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Human papillomavirus (HPV) type 16 can integrate into the host genome, thereby rendering the viral coding genes susceptible to epigenetic modification. Using bisulfite genomic sequencing, we determined the methylation status of all 110 CpG sites within the viral epigenome in advanced stage III/IV HPV-16–associated head and neck cancers. We found that the viral genome was hypomethylated in the majority of head and neck cancers, in particular within the viral regulatory region, long control region (LCR), which controls transcription of the E6 and E7 oncogenes. The hypomethylation status of LCR correlated with detectable levels of E6 and E7 expression, which suggests that the tumors may still be dependent on these viral oncogenes to maintain the malignant phenotype. In addition to the methylation status of LCR, we report other potential factors which may influence intratumoral E6 and E7 expression including viral copy number and integration site. We were able to detect the viral epigenetic alterations in sampled body fluids, such as serum and saliva, which correlated with the changes observed in the primary tumors. Because viral epigenetic changes occur in the setting of viral integration into the human genome, the detection of methylated HPV genes in the serum and/or saliva may have diagnostic potential for early detection strategies of viral integration and assessment of risk for cancer development in high-risk individuals. Our findings also support continued targeting of the E6 and/or E7 antigens through various vaccine strategies against HPV-associated cancers. Cancer Prev Res; 4(2); 207–17. ©2011 AACR.

Hypermethylation of gene promoters and the corresponding loss of gene expression are recognized as a hallmark of human cancer, and DNA methylation has emerged as a promising biomarker for the detection of human esophageal squamous cell carcinoma (ESCC). To identify novel genes methylated in ESCC, we screened 35 candidate genes identified from an oligonucleotide microarray. Among them, the heat shock protein B2 (HSPB2) was methylated in 95.7% (67/70) of primary ESCCs, whereas no methylation was found in normal esophageal tissues from ESCC patients (0%, 0/20). RT-PCR analysis revealed that HSPB2 expression was silenced or weakly expressed in most ESCC cell lines, and re-activated by the demethylating agent 5-aza-2'-deoxy-cytidine. These results indicate that promoter methylation of HSPB2 is one of the causal factors for loss or down-regulation of HSPB2 expression. mRNA expression of HSPB2 in ESCC tissues was significantly down-regulated compared to normal tissues. Our data suggest that promoter methylation of HSPB2 deserves further attention as a novel molecular biomarker in human ESCC.

In this paper, we present a design of Quasi-Delay-Insensitive Dual-Rail (QDI-DR) asynchronous FIR low-pass filter working in subthreshold region. The filter is implemented in 0.13μm standard CMOS technology and is able to operate with variations of voltages, temperatures and processes. Postlayout simulation shows the design achieves the lowest operating voltage of 170mV with a speed of 2.5kHz. The most energy-efficient point occurs at 250mV with a Power-Delay Product (PDP) of 2.53pJ.

Silencing of tumor suppressor genes plays a vital role in head and neck carcinogenesis. In this study, we aimed to evaluate to the utility of aberrant promoter hypermethylation for detection in a panel of 10 genes (KIF1A, EDNRB, CDH4, TERT, CD44, NISCH, PAK3, VGF, MAL and FKBP4) in head and neck squamous cell carcinoma (HNSCC) via a candidate gene approach. We investigated methylation of the gene promoters by bisulfite modification and quantitative methylation-specific PCR (Q-MSP) in a preliminary study of a limited cohort of salivary rinses from healthy subjects (n = 61) and patients with HNSCC (n = 33). The methylation status of 2 selected genes (EDNRB and KIF1A) were then analyzed in 15 normal mucosa samples from a healthy population, 101 HNSCC tumors and the corresponding salivary rinses from 71 out of the 101 HNSCC patients were collected before treatment. The promoter regions of CDH4, TERT, VGF, MAL, FKBP4, NISCH and PAK3 were methylated in normal salivary rinses while no methylation of CD44 was observed in either normal salivary rinses or tumor samples. However, KIF1A and EDNRB were methylated in 98 and 97% of primary HNSCC tissues respectively and were only methylated in 2 and 6.6% of normal salivary rinses. In addition, KIF1A and EDNRB were methylated in 38 and 67.6% of salivary rinses from HNSCC patients, respectively. Promoter hypermethylation of KIF1A and EDNRB is a frequent event in primary HNSCC, and these genes are preferentially methylated in salivary rinses from HNSCC patients. KIF1A and EDNRB are potential biomarkers for HNSCC detection.

