EN1 promotes lung metastasis of salivary adenoid cystic carcinoma by regulating the PI3K-AKT pathway and epithelial-mesenchymal transition.

BACKGROUND: Engrailed homeobox 1 (EN1) is a candidate oncogene that is epigenetically modified in salivary adenoid cystic carcinoma (SACC). We investigated the expression of EN1 in SACC tissues and cells, EN1 promoter methylation, and the role of EN1 in tumour progression in SACC. METHODS: Thirty-five SACC samples were screened for key transcription factors that affect tumour progression. In vitro and in vivo assays were performed to determine the viability, tumorigenicity, and metastatic ability of SACC cells with modulated EN1 expression. Quantitative methylation-specific polymerase chain reaction analysis was performed on SACC samples. RESULTS: EN1 was identified as a transcription factor that was highly overexpressed in SACC tissues, regardless of clinical stage and histology subtype, and its level of expression correlated with distant metastasis. EN1 promoted cell invasion and migration through epithelial-mesenchymal transition in vitro and enhanced SACC metastasis to the lung in vivo. RNA-seq combined with in vitro assays indicated that EN1 might play an oncogenic role in SACC through the PI3K-AKT pathway. EN1 mRNA levels were negatively correlated with promoter hypermethylation, and inhibition of DNA methylation by 5-aza-dC increased EN1 expression. CONCLUSIONS: The transcription factor EN1 is overexpressed in SACC under methylation regulation and plays a pivotal role in SACC progression through the PI3K-AKT pathway. These results suggest that EN1 may be a diagnostic biomarker and a potential therapeutic target for SACC.

Notch activation promotes bone metastasis via SPARC inhibition in adenoid cystic carcinoma.

OBJECTIVES: We aimed to investigate bone metastasis induced by Notch signalling pathway dysregulation and to demonstrate that SPARC is a potential therapeutic target in adenoid cystic carcinoma (AdCC) with Notch dysregulation. MATERIALS AND METHODS: This retrospective study enrolled 144 AdCC patients. RNA-sequencing and enrichment analyses were performed using 32 AdCC samples. Osteonectin/SPARC and the Notch activation indicator Notch intracellular domain (NICD) were detected using immunohistochemistry. Cell proliferation and migration assays were conducted using stably NICD over-expressing cells. The effect of SPARC on osteoclast differentiation in NICD cells was investigated using western blotting, quantitative reverse transcription PCR, tartrate-resistant acid phosphatase staining and resorption assays. RESULTS: RNA-sequencing analysis showed that genes down-regulated in Notch-mutant AdCCs, such as SPARC, were enriched in ossification and osteoblast differentiation. Most (75/110, 68.2%) Notch1-wild-type AdCCs showed SPARC over-expression, whereas 30 out of 34 (88.2%) Notch1-mutant tumours showed low SPARC expression. SPARC over-expression was then found negatively to be correlated with NICD expression in 144 AdCCs. NICD over-expression promoted cell growth, migration and osteoclast differentiation, which could be partly reversed by exogenous SPARC. CONCLUSIONS: Notch activation in AdCC contributes to bone metastasis through SPARC inhibition. The study results suggest that SPARC may represent a prognostic biomarker and potential therapeutic target.

Salivary gland papillary adenocarcinoma with intestinal-like features: Clinicopathologic, immunohistochemical, and genetic study of six cases.

BACKGROUND: Salivary gland tumors with papillary architecture and intestinal-like mucinous cytologic features are rare. Their clinicopathologic and genetic features are not fully understood, and whether they represent one separate entity remains unclear. METHODS: Six salivary adenocarcinomas with papillary architecture and intestinal-like mucinous cytologic features were reported. Immunostaining was done for CK7, CK20, CDX2, SOX10, S100, MUC1, MUC2, and MUC5AC. Tumor DNA samples were extracted for Sanger sequencing. Previously reported morphology-analogous cases were reviewed. RESULTS: Six cases involved the palate (2), retromolar region (1), submandibular region (1), tongue (1), and mandible (1). Five cases were followed up, with one case of recurrence 1 year after surgery, one death from cerebral infarction 7 days after surgery, and three cases without signs of recurrence or metastasis over 5 years. All cases had abundant mucinous production and presented a typical immunophenotype common to salivary primaries, CK7 & MUC1 positive, CK20 & CDX2 negative. Sanger sequencing demonstrated recurrent AKT1 E17K mutations in four cases (4/6, 66.7%). A review of reported salivary intestinal-like tumors revealed 3 out of 13 cases presented with papillary morphology and CDX2 negative. Some salivary papillary neoplasms with mucinous cytologic features termed as intraductal papillary neoplasms or mucinous adenocarcinomas were also reported with AKT1 E17K mutations. CONCLUSION: We describe 6 cases of salivary gland papillary adenocarcinoma with intestinal-like mucinous cytologic features, which are different from conventional intestinal-type adenocarcinoma, presenting a consistent immunophenotype of CK7 & MUC1 positive, CK20 & CDX2 negative and exhibiting recurrent AKT1 E17K mutations.

