Synergistic induction of in vivo angiogenesis by the combination of insulin-like growth factor-II and epidermal growth factor

Tumor growth is accelerated by induction of angiogenesis regulated by many cytokines and growth factors. In a tumor mass, various angiogenic factors and their receptors are simultaneously expressed and the overlapped expressions of various factors may contribute to the aggressive growth of tumor. However, the possible combined effect and mechanism of growth factors involved in angiogenesis are still under investigated. Insulin-like growth factor-II (IGF-II) has been identified as an angiogenic factor and highly expressed in solid tumors. Here we demonstrated that another angiogenic factor, epidermal growth factor (EGF), synergistically induced the angiogenic activity when co-treated with IGF-II in vivo. We performed mouse Matrigel plug assay. Cotreatment of IGF-II and EGF resulted in a significant induction of functional new vessels in mouse Matrigel plug more than additive amount of vessels induced by each growth factor. However, synergism of these two factors was not found in in vitro angiogenic assays, i.e., in migration and proliferation assays. The metalloproteinase-2 (MMP-2) protein level was enhanced by co-treatment of IGF-II and EGF, similar to that of individual treatment of them or PMA or bFGF. EGF down-regulated hypoxia-induced IGF-II binding protein-3 (IGFBP-3), which may contribute to the enhancement of free IGF-II accessibility to its receptors in vivo. Moreover, the combination of IGF-II with EGF significantly induced bFGF mRNA level, a potent angiogenic molecule, which may also contribute to synergistic effect in vivo. These results suggest that IGF-II and EGF may synergistically cooperate to induce angiogenesis in vivo by indirect mechanisms, i.e., synergistic induction of another angiogenic factor and modulation of IGF-II's bioavailability.     

Positive correlation of insulin-like growth factor-II with proliferating cell index in patients with colorectal neoplasia.

BACKGROUND: Insulin-like growth factor-II (IGF-II) stimulates cell proliferation and is considered a potential risk factor for colorectal cancer. Tumor levels of IGF-II seem to positively correlate with colorectal cancer cell proliferation. This investigation examined the association of circulating IGF-II to the proliferating cell index (PCI) of tumor and matched normal mucosa in patients with colorectal neoplasia. METHODS: Circulating IGF-II level (ng/mL) was determined in the peripheral blood plasma by ELISA. The proliferating cells in tumor or matched normal mucosa were immunohistochemically stained using the primary antibody against Ki-67. Computer image analysis was used and PCI was expressed as the percentage of Ki-67-positive cells/total counted cells. RESULTS: Sixty-four patients were evaluated; 45 had colorectal neoplasia (27 males/18 females; mean age, 66.8 +/- 11.8 years) and 19 had hyperplastic polyps (6 males and 13 females; mean age, 58.4 +/- 14.4 years). Among patients with colorectal neoplasia, blood IGF-II levels were positively correlated with PCI in the matched normal mucosa (r = 0.46, P < 0.05) but not in the tumor. In patients with hyperplastic polyps, blood IGF-II levels were not correlated with the PCI in the polyps. Blood IGF-II levels were higher in colorectal cancer patients with Dukes' C/D stage (P < 0.01) or with positive lymph nodes (P < 0.01). CONCLUSION: Circulating IGF-II positively correlated with PCI in normal colonic mucosa of patients with colorectal neoplasia, suggesting that IGF-II may have a role in initiating the carcinogenic pathway by stimulating cell proliferation. Blood IGF-II was increased in advanced colorectal cancer, indicating that it might enhance colorectal cancer progression and be a useful marker of poor prognosis.     

Autocrine inhibition of chemotherapy response in human liver tumor cells by insulin-like growth factor-II

Insulin-like Growth Factor (IGF)-II is frequently overexpressed in experimental and human hepatocellular carcinomas (HCCs) and has been correlated with increased tumor growth. We have analyzed, whether IGF-II affects chemotherapy response and apoptosis in human liver tumor cells. Three liver tumor cell lines highly expressed IGF-II and supported their growth in an autocrine manner by secreting excessive amounts of IGF-II. Neutralization of IGF-II significantly increased response to the chemotherapeutic agents cisplatin and etoposide especially at lower, cytostatic doses. While blocking of IGF-II did not increase spontaneous cell death in exponentially growing cultures, increased cell death was found under conditions of confluent growth and chemotherapy. Thus in HCC cells, IGF-II is a relevant protumorigenic growth factor that significantly reduces susceptibility to apoptosis and chemotherapeutic treatment. Therefore interference with IGF-II activity may improve response of HCCs to otherwise inefficient chemotherapeutic agents.     