The p53 family (p53, p73, p63) plays an instrumental role in the cellular stress response, including induction of cell cycle arrest and apoptosis in response to DNA damage (1, 2). In addition to p63 and p73 isotypes capable of transactivating downstream target gene expression (TA isotypes), both genes also express dominant negative inhibitory isoforms, such as ΔNp63α. ΔNp63α is degraded in response to DNA damaging agents, thereby enabling an effective cellular response to genotoxic agents. Here, we identify a key molecular mechanism underlying the regulation of ΔNp63α expression in response to extrinsic stimuli, such as chemotherapeutic agents or TNF-α. We show that ΔNp63α interacts with IκB kinase (IKK), a multisubunit protein kinase that consists of two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ (NEMO-NF-κB essential modifier). We find that IKKβ kinase promotes ubiquitin-mediated proteasomal degradation of ΔNp63α, whereas a kinase-deficient mutant IKKβ-K44A fails to do so. Cytokine- or chemotherapy-induced stimulation of IKKβ leads to degradation of ΔNp63α and augments trans-activation of p53 family-induced genes involved in the cellular response to DNA damage. Conversely, inhibition of IKKβ with a NEMO-binding peptide or siRNA-mediated silencing IKKβ expression attenuates cytokine- or chemotherapy induced degradation of ΔNp63α. These data demonstrate that IKKβ plays an essential role in regulating ΔNp63α in response to extrinsic stimuli. Our findings suggest that the activation of IKK may be a mechanism by which levels of ΔNp63α are reduced, thereby rendering the cells susceptible to cell death in the face of cellular stress or DNA damage.

Regulation of DeltaNp63alpha can be achieved at the transcriptional and post-translational levels, with the latter being greatly influenced by external stimuli such as DNA damaging agents. In this present study, we demonstrated that DeltaNp63alpha physical interacts with Yes-associated protein 1 (Yap1). Ectopic expression of Yap1 decreased the stability and the half-like of DeltaNp63alpha and resulted in ubiquitin-dependent degradation of DeltaNp63alpha. Further, the cisplatin mediated degradation of DeltaNp63alpha is attenuated with the downregulation of Yap1. Thus, our results strongly suggest that Yap1 plays a role in the regulation of endogenous DeltaNp63alpha levels and is likely to contribute to the regulation of DeltaNp63alpha, in physiological conditions.

The requirement for controlling lunar lander in the powered descending phase is analyzed in the paper. With suboptimal minimum fuel considered first, the expected non-iterative calculation formulas of the attitude angles are accquired. The explicit guidance project in relevance with the state variables and terminal restrict are deduced. According to deflection between the real attitude angles and the desired attitude angles, the attitude tracking system is designed by adopting the method of exponent reaching law and chattering reducing of boundary layers based on variable structure control with sliding mode. The simulation results show that the control system can complete flying mission in the powered descending phase, and has good robustness and adaptability as well.

DeltaNp63alpha, the dominant negative isoform of the p63 family is an essential survival factor in head and neck squamous cell carcinoma. This isoform has been shown to be down regulated in response to several DNA damaging agents, including cisplatin. But little is understood about the post-translational protein stability of DeltaNp63alpha. In this present study we demonstrate for the first time that DeltaNp63alpha physically interacts with U-box-type E4 ubiquitin ligase UFD2a. UFD2a stabilizes DeltaNp63alpha, and ubiquitylation of DeltaNp63alpha is attenuated by UFD2a both in the presence and absence of cisplatin. Ectopic expression of UFD2a increased the half-life of DeltaNp63alpha in association with a significant enhancement of the repressive transcriptional activity of DeltaNp63alpha. Downregulation of endogenous UFD2a by RNAi resulted in degradation of DeltaNp63alpha. Taken together, our current study provides an insight onto the regulation of DeltaNp63alpha protein levels in response to cisplatin and also suggests that UFD2a might play an important role in the regulation of cisplatin mediated cell death mediated by p63.

