Breast cancer progression is likely a multistep process involving the activation and inactivation of a number of genes. Previously, we showed that the mRNA coding for Fra-1, a FOS family member and an AP-1 transcription factor component, was highly expressed in the more invasive estrogen receptor negative (ERÀ) breast cancer cell lines. We used a tet-off system to stably overexpress Fra-1 in MCF7 ER þ cells and evaluate the impact of Fra-1 on this aggressive phenotype. Conversely, Fra-1 was silenced in highly invasive ERÀMDA-MB231 cells using RNA interference. We report that in both systems the Fra-1 expression level was positively associated with cell proliferation, cell motility and invasiveness assessed in vitro. In addition, Fra-1 inhibition in fibroblastoid ERÀ cells, which formed colonies with large stellate projections in Matrigel, resulted in morphological changes. Cells acquired an epithelioid shape and had a spherical appearance in Matrigel. Fra-1 regulated several genes, implicated in invasion, angiogenesis and cell proliferation independently of b1-integrin activation, and directly induced MMP-1 and MMP-9 promoter activity. These overall results show that high Fra-1 expression is associated with a more malignant cell phenotype and suggest that Fra-1 could have a pivotal role in breast cancer progression.

Aberrant constitutive expression of c-Rel, p65 and p50 NF-kappaB subunits has been reported in over 90% of breast cancers. Recently, we characterized a de novo RelB NF-kappaB subunit synthesis pathway, induced by the cytomegalovirus (CMV) IE1 protein, in which binding of p50-p65 NF-kappaB and c-Jun-Fra-2 AP-1 complexes to the RELB promoter work in synergy to potently activate transcription. Although RelB complexes were observed in mouse mammary tumours induced by either ectopic c-Rel expression or carcinogen exposure, little is known about RelB in human breast disease. Here, we demonstrate constitutive de novo RelB synthesis is selectively active in invasive oestrogen receptor alpha (ERalpha)-negative breast cancer cells. ERalpha signalling reduced levels of functional NF-kappaB and Fra-2 AP-1 and inhibited de novo RelB synthesis, leading to an inverse correlation between RELB and ERalpha gene expression in human breast cancer tissues and cell lines. Induction of Bcl-2 by RelB promoted the more invasive phenotype of ERalpha-negative cancer cells. Thus, inhibition of de novo RelB synthesis represents a new mechanism whereby ERalpha controls epithelial to mesenchymal transition (EMT).

Interleukin-6 (IL-6), involved in cancer-related inflammation, acts as an autocrine and paracrine growth factor, which promotes angiogenesis, metastasis, and subversion of immunity, and changes the response to hormones and to chemotherapeutics.

Fra-1, a transcription factor that is phylogenetically and functionally related to the proto-oncoprotein c-Fos, controls many essential cell functions. It is expressed in many cell types, albeit with differing kinetics and abundances. In cells reentering the cell cycle, Fra-1 expression is transiently stimulated albeit later than that of c-Fos and for a longer time. Moreover, Fra-1 overexpression is found in cancer cells displaying high Erk1/2 activity and has been linked to tumorigenesis. One crucial point of regulation of Fra-1 levels is controlled protein degradation, the mechanism of which remains poorly characterized. Here, we have combined genetic, pharmacological, and signaling studies to investigate this process in nontransformed cells and to elucidate how it is altered in cancer cells. We report that the intrinsic instability of Fra-1 depends on a single destabilizer contained within the C-terminal 30 to 40 amino acids. Two serines therein, S252 and S265, are phosphorylated by kinases of the Erk1/2 pathway, which compromises protein destruction upon both normal physiological induction and tumorigenic constitutive activation of this cascade. Our data also indicate that Fra-1, like c-Fos, belongs to a small group of proteins that may, under certain circumstances, undergo ubiquitin-independent degradation by the proteasome. Our work reveals both similitudes and differences between Fra-1 and c-Fos degradation mechanisms. In particular, the presence of a single destabilizer within Fra-1, instead of two that are differentially regulated in c-Fos, explains the much faster turnover of the latter when cells traverse the G 0 /G 1 -to-S-phase transition. Finally, our study offers further insights into the signaling-regulated expression of the other Fos family proteins.

We have recently reported that interleukin-8 (IL-8) expression was inversely correlated to estrogen receptor (ER) status and was overexpressed in invasive breast cancer cells. In the present study, we show that IL-8 overexpression in breast cancer cells involves a higher transcriptional activity of IL-8 gene promoter. Cloning of IL-8 promoter from MDA-MB-231 and MCF-7 cells expressing high and low levels of IL-8, respectively, shows the integrity of the promoter in both cell lines. Deletion and site-directed mutagenesis of the promoter demonstrate that NF-jB and AP-1 and to a lesser extent C/EBP binding sites play a crucial role in the control of IL-8 promoter activity in MDA-MB-231 cells. Knockdown of NF-jB and AP-1 activities by adenovirusmediated expression of an NF-jB super-repressor and RNA interference, respectively, decreased IL-8 expression in MDA-MB-231 cells. On the contrary, restoration of Fra-1, Fra-2, c-Jun, p50, p65, C/EBPa and C/EBPb expression levels in MCF-7 cells led to a promoter activity comparable to that observed in MDA-MB-231 cells. Our data constitute the first extensive study of IL-8 gene overexpression in breast cancer cells and suggest that the high expression of IL-8 in invasive cancer cells requires a complex cooperation between NF-jB, AP-1 and C/EBP transcription factors.

