IGF-1R Signaling Pathway Research Articles

Abstract 1757: MEDI-573, an IGF-I/II neutralizing human antibody inhibits both IGF-1R and IR-A signaling pathways

Abstract Insulin-like growth factors (IGF), IGF-I and IGF-II, are small polypeptides involved in regulating cell proliferation, survival, differentiation and transformation. IGF activities are mediated through binding and activation of IGF-1R or insulin receptor isoform A (IR-A). Overexpression of IGF-II and IR-A has been reported in multiple types of cancer, and has also been proposed as a potential mechanism for cancer cells to develop resistance to IGF-1R targeting therapy. MEDI-573 is a dual targeting human antibody that neutralizes both IGF-I and IGF-II. We have shown previously that this antibody inhibits IGF-1R activation by IGF and demonstrated potent in vivo tumor growth inhibition activity in IGF driven xenograft models. Here we show that MEDI-573 inhibits IGF-II activated IR-A signal pathways without cross reactivity to insulin, and therefore has minimum impact on glucose metabolism, which is mediated mainly by insulin/IR interaction. MEDI-573 blocks the binding of IGF-II to, and inhibits the subsequent phosphorylation of IR-A and IRS-1. MEDI-573 inhibited IGF-II-induced in vitro growth of IR-A overexpressing cells with or without the presence of up to 10uIU of insulin in the medium. In addition, MEDI-573 inhibited IGF-II-induced proliferation of IR-A-overexpressing cells as effectively as it inhibited the IGF-1R-overexpressing cells. The anti-proliferative activity of MEDI-573 is not changed across heterogeneous mixed cell populations with various ratios of IR-A- versus IGF-1R-expressing cells. In contrast, an IGF-1R specific antibody was most active at inhibiting the proliferation of a pure IGF-1R expressing homogenous population, but lost activity when the heterogeneous cell population contained increasing percentages (25-50%) of IR-A-expressing cells. We also examined the relative abundance of IR-A versus IR-B (insulin receptor isoform B) mRNA in multiple cancer cell lines using a previously published quantitative RT-PCR method. The results show that IR-A is frequently the dominant isoform overexpressed in these cancer cells. Taken together, these results demonstrate that by neutralizing IGF-I and IGF-II ligands, MEDI-573 is a dual targeting antibody that offers an effective approach to selectively target both the IGF-1R and IR-A signaling pathways and potentially overcome IGF-1R targeting resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1757. doi:10.1158/1538-7445.AM2011-1757

Abstract 1058: LKB1 negatively regulates IGF-1 induced ERK phosphorylation

Abstract The LKB1/AMPK metabolic checkpoint can be activated by various energetic stresses, and a common reagent to induced LKB1/AMPK signaling is 2-deoxyglucose (2-DG), a glycolytic inhibitor. We recently discovered a novel function of 2-DG because it is capable of disrupting the interaction between IGF-1 and IGFBP3 to release free IGF-1 and activate IGF1R signaling. Therefore, 2-DG activates LKB1/AMPK and IGF1R signaling pathways through two distinct mechanisms. Interestingly, while 2-DG induced IGF1R signaling activates PI3K/Akt function in most cell lines; the induction of MEK/ERK signaling was only observed in some cancer cells. Here, we demonstrated that 2-DG induced ERK phosphorylation in LKB1 mutant cells but suppressed ERK phosphorylation in cells with wild-type LKB1. The phenomenon is LKB1-dependent because restoration of LKB1 expression in LKB1-null cells attenuated IGF-1 induced ERK phosphorylation, and the depletion of LKB1 by RNAi in LKB1-wild type cells promoted ERK phosphorylation. Mechanistically, LKB1 mediates ERK suppression through its down-stream target, AMPK, as the inhibition of AMPK function either by RNAi or small molecular inhibitors promotes IGF-1 induced ERK phosphorylation. In summary, this study has demonstrated a novel function of LKB1/AMPK in the negative regulation of IGF-1 induced ERK signaling. (This study was supported by the Georgia Cancer Coalition Distinguished Cancer Scholar award, NIH/NCI PO1 CA116676-030002. SY Sun, FR Khuri and W Zhou are Georgia Cancer Coalition Distinguished Cancer Scholars. W Zhou is an American Cancer Society Research Scholar.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1058. doi:10.1158/1538-7445.AM2011-1058

