Oral Squamous Cell Carcinoma Tumor Progression Research Articles

Autoregulation of RANK ligand in oral squamous cell carcinoma tumor cells.

Oral squamous cell carcinoma (OSCC) is the most common malignancy among oral cancers and shows potent activity for local bone invasion. Receptor activator of nuclear factor κB (RANK) ligand (RANKL) is critical for bone-resorbing osteoclast formation. We previously demonstrated that OSCC tumor cells express high levels of RANKL. In this study, confocal microscopy demonstrated RANKL specific receptor, RANK expression in OSCC tumor cell lines (SCC1, SCC12, and SCC14a). We also confirmed the expression of RANK and RANKL in primary human OSCC tumor specimens. However, regulatory mechanisms of RANKL expression and a functional role in OSCC tumor progression are unclear. Interestingly, we identified that RANKL expression is autoregulated in OSCC tumor cells. The RANKL specific inhibitor osteoprotegerin (OPG) treatment to OSCC cells inhibits autoregulation of RANKL expression. Further, we showed conditioned media from RANKL CRISPR-Cas9 knockout OSCC cells significantly decreased osteoclast formation and bone resorption activity. In addition, RANKL increases OSCC tumor cell proliferation. RANKL treatment to OSCC cells demonstrated a dose-dependent increase in RANK intracellular adaptor protein, TRAF6 expression, and activation of IKK and IκB signaling molecules. We further identified that transcription factor NFATc2 mediates autoregulation of RANKL expression in OSCC cells. Thus, our results implicate RANKL autoregulation as a novel mechanism that facilitates OSCC tumor cell growth and osteoclast differentiation/bone destruction.

Abstract 3933: Characterization of oncogenic activity of interferon-induced protein with tetratricopeptide repeats 1 and 3 in human oral squamous cell carcinoma progression

Abstract Interferon-induced protein with tetratricopeptide repeats (IFITs) family has multiple tetratricopeptide repeats with helix-turn-helix structural motifs that mediate a variety of protein-protein interactions. There are four IFIT genes that have been identified namely, ISG56/IFIT1, ISG54/IFIT2, ISG60/IFIT3, and ISG58/IFIT5. IFIT proteins have been documented to involve in a variety of cellular functions, such as cell proliferation, migration, virus-induced translation initiation, replication and double-stranded RNA signaling. Our previous study has demonstrated that IFIT2 inhibits oral squamous cell carcinoma (OSCC) metastasis. However, little is known about IFIT1 and IFIT3 in the progression of OSCC. In the present study, we are investigating the functional role of IFIT1 and IFIT3 genes in OSCC tumor progression. Immunohistochemical analysis of 147 patients derived tumor specimens with OSCC revealed a strong correlation between IFIT1 and IFIT3 proteins (p<0.001). Clinicoplathological characteristics correlation of IFIT1 and IFIT3 expression levels were positively associated with regional lymph node invasion and nerve invasion. IFIT1 was also associated with venous invasion. Ectopic expression of IFIT1 and IFIT3 proteins in Ca9-22 and SAS cells inducing PKC, EGFR, and AKT activation, resulting in OSCC cells resistance to cisplatin but highly susceptible to tyrosine kinase inhibitor gefitinib, whereas IFIT1 and IFIT3 knockdown SCC25 cells have shown opposite effect. Interestingly, IFIT1 and IFIT3 expression is associated with enhanced cell invasion activity and altered expression of epithelial-mesenchymal transition markers. These data suggested that IFIT1 and IFIT3 may serve as potential biomarkers for the OSCC treatment. The study on how IFIT1 and IFIT3 cause PKC, EGFR, and AKT activation and resistance to DNA damaging agents is ongoing. Our further investigations may explain the molecular signaling mechanism of IFIT1 and IFIT3 in OSCC tumor progression. Citation Format: Vijaya Kumar Pidugu, Ai-Hsin Yen, Ying-Chen Chen, Mei-Maan Wu, Chung-Ji Liu, Te-Chang Lee. Characterization of oncogenic activity of interferon-induced protein with tetratricopeptide repeats 1 and 3 in human oral squamous cell carcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3933. doi:10.1158/1538-7445.AM2017-3933

Calreticulin is a novel independent prognostic factor for oral squamous cell carcinoma.

