Notch4 intracellular domain binding to Smad3 and inhibition of the TGF-beta signaling

We present evidence that Notch4ICD attenuates TGF-beta signaling. Cells expressing the activated form of the Notch4 receptor (ICD4) were resistant to the growth-inhibitory effects of TGF-beta. Notch4ICD was found to bind to Smad2, Smad3 and Smad4 but with higher affinity to Smad3. Deletion analysis showed that binding of Smad3 to ICD4 was mediated by its MH2 domain and was not dependent on the presence of the RAM23 region in ICD4. Using two TGF-beta/Activin reporter luciferase assays, RT-PCR and Western blot analysis, we demonstrate that ICD4 and ICD4 deltaRAM23 inhibit Smad-binding element and 3TP luciferase reporter activity and PAI-1 gene expression. MCF-7 human breast cancer cells express Notch4ICD (ICD4) and are resistant to the growth-inhibitory effects of TGF-beta. Blockage of Notch4 processing to ICD4 by gamma-secretase inhibitor renders MCF-7 cells sensitive to growth inhibition by TGF-beta. The interplay between these two signaling pathways may be a significant determinant during mammary tumorigenesis.     

Overview of cell death signaling pathways

Apoptosis plays an important role in development and maintenance of tissue homeostasis. Intensive efforts have been made to explore the molecular mechanisms of the apoptotic signaling pathways including the initiation, mediation, execution and regulation of apoptosis. Caspases are central effectors of apoptosis. Cells undergo apoptosis through two major pathways, namely the extrinsic pathway (death receptor pathway) or the intrinsic pathway (the mitochondrial pathway). Finally, the contents of dead cells are packaged into apoptotic bodies, which are recognized by neighboring cells or macrophages and cleared by phagocytosis. Cellular apoptosis is tightly controlled by a complex regulatory networks including balancing pro-survival signals. De-regulation of apoptosis may lead to pathological disorders such as developmental defects, autoimmune diseases, neurodegeneration or cancer. Increasing attention is being focused on alternative signaling pathways leading to cell death including necrosis, autophagy, and mitotic catastrophe. Understanding of cell death signaling pathways is relevant to understanding cancer and to developing more effective therapeutics.     

Smad3 deficiency inhibits v-ras-induced transformation by suppression of JNK MAPK signaling and increased farnesyl transferase inhibition

The ability of transforming growth factor-beta (TGF-beta) to modulate various effects on distinct cell lineages has been a central feature of its multi-faceted nature. The purpose of this study was to access the effects of deletion of a key TGF-beta signal transducer, Smad3, on MAPK activation and v-Ras(Ha)-transformation of primary mouse embryonic fibroblasts (MEFs). We observe reduced TGF-beta1 and v-ras(Ha) mediated activation of the JNK and ERK MAPK pathway upon ablation of Smad3. Further, Smad3-deficient MEFs demonstrate resistance to v-ras(Ha)-induced transformation while the absence of Smad3 results in increased inhibition of farnesyl transferase activity. Taken together, these observations demonstrate that the absence of Smad3 protects fibroblasts from oncogenic transformation by (i) augmenting farnesyl transferase inhibition and (ii) suppressing the Ras-JNK MAPK pathway. These results provide new insights into the molecular mechanisms involved in v-Ras(Ha) oncogene-induced mesenchymal phenotypic transformation.     

