IGF2 Promoter Research Articles

Restoring Wnt6 signaling ameliorates behavioral deficits in MeCP2 T158A mouse model of Rett syndrome

The methyl-CpG-binding protein 2 gene, MECP2, is an X chromosome-linked gene encoding the MeCP2 protein, and mutations of MECP2 cause Rett syndrome (RTT). Previous study has shown that re-expression of SUMO-modified MeCP2 in Mecp2-null neurons rescues synaptic and behavioral deficits in Mecp2 conditional knockout mice, whereas about 12-fold decrease in Wnt6 mRNA level was found in MeCP2K412R sumo-mutant mice. Here, we examined the role of Wnt6 in MeCP2 T158A mouse model of RTT. Results show that lentiviral delivery of Wnt6 to the amygdala ameliorates locomotor impairment and social behavioral deficits in these animals. MeCP2 T158A mice show decreased level of GSK-3β phosphorylation and increased level of β-catenin phosphorylation. They also show reduced level of MeCP2 SUMOylation. These alterations were also restored by lenti-Wnt6 transduction. Further, both BDNF and IGF-1 expressions are decreased in MeCP2 T158A mice. Overexpression of Wnt6 increases Bdnf and Igf-1 promoter activity in HEK293T cells in a dose-dependent manner. Lenti-Wnt6 transduction to the amygdala similarly increases the mRNA level and protein expression of BDNF and IGF-1 in MeCP2 T158A mice. Moreover, environmental enrichment (EE) similarly ameliorates the locomotor and social behavioral deficits in MeCP2 T158A mice. One of the mechanisms underlying EE is mediated through enhanced MeCP2 SUMOylation and increased Wnt6 expression in these animals by EE.

Prenatal smoke effect on mouse offspring Igf1 promoter methylation from fetal stage to adulthood is organ and sex specific.

Prenatal smoke exposure (PSE) is associated with reduced birth weight, impaired fetal development, and increased risk for diseases later in life. Changes in DNA methylation may be involved, as multiple large-scale epigenome-wide association studies showed that PSE is robustly associated with DNA methylation changes in blood among offspring in early life. Insulin-like growth factor-1 (IGF1) is important in growth, differentiation, and repair processes after injury. However, no studies investigated the organ-specific persistence of PSE-induced methylation change of Igf1 into adulthood. Based on our previous studies on the PSE effect on Igf1 promoter methylation in fetal and neonatal mouse offspring, we now have extended our studies to adulthood. Our data show that basal Igf1 promoter methylation generally increased in the lung but decreased in the liver (except for 2 persistent CpG sites in both organs) across three different developmental stages. PSE changed Igf1 promoter methylation in all three developmental stages, which was organ and sex specific. The PSE effect was less pronounced in adult offspring compared with the fetal and neonatal stages. In addition, the PSE effect in the adult stage was more pronounced in the lung compared with the liver. For most CpG sites, an inverse correlation was found for promoter methylation and mRNA expression when the data of all three stages were combined. This was more prominent in the liver. Our findings provide additional evidence for sex- and organ-dependent prenatal programming, which supports the developmental origins of health and disease (DOHaD) hypothesis.

Developmental Stage-Specific <i>IGF1</i> Gene Methylation in Infants with Intrauterine Growth Restriction