We focused on the expression of Calreticulin (CALR) in oral squamous cell carcinoma (OSCC) on the basis of proteomic differential display analysis data. We used QR-32 cells in this study which is a regressive murine fibrosarcoma cell clone; and QRsP-11, a progressive malignant tumor cell clone originated from QR-32. CALR is an endoplasmic reticulum luminal Ca2+-binding chaperone protein, which is thought to affect the tumor behavior of various malignancies. This study was aimed to determine the usefulness of CALR as a prognostic factor in patients with OSCC. We investigated the expression of CALR in tissue samples taken from 111 OSCC patients by immunohistochemistry, and we also analyzed the relationship between CALR expression and patients' clinicopathological characteristics as well as patient survival. Positive immunohistchemical staining of CALR was observed in the cancer cell cytoplasm. Among 111 patients, high expression of CALR was observed in 44 patients (39.6%), whereas low expression was observed in 67 patients (60.4%). Significant association was found between CALR expression and T classification (P=0.0027), N classification (P=0.0219), stage (P=0.0013), and patient outcome (P=0.0014). Log-rank test showed that, there is a significant difference (P<0.0001) in 5-year survival rates between patients showing CALR high-expression (59.1%) and CALR low-expression (86.6%). According to our Multivariate analysis, reduced term survival of patients was correlated to high levels of CALR expression (P<0.0001). Our findings suggest that elevated expression of CALR might play an important role in tumor progression in OSCC, and could be considered as a useful prognostic factor in OSCC patients.

Clinical Significance and Roles in Angiogenesis of Circulating Microparticles in Oral Cancer

Our recent study established the increased circulating microparticles (MPs) and their procoagulant activity in oral squamous cell carcinoma (OSCC). In the present study, we further evaluated different phenotypes of circulating MPs in OSCC patients and explored their clinical significance and effects on angiogenesis (a critical event in tumor progression). To conduct the study, circulating MPs in 45 OSCC patients and 18 healthy volunteers were characterized and quantified by transmission electron microscopy and flow cytometry. Correlations between circulating MPs and clinicopathologic data, microvessel density, and proangiogenic factor levels in patients with OSCC were analyzed by immunohistochemistry and Spearman rank correlation test. Additionally, the in vitro studies were performed with use of human umbilical vein endothelial cells. Our results showed that the levels of circulating MPs as well as the subsets of platelet-derived, endothelium-derived, and pan-leukocyte MPs in stages III to IV OSCC were significantly higher than stages I to II and healthy subjects. Moreover, these increased circulating MPs were significantly correlated with tumor size, TNM stages, microvessel density, and expression levels of vascular endothelial growth factor (VEGF) and matrix metallopeptidase 9 (MMP9) in OSCC patients. The in vitro studies revealed that circulating MPs isolated from OSCC patients could be effectively taken up by human umbilical vein endothelial cells and could promote the proliferation, migration, invasion, and tube formation of recipient endothelial cells, accompanied by increased expression of proangiogenic factors. In summary, circulating MPs play important roles in angiogenesis and local tumor progression of OSCC. Our results shed new light on the progression of OSCC and might be helpful to explore novel treatment strategies targeting tumor angiogenesis.

RANK Ligand Modulation of Autophagy in Oral Squamous Cell Carcinoma Tumor Cells.

