Targeting PADI2 as a potential therapeutic strategy against metastasis in oral cancer via suppressing EMT-mediated migration and invasion and CCL3/5-induced angiogenesis.

BackgroundLong noncoding RNAs (lncRNAs) are widely involved in cancer development and progression, but the functions of most lncRNAs have not yet been elucidated. Metastasis is the main factor restricting the therapeutic outcomes of various cancer types, including oral squamous cell carcinoma (OSCC). Therefore, exploring the key lncRNAs that regulate OSCC metastasis and elucidating their molecular mechanisms will facilitate the development of new strategies for effective OSCC therapy.MethodsWe analyzed the lncRNA expression profiles of tumor tissues from OSCC patients with and without cervical lymph node metastasis, and OSCC cell lines. We revealed high expression of oral squamous cell carcinoma metastasis-related lncRNA 1 (lncOCMRL1) in OSCC patient tumor tissues with lymph node metastasis and highly metastatic OSCC cell lines. The effects of lncOCMRL1 knockdown on the invasion, migration and proliferation abilities of OSCC cells were explored through qRT-PCR, Transwell, colony formation, and cell proliferation experiments. The mechanism by which lncOCMRL1 promotes OSCC metastasis and proliferation was explored through RNA pull-down, silver staining, mass spectrometry, RIP, and WB experiments. To increase its translational potential, we developed a reduction-responsive nanodelivery system to deliver siRNA for antitumor therapy.ResultsWe determined that lncOCMRL1 is highly expressed in OSCC metastatic tumor tissues and cells. Functional studies have shown that high lncOCMRL1 expression can promote the growth and metastasis of OSCC cells both in vivo and in vitro. Mechanistically, lncOCMRL1 could induce epithelial-mesenchymal transition (EMT) via the suppression of RRM2 ubiquitination and thereby promote the proliferation, invasion, and migration of OSCC cells. We further constructed reduction-responsive nanoparticles (NPs) for the systemic delivery of siRNAs targeting lncOCMRL1 and demonstrated their high efficacy in silencing lncOCMRL1 expression in vivo and significantly inhibited OSCC tumor growth and metastasis.ConclusionsOur results suggest that lncOCMRL1 is a reliable target for blocking lymph node metastasis in OSCC.

Metastasis, a powerful prognostic indicator of oral squamous cell carcinoma (OSCC), is chiefly responsible for poor cancer outcomes. Despite an increasing number of studies examining the mechanisms underlying poor outcomes, the development of potent strategies is hindered by insufficient characterization of the crucial regulators. Long noncoding RNAs (lncRNAs) have recently been gaining interest as significant modulators of OSCC metastasis; however, the detailed mechanisms underlying lncRNA-mediated OSCC metastasis remain relatively uncharacterized. Here, we identified a novel alternative splice variant of oral cancer overexpressed 1 (ORAOV1), named as ORAOV1-B, which was subsequently validated as an lncRNA and correlated with OSCC lymph node metastasis; significantly increased invasion and migration were observed in ORAOV1-B–overexpressing OSCC cells. RNA pulldown and mass spectrometry identified Hsp90 as a direct target of ORAOV1-B, and cDNA microarrays suggested TNFα as a potential downstream target of ORAOV1-B. ORAOV1-B was shown to directly bind to and stabilize Hsp90, which maintains the function of client proteins, receptor-interaction protein, and IκB kinase beta, thus activating the NF-κB pathway and inducing TNFα. Additionally, TNFα reciprocally enhanced p-NF-κB-p65 and the downstream epithelial-mesenchymal transition. ORAOV1-B effects were reversed by a TNFα inhibitor, demonstrating that TNFα is essential for ORAOV1-B–regulated metastatic ability. Consistent epithelial-mesenchymal transition in the ORAOV1-B group was demonstrated via an orthotopic model. In the metastatic model, ORAOV1-B significantly contributed to OSCC-related lung metastasis. In summary, the novel splice variant ORAOV1-B is an lncRNA, which significantly potentiates OSCC invasion and metastasis by binding to Hsp90 and activating the NF-κB-TNFα loop. These findings demonstrate the versatile role of ORAOV1 family members and the significance of genes located within 11q13 in promoting OSCC. ORAOV1-B might serve as an attractive OSCC metastasis intervention target.

