Salivary gland-type cancers: cross-organ demographics of a rare cancer.

BACKGROUND: Salivary gland-type cancers (SGTCs) are histologically heterogeneous and can affect organs other than the salivary glands. Some tumors outside the salivary glands are diagnosed on their unique histological characteristics. Comprehensive cross-organ studies on SGTCs are limited. METHODS: We retrospectively analyzed the data of patients with salivary duct carcinoma (SDC), adenoid cystic carcinoma (AdCC), mucoepidermoid carcinoma (MEC), epithelial-myoepithelial carcinoma (EMC), acinic cell carcinoma (AcCC), and polymorphous adenocarcinoma (PAC) who visited our institution between 2009 and 2019. The primary tumor sites were classified into four categories; major salivary glands, head/neck (H/N) excluding (exc) major salivary glands (MSG) regions, broncho-pulmonary regions, and "others". H/N exc MSG was further divided into three subcategories, nasal/paranasal sinus, oral and pharynx/larynx. RESULTS: We identified 173 patients with SGTCs, with SDC, AdCC, MEC, EMC, AcCC, and PAC accounting for 20%, 42%, 27%, 3%, 8%, and 1% of the cases, respectively. The most frequent primary site was the major salivary glands (64%), followed by H/N exc MSG regions (27%), broncho-pulmonary regions, and "others", thus non-salivary gland origins accounted for 9% of all cases. Patients with SDC, MEC, AcCC, or SGTC of the major salivary glands and broncho-pulmonary regions were more frequently treated by surgery. The overall survival time of the patients with MEC was significantly better than that of patients with SDC or EMC. CONCLUSIONS: This cross-organ study highlights the clinical significance of SGTCs, underscoring the need for developing novel therapies for this rare disease entity.

Preoperative sleep-disordered breathing and craniofacial abnormalities are risk factors for postoperative sleep-disordered breathing in patients undergoing skin-flap oropharyngeal reconstruction surgery for oral cavity cancer: a prospective case-control study.

PURPOSE: After oropharyngeal reconstruction surgery, excessive flap volume within the oral cavity may increase the risk of pharyngeal obstruction during sleep. This prospective observational study aimed to test a hypothesis that the skin-flap oropharyngeal reconstructive surgery increases nocturnal apnea-hypopnea index (nAHI, primary variable) after surgery. METHODS: Adult patients undergoing oropharyngeal reconstruction surgery participated in this study. The hypothesis was tested by comparing the results of portable type 4 sleep study and craniofacial assessments with lateral head and neck computed tomography scout image before and after surgery. Multiple linear regression analyses were performed to identify predictors for nAHI increase after the surgery. RESULTS: In 15 patients, a postoperative sleep study was performed at 41 (27, 59) (median (IQR)) days after the surgery. nAHI did not increase after the surgery (mean (95% CI), 13.0 (7.2 to 18.7) to 18.4 (10.2 to 26.6) events.hour-1, p = 0.277), while apnea index significantly increased after the surgery (p = 0.026). Use of the pedicle flap for the oropharyngeal reconstruction (p = 0.051), small mandible (p = 0.008), longer lower face (0.005), and larger tongue size (p = 0.008) were independent predictors for worsening of nAHI after surgery. Hospital stay was significantly longer in patients with the pedicle flap (n = 8) than in those with the free flap (n = 7) (p = 0.014), and the period of hospital stay was directly associated with increase of nAHI after surgery (r = 0.788, p < 0.001, n = 15). CONCLUSIONS: Oropharyngeal reconstruction surgery worsens sleep-disordered breathing in some patients with craniofacial and surgical risk factors. TRIAL REGISTRATION: UMIN Clinical Trial Registry (UMIN000036260, March 22, 2019), https://rctportal.niph.go.jp/s/detail/um?trial_id=UMIN000036260.

Lysyl hydroxylase 2 deficiency promotes filopodia formation and fibroblast migration.