Background: IGF1 is a key molecule in the regulation of growth and metabolism. Low IGF1 secretion is known to cause growth restriction in childhood, as well as deregulated lipid metabolism, cardiovascular disease, and diabetes in adulthood. The IGF1 gene P2 promoter is highly methylated, resulting in low secretion of IGF1 in small infants and children. However, it is unknown when this methylation occurs. The aim of study was to clarify the point when this epigenetic program occurs during intrauterine development. Methods: We analyzed 57 preterm infants born before 32 weeks of gestation, including 20 intrauterine growth restriction (IUGR) infants whose birth weights were lower than −2SD calculated by the Japanese datasets. We extracted genomic DNA from whole blood at birth; methylation of the IGF1 P2 promoter was analyzed by the bisulfite amplicon method using the MiSeq platform. Findings: In contrast to term infants and children, IGF1 promoter methylation levels were significantly reduced in infants with IUGR. Interpretation: These findings indicated that the IGF1 gene is epigenetically activated before 32 weeks of gestation in infants with IUGR, and that the activated gene may become suppressed after this time point. This study may provide new insights to prevent the onset of adult diseases and to aid in nutritional management for preterm birth infants in neonatal intensive care units. Funding Statement: This work was supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT)-Supported Program for the Strategic Research Foundation at Private Universities, 2015-2019, Japan (M.K.) and by Grants-in-Aid for General Scientific Research, No. 17k16309, Japan (N.I.). Declaration of Interests:The authors declare no competing interests. Ethics Approval Statement: This study was approved by the Juntendo University Ethics Committee and conducted in accordance with the principles of the Declaration of Helsinki.

Non‑toxic sulfur enhances growth hormone signaling through the JAK2/STAT5b/IGF‑1 pathway in C2C12 cells.

Insulin‑like growth factor‑1 (IGF‑1) regulates cell growth, glucose uptake and protein metabolism, and is required for growth hormone (GH) signaling‑mediated insulin production and secretion. IGF1 expression is associated with STAT5, which binds to a region (TTCNNNGAA) of the gene. Although sulfur is used in various fields, the toxicity of this element is a significant disadvantage as it causes indigestion, vomiting, diarrhea, pain and migraine. Therefore, it is difficult to conduct invitro experiments to directly determine the effects of dietary sulfur. Additionally, it is difficult to dissolve non‑toxic sulfur (NTS). The present study aimed to identify the role of NTS in GH signaling as a Jak2/STAT5b/IGF‑1 pathway regulator. MTT assay was used to identify an optimum NTS concentration for C2C12 mouse muscle cells. Western blotting, RT‑PCR, chromatin immunoprecipitation, overexpression and small interfering RNA analyses were performed. NTS was dissolved in 1mg/ml DMSO and could be used invitro. Therefore, the present study determined whether NTS induced mouse muscle cell growth via GH signaling. NTS notably increased STAT5b binding to the Igf1 promoter. NTS also promoted GH signaling by upregulating GH receptor expression, similar to GH treatment. NTS enhanced GH signaling by regulating Jak2/STAT5b/IGF‑1 signaling pathway factor expression in C2C12 mouse muscle cells. Thus, NTS may be used as a GH‑enhancing growth stimulator.

Grandmaternal betaine supplementation enhances hepatic IGF2 expression in F2 rat offspring through modification of promoter DNA methylation.

We reported previously that maternal betaine promotes hepatic insulin-like growth factor (IGF2) expression in F1 offspring rats through hypermethylation of the IGF2/H19 imprinting control region (ICR). It remains unknown whether this acquired trait can be transmitted to the F2 generation. This study aimed to determine whether dietary betaine supplementation to grand dams affects the hepatic IGF2 expression in F2 rat offspring and how it is related to alterations in DNA methylation. F2 rat offspring derived from grand dams fed basal or betaine-supplemented diet (10 g kg-1 ) were examined at weaning. Serum IGF2 concentration was measured with enzyme-linked immunosorbent assay (ELISA). Hepatic expression of IGF2, together with other proliferation and apoptosis markers, was determined by using quantitative polymerase chain reaction (qPCR), western blot, and immunohistochemistry. The methylation status of the IGF2/H19 ICR and the promoters of IGF2 gene were detected by methylated DNA immunoprecipitation quantitative polymerase chain reaction (MeDIP-qPCR). The maternal betaine-induced up-regulation of hepatic IGF2 expression in F1 rat offspring was transmitted to the F2 generation. The F2 rats from the betaine group demonstrated enhanced hepatic IGF2 expression at both mRNA and protein levels, in association with higher serum IGF2 concentration. No alterations were observed in the ICR methylation of the IGF2/H19 locus, and hypomethylation was detected in promoters of IGF2 gene in betaine group. These results indicate that maternal betaine enhances hepatic IGF2 expression in F2 rat offspring through modification of DNA methylation on IGF2 promoters. © 2019 Society of Chemical Industry.

