Loss Of Imprinting Of Insulin-like Growth Factor 2 Research Articles

Effects of IGF2 hP4 promoter targeted methylation on imprinting status of renal cell carcinoma

Objective To investigate the imprinting status of insulin-like growth factor 2 (IGF2) in renal cell carcinoma ACHN cell line, as well as the effect of methylated drug TCF1001 on methylation status of IGF2 hP4 promoter. Furthermore, the relationship between hP4 promoter methylation and imprinting status change of IGF2 gene was also examined. Methods The heterozygote status of IGF2 gene was detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The imprinting status of IGF2 was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and PCR-RFLP. ACHN cells were then treated with either the methylated drug TCF1001 or control drugs (CT010 or PBS). Bisulfite sequencing was used to study the methylation status of IGF2 hP4 promoter region. Meanwhile, PCR-RFLP was for analyzing the variation of IGF2 imprinting status. Results It was found that IGF2 of ACHN cells was in the status of loss of imprinting (LOI). TCF1001 could effectively induce methylation of IGF2 hP4 promoter. Furthermore, the LOI of IGF2 could be reversed when IGF2 hP4 promoter was partially methylated. Conclusions IGF2 hP4 promoter can be targeted methylated by TCF1001. IGF2 hP4 promoter targeted methylation can partially reverse the LOI of IGF2, which provides the theoretical basis of renal cell carcinoma genetic therapy. Key words: Kidney Neoplasms; Insulin-Like Growth Factor II; Genomic Imprinting; Methylation

Carcinoma of the colon and rectum with deregulation of insulin-like growth factor 2 signaling: clinical and molecular implications.

Loss of imprinting (LOI) of the insulin-like growth factor 2 (IGF2) is an early event in the development of colorectal cancer (CRC). Whether LOI of IGF2 denotes a molecular or clinical cancer subgroup is currently unknown. Tumor biopsies and paired normal mucosa from 399 patients with extensive clinical annotations were analyzed for LOI and IGF2 expression. LOI status in 140 informative cases was correlated with clinicopathologic parameters and outcome. LOI was frequent in normal mucosa and tumors and occurred throughout the large intestine. LOI was unrelated to microsatellite instability, KRAS mutation status, stage, and survival. However, CRC with LOI showed increased IGF2 protein levels and activation of AKT1. Gene expression analysis of tumors with and without LOI and knockdown of IGF2 in cell lines revealed that IGF2 induced distinct sets of activated and repressed genes, including Wnt5a, CEACAM6, IGF2BP3, KPN2A, BRCA2, and CDK1. Inhibition of AKT1 in IGF2-stimulated cells showed that the downstream effects of IGF2 on cell proliferation and gene expression were strictly AKT1-dependent. LOI of IGF2 is a frequent and early event in CRC that occurs both in the adenomatous polyposis coli (APC) gene-mutated and serrated route of carcinogenesis. LOI leads to overexpression of IGF2, activates IGF1R and AKT1, and is a powerful driver of cell proliferation. Moreover, our results suggest that IGF2 via AKT1 also contributes to non-canonical wnt signaling. Although LOI had no significant impact on major clinical parameters and outcome, its potential as a target for preventive and therapeutic interventions merits further investigation.

Loss of imprinting of IGF2 in fibroadenomas and phyllodes tumors of the breast.

Loss of imprinting (LOI) of insulin-like growth factor 2 (IGF2) is thought to be implicated in the pathogenesis of some tumors by upregulating IGF2 mRNA but its role in the pathogenesis of fibroadenomas (FAs) and phyllodes tumors (PTs) of the breast is yet to be studied. LOI of IGF2 was investigated in 25 FAs and 17 PTs which were heterozygous for Apa I polymorphism, and was found to be present in 13 FAs and 12 PTs. IGF2 mRNA expression was more upregulated in FAs and PTs than in paired surrounding normal tissues and laser microdissection showed that IGF2 mRNA expression was significantly higher in the stromal than the epithelial cells. LOI was not associated with upregulation of IGF2 mRNA, nor were MED12 mutations and methylation status of the differentially methylated region 0 (DMR0) of IGF2. These results demonstrate that IGF2 mRNA expression is more upregulated in FAs and PTs than in normal tissues, especially in their stromal cells, but such an upregulation is not related to LOI of IGF2, and that hypomethylation of DMR0 is unlikely to be involved in induction of LOI.

