Insulin-like Growth Factor-binding Protein 7 Levels Research Articles

Urine Biomarkers of Tubular Renal Cell Damage for the Prediction of Acute Kidney Injury After Cardiac Surgery—A Pilot Study

To evaluate the perioperative course of urine levels of the renal damage biomarkers tissue inhibitor of metalloproteinase 2 (TIMP-2) and insulin-like growth factor-binding protein 7 (IGFBP7) and to evaluate the predictive value of elevated TIMP-2 × IGFBP7 concentrations to predict acute kidney injury (AKI) early after cardiac on-pump surgery. Prospective, observational cohort study. University hospital. The study comprised 110 consecutive patients undergoing elective cardiac surgery with cardiopulmonary bypass (CPB) between January and March 2014. None. Urinary TIMP-2 × IGFBP7 levels were quantified using a commercially available kit at the following measurement points: before surgery, 1 hour after starting CPB, 4 hours after weaning from CPB, and 24 hours after weaning from CPB (time points 1-4). Postoperative AKI was defined according to Kidney Disease Improving Global Outcomes criteria. AKI after cardiac surgery was diagnosed in 9 patients (8%). The perioperative course of TIMP-2 × IGFBP7 was significantly different in patients with and without postoperative AKI (p < 0.001). TIMP-2 × IGFBP7 levels were significantly higher in patients with AKI 1 hour after CPB start and 24 hours after weaning from CPB (p < 0.05). TIMP-2 × IGFBP7 levels >0.40 (ng/mL)2/1,000 measured at 1 hour after starting CPB were found to be the optimal cut-off, with a sensitivity of 0.778 and a specificity of 0.641. The negative predictive value was 0.972. Urine levels of TIMP-2 × IGFBP7 are predictive for AKI at an early time point (1 hour after starting CPB). Renal damage biomarkers such as TIMP-2 and IGFBP7 might be recommended as a supplement to traditionally used criteria of AKI prediction.

Cisplatin nephrotoxicity is not detected by urinary cell-cycle arrest biomarkers in lung cancer patients.

Cisplatin is a chemotherapeutic agent with potential nephrotoxicity. Delayed increase in serum creatinine after cisplatin administration shows that serum creatinine may not be a sufficient marker for early detection of nephrotoxicity. Urinary insulin-like growth factor binding protein-7 (IGFBP7) and tissue inhibitor of metalloproteinases-2 (TIMP-2) which are cell-cycle arrest biomarkers have been proposed recently for early detection of acute kidney injury (AKI). Herein, we evaluated urinary TIMP-2/IGFBP7 levels before and after cisplatin administration to patients with lung cancer and their role was examined in the early diagnosis of AKI. Patients with glomerular filtration rate above 60mL/min who had cisplatin treatment were enrolled. Urinary TIMP-2/IGFBP7 and serum creatinine levels were measured before and at 24th hour after cisplatin administration. Serum creatinine level was also measured at 48th hour after treatment. Cisplatin-associated AKI was detected in 13 patients (28%) among the 45 patients enrolled. There was no difference between creatinine, IGFBP7 and (IGFBP7×TIMP-2)/1000 levels before and after treatment; urinary TIMP-2 level at 24th hour was significantly higher than the level before the treatment (p=0.02). (IGFBP7×TIMP-2)/1000 values were not different between patients with or without AKI. The area under the curve of (IGFBP7×TIMP-2)/1000 at 24th hour of the treatment was 0.46 (CI 0.26-0.67). Although urinary IGFBP7 and TIMP-2 levels are used as biomarkers for early detection of AKI for patients in intensive care units and after surgery, they seem not to be useful for early detection of AKI due to cisplatin.

Serum insulin-like growth factor-1 and its binding protein-7: potential novel biomarkers for heart failure with preserved ejection fraction.

