Insulin-like Growth Factor-1 Receptor Protein Research Articles

Protein expression and gene copy number changes of receptor tyrosine kinase in thymomas and thymic carcinomas

Insulin-like growth factor-1 receptor (IGF-1R), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor-type 2 (HER2), and c-Met are members of the receptor tyrosine kinases (RTKs). The associations between the RTK status [protein expression and gene copy number (GCN)] and patient characteristics and between the RTK status and prognosis remain undetermined. The study included 140 patients who underwent surgery for thymic tumors. Protein expression was evaluated by immunohistochemistry (IHC) and GCN was evaluated by bright-field in situ hybridization (BISH). The correlations between the RTK status and clinicopathological findings were examined. IGF-1R protein was frequently detected in thymic carcinoma (83.8%) and EGFR in thymic tumors (91.4%). Thirty-six and 39 tumors were BISH high for IGF-1R and EGFR, respectively: 28 and 25 exhibited high polysomy; 8 and 14 exhibited gene amplification. No tumor was positive for HER2 or c-Met by IHC and BISH. Multivariate analysis revealed that IGF-1R gene amplification (P = 0.027), thymic carcinoma histology, and higher tumor stage were significantly correlated with an adverse prognosis. Thymic epithelial tumors frequently express IGF-1R and/or EGFR proteins. IGF-1R gene amplification is suggested to define an unfavorable subset for thymic epithelial tumors.

Combination of acyclic retinoid with branched‐chain amino acids inhibits xenograft growth of human hepatoma cells in nude mice

Combination chemoprevention is a promising strategy to improve the prognosis of hepatocellular carcinoma (HCC). A malfunction of retinoid X receptor-α (RXR-α) due to phosphorylation by Ras/mitogen-activated protein kinase is closely associated with liver carcinogenesis and acyclic retinoid (ACR) can prevent HCC development by inhibiting RXR-α phosphorylation. The present study examined the possible combined effects of ACR plus branched-chain amino acids (BCAA), which can also prevent the development of HCC in obese patients with liver cirrhosis, in human HCC xenografts in nude mice. This study examined the effects of the combination of ACR plus BCAA on the growth of Huh7 human HCC xenografts in nude mice. The effects of the combination on the phosphorylation of RXR-α, extracellular signal-regulated kinase (ERK), Akt and insulin-like growth factor-1 receptor (IGF-1R) proteins, and on the expression levels of retinoic acid receptor-β (RAR-β) and p21(CIP1) mRNA, were also examined by western blot and real-time reverse transcription polymerase chain reaction analyses, respectively. The combined treatment with ACR plus BCAA significantly inhibited the growth of Huh7 xenografts. The combination of these agents caused a marked inhibition of the phosphorylation of RXR-α, ERK, Akt and IGF-1R proteins in the xenografts. In addition, the expression levels of RAR-β and p21(CIP1) mRNA significantly increased by these agents. The combination of ACR and BCAA restores the function of RXR-α by inhibiting its phosphorylation and increasing the level of RAR-β, a heterodimeric partner for RXR-α, and thus suppresses the growth of HCC xenografts. Therefore, this combination might be an effective regimen for the treatment and, probably, chemoprevention of HCC.

Overexpression of insulin‐like growth factor 1 receptor and frequent mutational inactivation of SDHA in wild‐type SDHB‐negative gastrointestinal stromal tumors

Approximately 15% of gastrointestinal stromal tumors (GISTs) in adults and 85% in children lack mutations in KIT and PDGFRA and are known as wild-type GISTs. Wild-type GISTs from adults and children express high levels of insulin-like growth factor 1 receptor (IGF1R) and exhibit stable genomes compared to mutant GISTs. Pediatric wild-type GISTs, GISTs from the multitumor Carney-Stratakis syndrome, and the Carney triad share other clinicopathological properties (e.g., early-onset, multifocal GISTs with epitheliod cell morphology), suggesting a common etiology. Carney-Stratakis is an inherited association of GIST and paragangliomas caused by germline mutations in succinate dehydrogenase (SDH) genes. The connection between defective cellular respiration and GIST pathology has been strengthened by the utilization of SDHB immunohistochemistry to identify SDH deficiency in pediatric GISTs, syndromic GISTs, and some adult wild-type GISTs. SDHB and IGF1R expression was examined in 12 wild-type and 12 mutant GIST cases. Wild-type GISTs were screened for coding-region alterations in SDH genes and for chromosomal aberrations using genome-wide single-nucleotide polymorphism and MIP arrays. SDHB-deficiency, identified in 11/12 wild-type GIST cases, was tightly associated with overexpression of IGF1R protein and transcript. Biallelic inactivation of the SDHA gene was a surprisingly frequent event, identified in 5 of 11 SDHB-negative cases, generally due to germline point mutations accompanied by somatic SDHA allelic losses. As a novel finding, inactivation of the SDHC gene from a combination of a heterozygous coding-region mutation and hypermethylation of the wild-type allele was found in one SDHB-negative case.

