Generation of Amelx-iCre Mice Supports Ameloblast-Specific Role for Stim1.

The identification and targeting of the molecular pathways regulating amelogenesis is an ongoing challenge in dental research, and progress has been restricted by the limited number of genetic tools available to study gene function in ameloblasts. Here, we generated 4 transgenic Cre-driver mouse lines that express improved Cre (iCre)-recombinase from the locus of the mouse ameloblast-specific gene amelogenin X (Amelx-iCre) with a large (250-kb) bacterial artificial chromosome DNA vector. All 4 Amelx-iCre transgenic lines were bred with ROSA26 reporter mice to characterize the iCre developmental pattern with the LacZ gene encoding ß-galactosidase enzyme activity assay and Cre protein immunohistochemistry. From the 4 generated transgenic lines, 2 were selected for further analysis because they expressed a high amount of Cre recombinase exclusively in ameloblasts and showed developmental stage- and cell-specific ß-galactosidase activity mimicking the endogenous amelogenin expression. To test the functionality of the selected transgenic models, we bred the 2 Amelx-iCre mice lines with stromal interaction molecule 1 (Stim1) floxed mice to generate ameloblast-specific Stim1 conditional knockout mice (Stim1 cKO). STIM1 protein serves as one of the main calcium sensors in ameloblasts and plays a major role in enamel mineralization and ameloblast differentiation. Amelx-iCre mice displayed exclusive CRE-mediated recombination in incisor and molar ameloblasts. Stim1 cKO mice showed a severely defected enamel phenotype, including reduced structural integrity concomitant with increased attrition and smaller teeth. The phenotype and genotype of the Amelx-iCre/Stim1 cKO showed significant differences with the previously reported Ker14-Cre/Stim1 cKO, highlighting the need for cell- and stage-specific Cre lines for an accurate phenotype-genotype comparison. Furthermore, our model has the advantage of carrying the entire Amelx gene locus rather than being limited to an Amelx partial promoter construct, which greatly enhances the stability and the specificity of our Cre expression. As such, the Amelx-iCre transgenic lines that we developed may serve as a powerful tool for targeting ameloblast-specific gene expression in future investigations.

Role of Runx2 phosphorylation in prostate cancer and association with metastatic disease.

The osteogenic transcription factor, Runx2, is abnormally expressed in prostate cancer (PCa) and associated with metastatic disease. During bone development, Runx2 is activated by signals known to be hyperactive in PCa including the RAS/MAP kinase pathway, which phosphorylates Runx2 on multiple serine residues including S301 and S319 (equivalent to S294 and S312 in human Runx2). This study examines the role of these phosphorylation sites in PCa. Runx2 was preferentially expressed in more invasive PCa cell lines (PC3>C4-2B>LNCaP). Furthermore, analysis using a P-S319-Runx2-specific antibody revealed that the ratio of P-S319-Runx2/total Runx2 as well as P-ERK/total ERK was highest in PC3 followed by C4-2B and LNCaP cells. These results were confirmed by immunofluorescence confocal microscopy, which showed a higher percentage of PC3 cells staining positive for P-S319-Runx2 relative to C4-2B and LNCaP cells. Phosphorylated Runx2 had an exclusively nuclear localization. When expressed in prostate cell lines, wild-type Runx2 increased metastasis-associated gene expression, in vitro migratory and invasive activity as well as in vivo growth of tumor cell xenografts. In contrast, S301A/S319A phosphorylation site mutations greatly attenuated these Runx2 responses. Analysis of tissue microarrays from 129 patients revealed strong nuclear staining with the P-S319-Runx2 antibody in primary PCas and metastases. P-S319-Runx2 staining was positively correlated with Gleason score and occurrence of lymph node metastases while little or no Runx2 phosphorylation was seen in normal prostate, benign prostate hyperplasia or prostatitis indicating that Runx2 S319 phosphorylation is closely associated with PCa induction and progression towards an aggressive phenotype. These studies establish the importance of Runx2 phosphorylation in prostate tumor growth and highlight its value as a potential diagnostic marker and therapeutic target.

Isolation and Characterization of Human Adult Epithelial Stem Cells from the Periodontal Ligament.

