Transcriptomic Analysis of Extracellular Vesicles in the Search for Novel Plasma and Thrombus Biomarkers of Ischemic Stroke Etiologies.

Accurate etiologic diagnosis provides an appropriate secondary prevention and better prognosis in ischemic stroke (IS) patients; still, 45% of IS are cryptogenic, urging us to enhance diagnostic precision. We have studied the transcriptomic content of plasma extracellular vesicles (EVs) (n = 21) to identify potential biomarkers of IS etiologies. The proteins encoded by the selected genes were measured in the sera of IS patients (n = 114) and in hypertensive patients with (n = 78) and without atrial fibrillation (AF) (n = 20). IGFBP-2, the most promising candidate, was studied using immunohistochemistry in the IS thrombi (n = 23) and atrium of AF patients (n = 13). In vitro, the IGFBP-2 blockade was analyzed using thromboelastometry and endothelial cell cultures. We identified 745 differentially expressed genes among EVs of cardioembolic, atherothrombotic, and ESUS groups. From these, IGFBP-2 (cutoff > 247.6 ng/mL) emerged as a potential circulating biomarker of embolic IS [OR = 8.70 (1.84-41.13) p = 0.003], which was increased in patients with AF vs. controls (p < 0.001) and was augmented in cardioembolic vs. atherothrombotic thrombi (p < 0.01). Ex vivo, the blockage of IGFBP-2 reduced clot firmness (p < 0.01) and lysis time (p < 0.001) and in vitro, diminished endothelial permeability (p < 0.05) and transmigration (p = 0.06). IGFBP-2 could be a biomarker of embolic IS and a new therapeutic target involved in clot formation and endothelial dysfunction.

The effects of IGF-1 and IGFBP-2 treatments on the atherosclerosis in the aorta and the coronary arteries of the high cholesterol diet-fed rabbits.

Several studies have demonstrated suppression of aortic atherosclerosis by insulin like growth factor-1 (IGF-1) in hypercholesterolemic rabbits. Though a recent study has reported that IGF-1 exerts anti-atherogenic effects in coronary arteries, the mechanisms of IGF-1 in coronary arteries need to be further verified. Studies about insulin like growth factor binding protein-2 (IGFBP-2) in atherosclerosis are rarely. The objective of this study is to examine the effects of IGF-1 and IGFBP-2 on the atherosclerosis development in the aorta and coronary arteries of the high-cholesterol diet (HCD)-fed rabbits. New Zealand white rabbits were fed either normal chow (n = 5) or a diet containing 1.0 % cholesterol (n = 18) for 12 weeks. Cholesterol-fed rabbits were given IGF-1 or IGFBP-2 or saline intravenously (each n = 6) for 10 weeks. The results revealed that IGF-1 decreased total cholesterol (TC) and low-density lipoprotein (LDL) levels (p < 0.05), whereas IGFBP-2 did not. IGF-1 significantly attenuated atherosclerotic lesions and reduced accumulated macrophages within the coronary artery plaques, whereas IGFBP-2 deteriorated these changes. Moreover, IGF-1 reduced serum platelet-activating factor acetylhydrolase levels, C reactive protein (CRP), and inhibited the protein expression levels of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). IGFBP-2 elevated serum 8-hydroxy-2'-deoxyguanosine levels, CRP, and promoted the protein expression levels of TNF-α and IL-6. In conclusion, IGF-1 can substantially suppress plaque formation in coronary arteries with a marked inhibition of macrophage accumulation likely via its anti-inflammatory properties, whereas IGFBP-2 plays an opposite effect on atherosclerosis. The present study highlighted a theoretical basis for pharmacological treatment of atherosclerosis.

Plasma IGFBP-2 levels reveal heterogeneity in hepatic fat content in adults with excess visceral adiposity.

