Oral Cancer Cell Invasion Research Articles

Surfactin from Bacillus subtilis attenuates ambient air particulate matter-promoted human oral cancer cells metastatic potential.

Recently, many studies have indicated that ambient air particulate matter (PM) can increase the risk of oral cancer. The most common malignant tumor in the oral cavity is oral squamous cell carcinoma (OSCC). Usually, cancer cell migration/invasion is the most important cause of cancer mortality. Matrix metalloproteinase-2 (MMP-2) and MMP-9 have been shown to play important roles in regulating metastasis and the tumor microenvironment. Here, we studied the anti-cancer effects of surfactin, a cyclic lipopeptide generated by Bacillus subtilis, on cancer cell migration and invasion. Surfactin suppressed PM-promoted cell migration and invasion and colony formation of SCC4 and SCC25 human oral squamous cell carcinoma cell lines. We observed that PM induced MMP-2 and MMP-9 expression, which was inhibited by surfactin. Transfection with p65, p50, c-Jun, c-Fos, p85, p110, Akt, mammalian target of rapamycin (mTOR), or interleukin-6 (IL-6) siRNA markedly inhibited PM-induced MMP-2 and MMP-9 expression. Moreover, surfactin could reduce Akt, mTOR, p65, and c-Jun activation and IL-6 secretion induced by PM. Finally, we proved that transfection with Akt, p65, or c-Jun siRNA significantly inhibited PM-induced IL-6 release. Taken together, these results suggest that surfactin functions as a suppressor of PM-induced MMP2/9-dependent oral cancer cell migration and invasion by inhibiting the activation of phosphoinositide 3-kinase (PI3K)/Akt/mTOR and PI3K/Akt/nuclear factor-κB (NF-κB) and activator protein-1 (AP-1)/IL-6 signaling pathways.

Association of ATG4B and Phosphorylated ATG4B Proteins with Tumorigenesis and Prognosis in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the major leading causes of cancer death worldwide due to the limited availability of biomarkers and therapeutic targets. Autophagy related protease 4B (ATG4B) is an essential protease for the autophagy machinery, and ATG4B phosphorylation at Ser383/392 increases its proteolytic activity. ATG4B expression and activation are crucial for cancer cell proliferation and invasion. However, the clinical relevance of ATG4B and phospho-Ser383/392-ATG4B for OSCC remains unknown, particularly in buccal mucosal SCC (BMSCC) and tongue SCC (TSCC). With a tissue microarray comprising specimens from 428 OSCC patients, including 179 BMSCC and 249 TSCC patients, we found that the protein levels of ATG4B and phospho-Ser383/392-ATG4B were elevated in the tumor tissues of BMSCC and TSCC compared with those in adjacent normal tissues. High protein levels of ATG4B were significantly associated with worse disease-specific survival (DSS) in OSCC patients, particularly in patients with tumors at advanced stages. In contrast, phospho-Ser383/392-ATG4B expression was correlated with poor disease-free survival (DFS) in TSCC patients. Moreover, ATG4B protein expression was positively correlated with phospho-Ser383/392-ATG4B expression in both BMSCC and TSCC. However, high coexpression levels of ATG4B and phospho-Ser383/392-ATG4B were associated with poor DFS only in TSCC patients, whereas they had no significant association with DSS in BMSCC and TSCC patients. In addition, silencing ATG4B with an antisense oligonucleotide (ASO) or small interfering RNA (siRNA) diminished cell proliferation of TW2.6 and SAS oral cancer cells. Further, knockdown of ATG4B reduced cell migration and invasion of oral cancer cells. Taken together, these findings suggest that ATG4B might be a biomarker for diagnosis/prognosis of OSCC and a potential therapeutic target for OSCC patients.

Repression of metastasis-associated protein 2 for inhibiting metastasis of human oral cancer cells by promoting the p-cofilin-1/ LC3-II expression.

