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1.  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
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  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
18.  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
19.  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
20.  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
21.  Growth, cell division and sporulation in mycobacteria 
Antonie Van Leeuwenhoek  2010;98(2):165-177.
Bacteria have the ability to adapt to different growth conditions and to survive in various environments. They have also the capacity to enter into dormant states and some bacteria form spores when exposed to stresses such as starvation and oxygen deprivation. Sporulation has been demonstrated in a number of different bacteria but Mycobacterium spp. have been considered to be non-sporulating bacteria. We recently provided evidence that Mycobacterium marinum and likely also Mycobacterium bovis bacillus Calmette–Guérin can form spores. Mycobacterial spores were detected in old cultures and our findings suggest that sporulation might be an adaptation of lifestyle for mycobacteria under stress. Here we will discuss our current understanding of growth, cell division, and sporulation in mycobacteria.
doi:10.1007/s10482-010-9446-0
PMCID: PMC2906719  PMID: 20437098
Mycobacteria; Bacterial cell division; Septum formation; Sporulation
22.  Aberrant Promoter Methylation of Multiple Genes during Pathogenesis of Bladder Cancer 
Purpose
The aims of our study were to elucidate the role of methylation of a large panel of genes during multistage pathogenesis of bladder cancer and to correlate our findings with patient age and other clinicopathologic features.
Experimental Design
We studied the methylation status of 21 genes by quantitative methylation-specific PCR in an evaluation set of 25 tumor and 5 normal samples. Based on methylation frequency in tumors and normals in gene evaluation set, we selected 7 candidate genes and tested an independent set of 93 tumors and 26 normals. The presence or absence of methylation was evaluated for an association with cancer using cross-tabulations and χ2 or Fisher’s exact tests as appropriate. All statistical tests were two-sided.
Results
Most primary tumors (89 of 93, 96%) had methylation of one or more genes of independent set; 53 (57%) CCNA1, 29 (31%) MINT1, 36 (39%) CRBP, 53 (57%) CCND2, 66 (71%) PGP9.5, 60 (65%) CALCA, and 78 (84%) AIM1. Normal uroepithelium samples from 26 controls revealed no methylation of the CCNA1 and MINT1 genes, whereas methylation of CRBP, CCND2, PGP9.5, and CALCA was detected at low levels. All the 7 genes in independent set were tightly correlated with each other and 3 of these genes showed increased methylation frequencies in bladder cancer with increasing age. PGP9.5 and AIM1 methylation correlated with primary tumor invasion.
Conclusion
Our results indicate that the methylation profile of novel genes in bladder cancers correlates with clinicopathologic features of poor prognosis and is an age-related phenomenon.
doi:10.1158/1055-9965.EPI-08-0192
PMCID: PMC2778751  PMID: 18843024
23.  Following Mitochondrial Footprints through a Long Mucosal Path to Lung Cancer 
PLoS ONE  2009;4(8):e6533.
Background
Mitochondrial DNA (mtDNA) mutations are reported in different tumors. However, there is no information on the temporal development of the mtDNA mutations/content alteration and their extent in normal and abnormal mucosa continuously exposed to tobacco smoke in lung cancer patients.
Methodology
We examined the pattern of mtDNA alteration (mtDNA mutation and content index) in 25 airway mucosal biopsies, corresponding tumors and normal lymph nodes obtained from three patients with primary lung cancers. In addition, we examined the pattern of mtDNA mutation in corresponding tumors and normal lymph nodes obtained from eight other patients with primary lung cancers. The entire 16.5 kb mitochondrial genome was sequenced on Affymetrix Mitochip v2.0 sequencing platform in every sample. To examine mtDNA content index, we performed real-time PCR analysis.
Principal Findings
The airway mucosal biopsies obtained from three lung cancer patients were histopathologically negative but exhibited multiple clonal mtDNA mutations detectable in the corresponding tumors. One of the patients was operated twice for the removal of tumor from the right upper and left lower lobe respectively within a span of two years. Both of these tumors exhibited twenty identical mtDNA mutations. MtDNA content increased significantly (P<0.001) in the lung cancer and all the histologically negative mucosal biopsies except one compared to the control lymph node.
