Insulin-like growth factor-II expression is down-regulated in TrkA-transfected SK-N-AS neuroblastoma cells.

Expression level of trkA tyrosine kinase receptor for nerve growth factor is a major prognostic determinant of neuroblastoma, suggesting that defective trkA-mediated signaling is responsible for the tumorigenesis of this childhood malignancy. We investigated the biologic effect of trkA, with special reference to its effect on insulin-like growth factor-II (IGF-II) expression, in SK-N-AS human neuroblastoma cells transfected with human trkA cDNA. Nerve growth factor treatment of trkA-transfected cells promoted growth and changed the morphologic phenotype into a substrate-adherent, flatter phenotype (S-type), and down-regulated the mRNA expression of IGF-II. The effects on both growth and the morphologic differentiation of SK-N-AS cells differed significantly from those of previous studies, and implied that trkA effects can be diverse, depending on the phenotype of the individual neuroblastoma cells. Immunohistochemical screening of trkA and IGF-II expression in adrenal neuroblastomas (n = 25) also favored the nonoverlapping pattern of trkA and IGF-II expression (p < 0.05). Because IGF-II is believed to play a significant role in the tumorigenesis of neuroblastoma, the inverse relationship between trkA and IGF-II strongly suggests that a low level of trkA can be a feature of the pathogenetic mechanism of IGF-II expressing adrenal neuroblastomas.     

A possible role of insulin-like growth factor-II C-peptide in regulating the function of steroidogenic cells in adult frog adrenal glands

The sole structural determinant for the differential ability of the insulin-like growth factors (IGF-I and IGF-II) to induce autophosphorylation of specific insulin receptor (IR) tyrosine residues and activate downstream signaling molecules is the C domain. The IR is structurally related to the type I insulin-like growth factor receptor (IGF-IR). This study aimed to identify the presence of IGF receptors by which the IGF-II C-peptide could mediate its effects in the frog (Rana ridibunda) adrenal glands and to observe whether injection of IGF-II C-peptide affects the function of adrenal steroidogenic cells using light and transmission electron microscopy and by the evaluation of the immunoreactivity of steroidogenic acute regulatory protein (StAR). After IGF-II C-peptide injection, there was a reduction of StAR protein immunoreactivity levels, an accumulation of large lipid droplets in close contact with each other, and an induction of proliferation of the steroidogenic cells. These results indicate a possible role of IGF-II C-peptide in steroidogenic cell function and in induction of steroidogenesis. The detection in this study of IGF-I receptor (IGF-IR) immunoreactivity in frog adrenal glands also indicates that the metabolic and mitogenic effects of IGF-II C-peptide in these glands may occur via the IGF-IR.     

Expression of insulin-like growth factor-II mRNA in fetal kidney and Wilms' tumor. An in situ hybridization study

The pattern of insulin-like growth factor II (IGF-II) mRNA expression in developing kidney and Wilms' tumor was examined with in situ hybridization. In developing kidney, IGF-II was primarily expressed in blastemal cells and lost with their differentiation. In triphasic Wilms' tumor, a similar relationship was found. But in a monomorphous Wilms', tumor cells with epithelial differentiation expressed IGF-II mRNA. These data suggest that IGF-II may be involved in fetal nephrogenesis, that its expression is inversely coupled to normal epithelial differentiation, and that this differentiation may be aberrantly regulated in Wilms' tumor.     

Neoplasms causing nonhyperinsulinemic hypoglycemia

Non-islet cell tumor hypoglycemia (NICTH) is uncommon. Many of the tumors associated with NICTH are mesenchymal tumors, although carcinomas are also involved in some cases. High serum levels of insulin-like growth factor II (IGF-II) have been associated with NICTH. Analysis of 4 pituitary tumors, 2 adrenocortical tumors, 42 solitary fibrous tumors, and 23 other mesenchymal tumors for IGF-II protein and mRNA showed that most mesenchymal tumors expressed IGF-II protein and mRNA, although only 4 of 48 patients had associated hypoglycemia. Tumor size was related to IGF-II production. Tumors less than 5 cm were usually negative for IGF-II mRNA, whereas 92.3% of tumors greater than 9 cm were positive for IGF-II mRNA. These results show that IGF-II mRNA and protein can be readily detected in many tumors, even when the tumors are not associated with clinical hypoglycemia. The expression and production of this growth factor cannot accurately predict patients with clinical evidence of hypoglycemia. Presented in part at the Endocrine Pathology Society Companion Meeting of the United States and Canadian Academy of Pathology Meeting, in San Francisco, CA, March 20, 1999     

