A chimeric humanized single-chain antibody against the type I insulin-like growth factor (IGF) receptor renders breast cancer cells refractory to the mitogenic effects of IGF-I

Insulin-like growth factors (IGFs) stimulate breast cancer proliferation, motility, and survival.The type I IGF receptor (IGF1R) mediates the effects of IGF-I. Thus, inhibition of IGF1R activation could inhibit IGF action in breast cancer cells. A single-chain antibody directed against IGF1R (IGF1R scFv-Fc) has been shown to partially inhibit xenograft growth of MCF-7 cells in athymic mice. In this study, we have examined the effects of scFv-Fc on IGF1R signaling in the estrogen receptor-positive (ER+) MCF-7 breast cancer cells in vitro and in vivo. The antibody stimulated IGF1R activation in vitro in MCF-7 cells and was unable to block IGF-I effects. The antibody also stimulated proliferation of MCF-7 cells in monolayer growth assays. To determine how scFv-Fc could stimulate in vitro growth yet inhibit in vivo tumor growth, we examined the effect of scFv-Fc on IGF1R expression. In MCF-7 cells, scFv-Fc down-regulated IGF1R levels after 2 h, and the levels were greatly reduced after 24 h. In contrast, IGF-I treatment over the same time period did not affect IGF1R levels. Twenty-four-h pretreatment of cells with scFv-Fc blocked IGF-I mediated phosphorylation of insulin receptor substrate-1 and subsequent extracellular signal-regulated kinase 1/extracellular signal-regulated kinase 2 and phosphatidylinositol 3'-kinase activation. In contrast, cells treated with 5 nM IGF-I for 24 h still retained the ability to further activate downstream signaling pathways in response to IGF-I. Moreover, pretreatment of MCF-7 cells with scFv-Fc rendered them refractory to further proliferation induced by additional IGF-I. Twenty-four-h pretreatment of cells with scFv-Fc also inhibited IGF-I stimulated anchorage-independent growth. scFv-Fc did not enhance antibody-dependent cell-mediated cytotoxicity. In vivo, treatment of mice bearing MCF-7 xenograft tumors with scFv-Fc resulted in near complete down-regulation of IGF1R. Our data show that scFv-Fc stimulates biochemical activation of IGF1R, then causes receptor down-regulation, making MCF-7 cells refractory to additional IGF-I exposure. These results indicate that such chimeric single-chain antibodies against IGF1R have future potential in breast cancer therapy by causing down-regulation of receptor.     

Insulin-like growth factor receptor expression and function in human breast cancer

The insulin-like growth factors IGF-I and IGF-II are potent mitogens for several breast tumor cell lines in culture. Additionally, both IGF-I and IGF-II mRNAs are easily detected in the majority of breast tumor specimens examined, while no breast cancer epithelial cell lines we have studied express authentic IGF-I mRNA, and few lines express IGF-II mRNA. Although receptors for insulin, IGF-I, and IGF-II have been described, there is significant cross-reactivity between the various receptors and ligands in the insulin/insulin-like growth factor family, and it is not clear which receptor or receptors are responsible for the biological effects of these growth factors in this system. Using an RNase protection assay, we examined breast tumor specimens and breast cancer epithelial cell lines for expression of mRNA encoding the type I and type II IGF receptors as well as the insulin receptor. Virtually all of the specimens examined expressed mRNA for all three receptors. We then examined estrogen-dependent MCF-7 cells for the mitogenic effects of IGF-I and II in the presence of antibodies to both the type I and type II receptors. alpha IR-3, a monoclonal antibody which blocks the type I receptor, abolished the mitogenic effects of both IGF-I and IGF-II. It did not, however, block the mitogenic effects of insulin. We conclude that type I and type II IGF receptors are ubiquitously expressed in breast cancer, and our experiments with MCF-7 cells suggest the mitogenic effects of both IGF-I and IGF-II are mediated via the type I IGF receptor.     