BackgroundInsulin-like growth factor binding protein-7 (IGFBP-7) modulates the biological activities of insulin-like growth factor-1 (IGF-1). Previous studies demonstrated the prognostic value of IGFBP-7 and IGF-1 among patients with systolic heart failure (HF). This study aimed to evaluate the IGF1/IGFBP-7 axis in HF patients with preserved ejection fraction (HFpEF).MethodsSerum IGF-1 and IGFBP-7 levels were measured in 300 eligible consecutive patients who underwent comprehensive cardiac assessment. Patients were categorized into 3 groups including controls with normal diastolic function (n = 55), asymptomatic left ventricular diastolic dysfunction (LVDD, n = 168) and HFpEF (n = 77).ResultsIGFBP-7 serum levels showed a significant graded increase from controls to LVDD to HFpEF (median 50.30 [43.1-55.3] vs. 54.40 [48.15-63.40] vs. 61.9 [51.6-69.7], respectively, P < 0.001), whereas IGF-1 levels showed a graded decline from controls to LVDD to HFpEF (120.0 [100.8-144.0] vs. 112.3 [88.8-137.1] vs. 99.5 [72.2-124.4], p < 0.001). The IGFBP-7/IGF-1 ratio increased from controls to LVDD to HFpEF (0.43 [0.33-0.56] vs. 0.48 [0.38-0.66] vs. 0.68 [0.55-0.88], p < 0.001). Patents with IGFB-7/IGF1 ratios above the median demonstrated significantly higher left atrial volume index, E/E’ ratio, and NT-proBNP levels (all P ≤ 0.02).ConclusionIn conclusion, this hypothesis-generating pilot study suggests the IGFBP-7/IGF-1 axis correlates with diastolic function and may serve as a novel biomarker in patients with HFpEF. A rise in IGFBP-7 or the IGFBP-7/IGF-1 ratio may reflect worsening diastolic function, adverse cardiac remodeling, and metabolic derangement.

Urine biomarkers give early prediction of acute kidney injury and outcome after out-of-hospital cardiac arrest.

BackgroundPost-resuscitation care after out-of-hospital cardiac arrest (OHCA) is challenging due to the threat of organ failure and difficult prognostication. Our aim was to examine whether urine biomarkers could give an early prediction of acute kidney injury (AKI) and outcome.MethodsThis was a prospective observational study of comatose OHCA patients at Oslo University Hospital Ullevål, Norway. Risk factors were clinical parameters and biomarkers measured in spot urine (cystatin C, neutrophil gelatinase-associated lipocalin (NGAL) and the product of tissue inhibitor of metalloproteinase 2 (TIMP-2) and insulin-like growth factor-binding protein 7 (IGFBP7)) at admission and day 3. Outcome variables were AKI within 3 days using the Kidney Disease Improving Global Outcomes definition, 6-month mortality, and poor neurological outcome (PNO) defined as cerebral performance category 3–5.ResultsAmong 195 included patients (85 % males, mean age 60 years), 88 (45 %) died, 96 (49 %) had PNO, and 88 (45 %) developed AKI. In univariate analysis, increased urine cystatin C and NGAL concentration sampled at admission and day 3 were independent risk factors for AKI, mortality and PNO. Increased urine TIMP-2 × IGFBP7 levels was associated with AKI only at admission. In multivariate analyses combining clinical parameters and biomarker concentrations, the area under the receiver operating characteristics curve (AuROC) with 95 % confidence interval (CI) were 0.774 (0.700–0.848), 0.812 (0.751–0.873), and 0.819 (0.759–0.878) for AKI, mortality and PNO, respectively.ConclusionsIn comatose OHCA patients, urine levels of cystatin C and NGAL at admission and day 3 were independent risk factors for AKI, 6-month mortality and PNO.Trial registrationClinicaltrials.gov NCT01239420. Registered 10 November 2010.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-016-1503-2) contains supplementary material, which is available to authorized users.

Alterations in the proliferative/apoptotic equilibrium in semen of adolescents with varicocele.

To verify if the presence of varicocele (grades II and III) with and without seminal alterations, using the 5th centile cutoff values in table A1.1 of the World Health Organization (WHO, 2010) manual, alters the seminal plasma levels of proteins DNASE1 (deoxyribonuclease-1) and IGFBP7 (Insulin-like growth factor-binding protein 7), which are related to apoptosis regulation and cell proliferation, respectively, demonstrating that these proteins are important for correct spermatogenesis. This cross sectional study was performed at the Sao Paulo Federal University Paulo between May 2014 and April 2016. A total of 61 male adolescents were included in this study, of which 20 controls without varicocele (C), 22 with varicocele and normal semen analysis (VNS) and 19 with varicocele and altered semen analysis (VAS). Seminal plasma from each patient was used for Western blotting analysis of individual protein levels. Values of each protein were normalized to a testicular housekeeping protein (PARK7-protein deglycase DJ-1). Levels of IGFBP7 protein are increased in varicocele. Levels of DNASE1 are progressively decreased in varicocele (lower in varicocele and normal semen analysis, lowest in varicocele and altered semen analysis) when compared to adolescents without varicocele. DNASE1 levels are positively correlated with sperm concentration and morphology (correlation values of 0.400 and 0.404, respectively; p values of 0.001 and 0.001, respectively). In conclusion, in adolescents, seminal plasma levels of IGFBP7, responsible for proliferative activity, are increased in varicocele grades II and III, and DNASE1, responsible for apoptosis regulation, are lower in varicocele, lowest in varicocele and low semen quality. These proteins demonstrate molecular alterations brought upon by varicocele. Moreover, DNASE1 is capable of discriminating a varicocele that causes alterations to semen quality from one that does not. We propose that the initial response of varicocele is to increase proliferative activity which, if followed by regulation of apoptosis, may lead to the ejaculation of a population of sperm that are in accordance with WHO cutoff values but, in the presence of dysregulated apoptosis, leads to lower sperm concentration and morphology.

