Expression of insulin-like growth factor-II and its receptor in pediatric and adult adrenocortical tumors

Background: Adrenocortical tumors are heterogeneous neoplasms with incompletely understood pathogenesis. IGF-II overexpression has been consistently demonstrated in adult adrenocortical carcinomas. Objectives: The objective of the study was to analyze expression of IGF-II and its receptor (IGF-IR) in pediatric and adult adrenocortical tumors and the effects of a selective IGF-IR kinase inhibitor (NVP-AEW541) on adrenocortical tumor cells. Patients: Fifty-seven adrenocortical tumors (37 adenomas and 20 carcinomas) from 23 children and 34 adults were studied. Methods: Gene expression was determined by quantitative real-time PCR. Cell proliferation and apoptosis were analyzed in NCI H295 cells and a new cell line established from a pediatric adrenocortical adenoma. Results: IGF-II transcripts were overexpressed in both pediatric adrenocortical carcinomas and adenomas. Otherwise, IGF-II was mainly overexpressed in adult adrenocortical carcinomas (270.5 +/- 130.2 vs. 16.1 +/- 13.3; P = 0.0001). IGF-IR expression was significantly higher in pediatric adrenocortical carcinomas than adenomas (9.1 +/- 3.1 vs. 2.6 +/- 0.3; P = 0.0001), whereas its expression was similar in adult adrenocortical carcinomas and adenomas. IGF-IR expression was a predictor of metastases in pediatric adrenocortical tumors in univariate analysis (hazard ratio 1.84; 95% confidence interval 1.28 -2.66; P = 0.01). Furthermore, NVP-AEW541 blocked cell proliferation in a dose-and time-dependent manner in both cell lines through a significant increase of apoptosis. Conclusion: IGF-IR overexpression was a biomarker of pediatric adrenocortical carcinomas. Additionally, a selective IGF-IR kinase inhibitor had antitumor effects in adult and pediatric adrenocortical tumor cell lines, suggesting that IGF-IR inhibitors represent a promising therapy for human adrenocortical carcinoma.

Structure-function analysis of the human TFIIB-related factor II protein reveals an essential role for the C-terminal domain in RNA polymerase III transcription.

The transcription factors TFIIB, Brf1, and Brf2 share related N-terminal zinc ribbon and core domains. TFIIB bridges RNA polymerase II (Pol II) with the promoter-bound preinitiation complex, whereas Brf1 and Brf2 are involved, as part of activities also containing TBP and Bdp1 and referred to here as Brf1-TFIIIB and Brf2-TFIIIB, in the recruitment of Pol III. Brf1-TFIIIB recruits Pol III to type 1 and 2 promoters and Brf2-TFIIIB to type 3 promoters such as the human U6 promoter. Brf1 and Brf2 both have a C-terminal extension absent in TFIIB, but their C-terminal extensions are unrelated. In yeast Brf1, the C-terminal extension interacts with the TBP/TATA box complex and contributes to the recruitment of Bdp1. Here we have tested truncated Brf2, as well as Brf2/TFIIB chimeric proteins for U6 transcription and for assembly of U6 preinitiation complexes. Our results characterize functions of various human Brf2 domains and reveal that the C-terminal domain is required for efficient association of the protein with U6 promoter-bound TBP and SNAP(c), a type 3 promoter-specific transcription factor, and for efficient recruitment of Bdp1. This in turn suggests that the C-terminal extensions in Brf1 and Brf2 are crucial to specific recruitment of Pol III over Pol II.

Expression and imprinting of insulin-like growth factor II (IGF2) and H19 genes in uterine leiomyomas

Genomic imprinting is defined as a gamete of origin-specific epigenetic modification of DNA leading to differential gene expression in the zygote. Several imprinted genes have been identified and some of them are associated with tumor development. We investigated the expression and the imprinting status of IGF2 and H19 genes in 47 uterine leiomyomas. Using allelic transcription assay, we detected the expression of the IGF2 gene in 10 of a total of 15 informative cases. No loss of imprinting, as determined by the finding of biallelic expression, was detected in any case. The expression of H19 gene was detected in 10 of 20 informative cases and the imprinting pattern was also maintained in all of them. Our data suggest that alterations in IGF2 and H19 genes expression by loss of imprinting do not occur in uterine leiomyomas. (C) 1999 Academic Press.

