Dominant-negative Receptor Mutant Research Articles

Irinotecan Upregulates Fibroblast Growth Factor Receptor 3 Expression in Colorectal Cancer Cells, Which Mitigates Irinotecan-Induced Apoptosis

BACKGROUND: Irinotecan (IRI) is an integral part of colorectal cancer (CRC) therapy, but response rates are unsatisfactory and resistance mechanisms are still insufficiently understood. As fibroblast growth factor receptor 3 (FGFR3) mediates essential survival signals in CRC, it is a candidate gene for causing intrinsic resistance to IRI. METHODS: We have used cell line models overexpressing FGFR3 to study the receptor's impact on IRI response. For pathway blockade, a dominant-negative receptor mutant and a small molecule kinase inhibitor were employed. RESULTS: IRI exposure induced expression of FGFR3 as well as its ligands FGF8 and FGF18 both in cell cultures and in xenograft tumors. As overexpression of FGFR3 mitigated IRI-induced apoptosis in CRC cell models, this suggests that the drug itself activated a survival response. On the cellular level, the antiapoptotic protein bcl-xl was upregulated and caspase 3 activation was inhibited. Targeting FGFR3 signaling using a dominant-negative receptor mutant sensitized cells for IRI. In addition, the FGFR inhibitor PD173074 acted synergistically with the chemotherapeutic drug and significantly enhanced IRI-induced caspase 3 activity in vitro. In vivo, PD173074 strongly inhibited growth of IRI-treated tumors. CONCLUSION: Together, our results indicate that targeting FGFR3 can be a promising strategy to enhance IRI response in CRC patients.

Prostaglandin F2α Inhibits Adipogenesis Via an Autocrine‐Mediated Interleukin‐11/Glycoprotein 130/STAT1‐Dependent Signaling Cascade

Prostaglandin F2α (PGF2α) is a potent inhibitor of adipocyte differentiation in vitro, that has also recently been implicated in the regulation of the adipogenic process in vivo, by opposing adipose tissue accretion and the subsequent development of obesity and its attendant metabolic consequences. In previous studies, we have demonstrated that PGF2α inhibits adipocyte differentiation by means of a calcium-dependent signaling pathway that is critically dependent upon the activity of the calcineurin phosphatase. In the current study, we have now extended these findings to further elucidate the mechanism by which the PGF2α/calcineurin-pathway inhibits the adipogenic process. We now report that the IL-11 cytokine, a member of the gp130 cytokine co-receptor-related family, is a downstream transcriptional target of this pathway in 3T3-L1 preadipocytes and is actively secreted in differentiating cells in response to PGF2α stimulation. Using a combined shRNA and dominant-negative receptor mutant approach, we provide evidence that IL-11/gp130-signaling is required to mediate the inhibitory effects of PGF2α on adipogenesis. Moreover, by taking advantage of a well-characterized panel of chimeric gp130 mutant receptors, we demonstrate that gp130 signaling is sufficient to inhibit adipocyte differentiation and specifically requires the activation of the STAT1 transcription factor. Conversely, we find that depleting endogenous STAT1 levels rescues adipogenesis in the presence of both IL-11/gp130 signaling and PGF2α. Collectively, our findings support a model in which PGF2α inhibits adipocyte differentiation by means of an IL-11 mediated autocrine negative feedback loop, that acts via gp130 to block adipogenesis through the essential actions of the STAT1 transcription factor.

Dominant-negative inhibition of the Axl receptor tyrosine kinase suppresses brain tumor cell growth and invasion and prolongs survival

Malignant gliomas remain incurable brain tumors because of their diffuse-invasive growth. So far, the genetic and molecular events underlying gliomagenesis are poorly understood. In this study, we have identified the receptor tyrosine kinase Axl as a mediator of glioma growth and invasion. We demonstrate that Axl and its ligand Gas6 are overexpressed in human glioma cell lines and that Axl is activated under baseline conditions. Furthermore, Axl is expressed at high levels in human malignant glioma. Inhibition of Axl signaling by overexpression of a dominant-negative receptor mutant (AXL-DN) suppressed experimental gliomagenesis (growth inhibition >85%, P < 0.05) and resulted in long-term survival of mice after intracerebral glioma cell implantation when compared with Axl wild-type (AXL-WT) transfected tumor cells (survival times: AXL-WT, 10 days; AXL-DN, >72 days). A detailed analysis of the distinct hallmarks of glioma pathology, such as cell proliferation, migration, and invasion and tumor angiogenesis, revealed that inhibition of Axl signaling interfered with cell proliferation (inhibition 30% versus AXL-WT), glioma cell migration (inhibition 90% versus mock and AXL-WT, P < 0.05), and invasion (inhibition 62% and 79% versus mock and AXL-WT, respectively; P < 0.05). This study describes the identification, functional manipulation, in vitro and in vivo validation, and preclinical therapeutic inhibition of a target receptor tyrosine kinase mediating glioma growth and invasion. Our findings implicate Axl in gliomagenesis and validate it as a promising target for the development of approaches toward a therapy of these highly aggressive but, as yet, therapy-refractory, tumors.

