Inhibition Of IGF1R Signaling Research Articles

Effect of antibiotic monensin on cell proliferation and IGF1R signaling pathway in human colorectal cancer cells

Background/Aims Colorectal cancer is the third leading cause of death in patients with cancers in America. Monensin has represented anti-cancer effect on various human cancer cells. We seek to investigate the effect of monensin on proliferation of human colorectal cancer cells and explore whether IGF1R signaling pathway is involved in anti-cancer mechanism of monensin. Methods Cell proliferation and migration were assessed by crystal violet staining and cell wounding assay respectively. Cell apoptosis was analyzed by Hoechst 33258 staining and flow cytometry. Cell cycle progression was detected with the use of flow cytometry. Cancer-associated pathways were assessed with the use of pathway-specific reporters. Gene expression was detected by touchdown-quantitative real-time PCR. Inhibition of IGF1R was tested by immunofluorescence staining. Inhibition of IGF1R signaling was accomplished by adenovirus-mediated expression of IGF1. Results We found that monensin not only effectively inhibited cell proliferation, cell migration as well as cell cycle progression, but also induced apoptosis and G1 arrest in human colorectal cancer cells. Monensin was shown to target multiple cancer-related signaling pathways such as Elk1, AP1, as well as Myc/max, and suppressed IGF1R expression via increasing IGF1 in colorectal cancer cells. Conclusion Monensin could suppressed IGF1R expression via increasing IGF1 in colorectal cancer cells. It has the potential to be repurposed as an anti-colorectal cancer agent, but further studies are still required to investigate the detailed mechanisms of monensin underlying its anti-cancer motion. Key Messages Monensin inhibits the cell proliferation and the migration, induces apoptosis and inhibits cell cycle progression in human colorectal cancer cells. Monensin may exert anti-cancer activity by targeting multiple signaling pathways, including the IGF1R signaling pathway. Monensin has the potential to be repurposed as an anti-colorectal cancer agent.

Abstract P3-11-05: A phase Ib multi-cohort study of xentuzumab and abemaciclib in patients (pts) with solid tumors and breast cancer (BC) - Initial report of four dose-finding cohorts