Abstract 874: The CRTC1/MAML2 oncogene induces a PGC-1a splice variant that sensitizes mucoepidermoid carcinoma cells to PPARg inhibition

Abstract Mucoepidermoid carcinoma (MEC) is the most common type of malignant salivary gland tumor. MECs frequently exhibit a recurrent t(11;19)(q21;p13) chromosomal translocation that fuses the transcriptional coactivators CRTC1 (CREB-Regulated Transcription Coactivator 1; C1) and MAML2 (Mastermind-Like 2; M2). Though many C1/M2-positive tumors are easily resected, some do progress to advanced, metastatic disease accompanied by poor 5-year survival rates. Thus, it is critical to understand the mechanisms by which C1/M2 drives tumor growth and progression in order to identify therapeutic vulnerabilities. Our lab recently identified a correlation between C1/M2 expression and increased levels of the growth hormone IGF-1. This hormone plays critical roles in the development and homeostasis of normal salivary glands, and its dysregulation has been implicated in several cancers including colon and lung cancers. Interestingly, we demonstrate that IGF-1 is not directly upregulated by C1/M2. Rather, we have discovered a novel C1/M2-driven signaling circuit whereby constitutive CREB activation drives the increased production of a minor PGC-1α (PPARγ Coactivator 1α) splice variant, PGC-1α4, which is responsible for the high IGF-1 levels observed in C1/M2-positive MEC cells. Notably, the PGC-1α4 splice variant is minimally expressed in normal tissues and C1/M2-negative cell lines but is selectively upregulated in C1/M2-positive cell lines. We show that C1/M2 directly binds CREB at the PGC-1α4 promoter to induce expression of this variant, resulting in increased IGF-1 expression. IGF-1 promoter analysis revealed a significant enrichment of PPARγ binding motifs and functional studies confirm that this promoter is regulated by PPARγ. Consequently, we demonstrate that a novel PPARγ inverse agonist is capable of repressing IGF-1 promoter activity and reducing growth and clonogenic potential of C1/M2-positive cell lines that exhibit high PGC-1α4 expression. Collectively, our results reveal a novel therapeutic vulnerability in salivary MEC cells that may extend more broadly to other cancers that exhibit dysregulated CREB activity. Citation Format: Adele M. Musicant, Kshitij P. Sharma, Erin C. Henry, Jason Tasoulas, Ricardo Padilla, Monideepa Sengupta, Colin Flaveny, Antonio L. Amelio. The CRTC1/MAML2 oncogene induces a PGC-1a splice variant that sensitizes mucoepidermoid carcinoma cells to PPARg inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 874.

The GC-IGF1 axis-mediated testicular dysplasia caused by prenatal caffeine exposure

Prenatal caffeine exposure (PCE) can induce testicular developmental toxicity. Here, we aimed to explore the underlying mechanism of this process in reference to its intrauterine origin. Pregnant rats were intragastrically administrated caffeine (30 and 120 mg/kg/day) from gestational days 9 to 20. The results showed that the male fetuses exposed to high dose of caffeine (120 mg/kg/day) had a decreased bodyweight and inhibited testosterone synthetic function. Meanwhile, their serum corticosterone concentration was elevated and their testicular insulin-like growth factor 1 (Igf1) expression was decreased. Moreover, the histone 3 lysine 14 acetylation (H3K14ac) level in the Igf1 promoter region was reduced. Low-dose (30 mg/kg/day) caffeine exposure, however, increased steroidogenic enzymes expression in male fetuses. After birth, the serum corticosterone concentration gradually decreased in the PCE (120 mg/kg/day) offspring rats, whereas the expression and H3K14ac level of Igf1 gradually increased, with obvious catch-up growth and testicular development compensation. Intriguingly, when we subjected the offspring to 2 weeks of chronic stress to elevate the serum corticosterone concentration, the expression of Igf1 and testosterone synthesis were inhibited again in the PCE (120 mg/kg/day) group, accompanied by a decrease in the H3K14ac level in the Igf1 promoter region. In vitro, corticosterone (rather than caffeine) was proved to inhibit testosterone production in Leydig cells by altering the H3K14ac level and the expression of Igf1. These observations suggested that PCE-induced testicular developmental toxicity is related to the negative regulation of corticosterone on H3K14ac levels and the expression of Igf1.

