Malignant mesothelioma metastatic to the oral region and latest topics (Review).

Malignant mesothelioma (MM) is a rare neoplasm with poor prognosis that usually develops after exposure to asbestos, and is characterised by aggressive local invasion and metastatic spread. While metastasis to the oral cavity is very rare, a total of 23 cases of MM metastasising to the oral cavity were identifed. Among those, the tongue was the most common site of metastasis (39.1%), and frequently involved the epithelioid MM cell type. Recent studies have elucidated the mechanisms underlying the development of MM. Chronic inflammation has been implicated in promoting MM growth and was shown to play a key role by driving the release of high mobility group box protein 1 following asbestos deposition. Inherited heterozygous germline mutations in the deubiquitylase BRCA-associated protein 1 were shown to increase the incidence of MM in some families. Infection by the simian virus 40 was also found to be associated with the occurrence of MM. Moreover, the increasing incidence rates of MM, together with its propensity to metastasise to the oral cavity, indicate that clinicians and pathologists should be highly aware of this disease. Furthermore, identification of novel serum biomarkers would enable better screening and treatment of MM, and improve the survival outcomes.

Curcumin inhibits epithelial-mesenchymal transition in oral cancer cells via c-Met blockade.

Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer. OSCC cells are highly invasive, a characteristic that involves epithelial-mesenchymal transition (EMT); the conversion of immotile epithelial cells into motile mesenchymal cells. EMT is involved in the progression of various types of cancer by promoting tumour cell scattering and conferring to these cells cancer stem cell (CSC)-like characteristics, such as self-renewal. Hepatocyte growth factor (HGF) signalling plays an important role in EMT induction and, therefore, contributes to cell invasion and metastasis in cancer. Due to its potential chemopreventative and anti-tumour activities, curcumin has attracted much interest and has been shown to act as a potent EMT inhibitor in various types of cancer. However, at present, the potential effects of curcumin on HGF-induced EMT in OSCC have not been investigated. Here, we demonstrated that HGF signalling could induce EMT in the HSC4 and Ca9-22 OSCC cell lines via the HGF receptor c-Met and downstream activation of the pro-survival ERK pathway. Notably, curcumin inhibited HGF-induced EMT and cell motility in HSC-4 and Ca9-22 cells via c-Met blockade. Therefore, these findings establish curcumin as a candidate drug for OSCC treatment. Furthermore, curcumin was able to effectively inhibit the HGF-induced increase in the levels of vimentin by downregulating the expression of phosphorylated c-Met, an ERK. In conclusion, the results of the present study demonstrated that curcumin was able to reverse HGF-induced EMT, possibly by inhibiting c-Met expression in oral cancer cells, providing a strong basis for the development of novel approaches for the treatment of oral cancer.

Ligand-Independent EGFR Activation by Anchorage-Stimulated Src Promotes Cancer Cell Proliferation and Cetuximab Resistance via ErbB3 Phosphorylation.

Activation of the epidermal growth factor receptor (EGFR) pathway plays an important role in the progression of cancer and is associated with a poor prognosis in patients. The monoclonal antibody cetuximab, which displays EGFR extracellular domain-specific binding, has proven effective in the treatment of locally advanced disease and relapsed/metastatic disease. However, the effects of cetuximab are weaker than those of EGFR tyrosine kinase inhibitors (TKIs). This study investigates differences in the effects on cell growth of cetuximab and EGFR TKI AG1478 at the molecular level using oral squamous cell carcinoma (OSCC) cell lines. First, we found that there were EGFR-inhibitor-sensitive (EIS) and EGFR-inhibitor-resistant cell lines. The EIS cell lines expressed not only EGFR but also ErbB3, and both were clearly phosphorylated. The levels of phosphorylated ErbB3 were unaffected by cetuximab but were reduced by AG1478. EGFR ligand treatment increased the levels of phosphorylated EGFR but not phosphorylated ErbB3. Moreover, when EIS cell lines that were only capable of anchorage-dependent growth were grown in suspension, cell growth was suppressed and the levels of phosphorylated focal adhesion kinase (FAK), Src, and ErbB3 were significantly reduced. The levels of phosphorylated ErbB3 were unaffected by the FAK inhibitor PF573228, but were reduced by Src inhibition. Finally, combining cetuximab and a Src inhibitor produced an additive effect on the inhibition of EIS cell line growth.

