Repression of caspase-3 and RNA-binding protein HuR cleavage by cyclooxygenase-2 promotes drug resistance in oral squamous cell carcinoma.

A well-studied RNA-binding protein Hu Antigen-R (HuR), controls post-transcriptional gene regulation and undergoes stress-activated caspase-3 dependent cleavage in cancer cells. The cleavage products of HuR are known to promote cell death; however, the underlying molecular mechanisms facilitating caspase-3 activation and HuR cleavage remains unknown. Here, we show that HuR cleavage associated with active caspase-3 in oral cancer cells treated with ionizing radiation and chemotherapeutic drug, paclitaxel. We determined that oral cancer cells overexpressing cyclooxygenase-2 (COX-2) limited the cleavage of caspase-3 and HuR, which reduced the rate of cell death in paclitaxel resistant oral cancer cells. Specific inhibition of COX-2 by celecoxib, promoted apoptosis through activation of caspase-3 and cleavage of HuR in paclitaxel-resistant oral cancer cells, both in vitro and in vivo. In addition, oral cancer cells overexpressing cellular HuR increased the half-life of COX-2 mRNA, promoted COX-2 protein expression and exhibited enhanced tumor growth in vivo in comparison with cells expressing a cleavable form of HuR. Finally, our ribonucleoprotein immunoprecipitation and sequencing (RIP-seq) analyses of HuR in oral cancer cells treated with ionizing radiation (IR), determined that HuR cleavage product-1 (HuR-CP1) bound and promoted the expression of mRNAs encoding proteins involved in apoptosis. Our results indicated that, cellular non-cleavable HuR controls COX-2 mRNA expression and enzymatic activity. In addition, overexpressed COX-2 protein repressed the cleavage of caspase-3 and HuR to promote drug resistance and tumor growth. Altogether, our observations support the use of the COX-2 inhibitor celecoxib, in combination with paclitaxel, for the management of paclitaxel resistant oral cancer cells.

Translational regulation of inhibin ßA by TGFß via the RNA-binding protein hnRNP E1 enhances the invasiveness of epithelial-to-mesenchymal transitioned cells.

The epithelial-to-mesenchymal transition (EMT) is a cellular process that functions during embryonic development and tissue regeneration, thought to be aberrantly activated in epithelial-derived cancer and has an important role in the process of metastasis. The transforming growth factor (TGF)-ß signaling pathway is a key inducer of EMT and we have elucidated a posttranscriptional mechanism by which TGFß modulates expression of select transcripts via the RNA-binding protein hnRNP E1 during EMT. One such transcript inhibin ßA is a member of the TGFß superfamily. Here, we show by polysome profiling that inhibin ßA is translationally regulated by TGFß via hnRNP E1. TGFß treatment or knockdown of hnRNP E1 relieves silencing of the inhibin ßA transcript, resulting in increased protein expression and secreted levels of the inhibin ßA homodimer, activin A. Our data indicate that the translational upregulation of inhibin ßA enhances the migration and invasion of cells that have undergone an EMT and promotes cancer progression in vivo.

Dab2 stabilizes Axin and attenuates Wnt/beta-catenin signaling by preventing protein phosphatase 1 (PP1)-Axin interactions.

Wnt/beta-catenin signaling plays a pivotal role in modulating cellular proliferation, differentiation, tissue organization and embryonic development. Earlier, we found that the endocytic adaptor disabled-2 (Dab2) could attenuate Wnt/beta-catenin signaling by stabilizing Axin and preventing its translocation to the membrane. Recently, protein phosphatase 1 (PP1) has been shown to interact with, and dephosphorylate Axin, leading to its destabilization. Here, we show that Dab2 functions upstream of PP1 to block the interaction between Axin and PP1, inhibiting Axin dephosphorylation and thereby stabilizing its expression, ultimately leading to inhibition of Wnt/beta-catenin. We show that Dab2 acts as a competitive inhibitor of PP1 by binding to the same C-terminal domain of Axin. Both PP1 and Axin bind to the N-terminus of Dab2 and a Dab2 truncation mutant consisting of the N-terminal phosphotyrosine binding domain blocks PP1-Axin interactions and inhibits Wnt signaling. We confirm the inhibitory effect of Dab2 on Wnt/beta-catenin signaling in zebrafish embryos, showing that its ectopic expression phenocopies Axin overexpression resulting in altered dorsoventral patterning. We conclude that Dab2 stabilizes Axin and attenuates Wnt/beta-catenin signaling by preventing PP1 from binding Axin.