Abstract Background: Insulin-like growth factor receptor-1 (IGF-1R) signaling is implicated in cancer progression, prognosis, and treatment resistance. Cross-talk between IGF-1R signaling and cyclin-dependent kinase (CDK) 4 & 6 was reported in preclinical studies, and synergy was shown with concurrent inhibition of IGF-1R signaling and CDK 4 & 6. The CDK 4 & 6 inhibitor, abemaciclib, is FDA approved for hormone receptor-positive [HR+], HER2- advanced BC (ABC) in combination with an aromatase inhibitor as initial endocrine-based therapy, with fulvestrant for progression following endocrine therapy (ET), and as monotherapy for progression following ET and prior chemotherapy in the metastatic setting. NCT03099174 is a prospective, open-label study investigating the safety and tolerability of concurrent inhibition of IGF/IGF-1R signaling with the IGF-1/2-neutralizing monoclonal antibody, xentuzumab, plus abemaciclib (provided by Eli Lilly), with or without ET. Methods: 4 dose-finding cohorts were evaluated in 2 parts using a Bayesian Logistic Regression Model with overdose control, fitted to toxicity outcomes. Part 1 (Cohort A) included pts with solid tumors who had failed/were intolerant of prior treatments, or had no other option available. The starting dose was xentuzumab 1000 mg i.v. weekly plus abemaciclib 150 mg orally (p.o.) every 12 h (Q12h). Part 1 determined the maximum tolerated dose (MTD) and/or recommended phase II dose (RP2D) prior to starting Part 2. Part 2 included postmenopausal women with HR+, HER2- BC who had received 0-2 prior lines of chemotherapy in the metastatic setting and were eligible for ET. One of 3 ETs was combined with xentuzumab plus abemaciclib (doses determined in Part 1): Cohort B, letrozole; Cohort C, anastrozole; Cohort D, fulvestrant. Previous CDK 4 & 6 inhibitor therapy was not permitted. Treatment was given until progression, lack of tolerability, or withdrawal. Primary endpoints were MTD and dose-limiting toxicities (DLTs) during the first 28-day cycle. Tumor response and progression-free survival (PFS) were also assessed. Results: 27 pts were treated (26 female [96.3%]; median age, 58 years [range 34-70]); 9 pts remained on treatment at the time of analysis. Cohorts A/B/C/D included 6/7/6/8 pts. Cohort A included pts with ABC (n=3), and lung cancer, sarcoma, and colorectal cancer (n=1 each). In total, 23 pts were evaluable for MTD. MTD was xentuzumab 1000 mg i.v. weekly plus abemaciclib 150 mg p.o. Q12h in Cohorts A, B, and D (Cohort C is ongoing). During Cycle 1, 4 DLTs were reported (Cohort A: grade 3 neutrophil count decrease, n=1; Cohort B: grade 3 neutrophil count decrease, n=1; Cohort C: grade 4 thrombocytopenia, n=1; Cohort D: grade 3 neutropenia, n=1). The most common drug-related AEs (DRAEs) in Cohort A were diarrhea and decreased appetite (66.7% each), and nausea, vomiting, and asthenia (50% each). The most common DRAEs in Cohorts B/C/D were diarrhea (90.5%), asthenia (66.7%), anemia (61.9%), neutropenia (57.1%), and nausea (52.4%). Most were reversible and grade 1/2. The most common grade 3/4 DRAEs were diarrhea (16.7/0%) in Cohort A, and neutropenia (19.0/14.3%) and diarrhea (14.3/0%) in Cohorts B/C/D. 4 pts had a best overall response of partial response (Cohorts A and B, n=1; Cohort D, n=2) and 10 pts had stable disease (Cohort A/B/C/D, n=1/3/1/5; duration currently <24 weeks). Median PFS (95% CI) was 1.7 (1-7.3) months in Cohort A and 12.4 (3.5-not calculable) months in Cohorts B/C/D. Conclusions: The MTD and RP2D of xentuzumab was 1000 mg i.v. weekly plus abemaciclib 150 mg p.o. Q12h with or without ET. The safety profile of xentuzumab in combination with abemaciclib, with or without ET, was tolerable and manageable, with no new safety signals. Expansion cohorts for xentuzumab plus abemaciclib and fulvestrant in HR+ HER2- BC are ongoing. Citation Format: Douglas Yee, Mafalda Oliveira, Hiroji Iwata, Anthony Gonçalves, Javier García-Corbacho, Marie Paule Sablin, Aleix Prat, Molly Catherine Hardebeck, Marta Puig, Dennis Chin-Lun Huang, Mingchi Hsu, Patricia LoRusso. A phase Ib multi-cohort study of xentuzumab and abemaciclib in patients (pts) with solid tumors and breast cancer (BC) - Initial report of four dose-finding cohorts [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-05.

Abstract 2432: GPER1 expression is modulation by D-glucose concentration in estrogen-responsive cancer and tumor cells