Lay summary: Obesity is frequently accompanied by a fatty liver. However, some individuals with high abdominal fat levels nevertheless have low levels of liver fat. Reasons for such discordant phenotypes are unclear. In this paper, we report that among asymptomatic individuals with high levels of visceral fat, low concentrations of IGFBP-2 in the circulation were associated with significantly higher hepatic fat content compared to those with high IGFBP-2 levels. We conclude that quantification of plasma IGFBP-2 concentrations may be useful to identify the early risk for liver fat accumulation in apparently healthy individuals without cardiovascular symptoms. Aim/hypothesis: Although excess visceral adiposity (VAT) is generally associated with increased liver fat (LF), recent evidence has revealed heterogeneity in LF content among adults with visceral obesity, potentially contributing to specific differences in cardiometabolic outcomes. Reasons for such discordant VAT-LF phenotypes are largely unknown. The present study aimed at assessing whether circulating levels of insulin growth-factor binding protein-2 (IGFBP-2) could be a useful biomarker in the identification of heterogenous and discordant VAT-LF phenotypes. Methods: A sample of 308 middle-aged Caucasian apparently healthy men and women without cardiovascular symptoms were studied for the present cross-sectional analyses. Fasting plasma glucose and lipid levels were assessed and an oral glucose tolerance test was performed. Hepatic fat fraction (HFF) was measured using magnetic resonance spectroscopy whereas VAT was assessed by magnetic resonance imaging. Plasma IGFBP-2 levels were quantified by ELISA. Participants were then classified on the basis of median VAT (81 mL) and IGFBP-2 levels (233 ng/mL). Results: Individuals with high levels of VAT were characterized by higher waist circumference, lower insulin sensitivity, as well as by higher plasma triglyceride and lower HDL-cholesterol levels. Plasma IGFBP-2 levels were inversely correlated with HFF (r = -0.39, p < 0.0001). Among men and women with high levels of VAT, those with low levels of IGFBP-2 had significantly higher HFF (7.5 ± 0.7%), compared to participants with high IGFBP-2 concentrations (3.2 ± 0.5%, p < 0.0001). Conclusion: In the presence of excess VAT, high IGFBP-2 concentrations are associated with low levels of LF. Although additional studies will be necessary to establish causality and further clarify the clinical implications of these observations, these findings are concordant with a novel function of IGFBP-2 in modulating susceptibility to non-alcoholic fatty liver disease (NAFLD) in the presence of visceral obesity.

Associations of insulin-like growth factor binding protein-2 with metabolic profile and hepatic fat deposition in asymptomatic men and women.

Low circulating levels of insulin-like growth-factor binding protein-2 (IGFBP-2) have been associated with increased adiposity and metabolic alterations such as insulin resistance, dyslipidemia, and nonalcoholic fatty liver disease in individuals with obesity. However, whether IGFBP-2 affects energy metabolism in the early stages of these disorders remains unclear. Herein, we hypothesized that plasma IGFBP-2 concentrations are inversely associated with early liver fat accumulation and alterations in lipid and glucose homeostasis in apparently healthy and asymptomatic men and women. Three hundred thirty-three middle-aged Caucasian men and women apparently healthy and without cardiovascular symptoms were enrolled for a cross-sectional cardiometabolic imaging study. Individuals with BMI ≥ 40 kg/m2, cardiovascular disease, dyslipidemia, hypertension, and diabetes were excluded. Fasting glucose and lipid profiles were measured and an oral glucose tolerance test was performed. Liver fat content was assessed by magnetic resonance spectroscopy. Volume of visceral adipose tissue (VAT) was evaluated by magnetic resonance imaging. Plasma IGFBP-2 levels were quantified by ELISA. Participants with low IGFBP-2 levels were characterized by a higher body fat mass (P < 0.0001), insulin resistance (P < 0.0001), higher plasma triglyceride (TG) (P < 0.0001), and lower HDL-cholesterol levels (P < 0.0001) in a sex-independent manner. IGFBP-2 levels were inversely correlated with hepatic fat fraction in both men (r = -0.36, P < 0.0001) and women (r = -0.40, P < 0.0001). IGFBP-2 concentrations were negatively associated with hepatic fat fraction independently of age and VAT in both men (R2 = 0.23, P = 0.012) and women (R2 = 0.27, P = 0.028). In conclusion, our findings show that even in asymptomatic, apparently healthy individuals, low IGFBP-2 levels are associated with a more deteriorated cardiometabolic risk profile and with a high hepatic fat content in a VAT-independent manner. However, IGFBP-2 does not appear to influence the established sexual dimorphism observed for metabolic variables and hepatic fat fraction. Additional studies are required to better understand the relationships between IGFBP-2 and liver fat content.NEW & NOTEWORTHY Faced with a paucity of reliable clinical etiologic markers for fatty liver, this research article demonstrates, for the first time, that low blood levels of the protein IGFBP-2 are associated with a more deteriorated cardiometabolic risk profile and with a high hepatic fat content independently of visceral fat volume and sex, even in asymptomatic, apparently healthy individuals.

IGFBP2 expressing midlobular hepatocytes preferentially contribute to liver homeostasis and regeneration.

