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Cell Growth & Differentiation Vol. 11, 655-664, December 2000
© 2000 American Association for Cancer Research

Articles

Inhibition of Mitogen-activated Protein Kinase Kinase Selectively Inhibits Cell Proliferation in Human Breast Cancer Cells Displaying Enhanced Insulin-like Growth Factor I-mediated Mitogen-activated Protein Kinase Activation 1

Ulrich Hermanto, Cong S. Zong and Lu-Hai Wang2

Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029-6574

Abstract

Mitogen-activated protein (MAP) kinase mediates cell proliferation, cell differentiation, and cell survival by regulating signaling pathways activated by receptor protein tyrosine kinases (RPTKs), including the insulin-like growth factor 1 receptor (IGF-IR). We analyzed the upstream signaling components of the MAP kinase pathway, including RPTKs, in human breast cancer cell lines and found that some of those components were overexpressed. Importantly, signaling molecules such as IGF-IR, insulin receptor, and insulin receptor substrate 1, leading to the MAP kinase pathway, were found to be concomitantly overexpressed within certain tumor lines, i.e., MCF-7 and T-47D. When compared with the nonmalignant and other breast tumor lines examined, MCF-7 and T-47D cells displayed a more rapid, robust, and sustained MAP kinase activation in response to insulin-like growth factor I (IGF-I) stimulation. By contrast, IGF-I treatment led to a sustained down-regulation of MAP kinase in those lines overexpressing ErbB2-related RPTKs. Interestingly, blocking the MAP kinase pathway with PD098059 had the greatest antiproliferative effect on MCF-7 and T-47D among the normal and tumor lines tested. Furthermore, addition of an IGF-IR blocking antibody to growth medium attenuated the ability of PD098059 to suppress the growth of MCF-7 and T-47D cells. Thus, our study suggests that concomitant overexpression of multiple signaling components of the IGF-IR pathway leads to the amplification of IGF-I-mediated MAP kinase signaling and resultant sensitization to PD098059. The enhanced sensitivity to PD098059 implies an increased requirement for the MAP kinase pathway in those breast cancer cells, making this pathway a potential target in the treatment of selected breast malignancies.

Introduction

The Ras/MAP3 kinase pathway has been shown to play an important role in mitogenic signaling in various types of cells (1, 2, 3) . This pathway is used by various integral membrane proteins including members of the RPTK superfamily and thus represents a convergence point for RPTK-mediated signaling (4) . Activation of a RPTK typically results in its oligomerization, autophosphorylation, and further stimulation of its intrinsic kinase activity (5) . The adapter molecules Shc, IRS-1, and IRS-2 link the activated RPTK to the Ras/MAP kinase pathway via their Src homology 2 or phosphotyrosine binding domains, which bind to specific phosphotyrosine residues in the cytoplasmic domain of the activated receptor (6, 7, 8, 9) . Subsequently, phosphorylation by the RPTK of specific tyrosine residues on Shc and IRS-1 generates binding sites necessary for the recruitment of the Grb2/Sos complex to the plasma membrane where Sos mediates Ras activation by stimulating Ras to exchange GDP for GTP (10, 11, 12, 13, 14, 15, 16) . Activation of Ras leads to the recruitment of Raf, a MAP kinase kinase kinase, to the membrane where it is subsequently activated by as yet unclear mechanisms (17 , 18) . Raf phosphorylates a MEK which, in turn, phosphorylates the MAP kinases (ERK1 and ERK2) on critical tyrosine and threonine residues (2 , 3) . Activation of MAP kinase leads to the phosphorylation of both cytoplasmic and nuclear targets, including the ribosomal subunit protein kinase p90RSK and transcription factors Elk-1, c-Myc, c-Jun, and c-Fos, which are important in mediating cell cycle progression and proliferation (2 , 19, 20, 21) . In addition to its established role in mitogenic signaling, the Ras/MAP kinase pathway has been shown to be involved in regulating cell differentiation and cell survival signaling (22 , 23) .

A large number of studies have provided evidence for the aberrant overexpression of RPTKs, their cognate growth factor ligands, or their downstream signaling molecules in human breast cancer and have implicated their involvement in malignant progression. Overexpression of the ErbB RPTK family, including the EGFR and ErbB2, occurs in 20–30% of primary breast cancers and is correlated with an increased resistance to hormone-based therapy and a negative clinical prognosis in this population of patients (24, 25, 26) . Similarly, in breast cancer patients, elevated levels of plasma IGF-I and the IGF-IR within primary tumor tissue have been documented (27, 28, 29) . IGF-IR overexpression has been correlated with an increased tumor recurrence after surgery and radiation therapy as well as a worse prognosis and reduced survival in the subgroup of patients with either node-positive or estrogen receptor-negative tumors (30 , 31) . Increased expression of IRS-1 in primary breast tumors was shown to be associated with an increased tumor recurrence in a subgroup of patients (32) . Recent studies have revealed the overexpression of Grb2 as well as increased phosphorylation and expression of MAP kinase in certain breast tumors, suggesting that upstream components of the MAP kinase pathway and MAP kinase itself may participate in breast cancer development (33 , 34) .

