Cynthia M. van Golen

Insulin-like growth factor-I signaling in human neuroblastoma cells

Neuroblastoma is a heterogeneous tumor consisting of N (neuronal) and S (stromal) cells. We report that more tumorigenic and motile N cells express higher levels of IGF-I receptor (IGF-IR) than less tumorigenic, more adherent S cells. Shc, one of the two major docking partners of IGF-IR, is equally expressed in N and S cell lines. IGF-I treatment phosphorylates Shc in N cells, but only weakly activates Shc in S cells. Expression of the second partner, insulin receptor substrate (IRS), is cell type specific. S cells exclusively express IRS-1 that undergoes sustained phosphorylation by IGF-I. In contrast, N cells express IRS-2 that is transiently phosphorylated by IGF-I. Downstream of IRS-2 and Shc, IGF-I treatment results in strong activation of Akt and MAPK in N cells and activation of both pathways is required for IGF-I-mediated differentiation. Only IGF-IR activation of phosphatidylinositol-3 kinase is required for tumor edge ruffling in N and S cells, with stimulation of focal adhesion kinase (FAK) and paxillin. This detailed understanding of the ‘biochemical signature’ of N and S cells provides the background needed to target and disrupt specific IGF signaling pathways in an attempt to develop more effective therapies.

Insulin-like growth factor I is the key growth factor in serum that protects neuroblastoma cells from hyperosmotic-induced apoptosis

Neuroblastoma is a childhood tumor of the peripheral nervous system that remains largely uncurable by conventional methods. Mannitol induces apoptosis in neuroblastoma cell types and insulin‐like growth factor I (IGF‐I) protects these cells from hyperosmotic‐induced apoptosis by affecting apoptosis‐regulatory proteins. In the current study, we investigate factors that enable SH‐SY5Y neuroblastoma cells to survive in the presence of an apoptotic stimulus. When SH‐SY5Y cells are exposed to high mannitol concentrations, more than 60% of the cells are apoptotic within 48 h. Normal CS prevents hyperosmotic‐induced apoptosis in a dose‐dependent manner, with 0.6% CS protecting 50% of the cells, and 3% CS rescuing more than 70% of the cells from apoptosis. Serum also delays the commitment point for SH‐SY5Y cells from 9 h to 35 h. A survey of several growth factors, including epidermal growth factor (EGF), platelet‐derived growth factor (PDGF), nerve growth factor (NGF), fibroblast growth factor (FGF), and IGF‐I reveals that IGF‐I is a component of serum necessary for protection of neuroblastoma cells from death. Mitochondrial membrane depolarization occurs in greater than 40% of the cells after mannitol exposure and caspase‐3 activation is increased in high mannitol conditions after 9 h. IGF‐I blocks both the mitochondrial membrane depolarization and caspase‐3 activation normally induced by hyperosmotic treatment in neuroblastoma cells. Our results suggest that (1) IGF‐I is a key factor in serum necessary for protection from death and (2) IGF‐I acts upstream from the mitochondria and the caspases to prevent apoptosis in human neuroblastoma. J. Cell. Physiol. 182:24–32, 2000.

Insulin-Like Growth Factor-I Receptor Expression Regulates Neuroblastoma Metastasis to Bone

Neuroblastoma is a pediatric tumor that preferentially metastasizes to bone. Patients with bone metastases have a mortality rate >93%, indicating a need for novel treatment targets. Our laboratory has shown that type I insulin-like growth factor receptor (IGF-IR) expression and activation regulate neuroblastoma cell proliferation, motility, invasion, and survival, and that expression of the IGF-IR correlates with neuroblastoma tumorigenicity. Bone expresses large amounts of IGF ligands, and the IGF system is required for normal bone physiology. The current study addresses the role of the IGF system in neuroblastoma metastasis to bone. Upon reaching the bone marrow through the circulation, neuroblastoma cells must dock at the bone marrow endothelium, extravasate into the bone microenvironment, and destroy bone tissue to allow for tumor growth. This report examines the effects of high IGF-IR expression on neuroblastoma cell interaction with bone. The current data show that neuroblastoma cells with high IGF-IR expression, either endogenously or through transfection, adhere to human bone marrow endothelial cells and subsequently migrate toward both IGF-I and human bone stromal cells. High IGF-IR-expressing neuroblastoma cells adhere tightly to bone stromal cells, flatten, and extend processes. When neuroblastoma cells are injected directly into the tibiae of mice, those cells with increased IGF-IR form both osteolytic lesions within the tibiae and secondary tumors within other sites. These results support the hypothesis that IGF-IR expression in neuroblastoma cells increases tumor cell interaction with the bone microenvironment, resulting in greater formation of metastases.

N-Myc and Bcl-2 coexpression induces MMP-2 secretion and activation in human neuroblastoma cells.