2-Methoxyestradiol (2-ME), an endogenous estrogen metabolite of 17beta-estradiol, is known to induce mitochondria-mediated apoptosis through several mechanisms. We sought to study the effect of mitochondrialy targeted hOGG1 (MTS-hOGG1) on HeLa cells exposed to 2-ME. MTS-hOGG1-expressing cells exposed to 2-ME showed increased cellular survival and had significantly less G(2)/M cell cycle arrest compared to vector-only-transfected cells. In addition, 2-ME exposure resulted in an increase in mitochondrial membrane potential, increased apoptosis, accompanied by higher activation of caspase-3, -9, cleavage of Bid to tBid and protein poly(ADP-ribose) polymerase (PARP) cleavage in HeLa cells lacking MTS-hOGG1. Fas inhibitors cerulenin or C75 inhibited 2-ME-induced caspase activation, PARP cleavage, apoptosis and reversed mitochondrial membrane hyperpolarization, thereby recapitulating the increased expression of MTS-hOGG1. Hence, MTS-hOGG1 plays an important protective role against 2-ME-mediated mitochondrial damage by blocking apoptosis induced through the Fas pathway.

To identify novel methylated gene promoters, we compared differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2'-deoxycytidine (5-aza-dC). Out of 1776 genes that were initially 'absent (that is, silenced)' by gene expression array analysis, we selected 163 genes that were increased after 5-aza-dC treatment in at least two of three CRC cell lines. The microarray results were confirmed by Reverse Transcription-PCR, and CpG island of the gene promoters were amplified and sequenced for examination of cancer-specific methylation. Among the genes identified, the deafness, autosomal dominant 5 gene, DFNA5, promoter was found to be methylated in primary tumor tissues with high frequency (65%, 65/100). Quantitative methylation-specific PCR of DFNA5 clearly discriminated primary CRC tissues from normal colon tissues (3%, 3/100). The mRNA expression of DFNA5 in four of five colon cancer tissues was significantly downregulated as compared to normal tissues. Moreover, forced expression of full-length DFNA5 in CRC cell lines markedly decreased the cell growth and colony-forming ability whereas knockdown of DFNA5 increased cell growth in culture. Our data implicate DFNA5 as a novel tumor suppressor gene in CRC and a valuable molecular marker for human cancer.

Purpose: To investigate whether the promoter methylation pattern in N-methyl-d-aspartate receptor 2B (NMDAR2B) is correlated with clinical features of human esophageal squamous cell carcinoma (ESCC), the methylation status of the gene was examined at three different sites (P1, P2, and P3) where two CpG islands reside within 1 kb upstream of the transcription start site. Experimental Design: Three independent modalities for methylation analysis (bisulfite sequencing, combined bisulfite restriction analysis, and TaqMan methylation-specific PCR) were done to analyze total 67 ESCC tissues that included 43 primary tumors with well-characterized clinicopathologic variables including patient outcome. Results: Using an optimized cutoff value based on quantitative methylation-specific PCR, we found that patients with higher NMDAR2B methylation ratio in the proximal region (P1) showed a worse 5-year disease-specific survival rate than those without NMDAR2B methylation (P < 0.006). A significant correlation was also seen between NMDAR2B promoter methylation and the presence of vascular permeation (P = 0.03). Conclusion: NMDAR2B promoter methylation could be a clinically applicable marker in ESCC.

NMDA receptor Type 2B (NMDAR2B) is a candidate TSG first identified in esophageal squamous cell carcinoma (ESCC). To evaluate NMDAR2B methylation in gastric cancer progression, we performed quantitative methylation-specific PCR (MSP), RT-PCR and immnunohistochemistry (IHC) in primary gastric tissues and colony formation assays in gastric cancer cell lines. We found that the expression of NMDAR2B was reactivated by the demethylating agent, 5-aza-2′-deoxycytidine, with or without trichostatin A in gastric cancer cell lines. Moreover, inactivation of NMDAR2B was found to be closely correlated with promoter methylation status in gastric cell lines and primary gastric tumors. IHC data also showed that NMDAR2B was specifically expressed in gastric epithelial cells and its expression was diminished or absent in gastric cancer epithelium. Quantitative analysis of NMDAR2B promoter methylation showed 61% (17/28) hypermethylation in primary gastric tumors versus 5% (1/20) in normal gastric tissues from nongastric cancer patients. Forced over-expression of NMDAR2B in gastric cancer cell lines significantly inhibited cell colony formation. Taken together, the above results suggest that NMDAR2B methylation is a common and important biologically relevant event in gastric cancer progression. © 2007 Wiley-Liss, Inc.

