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Differentially Methylated CpG Sites (2173 Sites) in 24 Nasopharyngeal Carcinoma (NPC) Tissues and 24 Non-cancer Nasopharyngitis Biopsy Tissues (NCNBT)

Kaplan-Meier curves estimation of disease-free survival (DFS) and overall survival (OS) for patients with high or low methylation levels stratified by tumor stage or the receipt of concurrent chemotherapy.

Supplementary Table S1: Clinicopathological Characteristics of 24 NPC and 24 NCNBT Supplementary Table S2: Clinical Characteristics of Nasopharyngeal Carcinoma Patients According to Gene Methylation Panel in Training, Validation, and Independent Cohorts. Supplementary Table S3: Primer Sequences of 28 Genes and GAPDH Supplementary Table S4: Primer Used for Pyrosequencing Methylation Analysis of Candidate Genes Supplementary Table S6: Data Analysis with PubMeth and NCBI PubMed Supplementary Table S7: Correlation Analysis between Methylation of Six Genes in the Training Cohort

Representative Pyrograms of Methylation Analysis by Bisulfite Pyrosequencing.

Supplementary Table S1. Clinical characteristics of 534 NPC patients. Supplementary Table S2. Sequences used for shRNA construction and siRNAs. Supplementary Table S3. Primers used for real-time PCR. Supplementary Table S4. Association of SPINK6 expression with clinicopathological characteristics of NPC patients.

Supplementary Materials and Methods

Purpose: Metastatic colorectal cancer patients with low epidermal growth factor receptor (EGFR) gene copy number are unlikely to respond to anti-EGFR monoclonal antibody (mAb) treatment. The objective of this study was to investigate EGFR fluorescence in situ hybridization (FISH) patterns of chromosome 7 disomy with efficacy of cetuximab therapy in metastatic colorectal cancer patients.Experimental Design: We detected the EGFR FISH patterns and KRAS status in 74 tumors from cetuximab-treated metastatic colorectal cancer patients and analyzed with response rate (RR) and progression-free survival (PFS).Results: One of the 16 (6.25%) patients with chromosome 7 homogeneous disomy (defined as FISH negative) had objective response to cetuximab. A total of 53(76.8%) patients with chromosome 7 pattern of variable ratios of disomy versus polysomy (defined as FISH positive) had a significantly higher RR (37.7% versus 6.25%; P = 0.01), a trend towards longer PFS (4.5 versus 2.9 months; P = 0.07). Among 54 KRAS wild-type patients, EGFR FISH-positive patients had significantly higher RR (51.3% versus 9%; P = 0.01) and longer PFS (5.0 versus 2.3 months; P = 0.02) than EGFR FISH-negative patients. However, among 20 KRAS mutant-type patients, there was no difference in RR (0% versus 0%) and PFS (2.5 versus 3.8 months; P = 0.51) between EGFR FISH-positive and -negative patients.Conclusion: Our results show firstly that patients with EGFR FISH pattern of chromosome 7 disomy have a very low chance to benefit from cetuximab-based therapy. EGFR FISH pattern of chromosome 7 disomy may be as a negative predicative factor for cetuximab response in KRAS wild-type metastatic colorectal cancer patients. Clin Cancer Res; 17(2); 382–90. ©2011 AACR.

Supplementary Tables 1-4. Supplementary Table 1. Odds ratios (ORs) and 95% confidence intervals (CIs) of nasopharyngeal carcinoma (NPC) associated with oral health among ever smokers in southern China (2010-2014). Supplementary Table 2. Stratified odds ratios (ORs) and 95% confidence intervals (CIs) of nasopharyngeal carcinoma (NPC) associated with number of teeth lost after age 20 years.* Supplementary Table 3. Stratified odds ratios (ORs) and 95% confidence intervals (CIs)of nasopharyngeal carcinoma (NPC) associated with frequency of tooth brushing.* Supplementary Table 4. Odds ratios (ORs) and 95% confidence intervals (CIs) of nasopharyngeal carcinoma (NPC) associated with oral health in southern China - restricted to cases interviewed within 30 days of diagnosis (2010-2014).

