Human Oral Squamous Cell Carcinoma Cell Lines Research Articles

Abstract 2339: Downregulation of cell cycle genes in HNSCC by erufosine

Abstract Head and neck cancer ranks amongst the sixth most prevalent cancers worldwide and oral squamous cell carcinomas (OSCC) constitute 90% thereof. Erufosine is an ether-lipid-derived synthetic compound belonging to alkylphosphocholines (APCs), which has been shown to inhibit the proliferation of OSCC cells. It simultaneously induces apoptosis and autophagy by modulating the Akt-mTOR signaling pathway, however, its exact mechanism of action is not fully understood. Here, we describe the activity of erufosine on the expression of cell cycle related genes in OSCC cells. The anti-proliferative effect of erufosine in cells of two human OSCC cell lines, HN-5 and SCC-61, was determined by MTT assay after 24h, 48h and 72h exposure. Based on these results, HN-5 cells were exposed to erufosine at concentrations corresponding to IC25, IC50 and IC75 concentrations, and then their mRNA was isolated and analyzed by Illumina Chip array. Gene expression modulation was confirmed qRT-PCR and Western blot. Gene set enrichment analysis was performed to identify core KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways and GO (Gene Ontology) terms. In addition, erufosine’s effect on cell cycle distribution and colony formation was determined as well as its antineoplastic effect on respective xenografts in nude mice. As shown from TCGA data, cell cycle deregulation in HNSCC ranks amongst the top three out of 24 different types of cancers. In line with this, the median expression of cell cycle genes was higher than that of other genes within HNSCC. We also looked at the median gene expression of all CDKs and cyclins in 519 HNSCC patient samples from TCGA and found that the expression levels were higher than the global gene expression. This was in agreement with IHC staining of CCND1 in tumors from 30 South East Asian HNSCC patients. Furthermore, increased CCND1 and CDK6 expression levels from TCGA data had negative implication on patient survival. In our experimental results erufosine caused a dose dependent growth inhibition of OSCC cell lines and negative enrichment of genes related to the cell cycle process. Our microarray findings revealed that cyclins and CDKs were downregulated in a dose dependent manner in response to erufosine exposure. These findings were verified at both, mRNA and protein levels. Erufosine not only caused a massive G2M block but also inhibition of colony formation thus preparing the OSCC cells to undergo apoptosis. We are the first to show that erufosine inhibited tumor growth in vivo in a HNSCC xenograft model and caused downregulation of cyclinD1, CDK4 and CDK6 in lesions of the animals. These findings collectively show the potential of erufosine to be used as a cell cycle inhibitor in HNSCC progression and support the future evaluation of erufosine as a therapeutic approach in cancer treatment alone, or in combination. Citation Format: Shariq S. Ansari, Ashwini K. Sharma, Michael Zepp, Frank Bergmann, Rainer König, Martin R. Berger. Downregulation of cell cycle genes in HNSCC by erufosine [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 2339. doi:10.1158/1538-7445.AM2017-2339

Loss of NDRG2 Expression Confers Oral Squamous Cell Carcinoma with Enhanced Metastatic Potential.

Loss of the tumor suppressor NDRG2 has been implicated in the development of oral squamous cell carcinoma (OSCC), acting by modulating PI3K/AKT-mediated dephosphorylation of PTEN at S380/S382/T383 (STT). Here, we show that the majority of OSCC tumors with lymph node metastasis, a major prognostic factor, exhibit high levels of phosphorylated AKT-S473 and PTEN-STT and low levels of NDRG2 expression. In Ndrg2-deficient mice, which develop a wide range of tumors, we developed a model of OSCC by treatment with the tobacco surrogate 4-nitroquinoline-1-oxide (4-NQO). In this model, both the number and size of OSCC tumors were increased significantly by Ndrg2 deficiency, which also increased invasion of cervical lymph nodes. 4-NQO treatment of human OSCC cell lines exhibiting low NDRG2 expression induced epithelial-mesenchymal transition via activation of NF-κB signaling. Conversely, ectopic expression of NDRG2 reversed the EMT phenotype and inhibited NF-κB signaling via suppression of PTEN-STT and AKT-S473 phosphorylation. Our results show how NDRG2 expression serves as a critical determinant of the invasive and metastatic capacity of OSCC. Cancer Res; 77(9); 2363-74. ©2017 AACR.