Notch activation leads to loss of myoepithelial differentiation and poor outcome in solid adenoid cystic carcinoma.

OBJECTIVE: We aimed to investigate Notch pathway dysregulation in solid adenoid cystic carcinoma (AdCC) and to define the association of Notch activation with cell differentiation and prognosis in AdCCs. MATERIALS AND METHODS: Notch1 mutations were detected from 125 AdCCs (62 cribriform-tubular; 63 solid). RNA-seq was performed in 16 AdCCs (6 Notch-mutant; 10 wild type). Notch activation indicator NICD and myoepithelial marker p63 were detected using immunohistochemistry and double-labelling immunofluorescence. The effect of exogenous NICD overexpression on p63 expression and cell proliferation was investigated using Western blotting and live-cell imaging. RESULTS: We identified 33 Notch1 activating mutations in 27 AdCCs including 26 solid and 1 cribriform-tubular subtypes. Six tumours harboured more than one Notch1 mutation, and 18 Notch1 mutations were novel. Most (47/63, 74.6%) solid AdCCs showed NICD overexpression, whereas 61 of 62 (98.4%) cribriform-tubular tumours were negative. NICD and p63 exhibited mutually exclusive expression, and exogenous NICD overexpression promoted cell proliferation and decreased p63 expression. NICD overexpression and Notch mutations were poor indicators for overall survival and metastasis, especially bone metastasis. CONCLUSIONS: Dysregulated Notch signalling plays a critical role in AdCC severity. Notch activation may contribute to loss of myoepithelial differentiation as well as high proliferation and metastasis rates in solid AdCC.

Interaction between oral squamous cell carcinoma cells and fibroblasts through TGF-ß1 mediated by podoplanin.

Podoplanin is upregulated in the invasive front of oral squamous cell carcinoma (OSCC). Carcinoma-associated fibroblasts (CAFs) may mediate podoplanin expression. However, the role of podoplanin in OSCC cell and fibroblast interaction remains elusive. In the present study, we found that positive podoplanin expression in OSCC cells correlated with smooth muscle actin (α-SMA) expression in CAFs. Using CAFs and normal mucosal fibroblasts (NFs), we established indirect and direct co-culture systems mimicking the structure of OSCC. Podoplanin-overexpressing OSCC cells promoted NF activation; in direct co-culture, but not in indirect co-culture, podoplanin-overexpressing OSCC cells increased fibroblast invasion via matrix metalloproteinase 2 (MMP-2), MMP-14, and αv/ß6 integrin receptor (ITGA5/ITGB6) signaling. CAFs also induced podoplanin expression through the transforming growth factor-ß1 (TGF-ß1)/Smad pathway. TGF-ß1 increased the podoplanin-dependent activation of epidermal growth factor receptor (EGFR), AKT, and extracellular signal-regulated kinase (ERK) signaling. Additionally, CAFs promoted OSCC cell invasion by upregulating MMP-2 and MMP-14 expression in both indirect and direct co-culture. Taken together, our findings indicate that podoplanin regulates the interaction between OSCC cells and CAFs via the mutual paracrine effects of TGF-ß1.

Myoepithelial differentiation in cribriform, tubular and solid pattern of adenoid cystic carcinoma: A potential involvement in histological grading and prognosis.

