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1.  Novel mechanism of MDA-7/IL-24 cancer-specific apoptosis through SARI induction 
Cancer research  2013;74(2):563-574.
Subtraction-hybridization combined with induction of cancer cell terminal differentiation in human melanoma cells identified melanoma differentiation associated gene-7 (mda-7/IL-24) and SARI (Suppressor of AP-1, induced by IFN) that display potent antitumor activity. These genes are not constitutively expressed in cancer cells and forced expression of mda-7/IL-24 (Ad.mda-7) or SARI(Ad.SARI) promotes cancer-specific cell death. Ectopic expression of mda-7/IL-24 induces SARI mRNA and protein in a panel of different cancer cells leading to cell death, without harming corresponding normal cells. Simultaneous inhibition of K-ras downstream extracellular regulated kinase 1/2 (ERK1/2) signaling in pancreatic cancer cells reverses the translational block of MDA-7/IL-24 and induces SARI expression and cell death. Using SARI-antisense-based approaches we demonstrate that SARI expression is necessary for mda-7/IL-24 antitumor effects. Secreted MDA-7/IL-24 protein induces antitumor ‘bystander’ effects by promoting its own expression. Recombinant MDA-7/IL-24 (His-MDA-7) induces SARI expression, supporting the involvement of SARI in the MDA-7/IL-24-driven autocrine loop culminating in antitumor effects. Moreover, His-MDA-7 after binding to its cognate receptors (IL-20R1/IL-20R2 or IL-22R/IL-20R2) induces intracellular signaling by phosphorylation of p38 MAPK leading to transcription of a family of growth arrest and DNA damage inducible (GADD) genes, culminating in apoptosis. Inhibition of p38 MAPK fails to induce SARI following Ad.mda-7 infection. These findings reveal the significance of the mda-7/IL-24-SARI axis in cancer-specific killing, and provide a potential strategy for treating both local and metastatic disease.
doi:10.1158/0008-5472.CAN-13-1062
PMCID: PMC3915776  PMID: 24282278
SARI; MDA-7/IL-24; apoptosis; IL-20/IL-22 receptors
2.  MDA-9/syntenin is a key regulator of glioma pathogenesis 
Neuro-Oncology  2013;16(1):50-61.
Background
The extraordinary invasiveness of human glioblastoma multiforme (GBM) contributes to treatment failure and the grim prognosis of patients diagnosed with this tumor. Consequently, it is imperative to define further the cellular mechanisms that control GBM invasion and identify promising novel therapeutic targets. Melanoma differentiation associated gene–9 (MDA-9/syntenin) is a highly conserved PDZ domain–containing scaffolding protein that promotes invasion and metastasis in vitro and in vivo in human melanoma models. To determine whether MDA-9/syntenin is a relevant target in GBM, we investigated its expression in tumor samples and involvement in GBM invasion and angiogenesis.
Materials
We assessed MDA-9/syntenin levels in available databases, patient tumor samples, and human-derived cell lines. Through gain-of-function and loss-of-function studies, we analyzed changes in invasion, angiogenesis, and signaling in vitro. We used orthotopic xenografts with GBM6 cells to demonstrate the role of MDA-9/syntenin in GBM pathogenesis in vivo.
Results
MDA-9/syntenin expression in high-grade astrocytomas is significantly higher than normal tissue counterparts. Forced overexpression of MDA-9/syntenin enhanced Matrigel invasion, while knockdown inhibited invasion, migration, and anchorage-independent growth in soft agar. Moreover, overexpression of MDA-9/syntenin increased activation of c-Src, p38 mitogen-activated protein kinase, and nuclear factor kappa-B, leading to elevated expression of matrix metalloproteinase 2 and secretion of interleukin-8 with corresponding changes observed upon knockdown. GBM6 cells that stably express small hairpin RNA for MDA-9/syntenin formed smaller tumors and had a less invasive phenotype in vivo.
Conclusions
Our findings indicate that MDA-9/syntenin is a novel and important mediator of invasion in GBM and a key regulator of pathogenesis, and we identify it as a potential target for anti-invasive treatment in human astrocytoma.
doi:10.1093/neuonc/not157
PMCID: PMC3870820  PMID: 24305713
MDA-9/syntenin; GBM; glioma; invasion; intracranial injection
3.  MDA-9/Syntenin regulates differentiation and angiogenesis programs in head and neck squamous cell carcinoma 
Oncoscience  2014;1(11):725-737.