Experimental evidence from in vitro and in vivo studies using cell lines suggest that RPTK signaling pathways play critical and essential roles in the proliferative and metastatic behavior of breast cancer cells. Inhibition of the IGF-IR by antisense RNA, dominant-negative mutant, or blocking antibody has been shown to suppress the monolayer and anchorage-independent proliferation of MCF-7 human breast cancer cells in culture and the growth of T 61 human breast cancer xenografts in mice (35, 36, 37) . Expression of a dominant-negative mutant of IGF-IR was also shown to suppress the ability of the malignant breast cancer lines MDA-MB-231 and MDA-MB-435 to adhere to several extracellular matrix proteins, to invade through a collagen matrix in vitro, and to metastasize from primary tumors in mice (38) . IRS-1 and Shc also appear to play important roles in breast cancer because the use of antisense RNA of these signaling molecules was shown to inhibit both the anchorage-dependent and -independent growth of MCF-7 cells (39) . Moreover, a dominant-negative mutant of Shc was shown to block cell cycle progression at G0-G1 and G2-M phases in breast cancer cells exhibiting high ErbB2 expression levels but not in a normal breast cell line (40) .

To further explore the role of the components of RPTK signaling pathways in breast cancer, specifically to examine how their overexpression might affect the signaling and overall behavior of mammary tumors, we used a panel of benign and malignant breast epithelial cell lines to establish a pattern of overexpression and activation of RPTK signaling molecules in breast cancer cells. In this context, we examined the activation of MAP kinase in those cell lines and explored the significance of MAP kinase signaling in cell proliferation by using an established pharmacological inhibitor of MEK called PD098059 (41 , 42) . Our results indicate that the activation of ERK1 and ERK2 in response to IGF-I was significantly higher in breast cancer lines exhibiting concomitant overexpression of multiple upstream signaling molecules of the MAP kinase pathway, including the IGF-IR, IR, and IRS-1, relative to other cell lines. In cell proliferation assays, breast cancer lines showing enhanced MAP kinase activation displayed an increased sensitivity to PD098059, which was attenuated when IGF-IR signaling was blocked in these cells. Our results suggest an increased role for the MAP kinase pathway in the growth of a subset of human breast cancers, making this pathway a potential target in the treatment of these breast malignancies.

Results

Elevated MAP Kinase Phosphorylation in Breast Cancer Cell Lines.
We first examined the extent of steady-state intracellular MAP kinase activation in a series of tumor-derived human breast epithelial cell lines growing exponentially in complete growth medium, i.e., DMEM containing 10% FBS and 5 µg/ml insulin. Total cell lysates were immunoblotted with an antibody (anti-phosphoERK1/ERK2) that is specific for the phosphorylated (Thr202/Tyr204) and thus activated forms of the p42 and p44 MAP kinases (ERK2 and ERK1, respectively; Fig. 1Citation ). When compared with the nonmalignant mammary lines HBL-100 and AB589, the phosphorylation of both ERK1 and ERK2 was increased in each of the nine breast tumor lines examined (Fig. 1Citation , upper panel). Although ERK phosphorylation in AB589 was undetectable at this exposure, phosphorylated ERK2 (and ERK1 to a lesser extent) was detected at exposures three times longer. A parallel SDS-PAGE gel immunoblotted with anti-ERK2 antibody demonstrated that MAP kinase expression levels were relatively uniform in the normal and tumor cell lines examined (Fig. 1Citation , lower panel). These results indicate that the increased phosphorylation of MAP kinase in breast cancer lines is attributable to specific activation. Furthermore, our findings suggest that MAP kinase signaling is frequently activated in breast cancer cell lines.



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  		Fig. 1. MAP kinase phosphorylation and expression in nonmalignant and malignant mammary cell lines. Nonmalignant human mammary epithelial cells and a panel of nine human breast cancer lines were grown exponentially in complete growth medium and lysed with RIPA buffer. Twenty µg of lysate protein from each line were fractionated by SDS-PAGE in duplicate gels and immunoblotted with anti-phosphoERK1/ERK2 (A) or anti-ERK2 (B). The anti-ERK2 antibody reacts strongly with ERK2 but less so with ERK1. *, nonmalignant.

 
Concomitant Overexpression of IGF-IR, IR, and IRS-1 in Breast Tumor Cells.
To explore upstream signaling molecules that may be responsible for the increased activation of MAP kinase in breast cancer cells, we examined the protein expression and activation of individual components of RPTK signaling pathways. Previous studies have demonstrated that members of the ErbB family are overexpressed in a significant proportion of breast cancer lines including BT-20, MDA-MB-231, SK-BR-3, BT-474, MDA-MB-361, and MDA-MB-453 when compared with normal and other breast cancer lines (43 , 44) .4 We examined in tumor lines the expression levels of IGF-IR and IR (Fig. 2)Citation . Immunoprecipitation and immunoblotting experiments with anti-IGF-IR showed that, in comparison with AB589, the expression level of IGF-IR was elevated by 2–4-fold in 50% of the tumor lines examined, including MCF-7, T-47D, BT-20, MDA-MB-361, and MDA-MB-453 (Fig. 2A)Citation , and is consistent with the reported frequent overexpression of the IGF-IR in primary breast tumors (27 , 28) . Activation of the IGF-IR in these cells was analyzed by performing in vitro autokinase assays with anti-IGF-IR immunoprecipitates from cells transiently treated with or without IGF-I. In unstimulated serum-starved conditions, the basal IGF-IR activity was elevated in MCF-7 cells in comparison with eight other cell lines, including the two nonmalignant lines, AB589 and MCF-10F (Fig. 2Citation B, upper panel). Consistent with the idea that ligand-independent oligomerization of overexpressed RPTKs can lead to constitutive receptor activation, IGF-IR overexpression in MCF-7 cells may underlie the increased total basal activity of this RPTK in these cells. After IGF-I treatment, IGF-IR activity was increased significantly in all cell lines examined, with the exception of Hs578T, and was the highest in MCF-7 cells (Fig. 2Citation B, lower panel). These results demonstrate that functionally active IGF-I receptors are overexpressed in a large proportion of breast cancer lines. Analysis of the IR expression level by immunoblotting total cell lysates with anti-IR demonstrated that, relative to AB589, the IR was overexpressed in six of nine tumor lines examined, including MCF-7, MDA-MB-231, ZR-75-1, and BT-474, and to a lesser extent MDA-MB-361 and MDA-MB453 (Fig. 2C)Citation . Together, these data indicate that IGF-IR and IR are commonly overexpressed in breast tumor cell lines, which may contribute to the activation of downstream signal transduction pathways.



