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.     

Progesterone Decreases Levels of the Adhesion Protein E-Cadherin and Promotes Invasiveness of Steroid Receptor Positive Breast Cancers

Progestins are reported to increase the risk of invasive breast cancers in postmenopausal women receiving hormone therapy with estrogen plus progestin. We report here that estrogen and progesterone receptor positive (ER+PR+) rat mammary tumors arising in the presence of estrogen and progesterone exhibit increased invasiveness and decreased expression of E-cadherin protein compared with tumors growing in the presence of estrogen alone. A similar decrease of E-cadherin expression was observed in human ER+PR+ invasive ductal carcinoma compared with ductal carcinoma in situ. In agreement with findings in the rat, estrogen plus progestin R5020 treatment decreased E-cadherin expression in vitro in T47D human breast cancer cells. Decrease of E-cadherin protein was mediated by progesterone receptor B (PRB) and dependent on the activation of the Wnt pathway. These results suggest that progesterone signaling via PRB contributes to tumor invasiveness and can provide an important therapeutic target for treatment of invasive ER+PR+ breast cancers.     

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.     

Re-expression of estrogen receptor alpha in estrogen receptor alpha-negative MCF-7 cells restores both estrogen and insulin-like growth factor-mediated signaling and growth

Estrogen can increase insulin-like growth factor-I receptor (IGF-IR) and insulin receptor substrate-1 (IRS-1) expression, two key components of IGF-I-mediated signaling. The result is sensitization of breast cancer cells to IGF-I and synergistic growth in the presence of estrogen and IGF-I. We hypothesized that loss of estrogen receptor alpha (ERalpha) would result in reduced IGF-mediated signaling and growth. To test this hypothesis, we examined IGF-I effects in MCF-7 breast cancer cell sublines that have been selected for loss of ERalpha (C4 and C4-12 cells are ERalpha-negative) by long-term estrogen withdrawal. C4 and C4-12 cells had reduced IGF-IR and IRS-1 mRNA and protein expression (compared with MCF-7 cells) that was not inducible by estrogen. Furthermore, C4 and C4-12 cells showed reduced IGF-I signaling and failed to show any growth response to either estrogen or IGF-I. To prove that loss of IGF and estrogen-mediated signaling and growth was a consequence of loss of ERalpha, we re-expressed ERalpha in C4-12 cells by stable transfection with HA-tagged ERalpha. Three independent C4-12 ERalpha-HA clones expressed a functional ERalpha that (a) was down-regulated by estrogen, (b) conferred estrogen-induction of cyclin D1 expression, and (c) caused estrogen-mediated increase in the number of cells in S phase. All of the effects were completely blocked by antiestrogens. Interestingly, ERalpha-HA expression in C4-12 cells did not restore estrogen induction of progesterone receptor expression. However, ERalpha-positive C4-12 cells now exhibited estrogen-induction of IGF-IR and IRS-1 levels and responded mitogenically to both estrogen and IGF-I. These data show that ERalpha is a critical requirement for IGF signaling, and to our knowledge this is the first report of functional ERalpha expression that confers estrogen-mediated growth of an ER-negative breast cancer cell line.     

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.     

Difference between R5020 and the antiprogestin RU486 in antiproliferative effects on human breast cancer cells

We have compared the effects of the progestin R5020 and the antiprogestin RU486 on the growth of the MCF-7 and T47D breast cancer cell lines. Differences between the two compounds were demonstrated in several parameters. 1. Estradiol was required for the efficient inhibition of cell growth of both lines by R5020 but not by RU486. Therefore in the total absence of estrogen (phenol-red free medium), the effects of the two drugs on cell growth were dissociated, RU486 remaining inhibitory while R5020 was inactive. 2. The proteins secreted by cells were differently affected, since R5020 induced a 48K protein and decreased the production of the estrogen-regulated 52K protein, while RU486 had no effect on these two parameters. 3. The morphology of cells treated by R5020 was more altered in the presence of estradiol than in its absence, while that of cells treated by RU486 was not affected whether or not estradiol was present. 4. There was a greater reduction of estrogen receptor sites in MCF-7 cells produced by R5020 than by RU486. Even though the two drugs appear to act through the same progesterone receptor and to inhibit total protein secretion, it is likely that they exert their antiproliferative effects on cultured breast cancer cells by different mechanisms. R5020 antagonizes the stimulation produced by estradiol. RU486 by contrast exerts a more direct progesterone receptor mediated inhibitory effect requiring no synergism by estradiol and therefore does not act through a partial progestin activity.     

