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Jihan K Osborne,
Jill E Larsen,
Misty D Shields,
Joshua X Gonzales, David S Shames,
Mitsuo Sato,
Ashwinikumar Kulkarni,
Ignacio I Wistuba,
Luc Girard,
John D Minna,
Melanie H Cobb
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ABSTRACT: Small-cell lung cancer and other aggressive neuroendocrine cancers are often associated with early dissemination and frequent metastases. We demonstrate that neurogenic differentiation 1 (NeuroD1) is a regulatory hub securing cross talk among survival and migratory-inducing signaling pathways in neuroendocrine lung carcinomas. We find that NeuroD1 promotes tumor cell survival and metastasis in aggressive neuroendocrine lung tumors through regulation of the receptor tyrosine kinase tropomyosin-related kinase B (TrkB). Like TrkB, the prometastatic signaling molecule neural cell adhesion molecule (NCAM) is a downstream target of NeuroD1, whose impaired expression mirrors loss of NeuroD1. TrkB and NCAM may be therapeutic targets for aggressive neuroendocrine cancers that express NeuroD1.
Proceedings of the National Academy of Sciences 04/2013; · 9.68 Impact Factor
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Mitsuo Sato,
Jill E Larsen,
Woochang Lee,
Han Sun, David S Shames,
Maithili P Dalvi,
Ruben D Ramirez,
Hao Tang,
J Michael Dimaio,
Boning Gao,
Yang Xie,
Ignacio I Wistuba,
Adi F Gazdar,
Jerry W Shay,
John D Minna
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ABSTRACT: We used CDK4/hTERT-immortalized normal human bronchial epithelial cells (HBECs) from several individuals to study lung cancer pathogenesis by introducing combinations of common lung cancer oncogenic changes (p53, KRAS, MYC) and followed the stepwise transformation of HBECs to full malignancy. This model demonstrated that: 1) the combination of five genetic alterations (CDK4, hTERT, sh-p53, KRASV12, and c-MYC) is sufficient for full tumorigenic conversion of HBECs; 2) genetically-identical clones of transformed HBECs exhibit pronounced differences in tumor growth, histology, and differentiation; 3) HBECs from different individuals vary in their sensitivity to transformation by these oncogenic manipulations; 4) high levels of KRASV12 are required for full malignant transformation of HBECs, however prior loss of p53 function is required to prevent oncogene-induced senescence; 5) over-expression of c-MYC greatly enhances malignancy but only in the context of sh-p53+KRASV12; 6) growth of parental HBECs in serum-containing medium induces differentiation while growth of oncogenically manipulated HBECs in serum increases in vivo tumorigenicity, decreases tumor latency, produces more undifferentiated tumors, and induces epithelial-to-mesenchymal transition (EMT); 7) oncogenic transformation of HBECs leads to increased sensitivity to standard chemotherapy doublets; 8) an mRNA signature derived by comparing tumorigenic vs. non-tumorigenic clones was predictive of outcome in lung cancer patients. Collectively, our findings demonstrate this HBEC model system can be used to study the effect of oncogenic mutations, their expression levels, and serum-derived environmental effects in malignant transformation, while also providing clinically translatable applications such as development of prognostic signatures and drug response phenotypes.
Molecular Cancer Research 02/2013; · 4.29 Impact Factor
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Momen Elshazley,
Mitsuo Sato,
Tetsunari Hase,
Ryo Yamashita,
Kenya Yoshida,
Shinya Toyokuni,
Futoshi Ishiguro,
Hirotaka Osada,
Yoshitaka Sekido,
Kohei Yokoi,
Noriyasu Usami, David S Shames,
Masashi Kondo,
Adi F Gazdar,
John D Minna,
Yoshinori Hasegawa
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ABSTRACT: Malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm arising from the mesothelial cells lining the parietal pleura and it exhibits poor prognosis. Although there has been significant progress in MPM treatment, development of more efficient therapeutic approaches is needed. BMAL1 is a core component of the circadian clock machinery and its constitutive overexpression in MPM has been reported. Here, we demonstrate that BMAL1 may serve as a molecular target for MPM. The majority of MPM cell lines and a subset of MPM clinical specimens expressed higher levels of BMAL1 compared to a nontumorigenic mesothelial cell line (MeT-5A) and normal parietal pleural specimens, respectively. A serum shock induced a rhythmical BMAL1 expression change in MeT-5A but not in ACC-MESO-1, suggesting that the circadian rhythm pathway is deregulated in MPM cells. BMAL1 knockdown suppressed proliferation and anchorage-dependent and independent clonal growth in two MPM cell lines (ACC-MESO-1 and H290) but not in MeT-5A. Notably, BMAL1 depletion resulted in cell cycle disruption with a substantial increase in apoptotic and polyploidy cell population in association with downregulation of Wee1, cyclin B and p21(WAF1/CIP1) and upregulation of cyclin E expression. BMAL1 knockdown induced mitotic catastrophe as denoted by disruption of cell cycle regulators and induction of drastic morphological changes including micronucleation and multiple nuclei in ACC-MESO-1 cells that expressed the highest level of BMAL1. Taken together, these findings indicate that BMAL1 has a critical role in MPM and could serve as an attractive therapeutic target for MPM.
