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Joseph W Briggs, Ling Ren,
Rachel Nguyen,
Kristi Chakrabarti,
Jessica Cassavaugh,
Said Rahim,
Gulay Bulut,
Ming Zhou,
Timothy D Veenstra,
Qingrong Chen,
Jun S Wei,
Javed Khan,
Aykut Uren,
Chand Khanna
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ABSTRACT: We previously associated the cytoskeleton linker protein, Ezrin, with the metastatic phenotype of pediatric sarcomas, including osteosarcoma and rhabdomyosarcoma. These studies have suggested that Ezrin contributes to the survival of cancer cells after their arrival at secondary metastatic locations. To better understand this role in metastasis, we undertook two noncandidate analyses of Ezrin function including a microarray subtraction of high-and low-Ezrin-expressing cells and a proteomic approach to identify proteins that bound the N-terminus of Ezrin in tumor lysates. Functional analyses of these data led to a novel and unifying hypothesis that Ezrin contributes to the efficiency of metastasis through regulation of protein translation. In support of this hypothesis, we found Ezrin to be part of the ribonucleoprotein complex to facilitate the expression of complex messenger RNA in cells and to bind with poly A binding protein 1 (PABP1; PABPC1). The relevance of these findings was supported by our identification of Ezrin and components of the translational machinery in pseudopodia of highly metastatic cells during the process of cell invasion. Finally, two small molecule inhibitors recently shown to inhibit the Ezrin metastatic phenotype disrupted the Ezrin/PABP1 association. Taken together, these results provide a novel mechanistic basis by which Ezrin may contribute to metastasis.
Neoplasia (New York, N.Y.) 04/2012; 14(4):297-310. · 5.48 Impact Factor
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ABSTRACT: The complexity of the process of metastasis is widely recognized. We report herein on a recurrent feature of high compared to low metastatic cells that is linked to their ability to survive early after their arrival at secondary sites. Using novel fluorescent-based imaging strategies that assess tumor cell interaction with the lung microenvironment, we have determined that most high and low metastatic cells can be distinguished within 6 hours of their arrival in the lung and further that this difference is defined by the ability of high metastatic cells to resist apoptosis at the secondary site. Despite the complexity of the metastatic cascade, the performance of cells during this critical window is highly defining of their metastatic proclivity. To explore mechanisms, we next evaluated biochemical pathways that may be linked to this survival phenotype in highly metastatic cells. Interestingly, we found no association between the Akt survival pathway and this metastatic phenotype. Of all pathways examined, only protein kinase C (PKC) activation was significantly linked to survival of highly metastatic cells. These data provide a conceptual understanding of a defining difference between high and low metastatic cells. The connection to PKC activation may provide a biologic rationale for the use of PKC inhibition in the prevention of metastatic progression.
Neoplasia (New York, N.Y.) 03/2012; 14(3):249-58. · 5.48 Impact Factor
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Ling Ren,
Sung-Hyeok Hong,
Qing-Rong Chen,
Joseph Briggs,
Jessica Cassavaugh,
Satish Srinivasan,
Michael M Lizardo,
Arnulfo Mendoza,
Ashley Y Xia,
Narayan Avadhani,
Javed Khan,
Chand Khanna
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ABSTRACT: Ezrin links the plasma membrane to the actin cytoskeleton where it plays a pivotal role in the metastatic progression of several human cancers; however, the precise mechanistic basis for its role remains unknown. Here, we define transitions between active (phosphorylated open) and inactive (dephosphorylated closed) forms of Ezrin that occur during metastatic progression in osteosarcoma. In our evaluation of these conformations we expressed C-terminal mutant forms of Ezrin that are open (phosphomimetic T567D) or closed (phosphodeficient T567A) and compared their biologic characteristics to full-length wild-type Ezrin in osteosarcoma cells. Unexpectedly, cells expressing open, active Ezrin could form neither primary orthotopic tumors nor lung metastases. In contrast, cells expressing closed, inactive Ezrin were also deficient in metastasis but were unaffected in their capacity for primary tumor growth. By imaging single metastatic cells in the lung, we found that cells expressing either open or closed Ezrin displayed increased levels of apoptosis early after their arrival in the lung. Gene expression analysis suggested dysregulation of genes that are functionally linked to carbohydrate and amino acid metabolism. In particular, cells expressing closed, inactive Ezrin exhibited reduced lactate production and basal or ATP-dependent oxygen consumption. Collectively, our results suggest that dynamic regulation of Ezrin phosphorylation at amino acid T567 that controls structural transitions of this protein plays a pivotal role in tumor progression and metastasis, possibly in part by altering cellular metabolism.