Interference of the IGF system as a strategy to inhibit breast cancer growth

Summary Experimental evidence suggests that human breast cancer cells can be regulated by the IGF-I and IGF-II present in the tumor stromal elements and/or by the endogenous tumor cell IGF-II in a paracrine or autocrine fashion. Thus, blockade of the receptor signalling pathway could lead to diminished tumor growth. Blockade of the type I IGF receptor by a monoclonal antibody (IR3) has been used as a strategy to demonstrate the importance of the IGF pathway. Although IR3 could not block serum-free growth of breast cancer cell lines, it could inhibit anchorage independent growth in most cell lines in the presence of serum. In vivo, IR3 administered at the time of tumor cell inoculation could inhibit MDA-MB-231 tumor formation in athymic mice; however, inhibition of established tumors was not seen. Moreover, IR3 could not inhibit tumor formation of the MCF-7 cell line in vivo. These results suggest that blockade of the type I IGF receptor can inhibit the growth of some breast cancer cells both in vitro and in vivo. Future anti-growth factor strategies include the combination of anti-IGF receptor antibodies with IGF neutralizing modalities, the dual blockade of growth factor receptors (epidermal growth factor receptor and type I IGF receptor), and combinations of steroid hormone antagonists and anti-growth factor treatments to maximize tumor inhibition.     

Type 1 IGF receptor in human breast diseases

Summary The first step of the action of IGF1 and IGF2 (IGFs) is their binding to membrane receptors. IGF binding sites have been characterized by competitive binding and cross-linking techniques in human breast cancer cell lines as well as in human breast cancers and in human benign breast diseases. IGF2 is a good competitor of¹²⁵I-IGF1 binding to IGF1-R; insulin competes but with a potency 1/100 lower than the IGF1 potency. Chemical cross-linking experiments revealed that the apparent molecular weight of the IGF1-binding sites is 130,000. Alpha IR-3, a murine monoclonal antibody against the IGF1-R, blocks IGF1-binding to this receptor. This antibody inhibits the IGF1-stimulated growth of breast cancer cells. Therefore, the IGF1 specific binding sites correspond to the previously described type 1 IGF receptors (IGF1-R) in normal tissues. Cross-linking experiments with labeled IGF2 resulted in a major band of apparent Mr 260,000–270,000 that was inhibited by unlabeled IGF2 but not by insulin, and corresponds to the type 2 IGF receptor; a second band of apparent Mr 130,000 was inhibited by excess IGFs and insulin (Type I receptor). The alpha-IR3 inhibition of the IGF2 mitogenic activity suggest that IGF1-R partially mediates the growth effect of IGF2 in these cells.We and others have demonstrated that most breast cancer cell lines contain IGF1-R. This is also the case in breast cancer biopsies in which histo-autoradiographic analyses allowed the localization of IGF1-R on the epithelial component. IGF1-R concentrations were positively correlated to estradiol and progesterone receptor concentrations. In our experience, the presence of IGF1-R is associated with a better prognosis. Finally, IGF1-R are found less frequently and at lower concentrations in benign breast diseases. These results suggest that IGF1-R could be a marker of the proliferative epithelial component within the tumor.     

Regulation of insulin-like growth factor-binding protein (IGFBP) expression by breast cancer cells: use of IGFBP-1 as an inhibitor of insulin-like growth factor action.

BACKGROUND: The insulin-like growth factors (IGFs) play an important role in normal growth and development. Evidence suggests they may also regulate the growth of several cancer cell types. This regulation is mediated by interactions between the receptors and ligands. There is now ample evidence to suggest that these interactions are also influenced by extracellular IGF-binding proteins (IGFBPs). Six different IGFBPs have been cloned. Some species may act to inhibit the mitogenic effects of the IGFs. Since breast cancer cells are responsive to the IGFs, it is possible that regulated expression of the IGFBPs affects tumor growth. Furthermore, inhibitory binding proteins could be used as neutralizers of IGF action. PURPOSE: We conducted this study to fully characterize the expression and hormonal regulation of IGF-binding protein expression in human MCF-7 breast cancer cells and to test the ability of purified IGFBP-1 to inhibit IGF-I action. METHODS: We used ribonuclease protection assays and Western ligand blotting to examine IGFBP expression in MCF-7 cells. The effect of IGF-I, IGFBP-1, and 17 beta-estradiol on serum-free cell growth was also studied. RESULTS: MCF-7 cells expressed IGFBP-2, IGFBP-4, and IGFBP-5 RNA and protein. These cells are dependent on estrogen for growth. In short-term culture, IGF-I can substitute for estrogen. Concomitant addition of IGF-I and estrogen enhanced stimulation above the level achieved by either factor alone. Estrogen also increased IGFBP production, making it unlikely that the IGFBPs induced by estrogen in MCF-7 cells could function as major inhibitors of IGF action. In contrast, exogenous addition of IGFBP-1 could block IGF-I-induced mitogenesis; this effect was reversible by excess IGF-I. CONCLUSIONS: The studies suggest that cancer cell growth may be regulated by endogenous IGFBP expression. Furthermore, the exogenous addition of the IGFBP-1 blocked IGF-I action and potentially could be used as a pharmacologic inhibitor of IGF action.     