The overexpression of metastasis-associated protein 2 (MTA2) contributes to human tumor progression and metastasis in various tumor cells. However, the role of MTA2 in human oral cancer progression remains unknown. MTA2 expression in human oral tumor tissues and cell lines was measured by immunohistochemistry and Western blotting. Cell proliferation and cell cycle were analyzed using MTT assay and flow cytometry. The effects of MTA2 on oral cell migration and invasion were investigated using migration and invasion assays. The expression of MTA2, p-cofilin-1, and MTA2-induced LC3-II levels were measured using Western blotting and an immunofluorescence assay. Based on the human oral cancer tissue array and TCGA database, we found that MTA2 was increased in oral cancer tissues than in non-tumor oral tissues (P<.01). Moreover, MTA2 is significantly associated with tumor grade (P<.01) and the overall survival rate of patients with grade III tumor (P<.05). MTA2 expression in oral cancer cells was markedly higher than that in normal oral cells. Cell proliferation and cell cycle were not significantly changed in the cells inhibited by MTA2. MTA2 knockdown can inhibit cell migration and invasion of human oral cancer cells. Furthermore, we suggest that MTA2 inhibition enhances p-cofilin and LC3-II expression, and the knockdown of LC3-II expression in cells inhibited by MTA2 had the opposite effect. These results indicate that MTA2 may serve as a candidate prognostic biomarker and that targeting autophagy is a potential therapeutic strategy for treating human oral cancer.

Effect of long chain non-coding RNA H19 on the migration and invasion of oral cancer cells and its molecular mechanism

To investigate the effect of the long chain non-coding RNA H19 (lncRNA H19) on the invasion and migration of oral cancer cells and its related molecular mechanism. The expression levels of lncRNA H19, miR-107, and cyclin-dependent kinase 6 (CDK6) in the immortalized oral epithelial cell line HIOEC and the oral cancer cell line CAL27 were detected by real-time quantitative polymerase chain reaction. CAL27 cells were transfected with siRNA H19, miR-107 mimics, pcDNA H19, or anti-miR-107, and the effects of H19 and miR-107 on the invasion and migration of cells were examined via Transwell assay. The TargetScan database predicted the targeting of H19, miR-107, and CDK6. Double luciferase reporter gene assay was performed to detect interactions among H19, miR-107, and CDK6. Western blot analysis was conducted to examine the effects of H19 and miR-107 on the protein level of the target gene CDK6. Compared with that in HIOEC cells, the expression of H19 was significantly increased in CAL27 cells (P<0.05). After transfection with siRNA H19, the expression of H19 decreased, and the invasion and migration ability of CAL27 cells were inhibited (P<0.05). H19 could bind specifically to the 3'-UTR of miR-107 to modulate the expression of miR-107. Compared with that in HIOEC cells, the expression of miR-107 significantly decreased in CAL27 cells (P<0.05). The expression of miR-107 increased after transfection with siRNA H19, and anti-mir-107 co-transfection could promote the invasion and migration ability of siRNA H19 in CAL27 cells (P<0.05). Compared with that in HIOEC cells, CDK6 expression significantly increased in CAL27 cells (P<0.05), and the expression level of the gene was coregulated by H19 and miR-107 (P<0.05). lncRNA H19 plays an important role in the development of oral cancer. It can regulate the invasion and migration of oral cancer cells by targeting the miR-107/CDK6 signaling axis.

Abstract 2860: RCP augments oral cancer cell invasion through Zeb1 and Slug expression

Abstract Background: Rab coupling protein (RCP), also called family interacting protein 1 (Rab11fip1) is a protein encoded by the RAB11FIP1 gene and has been known to augment cancer tumorigenesis, invasion, and metastasis. Recently, RCP was reported to aggravate cancer cell invasion and metastasis by stabilizing beta1 integrin and inducing epithelial-mesenchymal transition (EMT) in breast and ovarian cancer. Further, a high RCP expression was significantly correlated with shorter overall survival and disease-free survival of HNSCC patients. However, the molecule mechanisms by which RCP increases HNSCC invasion and metastasis are unclear. In the present study, we demonstrate the role of RCP in oral cancer cell invasiveness and its underlying mechanism. Methods: Oral cancer YD-10B and YD-38 cells were transiently transfected with RCP constructs. siRNAs were used to analyze the role of specific proteins for Slug and Zeb1 expression. Immunoblotting and immunofluorescence were used to analyze protein expression. Matrigel-coated in vitro insert and wound healing assay were used to analyze cancer cell invasion and migration, respectively. Results: We observed that ectopic expression of RCP induces invasion of oral cancer cells. Conversely, silencing of RCP expression increased E-cadherin expression and reduced oral cancer cell invasion. In addition, RCP upregulated Zeb1 and Slug expression that is important for RCP-induced oral cancer cell EMT and invasion. Furthermore, we identified that silencing of beta1 integrin efficiently attenuates RCP-induced Zeb1 and Slug expression. In addition, we observed that a selective inhibitor of EGFR kinase activity, Gefitinib, significantly reduces RCP-induced Zeb1 and Slug expression and that RasN17 recovers RCP-induced Zeb1 expression. Finally, RCP increased the viability of oral cancer cells against a chemotherapeutic agent cisplatin. Conclusion: Collectively, we demonstrate that RCP augments oral cancer cell EMT and invasiveness through a beta1 integrin/EGFR/Ras/Slug and Zeb1 signaling axis, providing potential biomarkers for oral cancer patients therapy. Citation Format: Jin Young Kim, Kyung Hwa Cho, Bo Young Jeong, Zi Hae Song, Chang Gyo Park, Hoi Young Lee. RCP augments oral cancer cell invasion through Zeb1 and Slug expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2860.