Differential roles of IRS-1 and SHC signaling pathways in breast cancer cells

Several polypeptide growth factors stimulate breast cancer growth and may be involved in tumor progression. However, the relative importance of diverse growth factor signaling pathways in the development and maintenance of the neoplastic phenotype is largely unknown. The activation of such growth factor receptors as the insulin-like growth factor I receptor (IGF-I R), erbB-type receptors (erbB Rs) and FGF receptors (FGF Rs) controls the phenotype of a model breast cancer cell line MCF-7. To evaluate the function of 2 post-receptor signaling molecules, insulin receptor substrate-1 (IRS-1) (a major substrate of the IGF-IR) and SHC (a common substrate of tyrosine kinase receptors), we developed several MCF-7-derived cell clones in which the synthesis of either IRS-1 or SHC was blocked by antisense RNA. In MCF-7 cells, down-regulation of IRS-1 by 80–85% strongly suppressed anchorage-dependent and -independent growth and induced apoptotic cell death under growth factor- and estrogen-reduced conditions. The reduction of SHC levels by approximately 50% resulted in the inhibition of monolayer and anchorage-independent growth but did not decrease cell survival. Importantly, cell aggregation and the ability of cells to survive on the extracellular matrix were inhibited in MCF-7/anti-SHC clones, but not in MCF-7/anti-IRS-1 clones. Cell motility toward IGF was not attenuated in any of the tested cell lines, but motility toward EGF was decreased in MCF-7/anti-SHC clones. Our results suggest that in MCF-7 cells: 1) both IRS-1 and SHC are implicated in the control of monolayer and anchorage-independent growth; 2) IRS-1 is critical to support cell survival; 3) SHC is involved in EGF-dependent motility; and 4) normal levels of SHC, but not IRS-1, are necessary for the formation and maintenance of cell-cell interactions. Int. J. Cancer 72:828–834, 1997. © 1997 Wiley-Liss, Inc.     

The type III TGF-beta receptor signals through both Smad3 and the p38 MAP kinase pathways to contribute to inhibition of cell proliferation

Transforming growth factor beta (TGFbeta) has an important role as a negative regulator of cellular proliferation. The type III transforming growth factor beta receptor (TbetaRIII) has an emerging role as both a TGFbeta superfamily co-receptor and in mediating signaling through its cytoplasmic domain. In L6 myoblasts, TbetaRIII expression enhanced TGFbeta1-mediated growth inhibition, with this effect mediated, in part, by the TbetaRIII cytoplasmic domain. The effects of TbetaRIII were not due to altered ligand presentation or to differences in Smad2 phosphorylation. Instead, TbetaRIII specifically increased Smad3 phosphorylation, both basal and TGFbeta-stimulated Smad3 nuclear localization and Smad3-dependent activation of reporter genes independent of its cytoplasmic domain. Conversely, SB431542, a type I transforming growth factor beta receptor (TbetaRI) inhibitor, as well as dominant-negative Smad3 specifically and significantly abrogated the effects of TbetaRIII on TGFbeta1-mediated inhibition of proliferation. TbetaRIII also specifically increased p38 phosphorylation, and SB203580, a p38 kinase inhibitor, specifically and significantly abrogated the effects of TbetaRIII/TGFbeta1-mediated inhibition of proliferation in L6 myoblasts and in primary human epithelial cells. Importantly, treatment with the TbetaRI and p38 inhibitors together had additive effects on abrogating TbetaRIII/TGFbeta1-mediated inhibition of proliferation. In a reciprocal manner, short hairpin RNA-mediated knockdown of endogenous TbetaRIII in various human epithelial cells attenuated TGFbeta1-mediated inhibition of proliferation. Taken together, these data demonstrate that TbetaRIII contributes to and enhances TGFbeta-mediated growth inhibition through both TbetaRI/Smad3-dependent and p38 mitogen-activated protein kinase pathways.     

The roles of JNK and apoptotic signaling pathways in PEITC-mediated responses in human HT-29 colon adenocarcinoma cells

Phenethyl isothiocyanate (PEITC) is a potential chemopreventive agent that is present naturally in widely consumed vegetables, especially in watercress. It has been extensively investigated for its anticancer activities against lung, forestomach and esophageal tumorigenesis. Here we investigated the pro-apoptotic effect of PEITC in HT-29 human colorectal carcinoma cell line, and the mechanism of apoptosis induced by PEITC. PEITC-induced apoptosis was determined by DNA fragmentation assay and diamidino-2-phenylindole (DAPI) staining technique. To understand the mechanisms of apoptosis induced by PEITC, we studied the role of caspases, mitochondria-cytochrome c release, and mitogen-activated protein kinase (MAPK) signaling pathways involved in PEITC-induced apoptosis in HT-29 cells. Both the caspase-3 and -9 activities were stimulated by PEITC. The release of cytochrome c from the mitochondrial inter-space was time- and dose-dependent, with a maximal release at 50 micro M after 10 h treatment. Three MAPKs [JNK (c-Jun N-terminal kinase), extracellular signal-regulated protein kinase (ERK) and p38 kinase] were activated shortly after PEITC treatment in HT-29 cells. Importantly, the SP600125 compound, an anthrapyrazolone inhibitor of JNK, but not the ERK and p38 inhibitor, suppressed apoptosis induced by PEITC. Similarly, this JNK inhibitor attenuated both cytochrome c release and caspase-3 activation induced by PEITC. In summary, this study shows that PEITC can induce apoptosis in HT-29 cells in a time- and dose-dependent manner via the mitochondria caspase cascade, and the activation of JNK is critical for the initiation of the apoptotic processes. This mechanism of PEITC may play an important role in the killing of cancerous cells and offer a potential mechanism for its anticancer action in vivo.     

