Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer.

Background: Oral cavity cancer is still an important public health problem throughout the world. Oral squamous cell carcinomas (OSCCs) can be quite aggressive and metastatic, with a low survival rate and poor prognosis. However, this is usually related to the clinical stage and histological grade, and molecular prognostic markers for clinical practice are yet to be defined. Heparanase (HPSE1) is an endoglycosidase associated with extracellular matrix remodeling, and although involved in several malignancies, the clinical implications of HPSE1 expression in OSCCs are still unknown. Methods: We sought to investigate HPSE1 expression in a series of primary OSCCs and further explore whether its overexpression plays a relevant role in OSCC tumorigenesis. mRNA and protein expression analyses were performed in OSCC tissue samples and cell lines. A loss-of-function strategy using shRNA and a gain-of-function strategy using an ORF vector targeting HPSE1 were employed to investigate the endogenous modulation of HPSE1 and its effects on proliferation, apoptosis, adhesion, epithelial-mesenchymal transition (EMT), angiogenesis, migration, and invasion of oral cancer in vitro. Results: We demonstrated that HPSE1 is frequently upregulated in OSCC samples and cell lines and is an unfavorable prognostic indicator of disease-specific survival when combined with advanced pT stages. Moreover, abrogation of HPSE1 in OSCC cells significantly promoted apoptosis and inhibited proliferation, migration, invasion, and epithelial-mesenchymal transition by significantly decreasing the expression of N-cadherin and vimentin. Furthermore, a conditioned medium of HPSE1-downregulated cells resulted in reduced vascular endothelial growth. Conclusion: Our results confirm the overexpression of HPSE1 in OSCCs, suggest that HPSE1 expression correlates with disease progression as it is associated with several important biological processes for oral tumorigenesis, and can be managed as a prognostic marker for patients with OSCC.

Polypodium leucotomos targets multiple aspects of oral carcinogenesis and it is a potential antitumor phytotherapy against tongue cancer growth.

Introduction: Oral cancer refers to malignant tumors, of which 90% are squamous cell carcinomas (OSCCs). These malignancies exhibit rapid progression, poor prognosis, and often mutilating therapeutical approaches. The determination of a prophylactic and/or therapeutic antitumor role of the polyphenolic extract Polypodium leucotomos(PL) would be relevant in developing new tools for prevention and treatment. Methods: We aimed to determine the antitumor effect of PL by treating OSCC cell lines with PL metabolites and evaluating its action during OSCC progression in vivo. Results: PL treatment successfully impaired cell cycling and proliferation, migration, and invasion, enhanced apoptosis, and modulated macrophage polarization associated with the tumoral immune-inflammatory response of tongue cancer cell lines (TSCC). PL treatment significantly decreased the expression of MMP1 (p < 0.01) and MMP2 (p < 0.001), and increased the expression of TIMP1 (p < 0.001) and TIMP2 (p < 0.0001) in these cells. The mesenchymal-epithelial transition phenotype was promoted in cells treated with PL, through upregulation of E-CAD (p < 0.001) and reduction of N-CAD (p < 0.05). PL restrained OSCC progression in vivo by inhibiting tumor volume growth and decreasing the number of severe dysplasia lesions and squamous cell carcinomas. Ki-67 was significantly higher expressed in tongue tissues of animals not treated with PL(p < 0.05), and a notable reduction in Bcl2 (p < 0.05) and Pcna (p < 0.05) cell proliferation-associated genes was found in dysplastic lesions and TSCCs of PL-treated mice. Finally, N-cad(Cdh2), Vim, and Twist were significantly reduced in tongue tissues treated with PL. Conclusion: PL significantly decreased OSCC carcinogenic processes in vitro and inhibited tumor progression in vivo. PL also appears to contribute to the modulation of immune-inflammatory oral tumor-associated responses. Taken together, these results suggest that PL plays an important antitumor role in processes associated with oral carcinogenesis and may be a potential phytotherapeutic target for the prevention and/or adjuvant treatment of TSCCs.

Primordial odontogenic tumor: report of 2 new cases.

