Chunbo Shao

TKTL1 Is Activated by Promoter Hypomethylation and Contributes to Head and Neck Squamous Cell Carcinoma Carcinogenesis through Increased Aerobic Glycolysis and HIF1α Stabilization

Purpose: This study aims to investigate the role of the aberrant expression of Transkelolase-like 1 (TKTL1) in head and neck squamous cell carcinoma (HNSCC) tumorigenesis and to characterize TKTL1 contribution to HNSCC tumorigenesis through aerobic glycolysis and HIF1α stabilization. Experimental Design: TKTL1 promoter hypomethylation and mRNA/protein aberrant expression were studied in human HNSCC tumor samples and normal mucosas. Oncogenic functions of TKTL1 were examined in HNSCC cell line panels and tumor xenograft models with TKTL1 expression construct. The metabolite levels of fructose-6-phosphate, glyceraldehydes-3-phosphate, pyruvate, lactate, and the levels of HIF1α protein and its downsteam glycolytic targets were compared between the TKTL1-expressing and vehicle-expressing HNSCC cells. Meanwhile, the effects of HIF1α/glycolytic inhibitors were evaluated on the TKTL1 transfectants. Results: TKTL1 exhibits high frequency of promoter hypomethylation in HNSCC tumors compared with the normal mucosas, correlating with its overexpression in HNSCC. Overexpression of TKTL1 in HNSCC cells promoted cellular proliferation and enhanced tumor growth in vitro and in vivo. Overexpression of TKTL1 increased the production of fructose-6-phosphate and glyceraldehyde-3-phosphate, in turn elevating the production of pyruvate and lactate, resulting in the normoxic stabilization of the malignancy-promoting transcription factor HIF1α and the upregulation of downstream glycolytic enzymes. Notably, the reduction of TKTL1 expression decreased HIF1α accumulation and inhibition with HIF1α and/or the glycolysis inhibitor could abrogate the growth effects mediated by TKTL1 overexpression. Conclusion: TKTL1 is a novel candidate oncogene that is epigenetically activated by aberrant hypomethlation and contributes to a malignant phenotype through altered glycolytic metabolism and HIF1α accumulation. Clin Cancer Res; 16(3); 857–66

Integrated, Genome-Wide Screening for Hypomethylated Oncogenes in Salivary Gland Adenoid Cystic Carcinoma

Purpose: Salivary gland adenoid cystic carcinoma (ACC) is a rare malignancy that is poorly understood. To look for relevant oncogene candidates under the control of promoter methylation, an integrated, genome-wide screen was conducted. Experimental Design: Global demethylation of normal salivary gland cell strains using 5-aza-2′-deoxycytidine (5-aza-dC) and trichostatin A (TSA), followed by expression array analysis was conducted. ACC-specific expression profiling was generated using expression microarray analysis of primary ACC and normal samples. Next, the two profiles were integrated to identify a subset of genes for further validation of promoter demethylation in ACC versus normal. Finally, promising candidates were further validated for mRNA, protein, and promoter methylation levels in larger ACC cohorts. Functional validation was then conducted in cancer cell lines. Results: We found 159 genes that were significantly re-expressed after 5-aza-dC/TSA treatment and overexpressed in ACC. After initial validation, eight candidates showed hypomethylation in ACC: AQP1, CECR1, C1QR1, CTAG2, P53AIP1, TDRD12, BEX1, and DYNLT3. Aquaporin 1 (AQP1) showed the most significant hypomethylation and was further validated. AQP1 hypomethylation in ACC was confirmed with two independent cohorts. Of note, there was significant overexpression of AQP1 in both mRNA and protein in the paraffin-embedded ACC cohort. Furthermore, AQP1 was upregulated in 5-aza-dC/TSA–treated SACC83. Finally, AQP1 promoted cell proliferation and colony formation in SACC83. Conclusions: Our integrated, genome-wide screening method proved to be an effective strategy for detecting novel oncogenes in ACC. AQP1 is a promising oncogene candidate for ACC and is transcriptionally regulated by promoter hypomethylation. Clin Cancer Res; 17(13); 4320–30. ©2011 AACR.

Molecular biology of adenoid cystic carcinoma

Adenoid cystic carcinoma (ACC) is an unusual salivary gland malignancy that remains poorly understood. Standard treatment, including surgery with postoperative radiation therapy, has attained reasonable local control rates, but the propensity for distant metastases has limited any improvement in survival over time. Our understanding of the molecular mechanisms driving ACC is quite rudimentary, due to the infrequent nature of its occurrence.

