Joseph Briggs

Transcriptional upregulation of SPARC, in response to c-Jun overexpression, contributes to increased motility and invasion of MCF7 breast cancer cells.

Overexpression of the c-Jun proto-oncogene in MCF7 breast cancer cells results in a variety of phenotype changes related to malignant progression including increased motility and invasion. Concurrent with these phenotypic effects are changes in the expression of multiple gene targets. We previously demonstrated that expression of the SPARC/osteonectin gene, while undetectable in the MCF7 cell line, is highly induced in response to stable c-Jun overexpression (c-Jun/MCF7). Because the SPARC gene product is associated with tumor cell invasion in a variety of different cancers, we have examined its role in mediating the phenotypic changes induced by c-Jun in MCF7 cells. We found that antisense mediated suppression of SPARC dramatically inhibits both motility and invasion in this c-Jun/MCF7 model. In contrast, stable overexpression of SPARC in the parental MCF7 cell line is not sufficient to stimulate cell motility or invasion. Examination of the promoter region of the human SPARC gene reveals three non-canonical AP-1 sites. We demonstrate that one of these sites binds c-Jun/Fra1 heterodimers in vitro, but that this and the other AP-1 like sites are dispensable with respect to c-Jun stimulated SPARC promoter activation. Deletion analysis identified a region between −120 and −70 as a c-Jun responsive element sufficient to induce maximal promoter activation. This region does not contain any AP-1 sites but does mediate binding by SP1 ‘like’ complexes. Furthermore, this region is necessary for SP1/SP3 responsiveness in Drosophila SL2 cells. These results demonstrate that SPARC plays an important role in stimulating motility and the invasive behavior of c-Jun/MCF7 cells and that SPARC promoter activation by c-Jun appears to occur through an indirect mechanism.

Beta4 integrin promotes osteosarcoma metastasis and interacts with ezrin

The development of pulmonary metastasis is the major cause of death in osteosarcoma, and its molecular basis is poorly understood. In this study, we show that β4 integrin is highly expressed in human osteosarcoma cell lines and tumor samples. Furthermore, highly metastatic MNNG-HOS cells have increased levels of β4 integrin. Suppression of β4 integrin expression by shRNA and disruption of β4 integrin function by transfection of dominant-negative β4 integrin was sufficient to revert this highly metastatic phenotype in the MNNG-HOS model without significantly affecting primary tumor growth. These findings suggest a role for β4 integrin expression in the metastatic phenotype in human osteosarcoma cells. In addition, we identified a previously uncharacterized interaction between β4 integrin and ezrin, a membrane-cytoskeletal linker protein that is implicated in the metastatic behavior of osteosarcoma. The β4 integrin–ezrin interaction appears to be critical for maintenance of β4 integrin expression. These data begin to integrate ezrin and β4 integrin expression into a model of action for the mechanism of osteosarcoma metastases.

Modeling metastasis biology and therapy in real time in the mouse lung

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.

Dysregulation of Ezrin Phosphorylation Prevents Metastasis and Alters Cellular Metabolism in Osteosarcoma

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.

A Compendium of Canine Normal Tissue Gene Expression

Background Our understanding of disease is increasingly informed by changes in gene expression between normal and abnormal tissues. The release of the canine genome sequence in 2005 provided an opportunity to better understand human health and disease using the dog as clinically relevant model. Accordingly, we now present the first genome-wide, canine normal tissue gene expression compendium with corresponding human cross-species analysis. Methodology/Principal Findings The Affymetrix platform was utilized to catalogue gene expression signatures of 10 normal canine tissues including: liver, kidney, heart, lung, cerebrum, lymph node, spleen, jejunum, pancreas and skeletal muscle. The quality of the database was assessed in several ways. Organ defining gene sets were identified for each tissue and functional enrichment analysis revealed themes consistent with known physio-anatomic functions for each organ. In addition, a comparison of orthologous gene expression between matched canine and human normal tissues uncovered remarkable similarity. To demonstrate the utility of this dataset, novel canine gene annotations were established based on comparative analysis of dog and human tissue selective gene expression and manual curation of canine probeset mapping. Public access, using infrastructure identical to that currently in use for human normal tissues, has been established and allows for additional comparisons across species. Conclusions/Significance These data advance our understanding of the canine genome through a comprehensive analysis of gene expression in a diverse set of tissues, contributing to improved functional annotation that has been lacking. Importantly, it will be used to inform future studies of disease in the dog as a model for human translational research and provides a novel resource to the community at large.

v-Jun downregulates the SPARC target gene by binding to the proximal promoter indirectly through sp1/3

Transformation of chick embryo fibroblasts by the v-Jun oncoprotein correlates with a downregulation of the extracellular matrix protein SPARC and repression of the corresponding mRNA. Repression of SPARC contributes to the oncogenic process by facilitating tumor development in vivo. A proximal promoter fragment, designated −124/+16, is responsible for high constitutive activity of the SPARC gene and is the target of repression by v-Jun. In this paper, using electrophoretic mobility shift and pull-down assays in vitro, and transient transfections and chromatin immunoprecipitation assays in Sp1/3-deficient Drosophila SL2 cells and in chick embryo fibroblasts, we show that (i) Sp1 and/or Sp3 is required for constitutive activation of SPARC transcription, by binding directly to the GGA-rich −92/−57 fragment; and (ii) v-Jun does not bind −124/+16 directly, but binds to the GGA-rich fragment indirectly, most likely through a physical interaction with Sp1/3. Moreover, a transactivation-proficient v-Jun derivative, designated v-Jun/cebp/glz, which cannot bind Jun DNA motifs anymore and cannot heterodimerize, is still capable of downregulating SPARC efficiently. Taken together, these data strongly suggest that v-Jun downregulates SPARC through the formation of a DNA–Sp1/3–v-Jun, chromatin-associated complex.