Ajoene-induced cell death in human promyeloleukemic cells does not require JNK but is amplified by the inhibition of ERK

Treatment of human promyeloleukemic HL-60 cells with the experimental antileukemic drug ajoene induces the activation of the mitogen-activated protein kinases (MAPKs) c-Jun NH(2)-terminal kinase (JNK), p38 and extracellular signal-regulated kinases (ERK) 1/2 as well as the survival kinase Akt. JNK activation occurred in HL-60/neo, HL-60/bcl-x(L), and in HL-60 cells pretreated with the pan-caspase inhibitor zVAD-fmk, indicating that JNK activation is not dependent on ajoene-induced mitochondria perturbation and subsequent caspase activation. Cells overexpressing a dominant-negative JNK showed no altered sensitivity towards ajoene suggesting that the activation of JNK is not necessary for ajoene-induced cell death. Inhibition of p38 MAPK by SB 203580 had no influence on ajoene-mediated apoptosis. In contrast, inhibition of ERK1/2 vastly enhanced ajoene-induced cell death. The survival kinase Akt, in contrast, did not participate in ajoene-induced death signaling as shown by the use of the phosphatidylinositol-3-kinase inhibitor wortmannin. Thus in contrast to the previous findings regarding stress-induced cell death, ajoene-mediated activation of JNK and p38 has no impact on ajoene-induced apoptosis in HL-60 cells. Blockade of ERK1/2 but not Akt pathways leads to sensitization of cells against ajoene-mediated apoptosis supporting the view that inhibition of ERK1/2 is a valuable strategy to increase the sensitivity of promyeloleukemic cells towards ajoene.     

Differential effects of RhoA signaling on anticancer agent-induced cell death

Substantial evidence exists to support a role for RhoA signaling in adhesion and cytoskeletal reorganization, while relatively less is known about the participation of RhoA on cell survival. We provide evidence that RhoA functions as a differential modulator of apoptosis induced by anticancer agents. Specifically, both RhoA and caRhoA induce statistically significant resistance to statin, etoposide, 5-FU and taxol while increasing sensitivity to vincristine (all p<0.001). The IC50 values for statin, etoposide, 5-fluorouracil (5-FU) and taxol in caRhoA transfectant were 8.70+/-0.74, 4.08+/-0.12, 4.12+/-0.12 microg/ml and 3.84+/-0.16 ng/ml, respectively, whereas the respective IC50 values in the mock-transfected control were 3.40+/-0.21, 1.12+/-0.06, 1.21+/-0.06 microg/ml and 2.84+/-0.15 ng/ml. This represented a 2.6-, 3.5-, 3.2- and 1.4-fold resistance to statin, etoposide, 5-FU and taxol, respectively. In contrast, caRhoA increased sensitivity to vincristine, decreasing IC50 values from 4.61+/-0.46 to 3.73+/-0.44 ng/ml (p<0.001). Western blot analysis demonstrated that RhoA mediates induction of E2F-1, Cdk2 and PCNA, accompanying concurrent reduction in p21 and p27. However, cleavage assays of poly (ADP-ribose) polymerase, BID, caspase-8 and caspase-3 indicate that the cell growth modulation mediated by RhoA in response to these anticancer agents occurs through the inhibition of apoptosis. Taken together, these results indicate that RhoA differentially modulates cancer cell death depending on the anticancer agent.     