Histone deacetylase 8 as a novel therapeutic target in oral squamous cell carcinoma.

The overexpression of histone deacetylases (HDACs) has been observed in many cancers and inhibition of specific HDAC has emerged as a new target for cancer therapy. The present study examined the expression of HDAC8 and the inhibitory effect of HDAC8 in oral squamous cell carcinoma (OSCC). The expression of HDAC8 was measured in human OSCC tissues and OSCC cell lines using immunohistochemistry and immunoblotting. HDAC8 was knocked down in OSCC cells by transfection with HDAC8 siRNAs and cell proliferation was quantified. Apoptosis and autophagy were measured using flow cytometry and immunoblotting. HDAC8 were overexpressed in OSCC tissues and OSCC cells, mainly localized in the cytoplasm. HDAC8 siRNAs effectively reduced the level of HDAC8 expression and HDAC8 silencing significantly inhibited the proliferation of OSCC cells. HDAC8 knockdown induced apoptotic cell death through caspases activation and pro-survival autophagy in OSCC cells. Combination with HDAC silencing and autophagy inhibition enhanced cell death by increasing apoptosis in OSCC cells. This study suggests that inhibition of HDAC8 might become a novel therapeutic strategy for OSCC.

SATB1 promotes tumor metastasis and invasiveness in oral squamous cell carcinoma.

Our aim is to evaluate the expression of SATB1 in human oral squamous cell carcinomas (OSCC) and its role in the invasiveness and metastasis of OSCC. A human OSCC tissue microarray was used to evaluate the expression pattern of SATB1. SATB1 mRNA knockdown was performed in human OSCC cell lines SCC25 and Cal27 to assess the function of SATB1 in the invasiveness and metastasis of OSCC. SATB1 is highly expressed in human OSCC determined by immunohistochemistry, and its nuclear/cytoplasmic ratio of histoscore is significantly correlated with patients' prognosis. Reduced cell motility, invasiveness, expression of epithelial to mesenchymal transition (EMT) markers (N-cadherin and β-catenin), and elevated expression of epithelial markers were observed in SATB1-knockdown cells in invitro studies. Depletion of SATB1 also restored a cobblestone-like morphology in TGF-β1-treated cells. These findings suggest SATB1 may play an important role in OSCC invasiveness and metastasis.

Antitumor activity of selenium compounds and its underlying mechanism in human oral squamous cell carcinoma cells: A preliminary study

ObjectiveOral cancer is an aggressive disease that infiltrates the adjacent tissues and frequently metastasizes to lymph nodes in the neck. Currently, no chemotherapy effectively prevents its metastasis. Selenium compounds have been recently scrutinized as chemotherapeutic agents for various cancers. In this study, we aimed to investigate the antitumor activity of selenium compounds and elucidate the underlying inhibitory mechanism of these agents in oral cancer cells. MethodsThe growth inhibitory effects of selenium compounds (sodium selenite, selenomethionine, and Se-methylselenocysteine) on human oral squamous cell carcinoma (HOSCC) cell lines (HSC-3, HSC-4, and SAS) were evaluated by MTT assay. Selenite-induced apoptosis, caspase activity, and endoplasmic reticulum (ER) stress in HSC-3 cells were evaluated by flow cytometry and western blot. Effects of selenite on Akt expression in HSC-3 cells were evaluated by ELISA and western blot. ResultsSelenium compounds significantly inhibited cell growth and induced apoptosis in HOSCC cell lines. HSC-3 cells, in particular, were highly sensitive to selenite. In selenite-treated HSC-3 cells, caspase-3, 8, and 9 were conspicuously activated; pretreatment with pan-caspase inhibitor or caspase-12 inhibitor dramatically reduced selenite-induced apoptosis; ER stress markers, caspase-12 and eIF-2α, were highly activated, but Akt activation in the Akt/phosphoinositide-3-kinase pathway was downregulated. ConclusionOur findings indicate that selenite induces apoptosis in HOSCC cell lines in a caspase-dependent manner through mitochondrial, death-receptor, and ER stress pathways. In addition, selenite could exhibit antitumor activity by downregulating Akt activation, which plays an important role in cell growth and chemotherapy resistance.

MiR-448 downregulates MPPED2 to promote cancer proliferation and inhibit apoptosis in oral squamous cell carcinoma.