Triple negative breast cancer has an extremely poor prognosis when chemotherapy is no longer effective. To overcome drug resistance, novel drug delivery systems based on nanoparticles have had remarkable success. We produced a novel nanoparticle component 'MDC' from milk-derived colloid. In order to evaluate the anti-cancer effect of MDC, we conducted in vitro and in vivo experiments on cancer cell lines and a primary tumor derived breast xenograft. Doxorubicin (Dox) conjugated to MDC (MDC-Dox) showed higher cancer cell growth inhibition than MDC alone especially in cell lines with high EGFR expression. In a mouse melanoma model, MDC-Dox significantly suppressed tumor growth when compared with free Dox. Moreover, in a primary tumor derived breast xenograft, one of the mice treated with MDC-Dox showed partial regression, while mice treated with free Dox failed to show any suppression of tumor growth. We have shown that a novel nanoparticle compound made of simple milk-derived colloid has the capability for drug conjugation, and serves as a tumor-specific carrier of anti-cancer drugs. Further research on its safety and ability to carry various anti-cancer drugs into multiple drug-resistant primary breast models is warranted.

Adenocarcinoma, the most common subtype of lung cancer, is notorious for eluding early detection. Thus, understanding the early steps of lung tumorigenesis at the genetic level might facilitate the development of more effective strategies for the prevention, early diagnosis, and treatment of lung cancer. Nearly 10% of lungs resected for adenocarcinomas harbored minute discrete foci of cytologically atypical bronchiolalveolar cells that are designated as atypical adenomatous hyperplasia (AAH). It has been postulated that these lesions represent an early stage in glandular neoplasia, which is now viewed as a progression from AAH to adenocarcinoma in situ (AIS) to minimally invasive adenocarcinoma (MIA), although, the biological nature of AAH is poorly understood and very controversial. To delineate tumor clonal heterogeneity as a function of tumor progression in early NSCLC neoplasias, we isolated DNA from different zones of histologic progression within the same lesion and performed targeted next generation sequencing which targets a panel of 134 well-characterized cancer and pharmacogenomics genes. We found that mutational landscape varies significantly between the three groups of patients. The most mutated genes in MIA were EGFR and TP53. 4 out of 5 MIA cases harbored multiple alternations in EGFR catalytic domain and inactivating mutations in TP53 were found in 3 out of 5 patients. Furthermore, mutations in Wnt pathway genes were found in 4 out of 5 cases. Notably, mutations/amplification in Notch family was detected in 3 out of 5 cases (predominantly in the tumor invasive zone). In contrast to MIA, in AIS, only one case out of 5 harbors mutated TP53 or EGFR. Unlike in MIA, only one AIS patient carried mutated APC gene, suggesting that Wnt signaling plays insignificant role in AIS carcinogenesis. On the contrary, we found that AIS carcinogenesis heavily relies on alternations of AKT pathway, since aberrations in KIT, KRAS, HRAS, IGF1R, FGFR3, MET or TSC2 genes were found in 4 out of 5 patients. For AAH cohort we sequenced 25 independent lesions from 6 patients. Sequencing revealed that RAS and AKT/ERK pathways were most frequently affected in these lesions. In 3 cases we were able to track a possible cancer driving mutation from AAH to the primary tumor. This observation provides for the first time a direct link between AAH lesion and primary adenocarcinoma. Most of the patients in all groups carried mutations in genes associated with DNA repair and chromatin remodeling, suggesting that deregulation of the DNA repair machinery is an early onset event of adenocarcinoma tumorigenesis. In all groups our analysis revealed mutations in genes that have been never implicated in lung adenocarcinoma. Our study helps to understand the potential role of heterogeneous clonal events in the progression of early glandular neoplasms including the transition from non-invasive to invasive disease. Citation Format: Eugene G. Izumchenko, Xiaofei Chang, Mariana Brait, William Westra, David Sidransky. Targeted sequencing for the assessment of intratumor heterogeneity. [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 1505. doi:10.1158/1538-7445.AM2014-1505

