IGF-I receptor (IGF-IR) signaling and functions are mediated through the activities of a complex molecular network of positive (e.g., type I IGF) and negative (e.g., the type II IGF receptor, IGF-IIR) effectors. Under normal physiological conditions, the balance between the expression and activities of these molecules is tightly controlled. Changes in this delicate balance (e.g., overexpression of one effector) may trigger a cascade of molecular events that can ultimately lead to malignancy. In recent years, evidence has been mounting that the IGF axis may be involved in human cancer progression and can be targeted for therapeutic intervention. Here we review old and more recent evidence on the role the IGF system in malignancy and highlight experimental and clinical studies that provide novel insights into the complex mechanisms that contribute to its oncogenic potential. Controversies arising from conflicting evidence on the relevance of IGF-IR and its ligands to human cancer are discussed. Our review highlights the importance of viewing the IGF axis as a complex multifactorial system and shows that changes in the expression levels of any one component of the axis, in a given malignancy, should be interpreted with caution and viewed in a wider context that takes into account the expression levels, state of activation, accessibility, and functionality of other interacting components. Because IGF targeting for anticancer therapy is rapidly becoming a clinical reality, an understanding of this complexity is timely because it is likely to have an impact on the design, mode of action, and clinical outcomes of newly developed drugs.

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The role of the IGF system in cancer growth and metastasis: overview and recent insights.

Glioblastomas, like other solid tumors, have extensive areas of hypoxia and necrosis. The importance of hypoxia in driving tumor growth is receiving increased attention. Hypoxia-inducible factor 1 (HIF-1) is one of the master regulators that orchestrate the cellular responses to hypoxia. It is a heterodimeric transcription factor composed of alpha and beta subunits. The alpha subunit is stable in hypoxic conditions but is rapidly degraded in normoxia. The function of HIF-1 is also modulated by several molecular mechanisms that regulate its synthesis, degradation, and transcriptional activity. Upon stabilization or activation, HIF-1 translocates to the nucleus and induces transcription of its downstream target genes. Most important to gliomagenesis, HIF-1 is a potent activator of angiogenesis and invasion through its upregulation of target genes critical for these functions. Activation of the HIF-1 pathway is a common feature of gliomas and may explain the intense vascular hyperplasia often seen in glioblastoma multiforme. Activation of HIF results in the activation of vascular endothelial growth factors, vascular endothelial growth factor receptors, matrix metalloproteinases, plasminogen activator inhibitor, transforming growth factors alpha and beta, angiopoietin and Tie receptors, endothelin-1, inducible nitric oxide synthase, adrenomedullin, and erythropoietin, which all affect glioma angiogenesis. In conclusion, HIF is a critical regulatory factor in the tumor microenvironment because of its central role in promoting proangiogenic and invasive properties. While HIF activation strongly promotes angiogenesis, the emerging vasculature is often abnormal, leading to a vicious cycle that causes further hypoxia and HIF upregulation.

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Hypoxia and the hypoxia-inducible-factor pathway in glioma growth and angiogenesis

Expression of vascular endothelial growth factor (VEGF), a key angiogenic protein, has been linked with pancreatic cancer progression. However, the molecular basis for VEGF overexpression remains unclear. Immunohistochemical studies have indicated that VEGF overexpression coincides with elevated Stat3 activation in human pancreatic cancer specimens. In our study, more than 80% of the human pancreatic cancer cell lines used exhibited constitutively activated Stat3, with Stat3 activation correlated with the VEGF expression level. Blockade of activated Stat3 via ectopic expression of dominant-negative Stat3 significantly suppressed VEGF expression, angiogenesis, tumor growth, and metastasis in vivo. Furthermore, constitutively activated Stat3 directly activated the VEGF promoter, whereas dominant-negative Stat3 inhibited the VEGF promoter. A putative Stat3-responsive element on the VEGF promoter was identified using a protein-DNA binding assay and confirmed using a promoter mutagenesis assay. These results indicate that Stat3 directly regulates VEGF expression and hence angiogenesis, growth, and metastasis of human pancreatic cancer, suggesting that Stat3 signaling may be targeted for treatment of pancreatic cancer.