Loss of imprinting of IGF2 correlates with hypomethylation of the H19 differentially methylated region in the tumor tissue of colorectal cancer patients

Expression of the imprinted genes insulin-like growth factor 2 (IGF2) and H19 depends on the methylation pattern of their differentially methylated region (DMR) located on chromosome 11p15. In the present study, we examined the imprinting status of the IGF2 gene in 120 human colorectal cancer (CRC) patients and 150 normal controls. In addition, we analyzed the DNA methylation of the sixth CTCF-binding site in the DMR of IGF2/H19 in 81 CRC patients using bisulfate sequencing. Of a total of 81 informative (heterozygous) samples, 51 samples showed bialleic IGF2 expression in tumor samples; however, only 15 of 69 informative samples showed loss of imprinting (LOI) of IGF2 in normal controls. Statistically significant differences in the methylation status between the retention of imprinting (ROI) and LOI groups (66.1±14.9 vs. 16.7±9.2, p=0.008) were observed. The results of the present study suggest that LOI of IGF2 is important in the carcinogenesis of CRC. Hypomethylation of the sixth CTCF-binding site in the DMR of IGF2/H19 is linked to LOI and the common IGF2-H19 enhancer competition model for IGF2 imprinting does not apply to human CRC.

Loss of imprinting and abnormal expression of the insulin‐like growth factor 2 gene in gastric cancer

This study examined the frequency of loss of imprinting (LOI) and expression of the insulin-like growth factor 2 (IGF2) gene, and their relationship to selected clinical and pathological factors, in a well defined series of 90 Chinese patients with gastric cancer (GC) and 90 matched patients (controls) diagnosed with nonmalignant conditions. Using peripheral blood and gastric tissue samples, polymerase chain reaction-based assays and restriction endonuclease (Apa I) digestion revealed 33 GC patients and 21 controls to be Apa I informative. LOI of IGF2 was positive in 48.5% (16/33) of primary GC tumor tissues, in 21.2% (7/33) of histologically normal adjacent gastric mucosa (AM) and in 12.1% (4/33) of distant gastric mucosa (DM), and in 15.2% (5/33) of peripheral blood lymphocytes (PBLs). The prevalence of IGF2 LOI in PBL was not statistically different between GC patients (5/33, 15.2%) and control subjects (2/21, 9.5%), P = 0.69. Although patients who were found to have LOI of IGF2 were more likely to have advanced stage gastric tumors (P = 0.04), no statistically significant differences in survival were found based on imprinting status. IGF2 LOI was associated with an increased expression of IGF2 level in both tumors (P < 0.01) and blood (P < 0.01). The results of this study implicate IGF2 LOI in the molecular pathogenesis of GC, most likely through increased IGF2 expression. Although the precise molecular mechanisms by which LOI of IGF2 increases GC risk require further study, LOI of IGF2 may be a potentially important clinical epigenetic marker to identify individuals at increased risk for gastric malignancy.

Loss of imprinting and marked gene elevation are 2 forms of aberrant IGF2 expression in colorectal cancer

Loss of imprinting (LOI) of insulin-like growth factor 2 (IGF2) is a common event in many cancers and typically activates the maternally silenced allele. The resulting biallelic IGF2 expression correlates strongly with the hypomethylation of a differentially methylated region (DMR) near its promoter. It has also been shown that IGF2 undergoes overexpression in human malignancies; nevertheless, this phenomenon and its link to aberrant DMR methylation have not been reported in colorectal cancer (CRC). The aim of this study was to determine the relationship between IGF2 LOI, overexpression and DMR hypomethylation in CRC. By analyzing IGF2 and H19 methylation in 97 primary CRC and 64 matched normal colorectal tissues, we have shown a significant correlation between IGF2 LOI and DMR hypomethylation of IGF2 and H19. Additionally, when analyzing Affymetrix expression data of 167 primary CRC tumors and 32 normal tissues, 15% of tumors showed marked IGF2 elevation. We further investigated if substantially elevated IGF2 levels were linked to IGF2 or H19 hypomethylation, but found no significant correlation. However, we demonstrated that noticeable IGF2 overexpression, rather than LOI, negatively correlated with CRC microsatellite instability. These observations indicate that IGF2 expression, particularly when transcribed at significantly high levels, is a result of mechanisms unrelated to LOI. Our results suggest that IGF2 participates in CRC tumorigenesis through 2 different forms of aberrant gene expression.

