Insulin-like Growth Factor-1 Receptor Gene Expression Research Articles

Blockade of IGF-1R with OSI-906 Overcomes Bortezomib-Resistance in Multiple Myeloma

Abstract 925▪▪This icon denotes a clinically relevant abstractBlockade of IGF-1R with OSI-906 Overcomes Bortezomib-resistance in Multiple MyelomaDeborah J. Kuhn, Hua Wang, Richard J. Jones, Chad C. Bjorklund, Robert Z. OrlowskiThe Department of Lymphoma & Myeloma, The University of Texas M. D. Anderson Cancer Center, Houston, TX Intro:Bortezomib (Velcade®) therapy is now one of the standards of care in the treatment of newly diagnosed and relapsed/refractory multiple myeloma. Unfortunately, like many other novel agents, the emergence of drug resistance often results in a reduced response to any subsequent therapies that contain bortezomib. Identifying the molecular signaling pathways predominant in bortezomib-resistance can lead to the discovery of therapies that can overcome or prevent the emergence of such resistance all together. Design:In order to improve our understanding of the mechanisms responsible for bortezomib-resistance, our group has developed cell line models of interleukin (IL)-6-dependent and –independent bortezomib-resistant multiple myeloma. Results:Gene expression profiling identified insulin-like growth factor (IGF-1) signaling as one pathway that was induced in bortezomib-resistant myeloma cell lines. Its role was validated in molecular studies that showed exogenous IGF-1 protected drug-naïve cells from bortezomib, while shRNA-mediated knockdown of the IGF-1 receptor (IGF-1R) in bortezomib-resistant models restored sensitivity to this proteasome inhibitor. We then evaluated whether targeting IGF-1R with the clinically relevant inhibitor OSI-906 (OSI Pharmaceuticals, Inc.) could be a valid strategy to overcome bortezomib-resistance. OSI-906 alone preferentially induced cell death in bortezomib-resistant cell lines, while drug-naïve cell populations were relatively spared. Simultaneous addition of bortezomib and increasing concentrations of OSI-906 enhanced the amount of cell death. Also, OSI-906 preferentially induced apoptosis as measured by Annexin V staining. Knockout of IGF-1R gene expression using lentiviral shRNAs in bortezomib-resistant cell lines decreased sensitivity to OSI-906 compared to their scrambled control counterparts, underscoring the importance of IGF-1R signaling in these cells. Conversely, lentiviral overexpression of IGF-1R in drug-naïve wild-type cell lines reduced apoptosis when these models were exposed to OSI-906. Next, we determined whether OSI-906 alone would have an effect on MDA-MM-002, a cell line developed from the pleural effusion of a patient with advanced myeloma. MDA-MM-002 cells, which are resistant to a number of chemotherapeutics, including bortezomib, showed no decrease in viable cell populations when treated with OSI-906 alone. However, when combined with bortezomib, there was a dose-dependent decrease in the viable cell population. Importantly, flank xenograft models of bortezomib-resistant myeloma cells in immunodeficient mice remained insensitive to bortezomib treatment, but showed a dose-dependent response to OSI-906 as evidenced by decrease tumor cell growth, and caspase-3 and PARP cleavage. Examination of synergy profiles using isobologram analysis demonstrated a high degree of synergy with OSI-906 and bortezomib over the use of either drug alone. Conclusions:In addition to the important role of IGF-1 in myeloma biology, the findings herein provide an excellent rationale for using OSI-906 to target IGF-1 signaling in combination with bortezomib as an approach to overcome, or possibly even prevent outgrowth of resistance to bortezomib in myeloma patients. Disclosures:No relevant conflicts of interest to declare.

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.