Autophagy is a cellular process to recycle nutrients and has been implicated in cancer treatment. Oral squamous cell carcinoma (OSCC) is the most common oral cancer which ranks 3% of cancers in men and 2% in women. In this study, immunohistochemical staining of OSCC tumor specimens from human subjects and an athymic mouse model demonstrated high levels of autophagy markers LC3-II and ATG5 expression. Further, we identified high levels LC3-II expression in OSCC tumor cell lines (SCC-1, SCC-12, and SCC-14a) compared to normal human epithelial (RWPE-1) cells. OSCC cells express high levels of RANK ligand (RANKL); however, a functional role in autophagy is unknown. Interestingly, RANKL stimulation significantly increased autophagosome-related gene expressions such as LC3, ATG5, BECN1, and PI3KC3 mRNA expression in OSCC cells. Further, Western blot analysis of total cell lysates demonstrated a dose-dependent increase in LC3-II and ATG5 expression in RANKL-stimulated cells. In addition, RANKL increased expression of LC3-I and LC3-II, essential for autophagosome formation. Confocal microscopy analysis of LC3-II and localization with lysosome further confirms autophagosome formation in response to RANKL treatment in OSCC cells. Collectively, our results indicate a novel function of RANKL to induce autophagosome formation, and could be a potential therapeutic target to control OSCC tumor progression.

Association of immunoexpression of the galectins-3 and -7 with histopathological and clinical parameters in oral squamous cell carcinoma in young patients.

An increasing incidence of oral squamous cell carcinoma (OSCC) in individuals younger than 45 years has been observed in recent years. OSCC in younger patients differs in terms of biological behavior and prognosis with the disease being more aggressive than in older patients. The aim of this study was to analyze the immunohistochemical expression of galectins-3 and -7 in 32 cases of OSCC in young patients and to correlate this expression with clinical and morphological parameters. All cases of OSCC of the sample were diagnosed at oncology referral hospitals in Paraíba, Brazil, between 2002 and 2012. Clinical data were obtained from the patient records. Histological malignancy grading systems proposed by Bryne et al. (J Pathol 166:375-381, 1992) and the World Health Organization (In: Pathology and genetics of head and neck tumours: Word Health Organization classification of tumours, 2005) were used for morphological analysis. Immunohistochemistry was performed by the streptavidin-biotin technique using anti-galectin-3 and -7 antibodies. The results were analyzed statistically by the Chi-squared and Fisher exact tests (p < 0.05). Immunoexpression of galectin-3 was observed in 65.6 % of the cases analyzed, but showed no significant association with any of the variables studied (clinical staging; histological malignancy grading systems). Immunoexpression of galectin-7 was observed in 96.9 % of cases and was significantly associated with histological malignancy grading systems (p < 0.05). In conclusion, the results suggest the use of galectin-7 as marker of biological behavior and tumor progression in OSCC in young patients.

MFG-E8 expression for progression of oral squamous cell carcinoma and for self-clearance of apoptotic cells

Milk fat globule—epidermal growth factor (EGF)—factor VIII (MFG-E8) is a secreted glycoprotein that promotes clearance of apoptotic cells by bridging phosphatidylserine on apoptotic cells and integrin αvβ3/5 on phagocytes. High expression of MFG-E8 has been reported in various types of cancer in humans. Apoptotic figures are frequently found in the surgical samples of oral squamous cell carcinoma (SCC) and carcinoma in situ, and we have often observed apoptotic carcinoma cells engulfed by macrophages or even by neighboring carcinoma cells. Thus we hypothesized that MFG-E8 might promote engulfment of apoptotic carcinoma cells by living carcinoma cells and that MFG-E8 expressed by carcinoma cells could contribute to tumor progression. The aim of this study was to elucidate the biological role of MFG-E8 in oral SCC. Fifty-three surgical specimens of oral SCC were used for immunohistochemistry for MFG-E8, and the expression profiles were correlated with clinicopathological properties. Also, we examined the MFG-E8 expression patterns and functions using three human oral SCC cell lines. Most of the cases had MFG-E8-positive SCC cells, and the expression of MFG-E8 was correlated with such clinicopathological features as tumor size, pathological stage, locoregional recurrence, scattering invasion pattern, and SCC cell figures engulfing apoptotic SCC cells. The MFG-E8 staining was enhanced in apoptotic SCC cells, some of which were apparently engulfed by the neighboring SCC cells. ZK-1 cells showed high MFG-E8 expression, and its localization was found in the cytoplasm and the cell surface. Transient MFG-E8 knockdown by siRNA in ZK-1 decreased cell proliferation and invasiveness and increased cell death. Thus we have demonstrated that MFG-E8 promotes tumor progression in oral SCC and that it might be involved in the clearance of apoptotic SCC cells by living SCC cells.