Abstract 2824: Modulation of oral cancer cells survival and invasiveness by fusobacteria

Abstract Oral cancer represents a significant health burden worldwide while periodontitis is a common chronic infection of the periodontium. Many studies have suggested the association between periodontal disease and oral cancer. Studies have shown that biofilms on oral cancer lesions are rich in anaerobic bacteria, known to be associated with periodontal disease. This establishes that periodontal bacteria are present in the oral cancer microenvironment and likely directly interact with cancer cells. Therefore, it is vital to understand the interactions between the oral bacteria and cancer cells in order to improve treatment outcomes. The aim of this study was to investigate the effect of periodontal bacteria on oral cancer cells. Three oral cancer cell lines (OQ01, BHY and HN) were polyinfected with 4 bacteria associated with chronic periodontitis (Tanneralla forsynthia, Treponema denticola, Porphyromonas gingivalis and Fusobacterium nucleatum). Likely because BHY and HN already have a high baseline level of IL-8, OQ01 alone showed significantly enhanced IL-8 secretion (P&amp;lt; 0.05) but enhanced TFG-β secretion was detected in all cell lines tested. Polybacterial infection of oral cancer cells also upregulated mRNA for MMP1 (40 fold) and MMP9 (more than 60 fold), which are known to enhance cancer cell invasiveness. In addition, the expression of ZEB1 (4 fold higher), an oncogene known to induce epithelial mesenchymal transition in cancer cells, and MYC, JAK1 and STAT3, oncogenes involved in cell survival, were all significantly enhanced in polybacterial infected cancer cells. Further analysis using OQ01 cells in a single bacterial infection showed that F. nucleatum alone had the same or greater effect (4 fold higher IL-8 secretion) as the polybacterial infection with all 4 bacteria. All three strains of F. nucleatum tested enhanced the invasiveness of the oral cancer cells in vitro. Interestingly, Fusobacterial supernatant alone was enough to induce the same invasive phenotype as live bacteria in OQ01. These results suggest that F. nucleatum is the main periodontal pathogen responsible for inducing an invasive phenotype in these oral cancer cells. For in vivo studies, we used a mouse model of infection-associated oral tumorigenesis, which included the induction of chronic periodontitis by P. gingivalis and F. nucleatum and the administration of an oral carcinogen 4-nitroquinoline-1-oxide (4NQO). The results demonstrated that infection with Fusobacteria and P. gingivalis promoted oral cancer progression and enhanced invasion compared to controls. Our data also showed that Fusobacteria enhanced both survival and invasiveness of oral cancer cells. Collectively, this is the first study revealing the interaction between oral cancer cells and multiple periodontal bacteria. The modulation of the tumor microenvironment by oral bacteria has the potential to identify target genes for cancer treatment and improve patients’ prognosis. Citation Format: Amani Harrandah, Sasanka S. Chukkapalli, William JR Dunn, Ann Progulske-Fox, Edward K. Chan. Modulation of oral cancer cells survival and invasiveness by fusobacteria [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2824.