Little is known about the molecular pathways regulating poor differentiation and invasion of head and neck squamous cell carcinoma (HNSCC). In the present study, we aimed to determine the role of MDA-9/Syntenin, a metastasis associated molecule in HNSCC tumorigenesis. Elevated MDA-9/Syntenin expression was evident in 67% (54/81) primary HNSCC tumors (p=0.001-0.002) and 69% (9/13) pre-neoplastic tissues (p=0.02-0.03). MDA-9/Syntenin overexpression was associated with the stage (p=0.001), grade (p=0.001) and lymph node metastasis (p=0.0001). Silencing of MDA-9/Syntenin in 3 poorly differentiated HNSCC cell lines induced squamous epithelial cell differentiation, disrupted angiogenesis and reduced tumor growth in vitro and in vivo. We confirmed SPRR1B and VEGFR1 as the key molecular targets of MDA-9/Syntenin on influencing HNSCC differentiation and angiogenesis respectively. MDA-9/Syntenin disrupted SPRR1B expression interacting through its PDZ1 domain and altered VEGFR1 expression in vitro and in vivo. VEGFR1 co-localized with MDA-9/Syntenin in HNSCC cell lines and primary tumor. Downregulation of growth regulatory molecules CyclinD1, CDK4, STAT3, PI3K and CTNNB1 was also evident in the MDA-9/Syntenin depleted cells, which was reversed following over-expression of MDA-9/Syntenin in immortalized oral epithelial cells. Our results suggest that early induction of MDA-9/Syntenin expression influences HNSCC progression and should be further evaluated for potential biomarker development.
PMCID: PMC4278274  PMID: 25593999
Head and neck suqamous cell carcinoma; MDA-9/Syntenin; differentiation; SPRR1B; VEGFR1
4.  Intracellular Locations of Replication Proteins and the Origin of Replication during Chromosome Duplication in the Slowly Growing Human Pathogen Helicobacter pylori 
Journal of Bacteriology  2014;196(5):999-1011.
We followed the position of the replication complex in the pathogenic bacterium Helicobacter pylori using antibodies raised against the single-stranded DNA binding protein (HpSSB) and the replicative helicase (HpDnaB). The position of the replication origin, oriC, was also localized in growing cells by fluorescence in situ hybridization (FISH) with fluorescence-labeled DNA sequences adjacent to the origin. The replisome assembled at oriC near one of the cell poles, and the two forks moved together toward the cell center as replication progressed in the growing cell. Termination and resolution of the forks occurred near midcell, on one side of the septal membrane. The duplicated copies of oriC did not separate until late in elongation, when the daughter chromosomes segregated into bilobed nucleoids, suggesting sister chromatid cohesion at or near the oriC region. Components of the replication machinery, viz., HpDnaB and HpDnaG (DNA primase), were found associated with the cell membrane. A model for the assembly and location of the H. pylori replication machinery during chromosomal duplication is presented.
doi:10.1128/JB.01198-13
PMCID: PMC3957694  PMID: 24363345
5.  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 knock down 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% (28/44) 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 non-tumorigenic HUC-1 cells increased proliferation (p=0.0012), invasion (p=0.0001) and EGFR, AKT, PI3K and c-Src expression. Alteration of Beta-catenin, E-Cadherin, Vimentin, Claudin-1, ZO-1 and TCF4 expression were also observed. MDA-9/Syntenin knock down in 3 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 co-localization 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 co-expression 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.
doi:10.1158/1078-0432.CCR-13-0585
PMCID: PMC3872137  PMID: 23873690
Urothelial cancer; MDA-9/Syntenin; invasion; EGFR signaling
6.  Expression patterns of MDA-9/syntenin during development of the mouse embryo 
Journal of molecular histology  2012;44(2):159-166.
MDA-9 (melanoma differentiation associated gene-9)/Syntenin is a PDZ domain-containing adaptor protein involved in multiple diverse cellular processes including organization of protein complexes in the plasma membrane, intracellular trafficking and cell surface targeting, synaptic transmission, and cancer metastasis. In the present study, we analyzed the expression pattern of MDA-9/syntenin during mouse development. MDA-9/syntenin was robustly expressed with tight regulation of its temporal and spatial expression during fetal development in the developing skin, spinal cord, heart, lung and liver, which are regulated by multiple signaling pathways in the process of organogenesis. Recent studies also indicate that MDA-9/syntenin is involved in the signaling pathways crucial during development such as Wnt, Notch and FGF. Taken together, these results suggest that MDA-9/syntenin may play a prominent role during normal mouse development in the context of cell proliferation as well as differentiation through modulating multiple signaling pathways as a crucial adaptor protein. Additionally, temporal regulation of MDA-9/syntenin expression may be required during specific stages and in specific tissues during development.
doi:10.1007/s10735-012-9468-1
PMCID: PMC3605205  PMID: 23180153
MDA-9/syntenin; development; mouse embryo; adaptor protein
7.  Targeting the Bcl-2 Family for Cancer Therapy 
Introduction
Programmed cell death is well-orchestrated process regulated by multiple pro-apoptotic and anti-apoptotic genes, particularly those of the Bcl-2 gene family. These genes are well documented in cancer with aberrant expression being strongly associated with resistance to chemotherapy and radiation.