To “Grow” or “Go”: TMEM16A Expression as a Switch between Tumor Growth and Metastasis in SCCHN

Purpose: Tumor metastasis is the leading cause of death in patients with cancer. However, the mechanisms that underlie metastatic progression remain unclear. We examined TMEM16A (ANO1) expression as a key factor shifting tumors between growth and metastasis. Experimental Design: We evaluated 26 pairs of primary and metastatic lymph node (LN) tissue from patients with squamous cell carcinoma of the head and neck (SCCHN) for differential expression of TMEM16A. In addition, we identified mechanisms by which TMEM16A expression influences tumor cell motility via proteomic screens of cell lines and in vivo mouse studies of metastasis. Results: Compared with primary tumors, TMEM16A expression decreases in metastatic LNs of patients with SCCHN. Stable reduction of TMEM16A expression enhances cell motility and increases metastases while decreasing tumor proliferation in an orthotopic mouse model. Evaluation of human tumor tissues suggests an epigenetic mechanism for decreasing TMEM16A expression through promoter methylation that correlated with a transition between an epithelial and a mesenchymal phenotype. These effects of TMEM16A expression on tumor cell size and epithelial-to-mesenchymal transition (EMT) required the amino acid residue serine 970 (S970); however, mutation of S970 to alanine does not disrupt the proliferative advantages of TMEM16A overexpression. Furthermore, S970 mediates the association of TMEM16A with Radixin, an actin-scaffolding protein implicated in EMT. Conclusions: Together, our results identify TMEM16A, an eight transmembrane domain Ca2+-activated Cl− channel, as a primary driver of the “Grow” or “Go” model for cancer progression, in which TMEM16A expression acts to balance tumor proliferation and metastasis via its promoter methylation. Clin Cancer Res; 20(17); 4673–88. ©2014 AACR.

Value of chest CT in the diagnosis and management of tracheobronchial foreign bodies

Background:  The aim of the present study was to investigate the value of chest multidetector computed tomography (CT) in the evaluation of children with suspected foreign body aspiration.

BORIS Binding to the Promoters of Cancer Testis Antigens, MAGEA2, MAGEA3, and MAGEA4, Is Associated with Their Transcriptional Activation in Lung Cancer

Purpose: Aim of this study was to determine whether BORIS (Brother of the Regulator of Imprinted Sites) is a regulator of MAGEA2, MAGEA3, and MAGEA4 genes in lung cancer. Experimental Design: Changes in expression of MAGEA genes upon BORIS induction/knockdown were studied. Recruitment of BORIS and changes in histone modifications at their promoters upon BORIS induction were analyzed. Luciferase assays were used to study their activation by BORIS. Changes in methylation at these promoters upon BORIS induction were evaluated. Results: Alteration of BORIS expression by induction/knockdown directly correlated with expression of MAGEA genes. BORIS was enriched at their promoters in H1299 cells, which show high expression of these cancer testis antigens (CTA), compared with normal human bronchial epithelial (NHBE) cells which show low expression of the target CTAs. BORIS induction in A549 cells resulted in increased amounts of BORIS and activating histone modifications at their promoters along with a corresponding increase in their expression. Similarly, BORIS binding at these promoters in H1299 correlates with enrichment of activating modifications, whereas absence of BORIS binding in NHBE is associated with enrichment of repressive marks. BORIS induction of MAGEA3 was associated with promoter demethylation, but no methylation changes were noted with activation of MAGEA2 and MAGEA4. Conclusions: These data suggest that BORIS positively regulates these CTAs by binding and inducing a shift to a more open chromatin conformation with promoter demethylation for MAGEA3 or independent of promoter demethylation in case of MAGEA2 and MAGEA4 and may be a key effector involved in their derepression in lung cancer. Clin Cancer Res; 17(13); 4267–76. ©2011 AACR.

Novel Role of MDA-9/Syntenin in Regulating Urothelial Cell Proliferation by Modulating EGFR Signaling

Purpose: Urothelial cell carcinoma (UCC) rapidly progresses from superficial to muscle-invasive tumors. The key molecules involved in metastatic progression and its early detection require clarification. The present study defines a seminal role of the metastasis-associated gene MDA-9/Syntenin in UCC progression. Experimental Design: Expression pattern of MDA-9/Syntenin was examined in 44 primary UCC and the impact of its overexpression and knockdown was examined in multiple cells lines and key findings were validated in primary tumors. Results: Significantly higher (P = 0.002–0.003) expression of MDA-9/Syntenin was observed in 64% (28 of 44) of primary tumors and an association was evident with stage (P = 0.01), grade (P = 0.03), and invasion status (P = 0.02). MDA-9/Syntenin overexpression in nontumorigenic HUC-1 cells increased proliferation (P = 0.0012), invasion (P = 0.0001), and EGF receptor (EGFR), AKT, phosphoinositide 3-kinase (PI3K), and c-Src expression. Alteration of β-catenin, E-cadherin, vimentin, claudin-1, ZO-1, and T-cell factor-4 (TCF4) expression was also observed. MDA-9/Syntenin knockdown in three UCC cell lines reversed phenotypic and molecular changes observed in the HUC-1 cells and reduced in vivo metastasis. Key molecular changes observed in the cell lines were confirmed in primary tumors. A physical interaction and colocalization of MDA-9/Syntenin and EGFR was evident in UCC cell lines and primary tumors. A logistic regression model analysis revealed a significant correlation between MDA-9/Syntenin:EGFR and MDA-9/Syntenin:AKT expressions with stage (P = 0.04, EGFR; P = 0.01, AKT). A correlation between MDA-9/Syntenin:β-catenin coexpression with stage (P = 0.03) and invasion (P = 0.04) was also evident. Conclusions: Our findings indicate that MDA-9/Syntenin might provide an attractive target for developing detection, monitoring, and therapeutic strategies for managing UCC. Clin Cancer Res; 19(17); 4621–33. ©2013 AACR.