Protein kinase C regulates ezrin–radixin–moesin phosphorylation in canine osteosarcoma cells

The development of metastasis is the most significant cause of death for both canine and human patients with osteosarcoma (OS). Ezrin has been associated with tumour progression and metastasis in human, canine and murine OS. Ezrin activation is dynamically regulated by protein kinase C (PKC) during metastatic progression in human and murine OS. To include the dog in the development of therapeutics that target ezrin biology, we characterized four new canine OS cell lines and confirmed the relationship between PKC and ezrin in these cells. Three of four cell lines formed tumours in mice that were histologically consistent with OS. All cell lines were markedly aneuploid and expressed ezrin and PKC. Finally, both ezrin phosphorylation and cell migration were inhibited using a PKC inhibitor. These data suggest that an association between PKC-mediated activation of ezrin and the metastatic phenotype in canine OS cells.

Murine osteosarcoma primary tumour growth and metastatic progression is maintained after marked suppression of serum insulin-like growth factor I

The insulin‐like growth factor I (IGF‐I) signaling pathway has been shown to play an important role in several aspects of cancer biology, including metastasis. The aim of this study was to define the contribution of serum (endocrine) and local (tumour microenvironment) IGF‐I on osteosarcoma tumour growth and metastasis, a cancer that is known to be dependent on the IGF‐I axis. To test this hypothesis, we evaluated the primary tumour growth and metastatic progression of K7M2 murine osteosarcoma cells injected to a genetically engineered mouse [liver‐specific IGF‐I deficient (LID)] in which serum IGF‐I levels are reduced by 75%, while maintaining expression of IGF‐I in normal tissues. We first demonstrated that IGF‐I in the tumour and the tumour‐microenvironment were maintained in the LID mice. Within this designed model, there was no difference in primary tumour growth or in pulmonary metastasis in LID mice compared to control mice. Furthermore, there was no difference in the number or localization of single metastatic cells immediately after their arrival in the lungs of LID mice and control mice, as analysed by single cell video microscopy. Collectively, these data suggest that marked reduction in serum IGF‐I is not sufficient to slow the progression of either primary or metastatic models of osteosarcoma.

Characterization of the metastatic phenotype of a panel of established osteosarcoma cells

Osteosarcoma (OS) is the most common bone tumor in pediatric patients. Metastasis is a major cause of mortality and morbidity. The rarity of this disease coupled with the challenges of drug development for metastatic cancers have slowed the delivery of improvements in long-term outcomes for these patients. In this study, we collected 18 OS cell lines, confirmed their expression of bone markers and complex karyotypes, and characterized their in vivo tumorgenicity and metastatic potential. Since prior reports included conflicting descriptions of the metastatic and in vivo phenotypes of these models, there was a need for a comparative assessment of metastatic phenotypes using identical procedures in the hands of a single investigative group. We expect that this single characterization will accelerate the study of this metastatic cancer. Using these models we evaluated the expression of six previously reported metastasis-related OS genes. Ezrin was the only gene consistently differentially expressed in all the pairs of high/low metatstatic OS cells. We then used a subtractive gene expression approach of the high and low human metastatic cells to identify novel genes that may be involved in OS metastasis. PHLDA1 (pleckstrin homology-like domain, family A) was identified as one of the genes more highly expressed in the high metastatic compared to low metastatic cells. Knocking down PHLDA1 with siRNA or shRNA resulted in down regulation of the activities of MAPKs (ERK1/2), c-Jun N-terminal kinases (JNK), and p38 mitogen-activated protein kinases (MAPKs). Reducing the expression of PHLDA1 also delayed OS metastasis progression in mouse xenograft models.

The T-cell leukemia-associated ribosomal RPL10 R98S mutation enhances JAK-STAT signaling

Several somatic ribosome defects have recently been discovered in cancer, yet their oncogenic mechanisms remain poorly understood. Here we investigated the pathogenic role of the recurrent R98S mutation in ribosomal protein L10 (RPL10 R98S) found in T-cell acute lymphoblastic leukemia (T-ALL). The JAK-STAT signaling pathway is a critical controller of cellular proliferation and survival. A proteome screen revealed overexpression of several Jak-Stat signaling proteins in engineered RPL10 R98S mouse lymphoid cells, which we confirmed in hematopoietic cells from transgenic Rpl10 R98S mice and T-ALL xenograft samples. RPL10 R98S expressing cells displayed JAK-STAT pathway hyper-activation upon cytokine stimulation, as well as increased sensitivity to clinically used JAK-STAT inhibitors like pimozide. A mutually exclusive mutation pattern between RPL10 R98S and JAK-STAT mutations in T-ALL patients further suggests that RPL10 R98S functionally mimics JAK-STAT activation. Mechanistically, besides transcriptional changes, RPL10 R98S caused reduction of apparent programmed ribosomal frameshifting at several ribosomal frameshift signals in mouse and human Jak-Stat genes, as well as decreased Jak1 degradation. Of further medical interest, RPL10 R98S cells showed reduced proteasome activity and enhanced sensitivity to clinical proteasome inhibitors. Collectively, we describe modulation of the JAK-STAT cascade as a novel cancer-promoting activity of a ribosomal mutation, and expand the relevance of this cascade in leukemia.