Abstract 1487: Identification of oral carcinoma miRNA signature and control OSCC tumorigenesis through Wnt/β-catenin signaling

Abstract Aberrant microRNAs (miRNAs) expressions are often happened in cancer. In order to identify miRNAs altered in oral carcinogenesis, we investigated miRNAs and cDNA expression profiles in 40 pair-wise oral squamous cell carcinoma (OSCC) specimens. Eighty-four miRNAs were differentially expressed in the OSCC specimens compared to the matched tissue. Interestingly, 19 down-regulated miRNAs are clustered in DLK-1/Meg3 imprinted domain of chromosome 14q32.2 region. Loss of 14q32.2-miRNAs expression also raised the 14q32.2-miRNA targets expression in OSCC. We identified one target protein, Wnt-7b, was highly expressed in OSCC and regulated by miR-329 and -410. Wnt-7b increased the Wnt/β-catenin signaling activity and promoted OSCC tumor progression. Furthermore we also identify the regulation mechanism that down-regulated 14q32.2 miRNAs. Hyper-methylation in meg-3 differential methylated region (DMR) was confirmed by bisulfate sequencing. The hyper-methylation of meg-3 DMR was also induced by betel nuts extract exposure. In summary, we identified the expression signature of aberrant miRNAs in OSCC and provide a novel molecular insight how betel quid contribute to oral carcinogenesis through the epigenetic silencing of tumor suppressor miRNAs targeting the Wnt/β-catenin signaling pathway. Note: This abstract was not presented at the meeting. Citation Format: Wei-Min Chang, Yuan-Ming Hsu, Sung-Tau Chou, Yi-Shing Shieh, Michael Hsiao, Jenn-Ren Hsiao, Jang-Yang Chang, Shine-Gwo Shiah. Identification of oral carcinoma miRNA signature and control OSCC tumorigenesis through Wnt/β-catenin signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1487. doi:10.1158/1538-7445.AM2014-1487

The expression and functional role of a FOXC1 related mRNA-lncRNA pair in oral squamous cell carcinoma.

The Fork head box C1 (FOXC1) gene is overexpressed in multiple malignant tumors and is functionally correlated with tumor progression. However, its’ role in oral squamous cell carcinoma (OSCC) is still unclear. Recent studies have revealed that many long non-coding RNA (lncRNAs) cooperate with adjacent coding genes and form a functional “lncRNA-mRNA pair”. In this study, we report a new lncRNA FOXC1 upstream transcript (FOXCUT) that was remarkably overexpressed in 23 OSCC patients, as was the adjacent FOXC1 gene. The expressions of FOXC1 and FOXCUT were positively correlated. When the expression of FOXCUT was down-regulated by small interfering RNA (siRNA), the expression of FOXC1 was also decreased. Moreover, in OSCC cells Tca8113 and SCC-9, down-regulation of either FOXC1 or FOXCUT by siRNA could inhibit cell proliferation and cell migration in vitro and was accompanied with a reduction of MMP2, MMP7, MMP9, and VEGF-A. In conclusion, FOXC1 may be co-amplified with FOXCUT in OSCC, and both of them may be functionally involved in the tumor progression of OSCC. This provides evidence that both FOXC1 and FOXCUT may serve as novel biomarkers and therapeutic targets in OSCC patients who overexpress this “lncRNA-mRNA pair”.

