Resveratrol Targets Urokinase-Type Plasminogen Activator Receptor Expression to Overcome Cetuximab-Resistance in Oral Squamous Cell Carcinoma.

Drug resistance to anti-cancer agents is a major concern regarding the successful treatment of malignant tumors. Recent studies have suggested that acquired resistance to anti-epidermal growth factor receptor (EGFR) therapies such as cetuximab are in part caused by genetic alterations in patients with oral squamous cell carcinoma (OSCC). However, the molecular mechanisms employed by other complementary pathways that govern resistance remain unclear. In the current study, we performed gene expression profiling combined with extensive molecular validation to explore alternative mechanisms driving cetuximab-resistance in OSCC cells. Among the genes identified, we discovered that a urokinase-type plasminogen activator receptor (uPAR)/integrin ß1/Src/FAK signal circuit converges to regulate ERK1/2 phosphorylation and this pathway drives cetuximab-resistance in the absence of EGFR overexpression or acquired EGFR activating mutations. Notably, the polyphenolic phytoalexin resveratrol, inhibited uPAR expression and consequently the signaling molecules ERK1/2 downstream of EGFR thus revealing additive effects on promoting OSCC cetuximab-sensitivity in vitro and in vivo. The current findings indicate that uPAR expression plays a critical role in acquired cetuximab resistance of OSCC and that combination therapy with resveratrol may provide an attractive means for treating these patients.

TEAD4-YAP interaction regulates tumoral growth by controlling cell-cycle arrest at the G1 phase.

TEA domain transcription factor 4 (TEAD4), which has critical functions in the process of embryonic development, is expressed in various cancers. However, the important role of TEAD4 in human oral squamous cell carcinomas (OSCCs) remain unclear. Here we investigated the TEAD4 expression level and the functional mechanism in OSCC using quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry. Furthermore, TEAD4 knockdown model was used to evaluate cellular proliferation, cell-cycle analysis, and the interaction between TEAD4 and Yes-associated protein (YAP) which was reported to be a transcription coactivator of cellular proliferation. In the current study, we found that TEAD4 expression increased significantly in vitro and in vivo and correlated with tumoral size in OSCC patients. TEAD4 knockdown OSCC cells showed decreased cellular proliferation resulting from cell-cycle arrest in the G1 phase by down-regulation of cyclins, cyclin-dependent kinases (CDKs), and up-regulation of CDK inhibitors. We also found that the TEAD4-YAP complex in the nuclei may be related closely to transcriptions of G1 arrest-related genes. Taken together, we concluded that TEAD4 might play an important role in tumoral growth and have potential to be a therapeutic target in OSCCs.

Cavin-2 in oral cancer: A potential predictor for tumor progression.

Cavin-2 (CVN2) affects formation of large caveolae, which are membrane-rich cholesterol domains associated with several functions in signal transduction. Accumulating evidence suggests that CVN2 is present in many cellular types; however, the molecular mechanisms of CVN2 in cancers and its clinical relevance are unknown. We proposed a mechanism by which CVN2 regulates caveolin-1 expression leading to slow cellular proliferation by inactivation of the extracellular regulated kinase (ERK) pathway. Quantitative reverse transcriptase-polymerase chain reaction and immunoblot analyses were used to assess the CVN2 regulation mechanism in oral squamous cell carcinoma (OSCC). Immunohistochemistry (IHC) was performed to analyze the correlation between CVN2 expression and clinical behavior in 115 patients with OSCC. A CVN2 overexpressed model of OSCC cells (oeCVN2 cells) was used for functional experiments. CVN2 expression was down-regulated significantly (P < 0.05) in OSCCs compared with normal counterparts in vitro and in vivo. In addition to the findings that a serum deprivation culture induced up-regulation of CVN2 and slowed cellular proliferation, oeCVN2 cell growth decreased because of cell-cycle arrest at the G1 phase resulting from up-regulated cyclin-dependent kinase inhibitors (p21(Cip1) and p27(Kip1) ) and down-regulated cyclins (cyclin D1, cyclin E) and cyclin-dependent kinases (CDK2, CDK4, and CDK6). Interestingly, CVN2 overexpression facilitated caveolin-1 recruitment and colocalization with each other. We also found decreased ERK phosphorylation levels, an upstream event in cell-cycle arrest. Clinically, IHC data from primary OSCCs showed high tumoral progression in CVN2-negative patients with OSCC. CVN2 may be a possible key regulator of OSCC progression via the CVN2/caveolin-1/ERK pathway and a potential therapeutic target for developing new treatments for OSCCs. © 2015 Wiley Periodicals, Inc.