Membrane progesterone receptors (mPRs) mediate progestin induced antimorbidity in breast cancer cells and are expressed in human breast tumors

Membrane progesterone receptors (mPRs) have been detected in breast cancer cells and tissues, but their roles in cancer progression remain unclear. Here, we demonstrate the localization, signaling, and antiapoptotic actions of mPRs in two nuclear progesterone receptor (PR)-negative breast cancer cell lines, SKBR3 and MDA-MB-468 (MB468), and mPR expression in human breast tumor biopsies. mPRα, mPRβ, and mPRγ subtypes were detected in both cell lines as well as in breast tumor tissues from 13 individuals irrespective of nuclear steroid receptor expression. Competitive receptor binding studies with a selective PR ligand, R5020, and an mPR agonist, Org OD 02-0 confirmed the presence of functional mPRs on both cancer cell lines. Progesterone treatment of either cell line caused rapid activation of an inhibitory G protein, as well as activation of p42/44 MAP kinase. Treatment with progesterone or Org OD 02-0 significantly decreased cell death and apoptosis in response to serum starvation, whereas testosterone, 17β-estradiol, dexamethasone, and R5020 and RU486 were ineffective. Progesterone treatment of MB468 cells also increased mitochondrial membrane potential and Akt activity, but no decrease in caspase 3 activity was observed. Knockdown of mPRα expression in MB468 cells by siRNA transfection blocked the inhibitory effects of progesterone on cell death. The results indicate that progesterone can act through mPRs to inhibit apoptosis in breast cancer cells. The involvement of mPRs in the development or progression of breast tumor growth through inhibition of cell death is an intriguing possibility and requires further investigation.     

Tyrosine kinase signalling in breast cancer: Insulin-like growth factors and their receptors in breast cancer

The insulin-like growth factor (IGF) system exerts pleiotropic effects on mammalian cells. This review focuses on type I IGF receptor (IGF1R)-mediated signal transduction and its relevance in breast cancer. Upon activation by the IGFs, IGF1R, a transmembrane tyrosine kinase receptor, undergoes autophosphorylation, and then binds and phosphorylates additional signaling molecules. These intermediates initiate a series of downstream signaling events that are involved in multiple physiologic processes for cells. Recent data demonstrate that the IGF receptor system actively interacts with the estrogen receptor and integrin receptor systems. Cross-talk among these pathways regulates breast cancer proliferation, protection from cell death, and metastasis. Better understanding of IGF biochemical signaling pathways is of utmost importance for developing therapies for breast cancer.     

The type-1 insulin-like growth factor receptor tyrosine kinase and breast cancer: biology and therapeutic relevance

The development of the mammary gland requires the coordinated expression of hormones and growth factors. Likewise, some transformed breast cells continue to respond to these same extracellular signals. Thus, understanding the mechanisms that control normal development of tissues can lead to new therapeutic targets. The insulin-like growth factor (IGF) system plays an important role in the normal development and function of the mammary gland. Accumulating evidence suggests that the IGFs are also key regulators of the malignant phenotype. The IGFs stimulate proliferation, promote survival, and enhance metastatic potential of breast cancer cells. Although multiple receptors for the IGFs have been identified, the IGFs primarily exert their biologic effects through ligation of the type I IGF receptor tyrosine kinase (IGF1R). IGF binding to the IGF1R initiates an intracellular signaling cascade that leads to changes in gene expression and cell biology. This review will focus on the evidence that the IGF1R is a relevant treatment target in breast cancer.     

Immunocytochemical localization of progesterone receptors in breast cancer with anti-human receptor monoclonal antibodies

Monoclonal antibodies (MAbs), recently produced against human progesterone receptors (PR), were used for immunocytochemical localization of PR. The specificity of the immunocytochemical assay for PR was demonstrated by incubation with control MAbs, preabsorption of MAbs with highly purified human PR, and by the cell and tissue distribution of the immunostaining reaction. With human breast cancer cell lines, immunoreactivity was confined to cells that contain PR by steroid-binding assay. Moreover, immunostaining was induced by estradiol in estrogen-responsive cells, MCF-7 and ZR-75-1. In a preliminary study with 33 breast carcinomas, a good correspondence was obtained between immunocytochemical staining and PR content assessed by conventional steroid-binding assay. Immunoperoxidase localization was also obtained with other human target tissues. In normal breast and benign breast disease, immunoreactivity was observed with nuclei of ductal epithelial cells and hyperplastic epithelium. In uterus, immunostaining of endometrium was localized to nuclei of stromal and glandular epithelial cells and in myometrium to nuclei of smooth muscle cells. The effect of the progestin agonist, R5020, and antagonist, RU 486, on PR localization was investigated with the PR-rich T47D human breast cancer cell line. In the absence of hormone, immunostaining was exclusively nuclear. This was true under a number of cell culture conditions designed to eliminate endogenous progestins from the culture medium. Exclusive nuclear localization of PR was not due to a failure of the MAbs to recognize unoccupied PR, since each MAb bound equally well in vitro with different receptor forms. These included liganded and unliganded cytosol PR, molybdate stabilized PR, and nuclear-transformed receptors. Nor was failure to detect cytoplasmic staining due to a selective destruction or loss of unoccupied PR from the cytoplasmic compartment as a result of cell fixation. This was assessed by dot blot immunoassay of PR antigen distribution in subcellular fractions of fixed and unfixed cells. Continuous exposure of cells to R5020 resulted in a transient (30-60 min) increase in nuclear staining intensity (without change in cytoplasmic reactivity), followed by a progressive decline in immunoreactivity. By 24 h of R5020 treatment, the vast majority of cells displayed no immunostaining reaction. These immunocytochemical data are consistent with progestins down regulating their own receptors due to a loss in cellular PR content and not to inactivation of receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Insulin receptor substrate 1 is a target for the pure antiestrogen ICI 182,780 in breast cancer cells