Supplementary Fig. S1. Expression of SPINK6 in condition medium of NPC cells. Supplementary Fig. S2. Cellular growth of NPC cells. Supplementary Fig. S3. Wound-healing ability of NPC cells upon SPINK6 knockdown. Supplementary Fig. S4. Migration and invasion of NPC cells upon silencing of KLKs. Supplementary Fig. S5. Promoted motility of NPC cells by recombinant SPINK6. Supplementary Fig. S6. Interaction between SPINK6 and EGFR. Supplementary Fig. S7. Phosphorylation of EGFR and downstream signaling molecules in NPC cells. Supplementary Fig. S8. Wound-healing ability of NPC cells. Supplementary Fig. S9. The expression of E-cadherin and vimentin in NPC cells. Supplementary Fig. S10. The expression of EGF, EGFR and KLKs in NPC and NP tissues.

Nasopharyngeal carcinoma has the highest rate of metastasis among head and neck cancers, and distant metastasis is the major reason for treatment failure. The underlying molecular mechanisms of nasopharyngeal carcinoma metastasis are not fully understood. Here, we report the identification of serine protease inhibitor Kazal-type 6 (SPINK6) as a functional regulator of nasopharyngeal carcinoma metastasis via EGFR signaling. SPINK6 mRNA was upregulated in tumor and highly metastatic nasopharyngeal carcinoma cells. Immunohistochemical staining of 534 nasopharyngeal carcinomas revealed elevated SPINK6 expression as an independent unfavorable prognostic factor for overall, disease-free, and distant metastasis–free survival. Ectopic SPINK6 expression promoted in vitro migration and invasion as well as in vivo lymph node metastasis and liver metastasis of nasopharyngeal carcinoma cells, whereas silencing SPINK6 exhibited opposing effects. SPINK6 enhanced epithelial–mesenchymal transition by activating EGFR and the downstream AKT pathway. Inhibition of EGFR with a neutralizing antibody or erlotinib reversed SPINK6-induced nasopharyngeal carcinoma cell migration and invasion. Erlotinib also inhibited SPINK6-induced metastasis in vivo. Notably, SPINK6 bound to the EGFR extracellular domain independent of serine protease–inhibitory activity. Overall, our results identified a novel EGFR-activating mechanism in which SPINK6 has a critical role in promoting nasopharyngeal carcinoma metastasis, with possible implications as a prognostic indicator in nasopharyngeal carcinoma patients. Cancer Res; 77(2); 579–89. ©2016 AACR.

Background: The association between oral health and risk of nasopharyngeal carcinoma (NPC) is largely unknown. Further understanding could shed light on potential pathogenic mechanisms and preventive measures.Methods: We conducted a population-based case–control study in southern China between 2010 and 2014. We enrolled 2,528 incident NPC cases, aged 20–74 years, and 2,596 controls, randomly selected from the total population registers, with frequency matching to the 5-year age and sex distribution of the cases by geographic region. We interviewed subjects using a structured questionnaire inquiring about oral health indicators and potential confounding factors. We used unconditional logistic regression to estimate multivariate-adjusted ORs with 95% confidence intervals (CI).Results: A higher number of filled teeth was associated with an elevated risk of NPC. Individuals with 1 to 3 and more than 3 teeth filled versus none had adjusted ORs of 1.25 (95% CI, 1.06–1.49) and 1.55 (95% CI, 1.13–2.12), respectively (Ptrend = 0.002). Conversely, the adjusted OR for those who brushed teeth twice or more per day versus once or less per day was 0.62 (95% CI, 0.55–0.70). We detected a borderline significant positive association with earlier age at first adult tooth loss.Conclusion: Our study suggested a positive association between some indicators of poor oral health and risk of NPC. Further studies are needed to confirm whether the findings are causal and, if so, to further explain the underlying mechanisms.Impact: Improvement of oral hygiene might contribute to reducing NPC risk. Cancer Epidemiol Biomarkers Prev; 25(8); 1201–7. ©2016 AACR.

Supplementary Table S1.

Supplementary Tables 1-3 from Serglycin Is a Theranostic Target in Nasopharyngeal Carcinoma that Promotes Metastasis

Supplementary Figures 1-2 from Serglycin Is a Theranostic Target in Nasopharyngeal Carcinoma that Promotes Metastasis

