Role of Ubiquitination in IGF-1 Receptor Signaling and Degradation

BACKGROUND: The insulin-like growth factor 1 receptor (IGF-1R) plays numerous crucial roles in cancer biology. The majority of knowledge on IGF-1R signaling is concerned with its role in the activation of the canonical phosphatidyl inositol-3 kinase (PI3K)/Akt and MAPK/ERK pathways. However, the role of IGF-1R ubiquitination in modulating IGF-1R function is an area of current research. In light of this we sought to determine the relationship between IGF-1R phosphorylation, ubiquitination, and modulation of growth signals. METHODOLOGY: Wild type and mutant constructs of IGF-1R were transfected into IGF-1R null fibroblasts. IGF-1R autophosphorylation and ubiquitination were determined by immunoprecipitation and western blotting. IGF-1R degradation and stability was determined by cyclohexamide-chase assay in combination with lysosome and proteasome inhibitors. PRINCIPAL FINDINGS: IGF-1R autophosphorylation was found to be an absolute requirement for receptor ubiquitination. Deletion of C-terminal domain had minimal effect on IGF-1 induced receptor autophosphorylation, however, ubiquitination and ERK activation were completely abolished. Cells expressing kinase impaired IGF-1R, exhibited both receptor ubiquitination and ERK phosphorylation, however failed to activate Akt. While IGF-1R mutants with impaired PI3K/Akt signaling were degraded mainly by the proteasomes, the C-terminal truncated one was exclusively degraded through the lysosomal pathway. CONCLUSIONS: Our data suggest important roles of ubiquitination in mediating IGF-1R signaling and degradation. Ubiquitination of IGF-1R requires receptor tyrosine kinase activity, but is not involved in Akt activation. In addition we show that the C-terminal domain of IGF-1R is a necessary requisite for ubiquitination and ERK phosphorylation as well as for proteasomal degradation of the receptor

Insulin-Like Growth Factors Promote Vasculogenesis in Embryonic Stem Cells

The ability of embryonic stem cells to differentiate into endothelium and form functional blood vessels has been well established and can potentially be harnessed for therapeutic angiogenesis. However, after almost two decades of investigation in this field, limited knowledge exists for directing endothelial differentiation. A better understanding of the cellular mechanisms regulating vasculogenesis is required for the development of embryonic stem cell-based models and therapies. In this study, we elucidated the mechanistic role of insulin-like growth factors (IGF1 and 2) and IGF receptors (IGFR1 and 2) in endothelial differentiation using an embryonic stem cell embryoid body model. Both IGF1 or IGF2 predisposed embryonic stem to differentiate towards a mesodermal lineage, the endothelial precursor germ layer, as well as increased the generation of significantly more endothelial cells at later stages. Inhibition of IGFR1 signaling using neutralizing antibody or a pharmacological inhibitor, picropodophyllin, significantly reduced IGF-induced mesoderm and endothelial precursor cell formation. We confirmed that IGF-IGFR1 signaling stabilizes HIF1α and leads to up-regulation of VEGF during vasculogenesis in embryoid bodies. Understanding the mechanisms that are critical for vasculogenesis in various models will bring us one step closer to enabling cell based therapies for neovascularization

Identification of the cathelicidin peptide LL-37 as agonist for the type I insulin-like growth factor receptor

The human cathelicidin antimicrobial protein-18 and its C terminal peptide, LL-37, displays broad antimicrobial activity that is mediated through direct contact with the microbial cell membrane. In addition, recent studies reveal that LL-37 is involved in diverse biological processes such as immunomodulation, apoptosis, angiogenesis and wound healing. An intriguing role for LL-37 in carcinogenesis is also beginning to emerge and the aim of this paper was to explore if and how LL-37 contributes to the signaling involved in tumor development. To this end, we investigated the putative interaction between LL-37 and growth factor receptors known to be involved in tumor growth and progression. Among several receptors tested, LL-37 bound with the highest affinity to insulin-like growth factor 1 receptor (IGF-1R), a receptor that is strongly linked to malignant cellular transformation. Furthermore, this interaction resulted in a dose-dependent phosphorylation and ubiquitination of IGF-1R, with downstream signaling confined to the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK)-pathway but not affecting phosphatidylinositol 3 kinase/Akt signaling. We found that signaling induced by LL-37 was dependent on the recruitment of β-arrestin to the fully functional IGF-1R and by using mutant receptors we demonstrated that LL-37 signaling is dependent on β-arrestin-1 binding to the C-terminus of IGF-1R. When analyzing the biological consequences of increased ERK activation induced by LL-37, we found that it resulted in enhanced migration and invasion of malignant cells in an IGF-1R/β-arrestin manner, but did not affect cell proliferation. These results indicate that LL-37 may act as a partial agonist for IGF-1R, with subsequent intra-cellular signaling activation driven by the binding of β-arrestin-1 to the IGF-1R. Functional experiments show that LL-37-dependent activation of the IGF-1R signaling resulted in increased migratory and invasive potential of malignant cells

