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Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia
Address all correspondence and requests for reprints to: Alison J. Butt, Ph.D., Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia. E-mail: a.butt{at}garvan.org.au.
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Introduction |
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IGFBP-3 is the major IGF-binding protein in serum and is expressed in many tissues, including normal and malignant breast epithelium (5). There is now accumulating evidence to suggest that IGFBP-3 may have intrinsic antiproliferative and proapoptotic effects on the growth of human cancer cells (6). For example, we have previously shown that IGFBP-3 expression is growth inhibitory and induces apoptosis in breast cancer cells in vitro, which are effects associated with a cell cycle arrest and induction of proapoptotic proteins such as Bax and Bad (7, 8). The mechanisms for these growth-inhibitory effects of IGFBP-3 remain unclear. Cell membrane receptors for IGFBP-3 have been postulated to be expressed by breast cancer cells (9), although a true signaling receptor for IGFBP-3 remains to be characterized. IGFBP-3 can also translocate to the nucleus in breast cancer cells (10), and Liu et al. (11) have shown its interaction with the nuclear retinoid X receptor-
, suggesting that this may be a mechanism for its proapoptotic effects. However, inhibiting the ability of IGFBP-3 to interact with the cell surface or translocate to the nucleus does not ablate its growth-inhibitory and proapoptotic effects (8), leading us to hypothesize that IGFBP-3 may initiate these growth effects in the cytoplasm.
There is also evidence that IGFBP-3 can have stimulatory effects on the growth of breast cancer cells. T47D cells stably transfected with human IGFBP-3 cDNA overcome their initial growth inhibition by IGFBP-3 after several passages in culture and become growth stimulated compared with controls (12). IGFBP-3 can enhance IGF-dependent mitogenesis in both normal (13) and malignant (4) breast cells, with some evidence that this may occur through enhanced signaling through the phosphatidylinositol (PI) 3-kinase pathway (13). We have also shown that malignant transformation of breast epithelial cells by transfection with oncogenic ras induces a state of resistance to the growth-inhibitory effects of IGFBP-3, and these cells are growth stimulated by low doses of IGFBP-3 (14). Furthermore, IGFBP-3 can potentiate the mitogenic effects of epidermal growth factor (EGF) in phenotypically normal MCF-10A breast epithelial cells (15).
Similar to the conflicting data in vitro, the role of IGFBP-3 in the development and progression of human breast cancer in vivo is also unclear. Clinical studies have shown that a high serum IGFBP-3 level is a negative risk factor for breast cancer (16), whereas a high tissue level of IGFBP-3 is associated with unfavorable prognostic features of the disease, including the formation of large, highly proliferative tumors (17, 18, 19). These data and our recent in vitro studies imply that there may be a switch in the response of breast cancer cells to IGFBP-3 from growth inhibition to growth stimulation and that IGFBP-3 expression may enhance the growth of breast tumors.
To further investigate this apparent dichotomy between the effects of IGFBP-3 in vitro and in clinical studies, we examined the effects of increased IGFBP-3 expression on the growth of breast cancer cells in vitro and in vivo. We observed that IGFBP-3 expression, although inhibitory to the growth of T47D cells in vitro at early passages post transfection, was associated with enhanced breast tumor growth in vivo. Furthermore, growth stimulation in IGFBP-3-expressing cells was associated with an up-regulation of EGF receptor (EGFR) expression in vivo and increased responsiveness to the mitogenic effects of EGF in vitro.
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Materials and Methods |
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The breast cancer cell line T47D was routinely maintained in RPMI-1640 supplemented with 10% fetal calf serum, 10 µg/ml insulin, and 2.92 mg/ml glutamine under standard conditions. T47D cells were stably transfected with a 0.9-kb human IGFBP-3 cDNA fragment in the expression vector pOP13 (Invitrogen Corp., Carlsbad, CA) or vector alone as described previously (12), then mixed populations of transfectants were selected and grown up for subsequent experiments.
Cell proliferation assays
Early (four to six passages post transfection) and late (12–15 passages post transfection) IGFBP-3 transfectants (T47D/BP-3) and vector controls (T47D/VEC) were seeded at 5 x 104 cells/well in 12-well plates in the presence of 10 µg/ml IGFBP-3 antiserum (R100) or rabbit IgG or in the presence or absence of the EGFR-specific protein kinase inhibitor, PD153035 (0.5 µM; Merck, Darmstadt, Germany) (20). At the indicated time points post seeding, cells were trypsinized, and the number of viable cells was determined by trypan blue exclusion and cell counting. Conditioned medium was also collected at each time point from cells treated with rabbit IgG and assayed for IGFBP-3 concentration by specific RIA (21). At each time point, fresh growth media with or without test reagents was added.