GATA6 regulates expression of annexin A10 (ANXA10) associated with epithelial–mesenchymal transition of oral squamous cell carcinoma

miR-223 regulates oral squamous cell carcinoma metastasis through the Wnt/β-catenin signaling pathway.

Cancer metastasis is a complex problem that presents clinical challenges and is the cause of most cancer-related deaths. Mechanistic studies on metastasis hold promise for finding better approaches to control cancer mortality. Oral cavity cancer brings over 200,000 deaths annually and is one of the top eight most frequently diagnosed cancers worldwide. However there is a lack of appropriate experimental models to address oral cancer metastasis. An ideal experimental system should recapitulate the complete process of cancer metastasis in vivo while maintaining molecular-level manipulability. Our current efforts are directed at development of a series of murine oral squamous cell carcinoma (OSCC) cell lines that will be amenable for syngeneic transplantation studies in immuno-competent mice. The cell lines were created from C57BL/6J mice, a strain frequently used in targeted gene manipulation. This strategy ensures that the cells also have utility in orthotopic xenograft cancer models using currently available knockout mice made with a syngeneic background. As abundant knockout mice are available on the C57BL/6J background, our new syngeneic cancer cell lines will greatly facilitate studies on the interrelationship between host and tumor during the process of metastasis. Cell lines were generated from primary murine tongue epithelial cells by hTERT induced immortalization and oncogene induced transformation. They were screened for tumorigenicity and metastasis in both immunocompetent and immunocompromised mice. Properties of in vitro and vivo growth, morphology and cytogenetics were characterized. As proof of concept, one type of gene knockout mouse was used to test transplantation of such new cellular models in vivo to observe the impact of gene loss in the host on the growth and metastasis of tumors. The completion of this project will establish solid and currently unavailable tools for the oral cancer metastasis research community. Citation Format: Zonggao Shi, Jeff Johnson, Yueying Liu, Sharon Stack. Generating new syngeneic models for oral squamous cell carcinoma metastasis study. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 92. doi:10.1158/1538-7445.AM2014-92

Oral squamous cell carcinoma (OSCC) is a common malignant cancer with unfavorable prognosis, and the epithelial-to-mesenchymal transition (EMT) is a critical contributor to OSCC metastasis. Recently, we have shown that sirtuin 7 (Sirt7) is associated with EMT and OSCC metastasis by acetylating small mother against decapentaplegic 4 (Smad4). Nonetheless, the mechanism of Sirt7 downregulation in OSCC cells remains unknown. This study analyzed the potential microRNAs that were predicted to regulate Sirt7 expression by online databases. We identified miR-770 as an upstream regulator of Sirt7 that targets its 3'-untranslated region. The expression of miR-770 was observed to be negatively correlated with the mRNA expression of Sirt7 in metastatic OSCC tumors, and higher miR-770 expression was correlated with poorer OSCC patient survival. Our in vitro data indicated that miR-770 promoted OSCC cell migration and invasion, and this process was dependent on Sirt7/Smad4 signaling. Furthermore, in vivo metastasis experiments indicated that miR-770 overexpression led to more prominent OSCC metastasis and downregulated Sirt7 expression. Collectively, our results revealed a new role of Sirt7 downregulation in metastatic OSCC and suggested that miR-770 is a potential target in counteracting OSCC metastasis.