Abstract 2211: Quantitative analysis of IGF-1R expression and signaling pathway activation in FFPE xenograft and human tumor tissues

Abstract The IGF-1R signal pathway is activated in many cancer types which has led to active clinical development of both antibody and small molecule IGF1R targeted therapeutics. Multiple clinical trials targeting IGF1R are currently underway, however after initial optimism in NSCLC and Ewing's sarcoma, several trials have failed to show efficacy or had severe toxicity. Pretreatment quantitation of tumor IGF1R protein expression remains a challenge due to high homology between IGF1R and the insulin receptor. Thus, there is a need for a specific and quantitative clinical assay for IGF-1R protein expression. The ability to quantify IGF1R expression and determine signaling pathway activation status directly in formalin-fixed paraffin-embedded (FFPE) patient tissue biopsies should help identify and select patients most likely to benefit from anti-IGF1R therapies. We have developed a quantitative IGF1R assay which can be performed directly in FFPE patient tissue. This approach is based on the Liquid Tissue®-SRM technology platform which enables relative and absolute quantification of proteins and their phosphorylation status directly in formalin fixed tissue. The IGF1R- specific SRM assay was developed and preclinically validated on cell lines expressing a range of IGF1R protein. The final assay has a LOD of <20 attomol/ug and an LOQ of ∼35 attomol/ug, values that are well in line with expected levels of expression in primary tumor sourced cell lines and xenograft explants. The expression levels of IGF1R in seven formalin fixed rhabdomyosarcoma (RMS) cell lines were measured by SRM mass spectrometry. We detected a range of IGF1R expression from 68 to 333 amol/ug for these cell lines, values that showed extremely high correlation with antibody based techniques (Cao et al, Cancer Res. 68:8039). We extended this quantitation study by measuring expression of IGF-1R in human rhabdomyosarcoma xenograft explants and a cohort of ∼40 embryonal and alveolar rhabdomyosarcoma tumors. Interestingly, in xenografts treated with a therapeutic IGF1R antibody, tumor regression was seen, and a loss of IGF1R expression was noted by SRM analysis. Our goal is to define expression levels of IGF1R in these tumors, and to validate this assay for use as a companion diagnostic in follow on clinical trials of IGF1R inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2211. doi:10.1158/1538-7445.AM2011-2211

Abstract LB-212: XGFR, an Fc-engineered dual signaling inhibitor targeting IGF-1R and EGFR

Abstract Background: Elevated signaling via the receptor tyrosine kinases IGF-1R and EGFR has been identified as common characteristic of multiple cancer type. IGF-1R and EGFR signal predominantly through the PI3K and MAPK signaling pathways and thereby mediate growth and survival signals crucial for the development and progression of cancer. There is strong cross talk on multiple levels between IGF-1R and EGFR dependent signaling pathways. Therefore, targeting IGF-1R and EGFR simultaneously is an attractive way to achieve maximal inhibition of signal transduction and to avoid resistance formation. Methods: Bispecific IGF1R-EGFR antibodies were engineered by linking scFv domains of an EGFR Mab (GA201) via Serine-Glycine linkers to an IgG1 IGF-1R Mab (RG1507). The functional properties of the bispecific antibodies were evaluated in cellular in vitro assays (IGF-1R/EGFR phosphorylation, downregulation, 3D proliferation and ADCC assays) and in in vivo xenograft models for tumor growth inhibition and survival. Results: Bispecific IGF-1R-EGFR antibodies (XGFR2, XGFR3, XGFR4) were successfully generated with yields and stability comparable to conventional IgG1 antibodies. XGFR antibodies effectively inhibited IGF-1R and EGFR phosphorylation and 3D proliferation in H322M tumor cells and induced strong downmodulation of IGF-1R and enhanced EGFR downmodulation compared to the parental EGFR antibody GA201. XGFR antibodies showed strong anti-tumor efficacy comparable to the combination of monospecific IGF-1R and EGFR Mabs in the BxPC3 and H322M xenograft models. To enhance the ADCC properties of XGFR, afucosylated, glycoengineered bispecific antibodies with enhanced affinity for FcγRIIIA were generated using the GlycoMab technology. Glycoengineered bispecific antibodies were shown to have superior ADCC properties in in vitro ADCC assays and XGFR4 significantly prolonged median and overall survival of mice in an ADCC competent in vivo model (A549 i.v.). Conclusions: Bispecific IGF-1R-EGFR antibodies represent an attractive therapeutic strategy to simultaneously target two key components of multiple cancer types (IGF-1R and EGFR), resulting in effective inhibition of the PI3K and MAPK signaling pathway and to avoid the formation of resistance to therapy. Having overcome issues of stability and productivity, bispecific antibodies may become an advantageous way to reduce costs and infusion times in cancer therapy, while at the same time, achieving maximal anti-tumor effects through inhibition of multiple targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-212. doi:10.1158/1538-7445.AM2011-LB-212

