Role of Stromal Fibroblast-Induced WNT7A Associated with Cancer Cell Migration Through the AKT/CLDN1 Signaling Axis in Oral Squamous Cell Carcinoma.

Wnt signaling plays a crucial role in the progression of various cancers, including oral squamous cell carcinoma (OSCC). However, the tumor microenvironment (TME) regulating Wnt signaling has not yet been fully elucidated. In this study, we investigated whether cancer-associated fibroblasts (CAFs), the primary components of the TME, activate Wnt signaling and promote tumor progression in OSCC. We conducted a Transwell coculture assay using human OSCC cell lines and normal human dermal fibroblasts (NHDFs). NHDFs stimulated WNT7A expression in several OSCC cell lines, especially HO-1-N-1 and HSC-5. An immunohistochemical study using 122 human OSCC samples indicated that high WNT7A expression in tumor cells was significantly associated with invasion depth and poor prognosis. Moreover, WNT7A expression in OSCC cells was positively correlated with α-smooth muscle actin expression in CAFs. WNT7A knockdown in OSCC cells demonstrated that OSCC cells cocultured with NHDFs significantly promoted tumor cell migration and invasion, which was dependent on WNT7A expression in OSCC cells. We also isolated HSC-5 cells from the coculture and conducted microarray analysis to investigate the factors that promote tumor progression induced by WNT7A. Among the various differentially expressed genes, we identified a downregulated gene encoding CLDN1 and confirmed that WNT7A negatively regulated CLDN1 expression in OSCC cells and CLDN1 knockdown in OSCC cells promoted their migration. Phosphokinase array analysis showed that WNT7A activates protein kinase B (AKT) phosphorylation. Activating AKT signaling using the SC79 agonist induced CLDN1 downregulation in OSCC cells. In the coculture assay, the AKT inhibitor MK2206 significantly recovered CLDN1 expression downregulated by WNT7A, resulting in OSCC cell migration suppression. These results suggest that CAFs stimulate OSCC cells to produce WNT7A, following CLDN1 expression downregulation by activating AKT signaling, promoting cancer cell migration. These findings highlight the importance of molecular therapies targeting the TME in OSCC.

IL-1 Generated by Oral Squamous Cell Carcinoma Stimulates Tumor-Induced and RANKL-Induced Osteoclastogenesis: A Possible Mechanism of Bone Resorption Induced by the Infiltration of Oral Squamous Cell Carcinoma.

We previously observed a novel osteoclastogenesis system that is induced by oral squamous cell carcinoma (OSCC) cells, which target osteoclast precursor cells (OPC) without upregulation of the master transcriptional factor of osteoclastogenesis, NFATc1. Here, we analyzed inflammatory cytokines that were preferentially expressed in one of the osteoclastogenic OSCC cell lines, namely NEM, compared with the subclone that had lost its osteoclastogenic properties. Based on a gene expression microarray and a protein array analyses, IL-1, IL-6, IL-8, and CXCL1 were chosen as candidates responsible for tumor-induced osteoclastogenesis. From the results of the in vitro osteoclastogenesis assay using OPCs cultured with OSCC cells or their culture supernatants, IL-1 was selected as a stimulator of both OSCC-induced and RANKL-induced osteoclastogenesis. The IL-1 receptor antagonist significantly attenuated osteoclastogenesis induced by NEM cells. The stimulatory effects of IL-1 for OSCC-induced and RANKL-induced osteoclastogenesis were effectively attenuated with cannabidiol and denosumab, respectively. These results suggest that IL-1 secreted from OSCC cells stimulates not only tumor-induced osteoclastogenesis targeting OPCs but also physiological RANKL-induced osteoclastogenesis, and this may be the biological mechanism of bone resorption induced by the infiltration of OSCC. These results also suggest that IL-1 inhibitors are candidates for therapeutic agents against bone resorption induced by OSCC.