Although midlobular hepatocytes in zone 2 are a recently identified cellular source for liver homeostasis and regeneration, these cells have not been exclusively fate mapped. We generated an Igfbp2-CreER knockin strain that specifically labels midlobular hepatocytes. During homeostasis over 1 year, zone 2 hepatocytes increased in abundance from occupying 21%-41% of the lobular area. After either pericentral injury with carbon tetrachloride or periportal injury with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), IGFBP2+ cells replenished lost hepatocytes in zones 3 and 1, respectively. IGFBP2+ cells also preferentially contributed to regeneration after 70% partial hepatectomy, as well as liver growth during pregnancy. Because IGFBP2 labeling increased substantially with fasting, we used single nuclear transcriptomics to explore zonation as a function of nutrition, revealing that the zonal division of labor shifts dramatically with fasting. These studies demonstrate the contribution of IGFBP2-labeled zone 2 hepatocytes to liver homeostasis and regeneration.

High baseline tumor burden-associated macrophages promote an immunosuppressive microenvironment and reduce the efficacy of immune checkpoint inhibitors through the IGFBP2-STAT3-PD-L1 pathway.

BACKGROUND: Several clinical studies have uncovered a negative correlation between baseline tumor burden and the efficacy of immune checkpoint inhibitor (ICI) treatment. This study aimed to uncover the specific mechanisms underlying the difference in sensitivity to ICI treatment between tumors with high (HTB) and low (LTB) tumor burden. METHODS: For in vivo studies, several mouse models of subcutaneous tumors were established, and transcriptome sequencing, immunohistochemistry, and flow cytometry assays were used to detect the immune status in these subcutaneous tumors. For in vitro experiments, co-culture models, cytokine antibody arrays, western blotting, flow cytometry, and enzyme-linked immunosorbent assays were used to explore the underlying molecular mechanisms RESULTS: We found that MC38 or B16 subcutaneous tumors from the HTB group did not show any response to anti-programmed cell death protein-1 (PD-1) therapy. Through flow cytometry assays, we found that the infiltration with CD8+ T cells was significantly decreased whereas M2-like macrophages were enriched in subcutaneous tumors of HTB groups compared with those of LTB group. These changes were not affected by the initial number of injected tumor cells or tumor age, nor could they be reversed by surgical tumor reduction. Intraperitoneal colony-stimulating factor 1 receptor (CSF-1R) inhibitor PLX3397 injection at different time points of tumor growth only had an effect when administered in the early tumor stage to maintain the "heat" of the tumor microenvironment during the process of tumor growth, thereby achieving a response to ICI treatment when the tumor grew to a large size. Mechanistically, we found that insulin-like growth factor binding protein 2 (IGFBP2) expression levels were significantly elevated in HTB tumor tissues. IGFBP2 promoted the programmed death-ligand 1 (PD-L1) expression in M2-like macrophages by activating signal transducer and activator of transcription 3 (STAT3), and PD-L1+ M2-like macrophages exerted an immunosuppressive effect by inhibiting the proliferation and activation of CD8+ T cells in a PD-L1-dependent fashion. CONCLUSIONS: This study suggested that the low efficacy of ICI treatment in HTB tumors is mainly attributed to the intratumoral accumulation of PD-L1+ M2-like macrophages via the IGFBP2-STAT3-PD-L1 signaling pathway and their substantial inhibitory effects on T cell proliferation and activation.

circSMARCA5 Is an Upstream Regulator of the Expression of miR-126-3p, miR-515-5p, and Their mRNA Targets, Insulin-like Growth Factor Binding Protein 2 (IGFBP2) and NRAS Proto-Oncogene, GTPase (NRAS) in Glioblastoma.