We report a novel method for the isolation of adult human epithelial stem cells (hEpiSCs) from the epithelial component of the periodontal ligament-the human epithelial cell rests of Malassez (hERM). hEpiSC-rich integrin-α6(+ve) hERM cells derived by fluorometry can be clonally expanded, can grow organoids, and express the markers of pluripotency (OCT4, NANOG, SOX2), polycomb protein RING1B, and the hEpiSC supermarker LGR5. They maintain the growth profile of their originating hERM in vitro. Subcutaneous cotransplantation with mesenchymal stem cells from the dental pulp on poly-l-lactic acid scaffolds in nude mice gave rise to perfect heterotopic ossicles in vivo with ultrastructure of dentin, enamel, cementum, and bone. These remarkable fully mineralized ossicles underscore the importance of epithelial-mesenchymal crosstalk in tissue regeneration using human progenitor stem cells, which may have already committed to lineage despite maintaining hallmarks of pluripotency. In addition, we report the clonal expansion and isolation of human LGR5(+ve) cells from the hERM in xeno-free culture conditions. The genetic profile of LGR5(+ve) cells includes both markers of pluripotency and genes important for secretory epithelial and dental epithelial cell differentiation, giving us a first insight into periodontal ligament-derived hEpiSCs.

Biological and bactericidal properties of Ag-doped bioactive glass in a natural extracellular matrix hydrogel with potential application in dentistry.

The aim of this study was the fabrication and evaluation of a novel bioactive and bactericidal material, which could have applications in dentistry by supporting tissue regeneration and killing oral bacteria. Our hypothesis was that a new scaffold for pulp-dentin tissue engineering with enhanced antibacterial activity could be obtained by associating extracellular matrix derived from porcine bladder with an antibacterial bioactive glass. Our study combines in vitro approaches and ectopic implantation in scid mice. The novel material was fabricated by incorporating a sol-gel derived silver (Ag)-doped bioactive glass (BG) in a natural extracellular matrix (ECM) hydrogel in ratio 1:1 in weight % (Ag-BG/ECM). The biological properties of the Ag-BG/ECM were evaluated in culture with dental pulp stem cells (DPSCs). In particular, cell proliferation, cell apoptosis, stem cells markers profile, and cell differentiation potential were studied. Furthermore, the antibacterial activity against Streptococcus mutans and Lactobacillus casei was measured. Moreover, the capability of the material to enhance pulp/dentin regeneration in vivo was also evaluated. Our data show that Ag-BG/ECM significantly enhances DPSCs' proliferation, it does not affect cell morphology and stem cells markers profile, protects cells from apoptosis, and enhances in vitro cell differentiation and mineralisation potential as well as in vivo dentin formation. Furthermore, Ag-BG/ECM strongly inhibits S. mutans and L. casei growth suggesting that the new material has also anti-bacterial properties. This study provides foundation for future clinical applications in dentistry. It could potentially advance the currently available options of dental regenerative materials.

Evaluation of CD44 variant expression in oral, head and neck squamous cell carcinomas using a triple approach and its clinical significance.

Cancer stem cells possess the qualities of self-renewal, tumorigenesis and the ability to recapitulate a heterogeneous tumor. Our group was the first to isolate head and neck squamous cell carcinoma (HNSCC) stem cells using the cell surface marker CD44. CD44 is a trans-membrane glycoprotein with a multitude of key-functions that regulate cancer cell proliferation and metastasis. The variety of CD44 functions is due to tissue-specific patterns of glycosylation of the extracellular portion, and to the multiple protein isoforms (CD44 variants, CD44v) generated by alternative splicing. This study investigates the expression pattern of CD44 variants in HNSCC. Ten cell lines from the most common HNSCC locations and representative of various clinical outcomes were assayed by quantitative realtime PCR, flow cytometry and immunofluorescence comparatively with normal oral keratinocytes. The CD44 v4 and v6 were exclusively abundant in HNSCC while the isoform v1,2 was expressed in normal oral keratinocytes. Of interest, the highest level of CD44v6 expression was detected in advanced metastatic HNSCC, suggesting a link between CD44v6 expression and HNSCC metastasis, while the highest CD44v4 was detected in a stage IV HNSCC refractory to chemotherapy which developed recurrence. Oral-derived HNSCC expressed the highest CD44v4 and v6, and levels corresponded with staging, showing also an increasing tendency with recurrence and metastasis. CD44v were detected predominantly in smaller cells (a characteristic that has been associated with stem cell properties) or cells with mesenchymal morphology (a characteristic that has been associated with the migratory and invasive potential of epithelial tumor cells), suggesting that CD44v differential expression in HNSCC may be representative of the morphological changes inherent during tumor progression towards a more aggressive potential, and thus contributing to the individual tumor biology. The mechanism of CD44 variant involvement in HNSCC progression and metastasis is under investigation.

The role of E-cadherin down-regulation in oral cancer: CDH1 gene expression and epigenetic blockage.

