Laminin γ2-enriched extracellular vesicles of oral squamous cell carcinoma cells enhance in vitro lymphangiogenesis via integrin α3-dependent uptake by lymphatic endothelial cells.

Oral squamous cell carcinoma (OSCC) LN1-1 cells previously showed greater capacities for lymphangiogenesis and lymph node metastasis compared to their parental OEC-M1 cells, in addition to an ability to enhance the migration and tube formation of lymphatic endothelial cells (LECs). Purified by a series of differential centrifugations and characterized using electron microscopy, dynamic light scattering and western blot, LN1-1 cell-derived extracellular vesicles (LN1-1 EVs) were shown to promote LEC migration, tube formation and uptake by LECs more effectively than did OEC-M1 cell-derived EVs (OEC-M1 EVs). Using stable isotope labeling with amino acids in cell culture/liquid chromatography-tandem mass spectrometry-based proteomic platform, the laminin-332 proteins, including laminin α3, ß3 and γ2, were validated as highly expressed proteins in LN1-1 EVs. Clinically, a higher level of laminin-332 was detected in plasma EVs from OSCC patients with lymph node metastasis than in both healthy controls and OSCC patients without lymphatic metastasis, suggesting EV-borne laminin-332 as a novel and noninvasive biomarker for the detection of lymph node metastasis in OSCC. The knockdown of laminin γ2 and inhibition by anti-laminin-332 neutralizing antibodies impaired LN1-1 EV-mediated LEC migration, tube formation and uptake by LECs. Importantly, laminin γ2-deficient EVs showed a reduced ability to drain into lymph nodes in comparison with the control EVs. In addition, the laminin 332/γ2-mediated EV uptake was dependent on integrin α3 but not ß1, ß4 or α6. Collectively, the uptake of laminin γ2-enriched EVs by LECs enhanced in vitro lymphangiogenesis and EV-borne laminin-332 is thus a viable biomarker for OSCC.

Reciprocal regulation of microRNA-99a and insulin-like growth factor I receptor signaling in oral squamous cell carcinoma cells.

BACKGROUND: MicroRNAs (miRNAs), small noncoding RNA molecules can function as oncogenes or tumor suppressors in tumorigenesis. Oral squamous cell carcinoma (OSCC) is one of the most prevalent cancers worldwide with a 5-year survival rate of approximately 50%. METHODS: The expression of microRNA-99a (miR-99a) in OSCC tissues and cell lines was investigated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. The functions of miR-99a in migration/invasion and lung colonization were determined by transwell and tail vein injection assays, respectively. Specific targets of miR-99a were determined by software prediction, correlation with target protein expression, and luciferase reporter assay. The signaling pathways involved in regulation of miR-99a were investigated using the kinase inhibitors. RESULTS: We observed reduced levels of miR-99a, identified as one of the most downregulated miRNA in OSCC and all tested OSCC cell lines compared to normal oral keratinocytes. Ectopic miR-99a expression in OSCC cells markedly reduced migration and invasion in vitro as well as lung colonization in vivo. When evaluating the specific targets of miR-99a, we found that ectopic miR-99a expression downregulates insulin-like growth factor 1 receptor (IGF1R) protein and that the expression of miR-99a correlates negatively with IGF1R protein in OSCC cells. Insertion of the 3'UTR of IGF1R mRNA into the 3'UTR of a reporter gene markedly reduced luciferase activity in OSCC cells expressing miR-99a, suggesting that miR-99a reduces luciferase activity by targeting the 3'UTR of IGF1R mRNA. When evaluating the mechanisms of miR-99a downregulation, we observed the upregulation of miR-99a expression in serum-starved conditions and its suppression in response to insulin-like growth factor (IGF1) stimulation. Inhibitors of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) kinase inhibited IGF1-induced suppression of miR-99a, suggesting the negative regulation of miR-99a expression by IGF1R signaling. CONCLUSION: Overall, results indicate that miR-99a functions as a tumor metastasis suppressor in OSCC cells and mutually regulates IGF1R expression in a reciprocal regulation.

Differentiated epithelial- and mesenchymal-like phenotypes in subcutaneous mouse xenografts using diffusion weighted-magnetic resonance imaging.