Strong suppression of tumor growth by insulin-like growth factor-binding protein-related protein 1/tumor-derived cell adhesion factor/mac25

Insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) has been shown to induce cellular senescence or apoptosis of breast and prostate cancer cell lines in vitro. To examine whether IGFBP-rP1 acts as a tumor-suppressive protein in vivo, we established two model systems. Expression of IGFBP-rP1 in the human bladder carcinoma cell line EJ-1 was blocked by RNA interference. Human colon cancer cell line DLD-1, which did not express endogenous IGFBP-rP1, was transfected with an IGFBP-rP1 expression vector. When injected intraperitoneally or subcutaneously into nude mice, the IGFBP-rP1-expressing EJ-1 and DLD-1 cell lines grew poorly, whereas the IGFBP-rP1 non-producers grew rapidly and produced large tumors. In monolayer culture the IGFBP-rP1 producers and non-producers grew similarly in each model, whereas in soft agar culture the former produced far less colonies than the latter. The IGFBP-rP1 producers had IGFBP-rP1 bound to the cell surface, and adhered more efficiently to fibronectin and laminin-5 than the respective non-producers. Expression of IGFBP-rP1 did not affect the efficiency of insulin signaling. These results demonstrate that IGFBP-rP1 strongly suppresses tumor growth by an insulin-independent or insulin-like growth factor-independent mechanism. Cell surface IGFBP-rP1 may reduce the anchorage-independent growth ability, leading to the marked loss of tumorigenicity.     

Histamine suppresses fibulin-5 and insulin-like growth factor-II receptor expression in melanoma

We previously showed that transgenic enhancement of histamine production in B16-F10 melanomas strongly supports tumor growth in C57BL/6 mice. In the present study, gene expression profiles of transgenic mouse melanomas, secreting different amounts of histamine, were compared by whole genome microarrays. Array results were validated by real-time PCR, and genes showing histamine-affected behavior were further analyzed by immunohistochemistry. Regulation of histamine-coupled genes was investigated by checking the presence and functional integrity of all four known histamine receptors in experimental melanomas and by administering histamine H1 receptor (H1R) and H2 receptor (H2R) antagonists to tumor-bearing mice. Finally, an attempt was made to integrate histamine-affected genes in known gene regulatory circuits by in silico pathway analysis. Our results show that histamine enhances melanoma growth via H1R rather than through H2R. We show that H1R activation suppresses RNA-level expression of the tumor suppressor insulin-like growth factor II receptor (IGF-IIR) and the antiangiogenic matrix protein fibulin-5 (FBLN5), decreases their intracellular protein levels, and also reduces their availability in the plasma membrane and extracellular matrix, respectively. Pathway analysis suggests that because plasma membrane-bound IGF-IIR is required to activate matrix-bound, latent transforming growth factor-beta1, a factor suggested to sustain FBLN5 expression, the data can be integrated in a known antineoplastic regulatory pathway that is suppressed by H1R. On the other hand, we show that engagement of H2R also reduces intracellular protein pools of IGF-IIR and FBLN5, but being a downstream acting posttranslational effect with minimal consequences on exported IGF-IIR and FBLN5 protein levels, H2R is rather irrelevant compared with H1R in melanoma.     

Loss of insulin-like growth factor-II imprinting and the presence of screen-detected colorectal adenomas in women

Loss of imprinting (LOI) of insulin-like growth factor-II (IGF-II) may be an inherited epigenetic trait that is polymorphic in the population, and its presence may predispose an individual to the development of colorectal cancer. We evaluated the association between LOI of IGF-II in normal colonic mucosal samples and adenomas in women participating in a colonoscopy screening study. Among 40 participants, 11 (27.5%) had LOI of IGF-II in their normal colonic mucosal tissue. After adjusting for body mass index and family history of colorectal cancer, LOI status was associated with a fivefold increased risk of adenoma formation (odds ratio = 5.2, 95% confidence interval = 1.0 to 26.7). On average, IGF-II expression was more than threefold higher among women with LOI of IGF-II than among women with normal imprinting status. Our findings support the hypothesis that LOI of IGF-II is an epigenetic trait polymorphic in the population and suggest that LOI of IGF-II may play a role in colorectal cancer. These findings are intriguing and need to be confirmed in larger studies.     