CAMP-induced differentiation of human neuronal progenitor cells is mediated by nuclear fibroblast growth factor receptor-1 (FGFR1).

Activation of cAMP signaling pathway and its transcriptional factor cyclic AMP response element binding protein (CREB) and coactivator are key determinants of neuronal differentiation and plasticity. We show that nuclear fibroblast growth factor receptor-1 (FGFR1) mediates cAMP-induced neuronal differentiation and regulates CREB and CREB binding protein (CBP) function in alpha-internexin-expressing human neuronal progenitor cells (HNPC). In proliferating HNPC, FGFR1 was associated with the cytoplasm and plasma membrane. Treatment with dB-cAMP induced nuclear accumulation of FGFR1 and caused neuronal differentiation, accompanied by outgrowth of neurites expressing MAP2 and neuron-specific neurofilament-L protein and enolase. HNPC transfected with nuclear/cytoplasmic FGFR1 or non-membrane FGFR1(SP-/NLS), engineered to accumulate exclusively in the cell nucleus, underwent neuronal differentiation in the absence of cAMP stimulation. In contrast, FGFR1/R4, with highly hydrophobic transmembrane domain of FGFR4, was membrane associated, did not enter the nucleus and failed to induce neuronal differentiation. Transfection of tyrosine kinase-deleted dominant negative receptor mutants, cytoplasmic/nuclear FGFR1(TK-) or nuclear FGFR1(SP-/NLS)(TK-), prevented cAMP-induced neurite outgrowth. Nuclear FGFR1 localized in speckle-like domains rich in phosphorylated histone 3 and splicing factors, regions known for active RNA transcription and processing, and activated the neurofilament-L gene promoter. FGFR1(SP-/NLS) transactivated CRE, up-regulated phosphorylation and transcriptional activity of CREB and stimulated the activity of CBP several-fold. Thus, cAMP-induced nuclear accumulation of FGFR1 provides a signal that triggers molecular events leading to neuronal differentiation.

A kinase-inactive type II TGFβ receptor impairs BMP signaling in human breast cancer cells

Dominant negative receptor mutants are often utilized in order to abrogate signaling induced by growth factors. We have previously shown that expression of a dominant negative type II TGFβ receptor (dnTβRII) in MDA-MB-231 breast cancer cells effectively abrogates TGFβ signaling. In this letter, we report that expression of dnTβRII also impairs BMP2-mediated Smad1 phosphorylation as well as BMP2-mediated Smad-dependent transcriptional responses, resulting in an attenuation of BMP-mediated anti-proliferative effects. The fact that dnTβRII not only abrogates TGFβ signaling but BMP signaling as well has important implications for the interpretation of data in which dominant negative mutants are utilized.

Inhibition of the type I insulin-like growth factor receptor expression and signaling: Novel strategies for antimetastatic therapy

The receptor for the type 1 insulin-like growth factor (IGF-1R) plays a critical role in the acquisition of the malignant phenotype. Using a highly metastatic murine lung carcinoma model, it was demonstrated that this receptor regulates several cellular functions that can impact on the metastatic potential of the cells, including cellular proliferation, anchorage-independent growth, cell migration, and invasion. The tumor model was used to develop several strategies for altering receptor expression and function as means of abrogating the metastatic potential of the cells. They include stable expression in the tumor cells of IGF-1R antisense RNA and dominant negative receptor mutants in which tyrosines in the kinase domain were substituted with phenylalanine. In addition, a novel strategy was used based on altering post ligand-binding receptor turnover. This led to inhibition of receptor re-expression and signaling and resulted in increased tumor cell apoptosis. When combined with the development of viral vectors designed to deliver genetic information with high efficiency, these strategies could form the basis for development of highly specific, antimetastatic therapy in tumors with known IGF-IR involvement.

L’inducteur céphalique Cerberus est un inhibiteur multivalent extracellulaire

Cerberus encodes for a secreted protein which when overexpressed ventrally in a Xenopus embryo induces head differentiation without trunk (Bouwmeester et al., 1996). We have recently shown that Cerberus can bind BMP-4 (Bone Morphogenetic Protein-4), Xnr-1 (Xenopus Nodal-related 1) and Xwnt-8 in the extracellular space (Piccolo et al., 1999). We present here studies showing that Cerberus does not have a receptor nor a dedicated transduction pathway but rather acts as an extracellular inhibitor. Our results suggest that the action of Cerberus in head induction can be explained by an inhibitory activity upstream of the Nodal-related and BMP-4 receptors. In addition, using dominant negative receptor mutants which block both the Xnr-1 and BMP-4 transduction pathways, we show that this double inhibition is sufficient for head induction in ventral mesoderm explants.

Dominant-negative mutations in the G-protein-coupled alpha-factor receptor map to the extracellular ends of the transmembrane segments.