BACKGROUND: The prognosis of the oral squamous cell carcinoma (OSCC) patients remains very poor, mainly due to their high propensity to invade and metastasize. E-cadherin reduced expression occurs in the primary step of oral tumour progression and gene methylation is a mode by which the expression of this protein is regulated in cancers. In this perspective, we investigated E-cadherin gene (CDH1) promoter methylation status in OSCC and its correlation with Ecadherin protein expression, clinicopathological characteristics and patient outcome. METHODS: Histologically proven OSCC and paired normal mucosa were analyzed for CDH1 promoter methylation status and E-cadherin protein expression by methylation-specific polymerase chain reaction and immunohistochemistry. Colocalization of E-cadherin with epidermal growth factor (EGF) receptor (EGFR) was evidenced by confocal microscopy and by immunoprecipitation analyses. RESULTS: This study indicated E-cadherin protein down-regulation in OSCC associated with protein delocalization from membrane to cytoplasm. Low E-cadherin expression correlated to aggressive, poorly differentiated, high grade carcinomas and low patient survival. Moreover, protein down-regulation appeared to be due to E-cadherin mRNA downregulation and CDH1 promoter hypermethylation. In an in vitro model of OSCC the treatment with EGF caused internalization and co-localization of E-cadherin with EGFR and the addition of demethylating agents increased E-cadherin expression. CONCLUSION: Low E-Cadherin expression is a negative prognostic factor of OSCC and is likely due to the hypermethylation of CDH1 promoter. The delocalization of E-cadherin from membrane to cytoplasm could be also due to the increased expression of EGFR in OSCC and the consequent increase of E-cadherin co-internalization with EGFR.

The circadian clock in oral health and diseases.

Most physiological processes in mammals display circadian rhythms that are driven by the endogenous circadian clock. This clock is comprised of a central component located in the hypothalamic suprachiasmatic nucleus and subordinate clocks in peripheral tissues. Circadian rhythms sustain 24-hour oscillations of a large number of master genes controlling the correct timing and synchronization of diverse physiological and metabolic processes within our bodies. This complex regulatory network provides an important communication link between our brain and several peripheral organs and tissues. At the molecular level, circadian oscillations of gene expression are regulated by a family of transcription factors called "clock genes". Dysregulation of clock gene expression results in diverse human pathological conditions, including autoimmune diseases and cancer. There is increasing evidence that the circadian clock affects tooth development, salivary gland and oral epithelium homeostasis, and saliva production. This review summarizes current knowledge of the roles of clock genes in the formation and maintenance of oral tissues, and discusses potential links between "oral clocks" and diseases such as head and neck cancer and Sjögren's syndrome.

In silico modeling of the molecular interactions of antacid medication with the endothelium: novel therapeutic implications in head and neck carcinomas.

Pathological acid reflux is a common event in patients afflicted with head and neck squamous cell carcinomas (HNSCCs), known to play a role in HNSCC etiology and contribute to complications after surgery or during radiation and chemotherapy. Antacid medications are commonly prescribed in HNSCC patients as part of their cancer treatment, and consist of two classes: histamine 2 receptor antagonist class (H2RA, with cimetidine as its prototypical drug) and proton pump inhibitors class (PPI, with omeprazole as its prototypical drug). Clinical evidence revealed a significant survival benefit of antacid usage in a large cohort of HNSCC patients treated in our Otolaryngology Department, with a median follow-up of over 5 years. Therefore, we postulate that one mechanism by which antacid intake enhances patient survival could involve modulation of tumor cell adhesion to endothelium, critical in the initiation of the metastatic dissemination. This study investigates the potential physical interactions between cimetidine and omeprazole with the endothelial E-selection (E-sel) and its ligand sialyl Lewis X (sLe(x)) using a molecular visualization energy-based program (AutoDock). Docking results were further analyzed with the PyMOL program, which allowed for measurements of the distances between the drugs and the closest interacting atoms or residues on E-sel and sLe(x) molecules. Our model predicts that omeprazole displays a stronger interaction with E-sel than cimetidine, as extrapolated from the calculated overall binding energies. However, the shorter distances existing between interacting atoms in the proposed E-sel/cimetidine complex are suggestive of more stable interactions. Neither antacid/E-sel complex overcame the stronger Autodock-calculated sLe(x)/E-sel interaction, suggesting competitive inhibition was not involved. This study provides the first in silico evidence of omeprazole and cimetidine ability to bind to adhesion molecules involved in tumor dissemination, underlining their therapeutic potential in the HNSCC clinical management.