Down-Regulation of Nucleolar and Spindle-Associated Protein 1 (NUSAP1) Expression Suppresses Tumor and Cell Proliferation and Enhances Anti-Tumor Effect of Paclitaxel in Oral Squamous Cell Carcinoma.

BACKGROUND: Nucleolar and spindle-associated protein 1 (NUSAP1) is an important mitotic regulator. In addition to its crucial function in mitosis, NUSAP1 has recently received attention due to the interesting roles in carcinogenesis. The aim of this study was to reveal functional mechanisms of NUSAP1 in oral squamous cell carcinoma (OSCC). METHODS: mRNA and protein expression levels of NUSAP1 in 9 OSCC-derived cells were analyzed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and immunoblotting analyses. The correlation between the NUSAP1 expression profile and the clinicopathological factors was evaluated by immunohistochemistry (IHC) in clinical OSCC samples (n = 70). The NUSAP1 knockdown cells were established with short hairpin RNA (shRNA) in OSCC cells, and functional assays were performed using these cells. In addition to the evaluation of cellular proliferation and cell cycle, we also investigated the potential role of NUSAP1 in paclitaxel (PTX)-induced cellular responses. RESULTS: mRNA and protein expression of NUSAP1 were significantly up-regulated in OSCC-derived cells compared with human normal oral keratinocytes (P < 0.05). IHC revealed that NUSAP-1 expression is closely associated with primary advanced T stage (P<0.05). Suppression of NUSAP1 expression levels led to significant (P < 0.05) inhibition of cellular proliferation. Furthermore, apoptosis induced by PTX was enhanced in NUSAP1 knockdown OSCC cells. CONCLUSIONS: NUSAP1 may be a crucial biomarker for OSCC. Moreover, down-regulated NUSAP1 expression suppresses tumor proliferation and also enhances anti-tumor effect of PTX by activating apoptotic pathways. Thus, the present study strongly suggests that regulating NUSAP1 expression should contribute to the therapy for OSCC.

Semaphorin7A Promotion of Tumoral Growth and Metastasis in Human Oral Cancer by Regulation of G1 Cell Cycle and Matrix Metalloproteases: Possible Contribution to Tumoral Angiogenesis.