Abstract G protein-coupled estrogen receptor 1 (GPER1, aka GPR30), is a 7-transmembrane receptor that mediates rapid cell signaling events stimulated by 17β-estradiol (E2) in cancer and tumor cells. In breast cancer cells, GPER1 mediates E2-induced CTGF-dependent cell migration and cyclin E expression resulting in cell cycle progression. GPER1 has also been shown to mediate antiproliferative cell signaling such as p53-dependent inhibition of cell cycle progression in breast cancer cells treated with the GPER1-specific agonist G1 and the inhibition of IGF-1R signaling in tamoxifen-treated breast cancer cells. In addition to E2, both natural and synthetic compounds have been shown to activate GPER1 in breast cancer cells. GPER1 has also been shown to regulate cell signaling in cells that lack ERα. For example, GPER1 is antiproliferative in triple-negative breast cancer cells. While the importance of GPER1 in the modulation of E2-responsive tumor and cancer cells as been demonstrated, there is very little information regarding the regulation of GPER1 expression in these cell types. The work presented here is aimed at determining the molecular mechanisms that regulate GPER1 expression. To determine if GPER1 expression is sensitive to D-glucose concentration in breast cancer cells, MCF-7 and T47D human breast cancer cells were cultured in media containing increasing concentrations of D-glucose and GPER1 expression was measured using real-time PCR and immunoblot. Data from these experiments showed that GPER1 expression was significantly increased in breast cancer cells cultured in media containing low D-glucose concentrations (0 and 2.5mM) and significantly reduced in media containing high D-glucose (25 mM). Since low D-glucose concentration is known to activate the energy-sensing AMP kinase (AMPK), the observed GPER1 induction in low D-glucose conditions was determined after pretreatment with the AMPK inhibitor compound C. GPER1 expression was inhibited in both MCF-7 and T47D breast cancer cells cultured in low D-glucose media when pretreated with compound C. Additionally, the AMPK activator metformin induced GPER1 expression in MCF-7 and T47D breast cancer cells cultured in high D-glucose (25mM) conditions. These data suggest that AMPK mediates GPER1 expression in cells cultured in low D-glucose. The D-glucose sensitivity of GPER1 expression was also determined in the Eker rat-derived uterine leiomyoma cells (ELT-3 and ELT-6) which are characterized by hyperactive AMPK. In both ELT-3 and ELT-6 cells, GPER1 expression was not sensitive to D-glucose concentration nor was the activation state of AMPK sensitive to D-glucose concentrations. However, the inhibition of AMPK by compound C in both ELT-3 and ELT-6 cells resulted in decreased GPER1 expression. These findings reveal a previously unknown mechanism that regulates GPER1 expression in E2-responsive tumor and cancer cells. Citation Format: Yan Zheng, Kevin D. Houston. GPER1 expression is modulation by D-glucose concentration in estrogen-responsive cancer and tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2432. doi:10.1158/1538-7445.AM2017-2432

Abstract 1848: Autocrine IGF1 signaling mediates the survival of pancreatic cancer cells following the ablation of oncogenes

Abstract Pancreatic cancer is an extremely lethal disease, which is mostly associated with the gain-of-function mutation in KRAS gene. Due to a very high frequency of activating mutations within the KRAS gene, mutant KRAS is considered a rational therapeutic target in pancreatic cancer. In line with the existing idea that oncogenic KRAS is required for the maintenance of pancreatic cancer, we show a complete macroscopic regression of both primary and orthotopically transplanted tumors in our ligand-regulatable (Doxycycline) mouse model of KRASG12D-driven pancreatic cancer. This finding indicate that the expression, and provides a strong rationale for the development of KRAS targeted therapy for the treatment of pancreatic cancer. However, in our current study, despite the complete macroscopic regression of PDACs upon downregulation of oncogenic KRAS, few cancer cells always survived and remained dormant for a protracted duration. These residual cancer cells were responsible for the rapid recurrence upon KRAS reactivation. Using a genome-wide gene expression (RNA-Seq) analysis of in vivo-derived bulk and residual cancer cells followed by biochemical analysis, we identified a significant increase in autocrine IGF-1/AKT signaling in residual cancer cells. We also identified IGF1 signaling as a common survival mechanism of residual cancer cells in c-MYC-driven pancreatic tumor model. Pharmacological inhibition of IGF-1R signaling significantly delayed the tumor recurrence in both the tumor models, suggesting the crucial role of IGF-1R signaling in the survival of cancer cells in the absence of oncogenic drivers. Our findings from animal models were also validated in human pancreatic cancer cell lines harboring KRAS mutations, by demonstrating a compensatory increase in IGF1 signaling in response to a conditional knockdown of KRAS in vitro and in vivo. Collectively, all our findings from mouse model and human cancer cell studies strongly indicate that residual cancer cells can survive in the absence of tumor driving oncogenes by upregulation of autocrine IGF1 signaling loop. Thus, targeting oncogenic drivers along with IGF-1R signaling might be an effective strategy for prevention of tumor recurrence in pancreatic cancer. Citation Format: Nirakar Rajbhandari. Autocrine IGF1 signaling mediates the survival of pancreatic cancer cells following the ablation of oncogenes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1848. doi:10.1158/1538-7445.AM2017-1848