Possible involvement of ΔNp63 downregulation in the invasion and metastasis of oral squamous cell carcinoma via induction of a mesenchymal phenotype

Epithelial-to-mesenchymal transition (EMT), an essential developmental program, is involved in tumor progression. ΔNp63, a homolog of p53, is associated with the EMT program, but the detailed mechanism remains to be elucidated. In this study, we investigated the role of ΔNp63 in EMT during progression of oral squamous cell carcinoma (OSCC). Five OSCC cell lines and specimens from 78 patients with OSCC were used. The expressions of ΔNp63, p63α, p63β and epithelial markers (cytokeratins 5 and 14) was detected in the OSCC cells, but not in SQUU-B cells (high metastatic potential). E-cadherin was expressed in all OSCC cells. Mesenchymal markers were strongly expressed in the SQUU-B cells. Knockdown of endogenous ΔNp63 in HSC-2 cells induced morphological changes to the spindle shape, decreased the expression of epithelial markers, increased the expression of mesenchymal markers, increased migration and reduced proliferation. By contrast, SQUU-B cells overexpressing ΔNp63β showed changed their morphology from stromal cell-like to epithelial cells. However, E-cadherin expression was not affected by ΔNp63 knockdown or overexpression. Immunohistochemical staining revealed that cancer cells expressing vimentin were found at the invasive front in the OSCC specimens. The intensity of ΔNp63 expression was also decreased in these cells. Interestingly, the vimentin positivity or decreased intensity of ΔNp63 was positively associated with metastases and poor prognosis in the OSCC patients. These results indicated that ΔNp63 downregulation in cancer cells induces a mesenchymal phenotype that is related to tumor progression of OSCC.

Depleting IFIT2 mediates atypical PKC signaling to enhance the migration and metastatic activity of oral squamous cell carcinoma cells

Interferon-induced protein with tetratricopeptide repeats 2 (IFIT2) is one of the most highly responsive interferon-stimulated genes, but its biological functions are poorly understood. In this study, we aimed to explore the underlying mechanisms by which depleting IFIT2 induces the migration of oral squamous cell carcinoma (OSCC) cells. Stable IFIT2-depleted cells underwent epithelial-mesenchymal transition (EMT) and exhibited enhanced cell motility and invasiveness compared with control cells. Furthermore, our results indicated that atypical protein kinase C (aPKC) was activated in IFIT2-depleted cells. Inhibition of aPKC using a specific myristoylated PKCζ pseudosubstrate or aPKC-targeting small interfering RNA (siRNA) abolished IFIT2 depletion-induced EMT, migration and invasion, indicating that the activation of aPKC has an essential role in regulating the cellular responses induced by IFIT2 depletion. Following tail-vein injection, IFIT2-depleted OSCC cells colonized not only the lungs but also the heart, head and neck, retroperitoneal, and peritoneal cavities; whereas control cells predominantly localized in the lungs. IFIT2 mRNA and protein expression was positively associated with E-cadherin expression in OSCC patient specimens. The loss of E-cadherin and IFIT2 expression was observed at the invasive front of OSCC tumors, suggesting that the loss of IFIT2 may induce EMT and lead to the metastasis of OSCCs. OSCC patients possessing reduced IFIT2-expression levels (IFIT2 <50%) exhibited greater rates of distant metastasis and poor prognoses compared with OSCC patients who expressed greater levels of IFIT2 (IFIT2 ≥50%). These results demonstrate that IFIT2 depletion activates the aPKC pathway and consequently induces EMT, cell migration and invasion. Most importantly, depleting IFIT2 may participate in OSCC tumor progression, particularly during metastasis. Taken together, our study demonstrates that IFIT2, a protein responsible for interferon stimulation, may prevent OSCC metastasis and serve as a valuable prognostic marker.