Abstract 5167: Cell autonomous and non-autonomous actions of CCL22 in oral carcinogenesis

Abstract Oral cancer, a subtype of head and neck cancer, has been the 4th leading cause of male cancer death in Taiwan for the past decade. Smoking, alcohol consumption, and betel quid chewing are major risk factors for oral cancer in Taiwan. The crosstalk between immunity and cancer cells can both inhibit and enhance tumor growth and is now classified as a cancer hallmark. The accumulation of regulatory T cells (Tregs) in several tumor types potentially plays a critical role in tumor evasion of immune surveillance. Oral cancer is also characterized by an increase in the number of infiltrating Tregs; however, the significance of the increase on the patient prognosis remains controversial. We found that the expression of CCL22, a chemokine for Treg recruitment, was not only positively with poor prognosis in oral cancer patients but also significantly correlated with that of FOXP3, a master regulator for Treg development and functions, in the same patients. The correlation is also validated in TCGA database analysis of head and neck cancer patients. The depletion of CCL22 expression reduced oral cancer cell proliferation, migration and invasion, suggesting an oncogenic role of CCL22 in oral cancer. Since CCR4 is the receptor for CCL22, we studied the in vitro and in vivo impact of deregulated CCL22-CCR4 signaling axis in oral carcinogenesis. We first confirmed the surface expression of CCR4 in oral cancer cells by flow cytometry. Ectopic CCL22 overexpression increased oral cancer cell migration and invasion without the effect on proliferation. Through injection of CCL22-altered oral cancer cells in both syngeneic and athymic backgrounds, the cell autonomous and non-autonomous actions of CCL22 and the mechanism responsible for the CCL22 deregulation in oral cancer will be discussed. This study will not only further the role of CCL22-CCR4 signaling axis in modulating the crosstalk between oral cancer and its microenvironment but also provide a novel therapeutic strategy for treating oral cancer. Citation Format: Li-Wha Wu. Cell autonomous and non-autonomous actions of CCL22 in oral carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5167.

Abstract 1816: Tumor suppressor LINC02487 inhibits oral cancer cell migration and invasion through directly binding protein USP17

Abstract Objective: The aim of this study is to explore the functions and mechanisms of long non-coding RNA LINC02487 in oral squamous cell carcinoma(OSCC). Materials and methods: Relative expression level of LINC02487 in OSCC cell lines and samples were examined by qRT-PCR. Intracellular localization was done using RNA fluorescence in situ hybridization. LINC02487 was cloned into pCMV-puro vector, and transfected into OSCC cell lines HN6 and HN30 by lentivirus. Cell functions as proliferation, apoptosis, cell cycle, migration and invasion were examined subsequently. RNA binding protein was explored by ChIRP-MS. And protein expression was determined by western-blotting assay. Results: Previous data showed a downregulation of LINC02487 in OSCCs. Here we confirmed the lower expression of LINC02487 in 6 OSCC cell lines comparing with immortalized normal oral epithelial cell lines, and in 50 OSCC samples comparing with paired adjacent normal tissue. LINC02487 was located in cytoplasm, aggregated around nuclei membrane. Overexpression of LINC02487 significantly suppressed cell migration and invasion, increased the ratio of cells in G1 phase, with a minor influence on cell proliferation and apoptosis. Furtherly, LINC02487 was found directly binding to USP17, a deubiquitinating enzyme, and regulating cell migration and invasion through USP17 expression. Conclusion: Our study confirmed that long non-coding RNA LINC02487 was downregulated in paired OSCC samples and cell lines. It acts as a tumor suppressor through its directly binding protein USP17. Citation Format: Lu Feng, Feng Qiu, Jianjun Zhang, Minglei Sun, Wantao Chen, Weiliu Qiu. Tumor suppressor LINC02487 inhibits oral cancer cell migration and invasion through directly binding protein USP17 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1816.

Pinosylvin reduced migration and invasion of oral cancer carcinoma by regulating matrix metalloproteinase-2 expression and extracellular signal-regulated kinase pathway

BackgroundPinosylvin possesses several biological properties, including anti-inflammatory, antitumor, and antioxidant characteristics. However, the effects of pinosylvin on the migration and invasion of human oral cancer cells and the underlying mechanisms remain unclear. Hypothesis/purposeIn this research, we investigated the outcome of different concentrations of pinosylvin (0–80 μM) on the metastatic and invasive abilities of SAS, SCC-9, and HSC-3 cells. Methods and resultsWestern blotting assay and Gelatin zymography assay indicated that pinosylvin inhibited the enzymatic activity of matrix metalloproteinase-2 (MMP-2) and reduced its protein level but increased the expression of tissue inhibitor of metalloproteinase-2 (TIMP-2). Additionally, the wound healing assay and Transwell method showed that pinosylvin reduced the migration of SAS, SCC-9 and HSC-3 oral cancer cells. Besides, pinosylvin decreased the phosphorylation of ERK1/2 protein experssion in both SAS and SCC-9 cells. ConclusionThese results indicate that pinosylvin is a potential anticancer agent for preventing oral cancer metastasis.