Lysyl hydroxylase 2 (LH2) regulates intermolecular cross-linking of collagen molecules. Accumulation of LH2-modified collagen, which is highly stable and resistant to collagenase cleavage, is one cause of fibrosis. We previously demonstrated that conventional LH2 knockout mice showed embryonic lethality. Here we established LH2 conditional knockout mice using a tamoxifen-inducible Cre system. Morphological analysis of LH2-deficient fibroblasts by microscopy showed a dramatic increase in the number of filopodia, the finger-like cell surface projections that enable cell movement. The tips and leading edges of these filopodia exhibited up-regulated expression of Myosin-X (Myo10), a regulator of filopodial integrity. Wound healing assays demonstrated that migration of LH2-deficient cells was significantly faster than that of control cells. Gene expression profiling data also supported this phenotype. Together these findings indicate that LH2 deficiency may prevent fibrosis through decreased accumulation of LH2-cross-linked collagen, and that fibroblasts with faster migration contribute to enhanced wound healing activity. In conclusion, our cellular models provide evidence that LH2 deficiency plays a critical role in cell migration mediated through filopodia formation. Understanding the precise role of this phenotype in LH2-deficient cells may be helpful to define the pathogenesis of fibrosis. As such, detailed analyses of fibrosis and wound healing using LH2-deficient mouse models are needed.

Assay for transposase-accessible chromatin with high-throughput sequencing reveals radioresistance-related genes in oral squamous cell carcinoma cells.

Radiotherapy is commonly used to treat oral squamous cell carcinoma (OSCC), and radioresistance is a critical factor resulting in poor outcomes. Several genes have been reported to be therapeutic targets for radioresistance; however, the involvement of chromatin accessibility in radioresistance has not been clarified in OSCC cells. Accordingly, in this study, we evaluated chromatin accessibility in radioresistant (HSC-3) and radiosensitive (KOSC-2) cells, identified from nine OSCC cell lines using clonogenic survival assays after irradiation. Chromatin accessibility in radioresistant OSCC cells was assessed using assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). Gene expression was evaluated by quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and immunoblot analysis. Viability was assessed by MTS assay. We found 1273 peaks (open chromatin regions by ATAC-seq) related to 8 Gy irradiation in HSC-3 but not KOSC-2 cells, among which 235 genes located around the chromatin open peaks were identified by ChIPpeakAnno analysis. Subsequently, 12 genes were selected as signal transduction-related genes by Gene Ontology analysis, and gene expression was confirmed by RT-qPCR. Among these genes, adenylate cyclase 2 (ADCY2) was significantly upregulated after treatment with irradiation in HSC-3 but not KOSC-2 cells. To further evaluate ADCY2 function in radioresistant cells, we performed ADCY2 knockdown by transfection of HSC-3 cells with small interfering RNA (siADCY2). Cell viability after irradiation was significantly decreased in siADCY2-transfected cells compared with that in control cells. These results suggested that ADCY2 expression was related to the open chromatin region in radioresistant OSCC cells and that ADCY2 may have therapeutic efficacy when used in combination with radiotherapy in patients with OSCC.

Multicenter retrospective study of nivolumab for recurrent/metastatic oral squamous cell carcinoma.

OBJECTIVES: Immunotherapy with nivolumab for patients with recurrent/metastatic oral squamous cell carcinoma has not been evaluated. Here, we aimed to examine the efficacy, safety, and prognostic factors of nivolumab in these patients. MATERIALS AND METHODS: This multicenter retrospective observational study involved patients who received nivolumab between April 2017 and June 2019. The patient characteristics were evaluated for association with progression-free and overall survival. Progression-free and overall survival rates were calculated; parameters that were significant in the univariate analysis were used as explanatory variables. Independent factors for progression-free and overall survival were identified using multivariate analysis. RESULTS: Totally, 143 patients were included. The overall response and disease control rates were 27.3% and 46.2%, respectively. The median, 1- and 2-year progression-free survival rates were 2.7 months, 25.4%, and 19.2%, respectively; those for overall survival were 11.2 months, 47.3%, and 33.6%, respectively. The independent factors affecting progression-free survival were performance status and immune-related adverse event occurrence, whereas those affecting overall survival were performance status, target disease, and number of previous lines of systemic cancer therapy. Eight patients reported grade ≥3 immune-related adverse events. CONCLUSION: Nivolumab was effective for recurrent/metastatic oral squamous cell carcinoma treatment and was well tolerated by patients.

Identification of Tumor-Suppressive miR-30e-3p Targets: Involvement of SERPINE1 in the Molecular Pathogenesis of Head and Neck Squamous Cell Carcinoma.