While sensitizing mutations in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) render tumors more sensitive to EGFR tyrosine kinase inhibitors (TKIs), these genetic alterations are present in only a minority of patients who respond to treatment and are rare in tumors other than NSCLCs. Thus, clinical benefit of TKIs in a subset of patients with wild-type EGFR may be underestimated due to the lack of appropriate predictive markers to stratify them from larger number of patients who do not response. It has long been observed that the response of anti-EGFR agents is associated with epithelial and mesenchymal transition (EMT) in multiple types of tumors without known EGFR mutations. However, little is known about the molecular mechanisms underlying this association, and no EMT-associated biomarkers of clinical utility have been successfully developed. Here we show that during the TGFβ-induced EMT, upregulation of mitogen-inducible gene 6 (Mig6), a negative regulator of EGFR, by microRNAs 200 (miR200) family is leading to reduced EGFR dependence, initiation of kinase switch in mesenchymal cells and subsequent insensitivity of these cells to EGFR inhibition. Moreover, we found that the ratio between mRNA levels of Mig6 and miR200c highly correlated with erlotinib sensitivity and EMT status in panel of 25 cancer cell lines of different tissue origins. Furthermore, analysis of directly xenografted human lung and pancreatic tumors (tumorgrafts) bearing wtEGFR demonstrated a higher response to EGFR inhibitor erlotinib for models with low Mig6 (mRNA)/miR200 ratio. Collectively, our data demonstrates that during the EMT-associated evolution of resistance to EGFR inhibitors, Mig6 expression is highly correlated with EMT status and identifies the ratio of Mig6 mRNA to miR200 as a novel, reliable and cost-effective predictor of biologic and clinical response to TKIs. Citation Format: Eugene G. Izumchenko, Xiaofei Chang, Christina Michailidi, Luciane Kagohara, Rajani Ravi, Mariana Brait, Atul Bedi, David Sidransky. Relative expression of miR200 microRNAs and ERBB family negative regulator Mig6 in estimation of EMT status and erlotinib sensitivity. [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 4382. doi:10.1158/1538-7445.AM2014-4382

Background: Locoregional recurrence occurs in as many as 40% of patients undergoing surgery for head and neck squamous cell carcinoma (HNSCC). We have been focused some molecular alterations exsiting even in histologically negative surgical margins. However, in head and neck cancer resection, surgical margins are hard to evaluate because the most of them usually consists of complicated muscular margins. In order to evaluate these deep surgical margins, before collecting some margin tissues, we often conduct tissue imprinting by using one layer nitrocellulose sheet which can collect proteins without hurting surgical samples. Method: Both 161 deep margin tissues and 166 deep margin imprints were collected from 74 histologically margin negative resections of primary HNSCC from May, 2009 to September, 2012. Bisulfited DNA from samples were amplified using quantitive methylation-specific polymerase chain reaction (QMSP) for 6 genes (DCC, EDNRB, HOXA9, KIF1A, NID2 and NR2B). The cut off value was calculated using QMSP data of tumors and normal muscles, and was applied to deep margin samples. The correlation between the QMSP status of deep margin samples and clinical outcome was evaluated. Results: The average QMSP of tumor tissues, that of normal muscles and the cut off value are 21.0, 0.6, 2.1 in DCC, 23.3, 0.6, 2.2 in EDNRB, 32.0, 0.7, 2.3 in HOXA9, 20.3, 0.4, 1.3 in KIF1A, 36.0, 3.0, 9.4 in NID2 and 10.7, 0.1, 0.4 in NR2B. Those who has the higher QMSP values of deep margin tissues than the cut off value tends to have a poor recurrence free survival (P=0.073 in DCC, P=0.018 in HOXA9, P=0.005 in KIF1A, P=0.011 in NID2) and a poor overall survival (P=0.076 in HOXA9, P=0.059 in NID2). Also, those who has the higher QMSP values of deep margin imprints than the cut off value tends to have a poor recurrence free survival (P=0.051 in HOXA9) and a poor overall survival (P=0.075 in HOXA9). Conclusion: The methylation status of HNSCC deep surgical margin samples is correlated with the local recurrence. Especially, hyper-methylated HOXA9 gene of both deep margin tissues and imprints is a significant biomarker of poor recurrence free survival and overall survival. Citation Format: Masamichi Hayashi, Gaosong Wu, Xiaofei Chang, Xiufeng Li, Julie Ahn, Zubair Khan, Belinda Akpeng, Jong–Lyel Roh, Wayne Koch. Correlation of methylation status of deep surgical margins with the local reccurence of head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 641. doi:10.1158/1538-7445.AM2013-641