The incidence of oral squamous cell carcinoma (OSCC) is continuously increasing while its survival rate has not notably improved. There is a pressing need for improved understanding of the genetic regulation of OSCC tumorigenesis and progression. In this study, the function of miR-448 in the regulation of OSCC growth and its putative target were thoroughly analyzed in vitro. The expression of miR-448 was detected in human OSCC specimens and OSCC cell lines (Cal-27 and Scc-9) by reverse transcription-quantitative polymerase chain reaction. The function of miR-448 was investigated in Cal-27 cells transfected with miR-448 inhibitor, and its putative target determined using a luciferase reporter assay. MTT and wound healing assays and flow cytometry were used to evaluate the effects of miR-448 on OSCC proliferation, metastasis and apoptosis. The level of miR-448 was significantly elevated in human OSCC tissues and the Cal-27 cell line. Suppression of miR-448 expression attenuated cell proliferation and migration, and induced apoptosis of Cal-27 cells. Furthermore, miR-448 bound with the 3'-untranslated region of metallophosphoesterase domain containing 2 (MPPED2) mRNA, thereby reducing the MPPED2 protein level. Thus, it appears that miR-448 acts as a tumor inducer, causing OSCC growth by inhibiting the expression of its target MPPED2. These results demonstrate that miR-448 plays a critical role in OSCC tumorigenesis, and is a potential therapeutic target.

Knockdown of Myosin 6 inhibits proliferation of oral squamous cell carcinoma cells.

Oral squamous cell carcinoma (OSCC) accounts for 95% of all oral cancer with higher mortality and morbidity rates worldwide. However, the potential molecular mechanism of OSCC remains largely unclear. Myosin VI (MYO6) is a unique actin motor and reported to be overexpressed in several cancers. This study aims to examine the functional relationship between OSCC and MYO6. The mRNA expression of MYO6 was firstly investigated by analyzing data derived from Oncomine database. On the basis of the results, the expression of MYO6 was knocked down using lentivirus-delivered RNA interference in human OSCC cell line CAL27, as confirmed by qPCR and Western blot analysis. Stable MYO6 knockdown cells were employed to determine the effects of MYO6-silencing on cell growth by MTT, colony formation and cell cycle distribution and apoptosis by flow cytometry assay. Moreover, the expressions of cell apoptotic proteins were examined by Western blot analysis. We first observed MYO6 was overexpressed in tongue squamous cell carcinoma TSCC belongs to OSCC, compared with normal tissues. For cellular analysis, shRNA sequences against MYO6 could efficiently reduce its expression in CAL27 cells. Knockdown of MYO6 significantly decreased cell proliferation, caused cell cycle arrest at G2/M phase, and promoted cell apoptosis. Moreover, cell apoptosis-associated proteins, caspase-3 and PARP, were obviously upregulated in CAL27 after MYO6-silencing. MYO6 could play an essential role in the growth of OSCC cells via regulation of cell cycle progression and apoptosis.

Abstract 1147: CDCA5 is a novel therapeutic target molecule in oral squamous cell carcinoma