BACKGROUND: Semaphorins (SEMAs) consist of a large family of secreted and membrane-anchored proteins that are important in neuronal pathfinding and axon guidance in selected areas of the developing nervous system. Of them, SEMA7A has been reported to have a chemotactic activity in neurogenesis and to be an immunomodulator; however, little is known about the relevance of SEMA7A in the behaviors of oral squamous cell carcinoma (OSCC). METHODS: We evaluated SEMA7A expression in OSCC-derived cell lines and primary OSCC samples using quantitative reverse transcriptase-polymerase chain reaction, immunoblotting, and semiquantitative immunohistochemistry (sq-IHC). In addition, SEMA7A knockdown cells (shSEMA7A cells) were used for functional experiments, including cellular proliferation, invasiveness, and migration assays. We also analyzed the clinical correlation between SEMA7A status and clinical behaviors in patients with OSCC. RESULTS: SEMA7A mRNA and protein were up-regulated significantly (P<0.05) in OSCC-derived cell lines compared with human normal oral keratinocytes. The shSEMA7A cells showed decreased cellular growth by cell-cycle arrest at the G1 phase, resulting from up-regulation of cyclin-dependent kinase inhibitors (p21Cip1 and p27Kip1) and down-regulation of cyclins (cyclin D1, cyclin E) and cyclin-dependent kinases (CDK2, CDK4, and CDK6); and decreased invasiveness and migration activities by reduced secretion of matrix metalloproteases (MMPs) (MMP-2, proMMP-2, pro-MMP-9), and expression of membrane type 1- MMP (MT1-MMP). We also found inactivation of the extracellular regulated kinase 1/2 and AKT pathways, an upstream molecule of cell-cycle arrest at the G1 phase, and reduced secretion of MMPs in shSEMA7A cells. sq-IHC showed that SEMA7A expression in the primary OSCCs was significantly (P = 0.001) greater than that in normal counterparts and was correlated with primary tumoral size (P = 0.0254) and regional lymph node metastasis (P = 0.0002). CONCLUSION: Our data provide evidence for an essential role of SEMA7A in tumoral growth and metastasis in OSCC and indicated that SEMA7A may play a potential diagnostic/therapeutic target for use in patients with OSCC.

Adenosine A2b receptor promotes progression of human oral cancer.

BACKGROUND: Adenosine A2b receptor (ADORA2B) encodes an adenosine receptor that is a member of the G protein-coupled receptor superfamily. This integral membrane protein stimulates adenylate cyclase activity in the presence of adenosine. Little is known about the relevance of ADORA2B to human malignancy including oral squamous cell carcinoma (OSCC). We aimed to characterize the expression state and function of ADORA2B in OSCC. METHODS: The ADORA2B expression levels in nine OSCC-derived cells were analyzed by quantitative reverse transcriptase-polymerase chain reaction and immunoblotting analyses. Using an ADORA2B knockdown model, we assessed cellular proliferation and expression of hypoxia-inducible factor1α (HIF-1α). We examined the adenosine receptor expression profile under both normoxic and hypoxic conditions in the OSCC-derived cells. In addition to in vitro data, the clinical correlation between the ADORA2B expression levels in primary OSCCs (n = 100 patients) and the clinicopathological status by immunohistochemistry (IHC) also was evaluated. RESULTS: ADORA2B mRNA and protein were up-regulated significantly (p < 0.05) in seven OSCC-derived cells compared with human normal oral keratinocytes. Suppression of ADORA2B expression with shRNA significantly (p < 0.05) inhibited cellular proliferation compared with the control cells. HIF-1α also was down-regulated in ADORA2B knockdown OSCC cells. During hypoxia, ADORA2B expression was induced significantly (p < 0.05) in the mRNA and protein after 24 hours of incubation in OSCC-derived cells. IHC showed that ADORA2B expression in primary OSCCs was significantly (p < 0.05) greater than in the normal oral counterparts and that ADORA2B-positive OSCCs were correlated closely (p < 0.05) with tumoral size. CONCLUSION: Our results suggested that ADORA2B controls cellular proliferation via HIF-1α activation, indicating that ADORA2B may be a key regulator of tumoral progression in OSCCs.

Quantitative detection of circulating tumor-derived mitochondrial NADH subunit variants as a potential prognostic biomarker for oral cancer.