Molecularly-targeted therapy for the oral cancer stem cells.

Human cancer tissues are heterogeneous in nature and become differentiated during expansion of cancer stem cells (CSCs). CSCs initiate tumorigenesis, and are involved in tumor recurrence and metastasis. Furthermore, data show that CSCs are highly resistant to anticancer drugs. Cetuximab, a specific anti-epidermal growth factor receptor (EGFR) monoclonal antibody, is used in cancer treatment. Although development of resistance to cetuximab is well recognized, the underlying mechanisms remain unclear. Lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR)/ErbB2, has antiproliferative effects and is used to treat patients with ErbB2-positive metastatic breast cancer. In this review, cetuximab and lapatinib-resistant oral squamous cell carcinoma (OSCC) cells proliferation and migration signal transduction passway is discussed by introducing our research.

Migration of oral squamous cell carcinoma cells are induced by HGF/c-Met signalling via lamellipodia and filopodia formation.

The activation of receptor tyrosine kinases (RTKs) results in cellular effects including cell proliferation, survival, migration and invasion; RTKs also play an important role in tumourigenesis. It has been reported that EGFR signalling controls the migration of oral squamous cell carcinoma (OSCC) SAS and HSC3 cells but not of HSC4 cells, although the proliferation of HSC4 cells is regulated by EGF/EGFR. In the present study, we investigated the roles of EGFR and the c-Met signalling pathway in cell migration via filopodia and lamellipodia formation, which may be prerequisites for migration. To explore the role of c-Met in cell migration, we inhibited c-Met RTK activity using the c-Met inhibitor SU11274 and activated c-Met using hepatocyte growth factor (HGF) in three OSCC cell lines HSC4, SAS and Ca9-22 and investigated migration potency using a wound healing assay. We showed that inhibition of c-Met significantly suppressed, and activation of c-Met significantly promoted, the migration of OSCC cells. Additionally, the migration of SAS and Ca9-22 cells was inhibited by the EGFR inhibitors AG1478 and cetuximab and promoted by EGF treatment. Moreover, migration potency was correlated with lamellipodia formation. Furthermore, western blot analyses demonstrated that SU11274 decreased and HGF increased lamellipodin protein levels as well as phosphorylated c-Met levels. Collectively, we demonstrated that c-Met signalling induced lamellipodia formation by upregulating lamellipodin, thereby promoting the migration of OSCC cells.

Regulation of cell migration via the EGFR signaling pathway in oral squamous cell carcinoma cells.

Cell migration potency is essential in cancer metastasis and is often regulated by extracellular stimuli. Oral squamous cell carcinoma cell lines include those that are sensitive, as well as resistant, to the effects of the epidermal growth factor receptor (EGFR) inhibitor cetuximab on cell migration. In the present study, the molecular differences in the EGFR response to cell migration between the SAS cetuximab-sensitive and HSC4 cetuximab-resistant cell lines was examined. Treatment with the EGFR inhibitors AG1478 and cetuximab reduced the migration potency of SAS cells, but not HSC4 cells. The migration of the two cell lines was inhibited under serum-free culture conditions, and the addition of EGF to the serum-free medium promoted the migration of SAS cells, but not HSC4 cells. In addition, SAS cell migration was reduced by the mitogen-activated protein kinase kinase and protein kinase B (Akt) inhibitors PD98059 and MK2206, whereas HSC4 cell migration was only inhibited by MK2206. EGF induced an increase in extracellular signal-regulated kinase phosphorylation levels in HSC4 cells, and stimulated Akt phosphorylation levels in SAS cells. Furthermore, the staining of actin filaments with phalloidin was significantly increased by the inhibition of EGFR in SAS cells, but was not observed as altered in HSC4 cells. Conversely, the addition of EGF to the culture medium decreased the accumulation of actin filaments in SAS cells. The results suggest that the EGF-EGFR signaling pathway has an important role in SAS cell migration via the modulation of actin dynamics, and that HSC4 cell migration is regulated by a serum component other than EGFR.