The involvement of non-coding RNAs (ncRNAs) in glioblastoma multiforme (GBM) pathogenesis and progression has been ascertained but their cross-talk within GBM cells remains elusive. We previously demonstrated the role of circSMARCA5 as a tumor suppressor (TS) in GBM. In this paper, we explore the involvement of circSMARCA5 in the control of microRNA (miRNA) expression in GBM. By using TaqMan® low-density arrays, the expression of 748 miRNAs was assayed in U87MG overexpressing circSMARCA5. Differentially expressed (DE) miRNAs were validated through single TaqMan® assays in: (i) U87MG overexpressing circSMARCA5; (ii) four additional GBM cell lines (A172; CAS-1; SNB-19; U251MG); (iii) thirty-eight GBM biopsies; (iv) twenty biopsies of unaffected brain parenchyma (UC). Validated targets of DE miRNAs were selected from the databases TarBase and miRTarbase, and the literature; their expression was inferred from the GBM TCGA dataset. Expression was assayed in U87MG overexpressing circSMARCA5, GBM cell lines, and biopsies through real-time PCR. TS miRNAs 126-3p and 515-5p were upregulated following circSMARCA5 overexpression in U87MG and their expression was positively correlated with that of circSMARCA5 (r-values = 0.49 and 0.50, p-values = 9 × 10-5 and 7 × 10-5, respectively) in GBM biopsies. Among targets, IGFBP2 (target of miR-126-3p) and NRAS (target of miR-515-5p) mRNAs were positively correlated (r-value = 0.46, p-value = 0.00027), while their expression was negatively correlated with that of circSMARCA5 (r-values = -0.58 and -0.30, p-values = 0 and 0.019, respectively), miR-126-3p (r-value = -0.36, p-value = 0.0066), and miR-515-5p (r-value = -0.34, p-value = 0.010), respectively. Our data identified a new GBM subnetwork controlled by circSMARCA5, which regulates downstream miRNAs 126-3p and 515-5p, and their mRNA targets IGFBP2 and NRAS.

Associations between insulin-like growth factor binding protein-2 and lipoprotein kinetics in men.

Low circulating concentrations of insulin-like growth factor binding protein-2 (IGFBP-2) have been associated with dyslipidemia, notably with high triglyceride (TG) levels. However, the determinants by which IGFBP-2 influences lipoprotein metabolism, especially that of TG-rich lipoproteins (TRLs), are poorly understood. Here, we aimed to assess the relationships between IGFBP-2 levels and lipoprotein production and catabolism in human subjects. Fasting IGFBP-2 concentrations were measured in the plasma of 219 men pooled from previous lipoprotein kinetics studies. We analyzed production rate and fractional catabolic rates of TRLapoB-48, and LDL-, IDL-, and VLDLapoB-100 by multicompartmental modeling of l-[5,5,5-D3] leucine enrichment data after a 12 h primed constant infusion in individuals kept in a constant nutritional steady state. Subjects had an average BMI of 30 kg/m2, plasma IGFBP-2 levels of 157 ng/ml, and TG of 2.2 mmol/l. After adjustments for age and BMI, IGFBP-2 levels were negatively associated with plasma TG (r = -0.29; P < 0.0001) and positively associated with HDL-cholesterol (r = 0.26; P < 0.0001). In addition, IGFBP-2 levels were positively associated with the fractional catabolic rate of VLDLapoB-100 (r = 0.20; P < 0.01) and IDLapoB-100 (r = 0.19; P < 0.05) and inversely with the production rate of TRLapoB-48 (r = -0.28; P < 0.001). These correlations remained statistically significant after adjustments for age, BMI, and the amount of fat given during the tracer infusion. These findings show that the association between low plasma IGFBP-2 and high TG concentrations could be due to both an impaired clearance of apoB-100-containing VLDL and IDL particles and an increased production of apoB-48-containing chylomicrons. Additional studies are necessary to investigate whether and how IGFBP-2 directly impacts the kinetics of TRL.

Circulating insulin-like growth factor system adaptations in hibernating brown bears indicate increased tissue IGF availability.

Brown bears conserve muscle and bone mass during 6 mo of inactive hibernation. The molecular mechanisms underlying hibernation physiology may have translational relevance for human therapeutics. We hypothesize that protective mechanisms involve increased tissue availability of insulin-like growth factors (IGFs). In subadult Scandinavian brown bears, we observed that mean plasma IGF-1 and IGF-2 levels during hibernation were reduced to 36 ± 10% and 56 ± 15%, respectively, compared with the active state (n = 12). Western ligand blotting identified IGF-binding protein (IGFBP)-3 as the major IGFBP in the active state, whereas IGFBP-2 was codominant during hibernation. Acid labile subunit (ALS) levels in hibernation were reduced to 41±16% compared with the active state (n = 6). Analysis of available grizzly bear RNA sequencing data revealed unaltered liver mRNA IGF-1, IGFBP-2, and IGFBP-3 levels, whereas ALS levels were significantly reduced during hibernation (n = 6). Reduced ALS synthesis and circulating levels during hibernation should prompt a shift from ternary IGF/IGFBP/ALS to smaller binary IGF/IGFBP complexes, thereby increasing IGF tissue availability. Indeed, size-exclusion chromatography of bear plasma demonstrated a shift to lower molecular weight IGF-containing complexes in the hibernating versus the active state. Furthermore, we note that the major IGF-2 mRNA isoform expressed in livers in both Scandinavian brown bears and grizzly bears was an alternative splice variant in which Ser29 is replaced with a tetrapeptide possessing a positively charged Arg residue. Homology modeling of the bear IGF-2/IGFBP-2 complex showed the tetrapeptide in proximity to the heparin-binding domain involved in bone-specific targeting of this complex. In conclusion, this study provides data which suggest that increased IGF tissue availability combined with tissue-specific targeting contribute to tissue preservation in hibernating bears.NEW & NOTEWORTHY Brown bears shift from circulating ternary IGF/IGFBP/ALS complexes in the active state to binary IGF/IGFBP complexes during hibernation, indicating increased tissue IGF-bioactivity. Furthermore, brown bears use a splice variant of IGF-2, suggesting increased bone-specific targeting of IGF anabolic signaling.