Recently, our studies revealed that some passenger strands of microRNAs (miRNAs) were closely involved in cancer pathogenesis. Analysis of miRNA expression signatures showed that the expression of miR-30e-3p (the passenger strand of pre-miR-30e) was significantly downregulated in cancer tissues. In this study, we focused on miR-30e-3p (the passenger strand of pre-miR-30e). We addressed target genes controlled by miR-30e-3p that were closely associated with the molecular pathogenesis of head and neck squamous cell carcinoma (HNSCC). Ectopic expression assays demonstrated that the expression of miR-30e-3p attenuated cancer cell malignant phenotypes (e.g., cell proliferation, migration, and invasive abilities). Our analysis of miR-30e-3p targets revealed that 11 genes (ADA, CPNE8, C14orf126, ERGIC2, HMGA2, PLS3, PSMD10, RALB, SERPINE1, SFXN1, and TMEM87B) were expressed at high levels in HNSCC patients. Moreover, they significantly predicted the short survival of HNSCC patients based on 5-year overall survival rates (p < 0.05) in The Cancer Genome Atlas (TCGA). Among these targets, SERPINE1 was found to be an independent prognostic factor for patient survival (multivariate Cox regression; hazard ratio = 1.6078, p < 0.05). Aberrant expression of SERPINE1 was observed in HNSCC clinical samples by immunohistochemical analysis. Functional assays by targeting SERPINE1 expression revealed that the malignant phenotypes (e.g., proliferation, migration, and invasion abilities) of HNSCC cells were suppressed by the silencing of SERPINE1 expression. Our miRNA-based approach will accelerate our understanding of the molecular pathogenesis of HNSCC.

Genome-Wide Super-Enhancer-Based Analysis: Identification of Prognostic Genes in Oral Squamous Cell Carcinoma.

Advanced-stage oral squamous cell carcinoma (OSCC) patients are treated with combination therapies, such as surgery, radiation, chemotherapy, and immunotherapy. However, OSCC cells acquire resistance to these treatments, resulting in local recurrence and distant metastasis. The identification of genes involved in drug resistance is essential for improving the treatment of this disease. In this study, we applied chromatin immunoprecipitation sequencing (ChIP-Seq) to profile active enhancers. For that purpose, we used OSCC cell lines that had been exposed to cetuximab for a prolonged period. In total, 64 chromosomal loci were identified as active super-enhancers (SE) according to active enhancer marker histone H3 lysine 27 acetylation (H3K27ac) ChIP-Seq. In addition, a total of 131 genes were located in SE regions, and 34 genes were upregulated in OSCC tissues by TCGA-OSCC analysis. Moreover, high expression of four genes (C9orf89; p = 0.035, CENPA; p = 0.020, PISD; p = 0.0051, and TRAF2; p = 0.0075) closely predicted a poorer prognosis for OSCC patients according to log-rank tests. Increased expression of the four genes (mRNA Z-score ≥ 0) frequently co-occurred in TCGA-OSCC analyses. The high and low expression groups of the four genes showed significant differences in prognosis, suggesting that there are clear differences in the pathways based on the underlying gene expression profiles. These data indicate that potential stratified therapeutic strategies could be used to overcome resistance to drugs (including cetuximab) and further improve responses in drug-sensitive patients.

Molecular Pathogenesis of the Coronin Family: CORO2A Facilitates Migration and Invasion Abilities in Oral Squamous Cell Carcinoma.

In humans, the coronin family is composed of seven proteins containing WD-repeat domains that regulate actin-based cellular processes. Some members of the coronin family are closely associated with cancer cell migration and invasion. The Cancer Genome Atlas (TCGA) analysis revealed that CORO1C, CORO2A, and CORO7 were significantly upregulated in oral squamous cell carcinoma (OSCC) tissues (p < 0.05). Moreover, the high expression of CORO2A was significantly predictive of the 5-year survival rate of patients with OSCC (p = 0.0203). Overexpression of CORO2A was detected in OSCC clinical specimens by immunostaining. siRNA-mediated knockdown of CORO2A suppressed cancer cell migration and invasion abilities. Furthermore, we investigated the involvement of microRNAs (miRNAs) in the molecular mechanism underlying CORO2A overexpression in OSCC cells. TCGA analysis confirmed that tumor-suppressive miR-125b-5p and miR-140-5p were significantly downregulated in OSCC tissues. Notably, these miRNAs bound directly to the 3'-UTR of CORO2A and controlled CORO2A expression in OSCC cells. In summary, we found that aberrant expression of CORO2A facilitates the malignant transformation of OSCC cells, and that downregulation of tumor-suppressive miRNAs is involved in CORO2A overexpression. Elucidation of the interaction between genes and miRNAs will help reveal the molecular pathogenesis of OSCC.