Clock genes show circadian rhythms in salivary glands.

Circadian rhythms are endogenous self-sustained oscillations with 24-hour periods that regulate diverse physiological and metabolic processes through complex gene regulation by "clock" transcription factors. The oral cavity is bathed by saliva, and its amount and content are modified within regular daily intervals. The clock mechanisms that control salivary production remain unclear. Our objective was to evaluate the expression and periodicity of clock genes in salivary glands. Real-time quantitative RT-PCR, in situ hybridization, and immunohistochemistry were performed to show circadian mRNA and protein expression and localization of key clock genes (Bmal1, Clock, Per1, and Per2), ion and aqua channel genes (Ae2a, Car2, and Aqp5), and salivary gland markers. Clock gene mRNAs and clock proteins were found differentially expressed in the serous acini and duct cells of all major salivary glands. The expression levels of clock genes and Aqp5 showed regular oscillatory patterns under both light/dark and complete-dark conditions. Bmla1 overexpression resulted in increased Aqp5 expression levels. Analysis of our data suggests that salivary glands have a peripheral clock mechanism that functions both in normal light/dark conditions and in the absence of light. This finding may increase our understanding of the control mechanisms of salivary content and flow.

Aberrant beta-catenin and LEF1 expression may predict the clinical outcome for patients with oropharyngeal cancer.

Beta-catenin, normally expressed on the epithelial cell surface, plays a crucial role in cadherin-mediated cell adhesion. Recent evidence suggests that beta-catenin is also involved in other functions such as intracellular signaling via the Wnt pathway by creating a nuclear complex with members of the Lymphoid-Enhancer-Factor/T-Cell-Factor (LEF/TCF) family of transcription factors, and gene regulation that it is implicated in the development of several tumors. Little information is available on beta-catenin expression and its main partner in the Wnt signaling pathway, LEF1, in oropharyngeal squamous cell carcinomas (OP-SCCs). The aim of this study is to investigate the expression of beta-catenin and LEF1 expression in human primary OP-SCCs and to evaluate their clinical and prognostic significance. OP-SCCs and normal peritumoral areas were analyzed by immunohistochemistry, Western-blot and RT-PCR. Beta-catenin was overexpressed in tumors in comparison to normal peritumoral areas and displayed predominantly intracellular (cytosolic/nuclear) localization in 62% of the tumors. Immunoreactivity was correlated with clinicopathological parameters and long-term follow-up, and a significant association was found between protein expression and development of local recurrences (P =0.03). The OP-SCCs with poor clinical outcome, which displayed intracellular beta-catenin expression, were also strongly positive for LEF1, with their co-expression statistically significant (P = 0.040). All (100%) advanced (stages 3+4) SCCs, 66.7% of the SCCs with positive lymph nodes and 80% of the SSCs that developed local recurrences were LEF1 positive. Cox regression analysis confirmed a poorer overall survival in cases with high expression of beta-catenin and LEF1. Our results suggest that assessing intracellular beta-catenin and LEF1 expression might help in patient risk stratification and outcome prediction, and serve as novel therapeutic targets in advanced OP-SCC.

Prognostic significance of N-Cadherin expression in oral squamous cell carcinoma.

BACKGROUND: N-Cadherin (CDH2) is a calcium-dependent adhesion protein, whose de novo expression, re-expression, up-regulation and down-regulation in human tumors has been demonstrated. The aim of the present work was to define the prognostic role of N-Cadherin in a large series of oral squamous cell carcinomas (OSCCs). MATERIALS AND METHODS: A total of 94 selected OSCCs were quantitatively and qualitatively analyzed by immunohistochemistry for N-Cadherin. The association between protein expression and clinico-pathological parameters was assessed by statistical analysis. RESULTS: In neoplastic tissue, N-Cadherin levels were more evident than in normal peritumoral epithelium (p<0.05). Protein staining was mainly detected in the neoplastic cells, and only focal nuclear positivity was observed. Expression of cytoplasmic N-Cadherin correlated significantly with poor histological differentiation (p<0.05). Furthermore, we have observed significant a statistical trend for stage and a correlation with worst patient outcome, also confirmed by Kaplan-Meier estimates. CONCLUSION: Our work has underlined the key-role of N-Cadherin in oral carcinogenesis and in the prognostic stratification of patients.

Double demonstration of oncogenic high risk human papilloma virus DNA and HPV-E7 protein in oral cancers.