Cisplatin is an effective anticancer drug used to treat many types of cancer, including non-small cell lung carcinoma (NSCLCs), but development of resistance is the primary impediment in cancer treatment. Insulin-like growth factor-binding protein 7 (IGFBP7) is a secreted tumor suppressor that is inactivated in human lung cancer. IGFBP7 is known to alter sensitivity to interferon-based anticancer therapy, and here, we examined loss of IGFBP7 as a potential contributor to chemo-resistance to cisplatin. The transcriptional level of IGFBP7 was decreased in cisplatin-resistant human cancer cell lines and NSCLC xenografts. IGFBP7 knock-down increased cellular resistance to cisplatin and increased the level of mitogen-activated protein kinase phosphatases (MKP) 3 levels. The expression of MKP3 increased in a cisplatin-resistant NSCLC cell line and lung xenografts. MKP3 knock-down increased IGFBP7 level, indicating that MKP3 regulates IGFBP7. These findings suggest a novel molecular mechanism responsible for the tumor suppressive function of IGFBP7 in cisplatin-resistant human lung cancer and could lead to the development of IGFBP7 as a cisplatin-sensitizing agent. 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 829. doi:1538-7445.AM2012-829

The human cysteine dioxygenase 1 (CDO1) gene is a non-heme structured, iron-containing metalloenzyme involved in the conversion of cysteine to cysteine sulfinate, and plays a key role in taurine biosynthesis. In our search for novel methylated gene promoters, we have analyzed differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2′-deoxycytidine. Among the genes identified, the CDO1 promoter was found to be differentially methylated in primary CRC tissues with high frequency compared to normal colon tissues. In addition, a statistically significant difference in the frequency of CDO1 promoter methylation was observed between primary normal and tumor tissues derived from breast, esophagus, lung, bladder and stomach. Downregulation of CDO1 mRNA and protein levels were observed in cancer cell lines and tumors derived from these tissue types. Expression of CDO1 was tightly controlled by promoter methylation, suggesting that promoter methylation and silencing of CDO1 may be a common event in human carcinogenesis. Moreover, forced expression of full-length CDO1 in human cancer cells markedly decreased the tumor cell growth in an in vitro cell culture and/or an in vivo mouse model, whereas knockdown of CDO1 increased cell growth in culture. Our data implicate CDO1 as a novel tumor suppressor gene and a potentially valuable molecular marker for human cancer. 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 LB-341. doi:1538-7445.AM2012-LB-341

BACKGROUND and PURPOSE: Salivary gland adenoid cystic carcinoma (ACC) is the second most common malignancy of the salivary glands. It is characterized by a heterogeneous morphology, perineural invasion, and a variable clinical course. The molecular mechanisms underlying ACC are still unknown. We previously developed a high-throughput integrative epigenomic screening method to discover novel proto-oncogene candidates in ACC. With this screening method, we found that cat eye syndrome chromosome region, candidate 1 (CECR1) was a promising oncogene candidate for ACC. CECR1 encodes a member of a subfamily of the adenosine deaminase protein family and acts as a growth factor through its adenosine deaminase activity. No previous studies have correlated CECR1 in human cancers. In the current study, we validated several features of CECR1 in primary ACC samples, including promoter methylation status and expression levels. METHODS and RESULTS: Bisulfite genomic sequencing was first employed to analyze the DNA methylation levels in the promoter of CECR1 in four ACC samples and four normal salivary gland tissues. Decreased methylation in CECR1 was found in three out of four ACC samples compared to zero out of four normal tissues. Secondly, quantitative methylation specific PCR (qMSP) was performed in a larger ACC cohort including 15 ACCs and 18 normal tissues. Consistent with the bisulfite genomic sequencing results, significant demethylation of CECR1 was detected in ACC samples (methylation levels ranged from 0.1 to 300.1; median, 19.8) compared to normal tissues (range from 10.2 to 600.0; median, 235.6), p < 0.05. Thirdly, we induced global demethylation in an ACC cell line, ACC83, by 5-aza-2′-deoxycytidine/ trichostatin A (5 Aza dC/TSA) treatment. Quantitative reverse transcription PCR (qRT-PCR) showed that CECR1 was significantly re-expressed in 5 Aza dC/TSA treated cell lines. Lastly, we analyzed the expression levels of CECR1 by qRT-PCR in the same ACC cohort. We found significant overexpression of CECR1 in ACCs compared to normal salivary tissue, p < 0.05. CONCLUSIONS: This is the first study to associate elevated CECR1 levels with salivary gland ACC. The transcription of CECR1 appears to be upregulated by promoter demethylation. Both overexpression and promoter hypomethylation of CECR1 are hallmarks of ACC. CECR1 might play an important role in salivary gland ACC. FUTURE DIRECTIONS: Functional studies of CECR1's role(s) in the carcinogenesis of salivary gland ACC are underway. 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 4802. doi:10.1158/1538-7445.AM2011-4802

