Search

Supplementary Figures S1-S4 from IFN-Induced Protein with Tetratricopeptide Repeats 2 Inhibits Migration Activity and Increases Survival of Oral Squamous Cell Carcinoma

The function of the IFN-stimulated gene family protein, IFN-induced protein with tetratricopeptide repeats 2 (IFIT2), is poorly understood. Here, we report that IFIT2 colocalizes with cytokeratin 18 in oral squamous cell carcinoma (OSCC) cells. Treatment of OSCC cells with IFN-β significantly increased the expression of IFIT2 and remarkably inhibited cell migration. To further explore the effect of IFIT2 on cell migration, IFIT2 expression was either silenced with a small interfering RNA or increased by ectopic expression. IFIT2 knockdown in OSCC cells led to a significantly higher level of migration in vitro (P < 0.05) compared with control cells; by contrast, IFIT2 overexpression led to a significantly lower level of migration in vitro (P < 0.05). Immunohistochemically, 71.4% of OSCC tissues had elevated IFIT2 protein levels compared with noncancerous matched tissues. Elevated IFIT2 protein expression was positively associated with tumor differentiation status and inversely associated with nodal stage in OSCC specimens (P < 0.05). Higher IFIT2 protein levels in tumor tissues were also associated with better patient survival (P < 0.01). Our present study shows an inverse correlation between IFIT2 expression and cell migration, suggesting that IFIT2 plays an important role in inhibiting this process and that its expression may be associated with better prognosis in patients with OSCC. (Mol Cancer Res 2008;6(9):1431–9)

Supplementary Figures 1-8, Tables 1-7 from miR-31 Ablates Expression of the HIF Regulatory Factor FIH to Activate the HIF Pathway in Head and Neck Carcinoma

Supplementary Materials and Methods. Table S1. Cell cultivation conditions. Table S2. siRNAs used in the present study. Table S3. TaqMan assay probes used in the present study. Table S4. Primers used in the present study. Table S5. shRNA clones used in the present study. Table S6. Paired OSCC samples for qRT-PCR and Western blot analysis. Table S7. OSCC samples in TMA for IHC and ISH analysis. Table S8. Primary antibodies or associated reagents used in the present study. Table S9. The complimentarity between miR-211 and the 3’UTR of TCF12 gene. Table S10. Jaspar prediction scores. Fig S1. Genotyping of K14-EGFP-miR-211 transgenic mouse model. Fig S2. GFP immunoreactivity and thickness in squamous epithelium. Fig S3. Immunohistochemistry of the cell proliferation and survival proteins in tongue epithelium. Fig S4. Induction of mouse esophageal tumorigenesis by 4NQO. Fig. S5. Representative histopathological sections of orthotopic xenografts and neck lymph nodes. Fig. S6. Analysis of pri-miR-211 expression in OSCC cells. Fig. S7. miR-211 staining and TCF12 immunoreactivity in mouse tongue tissues. Fig. S8. Analysis of TCF12 protein in subcellular fractions in FaDu cells. Fig. S9. Phenotypic analysis of OECM1 cell with TCF12 expression. Fig. S10. Correlation between miR-211 expression and TCF12 mRNA expression in OSCC tumors. Fig. S11. miR-211 staining and TCF12 immunoreactivity in OSCC. Fig. S12. TCF12 immunoreactivity in mouse multistep carcinogenesis. Fig. S13. Color diagram of Fig. 4A, a. Fig S14. Analysis of TCF12 associated biological processes and gene interaction networks. Fig. S15. Prediction of the potential regulation of TCF12 on the promoter activity of downstream genes using Jasper software. Fig. S16. qRT-PCR analysis to confirm the regulation of TCF12 on downstream genes across different OSCC cells. Fig. S17. Correlation between the expression of TCF12 and FAM213A in database. Fig. S18. FAM213A immunoreactivity. Fig. S19. Color diagram of Fig. 6D. Fig. S20. Kaplan-Meier survival analysis.

