Retinoic acid-induced growth arrest of MCF-7 cells involves the selective regulation of the IRS-1/PI 3-kinase/AKT pathway

In the MCF-7 breast cancer cell line, insulin-like growth factors (IGFs) are known to elicit antiproliferative actions via the insulin receptor substrate-1 (IRS-1)/PI 3-kinase/AKT pathway. All-trans retinoic acid (RA) is a potent inhibitor of MCF-7 cell proliferation, but the mechanism by which growth regulation is achieved remains unclear. We investigated the effects of RA on the regulation of the IGF-IR and its key signaling elements: IRS-1, IRS-2, and SHC. Treatment of MCF-7 cells with RA caused a significant reduction in IRS-1 protein and tyrosine phosphorylation levels at a concentration and time consistent with RA-mediated growth inhibition. IRS-1 regulation is selective, as RA did not influence IRS-2 or SHC levels. Downstream signaling events were also selectively reduced, as RA abrogated IGF-I-stimulated AKT activation but did not alter erk1/2 activation. To confirm the importance of IRS-1 regulation by RA, we examined the response to RA in MCF-7 cells overexpressing IGF-IR and IRS-1. RA resistance was observed in MCF-7 cells overexpressing IRS-1 but not IGF-IR. This suggests that RA-mediated growth inhibition requires the selective downregulation of IRS-1 and AKT. Therapeutic agents targeting the IRS-1/PI 3-kinase/AKT pathway may enhance the cytostatic effects of RA in breast cancer, since overexpression of IRS-1 and AKT have been reported in primary breast tumors.     

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.     

Differential insulin-like growth factor I receptor signaling and function in estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells

The insulin-like growth factor I receptor (IGF-IR) is a ubiquitous and multifunctional tyrosine kinase that has been implicated in breast cancer development. In estrogen receptor (ER)-positive breast tumors, the levels of the IGF-IR and its substrate, insulin-receptor substrate 1 (IRS-1), are often elevated, and these characteristics have been linked with increased radioresistance and cancer recurrence. In vitro, activation of the IGF-IR/IRS-1 pathway in ER-positive cells improves growth and counteracts apoptosis induced by anticancer treatments. The function of the IGF-IR in hormone-independent breast cancer is not clear. ER-negative breast cancer cells often express low levels of the IGF-IR and fail to respond to IGF-I with mitogenesis. On the other hand, anti-IGF-IR strategies effectively reduced metastatic potential of different ER-negative cell lines, suggesting a role of this receptor in late stages of the disease. Here we examined IGF-IR signaling and function in ER-negative MDA-MB-231 breast cancer cells and their IGF-IR-overexpressing derivatives. We demonstrated that IGF-I acts as a chemoattractant for these cells. The extent of IGF-I-induced migration reflected IGF-IR levels and required the activation of phosphatidylinositol 3-kinase (PI-3K) and p38 kinases. The same pathways promoted IGF-I-dependent motility in ER-positive MCF-7 cells. In contrast with the positive effects on cell migration, IGF-I was unable to stimulate growth or improve survival in MDA-MB-231 cells, whereas it induced mitogenic and antiapoptotic effects in MCF-7 cells. Moreover, IGF-I partially restored growth in ER-positive cells treated with PI-3K and ERK1/ERK2 inhibitors, whereas it had no protective effects in ER-negative cells. The impaired IGF-I growth response of ER-negative cells was not caused by a low IGF-IR expression, defective IGF-IR tyrosine phosphorylation, or improper tyrosine phosphorylation of IRS-1. Also, the acute (15-min) IGF-I activation of PI-3 and Akt kinases was similar in ER-negative and ER-positive cells. However, a chronic (2-day) IGF-I exposure induced the PI-3K/Akt pathway only in MCF-7 cells. The reactivation of this pathway in ER-negative cells by overexpression of constitutively active Akt mutants was not sufficient to significantly improve proliferation or survival (with or without IGF-I), which indicated that other pathways are also required to support these functions. Our results suggest that in breast cancer cells, IGF-IR can control nonmitogenic processes regardless of the ER status, whereas IGF-IR growth-related functions may depend on ER expression.     

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.     