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  		Fig. 2. Expression of IGF-IR and IR and IGF-IR kinase activity. A, nonmalignant and malignant mammary cell lines growing in complete growth medium were lysed with RIPA buffer. Five hundred µg of lysate protein were immunoprecipitated (IP) with anti-IGF-IR, resolved by 10% SDS-PAGE, and immunoblotted (IB) with anti-IGF-IR. B, cells were grown to confluence, serum-starved for 16 h, and incubated for 10 min with or without IGF-I. Cell lysates were prepared with RIPA buffer, and 500 µg of each lysate were immunoprecipitated with anti-IGF-IR. Immunoprecipitates were subjected to in vitro kinase assay and analyzed as described in "Materials and Methods." C, 20 µg of cell lysate from A was resolved by SDS-PAGE and immunoblotted with anti-IR. The positions of the molecular weight markers are indicated. *, nonmalignant.

 
We next analyzed the expression of IRS-1, a primary substrate for IR and IGF-IR, which couples these receptors to the PI 3-kinase and MAP kinase pathways by providing binding sites for the p85 regulatory subunit of PI 3-kinase and the adapter protein Grb2, respectively. Immunoprecipitation and immunoblotting with anti-IRS-1 showed that, relative to nonmalignant AB589 and MCF-10F cells, IRS-1 levels were increased in three of seven tumor lines examined (Fig. 3A)Citation . IRS-1 was overexpressed in MCF-7, T-47D, and MDA-MB-231 cells by 30-, 15-, and 2.5-fold, respectively, compared with AB589. We then determined whether IRS-1 was activated in these cells by assessing its tyrosine phosphorylation level with or without IGF-I stimulation (Fig. 3B)Citation . In unstimulated conditions, the basal level of IRS-1 tyrosine phosphorylation was detectable only in MCF-7 cells (Fig. 3B)Citation , consistent with its elevated basal IGF-IR activity (Fig. 2B)Citation and increased IRS-1 steady state level (Fig. 3A)Citation . IGF-I treatment led to a significant increase in IRS-1 phosphorylation in MCF-7 and T-47D cells, whereas IRS-1 phosphorylation was undetectable under those conditions in normal (MCF-10F) and tumor (BT-20 and BT-474) cells, which do not overexpress IRS-1 (Fig. 3B)Citation . These results indicate that the total levels of IGF-I-mediated IRS-1 tyrosine phosphorylation are greater in MCF-7 and T-47D cells, which may lead to its augmented downstream signaling. In addition, our results indicate that IRS-1, a major substrate for IR and IGF-IR, is overexpressed in a subset of breast cancer lines, including those which overexpress the IGF-IR (T-47D), the IR (MDA-MB-231), or both (MCF-7).



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  		Fig. 3. IRS-1 expression and tyrosine phosphorylation. A, nonmalignant and malignant mammary cell lines were grown in complete growth medium and were lysed with RIPA buffer. One mg of lysate protein was immunoprecipitated (IP) with anti-IRS-1, resolved by SDS-PAGE, and immunoblotted (IB) with anti-IRS-1. B, cells were grown to confluence, serum-starved overnight, and incubated for 10 min with or without IGF-I. Prior to lysis, each cell line was treated for 15 min with 40 µM phenylarsine oxide (phosphatase inhibitor) to inhibit phosphatases. One mg of RIPA-extracted lysate protein was immunoprecipitated with anti-IRS-1, resolved by SDS-PAGE, and immunoblotted with anti-phosphotyrosine (anti-pTyr). The positions of the molecular weight markers are indicated. *, nonmalignant.

 
Abnormal IGF-I-dependent Regulation of MAP Kinase Activity in Breast Cancer Cells.
To determine whether the increased expression of IRS-1 in mammary tumor lines that co-overexpress IGF-IR, IR, or both can potentially amplify downstream signaling pathways, e.g., the Ras/MAP kinase pathway, we investigated the activation of the MAP kinase pathway. After transient (30 min or less) or prolonged (>30 min) exposure of cells to IGF-I, MAP kinase phosphorylation was analyzed by immunoblotting total cell lysates with anti-phosphoERK1/ERK2 (Fig. 4)Citation . Our results revealed that in nonmalignant AB589 cells, ERK2 phosphorylation remained unchanged for up to 30 min and increased to 3-fold over the basal level after 6 h of treatment with IGF-I (Fig. 4A)Citation . Under those conditions, however, ERK1 phosphorylation was not detectable at any time point examined. In contrast, phosphorylation of both ERK1 and ERK2 in MCF-7 and T-47D breast cancer cells was rapid and robust, increasing to 29- and 17-fold, respectively, over the unstimulated levels within 10 min of treatment with IGF-I (Fig. 4A)Citation . Furthermore, phosphorylation stimulated by IGF-I in both cell lines was sustained above the unstimulated levels for at least 6 h. In Hs578T cells, phosphorylation of both ERK1 and ERK2 after IGF-I treatment was mild; ERK2 phosphorylation increased within 10 min to ~2-fold over the unstimulated level, remaining elevated for the next 6 h, and ERK1 phosphorylation increased within 30 min to 4.5-fold over the unstimulated level, also remaining sustained for at least 6 h (Fig. 4A)Citation . In MDA-MB-231 cells, the basal unstimulated level of ERK2 phosphorylation (Fig. 4A)Citation and in vitro kinase activity (data not shown) were both elevated significantly when compared with the other cell lines, and IGF-I did not further increase the phosphorylation and activity (data not shown) of ERK2, suggesting that the MAP kinase pathway is constitutively activated in these cells. The relative refractoriness of MAP kinase activation in response to IGF-I may also reflect that the modest overexpression of IRS-1 (Fig. 3A)Citation and the low IGF-IR expression (Fig. 2A)Citation in these cells may not be sufficient to augment MAP kinase signaling above constitutive levels, despite the fact that ERK1 appears to be modestly and transiently activated. These data, which were confirmed by in vitro kinase and gel mobility shift assays (data not shown), establish that MCF-7 and T-47D cells, which overexpress more than one component of the IGF-IR signaling pathway, possess an enhanced ability to respond to IGF-I, leading to amplified MAP kinase signaling.