Stat3 activation regulates the expression of vascular endothelial growth factor and human pancreatic cancer angiogenesis and metastasis

In pugilistic parlance, the one-two punch is a devastating combination of blows, with the first punch setting the stage and the second delivering the knock-out. This analogy can be extended to molecularly targeted cancer therapies, with oncogene addiction serving to set the stage for tumor cell killing by a targeted therapeutic agent. While in vitro and in vivo examples abound documenting the existence of this phenomenon, the mechanistic underpinnings that govern oncogene addiction are just beginning to emerge. Our current inability to fully exploit this weakness of cancer cells stems from an incomplete understanding of oncogene addiction, which nonetheless represents one of the rare chinks in the formidable armor of cancer cells.

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Oncogene addiction: setting the stage for molecularly targeted cancer therapy

Angiogenesis, the process of new blood vessel formation, plays a central role in both local tumor growth and distant metastasis in breast cancer. Extensive laboratory data suggest that angiogenesis plays an essential role in breast cancer development, invasion, and metastasis. Hyperplastic murine breast papillomas and histologically normal lobules adjacent to cancerous breast tissue support angiogenesis in preclinical models, suggesting that angiogenesis precedes transformation of mammary hyperplasia to malignancy. Transfection of tumor cells with angiogenic stimulatory peptides has been shown to increase tumor growth, invasiveness, and metastasis. Conversely, transfection of tumor cells with inhibitors of angiogenesis decreases growth and metastasis. The matrix metalloproteinase (MMP) family of enzymes degrades the basement membrane and extracellular matrix and is associated with a family of endogenous inhibitors, tissue inhibitors ofmetalloproteinases (TIMPs). Under normal physiologic conditions, the MMPs and TIMPs exist in anexquisitebalance.Thisbalance isdisrupted during active angiogenesis. Expression of MMPs increases with the progression from benign to preinvasive, invasive, and metastatic breast cancer and is associated with increasing histologic tumor grade. Microscopic metastases are growth restricted and remain dormant until they undergo an angiogenic switch, presumably a result of further mutation. This angiogenic switch often results in increased expression of MMPs. Hypoxia is a key signal for the induction of angiogenesis. Hypoxia-inducible factors (HIF-1 and HIF-2) are heterodimeric transcription factors consisting of and subunits. The subunit is constitutively expressed while the subunit is protected from degradation only under hypoxic condition. HIF-1 expression progressively increases from normal breast tissue to usual ductal hyperplasia to ductal carcinoma-insitu to invasive ductal carcinoma. HIF-1 expression is higher in poorly differentiated than in well-differentiated lesions and is associated with increased proliferation and expression of the estrogen receptor and vascular endothelial growth factor (VEGF). Similarly, expression of carbonic anhydrase IX, an HIF-1 -dependant enzyme important in pH regulation, is associated with worse relapse-free and overall survival in patients with invasive breast cancer.

https://ascopubs.org/doi/pdf/10.1200/JCO.2005.12.017

Angiogenesis of Breast Cancer

Over-expression of truncated epidermal growth factor receptor (EGFR) occurs in a variety of malignancies including glioblastoma multiforme, breast and lung cancer. The truncation deletes an extracellular domain and results in constitutive activation of the receptor. NIH3T3 cells were transfected with full length or truncated human EGFR and differences in growth rates in vivo and in vitro analysed. A growth advantage was seen for cells expressing mutant receptor compared to full length EGFR in vivo only. Administration of an anti-mutant EGFR antibody to mice transiently reduced the growth rates of mutant tumours, confirming that the mutant receptor itself was important in this enhanced tumorigenicity. This showed that stimuli present in vivo and not in vitro may be contributing to growth. We therefore analysed the regulation of the angiogenic factor vascular endothelial growth factor (VEGF). Although levels of secreted VEGF did not differ significantly between wild-type and mutant EGFR cell lines when grown in vitro under normoxic conditions, following exposure to 0.1% hypoxia levels of VEGF produced by mutant cells increased 3.5-6.6 fold compared to 2 or less for full length EGFR cells. The fold induction was influenced by experimental conditions, including cell confluence and percentage of fetal bovine serum, but was consistently higher for mutant cell lines. The increase in VEGF under hypoxic conditions was blocked by the addition of PI3 kinase inhibitors, indicating that the latter pathway is important in the hypoxic stress response. Basal levels were not affected. Addition of insulin-like growth factor-1 also increased levels of VEGF under normoxic conditions in the mutant cells and no further increase was seen when added to cells exposed to 0.1% oxygen, indicating that levels of VEGF were already maximally stimulated. These results show that the mutant EGFR interacts with other growth factors and hypoxia to regulate VEGF via a PI3 kinase pathway, and suggests a specific role for anti-mutant EGFR antibodies and PI3 kinase inhibitors as therapy of this specific tumour target.