International Journal of Cancer 04/2012; 131(12):2820-31. · 5.44 Impact Factor
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ABSTRACT: The epithelial-to-mesenchymal transition (EMT) is a developmental programme that regulates embryonic morphogenesis and involves significant morphological and molecular changes in cells. Experimental models have revealed that EMT also contributes to various malignant features of cancer cells, including motile, invasive, anti-apoptotic and stem-like phenotypes. Clinically, correlative studies have indicated that mesenchymal-like features of tumour cells are associated with poor tumour differentiation as well as worse patient prognosis. Nevertheless, due to its transitory nature, demonstration of an actual occurrence of EMT during human carcinogenesis is challenging, and most of the evidence to date has been limited to breast and colorectal cancers. However, recent studies suggest that EMT may occur during lung cancer development, although such evidence is still limited. We propose three approaches for obtaining direct evidence of EMT in human cancers and use these criteria to review the available data. We suggest that multiple intrinsic and extrinsic factors cooperatively induce EMT in lung cancer. Intrinsic factors include oncogenic genetic changes such as mutant K-RAS. Extrinsic factors are associated with a tumour microenvironment that is inflammatory and hypoxic. The induction of EMT is primarily mediated by various EMT-inducing transcription factors that suppress E-cadherin expression, including SLUG and ZEB1. miR-200 family expression can reverse EMT by suppressing EMT- inducing transcription factors. Obviously, more data demonstrating the clinical relevance of EMT in lung cancer are required, and further elucidation of how EMT is regulated in lung cancer will enable us to develop novel therapeutics that specifically target molecules with critical roles in EMT.
Respirology 03/2012; 17(7):1048-59. · 2.42 Impact Factor
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Noriaki Sunaga,
Hisao Imai,
Kimihiro Shimizu, David S. Shames,
Seiichi Kakegawa,
Luc Girard,
Mitsuo Sato,
Kyoichi Kaira,
Tamotsu Ishizuka,
Adi F. Gazdar,
John D. Minna,
Masatomo Mori
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ABSTRACT: The CXC chemokine interleukin-8 (IL-8) is an angiogenic growth factor that is overexpressed in various cancers, including non-small cell lung cancer (NSCLC). Previously, IL-8 was shown as a transcriptional target of RAS signaling, raising the possibility of its role in oncogenic KRAS-driven NSCLC. Using microarray analysis, we identified IL-8 as the most downregulated gene by shRNA-mediated KRAS knockdown in NCI-H1792 NSCLC cells where IL-8 is overexpressed. NSCLC cell lines harboring KRAS or EGFR mutations overexpressed IL-8, while IL-8 levels were more prominent in KRAS mutants compared to EGFR mutants. IL-8 expression was downregulated by shRNA-mediated KRAS knockdown in KRAS mutants or by treatment with EGFR tyrosine kinase inhibitors and EGFR siRNAs in EGFR mutants. In our analysis of the relationship of IL-8 expression with clinical parameters and mutation status of KRAS or EGFR in 89 NSCLC surgical specimens, IL-8 expression was shown to be significantly higher in NSCLCs of males, smokers, and elderly patients and those with pleural involvement and KRAS mutated adenocarcinomas. In KRAS mutant cells, the MEK inhibitor markedly decreased IL-8 expression, while the p38 inhibitor increased IL-8 expression. Attenuation of IL-8 function by siRNAs or a neutralizing antibody inhibited cell proliferation and migration of KRAS mutant/IL-8 overexpressing NSCLC cells. These results indicate that activating mutations of KRAS or EGFR upregulate IL-8 expression in NSCLC; IL-8 is highly expressed in NSCLCs from males, smokers, elderly patients, NSCLCs with pleural involvement, and KRAS-mutated adenocarcinomas; and IL-8 plays a role in cell growth and migration in oncogenic KRAS-driven NSCLC.