Areas covered
This review focuses on the resistance induced by the Bcl-2 family of anti-apoptotic proteins and current therapeutic interventions currently in preclinical or clinical trials that target this pathway. Major resistance mechanisms that are regulated by Bcl-2 family proteins and potential strategies to circumvent resistance are also examined. Although antisense and gene therapy strategies are used to nullify Bcl-2 family proteins, recent approaches use small molecule inhibitors and peptides. Structural similarity of the Bcl-2 family of proteins greatly favors development of inhibitors that target the BH3 domain, called BH3 mimetics.
Expert opinion
Strategies to specifically identify and inhibit critical determinants that promote therapy-resistance and tumor progression represent viable approaches for developing effective cancer therapies. From a clinical perspective, pretreatment with novel, potent Bcl-2 inhibitors either alone or in combination with conventional therapies hold significant promise for providing beneficial clinical outcomes. Identifying small molecule inhibitors with broader and higher affinities for inhibiting all of the Bcl-2 pro-survival proteins will facilitate development of superior cancer therapies.
doi:10.1517/14728222.2013.733001
PMCID: PMC3955095  PMID: 23173842
BH3 domain; apoptosis; Mcl-1; radiation resistance; chemotherapy resistance
8.  AEG-1/MTDH/LYRIC: Signaling Pathways, Downstream Genes, Interacting Proteins, and Regulation of Tumor Angiogenesis 
Advances in cancer research  2013;120:75-111.
Astrocyte elevated gene-1 (AEG-1), also known as metadherin (MTDH) and lysine-rich CEACAM1 coisolated (LYRIC), was initially cloned in 2002. AEG-1/MTDH/LYRIC has emerged as an important oncogene that is overexpressed in multiple types of human cancer. Expanded research on AEG-1/MTDH/LYRIC has established a functional role of this molecule in several crucial aspects of tumor progression, including transformation, proliferation, cell survival, evasion of apoptosis, migration and invasion, metastasis, angiogenesis, and chemoresistance. The multifunctional role of AEG-1/MTDH/LYRIC in tumor development and progression is associated with a number of signaling cascades, and recent studies identified several important interacting partners of AEG-1/MTDH/LYRIC in regulating cancer promotion and other biological functions. This review evaluates the current literature on AEG-1/MTDH/LYRIC function relative to signaling changes, interacting partners, and angiogenesis and highlights new perspectives of this molecule, indicating its potential as a significant target for the clinical treatment of various cancers and other diseases.
doi:10.1016/B978-0-12-401676-7.00003-6
PMCID: PMC3928810  PMID: 23889988
9.  MDA-9/Syntenin and IGFBP-2 Promote Angiogenesis in Human Melanoma 
Cancer research  2012;73(2):844-854.
Melanoma differentiation associated gene-9 (mda-9/syntenin) encodes an adapter scaffold protein whose expression correlates with and mediates melanoma progression and metastasis. Tumor angiogenesis represents an integral component of cancer metastasis prompting us to investigate a possible role of mda-9/syntenin in inducing angiogenesis. Genetic (gain-of-function and loss-of-function) and pharmacological approaches were employed to modify mda-9/syntenin expression in normal immortal melanocytes, early radial growth phase melanoma and metastatic melanoma cells. The consequence of modifying mda-9/syntenin expression on angiogenesis was evaluated using both in vitro and in vivo assays, including tube formation assays using human vascular endothelial cells, CAM assays and xenograft tumor animal models. Gain-of-function and loss-of-function experiments confirm that MDA-9/syntenin induces angiogenesis by augmenting expression of several pro-angiogenic factors/genes. Experimental evidence is provided for a model of angiogenesis induction by MDA-9/syntenin in which MDA-9/syntenin interacts with the ECM activating Src and FAK resulting in activation by phosphorylation of Akt, which induces HIF-1α. The HIF-1α activates transcription of Insulin Growth Factor Binding Protein-2 (IGFBP-2), which is secreted thereby promoting angiogenesis and further induces endothelial cells to produce and secrete VEGF-A augmenting tumor angiogenesis. Our studies delineate an unanticipated cell non-autonomous function of MDA-9/syntenin in the context of angiogenesis, which may directly contribute to its metastasis-promoting properties. As a result, targeting MDA-9/syntenin or its downstream-regulated molecules may provide a means of simultaneously impeding metastasis by both directly inhibiting tumor cell transformed properties (autonomous) and indirectly by blocking angiogenesis (non-autonomous).
doi:10.1158/0008-5472.CAN-12-1681
PMCID: PMC3548987  PMID: 23233738
mda-9/syntenin; melanoma; angiogenesis; IGFBP-2; HuVECs; CAM assay
10.  Draft Genome Sequence of Saccharopolyspora rectivirgula 
Genome Announcements  2014;2(1):e01117-13.
We have sequenced the genome of Saccharopolyspora rectivirgula, the causative agent of farmer’s lung disease. The draft genome consists of 182 contigs totaling 3,977,051 bp, with a GC content of 68.9%.
doi:10.1128/genomeA.01117-13
PMCID: PMC3886947  PMID: 24407634
11.  Identification of Genes Potentially Regulated by Human Polynucleotide Phosphorylase (hPNPaseold-35) Using Melanoma as a Model 
PLoS ONE  2013;8(10):e76284.