Clinical manifestations of diffuse large B-cell lymphoma that exhibits initial symptoms in the maxilla and mandible: a single-center retrospective study.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphatic tumor; however, extranodal DLBCLs that exhibit initial symptoms in the maxilla and mandible are rare. Moreover, DLBCL is clinically classified as a moderate to highly malignant lymphatic tumor that can progress rapidly; therefore, early diagnosis is crucial. However, diagnosis is difficult as the disease causes a diverse range of clinical symptoms with no characteristic imaging findings. We conducted a clinical investigation to clarify the clinical characteristics of DLBCL that exhibits initial manifestation in the maxilla and mandible. METHODS: Of the 2748 patients with malignant tumors of the oral and maxillofacial region examined at our hospital during a period of 11 years between January 2006 and December 2016, 27 primary cases diagnosed with DLBCL based on the chief complaint of symptoms in the gingiva and bone of the maxilla and mandible were enrolled in this study. Evaluations were based on sex, age, whether treatment was provided by a previous physician, symptoms, duration of disease until treatment was sought, clinical diagnosis, laboratory findings, and imaging results. RESULTS: There were 15 cases that involved the maxilla and 12 that involved the mandible. The median duration of disease until treatment was sought was 60 d (3-450 d). All cases exhibited a tumor or a mass, and hypoesthesia of the chin was confirmed in eight cases wherein the mandible was involved. The clinical stages were stage I in eight cases, stage II in ten cases, and stage IV in nine cases. Serum lactate dehydrogenase (LDH) levels were elevated in 13 of 22 patients. The overall survival rate was 63%. CONCLUSIONS: Symptoms associated with nontender swelling and numbness of the lip or chin in the absence of other findings such as dental infections should raise suspicions about DLBCL. Patients should be provided appropriate imaging and accurate biopsy assessments to improve prognosis.

Primordial odontogenic tumor occurred in the maxilla with unique calcifications and its crucial points for differential diagnosis.

Primordial odontogenic tumor (POT) is a newly classified, mixed epithelial and mesenchymal odontogenic tumor, with only 17 reported cases to date. Herein, we report a case of POT that occurred in the right maxilla of a 10-year-old boy and reveal unique features in comparison with those previously reported. Radiologically, the lesion presented as a well-defined, unilocular radiolucency with notable radiopaque foci on the periphery. Microscopically, the tumor was mainly composed of dental papilla-like myxoid fibrous connective tissue, largely surrounded by non-keratinized squamous epithelium with numerous calcified particles, and partly enclosed by inner enamel epithelium-like columnar cells and enamel organ-like structures accompanied with cuboidal and/or stellate reticulum-like cells. Immunohistochemically, the epithelium tested positive for cytokeratin 14 and 19. Moreover, amelogenin and ameloblastin, matrix proteins relating to enamel formation, were positive in the covering epithelium. The tumor was enucleated as a whole, and no recurrence was recorded thereafter. Although the presence of numerous calcified particles was unique, we diagnosed this lesion as POT based on the above-described features. Furthermore, we emphasize the importance of the differential diagnosis of POT and other odontogenic tumors that resemble corresponding tooth germ components.

Homeobox transcription factor engrailed homeobox 1 is a possible diagnostic marker for adenoid cystic carcinoma and polymorphous adenocarcinoma.

Diagnostic utility of a homeobox transcription factor, engrailed homeobox 1 (En1) in the histopathology of salivary gland neoplasms was studied. The expression of En1 was immunohistochemically examined in 51 cases of adenoid cystic carcinoma (AdCC) and 143 cases of other salivary gland neoplasms. In all 51 AdCCs, En1 was expressed in 30-100% of tumor cells. In eight of nine polymorphous adenocarcinomas (PACs), En1 was expressed in 40-100% of tumor cells. Less than 5% of tumor cells expressed En1 in three of 12 epithelial-myoepithelial carcinomas, one of 17 basal cell adenomas (BCAs), and one of 34 pleomorphic adenomas (PAs). Among 55 other carcinoma cases, 1-30% of tumor cells expressed En1 in three salivary duct carcinomas (SDCs) ex PA. None of the myoepitheliomas and Warthin tumors expressed En1. When the cut-off value of the percentage of En1-expressing cells was set to 25%, all 51 AdCCs, eight of nine PACs and one SDC ex PA were En1-positive and the others were En1-negative. En1 is expressed consistently in AdCCs, frequently in PACs, but rarely in other salivary gland neoplasms. En1 is a possible diagnostic marker for AdCC and PAC in the histopathology of salivary gland neoplasms.

Malignant transformation of oral lichen planus: a retrospective study of 565 Japanese patients.