Regulation of breast cancer cell motility by insulin receptor substrate-2 (IRS-2) in metastatic variants of human breast cancer cell lines.

Insulin-like growth factors (IGFs) regulate breast cancer cell proliferation, protect cells from apoptosis, and enhance metastasis. In this study, we examined the IGF signaling pathway in two breast cancer cell lines selected for metastatic behavior. LCC6 was selected for growth as an ascites tumor in athymic mice from parental MDA-MB-435 cells (435P). The MDA-231BO cell line was derived from osseous metastases that formed after intracardiac injection of the MDA-MB-231 cell line in athymic mice. Compared to the parental cell lines, IGF-I treatment enhanced IRS-2 phosphorylation over IRS-1 in the metastatic variants. IGF-I stimulated cell migration in the variant cells, but not in the parental cells. To determine the role for IRS-2 in IGF-mediated motility, we transfected MDA-231BO cells with an anti-sense IRS-2 construct. Transfected cells had decreased levels of IRS-2 with diminished IGF-mediated motility and anchorage independent growth when compared to control cells. However, adherence to fibronectin was enhanced in the transfected cells compared to MDA-231BO cells. Our data show that breast cancer cells selected for metastatic behavior in vivo have increased IRS-2 activation and signaling. In these cells, IGF-I enhances cell adhesion and motility suggesting that IRS-2 may mediate these aspects of the malignant phenotype.     

Growth-control of human ovarian-carcinoma cells by insulin-like growth-factors

The role of the insulin-like growth factors (IGFs) in 3 cultured human ovarian cancer cell lines (PEO1, PEO4, PEO14) was investigated. All three cell lines express mRNA for IGF-I and the PEO14 cell line expresses mRNA for IGF-II. Protein expression of IGF-II was demonstrated in the PEO14 and PEO4 cell lines. All 3 cell lines expressed mRNA for the IGF type I, IGF type II and insulin receptors; the presence of type I IGF receptors was confirmed by immuno-cytochemistry. IGF-I and insulin markedly stimulated the proliferation of PEO1 and PEO4 but not PEO14 cells while all 3 lines were insensitive to the addition of exogenous IGF-II.     

Insulin receptor expression and function in human breast cancer cell lines.

We have previously reported that insulin receptor expression is increased in human breast cancer specimens (V. Papa et al., J. Clin. Invest., 85:1503-1510, 1990). In the present study, in order to further understand the role of the insulin receptor in breast cancer, insulin receptor expression and function were characterized in three human breast cancer cell lines, MCF-7, ZR-75-1, and T-47D, and compared to a nonmalignant human breast epithelial cell line, 184B5. Insulin receptor content, measured by radioimmunoassay, was elevated 5- and 3-fold in MCF-7 and ZR-75-1 breast cancer cell lines, respectively, when compared to the nonmalignant cell line 184B5. In contrast, the insulin receptor content of T-47D cells was not increased. The increase in insulin receptor content in MCF-7 and ZR-75-1 cells was not due to amplification of the insulin receptor gene. Also, total insulin receptor mRNA content was not increased in breast cancer cells in respect to nonmalignantly transformed 184B5 breast epithelial cells. However, significant differences in the content of receptor mRNA species were observed. The insulin receptors in the breast cancer cell lines were functional: (a) In all 4 cell lines, high-affinity insulin-binding sites were detected, and, in concert with the insulin receptor radioimmunoassay data, binding capacity was highest in MCF-7 and then in ZR-75-1 cells. (b) In all cell lines, insulin stimulated insulin receptor tyrosine kinase activity. However, the effect of insulin was greater in breast cancer cell lines than in nonmalignant breast cells. (c) In all cell lines, insulin at concentrations of 1 nM or less stimulated [3H]thymidine incorporation. This effect of insulin was inhibited by 50% in MCF-7 cells and by 60% in 184B5 cells when alpha-IR3, a monoclonal antibody to the insulin-like growth factor I receptor, was present. In these cells, therefore, insulin was active via both its own receptor and the IGF-I receptor. In contrast, alpha-IR3 antibody was without effect in T-47D and ZR-75-1 cells, suggesting that in these cell lines insulin acted only via its receptor. In the breast cancer cells, MA-5, an agonist monoclonal antibody to the insulin receptor, stimulated [3H]thymidine incorporation. This present study indicates therefore that in breast cancer cell lines there are functional insulin receptors that regulate breast cancer cell growth.     