Adenoid cystic carcinoma (AdCC) is known as a biphasic tumor composed of ductal and myoepithelial cells. The present study aimed to evaluate the amount and distribution of the myoepithelial cells in cribriform, tubular and solid subtypes of AdCC and analyze their relationship with histological grading and prognosis. A panel of myoepithelial markers including CK5/6, p63, p40, D2-40, calponin, α-SMA, S-100, and vimentin, together with a luminal cell marker CK7, and Ki-67 were used for immunohistochemical study in 109 AdCCs that included 38 cribriform, 36 tubular and 35 solid subtypes. The myoepithelial cells were labeled and found lined cystic-like paces, located at the periphery of the cribriform arrangements, and presented at the nonluminal cells of the two-layered tubular structures, while absent or dispersed in the solid pattern. Meantime, the solid subtype presented a higher proliferation rate assessed by mitotic count and Ki-67 labeling index, followed by poorer overall survival and recurrent-free survival. Furthermore, CK7 expression was found higher in solid pattern than in cribriform-tubular subtype, which showed negative correlation with the myoepithelial markers including D2-40, Calponin, α-SMA, p63, p40 and vimentin. The solid pattern of AdCC showed gland differentiation but loss of myoepithelial differentiation with a higher proliferation and more aggressiveness as well as poorer prognosis compared with the cribriform-tubular subtypes, which implies that loss of MEC differentiation might contribute to the poor prognosis of the solid subtype of AdCC. However, further studies are required to clarify its exact role in AdCC progression.

Bmi-1 expression predicts prognosis in salivary adenoid cystic carcinoma and correlates with epithelial-mesenchymal transition-related factors.

Salivary adenoid cystic carcinoma (AdCC) is known for its high propensity to invade and metastasize. Bmi-1 acts as an oncogene by controlling cell cycle and self-renewal of adult stem cells, and its overexpression correlates with metastasis and poor prognosis in several cancers. Epithelial-mesenchymal transition (EMT) plays a central role in cancer metastasis. A key step in EMT is the down-regulation of E-cadherin that can be repressed by the transcriptional factors, such as Snail and Slug. In the present study, we investigated Bmi-1, Snail, Slug, and E-cadherin expression by immunohistochemistry in 102 patients with AdCC and analyzed statistically whether their expression correlated with clinicopathologic factors and prognosis. Reverse transcription-polymerase chain reaction was also performed in 22 tumor tissues and the adjacent noncancerous tissues to confirm Bmi-1 status in AdCCs. Our data demonstrated significant associations between the tumor metastasis and the expression of Bmi-1, Snail, Slug, and E-cadherin. Furthermore, a high level of Bmi-1 was not only correlated with the overexpression of Snail and Slug but also indicated an unfavorable metastasis-free survival and served as a high-risk marker for AdCC. In addition, Bmi-1 messenger RNA level was found much higher in AdCC tissues than in the adjacent noncancerous salivary gland tissues. Our results suggest that Bmi-1 may play a crucial role in AdCC progression by interaction with EMT-related markers and predict poor survival.

Snail regulates the motility of oral cancer cells via RhoA/Cdc42/p-ERM pathway.

The transcriptional factor Snail has been reported to possess properties related to cancer progression; however, the mechanism for it is not fully understood. Our data showed that Snail knockdown by small interfering RNA in two OSCC cell lines, WSU-HN6 and CAL27, significantly inhibited cell migration and invasion which also resulted in decreased cell motility, such as impaired cell spreading on type I collagen substrate, reduced filopodia, and premature assembly of stress fibers. In addition, Snail-silencing decreased Cdc42 activity but increased RhoA activity, accompanied by the downregulation in both p-ERM expression and cell motility. Meanwhile, endogenous p-ERM was found specifically co-precipitated with activated Cdc42, but not RhoA, and this co-association was decreased by Snail-silencing. The small molecule inhibitors of Rho-associated kinase (Y27632) markedly enhanced Cdc42 activity and the association of p-ERM with activated Cdc42, increasing cell motility remarkably. Using immunohistochemistry, Snail and p-ERM overexpressions were found in OSCC tissues correlated with nodal metastasis and shorter survival. Taken together, these results demonstrate that Snail regulates cell motility through RhoA/Cdc42/p-ERM pathway and may serve as a biomarker to predict prognosis for OSCC patients. Although RhoA and Cdc42 are concurrently regulated downstream of Snail, there is a direct interplay between them, which indicates RhoA has to be inactivated at some point in cell motility cycle.

Basal cell adenoma of salivary glands with a focal cribriform pattern: clinicopathologic and immunohistochemical study of 19 cases of a potential pitfall for diagnosis.