Dual IGF-I/II–Neutralizing Antibody MEDI-573 Potently Inhibits IGF Signaling and Tumor Growth

Insulin-like growth factors (IGF), IGF-I and IGF-II, are small polypeptides involved in regulating cell proliferation, survival, differentiation, and transformation. IGF activities are mediated through binding and activation of IGF-1R or insulin receptor isoform A (IR-A). The role of the IGF-1R pathway in promoting tumor growth and survival is well documented. Overexpression of IGF-II and IR-A is reported in multiple types of cancer and is proposed as a potential mechanism for cancer cells to develop resistance to IGF-1R-targeting therapy. MEDI-573 is a fully human antibody that neutralizes both IGF-I and IGF-II and inhibits IGF signaling through both the IGF-1R and IR-A pathways. Here, we show that MEDI-573 blocks the binding of IGF-I and IGF-II to IGF-1R or IR-A, leading to the inhibition of IGF-induced signaling pathways and cell proliferation. MEDI-573 significantly inhibited the in vivo growth of IGF-I- or IGF-II-driven tumors. Pharmacodynamic analysis demonstrated inhibition of IGF-1R phosphorylation in tumors in mice dosed with MEDI-573, indicating that the antitumor activity is mediated via inhibition of IGF-1R signaling pathways. Finally, MEDI-573 significantly decreased (18)F-fluorodeoxyglucose ((18)F-FDG) uptake in IGF-driven tumor models, highlighting the potential utility of (18)F-FDG-PET as a noninvasive pharmacodynamic readout for evaluating the use of MEDI-573 in the clinic. Taken together, these results demonstrate that the inhibition of IGF-I and IGF-II ligands by MEDI-573 results in potent antitumor activity and offers an effective approach to selectively target both the IGF-1R and IR-A signaling pathways.

The role of microRNAs in neural stem cell-supported endothelial morphogenesis

Functional signaling between neural stem/progenitor cells (NSPCs) and brain endothelial cells (ECs) is essential to the coordination of organized responses during initial embryonic development and also during tissue repair, which occurs following brain injury. In this study, we investigated the molecular mechanisms underlying this functional signaling, using primary mouse brain ECs and NSPCs from embryonic mouse brain. EC/NSPC co-culture experiments have revealed that neural progenitors secrete factors supporting angiogenesis, which induce noticeable changes in endothelial morphology. We demonstrate that NSPCs influence the expression of mTOR and TGF-β signaling pathway components implicated in the regulation of angiogenesis. Endothelial morphogenesis, an essential component of vascular development, is a complex process involving gene activation and the upregulation of specific cell signaling pathways. Recently identified small molecules, called microRNAs (miRNAs), regulate the expression of genes and proteins in many tissues, including brain and vasculature. We found that NSPCs induced considerable changes in the expression of at least 24 miRNAs and 13 genes in ECs. Three NSPC-regulated EC miRNAs were identified as the potential primary mediators of this NSPC/EC interaction. We found that the specific inhibition, or overexpression, of miRNAs miR-155, miR-100, and miR-let-7i subsequently altered the expression of major components of the mTOR, TGF-β and IGF-1R signaling pathways in ECs. Overexpression of these miRNAs in ECs suppressed, while inhibition activated, the in vitro formation of capillary-like structures, a process representative of EC morphogenesis. In addition, we demonstrate that inhibition of FGF, VEGF, and TGF-β receptor signaling abolished NSPC-promoted changes in the endothelial miRNA profiles. Our findings demonstrate that NSPCs induce changes in the miRNA expression of ECs, which are capable of activating angiogenesis by modulating distinct cell signaling pathways.