Targeting insulin-like growth factor pathways

Some cancer cells depend on the function of specific molecules for their growth, survival, and metastatic potential. Targeting of these critical molecules has arguably been the best therapy for cancer as demonstrated by the success of tamoxifen and trastuzumab in breast cancer. This review will evaluate the type I IGF receptor (IGF-IR) as a potential target for cancer therapy. As new drugs come forward targeting this receptor system, several issues will need to be addressed in the early clinical trials using these agents

Targeting the IGF-1R signaling and mechanisms for epigenetic gene silencing in human multiple myeloma

Multiple myeloma (MM) is a B cell malignancy characterized by the expansion of clonal plasmablast/plasma cells within the bone-marrow. It is well established that the bone-marrow microenvironment has a pivotal role in providing critical cytokines and cell–cell interactions to support the growth and survival of the MM tumor clone. The pathogenesis of MM is, however, only fragmentarily understood. Detailed genomic analysis reveals a heterogeneous and complex pattern of structural and numerical chromosomal aberrations. In this review we will discuss some of the recent results on the functional role and potential clinical use of the IGF-1R, one of the major mediators of growth and survival for MM. We will also describe some of our results on epigenetic gene silencing in MM, as it may indeed constitute a novel basis for the understanding of tumor initiation and maintenance in MM and thus may change the current view on treatment strategies for MM

Regulation of insulin-like growth factor-1 receptor expression and signaling

Insulin-like growth factor-1 receptor (IGF-1R), a member of the insulin receptor tyrosine kinase family is a broadly expressed transmembrane receptor that plays a key role in malignant cell growth. IGF-1R transmits information provided by extracellular stimuli into intracellular signaling pathways resulting in the subsequent regulation of various effector systems. Under normal cellular conditions IGF-1R signaling network is tightly regulated. The most prominent regulator of IGF-1R signal termination is desensitization of receptors by the removal of activated receptors from the cell surface mediated by accelerated endocytosis. For some membrane receptors the signal mediating receptor internalization/downregulation is constituted by ubiquitination. Recently, we showed that IGF-1R undergoes ubiquitination following ligand stimulation. The proto-oncogene MDM2 was identified as an E3 ligase involved in IGF-1R ubiquitination. Studies on new events involved in IGF-1R downregulation and intracellular signaling constitute the subject of the present thesis. â-arrestins are ubiquitously expressed cytosolic proteins generally known to be involved in the regulation of endocytosis and signaling elicited by G protein-coupled receptors (GCPRs). We provide evidence that the two widely co-expressed isoforms of â-arrestin, bind to the IGF-1R and, by serving as adaptor proteins bring the oncoprotein E3 ligase MDM2 to the receptor. Thus, â-arrestins promote ubiquitination but also degradation of the receptor. In this respect, â-arrestin 1 is more potent then isoform 2. Actually, â-arrestins are an absolute requirement for interaction between MDM2 and IGF-1R, indicating their relevance for cell growth and cancer. We also investigated the role of â-arrestin 1 and MDM2 in intracellular signaling. We found that both MDM2 and â-arrestin 1 also are necessary for IGF-1 stimulated phosphorylation of ERK1/2 but not of Akt. In addition, the modulating effect of MDM2 and â-arrestin 1 on ERK activation has consequences on cell cycle progression. Thus, MDM2 and â-arrestin 1 do not only induce ubiquitination and degradation of IGF-1R but also influence cell growth by modulating the activity of ERKs. The cyclolignan PPP is an inhibitor of phosphorylation of IGF-1R and activation of downstream molecules, without interfering with the highly homologous insulin receptor (IR). Further, PPP has well established anti-tumor effects on several in vivo tumor models. We could demonstrate that PPP also causes downregulation of IGF-1R. Furthermore, the PPP-induced downregulation of IGF-1R required the expression of wild type MDM2 E3 ligase, indicating that MDM2-dependent ubiquitination and degradation of IGF-1R represents an important mechanism in this respect. Our data also suggest that this effect of PPP plays a role in induction of apoptosis. Finally, we demonstrated that PPP in fact induces IGF-1R ubiquitination, but also temporarily activates ERK1/2. This effect is IGF-1R-specific since PPP does not affect ERK phosphorylation in IGF-1R negative cells. Moreover, in the absence of MDM2, PPP-induced activation of ERK did not occur. The temporary MDM2-dependent ERK phosphorylation induced by PPP may contribute to the apoptotic effect of this compound