Circulating tumor cells (CTCs) and/or their relating molecules are promising determinants during the course of cancer treatment, especially for post-therapeutic monitoring. We recently reported the clinical relevance of detecting circulating tumor-associated mutant mitochondrial DNAs (mut-mtDNAs) at three different regions including the displacement loop, 12S-rRNA and 16S-rRNA in oral squamous cell carcinomas (OSCCs). In the present study, to further investigate if the other mut-mtDNAs have novel efficiency for detecting potential tumoral micrometastasis, mut-mtDNAs on the ND2 and ND3 regions of the genome in 240 clinical samples from patients with OSCC were assessed in vitro and in vivo by quantitative real-time PCR combined with high-resolution melting curve analysis. Furthermore, the clinical relevance was evaluated by the area under the receiver operating characteristic curve (AUC) analysis. Three discrete sequence variations were identified in OSCC derived cell lines at the regions of ND2 (T:A to C:G at position 5108) and ND3 (A:T to G:C at position 10397 and C:G to T:A at position 10400), whereas no mutation was observed in normal control human normal oral keratinocytes. In OSCC patients examined, the presence of mut-mtDNAs in serum during the postoperative period accurately predicted poor prognoses (ND2 AUC, 0.761; ND3 AUC, 0.704). The data presented here provide a novel approach for detecting the circulating mut-mtDNAs that are promising molecular markers for evaluating tumoral micrometastasis in OSCCs.

Lysophosphatidylcholine acyltransferase1 overexpression promotes oral squamous cell carcinoma progression via enhanced biosynthesis of platelet-activating factor.

BACKGROUND: The relevance of lysophosphatidylcholine acyltransferase1 (LPCAT1), a cytosolic enzyme in the remodeling pathway of phosphatidylcholine metabolism, in oral squamous cell carcinoma (OSCC) is unknown. We investigated LPCAT1 expression and its functional mechanism in OSCCs. METHODS: We analyzed LPCAT1 mRNA and protein expression levels in OSCC-derived cell lines. Immunohistochemistry was performed to identify correlations between LPCAT1 expression levels and primary OSCCs clinicopathological status. We established LPCAT1 knockdown models of the OSCC-derived cell lines (SAS, Ca9-22) for functional analysis and examined the association between LPCAT1 expression and the platelet-activating factor (PAF) concentration and PAF-receptor (PAFR) expression. RESULTS: LPCAT1 mRNA and protein were up-regulated significantly (p<0.05) in OSCC-derived cell lines compared with human normal oral keratinocytes. Immunohistochemistry showed significantly (p<0.05) elevated LPCAT1 expression in primary OSCCs compared with normal counterparts and a strong correlation between LPCAT1-positive OSCCs and tumoral size and regional lymph node metastasis. In LPCAT1 knockdown cells, cellular proliferation and invasiveness decreased significantly (p<0.05); cellular migration was inhibited compared with control cells. Down-regulation of LPCAT1 resulted in a decreased intercellular PAF concentration and PAFR expression. CONCLUSION: LPCAT1 was overexpressed in OSCCs and correlated with cellular invasiveness and migration. LPCAT1 may contribute to tumoral growth and metastasis in oral cancer.

Interleukin-1 receptor antagonist (IL1RN) is associated with suppression of early carcinogenic events in human oral malignancies.

Inflammatory abnormalities have been implicated in the pathogenesis of various human diseases, including cancer. Interleukin-1 receptor antagonist (IL1RN) is a potent anti-inflammatory molecule that modulates the biological activity of the proinflammatory cytokine, interleukin-1. The aim of this study was to examine the expression of IL1RN in oral squamous cell carcinomas (OSCCs), and to determine its clinical significance. Expression levels of IL1RN in matched normal and tumor specimens from 39 OSCCs were evaluated using real-time quantitative polymerase chain reaction methods, and immunohistochemical analysis. Protein expression of IL1RN was also examined in 18 oral premalignant lesions (OPLs). Expression of IL1RN mRNA was significantly downregulated in OSCCs compared with normal tissues. Decreased expression of IL1RN protein was also observed in OPLs and OSCCs. The IL1RN expression level was lower in the OPL cases with severe dysplasia compared to those with mild/moderate dysplasia. Significantly downregulated IL1RN expression was observed in all OSCC lesion sites examined when compared with the matched normal tissues. However, the decreased level of IL1RN expression did not correspond with tumor progression. Noteworthy, IL1RN expression was higher in the advanced OSCC cases (T3/T4) compared to early cases (T1/T2). Among OSCC samples, relatively higher IL1RN expression was associated with active tumor development in the OSCCs occurring in the buccal mucosa, oral floor, fauces and gingiva, but not the tongue. These data suggest that IL1RN may exhibit opposing characteristics in oral malignancies depending on the stage of cancer development, suppressing early carcinogenic events, yet promoting tumor development in some lesion sites. Thus, IL1RN could represent a reliable biomarker for the early diagnosis of OSCCs. Furthermore, IL1RN may possess unknown and complex functions in the developed OSCC.