Insulin-like growth factor binding protein-1 (IGFBP-1) inhibits breast cancer cell motility

The breast cancer malignant phenotype is regulated by steroid hormones and peptide growth factors. We have shown previously that insulin-like growth factor-I (IGF-I) stimulates cell motility in a metastatic cell line, MDA-231BO. In this study, we show that neutralization of IGF action by a type I IGF receptor (IGFR1) blocking antibody or neutralization of IGF-I by IGFBP-1 reduced cell motility. However, in addition to inhibiting IGF effects, IGFBP-1 also diminished basal motility. Because IGFBP-1 contains a RGD motif important in binding of fibronectin to its alpha 5 beta 1 integrin receptor, we examined the effect of inhibiting integrin function on cell motility. As expected, disruption of fibronectin-integrin interactions interrupted basal motility in MDA-231BO cells. In addition, disruption of integrin function by an alpha 5 beta 1 blocking peptide also inhibited IGF stimulation of cell motility. To determine whether integrin function could interfere with IGF signaling, we used an alpha 5 beta 1 blocking peptide to show that in MDA-231BO cells integrin occupancy appeared necessary for phosphorylation of insulin receptor substrate-2 but not for IGFR1 activation. We conclude that IGFR1 and integrin action are linked in these breast cancer cells as disruption of integrin binding to its receptor influences IGF signaling pathways. Moreover, IGFBP-1 could have dual effects on cancer cell motility by disrupting both receptor systems.     

Cytotoxic activity of a recombinant fusion protein between insulin-like growth factor I and Pseudomonas exotoxin.

A chimeric toxin in which the cell binding domain of Pseudomonas exotoxin was replaced with mature human insulin-like growth factor I (IGF-I) was produced in Escherichia coli. This protein, IGF-I-PE40, was cytotoxic to human cell lines derived from a variety of tumor types, with a breast carcinoma line (MCF-7) and two hepatoma lines (HEP3B and HEPG2) showing the highest sensitivity to the toxin. The specificity of IGF-I-PE40 cytotoxicity was confirmed through competition with excess IGF-I and through blockage of toxin binding using an antibody specific to the type I IGF receptor. A potential interaction between the toxin and soluble IGF-binding proteins was also demonstrated. IGF-I-PE40 may be useful in the selective elimination of cells bearing the type I IGF receptor.     

Influence of IGF-IR stimulation or blockade on proliferation of human renal cell carcinoma cell lines

Several tumors secrete insulin-like growth factors (IGFs) for autocrine growth stimulation and protection from apoptosis. However, the mechanisms responsible for tumor growth in renal cell carcinoma (RCC) are unclear. In this study the biological role of exogenous IGFs in two malignant RCC cells (Caki-2 and SK-RC-52) were investigated in vitro, and compared to the breast cancer cell line MCF-7. IGFs but not the related epidermal growth factor stimulated both RCC cell lines. Caki-2 expressed higher levels of IGF-IR and proliferated more vigorously to added IGF-I, IGF-I analogues des(1-3)IGF-I, LongR3IGF-I and IGF-II compared to SK-RC-52. Neutralizing IGF-IR antibodies reduced the IGF driven proliferation in both cell lines. Interestingly, soluble IGF-I receptor resulted in strong growth inhibition of SK-RC-52, while no marked effect on Caki-2 was observed. Moreover, Caki-2 expressed a broad panel of IGFBPs, while SK-RC-52 more selectively secreted high levels of IGFBP-3. Exogenous IGFBP-3 strongly inhibited IGF-I driven proliferation in SK-RC-52, but worked in synergy with IGF-I in Caki-2. Both the IGF-IR and IGFBP-3 were present in respectively 4/4 and 4/8 human malignant renal tissues. In light of this and with the functional data presented in this study, interference with this growth factor system may provide a novel therapeutic approach in renal cancer therapy.     