IGF-1R inhibition : A tool for functional studies of insulin-like growth factor family in malignant cells

Cancer cells generally posses the capability of overusing normal extracellular signaling for proliferation and/or antiapoptosis to create growth advantage over the normal cells. Major players in extracellular signaling are the growth factor receptors. Among them, an activated IGF-1R is important for the establishment of a malignant cell phenotype. Interestingly, the targeting of IGF-1R can reverse the malignant phenotype in cancer cells and render them sensitive to apoptosis, without seriously affecting the biology of normal cells. For these reasons, IGF-1R seems to be a very promising target in cancer therapy. Recently, we demonstrated that the cyclolignan PPP efficiently inhibited phosphorylation of IGF-1R without interfering with insulin receptor activity. This thesis is centered on: (1) functional studies of IGF-1R using PPP as a tool, with focus on importance for survival and proliferation of malignant cells as well as possible mechanisms of PPP action; (2) possible caveats in clinical applications of PPP (e.g. resistance, side effects secondary to IGF-1R inhibition, effects on glucose uptake). Using a IGF-1R tyrosine kinase construct, isolated by immunoprecipitation and amplified in insect cells, we found that PPP decreased phosphorylation of tyrosine residue (Y) 1136 in the activation loop of the IGF-1R kinase domain. Studies using dominant-negative constructs of IGF-1R (in which specific tyrosine residues are replaced by phenylalanine) suggest that the inhibition of Y 1136 phosphorylation may be important for the inhibition of Akt phosphorylation seen in PPP treated cell cultures. Whether PPP directly or indirectly (e.g. by interfering with IGF-1R associated proteins) inhibits Y1136 phosphorylation is still unknown. It was confirmed that inhibitions of downstream reactions of the phosphatyl inositol-3 kinase/anti-apoptotic pathway (e.g. attenuated Bad phosphorylation, PARP cleavage, caspase activation) were a consequence of the PPP-induced inhibition of IGF-1R. (Paper 1) We demonstrated the presence and growth dependence of IGF-1R in primary cultured craniopharyngioma cells from a subset of affected patients (5 out of 9). Upon treatment with PPP, cells with high IGF-1R expression responded promptly with decreased Akt phosphorylation followed by cell growth inhibition, whereas these responses did not appear in cells with low receptor expression. Our data points to the possibility of using IGF-1R inhibitors (e.g. PPP) as a treatment modality to obtain complete tumor-free conditions before growth hormone substitution. (Paper 2) A general concern with antitumor agents is development of resistance. In light of this problem we aimed to investigate whether malignant cells may develop serious resistance to PPP. After trying to select several malignant cell lines, only two out of 10 survived an 80-week selection. We could observe a temporary and limited increase in IGF- I R expression but there were no rearrangements or amplification of the IGF-1R gene. The resistant cell lines did not exhibit cross-resistance to known cytostatic dugs. In conclusion, no or slight resistance to PPP occurred. (Paper 3) Finally, we confirmed that PPP does not inhibit activity of the highly related insulin receptor and induce diabetogenic effects (like high blood glucose). Instead, in vivo and in vitro studies showed that PPP treatment reduces the blood glucose levels in mice and induces increase of glucose uptake in cells expressing the insulin-dependent glucosetransporter GLUT-4. (Paper 4