Human Polynucleotide Phosphorylase (hPNPaseold-35 or PNPT1) is an evolutionarily conserved 3′→5′ exoribonuclease implicated in the regulation of numerous physiological processes including maintenance of mitochondrial homeostasis, mtRNA import and aging-associated inflammation. From an RNase perspective, little is known about the RNA or miRNA species it targets for degradation or whose expression it regulates; except for c-myc and miR-221. To further elucidate the functional implications of hPNPaseold-35 in cellular physiology, we knocked-down and overexpressed hPNPaseold-35 in human melanoma cells and performed gene expression analyses to identify differentially expressed transcripts. Ingenuity Pathway Analysis indicated that knockdown of hPNPaseold-35 resulted in significant gene expression changes associated with mitochondrial dysfunction and cholesterol biosynthesis; whereas overexpression of hPNPaseold-35 caused global changes in cell-cycle related functions. Additionally, comparative gene expression analyses between our hPNPaseold-35 knockdown and overexpression datasets allowed us to identify 77 potential “direct” and 61 potential “indirect” targets of hPNPaseold-35 which formed correlated networks enriched for cell-cycle and wound healing functional association, respectively. These results provide a comprehensive database of genes responsive to hPNPaseold-35 expression levels; along with the identification new potential candidate genes offering fresh insight into cellular pathways regulated by PNPT1 and which may be used in the future for possible therapeutic intervention in mitochondrial- or inflammation-associated disease phenotypes.
doi:10.1371/journal.pone.0076284
PMCID: PMC3797080  PMID: 24143183
12.  Selected Approaches for Rational Drug Design and High Throughput Screening to Identify Anti-Cancer Molecules 
Structure-based modeling combined with rational drug design, and high throughput screening approaches offer significant potential for identifying and developing lead compounds with therapeutic potential. The present review focuses on these two approaches using explicit examples based on specific derivatives of Gossypol generated through rational design and applications of a cancer-specific-promoter derived from Progression Elevated Gene-3. The Gossypol derivative Sabutoclax (BI-97C1) displays potent anti-tumor activity against a diverse spectrum of human tumors. The model of the docked structure of Gossypol bound to Bcl-XL provided a virtual structure-activity-relationship where appropriate modifications were predicted on a rational basis. These structure-based studies led to the isolation of Sabutoclax, an optically pure isomer of Apogossypol displaying superior efficacy and reduced toxicity. These studies illustrate the power of combining structure-based modeling with rational design to predict appropriate derivatives of lead compounds to be empirically tested and evaluated for bioactivity. Another approach to cancer drug discovery utilizes a cancer-specific promoter as readouts of the transformed state. The promoter region of Progression Elevated Gene-3 is such a promoter with cancer-specific activity. The specificity of this promoter has been exploited as a means of constructing cancer terminator viruses that selectively kill cancer cells and as a systemic imaging modality that specifically visualizes in vivo cancer growth with no background from normal tissues. Screening of small molecule inhibitors that suppress the Progression Elevated Gene-3-promoter may provide relevant lead compounds for cancer therapy that can be combined with further structure-based approaches leading to the development of novel compounds for cancer therapy.
PMCID: PMC3763986  PMID: 22931411
Progression Elevated Gene-3; Sabutoclax; Apogossypol; BI-97C1; Gossypol; AP-1; PEA3; ETV4; E1AF; c-fos; c-jun; Cancer Terminator Virus
13.  Detection of Mitochondrial DNA Alterations in Urine from Urothelial Cell Carcinoma Patients 
The present study aims at understanding the timing and nature of mitochondrial DNA (mtDNA) alterations in urothelial cell carcinoma (UCC) and their detection in urine sediments. The entire 16.5 kb mitochondrial genome was sequenced in matched normal lymphocytes, tumor and urine sediments from 31 UCC patients and compared with different clinical stages and histological grades. The mtDNA content index was examined in all the specimens. Sixty five percent (20/31) of the patients harbored at least 1 somatic mtDNA mutation. A total of 25 somatic mtDNA mutations were detected, which were more frequent in the respiratory complex coding regions (Complex-I, III, IV and V) of the mtDNA and significantly affected respiratory complex-III compared to the other complexes (P=0.021–0.039). Compared to stage Ta, mtDNA mutation was higher in stage T1 and significantly higher in stage T2 (P=0.01) patients. MtDNA mutation was also significantly higher (P=0.04) in stage T2 compared to stage T1 patients. Ninety percent (18/20) of the patients harboring mtDNA mutation in the tumor also had mutation in their urine sediments. Eighty percent (20/25) of the tumor-associated mtDNA mutations was detectable in the urine sediments. Compared to the normal lymphocytes, the mtDNA content increased significantly in the tumor (P=0.0013) and corresponding urine sediments (P=0.0025) in 19/25 (76%) patients analyzed. Our results indicate that mtDNA alterations occur frequently in progressive stages of UCC patients and are readily detectable in the urine sediments. MtDNA mutations appear to provide a promising tool for developing early detection and monitoring strategies for UCC patients.