Localization of insulin-like growth factor-II mRNA in human pituitary adenomas

Insulin-like growth factors (IGFs) have been reported to promote cell proliferation in many tumours, but their contribution to pituitary adenoma development and growth has not been characterized. We report the presence of insulin-like growth factor II (IGF-II) mRNA in pituitary adenomas using in situ hybridization (ISH). The intensity of IGF-II hybridization signal was correlated with adenoma type, and the presence of Ki-67. Among the 109 adenomas examined, 55 (50.4%) were positive for IGF-II mRNA. All acidophil stem cell, functioning corticotrophic and plurihormonal adenomas contained the message; a high incidence of signal was found among sparsely (7/8) and densely (4/6) granulated growth hormone (GH) cell adenomas, mixed GH cell–prolactin (PRL) cell adenomas (6/7), thyrotrophic (4/6) and null-cell (6/7) adenomas. Less frequently, IGF-II mRNA was localized in mammosomatotrophic, silent subtype 3, gonadotrophic, and oncocytic adenomas, whereas all sparsely granulated PRL cell adenomas and silent corticotrophic adenomas of subtypes 1 and 2 were negative. The MIB-1 labelling index was significantly higher in adenomas with a moderate to intense IGF-II signal than in adenomas with weak or no signal. The results suggest that IGF-II, when highly expressed, may have a role in pituitary adenoma proliferation.     

Rearrangements and increased expression of cyclin D1 (CCND1) in neuroblastoma

Cyclin D1 regulates G1 cell cycle progression by controlling the phosphorylation of the retinoblastoma protein. This pathway is frequently deregulated in many malignancies. In neuroblastoma, however, no consistent G1 cell cycle checkpoint aberrations have been found. We examined the possible deregulation of cyclin D1 (CCND1) in this tumor. mRNA expression profiles of neuroblastoma generated by SAGE (Serial Analysis of Gene Expression) revealed a high expression of CCND1 in a subset of neuroblastoma cell lines and tumors. The CCND1 expression level can be 0.3% of the total cellular mRNA. Northern blot analysis of CCND1 expression showed a relative overexpression in 16 of 23 neuroblastoma cell lines and 10 of 15 tumor samples. In the majority of cases, the high CCND1 mRNA levels also led to high CCND1 protein levels. In the search for mechanisms causing this relative overexpression, we screened for amplifications and rearrangements of CCND1. Five amplifications were found in 202 neuroblastoma tumors and cell lines. Analysis of the 3'-UTR of CCND1 showed a rearrangement in 1 of 96 tumors. These clonal aberrations of CCND1 together with the high expression suggest a role for deregulated CCND1 activity in neuroblastoma tumorigenesis.     

Co-localisation of insulin-like growth factor II and the proliferation marker MIB1 in hepatocellular carcinoma cells.

AIM: To assess the effect of insulin-like growth factor II (IGF-II) on proliferation of hepatocellular carcinoma (HCC) cells. METHODS: Expression of IGF-II mRNA and protein was detected in 10 archival HCC specimens using in situ hybridisation and immunohistochemistry, respectively. Expression of the Ki-67 antigen, a proliferation marker, was determined immunohistochemically on the same sections. RESULTS: Increased expression of IGF-II mRNA and protein was detected in five of the 10 HCCs in cells located at the periphery of tumour nests. The pattern of localisation of IGF-II was almost identical with that of Ki-67 antigen. CONCLUSIONS: Most of the Ki-67 antigen positive cells co-expressed IGF-II, suggesting that IGF-II may act as an autocrine or paracrine growth factor, and may play an important role in the development of HCC.     

Nerve growth factor receptor expression in peripheral and central neuroectodermal tumors, other pediatric brain tumors, and during development of the adrenal gland.

Nerve growth factor (NGF) is important to the survival, development, and differentiation of neurons. Its action is mediated by a specific cell surface transmembrane glycoprotein, nerve growth factor receptor (NGFR). In this study, NGFR expression by human fetal and adult adrenal medullary tissue, peripheral nervous system (PNS) neuroectodermal tumors (neuroblastoma, ganglioneuroblastoma, ganglioneuroma), pediatric primitive neuroectodermal tumors (PNETs) of the central nervous system (CNS), and CNS gliomas was examined by an immunohistochemical technique. Sixty-nine tumors in total were probed in this manner. Nerve growth factor receptor immunoreactivity was confined to nerve fibers and clusters of primitive-appearing cells in the fetal adrenal, and to nerve fibers and ganglion cells of the adult adrenal medulla; adrenal chromaffin cells were negative. In PNS neuroectodermal tumors, there was NGFR expression in tumor cells of 6 of 11 neuroblastomas and 6 of 6 ganglioneuroblastomas or ganglioneuromas. Thirteen of thirty-five CNS PNETs showed NGFR positivity. In most CNS PNETs, NGFR was restricted to scattered single or small groups of cells, but two tumors with astroglial differentiation showed much more extensive immunoreactivity. Most astrocytomas (11 of 14) and all ependymomas (3 of 3) were intensely NGFR positive.     