Abstract Molecularly targeted drugs are used in the treatment of a variety of malignant tumors, but this approach to developing novel therapies for oral squamous cell carcinoma (OSCC) has lagged behind the progress seen for other cancers. We attempted to find appropriate molecular targets for OSCC, and identified cell division cycle associated 5 (CDCA5) as a cancer-related gene which was overexpressed in 9 human OSCC cells tested by microarray analysis. CDCA5 was initially identified as a substrate of anaphase-promoting complex and as a regulator of sister chromatid cohesion in HeLa cells. In addition, CDCA5 has been reported to be overexpressed in the majority of human lung cancers and urothelial cancers and to play a critical role in carcinogenesis. However, function of CDCA5 in OSCC remains still unknown. In this study, we investigated the expression and function of CDCA5 in OSCC. First, we confirmed the expression level of CDCA5 in 4 human OSCC cell lines and an immortalized non-neoplastic human keratinocyte cell line, HaCaT, by quantitative RT-PCR (qRT-PCR) and Western blotting. The expression levels of CDCA5 mRNA and protein were markedly elevated in all human OSCC cell lines compared to HaCaT cells. Next, we tested the effect of synthetic small interfering RNAs specific for CDCA5 (siCDCA5) on the growth of human OSCC cells. The RNA interference effect of siCDCA5 was confirmed by Western blotting. Transfection of siCDCA5 suppressed the expression of its target protein in all types of cells. The growth inhibitory effect of siCDCA5 in OSCC cells was evaluated by WST-8 assay. The Knockdown of CDCA5 significantly inhibited the growth of OSCC cells in vitro. Subsequently, we assessed the effect of siCDCA5 on the in vivo growth of human OSCC cells, green fluorescent protein (GFP)-SAS. We administered siCDCA5/atelocollagen complexes into the subcutaneous spaces around the xenograft tumors every 3 days. We found that these complexes significantly reduced the size of subcutaneously xenografted GFP-SAS tumors, compared to the control group treated with synthetic siRNA specific for GFP (siGFP)/atelocollagen complexes. Next, we analyzed the influence of siCDCA5 on the cell cycle of OSCC cells by flow cytometry. Suppression of CDCA5 resulted in the decrease in percentage of cells in G0/G1 phase and the increase in G2 phase. This indicated that the anti-proliferative effect of siCDCA5 was due to G2 arrest. Finally, we investigated the clinical significance of CDCA5 expression in OSCC. We examined the expression of CDCA5 in OSCC tissues by immunohistochemistry, and then found a significant association between CDCA5 expression levels and overall survival. These results suggest that CDCA5 functions as a critical gene supporting OSCC progression and that targeting CDCA5 may be a useful therapeutic strategy for OSCC. Citation Format: Norihiko Tokuzen, Koh-ichi Nakashiro, Hiroshi Tanaka, Kazuki Iwamoto, Hitoshi Akiyama, Hiroyuki Hamakawa. CDCA5 is a novel therapeutic target molecule in oral squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1147.

Abstract 636: An orthotopic xenograft animal model for functional analysis of podoplanin in oral squamous cell carcinoma

Abstract Podoplanin (PDPN) is a marker for poor prognosis in human oral squamous cell carcinoma (OSCC). Subcutaneous or intravenous injection of OSCC cells has been used in a number of studies as the animal models to investigate the function of PDPN in OSCC cancer progression. However, these models usually overlook the potential interactions between cancer cells and the microenvironment of oral cavity. In this study, we aimed to establish an animal model for functional and mechanistic study of PDPN in OSCC by taking into account the influence of the microenvironment of oral cavity. Profiling of nine human OSCC cell lines revealed that PDPN was highly expressed in five of the cell lines, including OECM-1, OC2, TW2.6, HSC-3 and CAL27. Notably, OECM-1 cells were shown by immunofluorescence staining to compose of two different cell populations with highly expression (P+) or absence (P-) of PDPN. To preclude the potential artifacts associated with gene overexpression study, the P+ cells were isolated by fluorescence-activated cell sorting followed by establishing the sublines shLacZ or shPDPN that co-expressed luciferase and the short-hairpin RNA targeting on β-galactosidase or pdpn. In vitro analysis revealed no difference for the cell growth of shLacZ and shPDPN cells on day 5 (p = 0.79). Orthotopic xenograft animal model for functional analysis of PDPN in OSCC was established by inoculating shLacZ or shPDPN cells into the anterior neck region of athymic nude mice. Consistent with the in vitro assays, there is no difference in the growth of the tumors derived from shLacZ and shPDPN cells (p > 0.05). Notably, the survival of the mice bearing tumors derived from shPDPN cells was significantly prolonged when compared with the mice bearing shLacZ cells-derived tumors (p < 0.001). At day 50 after orthotopic inoculation of cancer cells, 10% (n = 10, 1/10) and 90% (n = 10, 9 /10) of the mice bearing tumors derived from shLacZ and shPDPN cells was alive, respectively. These results were consistent with the notion that PDPN is a poor prognosis factor for the patients with OSCC. In contrast to the orthotopic xenograft model, subcutaneous injection of shLacZ and shPDPN cells into the athymic nude mice did not result in a significant increase in tumor growth and tumor size. These data further demonstrate that the microenvironment of the oral cavity has significant effect on promoting the survival and growth of OSCC cells. The orthotopic xenograft mouse model we established in this study represents a useful model for functional and mechanistic study of PDPN in the cancer progression of OSCC. Citation Format: Hsing-Ying Lee, Yao-Wen Chang, Ju-Chien Cheng, Ching-Ping Tseng. An orthotopic xenograft animal model for functional analysis of podoplanin in oral squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 636.