Identification of Tumor Suppressive Genes Regulated by miR-31-5p and miR-31-3p in Head and Neck Squamous Cell Carcinoma.

We identified the microRNA (miRNA) expression signature of head and neck squamous cell carcinoma (HNSCC) tissues by RNA sequencing, in which 168 miRNAs were significantly upregulated, including both strands of the miR-31 duplex (miR-31-5p and miR-31-3p). The aims of this study were to identify networks of tumor suppressor genes regulated by miR-31-5p and miR-31-3p in HNSCC cells. Our functional assays showed that inhibition of miR-31-5p and miR-31-3p attenuated cancer cell malignant phenotypes (cell proliferation, migration, and invasion), suggesting that they had oncogenic potential in HNSCC cells. Our in silico analysis revealed 146 genes regulated by miR-31 in HNSCC cells. Among these targets, the low expression of seven genes (miR-31-5p targets: CACNB2 and IL34; miR-31-3p targets: CGNL1, CNTN3, GAS7, HOPX, and PBX1) was closely associated with poor prognosis in HNSCC. According to multivariate Cox regression analyses, the expression levels of five of those genes (CACNB2: p = 0.0189; IL34: p = 0.0425; CGNL1: p = 0.0014; CNTN3: p = 0.0304; and GAS7: p = 0.0412) were independent prognostic factors in patients with HNSCC. Our miRNA signature and miRNA-based approach will provide new insights into the molecular pathogenesis of HNSCC.

Impact of Oncogenic Targets by Tumor-Suppressive miR-139-5p and miR-139-3p Regulation in Head and Neck Squamous Cell Carcinoma.

We newly generated an RNA-sequencing-based microRNA (miRNA) expression signature of head and neck squamous cell carcinoma (HNSCC). Analysis of the signature revealed that both strands of some miRNAs, including miR-139-5p (the guide strand) and miR-139-3p (the passenger strand) of miR-139, were downregulated in HNSCC tissues. Analysis of The Cancer Genome Atlas confirmed the low expression levels of miR-139 in HNSCC. Ectopic expression of these miRNAs attenuated the characteristics of cancer cell aggressiveness (e.g., cell proliferation, migration, and invasion). Our in silico analyses revealed a total of 28 putative targets regulated by pre-miR-139 (miR-139-5p and miR-139-3p) in HNSCC cells. Of these, the GNA12 (guanine nucleotide-binding protein subunit alpha-12) and OLR1 (oxidized low-density lipoprotein receptor 1) expression levels were identified as independent factors that predicted patient survival according to multivariate Cox regression analyses (p = 0.0018 and p = 0.0104, respectively). Direct regulation of GNA12 and OLR1 by miR-139-3p in HNSCC cells was confirmed through luciferase reporter assays. Moreover, overexpression of GNA12 and OLR1 was detected in clinical specimens of HNSCC through immunostaining. The involvement of miR-139-3p (the passenger strand) in the oncogenesis of HNSCC is a new concept in cancer biology. Our miRNA-based strategy will increase knowledge on the molecular pathogenesis of HNSCC.

Growth suppression of human oral cancer cells by candidate agents for cetuximab-side effects.

Cetuximab, an inhibitor of the epidermal growth factor receptor that is used widely to treat human cancers including oral squamous cell carcinoma (OSCC), has characteristic side effects of skin rash and hypomagnesemia. However, the mechanisms of and therapeutic agents for skin rashes and hypomagnesemia are still poorly understood. Our gene expression profiling analyses showed that cetuximab activates the p38 MAPK pathways in human skin cells (human keratinocyte cell line [HaCaT]) and inhibits c-Fos-related signals in human embryonic kidney cells (HEK293). We found that while the p38 inhibitor SB203580 inhibited the expression of p38 MAPK targets in HaCaT cells, flavagline reactivated c-Fos-related factors in HEK293 cells. It is noteworthy that, in addition to not interfering with the effect of cetuximab by both compounds, flavagline has additive effect for OSCC growth inhibition in vivo. Collectively, our results indicate that combination of cetuximab and these potential therapeutic agents for cetuximab-related toxicities could be a promising therapeutic strategy for patients with OSCC.