10 Targeting the IGF-1R signalling pathway in non-small cell lung cancer (NSCLC)

10 Targeting the IGF-1R signalling pathway in non-small cell lung cancer (NSCLC)

Comprehensive analysis of expression patterns of insulin-like growth factor and Src pathway in patients with non-small cell lung cancer: Two large, independent series of tissue microarray.

10531 Background: IGF-1R and Src-family kinase (SFK) signaling pathways have been implicated in non-small cell lung cancer (NSCLC) and suggested to cooperate for enhanced tumor activities. Here we assessed the potential prognostic roles of the activation status of IGF-1R and Src pathways in patients with NSCLC. Methods: Expressions of the IGF-1R signaling axis, including phospho-IGF-1R/IR (pIGF-1R/IR), IGF- 1, IGF-2, IGF-1R and phospho-Src (pSrc) were evaluated in tissue microarrays of NSCLC from two independent databases (N=352, and 458, respectively). The expression levels of these biomarkers were correlated with clinicopathologic characteristics and patient survival. Univariate and multivariate analyses were performed to assess whether the expression levels of these biomarkers were independent prognostic factors. Results: pIGF-1R/IR (p=.037), IGF-1R (p<.0001), IGF-2 (p<.0001), and pSrc (p<.0001) expressions were significantly associated with squamous cell carcinoma (SCC), while IGF-1 (p<.0001) expression was significantly correlated with adenocarcinoma (ADC). pIGF-1R/IR (p=.009) and pSrc (p=.005) were positively correlated with smoking history. The expression of pSrc serves as a predictor of poor survival in these patients by multivariate analysis. Conclusions: Our data suggest that: 1) IGF-1R and Src are highly activated in patients with NSCLC, especially those with SCC or smoking history, and should be investigated as therapeutic targets in NSCLC patients; and 2) Src activity may counteract the antitumor action of targeting agents against IGF-1R, providing the rationale to integrate Src-targeting agents in treatment regimens with IGF-1R inhibitors for NSCLC patients. NIH grants R01 CA109520 and CA100816 (H-Y. Lee), and in part by DOD grant W81XWH-04-1-0142-01-VITAL (W.K. Hong). No significant financial relationships to disclose.

Development of cetuximab-resistant human nasopharyngeal carcinoma cell lines and mechanisms of drug resistance

This work aimed to explore the induction of cetuximab-resistant human nasopharyngeal carcinoma (hNPC) 5-8F/Erbitux cells and the mechanisms of drug resistance. The 5-8F cells with high EGFR expression and cetuximab sensitivity were screened and then induced by stepwise exposure to increasing doses of cetuximab. Western blot was conducted to detect protein levels. Our results are as follows: we successfully induced the cetuximab-resistant 5-8F/Erbitux hNPC cells. After treatment with cetuximab for 3 and 5 days, the RI was 1.2 and 1.1, respectively. The 5-8F/Erbitux cells showed cross-resistance to 5-FU ( P < 0.01) and some resistance to Taxol ( P > 0.05) as well as enhanced sensitivity to DDP ( P > 0.05). The cells had increased levels of P-gP, IGF-1R, P-IGF-1R, K-ras, H-ras, and PTEN protein expression ( P < 0.001), while survivin decreased ( P < 0.001). Through sequence alignments, gene mutations in the PTEN gene at exons 5, 7, and 8, as well as the H-ras and K-ras genes in codons 12, 13, 59, and 61, were not observed. After transfection with H-ras-shRNA plasmid, the 5-8F/Erbitux cells showed reduced levels of gene and protein expression of H-ras and elevated sensitivity to cetuximab. In conclusion, gene amplification and overexpression of H-ras was the major mechanism that caused resistance of 5-8F/Erbitux cells to cetuximab, while the overexpression of the H-ras gene was probably associated with the over-activity of the IGF-1R signaling pathway. Gene deletion or mutation of PTEN was not associated with resistance of 5-8F/Erbitux cells to cetuximab.

Targeting the Insulin-like Growth Factor Receptor-1R Pathway for Cancer Therapy

Signaling through the insulin-like growth factor receptor (IGF-1R) is required for neoplastic transformation by a number of oncogenes, and preclinical validation studies have suggested IGF-1R is an attractive target for anticancer therapy. A number of small molecules and antibodies targeting IGF-1R have entered clinical development, and early results have suggested that these agents have generally acceptable safety profiles as single agents. Some evidence of antitumor activity has also been reported. This review highlights key aspects of the IGF-1R signaling pathway that implicate it as an attractive therapeutic target in the management of cancer, as well as some key lessons that have emerged from early clinical development of anti-IGF-1R targeting agents. In addition, we consider the importance of selecting indications characterized by pathological alterations in the signaling pathway, rational selection of combinations based on signaling pathway interactions, and strategies for patient selection based on analysis of predictive biomarkers.