The pure antiestrogen ICI 182,780 inhibits insulin-like growth factor (IGF)-dependent proliferation in hormone-responsive breast cancer cells. However, the interactions of ICI 182,780 with IGF-I receptor (IGF-IR) intracellular signaling have not been characterized. Here, we studied the effects of ICI 182,780 on IGF-IR signal transduction in MCF-7 breast cancer cells and in MCF-7-derived clones overexpressing either the IGF-IR or its 2 major substrates, insulin receptor substrate 1 (IRS-1) or src/collagen homology proteins (SHC). ICI 182,780 blocked the basal and IGF-I-induced growth in all studied cells in a dose-dependent manner; however, the clones with the greatest IRS-1 overexpression were clearly least sensitive to the drug. Pursuing ICI 182,780 interaction with IRS-1, we found that the antiestrogen reduced IRS-1 expression and tyrosine phosphorylation in several cell lines in the presence or absence of IGF-I. Moreover, in IRS-1-overexpressing cells, ICI 182,780 decreased IRS-1/p85 and IRS-1/GRB2 binding. The effects of ICI 182,780 on IGF-IR protein expression were not significant; however, the drug suppressed IGF-I-induced (but not basal) IGF-IR tyrosine phosphorylation. The expression and tyrosine phosphorylation of SHC as well as SHC/GRB binding were not influenced by ICI 182,780. In summary, downregulation of IRS-1 may represent one of the mechanisms by which ICI 182,780 inhibits the growth of breast cancer cells. Thus, overexpression of IRS-1 in breast tumors could contribute to the development of antiestrogen resistance.     

Insulin Receptor Substrate Suppression by the Tyrphostin NT157 Inhibits Responses to Insulin-Like Growth Factor-I and Insulin in Breast Cancer Cells

Insulin and insulin-like growth factor (IGF) signaling systems regulate breast cancer growth, progression, and metastasis. The insulin receptor substrates 1 and 2 (IRS1/2) transduce signaling from the type I IGF receptor (IGF-IR) and insulin receptor (InR) to mediate the biological effects of receptor activation. In breast cancer, IRS-1 plays a critical role in cancer cell proliferation while IRS-2 is associated with motility and metastasis. NT157, a small-molecule tyrphostin, downregulates IRS proteins in several model systems. In breast cancer cells, NT157 treatment suppressed IRS protein expression in a dose-dependent manner. Exposure to NT157 inhibited the activation of downstream signaling mediated by the IRS proteins. NT157 induced a MAPK-dependent serine phosphorylation of IRS proteins which resulted in disassociation between IRS proteins and their receptors resulting in IRS degradation. In estrogen receptor-α-positive (ERα+) breast cancer cells (MCF-7 and T47D), NT157 also resulted in cytoplasmic ERα downregulation likely because of disruption of an IRS-1-IGF-IR/InR/ERα complex. NT157 decreased S phase fraction, monolayer, and anchorage-independent growth after IGF/insulin treatment in ERα+ breast cancer cells. NT157 downregulation of IRS protein expression also sensitized ERα+ breast cancer cells to rapamycin. Moreover, NT157 inhibited the growth of tamoxifen-resistant ERα+ breast cancer cells. Given that both IGF-IR and InR play a role in cancer biology, targeting of IRS adaptor proteins may be a more effective strategy to inhibit the function of these receptors.     