Fibroblast growth factor and insulin-like growth factor differentially modulate the apoptosis and G1 arrest induced by anti-epidermal growth factor receptor monoclonal antibody

DiFi human colon carcinoma cells are stimulated by the transforming growth factor-alpha (TGF-alpha)/epidermal growth factor (EGF) receptor autocrine loop. Exposure of DiFi cells to monoclonal antibody (mAb) 225, which blocks ligand-induced activation of the EGF receptor, induces G1 arrest and subsequent cell death via apoptosis. We investigated the signal pathways by which basic fibroblast growth factor (bFGF) and insulin-like growth factor-1 (IGF-1) modulate mAb 225-induced G1 arrest and apoptosis in DiFi cells. Both bFGF and IGF-1 activated the mitogen-activated protein kinase (MAPK) kinase (MEK) pathway in DiFi cells. Additionally, IGF-1 activated the phosphoinositide 3-kinase (PI-3K)/Akt pathway. Both bFGF and IGF-1 inhibited mAb 225-induced apoptosis; however, bFGF provided sustained protection against apoptosis, while the protection by IGF-1 was only temporary. Also, bFGF reversed the mAb 225-induced increase in the p27(Kip1) level, inhibition of cyclin-dependent kinase-2 (CDK-2) activity, dephosphorylation of the retinoblastoma (Rb) protein and the resultant G1 arrest of the cells. In contrast, IGF-1 did not reverse such effects by mAb 225. The prevention of mAb 225-induced G1 arrest and apoptosis in DiFi cells by bFGF was sensitive to the MEK/MAPK inhibitor PD98059 but not to the PI-3K inhibitor LY294002. In contrast, inhibition of apoptosis by IGF-1 in DiFi cells was sensitive only to LY294002 and not to PD98059. These results further our understanding of how mAb 225 induces apoptosis in DiFi cells.     

Matrix metalloproteinase-7 triggers the matricrine action of insulin-like growth factor-II via proteinase activity on insulin-like growth factor binding protein 2 in the extracellular matrix

Many growth factors and cytokines are immobilized on the extracellular matrix (ECM) by binding to glycosaminoglycans and are stored in an inactive form in the cellular microenvironment. However, the mechanisms of ECM-bound growth factor or cytokine activation have not been well documented. We showed that the insulin-like growth factor type-1 receptor (IGF-1R) was rapidly phosphorylated after the addition of matrix metalloproteinase (MMP)-7 to a serum-starved human colon cancer cell line (HT29) and that phosphorylation was completely inhibited by an IGF-II neutralizing antibody. In the ECM of this cell line, IGF-II and IGF binding protein (BP)-2 coexisted, but IGFBP-2 disappeared from the ECM fraction after treatment with MMP-7 or heparinase III. On the other hand, in a cell line in which IGF-1R was overexpressed, IGF-1R was phosphorylated by supernatant from the MMP-7-treated ECM fraction of HT29 but not by that from a heparinase-III-treated ECM fraction. We also demonstrated that MMP-7 degrades IGFBP-2 in vitro at three cleavage sites (peptide bonds E(151)-L(152), G(175)-L(176) and K(181)-L(182)), which have not been documented previously. Taken together, these results demonstrate that MMP-7 generates bioactive IGF-II by degrading the IGF-II/IGFBP-2 complex binding to heparan sulfate proteoglycan in the ECM, resulting in IGF-II-induced signal transduction. This evidence indicates that some ECM-associated growth factors enhance their ability to bind to their receptors by some proteases in the tumor microenvironment. This mechanism of action ('protease-triggered matricrine') represents an attractive model for understanding ECM-tumor interactions.     

Plasma levels of insulin-like growth factor-1 and insulin-like growth factor binding protein-3 in women with cervical neoplasia

Objective

The goal of this study was to investigate the relationship between plasma levels of insulin-like growth factors-1 (IGF-1) and IGF-binding protein-3 (IGFBP-3) and the risk for cervical intraepithelial neoplasia (CIN) and cervical cancer.

Methods

Plasma levels of IGF-1 and IGFBP-3 of 44 cervical cancer patients, 82 CIN patients and 40 neoplasm-free patients were investigated. Then the associations of the plasma levels of IGF-1 and IGFBP-3 with cervical neoplasm or its clinicopathologic parameters were analyzed.

Results

The mean IGF-1 concentrations were significantly different among the control, CIN, and cervical cancer groups; the levels were higher in the CIN group compared to the controls. According to the quartile category, the plasma IGF-1 level was significantly higher (p=0.0015) in the CIN group than in the controls. The IGFBP-3 level showed no association between the controls and CIN groups (p=0.842). Although the mean IGF-1/IGFBP-3 molar ratio had borderline significance (p=0.08) among the study population, the quartile comparison showed a significantly higher IGF-1/IGFBP-3 molar ratio in the CIN group compared to the control group (p=0.041).

Conclusion

Plasma levels of IGF-1 and the IGF-1/IGFBP-3 molar ratio might be useful for the development early detection of cervical lesions and used as an adjuvant diagnostic tool for cervical neoplasia after more larger scale research.     