miR-211 expression in human oral squamous cell carcinoma (OSCC) has been implicated in poor patient survival. To investigate the oncogenic roles of miR-211, we generated K14-EGFP-miR-211 transgenic mice tagged with GFP. Induction of oral carcinogenesis in transgenic mice using 4-nitroquinoline 1-oxide (4NQO) resulted in more extensive and severe tongue tumorigenesis compared with control animals. We found that 4NQO and arecoline upregulated miR-211 expression in OSCC cells. In silico and experimental evidence further revealed that miR-211 directly targeted transcription factor 12 (TCF12), which mediated suppressor activities in OSCC cells and was drastically downregulated in tumor tissues. We used GeneChip analysis and bioinformatic algorithms to identify transcriptional targets of TCF12 and confirmed through reporter and ChIP assays that family with sequence similarity 213, member A (FAM213A), a peroxiredoxin-like antioxidative protein, was repressed transcriptionally by TCF12. FAM213A silencing in OSCC cells diminished oncogenic activity, reduced the ALDH1-positive cell population, and increased reactive oxygen species. TCF12 and FAM213A expression was correlated inversely in head and neck carcinoma samples according to The Cancer Genome Atlas. OSCC patients bearing tumors with high FAM213A expression tended to have worse survival. Furthermore, 4NQO treatment downregulated TCF12 and upregulated FAM213A by modulating miR-211 both in vitro and in vivo. Overall, our findings develop a mouse model that recapitulates the molecular and histopathologic alterations of human OSCC pathogenesis and highlight a new miRNA-mediated oncogenic mechanism. Cancer Res; 76(16); 4872–86. ©2016 AACR.

Oral squamous cell carcinoma (OSCC) is one of the most prevalent malignancies worldwide. Despite the advances in diagnosis and treatment, the survival of OSCC remains to be improved. miR-211 is an oncogenic microRNA frequently disrupted in human OSCC and its high expression is a determinant of patient’s poor survival. Herein, we established four murine OSCC cell lines, designated MOC-L1 - MOC-L4 from the tongue tumor tissues induced by 4-nitroquinoline 1-oxide in K14-EGFP-miR-211 transgenic mice. All cell lines appear green fluorescence and express epithelial markers. The gene expression profiles among cell lines are complex and diverse, while MOC-L1 - MOC-L3 cells carry missense mutations in p53 gene. MOC-L1 exhibits tremendous epithelial-mesenchymal transition and the associated aggressive characteristics. On the contrary, MOC-L4 displays the least invasiveness. Both MOC-L1 and MOC-L2 are clonogenic in vitro and tumorigenic when implemented into dermis or tongue in syngeneic recipients. But, only MOC-L1 exhibits high potential for local regional and distal metastasis. Since the expression of miR-196b in MOC-L1 xenografts drastically decreased upon cisplatin treatment, targeting of miR-196b might facilitate tumor abrogation. As these cell lines originate from the C57BL/6 mouse, which is the strain most suitable for transgenic engineering, to approach the interplay of these OSCC cells with other type of genetically modified cells in immune-competent mice would bestow profound insight on OSCC pathogenesis. Note: This abstract was not presented at the meeting. Citation Format: Yi-Fen Chen, Chung-Ji Liu, Shou-Yen Kao, Shu-Chun Lin, Kuo-Wei Chang. Newly established mouse oral carcinoma cell lines and their syngeneic tumorigenesis models [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 4635.

The function of the IFN-stimulated gene family protein, IFN-induced protein with tetratricopeptide repeats 2 (IFIT2), is poorly understood. Here, we report that IFIT2 colocalizes with cytokeratin 18 in oral squamous cell carcinoma (OSCC) cells. Treatment of OSCC cells with IFN-β significantly increased the expression of IFIT2 and remarkably inhibited cell migration. To further explore the effect of IFIT2 on cell migration, IFIT2 expression was either silenced with a small interfering RNA or increased by ectopic expression. IFIT2 knockdown in OSCC cells led to a significantly higher level of migration in vitro (P < 0.05) compared with control cells; by contrast, IFIT2 overexpression led to a significantly lower level of migration in vitro (P < 0.05). Immunohistochemically, 71.4% of OSCC tissues had elevated IFIT2 protein levels compared with noncancerous matched tissues. Elevated IFIT2 protein expression was positively associated with tumor differentiation status and inversely associated with nodal stage in OSCC specimens (P < 0.05). Higher IFIT2 protein levels in tumor tissues were also associated with better patient survival (P < 0.01). Our present study shows an inverse correlation between IFIT2 expression and cell migration, suggesting that IFIT2 plays an important role in inhibiting this process and that its expression may be associated with better prognosis in patients with OSCC. (Mol Cancer Res 2008;6(9):1431–9)