BACKGROUND: Oral lichen planus (OLP) is a chronic inflammatory oral mucosa disease that is recognized as an oral potentially malignant disorder. However, the potentially malignant nature of OLP remains unclear. METHODS: We designed this study to examine the demographic and clinical characteristics of patients with OLP and evaluate the associated malignant transformation rate. A total of 565 patients with a clinical and histopathological diagnosis of OLP who presented at our department between 2001 and 2017 were retrospectively studied. Patients who had clinical and histopathological features of oral lichenoid lesions (OLLs) classified as oral lichenoid contact lesions, oral lichenoid drug reactions and oral lichenoid lesions of graft-versus-host disease were excluded. RESULTS: The study population included 123 men and 442 women aged 21-93 years (mean ± standard deviation, 60.5 ± 11.8). The 565 patients were followed up for a duration of 55.9 ± 45.3 months, during which 4 (0.7%) patients developed squamous cell carcinoma (SCC). In three of these 4 patients who developed SCC, the clinical type of OLP was the red type. CONCLUSIONS: Our results suggested that OLP was associated with a low risk of malignant transformation. We recommend regular follow-up for OLP patients and clear differentiation of oral epithelial dysplasia and OLLs to enable early detection of malignant transformation. Further investigation of the clinical risk factors associated with malignant transformation is necessary.

Intracellular claudin-1 at the invasive front of tongue squamous cell carcinoma is associated with lymph node metastasis.

Claudins are the major component of tight junctions, which form a primary barrier to paracellular diffusion and maintain cell polarity in normal epithelia and endothelia. In cancer cells, claudins play additional roles besides serving as components of the tight junctions, and participate in anoikis or invasion. Among the claudin family proteins, claudin-1 has the most promising potential, both diagnostically and prognostically, in many types of cancers, including oral, gastric, liver, and colon cancers. However, conflicting results have been reported in relation to the degree of claudin-1 expression and the prognosis, suggesting that the expression level of claudin-1 alone is not sufficient to analyze the relationship between claudin-1 and cancer progression. As endocytic trafficking of claudin-1 has been reported in several epithelial cell types in vitro, we aimed to determine whether intracellular localization of claudin-1 is the missing aspect between claudin-1 and cancer. We investigated the expression of claudin-1 in 83 tongue squamous cell carcinoma (TSCC) pathological specimens. Although the expression level of claudin-1 based on immunohistochemistry was not associated with TSCC progression, within the high claudin-1 expression group, the incidence of intracellular localization of claudin-1 was correlated with cervical lymph node metastasis. In an in vitro experiment, claudin-1 was constitutively internalized in TSCC-derived cells. Motility of TSCC-derived cells was increased by deficiency of claudin-1, suggesting that the decrease in cell-surface claudin-1 promoted the cell migration. Therefore, intracellular localization of claudin-1 at the invasion front may represent a promising diagnostic marker of TSCC.

Co-expression of EGFR and MET has a synergistic effect on the prognosis of patients with oral squamous cell carcinoma.

BACKGROUND: This study aimed to elucidate the correlation between gene amplification, protein expression of receptor tyrosine kinase, and prognosis of patients with oral squamous cell carcinoma (OSCC) using immunohistochemistry (IHC) and next-generation sequencing data. METHODS: We evaluated data pertaining to 208 patients with OSCC using IHC for epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor (MET). RESULTS: High expressions of EGFR and MET were detected in 60 and 41 patients, respectively. We evaluated the association of clinicopathological variables with high expressions of EGFR and/or MET. Distant metastasis was found in 9 of 41 patients (22.0%) and 6 of 15 patients (40.0%) with high expression of MET and high co-expressions of EGFR and MET, respectively; statistically significant differences were detected in both high expression of MET (P = .003) and high co-expressions of EGFR and MET (P = 3.41 × 10-5 ). The cumulative 5-year survival rate of patients with high and low expressions of EGFR or MET was approximately 65% and 85%, respectively. Conversely, among cases with high expressions of EGFR or MET, there was no additional decrease in the survival rate of patients harboring TP53 mutations. Moreover, the survival rate of patients with high co-expression of both EGFR and MET was very poor (22.0%) (P < 1.0 × 10-9 ). CONCLUSION: Our findings suggest that evaluation of protein expressions of EGFR and MET may facilitate prognostic assessment of patients with OSCC; in addition, patients with OSCC should be screened for enrollment in clinical trials of combination therapy with EGFR and MET inhibitors.

Genetic and histopathological analysis of a case of primary intraosseous carcinoma, NOS with features of both ameloblastic carcinoma and squamous cell carcinoma.