Loss of imprinting of insulin-like growth factor 2 is associated with increased risk of lymph node metastasis and gastric corpus cancer

BackgroundThe aim of this study was to determine the clinicopathological features of gastric cancers with loss of imprinting (LOI) of LIT1. Insulin-like growth factor 2 (IGF2) and H19 in Chinese patients.MethodsDNA and RNA from tumours were amplified and then digested with RsaI, ApaI and HinfI, and RsaI respectively to determine the LOI status. The demographic and clinicopathological characteristics in LOI positive and LOI negative patients were compared and tested with Statistical analysis.ResultsOf the 89 patients enrolled for analysis, 22, 40 and 35 were heterozygous and thus informative for LIT1, IGF2 and H19 LOI analyses respectively. The positive rate of LIT1, IGF2 and H19 LOI of gastric cancer tissues were 54.6% (12/22), 45% (18/40) and 8.6% (3/32) in Chinese patients. Gastric corpus cancer (8/10, 80%) were more likely to have LOI of IGF2 in tumours than antrum cancers (10/30, 33.3%){odds ratio (OR) = 8, 95% confidence intervals (CI) = 1.425-44.920, p = 0.018)}. LOI of IGF2 in tumours was also associated with the lymph node metastasis (LNM) (OR = 4.5, 95% CI = 1.084-18.689, p = 0.038).ConclusionIGF2 LOI is present in high frequency in Chinese gastric cancer patients, especially those with gastric corpus cancer.

Loss of imprinting of the insulin-like growth factor 2 gene and risk of colorectal cancer

The insulin-like growth factor 2 (IGF2) gene is the first gene discovered to be imprinted and expressed exclusively from the paternal allele in both humans and mice. Similarly, IGF2 is the first imprinted gene displaying loss of imprinting (LOI) in human cancers. LOI of IGF2 results in activation of the normally silent maternal allele, with increased IGF2 expression and possible increased cancer risk. LOI occurs in colonic cancer tissues, matched normal tissues, and peripheral blood lymphocytes. Human studies have found a significant, independent, positive association between LOI of IGF2 and personal and family history of colorectal neoplasia. Furthermore, animal studies using a murine model of LOI of IGF2 support an increase in intestinal neoplasia risk and abnormal colonic mucosal differentiation. LOI of IGF2 appears to be associated with a human colorectal cancer phenotype involving younger age at diagnosis, more advanced disease, right-side colonic location, and poorly differentiated or mucinous carcinoma.

Role of birthweight in the etiology of breast cancer

Breast cancer may originate in utero. We reviewed the available evidence on the association between birthweight and the risk of breast cancer. To date, 26 research papers addressing this issue have been published. The majority of studies identified a positive link between birthweight and premenopausal, but not postmenopausal, breast cancer. The relative risk estimate for breast cancer comparing women with high birthweight to women with low birthweight combining all studies including both pre- and postmenopausal breast cancer was 1.23 (95% confidence interval 1.13-1.34). The mechanisms underlying this association likely include elevated levels of growth factors that may increase the number of susceptible stem cells in the mammary gland or initiate tumors through DNA mutations. Loss of imprinting (LOI) of growth hormone genes relevant for intrauterine growth, such as insulin-like growth factor 2 (IGF2), leads to abnormally high levels of these hormones evidenced by high birthweight. LOI of IGF2 has also been found in mammary tumor tissue. The role of environmental factors that stimulate such epigenetic regulation of gene expression remains to be elucidated.

Loss of imprinting of IGF2 and H19 in osteosarcoma is accompanied by reciprocal methylation changes of a CTCF-binding site.

The adjacent insulin-like growth factor 2 (IGF2) and H19 genes are imprinted in most normal human tissues, but imprinting is often lost in tumors. The mechanisms involved in maintenance of imprinting (MOI) and loss of imprinting (LOI) are unresolved. We show here that osteosarcoma (OS) tumors with IGF2/H19 MOI exhibit allele-specific differential methylation of a CTCF-binding site upstream of H19. LOI of IGF2 or H19 in OS occurs in a mutually exclusive manner, and occurs with monoallelic expression of the other gene. Bisulfite sequencing reveals IGF2 LOI occurs with biallelic CpG methylation of the CTCF-binding site, while H19 LOI occurs with biallelic hypomethylation of this site. Our data demonstrate that IGF2 LOI and H19 LOI are accompanied by reciprocal methylation changes at a critical CTCF-binding site. We propose a model in which incomplete gain or loss of methylation at this CTCF-binding site during tumorigenesis explains the complex and often conflicting expression patterns of IGF2 and H19 in tumors.