Lapatinib-resistant cancer cells possessing epithelial cancer stem cell properties develop sensitivity during sphere formation by activation of the ErbB/AKT/cyclin D2 pathway.

Lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR)/ErbB2, has antiproliferative effects and is used to treat patients with ErbB2-positive metastatic breast cancer. In the present study, we examined the effects of lapatinib on growth of oral and prostate cancer cells. Oral squamous cell carcinoma (OSCC) cell lines HSC3, HSC4 and Ca9-22 were sensitive to the antiproliferative effects of lapatinib in anchorage-dependent culture, but the OSCC cell lines KB and SAS and the prostate cancer cell line DU145 were resistant to lapatinib. Phosphorylation levels of EGFR in all cell lines decreased during lapatinib treatment in anchorage­dependent culture. Furthermore, the phosphorylation levels of ErbB2, ErbB3 and Akt and the protein levels of cyclin D1 were decreased by lapatinib treatment of HSC3, HSC4 and Ca9-22 cells. ErbB3 was not expressed and cyclin D1 protein levels were not altered by lapatinib treatment in KB, DU145 and SAS cells. The phosphorylation of ErbB2 and AKT was not affected by lapatinib in SAS cells and was not detected in KB and DU145 cells. Lapatinib-resistant cell lines exhibited sphere-forming ability, and SAS cells developed sensitivity to lapatinib during sphere formation. The phosphorylation levels of ErbB2 and AKT and protein levels of cyclin D2 increased during sphere formation of SAS cells and decreased with lapatinib treatment. In addition, sphere formation of SAS cells was inhibited by the AKT inhibitor MK2206. AKT phosphorylation and cyclin D2 levels in SAS spheres were decreased by MK2206 treatment. SAS cells expressed E-cadherin, but not vimentin and KB cells expressed vimentin, but not E-cadherin. DU145 cells expressed vimentin and E-cadherin. These results suggested that phosphorylation of EGFR and ErbB2 by cell detachment from the substratum induces the AKT pathway/cyclin D2-dependent sphere growth in SAS epithelial cancer stem-like cells, thereby rendering SAS spheres sensitive to lapatinib treatment.

Usefulness of a fluorescence visualization system for the detection of oral precancerous and early cancerous lesions.

Early detection of precancerous and early cancerous lesions could greatly reduce both the mortality and morbidity of oral cancer. The objective of this study was to analyze a fluorescence visualization (FV) system for the detection of precancerous and early cancerous lesions in rat tongue carcinogenesis and human oral cancerous lesions using for the first time a 4NQO rat model and human tissue. Based on the results from the rat tongue carcinogenesis model, under direct FV, the normal oral mucosa emitted various shades of pale green autofluorescence. In the precancerous and early cancerous cases, the lesion appeared as an irregular dark area. Histological examination of the lesions showed that the VELscope system had a sensitivity of 95% and specificity of 100% in discriminating normal mucosa from dysplasia/carcinoma in situ (CIS) or invasive carcinoma. The proliferating cell nuclear antigen (PCNA) protein level was gradually increased with progression of carcinogenic transformation. Furthermore, the results of PCNA and FV loss (FVL) were correlated. Next, results from 17 patients were also presented. Histological examination of the lesions showed that the VELscope system had a sensitivity of 95% and specificity of 100% in discriminating normal mucosa from severe dysplasia/CIS or invasive carcinoma. There were no normal epithelium cells in any of the FVL regions. Furthermore, to clarify the usefulness of FV compared to vital staining with iodine, we investigated the surgical margins of early oral squamous cell carcinoma (OSCC) tissues and compared the FVL and iodine unstained area (IU). The percentage of various types of dysplasia were almost equal when comparing the FVL and IU. These results suggest that this direct FV device has the potential for simple, cost-effective screening, detection and margin determination of oral precancerous and early cancerous lesions.