BACKGROUND: Primary intraosseous carcinoma (PIOC), NOS is an odontogenic carcinoma with unknown etiology. Its diagnosis may be used when central jaw carcinoma cannot be categorized as any other type of carcinoma. Further information on this extremely rare tumor is needed to improve our understanding and evaluate the classification of odontogenic carcinomas. CASE PRESENTATION: We herein presented two patients with PIOC, NOS with different clinical and histopathological features and analyzed gene mutations in these patients using next-generation sequencing (NGS). The typical PIOC, NOS case had many histopathological similarities to oral squamous cell carcinoma (OSCC), including the missense point mutations of TP53 Glu285Val, KDR Gln472His, and APC Pro1433Leu, which are similar to those in other cancers; however, no mutations were detected in the other patient with an atypical presentation of PIOC, NOS, which was derived from a precursor cystic lesion with similarities to both ameloblastic carcinoma and OSCC. CONCLUSIONS: Genetic analysis suggested that these two PIOC, NOS cases have different features and can be subcategorized.

A new osteoclastogenesis pathway induced by cancer cells targeting osteoclast precursor cells.

The precise mechanism of osteolysis induced by tumors infiltrating into the bone remains unclear. The main hypothesis is that tumor cells generate receptor activator of nuclear factor kappa-B ligand (RANKL), tumor necrosis factor-alpha (TNF-α), or other molecules that activate the expression of RANKL in osteoblasts, osteocytes, or bone marrow stromal cells. Administration of bisphosphonates or anti-RANKL antibody drugs, which suppress systemic bone resorption, prevents osteolysis induced by tumors infiltrating into the bone. However, these therapeutic agents may cause medication-related osteonecrosis of the jaw. In this study, we found a novel tumor-associated osteoclastogenesis pathway in osteoclast precursor cells. Co-culture with human oral squamous cell carcinoma cells, 3A or NEM, or culture with each of their conditioned medium induced many osteoclasts from osteoclast precursor cells, which were generated by a 24-h pretreatment of RANKL or TNF-α. Osteoprotegerin, a decoy RANKL receptor, denosumab, an anti-RANKL antibody drug, and infliximab, an anti-TNF-α antibody drug, did not prevent this tumor-associated osteoclastogenesis. Quantitative RT-PCR analysis showed that the expression of NFATc1 was decreased in this tumor-associated osteoclastogenesis, which was suggested to be independent of NFATc1. These results revealed a novel pathway for tumor-associated osteoclastogenesis, which may be a new therapeutic target for osteolysis induced by tumors infiltrating into the bone without affecting systemic bone metabolism.

Loss of Notch1 predisposes oro-esophageal epithelium to tumorigenesis.

Notch signaling functions in diverse developmental and homeostatic processes, including stem cell self-renewal and cell fate determination. Notch1-inactivating mutations are frequently detected in skin and oro-esophageal cancers, suggesting a role for Notch1 as a tumor suppressor. Here, we clarify the contribution of Notch1 deficiency to oro-esophageal tumorigenesis using a physiological experimental model. Tongue and esophageal tumors induced in mice by 4-nitroquinoline-1-oxide (4-NQO) showed pathophysiological similarities to human tumors, including decreased Notch1 expression in the basal cells. We created mutant mice (N1cKO), in which the Notch1 gene was disrupted specifically in the squamous epithelium. The epithelium formed normally in N1cKO mice, and although multiple skin tumors were detected at 65 weeks, no tumors developed in the tongue and esophagus. However, 4-NQO-induced tumorigenesis assays revealed that tumor onset occurred earlier in N1cKO mice than in wild-type littermates, and the tumors arose preferentially from the Notch1-negative epithelium, indicating the tumor susceptibility of Notch1-deficient epithelium. Notch1 regulates the expression of TERT, and age-related telomere erosion was more rapid in Notch1-deficient basal cells. Our results indicated that although Notch1 deficiency had little effect on squamous epithelium formation, it predisposed the affected epithelium to tumor development, at least in part through accelerated telomere erosion.

Alpha-L-fucosidase-1 is a diagnostic marker that distinguishes mucoepidermoid carcinoma from squamous cell carcinoma.

