Patricia Pennisi

Insulin-Mediated Acceleration of Breast Cancer Development and Progression in a Nonobese Model of Type 2 Diabetes

Epidemiologic studies suggest that type 2 diabetes (T2D) increases breast cancer risk and mortality, but there is limited experimental evidence supporting this association. Moreover, there has not been any definition of a pathophysiological pathway that diabetes may use to promote tumorigenesis. In the present study, we used the MKR mouse model of T2D to investigate molecular mechanisms that link T2D to breast cancer development and progression. MKR mice harbor a transgene encoding a dominant-negative, kinase-dead human insulin-like growth factor-I receptor (IGF-IR) that is expressed exclusively in skeletal muscle, where it acts to inactivate endogenous insulin receptor (IR) and IGF-IR. Although lean female MKR mice are insulin resistant and glucose intolerant, displaying accelerated mammary gland development and enhanced phosphorylation of IR/IGF-IR and Akt in mammary tissue, in the context of three different mouse models of breast cancer, these metabolic abnormalities were found to accelerate the development of hyperplastic precancerous lesions. Normal or malignant mammary tissue isolated from these mice exhibited increased phosphorylation of IR/IGF-IR and Akt, whereas extracellular signal-regulated kinase 1/2 phosphorylation was largely unaffected. Tumor-promoting effects of T2D in the models were reversed by pharmacological blockade of IR/IGF-IR signaling by the small-molecule tyrosine kinase inhibitor BMS-536924. Our findings offer compelling experimental evidence that T2D accelerates mammary gland development and carcinogenesis,and that the IR and/or the IGF-IR are major mediators of these effects.

Serum complexes of insulin-like growth factor-1 modulate skeletal integrity and carbohydrate metabolism

Serum insulin‐like growth factor (IGF) ‐1 is secreted mainly by the liver and circulates bound to IGF‐binding proteins (IGFBPs), either as binary complexes or ternary complexes with IGFBP‐3 or IGFBP‐5 and an acid‐labile subunit (ALS). The purpose of this study was to genetically dissect the role of IGF‐1 circulatory complexes in somatic growth, skeletal integrity, and metabolism. Phenotypic comparisons of controls and four mouse lines with genetic IGF‐1 deficits— liver‐specific IGF‐1 deficiency (LID), ALS knockout (ALSKO), IGFBP‐3 (BP3) knockout, and a triply deficient LID/ALSKO/BP3 line—produced several novel findings. 1) All deficient strains had decreased serum IGF‐1 levels, but this neither predicted growth potential or skeletal integrity nor defined growth hormone secretion or metabolic abnormalities. 2) IGF‐1 deficiency affected development of both cortical and trabecular bone differently, effects apparently dependent on the presence of different circulating IGF‐1 complexes. 3) IGFBP‐3 deficiency resulted in increased linear growth. In summary, each IGF‐1 complex constituent appears to play a distinct role in determining skeletal phenotype, with different effects on cortical and trabecular bone compartments.— Yakar, S., Rosen, C. J., Bouxsein, M. L., Sun, H., Mejia, W., Kawashima, Y., Wu, Y., Emerton, K., Williams, V., Jepsen, K., Schaffler, M. B., Majeska, R. J., Gavrilova, O., Gutierrez, M., Hwang, D., Pennisi, P., Frystyk, J., Boisclair, Y., Pintar, J., Jasper, H., Domene, H., Cohen, P., Clemmons, D., LeRoith, D. Serum complexes of insulin‐like growth factor‐1 modulate skeletal integrity and carbohydrate metabolism. FASEB J. 23, 709–719 (2009)

Differential impact of simple childhood obesity on the components of the growth hormone-insulin-like growth factor (IGF)-IGF binding proteins axis.