Papillary oncocytic cystadenoma of a palatal minor salivary gland: A case report.

Papillary cystadenomas of the salivary gland are uncommon, benign, encapsulated or well-circumscribed, multicystic tumors with intracystic papillations. In a large review, papillary cystadenoma constituted 2% of all minor salivary gland tumors. The present study reports an extremely rare case of a papillary cystadenoma arising from the palate that demonstrated oncocytic features. A 60-year-old man was referred by his dentist to the Second Department of Oral and Maxillofacial Surgery at Osaka Dental University Hospital for the diagnostic evaluation of a mass of the left palate. An incisional biopsy was performed and the microscopic findings were interpreted as consistent with a papillary oncocytic cystadenoma. Therefore, the lesion was excised under general anesthesia. The post-operative course was uneventful and no recurrence had developed 5 years subsequent to surgery.

Cetuximab-resistant oral squamous cell carcinoma cells become sensitive in anchorage-independent culture conditions through the activation of the EGFR/AKT pathway.

We have previously shown that growth of the oral squamous cell carcinoma cell line SAS, is resistant to cetuximab in monolayer culture conditions, even though epidermal growth factor receptor (EGFR) was phosphorylated, but the growth of SAS aggregates was sensitive to cetuximab. In the present study, we demonstrate differences in the EGFR signaling pathways utilized by SAS cells in monolayer and suspension cultures at the molecular level. Cetuximab treatment of SAS cells in monolayer cultures inhibits the phosphorylation of EGFR and ERK, and reduces the cell migratory potency, but not cell proliferation. AG1478 treatment reduces the phosphorylation of EGFR, ERK and AKT, and affects cell growth in monolayer cultures. The phosphorylation levels of EGFR and AKT are significantly higher in SAS cell aggregates compared to monolayer cultures. Treatment with cetuximab and AG1478 reduces the growth of SAS aggregates and eliminates the phosphorylation of EGFR and AKT. Furthermore, proliferation of SAS aggregates is also inhibited by LY294002 and MK2206, which are inhibitors of PI3K and AKT, respectively. In addition, treatment with the lipid raft disruptor filipin III reduced the phosphorylation levels of EGFR and Akt in SAS aggregates, but not in SAS monolayer culture. These results suggest the possibility that ligands in the serum stimulate the phosphorylation of EGFR localized in lipid rafts leading to PI3K-AKT activation, which results in the growth of SAS aggregates, therefore resulting in the sensitivity of SAS aggregates to cetuximab.

Infiltrating angiolipoma of the lower lip: A case report and literature review.

Infiltrating angiolipoma (IAL) is a rare lesion and is a clinicopathological variant of angiolipoma. IAL occurs most commonly in the trunk and extremities, it is rarely found in the head and neck regions and extremely rare in the oral cavity. This study presents the case of a 74-year-old female with IAL of the lower lip. To the best of our knowledge, this is the first case of IAL arising in the lower lip to be reported. Microscopically, IAL was unencapsulated and mature lipocytes were separated by a branching network of proliferating small vessels that infiltrated the adjacent tissues. Therefore, complete excision was difficult to perform. Magnetic resonance imaging has been reported to be valuable in determining the extent of the tumor and asserting a preoperative diagnosis. According to previous studies, the recurrence rate of IAL following surgical extirpation is 35-50%. Furthermore, the levels of mRNA expression of the vascular endothelial growth factor (VEGF) family members in the tumor were investigated. VEGF-A and -B expression were detected, however, VEGF-C and -D were expressed at extremely low levels. Excisional biopsy was performed under local anesthesia. During four years of follow-up, no evidence of tumor recurrence had been identified. An operating microscope may be utilized for the total removal of an IAL to minimize damage to normal tissues. This report indicates that mast cell-derived VEGF may be responsible for the enhanced vascularity in the tumor. We would therefore consider careful extirpation with no wide safety margin to be the procedure of choice, except when the tumor invades irregularly into the muscles.