Epithelial-mesenchymal transition (EMT) is important for tumor metastasis. Detection of EMT protein expression and observation of morphological changes are commonly used to identify EMT. Diffusion-weighted magnetic resonance imaging (DW-MRI) and measuring apparent diffusion coefficient (ADC) values are noninvasive techniques for characterizing tumor microenvironments. We investigated the difference in ADC values between epithelial- and mesenchymal-like subcutaneous mouse xenografted tumors using DW-MRI. Epithelial-like MM189 PB-Klf4 and BL322 PB-Klf4 cells were generated from tumor suppressive Kruppel-like factor 4 (Klf4)-expressing mesenchymal-like MM189 and BL322 cells. The ADC values of xenografted tumors from epithelial-like MM189 PB-Klf4 and BL322 PB-Klf4 were significantly lower than those from their mesenchymal-like counterparts (p < 0.05 and p < 0.01, respectively). Our results suggested that DW-MRI is a potential tool for observing mesenchymal- or epithelial-like characteristics of subcutaneous xenografted tumors.

Pro-apoptotic role of Cdc25A: activation of cyclin B1/Cdc2 by the Cdc25A C-terminal domain.

Cdc25A is a dual specificity protein phosphatase that activates cyclin/cyclin-dependent protein kinase (Cdk) complexes by removing inhibitory phosphates from conserved threonine and tyrosine in Cdks. To address how Cdc25A promotes apoptosis, Jurkat cells were treated with staurosporine, an apoptosis inducer. Upon staurosporine treatment, a Cdc25A C-terminal 37-kDa fragment, designated C37, was generated by caspase cleavage at Asp-223. Thr-507 in C37 became dephosphorylated, which prevented 14-3-3 binding, as shown previously. C37 exhibited higher phosphatase activity than full-length Cdc25A. C37 with alanine substitution for Thr-507 (C37/T507A) that imitated the cleavage product during staurosporine treatment interacted with Cdc2, Cdk2, cyclin A, and cyclin B1 and markedly activated cyclin B1/Cdc2. The dephosphorylation of Thr-507 might expose the Cdc2/Cdk2-docking site in C37. C37/T507A also induced apoptosis in Jurkat and K562 cells, resulting from activating cyclin B1/Cdc2 but not Cdk2. Thus, this study reveals that Cdc25A is a pro-apoptotic protein that amplifies staurosporine-induced apoptosis through the activation of cyclin B1/Cdc2 by its C-terminal domain.

Ephrin A4-ephrin receptor A10 signaling promotes cell migration and spheroid formation by upregulating NANOG expression in oral squamous cell carcinoma cells.

Ephrin type-A receptor 10 (EPHA10) has been implicated as a potential target for breast and prostate cancer therapy. However, its involvement in oral squamous cell carcinoma (OSCC) remains unclear. We demonstrated that EPHA10 supports in vivo tumor growth and lymphatic metastasis of OSCC cells. OSCC cell migration, epithelial mesenchymal transition (EMT), and sphere formation were found to be regulated by EPHA10, and EPHA10 was found to drive expression of some EMT- and stemness-associated transcription factors. Among EPHA10 ligands, exogenous ephrin A4 (EFNA4) induced the most OSCC cell migration and sphere formation, as well as up-regulation of SNAIL, NANOG, and OCT4. These effects were abolished by extracellular signal-regulated kinase (ERK) inhibition and NANOG knockdown. Also, EPHA10 was required for EFNA4-induced cell migration, sphere formation, and expression of NANOG and OCT4 mRNA. Our microarray dataset revealed that EFNA4 mRNA expression was associated with expression of NANOG and OCT4 mRNA, and OSCC patients showing high co-expression of EFNA4 with NANOG or OCT4 mRNA demonstrated poor recurrence-free survival rates. Targeting forward signaling of the EFNA4-EPHA10 axis may be a promising therapeutic approach for oral malignancies, and the combination of EFNA4 mRNA and downstream gene expression may be a useful prognostic biomarker for OSCC.

Computed tomography-based differentiation of primary pulmonary lymphoepithelioma-like carcinoma and small-cell lung cancer.