BackgroundThe epithelial-to-mesenchymal transition (EMT) is a key process in carcinogenesis, invasion, and metastasis of oral squamous cell carcinoma (OSCC). In our previous studies, we found that neuropilin-1 (NRP1) is overexpressed in tongue squamous cell carcinoma and that this overexpression is associated with cell migration and invasion. Nuclear factor-kappa B (NF-κB) plays an essential role both in the induction and the maintenance of EMT and tumor metastasis. Therefore, we hypothesized that NRP1 induces EMT, and that NRP1-induced migration and invasion may be an important mechanism for promoting invasion and metastasis of OSCC through NF-κB activation.Methods/ResultsThe variations in gene and protein expression and the changes in the biological behavior of OSCC cell lines transfected with a vector encoding NRP1, or the corresponding vector control, were evaluated. NRP1 overexpression promoted EMT and was associated with enhanced invasive and metastatic properties. Furthermore, the induction of EMT promoted the acquisition of some cancer stem cell (CSC)-like characteristics in OSCC cells. We addressed whether selective inhibition of NF-κB suppresses the NRP1-mediated EMT by treating cells with pyrrolidinedithiocarbamate ammonium (PDTC), an inhibitor of NF-κB. Immunohistochemical analysis of NRP1 in OSCC tissue samples further supported a key mediator role for NRP1 in tumor progression, lymph node metastasis, and indicated that NRP1 is a predictor for poor prognosis in OSCC patients.ConclusionOur results indicate that NRP1 may regulate the EMT process in OSCC cell lines through NF-κB activation, and that higher NRP1 expression levels are associated with lymph node metastasis and poor prognosis in OSCC patients. Further investigation of the role of NRP1 in tumorigenesis may help identify novel targets for the prevention and therapy of oral cancers.

Targeted blockade of interleukin-8 negates metastasis and chemoresistance via Akt/Erk-NFκB axis in oral cancer

CD133 receptor mediated delivery of STAT3 inhibitor for simultaneous elimination of cancer cells and cancer stem cells in oral squamous cell carcinoma

Epithelial-mesenchymal transition (EMT) enables tumor cell invasion and metastasis. Many studies have demonstrated the critical role of EMT in lymph node metastasis in oral squamous cell carcinoma (OSCC). During EMT, epithelial cancer cells lose intercellular adhesion and apical-basal polarity and acquire mesenchymal properties such as motility and invasiveness. A significant feature of EMT is cadherin switching, involving the downregulation of E-cadherin and upregulation of N-cadherin. The TGF-β/SMAD pathway can also induce EMT. We aimed to evaluate EMT markers as predictors of lymph node metastasis in OSCC. We performed genetic profiling of 159 primary OSCCs from TCGA and analyzed the expression of EMT markers, including cadherin switch genes (CDH1, CDH2), and TGF-β/SMAD pathway genes. Samples were divided into advanced (stage III-IV) and early (stage I-II) stage groups. Differential expression analysis was performed, as well as an independent validation study containing fresh OSCC samples. TGF-β/SMAD pathway genes such as SMAD6 were upregulated in advanced stage tumors. N-cadherin and SNAIL2 were overexpressed in node-positive tumors. Keratins were downregulated in these groups. Our findings demonstrate that EMT marker expression correlates with lymph node metastasis in OSCC. Developing therapies targeting regulators such as N-cadherin may prevent metastasis and improve outcomes.

Neoadjuvant chemotherapy has been widely used for the treatment of solid tumors. However, clinical observations have shown that patients with oral squamous cell carcinoma (OSCC) who are receiving neoadjuvant chemotherapy with cisplatin still face issues such as a poor lymph node response and even lymph node progression, but the underlying mechanisms remain unidentified. In this work, it is found that low‐dose cisplatin promoted oral squamous cell carcinoma migration, invasion and lymph node metastasis, and gasdermin D (GSDMD) is identified as a potential regulator. GSDMD interacted with MMP14, promoting its expression and epithelial‒mesenchymal transition (EMT) activation without activating pyroptosis. Moreover, pH‐responsive nanoparticles (NPs) for the systemic delivery of a GSDMD siRNA (siGSDMD) is developed and showed that this NP‐delivered siGSDMD can effectively inhibit OSCC tumor growth and metastasis via the efficient silencing of GSDMD expression in vivo. This findings indicate that GSDMD can be a biomarker to predict the prognosis of OSCC patients receiving neoadjuvant chemotherapy and that NP‐mediated GSDMD silencing can be a promising strategy for the treatment of patients with advanced OSCC receiving neoadjuvant chemotherapy with cisplatin.