The inhibitory effects of Disabled-2 (Dab2) on Wnt signaling are mediated through Axin.

beta-Catenin-mediated Wnt signaling is essential in embryonic development and in adult tissues. Recent studies have demonstrated that Axin not only plays an important inhibitory role in coordinating beta-catenin degradation, but is itself degraded by the low-density-lipoprotein receptor-related protein (LRP)5/6 Wnt co-receptor. Here, we demonstrate that the endocytic adaptor molecule Disabled-2 (Dab2), which we have previously demonstrated to act as an inhibitor of beta-catenin signaling, interacts with Axin and prevents its interaction with and degradation by the LRP5 co-receptor, thereby increasing its half-life and stabilization. Dab2 levels induced during retinoic acid-induced differentiation of F9, or during transforming growth factor-beta-induced epithelial-mesenchymal transdifferentiation of mouse mammary epithelial cells result in the stabilization of Axin and concomitant inhibition of beta-catenin signaling. Ectopic expression of Dab2 in F9 cells as well as in transformed cell lines results in increased Axin expression and attenuation of Wnt-mediated signaling. We conclude that Dab2 may play an important role in the maintenance of the differentiated state and restrain Wnt-mediated proliferation through its association with and modulation of Axin.

Regulation of the Wnt signaling pathway by disabled-2 (Dab2).

The adaptor molecule Disabled-2 (Dab2) has been shown to link cell surface receptors to downstream signaling pathways. Using a small-pool cDNA screening strategy, we identify that the N-terminal domain of Dab2 interacts with Dishevelled-3 (Dvl-3), a signaling mediator of the Wnt pathway. Ectopic expression of Dab2 in NIH-3T3 mouse fibroblasts attenuates canonical Wnt/beta-catenin-mediated signaling, including accumulation of beta-catenin, activation of beta-catenin/T-cell-specific factor/lymphoid enhancer-binding factor 1-dependent reporter constructs, and endogenous cyclin D1 induction. Wnt stimulation leads to a time-dependent dissociation of endogenous Dab2-Dvl-3 and Dvl-3-axin interactions in NIH-3T3 cells, while Dab2 overexpression leads to maintenance of Dab2-Dvl-3 association and subsequent loss of Dvl-3-axin interactions. In addition, we find that Dab2 can associate with axin in vitro and stabilize axin expression in vivo. Mouse embryo fibroblasts which lack Dab2 exhibit constitutive Wnt signaling as evidenced by increased levels of nuclear beta-catenin and cyclin D1 protein levels. Based on these results, we propose that Dab2 functions as a negative regulator of canonical Wnt signaling by stabilizing the beta-catenin degradation complex, which may contribute to its proposed role as a tumor suppressor.

The adaptor molecule Disabled-2 links the transforming growth factor beta receptors to the Smad pathway.

Using a genetic complementation approach we have identified disabled-2 (Dab2), a structural homolog of the Dab1 adaptor molecule, as a critical link between the transforming growth factor beta (TGFbeta) receptors and the Smad family of proteins. Expression of wild-type Dab2 in a TGFbeta-signaling mutant restores TGFbeta-mediated Smad2 phosphorylation, Smad translocation to the nucleus and Smad-dependent transcriptional responses. TGFbeta stimulation triggers a transient increase in association of Dab2 with Smad2 and Smad3, which is mediated by a direct interaction between the N-terminal phosphotyrosine binding domain of Dab2 and the MH2 domain of Smad2. Dab2 associates with both the type I and type II TGFbeta receptors in vivo, suggesting that Dab2 is part of a multiprotein signaling complex. Together, these data indicate that Dab2 is an essential component of the TGFbeta signaling pathway, aiding in transmission of TGFbeta signaling from the TGFbeta receptors to the Smad family of transcriptional activators.