Simple childhood obesity is characterized by normal or even accelerated growth in spite of reduced growth hormone (GH) secretion. There are conflicting reports on the effects of obesity upon components of the GH-insulin-like growth factor-I (IGF-I)-IGF binding proteins (IGFBPs) system. In the present study we aimed to determine GH, IGF-I, IGFBP-3 and IGFBP-2 as well as some of the less explored components of this axis (IGFBP-3 proteolytic activity, IGFBP-3 plasma fragments, and total acid labile subunit [ALS]) in 22 obese and 17 age-matched control children. We also evaluated not only total GH binding protein (GHBP) serum levels but also GHBP bound to GH (complexed) in both groups. Obese and control groups strongly differed in BMI (obese: 4.7 +/- 0.36 vs control: 0.37 +/- 0.25 SDS, p <0.0001). In the obese group, we found lower GH serum levels, but normal serum levels of GH-GHBP complex, IGF-I, IGFBP-3, IGF-I/IGFBP-3 molar ratio, IGFBP-3 proteolytic activity, IGFBP-3 plasma fragments and total ALS. Obese children presented higher total circulating GHBP (6.0 +/- 0.44 vs 2.9 +/- 0.29 nmol/l, p <0.001) and insulin levels (10.5 +/- 1.5 vs 5.1 +/- 0.8 mU/l, p <0.001), while IGFBP-2 (4.6 +/- 0.5 vs 6.6 +/- 0.7%, p <0.05) and the ratio IGFBP-2/IGF-I (0.032 +/- 0.019 vs 0.095 +/- 0.01, p = 0.013) were lower than in controls. BMI and insulin were directly, and IGFBP-2 serum levels inversely, correlated to total GHBP serum levels when multiple regression analysis was performed (r = 0.74, p <0.001). By stepwise regression analysis, insulin (r = -0.37, p <0.05) and BMI (r = -0.52, p <0.01) inversely determined IGFBP-2. In summary, obese children present normal growth in spite of reduced GH secretion, probably because the combination of increased total GHBP and normal GH-GHBP complex serum levels (suggesting increased GH receptor [GHR] number and a normal serum GH reservoir, respectively) allow for the achievement of normal levels of IGF-I, IGFBP-3, IGFBP-3 proteolytic activity, IGFBP-3 plasma fragments and total ALS. Reduced IGFBP-2 serum levels and a lower ratio of IGFBP-2/IGF-I in obese children may suggest an increase of tissue IGF-I bioavailability, thus promoting its action. Normal IGF-I and GH availability may be contributing to maintain normal growth in obese children.

Clinical relevance of systemic and local IGF-I.

The insulin-like growth factor family of ligands, receptors and binding proteins are critical for many normal physiological functions. These include normal development during fetal and post-natal development and maintenance of organ function in adult life. Circulating IGF-I is produced primarily by the liver under GH control, whereas the production of tissue IGF-I has other controls. Recent studies have demonstrated that both circulating and tissue IGF-I are important for maintaining the normal structure-function of complex organs such as bone. Circulating IGF-I is important for maintaining ambient GH levels; in its absence GH elevation is seen leading to insulin resistance. In addition, low levels of circulating IGF-I retard the progression and metastatic potential of a number of cancers.

Effect of deflazacort versus methylprednisone on growth, body composition, lipid profile, and bone mass after renal transplantation