Plasma Proteomic Signature of Cellular Senescence and Markers of Biological Aging Among Postmenopausal Women.

We aimed to investigate the association of circulatory senescence-associated secretory phenotypes (SASPs) produced by senescent cells with chronological and menopausal age in women aged 45 years or more. The proteomic profiles for 32 SASP factors of plasma samples were measured in 76 healthy postmenopausal women aged 46-82 years from the Korean Genome and Epidemiology Study Cardiovascular Disease Association Study (KoGES-CAVAS). We assessed the association between the SASP factors and aging indicators (chronological age, menopausal age, and years since menopause) using single- and multiprotein models. First, we composed a profile of proteins associated with chronological age, menopausal age, and years since menopause. In a single-protein model, three proteins (growth differentiation factor 15 [GDF15], insulin-like growth factor binding protein-2 [IGFBP-2], and tumor necrosis factor-α [TNF-α]) are positively associated with chronological age. Menopausal age and years since menopause are interrelated with interleukin-8 (IL-8). The direction of association between menopausal age and monocyte chemoattractant protein-1 (MCP-1) was only negative, and IGFBP-2 and TNF-α were significant in all three aging factors. We also constructed parsimonious multiprotein models to confirm the association of the proteomic signature for aging after adjusting for covariates and the combination of proteomic signature of 13 proteins (GDF15, interferon-γ [IFN-γ], IGFBP-2, IGFBP-7, IL-15, IL-1ß, IL-17A, IL-8, MCP-1, tissue inhibitors of metalloproteinase-2 [TIMP-2], TNF-α, vascular endothelial growth factor-A [VEGF-A], and interferon-inducible protein 10 [IP-10]) appear to be associated with chronological age and menopausal state of individuals. Thus, by observing association between the selected SASPs and age-related markers among healthy postmenopausal women, we examine how menopause in women relates to proteomic indicators of aging and highlight the potential use of SASP factors as a marker to reflect the state of biological aging attributed by ovarian senescence.

IGF-dependent dynamic modulation of a protease cleavage site in the intrinsically disordered linker domain of human IGFBP2.

Functional regulation via conformational dynamics is well known in structured proteins but less well characterized in intrinsically disordered proteins and their complexes. Using NMR spectroscopy, we have identified a dynamic regulatory mechanism in the human insulin-like growth factor (IGF) system involving the central, intrinsically disordered linker domain of human IGF-binding protein-2 (hIGFBP2). The bioavailability of IGFs is regulated by the proteolysis of IGF-binding proteins. In the case of hIGFBP2, the linker domain (L-hIGFBP2) retains its intrinsic disorder upon binding IGF-1, but its dynamics are significantly altered, both in the IGF binding region and distantly located protease cleavage sites. The increase in flexibility of the linker domain upon IGF-1 binding may explain the IGF-dependent modulation of proteolysis of IGFBP2 in this domain. As IGF homeostasis is important for cell growth and function, and its dysregulation is a key contributor to several cancers, our findings open up new avenues for the design of IGFBP analogs inhibiting IGF-dependent tumors.

ADAMTS9-AS2 Promotes Angiogenesis of Brain Microvascular Endothelial Cells Through Regulating miR-185-5p/IGFBP-2 Axis in Ischemic Stroke.