Tspan15 plays a crucial role in metastasis in oral squamous cell carcinoma.

Tetraspanin 15 (Tspan15) is a member of the tetraspanin family, which is associated with various biological events and several diseases, however, its role in human oral squamous cell carcinoma (OSCC) remains unknown. The current study aimed to clarify the role of Tspan15 in OSCC. The mRNA and protein expression levels of Tspan15 were up-regulated in OSCC cases and OSCC-derived cell lines. Significant up-regulated Tspan15 expression was found in the advanced OSCC cases; primary tumoral size (P = 0.042), regional lymph node metastasis (P = 0.036) and TNM classification (P = 0.024). The decreased expression of Tspan15 did not significantly affect cellular proliferation, whereas tumoral invasion and migration activities were suppressed in Tspan15-down-regulated cells, suggesting that Tspan15 might activate metastasis-related signaling. Moreover, in the Tspan15-down-regulated cells, the expression of a disintegrin and metalloproteinase (ADAM) 10 was also down-regulated and the cells secreted less soluble N-cadherin compared with control cells. And weak immunoreactivity of ß-catenin in the nucleus was detected in Tspan15-down-regulated cells compared with the control cells. These findings suggested that overexpression of Tspan15 positively regulates development of OSCC, and that ADAM10, N-cadherin, ß-catenin might be involved in the Tspan15-mediated pathway. These unusual conditions of cell adhesion molecules may lead to high metastasis rate found in Tspan15-overexpressing cases.

Increased expression of tripartite motif (TRIM) like 2 promotes tumoral growth in human oral cancer.

Tripartite motif family-like 2 (TRIML2), a member of the TRIM proteins family, is closely related to Alzheimer's disease, however, no studies of TRIML2 have been published in the cancer research literature. In the current study, we investigated the expression level of TRIML2 and its molecular mechanisms in human oral squamous cell carcinoma (OSCC); reverse transcriptase-quantitative polymerase chain reaction, immunoblot analysis, and immunohistochemistry showed that TRIML2 is up-regulated significantly in OSCCs in vitro and in vivo. TRIML2 knockdown OSCC cells showed decreased cellular proliferation by cell-cycle arrest at G1 phase that resulted from down-regulation of CDK4, CDK6, and cyclin D1 and up-regulation of p21Cip1 and p27Kip1. Surprisingly, resveratrol, a polyphenol, led to not only down-regulation of TRIML2 but also cell-cycle arrest at G1 phase similar to TRIML2 knockdown experiments. Taken together, we concluded that TRIML2 might play a significant role in tumoral growth and that resveratrol may be a new drug for treating OSCC by interfering with TRIML2 function.

Deficiency of lysyl hydroxylase 2 in mice causes systemic endoplasmic reticulum stress leading to early embryonic lethality.

Lysyl hydroxylase 2 (LH2) is an endoplasmic reticulum (ER)-resident enzyme that catalyzes the hydroxylation of lysine residues in the telopeptides of fibrillar collagens. This is a critical modification to determine the fate of collagen cross-linking pathway that contributes to the stability of collagen fibrils. Studies have demonstrated that the aberrant LH2 function causes various diseases including osteogenesis imperfecta, fibrosis, and cancer metastasis. However, surprisingly, a LH2-deficient animal model has not been reported. In the current study, to better understand the function of LH2, we generated LH2 gene knockout mice by CRISPR/Cas9 technology. LH2 deficiency was confirmed by genotyping polymerase chain reaction (PCR), reverse transcriptase-PCR, and immunohistochemical analyses. Homozygous LH2 knockout (LH2-/-) embryos failed to develop normally and died at early embryonic stage E10.5 with abnormal common ventricle in a heart, i.e., an insufficient wall, a thin ventricular wall, and loosely packed cells. In the LH2-/- mice, the ER stress-responsive genes, ATF4 and CHOP were significantly up-regulated leading to increased levels of Bax and cleaved caspase-3. These data indicate that LH2 plays an essential role in cardiac development through an ER stress-mediated apoptosis pathway.