doi:10.1002/ijc.26357
PMCID: PMC3328657  PMID: 21826645
Urothelial cell carcinoma; mitochondria; mtDNA alteration; urine detection
14.  SH3GL2 is frequently deleted in non-small cell lung cancer and downregulates tumor growth by modulating EGFR signaling 
The purpose of this study was to identify key genetic pathways involved in non-small cell lung cancer (NSCLC) and understand their role in tumor progression. We performed a genome wide scanning using paired tumors and corresponding 16 mucosal biopsies from four follow-up lung cancer patients on Affymetrix 250K-NSpI array platform. We found that a single gene SH3GL2 located on human chromosome 9p22 was most frequently deleted in all the tumors and corresponding mucosal biopsies. We further validated the alteration pattern of SH3GL2 in a substantial number of primary NSCLC tumors at DNA and protein level. We also overexpressed wild-type SH3GL2 in three NSCLC cell lines to understand its role in NSCLC progression. Validation in 116 primary NSCLC tumors confirmed frequent loss of heterozygosity of SH3GL2 in overall 51 % (49/97) of the informative cases. We found significantly low (p=0.0015) SH3GL2 protein expression in 71 % (43/60) primary tumors. Forced over-expression of wild-type (wt) SH3GL2 in three NSCLC cell lines resulted in a marked reduction of active epidermal growth factor receptor (EGFR) expression and an increase in EGFR internalization and degradation. Significantly decreased in vitro (p=0.0015–0.030) and in vivo (p=0.016) cellular growth, invasion (p=0.029–0.049), and colony formation (p=0.023–0.039) were also evident in the wt-SH3GL2-transfected cells accompanied by markedly low expression of activated AKT(Ser473), STAT3 (Tyr705), and PI3K. Downregulation of SH3GL2 interactor USP9X and activated β-catenin was also evident in the SH3GL2-transfected cells. Our results indicate that SH3GL2 is frequently deleted in NSCLC and regulates cellular growth and invasion by modulating EGFR function.
doi:10.1007/s00109-012-0955-3
PMCID: PMC3691869  PMID: 22968441
Single nucleotide polymorphism array; Lung cancer; SH3GL2; Deletion
15.  Mitochondrial DNA Mutations in Respiratory Complex-I in Never-Smoker Lung Cancer Patients Contribute to Lung Cancer Progression and associated with EGFR gene mutation 
Journal of cellular physiology  2012;227(6):2451-2460.
Mitochondrial DNA (mtDNA) mutations were reported in different cancers. However, the nature and role of mtDNA mutation in never-smoker lung cancer patients including patients with EGFR and KRAS gene mutation are unknown. In the present study, we sequenced entire mitochondrial genome (16.5 kb) in matched normal and tumors obtained from 30 never-smoker and 30 current-smoker lung cancer patients, and determined the mtDNA content. All the patients’ samples were sequenced for KRAS (exon 2) and EGFR (exon 19 and 21) gene mutation. The impact of forced overexpression of a respiratory complex-I gene mutation was evaluated in a lung cancer cell line. We observed significantly higher (P=0.006) mtDNA mutation in the never-smokers compared to the current-smoker lung cancer patients. MtDNA mutation was significantly higher (P=0.026) in the never-smoker Asian compared to the current-smoker Caucasian patients’ population. MtDNA mutation was significantly (P=0.007) associated with EGFR gene mutation in the never-smoker patients. We also observed a significant increase (P=0.037) in mtDNA content among the never-smoker lung cancer patients. The majority of the coding mtDNA mutations targeted respiratory complex-I and forced overexpression of one of these mutations resulted in increased in vitro proliferation, invasion and superoxide production in lung cancer cells. We observed a higher prevalence and new relationship between mtDNA alterations among never-smoker lung cancer patients and EGFR gene mutation. Moreover, a representative mutation produced strong growth effects after forced overexpression in lung cancer cells. Signature mtDNA mutations provide a basis to develop novel biomarkers and therapeutic strategies for never-smoker lung cancer patients.
doi:10.1002/jcp.22980
PMCID: PMC3256258  PMID: 21830212
Lung cancer; never-smokers; MtDNA mutation; Respiratory Complex-I; EGFR mutation
16.  Identification of guanine nucleotide-binding protein γ-7 as an epigenetically silenced gene in head and neck cancer by gene expression profiling 
International Journal of Oncology  2013;42(4):1427-1436.