Abstract P345: Plasma Insulin Like Growth Factor Binding Protein-7 and Tissue Inhibitor of Metalloproteinase-2 are Associated with Reduced Renal Blood Flow and Change in Kidney Function After Revascularization in Human Atherosclerotic Renovascular Disease RVD

Background: Insulin-like growth factor binding protein7 (IGFBP-7) and tissue inhibitor of metalloproteinase-2 (TIMP-2) reflect G1-cell cycle arrest and are used as biomarkers for AKI. Recent studies show that these biomarkers rise in ischemic conditions and suggest that they actually may limit the severity of AKI. We tested the hypothesis that renal vein [IGFBP-7]*[TIMP-2] correlate with reductions in renal blood flow (RBF) and post-stent single kidney (SK)-GFR changes in patients with RVD undergoing contrast-based imaging and stent revascularization Methods: Inpatient studies were performed during 150 mEq Na+ intake and ACE/ARB Rx in patients with hemodynamically severe RVD (n=29, Doppler velocity = 318 ± 100cm/sec, and eGFR= 34.7 ± 11.7 mL/ min) scheduled for renal artery stenting, and compared to essential hypertensive (EH) healthy controls (n=32). Cortical and medullary RBFs (by multidetector CT) and renal vein levels of IGFBP-7 and TIMP-2 were measured before renal artery stenting and 3 months later Results: Pre-stenting IGFBP-7 and TIMP-2 levels were elevated in RVD compared to EH (18.5±2. vs 15.7±1.5 and 97.4±23.1vs 62.7±9.2 ng/mL respectively, P &lt;0.0001). Baseline renal vein levels of IGFBP-7 * TIMP-2 correlated inversely with pre-stent RBF (r= - 0.53, P=0.01) and directly with the change (%) in SK-GFR observed 3 months after stenting Conclusion: IGFBP-7 and TIMP-2 levels are elevated in patients with chronic RVD as a function of the baseline reduction in RBF, and associate with subsequent improvement in SK-GFR 3 months after revascularization. These data are consistent with renovascular occlusion inducing cell-cycle arrest that serves to protect kidney function.

Atherosclerotic renal artery stenosis is associated with elevated cell cycle arrest markers related to reduced renal blood flow and postcontrast hypoxia.

Atherosclerotic renal artery stenosis (ARAS) reduces renal blood flow (RBF), ultimately leading to kidney hypoxia and inflammation. Insulin-like growth factor binding protein-7 (IGFBP-7) and tissue inhibitor of metalloproteinases-2 (TIMP-2) are biomarkers of cell cycle arrest, often increased in ischemic conditions and predictive of acute kidney injury (AKI). This study sought to examine the relationships between renal vein levels of IGFBP-7, TIMP-2, reductions in RBF and postcontrast hypoxia as measured by blood oxygen level-dependent (BOLD) magnetic resonance imaging. Renal vein levels of IGFBP-7 and TIMP-2 were obtained in an ARAS cohort (n= 29) scheduled for renal artery stenting and essential hypertensive (EH) healthy controls (n = 32). Cortical and medullary RBFs were measured by multidetector computed tomography (CT) immediately before renal artery stenting and 3 months later. BOLD imaging was performed before and 3 months after stenting in all patients, and a subgroup (N = 12) underwent repeat BOLD imaging 24 h after CT/stenting to examine postcontrast/procedure levels of hypoxia. Preintervention IGFBP-7 and TIMP-2 levels were elevated in ARAS compared with EH (18.5 ± 2.0 versus 15.7 ± 1.5 and 97.4 ± 23.1 versus 62.7 ± 9.2 ng/mL, respectively; P< 0.0001); baseline IGFBP-7 correlated inversely with hypoxia developing 24 h after contrast injection (r = -0.73, P< 0.0001) and with prestent cortical blood flow (r = -0.59, P= 0.004). These data demonstrate elevated IGFBP-7 and TIMP-2 levels in ARAS as a function of the degree of reduced RBF. Elevated baseline IGFBP-7 levels were associated with protection against postimaging hypoxia, consistent with 'ischemic preconditioning'. Despite contrast injection and stenting, AKI in these high-risk ARAS subjects with elevated IGFBP-7/TIMP-2 was rare and did not affect long-term kidney function.