Smad7 is induced by CD40 and protects WEHI 231 B-lymphocytes from transforming growth factor-beta -induced growth inhibition and apoptosis.

Transforming growth factor-beta (TGF-beta) is a potent inducer of apoptosis in B-lymphocytes and is essential for immune regulation and maintenance of self-tolerance. Here we show that concomitant signaling through CD40 sustains proliferation and rescues the premature B cell line WEHI 231 from both TGF-beta-induced and anti-IgM-induced apoptosis. The anti-apoptotic effect of CD40 is associated with the transcriptional activation of the inhibitory Smad7 protein. The transactivation of Smad7 by CD40 is NFkappaB-dependent in that pharmacological inhibitors of this pathway, N-tosyl-l-phenylalanine chloromethyl ketone and pyrrolidine dithiocarbamate, abrogate CD40-induced Smad7 expression. Ectopic overexpression of Smad7 inhibited Smad2 activation, TGF-beta-mediated growth inhibition, and apoptosis in WEHI 231 cells. Consistent with this result, dominant negative interference with Smad2 and Smad3 function also inhibited TGF-beta-induced apoptosis. The inhibitory effects of Smad7 overexpression were specific to TGF-beta-induced apoptosis and were without effect on anti-IgM-induced cell death. These results suggest a mechanism of suppression of TGF-beta-induced apoptosis by CD40, mediated through activation of NF-kappaB and, consequently, induction of Smad7 expression.

Transforming growth factor beta induces caspase 3-independent cleavage of alphaII-spectrin (alpha-fodrin) coincident with apoptosis.

Transforming growth factor beta (TGF-beta) is a potent growth inhibitor and inducer of cell death in B-lymphocytes and is essential for immune regulation and maintenance of self-tolerance. In this report the mouse immature B cell line, WEHI 231, was used to examine the mechanisms involved in TGF-beta-mediated apoptosis. Induction of apoptosis is detected as early as 8 h after TGF-beta administration. Coincident with the onset of apoptosis, the cytoskeletal actin-binding protein, alphaII-spectrin (alpha-fodrin) is cleaved into 150-, 115-, and 110-kDa fragments. The broad spectrum caspase inhibitor (Boc-D-fmk (BD-fmk)) completely abolished TGF-beta-induced apoptosis and alphaII-spectrin cleavage. Caspase 3, although present in WEH1 231 cells, was not activated by TGF-beta, nor was its substrate, poly(ADP-ribose) polymerase. These results identify alphaII-spectrin as a novel substrate that is cleaved during TGF-beta-induced apoptosis. Our data provide the first evidence of calpain and caspase 3-independent cleavage of alphaII-spectrin during apoptosis and suggests that TGF-beta induces apoptosis and alphaII-spectrin cleavage via a potentially novel caspase. This report also provides the first direct evidence of caspase 3 activation in WEH1 231 cells and indicates that at least two distinct apoptotic pathways exist.

Transforming growth factor beta regulates clusterin gene expression via modulation of transcription factor c-Fos.

Transforming growth factor-beta (TGFbeta) induces gene expression of the glycoprotein clusterin in a variety of cell types via a consensus AP-1 binding site. Here, we demonstrate, by supershift analysis, that JunB, JunD, Fra1, Fra2, and c-Fos bound to AP-1 but that prior treatment of the cells with TGFbeta reduced dramatically c-Fos binding, suggesting that c-Fos might be playing a negative regulatory role in clusterin gene expression. Transient cotransfection assays in mink lung epithelial (CCL64) cells, using a human c-Fos expressing plasmid together with a clusterin promoter/reporter construct or the artificial TGFbeta-inducible reporter construct 3TPLux, revealed that c-Fos was indeed repressive for TGFbeta-induced promoter transactivation. Further, we demonstrate that in stable c-Fos-overexpressing cell lines, TGFbeta induction of endogenous clusterin mRNA, as well as clusterin promoter transactivation are blocked. Co-transfection with c-Fos deletion constructs revealed that the C-terminal region, including the homologue box 2 motif and the extreme C-terminal serine phosphorylation sites (Ser362 and Ser374) are required for repression of clusterin and 3TPLux transactivation. TGFbeta treatment of CCL64 cells resulted in the induction of c-Fos mRNA but caused no alternation in total c-Fos protein levels. The results suggest that the c-Fos represses clusterin gene expression, maintaining a low basal level in the absence of TGFbeta, and that TGFbeta, presumably through its effects on c-Fos protein synthesis and/or stability, abrogates the repression of c-Fos, thereby resulting in gene expression.