Alpha-L-fucose is a component of glycans on the cell surface. Alpha-L-fucose is correlated with tumorigenesis and malignancy, and alpha-L-fucosidase-1 (FUCA1), the enzyme that removes terminal α-L-fucose residues from glycoproteins, is downregulated in some high malignancy cancers. The expression profile of FUCA1 in head and neck tumors remains unknown, and we analyzed the expression profiles of FUCA1 and an upstream molecule p53 in mucoepidermoid carcinoma (MEC) and oral squamous cell carcinoma (OSCC). FUCA1 was expressed in most MECs irrespective of histopathological grading, whereas expression in OSCCs was low. High immunohistochemical intensity of p53 was detected in OSCCs at high frequency, but rarely detected in MECs. Genetic mutation analysis using next-generation sequencing revealed no significant mutation of TP53 in MECs. We further analyzed the expression profiles of FUCA1 in normal major and minor salivary glands and found strong expression in the intercalated duct, moderate expression in mucous acinar cells and no expression in serous acinar cells. These contrasting immunohistochemical profiles and anatomical distribution in normal salivary glands suggest that FUCA1 is a useful marker to distinguish MEC from OSCC, and many MECs have similar immunohistochemical phenotypes to intercalated duct and mucous acinar cells.

Metastasis of lower gingival squamous cell carcinoma to buccinator lymph node: case report and review of the literature.

BACKGROUND: Metastasis of oral cancer to the buccinator lymph nodes (BN) is uncommon. The antegrade lymphatic flow in patients with normal anatomy and physiology makes metastasis of lower gingival cancer to BN unlikely. CASE PRESENTATION: A 67-year-old woman presented with a 46 × 25-mm tumor on her lower gingiva, along with metastatic foci in BN and cervical lymph nodes. After neoadjuvant chemotherapy, she underwent radical resection of the primary tumor and BN, along with neck dissection. Following surgery, she received adjuvant chemoradiotherapy. Two years after treatment, there has been no evidence of tumor recurrence or metastasis. CONCLUSION: This is the first report of lower gingival squamous cell carcinoma with metastasis to BN. Metastasis to BN from lower gingival cancer is very rare but should be considered in patients with locally advanced tumors or tumors that metastasize to the submandibular node.

A Novel, Tumor-Induced Osteoclastogenesis Pathway Insensitive to Denosumab but Interfered by Cannabidiol.

Bone metabolism is strictly regulated, and impaired regulation caused by hormonal imbalances induces systemic bone loss. Local bone loss caused by tumor invasion into bone is suggested to be induced by the generation of cytokines, which affect bone metabolism, by tumor cells. The major cause of systemic and local bone losses is excess bone resorption by osteoclasts, which differentiate from macrophages by receptor activator of nuclear factor kappa-B ligand (RANKL) or tumor necrosis factor-alpha (TNF-α). We previously found a novel pathway for tumor-induced osteoclastogenesis targeting osteoclast precursor cells (OPCs). Tumor-induced osteoclastogenesis was resistant to RANKL and TNF-α inhibitors. In the present study, we confirmed that exosomes derived from oral squamous cell carcinoma (OSCC) cells induced osteoclasts from OPCs. We also showed that the depletion of exosomes from culture supernatants of OSCC cells partially interfered with osteoclastogenesis, and cannabidiol, an innoxious cannabinoid without psychotropic effects, almost completely suppressed tumor-induced osteoclastogenesis. Osteoclastogenesis and its interference by cannabidiol were independent of the expression of nuclear factor of T cell c1 (NFATc1). These results show that osteoclastogenesis induced by OSCC cells targeting OPCs is a novel osteoclastogenic pathway independent of NFATc1 expression that is partially caused by tumor-derived exosomes and suppressed by cannabidiol.

Homeobox transcriptional factor engrailed homeobox 1 is expressed specifically in normal and neoplastic sweat gland cells.