Silencing of tumor suppressor genes plays a vital role in head and neck carcinogenesis. Aberrant hypermethylation in the promoter region of some known or putative tumor suppressor genes occurs frequently during the development of various types of cancer including head and neck squamous cell carcinoma (HNSCC). In this study we used an expanded mRNA expression profiling approach followed by microarray expression analysis to identify epigenetically inactivated genes in HNSCC. Two HNSCC cell lines were treated with 5-aza-2′-deoxycytidine followed by microarray analysis to identify epigenetically silenced genes in HNSCC. We found 1,960, 614 and 427 genes were upregulated in the HNSCC cell lines JHU-012, JHU-011 and the combination of both cell lines, respectively. HNSCC tumor and normal mucosal samples were used for gene profiling by a 47K mRNA gene expression array and we found 7,140 genes were downregulated in HNSCC tumors compared to normal mucosa, as determined by microarray analysis, and were integrated with cell line data. Integrative analysis defined 126 candidate genes, of which only seven genes showed differential methylation in tumors and no methylation in normal mucosa after bisulfite sequencing. Following validation by QMSP, one gene, guanine nucleotide-binding protein γ-7 (GNG7), was confirmed to be highly methylated in tumors and unmethylated in normal mucosal and salivary rinse samples demonstrating cancer-specific methylation in HNSCC tissues. TXNIP and TUSC2 were partially methylated in tumors and normal salivary rinses but unmethylated in normal mucosa. We concluded that GNG7 is a highly specific promoter methylated gene associated with HNSCC. In addition, TXNIP and TUSC2 are also potential biomarkers for HNSCC.
doi:10.3892/ijo.2013.1808
PMCID: PMC3981008  PMID: 23403885
guanine nucleotide-binding protein γ-7; gene expression; silencing; head and neck squamous cell carcinoma; epigenetics
17.  Emerging Strategies for the Early Detection and Prevention of Head and Neck Squamous Cell Cancer 
Journal of cellular physiology  2012;227(2):467-473.
Despite significant improvements in therapeutic protocols, Head and Neck Squamous Cell Carcinoma (HNSCC) remains a major health problem worldwide. The 5-year post therapeutic survival rate is among the lowest of the major cancers with loco-regional relapse being the main cause of death. Moreover, in most instances, the quality of life of the afflicted patient is severely compromised. The poor prognosis for HNSCC is primarily due to disease detection at advanced stages. Accordingly, development of early detection and preventive strategies are essential. Recent advances in our understanding of the molecular biology and etiology of HNSCC should facilitate development of improved intervention and therapeutic approaches. The present review discusses the potential role of such factors for developing preventive and early diagnostic strategies for HNSCC management.
doi:10.1002/jcp.22767
PMCID: PMC3158829  PMID: 21465466
18.  Role of Excitatory Amino Acid Transporter-2 (EAAT2) and Glutamate in Neurodegeneration: Opportunities for Developing Novel Therapeutics 
Journal of Cellular Physiology  2011;226(10):2484-2493.
Glutamate is an essential excitatory neurotransmitter regulating brain functions. Excitatory amino acid transporter (EAAT)-2 is one of the major glutamate transporters expressed predominantly in astroglial cells and is responsible for 90% of total glutamate uptake. Glutamate transporters tightly regulate glutamate concentration in the synaptic cleft. Dysfunction of EAAT2 and accumulation of excessive extracellular glutamate has been implicated in the development of several neurodegenerative diseases including Alzheimer’s disease, Huntington’s disease, and amyotrophic lateral sclerosis. Analysis of the 2.5-kb human EAAT2 promoter showed that NF-κB is an important regulator of EAAT2 expression in astrocytes. Screening of approximately 1,040 FDA-approved compounds and nutritionals led to the discovery that many β-lactam antibiotics are transcriptional activators of EAAT2 resulting in increased EAAT2 protein levels. Treatment of animals with ceftriaxone (CEF), a β-lactam antibiotic, led to an increase of EAAT2 expression and glutamate transport activity in the brain. CEF has neuroprotective effects in both in vitro and in vivo models based on its ability to inhibit neuronal cell death by preventing glutamate excitotoxicity. CEF increases EAAT2 transcription in primary human fetal astrocytes (PHFA) through the NF-κB signaling pathway. The NF-κB binding site at −272 position was critical in CEF-mediated EAAT2 protein induction. These studies emphasize the importance of transcriptional regulation in controlling glutamate levels in the brain. They also emphasize the potential utility of the EAAT2 promoter for developing both low and high throughput screening assays to identify novel small molecule regulators of glutamate transport with potential to ameliorate pathological changes occurring during and causing neurodegeneration.
doi:10.1002/jcp.22609
PMCID: PMC3130100  PMID: 21792905
19.  Targeting Mcl-1 for the therapy of cancer 
Introduction
Human cancers are genetically and epigenetically heterogeneous and have the capacity to commandeer a variety of cellular processes to aid in their survival, growth and resistance to therapy. One strategy is to overexpress proteins that suppress apoptosis, such as the Bcl-2 family protein Mcl-1. The Mcl-1 protein plays a pivotal role in protecting cells from apoptosis and is overexpressed in a variety of human cancers.
Areas covered
Targeting Mcl-1 for extinction in these cancers, using genetic and pharmacological approaches, represents a potentially effectual means of developing new efficacious cancer therapeutics. Here we review the multiple strategies that have been employed in targeting this fundamental protein, as well as the significant potential these targeting agents provide in not only suppressing cancer growth, but also in reversing resistance to conventional cancer treatments.