Factor V Leiden and factor II G20210A mutations in patients with recurrent abortion

Recurrent abortion (RA) represents an intriguing problem in obstetric practice in which genetic and acquired factors may play a role. In the present investigation we sought to assess the possibility that inherited thrombophilia might determine the risk of RA. We therefore investigated the prevalence of two genetic abnormalities frequently associated with venous thrombosis [factor V Leiden (FVL) and factor II G20210A] in 56 patients with primary or secondary abortion and in 384 healthy control women. Polymerase chain reaction amplification followed by digestion with the restriction enzymes MnlI and HindIII was used to define the FVL and FII G20210A genotypes respectively. FVL was found in 4/56 patients (7.1%) and in 6/384 controls (1.6%), yielding an odds ratio (OR) for RA related to FVL of 4.9 [95% confidence interval (CI): 1.3-17.8]. FII G20210A was detected in 2/56 (3.6%) patients and in 4/384 (1%) controls (OR for RA: 3.5, CI: 0.6-19.7). In conclusion, FVL and FII G20210A mutations in patients with RA were more prevalent in comparison with controls. These data support a role for both mutations as determinants of the risk of RA and strengthen the notion that thrombophilia plays a role in this clinical entity.

Loss of the gene encoding mannose 6-phosphate/insulin-like growth factor II receptor is an early event in liver carcinogenesis

This report shows that loss of heterozygosity at the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) locus occurred in 5/8 (63%) dysplastic liver lesions and 11/18 (61%) hepatocellular carcinomas (HCCs) associated with the high risk factors of hepatitis virus infection and liver cirrhosis. Mutations in the remaining allele were detected in 6/11 (55%) HCCs, including deletions in a polydeoxyguanosine region known to be a target of microsatellite instability. M6P/IGF2R allele loss was also found in cirrhotic tissue of clonal origin adjacent to these dysplastic lesions and HCCs, demonstrating that M6P/IGF2R inactivation occurs early in liver carcinogenesis. In conclusion, HCCs frequently develop from clonal expansions of phenotypically normal, M6P/IGF2R-mutated hepatocytes, providing further support for the idea that M6P/IGF2R functions as a liver tumor-suppressor gene.

Epigenetic changes at the insulin-like growth factor II/H19 locus in developing kidney is an early event in Wilms tumorigenesis

Relaxation of imprinting at the insulin-like growth factor II (IFG-II)/H19 locus is a major mechanism involved in the onset of sporadic Wilms tumor and several other embryonal tumors. The high prevalence of histologically abnormal foci in kidney adjacent to Wilms tumors suggests that tumor-predisposing genetic/epigenetic lesion might also be found at high frequency in Wilms tumor-bearing kidneys. Focusing on Wilms tumors with relaxation of IFG-II imprinting, we determined the frequency of epigenetic change at the IFG-II/H19 locus in adjacent kidney. In all kidneys adjacent to these Wilms tumors, we detected substantial mosaicism for a population of cells with relaxation of IFG-II imprinting and biallelic H19 methylation, regardless of whether the patient had a tumor-predisposing syndrome or not. The high proportion of epigenetically modified cells among “normal” tissue indicates that the epigenetic error occurred very early in development, before the onset of Wilms tumor. Not only does this suggest that the major Wilms tumor-predisposing event occurs within the first few days of development, but it also suggests that sporadic Wilms tumor may represent one end of a spectrum of overgrowth disorders characterized by mosaic epigenetic change at the IFG-II/H19 locus.