Purpose: AXL receptor tyrosine kinase has been described as a relevant molecular marker and a key player in invasiveness, especially in triple-negative breast cancer (TNBC).Experimental Design: We evaluate the antitumor efficacy of the anti-AXL monoclonal antibody 20G7-D9 in several TNBC cell xenografts or patient-derived xenograft (PDX) models and decipher the underlying mechanisms. In a dataset of 254 basal-like breast cancer samples, genes correlated with AXL expression are enriched in EMT, migration, and invasion signaling pathways.Results: Treatment with 20G7-D9 inhibited tumor growth and bone metastasis formation in AXL-positive TNBC cell xenografts or PDX, but not in AXL-negative PDX, highlighting AXL role in cancer growth and invasion. In vitro stimulation of AXL-positive cancer cells by its ligand GAS6 induced the expression of several EMT-associated genes (SNAIL, SLUG, and VIM) through an intracellular signaling implicating the transcription factor FRA-1, important in cell invasion and plasticity, and increased their migration/invasion capacity. 20G7-D9 induced AXL degradation and inhibited all AXL/GAS6-dependent cell signaling implicated in EMT and in cell migration/invasion.Conclusions: The anti-AXL antibody 20G7-D9 represents a promising therapeutic strategy in TNBC with mesenchymal features by inhibiting AXL-dependent EMT, tumor growth, and metastasis formation.

Fibulin-1 is an extracellular matrix protein induced by estradiol in estrogen receptor (ER) positive ovarian cancer cell lines. Alternative splicing of ®bulin-1 mRNA results in four di erent variants named A, B, C and D that may have distinct biological functions. We studied the relative expression of ®bulin-1 mRNA variants and their estrogen regulation in human ovarian cancer cells. In ovarian tissues and cancer cell lines, ®bulin-1C and -1D are the predominant forms, whereas ®bulin-1A and -1B are weakly expressed. We developed a competitive PCR assay based on coampli®cation of ®bulin-1C and -1D to study the relative expression of these ®bulin-1 variants in human ovarian samples. In ovarian cancer cell lines and ovarian cancer samples, there was a marked increase in the ®bulin-1C:1D and ®bulin-1C:HPRT mRNA ratios as compared to normal ovaries. In the BG1 estrogen receptor positive ovarian cancer cell line, ®bulin-1C mRNA was induced by estradiol in a dose-and time-dependent manner. Since others and we have previously shown an increased expression of ERa as compared to ERb in ovarian cancer cells, we investigated whether ERa or ERb is involved in this induction. For this aim, MDA-MB-231 breast cancer cell line, which expresses both low basal levels of ERs and ®bulin-1, was infected with recombinant ERa or ERb encoding adenovirus and treated with estradiol. Fibulin-1C was induced by estradiol in ERa-but not ERb-infected cells, suggesting that ®bulin-1C induction is mediated through ERa. In ovarian tumors, a trend towards a correlation between ®bulin-1C and ERa expression levels was noted. In conclusion, this study showed an increased ®bulin-1C: -1D mRNA ratio in ovarian cancer cells as compared to normal ovaries. This ®nding suggests that the C variant may be involved in ovarian carcinogenesis. Fibulin-1C overexpression may thus be a clue for the understanding of a putative role of estrogens in ERa promoted ovarian tumor progression.

Aberrant constitutive expression of NF-B subunits, reported in more than 90% of breast cancers and multiple other malignancies, plays pivotal roles in tumorigenesis. Higher RelB subunit expression was demonstrated in estrogen receptor alpha (ER␣)-negative breast cancers versus ER␣-positive ones, due in part to repression of RelB synthesis by ER␣ signaling. Notably, RelB promoted a more invasive phenotype in ER␣-negative cancers via induction of the BCL2 gene. We report here that RelB reciprocally inhibits ER␣ synthesis in breast cancer cells, which contributes to a more migratory phenotype. Specifically, RelB is shown for the first time to induce expression of the zinc finger repressor protein Blimp1 (B-lymphocyte-induced maturation protein), the critical mediator of Band T-cell development, which is transcribed from the PRDM1 gene. Blimp1 protein repressed ER␣ (ESR1) gene transcription. Commensurately higher Blimp1/PRDM1 expression was detected in ER␣-negative breast cancer cells and primary breast tumors. Induction of PRDM1 gene expression was mediated by interaction of Bcl-2, localized in the mitochondria, with Ras. Thus, the induction of Blimp1 represents a novel mechanism whereby the RelB NF-B subunit mediates repression, specifically of ER␣, thereby promoting a more migratory phenotype.NF-B is a structurally and evolutionary conserved family of dimeric transcription factors with subunits having an N-terminal region of approximately 300 amino acids that shares homology with the v-Rel oncoprotein (17, 44). The conserved Rel homology domain is responsible for DNA binding, dimerization, nuclear translocation, and interaction with inhibitory proteins of NF-B (IBs). Mammals express five NF-B members, including c-Rel, RelB, RelA (p65), p50, and p52, which can form either homo-or heterodimers. RelB differs from the other members in that it only binds DNA as a heterodimer with either p52 or p50 and interacts only poorly with the inhibitory protein IB␣. In most untransformed cells, other than B lymphocytes, NF-B complexes are sequestered in the cytoplasm bound to specific IB proteins.