Quantitative Methylation Profiles for Multiple Tumor Suppressor Gene Promoters in Salivary Gland Tumors

Background Methylation profiling of tumor suppressor gene (TSGs) promoters is quickly becoming a powerful diagnostic tool for the early detection, prognosis, and even prediction of clinical response to treatment. Few studies address this in salivary gland tumors (SGTs); hence the promoter methylation profile of various TSGs was quantitatively assessed in primary SGT tissue to determine if tumor-specific alterations could be detected. Methodology DNA isolated from 78 tumor and 17 normal parotid gland specimens was assayed for promoter methylation status of 19 TSGs by fluorescence-based, quantitative methylation-specific PCR (qMSP). The data were utilized in a binary fashion as well as quantitatively (using a methylation quotient) allowing for better profiling and interpretation of results. Principal Findings The average number of methylation events across the studied genes was highest in salivary duct carcinoma (SDC), with a methylation value of 9.6, compared to the normal 4.5 (p<0.0003). There was a variable frequency and individual methylation quotient detected, depending on the TSG and the tumor type. When comparing normal, benign, and malignant SGTs, there was a statistically significant trend for increasing methylation in APC, Mint 1, PGP9.5, RAR-β, and Timp3. Conclusions/Significance Screening promoter methylation profiles in SGTs showed considerable heterogeneity. The methylation status of certain markers was surprisingly high in even normal salivary tissue, confirming the need for such controls. Several TSGs were found to be associated with malignant SGTs, especially SDC. Further study is needed to evaluate the potential use of these associations in the detection, prognosis, and therapeutic outcome of these rare tumors.

Suprabasin Is Hypomethylated and Associated with Metastasis in Salivary Adenoid Cystic Carcinoma

Background Salivary gland adenoid cystic carcinoma (ACC) is a rare cancer, accounting for only 1% of all head and neck malignancies. ACC is well known for perineural invasion and distant metastasis, but its underlying molecular mechanisms of carcinogenesis are still unclear. Principal Findings Here, we show that a novel oncogenic candidate, suprabasin (SBSN), plays important roles in maintaining the anchorage-independent and anchorage-dependent cell proliferation in ACC by using SBSN shRNA stably transfected ACC cell line clones. SBSN is also important in maintaining the invasive/metastatic capability in ACC by Matrigel invasion assay. More interestingly, SBSN transcription is significantly upregulated by DNA demethylation induced by 5-aza-2′-deoxycytidine plus trichostatin A treatment and the DNA methylation levels of the SBSN CpG island located in the second intron were validated to be significantly hypomethylated in primary ACC samples versus normal salivary gland tissues. Conclusions/Significance Taken together, these results support SBSN as novel oncogene candidate in ACC, and the methylation changes could be a promising biomarker for ACC.

Expression microarray analysis reveals alternative splicing of LAMA3 and DST genes in head and neck squamous cell carcinoma.

Purpose Prior studies have demonstrated tumor-specific alternative splicing events in various solid tumor types. The role of alternative splicing in the development and progression of head and neck squamous cell carcinoma (HNSCC) is unclear. Our study queried exon-level expression to implicate splice variants in HNSCC tumors. Experimental Design We performed a comparative genome-wide analysis of 44 HNSCC tumors and 25 uvulopalatopharyngoplasty (UPPP) tissue samples at an exon expression level. In our comparison we ranked genes based upon a novel score—the Maximum-Minimum Exon Score (MMES) – designed to predict the likelihood of an alternative splicing event occurring. We validated predicted alternative splicing events using quantitative RT-PCR on an independent cohort. Results After MMES scoring of 17,422 genes, the top 900 genes with the highest scores underwent additional manual inspection of expression patterns in a graphical analysis. The genes LAMA3, DST, VEGFC, SDHA, RASIP1, and TP63 were selected for further validation studies because of a high frequency of alternative splicing suggested in our graphical analysis, and literature review showing their biological relevance and known splicing patterns. We confirmed TP63 as having dominant expression of the short DeltaNp63 isoform in HNSCC tumor samples, consistent with prior reports. Two of the six genes (LAMA3 and DST) validated by quantitative RT-PCR for tumor-specific alternative splicing events (Students t test, P<0.001). Conclusion Alternative splicing events of oncologically relevant proteins occur in HNSCC. The number of genes expressing tumor-specific splice variants needs further elucidation, as does the functional significance of selective isoform expression.