International Journal of Cancer 08/2011; 130(8):1733 - 1744. · 5.44 Impact Factor
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Noriaki Sunaga,
Hisao Imai,
Kimihiro Shimizu, David S Shames,
Seiichi Kakegawa,
Luc Girard,
Mitsuo Sato,
Kyoichi Kaira,
Tamotsu Ishizuka,
Adi F Gazdar,
John D Minna,
Masatomo Mori
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ABSTRACT: The CXC chemokine interleukin-8 (IL-8) is an angiogenic growth factor that is overexpressed in various cancers, including non-small cell lung cancer (NSCLC). Previously, IL-8 was shown as a transcriptional target of RAS signaling, raising the possibility of its role in oncogenic KRAS-driven NSCLC. Using microarray analysis, we identified IL-8 as the most downregulated gene by shRNA-mediated KRAS knockdown in NCI-H1792 NSCLC cells where IL-8 is overexpressed. NSCLC cell lines harboring KRAS or EGFR mutations overexpressed IL-8, while IL-8 levels were more prominent in KRAS mutants compared to EGFR mutants. IL-8 expression was downregulated by shRNA-mediated KRAS knockdown in KRAS mutants or by treatment with EGFR tyrosine kinase inhibitors and EGFR siRNAs in EGFR mutants. In our analysis of the relationship of IL-8 expression with clinical parameters and mutation status of KRAS or EGFR in 89 NSCLC surgical specimens, IL-8 expression was shown to be significantly higher in NSCLCs of males, smokers, and elderly patients and those with pleural involvement and KRAS mutated adenocarcinomas. In KRAS mutant cells, the MEK inhibitor markedly decreased IL-8 expression, while the p38 inhibitor increased IL-8 expression. Attenuation of IL-8 function by siRNAs or a neutralizing antibody inhibited cell proliferation and migration of KRAS mutant/IL-8 overexpressing NSCLC cells. These results indicate that activating mutations of KRAS or EGFR upregulate IL-8 expression in NSCLC; IL-8 is highly expressed in NSCLCs from males, smokers, elderly patients, NSCLCs with pleural involvement, and KRAS-mutated adenocarcinomas; and IL-8 plays a role in cell growth and migration in oncogenic KRAS-driven NSCLC.
International Journal of Cancer 05/2011; 130(8):1733-44. · 5.44 Impact Factor
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Tetsunari Hase,
Mitsuo Sato,
Kenya Yoshida,
Luc Girard,
Yoshihiro Takeyama,
Mihoko Horio,
Momen Elshazley,
Tomoyo Oguri,
Yoshitaka Sekido, David S Shames,
Adi F Gazdar,
John D Minna,
Masashi Kondo,
Yoshinori Hasegawa
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ABSTRACT: Epithelial cell adhesion molecule (EpCAM) is overexpressed in a wide variety of human cancers including lung cancer, and its contribution to increased proliferation through upregulation of cell cycle accelerators such as cyclins A and E has been well established in breast and gastric cancers. Nevertheless, very little is known about its role in supporting the survival of cancer cells. In addition, the functional role of EpCAM in the pathogenesis of lung cancer remains to be explored. In this study, we show that RNAi-mediated knockdown of EpCAM suppresses proliferation and clonogenic growth of three EpCAM-expressing lung cancer cell lines (H3255, H358, and HCC827), but does not induce cell cycle arrest in any of these. In addition, EpCAM knockdown inhibits invasion in the highly invasive H358 but not in less invasive H3255 cells in a Transwell assay. Of note, the EpCAM knockdown induces massive apoptosis in the three cell lines as well as in another EpCAM-expressing lung cancer cell line, HCC2279, but to a much lesser extent in a cdk4/hTERT immortalized normal human bronchial epithelial cell line, HBEC4, suggesting that EpCAM could be a therapeutic target for lung cancer. Finally, EpCAM knockdown partially restores contact inhibition in HCC827, in association with p27(Kip1) upregulation. These results indicate that EpCAM could contribute substantially to the pathogenesis of lung cancer, especially cancer cell survival, and suggest that EpCAM targeted therapy for lung cancer may have potential.