A promising marker in early diagnosis of septic acute kidney injury of critically ill patients: urine insulin like growth factor binding protein-7

Aim: An ideal biomarker for early diagnosis of septic acute kidney injury (AKI) should reflect renal stress or damage at initiation point, at cellular level. The aim of this study was to assess the role of a urinary cell cycle arrest marker, insulin-like growth factor-binding protein 7 (IGFBP7) in early diagnosis of septic AKI in adult critical care patients.Methods: This was a single-center prospective cohort study. Patients without AKI, admitted to a medical intensive care unit (ICU) between January 2010 and March 2013, were included. According to ‘sepsis’ and ‘AKI’ development during their ICU stay, they were grouped as ‘sepsis-non AKI’, ‘sepsis-AKI’ and ‘non-sepsis-non AKI (control)’. Among these groups, urine IGFBP7 was studied and compared with Human ELISA Kit/96 Test/USCNK® first on admission and then on daily collected serial urine samples.Results: A total of 118 patients formed the cohort; 52 in sepsis-non AKI, 43 in sepsis-AKI, 23 in control group. Admission urine IGFBP7 predicted septic AKI development with 72% sensitivity and 70% specificity for a threshold level of 2.5 ng/mL with an area under the receiver operating characteristics curve (AUC) of 0.79 (95% CI: 0.70–0.88). No impact of sepsis was observed on urine IGFBP7 levels in the absence of AKI. In the septic AKI group urine IGFBP7 levels continuously increased up to the day of AKI development and high levels were suspended for 10 days further.Conclusion: Admission urine IGFBP7 levels and following its course in ICUs can be used as a promising new biomarker for the early diagnosis of septic AKI development without being affected by sepsis itself.

Abstract 2339: IGFBP7 eradicates leukemic stem and progenitor cells in acute myeloid leukemia

Abstract Despite high complete remission rates after chemotherapy treatment, only 30-40% of acute myeloid leukemia (AML) patients (&amp;lt; 60 years) survive five years after diagnosis. This poor prognosis is mainly due to relapse which is thought to be initiated by a subpopulation of leukemic cells with stem cell-like features that survived the treatment, often referred to as leukemic stem cells (LSC). The development of novel alternative therapies, specifically targeting these LSC, are needed to prevent relapse and improve AML outcome. To develop these anti-LSC therapies, identification of genes differentially expressed between LSC and the AML bulk and the normal HSC, still residing within the AML bone marrow, is critical. Studying these differences is most relevant in cell fractions obtained from the same AML bone marrow, taking into account the effects of the leukemic microenvironment on these cell compartments. Since LSC and HSC share the same immunophenotype (CD34+CD38-), we and others identified markers including CLL-1 as well as scatter properties, to distinguish leukemic from normal stem cells. By these properties we purified LSC, HSC and progenitors fractions from AML bone marrow. Gene expression profiling revealed the insulin-like growth factor binding-protein-7 (IGFBP7) as differentially expressed between LSC and HSC and between LSC and the AML bulk. IGFBP7 is a secreted tumor suppressor and based on our expression data we hypothesized that IGFBP7 might induce the eradication of LSC. Using a knockdown strategy, we show that decreased levels of IGFBP7 in AML cell lines result in decreased sensitivity to chemotherapy. Moreover, we show that both IGFBP7 overexpression and recombinant human IGFBP7 (rhIGFBP7) reduces the survival of leukemic stem and progenitor cells from AML patients. Incubation with rhIGFBP7 showed decreased survival of leukemic blasts while sparing healthy cells. Most importantly, IGFBP7 significantly reduces human leukemic engraftment of primary AML cells in immune-deficient NOD/SCID-IL2γ knockout (NSG) mice. Altogether, LSC have reduced levels of IGFBP7 which might be responsible for their decreased sensitivity to chemotherapy. The eradication of LSC and leukemic progenitors can be accomplished by enhancing IGFBP7 levels, suggesting that AML patients might benefit from a combination of rhIGFBP7 and chemotherapy. This combination therapy might prevent relapse and improve AML outcome. Citation Format: Han Verhagen, Marjon Smit, David de Leeuw, Arjo Rutten, Mei-Ling Tsui, Fedor Denkers, Monique Terwijn, Patrick Celie, Gert Ossenkoppele, Gerrit Jan Schuurhuis, Linda Smit. IGFBP7 eradicates leukemic stem and progenitor cells in acute myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2339. doi:10.1158/1538-7445.AM2015-2339