ANGPTL3 is a novel biomarker as it activates ERK/MAPK pathway in oral cancer.

Angiopoietin-like 3 (ANGPTL3), which is involved in new blood vessel growth and stimulation of mitogen-activated protein kinase (MAPK), is expressed aberrantly in several types of human cancers. However, little is known about the relevance of ANGPTL3 in the behavior of oral squamous cell carcinoma (OSCC). In this study, we evaluated ANGPTL3 mRNA and protein in OSCC-derived cell lines (n = 8) and primary OSCCs (n = 109) and assessed the effect of ANGPTL3 on the biology and function of OSCCs in vitro and in vivo. Significant (P < 0.05) ANGPTL3 upregulation was detected in the cell lines and most primary OSCCs (60%) compared with the normal counterparts. The ANGPTL3 expression level was correlated closely (P < 0.05) with tumoral size. In patients with T3/T4 tumors, the overall survival rate with an ANGPTL3-positive tumor was significantly (P < 0.05) lower than that of ANGPTL3-negative cases. In vitro, cellular growth in ANGPTL3 knockdown cells significantly (P < 0.05) decreased with inactivated extracellular regulated kinase (ERK) and cell-cycle arrest at the G1 phase resulting from upregulation of the cyclin-dependent kinase inhibitors, including p21(Cip1) and p27(Kip1) . We also observed a marked (P < 0.05) reduction in the growth in ANGPTL3 knockdown-cell xenografts with decreased levels of phosphorylated ERK relative to control-cell xenografts. The current data indicated that ANGPTL3 may play a role in OSCCs via MAPK signaling cascades, making it a potentially useful diagnostic/therapeutic target for use in patients with OSCC.

Decorin in human oral cancer: a promising predictive biomarker of S-1 neoadjuvant chemosensitivity.

We reported previously that decorin (DCN) is significantly up-regulated in chemoresistant cancer cell lines. DCN is a small leucine-rich proteoglycan that exists and functions in stromal and epithelial cells. Accumulating evidence suggests that DCN affects the biology of several types of cancer by directly/indirectly targeting the signaling molecules involved in cell growth, survival, metastasis, and angiogenesis, however, the molecular mechanisms of DCN in chemoresistance and its clinical relevance are still unknown. Here we assumed that DCN silencing cells increase chemosusceptibility to S-1, consisted of tegafur, prodrug of 5-fluorouracil. We first established DCN knockdown transfectants derived from oral cancer cells for following experiments including chemosusceptibility assay to S-1. In addition to the in vitro data, DCN knockdown zenografting tumors in nude mice demonstrate decreasing cell proliferation and increasing apoptosis with dephosphorylation of AKT after S-1 chemotherapy. We also investigated whether DCN expression predicts the clinical responses of neoadjuvant chemotherapy (NAC) using S-1 (S-1 NAC) for oral cancer patients. Immunohistochemistry data in the preoperative biopsy samples was analyzed to determine the cut-off point for status of DCN expression by receiver operating curve analysis. Interestingly, low DCN expression was observed in five (83%) of six cases with complete responses to S-1 NAC, and in one (10%) case of 10 cases with stable/progressive disease, indicating that S-1 chemosensitivity is dramatically effective in oral cancer patients with low DCN expression compared with high DCN expression. Our findings suggest that DCN is a key regulator for chemoresistant mechanisms, and is a predictive immunomarker of the response to S-1 NAC and patient prognosis.