Effect of STAT3 on Lysophosphatidic Acid-Induced Oral Cancer Cell Invasion

Background: Oral cancer has a high incidence worldwide and has been closely associated with smoking, alcohol, and infection by the human papillomavirus. Metastasis is highly important for oral cancer survival. Lysophosphatidic acid (LPA) is a bioactive lipid mediator that promotes various cellular processes, including cell survival, proliferation, metastasis, and invasion. Signal transducer and activator of transcription (STATs) are transcription factors that mediate gene expression. Among the seven types of STATs in mammals, STAT3 is involved in invasion and metastasis of numerous tumors. However, little is known about the role of STAT3 in oral tumor invasion. In the present study, we hypothesized that STAT3 mediates LPA-induced oral cancer invasion. Methods: Immunoblotting was performed to analyze LPA-induced STAT3 activation. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was performed to assess the survival rates of YD-10B cells. STAT3 levels in LPA-treated oral tumor cells were evaluated by performing in vitro invasion assay. Results: To the best of our knowledge, this is the first study to demonstrate that LPA enhances STAT3 phosphorylation in oral cancer. In addition, treatment with WP1066, a selective inhibitor of STAT3, at a concentration that does not cause severe reduction in cell viability, significantly attenuated LPA-induced YD-10B cancer cell invasion. Conclusion: The results suggested that LPA induces oral tumor cells with greater invasive potential via STAT3 activation. Our findings provided important insights into the mechanisms underlying mouth neoplasms.

Exosomes derived from microRNA-101-3p-overexpressing human bone marrow mesenchymal stem cells suppress oral cancer cell proliferation, invasion, and migration.

Dysregulation of microRNAs (miRNAs) has been found to disrupt the progression of oral cancer. However, which miRNAs are most effective against oral cancer and how these miRNAs should be delivered are major unanswered problems. We aimed at investigating if human bone marrow mesenchymal stem cells (hBMSCs)-derived exosomes affect oral cancer development, and the potential regulatory mechanism associated with COL10A1 and miR-101-3p. COL10A1 was upregulated, while miR-101-3p was downregulated in oral cancer, and miR-101-3p targeted COL10A1 as verified by dual-luciferase reporter gene assay. Meanwhile, exosomes derived from hBMSCs were isolated and then co-cultured with oral cancer cells to identify the role of exosomes, and the results suggested that hBMSCs-derived exosomes overexpressing miR-101-3p inhibited oral cancer progression. Furthermore, tumorigenicity assay in nude mice further confirmed the inhibitory effects of hBMSCs-derived exosomes, loaded with miR-101-3p, on oral cancer, which provides a new theoretical basis in the treatment of oral cancer.

The enigmatic role of matrix metalloproteinases in epithelial-to-mesenchymal transition of oral squamous cell carcinoma: Implications and nutraceutical aspects.

The most prevalent malignancy in the oral cavity is represented by oral squamous cell carcinoma, an aggressive disease mostly detected in low-income communities. This neoplasia is mostly diffused in older men particularly exposed to risk factors such as tobacco, alcohol, and a diet rich in fatty foods and poor in vegetables.In oral squamous cell carcinoma, a wide range of matrix-cleaving proteinases are involved in extracellular matrix remodeling of cancer microenvironment. In particular, matrix metalloproteinases (MMPs) represent the major and most investigated protagonists. Owing to their strong involvement in malignant pathologies, MMPs are considered the most promising new biomarkers in cancer diagnosis and prognosis. The interest in studying MMPs in oral cancer biology is also owing to their prominent role in epithelial-to-mesenchymal transition (EMT). EMT is an intricate process involving different complex pathways. EMT-related proteins are attractive diagnostic biomarkers that characterize the activation of biological events that promote cancer's aggressive expansion. Different antioncogenic natural compounds have been investigated to counteract oral carcinogenesis, with the scope of obtaining better clinical results and lower morbidity. In particular, we describe the role of different nutraceuticals used for the regulation of MMP-related invasion and proliferation of oral cancer cells.

MiR-1254 Functions as a Tumor Suppressor in Oral Squamous Cell Carcinoma by Targeting CD36.