Cancer Science 05/2011; 102(8):1493-500. · 3.33 Impact Factor
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Noriaki Sunaga, David S Shames,
Luc Girard,
Michael Peyton,
Jill E Larsen,
Hisao Imai,
Junichi Soh,
Mitsuo Sato,
Noriko Yanagitani,
Kyoichi Kaira,
Yang Xie,
Adi F Gazdar,
Masatomo Mori,
John D Minna
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ABSTRACT: Oncogenic KRAS is found in more than 25% of lung adenocarcinomas, the major histologic subtype of non-small cell lung cancer (NSCLC), and is an important target for drug development. To this end, we generated four NSCLC lines with stable knockdown selective for oncogenic KRAS. As expected, stable knockdown of oncogenic KRAS led to inhibition of in vitro and in vivo tumor growth in the KRAS-mutant NSCLC cells, but not in NSCLC cells that have wild-type KRAS (but mutant NRAS). Surprisingly, we did not see large-scale induction of cell death and the growth inhibitory effect was not complete. To further understand the ability of NSCLCs to grow despite selective removal of mutant KRAS expression, we conducted microarray expression profiling of NSCLC cell lines with or without mutant KRAS knockdown and isogenic human bronchial epithelial cell lines with and without oncogenic KRAS. We found that although the mitogen-activated protein kinase pathway is significantly downregulated after mutant KRAS knockdown, these NSCLCs showed increased levels of phospho-STAT3 and phospho-epidermal growth factor receptor, and variable changes in phospho-Akt. In addition, mutant KRAS knockdown sensitized the NSCLCs to p38 and EGFR inhibitors. Our findings suggest that targeting oncogenic KRAS by itself will not be sufficient treatment, but may offer possibilities of combining anti-KRAS strategies with other targeted drugs.
Molecular Cancer Therapeutics 02/2011; 10(2):336-46. · 5.23 Impact Factor
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Di Cai, David S Shames,
Maria Gabriela Raso,
Yang Xie,
Young H Kim,
Jonathan R Pollack,
Luc Girard,
James P Sullivan,
Boning Gao,
Michael Peyton,
Meera Nanjundan,
Lauren Byers,
John Heymach,
Gordon Mills,
Adi F Gazdar,
Ignacio Wistuba,
Thomas Kodadek,
John D Minna
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ABSTRACT: Steroid receptor coactivator-3 (SRC-3) is a histone acetyltransferase and nuclear hormone receptor coactivator, located on 20q12, which is amplified in several epithelial cancers and well studied in breast cancer. However, its possible role in lung cancer pathogenesis is unknown. We found SRC-3 to be overexpressed in 27% of non-small cell lung cancer (NSCLC) patients (n = 311) by immunohistochemistry, which correlated with poor disease-free (P = 0.0015) and overall (P = 0.0008) survival. Twenty-seven percent of NSCLCs exhibited SRC-3 gene amplification, and we found that lung cancer cell lines expressed higher levels of SRC-3 than did immortalized human bronchial epithelial cells (HBEC), which in turn expressed higher levels of SRC-3 than did cultured primary human HBECs. Small interfering RNA-mediated downregulation of SRC-3 in high-expressing, but not in low-expressing, lung cancer cells significantly inhibited tumor cell growth and induced apoptosis. Finally, we found that SRC-3 expression is inversely correlated with gefitinib sensitivity and that SRC-3 knockdown results in epidermal growth factor receptor tyrosine kinase inhibitor-resistant lung cancers becoming more sensitive to gefitinib. Taken together, these data suggest that SRC-3 may be an important oncogene and therapeutic target for lung cancer.
Cancer Research 08/2010; 70(16):6477-85. · 7.86 Impact Factor
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ABSTRACT: MFSD2A (major facilitator superfamily domain containing 2) gene maps on chromosome 1p34 within a linkage disequilibrium block containing genetic elements associated with progression of lung cancer.
Here we show that MFSD2A expression is strongly downregulated in non-small cell lung cancer cell lines of different histotypes and in primary lung adenocarcinomas. Experimental modulation of MFSD2A in lung cancer cells is associated with alteration of mRNA levels of genes involved in cell cycle control and interaction with the extracellular matrix. Exogenous expression of MFSD2A in lung cancer cells induced a G1 block, impaired adhesion and migration in vitro, and significantly reduced tumor colony number in vitro (4- to 27-fold, P < 0.0001) and tumor volume in vivo (approximately 3-fold, P < 0.0001). siRNA knockdown studies in normal human bronchial epithelial cells confirmed the role of MFSD2A in G1 regulation.
