C. Worrall

Selective recruitment of G protein-coupled receptor kinases (GRKs) controls signaling of the insulin-like growth factor 1 receptor

β-Arrestins are multifunctional proteins that play central roles in G protein-coupled receptor (GPCR) trafficking and signaling. β-Arrestin1 is also recruited to the insulin-like growth factor-1 receptor (IGF-1R), a receptor tyrosine kinase (RTK), mediating receptor degradation and signaling. Because GPCR phosphorylation by GPCR-kinases (GRKs) governs interactions of the receptors with β-arrestins, we investigated the regulatory roles of the four widely expressed GRKs on IGF-1R signaling/degradation. By suppressing GRK expression with siRNA, we demonstrated that lowering GRK5/6 abolishes IGF1-mediated ERK and AKT activation, whereas GRK2 inhibition increases ERK activation and partially inhibits AKT signaling. Conversely, β-arrestin–mediated ERK signaling is enhanced by overexpression of GRK6 and diminished by GRK2. Similarly, we demonstrated opposing effects of GRK2 and -6 on IGF-1R degradation: GRK2 decreases whereas GRK6 enhances ligand-induced degradation. GRK2 and GRK6 coimmunoprecipitate with IGF-1R and increase IGF-1R serine phosphorylation, promoting β-arrestin1 association. Using immunoprecipitation, confocal microscopy, and FRET analysis, we demonstrated β-arrestin/IGF-1R association to be transient for GRK2 and stable for GRK6. Using bioinformatic studies we identified serines 1248 and 1291 as the major serine phosphorylation sites of the IGF-1R, and subsequent mutation analysis demonstrated clear effects on IGF-1R signaling and degradation, mirroring alterations by GRKs. Targeted mutation of S1248 recapitulates GRK2 modulation, whereas S1291 mutation resembles GRK6 effects on IGF-1R signaling/degradation, consistent with GRK isoform-specific serine phosphorylation. This study demonstrates distinct roles for GRK isoforms in IGF-1R signaling through β-arrestin binding with divergent functional outcomes.

Something old, something new and something borrowed: emerging paradigm of insulin-like growth factor type 1 receptor (IGF-1R) signaling regulation.

The insulin-like growth factor type 1 receptor (IGF-1R) plays a key role in the development and progression of cancer; however, therapeutics targeting it have had disappointing results in the clinic. As a receptor tyrosine kinase (RTK), IGF-1R is traditionally described as an ON/OFF system, with ligand stabilizing the ON state and exclusive kinase-dependent signaling activation. Newly added to the traditional model, ubiquitin-mediated receptor downregulation and degradation was originally described as a response to ligand/receptor interaction and thus inseparable from kinase signaling activation. Yet, the classical model has proven over-simplified and insufficient to explain experimental evidence accumulated over the last decade, including kinase-independent signaling, unbalanced signaling, or dissociation between signaling and receptor downregulation. Based on the recent findings that IGF-1R “borrows” components of G-protein coupled receptor (GPCR) signaling, including β-arrestins and G-protein-related kinases, we discuss the emerging paradigm for the IGF-1R as a functional RTK/GPCR hybrid, which integrates the kinase signaling with the IGF-1R canonical GPCR characteristics. The contradictions to the classical IGF-1R signaling concept as well as the design of anti-IGF-1R therapeutics treatment are considered in the light of this paradigm shift and we advocate recognition of IGF-1R as a valid target for cancer treatment.

β-Arrestin–biased agonism as the central mechanism of action for insulin-like growth factor 1 receptor–targeting antibodies in Ewing’s sarcoma

