Timothy J. Bos

jun:Oncogene and Transcription Factor

Publisher Summary This chapter discusses the structure of the cellular and retroviral jun genes and their relationship to other transcriptional regulators. The interaction between the jun and fos proteins, and the control and oncogenic potential of jun are also presented. In the cell the fos protein is associated with a cellular protein, p 39. Together with p 39, the fos binds sequence specifically to DNA and can trans-activate genes. By itself, however, the fos protein does not show specific DNA binding. DNA affinity precipitation and gel retardation assays in conjunction with immunoblots of the bound proteins have shown that the target site for the fos – p 39 complex is the AP-1 consensus sequence. The p 39 precipitated from the cell with anti- jun sera and p 39 precipitated with anti- fos serum, as part of the fos – p 39 complex have identical tryptic peptide maps. Therefore, p 39 and jun are considered to be identical.

cJun overexpression in MCF-7 breast cancer cells produces a tumorigenic, invasive and hormone resistant phenotype

We have previously demonstrated decreased Jun/AP-1 activity in the breast cancer cell line MCF-7 when compared to normal or immortalized mammary epithelial cells. In this paper, we overexpress Jun in MCF-7 cells (MCF7Jun) and demonstrate that it results in diverse biologic and biochemical changes, which mimic those seen clinically in breast cancer. Overexpression of Jun causes significant alterations in the composition of AP-1, decreased junB and increased fra-1 expression and results in an increased biologic aggressiveness. MCF7Jun cells exhibit increased cellular motility, increased expression of a matrix degrading enzyme MMP-9, increased in vitro chemoinvasion and tumor formation in nude mice in the absence of exogenous estrogens. Furthermore, MCF7Jun cells are unresponsive to the growth stimulating effects of estrogen and growth inhibitory effects of tamoxifen. Analysis of the estrogen receptor (ER) expression and activity showed that the MCF7Jun cells have no detectable ER. MCF-7 cells overexpressing mutant forms of cJun were responsive to the growth stimulatory effects of estrogen indicating that full-length cJun is required to acquire the estrogen-independent phenotype in breast cancer cells.

v-jun encodes a nuclear protein with enhancer binding properties of AP-1

The jun oncogene of ASV17 is expressed as a 65 kd protein (p65gag-jun) that contains partial gag sequences at its amino terminus fused to jun sequences that make up the carboxy terminal two-thirds of the molecule. As a first step toward evaluating potential functional differences between the activated oncogene, v-jun, and its cellular counterpart, c-jun, we have characterized the biochemical properties of the gag-jun product of ASV17. Immunofluorescence studies revealed that the v-jun protein is localized in the nucleus of CEF transfected with ASV17 DNA. DNAase I foot-printing analysis indicates that p65gag-jun synthesized in bacteria binds to enhancer elements in SV40 that are recognition sites for the human transcription factor AP-1. Analysis of point mutants confirmed that v-jun protein binds with DNA sequence specificity of the mammalian enhancer factor AP-1 and the yeast transcription factor GCN4. These findings suggest that activation of the jun oncogene may not exclusively be the result of alterations in the DNA binding properties of the normal cellular protein.

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.

The oncogene jun and nuclear signalling

Jun is a transcription factor that can also induce oncogenic transformation. Its DNA-binding domain is conserved from yeast to man and shows homology to several other transcriptional regulators. Jun dimerizes with the fos protein through an alpha-helical domain termed the leucine zipper, and the jun-fos heterodimers bind to DNA and regulate transcription of numerous specific unlinked genes.

Alterations in the gene expression profile of MCF‐7 breast tumor cells in response to c‐Jun

MCF7 breast tumor cells overexpressing human c‐Jun exhibit a transformed phenotype characterized not only by increased tumorigenicity but also by enhanced motility and invasion. The cellular phenotypic response to c‐Jun overexpression is likely due, at least in part, to altered patterns of gene expression. In order to begin to understand the complexities by which elevated production of c‐Jun alters the state of the cell, we have profiled the expression of 588 different genes by comparative hybridization. By using this approach, we have identified a total of 21 upregulated or downregulated gene targets responsive to c‐Jun overexpression. Interestingly, 8 of these genes have been previously found associated with c‐Jun or AP‐1 activity and therefore provide internal validation for this approach to target gene discovery. The remaining 13 genes represent potential new c‐Jun regulated target genes. Genomic sequence information was available for 15 of the 21 genes identified in this screen. Analysis of these genomic sequences revealed the presence of AP‐1 or AP‐1‐like sequences in 12 of the 15 genes examined. Consistent with a direct mechanism of target regulation by c‐Jun, gel shift analysis of selected AP‐1‐containing promoter regions revealed elevated and specific binding by proteins present in nuclear extracts of c‐Jun expressing MCF7 cells. Int. J. Cancer 88:180–190, 2000.