Together these data suggest that MFSD2A is a novel lung cancer tumor suppressor gene that regulates cell cycle progression and matrix attachment.
Molecular Cancer 03/2010; 9:62. · 3.99 Impact Factor
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ABSTRACT: Vascular endothelial growth factor (VEGF) is a primary stimulant of angiogenesis and is a macrophage chemotactic protein. Inhibition of VEGF is beneficial in combination with chemotherapy for some breast cancer patients. However, the mechanism by which inhibition of VEGF affects tumor growth seems to involve more than its effect on endothelial cells. In general, increased immune cell infiltration into breast tumors confers a worse prognosis. We have shown previously that 2C3, a mouse monoclonal antibody that prevents VEGF from binding to VEGF receptor 2 (VEGFR2), decreases tumor growth, angiogenesis, and macrophage infiltration into pancreatic tumors and therefore hypothesized that r84, a fully human IgG that phenocopies 2C3, would similarly affect breast tumor growth and immune cell infiltration. In this study, we show that anti-VEGF therapy with bevacizumab, 2C3, or r84 inhibits the growth of established orthotopic MDA-MB-231 breast tumors in severe combined immunodeficiency (SCID) mice, reduces tumor microvessel density, limits the infiltration of tumor-associated macrophages, but is associated with elevated numbers of tumor-associated neutrophils. In addition, we found that treatment with r84 reduced the number of CD11b(+)Gr1(+) double-positive cells in the tumor compared with tumors from control-treated animals. These results show that selective inhibition of VEGFR2 with an anti-VEGF antibody is sufficient for effective blockade of the protumorigenic activity of VEGF in breast cancer xenografts. These findings further define the complex molecular interactions in the tumor microenvironment and provide a translational tool that may be relevant to the treatment of breast cancer.
Molecular Cancer Therapeutics 07/2009; 8(7):1761-71. · 5.23 Impact Factor
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Junichi Soh,
Naoki Okumura,
William W Lockwood,
Hiromasa Yamamoto,
Hisayuki Shigematsu,
Wei Zhang,
Raj Chari, David S Shames,
Ximing Tang,
Calum MacAulay,
Marileila Varella-Garcia,
Tõnu Vooder,
Ignacio I Wistuba,
Stephen Lam,
Rolf Brekken,
Shinichi Toyooka,
John D Minna,
Wan L Lam,
Adi F Gazdar
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ABSTRACT: Activating mutations in one allele of an oncogene (heterozygous mutations) are widely believed to be sufficient for tumorigenesis. However, mutant allele specific imbalance (MASI) has been observed in tumors and cell lines harboring mutations of oncogenes.
We determined 1) mutational status, 2) copy number gains (CNGs) and 3) relative ratio between mutant and wild type alleles of KRAS, BRAF, PIK3CA and EGFR genes by direct sequencing and quantitative PCR assay in over 400 human tumors, cell lines, and xenografts of lung, colorectal, and pancreatic cancers. Examination of a public database indicated that homozygous mutations of five oncogenes were frequent (20%) in 833 cell lines of 12 tumor types. Our data indicated two major forms of MASI: 1) MASI with CNG, either complete or partial; and 2) MASI without CNG (uniparental disomy; UPD), due to complete loss of wild type allele. MASI was a frequent event in mutant EGFR (75%) and was due mainly to CNGs, while MASI, also frequent in mutant KRAS (58%), was mainly due to UPD. Mutant: wild type allelic ratios at the genomic level were precisely maintained after transcription. KRAS mutations or CNGs were significantly associated with increased ras GTPase activity, as measured by ELISA, and the two molecular changes were synergistic. Of 237 lung adenocarcinoma tumors, the small number with both KRAS mutation and CNG were associated with shortened survival.
MASI is frequently present in mutant EGFR and KRAS tumor cells, and is associated with increased mutant allele transcription and gene activity. The frequent finding of mutations, CNGs and MASI occurring together in tumor cells indicates that these three genetic alterations, acting together, may have a greater role in the development or maintenance of the malignant phenotype than any individual alteration.