IGF-1 alleviates ox-LDL-induced inflammation <italic>via</italic> reducing HMGB1 release in HAECs

Atherosclerosis, a multifactorial chronic inflammatory response, is closely associated with oxidatively modified low-density lipoprotein (ox-LDL). High-mobility group box 1 (HMGB1) is a DNA-binding protein, which upon release from cells exhibits potent inflammatory action. Insulin-like growth factor 1 (IGF-1) can elicit a repertoire of cellular responses including proliferation and anti-apoptosis. However, the role of IGF-1 in inflammation is still unclear. In the present study, we aimed to investigate the role of IGF-1 in inflammation and the underlying mechanism. Human aortic endothelial cells were stimulated by ox-LDL (50 μg/ml) to induce inflammation. The expression of intercellular adhesion molecule 1 (ICAM-1) was assessed by western blot analysis and immunofluorescence. The release of HMGB1 was determined by enzyme-linked immunosorbent assay. IGF-1 receptor (IGF-1R) expression was assessed by reverse transcription-polymerase chain reaction and western blot analysis. IGF-1R phosphorylation was determined by western blot analysis. Ox-LDL stimulation reduced IGF-1R mRNA and protein expression but increased HMGB1 release. IGF-1 treatment decreased ox-LDL-induced ICAM-1 expression potentially through reducing HMGB1 release, while picropodophyllin, an IGF-1R specific inhibitor, increased the inflammatory response. In conclusion, IGF-1 can alleviate ox-LDL-induced inflammation by reducing HMGB1 release, suggesting an unexpected beneficial role of IGF-1 in inflammatory disease.

Short communication: Grain-induced subacute ruminal acidosis is associated with the differential expression of insulin-like growth factor-binding proteins in rumen papillae of lactating dairy cattle

The objective of this study was to characterize the mRNA expression of genes involved in the insulin-like growth factor (IGF) axis in the rumen epithelium during grain-induced ruminal acidosis. Eight lactating dairy cattle were randomly assigned to a control (38% concentrate) or a high-grain (HG; 57% concentrate) diet in a randomized study. Dry matter intake, milk production, ruminal pH, and rumen papillae gene expression were measured before treatment allocation (d 0) and on the fourth day of treatment. On d 4, no differences were observed in total feed intake and milk production; however, the cattle fed the HG diet displayed lower ruminal pH (587±130min/d below 5.6; mean ± SE) compared with cattle receiving the control diet (169±145min/d below 5.6). No change in the relative mRNA expression of IGF-1, IGF-1 receptor (IGF-1R), and IGF-binding protein 6 (IGFBP6) was detected between treatments. However, the relative expression value of IGF-binding protein 3 (IGFBP3) decreased (0.73±0.07 fold, mean ± SE), whereas IGF-binding protein 5 (IGFBP5) expression increased (1.53±0.20 fold). These results indicate that the IGF axis may play a role in rumen epithelial adaptation to HG diets.

Upregulation of the E3 ligase NEDD4-1 by Oxidative Stress Degrades IGF-1 Receptor Protein in Neurodegeneration

The importance of ubiquitin E3 ligases in neurodegeneration is being increasingly recognized. The crucial role of NEDD4-1 in neural development is well appreciated; however, its role in neurodegeneration remains unexplored. Herein, we report increased NEDD4-1 expression in the degenerated tissues of several major neurodegenerative diseases. Moreover, its expression is upregulated in cultured neurons in response to various neurotoxins, including zinc and hydrogen superoxide, via transcriptional activation likely mediated by the reactive oxygen species (ROS)-responsive FOXM1B. Reduced protein levels of the insulin-like growth factor receptor (IGF-1Rβ) were observed as a consequence of upregulated NEDD4-1 via the ubiquitin-proteasome system. Overexpression of a familial mutant form of superoxide dismutase 1 (SOD1) (G93A) in neuroblastoma cells resulted in a similar reduction of IGF-1Rβ protein. This inverse correlation between NEDD4-1 and IGF-1Rβ was also observed in the cortex and spinal cords of mutant (G93A) SOD1 transgenic mice at a presymptomatic age, which was similarly induced by in vivo-administered zinc in wild-type C57BL/6 mice. Furthermore, histochemistry reveals markedly increased NEDD4-1 immunoreactivity in the degenerating/degenerated motor neurons in the lumbar anterior horn of the spinal cord, suggesting a direct causative role for NEDD4-1 in neurodegeneration. Indeed, downregulation of NEDD4-1 by shRNA or overexpression of a catalytically inactive form rescued neurons from zinc-induced cell death. Similarly, neurons with a NEDD4-1 haplotype are more resistant to apoptosis, largely due to expression of higher levels of IGF-1Rβ.Together, our work identifies a novel molecular mechanism for ROS-upregulated NEDD4-1 and the subsequently reduced IGF-1Rβ signaling in neurodegeneration.