Enhanced cell motility and invasion of chicken embryo fibroblasts in response to JUN over‐expression

Malignant tumor cells exhibit a number of distinct properties involved not only with deregulated cell proliferation but also enhanced migration and invasion. The Jun oncogene has been well studied in regard to its role in cell proliferation. Many of the target genes deregulated by Jun encode matrix metalloproteases (MMPs) such as MMP1, MMP3 and MMP9. These targets implicate a prominent role for Jun in tumor cell invasion, in addition to its role in growth transformation. To investigate this possibility, we have examined the effect of over‐expression of transforming and non‐transforming versions of Jun on motility and invasion of chicken embryo fibroblasts (CEFs). We found that over‐expression of either form of Jun results in elevated intrinsic cellular motility as well as increased motility in response to several different chemo‐attractants, including 3T3‐conditioned media, basic fibroblast growth factor, hepatocyte growth factor and Matrigel. The capacity of these cells to invade through Matrigel is also elevated as a result of Jun over‐expression. In addition to these effects, CEFs expressing Jun secrete factors that stimulate the motility of a human tongue carcinoma cell line. Our results suggest that Jun plays an important role in the potentiation of cell motility and invasion through multiple mechanisms. Int. J. Cancer 81:404–410, 1999.

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.

The chicken c-Jun 5' untranslated region directs translation by internal initiation.

The 5′ untranslated region (UTR) of the chicken c-jun message is exceptionally GC rich and has the potential to form a complex and extremely stable secondary structure. Because stable RNA secondary structures can serve as obstacles to scanning ribosomes, their presence suggests inefficient translation or initiation through alternate mechanisms. We have examined the role of the c-jun 5′ UTR with respect to its ability to influence translation both in vitro and in vivo. We find, using rabbit reticulocyte lysates, that the presence of the c-jun 5′ UTR severely inhibits translation of both homologous and heterologous genes in vitro. Furthermore, translational inhibition correlates with the degree of secondary structure exhibited by the 5′ UTR. Thus, in the rabbit reticulocyte lysate system, the c-jun 5′ UTR likely impedes ribosome scanning resulting in inefficient translation. In contrast to our results in vitro, the c-jun 5′ UTR does not inhibit translation in a variety of different cell lines suggesting that it may direct an alternate mechanism of translational initiation in vivo. To distinguish among the alternate mechanisms, we generated a series of bicistronic expression plasmids. Our results demonstrate that the downstream cistron, in the bicistronic gene, is expressed to a much higher level when directly preceded by the c-jun 5′ UTR. In addition, inhibition of ribosome scanning on the bicistronic message, through insertion of a synthetic stable hairpin, inhibits translation of the first cistron but does not inhibit translation of the cistron downstream of the c-jun 5′ UTR. These results are consistent with a model by which the c-jun message is translated through cap independent internal initiation.

Apolipoprotein A-1 is a negative target of v-Jun overexpression.

The product of the Jun oncogene influences a variety of processes including cell proliferation and differentiation. Jun exerts its influence by binding to the promoter and enhancer regions of a number of different target genes resulting in their activation or repression. We describe here the isolation and characterization of a gene differentially downregulated upon overexpression of v-Jun but not c-Jun. DNA and amino acid homology search analysis revealed this gene to be identical to chicken apolipoprotein A-1, the major component of high density lipoprotein (HDL). The half life of apolipoprotein A-1 RNA remains constant in the presence or absence of v-Jun overexpression suggesting downregulation by v-Jun is at the level of promoter activity. Consistent with this hypothesis, apolipoprotein A-1 upstream promoter fragments active in normal and c-Jun expressing CEF are inactive in v-Jun transformed CEF. Analysis of expression of apolipoprotein A-1 in CEF overexpressing other oncogenes revealed a similar downregulation by Myc and v-Src but not c-Fos, v-Ha-Ras, c-Src or c-Ski. Our findings point to a potential regulatory affect on cholesterol metabolism by v-Jun, as a result of altered levels of apolipoprotein A-1 message expression.