Mannose-6-phosphate/insulin-like growth factor-II receptor expression levels during the progression from normal human mammary tissue to invasive breast carcinomas

The putative role of mannose-6-phosphate/insulin-like growth factor-II receptor (M6P/IGFII-R) as a tumour suppressor and its value as a prognostic marker of breast cancer was studied in 42 benign breast diseases (BBD), 61 in situ carcinomas (CIS) and 133 invasive carcinomas. The receptor was quantified by immunohistochemistry with a computerised image analyser, using specific polyclonal IGY antibodies. The M6P/IGFII-R level varied markedly according to the different patient samples, but median values and distributions were similar in lesions and normal adjacent glands. However, the receptor level was significantly increased in high-grade ductal carcinomas in situ (DCIS) and decreased in invasive carcinomas relative to adjacent normal tissue. The M6P/IGFII-R protein concentration in invasive breast carcinomas was mostly independent of prognostic parameters: tumour size, histological grade, lymph node (N) invasiveness and oestrogen receptor alpha (ERalpha) status. The only positive correlation was with cathepsin D, the progesterone receptor (PgR) and with patients aged >60 years. These results do not support the hypothesis of a frequent and early inactivation of the M6P/IGFII-R gene in breast cancer. Clinical follow-up of patients might reveal a prognostic value for one of the cathepsin receptors.     

RACK1 inhibits colonic cell growth by regulating Src activity at cell cycle checkpoints

Previously, we showed that Src tyrosine kinases are activated early in the development of human colon cancer and are suppressed as intestinal cells differentiate. We identified RACK1 as an endogenous substrate, binding partner and inhibitor of Src. Here we show (by overexpressing RACK1, depleting Src or RACK1 and utilizing cell-permeable peptides that perturb RACK1's interaction with Src) that RACK1 regulates growth of colon cells by suppressing Src activity at G(1) and mitotic checkpoints, and consequently delaying cell cycle progression. Activated Src rescues RACK1-inhibited growth of HT-29 cells. Conversely, inhibiting Src abolishes growth promoted by RACK1 depletion in normal cells. Two potential mechanisms whereby RACK1 regulates mitotic exit are identified: suppression of Src-mediated Sam68 phosphorylation and maintenance of the cyclin-dependent kinase (CDK) 1-cyclin B complex in an active state. Our results reveal novel mechanisms of cell cycle control in G(1) and mitosis of colon cells. The significance of this work lies in the discovery of a mechanism by which the growth of colon cancer cells can be slowed, by RACK1 suppression of an oncogenic kinase at critical cell cycle checkpoints. Small molecules that mimic RACK1 function may provide a powerful new approach to the treatment of colon cancer.     

Adhesion to fibronectin via beta1 integrins regulates p27kip1 levels and contributes to cell adhesion mediated drug resistance (CAM-DR)

The tumor cell environment may influence drug response through interactions with the extracellular matrix (ECM). We recently reported that adhesion of myeloma cells to fibronectin (FN) via beta1 integrins is associated with a cell adhesion mediated drug resistance (CAM-DR). Activation of beta1 integrins is known to influence both apoptosis and cell growth. We hypothesized that the FN mediated cytoprotection may be in part due to perturbations in cell cycle progression. In this report we demonstrate that adhesion of myeloma cells to FN results in a G1 arrest associated with increased p27kip1 protein levels and inhibition of cyclin A and E associated kinase activity. Disruption of cells from FN adhesion resulted in a rapid recruitment of cells into S phase, a decrease in p27kip1 levels, and reversion to a drug sensitive phenotype. Treatment of cells with p27Kip1 antisense oligonucleotides did not affect FN adhesion; however, p27Kip1 protein levels were reduced and cells became sensitive to cytotoxic drugs. These studies demonstrate that beta1 mediated adhesion of myeloma cells to FN regulates p27kip1 levels and that p27kip1 levels are causally related to CAM-DR. Disruption of beta1 integrin mediated FN adhesion may represent a potential target for the potentiation of drug induced apoptosis.     