Abstract Kidney function, growth velocity, weight/ height ratio, body composition, lipid profile, and bone mass were studied in a randomized, multicenter trial of deflazacort versus methylprednisone in 27 prepubertal patients with kidney transplantation. Methylprednisone (0.20±0.03) was replaced by deflazacort (13 patients, 0.30±0.03 mg/kg per day). After 12 months, creatinine clearance decreased significantly only during methylprednisone therapy. Growth velocity increased only in patients treated with deflazacort from 3.3±0.6 to 5.6±0.5 cm/year. Serum levels of several components of the insulin-like growth factor axis did not change. Weight/height ratio was increased in methylprednisone-treated patients (P<0.05) and decreased in deflazacort-treated patients (P<0.005). Lean body mass increased in both groups (P<0.005). Fat body mass and serum leptin increased only in methylprednisone-treated patients (P<0.025). Total cholesterol and low-density lipoprotein-cholesterol increased in methylprednisone-treated patients by 9.9% (P<0.05) and 12.5% (P<0.025). High-density lipoprotein-cholesterol increased by 21% (P<0.005) and apolipoprotein B decreased by 11% (P<0.005) in deflazacort-treated patients. Total skeleton and lumbar spine bone mineral density decreased in both groups, but at 1 year methylprednisone-treated patients had lost 50% more bone. Bone mineral content decreased only in methylprednisone-treated patients (P<0.01). Our data suggest that substituting deflazacort for maintenance methylprednisone might prevent height loss, excessive bone loss, and fat accumulation; and leads to an improvement in the lipoproteins of these children.

The growth hormone-insulin like growth factor axis revisited: lessons from IGF-1 and IGF-1 receptor gene targeting

We have created a liver-specific igf1 gene-deletion mouse model (LID) with markedly reduced circulating IGF-I levels. They demonstrate that while they have normal growth and development they develop insulin resistance secondary to the elevation of circulating growth hormone. When mated with an acid-labile subunit (ALS) gene-deleted mouse they also show osteopenia suggesting that circulating IGF-I levels play a significant role in bone formation. In a separate transgenic mouse we created a model of severe insulin resistance and type 2 diabetes by the overexpression of a dominant-negative IGF-I receptor in skeletal muscle. In this model we show that lipotoxicity plays a major role in the progression of the disease and is affected by treatment with a fibrate, which reverses the insulin resistance and diabetic state. These models are therefore very useful in studying human physiology and disease states.

Effects of deflazacort immunosuppression on long-term growth and growth factors after renal transplantation.

Abstract. Deflazacort is an oxazoline compound derived from prednisolone. We studied changes in kidney function, growth velocity, weight/height ratio, insulin-like growth factor (IGF-I), and IGF binding proteins before and after substitution of deflazacort for methylprednisone in 27 transplanted patients aged 3.1 – 20 years. Methylprednisone (mean±SEM 0.17±0.01 mg/kg per day) was replaced by deflazacort (0.29±0.01 mg/kg per day) for a period of 1 – 5 years. Calculated creatinine clearance did not change significantly during deflazacort treatment. Growth velocity increased from 2.6±0.5 cm/year to 5.2±0.7 cm/year (1st year) in 14 prepubertal patients. After 4 years of deflazacort treatment, height standard deviation score for chronological age did not change in 7 prepubertal patients. Mean weight/height ratio decreased by 50% (1st year) and remained reduced during follow-up. Serum IGF-I, IGF binding protein -3 (IGFBP3), IGF/IGFBP3 molar ratio, and IGF-I and -II binding capacities showed no significant change; however in 5 of 6 patients IGFBP2 decreased during deflazacort therapy. Our findings suggest that immunosuppressive treatment with deflazacort is as effective as methylprednisone and may lead to an improvement in the growth prognosis of children with renal transplantation.

IGF-I Regulates Pheochromocytoma Cell Proliferation and Survival In Vitro and In Vivo