Oncogenic HPVs are necessarily involved in cervical cancer but their role in oral carcinogenesis is debated. To detect HPV in oral cancer, 38 cases of formalin fixed-paraffin embedded OSCC were studied by both DNA genotyping (MY09/11 L1 consensus primers in combination with GP5-GP6 primer pair followed by sequencing) and immunohistochemistry (monoclonal Abs against capsid protein and HPV-E7 protein, K1H8 DAKO and clone 8C9 INVITROGEN, respectively). HPV-16 tonsil cancer was used as positive control. The overall prevalence of HPV infection in OSCCs was 10.5%. Amplification of DNA samples showed single HPV DNA infection in 3 cases (HPV16; HPV53; HPV70) and double infection in one case of cheek cancer (HPV31/HPV44). The overall HR-HPV prevalence was 7.5%. E-7 antigen was immunohistochemically detected in all HPV-positive cases. HPV+ OSCC cases showed an overall better outcome than HPV negative oral cancers, as evaluated by Kaplan-Meier curves. HPVs exert their oncogenic role after DNA integration, gene expression of E5, E6 and E7 loci and p53/pRb host proteins suppression. This study showed that HPV-E7 protein inactivating pRb is expressed in oral cancer cells infected by oncogenic HPV other than classical HR-HPV-16/18. Interestingly HPV-70, considered a low risk virus with no definite collocation in oncogenic type category, gives rise to the expression of HPV-E7 protein and inactivate pRb in oral cancer. HPV-70, as proved in current literature, is able to inactivates also p53 protein, promoting cell immortalization. HPV-53, classified as a possible high risk virus, expresses E7 protein in OSCC, contributing to oral carcinogenesis. We have identified among OSCCs, a subgroup characterized by HPV infection (10.5%). Finally, we have proved the oncogenic potential of some HPV virus types, not well known in literature.

A Troubling Diagnosis of Verrucous Squamous Cell Carcinoma ("the Bad Kind" of Keratosis) and the Need of Clinical and Pathological Correlations: A Review of the Literature with a Case Report.

Verrucous carcinoma (also known as Ackerman tumor) is an uncommon exophytic low-grade well-differentiated variant of squamous cell carcinoma. This neoplasm typically involves the oral cavity, larynx, genitalia, skin, and esophagus. It is well known for its locally aggressiveness and for its clinically slow-growing behaviour with minimal metastatic potential. Verrucous carcinoma of oral cavity is so closely aligned with the use of snuff and chewing tobacco that it has been called the "snuff dipper's cancer". Recent studies have proved the role of HPV. The typical clinical presentation of oral verrucous carcinoma has long been known, as its remarkably innocuous appearance and biological behaviour. In this work, we report a review of the scientific literature and describe a troublesome case of oral verrucous cancer.

WNT pathway in oral cancer: epigenetic inactivation of WNT-inhibitors.

Epigenetic DNA methylations plays an important role in oral carcinogenesis. The soluble frizzled receptor protein (SFRP) family together with WIF-1 and DKK-3 encodes antagonists of the WNT pathway. Silencing of these genes leads to constitutive WNT signalling. Because aberrant expression of beta-catenin might be associated with the epigenetic inactivation of WNT inhibitors, we analyzed, in a collection of primary OSCC with matched normal oral mucosa, the methylation status of a complete panel of genes, SFRP-1, SFRP-2, SFRP-4, SFRP-5, WIF-1, DKK-3, that are involved directly and indirectly in WNT pathway, in order to demonstrate WNT-pathway activation in the absence of beta-catenin and/or APC/Axin mutations during oral carcinogenesis. Methylation-specific PCR (MSP) was performed to study inactivation of SFRP-1, SFRP-2, SFRP-4, SFRP-5, WIF-1, DKK-3 genes in 37 cases of paraffin embedded oral cancer. This study showed that the methylation is an important epigenetic alteration in oral cancer. In particular, SFRP-2, SFRP-4, SFRP-5, WIF-1, DKK-3 revealed methylation status of their promoter in OSCC, whereas SFRP-1 showed demethylation in cancer. Fisher's exact test revealed statistically significant results (p<0.05) for all genes. The Wald test confirmed the statistically significant association between SFRP2-4-5 gene methylation and OSCC (p<0.05). SFRP-1 was also characterized by a different statistically significant epigenetic behaviour, because of it was demethylated in cancer (p<0.05). Statistical regression test showed high levels of sensitivity, specificity and accuracy for SFRP genes, while WIF-1 and DKK-3 have reportedly high specificity, moderate accuracy but low sensitivity. This study suggests that a cause of catenin delocalization in oral cancer could be due to WNT pathway activation, by epigenetic alterations of SFRP, WIF-1 and DKK-3 genes.