Isolation and propagation of neural crest stem cells from mouse embryonic stem cells via cranial neurospheres.

The developmental fate of the multipotent neural crest (NC) is determined along with the neural axis in which NC cells are generated. Only the cranial NC can differentiate into mesectodermal derivatives such as osteoblasts, chondrocytes, and adipocytes in vivo. Here, we attempted to selectively differentiate mouse embryonic stem (ES) cells into cranial NC stem cells and propagate them to explore their developmental potential to differentiate into mesectodermal derivatives. Using aggregation cultures in feeder- and serum-free neural induction medium (NIM) without serum replacement and l-glutamine, we obtained NIM neurospheres composed of neuroepithelium. The NIM neurospheres expressed the rostral markers Otx1 and Otx2, but not nonrostral markers Hoxb4, Hoxb9, Lbx1, and TH, which characterize cranial neurospheres. Subsequently, AP2α, Sox9, p75, Snail, Slug, and Twist-positive NC cells were differentiated in 4-day adhesion cultures of cranial neurospheres. In addition, sphere clusters in adhesion cultures were differentiated into osteoblasts, while migrating cells were not. By taking advantage of the sphere-formation capability, we isolated and propagated NC stem cells from the sphere clusters and confirmed their multipotency. NC stem cells expressed NC and stem cell markers, and they maintained differentiation potency in the NC derivatives. These results show that cranial NC stem cells were obtained reproducibly and efficiently without special inducing factors, gene transfection, or fluorescence-activated cell sorting selection.

A rare case of amelanotic malignant melanoma in the oral region: Clinical investigation and immunohistochemical study.

Amelanotic malignant melanoma (AMM) is rare in the oral region. The present study examined the clinical features of this tumor in an attempt to establish diagnostic criteria. The expression of three melanocytic differentiation markers, HMB-45, S-100 and Melan-A, was also measured in primary oral AMMs in order to determine whether the markers could be used to diagnose primary oral AMMs and to find out which marker was the most sensitive. It may be particularly difficult to correctly diagnose AMM that lacks a radial growth phase without immunohistochemical assistance. In the present study, mixtures of polygonal and spindle cells at different ratios were observed in the tumors with and without a radial growth phase. Immunohistochemistry was used to examine the HMB-45, S-100 and Melan-A expression in the formalin-fixed paraffin-embedded specimens of primary oral AMMs. Comparison of staining intensities (SIs) and labeling indices (LIs) of the markers was also performed. The immunostaining results revealed that the SI of Melan-A was significantly higher than that of S-100 (P=0.0011). HMB-45, S-100 and Melan-A also exhibited high positive rates and LIs in AMMs and, therefore, may be good markers for the immunohistochemical diagnosis of primary oral AMMs. Furthermore, Melan-A may be a more sensitive marker than S-100 and HMB-45, as it has a higher SI.

Metastasis of mesothelioma to the maxillary gingiva.

Malignant mesothelioma predominantly arises from the serosal surfaces of the pleural or peritoneal cavity. There is currently no effective standard treatment for mesothelioma and the prognosis for patients is poor; the majority of patients with malignant mesothelioma succumb between 12 and 17 months following diagnosis. The association of all forms of malignant mesothelioma with asbestos exposure has been well documented. However, metastasis to the oral gingiva is rare, as only four cases have previously been reported; two cases of metastasis to the tongue and four cases to the jaw bone. In the current report, the case of a 62-year-old male with metastatic mesothelioma is presented. To the best of our knowledge, this is the first report regarding the metastasis of this type of neoplasm to the maxillary gingiva.

Cyclin D1 expression is correlated with cell differentiation and cell proliferation in oral squamous cell carcinomas.