Multiple Signaling Pathways are Activated During Insulin-like Growth Factor-I (IGF-I) Stimulated Breast Cancer Cell Migration

In order to display the full metastatic phenotype, the cancer cell must acquire the ability to migrate. In breast cancer, we have previously shown that insulin-like growth factor I (IGF-I) enhances cell motility in the highly metastatic MDA-231BO cell line by activating the type I IGF receptor (IGF1R). This motility response requires activation of IRS-2 and integrin ligation. In order to identify the key molecules downstream of IRS-2, we examined several signaling pathways known to be involved in cell motility. Focal adhesion kinase (FAK) was not activated by IGF-I, but IGF-I caused redistribution of FAK away from focal adhesion plaques. IGF-I treatment of MDA-231BO cells activated RhoA and inhibition of Rho-kinase (ROCK) inhibited the IGF-mediated motility response. The mitogen activated protein kinase (MAPK), p38, was also activated by IGF-I and inhibition of p38 by SB203580 blocked IGF-I induced cell motility. ROCK inhibition with Y-27632 also inhibited p38 phosphorylation suggesting that p38 lies downstream of ROCK. Both Erk1,2 and phosphatidyl-3 kinase (PI3K) were required for IGF-I stimulated cell motility, but only PI3K appeared to be directly downstream of IGF-I. Thus, IGF-I activation of its receptor coordinates multiple signaling pathways required for cell motility. Defining the key molecules downstream of the type I IGF receptor may provide a basis for optimizing therapies directed at this target.     

Role of estrogen-induced insulin-like growth factors in the proliferation of human breast cancer cells

Insulin-like growth factor I (IGF-I) is the most potent growth factor for estrogen (E2)-dependent breast cancer cell lines. It has been reported that such cell lines produce an immunoreactive IGF-I-related protein in an E2-dependent fashion, while autonomously growing cell lines constitutively produce these factors, indicating that they might be involved in autocrine growth stimulation of breast cancer cells. We have studied the role of IGFs in autocrine growth stimulation of the E2-dependent breast cancer cell line MCF7 by using IGF-binding proteins (BPs) that were able to neutralize completely the mitogenic effect of IGF-I on this cell line. These BPs, however, did not have any inhibitory effect on E2-induced mitogenesis, suggesting that secretion of autocrine IGFs is not involved in growth stimulation by E2. To exclude the possibility that variants of IGF are produced by the cells that are not recognized by the BPs, we also studied the production of biologically active IGF using the same MCF7 cell line in a sensitive bioassay in which the various forms of IGF and insulin can be detected. Although the conditioned medium (CM) of human fibroblasts used as a control showed IGF-like activity in this assay, no such activity was detected in CM of both untreated and E2-stimulated MCF7 cells, while the CM of the E2-independent cell lines BT20 and Hs578T had only slight mitogenic activity or was even growth inhibitory, respectively. These data show that no significant secretion of biologically active IGFs by human breast cancer cells could be detected and do not support a possible autocrine function of IGFs in the proliferation of such cells. Because E2-dependent cells strongly react to externally added IGF-like factors produced by human fibroblasts, a role for IGFs in paracrine regulation of proliferation is suggested.     