Conclusions/Significance:
Our results document the extent of massive clonal patches that develop in lifetime smokers and ultimately give rise to clinically significant cancers. These observations shed light on the extent of disease in the airway of smokers traceable through mtDNA mutation. MtDNA mutation could be a reliable tool for molecular assessment of respiratory epithelium exposed to continuous smoke as well as disease detection and monitoring. Functional analysis of the pathogenic mtDNA mutations may be useful to understand their role in lung tumorigenesis.
doi:10.1371/journal.pone.0006533
PMCID: PMC2719062  PMID: 19657397
24.  A single-step method for purification of active His-tagged ribosomes from a genetically engineered Escherichia coli 
Nucleic Acids Research  2008;37(2):e15.
With the rapid development of the ribosome field in recent years a quick, simple and high-throughput method for purification of the bacterial ribosome is in demand. We have designed a new strain of Escherichia coli (JE28) by an in-frame fusion of a nucleotide sequence encoding a hexa-histidine affinity tag at the 3′-end of the single copy rplL gene (encoding the ribosomal protein L12) at the chromosomal site of the wild-type strain MG1655. As a result, JE28 produces a homogeneous population of ribosomes (His)6-tagged at the C-termini of all four L12 proteins. Furthermore, we have developed a single-step, high-throughput method for purification of tetra-(His)6-tagged 70S ribosomes from this strain using affinity chromatography. These ribosomes, when compared with the conventionally purified ones in sucrose gradient centrifugation, 2D-gel, dipeptide formation and a full-length protein synthesis assay showed higher yield and activity. We further describe how this method can be adapted for purification of ribosomal subunits and mutant ribosomes. These methodologies could, in principle, also be used to purify any functional multimeric complex from the bacterial cell.
doi:10.1093/nar/gkn992
PMCID: PMC2632923  PMID: 19074194
25.  Asymmetry of Chromosome Replichores Renders the DNA Translocase Activity of FtsK Essential for Cell Division and Cell Shape Maintenance in Escherichia coli 
PLoS Genetics  2008;4(12):e1000288.
Bacterial chromosomes are organised as two replichores of opposite polarity that coincide with the replication arms from the ori to the ter region. Here, we investigated the effects of asymmetry in replichore organisation in Escherichia coli. We show that large chromosome inversions from the terminal junction of the replichores disturb the ongoing post-replicative events, resulting in inhibition of both cell division and cell elongation. This is accompanied by alterations of the segregation pattern of loci located at the inversion endpoints, particularly of the new replichore junction. None of these defects is suppressed by restoration of termination of replication opposite oriC, indicating that they are more likely due to the asymmetry of replichore polarity than to asymmetric replication. Strikingly, DNA translocation by FtsK, which processes the terminal junction of the replichores during cell division, becomes essential in inversion-carrying strains. Inactivation of the FtsK translocation activity leads to aberrant cell morphology, strongly suggesting that it controls membrane synthesis at the division septum. Our results reveal that FtsK mediates a reciprocal control between processing of the replichore polarity junction and cell division.
Author Summary
In most bacteria, chromosomes consist of a single replication unit. Replication initiates at a single ori and terminates in the diametrically opposite zone, ter, defining two replication arms. These are also called replichores to account for base composition and sequence motif polarity biases from ori to ter. This organisation as symmetrical replichores is conserved in bacteria. Here, we have investigated the effect of replichore asymmetry in E. coli. We show that it blocks the cell cycle at a step posterior to chromosome replication. This phenotype is not corrected by restoration of the termination of replication opposite ori, strongly suggesting that it is due to the asymmetry of replichore polarity. FtsK, a DNA-translocase associated with the division septum that processes the terminal junction of replichore polarity, is essential for growth and for the controlled blockage of cell growth in cells with asymmetric replichores. These results reveal a reciprocal control between the processing of the terminal junction of the replichores by FtsK and the progression of the cell cycle.
doi:10.1371/journal.pgen.1000288
PMCID: PMC2585057  PMID: 19057667

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