Insulinlike Growth Factor 1 Gene Variation in Vertebrates.

IGF1-a small, single-chain, secreted peptide in mammals-is essential for normal somatic growth and is involved in a variety of other physiological and pathophysiological processes. IGF1 expression appears to be controlled by several different signaling mechanisms in mammals, with GH playing a key role by activating an inducible transcriptional pathway via the Jak2 protein kinase and the Stat5b transcription factor. Here, to understand aspects of Igf1 gene regulation over a substantially longer timeline than is discernible in mammals, Igf1 genes have been examined in 21 different nonmammalian vertebrates representing five different classes and ranging over ∼500 million years of evolutionary history. Parts of vertebrate Igf1 genes resemble components found in mammals. Conserved exons encoding the mature IGF1 protein are detected in all 21 species studied and are separated by a large intron, as seen in mammals; the single promoter contains putative regulatory elements that are similar to those functionally mapped in human IGF1 promoter 1. In contrast, GH-activated Stat5b-binding enhancers found in mammalian IGF1 loci are completely absent, there is no homolog of promoter 2 or exon 2 in any nonmammalian vertebrate, and different types of "extra" exons not present in mammals are found in birds, reptiles, and teleosts. These data collectively define properties of Igf1 genes and IGF1 proteins that were likely present in the earliest vertebrates and support the contention that common structural and regulatory features in Igf1 genes have a long evolutionary history.

A novel DNA methylation panel accurately detects colorectal cancer independently of molecular pathway

BackgroundColorectal cancer (CRC) is one of the most incident cancers, associated with significant morbidity and mortality, and usually classified into three main molecular pathways: chromosomal instability, microsatellite instability (MSI) and CpG island methylator phenotype (CIMP). Currently, available screening methods are either costly or of limited specificity, impairing global implementation. More cost-effective strategies, including DNA methylation-based tests, might prove advantageous. Although some are already available, its performance is suboptimal, entailing the need for better candidate biomarkers. Herein, we tested whether combined use of APC, IGF2, MGMT, RASSF1A, and SEPT9 promoter methylation might accurately detect CRC irrespective of molecular subtype.MethodsSelected genes were validated using formalin-fixed paraffin-embedded tissues from 214 CRC and 50 non-malignant colorectal mucosae (CRN). Promoter methylation levels were assessed using real-time quantitative methylation-specific PCR. MSI and CIMP status were determined. Molecular data were correlated with standard clinicopathological features. Diagnostic and prognostic performances were evaluated by receiver operator characteristics curve and survival analyses, respectively.ResultsExcept for IGF2, promoter methylation levels were significantly higher in CRC compared to CRN. A three-gene panel (MGMT, RASSF1A, SEPT9) identified malignancy with 96.6% sensitivity, 74.0% specificity and 91.5 positive predictive value (area under the curve: 0.97), independently of tumor location, stage, and molecular pathway.ConclusionsCombined promoter methylation analysis of MGMT/RASSF1A/SEPT9 displays a better performance than currently available epigenetic-based biomarkers for CRC, providing the basis for the development of a non-invasive assay to detect CRC irrespective of the molecular pathway.

Insulin-like growth factor 2 expression in prostate cancer is regulated by promoter-specific methylation.