Progesterone crosstalks with insulin-like growth factor signaling in breast cancer cells via induction of insulin receptor substrate-2

Both progesterone and the insulin-like growth factors (IGFs) are critically involved in mammary gland development and also in breast cancer progression. However, how the progesterone and IGF signaling pathways interact with each other to regulate breast cancer cell growth remains unresolved. In this study, we investigated progesterone regulation of IGF signaling components in breast cancer cells. We found that insulin receptor substrate-2 (IRS-2) levels were markedly induced by progesterone and the synthetic progestin R5020 in MCF-7 and other progesterone receptor (PR) positive breast cancer cell lines, whereas IRS-1 and the IGF-I receptor were not induced. The antiprogestin RU486 blocked the R5020 effect on IRS-2 expression. Ectopic expression of either PR-A or PR-B in C4-12 breast cancer cells (estrogen receptor and PR negative) showed that progestin upregulation of IRS-2 was mediated specifically by PR-B. The IRS-2 induction by R5020 occurred via an increase of IRS-2 mRNA levels. Furthermore, progestin treatment prior to IGF-I stimulation resulted in higher tyrosine-phosphorylated IRS-2 levels, increased binding of IRS-2 to Grb-2 and the PI3K regulatory subunit p85, and correspondingly enhanced ERK and Akt activation, as compared with IGF-I-only conditions. Taken together, our data suggest that IRS-2 may play an important role in crosstalk between progesterone and the IGFs in breast cancer cells.     

Interaction between hormone-dependent and hormone-independent human breast cancer cells

We developed two different models based on in vitro co-culture of hormone-dependent and hormone-independent cell lines to simulate the cell population heterogeneity of human breast cancer tumours. Oestrogen-dependent (MCF-7, ZR 75.1) and oestrogen-independent cell lines (MDAMB-231 BT-20) were grown under serum-free conditions. Co-culture of hormone-dependent and hormone-independent cell lines resulted in an increased cell yield compared to single cell cultures carried out at the same seeding ratios. Such an increase was not affected by addition of oestradiol and single growth factors (EGF, bFGF and IGF-I). These results allow us to conclude that in a heterogeneous cell population like human breast tumours, interaction between hormone-dependent and hormone-independent cell lines may result in a complex regulation of cell growth.     

Paracrine interaction in co-culture of hormone-dependent and independent breast cancer cells

Summary A permeable solid support (Transwell Coll.) was used to develop serum-free co-cultures allowing paracrine interactions between hormone-dependent (MCF-7, ZR75.1) and hormone-independent (MDAMB-231, BT20) breast cancer cell lines. Both hormone-independent cell lines were able to stimulate the growth of the hormone-dependent lines, whereas the opposite was observed only in the case of BT20 co-culture with ZR75.1 cells. The cell growth stimulation observed in co-cultures could be abolished by the addition to the culture medium of an excess of transforming growth factor alpha (TGF) or insulin-like growth factor I (IGF-I). Similarly, treatment with a neutralizing anti TGF antibody impaired the growth stimulation exerted by hormone-independent cells on hormone-dependent cells. These results confirm the important role of paracrine interactions in control of the growth of human heterogeneous breast tumors and suggest that the main growth factors involved in such interactions are TGF and probably some growth factors from the insulin-like growth factor family rather than IGF-I itself.     

Insulin-like Growth Factor-Dependent Proliferation and Survival of Triple-Negative Breast Cancer Cells: Implications for Therapy

Triple-negative breast cancers have a poor prognosis and are not amenable to endocrine- or HER2-targeted therapies. The prevailing view is that targeting the insulin-like growth factor (IGF) signal transduction pathway will not be beneficial for triple-negative breast cancers because their growth is not IGF-responsive. The present study investigates the importance of IGFs in the proliferation and survival of triple-negative breast cancer cells. Estrogen and progesterone receptors, HER2, type I IGF, and insulin receptors were measured by Western transfer analysis. The effects of IGF-1 on proliferation were assessed by DNA quantitation and on cell survival by poly (ADP-ribose) polymerase cleavage. The effect of IGF-1 on phosphorylation of the IGF receptors, Akt and mitogen-activated protein kinase, was measured by Western transfer analysis. Seven cell lines were identified as models of triple-negative breast cancer and shown to express IGF receptors at levels similar to those present in estrogen-responsive cell lines known to respond to IGFs. IGF-1 increased the proliferation and cell survival of all triple-negative cell lines. Proliferation was attenuated after reduction of type I IGF receptor expression. Cells that express higher levels of receptor were more sensitive to subnanomolar IGF-1 concentrations, but the magnitude of the effects was not correlated simply with the absolute amount or phosphorylation of the IGF receptors, Akt or mitogen-activated protein kinase. These results show that IGFs stimulate cell proliferation and promote cell survival in triple-negative breast cancer cells and warrant investigation of the IGF signal transduction pathway as a therapeutic target for the treatment of triple-negative breast cancer.     