PLoS ONE 01/2009; 4(10):e7464. · 4.09 Impact Factor
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ABSTRACT: Macrophages are an abundant inflammatory cell type in the tumor microenvironment that can contribute to tumor growth and metastasis. Macrophage recruitment into tumors is mediated by multiple cytokines, including vascular endothelial growth factor (VEGF), which is thought to function primarily through VEGF receptor (VEGFR) 1 expressed on macrophages. Macrophage infiltration is affected by VEGF inhibition. We show that selective inhibition of VEGFR2 reduced macrophage infiltration into orthotopic pancreatic tumors. Our studies show that tumor-associated macrophages express VEGFR2. Furthermore, peritoneal macrophages from tumor-bearing animals express VEGFR2, whereas peritoneal macrophages from non-tumor-bearing animals do not. To our knowledge, this is the first time that tumor-associated macrophages have been shown to express VEGFR2. Additionally, we found that the cytokine pleiotrophin is sufficient to induce VEGFR2 expression on macrophages. Pleiotrophin has previously been shown to induce expression of endothelial cell markers on macrophages and was present in the microenvironment of orthotopic pancreatic tumors. Finally, we show that VEGFR2, when expressed by macrophages, is essential for VEGF-stimulated migration of tumor-associated macrophages. In summary, tumor-associated macrophages express VEGFR2, and selective inhibition of VEGFR2 reduces recruitment of macrophages into orthotopic pancreatic tumors. Our results show an underappreciated mechanism of action that may directly contribute to the antitumor activity of angiogenesis inhibitors that block the VEGFR2 pathway.
Cancer Research 06/2008; 68(11):4340-6. · 7.86 Impact Factor
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04/2008: pages 61 - 83; , ISBN: 9780470696330
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Proceedings of the National Academy of Sciences 03/2008; 105(5):1389-90. · 9.68 Impact Factor
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Boning Gao,
Xian-Jin Xie,
Chunxian Huang, David S Shames,
Tina T-L Chen,
Cheryl M Lewis,
Aihua Bian,
Bifeng Zhang,
Olufunmilayo I Olopade,
Judy E Garber,
David M Euhus,
Gail E Tomlinson,
John D Minna
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ABSTRACT: The tumor suppressor gene RASSF1A regulates cell cycle progression, apoptosis, and microtubule stability and is inactivated by promoter methylation in approximately 50% of breast cancers. It has been shown previously that the polymorphism A133S in RASSF1A reduces its ability to regulate cell cycle progression and this polymorphism is associated with an increased risk of breast cancer. We analyzed the frequency of RASSF1A A133S in 190 Caucasian women without breast cancer and 653 patients with breast cancer including 138 BRCA1 and BRCA2 (BRCA1/2) mutation carriers, 395 non-BRCA1/2 mutations carriers, and 120 untested for BRCA1/2 mutations. Patients with breast cancer had a higher frequency of A133S than the controls [P = 0.017; odds ratios (OR), 1.71; 95% confidence intervals (95% CI), 1.10-2.66]. There is also a higher frequency of A133S in patients with higher familial breast cancer risk (P = 0.029; OR, 1.76; 95% CI, 1.06-2.92) and patients carrying BRCA1/2 mutations (P = 0.037, OR, 1.82; 95% CI, 1.04-3.18). Importantly, we found that the co-occurrence of a BRCA1 or BRCA2 mutation and A133S in RASSF1A was associated with earlier onset of breast cancer compared with those individuals with either a BRCA1/2 mutation or the A133S polymorphism alone (36.0 versus 42.0 years old, P = 0.002). Our data suggest that the presence of the RASSF1A A133S polymorphism is associated with breast cancer pathogenesis in general and modifies breast cancer age of onset in BRCA1/2 mutations carriers. Our results warrant a large-scale study to examine the effect of the A133S polymorphism in the development of breast and other types of cancers.
Cancer Research 02/2008; 68(1):22-5. · 7.86 Impact Factor
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ABSTRACT: Tumor-acquired changes in DNA methylation are the focus of research in an increasing number of basic, translational, and clinical laboratories around the world. In the laboratory, genome-wide technologies such as expression and DNA microarrays have been adapted to analyze patterns of DNA methylation and to screen for novel disease markers. Other technologies that are relatively inexpensive and highly sensitive such as methylation-specific PCR (MSP), or quantitative, such as quantitative MSP and pyrosequencing are widely used in retrospective studies and have potential in a diagnostic setting. In the near future, it may be possible to screen patients for common cancers using DNA methylation signatures as well as to measure patient responses to treatment, to identify patients at increased risk, or to monitor interventions designed to reduce cancer incidence. In this article, we review genome-wide and quantitative, high- resolution methods for methylation analysis that are used in the laboratory and clinic, and discuss their potential for use in a clinical setting.