Loss of genomic imprinting of insulin-like growth factor 2 is strongly associated with cellular proliferation in normal hematopoietic cells.

The human insulin-like growth factor 2 (IGF2) gene was thought to be imprinted and expressed only from the paternal allele in normal tissue. Initially, we analyzed the imprinting status of IGF2 in bone marrow cells from 49 patients with myelodysplastic syndromes (MDS) utilizing the Apa I polymorphism of IGF2. Thirteen bone marrow and 14 peripheral blood samples from normal individuals served as controls. We utilized normal peripheral blood T lymphocytes to examine the relationship between genomic imprinting and cell proliferation. Expression of IGF2 was quantified by real-time PCR and proliferation of T cells was measured by 3H-thymidine incorporation. Furthermore, methylation status of the imprinting controlling region (ICR) was analyzed by subcloning and sequencing of genomic DNA after sodium bisulfite modification. Among 24 patients who were heterozygous for IGF2, loss of imprinting (LOI) occurred in 22 cases (92%). Surprisingly, LOI of IGF2 occurred in the normal bone marrow cells, but the normal peripheral blood cells showed retention of imprinting (ROI). Unstimulated normal T cells showed ROI. After 24 hours of exposure to PHA, these cells changed their IGF2 imprinting status from ROI to LOI. Concomitantly, their IGF2 RNA levels increased up to sixfold and their proliferation increased 10- to 20-fold. In contrast, normal T cells not stimulated with PHA did not develop LOI of IGF2, had negligible levels of IGF2 RNA, and did not increase their proliferation. In unstimulated T cells, the CpG islands of the ICR were completely methylated on one allele and nearly completely unmethylated on the other allele. After PHA stimulation, the CpG islands at the ICR became completely methylated on both alleles. LOI of IGF2 is strongly associated with cell proliferation and is not limited to cancer cells.

Loss of Imprinting of Long QT Intronic Transcript 1 in Colorectal Cancer

Loss of imprinting (LOI) of the insulin-like growth factor 2 (IGF2) and H19 genes on human chromosome 11 has been found not only in childhood tumors but also in common adult cancers including colorectal cancer. Recently, a transcript called LIT1 (long QT intronic transcript 1) has been identified within the KvLQT1 locus on chromosome 11. LIT1 is expressed preferentially from the paternal allele and is transcribed in most human tissues. LOI of LIT1 was found in a considerable number of Beckwith-Wiedemann syndrome (BWS) patients, suggesting that it is associated with the etiology of BWS. Since LOI of IGF2 was observed in association with overexpression of IGF2 in colorectal cancer in our previous study, we examined the status of genomic imprinting of LIT1 and H19 in comparison with IGF2 in colorectal cancer. We examined 44 surgically dissected colorectal cancer tissues. Ten of them represented informative cases for LIT1. None of these patients exhibited loss of heterozygosity (LOH) of LIT1, and LOI of LIT1 was observed in 4 of the 10 (40%) informative patients, but not in non-cancerous tissues. Neither LOH nor LOI of H19 was observed. LOI of IGF2 was observed in 4 of 18 (22%) informative patients. These results suggest that LOI of LIT1 is frequently observed in colorectal cancer and may be a useful marker for diagnosis of colorectal cancer.

Analysis of Genomic Imprinting of Insulin-Like Growth Factor 2 in Colorectal Cancer

Genomic imprinting of insulin-like growth factor 2 (IGF2) has been shown to play an important role in the development of Wilms’ tumor and adult cancers including lung and esophageal cancer. Although IGF2 has been reported to be overexpressed in colorectal cancer, the status of this gene has not been fully elucidated. To clarify genomic imprinting of IGF2 in colorectal cancer, we examined loss of imprinting (LOI) and loss of heterozygosity (LOH) in surgically resected tissues by utilizing Apa1 polymorphism. Of 46 patients with colorectal cancer, 2 exhibited (2/15, 13%) LOH among 15 patients who were heterozygous for IGF2 DNA in nontumorous tissues. Four (4/12, 33%) patients showed LOI of IGF2 gene among informative patients. In these patients, the reverse transcription polymerase chain reaction and immunohistochemistry revealed that IGF2 mRNA is overexpressed in tumorous tissues, compared to nontumorous tissues. Of 15 patients in whom IGF2 was immunohistochemically more highly expressed in tumorous than in normal tissues, there were 2 with LOH and 4 with LOI. The results of the present study suggest that LOI of IGF2 plays an important role in the carcinogenesis of colorectal cancer.

