The AP-1 transcription factor JunB is essential for multiple myeloma cell proliferation and drug resistance in the bone marrow microenvironment.

Despite therapeutic advances, multiple myeloma (MM) remains an incurable disease, predominantly because of the development of drug resistance. The activator protein-1 (AP-1) transcription factor family has been implicated in a multitude of physiologic processes and tumorigenesis; however, its role in MM is largely unknown. Here we demonstrate specific and rapid induction of the AP-1 family member JunB in MM cells when co-cultured with bone marrow stromal cells. Supporting a functional key role of JunB in MM pathogenesis, knockdown of JUNB significantly inhibited in vitro MM cell proliferation and survival. Consistently, induced silencing of JUNB markedly decreased tumor growth in a murine MM model of the microenvironment. Subsequent gene expression profiling revealed a role for genes associated with apoptosis, DNA replication and metabolism in driving the JunB-mediated phenotype in MM cells. Importantly, knockdown of JUNB restored the response to dexamethasone in dexamethasone-resistant MM cells. Moreover, 4-hydroxytamoxifen-induced activation of a JunB-ER fusion protein protected dexamethasone-sensitive MM cells against dexamethasone- and bortezomib-induced cytotoxicity. In summary, our results demonstrate for the first time a specific role for AP-1/JunB in MM cell proliferation, survival and drug resistance, thereby strongly supporting that this transcription factor is a promising new therapeutic target in MM.

Epidermal-specific deletion of CD44 reveals a function in keratinocytes in response to mechanical stress.

CD44, a large family of transmembrane glycoproteins, plays decisive roles in physiological and pathological conditions. CD44 isoforms are involved in several signaling pathways essential for life such as growth factor-induced signaling by EGF, HGF or VEGF. CD44 is also the main hyaluronan (HA) receptor and as such is involved in HA-dependent processes. To allow a genetic dissection of CD44 functions in homeostasis and disease, we generated a Cd44 floxed allele allowing tissue- and time-specific inactivation of all CD44 isoforms in vivo. As a proof of principle, we inactivated Cd44 in the skin epidermis using the K14Cre allele. Although the skin of such Cd44Δker mutants appeared morphologically normal, epidermal stiffness was reduced, wound healing delayed and TPA induced epidermal thickening decreased. These phenotypes might be caused by cell autonomous defects in differentiation and HA production as well as impaired adhesion and migration on HA by Cd44Δker keratinocytes. These findings support the usefulness of the conditional Cd44 allele in unraveling essential physiological and pathological functions of CD44 isoforms.

Fra-1/AP-1 induces EMT in mammary epithelial cells by modulating Zeb1/2 and TGFß expression.

Epithelial-to-mesenchymal transition (EMT) is essential for embryonic morphogenesis and wound healing and critical for tumour cell invasion and dissemination. The AP-1 transcription factor Fra-1 has been implicated in tumorigenesis and in tumour-associated EMT in human breast cancer. We observed a significant inverse correlation between Fra-1 mRNA expression and distant-metastasis-free survival in a large cohort of breast cancer patients derived from multiple array data sets. This unique correlation among Fos genes prompted us to assess the evolutionary conservation between Fra-1 functions in EMT of human and mouse cells. Ectopic expression of Fra-1 in fully polarized, non-tumourigenic, mouse mammary epithelial EpH4 cells induced a mesenchymal phenotype, characterized by a loss of epithelial and gain of mesenchymal markers. Proliferation, motility and invasiveness were also increased in the resulting EpFra1 cells, and the cells were tumourigenic and efficiently colonized the lung upon transplantation. Molecular analyses revealed increased expression of Tgfß1 and the EMT-inducing transcription factors Zeb1, Zeb2 and Slug. Mechanistically, Fra-1 binds to the tgfb1 and zeb2 promoters and to an evolutionarily conserved region in the first intron of zeb1. Furthermore, increased activity of a zeb2 promoter reporter was detected in EpFra1 cells and shown to depend on AP-1-binding sites. Inhibiting TGFß signalling in EpFra1 cells moderately increased the expression of epithelial markers, whereas silencing of zeb1 or zeb2 restored the epithelial phenotype and decreased migration in vitro and tumorigenesis in vivo. Thus Fra-1 induces changes in the expression of genes encoding EMT-related transcription factors leading to the acquisition of mesenchymal, invasive and tumorigenic capacities by epithelial cells. This study defines a novel function of Fra-1/AP-1 in modulating tgfb1, zeb1 and zeb2 expression through direct binding to genomic regulatory regions, which establishes a basis for future in vivo genetic manipulations and preclinical studies using mouse models.

Loss of JUNB/AP-1 promotes invasive prostate cancer.