Expression of IGF1R Is Associated with Tumor Differentiation and Survival in Patients with Lung Adenocarcinoma

The insulin-like growth factor 1 receptor (IGF1R) is widely expressed in normal tissues and many malignancies in humans. We investigated the clinical significance of the expression of the IGF1R gene in human lung adenocarcinoma. A total of 238 patients with lung adenocarcinoma were investigated. Quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) assays were performed to evaluate the gene expression of IGF1R, and immunohistochemical staining was done to evaluate the protein expression of IGF1R. Among the 238 patients with lung adenocarcinoma, 107 tumors (45.0%) were IGF1R-low and 131 tumors (55.0%) were IGF1R-high. The IGF1R gene expression ratio was significantly lower in moderately to poorly differentiated adenocarcinomas than in well-differentiated adenocarcinomas (P = 0.0388). Gene expression of IGF1R was significantly correlated with protein expression of IGF1R (r = 0.7163, P < 0.0001). Regarding patient survival, overall survival was significantly lower in patients with IGF1R-low tumors than in those with IGF1R-high tumors (63.2% versus 76.1% 5-year survival, P = 0.0188). Multivariate analysis using a Cox proportional-hazards model demonstrated that IGF1R gene status was a significant prognostic factor predicting overall survival of patients with lung adenocarcinoma (hazard ratio 1.800; P = 0.0321). Moreover, the disease-free survival rate was significantly lower in patients with IGF1R-low tumors than in those with IGF1R-high tumors (49.2% versus 64.6% 5-year survival, P = 0.0084). The present study suggests the level of IGF1R expression to be a useful prognostic marker for patients with dedifferentiated lung adenocarcinoma.

Biochemische und immunologische Parameter der Alterung

Growth hormone and IGF-1 regulate the brain function. With the ageing of the brain, IGF-1 decreases. In animal experiments it was possible to improve the utilization of glucose in the brain by supplying IGF-1 when a deficiency of growth hormone and IGF-1 was present. IGF-1 and the IGF-1 receptor gene expression are increased by zinc supplementation. IL-2 is the most important cytokine which improves the cell proliferation. With ageing, the secretion of IL-2 is reduced, especially with people suffering from dementia. By supplying zinc, the reduction of IL-2 can be normalized again. Oxidative stress is of pathogenic significance. Enzymatic mechanisms are necessary for the detoxification of radicals. In this case, zinc has an anti-inflammatory and membrane stabilizing effect.

Suppression of insulin-like growth factor-1 receptor by RNA interference inhibits cell growth in vitro and induces chemosensitization of HO8910PM cell to cisplatin

To assess the effect of suppression of insulin-like growth factor-1 receptor (IGF1R) in HO8910PM cell line by small interference RNA (siRNA). Transfection of siRNA using lipofectamine 2000 was conducted to silence IGF1R gene expression, the expression levels of IGF1R mRNA and protein were evaluated, and the effects on the cell cycles at 48 hours of transfection were assessed by real-time PCR, western blot and flow cytometry (FCM) assay respectively. The cell growth was detected by cell counting kit-8 (CCK-8) at 24, 48, 72, 96 hours of transfection. After 24 hours of transfection, the cells were cultured with different concentrations of cisplatin (DDP) for 24 hours, the cell growth inhibition rate was evaluated by CCK-8. Following incubation with 10 microg/ml DDP for 24 hours after 24 hours of transfection, the apoptosis cells and the protein expression level of apoptosis-related gene, B cell leukemia/lymphoma 2 (Bcl-2), were identified by FCM and western blot respectively. (1) Expression levels of IGF1R mRNA and protein were markedly decreased respectively at 48 hours of transfection IGF1R siRNA. (2) Suppression of IGF1R accompanied the reduction of cell growth at 48, 72, 96 hours of transfection with IGF1R siRNA, absorbance were 1.71+/-0.13, 2.32+/-0.23, 2.79+/-0.28 respectively (P<0.01). (3) IGF1R siRNA induces arrest of G2 phase, the G2 phase rate of cells were 24.37% (P<0.05). (4) Following treatment with 2.5, 5, 10, 20 microg/ml DDP for 24 hours after 24 hours of transfection, the cell growth inhibition rates were (25.94+/-0.08)%, (40.25+/-0.05)%, (59.48+/-0.03)% and (74.18+/-0.08)% respectively (P<0.01). (5) Treatment with 10 microg/ml DDP for 24 hours after 24 hours of transfection, induces 17.95% of cells apoptosis (P<0.05), and decreases Bcl-2 protein level. RNA interference of IGF1R gene induces the IGF1R silence in HO8910PM cell line significantly, inhibits cell growth in vitro, arrests the G2 phase, and enhances the chemosensitization to DDP.