Abstract 4587: Quantitative analysis of IGF-1R signaling pathway activation in FFPE tissue

Abstract Multiple Ewing's sarcoma clinical trials targeting insulin-like growth factor receptor (IGF-1R) with IGF-1R small molecules and antibody inhibitors are currently underway. The IGF-1R signal pathway is commonly activated in many cancer types so this targeted therapeutic approach holds great promise for not only treating Ewing's sarcoma but other types of cancers as well. There is currently no good method to quantitatively measure the IGF-1R protein, its phosphorylation status, or the immediate downstream signal pathway target IRS-1. Being able to quantify these proteins and determine their phosphorylation status directly in patient tissue biopsies could help identify and select patients most likely to benefit from anti-IGF-1R therapies. We have developed an approach to determine both absolute IGF-1R and IRS-1 levels and the phosphorylation status of both proteins directly in formalin-fixed paraffin-embedded (FFPE) patient tissue. This approach is based on the Liquid Tissue®-SRM technology platform, a combination of tissue microdissection, Liquid Tissue® processing which turns dissected tissue to a complete solubilized tryptic digest, and mass spectrometry-based selected reaction monitoring (SRM). This approach enables relative and absolute quantification of proteins and their phosphorylation status directly in formalin fixed tissue. This approach was used to measure the IGF-1R protein and its phosphorylation sites pY1131, pY1135, and pY1136 as well as the IRS-1 protein and its phosphorylation sites pY612 and pY896 in formalin fixed tissue culture cells and xenograft tumors. Results indicate the potential to detect and quantify the IGF-1R and IRS-1 proteins and to monitor their phosphorylation status directly in FFPE tumor tissue which could be used to identify and stratify patients most likely to benefit from anti-IGF-1R therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4587.

Abstract 616: R1507, the human monoclonal antibody targeting the insulin-like growth factor receptor (IGF-1R), in combination with cetuximab, can reverse acquired resistance to cetuximab in pancreatic and colon cancer models

Abstract Background: Cetuximab, the chimeric monoclonal antibody (mAb) directed against the human epidermal growth factor receptor (EGFR) is widely used for the treatment of colorectal cancer and is also effective in pancreatic cancer preclinical models. Acquired resistance to cetuximab is common and the IGF-1R signaling pathway is critically involved in this process. We hypothesized that the combined activation of EGFR and IGF1-R is responsible for the constitutive activation of the transcriptional factor STAT-3 which in turn results in acquired resistance to cetuximab in colorectal and pancreatic tumors. Targeting this activation of EGFR and IGF1-R using the combination of cetuximab and R1507 could potentially reverse this resistance. Methods: A model of pancreatic cancer (PC) cell line resistant in vivo to cetuximab was established by injecting orthotopically, luciferase- and GFP- expressing AsPC-1 human PC cells into nude mice. Once established these tumors in vivo, the mice were treated with cetuximab 500 µg ip bi-weekly until the 34th week, when tumor growth occurred despite cetuximab due to acquired resistance. The tumors were rapidly excised and the GFP- positive cells were selected to establish the cetuximab- resistant clone AsPC-1-CR. Two models of colon cancer (CRC) cell line with acquired resistance in vivo to cetuximab, SW480-CR and GEO-CR, were established as previously described (Bianco et al. Clin Cancer Res. 2008 14(16):5069-80). MTT assay was used to assess the antitumor activity of cetuximab, R1507, and the combination in vitro. Synergy was assessed according the combination-index method of Chou and Talalay. The expression of IGF1-R was studied by Western blot. STAT-3 activation was analyzed by EMSA. Inhibition of directional cell migration was studied in an in vitro wound-healing model. Results: The cetuximab- resistant PC and CRC cell lines, showed a significantly lower in vitro sensitivity to cetuximab when compared to the respective control cell lines. AsPC-1-CR and SW480-CR cells demonstrated a higher expression of IGF1-R. STAT-3 DNA binding activity was significantly increased in all of the cetuximab- resistant cells as compared with control cell lines. The addition of R1507 did not improve the antitumor activity of cetuximab in the control cell lines. However, the combination of cetuximab + R1507 reversed the resistance to cetuximab in the AsPC-1-CR and SW480-CR cell lines, thereby demonstrating synergistic antitumor activity in the setting of acquired resistance. R1507+cetuximab also significantly inhibited the migration of cetuximab- resistant CRC cells. Conclusion: IGF-1R inhibition using R1507 is a promising approach to restore the sensitivity to cetuximab in PC and CRC cells that have developed acquired resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 616.