Primordial odontogenic tumor (POT) is a recently described benign odontogenic tumor, with only 16 cases reported in the literature. We present 2 new cases of POT affecting the mandible. Case 1 is that of 12-year-old girl with an asymptomatic, slow-growing mass, causing facial asymmetry. Radiography showed a well-defined unilocular radiolucency surrounding an impacted second premolar. Case 2 is that of a 13-year-old girl with a mass involving the crown of the unerupted third molar and showing similar radiographic features. Microscopically, both lesions were composed of variably cellular fibromyxoid tissue surrounded by thin ameloblastic epithelium, with stellate reticulum-like areas, but no mineralized tissue, yielding the diagnosis of POT. Immunohistochemical analysis showed diffuse expression of CK14 in epithelial cells, whereas CK19 was expressed mainly in the basal layer. Syndecan-1 (CD138) was expressed in the stellate-like regions and in the subepithelial zone. Both patients were treated surgically, with no signs of recurrence seen after 15 and 60 months, respectively.

Eukaryotic translation elongation factor 1δ, N-terminal propeptide of type I collagen and cancer-associated fibroblasts are prognostic markers of oral squamous cell carcinoma patients.

OBJECTIVE: Identifying markers that influence oral squamous cell carcinoma (OSCC) prognosis is a fundamental strategy to improve the overall survival of patients. Markers such as eukaryotic translation elongation factor 1δ (EEF1D), fascin, N-terminal propeptide of type I collagen (PINP), and cancer-associated fibroblasts (CAFs) have been noticed in OSCCs and their levels are closely related to the prognosis of tumors. Our aim was to confirm the role of those markers in OSCC prognosis. STUDY DESIGN: Immunohistochemistry was performed in 90 OSCC specimens. The associations between clinicopathologic features and expression of markers were assessed by χ2 test. Kaplan-Meier curves and univariate and multivariate Cox regression models were used for survival analysis. Markers were analyzed individually and in combination. RESULTS: High expression of EEF1D (P = .017) and PINP (P = .02) and abundant density of CAFs in tumor stroma (P = .005) predicted significantly poor survival in OSCC patients. Multivariate analysis revealed that all 3 parameters are individually independent prognostic factors of OSCC patients, and their combination improved the discrimination of patients at high risk for poor survival. CONCLUSIONS: Our results suggested that the expression of EEF1D and PINP and the density of CAFs might influence the survival of patients with OSCC.

Synthesis and evaluation of the antileishmanial activity of silver compounds containing imidazolidine-2-thione.

A new series of silver compounds could be of interest on designing new drugs for the treatment of leishmaniasis. The compounds [Ag(phen)(imzt)]NO3(1), [Ag(phen)(imzt)]CF3SO3(2), [Ag(phen)2](BF4)·H2O (3), [Ag2(imzt)6](NO3)2(4), and imzt have been synthesized and evaluated in vitro for antileishmanial activity against Leishmania. (L.) amazonensis (La) and L. (L.) chagasi (Lc), and two of them were selected for in vivo studies. In addition to investigating the action on Leishmania, their effects on the hydrogen peroxide production and cysteine protease inhibition have also been investigated. As for antileishmanial activity, compound (4) was the most potent against promastigote and amastigote forms of La (IC50 = 4.67 and 1.88 µM, respectively) and Lc (IC50 = 9.35 and 8.05 µM, respectively); and comparable to that of amphotericin B, reference drug. Beside showing excellent activity, it also showed a low toxicity. In the in vivo context, compound (4) reduced the number of amastigotes in the liver and spleen when compared to the untreated group. In evaluating the effect of the compounds on Leishmania, the level of hydrogen peroxide production was maintained between the lag and log phases; however, in the treatment with compound (4) it was possible to observe a reduction of 25.44 and 49.13%, respectively, in the hydrogen peroxide rates when compared to the lag and log phases. It was noticed that the presence of a nitrate ion and imzt in compound (4) was important for the modulation of the antileishmanial activity. Thus, this compound can represent a potentially new drug for the treatment of leishmaniasis.

Extracellular vesicles derived from cancer-associated fibroblasts induce the migration and invasion of oral squamous cell carcinoma.