Mechanism involved in genistein activation of insulin-like growth factor 1 receptor expression in human breast cancer cells

Our previous studies have shown that genistein can enhance the insulin-like growth factor (IGF)-1 receptor signalling pathway via an oestrogen receptor (ER) in human breast cancer MCF-7 cells. The present study aims to investigate how genistein regulates IGF-1 receptor expression in human MCF-7 cells. Genistein at 1 microm stimulated the growth of MCF-7 cells and this effect could be completely blocked by the IGF-1 receptor antagonist JB-1, suggesting that IGF-1 receptor is essential for mediating the proliferative effects of genistein in MCF-7 cells. Genistein increased IGF-1 receptor promoter activity. This effect could be completely abolished by co-treatment of MCF-7 cells with ICI 182,780 (10- 6 m). Genistein increased IGF-1 receptor gene expression and this effect could be completely blocked by the IGF-1 receptor antagonist JB-1. Co-treatment of MCF-7 cells with cycloheximide (5 microg/ml) completely blocked the induction of IGF-1 receptor protein and mRNA expression by genistein. The results indicated that the induction of IGF-1 receptor promoter activity by genistein required the action of ER while the stimulatory actions of genistein on IGF-1 receptor expression required the activity of the IGF-1 receptor and de novo protein synthesis. These data provide evidence to support the hypothesis that the inductive effects of genistein on IGF-1 receptor expression require the cross-talk between IGF-1 receptor and the ER-dependent pathways.

1A-4 IUGR alters IGF1 receptor gene expression, DNA methylation and histones acetylation in the brain

1A-4 IUGR alters IGF1 receptor gene expression, DNA methylation and histones acetylation in the brain

The potentiation of curcumin on insulin-like growth factor-1 action in MCF-7 human breast carcinoma cells

Curcumin has anticarcinogenic and chemopreventive properties in a variety of experimental cancer models. Our in vitro studies have shown that curcumin inhibits cell growth and induces apoptosis in MCF-7, a human breast carcinoma cell line. The insulin-like growth factor-1 (IGF-1) system, including IGFs (IGF-1 and IGF-2), IGF-1R (IGF-1 receptor) and IGFBPs (IGF binding proteins), has been implicated to play a critical role in the development of breast cancer. The aim of the present study was to investigate whether the growth inhibitory effects of curcumin were related to changes of the IGF-1 system in MCF-7 cells. IGF-1 at 50 μg/l in serum-free medium produced maximum proliferation and minimized apoptosis. However, curcumin exhibited a potent ability to blunt IGF-1-stimulated MCF-7 cell growth and reverse the IGF-1-induced apoptosis resistance. To determine whether curcumin intervenes in IGF-1 or IGFBP-3 secretion, MCF-7 cells were incubated in serum-free medium in the presence of various concentrations of curcumin for indicated time periods. Curcumin decreased the secretion of IGF-1 with a concomitant increase of IGFBP-3 in a dose-dependent manner. Receptor tyrosine kinase assays revealed that IGF-1-stimulated IGF-1R tyrosine kinase activation was also abrogated by curcumin in a dose-dependent manner. Real-time fluorescence quantitative reverse transcriptase-polymerase chain reaction (RFQ-RT-PCR) further revealed that curcumin suppressed IGF-1R gene expression at transcriptional level. In conclusion, the inhibition of cell growth and induction of apoptosis by curcumin in MCF-7 cells might be mediated, at least partially, by its ability to down-regulate the IGF-1 axis.