Inhibition of IGF-1R Signaling Enhances the Head and Neck Tumor Radioresponse but Fails to Further Improve Tumor Response to Cetuximab and Radiation Therapy In Vivo

Purpose/Objective(s): Interaction between the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGF-1R) has been well established inmany cancer types. Ligands of these receptors are capable of activating both pathways. We investigated the effects of cetuximab (EGFR antibody) and IMC-A12 (IGF-1R antibody) on the response of head and neck squamous cell carcinoma (HNSCC) to radiation therapy (IR). Materials/Methods: SixHNSCCmodels expressing different levels of EGFR and IGF-1R were studied. The effects of cetuximab and IMC-A12 on cell viability (MTS assay) and radiosensitivity (clonogenic survival assay) were determined. Formation of nuclear g-H2AX and 53BP1 foci was monitored by immunofluorescence to assess the IR-induced DNA damage and repair kinetics. Alterations in target signaling were analyzed by Western blots. In vivo tumor growth delay assay was performed to determine the efficacy of IMCA12 in enhancing tumor response to cetuximab and radiation using two HNSCC tumor xenograft models generated in mice. Results: In vitro studies in six HNSCC lines showed that exposure to cetuximab resulted in increased cell radiosensitivity in two cell lines. EGFR inhibition suppressed DNA repair that was evident by the prolonged presence of nuclear g-H2AX and 53BP1 foci. Western blot analyses showed that EGFR inhibition increased IGF-1R expression levels and also the association between EGFR and IGF-1R. When IMC-A12 was added concurrently to the treatment regimen along with cetuximab, a further increase in radiosensitivity was observed in one of the three cell lines tested. In vivo tumor growth delay data in two of the HNSCC tumor models showed that tumor xenografts exhibited enhanced tumor response to RT in the presence of either cetuximab or IMC-A12 depending on the tumor type. Addition of IMC-A12 to cetuximab and IR treatment regimen failed to further enhance the tumor response to cetuximab as well as to IR. Conclusions: Taken together our data suggest that concomitant inhibition of both EGFR and IGF-1R pathways using their respective antibodies did not yield additional therapeutic benefit in overcoming resistance to RT. Author Disclosure: U. Raju: None. D.P. Molkentine: None. D.R. Valdecanas: None. A.A. Deorukhkar: None. K.A. Mason: None. T.A. Buchholz: None. R.E. Meyn: None. K.K. Ang: None. H. Skinner: None.

Igf Signaling is Required for Cardiomyocyte Proliferation during Zebrafish Heart Development and Regeneration

Unlike its mammalian counterpart, the adult zebrafish heart is able to fully regenerate after severe injury. One of the most important events during the regeneration process is cardiomyocyte proliferation, which results in the replacement of lost myocardium. Growth factors that induce cardiomyocyte proliferation during zebrafish heart regeneration remain to be identified. Signaling pathways important for heart development might be reutilized during heart regeneration. IGF2 was recently shown to be important for cardiomyocyte proliferation and heart growth during mid-gestation heart development in mice, although its role in heart regeneration is unknown. We found that expression of igf2b was upregulated during zebrafish heart regeneration. Following resection of the ventricle apex, igf2b expression was detected in the wound, endocardium and epicardium at a time that coincides with cardiomyocyte proliferation. Transgenic zebrafish embryos expressing a dominant negative form of Igf1 receptor (dn-Igf1r) had fewer cardiomyocytes and impaired heart development, as did embryos treated with an Igf1r inhibitor. Moreover, inhibition of Igf1r signaling blocked cardiomyocyte proliferation during heart development and regeneration. We found that Igf signaling is required for a subpopulation of cardiomyocytes marked by gata4:EGFP to contribute to the regenerating area. Our findings suggest that Igf signaling is important for heart development and myocardial regeneration in zebrafish.