Autocrine function for insulin-like growth factor I in human small cell lung cancer cell lines and fresh tumor cells

We showed previously that insulin-like growth factor I (IGF-I) is detectable in small cell lung cancer (SCLC) tumor biopsies and cell lines and that recombinant human IGF-I stimulates DNA synthesis in SCLC cells. Here we report further studies on the role of IGF-I in 2 SCLC cell lines: HC12, classic; and ICR-SC17, variant. Immunoreactive IGF-I was detected in medium conditioned by HC12 but not ICR-SC17. Both HC12 and ICR-SC17 bound IGF-I with 100-fold greater affinity than insulin. Scatchard analysis revealed two classes of IGF-I binding site of high (Kd 0.1 nM, n = 2,300) and lower (Kd 3 nM, n = 28,000) affinity. In both cell lines [3H]thymidine incorporation was enhanced by recombinant human IGF-I, 100-1000 ng/ml. ICR-SC17 also showed growth enhancement as measured by increase in cell numbers. There was no response in HC12, probably due to endogenous IGF-I production. 125I-IGF-I binding and basal and IGF-I-stimulated mitogenesis were inhibited by monoclonal antibodies to IGF-I (SM1.20B, SM1.25) or the type I IGF receptor alpha IR3 but not an isotypic control monoclonal antibody. Antiproliferative effects were manifest in [3H]thymidine incorporation assays in serum-free conditions and growth of serum-supplemented liquid cultures. We also tested fresh or newly cultured tumor cells obtained by fine needle aspiration of metastases in three previously untreated and four relapsed patients with SCLC. IGF-I binding sites were demonstrable on fresh SCLC cells, and specific binding was inhibited by SM1.20B. All seven samples showed stimulation of [3H]thymidine incorporation in the presence of recombinant human IGF-I, 100-500 ng/ml. As in cultured cells, basal and IGF-I-stimulated DNA synthesis was inhibited by monoclonal antibodies SM1.20B, SM1.25, and alpha IR3 but not the isotypic control. These results confirm the findings of previous studies and suggest that IGF-I can function as an autocrine growth factor in SCLC in vitro and possibly also in vivo.     

Mitogenic Properties of Insulin-Like Growth Factors I and II, Insulin-like Growth Factor Binding Protein-3 and Epidermal Growth Factor on Human Breast Stromal Cells in Primary Culture

Insulin-like growth factors I and II (IGF-I and IGF-II) are growth factors implicated in both normal mammary gland development and breast cancer. We have previously reported on the effects of components of the IGF system on breast epithelial cells. Since data suggests that stromal-epithelial interactions play a crucial role in breast cancer, we have now investigated the mitogenic properties of IGF-I, IGF-II, insulin-like growth factor binding protein-3 (IGFBP-3) and epidermal growth factor (EGF) on human breast stromal cells in primary culture. We show that, under serum-free conditions, stromal cells are stimulated to grow in response to IGF-I and IGF-II in a dose-dependent manner. IGF-I and EGF, a potent stimulator of human breast epithelial cell growth in primary culture and also associated with breast cancer, appear to stimulate stromal cell growth in a synergistic manner. IGFBP-3 does not inhibit the stimulation of growth by IGF-I, or IGF-I plus EGF. However, IGFBP-3 does inhibit the stimulation of growth by IGF-II. In contrast to our previous results with human breast epithelial cells, IGFBP-3 does not have an IGF-independent inhibitory effect on stromal cell growth. This study is the first to address the effects of IGF-I, IGF-II and IGFBP-3 alone and in combination with EGF on human breast stromal cell growth in primary culture. Characterizing the role of the IGF system in both normal breast epithelial cells and stromal cells will aid in our understanding of the mechanisms behind the role of the IGF system in breast cancer.