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  		Fig. 4. Activation of MAP kinase by IGF-I. Nonmalignant and malignant mammary cell lines were grown to confluence, serum-starved overnight, and treated with IGF-I for 10, 30, or 360 min except for BT-474 and BT-20, which were incubated for 15, 30, 45, 90, or 180 min. Unstimulated cells were mock-treated without IGF-I for 30 min. Twenty µg of each RIPA-extracted cell lysate were fractionated by SDS-PAGE and immunoblotted with anti-phosphoERK1/ERK2. Arrows, the phosphorylated forms of p44 (ERK1) and p42 (ERK2) MAP kinase. Although ERK2 phosphorylation in AB589 cells was detected with ECL exposures >2 min, ERK1 phosphorylation in these cells was not detectable at any exposure up to 25 min. Exposure times of <30 s were required to detect phosphorylation in the other cell lines. *, nonmalignant.

 
Unexpectedly, treatment with IGF-I did not stimulate MAP kinase phosphorylation but, instead, led to a down-regulation of the basal MAP kinase activity in MDA-MB-453, SK-BR-3, BT-474, and BT-20 breast cancer cells (Fig. 4B)Citation . Relative to unstimulated levels, phosphorylation of ERK1 and ERK2 was markedly reduced within 30 min of IGF-I treatment in each of these tumor lines. In addition, MAP kinase phosphorylation did not return to basal levels after prolonged treatment with IGF-I (6 h for MDA-MB-453 and SK-BR-3 cells and 3 h for BT-474 and BT-20 cells). These experiments were corroborated by in vitro kinase assays, which show a similar suppression of MAP kinase activity (data not shown). Previous studies, including our own, have demonstrated the overexpression of EGFR in BT-20 cells and the overexpression of ErbB2 in MDA-MB-453, SK-BR-3, and BT-474 cells (43 , 44) .4 Thus, our results demonstrate that IGF-I causes a sustained down-regulation of MAP kinase phosphorylation and kinase activity in a subset of breast cancer lines, i.e., those that overexpress ErbB RPTK family members.

Blockade of the MAP Kinase Pathway by PD098059 Selectively Inhibits Growth.
The activated and enhanced MAP kinase signaling in breast cancer lines, as suggested by the increased responsiveness of MAP kinase to IGF-I, led us to explore the functional role of MAP kinase in these tumor cells. PD098059, an established inhibitor of MEK1 and MEK2 (41 , 42) , was used to assess the role of this pathway in regulating cellular proliferation under standard monolayer growth conditions, i.e., in complete growth medium containing FBS and insulin. Four days after plating at equivalent subconfluent densities, we analyzed the growth of tumor and nonmalignant cells with or without PD098059 treatment (Fig. 5A)Citation . Our result showed that PD098059 most severely affected proliferation of MCF-7 and T-47D cells, because growth was reduced to 41 and 43%, respectively. Proliferation of Hs578T and MDA-MB-231 tumor cells was modestly reduced to 74 and 71%, respectively. However, the remaining malignant and nonmalignant breast epithelial cell lines were not significantly affected by the drug, with cell growth of 88–98% relative to their respective untreated controls. In untreated conditions, the population doubling times of normal HBL-100 and AB589 cells and malignant MCF-7, MDA-MB-231, and Hs578T cells were comparable (22–26 h), whereas that of T-47D cells was longer (43 h). Therefore, the sensitivity of these breast cancer lines to PD098059 is unlikely to be a reflection of the differences in the rates of proliferation among these lines. Together, these data demonstrate that under standard monolayer growth conditions, treatment with 25 µM PD098059 is effective in selectively suppressing the proliferative capacity of MCF-7 and T-47D cells and, to a lesser extent, Hs578T and MDA-MB-231 cells, all of which display, to various extents, IGF-I-dependent MAP kinase activation (Fig. 4A)Citation , when compared with the normal and other tumor lines examined.



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  		Fig. 5. Effect of PD098059 on cellular proliferation and MAP kinase phosphorylation. A, nonmalignant and malignant mammary cell lines were plated at a density of 1 x 104 cells/cm2 in 3.5-cm dishes and incubated in complete growth medium supplemented with PD098059 or with DMSO as vehicle control. Duplicate dishes were counted after 3–4 days. The number of cells in PD098059-treated dishes was expressed as a percentage of the number of cells in the respective control dishes. Results from at least three separate experiments were used to calculate the mean; bars, SE. The Student’s t test was used to determine that growth in the first four malignant lines was significantly different when compared with each of the other nonmalignant and malignant cell lines. *, P < 0.05. B, cells plated at equivalent subconfluent densities were treated as in A. After 3 h of treatment, cell lysates were prepared with RIPA buffer, and 10 µg of lysate protein were fractionated by SDS-PAGE and immunoblotted with anti-phosphoERK1/ERK2. The membrane was stripped and reprobed with anti-ERK2 to confirm that equivalent amounts of protein were present.