Retinoic acid alters the intracellular trafficking of the mannose-6-phosphate/insulin-like growth factor II receptor and lysosomal enzymes

Previously, we showed that retinoic acid (RA) binds to the mannose-6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) with high affinity, suggesting that M6P/IGF2R may be a receptor for RA. Here, we show that RA, after 2–3 h of incubation with cultured neonatal-rat cardiac fibroblasts, dramatically alters the intracellular distribution of M6P/IGF2R as well as that of cathepsin B (a lysosomal protease bearing M6P). Immunofluorescence techniques indicate that this change in intracellular distribution is characterized by a shift of the proteins from the perinuclear area to cytoplasmic vesicles. The effect of RA was neither blocked by an RA nuclear receptor antagonist (AGN193109) nor mimicked by a selective RA nuclear-receptor agonist (TTNPB). Furthermore, the RA-induced translocation of cathepsin B was not observed in M6P/IGF2R-deficient P388D1 cells but occurred in stably transfected P388D1 cells expressing the receptor, suggesting that the effect of RA might be the result of direct interaction with M6P/IGF2R, rather than the result of binding to the nuclear receptors. These observations not only support the idea that M6P/IGF2R mediates an RA-response pathway but also indicate a role for RA in control of intracellular trafficking of lysosomal enzymes. Therefore, our observations may have important implications for the understanding of the diverse biological effects of retinoids.

Internalization of CD26 by mannose 6-phosphate/insulin-like growth factor II receptor contributes to T cell activation

CD26 is a T cell activation antigen known to bind adenosine deaminase and have dipeptidyl peptidase IV activity. Cross-linking of CD26 and CD3 with immobilized mAbs can deliver a costimulatory signal that contributes to T cell activation. Our earlier studies revealed that cross-linking of CD26 induces its internalization, the phosphorylation of a number of proteins involved in the signaling pathway, and subsequent T cell proliferation. Although these findings suggest the importance of internalization in the function of CD26, CD26 has only 6 aa residues in its cytoplasmic region with no known motif for endocytosis. In the present study, we have identified the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIR) as a binding protein for CD26 and that mannose 6-phosphate (M6P) residues in the carbohydrate moiety of CD26 are critical for this binding. Activation of peripheral blood T cells results in the mannose 6 phosphorylation of CD26. In addition, the cross-linking of CD26 with an anti-CD26 antibody induces not only capping and internalization of CD26 but also colocalization of CD26 with M6P/IGFIIR. Finally, both internalization of CD26 and the T cell proliferative response induced by CD26-mediated costimulation were inhibited by the addition of M6P, but not by glucose 6-phosphate or mannose 1-phosphate. These results indicate that internalization of CD26 after cross-linking is mediated in part by M6P/IGFIIR and that the interaction between mannose 6-phosphorylated CD26 and M6P/IGFIIR may play an important role in CD26-mediated T cell costimulatory signaling.

Mannose-6-phosphate/insulin-like growth factor-II receptor is a receptor for retinoic acid

Retinoic acid (RA) exerts diverse biological effects in the control of cell growth in embryogenesis and oncogenesis. These effects of RA are thought to be mediated by the nuclear retinoid receptors. Mannose-6-phosphate (M6P)/insulin-like growth factor-II (IGF-II) receptor is a multifunctional membrane glycoprotein that is known to bind both M6P and IGF-II and function primarily in the binding and trafficking of lysosomal enzymes, the activation of transforming growth factor-β, and the degradation of IGF-II. M6P/IGF-II receptor has recently been implicated in fetal development and carcinogenesis. Despite the functional similarities between RA and the M6P/IGF-II receptor, no direct biochemical link has been established. Here, we show that the M6P/IGF-II receptor also binds RA with high affinity at a site that is distinct from those for M6P and IGF-II, as identified by a photoaffinity labeling technique. We also show that the binding of RA to the M6P/IGF-II receptor enhances the primary functions of this receptor. The biological consequence of the interaction appears to be the suppression of cell proliferation and/or induction of apoptosis. These findings suggest that the M6P/IGF-II receptor mediates a RA response pathway that is important in cell growth regulation. This discovery of the interaction of RA with the M6P/IGF-II receptor may have important implications for our understanding of the roles of RA and the M6P/IGF-II receptor in development, carcinogenesis, and lysosomal enzyme-related diseases.

Insulin-like growth factor II mediates epidermal growth factor-induced mitogenesis in cervical cancer cells.