Insulin-like growth factor II acts through an endogenous growth pathway regulated by imprinting in early mouse embryos.

We present evidence that insulin-like growth factor II (IGF-II) mediates growth in early mouse embryos and forms a pathway in which imprinted genes influence development during preimplantation stages. mRNA and protein for IGF-II were expressed in preimplantation mouse embryos, but the related factors IGF-I and insulin were not. IGF-I and insulin receptors and the IGF-II/mannose-6-phosphate receptor were expressed. Exogenous IGF-II or IGF-I increased the cell number in cultured blastocysts, but a mutant form of IGF-II that strongly binds only the IGF-II receptor did not. Reduction of IGF-II expression by antisense IGF-II oligonucleotides decreased the rate of progression to the blastocyst stage and decreased the cell number in blastocysts. Preimplantation parthenogenetic mouse embryos expressed mRNA for the IGF-II receptor but not for either IGF-II ligand or the IGF-I receptor, indicating that the latter genes are not expressed when inherited maternally. These data imply that some growth factors and receptors, regulated by genomic imprinting, may control cell proliferation from the earliest stages of embryonic development.     

Biological role of anaplastic lymphoma kinase in neuroblastoma

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor originally identified as part of the chimeric nucleophosmin-ALK protein in the t(2;5) chromosomal rearrangement associated with anaplastic large cell lymphoma. We recently demonstrated that the ALK kinase is constitutively activated by gene amplification at the ALK locus in several neuroblastoma cell lines. Forming a stable complex with hyperphosphorylated ShcC, activated ALK modifies the responsiveness of the mitogen-activated protein kinase pathway to growth factors. In the present study, the biological role of activated ALK was examined by suppressing the expression of ALK kinase in neuroblastoma cell lines using an RNA interference technique. The suppression of activated ALK in neuroblastoma cells by RNA interference significantly reduced the phosphorylation of ShcC, mitogen-activated protein kinases, and Akt, inducing rapid apoptosis in the cells. By immunohistochemical analysis, the cytoplasmic expression of ALK was detected in most of the samples of neuroblastoma tissues regardless of the stage of the tumor, whereas significant amplification of ALK was observed in only 1 of 85 cases of human neuroblastoma samples. These data demonstrate the limited frequency of ALK activation in the real progression of neuroblastoma.     

Atypical teratoid/rhabdoid tumor of the CNS: cytopathology and immunohistochemistry of insulin-like growth factor-II, insulin-like growth factor receptor type 1, cathepsin D, and Ki-67.

Insulin-like growth factor (IGF)-II is a potent growth factor, normally controlled by a number of other factors, including IGF binding proteins and IGF binding protein proteases. In general, the latter increase the bioavailability of IGF by cleaving IGF binding proteins. Cathepsin D (an IGF binding protein protease) was also implicated in tumor invasion. Although IGF-II was implicated in the pathogeneses of various childhood neoplasms, its significance in the pathogenesis of atypical teratoid/rhabdoid tumor of the central nervous system (ATRT-CNS) was not studied to date. We present clinicopathologic features of two cases of ATRT-CNS. In addition, formalin-fixed, paraffin-embedded tissue sections were stained immunohistochemically for IGF-II, IGF receptor type 1, cathepsin D, and Ki-67. Both tumors demonstrated diffuse strong cytoplasmic positivity for IGF-II, diffuse cytoplasmic and focal membranous positivity for IGF receptor type I, and diffuse cytoplasmic positivity for cathepsin D. The Ki-67 labeling indices were 10.0% and 1.4%. We conclude that ATRT-CNS cells express both IGF-II and IGF receptor type 1, supporting the hypothesis that autocrine/paracrine stimulation of cell growth by IGF-II might be one mechanism involved in ATRT-CNS tumorigenesis. Cathepsin D expressed by the tumor cells might also be involved in both tumor cell invasion and growth. The exact pathogenesis of ATRT-CNS remains to be elucidated.     

Synthesis and secretion of insulin-like growth factor II by a leiomyosarcoma with associated hypoglycemia