Owing to its essential role in cancer, insulin-like growth factor type 1 receptor (IGF-1R)–targeted therapy is an exciting approach for cancer treatment. However, when translated into clinical trials, IGF-1R–specific antibodies did not fulfill expectations. Despite promising clinical responses in Ewing’s sarcoma (ES) phase I/II trials, phase III trials were discouraging, requiring bedside-to-bench translation and functional reevaluation of the drugs. The anti-IGF-1R antibody figitumumab (CP-751,871; CP) was designed as an antagonist to prevent ligand–receptor interaction but, as with all anti-IGF-1R antibodies, it induces agonist-like receptor down-regulation. We explored this paradox in a panel of ES cell lines and found their sensitivity to CP was unaffected by presence of IGF-1, countering a ligand blocking mechanism. CP induced IGF-1R/β-arrestin1 association with dual functional outcome: receptor ubiquitination and degradation and decrease in cell viability and β-arrestin1–dependent ERK signaling activation. Controlled β-arrestin1 suppression initially enhanced CP resistance. This effect was mitigated on further β-arrestin1 decrease, due to loss of CP-induced ERK activation. Confirming this, the ERK1/2 inhibitor U0126 increased sensitivity to CP. Combined, these results reveal the mechanism of CP-induced receptor down-regulation and characteristics that functionally qualify a prototypical antagonist as an IGF-1R–biased agonist: β-arrestin1 recruitment to IGF-1R as the underlying mechanism for ERK signaling activation and receptor down-regulation. We further confirmed the consequences of β-arrestin1 regulation on cell sensitivity to CP and demonstrated a therapeutic strategy to enhance response. Defining and suppressing such biased signaling represents a practical therapeutic strategy to enhance response to anti-IGF-1R therapies.

Molecular Characterization of Acquired Tolerance of Tumor Cells to Picropodophyllin (PPP)

Background Picropodophyllin (PPP) is a promising novel anti-neoplastic agent that efficiently kills tumor cells in vitro and causes tumor regression and increased survival in vivo. We have previously reported that PPP treatment induced moderate tolerance in two out of 10 cell lines only, and here report the acquired genomic and expression alterations associated with PPP selection over 1.5 years of treatment. Methodology/Principal Findings Copy number alterations monitored using metaphase and array-based comparative genomic hybridization analyses revealed largely overlapping alterations in parental and maximally tolerant cells. Gain/ amplification of the MYC and PVT1 loci in 8q24.21 were verified on the chromosome level. Abnormalities observed in connection to PPP treatment included regular gains and losses, as well as homozygous losses in 10q24.1-q24.2 and 12p12.3-p13.2 in one of the lines and amplification at 5q11.2 in the other. Abnormalities observed in both tolerant derivatives include amplification/gain of 5q11.2, gain of 11q12.1-q14.3 and gain of 13q33.3-qter. Using Nexus software analysis we combined the array-CGH data with data from gene expression profilings and identified genes that were altered in both inputs. A subset of genes identified as downregulated (ALDH1A3, ANXA1, TLR4 and RAB5A) or upregulated (COX6A1, NFIX, ME1, MAPK and TAP2) were validated by siRNA in the tolerant or parental cells to alter sensitivity to PPP and confirmed to alter sensitivity to PPP in further cell lines. Conclusions Long-term PPP selection lead to altered gene expression in PPP tolerant cells with increase as well as decrease of genes involved in cell death such as PTEN and BCL2. In addition, acquired genomic copy number alterations were observed that were often reflected by altered mRNA expression levels for genes in the same regions.

Chapter Seven - When Phosphorylation Encounters Ubiquitination: A Balanced Perspective on IGF-1R Signaling

Cell-surface receptors govern the critical information passage from outside to inside the cell and hence control important cellular decisions such as survival, growth, and differentiation. These receptors, structurally grouped into different families, utilize common intracellular signaling-proteins and pathways, yet promote divergent biological consequences. In rapid processing of extracellular signals to biological outcomes, posttranslational modifications offer a repertoire of protein processing options. Protein ubiquitination was originally identified as a signal for protein degradation through the proteasome system. It is now becoming increasingly recognized that both ubiquitin and ubiquitin-like proteins, all evolved from a common ubiquitin structural superfold, are used extensively by the cell and encompass signal tags for many different cellular fates. In this chapter we examine the current understanding of the ubiquitin regulation surrounding the insulin-like growth factor and insulin signaling systems, major members of the larger family of receptor tyrosine kinases (RTKs) and key regulators of fundamental physiological and pathological states.