Co-targeting ALK and EGFR parallel signaling in oral squamous cell carcinoma

Squamous cell carcinoma (SCC) comprises 90% of all head and neck cancers and has a poor survival rate due to late-stage disease that is refractive to traditional therapies. Epidermal growth factor receptor (EGFR) is over-expressed in greater than 80% of head and neck SCC (HNSCC). However, EGFR targeted therapies yielded little to no efficacy in clinical trials. This study investigated the efficacy of co-targeting EGFR and the anaplastic lymphoma kinase (ALK) whose promoter is hypomethylated in late-stage oral SCC (OSCC). We observed increased ALK activity in late-stage human OSCC tumors and invasive OSCC cell lines. We also found that while ALK inhibition alone had little effect on proliferation, co-targeting ALK and EGFR significantly reduced OSCC cell proliferation in vitro. Further analysis showed significant efficacy of combined treatment in HSC3-derived xenografts resulting in a 30% decrease in tumor volumes by 14days (p<0.001). Western blot analysis showed that co-targeting ALK and EGFR significantly reduced EGFR phosphorylation (Y1148) in HSC3 cells but not Cal27 cells. ALK and EGFR downstream signaling interactions are also demonstrated by Western blot analysis in which lone EGFR and ALK inhibitors attenuated AKT activity whereas co-targeting ALK and EGFR completely abolished AKT activation. No effects were observed on ERK1/2 activation. STAT3 activity was significantly induced by lone ALK inhibition in HSC3 cells and to a lower extent in Cal27 cells. Together, these data illustrate that ALK inhibitors enhance anti-tumor activity of EGFR inhibitors in susceptible tumors that display increased ALK expression, most likely through abolition of AKT activation.

Transforming growth factor-β1 activates ΔNp63/c-Myc to promote oral squamous cell carcinoma

During the development of oral squamous cell carcinoma (OSCC), the transformed epithelial cells undergo increased proliferation resulting in tumor growth and invasion. Interestingly, throughout all phases of differentiation and progression to OSCC, transforming growth factor-β1 (TGF)-β1 induces cell cycle arrest or apoptosis; however, the role of TGF-β1 in promoting cancer cell proliferation has not been explored in detail. The purpose of this study was to identify the effect of TGF-β1 on OSCC cell proliferation. Using both human OSCC samples and cell lines (UMSCC38 and UMSCC11B), we assessed protein, mRNA, gene expression, and protein-DNA interactions during OSCC progression. Our results showed that TGF-β1 increased OSCC cell proliferation by upregulating the expression of ΔNp63 and c-Myc oncogenes. Although the basal OSCC cell proliferation is sustained by activating ΔNp63, increased induction of c-Myc causes unregulated OSCC cell proliferation. Following induction of the cell cycle by ΔNp63 and c-Myc, cancer cells that halt c-Myc activity undergo epithelial mesenchymal transition or invasion while those that continue to express ΔNp63/c-Myc undergo unlimited progression through the cell cycle. OSCC proliferation is manifested by the induction of c-Myc in response to TGF-β1 signaling, which is essential for OSCC growth. Our data highlight the potential role of TGF-β1 in the induction of cancer progression and invasion of OSCC.

Clinicopathological implications of vascular endothelial growth factor 165b expression in oral squamous cell carcinoma stroma.

Vascular endothelial growth factor (VEGF) is one of the most important angiogenic factors. VEGF165b was recently isolated as the anti-angiogenic VEGF splice variant. In the present study, we examined the association between VEGF165b expression and clinicopathological characteristics in order to determine how VEGF165b produced from oral squamous cell carcinoma (OSCC) affects the stromal cell biological activity. We examined the relationships between the expressions of both VEGF isoforms in normal human dermal fibroblasts (NHDFs) and OSCC cell lines (HSC2, 3, 4 and SAS). Our analyses indicated that both the mRNA and protein expression levels of VEGF165b in the HSC2 and SAS cells were higher than those in the NHDFs. VEGF165b did not promote cell growth or invasive capabilities, but it induced the cell adhesive capabilities to ECM. Although strong expression of the VEGF165 isoforms in tumor cells of OSCC tissues was observed, there was no significant difference in the VEGF165b expression level among the various degrees of malignancy. OSCC cells secrete VEGF165b into the stroma, and this factor may contribute to the process of anti-angiogenesis by inhibiting gelatinase-expressing cells and activating cell adhesive capabilities to ECM, such as that of fibroblasts surrounding tumor cells.