Persephin: A potential key component in human oral cancer progression through the RET receptor tyrosine kinase-mitogen-activated protein kinase signaling pathway.

Persephin (PSPN) is a neurotrophic factor of the glial cell line-derived neurotrophic factor (GDNF) family that promotes survival of multiple populations of neurons. Little is known about the relevance of PSPN in human malignancy including oral squamous cell carcinoma (OSCC). This study was undertaken to evaluate PSPN mRNA and protein expression by analyzing cellular proliferation and the cell cycle in PSPN knockdown cells in vitro. PSPN mRNA and protein were significantly (P < 0.05) up-regulated in OSCC-derived cells compared with human normal oral keratinocytes (n = 7). Cellular proliferation decreased significantly (P < 0.05) in PSPN knockdown cells with reduced receptor tyrosine kinase (RTK) signaling, and cell-cycle arrest at the G1 phase resulted from up-regulation of the cyclin-dependent kinase inhibitors (p21(Cip1) , p27(Kip1) , p15(INK4B) , and p16(INK4A) ). Furthermore, the PSPN protein expression in 101 primary OSCCs was significantly (P < 0.05) higher than in normal counterparts. Among the clinical variables analyzed, overexpression of PSPN also was related closely (P < 0.05) to tumoral size. Our results suggested that PSPN is a possible key regulator of OSCC progression via PSPN-RET-mitogen-activated protein kinase activation and that PSPN overexpression may have diagnostic potential for OSCC.

Controlling distant metastasis and surgical treatment are crucial for improving clinical outcome in uncommon head and neck malignancies, such as non-squamous cell carcinoma.

The objective of this study was to elucidate the clinical characteristics of uncommon head and neck malignancies, such as non-squamous cell carcinoma (SCC), in order to improve patient outcomes. A total of 463 head and neck malignancies were retrospectively analyzed, with 43 cases (9.3%) diagnosed as non-SCC. The overall survival rate of patients with adenoid cystic carcinoma was significantly worse compared to that of patients with SCC. The 5-year survival rates were <50% for patients with malignant melanoma, adenocarcinoma, small-cell carcinoma and sarcomas. Distant metastasis to the lung was frequently observed in cases with a poor outcome. Non-SCC malignancies treated without surgery were associated with a worse outcome. Some non-SCC patients had a poor prognosis and distant metastasis was associated with an unsatisfactory outcome. Timely treatment and control of distant metastasis are essential and surgical treatment should be prioritized in non-SCC cases to improve patient outcomes.

Suppression of metastasis by mirtazapine via restoration of the Lin-7C/ß-catenin pathway in human cancer cells.

No definitive therapy exists to treat human metastatic tumors. We reported previously that down-regulation of Lin-7C is essential for metastasis of human squamous cell carcinomas (hSCCs). In this study, we investigated the chemical restoration of Lin-7C expression and demonstrated its effectiveness for suppressing the metastatic potential in human cancer cells. Ingenuity Pathway Analysis (IPA) identified candidate chemical agents, i.e., apomorphine, caffeine, risperidone, quetiapine, and mirtazapine. Among them, mirtazapine, an antagonist of HTR2C, an upstream molecule of Lin-7C, caused substantial up-regulation of the Lin-7C/ß-catenin pathway in a metastatic hSCC cell line and human melanoma-derived cell line in vitro, and up-regulation did not contribute to cellular proliferation. Moreover, the antimetastatic effect of mirtazapine in these metastatic cell lines in vivo also was evident in multiple organs of immunodeficient mice with no marked side effects. The current data offer novel information for further study of antimetastatic activity in association with enhanced Lin-7C/ß-catenin pathway activation with mirtazapine.

Treatment of an extensive maxillary cyst using nasal airway and balloon catheter devices.