Enhanced response of melanoma cells to MEK inhibitors following unbiased IGF-1R down-regulation

Due to its ability to compensate for signals lost following therapeutic MAPK-inhibition, insulin-like growth factor type 1 receptor (IGF-1R) co-targeting is a rational approach for melanoma treatment. However IGF-1R conformational changes associated with its inhibition can preferentially activate MAPK-pathway in a kinase-independent manner, through a process known as biased signaling. We explored the impact of biased IGF-1R signaling, on response to MAPK inhibition in a panel of skin melanoma cell lines with differing MAPK and p53 mutation statuses. Specific siRNA towards IGF-1R down-regulates the receptor and all its signaling in a balanced manner, whilst IGF-1R targeting by small molecule Nutlin-3 parallels receptor degradation with a transient biased pERK1/2 activity, with both strategies synergizing with MEK1/2 inhibition. On the other hand, IGF-1R down-regulation by a targeted antibody (Figitumumab) induces a biased receptor conformation, preserved even when the receptor is exposed to the balanced natural ligand IGF-1. This process sustains MAPK activity and competes with the MEK1/2 inhibition. Our results indicate that IGF-1R down-regulation offers an approach to increase the sensitivity of melanoma cells to MAPK inhibition, and highlights that controlling biased signaling could provide greater specificity and precision required for multi-hit therapy.Due to its ability to compensate for signals lost following therapeutic MAPK-inhibition, insulin-like growth factor type 1 receptor (IGF-1R) co-targeting is a rational approach for melanoma treatment. However IGF-1R conformational changes associated with its inhibition can preferentially activate MAPK-pathway in a kinase-independent manner, through a process known as biased signaling. We explored the impact of biased IGF-1R signaling, on response to MAPK inhibition in a panel of skin melanoma cell lines with differing MAPK and p53 mutation statuses. Specific siRNA towards IGF-1R down-regulates the receptor and all its signaling in a balanced manner, whilst IGF-1R targeting by small molecule Nutlin-3 parallels receptor degradation with a transient biased pERK1/2 activity, with both strategies synergizing with MEK1/2 inhibition. On the other hand, IGF-1R down-regulation by a targeted antibody (Figitumumab) induces a biased receptor conformation, preserved even when the receptor is exposed to the balanced natural ligand IGF-1. This process sustains MAPK activity and competes with the MEK1/2 inhibition. Our results indicate that IGF-1R down-regulation offers an approach to increase the sensitivity of melanoma cells to MAPK inhibition, and highlights that controlling biased signaling could provide greater specificity and precision required for multi-hit therapy.

IRS-2 deubiquitination by USP9X maintains anchorage-independent cell growth via Erk1/2 activation in prostate carcinoma cell line

Insulin-like growth factors (IGFs) have been shown to induce proliferation of many types of cells. Insulin receptor substrates (IRSs) are major targets of IGF-I receptor (IGF-IR) tyrosine kinase activated by IGFs, and are known to play important roles in the activation of downstream signaling pathways, such as the Erk1/2 pathway. Dysregulation of IGF signaling represents a central tumor promoting principle in human carcinogenesis. Prostate carcinoma is highly dependent on the IGF/IGF-IR/IRS axis. Here we identified the deubiquitinase, ubiquitin specific peptidase 9X (USP9X) as a novel binding partner of IRS-2. In a human prostate carcinoma cell line, small interfering RNA (siRNA)-mediated knockdown of USP9X reduced IGF-IR as well as IRS-2 protein levels and increased their ubiquitination. Knockdown of USP9X suppressed basal activation of the Erk1/2 pathway, which was significantly restored by exogenous expression of IRS-2 but not by IGF-IR, suggesting that the stabilization of IRS-2 by USP9X is critical for basal Erk1/2 activation. Finally, we measured anchorage-independent cell growth, a characteristic cancer feature, by soft-agar colony formation assay. Knockdown of USP9X significantly reduced anchorage-independent cell growth of prostate carcinoma cell line. Taken all together, our findings indicate that USP9X is required for the promotion of prostate cancer growth by maintaining the activation of the Erk1/2 pathway through IRS-2 stabilization.

P03-23 Effects of targeting p53/Mdm2/IGF-1R axis on skin melanoma migration, invasion and metastasis