Abstract 5402: Pre-clinical treatment of adrenal cortical carcinoma with a combination of small molecule inhibitors to IGF-1R and mTOR

Abstract Adrenocortical carcinoma (ACC) is a rare, aggressive cancer of the adrenal cortex. The five-year survival rate is 10-20% due to late diagnosis and lack of effective systemic therapy. The only treatment options available are surgical resection and chemotherapy in the form of mitotane; however, up to 95% of patients are not responsive to treatment, so new therapies are needed. Research has shown that the mitogenic hormone insulin-like growth factor 2 (IGF-2) is overexpressed in 93% of sporadic cases of ACC. IGF-2 binds to the receptor tyrosine kinase IGF receptor 1 (IGF-1R). Inhibition of IGF-1R has been shown to be effective against ACC in animal studies using IGF-1R targeted therapies. The major IGF-1R signaling pathway is phosphotidylinositol-3′-kinase (PI3′K), which activates AKT leading to the activation of mTOR, the mammalian target of rapamycin. Recent research has shown that mTOR may be a viable target for inhibition to treat tumors. Work in our lab has shown that a combination of mTOR inhibition by rapamycin and IGF1-R inhibition by either PQ401 or NVP-AEW54 at sub optimal doses blocks proliferation of human ACC H295R cells compared to application of either drug alone. Further investigation will be conducted in a mouse model system to determine the effects of the mTOR inhibitor everlimus (RAD001) alone and in combination with IGF1-R inhibitor OSI-906 to determine possible synergistic effects of the drugs that can be clinically applicable to treat ACC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5402.

Autocrine IGF-1/IGF-1R signaling is responsible for constitutive PI3K/Akt activation in acute myeloid leukemia: therapeutic value of neutralizing anti-IGF-1R antibody

Alterations in the PI3K/Akt pathway are found in a wide range of cancers and the development of PI3K inhibitors represents a promising approach to cancer therapy. Constitutive PI3K activation, reflecting an intrinsic oncogenic deregulation of primary blast cells, is detected in 50% of patients with acute myeloid leukemia. However, the mechanisms leading to this activation are currently unknown. As we previously reported IGF-1 autocriny in acute myeloid leukemia cells, we investigated whether IGF-1 signaling was involved in the constitutive activation of PI3K. We analyzed the IGF-1/IGF-1R signaling pathway and PI3K activity in 40 acute myeloid leukemia bone marrow samples. Specific inhibition of IGF-1/IGF-1R signaling was investigated using neutralizing anti-IGF-1R, anti-IGF-1 antibodies or IGF-1 short interfering RNA. The anti-leukemic activity of the neutralizing anti-IGF-1R was tested by analyzing its effects on leukemic progenitor clonogenicity, blast cell proliferation and survival. In all samples tested, we found that functional IGF-1R was constantly expressed in leukemic cells. In the acute myeloid leukemia samples with PI3K activation, we found that the IGF-1R was constitutively phosphorylated, although no IGF-1R activating mutation was detected. Specific inhibition of IGF-1R signaling with neutralizing anti-IGF-1R strongly inhibited the constitutive phosphorylation of both IGF-1R and Akt in 70% of the PI3K activated samples. Moreover, both incubation with anti-IGF-1 antibody and IGF-1 short interfering RNA inhibited Akt phosphorylation in leukemic cells. Finally, neutralizing anti-IGF-1R treatment decreased the clonogenicity of leukemic progenitors and the proliferation of PI3K activated acute myeloid leukemia cells. Our current data indicate a critical role for IGF-1 autocriny in constitutive PI3K/Akt activation in primary acute myeloid leukemia cells and provide a strong rationale for targeting IGF-1R as a potential new therapy for this disease.