Cribriform type of salivary basal cell adenoma (cBCA) is relatively rare and problematic in distinction from adenoid cystic carcinoma (AdCC). The aim of this study was to investigate the clinicopathology and immunoprofile of cBCA. Nineteen cases of cBCA with at least a 30% area of cribriform structure under microscope were analyzed by the description of their histopathologic and immunohistochemical features using the antibodies of matrix metalloproteinase-9 (MMP9), CK8&18, calponin, SMA, S100, P63, CD117, and laminin. The patients of cBCA ranged from 24 to 71 years with a distinct predilection for females (79%). The tumor was well-circumscribed and had no recurrent tendency after a local excision followed by a median of 67 months. Enhanced computed tomography (CT) showed that the tumor was rich in blood supply. Microscopically, it was mainly composed by the basaloid cells with the peripheral palisading. The cells around the cribriform pattern expressed P63 protein and had almost no immunoreactivity for calponin, SMA, S100, or CK8&18. The expression level of MMP9, laminin, and CD117 were significantly lower in cBCA than those in AdCC. Good circumscription, lack of infiltrative properties, and absence of MMP9, laminin, CD117, and myoepithelial marker (SMA, S100 and calponin) in the cells around the cribriform spaces, are the most reliable points for differential diagnosis of cBCA from AdCC.

Familial gigantiform cementoma with recurrent ANO5 p.Cys356Tyr mutations: Clinicopathological and genetic study with literature review.

BACKGROUND: Familial gigantiform cementoma (FGC) is a rare tumor characterized by the early onset of multi-quadrant fibro-osseous lesions in the jaws, causing severe maxillofacial deformities. Its clinicopathological features overlap with those of other benign fibro-osseous lesions. FGC eventually exhibits progressively rapid growth, but no suspected causative gene has been identified. METHODS: In this study, three patients with FGC were recruited, and genomic DNA from the tumor tissue and peripheral blood was extracted for whole-exome sequencing. RESULTS: Results showed that all three patients harbored the heterozygous mutation c.1067G > A (p.Cys356Tyr) in the ANO5 gene. Furthermore, autosomal dominant mutations in ANO5 at this locus have been identified in patients with gnathodiaphyseal dysplasia (GDD) and are considered a potential causative agent, suggesting a genetic association between FGC and GDD. In addition, multifocal fibrous bone lesions with similar clinical presentations were detected, including five cases of florid cemento-osseous dysplasia, five cases of polyostotic fibrous dysplasia, and eight cases of juvenile ossifying fibromas; however, none of them harbored mutations in the ANO5 gene. CONCLUSION: Our findings indicate that FGC may be an atypical variant of GDD, providing evidence for the feasibility of ANO5 gene testing as an auxiliary diagnostic method for complex cases with multiple quadrants.

Colloid Pattern of Salivary Mucinous Adenocarcinomas With Recurrent BRAF V600E Mutations.

The relationship between various patterns of mucin-producing salivary adenocarcinomas, including invasive salivary adenocarcinomas with mucinous differentiation, such as colloid and papillary carcinomas, remains unclear. Herein, we aimed to describe the clinicopathologic characteristics, immunophenotypes, molecular underpinnings, and clinical behavior of salivary mucinous adenocarcinomas (MA) to clarify their classification. We described a broad series of colloid and papillary patterns of MAs, indicating that papillary pattern presented papillary cystic proliferation of mucinous columnar cells as salivary intraductal papillary mucinous neoplasms with recurrent AKT1 E17K mutations, whereas colloid adenocarcinomas containing large mucinous pools or lakes around the malignant epithelial nests or islands harbored BRAF V600E mutations with worse prognosis. Typical morphologic structures, CK7(+), CK20(-), CDX2(-), p63(-), p40(-), MAML2 fluorescence in situ hybridization (-), AR(-), TTF-1(-), S100(-), mammaglobin(-), or S100/mammaglobin(+) with ETV6 fluorescence in situ hybridization (-) immunophenotype, and recurrent AKT1 E17K or BRAF V600E mutations may be defined. To our knowledge, this small series represents the first genetic study on a typical colloid pattern of MA, and our study with the spectrum documentation for MA in clinicopathologic characteristics, histologic and immunophenotypes, molecular features, and clinical behavior will allow for a better understanding of these rare but distinctive tumors.

PON3::LCN1 and HTN3::MSANTD3 Gene Fusions With NR4A3/NR4A2 Expression in Salivary Acinic Cell Carcinoma.