High expression of IGFBP7 in fibroblasts induced by colorectal cancer cells is co-regulated by TGF-β and Wnt signaling in a Smad2/3-Dvl2/3-dependent manner.

Fibroblasts in the tumor microenvironment are a key determinant in cancer progression and may be a promising target for cancer therapy. Insulin-like growth factor binding protein 7 (IGFBP7) is known as a tumor suppressor in colorectal cancer (CRC). The present study investigated the inductive mechanism of IGFBP7 expression in fibroblasts by supernatant from the CRC cell line, SW620. The results showed that the expression of IGFBP7 was up-regulated in the fibroblasts when treated with SW620 supernatant and exogenous TGF-β1. The IGFBP7 induced by SW620 supernatant or TGF-β1 was partially inhibited by the TGF-β1 specific antibody AF and TGF-β1 receptor antagonist SB431542. The Wnt signaling-targeted genes, c-Myc, CCND1 and the proteins Dvl2/3, were all up-regulated in fibroblasts expressing high levels of IGFBP7, and the up-regulation could be inhibited both by the Wnt signaling antagonist Dickkopf-1 (DKK1) and by the TGF-β1 receptor antagonist SB431542. In conclusion, CRC cells promote the high expression of IGFBP7 in fibroblasts, most likely through the co-regulation of TGF-β and Wnt signaling in a Smad2/3-Dvl2/3 dependent manner. Taken together, these data suggest that the fibroblasts could be a novel therapeutic target in tumor therapy.

Evaluation of IGFBP-7 DNA methylation changes and serum protein variation in Swedish subjects with and without type 2 diabetes

BackgroundInsulin-like growth factor-binding protein 7 (IGFBP-7) is able to interact with insulin-like growth factor 1 (IGF-1) as well as insulin. Previous studies have suggested that serum IGFBP-7 levels may be associated with insulin resistance in type 2 diabetes (T2D). This study aimed to evaluate IGFBP-7 serum protein and IGFBP7 DNA methylation levels in the subjects with and without T2D.ResultsA total of 340 Swedish subjects including 100 newly diagnosed T2D patients (50 women/50 men), 100 age-matched nondiabetic control subjects (50/50) and 140 treated T2D patients (54/86) were studied. Serum IGFBP-7 levels were measured with a novel ELISA. IGF1, IGFBP-1, and insulin were determined by in-house radioimmunoassays. DNA methylation levels in the IGFBP7 gene were analyzed with a bisulfite-pyrosequencing technique. Serum IGFBP-7 protein levels were similar among nondiabetic subjects, newly diagnosed, and treated T2D patients and were not correlated with IGFBP7 DNA methylation. However, IGFBP7 DNA methylation was increased in men with newly diagnosed T2D compared with nondiabetic controls (17.6% vs. 12.5%, P < 0.01). Serum IGFBP-7 levels correlated (r = 0.331, P = 0.019) with serum IGFBP-1 levels, a marker of insulin production, in men but not women with newly diagnosed T2D.ConclusionsThis study demonstrates for the first time that IGFBP7 DNA methylation levels are increased in Swedish men with newly diagnosed T2D. The correlation between IGFBP-7 and IGFBP-1 suggests that low IGFBP-7 may be associated with insulin resistance in T2D.