Epigenetic modification after inhibition of IGF-1R signaling in human central nervous system atypical teratoid rhabdoid tumor (AT/RT)

This study investigated epigenetic modifications in human central nervous system atypical teratoid rhabdoid tumors (AT/RTs), in response to inhibition of insulin-like growth factor receptor 1 (IGF-1R). Tumor tissue was obtained from two pediatric patients, tissue was dissociated, and primary cultures were established. Cultured cells were treated with picropodophyllin (PPP; 0, 1, and 2 μM for 48 h), a selective IGF-1R inhibitor. Histone acetylation and methylation patterns (H3K9ac, H3K18ac, H3K4me3, H3K27me3) and levels of histone deacetylases (HDACs; HDAC1, HDAC3, and SirT1) and histone acetyl transferases (GCN5 and p300) were examined. H3K9ac and H3K18ac decreased in response to treatment with PPP. HDAC levels showed a biphasic response, increasing with 1 μM PPP, but then decreasing with 2 μM PPP. Inhibition of IGF-1R modified epigenetic status in AT/RT. Determining the mechanisms behind these modifications will guide the development of novel therapeutic targets for this malignant embryonal cancer.

Expression of the Receptor for Type I Insulin-like Growth Factor (IGF1R) in Gastrointestinal Stromal Tumors

A majority of gastrointestinal stromal tumors (GISTs) carry gain-of-function KIT or platelet-derived growth factor receptor α (PDGFRA) mutations. However, no mutational activation of KIT or PDGFRA has been identified in pediatric gastric GISTs, neurofibromatosis-1-associated GISTs, and a small subset of sporadic GISTs in adults [so-called wild-type (WT) GISTs]. Recently, pediatric gastric GISTs and some adult WT gastric GISTs have been found to have losses of the succinate dehydrogenase (SDH) complex, a Krebs cycle/electron transport chain interface protein, as seen by immunohistochemical loss of SDH subunit B (SDHB) expression. Moreover, recently, expression of the receptor for type I insulin-like growth factor (IGF1R) has been detected in pediatric and WT GISTs, although only a small number of cases have been analyzed. In this study, IGF1R expression was examined immunohistochemically in 1078 well-characterized GISTs representing different clinicogenetic categories and in 103 non-GIST gastrointestinal tumors. IGF1R expression was detected in 71/80 of SDH-deficient GISTs (SDHB-negative GISTs) but only in 9/625 (1%) of the SDHB-positive gastric GISTs. The latter often carried KIT or PDGFRA mutations and generally occurred in older patients. None of the 373 intestinal GISTs was IGF1R positive, whereas many primary intestinal sarcomas, including clear cell sarcomas, leiomyosarcomas, and undifferentiated sarcomas, were IGF1R positive. The consistent lack of IGF1R expression in intestinal GISTs should be considered an additional immunohistochemical marker in the differential diagnosis between GISTs and non-GIST sarcomas. Because inhibition of IGF1R signaling might become a therapeutic target in GISTs, screening for IGF1R expression may become important in the near future.

P2-01-03: Insulin-Like Growth Factor I (IGF) Induced Metastasis Signature Correlates with Poor Prognosis and Decreased Distant Metastasis-Free Survival in Human Breast Cancer.