As one of the most abundant constituents of the tumour microenvironment (TME), cancer-associated fibroblasts (CAF) display critical roles during tumour progression and metastasis. Multiple classes of molecules including growth factors, cytokines, proteases and extracellular matrix proteins, are produced by CAF to act as mediators of the stroma-tumour interactions. One of the main channels for this communication is associated with extracellular vesicles (EV), which are secreted particles loaded with protein and genetic information. In this study, we evaluated the effects of EV derived from CAF primary human cell lines (n = 5) on proliferation, survival, migration, and invasion of oral squamous cell carcinoma (OSCC) cells. As controls, EV from human primary-established normal oral fibroblasts (NOF, n = 5) were used. Our in vitro assays showed that CAF-EV significantly induces migration and invasion of OSCC cells and promote a disseminated pattern of HSC-3 cell invasion in the 3D organotypic assay. Furthermore, gene expression analysis of EV-treated cancer cells revealed changes in the pathways associated with tumour metabolism and up-regulation of tumour invasion genes. Our findings suggest a significant role of CAF-EV in promoting the migration and invasion of OSCC cells, which are related to the activation of cancer-related pathways.

ST2/IL-33 signaling promotes malignant development of experimental squamous cell carcinoma by decreasing NK cells cytotoxicity and modulating the intratumoral cell infiltrate.

Squamous cell carcinoma (SCC) is the second most common form of skin cancer and the mechanism(s) involved in the progression of this tumor are unknown. Increases in the expression of IL-33/ST2 axis components have been demonstrated to contribute to neoplastic transformation in several tumor models and interleukin-33 is correlated with poor prognosis of patients with squamous cell carcinoma of the tongue. Based on these observations, we sought to determine the role of the IL-33/ST2 pathway during the development of SCC. Our findings show that ST2-deficiency led to a marked decrease in the severity of skin lesions, suggesting that ST2 signaling contributed to tumor development. An analysis of tumor lesions in wild-type and ST2KO mice revealed that a lack of ST2 was associated with specific and significant reductions in the numbers of CD4+ T cells, CD8+ T cells, dendritic cells, and macrophages. In addition, NK cells that were isolated from ST2KO mice exhibited higher cytotoxic activity than cells isolated from wild-type mice. Notably, ST2 deficiency resulted in lower IFN-γ, TNF-α, IL-10, and IL-17 production in tumor samples. Our findings indicate that the IL-33/ST2 pathway contributes to the development of SCC by affecting leukocyte migration to tumor microenvironment and impairing NK cytotoxic activity.

Recognition of Candida albicans by gingival fibroblasts: The role of TLR2, TLR4/CD14, and MyD88.

Recent evidence indicates that nonprofessional immune cells such as epithelial cells, endothelial cells, and fibroblasts also contribute to innate immunity via secretion of cytokines. Fibroblasts are the principal type of cell found in the periodontal connective tissues and they are involved in the immune response during periodontal disease. The role of fibroblasts in the recognition of pathogens via Toll-like receptors (TLRs) has been established; however, few studies have been conducted concerning the involvement of innate immune receptors in the recognition of Candida albicans by gingival fibroblast. In the current study, we investigate the functional activity of TLR2, cluster of differentiation 14 (CD14), and myeloid differentiation primary response gene 88 (MyD88) molecules in the recognition of C. albicans by gingival fibroblast. First, we identified that gingival fibroblasts expressed TLR2, TLR3, and TLR4. Our results showed that TLR agonists had no effect on these receptors' expression by TLR2, MyD88, and CD14-deficient cells. Notably, C. albicans and a synthetic triacylated lipoprotein (Pam3CSK4) induced a remarkable increase of TLR3 expression on MyD88-deficient gingival fibroblasts. TLR4 expression levels were lower than TLR2 and TLR3 levels and remained unchanged after TLR agonist stimulation. Gingival fibroblasts presented morphological similarities; however, TLR2 deficiency on these cells leads to a lower proliferative response, whereas the deficiency on CD14 expression resulted in lower levels of type I collagen by these cells. In addition, the recognition of C. albicans by gingival fibroblasts had an effect on the secretion of cytokines and it was dependent on a specific recognition molecule. Specifically, tumor necrosis factor-α (TNF-α) production after the recognition of C. albicans was dependent on MyD88, CD14, and TLR2 molecules, whereas the production of interleukin-1ß (IL-1ß) and IL-13 was dependent on TLR2. These findings are the first to describe a role of gingival fibroblast in the recognition of C. albicans and the pathways involved in this process. An understanding of these pathways may lead to alternative treatments for patients with periodontal disease.

CCR5-Dependent Homing of T Regulatory Cells to the Tumor Microenvironment Contributes to Skin Squamous Cell Carcinoma Development.

