The Key Molecules Modulating IGF-1R Signaling Pathway In Careinoma Cells

Cancer is called by a joint name of various diseases, all involving unregulated cell growth. Cancer cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may spread to distant parts of the body through the lymphatic system or bloodstream. Determining what causes cancer is rather complex. Many factors are known to increase the risk of cancer, including tobacco, infections, radiation, lack of physical activity, poor diet and obesity, and environmental pollutants. These can directly damage genes or combine with existing genetic faults within cells to result in the disease. Approximately5%-10%of cancers are entirely hereditary.Research about cancer causes focuses on the following issues:a) Agents and events which cause or facilitate genetic changes in cells destined to become cancer. b) The nature of the genetic damages, and the genes that are affected by it. c) The consequences of those genetic changes on the cell biology, both in generating the defining properties of cancer cell, and in facilitating additional genetic events that lead to further progression of cancer.The Insulin-like Growth Factor1Receptor (IGF-1R) is a transmembrane receptor that is activated by IGF-1and by the related growth factor IGF-2. It belongs to the family of tyrosine kinase receptors. IGF-1plays an important role in growth and continues to have anabolic effects in adults, which means that it induces hypertrophy of skeletal muscle and other target tissues. Mice lacking the IGF-1receptor die late in development, and present a dramatic reduction in body mass, testifying the strong growth-promoting effect of the receptor.The IGF-1R is implicated in several cancers, including lung, prostate, and breast cancers. In some instances its anti-apoptotic properties allows cancerous cells to resist the cytotoxic properties of chemotheraputic drugs or radiotherapy. In breast cancer, where EGFR inhibitors such as erlotinib are being used to inhibit the EGFR signaling pathway, IGF-1R confers resistance by forming one half of a heterodimer, allowing EGFR signaling to resume even in the presence of a suitable inhibitor. The process is referred to as crosstalk between EGFR and IGF-1R. It is further implicated in breast cancer by increasing the metastatic potential of the original tumour by inferring the ability to promote angiogenosis.Increased levels of the IGF-IR are expressed in the majority of primary and metastatic prostate cancers. Evidence suggests that IGF-IR signaling is required for survival and growth when prostate cancer cells progress to androgen independence. In addition, when immortalized prostate cancer cells mimicking advanced disease are treated with the IGF-1R ligand, IGF-1, the cells become more motile.This study will focus on the problems in the targeting IGF-1R signaling pathway, in-depth research the role of IGF-1R in tumor development; and actively looking for new and more effective therapeutic targets in this signaling pathway. Hopefully providing theoretical support for the targeting to IGF-1R, and predictive markers for tumor targeted therapy; and improve the existing problems in the cancer targeted therapy.CHAPTER I Signalling and trafficking of the Insulin-like Growth Factor1Receptor modulated by Selective recruitment of G protein coupled receptor kinases (GRKs) Background and Objective:G Protein Coupled Receptors (GPCR) and Receptor Tyrosine Kinases (RTK) are major transducer of signals across the plasma membrane. Each cell surface receptor family possesses unique structural characteristics and leads to specific signaling outcomes in the cell. However, there is extensive overlap in the signaling proteins and pathways used to produce these effects. Among them, β-arrestins, molecules previously considered to be associated exclusively with GPCRs are also involved in modulating signaling through a classical RTK, Insulin-like growth Factor type1(IGF-1R), with the same major outcomes as for GPCRs:they shut down the receptor and redirect signaling to MAPK/ERK pathway.Methods and Results:In the present they shut down the receptor and redirect signaling to MAPK/ERK pathway. In the present study, we investigated the β-arr-IGF-1R binding mechanism and reveal the missing links that would functionally portray a prototypical RTK, the IGF-1R, as a GPCR:GRK dependent phosphorylation of IGF-1R serine residues as the underlying mechanism for β-arr binding. By using bioinformatics tools and mutation analysis we could identify the specific serine residues responsible for this interaction. Finally, we confirmed the functional consequences of GRK regulation of β-arr recruitment on IGF-1R trafficking and signaling and found that while GRK6is targeting the receptor for degradation, GRK2mediated phosphorylation results in receptor endocytosis followed by recycling to the plasma membrane.Conclusion:While highlighting the cross-talk between the IGF-1R and GPCR at the level of GRKs, these results have led us to propose a novel concept in which a classical RTK operate as a GPCR. Far from being a simple philosophical exercise we believe that such an approach might have fundamental implications for cancer treatment.CHAPTER Ⅱ Molecular mechanism of IGF-1R inhibitors down--regulating the receptorPART Ⅰ IGF-1R down-regulated and ubiquinated by CP-751871in Ewing's sarcomaBackground and Objective:Figitumumab (CP-751871) is a selective fully human IgG2monoclonal antibody against the IGF-1R. It is believed that through this inhibition, figitumumab may block one of the key signaling pathways in cancer cells that leads to uncontrolled growth and survival of tumor cells. However, it is not clear that the mechanism of CP-751871inducing IGF-1R down-regulation and the signal transduction.In present study, we focus on the effects of CP-751871on IGF-1R, and how the CP-751871mediating the IGF-1R degradation. We also investigated why some cells was resistant to the mono-antibody. It is hopeful to find out one molecular target to solve the