Abstract The insulin-like growth factors (IGFs) acting via the type I insulin-like growth factor receptor (IGF-1R) regulate tumor growth, survival and metastasis of multiple types of cancers. Agents targeting IGF-1R are currently being tested in phase II/III clinical trials in human cancers. We have previously reported that IGF1R can regulate metastasis independently of primary tumor growth in a model of high risk metastatic breast cancer using the triple negative MDA435/LCC6 metastatic cancer cells, which form spontaneous lung metastases when injected into the mammary fat pad of mice. We have shown that disruption of IGF signaling either with an antibody against IGF1R, AVE1642, or a dominant negative IGF-1R construct, does not affect growth in the mammary fat pad but inhibits pulmonary metastases and colonization of the lungs. Regulation of such a phenotype will not be measured in clinical trials and these findings imply that clinical trials of this therapeutic strategy may need to adjust their endpoints for assessing benefit by taking into account that inhibition of IGF1R signaling may inhibit metastasis but not primary tumor growth. Here, we sought to develop an IGF induced metastasis signature that may be useful in selecting patients that could benefit from IGF1R targeted therapy and determine if changes in expression of the genes in the signature can be useful in monitoring response. Serum starved LCC6-WT cells (with wild-type functional IGF1R) and LCC6-DN (cells with dominant negative IGF1R) were treated with IGF-I, AVE1642 (fully human antibody against IGF1R that inhibits metastasis but not tumor growth in the mammary fat pad), or AVE1642+ IGF-I for 4 and 24 hours and microarray analysis was performed using the U133 Plus 2.0 human arrays with 47,000 transcripts. IGF-I regulated over 100 transcripts in LCC6-WT cells compared to IGF-I in LCC6-DN cells. Over 400 transcripts were basally differentially expressed in LCC6-WT cells compared to LCC6-DN cells. Array data were also analyzed to determine genes regulated by IGF-I signaling in LCC6-WT cells compared to untreated LCC6-WT cells and if disruption of signaling with AVE1642 affected genes whose expression was regulated by IGF-I signaling. A total of 263 transcripts were regulated by IGF signaling in LCC6-WT cells compared to untreated cells and AVE1642 treatment reversed the regulation of all transcripts induced by IGF-I in LCC6-WT cells. 53 of the most significantly regulated genes were used to query two different human breast tumor datasets to determine if genes in IGF signaling induced metastasis signature are associated with lymph node metastasis and poor prognosis in human tumors. IGF regulated metastasis signature genes were expressed in human tumors in the van't Veer and Wang datasets. Further, IGF activated metastasis signature was correlated to decreased survival and decreased distant metastasis free survival in breast cancer patients. Our results suggest that presence of IGF induced metastasis signature in patients confers prognostic value and identifying IGF driven metastasis signatures may be useful to identify and monitor patients who could benefit from IGF1R targeted therapy for inhibition of metastatic disease. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-03.

Combined inhibition of insulin-like growth factor-1 receptor enhances the effects of gefitinib in a human non-small cell lung cancer resistant cell line

The type I insulin-like growth factor receptor (IGF-1R) signaling pathway is an important growth-regulatory pathway that is prevalent in a variety of cancer types, including human non-small cell lung cancer (NSCLC). To observe the combined effects of gefitinib and AG1024-induced IGF-1R inhibition on the growth of NSCLC, PC9/G cells, a NSCLC cell line with acquired resistance to gefitinib, were treated with AG1024 and gefitinib, alone or in combination. The proliferative activity of PC9/G cells upon different treatments was assessed by CCK-8, and the median-effects principle was used to assess the effect of the combined treatment. Apoptotic rates of the PC9/G cells for the different treatment groups were analyzed by flow cytometry. The expression of phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated-Akt (p-Akt), and phosphorylated extracellular signal-regulated kinase (p-ERK) in PC9/G cells was examined by Western blotting. PC9/G cells exhibited apoptotic features after treatment with AG1024 and gefitinib alone, and their proliferation rates were inhibited to different degrees. The treatment of AG1024 combined with gefitinib resulted in a synergistic effect in inducing apoptosis, inhibiting cell proliferation and decreasing the expression of p-EGFR, p-Akt and p-ERK. In conclusion, combined inhibition of IGF-1R signaling enhances the anti-proliferative and pro-apoptotic effects of gefitinib in NSCLC gefinitib-resistant cells. Moreover, the addition of an anti-IGF-1R strategy to gefitinib treatment may be more effective than a single-agent approach.