An inhibitory function for JNK in the regulation of IGF-I signaling in breast cancer

Insulin-like growth factor-I receptor (IGF-IR) is frequently overexpressed in a variety of cancer types. Since many breast tumors and cancer cell lines overexpress IGF-IR, we tested IGF-I effects on chemotherapy-treated breast cancer cells. IGF-I protects from chemotherapy-induced apoptosis, suggesting that overlapping signaling pathways modulate IGF-I and chemotherapy treatment outcomes. Taxol and other chemotherapy drugs induce c-Jun N-terminal kinase (JNK), a kinase that conveys cellular stress and death signals. Notably, in this paper we show that IGF-I alone induces a potent JNK response and this activity is reversed by inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) with LY294002 in MCF-7 but not T47D cells. Cotreatment of cells with chemotherapy and IGF-I leads to additive JNK responses. Using cells overexpressing Akt, we confirm that IGF-I-mediated survival is Akt dependent. In contrast, overexpression of JNK significantly enhances Taxol-induced apoptosis and inhibits IGF-I survival effects. Further, JNK attenuates anchorage-independent growth of MCF-7 cells. The inhibitory effect of JNK appears to be mediated by serine phosphorylation of IRS-1 (insulin receptor substrate) since both Taxol and IGF-I treatment enhanced Ser(312) IRS-1 phosphorylation, while LY294002 blocked IGF-I-mediated phosphorylation. Taken together, these data provide a mechanism whereby stress or growth factors activate JNK to reduce proliferation and/or survival in breast cancer cells.     

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.     

Role of estrogen receptor alpha in modulating IGF-I receptor signaling and function in breast cancer

The insulin-like growth factor I (IGF-I) receptor (IGF-IR) is a multifunctional transmembrane tyrosine kinase that has been implicated in neoplastic transformation. The tumorigenic potential of IGF-IR relies on its strong anti-apoptotic and mitogenic activity. The growth and survival signals of IGF-IR are mediated through multiple intracellular pathways, many of which emanate from insulin receptor substrate 1 (IRS-1). In hormone-dependent breast cancer cells, IGF-IR and IRS-1 are often co-expressed with the estrogen receptor alpha (ERalpha), and IGF-I and ER systems are engaged in a powerful functional cross-talk. Most notably, activation of ERalpha upregulates the expression of IRS-1, IGF-IR, and IGF-1, which results in amplification of IGF-I responses. Reciprocally, stimulation of IGF-IR increases the phosphorylation and activity of ERalpha. In contrast, in ERalpha-negative breast cancer cells and tumors, the levels of IGF-IR and IRS-1 are often decreased and IGF-I is non-mitogenic. Our data suggest that defective IGF-IR signaling in ERalpha-negative cells is related, at least in part, to improper activation of the IRS-1/PI-3K/Akt/GSK-3 pathway and lack of Rb1 phosphorylation. These defects are partially reversed by re-expression of ERalpha. Interestingly, some non-mitogenic IGF-I responses, such as migration and invasion are retained in the absence of ERalpha, suggesting that IGF-IR function in breast cancer cells might depend on the ERalpha status. The understanding of how ERalpha may dictate IGF-I responses will help in devising rational anti-IGF-IR strategies for breast cancer treatment.     

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.     

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.     

Down-regulation of insulin-like growth factor-I receptor and insulin receptor substrate-1 expression in advanced human breast cancer

The ligands, receptors and related signaling proteins of the insulin-like growth factor family are involved in the regulation of breast-cancer cell growth. We investigated the expression pattern of insulin-like growth factor-I receptor (IGF-IR), insulin receptor (IR) and insulin receptor substrate-1 (IRS-1), a core downstream signaling protein, in 69 primary breast-cancer specimens of different grades and in 21 control tissues by immunohistochemistry. In addition, cell proliferation (percentage of Ki67(+) nuclei) and estrogen receptor (ER) expression were determined. IGF-IR, IRS-1 and IR were expressed mainly in epithelial cells. IRS-1 and IGF-IR were expressed at high levels in control tissues and in well and moderately differentiated carcinomas but at low levels in poorly differentiated breast cancers. IR expression did not show a significant correlation with the differentiation grade of the tissues investigated. Statistical analysis (ROC analysis for tumor grade) demonstrated that down-regulation of IGF-IR and IRS-1 correlated better with tumor progression than reduction of ER expression or increase in cell proliferation, IGF-IR showing the best correlation, followed by IRS-1 and, less significant, ER and Ki67. Our findings clearly show that progression of breast cancer is accompanied by a reduction of IGF-IR/IRS-1 expression and that IGF-IR/IRS-1 expression inversely correlates with high proliferation rate in dedifferentiated breast cancers. The strong correlation of IGF-IR and IRS-1 down-regulation with tumor progression suggests the use of IGF-IR and IRS-1 as a novel set of marker proteins for tumor grading.     