BACKGROUND: Primary pulmonary lymphoepithelioma-like carcinoma (LELC) is a rare subtype of lung cancer. Both small-cell lung cancer (SCLC) and LELC often manifest as a centrally located tumor with lymphadenopathy. This retrospective study investigated and compared the initial computed tomography (CT) features and subsequent survival outcomes of LELC and SCLC. METHODS: A total of 50 patients with a confirmed diagnosis of LELC were enrolled and matched at a ratio of 1:1 with patients with SCLC according to the tumor stage. Utilizing a consensus approach, two radiologists reviewed pretreatment CT images. Survival outcomes were analyzed. RESULTS: Well-defined tumors were significantly more common in the LELC group (LELC: 42% vs SCLC: 24%, p = 0.005). Based on the comparisons of the primary tumor with the muscles, LELC tumors exhibited a significantly higher percentage of attenuation on contrast-enhanced CT scans (21.6% ± 29% vs -14.2% ± 37%, p < 0.001). The prevalence of vascular or bronchial encasement (18% vs 40%, p = 0.028), background emphysematous changes (10% vs 60%, p < 0.001), and tumors located in upper lobes (18% vs 64%, p < 0.001) was significantly lower in the LELC group. Female gender (70% vs 12%, p < 0.001), younger age (57.6 ± 12.0 years vs 68.0 ± 11.0 years, p < 0.001), and without a history of smoking (16% vs 88%, p < 0.001) were factors more commonly found in the LELC group. The patients with LELC had a better prognosis with significantly longer median survival than did the patients with SCLC (23.4 months vs 17.3 months, p = 0.01). CONCLUSION: Because SCLC demonstrated a more aggressive disease progression, differentiating LELC from SCLC is crucial. In Epstein-Barr virus-endemic areas, the diagnosis of LELC should be considered when approaching a patient with the above-mentioned CT and clinical features.

Interferon-stimulated gene 15 modulates cell migration by interacting with Rac1 and contributes to lymph node metastasis of oral squamous cell carcinoma cells.

In an effort to understand the underlying mechanisms of lymph node metastasis in oral squamous cell carcinoma (OSCC), through in vivo selection, LN1-1 cells were previously established from OEC-M1 cells and showed enhanced lymphangiogenesis and lymphatic metastasis capabilities. In the current study, we use a stable isotope labeling with amino acids in cell culture (SILAC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic platform to compare LN1-1 to OEC-M1 cells. Interferon-stimulated gene 15 (ISG15) was found highly expressed in LN1-1 cells. Immunohistochemical analysis and meta-analysis of publicly available microarray datasets revealed that the ISG15 level was increased in human OSCC tissues and associated with poor disease outcome. Knockdown of ISG15 had minimal effects on tumor growth but did decrease tumor lymphangiogenesis and lymphatic metastasis of LN1-1 cells. Consistent with the in vivo assay, ISG15 knockdown did not impair cell growth but diminished cell migration, invasion, and transendothelial migration in vitro. ISG15-induced cell migration was independent of ISGylation and associated with membrane protrusion. Ectopic expression of ISG15 increased Rac1 activity and knockdown of Rac1 impaired ISG15-enhanced migration. Furthermore, Rac1 colocalized with ISG15 to a region of membrane protrusion and ISG15 coimmunoprecipitated with Rac1, especially with the Rac1-GDP form. Importantly, as shown by proximity ligation assays, ISG15 and Rac1 physically interacted with each other. Our results indicated that ISG15 affects cell migration by interacting with Rac1 and regulating Rac1 activity and contributes to lymphatic metastasis in OSCC.

ERK Activation Modulates Cancer Stemness and Motility of a Novel Mouse Oral Squamous Cell Carcinoma Cell Line.

We established the NHRI-HN1 cell line from a mouse tongue tumor induced by 4-nitroquinoline 1-oxide (4-NQO)/arecoline, with further selection for cell stemness via in vitro sphere culture, to evaluate potential immunotherapies for oral squamous cell carcinoma (OSCC) in East and Southeast Asia. In vivo and in vitro phenotypic characterization, including tumor growth, immune modulator administration, gene expression, morphology, migration, invasion, and sphere formation assays, were conducted. NHRI-HN1 cells are capable of generating orthotopic tumors in syngeneic mice. Interestingly, immune stimulation via CpG oligodeoxynucleotide (CpG-ODN) dramatically reduced the tumor growth in NHRI-HN1 cell-injected syngeneic mice. The pathways enriched in genes that were differentially expressed in NHRI-HN1 cells when compared to non-tumorigenic cells were similar to those that were identified when comparing human OSCC and non-tumorous tissues. NHRI-HN1 cells have characteristics of epithelial-mesenchymal transition (EMT), including enhanced migration and invasion. NHRI-HN1 cells showed aggressive cell growth and sphere formation. The blockage of extracellular signal-regulated kinase (ERK) activation suppressed cell migration and reduced stemness characteristics in NHRI-HN1 cells, similar to human OSCC cell lines. Our data suggest that NHRI-HN1 cells, showing tumorigenic characteristics of EMT, cancer stemness, and ERK activation, are sufficient in modeling human OSCC and also competent for use in investigating oral cancer immunotherapies.