Aberrant methylation of gene promoters and corresponding loss of gene expression plays a critical role in the initiation and progression of colorectal cancer. An IL-6-type cytokine receptor, leukemia inhibitory factor receptor (LIFR), is a component of cell-surface receptor complexes for multifunctional cytokines such as LIF. Herein, we report that LIFR is methylated in human colon cancer. LIFR promoter was methylated in primary tumor tissues with high frequency (65%, 52/80). Quantitative methylation-specific PCR (TaqMan-MSP) demonstrated differential promoter methylation of LIFR in primary colorectal cancer tissues as compared to normal colon tissues (5%, 4/80). LIFR methylation was not detectable in 13 normal colon mucosa samples obtained from patients without cancer. The mRNA expression of LIFR was significantly down-regulated in colon cancer tissues as compared to corresponding normal tissues. A strong expression of LIFR protein was observed in all non-malignant normal and adjacent normal colon mucosa tissues whereas down-regulated LIFR protein expression was observed in primary tumors. These results implicate that cancer-specific methylation and a specific decrease of LIFR expression are a common inactivation event in colon cancer development. 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 4799. doi:10.1158/1538-7445.AM2011-4799

Data from a variety of tissue types and experimental systems indicate that the transcriptional regulator SRY-related HMG box 9 (SOX9) can be induced by injury and promote migration as well as tumor growth. We hypothesize that in cancers associated with chronic injury, such as urothelial carcinoma (UroCa), SOX9 may mediate epithelial injury repair. We found that urothelial SOX9 was induced during times of rapid proliferation during bladder organogenesis and injury repair. High levels of epidermal growth factor (EGF) ligands have been noted in urine and represent a potential trigger for urothelial injury repair programs. Using specific antagonists, we found that EGF receptor activation by cognate ligands heparain binding EGF-like growth factor (HB-EGF) and/or amphiregulin was responsible for Sox9 induction in urothelial cells and tissues. As expected, EGFR activation occurred through the mitogen associated protein kinase (MAPK) pathway. With chronic breaches of urothelial integrity, EGFR-Sox9 stimulation may promote urothelial carcinogenesis, as Sox9 expression was significantly upregulated in invasive human UroCa samples and cell lines. When expressed in established tumors, SOX9 may confer a selective advantage to cancer cells by promoting their ability to invade and colonize new sites. As support for this idea, small interfering RNA inhibition of SOX9 levels abrogated in vitro wound healing and invasion, suggesting a role for SOX9 in tumor spread. These results indicate a potential mechanism for urothelial carcinogenesis through an EGFR-MAPK-SOX9 cascade, and a further function for this axis in established cancers. Future studies will examine the potential mechanisms supporting perseverative SOX9 expression in cancer and opportunities for disrupting this pathway as a therapeutic strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5171.