Prostate cancer is a frequent cause of male death in the Western world. Relatively few genetic alterations have been identified, likely owing to disease heterogeneity. Here, we show that the transcription factor JUNB/AP-1 limits prostate cancer progression. JUNB expression is increased in low-grade prostate cancer compared with normal human prostate, but downregulated in high-grade samples and further decreased in all metastatic samples. To model the hypothesis that this downregulation is functionally significant, we genetically inactivated Junb in the prostate epithelium of mice. When combined with Pten (phosphatase and tensin homologue) loss, double-mutant mice were prone to invasive cancer development. Importantly, invasive tumours also developed when Junb and Pten were inactivated in a small cell population of the adult anterior prostate by topical Cre recombinase delivery. The resulting tumours displayed strong histological similarity with human prostate cancer. Loss of JunB expression led to increased proliferation and decreased senescence, likely owing to decreased p16(Ink4a) and p21(CIP1) in epithelial cells. Furthermore, the tumour stroma was altered with increased osteopontin and S100 calcium-binding protein A8/9 expression, which correlated with poor prognoses in patients. These data demonstrate that JUNB/AP-1 cooperates with PTEN signalling as barriers to invasive prostate cancer, whose concomitant genetic or epigenetic suppression induce malignant progression.

Pancreatic ß-cells activate a JunB/ATF3-dependent survival pathway during inflammation.

Destruction of insulin-producing pancreatic ß-cells by local autoimmune inflammation is a hallmark of type 1 diabetes. Histochemical analysis of pancreases from non-obese diabetic mice indicated activation of the transcription factor JunB/AP-1 (activator protein-1) after autoimmune infiltration of the islets. In vitro studies demonstrated that the cytokines tumor necrosis factor (TNF)-α and interferon (IFN)-γ induce JunB expression as a protective mechanism against apoptosis in both human and rodent ß-cells. The gene network affected was studied by microarray analysis showing that JunB regulates nearly 20% of the cytokine-modified ß-cell genes, including the transcription factor ATF3. Direct transcriptional induction of ATF3 by JunB is a key event for ß-cell survival after TNF-α+IFN-γ treatment. Moreover, pharmacological upregulation of JunB/ATF3 via increased cAMP protected rodent primary ß-cells and human islet cells against pro-inflammatory mediators. These results were confirmed in genetically modified islets derived from Ubi-JunB transgenic mice. Our findings identify ATF3 as a novel downstream target of JunB in the survival mechanism of ß-cells under inflammatory stress.

Functions of Fos phosphorylation in bone homeostasis, cytokine response and tumourigenesis.

Mice lacking c-fos develop osteopetrosis due to a block in osteoclast differentiation. Carboxy-terminal phosphorylation of Fos on serine 374 by ERK1/2 and serine 362 by RSK1/2 regulates Fos stability and transactivation potential in vitro. To assess the physiological relevance of Fos phosphorylation in vivo, serine 362 and/or serine 374 was replaced by alanine (Fos362A, Fos374A and FosAA) or by phospho-mimetic aspartic acid (FosDD). Homozygous mutants were healthy and skeletogenesis was largely unaffected. Fos C-terminal phosphorylation, predominantly on serine 374, was found important for osteoclast differentiation in vitro and affected lipopolysaccharide (LPS)-induced cytokine response in vitro and in vivo. Importantly, skin papilloma development was delayed in FosAA, Fos362A and Rsk2-deficient mice, accelerated in FosDD mice and unaffected in Fos374A mutants. Furthermore, the related Fos protein and putative RSK2 target Fra1 failed to substitute for Fos in papilloma development. This indicates that phosphorylation of serines 362 and 374 exerts context-dependent roles in modulating Fos activity in vivo. Inhibition of Fos C-terminal phosphorylation on serine 362 by targeting RSK2 might be of therapeutic relevance for skin tumours.

Targeting c-Jun and JunB proteins as potential anticancer cell therapy.

The activating protein-1 transcription factor, in particular the Jun proteins play critical roles in the regulation of cell proliferation and tumor progression. To study the potential clinical relevance of interfering with JunB expression, we generated retroviruses expressing short hairpin RNA. Reduction of JunB levels causes increased proliferation and tumorigenicity in wild-type murine fibroblasts, whereas in c-Jun knockout cells p53-independent cell cycle arrest and apoptosis are induced. Using melanoma-derived B16-F10 cancer cells the combination of JunB knockdown and c-Jun/JNK inactivation leads to cell cycle arrest and apoptosis-inducing factor-dependent apoptosis. Furthermore, the combined treatment extends survival of mice inoculated with the tumor cells. These results indicate that in the absence of c-Jun, JunB can act as a tumor promoter and inactivation of both, c-Jun and JunB, could provide a valuable strategy for antitumor intervention.

Functional cooperation between JunD and NF-kappaB in rat hepatocytes.

AP-1 and NF-kappaB are rapidly activated during liver regeneration. Whether these parallel inductions have potential functional implications is not known. Isolated rat hepatocytes were stimulated with two mitogens, epidermal growth factor or hepatocyte growth factor and with tumor necrosis factor alpha, a cytokine involved in the liver regenerative response in vivo and a strong inducer of NF-kappaB. All three cytokines increased AP-1 and NF-kappaB binding to their cognate cis-element and induced a 2.5-fold activation of NF-kappaB-dependent transcription. Inactivation of AP-1 by TAM67, a dominant negative mutant of AP-1 drastically inhibited basal and cytokine-induced NF-kappaB transactivation. Overexpression of Jun D, but not of the other Jun or Fos proteins increased by threefold NF-kappaB transactivation. Functional cooperation between JunD and p65 was demonstrated in a simple Gal-hybrid system. Finally, a twofold decrease in NF-kappaB transactivation was found in hepatocytes isolated from JunD(-/-) mice compared with hepatocytes from JunD(+/+) mice. Altogether these data demonstrate a functional cooperation of p65 with JunD, a major constituent of AP-1 in normal hepatocytes.