MicroRNA profiling of cisplatin-resistant oral squamous cell carcinoma cell lines enriched with cancer-stem-cell-like and epithelial-mesenchymal transition-type features.

Oral cancer is of major public health problem in India. Current investigation was aimed to identify the specific deregulated miRNAs which are responsible for development of resistance phenotype through regulating their resistance related target gene expression in oral squamous cell carcinoma (OSCC). Cisplatin-resistant OSCC cell lines were developed from their parental human OSCC cell lines and subsequently characterised. The resistant cells exhibited enhanced proliferative, clonogenic capacity with significant up-regulation of P-glycoprotein (ABCB1), c-Myc, survivin, β-catenin and a putative cancer-stem-like signature with increased expression of CD44, whereas the loss of E-cadherin signifies induced EMT phenotype. A comparative analysis of miRNA expression profiling in parental and cisplatin-resistant OSCC cell lines for a selected sets (deregulated miRNAs in head and neck cancer) revealed resistance specific signature. Moreover, we observed similar expression pattern for these resistance specific signature miRNAs in neoadjuvant chemotherapy treated and recurrent tumours compared to those with newly diagnosed primary tumours in patients with OSCC. All these results revealed that these miRNAs play an important role in the development of cisplatin-resistance mainly through modulating cancer stem-cell-like and EMT-type properties in OSCC.

Neuropilin 1 Receptor Is Up-Regulated in Dysplastic Epithelium and Oral Squamous Cell Carcinoma

Neuropilins are receptors for disparate ligands, including proangiogenic factors such as vascular endothelial growth factor and inhibitory class 3 semaphorin (SEMA3) family members. Differentiated cells in skin epithelium and cutaneous squamous cell carcinoma highly express the neuropilin-1 (NRP1) receptor. We examined the expression of NRP1 in human and mouse oral mucosa. NRP1 was significantly up-regulated in oral epithelial dysplasia and oral squamous cell carcinoma (OSCC). NRP1 receptor localized to the outer suprabasal epithelial layers in normal tongue, an expression pattern similar to the normal skin epidermis. However, dysplastic tongue epithelium and OSCC up-regulated NRP1 in basal and proliferating epithelial layers, a profile unseen in cutaneous squamous cell carcinoma. NRP1 up-regulation is observed in a mouse carcinogen-induced OSCC model and in human tongue OSCC biopsies. Human OSCC cell lines express NRP1 protein in vitro and in mouse tongue xenografts. Sites of capillary infiltration into orthotopic OSCC tumors correlate with high NRP1 expression. HSC3 xenografts, which express the highest NRP1 levels of the cell lines examined, showed massive intratumoral lymphangiogenesis. SEMA3A inhibited OSCC cell migration, suggesting that the NRP1 receptor was bioactive in OSCC. In conclusion, NRP1 is regulated in the oral epithelium and is selectively up-regulated during epithelial dysplasia. NRP1 may function as a reservoir to sequester proangiogenic ligands within the neoplastic compartment, thereby recruiting neovessels toward tumor cells.

THBS1 is induced by TGFB1 in the cancer stroma and promotes invasion of oral squamous cell carcinoma.

THBS1 (thrombospondin-1) is the extracellular matrix (ECM) protein that affects diverse cellular activities. It constitutes the tumor stroma, but the role of THBS1 in oral squamous cell carcinoma (OSCC) development is unclear. The aim of this study was to clarify the relevance of THBS1 in the pathogenesis of OSCC. The expression of THBS1 was examined in 44 OSCC by immunohistochemical analysis and in 43 OSCC by cDNA microarray analysis. Cell culture experiments were conducted using human OSCC cell lines HSC3 and HO1N1 and mouse fibroblast ST2 cells to examine the effect of TGFB1 on THBS1 expression, and the effect of THBS1 on cellular behaviors. THBS1 was specifically induced in the tumor microenvironment of OSCC. THBS1 appeared to be produced mainly by the stromal cells, but also by OSCC cells. TGFB1 stimulated THBS1 expression in ST2, primary fibroblasts, and the OSCC cells. THBS1 promoted migration and invasion of HSC3 and HO1N1 in transwell migration assays. THBS1 stimulated the expression of MMP3 (matrix metalloprotease 3), MMP9, MMP11, and MMP13 in ST2 cells and MMP3, MMP11, and MMP13 in HO1N1 cells. The RGD peptide suppressed the THBS1-stimulated migration and upregulation of MMP11 and MMP13. THBS1 is a tumor-specific ECM protein that is induced by TGFB1 and promotes migration of cancer cells and stimulates the expression of MMPs partly through the integrin signaling, thereby favoring OSCC invasion.