Squamous cell carcinoma (SCC) is one of the most common human cancers worldwide. Recent studies show that regulatory T cells (Treg) have a critical role in the modulation of an antitumor immune response, and consequently the SCC development. Because the accumulation of Tregs at the tumor site is, in part, due to selective recruitment through CCR5- and CCR5-associated chemokines, we investigated the role of CCR5 in the SCC development. Our findings showed that CCR5-deficient mice (CCR5KO) were efficient in controlling papilloma's incidence when compared with wild-type mice. Analysis of tumor lesions in wild-type (WT) and CCR5KO mice revealed that lack of CCR5 lead to significant reduction in frequency of Tregs and increased of CD4 T cells into the tumors. Moreover, the adoptive transfer of naturally occurring Tregs CD4+CD25+CCR5+, CD4+CD25-CCR5+ or CD8+CCR5+ conventional T cells to CCR5KO mice resulted in an increased papilloma incidence. Interestingly, adoptive transfer of WT CD4+CD25+CCR5+ cells to CCR5KO mice induced more undifferentiated SCC lesions, characterized by higher infiltration of macrophages and dendritic cells. In this study, we also demonstrated that Treg migration to the tumor microenvironment is mediated by CCR5, and these cells are promoting tumor growth via inhibition of antitumor cells such as cytotoxic CD8+ T cells. Our findings reinforce the therapeutic potential of CCR5 inhibition for cancer treatment, and indicate an attractive approach for SCC treatment. Mol Cancer Ther; 16(12); 2871-80. ©2017 AACR.

Brazilian multicenter study of association between polymorphisms in CRISPLD2 and JARID2 and non-syndromic oral clefts.

BACKGROUND: Variants in the cysteine-rich secretory protein LCCL domain containing 2 gene (CRISPLD2) and in the jumonji, AT-rich interaction domain 2 gene (JARID2) were previously shown to influence non-syndromic oral cleft susceptibility. Herein, we performed a case-control study to examine the potential association of single-nucleotide polymorphisms (SNPs) in CRISPLD2 and JARID2 with non-syndromic cleft lip and/or palate (NSCL/P) in the Brazilian population. Given the ethnicity-dependent genetic predisposition to NSCL/P, we performed a structured analysis taking into account the genomic ancestry variation of each individual. METHODS: Four SNPs in CRISPLD2 (rs1546124, rs8061351, rs2326398, and rs4783099) and four in JARID2 (rs915344, rs2299043, rs2237138, and rs2076056), that were previously reported to be associated with NSCL/P, were genotyped in 785 Brazilian patients with NSCL/P (549 with cleft lip with or without cleft palate-NSCL ± P, and 236 with cleft palate only-NSCPO) and 693 unaffected Brazilian controls. Genomic ancestry was assessed with a set of 40 biallelic short insertion/deletion variants previously validated as ancestry informative markers of the Brazilian population. RESULTS: After adjustment of ancestry variations, allelic analysis revealed marginal associations between the CRISPLD2 rs4783099 T allele and increased risk for NSCPO (OR: 1.31, 95% CI: 1.05-1.62, P = 0.01) and between JARID2 rs2237138 and decreased NSCL ± P risk (OR: 0.80, 95% CI: 0.67-0.97, P = 0.02). Haplotype analysis indicated a lack of association between JARID2 haplotypes and non-syndromic oral cleft risk. CONCLUSIONS: Our results suggest that CRISPLD2 rs4783099 may represent a risk factor for NSCPO while JARID2 rs2237138 shows a protective effect against NSCL ± P in the Brazilian population.

CCR5-dependent homing of T Regulatory Cells to the tumor microenvironment contributes of skin squamous cell carcinoma develpment

Squamous cell carcinoma (SCC) is one of the most common human cancers worldwide. Recent studies show that regulatory T cells (Treg) have a critical role in the modulation of an antitumor immune response, and consequently the SCC development. Because the accumulation of Tregs at the tumor site is, in part, due to selective recruitment through CCR5- and CCR5-associated chemokines, we investigated the role of CCR5 in the SCC development. Our findings showed that CCR5-deficient mice (CCR5KO) were efficient in controlling papilloma's incidence when compared with wild-type mice. Analysis of tumor lesions in wild-type (WT) and CCR5KO mice revealed that lack of CCR5 lead to significant reduction in frequency of Tregs and increased of CD4 T cells into the tumors. Moreover, the adoptive transfer of naturally occurring Tregs CD4+CD25+CCR5+, CD4+CD25-CCR5+ or CD8+CCR5+ conventional T cells to CCR5KO mice resulted in an increased papilloma incidence. Interestingly, adoptive transfer of WT CD4+CD25+CCR5+ cells to CCR5KO mice induced more undifferentiated SCC lesions, characterized by higher infiltration of macrophages and dendritic cells. In this study, we also demonstrated that Treg migration to the tumor microenvironment is mediated by CCR5, and these cells are promoting tumor growth via inhibition of antitumor cells such as cytotoxic CD8+ T cells. Our findings reinforce the therapeutic potential of CCR5 inhibition for cancer treatment, and indicate an attractive approach for SCC treatment.