Cancer Research 12/2011; 72(4):1001-12. · 7.86 Impact Factor
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ABSTRACT: The ability to define osteosarcoma (OS) patients at greatest risk for metastatic progression and nonresponsiveness to conventional therapy is currently not possible. Such biomarkers are needed to predict overall prognosis, probability of metastases at diagnosis, and response to chemotherapy. The tissue microarray (TMA) serves as a powerful tool for detecting and validating protein biomarkers across a variety of patients. We constructed a novel outcome-linked TMA to add to and address shortcomings of currently available OS tissue resources. To test the use of our TMA, we surveyed the expression of eukaryotic initiation factor 4E (eIF4E) in OS patients using immunohistochemistry. Aberrant regulation of translation initiation is a feature of many cancers. eIF4E is central to initiation of protein synthesis. Its expression and activity have been implicated in tumor formation and potentially malignant and/or metastatic progression in some carcinomas. We found that eIF4E was uniformly expressed in OS patient samples. No association was found between eIF4E and outcome in OS patients. This novel OS TMA provided a facile mechanism to assess the role of a relevant protein biomarker in OS.
Journal of Pediatric Hematology/Oncology 10/2011; 33(7):524-8. · 1.16 Impact Factor
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Arnulfo Mendoza,
Sung-Hyeok Hong,
Tanasa Osborne,
Mohammed A Khan,
Kirk Campbell,
Joseph Briggs,
Ananth Eleswarapu,
Lauren Buquo, Ling Ren,
Stephen M Hewitt,
El Habib Dakir,
El-H Dakir,
Susan Garfield,
Renard Walker,
Glenn Merlino,
Jeffrey E Green,
Kent W Hunter,
Lalage M Wakefield,
Chand Khanna
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ABSTRACT: Pulmonary metastasis remains the leading ca use of death for cancer patients. Opportunities to improve treatment outcomes for patients require new methods to study and view the biology of metastatic progression. Here, we describe an ex vivo pulmonary metastasis assay (PuMA) in which the metastatic progression of GFP-expressing cancer cells, from a single cell to the formation of multicellular colonies, in the mouse lung microenvironment was assessed in real time for up to 21 days. The biological validity of this assay was confirmed by its prediction of the in vivo behavior of a variety of high- and low-metastatic human and mouse cancer cell lines and the discrimination of tumor microenvironments in the lung that were most permissive to metastasis. Using this approach, we provide what we believe to be new insights into the importance of tumor cell interactions with the stromal components of the lung microenvironment. Finally, the translational utility of this assay was demonstrated through its use in the evaluation of therapeutics at discrete time points during metastatic progression. We believe that this assay system is uniquely capable of advancing our understanding of both metastasis biology and therapeutic strategies.
The Journal of clinical investigation 08/2010; 120(8):2979-88. · 15.39 Impact Factor
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Su Young Kim,
Chih Hung Lee,
Brieanne V Midura,
Choh Yeung,
Arnulfo Mendoza,
Sung Hyeok Hong, Ling Ren,
Donald Wong,
Walter Korz,
Ahmed Merzouk,
Hassan Salari,
Hong Zhang,
Sam T Hwang,
Chand Khanna,
Lee J Helman
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ABSTRACT: Metastasis continues to be the leading cause of mortality for patients with cancer. High expression of the chemokine receptor CXCR4 correlates with poor prognosis in many cancers, including osteosarcoma and melanoma. CXCL12, the ligand for CXCR4, is expressed at high levels in the lung and lymph node, which are the primary sites to which these tumors metastasize respectively. These findings suggest that therapy aimed at disruption of this specific receptor/ligand complex may lead to a decrease in metastases. CTCE-9908, a small peptide CXCR4 antagonist was utilized in two murine metastasis models to test this hypothesis. Treatment of osteosarcoma cells in vitro with CTCE-9908 led to the following changes: decreased adhesion, decreased migration, decreased invasion, and decreased growth rate. Following tail vein injection of osteosarcoma cells, mice that were treated with CTCE-9908 had a 50% reduction in the number of gross metastatic lung nodules and a marked decrease in micro-metastatic disease. Similar findings were observed following injection of melanoma cells and treatment with CTCE-9908. However, these results could only be consistently reproduced when the cells were pre-treated with the inhibitor. A novel ex vivo luciferase assay showed decreased numbers of cells in the lung immediately after injection into mice, when treated with CTCE-9908, suggesting the importance of interactions between the receptor and the ligand. Our findings show that inhibition of the CXCR4/CXCL12 pathway decreases metastatic disease in two murine tumor models and expands on previous reports to describe potential mechanisms of action.