Genomic imprinting of insulin-like growth factor-2 in infant leukemia and childhood neuroblastoma

Loss of imprinting (LOI) of insulin-like growth factor-2 (IGF-2) has been implicated in the pathogenesis of certain human cancers and tumor-predisposing overgrowth disorders, such as Beckwith-Wiedemann syndrome. In a previous study, the authors revealed that certain patients with childhood acute leukemia and neuroblastoma had had rapid somatic growth after birth, suggesting the involvement of growth factor(s) in tumorigenesis. In the current study, the authors examined whether relaxation of IGF-2 imprinting occurred in infant leukemia and childhood neuroblastoma. The genomic DNA of infant leukemia, childhood neuroblastoma, and control individuals was amplified by polymerase chain reaction (PCR). Patients who had heterozygous genotype were selected as informative cases using Apa I polymorphism in exon 9 of the IGF-2 gene. Total RNA was isolated from informative cases, followed by cDNA synthesis. cDNA was amplified by PCR, and direct sequence was performed for determining allele specific transcription. Twenty of 22 infant leukemia blasts and all of 16 neuroblastoma cells showed normal monoallelic expression of IGF-2 as well as 23 controls. The height and weight of two acute lymphoblastic leukemia patients with LOI were within normal ranges for Japanese children. The current study revealed that the imprinting status of IGF-2 was generally maintained in infant leukemia and confirmed that it was maintained in childhood neuroblastoma. The results suggest that LOI of IGF-2 does not play a major role in the carcinogenesis of these diseases or in rapid physical growth of the patients.

Genomic imprinting and carcinogenesis.

The Mendelian inheritance is based on the fundamental rule in which mammalian genes are expressed equally from two homologous biparental alleles. Recently a small number of genes have been identified to show an exception to this rule in that homologous alleles can function differently in somatic cells depending on whether they come from the mother or the father. This intriguing biological phenomenon is called as genomic imprinting which does not conform classical Mendelian inheritance and has potentially far reaching implications for genetics, evolution, developmental biology and pathology including cancer. The gene encoding insulin-like growth factor 2 (IGF2) harbors at 11p15.5 and serves as paradigm for an imprinted gene. The IGF2 gene has been demonstrated to be imprinted with the paternal allele expressed and the maternal being silent which is evolutionally conserved between mice and human. Loss of imprinting (LOI) of IGF2 has been demonstrated in a dozen of tumor types including Wilms tumor (WT) with a promise of many more to come. The LOI of IGF2 may induce increased or deregulated IGF2 expression which could initiate the onset of WT. Thus the LOI of IGF2 may provide a novel mechanism of gene activation and play a role in the development of a wide range of tumors. This review also discusses other imprinted genes on 11p15 which may have a role in WT or other diseases. Finally molecular mechanisms of genomic imprinting are discussed.

Imprinting of IGF2 and H19: lack of reciprocity in sporadic Beckwith-Wiedemann syndrome.

Genomic imprinting is a novel form of control of gene expression in which the transcription of each allele of an imprinted gene is dependent on the sex of the gamete from which it was derived; to date > 15 genes have been demonstrated to show imprinting. The maintenance of a normal imprinting pattern in many loci has been shown to be essential for normal development and adult life. Many tumours, and some developmental disorders, exhibit loss of imprinting (LOI) in key genes such as insulin-like growth factor 2 (IGF2) which often results in hyperplasia and is associated with cancer. The mechanism by which the genomic imprint is first established, then maintained, is not understood. However, in the case of IGF2, the expression of a neighbouring gene, H19, has been suggested to influence its transcription by competition for a common enhancer, thereby generating a mutually exclusive and allele-specific pattern of gene expression. Associated changes in CpG methylation in discrete areas of both genes have been implicated in maintenance of the imprint. We have examined the allele-specific expression of IGF2 and H19 in fibroblasts derived from patients with sporadic Beckwith-Wiedemann syndrome (BWS), a fetal overgrowth syndrome associated with an imprinted locus on 11p15.5. We report that the majority of karyotypically normal patients show LOI of IGF2 with biallelic expression. In a proportion of these patients, loss of IGF2 imprinting was associated with complete suppression of H19 expression, as predicted by the enhancer competition model. However, in a significant number of cases, IGF2 showed biallelic expression even though H19 expression and methylation status were normal. This indicates that there must be an alternative H19-independent pathway by which allele-specific IGF2 expression is established or maintained.