Upregulation of CISD2 augments ROS homeostasis and contributes to tumorigenesis and poor prognosis of lung adenocarcinoma.

CISD2 is a redox-sensitive gene critical for normal development and mitochondrial integrity. CISD2 was known to have aberrant expression in several types of human cancers. However, its relation with lung cancer is still not clear. In this study we found CISD2 mRNA was significantly upregulated in lung adenocarcinoma (ADC) samples, compared with their adjacent normal counterparts, and was correlated with tumor stage, grade, and prognosis based on analysis of clinical specimens-derived expression data in public domain and our validation assay. Cell based assay indicated that CISD2 expression regulated accumulation of reactive oxygen species (ROS), polarization of mitochondrial membrane potential, as well as cell viability, apoptosis, invasiveness, and tumorigenicity. In addition, CISD2 expression was found significantly correlated with stress response/redox signaling genes such as EGR1 and GPX3, while such correlations were also found valid in many public domain data. Taken together, upregulation of CISD2 is involved in an increased antioxidant capacity in response to elevated ROS levels during the formation and progression of lung ADC. The molecular mechanism underlying how CISD2 regulates ROS homeostasis and augments malignancy of lung cancer warrants further investigations.

Correction: Krüppel-like Factor 4, a Tumor Suppressor in Hepatocellular Carcinoma Cells Reverts Epithelial Mesenchymal Transition by Suppressing Slug Expression.

[This corrects the article DOI: 10.1371/journal.pone.0043593.].

Endothelial cell colony forming units derived from malignant breast diseases are resistant to tumor necrosis factor-α-induced apoptosis.

Mobilisation of endothelial progenitor cells (EPCs) from the bone marrow is a crucial step in the formation of de novo blood vessels, and levels of peripheral blood EPCs have been shown to be elevated in certain malignant states. Using flow cytometry and a Hill-based colony forming unit (CFU) assay, the present study indicated that higher levels of CD34 and vascular endothelial growth factor receptor 2 (VEGFR2) double-positive EPCs, as well as increased formation of endothelial cell colony-forming units (EC-CFUs) are associated with benign and malignant breast diseases, providing possible indicators for breast disease detection. Gene expression profiles revealed a genetic difference between CD34+ VEGFR2+ EPCs and EC-CFUs. Decreased expression of tumour necrosis factor receptor 2 (TNFR2) signalling-related genes and inhibition of tumour necrosis factor (TNF)-induced signalling were demonstrated in EC-CFUs derived from patients with malignant breast disease in comparison with those from healthy controls. Interestingly, our data provided the first evidence that EC-CFUs derived from patients with malignant breast disease were resistant to TNF-α-induced apoptosis, indicating a plausible target for future therapeutic interventions.

Insulin-like growth factor-independent insulin-like growth factor binding protein 3 promotes cell migration and lymph node metastasis of oral squamous cell carcinoma cells by requirement of integrin ß1.