The present study conducted an immunohistochemical investigation of cyclin D1 and Ki-67 expression in oral squamous cell carcinoma (SCC) to evaluate the correlations between cell differentiation, cell proliferation and metastasis, and the effect of anticancer drug medication and cyclin D1 expression. Cyclin D1 and Ki-67 were detected clearly in the nuclei of 35 SCC samples. No correlation between cyclin D1 protein expression and oral SCC differentiation was found. By contrast, the majority of metastatic foci (90%) exhibited strong cyclin D1 expression, whereas weak expression was observed in metastatic foci with pre-operative adjuvant therapy. Additionally, cyclin D1 and Ki-67 were expressed in basal to suprabasal cells of well-differentiated oral SCC, whereas cyclin D1-positive and Ki-67-negative cells were present in the highly-differentiated region, according to a double-immunostaining method. These results indicate that the expression of cyclin D1 protein plays a role in cell differentiation and cell proliferation in well-differentiated oral SCC.

Resistance of oral squamous cell carcinoma cells to cetuximab is associated with EGFR insensitivity and enhanced stem cell-like potency.

Cetuximab, a specific anti-epidermal growth factor receptor (EGFR) monoclonal antibody, is used in cancer treatment. Although development of resistance to cetuximab is well recognized, the underlying mechanisms remain unclear. In the present study, we characterized cetuximab-resistant oral squamous cell carcinoma (OSCC) cell lines. The human OSCC cell lines HSC3, HSC4 and SAS were used in the present study. Effects of inhibitors including cetuximab on growth in cells were assessed by MTT assays. Southern blotting and immunofluorescence analysis were performed to examine protein expression and localization. Sphere formation was used to characterize stem cell-like properties. Floating aggregation culture was used for anchorage-independent growth. Cetuximab inhibited proliferation of HSC3 and HSC4 cells, but not SAS cells. Proliferation of all three cell lines was inhibited by the EGFR/ErbB2/ErbB4 inhibitor II. The EGFR inhibitor AG1478 strongly inhibited HSC3 and HSC4 proliferation, but that of SAS cells only moderately. EGFR proteins were localized on cell surface and phosphorylated in all three cell lines. SAS cells could proliferate in serum-free monolayer culture and formed spheres from single cells in floating culture. HSC3 and HSC4 could not proliferate under serum-free culture conditions and could not form spheres. Growth of SAS spheres required serum, and was inhibited by both AG1478 and cetuximab. Thus, cetuximab-resistant SAS cells not only engaged in EGFR-independent growth but also exhibited stem cell-like properties. However, growth was EGFR-dependent in aggregation culture, and the SAS cell aggregates became cetuximab-sensitive. This suggests that cetuximab sensitivity is not only cell-type-dependent but is also affected by the growth microenvironment.

Effects of epidermal growth factor on the invasive activity and cytoskeleton of oral squamous cell carcinoma cell lines.

Epidermal growth factor (EGF) is present at high concentrations in human saliva and modulates the growth and differentiation of various cancer cells. To elucidate the molecular mechanisms by which EGF affects oral cancer proliferation and invasion, the current study analyzed the Matrigel invasion activity of cultured oral cancer cell lines. Cell proliferation under the influence of EGF was subjected to Matrigel invasion assays, and cell proliferation in the absence of EGF was used as control. Northern blot analyses quantified the invasiveness and tumorigenicity. Chloramphenicol acetyltransferase assay determined the EGF stimulation of matrix metalloproteinase (MMP) 1 expression. EGF increased the number of cells penetrating the Matrigel membrane. Northern blot analysis revealed that MMP1 and cytokeratin 19 expression correlate with EGF. In addition, the morphology of HSC-3 and SAS cells changed following the addition of EGF to the culture medium. A transient transfection assay revealed that EGF increases the promoter activities of MMP1 in HSC-3 cells. These observations suggested that EGF increases the invasive activity of oral cancer cells, partly by increasing MMP1, and morphological changes may be induced by altering the composition of cytoskeletal proteins.

SOX4 expression is closely associated with differentiation and lymph node metastasis in oral squamous cell carcinoma.