The p53 family (p53, p73, p63) plays an instrumental role in the cellular stress response, including induction of cell cycle arrest and apoptosis in response to DNA damage. In addition to p63 and p73 isotypes capable of transactivating downstream target gene expression (TA isotypes), both genes also express dominant negative inhibitory isoforms, such as ΔNp63α. ΔNp63 is degraded in response to DNA damaging agents, thereby enabling an effective cellular response to genotoxic agents. Here, we identify a key molecular mechanism underlying the regulation of ΔNp63α expression in response to extrinsic stimuli, such as chemotherapeutic agents or TNF-α. We show that ΔNp63α interacts with IκB kinase (IKK), a multisubunit protein kinase that consists of two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ (NEMO-NF-κB essential modifier). We find that IKK kinase promotes ubiquitin-mediated proteasomal degradation of ΔNp63α, whereas a kinase-deficient mutant IKK -K44A fails to do so. Cytokine- or chemotherapy-induced stimulation of IKKβ leads to degradation of ΔNp63α and augments trans-activation of p53 family-induced genes involved in the cellular response to DNA damage. Conversely, inhibition of IKKβ with a NEMO-binding peptide or siRNA-mediated silencing IKKβ expression attenuates cytokine- or chemotherapy induced degradation of ΔNp63α. These data demonstrate that IKKβ plays an essential role in regulating ΔNp63α in response to extrinsic stimuli. Our findings suggest that the activation of IKK may be a mechanism by which levels of ΔNp63α are reduced, thereby rendering the cells susceptible to cell death in the face of cellular stress or DNA damage. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-25.

We sought to elucidate mechanisms of resistance to epidermal growth factor receptor (EGFR)-targeted therapies and to identify markers that could better distinguish resistant from sensitive patients. Here we show that mitogen-inducible gene 6 (Mig6), a negative regulator of EGFR, was selectively upregulated during the evolution of resistance to the EGFR tyrosine kinase inhibitor (TKI) erlotinib in lung and head and neck cancer cell lines. Mig6 was a major determinant of EGFR activity in panels of cancer cell lines of different tissue origins, and in direct validation of approved human use, in a unique collection of directly xenografted human lung and pancreas tumors. Our work highlights the importance of cellular negative regulators of EGFR in predicting and subverting TKI responsiveness, and identifies the ratio of Mig6 to EGFR, rather than the absolute expression of either protein alone, to be a predictor of tumor cell response to erlotinib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2703.

Aerobic glycolysis and mitochondrial dysfunction are common features of aggressive cancer growth. We observed promoter methylation and loss of expression in neurofilament heavy polypeptide (NEFH) in a significant proportion of primary esophageal squamous cell carcinoma (ESCC) samples that were of a high tumor grade and advanced stage. RNA interference-mediated knockdown of NEFH accelerated ESCC cell growth in culture and increased tumorigenicity in vivo, whereas forced expression of NEFH significantly inhibited cell growth and colony formation. Loss of NEFH caused up-regulation of pyruvate kinase-M2 type and down-regulation of pyruvate dehydrogenase, via activation of the Akt/β-catenin pathway, resulting in enhanced aerobic glycolysis and mitochondrial dysfunction. The acceleration of glycolysis and mitochondrial dysfunction in NEFH-knockdown cells was suppressed in the absence of β-catenin expression, and was decreased by the treatment of 2-Deoxyglucose, a glycolytic inhibitor, or API-2, an Akt inhibitor. Loss of NEFH activates the Akt/β-catenin pathway and increases glycolysis and mitochondrial dysfunction. Cancer cells with methylated NEFH can be targeted for destruction with specific inhibitors of deregulated downstream pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5067.

Aerobic glycolysis and mitochondrial dysfunction are common features of aggressive cancer growth. We observed promoter methylation and loss of expression in neurofilament heavy polypeptide (NEFH) in a significant proportion of primary esophageal squamous cell carcinoma (ESCC) samples that were of a high tumor grade and advanced stage. RNA interference-mediated knockdown of NEFH accelerated ESCC cell growth in culture and increased tumorigenicity in vivo, whereas forced expression of NEFH significantly inhibited cell growth and colony formation. Loss of NEFH caused up-regulation of pyruvate kinase-M2 type and down-regulation of pyruvate dehydrogenase, via activation of the Akt/beta-catenin pathway, resulting in enhanced aerobic glycolysis and mitochondrial dysfunction. The acceleration of glycolysis and mitochondrial dysfunction in NEFH-knockdown cells was suppressed in the absence of beta-catenin expression, and was decreased by the treatment of 2-Deoxyglucose, a glycolytic inhibitor, or API-2, an Akt inhibitor. Loss of NEFH activates the Akt/beta-catenin pathway and increases glycolysis and mitochondrial dysfunction. Cancer cells with methylated NEFH can be targeted for destruction with specific inhibitors of deregulated downstream pathways.