Cyclolignans as inhibitors of the insulin-like growth factor-1 receptor and malignant cell growth

The insulin-like growth factor-1 receptor (IGF-1R) plays a pivotal role in transformation, growth, and survival of malignant cells, and has emerged as a general and promising target for cancer treatment. However, no fully selective IGF-1R inhibitors have thus far been found. This is explained by the fact that IGF-1R is highly homologous to the insulin receptor, coinhibition of which may cause diabetic response. The receptors are both tyrosine kinases, and their ATP binding sites are identical, implying that ATP inhibitors cannot discriminate between them. Therefore, the current strategy has been to identify compounds interfering with receptor autophosphorylation at the substrate level. In this study we investigated the effects of cyclolignans and related molecules on IGF-1R activity. We report that certain cyclolignans are potent and selective inhibitors of tyrosine phosphorylation of the IGF-1R. Of particular interest was picropodophyllin (PPP), which is almost nontoxic (LD(50) >500 mg/kg in rodents). PPP efficiently blocked IGF-1R activity, reduced pAkt and phosphorylated extracellular signal regulated kinase 1 and 2 (pErk1/2), induced apoptosis in cultured IGF-1R-positive tumor cells, and caused complete tumor regression in xenografted and allografted mice. PPP did not affect the insulin receptor or compete with ATP in an in vitro kinase assay, suggesting that it may inhibit IGF-1R autophosphorylation at the substrate level. This is also in agreement with our molecular model of how the cyclolignans may act on the IGF-1R kinase. Our results open the possibility to use PPP or related compounds with inhibitory effects on IGF-1R as lead compounds in development of anticancer agents.     

Decreased insulin-like growth factor-II/mannose 6-phosphate receptor expression enhances tumorigenicity in JEG-3 cells

The insulin-like growth factor-II/mannose-6 phosphate receptor (IGF-II/M6PR) is believed to bind and degrade the potent mitogen IGF-II, a growth factor for many tumors. This receptor has been shown to be mutated and/or lost in a significant percentage of a variety of tumors, implying that it may act as a negative regulator of cell growth. In this study, we demonstrate that down-regulation of this receptor, mediated by antisense IGF-II/M6PR cDNA transfection into JEG-3 choriocarcinoma cells, results in increased growth rate in vitro and increased tumor growth rate in vivo. These findings demonstrate that a decrease in IGF-II/M6PR expression results in a growth advantage in JEG-3 cells and are consistent with the hypothesis that the IGF-II/M6PR is an inhibitor of tumor growth.     

Rack1 promotes epithelial cell-cell adhesion by regulating E-cadherin endocytosis

E-cadherin and its cytoplasmic partners, catenins, mediate epithelial cell-cell adhesion. Disruption of this adhesion allows cancer cells to invade and metastasize. Aberrant activation of the Src tyrosine kinase disrupts cell-cell contacts through an E-cadherin/catenin-dependent mechanism. Previously we showed that Rack1 regulates the growth of colon cells by suppressing Src activity at G(1) and mitotic checkpoints, and in the intrinsic apoptotic and Akt cell survival pathways. Here we show that Rack1, partly by inhibiting Src, promotes cell-cell adhesion and reduces the invasive potential of colon cancer cells. Rack1 stabilizes E-cadherin and catenins at cell-cell contacts by inhibiting the Src phosphorylation of E-cadherin, the ubiquitination of E-cadherin by the E3 ligase Hakai and the endocytosis of E-cadherin. Upon depletion and restoration of extracellular calcium, Rack1 facilitates the re-assembly of E-cadherin-containing cell-cell contacts. Rack1 also blocks HGF-induced endocytosis of E-cadherin, disruption of cell-cell contacts and cell scatter. Our results uncover a novel function of Rack1 in maintaining the junctional homeostasis of intestinal epithelial cells by regulation of the Src- and growth factor-induced endocytosis of E-cadherin.     

Lifestyle factors and insulin-like growth factor 1 levels among elderly men.

Insulin-like growth factor 1 (IGF-1) is a potentially important determinant of disease; hence epidemiological identification of factors that influence circulating IGF-1 is merited. We therefore analysed data collected in Greece to determine the relationship between anthropometric, lifestyle and dietary variables and serum levels of IGF-1 among elderly men. We identified 51 men with prostate cancer, 50 men with benign prostatic hyperplasia, and 52 apparently healthy elderly men (controls), all matched for age (+/- 1 year). These 153 men provided blood specimens and were interviewed using a validated lifestyle and food frequency questionnaire. We performed multivariate linear regression to identify potential predictors of circulating IGF-1. After controlling for age, body mass index, smoking habits, alcohol drinking and coffee consumption, each 5 cm increase in height predicted a 13.0% increase in IGF-1 (95% CI 0.4-27.2%) among the controls and a 11.3% increase in IGF-1 (95% CI 4.5-18.6%) among the entire study group. None of the investigated dietary factors (total fat, carbohydrate, protein, dairy products, tomatoes, calcium) were strongly related to IGF-1 levels. The positive association between IGF-1 and height integrates the empirical evidence linking IGF-1 and height with prostate cancer risk.