Tectorigenin and other phytochemicals extracted from leopard lily Belamcanda chinensis affect new and established targets for therapies in prostate cancer

Isoflavones have been shown to exert antiproliferative effects on cancer cells by steroid receptor signaling. In this study, we demonstrate the potential of plant constituents extracted from Belamcanda chinensis as anticancer drugs, which regulate the aberrant expression of genes relevant in proliferation, invasion, immortalization and apoptosis. LNCaP cells were treated with B.chinensis extract, tectorigenin or other isoflavones and mRNA expression was quantified by using real time RT-PCR. In addition, ELISA, TRAP assays and western blots were used to measure protein expression or activity. Male nude mice (n=18) were injected subcutaneously with LNCaP cells and were fed with extracts from B.chinensis, and tumor development was monitored versus a control animal group (n=18). Tectorigenin and several other phytochemicals downregulated PDEF, PSA and IGF-1 receptor mRNA expression in vitro. Furthermore, PSA secretion and IGF-1 receptor protein expression were diminished, and hTERT mRNA expression and telomerase activity decreased after tectorigenin treatments. However, TIMP-3 mRNA was upregulated on tectorigenin treatment. Growth of subcutaneous tumors in nude mice was delayed and diminished in animals fed with extracts from B.chinensis. The downregulation of PDEF, PSA, hTERT and IGF-1 receptor gene expression by tectorigenin demonstrates the antiproliferative potential of these agents. The upregulation of TIMP-3 gene expression indicates a pro-apoptotic function of the drug and a reduction of the invasiveness of tumors. The animal experiments demonstrate that B.chinensis markedly inhibited the development of tumors in vivo. Thus, these compounds may be useful for the prevention or treatment of human prostate cancer.

Cell-type specific expression of IGF-1R in porcine islet cells

Insulin-like growth factor-1 (IGF-1) functions as a growth factor regarding physiological regulations of cellular metabolism, regeneration and growth. In pancreas islets their potential function is unclear and only little information is available on occurrence and distribution of the corresponding insulin-like growth factor-1 receptor (IGF-1R) in islet cells. Therefore, we investigated the localization of IGF-1R by immunohistochemical techniques and its possible co-localization with other islet hormones. Further, we applied molecular biology techniques to determine the present of local gene expression of IGF-1R and IGF-1. Immunostaining on serial sections with anti-insulin, anti-glucagon and anti-somatostatin antibodies shows, IGF-1R was selectively expressed in insulin-producing B-cells and additionally more pronounced in somatostatin-containing D-cells, which are located in the periphery of porcine pancreatic islets. Furthermore, the RT-PCR experiment demonstrates clearly that IGF-1 and IGF-1R was expressed together in the porcine pancreas. The high expression of IGF-1R in porcine D-cells indicates that mammalian IGF-1R genes are regulated in a different manner since it was shown that in all other species IGF-1R was expressed in B- and A-cells but not in D-cells.

Insulin-like growth factor-1 expression in reflux nephropathy.