TGF-beta signaling in T cells: roles in lymphoid and epithelial neoplasia

Transforming growth factor-beta (TGF-beta) plays an essential role in regulating the homeostasis of cells in the lymphoid lineage. TGF-beta signaling is not required for normal thymopoiesis, but is essential for regulating the expansion, activation, and effector function of the mature CD4+ and CD8+ T cells in the peripheral lymphoid organs and target tissues. Recent studies in both mice and humans have elucidated an important and complex role for TGF-beta in regulatory T-cell biology. Disruption of TGF-beta signaling in T cells impairs the maintenance of regulatory T cells, results in the expansion of activated effector T cells, and is associated with the production of cytokines that have major effects on cells in their environment. While autoimmunity and inflammation are the principal phenotypes associated with the abrogation of TGF-beta signaling in T cells in mice, emerging evidence now also directly links Smad-dependent TGF-beta signaling in T cells to the suppression of epithelial neoplasia. The TGF-beta receptor-activated Smad3 plays a critical role in mediating many of the inhibitory effects of TGF-beta signaling in T cells, and has now been established as an important suppressor of leukemogenesis. These studies are increasing our awareness of the many complex mechanisms through which TGF-beta signaling controls the pathogenesis of cancer.     

Anti-Cripto Mab inhibit tumour growth and overcome MDR in a human leukaemia MDR cell line by inhibition of Akt and activation of JNK/SAPK and bad death pathways

Doxorubicin (DOX) selection of CCRF-CEM leukaemia cell line resulted in multidrug resistance (MDR) CEM/A7R cell line, which overexpresses MDR, 1 coded P-glycoprotein (Pgp). Here, we report for the first time that oncoprotein Cripto, a founding member of epidermal growth factor-Cripto-FRL, 1-Criptic family is overexpressed in the CEM/A7R cells, and anti-Cripto monoclonal antibodies (Mab) inhibited CEM/A7R cell growth both in vitro and in an established xenograft tumour in severe combined immunodeficiency mice. Cripto Mab synergistically enhanced sensitivity of the MDR cells to Pgp substrates epirubicin (EPI), daunorubicin (DAU) and non-Pgp substrates nucleoside analogue cytosine arabinoside (AraC). In particular, the combination of anti-Cripto Mab at less than 50% of inhibition concentrations with noncytotoxic concentrations of EPI or DAU inhibited more than 90% of CEM/A7R cell growth. Cripto Mab slightly inhibited Pgp expression, and had little effect on Pgp function, indicating that a mechanism independent of Pgp was involved in overcoming MDR. We demonstrated that anti-Cripto Mab-induced CEM/A7R cell apoptosis, which was associated with an enhanced activity of the c-Jun N-terminal kinase/stress-activated protein kinase and inhibition of Akt phosphorylation, resulting in an activation of mitochondrial apoptosis pathway as evidenced by dephosphorylation of Bad at Ser136, Bcl-2 at Ser70 and a cleaved caspase-9.     

Sigma-2 ligands induce tumour cell death by multiple signalling pathways

Background:

The sigma-2 receptor has been identified as a biomarker of proliferating cells in solid tumours. In the present study, we studied the mechanisms of sigma-2 ligand-induced cell death in the mouse breast cancer cell line EMT-6 and the human melanoma cell line MDA-MB-435.

Methods:

EMT-6 and MDA-MB-435 cells were treated with sigma-2 ligands. The modulation of multiple signaling pathways of cell death was evaluated.

Results:

Three sigma-2 ligands (WC-26, SV119 and RHM-138) induced DNA fragmentation, caspase-3 activation and PARP-1 cleavage. The caspase inhibitor Z-VAD-FMK partially blocked DNA fragmentation and cytotoxicity caused by these compounds. These data suggest that sigma-2 ligand-induced apoptosis and caspase activation are partially responsible for the cell death. WC-26 and siramesine induced formation of vacuoles in the cells. WC-26, SV119, RHM-138 and siramesine increased the synthesis and processing of microtubule-associated protein light chain 3, an autophagosome marker, and decreased the expression levels of the downstream effectors of mammalian target of rapamycin (mTOR), p70S6K and 4EBP1, suggesting that sigma-2 ligands induce autophagy, probably by inhibition of the mTOR pathway. All four sigma-2 ligands decreased the expression of cyclin D1 in a time-dependent manner. In addition, WC-26 and SV119 mainly decreased cyclin B1, E2 and phosphorylation of retinoblastoma protein (pRb); RHM-138 mainly decreased cyclin E2; and 10 μ siramesine mainly decreased cyclin B1 and pRb. These data suggest that sigma-2 ligands also impair cell-cycle progression in multiple phases of the cell cycle.

Conclusion:

Sigma-2 ligands induce cell death by multiple signalling pathways.