Hormonal modulation of HER-2/neu protooncogene messenger ribonucleic acid and p185 protein expression in human breast cancer cell lines

Recent work has suggested that overexpression of the HER-2/neu protooncogene may play a role in the aggressive clinical behavior of some breast tumors. Since hormones are also known to change the proliferation rate and invasiveness of these cells, we have studied the effect of sex steroid hormones and antihormones on levels of the HER-2/neu mRNA and protein in human breast cancer cell lines using complementary DNA and antibody probes. In MCF-7 cells, which contain high levels of estrogen receptor and an estradiol (E2)-inducible progesterone receptor (PR), 1 nM E2 caused a rapid drop in HER-2/neu mRNA (4.8 kilobases), to 40% of control values by 6 h, and a more gradual decrease in HER-2/neu protein, to 50% by 24 h. HER-2/neu protein and mRNA levels remained reduced throughout 1 week of E2 treatment. The effect of E2 was dose dependent, with the maximal effect seen with concentrations of 10(-10) M E2 and above, and antiestrogen partly reversed the E2-induced decrease in HER-2/neu expression. These characteristics suggest that the observed modulation of HER-2/neu is an estrogen receptor-mediated process. In contrast, progestins did not change HER-2/neu mRNA or protein levels in E2-primed MCF-7 cells that contain high levels of PR; in T47D cells, which contain low levels of ER and high levels of PR, addition of E2 or the progestin R5020 or the antiprogestin RU38,486 had no significant effect on HER-2/neu mRNA or protein levels over 6 days of treatment. These results indicate that estrogen but not progestin modulates HER-2/neu protooncogene expression in these breast cancer cell lines and suggest that aggressiveness associated with high levels of HER-2/neu mRNA and protein may be uncoupled from estrogen-stimulated proliferation in these cells.     

Inhibition of insulin-like growth factor I receptor signaling by the vitamin D analogue EB1089 in MCF-7 breast cancer cells: A role for insulin-like growth factor binding proteins.

Insulin-like growth factors I and II (IGF-I and IGF-II) are potent mitogens involved in growth regulation of breast epithelial cells and are implicated in the pathophysiology of breast cancer. Their bioactivity is enhanced or inhibited by specific IGF-binding proteins (IGFBPs). Vitamin D-related compounds (VDRCs) have been shown to inhibit proliferation and induce apoptosis of MCF-7 breast carcinoma cells. We have previously demonstrated that VDRCs antagonize the growth-promoting activity of IGF-I by stimulating autocrine production of IGFBP-5 in MCF-7 cells, but the effect of VDRCs on IGF-I receptor (IGF-IR) intracellular signaling has not been elucidated. We report here that the vitamin D analogue EB1089 interferes with the IGF-IR signaling pathway by attenuating IGF-I-induced tyrosine phosphorylation of IRS-1, and to a lesser extent, IRS-2. It does not affect protein levels of IRS-1, IRS-2 or IGF-IR. However, EB1089 does not inhibit tyrosine phosphorylation of IRS-1 induced by des(1-3) IGF-I, an IGF-I analogue with greatly reduced affinity for IGFBPs. Furthermore, we demonstrate that an antisense IGFBP-5 oligodeoxynucleotide attenuates EB1089-induced inhibition of IGF-I-stimulated tyrosine phosphorylation of IRS-1 and EB1089-induced IGFBP-5 accumulation. These data strongly suggest that IGFBP-5 plays a functional role in the interfering action of EB1089 with the IGF-IR signal transduction pathway.     

Differential roles of IRS-1 and SHC signaling pathways in breast cancer cells

Several polypeptide growth factors stimulate breast cancer growth and may be involved in tumor progression. However, the relative importance of diverse growth factor signaling pathways in the development and maintenance of the neoplastic phenotype is largely unknown. The activation of such growth factor receptors as the insulin-like growth factor I receptor (IGF-I R), erbB-type receptors (erbB Rs) and FGF receptors (FGF Rs) controls the phenotype of a model breast cancer cell line MCF-7. To evaluate the function of 2 post-receptor signaling molecules, insulin receptor substrate-1 (IRS-1) (a major substrate of the IGF-IR) and SHC (a common substrate of tyrosine kinase receptors), we developed several MCF-7-derived cell clones in which the synthesis of either IRS-1 or SHC was blocked by antisense RNA. In MCF-7 cells, down-regulation of IRS-1 by 80–85% strongly suppressed anchorage-dependent and -independent growth and induced apoptotic cell death under growth factor- and estrogen-reduced conditions. The reduction of SHC levels by approximately 50% resulted in the inhibition of monolayer and anchorage-independent growth but did not decrease cell survival. Importantly, cell aggregation and the ability of cells to survive on the extracellular matrix were inhibited in MCF-7/anti-SHC clones, but not in MCF-7/anti-IRS-1 clones. Cell motility toward IGF was not attenuated in any of the tested cell lines, but motility toward EGF was decreased in MCF-7/anti-SHC clones. Our results suggest that in MCF-7 cells: 1) both IRS-1 and SHC are implicated in the control of monolayer and anchorage-independent growth; 2) IRS-1 is critical to support cell survival; 3) SHC is involved in EGF-dependent motility; and 4) normal levels of SHC, but not IRS-1, are necessary for the formation and maintenance of cell-cell interactions. Int. J. Cancer 72:828–834, 1997. © 1997 Wiley-Liss, Inc.