Deregulation of the insulin‐like growth factor (IGF) axis and dysbalance of components of the IGF system as potential therapeutic targets have been described in different tumor types. IGF2 is a major embryonic growth factor and an important activator of IGF signaling. It is regulated by imprinting in a development‐ and tissue‐dependent manner and has been implicated in a broad range of malignancies including prostate cancer (PCa). Loss of imprinting (LOI) usually results in bi‐allelic gene expression and increased levels of IGF2. However, the regulatory mechanisms and the pathophysiological impact of altered IGF2 expression in PCa remain elusive. Here, we show that in contrast to many other tumors, IGF2 mRNA and protein levels were decreased in 80% of PCa in comparison with non‐neoplastic adjacent prostate and were independent of LOI status. Instead, IGF2 expression in both tumors and adjacent prostate depended on preferential usage of the IGF2 promoters P3 and P4. Decreased IGF2 expression in tumors was strongly related to hypermethylation of these two promoters. Methylation of the A region in promoter P4 correlated specifically with IGF2 expression in the 20% of PCa where IGF2 was higher in tumors than in adjacent prostate. We conclude that IGF2 is downregulated in most PCa and may be particularly relevant during early stages of tumor development or during chemotherapy and androgen deprivation. PCa differs from other tumors in that IGF2 expression is mainly regulated through methylation of promoter‐specific and not by imprinting. Targeting of promoter‐specific regions may have relevance for the adjuvant treatment of PCa.

Epigenetic alterations of the Igf2 promoter and the effect of miR‑483‑5p on its target gene expression in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most widespread malignancies in China. MicroRNAs (miRNAs/miRs) are endogenous evolutionarily-conserved small non-coding RNAs that are able to regulate ESCC formation and deterioration by negatively regulating specific target genes. In the present study, the expression levels of miR-483-5p and its associated mRNAs were measured by quantitative polymerase chain reaction (PCR) analysis, and the methylation levels of the insulin-like growth factor 2 (Igf2) promoter were detected via the methylation-specific PCR method in serum and tissues from patients with ESCC. The results demonstrated that the expression level of miR-483-5p was significantly upregulated in preoperative serum and cancer tissues from patients with ESCC (P<0.01), and the miR-483-5p expression levels were correlated with the tumor, node, metastasis stage (P<0.05) and lymph node metastasis (P<0.05). In addition, the mRNA levels of miR-483-5p target genes (Rho GDP dissociation inhibitor α, activated leukocyte cell adhesion molecule, and suppressor of cytokine signaling 3) in cancer tissues were significantly decreased compared with adjacent non-cancerous tissues. These results indicated that miR-483-5p and its target genes may be involved in the developmental process of ESCC. The Igf2 levels in cancer tissues were significantly increased compared with adjacent non-cancerous tissues (P<0.01). Additionally, the methylation levels of the Igf2 promoter region were 31.82 and 54.55% in cancer tissues and adjacent non-cancerous tissues, respectively, suggesting that low methylation of the Igf2 gene promoter region may promote the expression of Igf2 and miR-483-5p; this, in turn, induces the degradation of miR-483-5p target genes, and leads to the upregulation of oncogenes and the downregulation of tumor suppressors, which promotes the development of ESCC.

The mechanism of transactivation regulation due to polymorphic short tandem repeats (STRs) using IGF1 promoter as a model.

Functional short tandem repeats (STR) are polymorphic in the population, and the number of repeats regulates the expression of nearby genes (known as expression STR, eSTR). STR in IGF1 promoter has been extensively studied for its association with IGF1 concentration in blood and various clinical traits and represents an important eSTR. We previously used an in-vitro luciferase reporter model to examine the interaction between STRs and SNPs in IGF1 promoter. Here, we further explored the mechanism how the number of repeats of the STR regulates gene transcription. An inverse correlation between the number of repeats and the extent of transactivation was found in a haplotype consisting of three promoter SNPs (C-STR-T-T). We showed that these adjacent SNPs located outside the STR were required for the STR to function as eSTR. The C allele of rs35767 provides a binding site for CCAAT/enhancer-binding-protein δ (C/EBPD), which is essential for the gradational transactivation property of eSTR and FOXA3 may also be involved. Therefore, we propose a mechanism in which the gradational transactivation by the eSTR is caused by the interaction of one or more transcriptional complexes located outside the STR, rather than by direct binding to a repeat motif of the STR.

P7007 Targeted IGF1 promoter modification in mice using small intestine-specific regulatory element binding sites

P7007 Targeted IGF1 promoter modification in mice using small intestine-specific regulatory element binding sites

CRISPR Cas9-guided chromatin immunoprecipitation identifies miR483 as an epigenetic modulator of IGF2 imprinting in tumors.