TGF-beta induces fibronectin synthesis through a c-Jun N-terminal kinase-dependent, Smad4-independent pathway.

Transforming growth factor-beta (TGF-beta) exerts its effects on cell proliferation, differentiation and migration in part through its modulation of extracellular matrix components, such as fibronectin and plasminogen activator inhibitor-1 (PAI-1). Although the SMAD family of proteins recently has been shown to be a key participant in TGF-beta signaling, other signaling pathways have also been shown to be activated by TGF-beta. We report here that c-Jun N-terminal kinase (JNK), a member of the MAP kinase family, is activated in response to TGF-beta in the human fibrosarcoma HT1080-derived cell line BAHgpt. Stable expression of dominant-negative forms of JNK1 and MKK4, an upstream activator of JNK, results in loss of TGF-beta-stimulated fibronectin mRNA and protein induction, while having little effect on TGF-beta-induced levels of PAI-1. The human fibronectin promoter contains three CRE elements, one of which has been shown to bind a c-Jun-ATF-2 heterodimer. Utilizing a GAL4 fusion trans-reporting system, we demonstrate a decrease in transactivating potential of GAL4-c-Jun and GAL4-ATF-2 in dominant-negative JNK1- and MKK4-expressing cells. Finally, we show that TGF-beta-induced fibronectin synthesis is independent of Smad4. These results demonstrate that TGF-beta-mediated fibronectin induction requires activation of JNK which in turn modulates the activity of c-Jun and ATF-2 in a Smad4independent manner.

Caspase inhibitor BD-fmk distinguishes transforming growth factor beta-induced apoptosis from growth inhibition.

Transforming growth factor beta (TGFbeta) is essential for immune regulation and growth control. TGFbeta has previously been shown to inhibit cell growth as well as induce cell death in lymphocytes, although the mechanisms controlling these processes are not well understood. The mouse pre-B lymphoma cell line WEHI 231 was used to examine the mechanisms of TGFbeta-mediated growth inhibition and apoptosis. TGFbeta inhibits the growth of WEHI 231 cells in a dose-dependent manner; however, a decrease in viability is also observed, which is indicative of increased cell death. TGFbeta induces oligonucleosomal DNA ladder formation in a dose-dependent manner, indicating the mechanism of cell death is via apoptosis. The broad spectrum caspase inhibitor BD-fmk, but not tetrapeptide inhibitors of specific caspases, completely blocks TGFbeta-induced apoptosis while maintaining cellular viability. BD-fmk, however, has no effect on TGFbeta-induced growth inhibition. The ability of TGFbeta to down-regulate cyclin A-associated kinase activity, postulated to mediate TGFbeta growth inhibition, is not affected by the caspase inhibitor BD-fmk. These results suggest that caspases do not regulate cell cycle mediators of growth and provide the first demonstration that a pharmacological inhibitor of TGFbeta-induced apoptosis is capable of distinguishing growth inhibitory from apoptotic pathways.

Mechanisms of TGF-beta-induced cell cycle arrest.

Mitogenic growth factors stimulate cell growth by initiating a signaling cascade leading to the activation of the cyclin-dependent kinases (cdks), phosphorylation of pRb, and subsequent entry of the cell into the S phase. Transforming growth factor-beta (TGF-beta) is a potent antimitogen in a wide variety of cells and is postulated to inhibit cell cycle progression by blocking the late G1 activation of the cdks, thereby preventing pRb phosphorylation and S phase entry. The loss of TGF-beta sensitivity in many transformed cells coupled with recent data demonstrating a deregulation of cyclins, cdks, and cdk inhibitors in many types of cancer has attracted much attention to the molecular mechanism of TGF-beta-mediated growth arrest. Despite these recent advances, further research is required to elucidate how these effects of TGF-beta on the cyclins, cdks, and cdk inhibitors are linked to the TGF-beta receptor complex and the Smad proteins.