SOX4 is a member of the SOX family of transcription regulators. In recent years, SOX4 was shown to be overexpressed in cancers of various organs and related to epithelial-mesenchymal transition, which is a metastatic factor. This study was the first to investigate correlations between SOX4 expression levels and the clinicopathologic factors of oral squamous cell carcinoma (OSCC). We analyzed SOX4 expression levels in 50 patients with OSCC using immunohistochemistry. All samples expressed the SOX4 protein and elevated SOX4 expression was significantly correlated with gender, T status, and stage levels. The expression level of SOX4 in primary foci of poorly differentiated OSCC was higher than that of well differentiated OSCC, which indicated that SOX4 expression is associated with the differentiation of OSCC. However, regardless of the differentiation level in the primary focus, SOX4 expression levels were found to be very high in the metastatic focus. Furthermore, SOX4 expression in metastatic foci was significantly suppressed by neoadjuvant therapy. These results indicate that undifferentiated OSCC cells expressing SOX4 are more likely to metastasize and neoadjuvant therapy including chemoradiation therapy may have some effect in metastatic prevention.

NANOG expression correlates with differentiation, metastasis and resistance to preoperative adjuvant therapy in oral squamous cell carcinoma.

NANOG protein, a transcription factor expressed in embryonic stem cells, is overexpressed in tumor development. Although studies investigating the function of NANOG in cancer have shown that it plays several roles, such as in cell proliferation, invasion and metastasis, the overall function of NANOG in cancer cells has remained elusive. In the present study, NANOG expression in oral squamous cell carcinoma (OSCC) was examined to determine its potential clinical significance. The expression of NANOG protein was assessed in 60 patients with OSCC by immunohistochemistry, and its correlation with clinicopathological factors and metastasis was evaluated. NANOG protein levels in human OSCC cell lines were determined by western blotting and immunofluorescence staining. NANOG protein expression was identified in 52 cases (86.7%) and expression levels were higher in primary foci of poorly differentiated OSCC than in those of well-differentiated OSCC, indicating that NANOG expression is associated with OSCC differentiation. Regardless of the differentiation levels of primary foci, NANOG expression levels in metastatic foci were extremely high. In addition, NANOG expression in metastatic foci was maintained at high levels following preoperative adjuvant therapy. Furthermore, NANOG protein was detected at an identical level in human OSCC cell lines. These data indicate that NANOG-expressing OSCC cells tend to metastasize and that metastatic tumors expressing NANOG may be resistant to preoperative adjuvant therapy, including chemoradiation. Thus, assessment of NANOG expression may assist the strategy for treatment of OSCC metastasis.

Effects of CRM197, a specific inhibitor of HB-EGF, in oral cancer.

CRM197, a nontoxic mutant of diphtheria toxin, is a specific inhibitor of heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), which belongs to the EGF family that has been implicated in the increased progression, proliferation, and metastasis of oral cancer. In this study, we analyzed the antitumor effects of CRM197, which represent possible chemotherapeutic agents for oral cancer. In the experiment, we used the oral squamous cell carcinoma cell lines HSC3 and SAS. Cells treated with CRM197 were analyzed based on cell viability, MTT assay, invasion assay, Western blot, and zymography. HSC3 cells were injected subcutaneously into female BALB/c nu/nu mice at 5 weeks of age. CRM197 and/or CDDP were injected intraperitoneally into tumor-bearing mice, and tumor volume was measured over time. HB-EGF expression in HSC3 and SAS cells treated with CRM197 was significantly reduced and cell proliferation was inhibited. The invasiveness of CRM197-treated cells was relatively low. MMP-9 and VEGF were suppressed in HSC3 treated with CRM197 on zymography and Western blot. Further, tumor growth in xenografted mice was suppressed by CRM197 or CDDP at 1 mg/kg/day. Also, the coadministration of CDDP and CRM197 at 1 mg/kg/day completely inhibited tumor formation. These results suggest that HB-EGF is a target for oral cancer and that CRM197 is effective in oral cancer therapy.