The normally imprinted insulin-like growth factor II (IGF2) gene is aberrantly upregulated in a variety of human malignancies, yet the mechanisms underlying this dysregulation are still poorly defined. In this report, we used a CRISPR Cas9-guided chromatin immunoprecipitation assay to characterize the molecular components that participate in the control of IGF2 gene expression in human tumor cells. We found that miR483, an oncogenic intronic miRNA, binds to the most upstream imprinted IGF2 promoter, P2. Ectopic expression of miR483 induced upregulation of IGF2 expression, in parallel with an increase in tumor cell proliferation, migration, invasion, and tumor colony formation. miR483 induced loss of IGF2 imprinting by altering the epigenotype at P2, with reduction in histone H3K27 methylation and a decrease in chromatin binding of two imprinting regulatory factors, CTCF and SUZ12. This study identifies a new role for miR483 in the regulation of IGF2 gene expression through the alteration of the promoter epigenotype.

Association of insulin-like growth factor (IGF)-1 gene polymorphisms with plasma levels of IGF-1 and acne severity

Polymorphisms of the insulin-like growth factor (IGF)-1 gene consisting of variable cytosine adenosine repeats in the promoter region may directly influence the expression of IGF-1. We sought to assess the role of IGF-1 gene polymorphisms in determination of plasma IGF-1 levels, acne, and its severity. In this case-control study, 80 patients with acne vulgaris of 4 severity grades as per Global Acne Grading System and 80 age- and gender-matched control subjects without acne were studied. All the study subjects were without any disorder or a history of drug intake likely to affect IGF-1 level within a year before the study inclusion date. IGF-1 polymorphism was determined by polymerase chain reaction and plasma levels of IGF-1 by enzyme-linked immunosorbent assay. Acne severity was assessed by Global Acne Grading System. Mean plasma IGF-1 level in acne cases was significantly higher than in non-acne controls (P=.04). Plasma IGF-1 level positively correlated with severity of acne (P=.01). Individuals homozygous for the 192-base pair (bp) allele had 4.29 times odds risk (95% confidence interval 1.38-13.33) of having acne and a significantly higher mean level of IGF-1 compared with non-192/non-192 participants. Individuals homozygous for the 192-bp allele had 3.08 times odds risk (95% confidence interval 1.15- 8.31) of having higher severity grade of acne compared with non-192/non-192 participants. A relatively small number of participants were studied. Plasma IGF-1 levels positively correlate with severity of acne. The 192/192 homozygotes had higher risk of acne and higher severity grade of acne. Functional studies showing the relationship between IGF-1 promoter level polymorphism and actual gene expression in skin are warranted.

Effect of in ovo folic acid injection on hepatic IGF2 expression and embryo growth of broilers.

BackgroundInsulin-like factor 2 (IGF2) plays an important role in embryonic growth process by modulating intermediary metabolism and cell proliferation. Folic acid is involved in one carbon metabolism and contributes to DNA methylation which is related to gene expression. The purpose of this study was to explore whether folic acid could regulate IGF2 expression via epigenetic mechanism and further promote embryonic growth of new-hatched broilers.MethodsIn the present study, 360 fertile eggs were selected and randomly assigned to four treatments. On 11 embryonic day of incubation (E11), 0, 50, 100 and 150 μg folic acid were injected into eggs respectively. After hatched, growth performance of broilers were calculated. Hepatic IGF2 expression, methylation level and chromatin structure of promoter region were analyzed.ResultsResults have showed that IGF2 expression was up-regulated in 150 μg folic acid group (P < 0.05) and other two dose of folic acid did not affect gene expression (P > 0.05). Meanwhile, methylation level of IGF2 promoter were lower in 100 and 150 μg groups, which was consistent with lower expression of DNA methyltransferase 1 (DNMT1) (P < 0.05). What’s more, chromatin looseness of IGF2 promoter was higher in 150 μg group than control group (P < 0.05). Further, birth weight (BW), liver and bursa index of new-hatched chickens in 150 μg folic acid group were higher than the other groups (P < 0.05). There were positive correlations between hepatic IGF2 expression and BW and organs index (P < 0.05).ConclusionIn conclusion, our data have demonstrated that 150 μg folic acid injection on E11 could up-regulate IGF2 expression by modulating DNA hypomethylation and improving chromatin accessibility in the gene promoter region, and ulteriorly facilitate embryonic growth and organ development of broilers.Electronic supplementary materialThe online version of this article (doi:10.1186/s40104-016-0099-3) contains supplementary material, which is available to authorized users.