Neuroblastoma is a peripheral nervous system tumor that accounts for 8–10% of all solid childhood tumors. N-Myc is the most reliable prognostic indicator for neuroblastoma. Bcl-2 is detected in 40–60% of primary neuroblastoma tumors and demonstrates anti-apoptotic action by conferring resistance to chemotherapy and radiation treatment. In neuroblastoma cell lines, the coexpression of N-Myc and Bcl-2 leads to increased tumorigenic properties. Matrix metalloproteinases (MMPs) are endopeptidases that degrade a wide range of basement membrane components, a process important for tumor invasion. This study investigates the effect of N-Myc and Bcl-2 on MMP expression and activation. MMP-2 expression and secretion are increased in SHEP neuroblastoma cells expressing Bcl-2 alone (SHEP/Bcl-2 cells) or both N-Myc and Bcl-2 (SHEP/N-Myc/Bcl-2 cells). MMP-2 activity is increased in the SHEP/N-Myc/Bcl-2 cells yet remains unchanged in SHEP/Bcl-2 cells. TIMP-2 expression is high in SHEP/Bcl-2 cells, which likely inhibits MMP-2 activity, and absent in SHEP/N-Myc/Bcl-2 cells, allowing MMP-2 activity. Invasion is increased in SHEP/N-Myc/Bcl-2 cells and prevented by the use of a pharmacologic MMP-2 inhibitor. These data imply that N-Myc and Bcl-2 cooperate to increase the expression, secretion, and activation of MMP-2, which likely leads to a more tumorigenic phenotype due to increased MMP-2 mediated invasion.

N-Myc overexpression leads to decreased β1 integrin expression and increased apoptosis in human neuroblastoma cells

Neuroblastoma is a childhood tumor thought to arise through improper differentiation of neural crest cells. Increased N-Myc expression in neuroblastoma indicates highly malignant disease and poor patient prognosis. N-myc enhances cell growth, insulin-like growth factor type I receptor (IGF-IR) expression, and tumorigenicity in combination with Bcl-2. Despite these effects, N-Myc overexpression in SHEP neuroblastoma cells (SHEP/N-Myc cells) increases serum-withdrawal and mannitol-induced apoptosis. Although we have previously shown a protective effect of IGF-I in SHEP cells, in SHEP/N-Myc cells IGF-I rescue from mannitol-induced apoptosis is prevented. N-Myc overexpression has little effect on IGF-IR signaling pathways, but results in increased Akt phosphorylation when Bcl-2 is coexpressed. A loss of integrin-mediated adhesion promotes apoptosis in many systems. SHEP/N-Myc cells have dramatically less β1 integrin expression than control cells, consistent with previous reports. β1 integrin expression is decreased in more tumorigenic neuroblastoma cells lines, including IMR32 and SH-SY5Y cells. Reintroduction of β1 integrin into the N-Myc-overexpressing cells prevents mannitol-mediated apoptosis. We speculate that N-Myc repression of β1 integrin expression leads to a less differentiated phenotype, resulting in increased growth and tumorigenesis if properly supported or apoptosis if deprived of growth sustaining molecules.

Degradation and Dephosphorylation of Focal Adhesion Kinase During Okadaic Acid-Induced Apoptosis in Human Neuroblastoma Cells

Focal adhesion kinase (FAK) prevents apoptosis in many cell types. We have reported that tyrosine residues in FAK are dephosphorylated and FAK is degraded during mannitol-induced apoptosis in human neuroblastoma cells. Several studies suggest that FAK dephosphorylation and degradation are separate events. The current study defines the relationship between FAK dephosphorylation and degradation in neuroblastoma cells using okadaic acid (OA). OA, a serine phosphatase inhibitor, promotes serine/threonine phosphorylation, which in turn blocks tyrosine phosphorylation. OA induced focal adhesion loss, actin cytoskeleton disorganization, and cellular detachment, which corresponded to a loss of FAK Tyr397 phosphorylation. These changes preceded caspase-3 activation, Akt and MAP kinase activity loss, protein ubiquitination, and cellular apoptosis. Insulin-like growth factor-I prevented mannitol-induced, but not OA-induced, substrate detachment and FAK Tyr397 dephosphorylation, and the effects of OA on FAK Tyr397 phosphorylation were irreversible. The proteolytic degradation of FAK is temporally distinct from its tyrosine dephosphorylation, occurring when apoptotic pathways are already initiated and during a generalized destruction of signaling proteins. Therefore, agents resulting in the dephosphorylation of FAK may be beneficial for therapeutic treatment, irrespective of FAK protein levels, as this may result in apoptosis, which cannot be prevented by growth factor signaling.

Inflammatory Breast Cancer Stem Cells: Contributors to Aggressiveness, Metastatic Spread and Dormancy

Cancer stem cell populations have been identified for several types of cancers and suggest a way for tumor cells to be resistant to therapies. Further, because of the longevity, endurance and replicative potential of cancer cells with stem-like properties, other malignant attributes such as recurrence after long periods of dormancy can also be explained. Inflammatory breast cancer (IBC) is a unique and aggressive form of breast cancer that has a clinical course unlike other forms of breast cancer. The main hallmark of IBC is prolific invasion of the dermal lymphatic vessels by tumor emboli leading to rapid metastasis of the disease. Despite an extremely aggressive treatment approach, the majority of women with IBC present with disease recurrence suggesting the presence of chemo resistant and/or dormant breast cancer cells. Current evidence suggests that IBC tumor emboli contain distinct populations of cells with stem cell-like properties. Thus, specific targeting of these stem cell-like cancer cells may be the key to effectively treating IBC.