Insulin growth factor binding protein 7 is a novel target to treat dementia

Alzheimer's disease (AD) is the most common form of dementia in the elderly but effective therapeutic strategies to treat AD are not yet available. This is also due to the fact that the pathological mechanisms that drive the pathogenesis of sporadic AD are still not sufficiently understood and may differ on the individual level. Several risk factors such as altered insulin-like peptide (ILP) signaling have been linked to AD and modulating the ILP system has been discussed as a potential therapeutic avenue. Here we show that insulin-like growth factor binding protein 7 (IGFBP7), a protein that attenuates the function of ILPs, is up-regulated in the brains of AD patients and in a mouse model for AD via a process that involves altered DNA-methylation and coincides with decreased ILP signaling. Mimicking the AD-situation in wild type mice, by increasing hippocampal IGFBP7 levels leads to impaired memory consolidation. Consistently, inhibiting IGFBP7 function in mice that develop AD-like memory impairment reinstates associative learning behavior. These data suggest that IGFBP7 is a critical regulator of memory consolidation and might be used as a biomarker for AD. Targeting IGFBP7 could be a novel therapeutic avenue for the treatment of AD patients.

Aberrant methylation of LINE-1, SLIT2, MAL and IGFBP7 in non-small cell lung cancer

Genome-wide DNA hypomethylation and gene hypermethylation play important roles in instability and carcinogenesis. Methylation in long interspersed nucleotide element 1 (LINE-1) is a good indicator of the global DNA methylation level within a cell. Slit homolog 2 (SLIT2), myelin and lymphocyte protein gene (MAL) and insulin-like growth factor binding protein 7 (IGFBP7) are known to be hypermethylated in various malignancies. The aim of the present study was to assess the precise methylation levels of LINE-1, SLIT2, MAL and IGFBP7 in non-small cell lung cancer (NSCLC) using a pyrosequencing assay. Methylation of all regions was examined in 56 primary NSCLCs using a pyrosequencing assay. Changes in mRNA expression levels of SLIT2, MAL and IGFBP7 were measured before and after treatment with a demethylating agent. Methylation of these genes was also examined in 9 lung cancer cell lines using RT-PCR and a pyrosequencing assay. Frequencies of hypomethylation of LINE-1 and hypermethylation of SLIT2, MAL and IGFBP7, defined by predetermined cut off values, were 55, 64, 46 and 54% in NSCLCs, respectively and exhibited tumor-specific features. The hypermethylation of all genes was well correlated with changes in expression. The methylation level and frequency of MAL were significantly higher in smokers and in patients without EGFR mutations. Through accurate measurement of methylation levels using pyrosequencing, hypomethylation of LINE-1 and hypermethylation of SLIT2, MAL and IGFBP7 were frequently detected in NSCLCs and associated with various clinical features. Analysis of the methylation profiles of these genes may, therefore, provide novel opportunities for the therapy of NSCLCs.

Abstract 829: Down-regulation of insulin-like growth factor-binding protein 7 in cisplatin-resistant non-small cell lung cancer

Abstract Cisplatin is an effective anticancer drug used to treat many types of cancer, including non-small cell lung carcinoma (NSCLCs), but development of resistance is the primary impediment in cancer treatment. Insulin-like growth factor-binding protein 7 (IGFBP7) is a secreted tumor suppressor that is inactivated in human lung cancer. IGFBP7 is known to alter sensitivity to interferon-based anticancer therapy, and here, we examined loss of IGFBP7 as a potential contributor to chemo-resistance to cisplatin. The transcriptional level of IGFBP7 was decreased in cisplatin-resistant human cancer cell lines and NSCLC xenografts. IGFBP7 knock-down increased cellular resistance to cisplatin and increased the level of mitogen-activated protein kinase phosphatases (MKP) 3 levels. The expression of MKP3 increased in a cisplatin-resistant NSCLC cell line and lung xenografts. MKP3 knock-down increased IGFBP7 level, indicating that MKP3 regulates IGFBP7. These findings suggest a novel molecular mechanism responsible for the tumor suppressive function of IGFBP7 in cisplatin-resistant human lung cancer and could lead to the development of IGFBP7 as a cisplatin-sensitizing agent. 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 829. doi:1538-7445.AM2012-829

Downregulation of insulin-like growth factor-binding protein 7 in cisplatin-resistant non-small cell lung cancer

Cisplatin is an effective anticancer drug used to treat many types of cancer, including non-small cell lung carcinoma (NSCLCs), but development of resistance is the primary impediment in cancer treatment. Insulin-like growth factor-binding protein 7 (IGFBP7) is a secreted tumor suppressor that is inactivated in human lung cancer. IGFBP7 is known to alter sensitivity to interferon-based anticancer therapy, and here, we examined loss of IGFBP7 as a potential contributor to chemo-resistance to cisplatin. The transcriptional level of IGFBP7 was decreased in cisplatin-resistant human cancer cell lines and NSCLC xenografts. IGFBP7 knock-down increased cellular resistance to cisplatin and increased the level of mitogen-activated protein kinase phosphatases (MKP) 3 levels. The expression of MKP3 increased in a cisplatin-resistant NSCLC cell line and lung xenografts. MKP3 knock-down increased IGFBP7 level, indicating that MKP3 regulates IGFBP7. These findings suggest a novel molecular mechanism responsible for the tumor suppressive function of IGFBP7 in cisplatin-resistant human lung cancer and could lead to the development of IGFBP7 as a cisplatin-sensitizing agent.