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Mutant epidermal growth factor receptor enhances induction of vascular endothelial growth factor by hypoxia and insulin-like growth factor-1 via a PI3 kinase dependent pathway.

Abstract We have used Basic Fibroblast Growth Factor (FGF2) transgenic mice as experimental models for human X-linked hypophosphatemia (XLH)-related degenerative osteoarthritis (OA) to investigate the pathogenesis of the disease and to test potential pharmacotherapies for treatment. This study tested the efficacy of BJG398, a small molecule fibroblast growth factor receptor tyrosine kinase (FGFRTK) inhibitor, to rescue the knee joint osteoarthritis phenotype in High Molecular Weight fibroblast growth factor 2 transgenic (HMWTgFGF2) mice. BJG398 was administered in vivo to 8-month-old female HMWTgFGF2 mice for six weeks. Histomorphometry, immunohistochemistry and micro-CT were used to examine the knee joints in BGJ398-treated and control mice. We assessed: Fibroblast Growth Factor 23 (FGF23) expression and FGFR1 activity; Matrix metalloproteinase 13 (MMP13) and Aggrecanase2 (ADAMTS5) expression; then signaling by SMAD1/5/8-pSMAD6, pERK1/2 and Runt-related transcription factor 2 (RUNX2). Using PrimePCR arrays, we identified a contributing role for major target genes in the TGFB/BMP2 signaling pathway that were regulated by BGJ398. BGJ398 inhibited HMWFGF2/FGF23-induced increase in bone morphogenic protein receptor-1, bone morphogenic protein-2 and 4 and Serine peptidase inhibitor, clade E, member 1. The results from Micro-CT and histology show BGJ398 treatment rescued the OA changes in subchondral bone and knee articular cartilage of HMWTgFGF2 mice. The gene expression and signal transduction results provide convincing evidence that HMWFGF2 generates OA through FGFRTK with characteristic downstream signaling that defines OA, namely: increased FGF23-FGFR1 activity with BMP-BMPR, activation of pSMAD1/5/8-RUNX2 and pERK signaling pathways, then upregulation of MMP13 and ADAMTS5 to degrade matrix. BGJ398 treatment effectively reversed these OA molecular phenotypes, providing further evidence that the OA generated by HMWFGF2 in the transgenic mice is FGFR-mediated and phenocopies the OA found in the Hyp mouse homolog of XLH with a spontaneous mutation in the Phex (phosphate regulating endopeptidase on the X chromosome) gene and human XLH-OA. Overall, the results obtained here explain how the pleotropic effects of FGF2 emanate from the different functions of HMW protein isoforms for cartilage and bone homeostasis, and the pathogenesis of XLH-degenerative osteoarthropathy. BGJ398 inhibits HMWFGF2-induced osteoarthritis via multiple mechanisms. These results provided important scientific evidence for the potential application of BGJ398 as a therapeutic agent for osteoarthritis in XLH. 

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FGF receptor inhibitor BGJ398 partially rescues osteoarthritis-like phenotype in older high molecular weight FGF2 transgenic mice via multiple mechanisms

K. Clarke

Papers

Mutant epidermal growth factor receptor enhances induction of vascular endothelial growth factor by hypoxia and insulin-like growth factor-1 via a PI3 kinase dependent pathway.

FGF receptor inhibitor BGJ398 partially rescues osteoarthritis-like phenotype in older high molecular weight FGF2 transgenic mice via multiple mechanisms