MicroRNA-497 targets insulin-like growth factor 1 receptor and has a tumour suppressive role in human colorectal cancer

Past studies have shown that amplified insulin-like growth factor 1 (IGF1)/IGF1 receptor (IGF1-R) signalling has an important role in colorectal cancer (CRC) development, progression and resistance to treatment. In this report, we demonstrate that downregulation of microRNA-497 (miR-497) as a result of DNA copy number reduction is involved in upregulation of IGF1-R in CRC cells. MiR-497 and miR-195 of the miR-15/16/195/424/497 family that share the same 3′ untranslated region (3′UTR) binding seed sequence and are predicted to target IGF1-R were concurrently downregulated in the majority of CRC tissues relative to paired adjacent normal mucosa. However, only overexpression of miR-497 led to suppression of the IGF1-R 3′UTR activity and downregulation of the endogenous IGF1-R protein in CRC cells. This was associated with inhibition of cell survival, proliferation and invasion, and increased sensitivity to apoptosis induced by various stimuli including the chemotherapeutic drugs cisplatin and 5-fluorouracil, and the death ligand tumour necrosis factor-related apoptosis-inducing ligand. The biological effect of miR-497 on CRC cells was largely mediated by inhibition of phosphatidylinositol 3-kinase/Akt signalling, as overexpression of an active form of Akt reversed its impact on cell survival and proliferation, recapitulating the effect of overexpression of IGF1-R. Downregulation of miR-497 and miR-195 appeared to associate with copy number loss of a segment of chromosome 17p13.1, where these miRs are located at proximity. Similarly to miR-195, the members of the same miR family, miR-424 that was upregulated, and miR-15a, miR-15b and miR-16 that were unaltered in expression in CRC tissues compared with paired adjacent normal mucosa, did not appear to have a role in regulating the expression of IGF1-R. Taken together, these results identify downregulation of miR-497 as an important mechanism of upregulation of IGF1-R in CRC cells that contributes to malignancy of CRC.

Abstract 836: Insulin-like growth factor-I receptor (IGF-IR) dowregulation in cisplatin-resistant human ovarian cancer cells

Abstract Introduction: Ovarian cancer is the most lethal of the gynecological malignancies and the 5th leading cause of cancer related deaths amongst women in the US. It is a chemotherapy-sensitive disease, and about 85% of patients respond to first line treatment with platinum-based chemotherapeutic agents. However, 15% of all patients present with primary platinum-resistant disease, and those with recurrent ovarian cancer develop resistance. Resistance to platinum-based drugs is a major obstacle in ovarian cancer treatment, and mechanisms of platinum resistance are not well understood. Insulin-like growth factor-1 receptor (IGF-1R) signaling has not only been implicated in ovarian cancer development, but has also been suggested to mediate platinum-resistance. The IGF-1R has been proposed as a potential molecular target for ovarian cancer, and clinical trials evaluating the use of small molecule kinase inhibitors or humanized monoclonal antibodies against the IGF-1R are currently under way. This study examines the role of IGF-1R in cisplatin resistance in human ovarian carcinoma. Experimental Procedures: Differential gene expression in two pairs of cisplatin-resistant cell lines (SKOV3-CisR and OVCAR-CisR) and their syngeneic cisplatin-sensitive counterpart (SKOV3 and OVCAR5, respectively) was analyzed using the Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. IGF-1R mRNA transcripts and protein levels were verified via qRT-PCR and immunoblotting, respectively. Protein expression levels of IGF-1Rα and IGF-1Rα (immunoblotting) were correlated with cisplatin IC50 (XTT cell viability) in a panel of 12 ovarian cancer cell lines. IGF-1R was overexpressed (stable transfection) in SKOV3-CisR cells and the effect on cisplatin sensitivity evaluated. The effect of short-term cisplatin treatment on IGF-1Rα and IGF-1Rα protein levels was studied in SKOV3 and OVCAR5 cells. Results: Cisplatin-resistant SKOV3-CisR and OVCAR5-CisR cells exhibited 2-3 fold lower levels of IGF-1R mRNA transcripts compared to cisplatin-sensitive SKOV3 and OVCAR5 cells in the DNA microarray and via qRT-PCR. Correspondingly, decreased levels of IGF-1Rα and IGF-1Rα protein were found in immunoblot analyses. IGF-1Rα (r = −0.6118; p = 0.0345) and IGF-1Rα (r = −0.5839; p = 0.0462) protein levels and cisplatin IC50were negatively correlated. IGF-1R overexpression in SKOV3-CisR cells sensitized the cells to cisplatin, from 5 μg/ml (SKOV3-CisR) to 10-12 μg/ml (SKOV3-CisR-IGF-1R). Cisplatin induced a decrease in IGF-1Rα and IGF-1Rα levels after 24 and 48 hours treatment. Conclusions: Cisplatin treatment of ovarian cancer cells results in IGF-1R downregulation, which in turn decreases cisplatin sensitivity. Our results have important implications in the design of IGF-1R targeting strategies in clinical trials for ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 836. doi:1538-7445.AM2012-836