Ischemic stroke is a common disease threatening human health. ADAMTS9-AS2 is a lncRNA that has been widely studied in tumors, but not in ischemic stroke. The purpose of this study was to investigate the role and potential molecular mechanism of ADAMTS9-AS2 in endothelial cell function after ischemic stroke in vivo and in vitro. The results showed that ADAMTS9-AS2 was decreased in the plasma of acute ischemic stroke (AIS) patients and in the brain tissue and plasma of MCAO mice, and the low expression of ADAMTS9-AS2 was associated with the increase in infarct size. Besides, compared with the control group, MCAO treatment slightly promoted angiogenesis, which was enhanced by the overexpression of ADAMTS9-AS2. Molecular mechanism results indicated that ADAMTS9-AS2 and miR-185-5p affected the pathological process of ischemic stroke through ceRNA mechanism, and IGFBP-2 was a downstream target gene of miR-185-5p. These findings demonstrated that ADAMTS9-AS2 promoted angiogenesis through regulating miR-185-5p/IGFBP-2 axis, suggesting that ADAMTS9-AS2 may be a potential marker for the diagnosis and treatment of neurological diseases.

Insulin-like Growth Factor Binding Protein-2 (IGFBP2) Is a Key Molecule in the MACC1-Mediated Platelet Communication and Metastasis of Colorectal Cancer Cells.

Tumor cell crosstalk with platelets and, subsequently, their activation are key steps in hematogenous tumor metastasis. MACC1 is an oncogene involved in molecular pathogenesis of colorectal cancer (CRC) and other solid tumor entities, mediating motility and metastasis, making MACC1 an accepted prognostic biomarker. However, the impact of MACC1 on platelet activation has not yet been addressed. Here, we investigated the activation of platelets by human CRC cells upon MACC1 modulation, indicated by platelet aggregation and granule release. These approaches led to the identification of insulin-like growth factor binding protein-2 (IGFBP2) as a functional downstream molecule of MACC1, affecting communication with platelets. This was confirmed by an shRNA-mediated IGFBP2 knockdown, while maintaining MACC1 activity. Although IGFBP2 displayed an attenuated platelet activation potential, obviously by scavenging IGF-I as a platelet costimulatory mediator, the MACC1/IGFBP2 axis did not affect the thrombin formation potential of the cells. Furthermore, the IGFBP2/MACC1-driven cell migration and invasiveness was further accelerated by platelets. The key role of IGFBP2 for the metastatic spread in vivo was confirmed in a xenograft mouse model. Data provide evidence for IGFBP2 as a downstream functional component of MACC1-driven metastasis, linking these two accepted oncogenic biomarkers for the first time in a platelet context.

Anticancer properties of dried-pericarp water extracts of Camellia japonica L. fermented with Aspergillus oryzae through regulation of IGFBP-2/mTOR pathway.

This study aimed to investigate the anticancer activity of dried-pericarp water extract of fermented C. japonicus (CJ). The dried-pericarp water extracts of CJ were fermented using Aspergillus oryzae and Saccharomyces cerevisiae at 30 °C and 35 °C. The anticancer activities of both water extracts fermented at 30 °C and 35 °C using A. oryzae against FaDu cells were remarkably changed compared with unfermented dried-pericarp water extract of CJ, which has no anticancer activity. Cleaved-PARP, caspase 3, and apoptotic cells stained with annexin V/PI were significantly increased by treatment with A. oryzae extracts fermented at 30 °C. The insulin-like growth factor-binding protein 2 (IGFBP-2) protein level and mTOR phosphorylation by A. oryzae fermented extracts (AOFE) were dramatically reduced, and the expression levels of IGFBP-2 and phosphorylated mTOR were significantly increased depending on the glucose concentrations in FaDu cells. These results suggested that the cell viabilities in AOFE were restored as the glucose concentrations increased. Furthermore, it was confirmed LC/MS/MS that the content of gallic acid was increased by fermentation of Aspergillus oryzae (5.596 ± 0.1746 µg/mg) compared to the unfermented extract (1.620 ± 0.0432 µg/mg). Based on these results, the anticancer effect of AOFE was achieved through inhibition of the IGFBP-2/mTOR signaling pathway. These results suggest that AOFE may be a potential treatment for head and neck cancer.

SNAIL2 contributes to tumorigenicity and chemotherapy resistance in pancreatic cancer by regulating IGFBP2.