Insulin like growth factor binding protein-7 reduces growth of human breast cancer cells and xenografted tumors

Previously, we have shown that insulin-like growth factor binding protein-7 (IGFBP-7) expression is inversely correlated with disease progression in breast cancer and is associated with poor outcome. To further investigate the role of IGFBP-7 in the growth and metastatic behavior of breast cancer, primary breast tumors and metastatic tumors derived from the same patients were analyzed for IGFBP-7 expression. Immunohistochemical analysis revealed that IGFBP-7 is downregulated in half of the human metastatic breast tumors tested. IGFBP-7 has been linked to suppression of oncogenic pathways and can directly restore cellular senescence in melanomas, leading to their regression. It is possible that breast tumors with metastatic potential have escaped from IGFBP-7-induced suppression by its down-regulation. Twenty-two human primary breast tumor specimens were transplanted into human-bone NOD/SCID mice. One of the two triple negative primary breast tumors was serially xenotransplanted more than five times. Each serial transplant resulted in increased tumor take and rate of growth. Expression of IGFBP-7 was downregulated upon each serial implantation. To investigate the role of IGFBP-7 in breast tumor suppression, IGFBP-7 was overexpressed in the triple negative MDA-MB-468 human breast cancer line by stable transfection of a pSec-tag2-IGFBP-7 vector. The parental MDA-MB-468 breast cancer cells expressed extremely low levels of endogenous IGFBP-7. The production of IGFBP-7 protein by the MDA-MB-468 cells stably transfected with IGFBP-7 was confirmed by immunoblotting with anti-IGFBP-7 antibody. Ectopic overexpression of IGFBP-7 significantly reduced the growth of the IGFBP-7 transfected MDA-MB-468 cells compared to the parental MDA-MB-468 cells. We also assessed the role of IGFBP-7 on cell migration, a key determinant of malignant progression and metastasis. When parental MDA-MB-468 cells were treated with various amounts of conditioned medium derived from the IGFBP-7 overexpressing cell line, a significant difference in cell migration rate was observed between untreated and treated cells. IGFBP-7 strongly suppressed the phosphorylation of the mitogen-activated protein kinases (MAPK) ERK-1/2, suggesting that IGFBP-7 mediates its anti-proliferative effects through negative feedback signaling. Levels of phospho-ERK-1/2 were higher in the parental MDA-MB-468 than in IGFBP-7-expressing cells derived from it. When injected subcutaneously into NOD/SCID mice, the increased expression of IGFBP-7 in the MDA-MB-468 transfected cells reduced the rate of tumor growth in comparison to the parental MDA-MB-468 controls. These results suggest that the growth of breast cancer could be prevented by the forced expression of IGFBP-7 protein.

Gene Expression Profiling Identifies IGFBP7 as a BAALC Co-Expressed Gene with a Functional Role in Acute Leukemia.