Abstract 342: Silencing of IGF1R induces mesenchymal-to-epithelial transition and inhibits the metastatic properties of triple negative breast cancer cells

Abstract Next to skin cancer, breast cancer is the second most common cancer diagnosed among American women and affects nearly 1.3 million women worldwide each year. Aggressive, metastatic disease is directly responsible for the majority of breast cancer-related deaths. Breast tumors lacking expression of three receptors, estrogen receptor (ER), progesterone receptor (PR), and HER2, are commonly referred to as triple negative breast cancers (TNBCs) and make up 15-20% of all diagnosed breast cancers cases. Disparities exist in terms of survival following diagnosis of TNBC, and a devastating reality is that this diagnosis is highly correlated with incurable, metastatic disease. Despite tremendous advantages in treatments for hormone-sensitive breast tumors, a significant number of women with TNBC experience disease progression due to the unavailability of targeted therapies. Insulin-like growth factor-1 receptor (IGF1R) plays a role in breast cancer proliferation, adhesion, invasion, and migration. Overexpression of IGF1R has been noted in 50% of primary breast tumors and in nearly 65% of TNBCs. Consequently, high IGF1R expression in breast cancers correlates well with, high tumor grade, shorter disease-free survival, and poor prognosis. We report the in vitro effects of IGF1R inhibition on the metastatic properties of mesenchymal TNBC cells lines using two methods: (1) shRNA lentiviral system and (2) picropodophyllin (PPP), an IGF1R tyrosine kinase inhibitor. Stably transfected mesenchymal TNBC cells showed down-regulation of IGF1R protein levels, which resulted in mesenchymal-epithelial-transition (MET), as confirmed by up-regulation of epithelial marker, E-cadherin, and down-regulation of mesenchymal marker, vimentin. Importantly, this MET was accompanied by reduced colony formation, cell motility, and invasion. Cell survival assays and fluorescence-activated cell sorting (FACS) analysis revealed that PPP also effectively decreased cell viability and induced cell death, respectively, in TNBC cells. Furthermore, PPP dramatically inhibited cell invasion and migration. Our findings indicate that inhibitors of IGF1R exhibit promising anti-cancer activity in TNBC cells of mesenchymal origin by triggering MET, inducing cell death, and reducing invasive and metastatic properties. Given the very poor outcomes for patients with metastatic TNBCs, we believe the use of IGF1R inhibitors warrant further investigation as prospective anti-cancer options for treating a subset of mesenchymal TN breast carcinomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 342. doi:1538-7445.AM2012-342

35 THERAPEUTIC EFFECTS OF INSULIN-LIKE GROWTH FACTOR-1 ON STRESS URINARY INCONTINENCE IN RATS WITH SIMULATED CHILD BIRTH TRAUMA INJURY

You have accessJournal of UrologyUrodynamics/Incontinence/Female Urology: Basic Research I1 Apr 201235 THERAPEUTIC EFFECTS OF INSULIN-LIKE GROWTH FACTOR-1 ON STRESS URINARY INCONTINENCE IN RATS WITH SIMULATED CHILD BIRTH TRAUMA INJURY Yasuhiro Sumino, Satoru Yoshikawa, Hiromitsu Mimata, and Naoki Yoshimura Yasuhiro SuminoYasuhiro Sumino Pittsburgh, PA More articles by this author , Satoru YoshikawaSatoru Yoshikawa Pittsburgh, PA More articles by this author , Hiromitsu MimataHiromitsu Mimata Oita, Japan More articles by this author , and Naoki YoshimuraNaoki Yoshimura Pittsburgh, PA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.078AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Insulin-like growth factor-1 (IGF-1) plays an important role in cell proliferation, survival and regeneration in various tissues. However, the therapeutic potential of IGF-1 for stress urinary incontinence (SUI) has not been explored. We examined the effect of IGF-1 in a rat model of SUI induced by stimulated birth trauma. METHODS Simulated birth trauma was induced by vaginal distension (VD) with an inflated balloon catheter for 4 hrs in the vagina of female SD rats. At 4, 7, 14 and 28 days after VD, (1) functional assessments were performed by measuring leak point pressure (LPP), urethral baseline pressure (UBP) and urethral responses during passive increment in intravesicular pressure and (2) the expression of IGF-1 and IGF-1 receptor (IGF1R) mRNA and protein in damaged tissues was examined by real-time RT-PCR and immunohistochemistry. Thereafter, human recombinant IGF-1 (hrIGF-1) (50 and 150μg/kg/day) or vehicle (saline) was continuously delivered from 1 day before VD using subcutaneous osmotic pumps. At 4 and 7 days after VD, the effect of IGF-1 treatment was examined by ELISA measurement of serum hrIGF-1, functional analyses (LPP, UBP and urethral response), and Western blotting analysis of Akt signal transduction pathways in urethral tissues. RESULTS After 4 and 7 days, VD rats had significantly reduced LPP, UBP and urethral responses, with a recovery to the normal level 14 and 28 days after VD. mRNA and protein levels of IGF-1 and IGF1R were significantly increased in the urethra and pudendal nerves 4 and 7 days after VD. IGF-1 treatment (50 and 150μg/kg/day) increased serum hrIGF-1 levels to 17.8±5.3 and 40.4±12.5 ng/ml, respectively. These IGF-1-treated groups showed significant improvement in LPP, UBP and urethral responses 4 and 7 days after VD (Figure). Moreover, phosphorylated Akt was highly expressed in the IGF-1-treated urethra. CONCLUSIONS IGF-1 treatment accelerates recovery from SUI induced by stimulated child birth trauma in rats, possibly through activation of the Akt signal transduction pathway, suggesting that IGF-1 has an important role in the recovery from childbirth-related SUI. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e14-e15 Peer Review Report Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yasuhiro Sumino Pittsburgh, PA More articles by this author Satoru Yoshikawa Pittsburgh, PA More articles by this author Hiromitsu Mimata Oita, Japan More articles by this author Naoki Yoshimura Pittsburgh, PA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The effects of maternal diabetes on expression of insulin-like growth factor-1 and insulin receptors in male developing rat hippocampus