Frequent metastasis to the cervical lymph nodes leads to poor survival of patients with oral squamous cell carcinoma (OSCC). To understand the underlying mechanisms of lymph node metastasis, two sublines were successfully isolated from cervical lymph nodes of nude mice through in vivo selection, and identified as originating from poorly metastatic parental cells. These two sublines specifically metastasized to cervical lymph nodes in 83% of mice, whereas OEC-M1 cells did not metastasize after injection into the oral cavity. After gene expression analysis, we identified insulin-like growth factor binding protein 3 (IGFBP3) as one of the significantly up-regulated genes in the sublines in comparison with their parental cells. Consistently, meta-analysis of the public microarray datasets and IGFBP3 immunohistochemical analysis revealed increased both levels of IGFBP3 mRNA and protein in human OSCC tissues when compared to normal oral or adjacent nontumorous tissues. Interestingly, the up-regulated IGFBP3 mRNA expression was significantly associated with OSCC patients with lymph node metastasis. IGFBP3 knockdown in the sublines impaired and ectopic IGFBP3 expression in the parental cells promoted migration, transendothelial migration and lymph node metastasis of orthotopic transplantation. Additionally, ectopic expression of IGFBP3 with an IGF-binding defect sustained the IGFBP3-enhanced biological functions. Results indicated that IGFBP3 regulates metastasis-related functions of OSCC cells through an IGF-independent mechanism. Furthermore, exogenous IGFBP3 was sufficient to induce cell motility and extracellular signal-regulated kinase (ERK) activation. The silencing of integrin ß1 was able to impair exogenous IGFBP3-mediated migration and ERK phosphorylation, suggesting a critical role of integrin ß1 in IGFBP3-enchanced functions.

Downregulated miR329 and miR410 promote the proliferation and invasion of oral squamous cell carcinoma by targeting Wnt-7b.

microRNA (miRNA) dysregulation contributes widely to human cancer but has not been fully assessed in oral cancers. In this study, we conducted a global microarray analysis of miRNA expression in 40 pairs of betel quid-associated oral squamous cell carcinoma (OSCC) specimens and their matched nontumorous epithelial counterparts. Eighty-four miRNAs were differentially expressed in the OSCC specimens compared with the matched tissue. Among these downregulated miRNAs, 19 miRNAs were found and mapped to the chromosome 14q32.2 miRNA cluster region, which resides within a parentally imprinted region designated as Dlk-Dio3 and known to be important in development and growth. Bioinformatic analysis predicted two miRNAs from the cluster region, miR329 and miR410, which could potentially target Wnt-7b, an activator of the Wnt-ß-catenin pathway, thereby attenuating the Wnt-ß-catenin signaling pathway in OSCC. Stable ectopic expression of Wnt-7b in OSCC cells overexpressing miR329 or miR410 restored proliferation and invasion capabilities abolished by these miRNA. Combining a demethylation agent and a histone deacetylase inhibitor was sufficient to reexpress miR329, miR410, and Meg3, consistent with epigenetic regulation of these miRNA in human OSCC. Specifically, arecoline, a major betel nut alkaloid, reduced miR329, miR410, and Meg3 gene expression. Overall, our results provide novel molecular insights into how betel quid contributes to oral carcinogenesis through epigenetic silencing of tumor-suppressor miRNA that targets Wnt-ß-catenin signaling.

Krüppel-like factor 4, a tumor suppressor in hepatocellular carcinoma cells reverts epithelial mesenchymal transition by suppressing slug expression.

Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor that plays an important role in differentiation and pathogenesis. KLF4 has been suggested to act as an oncogene or tumor suppressor in different tumor types. However, the role of KLF4 in hepatocellular carcinoma (HCC) remains unclear. Here, we demonstrate that forced expression of Klf4 in murine HCC cell lines reduced anchorage-independent growth in soft agar as well as cell migration and invasion activities in vitro. Ectopic Klf4 expression impaired subcutaneous tumor growth and lung colonization in vivo. By contrast, Klf4 knockdown enhanced HCC cell migration. Interestingly, ectopic expression of Klf4 changed the morphology of murine HCC cells to a more epithelial phenotype. Associated with this, we found that expression of Slug, a critical epithelial mesenchymal transition (EMT)-related transcription factor, was significantly down-regulated in Klf4-expressing cells. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays showed that Klf4 is able to bind and repress the activity of the Slug promoter. Furthermore, ectopic Slug expression partially reverts the Klf4-mediated phenotypes. Consistent with a role as a tumor suppressor in HCC, analysis of the public microarray databases from Oncomine revealed reduced KLF4 expression in human HCC tissues in comparison with normal liver tissues in 3 out of 4 data sets. By quantitative reverse transcription-polymerase chain reaction (qRT-PCR), we found reduced KLF4 mRNA in 50% of HCC tissues. Importantly, an inverse correlation between the expression of KLF4 and SLUG was found in HCC tissues. Our data suggest that KLF4 acts as a tumor suppressor in HCC cells, in part by suppressing SLUG transcription.