Expert Opinion
We discuss the potential issues that arise in targeting Mcl-1 and other Bcl-2 anti-apoptotic proteins, as well problems with acquired resistance. The application of combinatorial approaches that involve inhibiting Mcl-1 and manipulation of additional signaling pathways to enhance therapeutic outcomes is also highlighted. The ability to specifically inhibit key genetic/epigenetic elements and biochemical pathways that maintain the tumor state represent a viable approach for developing rationally based, effective cancer therapies.
doi:10.1517/13543784.2011.609167
PMCID: PMC3205956  PMID: 21851287
20.  An epigenetic marker panel for detection of lung cancer using cell-free serum DNA 
PURPOSE
We investigated the feasibility of detecting aberrant DNA methylation of some novel and known genes in the serum of lung cancer patients.
EXPERIMENTAL DESIGN
To determine the analytical sensitivity, we examined the tumor and the matched serum DNA for aberrant methylation of fifteen gene promoters from 10 patients with primary lung tumors by using Quantitative methylation specific PCR. We then tested this 15 gene set to identify the more useful DNA methylation changes in the serum of a limited number of lung cancer patients and controls. In an independent set, we tested the six most promising genes (APC, CDH1, MGMT, DCC, RASSF1A and AIM) for further elucidation of the diagnostic application of this panel of markers.
RESULTS
Promoter hypermethylation of at least one of the genes studied was detected in all 10 lung primary tumors. In majority of cases, aberrant methylation in serum DNA was accompanied by methylation in the matched tumor samples. In the independent set, using a single gene that had 100% specificity (DCC), 35.5% (95% CI 25%, 47%) of the 76 lung cancer patients were correctly identified. For patients without methylated DCC, addition of a logistic regression score that was based on the five remaining genes improved sensitivity from 35.5% to 75% (95% CI: 64%, 84%) but decreased the specificity from 100% to 73% (95% CI:54%, 88%).
CONCLUSION
This approach needs to be evaluated in a larger test set to determine the role of this gene set in early detection and surveillance of lung cancer.
doi:10.1158/1078-0432.CCR-10-3436
PMCID: PMC3131425  PMID: 21610147
DNA methylation/epigenetics; serum; lung cancer
21.  Mitochondrial Subversion in Cancer 
Mitochondria control essential cellular activities including generation of ATP via oxidative phosphorylation. Mitochondrial DNA (mtDNA) mutations in the regulatory D-loop region and somatic mtDNA mutations are common in primary human cancers. The biological impact of a given mutation may vary, depending on the nature of the mutation and the proportion of mutant mtDNAs carried by the cell. Identification of mtDNA mutations in precancerous lesions supports their early contribution to cell transformation and cancer progression. Introduction of mtDNA mutations in transformed cells has been associated with increased ROS production and tumor growth. Studies reveal that increased and altered mtDNA plays a role in the development of cancer but further work is required to establish the functional significance of specific mitochondrial mutations in cancer and disease progression. This review offers some insight into the extent of mtDNA mutations, their functional consequences in tumorigenesis, mitochondrial therapeutics, and future clinical application.
doi:10.1158/1940-6207.CAPR-10-0326
PMCID: PMC3298745  PMID: 21543342
22.  Genetic and epigenetic analysis of erbB signaling pathway genes in lung cancer 
Prognosis for patients with non-small cell lung cancer (NSCLC) is poor. The potential value of modulating EGFR for treatment is reflected by the recent approval of specific drugs that inhibit its activity. Mutations in EGFR were reported in lung cancer and generated interest, once they enable the identification of lung cancers likely to respond to various targeted small molecules.
We tested 3 key genetic and epigenetic alterations (EGFR, RASSF1A, and BRAF) of this pathway on a series of primary NSCLC [Total 111; adenocarcinoma 49, squamous cell carcinoma (SCC) 48 and others 14]. The mutational status of KRAS (and p53) was known for these samples. The purpose of this study was to define the pattern of erbB pathway alterations in NSCLC and to test for associations with clinical parameters.
Five EGFR mutations were identified: 3 in adenocarcinoma (6 %), 1 in SCC (2%) and 1 in adenocarcinoma with bronchoalveolar component tumor (7%). EGFR mutations included 3 in-frame deletions in exon 19 and 2 point mutations in exon 21. Promoter methylation of RASSF1A was detected in 25 of 45 adenocarcinomas and 18 of 46 SCC. Mutations of EGFR, BRAF and KRAS in adenocarcinoma were mutually exclusive and inversely correlated with RASSF1A methylation (p = −0.394; p=0.007). Overall, genetic and/or epigenetic alterations of erbB pathway genes were detected in 80% (39/49) of adenocarcinomas.