G-protein-coupled receptors (GPCRs) transduce the signals for a wide range of hormonal and sensory stimuli by activating a heterotrimeric guanine nucleotide-binding protein (G protein). The analysis of loss-of-function and constitutively active receptor mutants has helped to reveal the functional properties of GPCRs and their role in human diseases. Here we describe the identification of a new class of mutants, dominant-negative mutants, for the yeast G-protein-coupled alpha-factor receptor (Ste2p). Sixteen dominant-negative receptor mutants were isolated based on their ability to inhibit the response to mating pheromone in cells that also express wild-type receptors. Detailed analysis of two of the strongest mutant receptors showed that, unlike other GPCR interfering mutants, they were properly localized at the plasma membrane and did not alter the stability or localization of wild-type receptors. Furthermore, their dominant-negative effect was inversely proportional to the relative amount of wild-type receptors and was reversed by overexpressing the G-protein subunits, suggesting that these mutants compete with the wild-type receptors for the G protein. Interestingly, the dominant-negative mutations are all located at the extracellular ends of the transmembrane segments, defining a novel region of the receptor that is important for receptor signaling. Altogether, our results identify residues of the alpha-factor receptor specifically involved in ligand binding and receptor activation and define a new mechanism by which GPCRs can be inactivated that has important implications for the evaluation of receptor mutations in other G-protein-coupled receptors.

Tumor angiogenesis: the pivotal role of vascular endothelial growth factor.

There is considerable evidence that vascular endothelial growth factor is involved in the vascularization and growth of primary tumors and in the formation of metastases. The expression of vascular endothelial growth factor depends on activated oncogenes and inactivated tumor-suppressor genes as well as several other factors, e.g., growth factors, hypoxia, and tumor promoters. Substantial expression of vascular endothelial growth factor receptors is mainly restricted to tumor vessels. The causal involvement of this angiogenic factor in the progression of the disease has been successfully evaluated using monoclonal antibodies against vascular endothelial growth factor, dominant negative receptor mutants, and antisense oligonucleotides against the messenger RNA of vascular endothelial growth factor. Thus, the vascular endothelial growth factor-signaling system seems to be an appropriate target for inhibition of tumor angiogenesis and metastasis formation.

VEGF-mediated tumour angiogenesis: a new target for cancer therapy

Considerable evidence is gathering for the involvement of vascular endothelial growth factor (VEGF) in the vascularization and growth of primary tumours as well as in the formation of metastases. The expression of VEGF depends on activated oncogenes and inactivated tumour suppressor genes as well as several other factors (e.g. growth factors, tumour promoters and hypoxia). Substantial expression of the receptors for VEGF is restricted mainly to the tumour blood vessels. The causal involvement of this angiogenic factor in the progression of disease has been successfully evaluated by means of monoclonal antibodies against VEGF, dominant-negative receptor mutants and the use of antisense oligonucleotides against the VEGF mRNA. Thus, the VEGF signalling system seems to be an appropriate target to inhibit tumour angiogenesis and metastases formation.

Report of a Meeting; Molecular Basis of Cancer Therapy

There has been an explosive increase in information relevant to the pathways that determine growth signal transduction, regulation of the cell cycle, mechanism of action of oncogenes and tumor suppressors, and mechanisms of programmed cell death (apoptosis). Additional information is needed to determine the targets for anticancer therapy that are most likely to lead to cancer cell death and/or growth cessation. Current experimental clinical approaches are directed toward killing cells with unique cancer-related phenotypes, such as cell surface antigens or growth factor receptors, or altering the host immune system to attack cancer cells. The following major therapeutic targets were identified during the course of this conference: 1) Reduce activity of gene products associated with stimulation of cell growth and increase activity of gene products that inhibit growth. The major principle here is that genes known to be sufficient for malignant transformation (such as Ras, Raf, and Bcr-Abl) and genes whose expression is necessary, but not sufficient, for malignant transformation (such as some cyclins) both may be important targets for anticancer drugs. The reason genes necessary but not sufficient for cell growth are targets is that progression through the cell cycle is based on a series of "on-off" switches whose activation depends on critical levels of specific kinases and phosphatases. Subtle differences in concentration or activity of these regulators, as may be found in cancer cells, could profoundly influence the position of the switch. There are many ways to affect activity of gene products, including use of anti-sense or ribozyme targeting of mRNAs; manipulation of regulatory controls (i.e., state of phosphorylation of Raf and p53; effect of SOS and GAP on Ras, etc.); alteration of essential covalent modifications (i.e., farnesylation of Ras which is essential for its association with the plasma membrane); and various forms of gene therapy to introduce genes (i.e., addition of wild-type p53) or to reduce activity of genes essential for growth (i.e., dominant negative receptor mutants). 2) Interfere with protein-protein or DNA-protein interactions that are needed for the activity of oncogenes and/or growth factors or the transcription factors essential for cell growth. This approach has been demonstrated to work in vitro to interfere with SH2-tyrosine phosphate interactions (i.e., Grb-2 and EGF receptor) and Ras-Raf interactions using specific peptides (J. Downward), but to be useful therapeutically it must be possible to introduce stable low-molecular-weight drugs into cells to affect these interactions.(ABSTRACT TRUNCATED AT 400 WORDS)