There is increasing evidence that activation of the insulin-like growth factor I (IGF-I) receptor plays a major role in the control of cellular proliferation of many cell types. We studied the mitogenic effects of IGF-I, IGF-II, and epidermal growth factor (EGF) on growth-arrested HT-3 cells, a human cervical cancer cell line. All three growth factors promoted dose-dependent increases in cell proliferation. In untransformed cells, EGF usually requires stimulation by a "progression" factor such as IGF-I, IGF-II, or insulin (in supraphysiologic concentrations) in order to exert a mitogenic effect. Accordingly, we investigated whether an autocrine pathway involving IGF-I or IGF-II participated in the EGF-induced mitogenesis of HT-3 cells. With the RNase protection assay, IGF-I mRNA was not detected. However, IGF-II mRNA increased in a time-dependent manner following EGF stimulation. The EGF-induced mitogenesis was abrogated in a dose-dependent manner by IGF-binding protein 5 (IGFBP-5), which binds to IGF-II and neutralizes it. An antisense oligonucleotide to IGF-II also inhibited the proliferative response to EGF. In addition, prolonged, but not short-term, stimulation with EGF resulted in autophosphorylation of the IGF-I receptor, and coincubations with both EGF and IGFBP-5 attenuated this effect. These data demonstrate that autocrine secretion of IGF-II in HT-3 cervical cancer cells can participate in EGF-induced mitogenesis and suggest that autocrine signals involving the IGF-I receptor occur "downstream" of competence growth factor receptors such as the EGF receptor.

The role of insulin-like growth factor II and its receptor in mouse preimplantation development

Insulin-like growth factor II (IGF-II) and its receptor, the IGF-II/mannose-6-phosphate (IGF-II/M6P) receptor, are first expressed from the zygotic genome at the two-cell stage of mouse development. However, their role is not clearly defined. Insulin-like growth factor II is believed to mediate growth through the heterologous type 1 IGF and insulin receptors, whereas the IGF-II/M6P receptor is believed to act as a negative regulator of somatic growth by limiting the availability of excess levels of IGF-II. These studies demonstrate that IGF-II does have a role in growth regulation in the early embryo through the IGF-II/M6P receptor. Insulin-like growth factor II stimulated cleavage rate in two-cell embryos in vitro. Moreover, this receptor is required for the glycaemic response of two-cell embryos to IGF-II and for normal progression of early embryos to the blastocyst stage. Improved development of embryos in crowded culture supports the concept of an endogenous embryonic paracrine activity that enhances cell proliferation. These responses indicate that the IGF-II/M6P receptor is functional and likely to participate in such a regulatory circuit. The functional role of IGF-II and its receptor is discussed with reference to regulation of early development.

Insulin-like growth factor 2 cDNA cloning and ontogeny of gene expression in the liver of the marsupial brushtail possum (Trichosurus vulpecula)

The cDNA sequence for insulin-like growth factor 2 (IGF-2) was determined from the liver of the marsupial brushtail possum (Trichosurus vulpecula) using reverse transcription followed by polymerase chain reaction (RT-PCR) with gene-specific primers. The 359 bp of possum sequence encompassed the mature peptide, 27 bp of the signal peptide, and 125 bp of the E-peptide. Alignment of the deduced amino acid sequence with those from other species indicated that the mature peptide was 71 amino acids in length, 4 amino acids longer than most other mammals. At both the nucleotide and amino acid levels there was a high degree of sequence identity with IGF-2 from other mammalian and nonmammalian species. Amino acid identity ranged from 94.4% with a variant form of human IGF-2 to 80.3% with zebrafinch IGF-2. Northern analysis revealed that radiolabeled possum IGF-2, cDNA hybridized to multiple transcripts in the liver of both adult possums and 150-day-old pouch young and that the overall level of expression was greater in pouch young. Semiquantitative RT-PCR with total RNA from liver samples of pouch young aged 12 to 150 days postpartum and adults confirmed that IGF-2 gene expression was two to three times more abundant in pouch young than in adults but there was no significant change in the level of expression during pouch life. Unlike other mammalian species, in which there is a decline in levels of liver IGF-2 gene expression around the time of birth, levels in the marsupial brushtail possum remain elevated for at least 150 days after birth. This suggests that the decline in liver IGF-2 expression in marsupials and eutherians occurs at a similar stage of development and may reflect a role for this growth factor during the postnatal growth and development of the marsupial, (C) 2001 Academic Press.