GLP-1 protects β-cells against apoptosis by enhancing the activity of an IGF-2/IGF1-receptor autocrine loop

GLP-1 protects β-cells against apoptosis by still incompletely understood mechanisms. In a recent study, we searched for novel anti-apoptotic pathways by performing comparative transcriptomic analysis of islets from Gipr-/-;Glp-1r-/- mice, which show increased susceptibility to cytokine-induced apoptosis. We observed a strong reduction in IGF-1R expression in the knockout islets suggesting a link between the gluco-incretin and IGF-1R signaling pathways. Using MIN6 and primary islet cells, we demonstrated that GLP-1 strongly stimulates IGF-1R expression and that activation of the IGF-1R/Akt signaling pathway required active secretion of IGF-2 by the β-cells. We showed that inactivation of the IGF-1 receptor gene in β-cells or preventing its up-regulation by GLP-1, as well as suppressing IGF-2 expression or action, blocked the protective effect of GLP-1 against cytokine-induced apoptosis. Thus, an IGF-2/IGF-1 receptor autocrine loop operates in β-cells and GLP-1 increases its activity by enhancing IGF-1R expression and by stimulating IGF-2 secretion. This mechanism is required for GLP-1 to protect β-cells against apoptosis.

Stable interference of EWS–FLI1 in an Ewing sarcoma cell line impairs IGF-1/IGF-1R signalling and reveals TOPK as a new target

BACKGROUND: Ewing sarcoma is a paradigm of solid tumour -bearing chromosomal translocations resulting in fusion proteins that act as deregulated transcription factors. Ewing sarcoma translocations fuse the EWS gene with an ETS transcription factor, mainly FLI1. Most of the EWS–FLI1 target genes still remain unknown and many have been identified in heterologous model systems.METHODS: We have developed a stable RNA interference model knocking down EWS–FLI1 in the Ewing sarcoma cell line TC71. Gene expression analyses were performed to study the effect of RNA interference on the genetic signature of EWS–FLI1 and to identify genes that could contribute to tumourigenesis.RESULTS: EWS–FLI1 inhibition induced apoptosis, reduced cell migratory and tumourigenic capacities, and caused reduction in tumour growth. IGF-1 was downregulated and the IGF-1/IGF-1R signalling pathway was impaired. PBK/TOPK (T-LAK cell-originated protein kinase) expression was decreased because of EWS–FLI1 inhibition. We showed that TOPK is a new target gene of EWS–FLI1. TOPK inhibition prompted a decrease in the proliferation rate and a dramatic change in the cell's ability to grow in coalescence.CONCLUSION: This is the first report of TOPK activity in Ewing sarcoma and suggests a significant role of this MAPKK-like protein kinase in the Ewing sarcoma biology.

Analysis of IGF1R gene copy number by silver in situ hybridization (SISH) in non-small cell lung cancer (NSCLC)

7525 Background: IGF1R is a potential biomarker for predicting outcome of NSCLC patients (pts) treated with therapeutics targeting the IGF1R signaling pathway. SISH can be expeditiously analyzed on a bright field microscope given that the morphology of the tissue is observed concurrent to the scoring of discrete gene signals. Methods: An experimental SISH probe designed by Ventana Medical Systems (Tucson, AZ) was used to evaluate IGF1R gene copy number on a tissue microarray containing triplicate samples from 189 pts surgically treated for NSCLC (median follow-up 4 years and 5-year survival probability of 40%). Valid results, at least one core with sufficient tumor content for assessment, were obtained for 166 patients. There were 128 males; 93 squamous cell carcinomas (SCC), 47 adenocarcinomas, 5 large cell carcinomas and 21 other histologies. The pathological stages were I: 68, II: 37 and III/IV: 60. The mean number of IGF1R gene copies/nuclei/core was determined by a board certified pathologist counting 50 nuclei in each core. The mean number of IGF1R gene copies/nuclei/patient was determined by using, within the triplicates, the core with the highest gene copy number/nuclei. Results: The mean number of IGF1R gene copies/nuclei per patient was 2.26 (range: 1.12 - 7.56; standard deviation 0.81). The median copy number was 2.11. The pts were divided into two groups, those with 2.1 genes/nuclei or less and those with greater than 2.1 genes/nuclei. There was no statistical difference related to gender (p=0.422) or between pathological stages, (p=0.221). However, there was a highly significant difference between the two categories when considering histological pattern. Among patients with SCC, 66.3% had high copy number compared to 33.7 % in non-SCC histologies (p=0.008). Analysis of overall survival comparing pts with low vs. high IGF1R gene copy number revealed no statistical difference in the median survival: 1.5 yrs (95% CI 0–3.7 yrs) vs. 3.3 yrs (2.0–4.5 yrs) or the 3-year survival: 46% (35–57%) vs. 52% (41–63%). Conclusions: IGF1R gene copy number detected by SISH is higher in SCC than in other histologies of NSCLC, but does not associate with gender, pathological stage or survival. IGF1R SISH should be further explored as a predictive biomarker for IGF1R therapeutics. [Table: see text]