Cancer Letters 07/2007; 251(2):187-98. · 4.24 Impact Factor
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ABSTRACT: Molecular genetic studies of lung cancer have revealed that clinically evident lung cancers have multiple genetic and epigenetic abnormalities, including DNA sequence alterations, copy number changes, and aberrant promoter hypermethylation. Together, these abnormalities result in the activation of oncogenes and inactivation of tumor-suppressor genes. In many cases these abnormalities can be found in premalignant lesions and in histologically normal lung bronchial epithelial cells. Findings suggest that lung cancer develops through a stepwise process from normal lung epithelial cells towards frank malignancy, which usually occurs as a result of cigarette smoking. Lung cancer has a high morbidity because it is difficult to detect early and is frequently resistant to available chemotherapy and radiotherapy. New, rationally designed early detection, chemoprevention, and therapeutic strategies based on the growing understanding of the molecular changes important to lung cancer are under investigation. For example, methylated tumor DNA sequences in sputum or blood are being investigated for early detection screening, and new treatments that specifically target molecules such as vascular endothelial growth factor and the epidermal growth factor receptor are becoming available. Meanwhile, global gene expression signatures from individual tumors are showing potential as prognostic and therapeutic indicators, such that molecular typing of individual tumors for therapy selection is not far away. Finally, the recent development of a model system of immortalized human bronchial epithelial cells, along with a paradigm shift in the conception of cancer stem cells, promises to improve the situation for patients with lung cancer. These advances highlight the translation of molecular discoveries on lung cancer pathogenesis from the laboratory to the clinic.
Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer 05/2007; 2(4):327-43. · 4.55 Impact Factor
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ABSTRACT: RRAD, a small Ras-related GTPase, is highly expressed in human skeletal muscle, lung, and heart. Although loss of expression of RRAD in breast cancer cells has been reported and it may act as an oncogene, the mechanism of silencing is unknown.
We examined (1) mRNA expression of RRAD in lung and breast cancer cell lines using RT-PCR and (2) methylation status of lung and breast cancers.
Loss of RRAD expression was found in 14 of 20 (70%) NSCLC cell lines, 11 of 11 (100%) SCLC cell lines, and 8 of 10 (80%) breast cancer cell lines; expression was not affected in normal bronchial and mammary epithelial cells. Treatment of 23 expression-negative cell lines with a demethylating agent restored expression in all cases. We developed a methylation-specific assay from the analysis of bisulfite sequencing of the 5' region of RRAD in expression-negative and positive cell lines, which resulted in good concordance between methylation and expression. Primary lung and breast cancers showed hypermethylation in 89 of 214 (42%) and 39 of 63 (62%) cases, respectively. RRAD hypermethylation correlated with smoking history and poorer prognosis in lung adenocarcinomas.
We conclude that epigenetic silencing of RRAD is a frequent event in lung and breast cancers, and analysis of it may provide novel opportunities for prognosis and therapy of these cancers.
Annals of Surgical Oncology 05/2007; 14(4):1397-404. · 4.17 Impact Factor
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ABSTRACT: The spatial arrangement and three-dimensional structure of DNA in the nucleus is controlled through the interdigitation of DNA binding proteins such as histones and their modifiers, the Polycomb-Trithorax proteins, and the DNA methyltransferase enzymes. DNA methylation forms the foundation of chromatin and is crucial to epigenetic gene regulation in mammals. Disease pathogenesis mediated through infectious agents, inflammation, aging, or genetic damage often involves changes in gene expression. In particular, cellular transformation coincides with multiple changes in chromatin architecture, many of which appear to affect genome integrity and gene expression. Infectious agents, such as viruses directly affect genome structure and induce methylation of particular sequences to suppress host immune responses. Hyperproliferative tissues such as those in the gastrointestinal tract and colon have been shown to gradually acquire aberrant promoter hypermethylation. Here we review recent findings on altered DNA methylation in human disease, with particular focus on cancer and the increasingly large number of genes subject to tumor-specific promoter hypermethylation and the possible role of aberrant methylation in tumor development.
Current Molecular Medicine 03/2007; 7(1):85-102. · 5.10 Impact Factor