Abstract 297: Epigenetic activation of IGF1R signaling promotes resistance to momelotinib and MEK inhibition in KRAS-driven lung cancer

Abstract KRAS is the most frequently mutated oncogene in non-small cell lung cancer (NSCLC), yet remains resistant to targeted therapy. Previously, we reported that RAL-TBK1 activation downstream of KRAS promotes an autocrine cytokine circuit that fuels tumorigenesis. Interruption of this signaling by treatment with momelotinib, an inhibitor of TBK1/IKKϵ and JAK kinases, together with MEK inhibition, was effective in the aggressive Kras-p53 (KP) mouse lung cancer model. These findings have prompted a human clinical trial of momelotinib and trametinib in advanced refractory KRAS mutant NSCLC. Because this combination leads to acquired resistance in mice, we sought to understand additional pathways that could compensate for TBK1/JAK and MEK inhibition. Receptor tyrosine kinase (RTK) profiling of A549 cells treated with momelotinib revealed delayed activation of IGF1 receptor (IGF1R) signaling. Indeed, IGF1 supplementation partially rescued momelotinib-induced growth arrest of A549 cells, while combination of momelotinib with the dual IGF1R/insulin receptor (IR) inhibitor linsitinib cooperated to impair cell viability, which was more pronounced in KRAS/STK11 (encoding LKB1) mutant cell lines compared with LKB1 wild type cell lines. Triple momelotinib/trametinib/linsitinib therapy also improved tumor shrinkage in Kras-Lkb1 (KL) mice compared with KP mice. However, chronic treatment with this triple combination required pulse therapy due to toxicity, and was inferior to momelotinib/trametinib dosed daily, which itself promoted deep responses in KL mice lasting 6 weeks. In parallel, we analyzed mediators of acquired resistance to chronic momelotinib and MEK inhibitor exposure in A549 cells. Interestingly, IGF1 expression was dramatically upregulated in resistant cells, in contrast to IGF2, and enhanced autocrine activation of IGF1R signaling. Importantly, both IGF1 upregulation and inhibitor resistance were completely reversible after drug withdrawal for several passages, yet reappeared quickly upon drug readdition, suggesting epigenetic reprogramming. Consistent with this hypothesis, whereas global H3K27 histone acetylation was decreased in resistant cells, this epigenetic mark was strongly enriched at the IGF1 promoter and linked tightly with both IGF1 levels and drug resistance. Treatment of resistant A549 cells with the BRD4 inhibitor JQ1 completely suppressed IGF1 expression and downstream IGF1R activation, and impaired cell viability. Combination JQ1 treatment also prevented momelotinib/MEK inhibitor induction of IGF1 in A549 cells, synergized to reduce outgrowth of resistant cells, and was well tolerated as daily triple combination therapy in mice. Together, these findings suggest the potential of BET inhibition to synergize with and prevent the development of acquired resistance to momelotinib and MEK inhibitor treatment in KRAS mutant NSCLC. Citation Format: Shunsuke Kitajima, Hajime Asahina, Jillian D. Cavanaugh, Ting Chen, Ashley A. Merlino, Thai C. Tran, Kwonk-Kin Wong, David A. Barbie. Epigenetic activation of IGF1R signaling promotes resistance to momelotinib and MEK inhibition in KRAS-driven lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 297.

Loss of insulin-like growth factor II imprinting is a hallmark associated with enhanced chemo/radiotherapy resistance in cancer stem cells.