UNC93B1 promotes tumoral growth by controlling the secretion level of granulocyte macrophage colony-stimulating factor in human oral cancer.

Unc-93 homolog B1 (UNC93B1), a transmembrane protein, is correlated with immune diseases, such as influenza, herpes simplex encephalitis, and the pathogenesis of systemic lupus erythematosus; however, the role of UNC93B1 in cancers including human oral squamous cell carcinomas (OSCCs) remains unknown. In the current study, we investigated the UNC93B1expression level in OSCCs using quantitative reverse transcription-polymerase chain reaction, immunoblot analysis, and immunohistochemistry. Our data showed that UNC93B1 mRNA and protein expressions increased markedly (p < 0.05) in OSCCs compared with normal cells and tissues and that high expression of UNC93B1 in OSCCs was related closely to tumoral size. UNC93B1 knockdown (shUNC93B1) OSCC cells showed decreased cellular proliferation by cell-cycle arrest in the G1 phase with up-regulation of p21Cip1 and down-regulation of CDK4, CDK6, cyclin D1, and cyclin E. We also found that granulocyte macrophage colony-stimulating factor (GM-CSF) was down-regulated significantly (p < 0.05) in shUNC93B1 OSCC cells. Moreover, inactivation of GM-CSF using neutralization antibody led to cell-cycle arrest at the G1 phase similar to the phenotype of the shUNC93B1 cells. The current findings indicated that UNC93B1 might play a crucial role in OSCC by controlling the secretion level of GM-CSF involved in tumoral growth and could be a potential therapeutic target for OSCCs.

Multiple coagulation factor deficiency protein 2 as a crucial component in metastasis of human oral cancer.

Multiple coagulation factor deficiency protein 2 (MCFD2), a binding partner of lectin mannose binding 1 (LMAN1), causes combined deficiencies of coagulation factors V and VIII. MCFD2 function in inherited hematologic disorders is well elucidated; however, little is known about its role in human tumorigenesis. The aim of the current study was to investigate the states of MCFD2 in oral squamous cell carcinoma (OSCC). The expression of MCFD2 was up-regulated significantly in all cell lines examined. Evaluation of the cellular functions associated with tumoral metastasis showed that MCFD2 knockdown (shMCFD2) cells exhibited significantly lower cellular invasiveness and migration and higher cellular adhesion compared with shControl cells. Of note, shMCFD2 cells also showed weak immunoreactivity of LMAN1 and a lower secretion level of galactoside-binding soluble 3 binding protein (LGALS3BP). In addition to in vitro validation, clinical data on 70 patients with OSCC indicated that state of MCFD2 expression level is associated with regional lymph node metastasis. Altogether, we have demonstrated that MCFD2 promotes cancer metastasis by regulating LMAN1 and LGALS3BP expression levels. Hence, MCFD2 may represent a promising candidate for a novel therapeutic target for patients with metastatic OSCCs.

Diacylglycerol lipase alpha promotes tumorigenesis in oral cancer by cell-cycle progression.

Diacylglycerol lipase alpha (DAGLA), which catalyzes the hydrolysis of diacylglycerol to 2-arachidonoylglycerol and free fatty acid, is required for axonal growth during the brain development and for retrograde synaptic signaling at mature synapses. So far, no information was found regarding the possible role of DAGLA in human tumorigenesis. Thus, the current study sought to clarify the contribution of DAGLA in oral squamous cell carcinomas (OSCCs) and assess the clinical possibilities for OSCC treatment. Using real-time quantitative reverse transcription-polymerase chain reaction, immunoblotting, and immunohistochemistry, we found a significant up-regulation of DAGLA in OSCCs compared with normal cells and tissues both at mRNA and protein expression levels. Knockdown models in OSCC-derived cell lines for DAGLA (siDAGLA) and treatment with a lipase inhibitor (orlistat) showed several depressed cellular functions, including cellular proliferation and migratory activities through cell-cycle arrest at G1 phase. Furthermore, we found that DAGLA-positive OSCC samples were correlated highly with the primary tumoral size. We concluded that DAGLA may be a key determinant in tumoral progression and might be a therapeutic target for OSCCs.