 
To evaluate the effect of the MEK inhibitor on MAP kinase activity in those breast cancer cells, we examined the steady-state MAP kinase phosphorylation of exponentially growing cells in monolayer after transient treatment with PD098059 (Fig. 5B)Citation . In agreement with the established roles of MEK1 and MEK2 as upstream activators of ERK1 and ERK2, treatment with PD098059 dramatically diminished the phosphorylation of ERK1 and ERK2 in both nonmalignant and tumor cells (Fig. 5Citation B, upper panel), consistent with abrogation of ERK2 in vitro kinase activity (data not shown). Immunoblotting with anti-ERK2 confirmed that there were equivalent amounts of ERK2 in treated and untreated cells (Fig. 5Citation B, lower panel). These data collectively indicate that blockade of the MAP kinase pathway by PD098059 results in the selective attenuation of the proliferation of some but not all breast cancer cell lines.

Sensitivity to PD098059-mediated Growth Inhibition in MCF-7 and T-47D Cells Is Attenuated upon Inhibition of IGF-IR-mediated Signaling.
Our findings indicate that MCF-7 and T-47D breast cancer cells overexpress multiple components of the IGF-IR signaling pathway and strongly suggest that they may lead to increased IGF-I-mediated MAP kinase activation. To investigate whether the increased sensitivity to PD098059 is dependent on IGF-IR-mediated signaling in MCF-7 and T-47D cells, we added IGF-IR blocking antibody into complete growth medium to inhibit the signaling by the IGF-IR and evaluated the effect of PD098059 on cell growth under these conditions (Fig. 6)Citation . The neutralizing antibody we used ({alpha}IR-3) has been shown to block the binding of IGF-I to IGF-IR (IC50, 100 ng/ml) and inhibit the activity of IGF-I on the growth of MCF-7 cells (45) . Initial experiments using 1 µg/ml of anti-IGF-IR resulted in maximal inhibition of cell proliferation of both cell lines without further inhibition by PD098059. Therefore, we titrated the amount of anti-IGF-IR antibody added to the medium to a range in which the difference in cell growth between anti-IGF-IR antibody alone and isotype-matched control antibody alone was minimized. At a concentration of 400 ng/ml of anti-IGF-IR antibody alone, cell growth was marginally reduced (6 and 11% in MCF-7 and T-47D, respectively) when compared with control antibody alone (Table 1)Citation . In the presence of this amount of control antibody, treatment with PD098059 inhibited cell growth to 42–44% of the respective DMSO-treated controls (Table 1Citation ; Fig. 6ACitation ), consistent with results from previous experiments performed without addition of antibody to the medium (Fig. 5A)Citation . However, in the presence of an equivalent amount of anti-IGF-IR antibody, treatment with PD098059 suppressed the growth of MCF-7 and T-47D cells to only 67–68% of the respective DMSO-treated controls (Table 1Citation ; Fig. 6ACitation ). We found that using 100–500 ng/ml of antibody yielded similar and consistent results in repeated experiments.



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  		Fig. 6. Role of IGF-IR in the sensitization of MCF-7 and T-47D cells to PD098059. A, MCF-7 and T-47D cells were plated in triplicate in 24-well plates at densities of 1 x 104 and 2.5 x 104 cells/cm2, respectively. Cells subsequently were incubated in complete growth medium supplemented with control antibody/DMSO, control antibody/PD098059, anti-IGF-IR antibody/DMSO, or anti-IGF-IR antibody/PD098059. After 4 days, triplicate wells were counted for each treatment (Table 1)Citation . The number of cells in antibody/PD098059-treated wells was expressed as a percentage of the number of cells in the respective antibody/DMSO-treated wells. Means are shown; bars, SD. The Student’s paired t test was used to determine that PD098059-inhibited growth in the presence of anti-IGF-IR blocking antibody was significantly different from PD098059-inhibited growth with control IgG antibody. *, P < 0.05. Data shown are representative of three similar experiments. B, MCF-7 and T-47D cells were plated at equivalent subconfluent densities and were treated as in A or were left untreated. After 3 h, cell lysates were prepared with RIPA buffer, and 750 µg of lysate protein were immunoprecipitated (IP) with polyclonal anti-IGF-IR, resolved by SDS-PAGE, and immunoblotted with anti-phosphotyrosine (anti-pTyr). From the same lysates, 20 µg of protein were fractionated by SDS-PAGE and immunoblotted with anti-phosphoERK1/ERK2. The membranes were stripped and reprobed with anti-IGF-IR or anti-ERK2 to confirm that equivalent amounts of protein were present in each lane.

 

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  		Table 1 Effect of PD098059 on cell proliferation in the presence of anti-IGF-IR antibody or isotype-matched control antibodya

 
To confirm that MAP kinase signaling was inhibited in the presence of IGF-IR blocking antibody, we examined the tyrosine phosphorylation of the IGF-IR and MAP kinase phosphorylation under similar conditions used to analyze cell growth (Fig. 6B)Citation . Treatment with PD098059 in parallel was included in this experiment for comparison. IGF-IR tyrosine phosphorylation levels in both MCF-7 and T-47D cells were clearly diminished but not totally abolished in the presence of anti-IGF-IR antibody as compared with control IgG. Phosphorylation of MAP kinase was reduced by at least 40% with treatment of anti-IGF-IR antibody alone but was unaffected using the control antibody, demonstrating that anti-IGF-IR neutralizing antibody at this concentration (i.e., 400 ng/ml) partially blocks IGF-IR-dependent signaling and downstream MAP kinase activation under these conditions (Fig. 6B)Citation . As expected, treatment with PD098059 did not affect IGF-IR phosphorylation; however, PD098059 treatment with either the control or anti-IGF-IR antibody reduced MAP kinase phosphorylation to <5% of the levels in the respective DMSO-treated controls. These observations collectively show that partial blockade of IGF-IR signaling mitigates the inhibitory effect of PD098059 on cell proliferation, indicating that IGF-IR signaling in MCF-7 and T-47D cells contributes, at least, in part to their increased sensitization to PD0908059.