Effects of budlein A on human neutrophils and lymphocytes.

OBJECTIVE: In this study, we evaluated whether budlein A modulates the activation of innate and adaptive immune cells such as neutrophils and lymphocytes. MATERIAL AND METHODS: Our research group has investigated several plant species and several compounds have been isolated, identified, and their medical potential evaluated. Budlein A is a SL isolated from the species Aldama buddlejiformis and A. robusta (Asteraceae) and shows anti-inflammatory and anti-nociceptive activities. Advances in understanding how plant-derived substances modulate the activation of innate and adaptive immune cells have led to the development of new therapies for human diseases. RESULTS: Budlein A inhibited MPO activity, IL-6, CXCL8, IL-10, and IL-12 production and induces neutrophil apoptosis. In contrast, budlein A inhibited lymphocyte proliferation and IL-2, IL-10, TGF-ß, and IFN-γ production, but it did not lead to cell death. CONCLUSIONS: Collectively, our results indicate that budlein A shows distinct immunomodulatory effects on immune cells.

Effects of budlein A on human neutrophils and lymphocytes

ABSTRACT Sesquiterpene lactones (SLs) are the active constituents of a variety of medicinal plants used in traditional medicine for the treatment of inflammatory diseases and other ailments. Objective In this study, we evaluated whether budlein A modulates the activation of innate and adaptive immune cells such as neutrophils and lymphocytes. Material and Methods Our research group has investigated several plant species and several compounds have been isolated, identified, and their medical potential evaluated. Budlein A is a SL isolated from the species Aldama buddlejiformis and A. robusta (Asteraceae) and shows anti-inflammatory and anti-nociceptive activities. Advances in understanding how plant-derived substances modulate the activation of innate and adaptive immune cells have led to the development of new therapies for human diseases. Results Budlein A inhibited MPO activity, IL-6, CXCL8, IL-10, and IL-12 production and induces neutrophil apoptosis. In contrast, budlein A inhibited lymphocyte proliferation and IL-2, IL-10, TGF-β, and IFN-γ production, but it did not lead to cell death. Conclusions Collectively, our results indicate that budlein A shows distinct immunomodulatory effects on immune cells.

Secretome profiling of oral squamous cell carcinoma-associated fibroblasts reveals organization and disassembly of extracellular matrix and collagen metabolic process signatures.

An important role has been attributed to cancer-associated fibroblasts (CAFs) in the tumorigenesis of oral squamous cell carcinoma (OSCC), the most common tumor of the oral cavity. Previous studies demonstrated that CAF-secreted molecules promote the proliferation and invasion of OSCC cells, inducing a more aggressive phenotype. In this study, we searched for differences in the secretome of CAFs and normal oral fibroblasts (NOF) using mass spectrometry-based proteomics and biological network analysis. Comparison of the secretome profiles revealed that upregulated proteins involved mainly in extracellular matrix organization and disassembly and collagen metabolism. Among the upregulated proteins were fibronectin type III domain-containing 1 (FNDC1), serpin peptidase inhibitor type 1 (SERPINE1), and stanniocalcin 2 (STC2), the upregulation of which was validated by quantitative PCR and ELISA in an independent set of CAF cell lines. The transition of transforming growth factor beta 1 (TGF-ß1)-mediating NOFs into CAFs was accompanied by significant upregulation of FNDC1, SERPINE1, and STC2, confirming the participation of these proteins in the CAF-derived secretome. Type I collagen, the main constituent of the connective tissue, was also associated with several upregulated biological processes. The immunoexpression of type I collagen N-terminal propeptide (PINP) was significantly correlated in vivo with CAFs in the tumor front and was associated with significantly shortened survival of OSCC patients. Presence of CAFs in the tumor stroma was also an independent prognostic factor for OSCC disease-free survival. These results demonstrate the value of secretome profiling for evaluating the role of CAFs in the tumor microenvironment and identify potential novel therapeutic targets such as FNDC1, SERPINE1, and STC2. Furthermore, type I collagen expression by CAFs, represented by PINP levels, may be a prognostic marker of OSCC outcome.