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world, and is highly mortal due to tumor recurrence and metastasis. Invasion of tumor cells into lymph nodes is one of the characters of regional metastasis and DNA hypermethylation is characterized in all types of human cancers. We therefore assessed whether epigenetic modification of DNA methylation is correlated to HNSCC metastasis, and evaluated the potential clinical implications of DNA methylation-based biomarker. Methylation array (Illumina infinium beadsarray) was applied to profile aberrant methylated CpG loci in HNSCC with lymph node invasiveness. Fourteen HNSCC tumors and two normal oral tissues were included. ≤ value, the indicator of methylation status of each tested CpG locus, was derived with normalization. A cluster of 111 CpG loci was selected based on the difference between the mean ≤ value of HNSCC tumors with and without regional metastasis (Δβmetz minus non-metz ≥ 0.2 or ≤ −0.2). Hierarchical diagram revealed distinct methylation signature was observed between tumors with or without regional metastasis. Ingenuity pathway analysis showed that the major diseases associated were neurological disorder and endocrine system disorder; and the top canonical pathway was Huntington's disease signaling. Of the top aberrant hypermethylated alleles, p16 and brain-derived neurotrophic factor (BDNF) were selected for further investigation on clinical implication. The methylation status of p16 (n=107) and BDNF (n=88) in HNSCC tumors was assayed by methylation-specific PCR. The primer sequences of both alleles were adopted from published report. Statistic analysis (t-test, χ2 or Fisher's exact, logistic regress, Kaplan-Meier survival, and Cox regression) was performed using SPSS. Hypermethylation of p16 or BDNF were not associated with stage, metastasis at diagnosis, and differentiation. Hypermethylation of p16 was significantly associated with age (p=0.008, t-test) and lymph node invasion (p=0.021, χ2-test), in contrast to distant metastasis of follow-up (p=0.061, χ2-test). However, hypermethylation of BDNF was associated with distant metastasis of follow-up (odds ratio: 4.96, 95% CI: 1.28-18.87, p=0.002), and combination with p16 improved the prediction (odds ratio: 10.31, 95% CI: 1.68-62.5, p=0.012). Hypermethylation of BDNF was associated with poor survival of patients (odds ratio: 2.80, 95% CI: 1.17-6.71, p=0.021), and hypermethylation of both alleles increased the risk (odds ratio: 3.81, 95% CI: 1.71-8.49, p=0.001). Together, our data suggest hypermethylation of p16 and BDNF may be potential biomarker associate with poor prognosis of HNSCC. The results also show the association of p16 and BDNF in gain of metastatic properties, which warrants further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4038. doi:1538-7445.AM2012-4038

Background: Oral squamous cell carcinoma (OSCC) is the sixth common cancer and is highly mortal due to tumor recurrence and metastasis. Invasion of tumor cells into lymph nodes is one of the characters of regional metastasis. Here we assessed whether epigenetic modification of DNA methylation is correlated to OSCC regional metastasis and evaluated the potential clinical implications of DNA methylation-based biomarker. Methods: A case-control study of OSCC patients with lymph node invasion (N=8), OSCC patients without lymph node invasion (N=6), and normal oral tissues (N=2) was performed. To characterize aberrant DNA methylation in OSCC tumors with regional metastasis at diagnosis, Illumina infinium beadsarrays containing 27,578 CpG loci which cover 14,475 genes were applied. The genomic DNA of test samples was individually bisulfite-converted and subjected to beadsarray analysis. β value, the indicator of methylation status of each tested CpG locus, was derived with proper normalization. Results: Methylation β values allocated tested tissues into regional metastatic tumor, non-metastatic tumor, and control groups. A cluster of 111 CpG loci was selected based on the difference between the mean β value of OSCC tumors with and without regional metastasis. There were 69 loci and 42 loci that were hypermethylated (Δβ value ≫ 0.2) and hypomethylated (Δβ value ≪ −0.2), respectively, in OSCC with regional metastasis. Differentially methylated genes appeared to be mainly related to organ development, cell death, cell signaling, behavior, nucleic acid metabolism, and molecular transport. Several genes were further investigated for their methylation statuses by genomic bisulfite sequencing and DNA methyltransferase inhibitor reactivation in oral carcinoma cell lines with or without invasiveness. Characterization the association of hypermethylation of metastatic-associated genes and clinical pathological variables, we found combination of the hypermethylation status of a candidate gene and p16INK4A improved the prediction of lymph node invasiveness (p=0.003) and survival rate (p=0.010). Our results show the feasibility of this beadsarray analysis to identify aberrant methylation of metastatic-associated genes in OSCC as well as potential risk assessment application. The cohort for the risk assessment assay will be increased and the biological activities of candidate genes in carcinogenesis will be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 103. doi:10.1158/1538-7445.AM2011-103