Fascin overexpression promotes neoplastic progression in oral squamous cell carcinoma

BackgroundFascin is a globular actin cross-linking protein, which plays a major role in forming parallel actin bundles in cell protrusions and is found to be associated with tumor cell invasion and metastasis in various type of cancers including oral squamous cell carcinoma (OSCC). Previously, we have demonstrated that fascin regulates actin polymerization and thereby promotes cell motility in K8-depleted OSCC cells. In the present study we have investigated the role of fascin in tumor progression of OSCC.MethodsTo understand the role of fascin in OSCC development and/or progression, fascin was overexpressed along with vector control in OSCC derived cells AW13516. The phenotype was studied using wound healing, Boyden chamber, cell adhesion, Hanging drop, soft agar and tumorigenicity assays. Further, fascin expression was examined in human OSCC samples (N = 131) using immunohistochemistry and level of its expression was correlated with clinico-pathological parameters of the patients.ResultsFascin overexpression in OSCC derived cells led to significant increase in cell migration, cell invasion and MMP-2 activity. In addition these cells demonstrated increased levels of phosphorylated AKT, ERK1/2 and JNK1/2. Our in vitro results were consistent with correlative studies of fascin expression with the clinico-pathological parameters of the OSCC patients. Fascin expression in OSCC showed statistically significant correlation with increased tumor stage (P = 0.041), increased lymph node metastasis (P = 0.001), less differentiation (P = 0.005), increased recurrence (P = 0.038) and shorter survival (P = 0.004) of the patients.ConclusionIn conclusion, our results indicate that fascin promotes tumor progression and activates AKT and MAPK pathways in OSCC-derived cells. Further, our correlative studies of fascin expression in OSCC with clinico-pathological parameters of the patients indicate that fascin may prove to be useful in prognostication and treatment of OSCC.

Investigation of trefoil factor expression in saliva and oral mucosal tissues of patients with oral squamous cell carcinoma

The aims of our study were to determine levels of trefoil factor (TFF) peptides in saliva and oral mucosal tissues from patients with oral squamous cell carcinoma (OSCC), and to evaluate whether individual members of TFFs (TFF1, TFF2, and TFF3) might act as biomarkers of disease. Saliva samples were from 23 healthy subjects and 23 OSCC patients. Tissue samples were collected from 32 normal oral mucosa (NOM) and 32 OSCC biopsy specimens. ELISA and immunohistochemical methods were used to evaluate the expression of TFF1, TFF2, and TFF3 in saliva and oral mucosal tissues, respectively. Expression of TFF2 and TFF3 in oral mucosal tissues of OSCC patients was strongly downregulated when compared to healthy subjects (p < 0.001 and p = 0.002, respectively). However, there were no differences in levels of salivary TFF concentrations between OSCC patients and healthy subjects. The present study extends previous observations, demonstrating the reduction of TFF2 and TFF3 expression in oral mucosal tissues of OSCC patients. These findings suggest the clinical significance of TFF2 and TFF3 molecules as negative markers of tumor progression in OSCC. Quantification of TFF levels in saliva may not be optimal in terms of diagnostic or predictive value for OSCC derived from oral mucosa.

Abstract 1634: Targeting L1 cell adhesion molecule using lentivirus-mediated short hairpin RNA interference reverses aggressiveness of oral squamous cell carcinoma

