therapeutic efficacy of splenocytes from tumor-free mice. These data demonstrate that large established tumors can be treated by a combination of tumor-specific CD8(+) and CD4(+) T cells. Additionally, recurrent tumors exhibited decreased Ag expression, which was accompanied by conversion of the therapeutic tumor-specific CD4(+) T cell population to a Foxp3(+)CD4(+) regulatory T cell population.     

Cytotoxic T cell responses to Streptococcus are associated with improved prognosis of oral squamous cell carcinoma

Several species of Streptococcus, such as S. salivarius, S. mitis, and S. anginosus, are found to extensively colonize the oral cavity and the upper respiratory tract, and have been shown to increase in patients with oral squamous cell carcinoma (OSCC). Accumulating evidence have revealed that commensal bacteria are involved in antitumor immunity via T cell-mediated mechanisms, but the role of Streptococcus enrichment in OSCC is yet unclear. In this study, we stimulated peripheral blood mononuclear cells from non-cancer controls (NCs) and OSCC patients with S. salivarius, S. mitis, and S. anginosus. We observed that compared to NC subjects, OSCC patients at earlier stages had higher frequencies of granzyme B-expressing CD8 T cells for all Streptococcus species tested, while OSCC patients at more advanced stages had higher frequencies of granzyme B-expressing CD8 T cells for S. anginosus but not other Streptococcus species. In OSCC patients, the Streptococcus-reactive CD8 T cells presented significantly lower levels of PD-1 and TIM-3 expression than Streptococcus-nonreactive CD8 T cells. The clinical outcomes of OSCC patients in our cohort were tracked for 24 months after the resection of the primary tumor. In patients that did not present tumor recurrence, the frequencies of S. salivarius-reactive and S. mitis-reactive CD8 T cells were significantly higher than that in patients that developed recurrent tumor. Furthermore, in patients with tumor recurrence, the duration between primary tumor resection and tumor recurrence was positively associated with the frequencies of S. salivarius-reactive and S. anginosus-reactive CD8 T cells. Together, we demonstrated that Streptococcus-reactive CD8 T cell responses might contribute to antitumor immunity in OSCC patients.     

Epigenetic inactivation of SPINT2 is associated with tumor suppressive function in esophageal squamous cell carcinoma

Hepatocyte growth factor activator inhibitor type 2 (SPINT2), a Kunitz-type serine proteinase inhibitor, has been identified as a putative tumor suppressor gene silenced by promoter methylation. We aimed to investigate whether SPINT2 might act as an esophageal squamous cell carcinoma (ESCC) tumor suppressor gene. Four ESCC cell lines, Fifty-two ESCC tissues and twenty-nine neighboring non-cancerous tissues were included in this study. The expression of SPINT2 was monitored by real time PCR. Bisulfite genomic sequencing and methylation-specific PCR were used to analyze methylation status. The effect of SPINT2 on cell proliferation and apoptosis in EC109 and EC9706 cells was observed by CCK-8 assay and flow cytometric analysis. We found that silencing of SPINT2 was associated with promoter methylation in ESCC cell lines. The densely methylated SPINT2 promoter region was confirmed by bisulfite genomic sequencing. Ectopic expression of SPINT2 inhibited cell proliferation through inducing cell apoptosis in vitro. Furthermore, methylation-specific PCR analysis revealed that SPINT2 promoter methylation was prominent in carcinoma tissues (52.08%) compared with neighboring non-cancerous tissues (22.58%). Kaplan–Meier analysis showed that patients with SPINT2 hypermethylation had shorter survival time. The tumor suppressor gene of SPINT2 is commonly silenced by promoter hypermethylation in human ESCC and SPINT2 hypermethylation is correlated with poor overall survival, implicating SPINT2 is an underlying prognostic marker for human ESCC.     

Germinal center helper T cells are dual functional regulatory cells with suppressive activity to conventional CD4+ T cells

Germinal center (GC) reaction is a T cell-dependent process in which activated B cells mature to produce high-affinity Abs and differentiate into memory B cells. The GC microenvironment is almost exclusively reserved for the optimal Ag-specific B cell clonal expansion, selection, and maturation, but lack significant conventional CD4(+) T cell responses. The mechanisms that ensure such a focused B cell response in the GC are not known. In this study, we report that human CD4(+)CD57(+) T cells, which are the major helper T cells in GCs, actively suppress the activation of conventional CD4(+) T cells, particularly Th1 cells, via a direct contact-dependent mechanism and soluble mediators. Our findings demonstrate that GC T cells are unique regulatory cells that provide critical help signals for B cell response but suppress conventional effector T cells in the same local environment.     