Introduction: Uveal melanoma

Introduction: Uveal melanoma

Curcumin enhances the effects of 5‐fluorouracil and oxaliplatin in mediating growth inhibition of colon cancer cells by modulating EGFR and IGF‐1R

Curcumin (diferuloylmethane), which has been shown to inhibit growth of transformed cells, has no discernible toxicity and achieves high levels in colonic mucosa. 5-fluorouracil (5-FU) or 5-FU plus oxaliplatin (FOLFOX) remains the backbone of colorectal cancer chemotherapeutics, but with limited success. The present investigation was, therefore, undertaken to examine whether curcumin in combination with conventional chemotherapeutic agent(s)/regimen will be a superior therapeutic strategy for colorectal cancer. Indeed, results of our in vitro studies demonstrated that curcumin together with FOLFOX produced a significantly greater inhibition (p < 0.01) of growth and stimulated apoptosis (p < 0.001) of colon cancer HCT-116 and HT-29 cells than that caused by curcumin, 5-FU, curcumin + 5-FU or FOLFOX. These changes were associated with decreased expression and activation (tyrosine phosphorylation) of EGFR, HER-2, HER-3 (72-100%) and IGF-1R (67%) as well as their downstream effectors such as Akt and cycloxygenase-2 (51-97%). Furthermore, while these agents produced a 2-3-fold increase in the expression of IGF-binding protein-3 (IGFBP-3), curcumin together with FOLFOX caused a 5-fold increase in the same, when compared to controls. This in turn led to increased sequestration of IGF by IGFBP-3 rendering IGF-1 unavailable for binding to and activation of IGF-1R. We conclude that the superior effects of the combination therapy of curcumin and FOLFOX are due to attenuation of EGFRs and IGF-1R signaling pathways. We also suggest that inclusion of curcumin to the conventional chemotherapeutic agent(s)/regimen could be an effective therapeutic strategy for colorectal cancer.

Mechanisms of Curcumin- and EGF-Receptor Related Protein (ERRP)-Dependent Growth Inhibition of Colon Cancer Cells

: Numerous dietary and pharmacological agents have been proposed as alternative strategies for treatment and prevention of colorectal cancer. Curcumin, an active ingredient of turmeric, that inhibits growth of malignant neoplasms, has a promising role in the prevention and treatment of colorectal cancer. EGF-R related protein (ERRP), a recently identified pan-erbB inhibitor, is a potential therapeutic agent for colorectal cancer. Here we examine whether curcumin together with ERRP will cause a greater inhibition of growth of colon cancer cells than either agent alone and the mechanisms of this inhibition. Human colon cancer HCT-116 or HT-29 cells were incubated with increasing doses of curcumin (up to 10 μM) or ERRP (up to 5 μg/ml), or a combination of both for 48 h. We observed that the cell growth inhibition and stimulation of apoptosis in response to the combinatorial treatment was significantly greater than that caused by either agent alone. These changes were associated with decreased activation (tyrosine phosphorylation) of EGFR, ErbB-2, ErbB-3, and/or IGF-1R. Whereas curcumin inhibited constitutive activation of both EGFR and IGF-1R, ERRP decreased activation of EGFR, ErbB-2, and ErbB-3 but had no effect on IGF-1R. Further, the combination therapy caused a greater attenuation of downstream effectors such as NF-κB, Akt and BAD activation, and down-regulation of procaspase-3 than that noted with either agent alone. The superior effects of the combinatorial treatment could partly be attributed to inhibition of constitutive activation of EGFRs and IGF-1R signaling pathways.