CP-751871resistance.Methods and Results:Phosphorylation of IGF-1R, Akt and ERK were initiated by50ng/ml IGF-1for5mins in ES1, RDES, LAP35and SKNMC but not in SKBR3. In Ewing's sarcoma cells such as ES1, RDES, LAP35and SKNMC, IGF-1R is ubiquitinated after stimulated with IGF-1, however, no ubiquitination was observed in the IGF-1R deficient SKBR3cells.There were obvious differences between CP-751871and IGF-1stimulation. IGF-1R, Akt and ERK were phosphorylated in all cell lines by IGF-1but not CP-751871. Only the ERK is phosphorylated in ES1cells by CP-751871. IGF-1R were obviously down regulated in all four Ewing's sarcoma cell lines by CP-751871and the LAP35is the most sensitive cells, nearly no IGF-1R left after treated24hours. CP-751871is more effective than IGF-1on IGF-1R degradation in four cell lines, however, there were significant differences in ES1and LAP35cells (p<0.05).IGF-1R was ubiquitinated by both IGF-1and CP-751871, however, CP-751871is more effective. We also found out that (3-arrestinl involved in the receptor ubiquitination, even more association in CP-751871comparing with IGF-1.BRET1was used to detect different residues involved in ubiquitination of IGF-1R in living cell in real time. In ES1LAP35and SKNMC cell lines, IGF-1could intiate both Lysine48and63linked ubiquitination, and maximum at5to10mins. However, CP-751871could only intiate the Lysine48-linked poly-ubiquitins in LAP35cells. We compared the β-arrestinl association with IGF-1R between IGF-1and CP-751871using the BRET1measurement. We could detect the most β-arrestinl association in ES1cells stimulated with IGF-1, which matched with BRET ratio of ubiquitination that is longer and stronger than in SKNMC and LAP35cells. However, the most β-arrestinl association stimulated with CP-751871was shown in LAP35, which also is matched with ubiquitination association and cell viability.Conclusion:CP-751871not only blocks ligands/receptor association, but also active signal pathway. CP-751871mediates K48-poly-ubiquitiation of IGF-1R depending on β-arrestinl. When applying the antibody as target therapy on clinic, we should pay more attention on personal treatment.PART Ⅱ IGF-1receptor is down-regulated by sunitinib induces MDM2-dependent ubiquitinationBackground and Objective:The insulin like growth factor receptor subtype1(IGF-1R) plays an important role in cancers transformationand progression. The aimis to investigate the effects of sunitinib onIGF-1R cell signaling transduction, especially on receptor phosphorylation and ubiquitination.Methods and Results:In HEK293cells, IGF-1R signaling pathways are activated inresponse to IGF-1,which induces obvious phosphorylations of receptor tyrosine and Akt, ERK. However, the phosphorylations of receptor tyrosine, Akt and ERK were significant inhibited by sunitinib. We found that both IGF-1and sunitinib obviously down regulated the IGF-1R expression. For analysis the ubiquitination, HEK293cells were simulated with100ng/ml IGF-1or10nM sunitinib for10min after serum starvation for24h. Both IGF-1and sunitinib could obviously induce the IGF-1R ubiquitination at10min compared with control (only serumfree, no stimulation), indicating IGF-1and sunitinib down-regulate the IGF-1R by increasing the receptor degradation through ubiquitination dependent proteasome pathway. We also found that MDM2combined to IGF-1R in response to sunitinib stimulation. To confirmit, HEK293cellswere transfected with human HA-MDM2(+MDM2) or siRNA toMDM2(-MDM2). Following24h serumstarvation, cells were stimulated with10nM sunitinib for10min. In over-expressed MDM2cells, IGF-1R wasmore ubiquitinated than that in mock-transfected cells (control), and no ubiquitination in-MDM2cells. These results mean that sunitinib mediates ubiquitination of IGF-1R dependent on MDM2.Conclusion:sunitinib blocks signal transduction and mediates degradation of IGF-1R.CHAPTER Ⅲ p53signaling associates with IGF-1R signal pathwayObjective:(1) clarify the sensitivity to IGF-1R mono-antibody, CP-751871, dependent on p53status.(2) explicit the effect of nutlin-3a sensitizing paclitaxel rest on p53status.Methods:(1) detect cell cycles and cell apoptosis by flow cytometry.(2) SDS-PAGE and Western bloting were used to determine proteins expression.(3) cell viability and drugs toxity were analyzed by MTT assay.Results:(1)There were higher β-arrestinl experssions in LAP35and ES1, which were more sensitive to CP-751871. The inhibition of LAP35is almost60%and ES1is nearly40%. If we knocked down the β-arrestinl in ES1and LAP35, they were more resistent to CP-751871, nearly20%increasing. On the other hand, we put β-arrestinl into the lower expressing cells, RDES and SKNMC, which will be more sensitive to CP-751871, nearly25%increasing.P53status was confirmed in the four cell lines. ES1is rearranged at t(11;22), Cys176Phe; RDES is at t(11,22), Arg273Cys; LAP35is wild type p53and SKNMC is p53deficient. We found out that wild type p53LAP35is the most sensitive and p53deficient SKNMC is the most resistant. If we treated the cell lines with Nutlin-3a, MDM2anagoist, combining with CP-751871, there was synergetic effect only in LAP35, nearly20%increasing.(2) A549cells treated with Nutlin-3a plus paclitaxel showed a significant increase in MDM2and wild-type p53protein, a marked increase in the number of cells in the GO-Gl and G2-M phases, and a significant decrease in the percentage of cells in the S phase. The percentage of apoptotic A549cells treated with10μmol/L Nutlin-3a plus10nmol/L paclitaxel was significantly lower than those treated with paclitaxel alone, and was also lower than that observed in H1299cells. MTT assays demonstrated that Nutlin-3a plus paclitaxel also significantly reduced the sensitivity of A549cells to paclitaxel compared with that of H1299cells. In conclusion,Conclusion:(1) CP-751871is more sensitive to wild type p53and higher β-arrestinl expressed cells.(2) Nutlin-3a mediates the cytotoxic effect of paclitaxel depending on p53status. It may also protect wild-type p53cells from mitotic chemotherapeutics.