Pancreatic cancer has an extremely poor prognosis because of its resistance to conventional therapies. Cancer stem cell (CSC)-targeted therapy is considered a promising approach for this disease. Epithelial-mesenchymal transition-inducing transcription factors (EMT-TFs) contribute to CSC properties in some solid tumors; however, this mechanism has not been fully elucidated in pancreatic cancer. Zinc finger protein, SNAIL2 (also known as SLUG), is a member of the SNAIL superfamily of EMT-TFs and is commonly overexpressed in pancreatic cancer. Patients exhibiting high SNAIL2 expression have a poor prognosis. In this study, we showed that the suppression of SNAIL2 expression using RNA interference decreased tumorigenicity in vitro (sphere formation assay) and in vivo (xenograft assay) in 2 pancreatic cancer cell lines, KLM1 and KMP5. In addition, SNAIL2 suppression resulted in increased sensitivity to gemcitabine and reduced the expression of CD44, a pancreatic CSC marker. Moreover, experiments on tumor spheroids established from surgically resected pancreatic cancer tissues yielded similar results. A microarray analysis revealed that the mechanism was mediated by insulin-like growth factor (IGF) binding protein 2. These results indicate that IGFBP2 regulated by SNAIL2 may represent an effective therapeutic target for pancreatic cancer.

Results of TRIO-14, a phase II, multicenter, randomized, placebo-controlled trial of carboplatin-paclitaxel versus carboplatin-paclitaxel-ganitumab in newly diagnosed epithelial ovarian cancer.

PURPOSE: Insulin-like growth factor (IGF) signaling is implicated in pathogenesis and chemotherapy resistance of epithelial ovarian cancer (EOC). We explored efficacy and safety of adding ganitumab, a monoclonal antibody targeting IGF-1R, to carboplatin/paclitaxel (CP) chemotherapy in patients with primary EOC. DESIGN: Patients were randomly assigned to receive CP/ganitumab (18 mg/kg q3w) or CP/placebo for 6 cycles followed by 6 cycles of single agent ganitumab/placebo maintenance therapy as front-line therapy. Primary endpoint was progression free survival. Secondary endpoints were time to progression and overall survival. Pretreatment samples were prospectively collected for retrospective biomarker analyses. RESULTS: 170 patients enrolled. 165 patients assessable for toxicity. Median PFS was 15.7 months with CP/ganitumab and 16.7 months with CP/placebo (HR 1.23; 95% CI 0.82-1.83, P = 0.313). All grade neutropenia (84.1% vs 71.4%), thrombocytopenia (75.3% vs 57.1%) and hyperglycemia (15.9% vs 2.6%) were more common in the ganitumab group compared to the placebo group. Ganitumab/placebo related serious adverse events were reported in 26.1% of the patients with ganitumab and in 6.5% with placebo. Non-progression related fatal events were more common with ganitumab (5 versus 2 patients). The ganitumab group experienced more dose delays which resulted in lower relative dose intensity of chemotherapy in the experimental group. In an exploratory model IGFBP2 expression was predictive of ganitumab response (treatment interaction; PFS, P = 0.03; OS, P = 0.01). CONCLUSION: Addition of ganitumab to CP chemotherapy in primary EOC did not improve PFS. Our results do not support further study of ganitumab in unselected EOC patients.

Immunization with a Plasmid DNA Vaccine Encoding the N-Terminus of Insulin-like Growth Factor Binding Protein-2 in Advanced Ovarian Cancer Leads to High-level Type I Immune Responses.

PURPOSE: Cancer vaccines targeting nonmutated proteins elicit limited type I T-cell responses and can generate regulatory and type II T cells. Class II epitopes that selectively elicit type I or type II cytokines can be identified in nonmutated cancer-associated proteins. In mice, a T-helper I (Th1) selective insulin-like growth factor binding protein-2 (IGFBP-2) N-terminus vaccine generated high levels of IFNγ secreting T cells, no regulatory T cells, and significant antitumor activity. We conducted a phase I trial of T-helper 1 selective IGFBP-2 vaccination in patients with advanced ovarian cancer. PATIENTS AND METHODS: Twenty-five patients were enrolled. The IGFBP-2 N-terminus plasmid-based vaccine was administered monthly for 3 months. Toxicity was graded by NCI criteria and antigen-specific T cells measured by IFNγ/IL10 ELISPOT. T-cell diversity and phenotype were assessed. RESULTS: The vaccine was well tolerated, with 99% of adverse events graded 1 or 2, and generated high levels of IGFBP-2 IFNγ secreting T cells in 50% of patients. Both Tbet+ CD4 (P = 0.04) and CD8 (P = 0.007) T cells were significantly increased in immunized patients. There was no increase in GATA3+ CD4 or CD8, IGFBP-2 IL10 secreting T cells, or regulatory T cells. A significant increase in T-cell clonality occurred in immunized patients (P = 0.03, pre- vs. post-vaccine) and studies showed the majority of patients developed epitope spreading within IGFBP-2 and/or to other antigens. Vaccine nonresponders were more likely to have preexistent IGFBP-2 specific immunity and demonstrated defects in CD4 T cells, upregulation of PD-1, and downregulation of genes associated with T-cell activation, after immunization. CONCLUSIONS: IGFBP-2 N-terminus Th1 selective vaccination safely induces type I T cells without evidence of regulatory responses.