Insulin-like growth factor II (IGF2) is maternally imprinted in most tissues, but the epigenetic regulation of the gene in cancer stem cells (CSCs) has not been defined. To study the epigenetic mechanisms underlying self-renewal, we isolated CSCs and non-CSCs from colon cancer (HT29, HRT18, HCT116), hepatoma (Hep3B), breast cancer (MCF7) and prostate cancer (ASPC) cell lines. In HT29 and HRT18 cells that show loss of IGF2 imprinting (LOI), IGF2 was biallelically expressed in the isolated CSCs. Surprisingly, we also found loss of IGF2 imprinting in CSCs derived from cell lines HCT116 and ASPC that overall demonstrate maintenance of IGF2 imprinting. Using chromatin conformation capture (3C), we found that intrachromosomal looping between the IGF2 promoters and the imprinting control region (ICR) was abrogated in CSCs, in parallel with loss of IGF2 imprinting in these CSCs. Loss of imprinting led to increased IGF2 expression in CSCs, which have a higher rate of colony formation and greater resistance to chemotherapy and radiotherapy in vitro. These studies demonstrate that IGF2 LOI is a common feature in CSCs, even when the stem cells are derived from a cell line in which the general population of cells maintain IGF2 imprinting. This finding suggests that aberrant IGF2 imprinting may be an intrinsic epigenetic control mechanism that enhances stemness, self-renewal and chemo/radiotherapy resistance in cancer stem cells.

Developmental changes of Insulin-like growth factors in the liver and muscle of chick embryos

The insulin-like growth factors (IGFs) are synthesized in tissues and play an important role in embryonic development of avian via autocrine/paracrine mechanisms. In the study, mRNA expression of IGFs were detected by real-time PCR in the muscle and liver from d 10 to 20 of chick embryo (E10 to E20). Methylation of IGF1 promoter in the muscle was analyzed by bisulfite sequencing PCR as well as IGF2 promoter in the liver. These results showed that there was obviously IGF1 expression in liver at E19 and E20. The higher IGF1 expression in muscle was found during E15 to E18 with the peak on E17, and then declined. Correspondingly, the lowest methylation level of IGF1 promoter was detectable on the same embryonic d 17. Expression of IGF2 in muscle increased gradually during embryonic growth and showed higher level in the later stages (E17 to E20) when IGF1 expression began to decrease. IGF2 expression in liver reached the first peak on E14, then declined but gradually elevated from E17. IGF2 promoter methylation in liver showed gradual decline on d 12, 15, 17 and 19 of incubation, meanwhile IGF2 expression of liver increased gradually. These results suggested that IGF1 and IGF2 might separately be more important for muscle and liver growth in chick embryonic development. Variation of IGFs expression during the incubation might be concerned with the methylation of gene promoter. The profile of IGFs expression in chick embryonic tissues may be meaningful for understanding organ growth and embryonic development in chick.

A polymorphic repeat in the IGF1 promoter influences the risk of endometrial cancer.

Due to the lack of high-throughput genetic assays for tandem repeats, there is a paucity of knowledge about the role they may play in disease. A polymorphic CA repeat in the promoter region of the insulin-like growth factor 1 gene (IGF1 has been studied extensively over the past 10 years for association with the risk of developing breast cancer, among other cancers, with variable results. The aim of this study was to determine if this CA repeat is associated with the risk of developing breast cancer and endometrial cancer. Using a case–control design, we analysed the length of this CA repeat in a series of breast cancer and endometrial cancer cases and compared this with a control population. Our results showed an association when both alleles were considered in breast and endometrial cancers (P=0.029 and 0.011, respectively), but this did not pass our corrected threshold for significance due to multiple testing. When the allele lengths were analysed categorically against the most common allele length of 19 CA repeats, an association was observed with the risk of endometrial cancer due to a reduction in the number of long alleles (P=0.013). This was confirmed in an analysis of the long alleles separately for endometrial cancer risk (P=0.0012). Our study found no association between the length of this polymorphic CA repeat and breast cancer risk. The significant association observed between the CA repeat length and the risk of developing endometrial cancer has not been previously reported.