Comparison of oral microbiota in tumor and non-tumor tissues of patients with oral squamous cell carcinoma

ABSTRACT: BACKGROUND: Bacterial infections have been linked to malignancies due to their ability to induce chronicinflammation. We investigated the association of oral bacteria in oral squamous cellcarcinoma (OSCC/tumor) tissues and compared with adjacent non-tumor mucosa sampled 5cm distant from the same patient (n = 10). By using culture-independent 16S rRNAapproaches, denaturing gradient gel electrophoresis (DGGE) and cloning and sequencing, weassessed the total bacterial diversity in these clinical samples. RESULTS: DGGE fingerprints showed variations in the band intensity profiles within non-tumor andtumor tissues of the same patient and among the two groups. The clonal analysis indicatedthat from a total of 1200 sequences characterized, 80 bacterial species/phylotypes weredetected representing six phyla, Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria,Actinobacteria and uncultivated TM7 in non-tumor and tumor libraries. In combined library,12 classes, 16 order, 26 families and 40 genera were observed. Bacterial species,Streptococcus sp. oral taxon 058, Peptostreptococcus stomatis, Streptococcus salivarius,Streptococcus gordonii, Gemella haemolysans, Gemella morbillorum, Johnsonella ignavaand Streptococcus parasanguinis I were highly associated with tumor site where asGranulicatella adiacens was prevalent at non-tumor site. Streptococcus intermedius waspresent in 70% of both non-tumor and tumor sites. CONCLUSIONS: The underlying changes in the bacterial diversity in the oral mucosal tissues from non-tumorand tumor sites of OSCC subjects indicated a shift in bacterial colonization. These mostprevalent or unique bacterial species/phylotypes present in tumor tissues may be associatedwith OSCC and needs to be further investigated with a larger sample size.     

Expansion and characteristics of human T regulatory type 1 cells in co-cultures simulating tumor microenvironment

Objective Chronic inflammation and cancer development are associated with dysregulated immune responses and the presence of regulatory T cells (T_reg). To study the role of T_reg in tumor cell escape from immune surveillance, an in vitro model simulating the tumor microenvironment and promoting the induction and expansion of IL-10⁺ T_reg type 1 (Tr1) was established. Methods An in vitro co-culture system (IVA) included an irradiated head and neck squamous cell carcinoma cell line, immature dendritic cells (iDC), CD4⁺CD25⁻ T cells and cytokines, IL-2 (10 IU/ml), IL-10 (20 IU/ml), IL-15 (20 IU/ml) ± 1 nM rapamycin. Autologous iDC and CD4⁺CD25⁻ T cells were obtained from the peripheral blood of 15 normal donors. Co-cultures were expanded for 10 days. Proliferating lymphocytes were phenotyped by multi-color flow cytometry. Their suppressor function was measured in CFSE inhibition assays ± neutralizing anti-IL-10 mAb and using transwell cultures. Culture supernatants were tested for IL-4, IL-10, TGF-β and IFN-γ in ELISA. Results In the IVA, low doses of IL-2, IL-10 and IL-15 promoted induction and expansion of CD3⁺CD4⁺CD25⁻IL2Rβ⁺IL2Rγ⁺FoxP3⁺CTLA-4⁺IL-10⁺ cells with suppressor activity (mean suppression ± SD = 58 ± 12%). These suppressor cells produced IL-10 (mean ± SD = 535 ± 12 pg/ml) and TGF-β (mean ± SD = 512 ± 38 pg/ml), but no IL-4 or IFN-γ. Suppressor function of co-cultures correlated with the percent of expanding IL-10⁺ Tr1 cells (r ² = 0.9; P < 0.001). The addition of rapamycin enriched Tr1 cells in all co-cultures. Neutralizing anti-IL-10 mAb abolished suppressive activity. Suppression was cell-contact independent. Conclusion The tumor microenvironment promotes generation of Tr1 cells which have the phenotype distinct from that of CD4⁺CD25^highFoxP3⁺ nTreg and mediate IL-10 dependent immune suppression in a cell-contact independent manner. Tr1 cells may play a critical role in cancer progression.     