Discussion

The MAP kinase signaling pathway has been established to play an important role in the processes of cell proliferation, cell transformation, and tumorigenicity in mammalian cells (46 , 47) . In human cancers, the constitutive activation of MAP kinase and its upstream signaling components, including Raf-1 and MEK, were shown to occur at higher frequencies in cell lines and primary tissue derived from tumors of the colon, lung, and kidney (48) . Requirement for activation of the MAP kinase pathway in the progression of these malignancies is supported by a recent study demonstrating the in vivo growth suppression of colon tumor cells after administration of the MEK-specific inhibitor PD184352 to mice (49) . Our data show that the steady-state phosphorylation of ERK1 and ERK2 in all breast cancer lines examined here is increased relative to nonmalignant breast epithelial cells, suggesting that activation of MAPK signaling is a frequent occurrence in breast cancer cells. This is consistent with previous work documenting the increased expression, phosphorylation, and activation of MEK and MAP kinase in malignant breast tissues (33 , 50) . These established breast epithelial lines, therefore, could serve as an in vitro model system to examine the functional significance of activated MAP kinase signaling in human breast cancer.

Our analyses have identified within the same tumor lines the overexpression of several functionally active signaling components lying upstream of the MAP kinase pathway. Furthermore, these same tumor cells displayed a more rapid, robust, and sustained activation of MAP kinase. An enhanced response to IGF-I was particularly evident in T-47D cells, which overexpress IGF-IR and IRS-1, and in MCF-7 cells, which overexpress IGF-IR, IR, and IRS-1 but not in other lines of malignant and nonmalignant breast epithelial cells that do not overexpress more than one of these signaling molecules. The overexpression of IR and IRS-1 in MDA-MB-231 cells may contribute to the constitutive activation of MAP kinase observed in these cells. Interestingly, the response of MAP kinase to insulin treatment in MDA-MB-231 cells paralleled that of IGF-I, i.e., the activation of MAP kinase over basal levels was minimal and transient.5 Others have reported amplification and overexpression of the Grb2 gene in primary neoplastic breast tissue as well as in cell lines including MCF-7 (34 , 51) . Thus, our study supports the idea that overexpression of multiple signaling molecules in a given RPTK pathway could provide breast cancer cells a mechanism to amplify specific signaling pathways, including but not limited to the MAP kinase pathway. Furthermore, we speculate that amplification of IGF-IR-mediated signaling, as we have shown here, increases the sensitivity of breast cancer cells to low or transient levels of IGF-I and, therefore, could provide a selective growth advantage during the progression of these cancers in vivo.

Despite the consistently elevated steady-state levels of MAP kinase phosphorylation observed in all of the breast cancer lines examined here, the MEK inhibitor PD098059 selectively and significantly inhibited the proliferation of only a subset of these cell lines. Interestingly, cell growth was inhibited only in breast cancer cells displaying modest (Hs578T), enhanced (MCF-7 and T-47D), or constitutive (MDA-MB-231) activation of MAP kinase. The amplified IGF-I-dependent signaling in MCF-7 and T-47D cells appears to contribute to their increased sensitivity to drugs that inhibit the MAP kinase pathway, as suggested by the reduced ability of PD098059 to inhibit the growth of these cells when IGF-IR-dependent signaling was attenuated. Therefore, the increased sensitization to PD098059 may reflect an increased dependence of these tumor cells on the MAP kinase pathway, as a consequence of the amplification of IGF-IR signaling. Although a modest sensitivity to PD098059 is consistent with the mild activation of MAP kinase by IGF-I observed in Hs578T cells, a greater degree of growth inhibition might be expected for MDA-MB-231 cells, which possess constitutively elevated levels of MAP kinase activity that is refractory to IGF-I. Our data indicate that the degree of sensitivity to PD098059 corresponds more closely to the degree of responsiveness of MAP kinase to IGF-I, which can be enhanced by overexpression of components of the IGF-IR pathway, than to activation of MAP kinase signaling per se. Furthermore, it is currently unclear which molecule is responsible for the constitutive activation of the MAP kinase pathway in MDA-MB-231 cells. Therefore, it remains plausible that other important mitogenic signaling pathway(s) are constitutively activated by this molecule as well, effectively decreasing the relative role played by the MAP kinase pathway in the proliferation of these cancer cells.