Diabetes during pregnancy causes neurodevelopmental and neurocognitive abnormalities in offspring. Insulin and insulin-like growth factor-1 (IGF-1) are important regulators of developmental and cognitive functions in the central nervous system. We examined the effects of maternal diabetes on insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor (InsR) expression in the developing rat hippocampus. Female rats were maintained diabetic from a week before pregnancy through parturition and male offspring was killed at P0, P7, and P14. We found a significant bilateral upregulation of both IGF-1R and InsR transcripts in the hippocampus of pups born to diabetic mothers at P0, as compared to controls. However, at the same time point, the results of western blot analysis revealed only a slight change in their protein levels. At P7, there was a marked bilateral reduction in mRNA expression and protein levels of IGF-1R, although not of InsR in the diabetic group. We also found a downregulation in IGF1-R transcripts, especially in left hippocampus of the diabetic group at P14. Moreover, at the same time point, InsR expression was significantly decreased in both hippocampi of diabetic newborns. When compared with controls, we did not find any difference in hippocampal IGF-1R or InsR mRNA and protein levels in the insulin-treated group. The present study revealed that diabetes during pregnancy strongly influences the regulation of both IGF-1R and InsR in the right/left developing hippocampi. Furthermore, the rigid control of maternal glycaemia by insulin administration normalized these effects.

Genomic and Molecular Characterization of Malignant Peripheral Nerve Sheath Tumor Identifies the IGF1R Pathway as a Primary Target for Treatment

Malignant peripheral nerve sheath tumor (MPNST) is a rare sarcoma that lacks effective therapeutic strategies. We gain insight into the most recurrent genetically altered pathways with the purpose of scanning possible therapeutic targets. We conducted a microarray-based comparative genomic hybridization profiling of two cohorts of primary MPNST tissue samples including 25 patients treated at The University of Texas MD Anderson Cancer Center and 26 patients from Tianjin Cancer Hospital. Immunohistochemistry (IHC) and cell biology detection and validation were carried out on human MPNST tissues and cell lines. Genomic characterization of 51 MPNST tissue samples identified several frequently amplified regions harboring 2,599 genes and regions of deletion including 4,901 genes. At the pathway level, we identified a significant enrichment of copy number-altering events in the insulin-like growth factor 1 receptor (IGF1R) pathway, including frequent amplifications of the IGF1R gene itself. To validate the IGF1R pathway as a potential target in MPNSTs, we first confirmed that high IGF1R protein correlated with worse tumor-free survival in an independent set of samples using IHC. Two MPNST cell lines (ST88-14 and STS26T) were used to determine the effect of attenuating IGF1R. Inhibition of IGF1R in ST88-14 cells using siRNAs or an IGF1R inhibitor, MK-0646, led to significant decreases in cell proliferation, invasion, and migration accompanied by attenuation of the PI3K/AKT and mitogen-activated protein kinase pathways. These integrated genomic and molecular studies provide evidence that the IGF1R pathway is a potential therapeutic target for patients with MPNST.

P1-02-06: Silencing of IGF-1R Has Paradoxical Effects in Triple Negative Breast Cancer Phenotypes.