Abstract 2629▪▪This icon denotes an abstract that is clinically relevant.Poster Board II-605The human gene BAALC (Brain And Acute Leukemia, Cytoplasmic) is a molecular marker of immature hematopoietic cells. In normal hematopoiesis, BAALC is highly expressed in CD34 positive progenitors and down-regulated with cell differentiation. Moreover, high mRNA expression levels of BAALC have shown to be an adverse risk factor and were associated with chemotherapy resistance in adult patients with cytogenetically normal acute myeloid leukemia (CN-AML) and acute T-lymphoblastic leukemia (T-ALL). While the prognostic value of BAALC expression has been validated, the function of BAALC in leukemia remains unknown. The aim of this study was to identify genes correlated with BAALC to gain further insights into the underlying pathways of the BAALC-associated chemotherapy resistant leukemic phenotype. Therefore, we generated gene expression profiles (GEP) using pre-treatment T-ALL samples (n=86) that differentiated high versus low BAALC expressers (HG-U133 Plus 2.0, Affymetrix). Samples were divided into quartiles (Q1-Q4) according to BAALC expression and QB1-3 was defined as low BAALC and QB4 as high BAALC. Differentially expressed genes between QB1–3 and QB4 were defined by a minimum expression change of three-fold (P'0.05). In order to identify BAALC-associated genes common to T-ALL, CN-AML and normal hematopoiesis we compared these BAALC-derived GEP in T-ALL with BAALC-associated profiles of CN-AML (Langer C et al, Blood 2008) and of normal CD34 positive progenitors (Komor et al, Stem Cells 2005). Only four genes (CD34, CD133, NPR3, IGFBP7) were common to the BAALC-associated GEP of all three entities (T-ALL, CN-AML, and CD34 positive progenitors). Of these genes, the human gene Insulin-like Growth Factor Binding Protein 7 (IGFBP7) was further investigated as the Insulin-like growth factor signaling plays an important role in various human cancers. Next we determined IGFBP7 expression levels by real-time RT-PCR in an independent cohort of 219 adults with newly diagnosed T-ALL, registered within the German Multicenter Acute Lymphoblastic Leukemia Study 06/99 and 07/03 protocols. In T-ALL samples, IGFBP7 showed a heterogeneous expression pattern compared to normal bone marrow (expression range: 0–79.1 vs 0.6–2.3). For correlation of IGFBP7 expression with clinical and molecular features, samples were divided into quartile groups according to IGFBP7 expression and defined as low IGFBP7 with expression levels in QI1 to QI3 (n=158) and as high IGFBP7 with expression levels in QI4 (n=53). High expression levels of IGFBP7 were associated with an immature phenotype of early T-ALL (62% of patients in QI4 showed an early T-ALL immunophenotype as compared to only 15% in QI1–3; P<0.001), high cell surface expression of CD34 (mean: 36% vs 15%; P<0.001) and CD33 (mean: 24% vs 3%; P<0.001). Moreover, high IGFBP7 expression significantly predicted a higher frequency of refractory disease (11% vs 3%; P=0.03) and an inferior overall survival (OS; 4-year OS: IGFBP7 QI4: 42% vs IGFBP7 QI1–3: 55%; P=0.03). In vitro, studies revealed that treatment with rIGFBP7 significantly inhibited proliferation of KG1a leukemic cells (65% reduction of proliferation compared to untreated cells; P<0.001) determined by Cell Proliferation Reagent WST-1. Moreover, a 26% reduction (P<0.001) of the DNA synthesis phase was detected by BrdU incorporation after rIGFBP7 treatment of KG1a cells. Taken together, we identified IGFBP7 as a lineage-independent, BAALC co-expressed gene involved in leukemia. High IGFBP7 expression was associated with stem cell features and treatment failure in T-ALL. In contrast to BAALC which likely represents only a surrogate marker of treatment failure in acute leukemias, IGFBP7 might play functional role and contribute to the molecular basis of BAALC-associated drug resistance. As rIGFBP7 inhibits proliferation of leukemic cells, secreted IGFBP7 protein may therefore promote resistance to cell cycle dependent cytostatic drugs targeting highly proliferate blast cells. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Decreased expression of insulin-like growth factor binding protein 7 in human colorectal carcinoma is related to DNA methylation

Insulin-like growth factor binding protein 7 (IGFBP-7) is considered a tumor suppressor in various cancers, but its role in colorectal cancer (CRC) is still uncertain. The aims of this study were to analyze the IGFBP-7 expression, and explore the mechanism responsible for the inactivation of IGFBP-7 in CRC. mRNA expression was studied by RT-PCR and Northern blot analysis of cultured cells. Methylation status was analyzed by treatment with 5-aza-2'-deoxycytidine followed by sequencing of PCR products of sodium bisulfite-treated genomic DNA. IGFBP-7 protein expression was evaluated by immunohistochemistry (IHC) on tissue microarrays. mRNA expression was lost in six out of eight CRC cell lines as compared to normal colon cells. DNA methylation was found in the region of exon 1 and intron 1 of IGFBP-7. In tumor tissue, 107 out of 279 samples showed a negative expression of IGFBP-7 by IHC, which was significantly associated with poor prognosis. The analysis of 37 paired cancerous and normal mucosa samples confirmed the downregulation in the tumors, but revealed variable basal expression levels of IGFBP-7 in normal mucosal samples. DNA methylation is a mechanism responsible for IGFBP-7 gene silencing providing a target for therapeutic intervention of this tumor suppressor gene.