Critical role of deoxynucleotidyl transferase terminal interacting protein 1 in oral cancer.

Deoxynucleotidyl transferase terminal interacting protein 1 (DNTTIP1) forms a complex with histone deacetylase (HDAC); however, the relevance of DNTTIP1 in cancer remains unknown. The aim of this study was to examine DNTTIP1 expression and its functional mechanisms in oral squamous cell carcinomas (OSCCs). DNTTIP1 expression was analyzed by quantitative reverse transcriptase-polymerase chain reaction, immunoblotting analysis, and immunohistochemistry. The expression of DNTTIP1 was upregulated significantly in vitro and in vivo, and in patients with OSCC in whom DNTTIP1 was overexpressed and the expression level was correlated significantly (P < 0.05) with tumoral growth. DNTTIP1 knockdown (siDNTTIP1) cells showed depressed cellular proliferation by cell-cycle arrest at the G1 phase with high acetylation of p53 and upregulation of p21Cip1. Moreover, resveratrol, a HDAC inhibitor, controlled not only acetylated p53 status but also DNTTIP1 expression, leading to a similar phenotype of siDNTTIP1 cells. A marked (P < 0.05) reduction of tumoral growth in mouse xenograft models was observed with lower DNTTIP1 expression under the presence of this chemical reagent. Taken together, our results suggested that DNTTIP1-HDAC interaction promotes tumoral growth through deacetylation of p53 and that DNTTIP1 might be a critical therapeutic target in OSCCs.

Evaluation of tryptophan-aspartic acid repeat-containing protein 34 as a novel tumor-suppressor molecule in human oral cancer.

Tryptophan-aspartic acid (WD) repeat-containing protein 34 (WDR34), one of the WDR protein superfamilies with five WD40 domains, inhibits a transforming growth factor-beta (TGF-ß) activated kinase 1 (TAK1)-associated NF-κB activation pathway. Nevertheless, little is known about the roles of WDR34 in cancer. The current study sought to elucidate the clinical relevance of WDRsfb34 in oral squamous cell carcinoma (OSCC). We found WDR34 down-regulation in OSCCs compared with normal control tissues using real-time quantitative reverse transcription-polymerase chain reaction, immunoblotting, and immunohistochemistry. Models of overexpression of WDR34 (oeWDR34) showed depressed cellular growth through cell-cycle arrest at the G1 phase. To investigate the inhibitory function of WDR34, we challenged oeWDR34 cells with interleukin (IL)-1, a ligand for activation of the TAK1-NF-κB pathway and assessed the expression of a target gene of the pathway. oeWDR34 strongly inhibited IL-6 expression, which is closely related to tumoral growth, compared with control cells, suggesting that WDR34 would be a critical molecule for control of tumoral progression. In addition to the in vitro experiments, WDR34 negativity was correlated with tumoral growth of OSCCs. Our findings suggested that WDR34 inhibits OSCC progression and might be a potential tumor-suppressor molecule in OSCCs.

FBLIM1 enhances oral cancer malignancy via modulation of the epidermal growth factor receptor pathway.

Filamin-binding LIM protein 1 (FBLIM1) is related to regulation of inflammatory responses, such as chronic recurrent multifocal osteomyelitis; however, the relevance of FBLIM1 in oral squamous cell carcinoma (OSCC) is unknown. The aim of the current study was to elucidate the possible role of FBLIM1 in the carcinogenesis of OSCC. We analyzed FBLIM1 expression using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), immunoblot analysis, and immunohistochemistry. The expression levels of FBLIM1 were up-regulated significantly (P < 0.05) in OSCC-derived cell lines and primary OSCCs specimens compared with normal counterparts. FBLIM1 expression also was correlated with the primary tumoral size (P < 0.05) and vascular invasion (P < 0.05). We then assessed tumoral progression after treatment with FBLIM1 siRNA and clopidogrel, an antiplatelet agent. Similar to the FBLIM1 knockdown effect, clopidogrel-treated cells had attenuated functions of proliferation, migration, and invasiveness. Interestingly, clopidogrel treatment led to down-regulation of epidermal growth factor receptor (EGFR) and FBLIM1. These findings identify FBLIM1 as a putative therapeutic target by using clopidogrel for inhibiting over activation of EGFR signaling to prevent OSCC malignancy.