Abstract Background: Triple negative (TN) breast cancers are a heterogeneous group of breast cancers with a poor prognosis in part due to a lack of effective targeted agents. Insulin-like growth factor-1 receptor (IGF-1R) has been shown to play a role in breast cancer cell proliferation, adhesion, invasion, and migration and is overexpressed in more than a third of TN breast tumors. We hypothesized that IGF-1R could be a therapeutic target for a subset of TN breast cancers. Methods: We evaluated the effects of IGF-1R silencing on the metastatic properties of TN breast cancer cells by knock down in two morphologically distinct TN breast cancer cell lines using shRNA lentiviral techniques. Reverse-transcription polymerase chain reaction (RT-PCR) and immunoblotting were used to detect mRNA and protein expression levels, respectively, of IGF-1R signaling molecules. Anchorage-dependent growth and Matrigel chamber assays were performed to assess the effects of IGF-1R silencing on colony formation and invasion of TN breast cancer cells, respectively; wound-healing and spheroid migration were also performed to assess the effects of IGF-1R inhibition on TN breast cancer cell motility. Results: Stably transfected mesenchymal MDA-MB-231 TN cells showed effective downregulation of IGF-1R protein expression, which resulted in mesenchymal-to-epithelial transition (MET), confirmed by upregulation of the epithelial marker E-cadherin and downregulation of the mesenchymal marker vimentin. Importantly, this MET resulted in reduced colony formation (p<0.0042) and cell motility and dramatically reduced invasion (p<0.0001). Conversely, silencing of IGF-1R in epithelial MDA-MB-468 TN cells induced epithelial-to-mesenchymal transition (EMT), confirmed by downregulation of E-cadherin and upregulation of vimentin expression, with resultant increased colony formation (p<0.006), cell motility, and invasion (p<0.0001). Conclusion: Collectively, these results demonstrate a paradoxical effect of targeting IGF-1R in TN breast cancers of mesenchymal and epithelial origin. Targeting IGF-1R in TN breast cancers with a mesenchymal phenotype decreases invasion and metastatic potential. In contrast, targeting IGF-1R in TN breast cancers with an epithelial phenotype could have potentially detrimental effects. Our data suggest that IGF-1R inhibition should be explored as a therapeutic modality in TN breast cancers with a mesenchymal phenotype. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-06.

Abstract P258: Effect of MicroRNA-223 on Hypoxic Pulmonary Hypertension

Chronic hypoxia plays a key role in pulmonary hypertension and remodeling by inducing marked changes in gene expression. However, the regulatory effect of chronic hypoxia on microRNA (miR) expression in the lung has not yet been investigated. The purpose of this study was to determine which miRs are regulated by chronic hypoxia in the mouse lung and to determine their functional significance. We identified miRs altered in mouse lung after 3 weeks chronic hypoxia (10% O 2 (10 in 100) ) treatment by Microarray screening, verified miR target genes by reporter gene assays and Western blotting. Furthermore, we analyzed in vitro and in vivo functional significance for mouse pulmonary vascular artery smooth muscle cells. Among the miRs regulated, miR-223 was the most significantly decreased, a phenomenon verified by RT-qPCR. The insulin-like growth factor 1 receptor (IGF1R) is a known target of miR-223 and the binding of miR-223 to the 3’UTR of IGF1R was demonstrated. We found that IGF1R protein levels (but not RNA) were increased in lungs from hypoxic mice via a mechanism involving HIF1α/2α and reduced C/EBPα expression and transcription activity. Moreover, the expression of IGF1R was decreased following introduction of pre-mir-223 into pulmonary artery smooth muscle cells, an phenomenon coupled to attenuated IGF1-induced Akt phosphorylation, cell migration and proliferation. Decreasing endogenous miR-223 levels using a specific antagomir (3 weeks), increased pulmonary artery pressure and vessel muscularization. A similar phenomenon was observed in miR-223 -/y mice, which also demonstrated a more severe response to hypoxia than wild-type mice. In lungs from primary pulmonary hypertension (PPH) patients we also detected a decrease in miR-223 and increase in IGF1R expression. These data indicate that the downregulation of miR-223 may contribute to the IGF1-induced pulmonary smooth muscle migration by regulating the expression of its receptor and thus contributing to pulmonary remodeling and the development of pulmonary hypertension.

IGF-1 and Its Receptor in Esophageal Cancer: Association with Adenocarcinoma and Visceral Obesity

The insulin-like growth factor (IGF) pathway and visceral obesity have been independently linked with esophageal cancer. This study aimed to delineate the differential and interlinked role of visceral obesity and the IGF-1 system in esophageal adenocarcinoma and esophageal squamous-cell carcinoma (SCC). IGF-1 receptor (IGF-1R) mRNA and protein were examined in esophageal SCC (KYSE 410, OE21) and esophageal adenocarcinoma (OE19, OE33) cell lines by western blotting. Tumor cell proliferation in response to IGF-1 was assessed by bromodeoxyuridine incorporation assay. In esophageal tumor sections, expression of IGF-1R and CD68(+) cell numbers were assessed by immunohistochemistry. IGF-1 was measured in serum from esophageal cancer patients, Barrett's esophagus patients, and healthy controls by enzyme-linked immunosorbent assay. Higher IGF-1R protein expressions were observed in SCC cells compared with esophageal adenocarcinoma cells however only adenocarcinoma cell lines significantly increased proliferation in response to IGF-1 (P<0.01). Serum IGF-1 levels were highest in esophageal adenocarcinoma patients (P<0.01) and higher in viscerally obese vs. nonobese (P<0.05) patients. In resected esophageal cancer, increased expression of IGF-1R was observed in the tumor and invasive edge compared with tumor-associated stroma (P<0.05), which coincided with increased CD68(+) cells in stromal tissue surrounding invasive tumor edge (P<0.01). This novel study examined the differential role of the IGF system in esophageal adenocarcinoma and SCC, and its association with visceral obesity. These results indicate that the IGF-1 axis has a key role in malignant progression of esophageal cancer, and represents a plausible mechanism through which visceral obesity impacts on esophageal adenocarcinoma risk and tumor biology.