HMGA2 contributes to vascular development and sprouting angiogenesis by promoting IGFBP2 production.

Angiogenesis is the process by which new blood vessels form from preexisting vessels and regulates the processes of embryonic development, wound healing and tumorigenesis. HMGA2 is involved in the occurrence of several cancers, but its biological role and the exact downstream genes involved in vascular development and sprouting angiogenesis remain largely unknown. Here, we first found that HMGA2 knockdown in zebrafish embryos resulted in defects of central artery formation. RNA sequencing revealed that IGFBP2 was significantly downregulated by interference with HMGA2, and IGFBP2 overexpression reversed the inhibition of brain vascular development caused by HMGA2 deficiency. In vitro, we further found that HMGA2 knockdown blocked the migration, tube formation and branching of HUVECs. Similarly, IGFBP2 protein overexpression attenuated the impairments induced by HMGA2 deficiency. Moreover, the promotion of angiogenesis by HMGA2 overexpression was verified in a Matrigel plug assay. We next found that HMGA2 bound directly to a region in the IGFBP2 promoter and positively regulated IGFBP2 expression. Interestingly, the mRNA expression levels of HMGA2 and IGFBP2 were increased significantly in the peripheral blood of hemangioma patients, indicating that overexpression of HMGA2 and IGFBP2 results in vessel formation, consistent with the results of the in vivo and in vitro experiments. In summary, our findings demonstrate that HMGA2 promotes central artery formation by modulating angiogenesis via IGFBP2 induction.

COL11A1 activates cancer-associated fibroblasts by modulating TGF-ß3 through the NF-κB/IGFBP2 axis in ovarian cancer cells.

Ovarian cancer has a unique tumor microenvironment (TME) that enables cancer-associated fibroblasts (CAFs) to interact with cellular and matrix constituents and influence tumor development and migration into the peritoneal cavity. Collagen type XI alpha 1 (COL11A1) is overexpressed in CAFs; therefore this study examines its role during CAF activation in epithelial ovarian cancer (EOC). Coculturing human ovarian fibroblasts (HOFs) with high COL11A1-expressing EOC cells or exposure to the conditioned medium of these cells prompted the expression of COL11A1 and CAF phenotypes. Conversely, coculturing HOFs with low COL11A1-expressing EOC cells or COL11A1-knockdown abrogated COL11A1 overexpression and secretion, in addition to CAF activation. Increased p-SP1 expression attributed to COL11A1-mediated extracellular signal-regulated kinase activation (ERK) induced p65 translocation into the nucleus and augmented its binding to the insulin-like growth factor binding protein 2 (IGFBP2) promoter, ultimately inducing TGF-ß3 activation. The CAF-cancer cell crosstalk triggered interleukin-6 release, which in turn promoted EOC cell proliferation and invasiveness. These in vitro results were confirmed by in vivo findings in a mouse model, showing that COL11A1 overexpression in EOC cells promoted tumor formation and CAF activation, which was inhibited by TGF-ß3 antibody. Human tumors with high TGF-ß3 levels showed elevated expression of COL11A1 and IGFBP2, which was associated with poor survival. Our findings suggest the possibility that anti-TGF-ß3 treatment strategy may be effective in targeting CAFs in COL11A1-positive ovarian tumors.

IGFBP-2 partly mediates the early metabolic improvements caused by bariatric surgery.

Insulin-like growth factor-binding protein (IGFBP)-2 is a circulating biomarker of cardiometabolic health. Here, we report that circulating IGFBP-2 concentrations robustly increase after different bariatric procedures in humans, reaching higher levels after biliopancreatic diversion with duodenal switch (BPD-DS) than after Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG). This increase is closely associated with insulin sensitization. In mice and rats, BPD-DS and RYGB operations also increase circulating IGFBP-2 levels, which are not affected by SG or caloric restriction. In mice, Igfbp2 deficiency significantly impairs surgery-induced loss in adiposity and early improvement in insulin sensitivity but does not affect long-term enhancement in glucose homeostasis. This study demonstrates that the modulation of circulating IGFBP-2 may play a role in the early improvement of insulin sensitivity and loss of adiposity brought about by bariatric surgery.