Oral squamous cell carcinoma is one of the most common cancers around the world. The patients with oral squamous cell carcinoma are often diagnosed at late stages, leading to unfavorable prognosis. MicroRNAs might function as oncogenes or tumor suppressor genes in the tumorigenesis of cancer. This study aimed to explore the role of miR-1254 in oral squamous cell carcinoma. We examined the expression levels of miR-1254 in oral squamous cell carcinoma tissue samples and cell line.Proliferation and invasion assays were performed in oral squamous cell carcinoma cells with miR-1254 overexpression or underexpression. The potential regulatory mechanisms were also explored. We found that miR-1254 was significantly reduced in oral squamous cell carcinoma tissues and cell lines. In addition, downregulation of miR-1254 in oral squamous cell carcinoma tumor tissues was closely associated with cancer staging and lymph node metastasis. Enforced expression of miR-1254 significantly inhibited proliferation and invasion in oral cancer cells, and downregulation of miR-1254 promoted the oncogenic activities of oral cancer cells. CD36 was identified as a direct downstream target of miR-1254 by the luciferase reporter assay. Overexpression of CD36 partially restored the proliferation and invasion capacity inhibited by miR-1254. CD36 expression was inversely correlated with miR-1254 expression in the oral squamous cell carcinoma tissues. Taken together, our study provided the compelling evidence that miR-1254 might inhibit the progression of OSCC by partially downregulating CD36, and restoration of miR-1254 may represent an effective strategy for treating oral squamous cell carcinoma.

Antimetastatic Potential of Garcinone E in Human Oral Cancer Cells

Objective:Oral cancer presents as a devastating type of malignancy. It is predominant in populations with high use of alcohol and various forms of tobacco as well as poor diets with low intake of fruits and vegetables. The present study focused on the potential of Garcinone E to inhibit HSC-4 oral cancer cell proliferation, migration and invasion.Methods:MTT and colony forming assays were performed to study antiproliferative effects of Garcinone E. Hoechst staining was used to determine levels of apoptosis, with cell invasion and scratch assays conducted for migration and invasion characteristics. The levels of MMPs and cytokines were quantified in Garcinone E treated cells by ELISA.Results:Garcinone E inhibited the proliferation and colony forming potential of HSC-4 cells. It also suppressed migration and invasion with inhibition of MMP-2 and MMP-9 expression. Moreover, it elevated IL-2 and reduced IL-6 expression in HSC-4 cells.Conclusion:Our results demonstrate for the first time that Garcinone E might inhibit metastasis of an oral cancer cell line by blocking invasion, migration and MMP production.

Targeted disruption of PI3K/Akt/mTOR signaling pathway, via PI3K inhibitors, promotes growth inhibitory effects in oral cancer cells.

The phosphoinositide-3-kinase (PI3K) pathway is the frequently altered in human cancer. This has led to the development and study of novel PI3K inhibitors for targeted therapy and also to overcome resistance to radiotherapy. The anti-tumour effects of PI3K inhibitors (PI-828, PI-103 and PX-866) in terms of cell proliferation, colony formation, induction of apoptosis, cell cycle arrest, invasion, autophagy, and pNF-κB/p65 translocation in SCC-4, SCC-9 and SCC-25 cells were studied by performing MTT, clonogenic, DAPI staining, propidium iodide staining, annexin-V binding, matrigel invasion, acridine orange staining and immuno-fluorescence assay. Western blot assay was performed to assess the alteration in the expression of various proteins. PI-828 and PI-103 treatment exhibited dose-dependent inhibition of growth and proliferation of OSCC cells with a concomitant induction of apoptosis, altered cell cycle regulation and decreased invasiveness (p < 0.01). PX-866 induced apoptosis, cell cycle arrest, autophagy and a significant decrease in the invasiveness of oral cancer cells as compared to untreated cells (p < 0.01). These compounds significantly reduced expression of COX-2, cyclin-D1 and VEGF in the treated cells besides cytoplasmic accumulation of pNF-κB/p65 protein. In addition to PI3Kα, inactivation of downstream components, i.e. Akt and mTOR was seen. PI3K inhibitors such as PI-103, PI-828 and PX-866 may be developed as potential therapeutic agents for effective treatment of oral squamous cell carcinoma (OSCC) patients, associated with activated PI3K/Akt pathway.