Quantitative determination of insulin-like growth factor 1 receptor mRNA in formalin-fixed paraffin-embedded tissues of invasive breast cancer

Insulin-like growth factor 1 receptor (IGF1R) has recently received much attention due to its role in initiation and progression of breast cancer. Previously analysis of its gene expression has been restricted to fresh-frozen (FF) samples, but application of this technique to routinely processed formalin-fixed paraffin-embedded (FFPE) samples could facilitate larger retrospective studies correlating IGF1R expression with prognosis and therapeutic response. A series of 77 paired FFPE and FF specimens of breast tumors was used to evaluate the possibility of quantifying IGF1R gene expression with FFPE samples and to compare the results obtained from FFPE and FF samples. The feasibility and prognostic value of analyzing IGF1R gene expression using FFPE samples was evaluated in a cohort of 260 primary breast tumors. Total RNA was extracted from 95.4% of the FFPE samples with concentration at least 30ng/μL. Real-time PCR based on Taqman methodology was successful in 90% of the FFPE samples. IGF1R gene expression showed strong correlation not only between FFPE and FF (Spearman ρ=0.74), but also with IGF1R protein expression in both types of specimen. Kaplan-Meier analysis showed that higher IGF1R mRNA expression was associated with longer recurrence-free survival (P=0.009) and breast cancer-specific survival (P=0.0002). Quantitative analysis of IGF1R gene expression in FFPE tissues can be feasibly and reliably conducted, and provides information relevant to the characteristics and outcome of invasive breast cancer.

A Phase I Pharmacokinetic and Pharmacodynamic Study of Dalotuzumab (MK-0646), an Anti-Insulin-like Growth Factor-1 Receptor Monoclonal Antibody, in Patients with Advanced Solid Tumors

Insulin-like growth factor-1 receptor (IGF-1R) mediates cellular processes in cancer and has been proposed as a therapeutic target. Dalotuzumab (MK-0646) is a humanized IgG1 monoclonal antibody that binds to IGF-1R preventing receptor activation. This study was designed to evaluate the safety and tolerability of dalotuzumab, determine the pharmacokinetic (PK) and pharmacodynamic (PD) profiles, and identify a recommended phase II dose. Patients with tumors expressing IGF-1R protein were allocated to dose-escalating cohorts of three or more patients each and received intravenous dalotuzumab weekly, every 2 or 3 weeks. Plasma was collected for PK analysis. Paired baseline and on-treatment skin and tumor biopsy samples were collected for PD analyses. Eighty patients with chemotherapy-refractory solid tumors were enrolled. One dose-limiting toxicity was noted, but a maximum-tolerated dose was not identified. Grade 1 to 3 hyperglycemia, responsive to metformin, occurred in 15 (19%) patients. At dose levels or more than 5 mg/kg, dalotuzumab mean terminal half-life was 95 hours or more, mean C(min) was more than 25 μg/mL, clearance was constant, and serum exposures were approximately dose proportional. Decreases in tumor IGF-1R, downstream receptor signaling, and Ki67 expression were observed. (18)F-Fluorodeoxy-glucose positron emission tomography metabolic responses occurred in three patients. One patient with Ewing's sarcoma showed a mixed radiologic response. The recommended phase II doses were 10, 20, and 30 mg/kg for the weekly, every other week, and every third week schedules, respectively. Dalotuzumab was generally well-tolerated, exhibited dose-proportional PK, inhibited IGF-1R pathway signaling and cell proliferation in treated tumors, and showed clinical activity. The low clearance rate and long terminal half-life support more extended dosing intervals.

Abstract A35: A role for drug resistance in hepatocellular carcinoma: Upregulatation of CD133, activation of IGF-1R signaling, and IGF-1R nuclear translocation