Regulation of clusterin gene expression by transforming growth factor beta.

Transforming growth factor beta (TGFbeta) induces the expression of a wide variety of genes in many cell types. Our previous studies have shown that TGFbeta stimulates both clusterin mRNA and protein levels, and induces its accumulation in the nucleus of CCL64 cells. To further investigate the molecular mechanism of clusterin mRNA induction by TGFbeta, we created a 1.3-kilobase rat clusterin promoter/luciferase reporter construct. We demonstrate that TGFbeta enhances luciferase activity 2.5-6-fold in transient transfection assays of epithelial, endothelial, and fibroblast cell lines. Deletional analysis reveals that an AP-1-binding site (5'-TGAGTCA) in the minimal promoter region is necessary for initiating transactivation by TGFbeta. A single T to G base mutation in the AP-1 site (5'-TGAGGCA) abolishes TGFbeta-induced clusterin promoter transactivation. In transcription factor decoy experiments, 23-mer oligonucleotides of wild type AP-1 reduce TGFbeta induction of clusterin mRNA levels and promoter transactivation, while an oligonucleotide containing the mutated AP-1 site has no effect. Two specific protein kinase C inhibitors, GF109203X and calphostin C, block TGFbeta-induced clusterin mRNA levels and promoter transactivation. Together these results indicate that TGFbeta regulates clusterin gene expression through an AP-1 site and its cognate transcription factor AP-1, and requires the involvement of protein kinase C.

TGF-beta suppresses IFN-gamma induction of class II MHC gene expression by inhibiting class II transactivator messenger RNA expression.

Recently, a non-DNA binding protein, class II transactivator (CIITA), has been shown to be required for constitutive and IFN-gamma-inducible class II MHC transcription. The cytokine TGF-beta inhibits IFN-gamma-induced class II MHC expression at the transcriptional level. In this study, we provide evidence that TGF-beta blocks IFN-gamma-induced CIITA mRNA accumulation. TGF-beta down-regulates class II MHC and CIITA mRNA accumulation in human astroglioma and fibrosarcoma cell lines, but TGF-beta does not destabilize the CIITA message, suggesting an effect at the transcriptional level. In cells that stably overexpressed CIITA, leading to a constitutive class II MHC-positive phenotype, the inhibitory effect of TGF-beta on class II MHC was abrogated, but the cells remained responsive for expression of TGF-beta-inducible genes. Cell lines that possessed defects in TGF-beta signaling also became refractory to inhibition of IFN-gamma-induced CIITA and class II MHC expression. Our data indicate that TGF-beta suppresses IFN-gamma-induced class II MHC expression by inhibiting accumulation of CIITA mRNA.

Isolation and characterization of mutant cell lines defective in transforming growth factor beta signaling.

To isolate and characterize effector molecules of the transforming growth factor beta (TGFbeta) signaling pathway we have used a genetic approach involving the generation of stable recessive mutants, defective in their TGFbeta signaling, which can subsequently be functionally complemented to clone the affected genes. We have generated a cell line derived from a hypoxanthine-guanine phosphoribosyltransferase negative (HPRT-) HT1080 clone that contains the selectable marker Escherichia coli guanine phosphoribosyltransferase (gpt) linked to a TGFbeta-responsive promoter. This cell line proliferates or dies in the appropriate selection medium in response to TGFbeta. We have isolated three distinct TGFbeta-unresponsive mutants following chemical mutagenesis. Somatic cell hybrids between pairs of individual TGFbeta-unresponsive clones reveal that each is in a distinct complementation group. Each mutant clone retains all three TGFbeta receptors yet fails to induce a TGFbeta-inducible luciferase reporter construct or TGFbeta-mediated plasminogen activator inhibitor-1 (PAI-1) expression. Two of the three have an attenuated TGFbeta-induced fibronectin response, whereas in the other mutant the fibronectin response is intact. These TGFbeta-unresponsive cells should allow selection and identification of signaling molecules through functional complementation.