Cutting edge: Th17 and regulatory T cell dynamics and the regulation by IL-2 in the tumor microenvironment

Th17 cells play an active role in inflammation and autoimmune diseases. However, the nature and regulation of Th17 in the context of tumor immunity remain unknown. In this study, we show that parallel to regulatory T (Treg) cells, IL-17(+) CD4(+) and CD8(+) T cells are kinetically induced in multiple tumor microenvironments in mice and humans. Treg cells play a crucial role in tumor immune pathogenesis and temper immune therapeutic efficacy. IL-2 is crucial for the production and function of Treg cells. We now show that IL-2 reduces IL-17(+) T cell differentiation in the tumor microenvironment accompanied with an enhanced Treg cell compartment in vitro and in vivo. Altogether, our work demonstrates a dynamic differentiation of IL-17(+) T cells in the tumor microenvironment, reveals a novel role for IL-2 in controlling the balance between IL-17(+) and Treg cells, and provides new insight of IL-17(+) T cells in tumor immune pathology and therapy.     

Increased salivary microvesicles are associated with the prognosis of patients with oral squamous cell carcinoma

Microvesicles (MVs), which are cell‐derived membrane vesicles present in body fluids, are closely associated with the development of malignant tumours. Saliva, one of the most versatile body fluids, is an important source of MVs. However, the association between salivary MVs (SMVs) and oral squamous cell carcinoma (OSCC), which is directly immersed in the salivary milieu, remains unclear. SMVs from 65 patients with OSCC, 21 patients with oral ulcer (OU), and 42 healthy donors were purified, quantified and analysed for their correlations with the clinicopathologic features and prognosis of OSCC patients. The results showed that the level of SMVs was significantly elevated in patients with OSCC compared to healthy donors and OU patients. Meanwhile, the level of SMVs showed close correlations with the lymph node status, and the clinical stage of OSCC patients. Additionally, the ratio of apoptotic to non‐apoptotic SMVs was significantly decreased in OSCC patients with higher pathological grade. Consistently, poorer overall survival was observed in patients with lower ratio of apoptotic to non‐apoptotic SMVs. In conclusion, the elevated level of SMVs is associated with clinicopathologic features and decreased survival in patients with OSCC, suggesting that SMVs are a potential biomarker and/or regulator of the malignant progression of OSCC.     

Peripheral blood CD4+CD25+CD127low regulatory T cells are significantly increased by tocilizumab treatment in patients with rheumatoid arthritis: increase in regulatory T cells correlates with clinical response

IntroductionTocilizumab (TCZ), an anti-interleukin-6 receptor antibody, is clinically effective against rheumatoid arthritis (RA), and several reports have indicated how TCZ influences a number of mechanisms underlying RA pathogenesis. However, it is still unclear whether TCZ affects inflammatory cells in peripheral blood and whether any such changes are associated with clinical response. We evaluated associations between proportions of subsets of peripheral immune cells and clinical response in patients with RA treated with TCZ.MethodsThirty-nine consecutive patients with RA who started to receive TCZ as their first biologic between March 2010 and April 2012 were enrolled. The proportions of several subsets of peripheral cells with their levels of expression of differentiation markers, activation markers and costimulatory molecules were measured sequentially from baseline to week 52 by flow cytometry analysis.ResultsClinical Disease Activity Index (CDAI) remission was achieved in 53.8% of patients at week 52 of TCZ therapy. The proportions of CD4⁺CD25⁺CD127^low regulatory T cells (T_reg) and HLA-DR⁺ activated T_reg cells significantly increased with TCZ therapy (P < 0.001 and P < 0.001, respectively), whereas proportions of CD3⁺CD4⁺CXCR3⁻CCR6⁺CD161⁺ T helper 17 cells did not change over the 52 weeks. The proportions of CD20⁺CD27⁺ memory B cells, HLA-DR⁺CD14⁺ and CD69⁺CD14⁺ activated monocytes, and CD16⁺CD14⁺ monocytes significantly decreased (P < 0.001, P < 0.001, P < 0.001 and P < 0.001, respectively). Among them, only the change in T_reg cells was inversely correlated with the change in CDAI score (ρ = −0.40, P = 0.011). The most dynamic increase in T_reg cells was observed in the CDAI remission group (P < 0.001).ConclusionThis study demonstrates that TCZ affected proportions of circulating immune cells in patients with RA. The proportion of T_reg cells among CD4⁺ cells correlated well with clinical response.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0526-4) contains supplementary material, which is available to authorized users.     