Abstract Oral cancer consistently ranks as one of the ten most frequently diagnosed cancers in the world. Despite multidisciplinary treatment with surgery, chemotherapy, and radiation, more than 50% of patients with oral squamous cell carcinoma (OSCC) receiving therapy will eventually develop recurrent and metastatic disease, which carries a particularly poor prognosis. Characterization of the specific molecular alternations associated with OSCC cell growth, invasion, and metastasis can advance our understanding of the molecular mechanisms underlying cancer induction and progression as well provide a basis for the development of new and effective therapeutic treatments for cancer patients. The L1 cell adhesion molecule (L1CAM) has been implicated in tumor progression of many types of cancers, but its role in OSCC has not been investigated. In the present study, we demonstrated overexpression of L1CAM in OSCC cells, but not in normal keratinocytes, using both clinical specimens and cell lines. This overexpression demonstrated a strong correlation with less differentiation and a higher invasion potential of cancer cells, supporting the significance of L1CAM in human OSCC tumor progression. Targeting L1CAM gene expression in SCC4 cells overexpressing L1CAM using a lentivirusmediated small hairpin RNA (shRNA) led to a significant reduction in cell proliferation in vitro via retardation of cell cycle at the G1 phase. In addition, shRNA knockdown of L1CAM strongly attenuated the migration and invasion of SCC4 cells, and this was also observed to parallel increased E-cadherin levels and decreased levels vimentin, fibronectin, and Snail-family transcription factors, indicating that L1CAM expression was related to the epithelial-mesenchymal transition. Furthermore, while mice receiving orthotopically placed control SCC4 cells died within 40 days due to invasive tumor growth and regional lymph node metastasis, prolonged animal survival and complete suppression of tumor progression was observed in mice implanted with L1CAM-deficent SCC4 cells, further substantiating the fundamental importance of L1CAM in OSCC pathophysiology. Our findings suggested that L1CAM is a critical mediator of tumor progression in OSCC, and targeting L1CAM using lentivirus-mediated shRNA may be a useful molecular pharmaceutical approach for the treatment of advanced OSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1634. doi:10.1158/1538-7445.AM2011-1634

Targeting L1 Cell Adhesion Molecule Using Lentivirus-Mediated Short Hairpin RNA Interference Reverses Aggressiveness of Oral Squamous Cell Carcinoma

The L1 cell adhesion molecule (L1CAM) has been implicated in tumor progression of many types of cancers, but its role in oral squamous cell carcinoma (OSCC) has not been investigated. In the present study, we demonstrated overexpression of L1CAM in OSCC cells, but not in normal keratinocytes, using both clinical specimens and cell lines. This overexpression demonstrated a strong correlation with less differentiation and a higher invasion potential of cancer cells, supporting the significance of L1CAM in human OSCC tumor progression. Targeting L1CAM gene expression in SCC4 cells overexpressing L1CAM using a lentivirus-mediated small hairpin RNA (shRNA) led to a significant reduction in cell proliferation in vitro via retardation of cell cycle at the G1 phase. In addition, shRNA knockdown of L1CAM strongly attenuated the migration and invasion of SCC4 cells, and this was also observed to parallel increased E-cadherin levels and decreased levels of vimentin, fibronectin, and Snail-family transcription factors, indicating that L1CAM expression was related to the epithelial-mesenchymal transition. Furthermore, while mice receiving orthotopically placed control SCC4 cells died within 40 days due to invasive tumor growth and regional lymph node metastasis, prolonged animal survival and complete suppression of tumor progression was observed in mice implanted with L1CAM-deficent SCC4 cells, further substantiating the fundamental importance of L1CAM in OSCC pathophysiology. Our findings suggested that L1CAM is a critical mediator of tumor progression in OSCC, and targeting L1CAM using lentivirus-mediated shRNA may be a useful molecular pharmaceutical approach for the treatment of advanced OSCC.

ADAM-17 associated with CD44 cleavage and metastasis in oral squamous cell carcinoma