Introduction. Large maxillary cysts occasionally expand into the maxilla and erode the maxillary sinus and nasal cavity. The Caldwell-Luc procedure is the recommended treatment for large maxillary sinus cysts. However, it is hard to preserve the nasal space in the case of large maxillary sinus cysts that penetrate into the nasal cavity. Methods. A 22-year-old man who had large maxillary sinus cysts was referred to our department for a surgical treatment. After removing the cyst from the maxillary sinus using the Caldwell-Luc procedure, we used nasal airway and balloon catheter devices to preserve the space of the inferior nasal meatus and maxillary sinus. These devices were removed 10 days postoperatively. Insertion and removal of both devices were simple and painless. Findings. The nasal airway and balloon catheter devices were useful for performing maxillary sinus surgery to remove large cysts. Our method was satisfactorily safe and was an effective minimally invasive treatment that preserved the space of the inferior nasal meatus and maxillary sinus.

Decreased expression of kallikrein-related peptidase 13: possible contribution to metastasis of human oral cancer.

The human kallikrein-related peptidase family is comprised of 15 serine protease genes on chromosome 19q13.4. Our previous microarray analyses showed that the gene kallikrein-related peptidase 13 (KLK13) was down-regulated in oral squamous cell carcinoma (OSCC) cell lines. We evaluated the expression status of KLK13 in primary OSCCs and performed functional molecular experiments in OSCC cell lines. In 102 primary tumors studied, KLK13 expression significantly (P < 0.05) decreased compared with matched normal counterparts. Interestingly, KLK13-negative cases correlated significantly (P < 0.05) with regional lymph node metastasis. In vitro, cells overexpressing KLK13 (oeKLK13) had decreased invasiveness and motility and up-regulation of adhesion molecules (E-cadherin, α-catenin, ß-catenin, junction plakoglobin, plakophilin4, desmocollin2, desmoglein3, and desmoplakin) compared with control cells. A rescue experiment that transfected oeKLK13 cells with siRNA against KLK13 restored invasiveness and migration activities with down-regulated adhesion molecules. Based on our results, we concluded that KLK13 may play an important role in regulating cellular migration and invasiveness, making the loss of KLK13 a potential biomarker for early detection of lymph node metastasis in OSCCs.

WWP2 is overexpressed in human oral cancer, determining tumor size and poor prognosis in patients: downregulation of WWP2 inhibits the AKT signaling and tumor growth in mice.

The WW domain containing E3 ubiquitin protein ligase 2 (WWP2) encodes a member of the Nedd4 family of E3 ligases, which catalyzes the final step of the ubiquitination cascade. WWP2 is involved in tumoral growth with degradation of the tumor suppressor phosphatase and tensin homologue deleted on chromosome TEN (PTEN). However, little is known about the mechanisms and roles of WWP2 in human malignancies including oral squamous cell carcinomas (OSCCs). We found frequent WWP2 overexpression in all OSCC-derived cell lines examined that was associated with cellular growth by accelerating the cell cycle in the G1 phase via degradation of PTEN and activation of the PI3K/AKT signaling pathway. Our in vivo data of WWP2 silencing showed dramatic inhibition of tumoral growth with increased expression of PTEN. Our 104 primary OSCCs had significantly higher expression of WWP2 than their normal counterparts. Moreover, among the clinical variables analyzed, enhanced WWP2 expression was correlated with primary tumoral size and poor prognosis. These data suggested that WWP2 overexpression contributes to neoplastic promotion via the PTEN/PI3K/AKT pathway in OSCCs. WWP2 is likely to be a biomarker of tumoral progression and prognosis and a potential therapeutic target for development of anticancer drugs in OSCCs.

Glutamate acid decarboxylase 1 promotes metastasis of human oral cancer by ß-catenin translocation and MMP7 activation.