The resultant suppression of MAP kinase phosphorylation after IGF-I treatment in a subset of breast cancer lines is both interesting and perplexing. IGF-I-mediated regulatory mechanisms to control MAP kinase activity appear to be altered in EGFR- or ErbB2-overexpressing breast cancer cells. The underlying mechanism of this suppressed modulation of IGF-IR-mediated MAP kinase activation is unclear. However, in a related study,5 we observed that MAP kinase phosphorylation in cells of the same ErbB2- and EGFR-overexpressing tumor lines increased dramatically above basal levels in response to EGF treatment, indicating that the suppression of MAP kinase signaling observed here is not generalizable to all mitogenic factors and may be specific to IGF-I. Interestingly, IGF-I was capable of stimulating the monolayer proliferation of BT-20 cells, which overexpress the EGFR,5 indicating that the IGF-I-dependent suppression of MAP kinase activity may be, at least in part, permissive for mitogenesis in these cells. The precise mechanism of the MAP kinase inhibition is unclear. However, it is possible that IGF-I mediates the activation of a MAP kinase phosphatase, e.g., MKP-1, or induces the degradation of MAP kinase to tightly control the total MAP kinase activity in those cancer cells. One implication is that the continued activation of MAP kinase by growth factors such as IGF-I would be incompatible with a mitogenic signal and/or deleterious in those cells. If this model holds, two predictions can be made: (a) there could be an inordinate increase in the activity of a MAP kinase phosphatase after IGF-I treatment; and (b) the inappropriate expression of a constitutively activated MAP kinase would lead to cell cycle arrest or apoptosis in those cells. Indeed, there is evidence to suggest that activation of the MAP kinase pathway blocks cell cycle progression and causes growth arrest in certain cells, including SK-BR-3 cells (52 , 53) . We are currently investigating this hypothesis. A recent report demonstrated that transfection of a dominant-negative mutant form of Shc, which couples various RPTKs to the initiating components of the MAP kinase pathway, inhibited the proliferation of ErbB2-overexpressing breast cancer cells but not the growth of normal HBL-100 cells (40) . Because our results showed that MEK inhibition did not significantly affect the proliferation of BT-474 and SK-BR-3, a Shc-dependent but MAP kinase-independent pathway may play an important role in the growth of ErbB2-overexpressing breast cancer cells. Consistent with this, a role for Shc in activating a mitogenic pathway involving Grb2, Gab2, PI 3-kinase, and Akt has been described recently (54) .

In conclusion, our study has shown that multiple signaling components of the IGF-IR pathway are concomitantly overexpressed in certain breast cancer lines, and overexpression in these cells may underlie the enhanced responsiveness of MAP kinase to IGF-I. Moreover, our results suggest that the amplification of signaling pathways, including the MAP kinase pathway, renders those cancer cells highly dependent on such pathways and predisposes the cells to an increased susceptibility to growth-inhibition by the blockade of pathways with inhibitors such as PD098059. This finding is supported by a parallel study in which we observed that several ErbB2-overexpressing breast cancer lines with activated PI 3-kinase signaling displayed an increased sensitivity to wortmannin, LY294002, and rapamycin, inhibitors of the PI 3-kinase pathway.4 These observations raise the possibility of targeting particular signaling components in the treatment of certain breast tumors that have specifically amplified signaling pathways. Indeed, certain groups of breast cancer patients with increased levels of IGF-IR, IRS-1, ErbB2, or EGFR in tumors have a poorer clinical prognosis than patients with lower levels (25 , 26 , 30, 31, 32) and may benefit from therapeutic regimens that target molecules critical for the growth of these tumors. Further elucidation of the molecular signaling pathways important to breast cancer cell behavior will lead to a better understanding of tumor development and may identify additional therapeutic targets for human breast cancer treatment.

Materials and Methods

Reagents.
FBS was from Life Technologies, Inc., and donor horse serum was from Gemini. EGF and IGF-I were purchased from Intergen. PD098059 and mouse monoclonal anti-IGF-IR (clone {alpha}IR-3) and anti-v-Src (clone 327) antibodies were purchased from Calbiochem. Anti-pTyr (RC20-HRP), anti-mouse IgG-HRP, and anti-rabbit IgG-HRP antibodies were purchased from Transduction Laboratories. Rabbit polyclonal anti-ERK2 and anti-IR antibodies were purchased from Santa Cruz Biotechnology, Inc. Anti-phosphoERK1/ERK2 antibody (T202/Y204) was purchased from New England Biolabs. Antibodies were used according to the manufacturers’ recommendations. Generation of rabbit polyclonal anti-IGF-IR and anti-IRS-1 antibodies have been described previously (55 , 56) and were used at 1:500 and 1:250 dilutions, respectively. Unless noted, other reagents were from Sigma Chemical Co.

Cells.
Nonmalignant human mammary cell lines (AB589, MCF-10F, and HBL-100) and human breast cancer cell lines (BT-20, BT-474, Hs578T, MCF-7, MDA-MB-231, MDA-MB-361, MDA-MB-453, SK-BR-3, T47D, and ZR-75-1) were obtained originally from American Type Culture Collection except for AB589 described by Welsh et al. (57) , which was obtained from Dr. X. Y. Fu (Department of Pathology, Yale University School of Medicine, New Haven, CT). Except where noted, cell lines were maintained at 37°C and 5% CO2 in a humidified incubator. All breast cancer lines and AB589 cells were routinely grown in DMEM supplemented with 10% heat inactivated FBS, 5 µg/ml insulin, and routine antibiotics. HBL-100 cells were grown in the same medium but at 10% CO2. MCF-10F cells were grown in a 1:1 mixture of Ham’s F-12 medium and DMEM, supplemented with 5% heat-inactivated donor horse serum, 10 µg/ml insulin, 0.5 mg/ml hydrocortisone, 20 ng/ml EGF, and routine antibiotics.

IGF-I Treatment.
Prior to experiments, cells were grown to confluence in the complete growth medium described above. Cells were incubated in serum-free DMEM for 16–20 h and were then incubated in serum-free DMEM supplemented with 100 ng/ml IGF-I for the times indicated. Cells in untreated control dishes were mock-incubated in fresh serum-free DMEM in parallel.