AMPK-induced activation of Akt by AICAR is mediated by IGF-1R dependent and independent mechanisms in acute lymphoblastic leukemia

BackgroundChildren with Acute Lymphoblastic Leukemia (ALL) diagnosed with resistant phenotypes and those who relapse have a dismal prognosis for cure. In search for novel treatment strategies, we identified the AMP activated protein kinase (AMPK) as a potential drug target based on its effects on cell growth and survival. We have shown previously that AICAR-induced AMPK activation also induced a compensatory survival mechanism via PI3K/Akt signaling.ResultsIn the present study, we further investigated the downstream signaling induced by AMPK activation in ALL cells. We found that AICAR-induced AMPK activation resulted in up-regulation of P-Akt (Ser473 and Thr308) and decrease of P-mTOR (Ser2448) expression and downstream signaling. We determined that activation of P-Akt (Thr308) was mediated by AMPK-induced IGF-1R activation via phosphorylation of the insulin receptor substrate-1 (IRS-1) at Ser794. Inhibition of IGF-1R signaling using the tyrosine kinase inhibitor HNMPA(AM)3 resulted in significant decrease in P-IRS-1 (Ser794) and P-Akt (Thr308). Co-treatment of AICAR plus HNMPA(AM)3 prevented AMPK-induced up-regulation of P-Akt (Thr308) but did not alter the activation of P-Akt (Ser473). Inhibition of AMPK using compound-C resulted in decreased P-Akt expression at both residues, suggesting a central role for AMPK in Akt activation. In addition, inhibition of IGF-1R signaling in ALL cells resulted in cell growth arrest and apoptosis. Additional Western blots revealed that P-IGF-1R (Tyr1131) and P-IRS-1 (Ser794) levels were higher in NALM6 (Bp-ALL) than CEM (T-ALL), and found differences in IGF-1R signaling within Bp-ALL cell line models NALM6, REH (TEL-AML1, [t(12;21)]), and SupB15 (BCR-ABL, [t(9;22)]). In these models, higher sensitivity to IGF-1R inhibitors correlated with increased levels of IGF-1R expression. Combined therapy simultaneously targeting IGF-1R, AMPK, Akt, and mTOR pathways resulted in synergistic growth inhibition and cell death.ConclusionsOur study demonstrates that AMPK activates Akt through IGF-1R dependent and independent mechanisms. Co-targeting IGF-1R and related downstream metabolic and oncogenic signaling pathways represent a potential strategy for future translation into novel ALL therapies.

Abstract 3295: The type I insulin-like growth factor receptor (IGF1R) pathway driven metastasis signature correlates with poor prognosis and decreased distant metastasis-free survival in human breast cancer

Abstract The insulin-like growth factors (IGFs) acting via the type I IGF receptor (IGF1R) regulate growth and metastasis of multiple types of cancers. Agents targeting IGF1R are currently being tested in phase II/III clinical trials. We have previously shown that IGF1R can regulate metastasis independently of primary tumor growth. In a model of high risk metastatic cancer using the MDA435/LCC6 metastatic cancer cells, we have shown that disruption of IGF signaling either with an antibody against IGF1R, AVE1642, or a dominant negative IGF-1R construct, does not affect growth in the mammary fat pad but inhibits pulmonary metastases and colonization of the lungs. Regulation of such a phenotype will not be measured in clinical trials and these findings imply that clinical trials of this therapeutic strategy may need to adjust their endpoints for assessing benefit by taking into account that inhibition of IGF1R signaling may inhibit metastasis but not primary tumor growth. Here, we sought to develop an IGF1R driven metastasis signature that may be useful in selecting patients that could benefit from IGF1R targeted therapy and determine if changes in expression of the genes in the signature can be useful in monitoring response. Serum starved LCC6-WT cells (with wild-type functional IGF1R) and LCC6-DN (cells with dominant negative IGF1R) were treated with IGF-I, AVE1642 (antibody against IGF1R that inhibits metastasis but not tumor growth in the mammary fat pad), or AVE1642+ IGF-I for 4 and 24 hours and microarray analysis was performed using the U133 Plus 2.0 human arrays with 47,000 transcripts. Array data were analyzed to determine genes regulated by IGF1R signaling in these cells and if disruption of signaling with AVE1642 affected genes whose expression was regulated by IGF-I signaling. A total of 206 transcripts were regulated by IGF signaling in LCC6-WT cells and AVE1642 treatment reversed the effects of IGF1R signaling. 53 of the most significantly regulated genes were used to query two different human breast tumor datasets to determine if genes regulated by activation of IGF1R are associated with lymph node metastasis and poor prognosis in human tumors. IGF1R regulated metastasis signature genes were expressed in human tumors and were correlated to decreased survival and decreased distant metastasis free survival in breast cancer patients. Our results suggest that presence of IGF1R metastasis signature in patients confers prognostic value and identifying IGF1R driven metastasis signatures may be useful to identify and monitor patients who could benefit from IGF1R targeted therapy for inhibition of metastatic disease. 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 3295.