Clinical and Experimental Metastasis 02/2008; 25(3):201-11. · 3.52 Impact Factor
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Benjamin Bruce,
Gaurav Khanna, Ling Ren,
Goran Landberg,
Karin Jirström,
Charles Powell,
Alain Borczuk,
Evan T Keller,
Kirk J Wojno,
Paul Meltzer,
Kristin Baird,
Andrea McClatchey,
Anthony Bretscher,
Stephen M Hewitt,
Chand Khanna
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ABSTRACT: Expression of the metastasis-associated protein, ezrin, in over 5,000 human cancers and normal tissues was analyzed using tissue microarray immunohistochemistry. Ezrin staining was compared between cancers and their corresponding normal tissues, between cancers of epithelial and mesenchymal origin, in the context of the putative inhibitor protein, merlin, and against clinicopathological data available for breast, lung, prostate cancers and sarcomas. Ezrin was found in most cancers and normal tissues at varying levels of intensity. In general ezrin was expressed at higher levels in sarcomas than in carcinomas. By normalizing the expression of ezrin in each cancer using ezrin expression found in the corresponding normal tissue, significant associations between ezrin were found in advancing histological grade in sarcomas (P = 0.02) and poor outcome in breast cancer (P = 0.025). Clinicopathologic associations were not changed by simultaneous assessment of ezrin and merlin in each patient sample for the cancer types examined. These data support a role for ezrin in the biology of human cancers and the need for additional studies in breast cancer and sarcoma patients that may validate ezrin as a marker of cancer progression and as a potential target for cancer therapy.
Clinical and Experimental Metastasis 02/2007; 24(2):69-78. · 3.52 Impact Factor
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ABSTRACT: We have constructed an enhancer cloning vehicle for higher plants, which is based on Agrobacterium tumefaciens T-DNA-mediated transformation of Nicotiana tabacum leaf disk segments. The binary vector plasmid, pROA97, contains two genes in a divergent transcription pattern, which are separated by a unique Bg/II cloning site. Both genes carry abbreviated TATA regions from the cauliflower mosaic virus 35S promoter region. The neomycin phosphotransferase (NPT II) gene is used as a selectable marker in callus tissue cultured cells. Expression from the other gene encoding -glucuronidase (GUS) can be monitored in regenerated plant tissues by fluorimetric and histochemical assays. Here we describe the transformation properties of the empty vector and control constructs with either the 35S enhancer or the nopaline synthetase promoter region inserted into the unique Bg/II site. A construct carrying the 35 S – 46 to + 8 TATA region is incapable of allowing callus production on selectable media; a construct carrying the 35 S –90 to + 8 TATA region linked to the NPT II gene coding sequence is sufficient to give transformed callus cells and regenerated transgenic plants. There is a high correlation between expression levels of the two genes in this bidirectional system. The vectors were designed so that the enhancer sequences could be rescued from the plant genome by a rapid marker rescue protocol.
MGG - Molecular and General Genetics 02/1990; 221(1):121-124.
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ABSTRACT: We have analyzed expression conferred by two domains from the cauliflower mosaic virus (CaMV) 35S promoter and found different patterns in seeds, seedlings and seven week old plants. Expression from domain A (-90 to +8) is strongest in the radicle of the embryo, the radicle pole of the endosperm and in root tissue of seedlings and mature plants. Expression from domain B (-343 to -90) is strongest in the cells adjacent the cotyledon of the endosperm, in the cotyledons of the embryo and seedings and in the leaves and stem of mature plants. When both domain A and domain B are present expression is detectable in most tissues at all stages of development. Thus analysis of a constitutive promoter in transgenic plants can be used to identify cis elements that confer tissue specific and developmentally regulated expression.
The EMBO Journal 09/1989; 8(8):2195-202. · 9.20 Impact Factor