ADAM-17 (a disintegrin and metalloproteinase 17) is a membrane-anchored protein, which can cleave the ectodomain in a variety of transmembrane proteins. In the in vitro experiments with tumor cells, ADAM-17 is reported to cleave CD44, an adhesion molecule that interacts with hyaluronic acid, to promote tumor cell migration. In the present study, we examined ADAM-17 expression and CD44 cleavage in specimens from 50 patients diagnosed to have oral squamous cell carcinoma (SCC). Each specimen was divided into two pieces, one was studied by immunohistochemistry and the other was subjected to a Western blot. By coordinating the results of both analyses, ADAM-17 expression was evaluated to be high in 23 cases (46%). When CD44 cleavage was also studied by immunohistochemical staining as well as with Western blotting, CD44 cleavage was judged to be positive in 29 cases (58%). When the ADAM-17 expression level was compared with the CD44 cleavage state, most of the cases expressing high levels of ADAM-17 (87%) showed positive CD44 cleavage. The level of ADAM-17 expression was significantly correlated to the nodal metastasis and local recurrence in oral SCC. Our findings suggest that ADAM-17 is involved in CD44 cleavage and contributes to tumor progression in oral SCC.

Comparison between immunohistochemical expression of cyclin D1 and p21 and histological malignancy graduation of oral squamous cell carcinomas

The objective of the present study was to correlate the expression of cyclin D1 with the expression of p21 in 28 cases of oral squamous cell carcinoma (OSCC), as well as to compare the expression of both with a histological graduation of this neoplasm. Immunohistochemistry was used to obtain the expression of cyclin D1 and p21. The results of statistical analysis showed no correlation between the expression of cyclin D1 and p21. Also, there was no correlation between the mean numbers of cyclin D1 positive nuclei and p21 positive nuclei and the histological scores of malignancy. However, the marked expression of cyclin D1 in high-grade tumors supports its role in proliferative activity. In contrast, p21 seems unable to arrest tumor progression in OSCC.

Isolation of differentiated squamous and undifferentiated spindle carcinoma cell lines with differing metastatic potential from a 4-nitroquinoline N-Oxide-induced tongue carcinoma in a F344 rat.

One differentiated squamous cell carcinoma (SCC) cell line (RSC3‐E2) and two undifferentiated tumor cell lines (RSC3‐LM and RSC3‐E2R) with different metastatic potential were established from a 4‐nitroquinoline N‐oxide (4NQO)‐induced differentiated SCC in F344 rat tongue. The RSC3‐E2 subline was isolated from a parental cell line (RSC3‐P) by single cell cloning in vitro, whereas the RSC3‐LM subline was isolated from a lung metastatic focus after subcutaneous (s.c.) injection of RSC3‐P cells. The RSC3‐E2R cell line was isolated from a lung metastatic focus following s.c. injection of RSC3‐E2 cells after X‐irradiation in vitro. The RSC3‐E2 cell line is keratinpositive and grows as a keratinizing tumor in nude mice, whereas RSC3‐LM and RSC3‐E2R cells are keratin‐negative, vimentin‐positive and form undifferentiated tumors. When s.c. injected into nude mice, the RSC3‐E2 cell line proved to be non‐metastatic, while the RSC3‐LM cell line was metastatic by both hematogenous and lymphogenous routes, and the RSC3‐E2R cell line was metastatic only hematogenously. In vitro relative growth rates and in vitro invasion activity of these cell lines were in the order RSC3‐LM>RSC3‐E2R>RSC3‐E2. Chromosome analysis revealed two peaks with modal chromosome numbers of 83 and 78 for RSC3‐P cells and single peaks at 83, 78 and 56 for RSC3‐LM, RSC3‐E2 and RSC3‐E2R cell lines, respectively. Common structural abnormalities on chromosome 11 were shared by all cell lines. Mutation analysis of the p53 gene using a yeast functional assay demonstrated RSC3‐LM cell line to have a point mutation at codon 269, whereas RSC3‐E2 and RSC3‐E2R had double mutations at codons 106 and 170 on each allele. These results suggest that the two undifferentiated RSC3‐LM and RSC3‐E2R tumor cell lines with different metastatic potential were generated from differentiated SCC cells via different genetic pathways as a consequence of tumor progression in vivo and in vitro, respectively. These cell lines should provide a useful model for understanding mechanisms of hematogenous and lymphogenous metastasis, as well as tumor progression of oral SCCs.