Nearly half of primary adenocarcinoma harbor molecular alterations of the erbB pathway. Careful characterization of these alterations and response to anti-EGFR therapies is warranted to determine better and accurate determinants of clinical response.
doi:10.1097/JTO.0b013e3181f77a53
PMCID: PMC3057519  PMID: 21102258
EGFR mutation; DNA methylation/epigenetics; RASSF1A
23.  Mitochondrial DNA Mutation in Normal Margins and Tumors of Recurrent Head and Neck Squamous Cell Carcinoma Patients 
Mitochondrial DNA (mtDNA) mutations were reported in primary head and neck squamous cell carcinoma (HNSCC) patients. However, very little information is available on the mtDNA mutation pattern in the histologically negative surgical margins and tumors of HNSCC patients who experienced tumor recurrence. The present study aimed at understanding the nature and timing of mtDNA mutation in histologically negative margins, and tumors in HNSCC patients who developed local recurrence during the follow ups. The entire 16.5-kb mitochondrial genome was sequenced in matched normal lymphocytes, histologically normal margins, and tumors of 50 recurrent HNSCC patients. The mtDNA mutations were then compared with clinical parameters. Forty-eight percent (24 of 50) patients harbored at least one somatic mtDNA mutation in the tumor, and a total of 37 somatic mtDNA mutations were detected. The mtDNA mutations were mostly heteroplasmic in nature and nucleotide transitions (A↔G; T↔C). Forty-six percent of the mutations (17 of 37) were detected in the tumors and were also detectable in the corresponding histologically normal margin of the patients. The mtDNA mutations involved both coding and noncoding regions of the mtDNA. Majority (9 of 17, 53%) of the noncoding mutations involved tRNAs. Seventy-five percent (15 of 20) of the coding mtDNA mutations were nonsynonymous in nature and mainly affected cytochrome c oxidase (Complex IV), frequently altered in different human mitochondrial diseases including cancer. Analysis of mtDNA mutation could be an invaluable tool for molecular assessment of histologically negative margins and as well for monitoring HNSCC patients with locoregional recurrences.
doi:10.1158/1940-6207.CAPR-10-0018
PMCID: PMC3040952  PMID: 20660573
24.  Profiling the expression pattern of GPI transamidase complex subunits in human cancer 
The glycosylphosphatidylinositol transamidase complex (GPIT) consists of five subunits: PIG-U, PIG-T, GPAA1, PIG-S and GPI8, and is important in attaching GPI anchors to target proteins. On the basis of our previous reports incriminating PIG-U as an oncogene in bladder cancer and PIG-T and GPAA1 as oncogenes in breast cancer, we evaluated the expression pattern of the GPIT subunits in 19 different human cancers at both mRNA and protein levels. In general, our results demonstrate a more frequent expression of GPIT subunits in cancers than in normal. Among the 19 anatomic sites compared; breast, ovary and uterus showed consistent evidence of overexpression of specific GPIT subunits. There was also overexpression of PIG-U and GPI8 in lymphoma. In addition, non-small cell lung carcinoma showed significant overexpression of the GPIT subunits as compared to small cell lung carcinoma and normal lung tissue. Also, deregulation of specific GPIT subunits was seen in various other cancers. Forced overexpression of two GPIT subunits; PIG-S and GPI8 alone or in combination induced increased proliferation and invasion of breast cancer cells. Collectively, our study defines a trend involving the deregulated expression and the functional contribution of the GPIT subunits in various cancers with potential implications in diagnosis, prognosis and therapeutic intervention.
doi:10.1038/modpathol.2008.76
PMCID: PMC3082921  PMID: 18487995
GPIT complex; gene expression; cancer
25.  Forced Cytochrome B gene mutation expression induces mitochondrial proliferation and prevents apoptosis in human uroepithelial SV-HUC-1 cells 
Mitochondria encoded Cytochrome B (CYTB) gene mutations were reported in tumors of different anatomic origin but the functional significance of these mutations are not well studied. Earlier, we found a 7-amino acid deletion mutation in the CYTB gene in a primary bladder cancer patient. In the present study, we overexpressed this 7-amino acid deletion mutation of CYTB gene in SV-40 transformed human uroepithelial HUC-1 cells. The nuclear transcribed mitochondrial CYTB (mtCYTB) was targeted into the mitochondria and generated increased copies of mitochondria and mitochondrial COX-I protein in the transfected HUC-1 cells. The pro-apoptotic protein Bax largely remained confined to the cytoplasm of the mtCYTB transfected HUC-1 cells without release of Cytochrome C. The downstream apoptotic proteins PARP also remained uncleaved along with increased Lamin B1 in the mtCYTB transfected cells. Our results demonstrate that forced overexpression of mtCYTB in transformed human uroepithelial HUC-1 cells triggered mitochondrial proliferation and induction of an anti-apoptotic signaling cascade favoring sustained cellular growth. Coding mitochondrial DNA mutations appear to have significant functional contribution in tumor progression.
doi:10.1002/ijc.24701
PMCID: PMC2988469  PMID: 19569044
Mitochondria; Cytochrome B mutation; apoptosis; mtDNA content

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