Abstract Introduction: Hepatocellular carcinoma (HCC) is an aggressive neoplasm that is resistant to chemotherapy with mechanisms underlying this drug resistance not well understood. CD133 is a known cancer stem cell (CSC) marker whose high expression is associated with aggressive tumor behavior and poor prognosis. The insulin-like growth factor 1 receptor (IGF-1R) signaling pathway has been shown to play a significant role in tumor behavior. To examine possible mechanisms of drug resistance in HCC, we investigated the role of CD133 expression and IGF-1R signaling activation using geftinib. Methods: We evaluated and separated five different human HCC cell lines (Mahlavu, Huh7, HepG2, Hep3B, PLC/PRF/5) using based on CD133 expression. We subjected these cells to geftinib at different doses, and determined IGF-1R signaling pathway activation using immunoblotting against relevant proteins in the pathway. CD133 mRNA level was examined using quantitative real-time PCR and its expression level was detected using immunofluorescence imaging and flow cytometry after drug treatment. Cell viability and sensitivity to drug treatment were measured using the MTT assay. Results: HCC cell lines showed variable percentages of total cell populations expressing CD133: Mahlavu 0.12 %, HepG2 46 %, Huh7 80.2 %, Hep3B 98.2 %, and PLC/PRF/5 83.5 %. Mahlavu and PLC/PRF/5 were found to be the most resistant cell lines even at higher doses (17.5 uM). Since Mahlavu cells displayed reduced CD133 expression, experiments were focused on this cell line to evaluate the CD133 expression changes after geftinib treatment. After three rounds (9 days) of high dose geftinib treatment, CD133 expression was increased in a dose dependent manner; the percentages of total cell populations expressing CD133 increased to 43.9 % at 10 uM concentration and 52.9 % at 15 uM concentration as compared to 0.12 % in untreated cells. In addition, we found a significant increase in IGF-1R and AKT phosphorylation as well as IGFBP3 expression in those geftinib treated cells. Furthermore, after cell sorting of Huh7 cells by CD133 expression using flow cytometry, we observed that CD133 positive cells showed more resistant to geftinib treatment. Immunofluorescence imaging analysis demonstrated that increased IGF-1R protein translocated to nuclei after geftinib treatment. Conclusion: Geftinib treatment enhanced CD133 expression level and activated IGF-1R signaling in HCC Mahlavu cells, one of the possible mechanism could be the nuclear translocation of IGF-1R. Our findings underscore the importance of a combined therapeutic approach with geftinib and IGF-1R blockage in the treatment of human HCC. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A35.

Prognostic impact of insulin receptor expression on survival of patients with nonsmall cell lung cancer

The purpose of this study was to characterize insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF-1R) expression in patients with nonsmall cell lung cancer (NSCLC). A total of 459 patients who underwent curative resection of NSCLC were studied (median follow-up duration, 4.01 years). Expression of the IR and IGF-1R protein in tumor specimens was assessed immunohistochemically using tissue microarrays. The cytoplasmic IR score was higher in patients with adenocarcinoma (ADC) than in those with squamous cell carcinoma (SCC), whereas cytoplasmic IGF-1R score was higher in patients with SCC than those with ADC. Neither IR nor IGF-1R expression was associated with sex, smoking history, or clinical stage. Patients with positive IR or IGF-1R expression levels had poor recurrence-free (RFS) (3.8 vs 3.3 years; 3.8 vs 2.0 years, respectively), but similar overall survival (OS). Patients with high expression levels of IR and IGF-1R had shorter RFS and OS compared with those with low levels of IR and/or IGF-1R expression. Finally, a multivariate analysis revealed the impact of IR, but not of IGF-1R, as an independent predictive marker of NSCLC survival: hazard ratio (HR) for OS, 1.005 (95% confidence interval [CI], 1.001-1.010], HR for RFS, 1.005 (95% CI, 1.001-1.009), when IR score was tested as a continuous variable. Overexpression of IR predicts a poor survival among patients with NSCLC, especially those with SCC. These results might serve as future guidance to the clinical trials involving IR or IGR-1R targeting agents.

Increased expression of insulin‐like growth factor‐1 receptor is correlated with tumor metastasis and prognosis in patients with osteosarcoma

The aim of this study was to investigate the association of insulin-like growth factor-1 receptor (IGF-1R) with metastasis and prognosis of osteosarcoma patients. RT-PCR and Western blot assays were performed to detect IGF-1R mRNA and protein expression in 26 osteosarcoma and noncancerous bone tissues. Immunohistochemistry was performed to analyze the correlation of IGF-1R expression in 84 osteosarcoma tissues with clinicopathological factors or survival of patients. Lentivirus-mediated RNA interference system was employed to downregulate IGF-1R expression and analyze the effects of IGF-1R downregulation on invasion and metastasis of osteosarcoma cells. The relative levels of IGF-1R mRNA and protein expression were significantly higher in osteosarcoma tissues than in corresponding noncancerous bone tissues. The expression of IGF-1R protein was closely associated with surgical stage and distant metastasis of osteosarcoma patients. Osteosarcoma patients with high IGF-1R expression had poorer survival, and multivariate Cox analyses showed that high IGF-1R expression was an independent prognostic maker. Lentivirus-mediated targeting IGF-1R could significantly inhibit adhesion, migration, invasion, and metastasis of osteosarcoma cells, which might be correlated with of inactivation of Akt signaling pathway. IGF-1R is an independent prognostic marker for osteosarcoma patients and increased expression of this molecular is correlated with metastasis of osteosarcoma.