BACKGROUND: Glutamate decarboxylase 1 (GAD1), a rate-limiting enzyme in the production of γ-aminobutyric acid (GABA), is found in the GABAergic neurons of the central nervous system. Little is known about the relevance of GAD1 to oral squamous cell carcinoma (OSCC). We investigated the expression status of GAD1 and its functional mechanisms in OSCCs. METHODS: We evaluated GAD1 mRNA and protein expressions in OSCC-derived cells using real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and immunoblotting analyses. To assess the critical functions of GAD1, i.e., cellular proliferation, invasiveness, and migration, OSCC-derived cells were treated with the shRNA and specific GAD1 inhibitor, 3-mercaptopropionic acid (3-MPA). GAD1 expression in 80 patients with primary OSCCs was analyzed and compared to the clinicopathological behaviors of OSCC. RESULTS: qRT-PCR and immunoblotting analyses detected frequent up-regulation of GAD1 in OSCC-derived cells compared to human normal oral keratinocytes. Suppression of nuclear localization of ß-catenin and MMP7 secretion was observed in GAD1 knockdown and 3-MPA-treated cells. We also found low cellular invasiveness and migratory abilities in GAD1 knockdown and 3-MPA-treated cells. In the clinical samples, GAD1 expression in the primary OSCCs was significantly (P < 0.05) higher than in normal counterparts and was correlated significantly (P < 0.05) with regional lymph node metastasis. CONCLUSIONS: Our data showed that up-regulation of GAD1 was a characteristic event in OSCCs and that GAD1 was correlated with cellular invasiveness and migration by regulating ß-catenin translocation and MMP7 activation. GAD1 might play an important role in controlling tumoral invasiveness and metastasis in oral cancer.

Targeting phosphodiesterase 3B enhances cisplatin sensitivity in human cancer cells.

We previously reported that human squamous cell carcinoma (SCC) cell lines refractory to cis-diaminedichloro-platinum II (cisplatin [CDDP]) had significant upregulation of the phosphodiesterase 3B gene (PDE3B), suggesting that inhibiting PDE3B suppresses CDDP resistance. shRNA-mediated PDE3B depletion in CDDP-resistant cells derived from SCC cells and Hela cells and induced CDDP sensitivity and inhibited tumor growth with elevated cyclic GMP induction resulting in upregulation of the multidrug-resistant molecule, but this did not occur in the 5-fluorouracil-resistant hepatocellular carcinoma cell lines. Furthermore, the antitumor growth effect of the combination of a PDE3B inhibitor (cilostazol) and CDDP in vivo was also greater than with either cilostazol or CDDP alone, with a significant increase in the number of apoptotic and cell growth-suppressive cancer cells in CDDP-resistance cell lines. Our results provided novel information on which to base further mechanistic studies of CDDP sensitization by inhibiting PDE3B in human cancer cells and for developing strategies to improve outcomes with concurrent chemotherapy.

Protein O-fucosyltransferase 1: a potential diagnostic marker and therapeutic target for human oral cancer.

Protein O-fucosyltransferase 1 (POFUT1) is the enzyme that adds O-fucose through O-glycosidic linkage to conserved serine or threonine residues in the epidermal growth factor-like repeats of a number of cellular surface and secreted proteins. Our previous study using microarray technology showed that significant upregulation of POFUT1 occurs in oral squamous cell carcinoma (OSCC)-derived cell lines compared to human normal oral keratinocytes. The aim of the present study was to examine the status of POFUT1 mRNA and protein expression in OSCC-derived cell lines and human primary OSCCs. POFUT1 mRNA was upregulated significantly (P<0.05 for both comparisons) in five OSCC-derived cell lines and primary OSCCs using quantitative reverse transcriptase-polymerase chain reaction. Immunohistochemistry data indicated that POFUT1 protein expression levels were consistent with mRNA expression status in OSCC-derived cell lines and primary OSCCs. Furthermore, POFUT1 expression status was correlated significantly (P=0.048) with the primary tumor size. The proliferation of POFUT1 knockdown cells was inhibited significantly compared with that of control cells. These results indicated that POFUT1 expression can contribute to cancer progression and that POFUT1 may serve as a diagnostic marker and a therapeutic target for OSCCs.