Reflux nephropathy (RN) is recognised as a major cause of end-stage renal failure in children and young adults. Insulin-like growth factor-1 (IGF-1), a peptide growth factor produced by collecting ducts, and its receptor, insulin-like growth factor-1 receptor (IGF-1R), are present in the glomeruli and basolateral membrane of renal proximal tubular cells. Exogenous IGF-1 has been shown to enhance proliferation and reduce apoptosis of tubular cells following renal injury. We designed this study to investigate the expression of IGF-1 in RN. The kidney specimens from 15 children with RN were obtained at the time of nephrectomy. Control material included normal kidney specimens obtained from adult patients during partial nephrectomy for an incidentaloma. Single-label immunofluorescence histochemistry was carried out using polyclonal antibodies to IGF-1 and IGF-1R employing laser scanning confocal microscopy. Double-label immunofluorescence histochemistry was carried out using monoclonal antibodies to vimentin and clusterin to assess tubulointerstitial fibrosis. IGF-1 and IGF-1R gene expression were evaluated by in situ hybridisation (ISH). The TUNEL method was utilised to assess tubular apoptosis. In the normal kidney there was strong IGF-1 and IGF-1R immunoreactivity in the proximal tubules, whereas IGF-1 and IGF-1R immunoreactivity was markedly reduced in RN specimens. Strong IGF-1 and IGF-1R mRNA expression was observed in the proximal tubules in normal kidneys, whereas IGF-1 and IGF-1R mRNA expression was undetectable in RN. Renal tubulointerstitial expression of vimentin and clusterin was markedly increased in RN kidneys. Decreased IGF-1 and IGF-1R expression in RN strongly correlated with severity of tubular apoptosis in RN compared with controls. These data suggest that the downregulation of IGF-1 and IGF-1R may play an important role in the pathogenesis of RN, at least in part by increasing interstitial collagen deposition and tubular apoptosis.

Expression of the insulin-like growth factor-1 gene and its receptor in preimplantation mouse embryos; is it a marker of embryo viability?

Recent studies have demonstrated the importance of insulin-like growth factors (IGF) in mouse preimplantation development. We examined IGF-1 and IGF-1 receptor (IGF-1R) gene expression in a single blastomere of an early mouse embryo and compared it with subsequent embryo development in culture. Fertilized eggs and 2-cell embryos were obtained by tubal flushing in superovulated and mated female mice. Single cells were removed from embryos at cleavage stage between 3 and 8 cells using the standard embryo biopsy techniques. Individual blastomeres from each embryo were then assayed for the presence of IGF-1 and IGF-1R mRNA using reverse transcription-polymerase chain reaction. The biopsied embryos were washed in medium and placed in co-culture with murine endometrial cells. Embryonic development in culture was assessed and blastocyst grading was performed. IGF-1 gene expression was then examined for an association with in-vitro development. Eighty-seven embryos were biopsied. IGF-1R gene expression was detected in the majority of embryos tested and IGF-1 gene expression was detected in 34 of 81 (42%) embryos. A significant association between IGF-1 expression and blastocyst formation in vitro was found (P < 0.01). There was no association between IGF-1R expression and subsequent embryo development. We conclude that IGF-1 gene expression could potentially be used as a marker of embryo quality.

Insulin-like growth factor-1 and insulin resistance in skeletal muscles of adult and old rats.

A study was designed to compare the effects, in vitro, of insulin-like growth factor-1 (IGF-1) and insulin on rat epitrochlearis muscle metabolism during aging (1, 6-8, or 18-20 months). Our results showed that in young epitrochlearis, IGF-1 was equipotent to insulin in stimulating 2-deoxy-glucose and aminoisobutyric acid transport but more potent in increasing tyrosine incorporation into protein. Both insulin and IGF-1 action on glucose transport was decreased in adult compared with young muscle. Whereas an insulin resistance of amino acid transport and protein synthesis was also recorded in adult rat muscle, the stimulatory effect of IGF-1 on these processes was abolished. Thus the degree of resistance observed varied both with the agonist and with the subsequent metabolic process observed. Whereas modifications of IGF-1 action in mature animals may be correlated in part to the dramatic decrease of IGF-1 receptors (80%), no similar observations were recorded for the insulin receptor. Since muscle IGF-1 receptor gene expression did not decrease in parallel with receptor number, an alteration in IGF-1 receptor messenger RNA (mRNA) translation or receptor degradation may be hypothetized. We concluded that: 1) In contrast to glucose transport, intracellular IGF-1 and insulin postreceptor pathways leading to amino acid uptake and protein metabolism differ. 2) Modification in postbinding events might be involved in decreased insulin- and IGF-1-stimulated muscle metabolism during aging.