Acinic cell carcinoma of the salivary gland (AciCC) is a low-grade carcinoma characterized by the overexpression of the transcription factor nuclear receptor subfamily 4 group A member 3 (NR4A3). AciCC has been the subject of a few molecular research projects. This study delves into AciCC's molecular landscape to identify additional alterations and explore their clinical implications. RNA sequencing and immunohistochemical staining for markers NR4A3/NR4A2, DOG-1, S100, and mammaglobin were utilized on 41 AciCCs and 11 secretory carcinoma (SC) samples. NR4A3 was evident in 35 AciCCs, while the residual 6 were NR4A3-negative and NR4A2-positive; SC samples were consistently NR4A3-negative. A novel fusion, PON3 exon 1- LCN1 exon 5, was detected in 9/41 (21.9%) AciCCs, exhibiting a classical histologic pattern with serous cell components growing in solid sheets alongside the intercalated duct-like component. Clinical follow-up of 39 patients over a median of 59 months revealed diverse prognostic outcomes: 34 patients exhibited no disease evidence, whereas the remaining 5 experienced poorer prognosis, involving local recurrence, lymph node, and distant metastasis, and disease-associated death, 4 of which harbored the PON3::LCN1 fusion. In addition, the HTN3::MSANTD3 fusion was recurrently identified in 7/41 AciCC cases. SC patients lacked both fusions. Immunohistochemistry uncovered differential expression of DOG-1, S100, and mammaglobin across samples, providing nuanced insights into their roles in AciCC. This study accentuates PON3::LCN1 and HTN3::MSANTD3 fusions as recurrent molecular events in AciCC, offering potential diagnostic and prognostic utility and propelling further research into targeted therapeutic strategies.

Erratum: Podoplanin promotes the invasion of oral squamous cell carcinoma in coordination with MT1-MMP and Rho GTPases.

[This corrects the article on p. 514 in vol. 5, PMID: 25973294.].

[A clinicopathologic study on oncocytoma and oncocytic carcinoma of the salivary glands].

OBJECTIVE: To investigate the clinicopathologic features, differential diagnosis, treatment and prognosis of oncocytoma and oncocytic carcinoma of the salivary glands. METHODS: The clinicopathologic features were studied in 23 cases of oncocytomas and 15 cases of oncocytic carcinomas, and immunohistochemical staining as well as electron microscopy were performed. RESULTS: Most oncocytomas occurred in the parotid gland (95.6%) with no recurrence. The diagnosis was made based on histopathological features. Oncocytic carcinomas were high-grade tumors, mainly occurring in the parotid gland. The diagnosis was based not only on histopathology but also on ultrastructural findings, phosphotungstic acid hematoxylin (PTAH), and immunohistochemistry. Of the 14 cases with follow-up information, 7 cases recurred. Regional or distant metastases were found in 7 and 4 cases, respectively. CONCLUSION: Oncocytoma is a rare tumor with well prognosis, whereas oncocytic carcinoma of the salivary glands is a high-grade tumor, with frequent local recurrence, regional or distant metastasis, the diagnosis of which should be based on a combination of clinical and histopathological features. Immunohistochemistry for mitochondria is considered as a practical and helpful adjuvant for diagnosis.

High Prevalence of BRAF V600E Mutations in Langerhans Cell Histiocytosis of Head and Neck in Chinese Patients.

Langerhans cell histiocytosis (LCH) is characterized by clonal proliferation of Langerhans cells and has been classified as a hematolymphoid tumor. BRAF V600E mutation was found to be frequent in LCH; however, it has also been reported that Asia patients with LCH tend to show a lower rate of BRAF V600E mutation. In this study, we found LCH from the head and neck region often involved bone especially the posterior of the mandible and presented a high prevalence of BRAF V600E mutation in Chinese patients. Our findings also showed immunohistochemical detection correlated very well to DNA sequencing of BRAF alterations, which may be useful in the diagnosis of LCH, especially in cases with a low proportion of Langerhans cells, and BRAF inhibitors might be a treatment option for patients with LCH harboring BRAF V600E mutation.

A clinicopathologic study on central odontogenic fibroma: with special reference to amyloid variant.