CD44v3+/CD24- cells possess cancer stem cell-like properties in human oral squamous cell carcinoma.

Cancer stem cells (CSCs) or cancer stem cell-like cells (CSC-LCs) are a minority population of cells that relate to tumor progression, metastasis and drug resistance. To identify CSC-LCs in oral squamous cell carcinoma (OSCC), we used two OSCC cell lines, SAS and OSC20, and cell surface markers, CD44v3 and CD24. In addition, we examined CD44v3 and CD24 expression immunohistochemically and evaluated the relationship between the expression and clinicopathological parameters in 50 OSCC tissues. In SAS and OSC20, CD44v3+/CD24− cells showed a higher sphere forming ability than the other fractions, i.e., CD44v3+/CD24+, CD44v3−/CD24− and CD44v3−/CD24+ cells. The proportion of CD44v3+/CD24− cells in SAS and OSC20 was 10.7 and 24.1%, respectively. Regarding SAS, CD44v3+/CD24− cells also showed a higher drug resistance for CDDP, 5-FU and cetuximab and expressed higher mRNA levels of CSC property-related genes than the other cell fractions. The tumorigenicity of CD44v3+/CD24− cells was not significantly different from the other fractions in SAS. An immunohistochemical study revealed a significant correlation between CD44v3 expression in the invasive portion and lymph node metastasis. Kaplan Meier analysis revealed cases with CD44v3 expression in the invasive portion tended to show poor overall survival (OS) compared with those without CD44v3, and there was a significant difference in OS between CD44v3+/CD24− and CD44v3−/CD24− immunophenotypes in the invasive portion. In conclusion, the results suggest that the CD44v3+/CD24− cell population displays CSC-LC properties in a human OSCC cell line. Additionally, we present evidence that CD44v3 immunoexpression and CD44v3+/CD24− immunophenotypes could give prognostic information associated with unfavorable clinical outcomes.

Therapeutic potential of targeting cell division cycle associated 5 for oral squamous cell carcinoma.

Molecularly targeted drugs are used in the treatment of a variety of malignant tumors, but this approach to developing novel therapies for oral squamous cell carcinoma (OSCC) has lagged behind the progress seen for other cancers. We have attempted to find appropriate molecular targets for OSCC and identified cell division cycle associated 5 (CDCA5) as a cancer-related gene which was overexpressed in all the human OSCC cells tested by microarray analysis. In this study, we investigated the expression and function of CDCA5 in OSCC. First, we confirmed that CDCA5 was overexpressed in 4 human OSCC cell lines by quantitative RT-PCR and Western blotting. We then tested the effect of synthetic small interfering RNAs specific for CDCA5 on the growth and invasion of human OSCC cells. Knockdown of CDCA5 markedly inhibited the growth of OSCC cells in vitro and in vivo. We also examined the expression of CDCA5 protein in 80 cases of OSCC immunohistochemically and found a significant association between CDCA5 expression levels and overall survival. These results suggest that CDCA5 functions as a critical gene supporting OSCC progression and that targeting CDCA5 may be a useful therapeutic strategy for OSCC.

Establishment of a xenograft model to explore the mechanism of bone destruction by human oral cancers and its application to analysis of role of RANKL.

The molecular mechanism underlying bone invasion caused by oral squamous cell carcinoma (OSCC) is not well understood. To elucidate the molecular mechanism, the development of more suitable xenograft models mimicking human mandibular bone destruction by OSCC has been required. Human OSCC cell lines, HSC3, HSC3-C1, and HSC3-R2, were injected in the periosteal region of the mandible in athymic mice, and the bone destruction was analyzed. Receptor activators of nuclear factor κ-B ligand (RANKL) mRNA and protein expression levels were measured in the OSCC cell lines. Antibody that specifically neutralizes mouse RANKL and human RANKL, respectively, was injected into HSC3-cell-transplanted mice. Transplantation of HSC3 cells induced mandibular bone destruction. Histological examination revealed numerous osteoclasts on the bone destruction surface. Fibroblastic cell intervention between the cancer nests and resorbing bone surface was observed in a similar fashion to those observed in human OSCC cases. The number of osteoclasts and fibroblasts was significantly correlated. Bone destruction induced by the transplantation of HSC3 cells was reduced by injection of an antibody that specifically neutralizes mouse RANKL. Transplantation of HSC3-R2 cells, which overexpresses RANKL, induced advanced bone destruction compared to that of HSC3-C1 cells, which only overexpress the empty vector. We established a useful xenograft model for investigating the molecular mechanism underlying the bone destruction induced by OSCC in the jaw. This model will be used to investigate the precise roles of several cytokines synthesized by both cancer cells and fibroblastic cells in OSCC-associated bone destruction in the jaw.