Insulin receptor substrate-1 regulates the transformed phenotype of BT-20 human mammary cancer cells

Although originating from a human breast cancer, BT-20 cells do not form colonies in soft agar. BT-20 cells do not express insulin receptor substrate-1 (IRS-1), which is known to promote both normal and abnormal growth and to inhibit differentiation. Stable expression of IRS-1 confers to BT-20 cells the ability to form colonies in soft agar. BT-20 cells form tumors in xenografts in mice, but the size of tumors is twice as large when the cells express IRS-1. The increased transformed phenotype is characterized by occupancy of the rDNA and cyclin D1 promoters by IRS-1 and the activation of the cyclin D1, c-myc, and rDNA promoters. In addition, the retinoblastoma protein, which is detectable in the rDNA promoter of quiescent BT-20/IRS-1 cells, is replaced by IRS-1 after insulin-like growth factor-I stimulation. Our results indicate that in BT-20 human mammary cancer cells, expression of IRS-1 activates promoters involved in cell growth and cell proliferation, resulting in a more transformed phenotype. Targeting of IRS-1 could be effective in inhibiting the proliferation of mammary cancer cells.     

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.     

The putative tumor suppressor gene PTPN13/PTPL1 induces apoptosis through insulin receptor substrate-1 dephosphorylation

The protein tyrosine phosphatase (PTP) PTPL1/PTPN13 is a candidate tumor suppressor gene. Indeed, PTPL1 activity has been reported recently to be decreased through somatic mutations, allelic loss, or promoter methylation in some tumors. We showed previously that its expression was necessary for inhibition of Akt activation and induction of apoptosis by antiestrogens in breast cancer cells. Implications of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in cancer progression are now well established, and our study was therefore designed to define whether PTPL1 is sufficient to inhibit this pathway and, if so, to identify a direct substrate of this PTP, which may trigger a proapoptotic effect. We first show by complementary approaches that PTPL1 specifically dephosphorylates insulin receptor substrate-1 (IRS-1) in vitro and in cellulo. Next, our experiments using a dominant-negative mutant and RNA interference confirm the crucial role of PTPL1 in IRS-1 dephosphorylation. Finally, we report that PTPL1 expression is sufficient to block the IRS-1/PI3K/Akt signaling pathway, to inhibit the insulin-like growth factor-I effect on cell survival, and to induce apoptosis. Altogether, these data provide the first evidence for a direct positive role of the putative tumor suppressor gene PTPL1/PTPN13 on apoptosis and identify its target in the IRS-1/PI3K/Akt signaling pathway.     

Clinical significance of insulin receptor substrate-I down-regulation in non-small cell lung cancer

Insulin receptor substrate-1 (IRS-1) is an adaptor protein for insulin-like growth factor (IGF) signaling and it is presumed associated with cancer development, progression or clinical outcome of patients harboring solid tumors. Therefore, we investigated by immunohistochemistry, the expression of IRS-1 in the tumor tissues from 94 patients who were diagnosed as stage I non-small cell lung cancer (NSCLC) and had undergone a curative lung resection. The relationships between its intratumoral expression and various clinical parameters were explored. IRS-1 is consistently expressed in the cytoplasm of intrapulmonary bronchial and bronchiolar epithelial cells comprising normal appearing adjacent lung tissues. Forty-one (43.6%) of 94 specimens showed loss of IRS-1 expression. In a subset analysis, IRS-1 was more frequently lost in stage IB than in IA tumors (50.0 vs. 22.7%, p=0.024, chi(2) test), which was reflected by the facts that tumors which showed down-regulation of IRS-1 had larger area than those with IRS-1 expression (18.1 vs. 12.1 cm(2), p=0.044, t-test). Down-regulation of IRS-1 is more frequently observed in squamous cell carcinoma than other cell type lung cancer (p=0.002, chi(2) test) and its expression was not affected by histological grade of differentiation. Comparing pack-years (P.Y.) between groups of smokers whose tumor expressed IRS-1 and those that did not, smokers whose tumor showed loss of IRS-1 expression had higher P.Y. than those whose tumor did express IRS-1 (39.2+/-23.67 vs. 25.6+/-26.61 P.Y., p=0.034, t-test). Intratumoral expression of IRS-1 did not influence disease-free survival, disease-specific survival or overall survival of stage I NSCLC patients, whose median follow-up duration is 7.5 years (95% CI; 7.21-7.86 years). These results suggest that loss of IRS-1 might rather be an early event in NSCLC development than a prognostic factor and that it is more strongly related with squamous cell carcinoma and with smoking.