Systematic analysis of genes involved in oral cancer metastasis to lymph nodes

Oral cancer remains a deadly disease worldwide. Lymph node metastasis and invasion is one of the causes of death from oral cancer. Elucidating the mechanism of oral cancer lymph node metastasis and identifying critical regulatory genes are important for the treatment of this disease. This study aimed to identify differentially expressed genes (gene signature) and pathways that contribute to oral cancer metastasis to lymph nodes. The GSE70604-associated study compared gene profiles in lymph nodes with metastasis of oral cancer to those of normal lymph nodes. The GSE2280-associated study compared gene profiles in primary tumor of oral cancer with lymph node metastasis to those in tumors without lymph node metastasis. There are 28 common differentially expressed genes (DEGs) showing consistent changes in both datasets in overlapping analysis. GO biological process and KEGG pathway analysis of these 28 DEGs identified the gene signature CCND1, JUN and SPP1, which are categorized as key regulatory genes involved in the focal adhesion pathway. Silencing expression of CCND1, JUN and SPP1 in the human oral cancer cell line OECM-1 confirmed that those genes play essential roles in oral cancer cell invasion. Analysis of clinical samples of oral cancer found a strong correlation of these genes with short survival, especially JUN expression associated with metastasis. Our study identified a unique gene signature – CCND1, JUN and SPP1 – which may be involved in oral cancer lymph node metastasis.

RETRACTED: MiR-650 regulates the proliferation, migration and invasion of human oral cancer by targeting growth factor independent 1 (Gfi1)

Oral cancer being one of the lethal cancers is generally detected at advanced stages and causes significant mortality world over. The unavailability of the reliable biomarkers and therapeutic targets/agents forms a bottleneck in the treatment of oral cancer. MicroRNAs are considered of immense therapeutic potential for the treatment of cancer. Consistently, in this study the role and therapeutic potential of miR-650 was explored in oral cancer. The analysis of miR-650 expression by qRT-PCR revealed significant (p < 0.05) upregulation of miR-650 in oral cancer cell lines. Cell cycle analysis by flow cytometery revealed that suppression of miR-650 significantly (p < 0.05) inhibits the proliferation of the SCC-25 cells by prompting Sub-G1 cell cycle arrest. Further, miR-650 suppression also inhibited the migration and invasion of the SCC-25 oral cancer cells as revealed by transwell assays. TargetScan analysis showed that miR-650 targets Growth factor independent 1 (Gfi1). Moreover, the results of western blot analysis showed that miR-650 suppression inhibits the expression of Gfi1. Interestingly, suppression of Gfi1 exhibited similar effects on cell proliferation, migration and invasion of the oral cancer cells as that of miR-650 suppression. Nonetheless, miR-650 promoted the proliferation, migration and invasion of the SCC-25 cells by upregulating the expression of Gfi1. Moreover, overexpression of miR-650 could not rescue the effects of Gfi1 silencing on SCC-25 oral cancer cells. Conversely, overexpression of Gfi1 could rescue the effects of miR-650 inhibition on SCC-25 cell proliferation, migration and invasion. Additionally, miR-650 suppression could also inhibit the xenografted tumor growth invivo by inhibiting the expression of Gfi1. Taken together, miR-650 may prove to be an important therapeutic target for the management of oral cancers.

Matrix metalloproteinase-13is a target gene of high-mobility group box-containing protein 1 in modulating oral cancer cell invasion.

Transcription factor high-mobility group box-containing protein 1 (HBP1) may function as a tumor suppressor in various types of cancer. In a previous study, we demonstrated that HBP1 suppressed cell invasion in oral cancer. To further understand the underlying mechanism, the current study is aimed at investigating how HBP1 exerts its antimetastatic potential in oral cancer. In a cell model, ectopic expression of HBP1 potently suppressed epithelial-mesenchymal transition, cellular migration, and invasion; conversely, HBP1 knockdown promoted these malignant phenotypes. The matrix metalloproteinase (MMP) family is highly implicated in tumor metastasis. Therefore, we examined the effect of HBP1 on the activation of the MMP members, MMP-2, -9, and -13 that are highly associated with the aggressiveness of oral cancer. Ectopic expression of HBP1 resulted in a mild reduction in the expression and activity of MMP-2 and -9, yet it had a potent inhibitory effect on MMP-13. In contrast, HBP1 knockdown strongly enhanced the activation of MMP-13. Further, we demonstrated that MMP-13 is a target of HBP1 transcription repression as evidenced by the identification of an HBP1 binding site in the cis proximal region of the MMP-13 promoter. More important, MMP-13 knockdown significantly alleviated HBP1 small interfering RNA-mediated promotion in cell invasion. Analysis of oral tumor specimens revealed that the low HBP1 (<0.3-fold)/high MMP-13 (>3-fold) status was associated with metastatic potential. All told, our study provides evidence supporting the idea that the HBP1-MMP-13 axis is a key regulator of the aggressiveness in oral cancer.