Preparation of Cell Lysates and Immunoprecipitation.
Cells were placed on ice and were washed twice with Tris-Glu buffer [25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM KCl, 1 mM sodium phosphate, and 0.1% glucose]. Cell lysates were made by lysing cells with ice-cold RIPA buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 5 mM EDTA, 1% aprotinin, 1 mM phenylmethylsulfonylfluoride, 1 mM sodium orthovanadate, 0.4 mM phenylarsine oxide, and 25 mM sodium fluoride]. The cell lysate was clarified by centrifugation at 12,000 rpm for 10 min at 4°C. The protein concentration of the supernatants was determined by Bradford assay, and normalized cell lysates were used for immunoprecipitation or were analyzed directly by SDS-PAGE. For immunoprecipitations, cell lysates were incubated with anti-IGF-IR or anti-IRS-1 for 16 h at 4°C. Immunoprecipitates were pulled down by adding 25 µl of protein A-Sepharose beads (Repligen) to cell lysates, further incubated for 2 h at 4°C, and centrifuged. Immunoprecipitate pellets were washed three times by gentle vortexing in fresh cold RIPA buffer and brief (10–15 s) centrifugation at maximum speed. The washed immunoprecipitates were subjected to SDS-PAGE and immunoblotting analysis.

In Vitro Kinase Assay.
Anti-IGF-IR immunoprecipitates were washed three times in high-salt RIPA buffer [50 mM Tris-HCl (pH 7.4), 300 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 25 mM EDTA, 10% glycerol, 1% aprotinin, and 1 mM sodium orthovanadate], once in low-salt RIPA buffer [50 mM Tris-HCl (pH 7.4), 10 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 25 mM EDTA, 10% glycerol, 1% aprotinin, and 1 mM sodium orthovanadate], and twice in kinase buffer [50 mM Tris-HCl (pH 8.0), 10 mM MnCl2]. Immunoprecipitates were resuspended in 50 µl of kinase buffer, and reactions were initiated by addition of 1 µl (10 µCi) of [{gamma}-32P]ATP and incubated at 22°C for 10 min. Reactions were terminated by the addition of high-salt RIPA buffer and then washed once with high-salt RIPA buffer and once with low-salt RIPA buffer. Samples were boiled in 6x SDS-PAGE sample buffer, fractionated by 10% SDS-PAGE, and electroblotted to nitrocellulose membranes according to standard procedures. Phosphorylated proteins were detected by exposure of membranes to X-ray film and quantitated by densitometric scanning.

SDS-PAGE and Immunoblotting.
Protein samples were resolved in 10% SDS polyacrylamide gels, followed by electroblotting onto a nitrocellulose membrane according to standard procedures. The membrane was blocked in TBS-Tween [10 mM Tris-HCl (pH 7.4), 50 mM NaCl, and 0.1% Tween 20] containing 5% (w/v) nonfat dry milk [or 3% (w/v) BSA for anti-phosphotyrosine] and was probed with the indicated antibody, followed by incubation with a HRP-conjugated secondary antimouse or antirabbit antibody. After washing with TBS-Tween, the blot was developed by the enhanced chemiluminescence (ECL) method according to the manufacturer’s instructions (Amersham). Where indicated, the membrane was stripped of antibody by washing three times in glycine buffer (0.2 M glycine, pH 2.0) and three times in TBS-Tween and then reprobed with another antibody by immunoblotting as described above.

Cell Proliferation.
Exponentially growing cells were trypsinized, counted by a hemacytometer, and plated at equivalent subconfluent cell densities. Twenty-four to 36 h after plating, cells were incubated in complete growth medium containing 25 µM PD098059 or 0.1% DMSO (vehicle control). When blocking antibody was used, 400 ng/ml of anti-IGF-IR IgG1{kappa} antibody or 400 ng/ml of control anti-v-Src IgG1{kappa} antibody were included together with PD098059 or DMSO. Incubations were carried out at 37°C for 3–4 days. For the proliferation assay, cells in duplicate dishes were trypsinized and counted by hemacytometer for each cell line. For the blocking antibody experiments, cells in triplicate wells were trypsinized and counted in each treatment group.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by NIH Grants CA29339 and CA55054. U. H. was also supported by Department of Defense Breast Cancer Predoctoral Training Program Grant DAMD17-94-J-4111 and the Mount Sinai School of Medicine Medical Scientist Training Program, National Institute of General Medical Sciences Grant T32GM07280. Back

2 To whom requests for reprints should be addressed, at Department of Microbiology, One Gustave L. Levy Place, Box 1124, New York, NY 10029-6574. Phone: (212) 241-3795; Fax: (212) 534-1684; E-mail: lu-hai.wang{at}mssm.edu Back

3 The abbreviations used are: MAP, mitogen-activated protein; RPTK, receptor protein tyrosine kinase; IGF-I, insulin-like growth factor I; IGF-IR, IGF-I receptor; IR, insulin receptor; IRS, IR substrate; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; PI 3-kinase, phosphatidylinositol 3-kinase; FBS, fetal bovine serum; HRP, horseradish peroxidase; MEK, MAP kinase kinase. Back

4 U. Hermanto, C. S. Zong, and L-H. Wang. ErbB2-overexpressing human mammary carcinoma cells display an increased requirement for the phosphoinositide 3-kinase signaling pathway in anchorage-independent growth, submitted for publication. Back

5 U. Hermanto and L-H. Wang, unpublished observations. Back

Received for publication 8/24/00. Revision received 10/30/00. Accepted for publication 10/31/00.

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The Mitogenic Action of Insulin-like Growth Factor I in Normal Human Mammary Epithelial Cells Requires the Epidermal Growth Factor Receptor Tyrosine Kinase
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