Identification of Akt1 as a potent therapeutic target for oral squamous cell carcinoma.

Oncogene addiction can provide therapeutic opportunities in human malignancies. In this study, we aimed to identify critical oncogenes for oral squamous cell carcinoma (OSCC) development and progression. We determined gene expression profiles in 10 primary OSCCs and 10 human OSCC cell lines using Applied Biosystems Human Genome Survey Arrays. Akt1 was the only gene identified that was expressed in all OSCC tissues and cultured cells, but not in non-neoplastic tissues and cells. Subsequently, western blot analysis showed that Akt1 protein was overexpressed in OSCC tissues and cell lines. Immunohistochemistry also showed Akt1 protein expression in 59 of 63 (94%) primary OSCCs. To clarify the oncogenic function of Akt1 in human OSCC cells, we used RNA interference. We designed and synthesized 5 small interfering RNAs specific for Akt1 (siAkt1). Transfecting human OSCC cells with siAkt1 in vitro markedly suppressed their expression of Akt1 protein and significantly reduced their growth rate. Furthermore, the growth of human OSCC tumors which had been subcutaneously xenografted in athymic nude mice lacking interferon responses was markedly inhibited by atelocollagen-mediated systemic siAkt1 administration. We also found that synthetic siAkt1 had an inhibitory effect on the growth of primary cultured OSCC cells. Finally, we investigated the molecular mechanisms involved in the growth inhibitory effect of Akt1 suppression using microarray analysis of human OSCC cells transfected with siAkt1. Knockdown of Akt1 induced the expression of CDKN2B, a tumor suppressor gene, and reduced the expression of TGFBR1, which supports malignant phenotypes. These results suggest that Akt1 functions as a critical oncogene in human OSCC cells and may therefore be an appropriate target for novel OSCC therapies.

Daxx and TCF4 interaction links to oral squamous cell carcinoma growth by promoting cell cycle progression via induction of cyclin D1 expression.

ObjectivesDeath domain-associated protein (Daxx) has been recently implicated as a positive factor in ovarian cancer and prostate cancer, but the role of Daxx in oral squamous cell carcinoma (OSCC) has never been addressed. Herein, we investigate the expression and function of Daxx in OSCC.Materials and methodsRT-quantitative PCR, Western blotting, and immunohistochemistry were used to evaluation of the expression of Daxx in human OSCC cell lines and clinical surgical specimens. Short hairpin RNA targeting Daxx was transduced by lentivirus infection to knockdown the expression of Daxx in SAS and SCC25 cell lines, and the influence of this knockdown was evaluated by analyzing the growth and the cell cycle in transduced cells. Immunoprecipitation and sequential chromatin immunoprecipitation-quantitative PCR were used to analyze the associations between Daxx, TCF4, and cyclin D1 promoter. Xenograft tumor model was used to evaluate the in vivo tumorigenicity of Daxx in OSCC.ResultsDaxx mRNA and protein expression are elevated in several OSCC cell lines and human OSCC samples in comparison to those in normal tissue. We further find that depletion of Daxx decreases OSCC cell growth activity through G1 cell cycle arrest. Daxx silencing reduces cyclin D1 expression via a Daxx-TCF4 interaction, whereas the Daxx depletion-mediated G1 arrest can be relieved by ectopic expression of cyclin D1. Moreover, we show that in OSCC clinical samples, the expression of Daxx is significantly correlated with that of cyclin D1.ConclusionOur data demonstrate the importance of Daxx in regulation of cyclin D1 expression and provide the first evidence that Daxx exhibits tumor-promoting activity in OSCC.Clinical relevanceDaxx plays an important role in malignant transformation of OSCC and may serves as a target for cancer prevention and treatment.