Differential control of T regulatory cell proliferation and suppressive activity by mature plasmacytoid versus conventional spleen dendritic cells

Anergy and suppression are cardinal features of CD4(+)CD25(+)Foxp3(+) T cells (T regulatory cells (Treg)) which have been shown to be tightly controlled by the maturation state of dendritic cells (DC). However, whether lymphoid organ DC subsets exhibit different capacities to control Treg is unclear. In this study, we have analyzed, in the rat, the role of splenic CD4(+) and CD4(-) conventional DC and plasmacytoid DC (pDC) in allogeneic Treg proliferation and suppression in vitro. As expected, in the absence of exogenous IL-2, Treg did not expand in response to immature DC. Upon TLR-induced maturation, all DC became potent stimulators of CD4(+)CD25(-) T cells, whereas only TLR7- or TLR9-matured pDC induced strong proliferation of CD4(+)CD25(+)Foxp3(+) T cells in the absence of exogenous IL-2. This capacity of pDC to reverse Treg anergy required cell contact and was partially CD86 dependent and IL-2 independent. In suppression assays, Treg strongly suppressed proliferation and IL-2 and IFN-gamma production by CD4(+)CD25(-) T cells induced by mature CD4(+) and CD4(-) DC. In contrast, upon stimulation by mature pDC, proliferating Treg suppressed IL-2 production by CD25(-) cells but not their proliferation or IFN-gamma production. Taken together, these results suggest that anergy and the suppressive function of Treg are differentially controlled by DC subsets.     

Kallikrein-related peptidase 4 contributes to the tumor metastasis of oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a disfiguring malignancy and significantly impacts the quality of patient’s life. Kallikrein-related peptidase 4 (KLK4), which is closely related to cancers, is highly expressed in OSCC. To explore the biological function of KLK4 in OSCC, a KLK4-specific shRNA was used to silence its endogenous expression, and then the migration and invasion of OSCC cells were explored. Results of our study showed that silencing KLK4 inhibited the migration and invasion of OSCC cells. The protein levels of epithelial mesenchymal transition-associated markers and proteases were also altered by KLK4 silencing. Further study showed that the phosphatidylinositol 3-kinase (PI3 K)/protein kinase B (AKT) signaling pathway was involved in the function of KLK4. Treatment with a PI3 K/AKT activator reversed the migration-inhibitory effect of KLK4 shRNA. Our study suggests that KLK4 may contribute to the metastasis of OSCC through the PI3 K/AKT signaling pathway.     

Salivary analytes in patients with oral squamous cell carcinoma

Literature data indicates that measurement of certain salivary constituents might serve as a useful diagnostic/prognostic tool in the patients with oral squamous cell carcinoma (OSCC). In 24 patients with OSCC (60 +/- 2.5 yrs) and in 24 controls (24 +/- 3.7 yrs) we have determined levels of salivary magnesium, calcium, copper, chloride, phosphate, potassium, sodium, total proteins and amylase. Sodium, potassium and chloride were determined by indirect potentiometry whereas copper, magnesium and phosphate were determined by atomic absorption spectrophotometry. Total proteins were determined by pyrogalol colorimetric method. Amylase levels were determined by continued colorimetric method. Statistical analysis was performed by use of chi2 test and Spearman's correlation test. The results of this study indicate that the concentrations of sodium and chloride were significantly elevated in patients with OSCC when compared to the controls. However, level of total protein was significantly decreased when compared to the healthy controls. Furthermore, there was a negative correlation between alcohol consumption and total protein concentration in patients with oral carcinoma. We might conclude that in patients with OSCC increased salivary sodium and chloride might reflect their overall dehydration status due to alcohol consumption rather than consequence of OSCC itself.     

Expression of Ubiquitin-specific protease 7 in oral squamous cell carcinoma promotes tumor cell proliferation and invasion

Oral Squamous Cell Carcinoma (OSCC) is the most common malignant cancer affecting oral cavity. Recent studies have demonstrated that Ubiquitin-specific protease 7 (USP7) was upregulated in several types of cancers. USP7 expression was associated with various proto-oncogenes and tumor suppressor genes. However, USP7 expression level and its functional role in OSCC is unclear. In the current study, we showed that USP7 expression in OSCC tissues was generally upregulated compared to normal adjacent tissues by using IHC. Furthermore, statistical analysis uncovered that USP7 expression was positively correlated with Ki-67, MMP2, VEGF in OSCC tissues. Importantly, high USP7 expression was significantly correlated with lymph node metastasis and histological differentiation in OSCC patients. So, our hypothesis is that USP7 plays a tumor-promoting role in OSCC. Knocking down of USP7 in tumor cells not only suppressed HSC3 cells proliferation, migration and invasion, but also promoted cell apoptosis. Moreover, USP7 siRNA blocked the activation of Akt/ERK signaling pathway. In conclusion, data presented here suggests that USP7 promotes the progression of OSCC. USP7 may be used as a new therapeutic target for OSCC diagnosis and treatment.Keywords: Oral Squamous Cell Carcinoma, USP7, siRNA, proliferation, invasion