Nasopharyngeal carcinoma (NPC) is known for its high-metastatic potential. Here we report the identification of the proteoglycan serglycin as a functionally significant regulator of metastasis in this setting. Comparative genomic expression profiling of NPC cell line clones with high- and low-metastatic potential revealed the serglycin gene (SRGN) as one of the most upregulated genes in highly metastatic cells. RNAi-mediated inhibition of serglycin expression blocked serglycin secretion and the invasive motility of highly metastatic cells, reducing metastatic capacity in vivo. Conversely, serglycin overexpression in poorly metastatic cells increased their motile behavior and metastatic capacity in vivo. Growth rate was not influenced by serglycin in either highly or poorly metastatic cells. Secreted but not bacterial recombinant serglycin promoted motile behavior, suggesting a critical role for glycosylation in serglycin activity. Serglycin inhibition was associated with reduced expression of vimentin but not other epithelial–mesenchymal transition proteins. In clinical specimens, serglycin expression was elevated significantly in liver metastases from NPC relative to primary NPC tumors. We evaluated the prognostic value of serglycin by immunohistochemical staining of tissue microarrays from 263 NPC patients followed by multivariate analyses. High serglycin expression in primary NPC was found to be an unfavorable independent indicator of distant metastasis-free and disease-free survival. Our findings establish that glycosylated serglycin regulates NPC metastasis via autocrine and paracrine routes, and that it serves as an independent prognostic indicator of metastasis-free survival and disease-free survival in NPC patients. Cancer Res; 71(8); 3162–72. ©2011 AACR.

Supplementary Materials and Methods from Serglycin Is a Theranostic Target in Nasopharyngeal Carcinoma that Promotes Metastasis

Supplementary Figures 1-5 from miR-125b Is Methylated and Functions as a Tumor Suppressor by Regulating the ETS1 Proto-oncogene in Human Invasive Breast Cancer

Background/Aims: Colorectal cancer (CRC) is a common cancer worldwide with increasing morbidity in China. Approximately 5% CRC are caused by well-defined hereditary CRC syndromes, whose inherited susceptibility account for nearly one-third of CRC predisposition. Most of hereditary CRC syndromes were diagnosed at the age of lower than 70, whereas little is known about the mutational profiles Chinese patients and their correlations with clinical features. Method: In our present research, we retrospectively collected 731 patients who were diagnosed as CRC from Sun Yat-sen University Cancer Center, with ages lower than 70. All of them received genetic mutation detection consisting of a set of 14 specific inherited genes, which used target area capture and the second-generation high throughput sequencing technology. The characteristics of he patients with or without mutations were compared. We applied several comparison analysis to investigate the germline mutations distribution of genes that associated with the defined hereditary CRC syndromes in Chinese cohort. Results: 397 patients (54.3%) harbored functionally variants of the 14 genes in CRC patients that diagnosis lower than 70. Intriguingly, we observed no statistical difference for clinical factors such as age, sex, smoke, drink, family history, pathology grade, clinic stage and overall survival between groups. In Mut group, the mutation frequency of each genes are MLH1(21.4%), MSH2(17.6%), MSH6(15.1%), PMS2(9.3%), EPCAM(8.3%), MLH3(8.1%), AXIN2(7.1%), PMS1(4.0%), MUTYH(12.8%), APC(12.3%), STK11(3.5%), BMPR1A(1.8%), SMAD4(0.3%), PTEN(1.0%). Genome mutual exclusivity analysis has identified a set of genes such as MLH1, MSH2, MUTYH, MSH6, APC and PMS2, for which most pairwise combinations are found rarely cooccurred. In correlation analysis between mutational genes and clinical features, pathology grade correlates strongly with PMS1 and SMAD4(P Conclusion: The clinic features of two groups have no statistic difference, which suggested other genes undiscovered may play a partial role in pathogenesis of patients of non-MUT group. In summary, we conducted a retrospective study to lead a comprehensive comparison of mutational profiles between MUT and non-MUT hereditary CRC patients, which supplemented the limited data of hereditary CRC in China. Citation Format: Feng Wang, Qi Zhao, Teng-jia Jiang, Ying-nan Wang, Fu-rong Liu, Jia-jia Hu, Pei-rong Ding, Zhi-zhong Pan, Jian-yong Shao, Rui-hua Xu. Germline mutational profileof Chinese patients with colorectal cancer and diagnosed lower than 70 years [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 4161.

The microRNA miR-125b is dysregulated in various human cancers but its underlying mechanisms of action are poorly understood. Here, we report that miR-125b is downregulated in invasive breast cancers where it predicts poor patient survival. Hypermethylation of the miR-125b promoter partially accounted for reduction of miR-125b expression in human breast cancer. Ectopic restoration of miR-125b expression in breast cancer cells suppressed proliferation, induced G1 cell-cycle arrest in vitro, and inhibited tumorigenesis in vivo. We identified the ETS1 gene as a novel direct target of miR-125b. siRNA-mediated ETS1 knockdown phenocopied the effect of miR-125b in breast cell lines and ETS1 overexpression in invasive breast cancer tissues also correlated with poor patient prognosis. Taken together, our findings point to an important role for miR-125b in the molecular etiology of invasive breast cancer, and they suggest miR-125b as a potential theranostic tool in this disease. Cancer Res; 71(10); 3552–62. ©2011 AACR.