Ultraweak photon emission in assessing bone growth factor efficiency using fibroblastic differentiation.

Photons participate in many atomic and molecular interactions and changes. Recent biophysical research has shown the induction of ultraweak photons in biological tissue. It is now established that plants, animal and human cells emit a very weak radiation which can be readily detected with an appropriate photomultiplier system. Although the emission is extremely low in mammalian cells, it can be efficiently induced by ultraviolet light. In our studies, we used the differentiation system of human skin fibroblasts from a patient with Xeroderma Pigmentosum of complementation group A in order to test the growth stimulation efficiency of various bone growth factors at concentrations as low as 5 ng/ml of cell culture medium. In additional experiments, the cells were irradiated with a moderate fluence of ultraviolet A. The different batches of growth factors showed various proliferation of skin fibroblasts in culture which could be correlated with the ultraweak photon emission. The growth factors reduced the acceleration of the fibroblast differentiation induced by mitomycin C by a factor of 10-30%. In view that fibroblasts play an essential role in skin aging and wound healing, the fibroblast differentiation system is a very useful tool in order to elucidate the efficacy of growth factors.

Glucagon-like peptide-1 increases beta-cell glucose competence and proliferation by translational induction of insulin-like growth factor-1 receptor expression.

Glucagon-like peptide-1 (GLP-1) protects beta-cells against apoptosis, increases their glucose competence, and induces their proliferation. We previously demonstrated that the anti-apoptotic effect was mediated by an increase in insulin-like growth factor-1 receptor (IGF-1R) expression and signaling, which was dependent on autocrine secretion of insulin-like growth factor 2 (IGF-2). Here, we further investigated how GLP-1 induces IGF-1R expression and whether the IGF-2/IGF-1R autocrine loop is also involved in mediating GLP-1-increase in glucose competence and proliferation. We show that GLP-1 up-regulated IGF-1R expression by a protein kinase A-dependent translational control mechanism, whereas isobutylmethylxanthine, which led to higher intracellular accumulation of cAMP than GLP-1, increased both IGF-1R transcription and translation. We then demonstrated, using MIN6 cells and primary islets, that the glucose competence of these cells was dependent on the level of IGF-1R expression and on IGF-2 secretion. We showed that GLP-1-induced primary beta-cell proliferation was suppressed by Igf-1r gene inactivation and by IGF-2 immunoneutralization or knockdown. Together our data show that regulation of beta-cell number and function by GLP-1 depends on the cAMP/protein kinase A mediated-induction of IGF-1R expression and the increased activity of an IGF-2/IGF-1R autocrine loop.

Progression to Adrenocortical Tumorigenesis in Mice and Humans through Insulin-Like Growth Factor 2 and beta-Catenin

Dysregulation of the WNT and insulin-like growth factor 2 (IGF2) signaling pathways has been implicated in sporadic and syndromic forms of adrenocortical carcinoma (ACC). Abnormal beta-catenin staining and CTNNB1 mutations are reported to be common in both adrenocortical adenoma and ACC, whereas elevated IGF2 expression is associated primarily with ACC. To better understand the contribution of these pathways in the tumorigenesis of ACC, we examined clinicopathological and molecular data and used mouse models. Evaluation of adrenal tumors from 118 adult patients demonstrated an increase in CTNNB1 mutations and abnormal beta-catenin accumulation in both adrenocortical adenoma and ACC. In ACC, these features were adversely associated with survival. Mice with stabilized beta-catenin exhibited a temporal progression of increased adrenocortical hyperplasia, with subsequent microscopic and macroscopic adenoma formation. Elevated Igf2 expression alone did not cause hyperplasia. With the combination of stabilized beta-catenin and elevated Igf2 expression, adrenal glands were larger, displayed earlier onset of hyperplasia, and developed more frequent macroscopic adenomas (as well as one carcinoma). Our results are consistent with a model in which dysregulation of one pathway may result in adrenal hyperplasia, but accumulation of a second or multiple alterations is necessary for tumorigenesis. (Ant J Pathol 2012, 181:1017-1033; http://dx.doi.org/10.1016/j.ajpath.2012.05.026)