Cell cycle-dependent variations in c-Jun and JunB phosphorylation: a role in the control of cyclin D1 expression.

The transcription factor AP-1, composed of Jun and Fos proteins, is a major target of mitogen-activated signal transduction pathways. However, little is known about AP-1 function in normal cycling cells. Here we report that the quantity and the phosphorylation state of the c-Jun and JunB proteins vary at the M-G(1) transition. Phosphorylation of JunB by the p34(cdc2)-cyclin B kinase is associated with lower JunB protein levels in mitotic and early G(1) cells. In contrast, c-Jun levels remain constant while the protein undergoes N-terminal phosphorylation, increasing its transactivation potential. Since JunB represses and c-Jun activates the cyclin D1 promoter, these modifications of AP-1 activity during the M-G(1) transition could provide an impetus for G(1) progression by a temporal increase in cyclin D1 transcription. These findings constitute a novel example of a reciprocal connection between transcription factors and the cell cycle machinery.

Stress-activated protein kinases are negatively regulated by cell density.

Stimulation by UV irradiation, TNFalpha, as well as PDGF or EGF activates the JNK/SAPK signalling pathway in mouse fibroblasts. This results in the phosphorylation of the N-terminal domain of c-Jun, increasing its transactivation potency. Using an antibody that specifically recognizes c-Jun phosphorylated at Ser63, we show that culture confluency drastically inhibited c-Jun N-terminal phosphorylation due to the inhibition of the JNK/SAPK pathway. Transfection experiments demonstrate that the inhibition occurs at the same level as, or upstream of, the small G-proteins cdc42 and Rac1. In contrast, the classical MAPK pathway was insensitive to confluency. The inhibition of JNK/SAPK activation depended on the integrity of the actin microfilament network. These results were confirmed and extended in monolayer wounding experiments. After PDGF, EGF or UV stimulation, c-Jun was predominantly phosphorylated in cells bordering the wound, which are the cells that move to occupy the wounded area. Thus, modulation of the stress-dependent signal cascade by confluency will restrict c-Jun N-terminal phosphorylation in response to mitogenic or chemotactic agents to cells that border a wounded area.

Induction of apoptosis by the transcription factor c-Jun.

c-Jun, a signal-transducing transcription factor of the AP-1 family, normally implicated in cell cycle progression, differentiation and cell transformation, recently has also been linked to apoptosis. To explore further the functional roles of c-Jun, a conditional allele was generated by fusion of c-Jun with the hormone-binding domain of the human estrogen receptor (ER). Here we demonstrate that increased c-Jun activity is sufficient to trigger apoptotic cell death in NIH 3T3 fibroblasts. c-Jun-induced apoptosis is evident at high serum levels, but is enhanced further in factor-deprived fibroblasts. Furthermore, apoptosis by c-Jun is not accompanied by an increase in DNA synthesis. Constitutive overexpression of the apoptosis inhibitor protein Bcl-2 delays the c-Jun-mediated cell death. The regions of c-Jun necessary for apoptosis induction include the amino-terminal transactivation and the carboxy-terminal leucine zipper domain, suggesting that c-Jun may activate cell death by acting as a transcriptional regulator. We further show that alpha-fodrin, a substrate of the interleukin 1beta-converting enzyme (ICE) and CED-3 family of cysteine proteases, becomes proteolytically cleaved in cells undergoing cell death by increased c-Jun activity. Moreover, cell-permeable irreversible peptide inhibitors of the ICE/CED-3 family of cysteine proteases prevented the cell death.

Identification of a novel 135-kDa Grb2-binding protein in osteoclasts.

The tyrosine kinase receptor for macrophage colony-stimulating factor and the non-receptor tyrosine kinase c-Src play critical roles in osteoclast differentiation and function. Since the ubiquitously expressed adaptor protein Grb2 plays an important role in several tyrosine kinase signal transduction pathways, we used a filter binding assay to identify osteoclast proteins that bind to Grb2. In osteoclasts, there were three major Grb2-binding proteins, two of which, mSos and c-Cbl (p120), have been previously identified as Grb2-binding proteins in many cell types. The third protein, p135, had a restricted pattern of expression and was present at high levels in authentic osteoclasts and osteoclast-like cells formed in an in vitro co-culture system. In addition to binding Grb2 in the filter binding assay, p135 was isolated in complexes with endogenous Grb2 from osteoclast cell extracts. The association of p135 and Grb2 was dependent on an intact Src homology 3 domain and furthermore, was shown to preferentially interact with the N-terminal Src homology 3 domain of Grb2, which is similar to the interaction of mSos and Grb2 in other cell types. p135 was not recognized by antibodies against several known Grb2-binding proteins and thus may be a novel Grb2-binding protein.