OBJECTIVE: The aim of this study was to clarify the clinicopathologic features of central odontogenic fibroma (OF), especially the amyloid variant, and to discuss its association with the Langerhans cell variant of calcifying epithelial odontogenic tumor (CEOT). STUDY DESIGN: The clinicopathologic features and immunophenotypes of 17 OFs, including 6 amyloid variants, were analyzed. The Langerhans cell variant of CEOT is reviewed, and its relationship with OF is discussed. RESULTS: Most OFs (13 of 17) were located at the anterior region of the jaws, often with root resorption. The amyloid variant exhibited the typical clinicopathologic features of OFs, characterized by dispersed small epithelial nests embedded in a fibrous stroma. Immunohistochemically, the epithelial component in all central OFs, including the amyloid variants, exhibited dispersed staining for CK10/13 but was negative for CK7 and CK8/18. Langerhans cells were positive for S-100 and Langerin in the epithelium of OFs, including the amyloid variants. CONCLUSIONS: The amyloid variant of OF is a rare benign tumor exhibiting the typical clinicopathologic features of conventional OFs and should not be diagnosed as CEOT even in the presence of amyloid deposits. Previously reported cases described as "Langerhans cell variant of CEOT" should be classified as the "amyloid variant of OF," given that it shares features more in common with OFs than with CEOTs.

Frequent genetic alterations and reduced expression of the Axin1 gene in oral squamous cell carcinoma: involvement in tumor progression and metastasis.

The aims of this study were to analyze the genetic alterations and expression levels of the Axin1 gene in oral squamous cell carcinoma (OSCC), to evaluate its clinical importance and to clarify whether the Axin1 gene is involved in the pathogenesis of OSCC. Mutation analysis of the Axin1 gene was performed by denaturing high performance liquid chromatography (DHPLC) and DNA sequencing in 44 OSCC samples. Meanwhile, Axin1 protein expression was investigated by immunohistochemistry in these samples. Aberrant profiles were detected by DHPLC screening in 26 different OSCC cases. After sequencing analysis, four mutations and five polymorphisms were identified. One case of poorly differentiated OSCC with metastasis contained two mutations: one revealed a T>G substitution at nucleotide 324 in exon 1, resulting in a glycine to stop codon substitution at amino acid residue 108; the other revealed an A>G heterozygous mutation in intron 7, located very near to exon 8. In another two patients with moderately differentiated OSCC and metastasis, a G>T heterozygous mutation at codon 488 in exon 5 and a C>G substitution at the intron 5+26 position was detected, respectively. Five polymorphisms were all frequent and localized at positions of codon 254 (GAT--> GAC), codon 429 (GTC-->ATC), codon 525 (GAC-->GAT), codon 609 (GCT--> GCC), and intron 4+17 (G>A), with a frequency of 39%, 8%, 6%, 13% and 9%, respectively. Immunoreactivity for Axin1 was strongly positive in normal stratified squamous epithelium but significantly reduced expression of Axin1 was shown in most of the 44 tumor specimens (35/44), especially in poorly differentiated tumors with metastasis. These results suggest that mutational inactivation and reduced expression of the Axin1 gene may play a pivotal role in OSCC carcinogenesis and metastasis.

Aberrant expression of beta-catenin, Pin1 and cylin D1 in salivary adenoid cystic carcinoma: relation to tumor proliferation and metastasis.

The aims of this study were to investigate the expression levels of beta-catenin, Pin1 and cyclin D1 in salivary adenoid cystic carcinomas (SACC ) and to evaluate its clinical importance, furthermore, to elucidate whether beta-catenin expression was aberrant in SACC and whether Pin1 was involved in aberrant beta-catenin and cyclin D1 expression. The expression of Pin1, beta-catenin and cyclin D1 were examined in the specimens of 65 patients with SACC by immunohistochemistry, protein and mRNA expressions were detected by western blotting and RT-PCR in four SACC cell lines. Pin1 was overexpressed in 51 cases of SACC (78%), and high levels of Pin1 expression correlated with cyclin D1 positive expression (p = 0.02). Fourteen (22%) cases showed positive immunoreactivity for beta-catenin protein in the nuclear/cytoplasmic fraction in tumor tissues, which was defined as cytoplasm/nucleus staining, among which quite evident nuclear expression of beta-catenin was detected in six cases (9%), while cyclin D1 positive expression was detected in 41 cases of SACC (63%). Reduced membranous expression of beta-catenin was detected in the cases with metastasis (11/14). Theses results suggest that Pin1 and Wnt signalling pathway are activated in SACC and may play a pivotal role in SACC carcinogenesis and metastasis.

Moesin regulates the motility of oral cancer cells via MT1-MMP and E-cadherin/p120-catenin adhesion complex.