BackgroundMetastatic disease remains the primary cause of death in patients with oral squamous cell carcinoma (OSCC), especially those who use betel nut. The different steps of the metastatic cascade rely on reciprocal interactions between cancer cells and the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are regarded as a significant component in the TME of OSCC. However, the precise mechanisms regulating CAFs in OSCC are poorly understood.MethodsThirteen genes related to the arecoline were analyzed to explore the significant ones involved in arecoline-related OSCC metastasis. The GSE139869 (n = 10) and The Cancer Genome Atlas (TCGA)-OSCC data (n = 361) were mined for the identification of the differentially expressed genes. The least absolute shrinkage and selection operator (LASSO) regression was performed to identify the independent prognostic signatures. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted to explore the functional enrichment of selected genes, and gene set enrichment analysis of cuproptosis-related genes was completed. Spearman’s analysis and Tumor Immune Estimation Resource (TIMER) were used to visualize the correlation between the infiltration of CAFs and the gene expression. The correlation analysis of the cells and different genes, including CAF infiltration and transcripts per million expression, was assessed. The relationship between arecoline and CAFs was confirmed by cell counting kit-8 assay (CCK-8). CancerSEA was searched to identify the single-cell phenotype.ResultArecoline-associated fibrosis-related OSCC differentially expressed genes (AFOC-DEGs), namely, PLAU, IL1A, SPP1, CCL11, TERT, and COL1A2, were screened out and selected from the Gene Expression Omnibus (GEO) database and TCGA database. AFOC-DEGs were highly expressed in OSCC, which led to poor survival of patients. Functional enrichment analysis, protein–protein interaction network construction, and Spearman’s correlation analysis all suggested that AFOC-DEGs were closely associated with cuproptosis. Cellular experiments demonstrated that arecoline stimulation could significantly increase the cell viability of CAFs. Single-sample Gene Set Enrichment Analysis (ssGSEA) results showed that GLS and MTF1 were highly expressed when fibroblasts proliferated at high enrichment levels. In addition, analysis of single-cell sequencing results suggested that OSCC cells with high expression of AFOC-DEGs were associated with OSCC metastasis.ConclusionWe found a close association between arecoline, cuproptosis, and CAFs, which might play an important role in the metastasis of OSCC.

BackgroundThe epithelial-to-mesenchymal transition (EMT) process results in a loss of cell-cell adhesion, increased cell mobility, and is crucial for enabling the metastasis of cancer cells. Recently, the enzyme SIRT1 has been implicated in a variety of physiological processes; however, its role in regulating oral cancer metastasis and EMT is not fully elucidated. Here, we propose a mechanism by which the enzyme sirtuin1 (SIRT1) regulates the EMT process in oral cancer by deacetylating Smad4 and repressing the effect of TGF-β signaling on matrix metalloproteinase-7 (MMP7).MethodsThe roles of SIRT1 in tumor cell migration/invasion and metastasis to the lungs were investigated using the Boyden chamber assay and orthotopic injections, respectively. RNA interference was used to knockdown either SIRT1 or Smad4 expression in oral squamous cell carcinoma (OSCC) cell lines. Immunoblotting, zymographic assays, and co-immunoprecipitation were used to examine the effects of SIRT1 overexpression on MMP7 expression and activity, as well as on SIRT1/ Smad4 interaction.ResultsWe found that compared with normal human oral keratinocytes (HOKs), SIRT1 was underexpressed in OSCC cells, and also in oral cancer tissues obtained from 14 of 21 OSCC patients compared with expression in their matched normal tissues. Overexpression of SIRT1 inhibited migration of OSCC cells in vitro, as well as their metastasis to the lung in vivo. Furthermore, up-regulation of SIRT1 in metastatic OSCCs significantly inhibited the migration and invasion abilities of OSCC cells, while concomitantly increasing the expression of E-cadherin, and decreasing the expressions of mesenchymal markers. We also identified Smad4, a TGF-β-activated transcription factor, as a direct target protein for SIRT1. Overexpression of SIRT1 in OSCC cells led to decreased levels of acetylated Smad4, and inhibition of TGF-β-induced signaling. By associating and deacetylating Smad4, SIRT1 enzyme can influence MMP7 expression, MMP enzyme activity, and consequently, cell migration, invasion, and tumor metastasis in OSCCs.ConclusionsThese findings provide a valuable insight into the potential role of the SIRT1 enzyme in regulating cell migration and invasion in oral squamous cell carcinoma. Our findings suggest the SIRT1/Smad4/MMP7 pathway as a target for oral cancer driven by EMT.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-254) contains supplementary material, which is available to authorized users.