AIMS: A number of homeobox transcriptional factors are utilised as organ-specific markers in the histopathological diagnosis of neoplasms. We have screened a homeobox gene that is expressed specifically in normal sweat gland cells and is useful for the histopathological diagnosis of sweat gland neoplasms. METHODS AND RESULTS: By screening an open database resource of The Human Protein Atlas, 37 genes among the 235 homeobox transcriptional factors were found to be expressed specifically in the skin. Among those 37 genes, the engrailed homeobox 1 (En1) was expressed in normal eccrine glands but not in the epidermal keratinocytes. Expression of En1 was found throughout the eccrine glands, but not in the apocrine secretory coils, sebaceous glands or hair follicles. Expression of En1 was examined immunohistochemically in 111 cases of cutaneous epithelial neoplasms. All nine cases of poroma, seven cases of spiradenoma and six cases of syringoma, which are considered to differentiate towards eccrine glands, showed positive nuclear staining in most of the tumour cells. Sebaceous gland and hair follicle tumours were immunonegative. En1 was expressed focally in the epidermal neoplasms of seborrheic keratosis and squamous cell carcinoma. CONCLUSION: Engrailed homeobox 1 was expressed specifically in normal eccrine glands and was expressed in most of the tumour cells of sweat gland neoplasms with eccrine gland differentiation. En1 was expressed focally in epidermal neoplasms; however, it was absent in sebaceous or hair follicle neoplasms. These findings will help in the histopathological diagnosis as well as understanding of the histogenesis of sweat gland neoplasms.

A lesion categorized between ghost cell odontogenic carcinoma and dentinogenic ghost cell tumor with CTNNB1 mutation.

Ghost cell odontogenic carcinoma (GCOC) is a rare malignant neoplasm characterized by the presence of ghost cells. It is considered to arise either de novo or from a preexisting benign precursor, calcifying odontogenic cyst (COC), or dentinogenic ghost cell tumor (DGCT). We report a case of a 44-year-old Japanese male with a left maxillary tumor. The patient received treatment to resect the left maxillary cyst 25 years prior; however, the details were uncertain. The tumor was resected with clear margins. Taken together with the results of histological and immunohistochemical examinations, the tumor was categorized between GCOC and DGCT, and we diagnosed the tumor as GCOC suggesting similarity to DGCT. Further, we focused on CTNNB1, which encodes ß-catenin and is frequently mutated in COCs. In this tumor, we identified CTNNB1 Ser33Cys, one of the mutations typically found in COCs. This finding suggests that CTNNB1 is a common target for the pathogenesis of tumors accompanied by ghost cells.

Receptor tyrosine kinase amplification is predictive of distant metastasis in patients with oral squamous cell carcinoma.

This study aimed to clarify the genomic factors associated with the diagnosis and prognosis of oral squamous cell carcinoma via next-generation sequencing. We evaluated data from 220 cases of oral squamous cell carcinoma. Genomic DNA was eluted using formalin-fixed, paraffin-embedded samples, and targeted resequencing of 50 cancer-related genes was performed. In total, 311 somatic mutations were detected in 220 patients, consisting of 68 synonymous mutations and 243 non-synonymous mutations. Genes carrying mutations included TP53, CDKN2A, and PIK3CA in 79 (35.9%), 35 (15.9%), and 19 patients (8.6%), respectively. Copy number analysis detected amplification of PIK3CA and AKT1 in 38 (17.3%) and 11 patients (5.0%), respectively. Amplification of receptor tyrosine kinases was found in 37 patients (16.8%). Distant metastasis was noted in nine of 37 patients (24%) with receptor tyrosine kinase amplification, accounting for 43% of the 21 cases of distant metastasis. The cumulative 5-year survival rate was 64.6% in the receptor tyrosine kinase amplification group vs 85.2% in the no receptor tyrosine kinase amplification group. Moreover, we identified significantly poorer prognosis in the TP53 mutation/receptor tyrosine kinase amplification group, for which the cumulative 5-year survival rate was 41.6%. In conclusion, the results of this study demonstrated that receptor tyrosine kinase amplification is a prognostic factor for distant metastasis of oral squamous cell carcinoma, indicating the necessity of using next-generation sequencing in clinical sequencing.

Cortical Actin Alteration at the Matrix-Side Cytoplasm in Lung Adenocarcinoma Cells and Its Significance in Invasion.