PO-148 Activin a upregulation mediated by P63 promotes migration and invasion of oral cancer cells

IntroductionTGF-β family proteins, Activins are homo/heterodimers of β subunits βA, βB, βC and βE, while Inhibins are αβ heterodimers. Relative abundance of each monomer dictates which dimer is predominantly formed. Activins bind to typeII receptor on cell membrane and trigger their tetramerization with typeI receptor, ALK4 phosphorylating SMAD 2/3 which translocate to nucleus and regulate transcription. Activins are reported to be up regulated in various cancers including HNSCC; however, these are transcriptome-scale studies that have indicated altered βA expression, without considering other subunits’ expression or their dimerization. An oncogenic role for Activins has been demonstrated in cancer; albeit, the precise mechanisms downstream of Activin or molecules regulating its expression/function are vaguely understood. This study evaluated the expression of Activins and phenotypes that are affected by its altered expression in oral cancer; with emphasis on Activin-SMAD 2/3 signalling as the downstream mechanism. We also tested the possible role of p53 family proteins in regulating Activin gene expression and functions.Material and methodsActivin subunits’ expression was assessed in oral cancer celllines and tissues by qRT-PCR, IHC and Western blot. Wound healing, Matrigel Invasion, and MTT assays were performed post knockdown of Activin βA and SMAD 2/3 using siRNA and post treatment with recombinant Activin A. We tested the regulation of Activin βA expression by p53 and p63 using ChIP assay and analysed the effect of p63 knockdown in context of Activin expression on oral cancer cells.Results and discussionsActivin βA was significantly overexpressed in oral cancer cells and tissues compared to normal counterparts at transcript and protein level. Knockdown of βA resulted in decreased, while treatment with recombinant Activin A resulted in increased migration and invasion of oral cancer cells. Knockdown of SMAD 2/3 also led to similar effects, indicating that Activin A contributes to these phenotypes through SMAD2/3 signalling pathway. The ChIP experiments demonstrated that βA promoter could be bound by p63, a p53 family member with established oncogenic functions. Recombinant Activin A treatment could rescue the loss of migration upon p63 knockdown suggesting that p63 could be an upstream regulator of Activin expression and function in oral cancer.ConclusionThe study revealed a novel role of p63 in regulating Activin A expression. We also deciphered the mechanism downstream of p63-Activin A axis that contributes to migration and invasion of oral cancer.

PO-093 Lipocalin 2 suppress metastases of oral cancer through miRNA-29A-regulated dipeptidyl peptidase-4

IntroductionLipocalin 2 (LCN2), a secreted glycoprotein, is up- or downregulated in different human cancers and it have been found to play a different role in tumorigenicity. Dipeptidyl peptidase IV (DPP4), a membrane-bound peptidase on the cell surface of a wide variety of cell types and plays an important role through enzyme activity. Several recent studies have shown that DPP4 affects tumour progression and invasion in several human malignancies. However, how LCN2 and DPP4 involved in tumour progression and metastasis in oral cancer have not been studies yet.Material and methodsDPP4 expressed in LCN2 overexpression OSCC cells by protease array assay. MicroRNA array analysis and microRNA target prediction (TargetScan and PiTar) reveal that DPP4 is one of the target gene of miR-29 family.Results and discussionsOverexpression of LCN2 in oral cancer cell lines reduced in vitro migration/invasion. Mechanistically, LCN2 inhibited the cell motility of oral cancer cells through transcriptional expression of the DPP4. Knockdown DPP4 in LCN2 overexpressed cell line significantly increased cell invasion and migration. Moreover, in oral cancer cells, LCN2 significantly decreased the levels of miRNA-29a, which increased the DPP4 expression. Overexpression of miRNA-29a significantly suppressed DPP4 expression and increased OSCC cell migration and invasion (p<0.05).ConclusionWe concluded that LCN2 suppresses oral cancer cell invasion and migration through miRNA-29a-regulated DPP4.