Simple SummaryThe treatment of oral squamous cell carcinoma (OSCC) represents a significant problem worldwide. Among cancers with the highest incidence, OSCC renders one of the worst prognoses. Therefore, novel prognostic biomarkers and therapeutic tools to tackle OSCC are urgently needed. Somatostatin-analogues (SSA) are an invaluable therapeutic option in the treatment of several cancers. We aimed to determine the expression levels of all somatostatin-receptors (SSTs) in OSCC, compared to adjacent healthy control tissues, to analyze the relationship of SSTs expression with key clinical and histopathological data, and to explore the direct in vitro effect of different SSAs on OSCC cancer cells. Our findings highlight a potential role of SST2 as a good prognostic biomarker for recurrence and metastasis in OSCC and unveil that SSA exerts antitumoral effects on OSCC cells, providing a relevant clinical conclusion, which should be soon tested for their use in humans.Oral squamous cell carcinoma (OSCC) incidence has increased by 50% over the last decade. Unfortunately, surgery and adjuvant radiotherapy and chemotherapy are still the mainstream modality of treatment, underscoring the need for alternative therapies. Somatostatin-analogues (SSA) are efficacious and safe treatments for a variety of tumors, but the presence of somatostatin-receptors (SSTs) and pharmacological effects of SSA on OSCC are poorly known. In this study, we demonstrated that SST2 and SST3 levels were significantly higher in OSCC, compared to adjacent healthy control tissues. SST2 expression was associated with less regional metastasis and a lower recurrence rate. Moreover, SST2 was elevated in OSCC and associated with histopathological good prognosis factors, such as high peritumoral inflammation, smaller depth of invasion, and expansive vs. infiltrative front of tumor invasion. Importantly, treatment with different SSA (octreotide, lanreotide, and pasireotide) significantly reduced cell-proliferation in OSCC primary cell cultures. Altogether, this study demonstrated that SST2 is overexpressed in OSCC vs. healthy tissues and could represent a novel prognostic biomarker, since its expression is associated with tumors that show better prognostic factors and less recurrent rate. Moreover, our data unveil clear antitumoral effects of SSAs on OSCC, opening new avenues to explore their potential as targeting therapy to OSCC.

Activin B, a homodimer of inhibin beta b (INHBB), is a multifunctional cytokine belonging to the transforming growth factor-β (TGF-β) family. However, the molecular functions and clinical relevance of activin B have not been determined in oral cancer. We investigated the critical roles of activin B in oral squamous cell carcinoma (OSCC). We performed quantitative reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry to study INHBB expression in OSCC-derived cell lines and OSCC clinical samples. The INHBB expression levels were significantly (P < 0.05) overexpressed in OSCCs compared to normal counterparts in vitro and in vivo. Activin B-positivity in OSCC cases was significantly (P < 0.05) correlated with regional lymph node metastasis. The INHBB knockdown (shINHBB) cells promoted cellular adhesion and suppression of cellular invasiveness and migration. After treatment of shINHBB cells with activin B, those activities were restored similar to the shMock cells. In the processes of invasiveness and metastasis, the cells cause epithelial-mesenchymal transition (EMT). TGF-β and its family members are promoters of the EMT process. To investigate whether activin B is related to EMT, we examined the expressions of EMT-related genes and found that INHBB was related closely to EMT. Our results suggested for the first time that activin B indicates tumoral metastasis in OSCCs and might be a useful biomarker for OSCC metastasis.