OBJECTIVES: Cortical actin is a thin layer of filamentous (F-)actin that lies beneath the plasma membrane, and its role in pathophysiology remains unclear. We investigated the subcellular localization of cortical actin by the histopathological and experimental studies of lung adenocarcinomas. MATERIALS AND METHODS: The subcellular localization of cortical actin was studied in surgically resected lung adenocarcinomas tissues and in 3-dimensionally cultured lung adenocarcinoma A549 cells. RESULTS: In normal type II alveolar cells and the bronchiolar epithelium, cortical actin was localized to the apical-side cytoplasm. In invasive adenocarcinoma cells, cortical actin was frequently localized to the matrix side. The degree of cortical actin localized to the matrix side was associated with the loss of basement membrane and a poor prognosis. In A549 cell spheroids cultured in a type I collagen and basement membrane extract Matrigel™ mixed gel, cortical F-actin was localized to the matrix side with phosphorylated myosin light chain. Super-resolution and electron microscopy results suggest that compact wrinkling of the plasma membrane by myosin-mediated F-actin contraction is an explanation for cortical actin accumulation at the matrix side. The myosin II inhibitor blebbistatin suppressed the 3-dimensional collective migration of A549 cells induced by constitutively active Cdc42 and MT1-MMP. CONCLUSION: Cortical actin accumulation at the matrix-side cytoplasm of cancer cells occurs in invasive lung adenocarcinomas and it possibly participates in the migration of cancer cells through myosin-mediated contraction.

LAMC2 is a predictive marker for the malignant progression of leukoplakia.

BACKGROUND: LAMC2 plays an important role in cancer invasion. The aim of this study was to (i) compare the immunoexpression of LAMC2 in different stages of oral squamous cell carcinoma (OSCC), early and advanced, and (ii) to evaluate LAMC2 as a marker of malignant transformation in leukoplakia. MATERIALS AND METHODS: The expression of LAMC2 was examined by immunohistochemistry in 50 surgical specimens of advanced OSCC assembled as tissue microarrays, and by cDNA microarray in 43 surgical specimens of advanced OSCC. LAMC2 expression was further examined in 39 surgical specimens of early OSCC and in 93 incisional biopsy specimens of leukoplakia of the tongue, which exhibited epithelial dysplasia. The relationship of LAMC2 expression score with clinico-pathological characteristics was analyzed. RESULTS: LAMC2 was remarkably upregulated in OSCC at the cancer-stroma interface. The grade of LAMC2 expression was significantly associated with the pattern and depth of invasion of OSCC. Foci of LAMC2-positive cells were observed in some cases of leukoplakia. The number and size of LAMC2-positive foci were significantly associated with the grade of dysplasia. The presence of LAMC2-positive foci was a significant predictive factor for the malignant progression of leukoplakia. LAMC2-positive leukoplakia had an approximately 11-fold increased risk of malignancy compared with LAMC2-negative leukoplakia. CONCLUSIONS: The results of this study highlight the value of LAMC2 as a marker of cancer invasion. LAMC2-positive foci in leukoplakia suggest an imminent risk of cancer. LAMC2 immunostaining is expected to contribute to a more precise assessment of the malignancy of leukoplakia.

CD163-Positive Macrophages Within the Tumor Stroma Are Associated With Lymphangiogenesis and Lymph Node Metastasis in Oral Squamous Cell Carcinoma.

PURPOSE: Increasing evidence shows that tumor stromal components, particularly tumor-associated macrophages (TAMs), play an important role in the tumor progression of various solid malignant tumor types. However, their roles in oral squamous cell carcinoma (OSCC) have not been fully elucidated. MATERIALS AND METHODS: Seventy human tongue OSCC samples were analyzed in the present study. Immunohistochemistry was used to investigate the correlations between the densities of CD68-, CD163-, and CD204-positive TAMs and clinicopathologic parameters. Lymphatic vessel density (LVD) was estimated using the D2-40 antibody. In vitro studies also were conducted to investigate the effect of conditioned medium (CM) derived from OSCC cell lines on cytokine and chemokine expression in RAW264.7 mouse monocytic leukemia cells. RESULTS: Increased densities of CD68-, CD163-, and CD204-positive TAMs were significantly correlated with lymph node metastasis (P = .035, .0082, and .038, respectively). Higher LVD occurred considerably more frequently in patients with nodal metastasis than in those without such metastasis. Moreover, LVD was considerably increased in patients with higher CD163-positive TAM densities. Studies using immunofluorescence showed that vascular endothelial growth factor (VEGF)-C was expressed in 52 of 70 patients with CD163-positive TAMs (74.2%). Moreover, CM derived from OSCC cell lines stimulated the expression of Il-10, Ccl22, Vegf-a, and Vegf-c in RAW264.7 cells; however, Il-12p35 expression levels were not changed. CONCLUSION: CD163-positive TAMs promote lymphangiogenesis through VEGF-C expression, which contributes to regional lymph node metastasis in OSCC.