A novel human leiomyoma tissue derived matrix for cell culture studies.

BACKGROUND: The composition of the matrix molecules is important in in vitro cell culture experiments of e.g. human cancer invasion and vessel formation. Currently, the mouse Engelbreth-Holm-Swarm (EHS) sarcoma-derived products, such as Matrigel®, are the most commonly used tumor microenvironment (TME) mimicking matrices for experimental studies. However, since Matrigel® is non-human in origin, its molecular composition does not accurately simulate human TME. We have previously described a solid 3D organotypic myoma disc invasion assay, which is derived from human uterus benign leiomyoma tumor. Here, we describe the preparation and analyses of a processed, gelatinous leiomyoma matrix, named Myogel. METHODS: A total protein extract, Myogel, was formulated from myoma. The protein contents of Myogel were characterized and its composition and properties compared with a commercial mouse Matrigel®. Myogel was tested and compared to Matrigel® in human cell adhesion, migration, invasion, colony formation, spheroid culture and vessel formation experiments, as well as in a 3D hanging drop video image analysis. RESULTS: We demonstrated that only 34% of Myogel's molecular content was similar to Matrigel®. All test results showed that Myogel was comparable with Matrigel®, and when mixed with low-melting agarose (Myogel-LMA) it was superior to Matrigel® in in vitro Transwell® invasion and capillary formation assays. CONCLUSIONS: In conclusion, we have developed a novel Myogel TME matrix, which is recommended for in vitro human cell culture experiments since it closely mimics the human tumor microenvironment of solid cancers.

Low miR-143/miR-145 Cluster Levels Induce Activin A Overexpression in Oral Squamous Cell Carcinomas, Which Contributes to Poor Prognosis.

Deregulated expression of activin A is reported in several tumors, but its biological functions in oral squamous cell carcinoma (OSCC) are unknown. Here, we investigate whether activin A can play a causal role in OSCCs. Activin A expression was assessed by qPCR and immunohistochemistry in OSCC tissues. Low activin A-expressing cells were treated with recombinant activin A and assessed for apoptosis, proliferation, adhesion, migration, invasion and epithelial-mesenchymal transition (EMT). Those phenotypes were also evaluated in high activin A-expressing cells treated with follistatin (an activin A antagonist) or stably expressing shRNA targeting activin A. Transfections of microRNA mimics were performed to determine whether the overexpression of activin A is regulated by miR-143/miR-145 cluster. Activin A was overexpressed in OSCCs in comparison with normal oral mucosa, and high activin A levels were significantly associated with lymph node metastasis, tumor differentiation and poor survival. High activin A levels promoted multiple properties associated with malignant transformation, including decreased apoptosis and increased proliferation, migration, invasion and EMT. Both miR-143 and miR-145 were markedly downregulated in OSCC cell lines and in clinical specimens, and inversely correlated to activin A levels. Forced expression of miR-143 and miR-145 in OSCC cells significantly decreased the expression of activin A. Overexpression of activin A in OSCCs, which is controlled by downregulation of miR-143/miR-145 cluster, regulates apoptosis, proliferation and invasiveness, and it is clinically correlated with lymph node metastasis and poor survival.

HOXA10 controls proliferation, migration and invasion in oral squamous cell carcinoma.

Although HOX genes are best known for acting in the regulation of important events during embryogenesis, including proliferation, differentiation and migration, alterations in their expression patterns have been frequently described in cancers. In previous studies we analyzed the expression profile of the members of the HOX family of homeobox genes in oral samples of normal mucosa and squamous cell carcinoma (OSCC) and identified differently expressed genes such as HOXA10. The present study aimed to validate the increased expression of HOXA10 in OSCCs, and to investigate the effects arising from its knockdown in OSCC cells. The levels of HOXA10 mRNA were determined in human OSCC samples and cell lines by quantitative PCR, and HOXA10-mediated effects on proliferation, apoptosis, adhesion, epithelial-mesenchymal transition (EMT), migration and invasion were studied in HSC-3 tongue carcinoma cells by using retrovirus-mediated RNA interference. Higher expression of HOXA10 mRNA was observed in OSCC cell lines and in tumor tissues compared to normal controls. HOXA10 knockdown significantly reduced the proliferation of the tumor cells which was accompanied by increased levels of p21. HOXA10 silencing also significantly induced the expression of EMT markers and enhanced the adhesion, migration and invasion of HSC-3 cells. No effects on cell death were observed after HOXA10 knockdown. The results of the current study confirm the overexpression of HOXA10 in OSCCs, and further demonstrate that its expression is functionally associated with several important biological processes related to oral tumorigenesis, such as proliferation, migration and invasion.