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.

IGF Signaling Is Critical for Growth and Survival of T-Cell Acute Lymphoblastic Leukemia Cells and Is Potentiated by Notch Upregulation of IGF1R

T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T cell progenitors in which we described activating mutations of Notch1 to occur in over 50% of cases. As well, others have identified loss-of-function mutations in Sel10/Fbw7 to occur in 8–16% of cases, which also enhance Notch signaling. Notably, inhibition of Notch signaling in these cells induces growth arrest and in some cases apoptosis as well. Subsequent studies have characterized c-myc as a critical downstream target of Notch signaling in this context. More recently, mutations in PTEN (occurring in 17% of cases) were shown to potentiate PI3K/Akt signaling and proposed to confer resistance to Notch signaling inhibition, but then impose “addiction” to PI3K/Akt. Most leukemias likely derive cooperative growth/survival signals from Notch and PI3K/Akt pathways, as evidenced by synergistic effects of gamma-secretase inhibitors (GSI) which block Notch signaling and rapamycin which blocks mTOR downstream of PI3K/Akt. In mouse models, combined activation of c-myc and b-catenin with inactivation of PTEN elicited T-ALL which was devoid of Notch mutations, suggesting Notch signaling coordinately activates c-myc, Wnt, and PI3K/Akt signaling pathways. In studying the mechanism for PI3K/Akt activation in T-ALL with activated Notch signaling, we examined insulin-like growth factor receptor-1 (IGF1R) as a candidate upstream initiator of PI3K/Akt activation. We observed IGF1R expression consistently in T-ALL cells from primary human leukemias and cell lines, as well as in primary mouse leukemias derived experimentally by retroviral transduction of marrow with activated forms of Notch1. In all cases, inhibition of IGF1R signaling either with blocking antibody or small molecule kinase inhibitors resulted in growth suppression and apoptosis of leukemia cells. We further observed that inhibition of Notch signaling either by small molecule GSI or transduction with a dominant negative coactivator, Mastermind, resulted in a 2–3 fold decrease in IGF1R expression. Given that the PI3K/Akt/mTOR pathway is known to be important for growth/survival of T-ALL leukemia cells, we hypothesized that Notch might be potentiating activation of this pathway by upregulating IGF1R expression. In fact, we found leukemia cells with active Notch and higher IGF1R levels showed 20-fold greater sensitivity to IGF-1 ligand induced Akt activation as compared to leukemia cells with inactive Notch and lower IGF1R levels. This enhanced signaling output cannot be explained by Notch suppression of PTEN as this effect was noted in both PTEN wild-type and PTEN null leukemia cells. Additionally, cells with wild type PTEN demonstrated only minimal if any changes in PTEN protein levels after Notch inhibition as measured by a highly quantitative flow cytometry assay. In sum, we have identified IGF-1 signaling as being critical to growth and survival of T-ALL leukemia cells, and provide evidence that Notch may potentiate PI3K/Akt signaling by upregulating expression of IGF1R. These data are of immediate clinical relevance as several IGF1R inhibitors are currently in Phase 3 clinical trial and hopefully will provide additional therapeutic options to refractory/relapsed patients and/or in combination with front-line therapy in newly diagnosed patients.