IGFs are involved in malignant transformation and growth of several tissues, including the adrenal medulla. The present study was designed to evaluate the impact of IGF-I on pheochromocytoma development. We used a murine pheochromocytoma (MPC) cell line (MPC4/30) and an animal model with a reduction of 75% in circulating IGF-I levels [liver-IGF-I-deficient (LID) mice] to perform studies in vitro and in vivo. We found that, in culture, IGF-I stimulation increases proliferation, migration, and anchorage-independent growth, whereas it inhibits apoptosis of MPC cells. When injected to control and to LID mice, MPC cells grow and form tumors with features of pheochromocytoma. Six weeks after cell inoculation, all control mice developed sc tumors. In contrast, in 73% of LID mice, tumor development was delayed to 7-12 wk, and the remaining 27% did not develop tumors up to 12 wk after inoculation. LID mice harboring MPC cells and treated with recombinant human IGF-I (LID+) developed tumors as controls. Tumors developed in control, LID, and LID+ mice had similar histology and were similarly positive for IGF-I receptor expression. The apoptotic index was higher in tumors from LID mice compared with those from control mice, whereas vascular density was decreased. In summary, our work demonstrates that IGF-I has a critical role in maintaining tumor phenotype and survival of already transformed pheochromocytoma cells and is required for the initial establishment of these tumors, providing encouragement to carry on research studies to address the IGF-I/IGF-I receptor system as a target of therapeutic strategies for pheochromocytoma treatment in the future.

Type 1 IGF Receptor Localization in Paediatric Gliomas: Significant Association with WHO Grading and Clinical Outcome

Nuclear localization of insulin-like growth factor receptor type 1 (IGF-1R) has been described as adverse prognostic factor in some cancers. We studied the expression and localization of IGF-1R in paediatric patients with gliomas, as well as its association with World Health Organization (WHO) grading and survival. We conducted a single cohort, prospective study of paediatric patients with gliomas. Samples were taken at the time of the initial surgery; IGF-1R expression and localization were characterized by immunohistochemistry (IHC), subcellular fractionation and western blotting. Tumours (47/53) showed positive staining for IGF-1R by IHC. IGF-1R nuclear labelling was observed in 10/47 cases. IGF-1R staining was mostly non-nuclear in low-grade tumours, while IGF-1R nuclear labelling was predominant in high-grade gliomas (p = 0.0001). Survival was significantly longer in patients with gliomas having non-nuclear IGF-1R localization than in patients with nuclear IGF-1R tumours (p = 0.016). In gliomas, IGF-1R nuclear localization was significantly associated with both high-grade tumours and increased risk of death. Based on a prospective design, we provide evidence of a potential usefulness of intracellular localization of IGF-1R as prognostic factor in paediatric patients with gliomas.

VHL Germline Mutations in Argentinian Patients with Clinical Diagnoses or Single Typical Manifestations of Type 1 von Hippel–Lindau Disease

AIMS von Hippel-Lindau (VHL) disease is caused by mutations in the VHL tumor suppressor gene. As tumors that develop in the context of VHL also occur in a sporadic context, the frequency of this syndrome may be underestimated. Our aim was to identify VHL gene mutations in Argentinian patients who fulfilled the clinical criteria for type 1 VHL disease and in patients with VHL-associated manifestations that did not meet these criteria. METHODS We performed a retrospective cohort study, including patients who met current diagnostic criteria for type 1 VHL (Group 1, n = 19) and patients with VHL-associated manifestations that did not meet these criteria (Group 2, n = 21). Genomic DNA was extracted from peripheral blood leukocytes. Mutation analysis involved DNA sequencing, while large deletions were determined by universal primer quantitative fluorescent multiplex polymerase chain reaction (UPQFM-PCR) and multiplex ligation-dependent probe amplification (MLPA) analysis. RESULTS VHL mutations were detected in 16/19 (84.2%) patients in Group 1 and included: gross deletions (4/16); nonsense mutations (6/16); frameshift mutations (4/16); missense mutations (1/16); and splicing mutations (1/16). Three of these mutations were novel. No alterations were found in 3 of 19 VHL patients. In Group 2, one nonsense VHL mutation was detected in a young patient with a solitary central nervous system hemangioblastoma without familial history. A study of 30 first-degree relatives revealed four carriers with VHL mutations. CONCLUSIONS We found three novel mutations in the VHL gene in our population. Our results emphasize the importance of a complete genetic study of VHL to confirm type 1 VHL disease, not only in patients with clinical diagnostic criteria but also in those presenting a single typical manifestation.