We describe a case of recurrent hypoglycemia apparently caused by secretion of insulin-like growth factor II (IGF-II) by a leiomyosarcoma. A 67-year-old woman presented with recurrent severe hypoglycemia and a large mass in the thorax. During hypoglycemia, plasma cortisol was elevated, but insulin and growth hormone levels were low. After resection of a large leiomyosarcoma, the hypoglycemia resolved. After an eight-year remission, both the tumor and symptomatic hypoglycemia recurred. During a second operation a second large tumor was removed, with relief of the patient's hypoglycemia. The tumor contained high concentrations of IGF-II mRNA and 2100 ng of IGF-II immunoreactive peptide per gram. Filtration through a BioGel P-60 gel column established that 77 percent of the IGF-II was present as a larger molecule, demonstrating incomplete processing of the pro-IGF-II peptides. A similar fraction of high-molecular-weight IGF-II was present in the serum, indicating that the tumor was the chief source of IGF-II. The high-molecular-weight IGF-II found in both the tumor and serum was fully reactive with the IGF-II receptor. Radioimmunoassay showed that the concentrations of insulin-like growth factor I (IGF-I) in tumor and serum were low, suggesting feedback inhibition of growth hormone secretion by IGF-II. Eight months after reoperation, plasma concentrations of IGF-I and IGF-II were normal, and high-molecular-weight IGF-II was virtually undetectable. We conclude that the most likely cause of this patient's recurrent hypoglycemia was IGF-II produced by the leiomyosarcoma.     

Pituitary Insulin: Insulin-Like Growth Factors

Insulin-like growth factor (IGF)-I and IGF-II peptides as well as their mRNAs are produced in many organs, including the pituitary. Although IGF-I and IGF-II peptides are localized in endocrine cells of the anterior pituitary, IGF-I mRNA can be detected throughout the adenohypophysis, and IGF-II mRNA is abundant in intermediate and neural lobes. It is well-established that both circulating and intrinsic IGF-I are negative regulators of pituitary GH production. Other functions of intrinsic IGFs in normal and tumorous pituitary are just emerging. IGF-I may play a role in the stimulation of PRL synthesis and mediation of proliferative effects of estrogen on lactotroph. Compared with IGF-I, the function of IGF-II has not been clarified so far. The growth-promoting actions of IGFs are mediated by IGF-I receptor. The role of local and circulating IGFBPs in pituitary are not yet documented. IGFBPs in other tissues have inhibitory and stimulatory effects on IGFs, and can act independently from the IGFs as well. IGFs have been reported to promote cell proliferation in many tumors. However, the extent to which IGFs contribute to pituitary tumor development and growth remains obscure.     

Immunocytochemical demonstration of IGF-II immunoreactivity in human phaeochromocytoma and extra-adrenal abdominal paraganglioma.

Insulin-like growth factor (IGF)-II immunoreactivity was demonstrated in 14 adrenal glands, six retroperitoneal paraganglia, 18 phaeochromocytomas, and six extra-adrenal abdominal paragangliomas through the use of a monoclonal antibody against rat IGF-II which cross-reacts with human IGF-II. A subpopulation of parenchymal cells in normal adrenal medulla and in retroperitoneal paraganglia was positively immunolabelled. Eighteen cases with phaeochromocytoma including two malignant and four multiple endocrine neoplasias all contained numerous IGF-II-immunoreactive tumour cells. All six extra-adrenal abdominal paragangliomas contained numerous immunoreactive tumour cells. Almost all the other normal human adult tissues examined failed to immunolabel. These results indicate that IGF-II-like immunoreactivity is widely expressed in normal and neoplastic human paraganglionic tissues, although its biological significance in these tissues has not been determined.     

Up-regulation of insulin-like growth factor axis components in human primary prostate cancer correlates with tumor grade

There is an evidence that components of the insulin-like growth factor (IGF)-signaling pathway are involved in the development and progression of prostate cancer. The aim of the present study was to provide a comprehensive analysis of the expression levels of proteins of the IGF axis in prostate cancer. We studied expression of the ligands IGF-I and IGF-II, the inhibitory IGF binding protein-3, the type I IGF receptor (IGF-IR), and the downstream mediator insulin receptor substrate-1 by immunohistochemistry in 56 tissue specimens (28 low-grade and 28 high-grade prostate adenocarcinomas). Protein expression in tumor areas, prostatic intraepithelial neoplasias (PINs), and adjacent benign prostatic tissue were evaluated regarding staining intensity and fraction of positive cells. An immunoreactivity score was established from staining intensity and fraction of positive cells, and correlated with the prognostic clinicopathologic parameters prostate-specific antigen serum levels, Gleason score, and TNM stage. The expression levels of all proteins investigated, except IGF binding protein-3, were up-regulated in PIN and in cancer. IGF-I and IGF-II expression showed a higher expression in high-grade compared with low-grade tumor areas. IGF-I and IGF-II and insulin receptor substrate-1 immunoreactivity was higher in tumors from patients with preoperative prostate-specific antigen serum levels 10 ng/mL or greater, and IGF-II expression was correlated with Gleason score. The data indicate significant alterations in the IGF system as prostate cancer develops. Differential expression of growth-stimulating components of the IGF system may be associated with the malignant phenotype and more aggressive tumor behavior. Expression of IGFs, especially IGF-II, may be predictors of the outcome of prostate cancer.