OBJECTIVE: The present study aimed to clarify the role of Moesin in oral squamous cell carcinoma (OSCC) progression, especially in regulation of cell motility. MATERIALS AND METHODS: Immunohistochemistry and western blotting were used to investigate the expression of Moesin, E-cadherin, p120-catenin and MT1-MMP in normal epithelia, dysplasia and OSCCs. Then, Moesin was knockdown by siRNA in OSCC cell lines, WSU-HN6 and CAL27, and the biological role of Moesin in cell adhesion and motility was evaluated by transwell system, cell spreading and aggregation assays. The interactions between Moesin, MT1-MMP and E-cadherin/p120-catenin complex were determined by co-immunoprecipitation and immunofluorescence. RESULTS: Moesin expression was found decreased in the membrane and increased in cytoplasm during the malignant transformation of oral epithelia, and cytoplasmic overexpression of Moesin correlated with nodal metastasis and poor prognosis of OSCCs. Furthermore, Moesin-silencing induced an increased cell-cell adhesion but decreased invasiveness, which was subsequently demonstrated might due to Moesin-mediated E-cadherin and p120-catenin interaction. Meantime, Moesin-silencing significantly down-regulated MT1-MMP expression, accompanied by reduced cell motility and impaired filopodia formation, which was also observed when MT1-MMP knockdown by RNAi or tissue inhibitor (TIMP2), indicating the involvement of MT1-MMP in Moesin-mediated cell motility. Finally, the relationship between Moesin, E-cadherin and MT1-MMP was confirmed in OSCC tissue samples. CONCLUSION: Taken together, our results indicate Moesin may regulate cell motility through its interactions with MT1-MMP and E-cadherin/p120-catenin adhesion complex and cytoplasmic expression of Moesin correlates with nodal metastasis and poor prognosis of OSCCs, indicating Moesin may be a potential candidate for targeted gene therapy for OSCCs.

Podoplanin promotes the invasion of oral squamous cell carcinoma in coordination with MT1-MMP and Rho GTPases.

Podoplanin overexpression has been reported in various cancers, however, the precise mechanism for podoplanin to promote tumor progression remains elusive. In the present study, podoplanin overexpression was found associated with invasiveness both in OSCC tissues and cell lines. Moreover, the cell invasiveness increased with forced podoplanin expression and decreased when podoplanin was knockdown, indicating podoplanin-mediated cell invasion during OSCC progression. To further identify the role of podoplanin in tumor invasion, cell spreading and immunofluorescence assay were performed firstly. It was found that podoplanin knockdown caused an impaired cell spreading with reduced filopodia and the premature assembly of stress fibers while podoplanin overexpression induced an increase in cellular protrusions and stress fibers with extensive parallel bundles. Then, pull-down assays revealed forced podoplanin expression increased Cdc42 activity and reduced RhoA activity while podoplanin knockdown decreased Cdc42 and increased RhoA markedly. Moreover, a hierarchy of crosstalk between RhoA and Cdc42 was confirmed in podoplanin-mediated cell motility. On the other hand, a significant correlation between podoplanin and MT1-MMP expression in OSCCs was found both in vivo and in vitro, co-located in invasive cells and cellular protrusions. Furthermore, our data showed MT1-MMP knockdown significantly blocked the upregulation of cell motility by forced podoplanin expression, indicating that MT1-MMP played a role in podoplanin-mediated tumor invasion. To further confirm the interaction between RhoA/Cdc42 complex, MT1-MMP and podoplanin, co-precipitation experiments were performed. Both the co-precipitation of podoplanin with MT1-MMP and the podoplanin-induced specific binding of MT1-MMP to Cdc42 were found, and immunofluorescence revealed the co-location of podoplanin, MT1-MMP and Cdc42 at the plasma membrane and filopodia induced an increase in cellular protrusion and stress fibers formation. Moreover, MT1-MMP inhibition could partly rescue the increase of Cdc42 activity caused by forced podoplanin expression. Taken together, our data demonstrated a hierarchy of crosstalk between RhoA and Cdc42 was involved in podoplanin-mediated cytoskeleton remodeling and invasion; the co-location and co-ordination of podoplanin, Cdc42 and MT1-MMP in the invadopodia might induce cytoskeleton remodeling, ECM degradation and tumor invasion, while podoplanin-induced EMT may not be indispensible during OSCC progression.