Nasopharyngeal carcinoma (NPC) is known for its high-metastatic potential. Here we report the identification of the proteoglycan serglycin as a functionally significant regulator of metastasis in this setting. Comparative genomic expression profiling of NPC cell line clones with high- and low-metastatic potential revealed the serglycin gene (SRGN) as one of the most upregulated genes in highly metastatic cells. RNAi-mediated inhibition of serglycin expression blocked serglycin secretion and the invasive motility of highly metastatic cells, reducing metastatic capacity in vivo. Conversely, serglycin overexpression in poorly metastatic cells increased their motile behavior and metastatic capacity in vivo. Growth rate was not influenced by serglycin in either highly or poorly metastatic cells. Secreted but not bacterial recombinant serglycin promoted motile behavior, suggesting a critical role for glycosylation in serglycin activity. Serglycin inhibition was associated with reduced expression of vimentin but not other epithelial–mesenchymal transition proteins. In clinical specimens, serglycin expression was elevated significantly in liver metastases from NPC relative to primary NPC tumors. We evaluated the prognostic value of serglycin by immunohistochemical staining of tissue microarrays from 263 NPC patients followed by multivariate analyses. High serglycin expression in primary NPC was found to be an unfavorable independent indicator of distant metastasis-free and disease-free survival. Our findings establish that glycosylated serglycin regulates NPC metastasis via autocrine and paracrine routes, and that it serves as an independent prognostic indicator of metastasis-free survival and disease-free survival in NPC patients. Cancer Res; 71(8); 3162–72. ©2011 AACR.

Serglycin is a proteoglycan consisting of a core protein to which negatively charged glycoaminoglycan (GAG) chains of either chondroitin sulfate or heparin are attached. Serglycin was first shown to be essential for the maturation of mast cell secretory granules, but it has recently been shown to be present within multiple other cell types. In this study we describe for the first time the role of serglycin as a potential metastasis regulator in nasopharyngeal carcinoma (NPC), a tumor that has the highest incidence of metastasis among head and neck cancers. The recognition that serglycin plays an important role in NPC comes from our earlier genomic expression profiles comparing clones derived from the NPC cell line CNE-2, where serglycin (SRGN) was identified as one of the most up-regulated genes in the high-metastasis Clone 18. The genomic expression profiling was performed on samples collected in cultured cells and in xenograft tumors generated from Clone-18, as well as low-metastasis clones (Clone-22, Clone-26) and their parental line, CNE-2. A popliteal lymph node model was used to evaluate the metastasis ability of different cellular populations. A tissue microarray was constructed from 330 NPC tissues and a total of 263 cases were informative for assessing the patients’ survival duration. A deglycosylation assay, lentiviral transduction, quantitative PCR, proliferation assay, Transwell assay, and wound-healing assay were used for the functional studies. The serglycin protein was shown to be secreted by the high-metastasis clone, but not by any of the low-metastasis clones. Suppression of serglycin by shRNA diminished serglycin secretion and subsequently inhibited migration and invasion by the high-metastasis clone, and also significantly reduced its metastasis rate in vivo. Overexpression of serglycin in low-metastasis cells resulted in a significantly increased metastasis rate in vivo. Moreover, secreted serglycin promoted cellular motility in the wild-type low-metastasis cells. Interestingly, suppression of serglycin reduced the protein level of vimentin but did not influence the level of E-cadherin in the high-metastasis clone. Proliferation was not influenced by serglycin in either the high- or low-metastasis clones. Importantly, in direct tumor samples, serglycin expression was significantly elevated in liver metastases from NPC relative to its expression in primary tumors. In conclusion, serglycin appears to play a potentially pivotal role in regulating NPC metastasis by way of enhancing cellular migration, cellular invasiveness, vimentin expression level, and the in vivo spread of cancer cells. Moreover, a high level of serglycin expression can potentially be used to predict shorter disease-free survival and shorter metastasis-free survival of NPC patients. Targeting serglycin could be a novel option for the prevention of NPC metastasis. 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 1476. doi:10.1158/1538-7445.AM2011-1476