mals. A plasma progesterone concentration of 1ng/ml or more was taken as evidence of ovarian activity. Based on the resumption of ovarian activity, cows were classified as early cycling (EC; ≤ 45 days), late cycling (LC; >45 days), non-cycling (NC) during the period of study (10 weeks postpartum). An ANOVA (with repeated measures) procedure from SAS was used. Plasma concentration of IGF-I was higher (P < 0.001) in Sanga than in Friesian-Sanga cows (18.7±0.7 vs 14.8±0.7 ng/ml) from week 1 to 10 postpartum. Also, Sanga cows had higher (P < 0.001) plasma concentration of leptin than FriesianSanga cows (1.12±0.01 vs 0.96±0.01ng/ml). The concentration of IGF-I in plasma was higher (P < 0.05) in EC (21.9±1.51ng/ml) than in NC (17.1±1.10ng/ml) Sanga cows. Similarly plasma leptin concentration in the Sanga cows was higher (P< 0.001) in EC (1.23±0.02) than LC (1.10±0.03) or NC Sanga cows (1.06±0.01ng/ml). The EC Friesian-Sanga crossbred cows had higher (P< 0.001) plasma IGF-I concentration (20.51±1.03ng/ml) than LC (14.7±1.39ng/ml) or NC (12.60±0.64ng/ml) Friesian-Sanga cows. Also the EC Friesian-Sanga cows had higher (P < 0.01) plasma leptin concentrations than NC Friesian-Sanga cows (1.03±0.03 vs 0.92±0.02ng/ml). The results indicate that systemic IGF-I and leptin vary with breed type, being higher in Sanga than in Friesian-Sanga crossbred cows. Higher concentrations of these hormones are associated with early resumption of ovarian cyclicity in Sanga and Friesian-Sanga crossbred cows.

P44 Functional study on beta-arrestins involvement in Ras induced transformation of mammalian cells

Background: Considerable cross-talk exists between the insulinlike growth factor (IGF) axis and estrogens; both are linked to breast cancer progression. Breast tumours expressing estrogen receptors (ERs) respond well to therapeutic strategies targeting this receptor. However, breast cancer progression is associated with loss of estrogen receptor expression, generally without deletion or mutation, but often due to epigenetic silencing. In screening tumours for aberrant DNA methylation, IGFBPs particularly IGFBP-3 have consistently been among the most prevalent genes identified. IGFBP-3 is the main IGFBP found in human serum and in addition to modulating the actions of the IGFs, IGFBP-3 can also act intrinsically in either a positive or negative manner depending on the context. In keeping with these in vitro data, epidemiology studies have also reported both positive and inverse associations of IGFBP-3 with pre-menopausal breast cancer. Aims: To treat cells with the demethylating agent, 5-Aza-2’deoxycytidine (AZA) to establish re-expression of the estrogen receptor with a view to assessing any coincident changes in cell growth and abundance of IGFBP-3 and any potential involvement of IGFBP-3 in the effects of AZA on the cells. Methods: ERa negative breast cancer cells, MDA-MB-231 were dosed with AZA (1mM) for 72 hrs +/− estradiol (10nM) for 24 hrs. DNA methylation status of the ERa was assessed using combined bisulfite restriction analysis (COBRA), a quantitative technique to determine DNA methylation levels at specific gene loci. Cell growth was evaluated by cell counting and tritiated thymidine incorporation. Abundance of ERa and IGFBP-3 were assessed using Western immunoblotting and concentrations of IGF-I, IGF-II and IGFBP-3 were measured by radioimmunoassay. Silencing of IGFBP-3 was achieved using siRNA with a non-silencing siRNA used as a control. Results: AZA caused a 36% decrease in methylation of the ERa promoter resulting in re-expression of ERa that was functional, as estradiol was then able to stimulate cell growth. The concentrations of IGF-I and IGF-II also increased slightly but despite these increases and the re-expression of ERa, AZA resulted in a 47% decrease in cell growth which was only partially negated by estradiol. AZA also caused an approximate 30% increase in IGFBP-3 abundance and preventing this increase using siRNA then negated the growth inhibitory effect of AZA. COBRA results indicated that the AZAinduced increase in IGFBP-3 was not due to DNA-demethylation, suggesting an indirect up-regulation of IGFBP-3 by AZA. In the presence of AZA, but with IGFBP-3 silenced, the cells responded even better to estradiol. Conclusion: AZA inhibited the growth of ER-negative breast cancer cells despite inducing the re-expression of a functional ERa and increases in IGF-I/-II. AZA also indirectly caused a coincident increase in the abundance of IGFBP-3, without affecting the methylation status of IGFBP-3. This increase in IGFBP-3 mediated the observed over-riding growth inhibition induced by AZA. P44 Functional study on beta-arrestins involvement in Ras induced transformation of mammalian cells H. Zheng, N. Natalishvili, C. Worrall, A. Girnita, L. Girnita. Department of Oncology-Pathology, Karolinska Institute, Stockholm, Sweden