Inflammasome activation is critical to the protective immune response during chemically induced squamous cell carcinoma.

Chronic inflammation affects most stages of tumorigenesis, including initiation, promotion, malignant differentiation, invasion and metastasis. Inflammasomes have been described as involved with persistent inflammation and are known to exert both pro and antitumour effects. We evaluated the influence of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase (CASP)-1 in the antitumor immune response using a multistage model of squamous cell carcinoma (SCC) development. Absence of ASC and CASP-1 resulted in an earlier incidence and increased number of papilloma. Loss of inflammassome function in mice resulted in decreased presence of natural killer (NK), dendritic (DC), CD4(+), CD8(+) and CD45RB(+) T cells in the tumor lesions as well as in lymph nodes (LN) compared with WT mice. Increased percentage of CD4(+)CD25(+)Foxp3(+) T cells was associated with association with inflammasome loss of function. Moreover, significant differences were also found with neutrophils and macrophage infiltrating the lesions. Myeloperoxidase (MPO), but not elastase (ELA), activity oscillated among the groups during the SCC development. Levels of proinflammatory cytokines IL-1ß, IL-18, Tumor Necrosis Factor (TNF)-α and Interferon (IFN)-γ were decreased in the tumor microenvironment in the absence of inflammasome proteins. These observations suggest a link between inflammasome function and SCC tumorigenesis, indicating an important role for inflammasome activation in the control of SCC development.

PD-1 blockage delays murine squamous cell carcinoma development.

Engagement of programmed death-1 (PD-1) with its two ligands [programmed death ligand-1 (PD-L1) and PD-L2] has been associated with the suppression of tumor-reactive T cells; however, the underlying mechanism for this T-cell dysfunction is not clear. We hypothesized that PD-1 and PD-L1 signals are, in part, responsible for squamous cell carcinoma (SCC) escape from immune antitumor regulation by modulation of the tumor environment. In the present study, we used a multistage model of SCC to examine the role of PD-1/PD-L1 activation during tumor development. Tumor sites presented an increased percentage of CD4(+) and CD8(+) T cells expressing PD-1 when compared with non-tumorigenic control mice, whereas the expression of PD-L1 was particularly increased in F4/80(+) macrophages in tumor sites. Further, the systemic immune neutralization of PD-1 resulted in a decreased number and delayed incidence rate of papillomas followed by a differential expression of cytokeratins, suggesting that the PD-1-PD-L1 interaction contributes to the progression of SCC by downregulation of antitumor responses. In fact, blocking PD-1 increased the percentage of CD8(+) and CD4(+) T cells, and the levels of interferon-γ in the tumor sites. Our results indicated involvement of PD-1(+) T cells in SCC development and in the modulation of the inflammatory immune response.

Activation profile of CXCL8-stimulated neutrophils and aging.

Neutrophils are pivotal effector cells of innate immunity representing the first line of defense against aggression. They are the first cells to arrive at the site of the aggression, where they can directly eliminate the invading microorganisms. Their activation and recruitment into peripheral tissues is indispensable for host defense. With aging, there are alterations of the receptor by driven functions of human neutrophils as a decrease in the functional changes in signaling elicited by specific receptors, as CXCR1. We investigated the activation of neutrophils from elderly after the cells were cultivated with CXCL8. Although, CXCL8 induced elastase (ELA) secretion, data showed neither myeloperoxidase (MPO) activity nor production of IL-6, IL-10, GM-CSF by neutrophils from elderly compared with young individuals. On the other hand, in the presence of only LPS or LPS associated with CXCL8 neutrophils from elderly individuals, there were significant levels of IL-6, IL-10, GM-CSF but not MPO. These results indicate that neutrophils from elderly do not respond to CXCL8 stimulus, but they are activated by LPS to produce cytokines. However, MPO activity from elderly individuals was not different in the presence or absence of LPS and CXCL8.