Tumor growth in vivo
T47D cells are estrogen-receptor (ER) positive and require the presence of estrogen for optimal growth in vivo. Pellets containing ß-estradiol (1.5–2 mg; Sigma Chemical Co., St. Louis, MO) were inserted sc into the left flank of 12 athymic, nu/nu female mice. Three days later, T47D/VEC and T47D/BP-3 cells (passage 4 post transfection) were trypsinized and resuspended at 1 x 107 viable cells/100 µl in serum-free/insulin-free (SF) medium. Cell suspensions were mixed with an equal volume of Matrigel (BD Biosciences, Bedford, MA) as a support matrix for initial cell growth. Two groups (T47D/VEC and T47D/BP-3) of six mice were injected with 5 x 106 cells sc at the dorsal neck. Tumor growth was monitored weekly by measuring tumor length and width using calipers, and tumor volume (cm3) was calculated using the following formula: length x width2 x 0.5. Experiments were terminated 10 wk post injection. Blood samples were taken from all animals at termination, and tumors were frozen in liquid nitrogen for RNA analysis or fixed in formalin for section preparation. Results were pooled from three separate experiments. All experiments were carried out with the approval of the Institutional Animal Care and Ethics Committee.
RNA extraction and Northern blotting
Total RNA extraction and Northern blotting analysis were performed essentially as previously described (22). IGFBP-3 cDNA probe (Dr. S. Shimasaki, University of California San Diego, San Diego, CA) was labeled using a HexaLabel DNA labeling kit (MBI Fermentas, Vilnius, Lithuania) and [-32P]deoxy-CTP. Filters were hybridized at 42 C overnight, and then washed in 1 x standard saline citrate at 42 C, with an additional wash in 0.1 x standard saline citrate if required. The ribosomal phosphoprotein, 36B4, was used as a loading control. Filters were quantitated using a PhosphorImager FLA3000 (Fuji, Tokyo, Japan), and data were normalized by expressing the ratio of target mRNA to 36B4 mRNA.
Immunohistochemical staining
Paraffin-embedded sections were prepared from T47D/BP-3 and T47D/VEC tumors and immunostained for IGFBP-3 (R100, 1:2000 dilution) or EGFR (1:100 dilution; Genesearch, Arundel, Queensland, Australia) using Envision immunohistochemical staining kit (Dako, Carpinteria, CA) and following the manufacturer’s instructions.
[3H]thymidine incorporation
DNA synthesis was determined using incorporation of [3H]thymidine, essentially as previously described (14), after addition of EGF (Sigma) as indicated for 24 h in phenol red-free/SF medium.
Flow cytometry
Cells were incubated in SF medium for 24 h and then treated with concentrations of EGF for 30 mins, detached with 5 mM EDTA, and fixed in 4% formaldehyde at room temperature (RT) for 20 mins. Fixed cells were washed in PBS and permeabilized in wash solution (3% BSA and 0.05% saponin in PBS) for 1 h at RT. Cells were then incubated in wash solution with 1:50 dilution of phospho-EGFR antibody for 1 h at RT and then washed twice in wash solution before a further 1-h incubation with fluorescein isothiocyanate-conjugated secondary antibody. After two further washes, cells were analyzed by flow cytometry.
EGF binding to cell monolayers
EGF was radiolabeled with [125I]sodium iodide using chloramine T to a specific activity of more than 80 Ci/mmol. For analysis of EGF binding to cell monolayers, cells in 48-well plates (100,000 cells/well) were serum-starved in phenol red-free/SF medium for 24 h and then incubated with concentrations of radiolabeled EGF overnight at 4 C. Cells were washed twice with saline (0.15 M NaCl) and solubilized in 0.5 M NaOH before 
-counting.
Statistical analysis
Statistical analysis was carried out using StatView 4.02 (Abacus Concepts, Inc., Berkeley, CA). Differences between groups were evaluated by Fisher’s protected least significant difference test after ANOVA using repeated measures or factorial analysis when appropriate.
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Results |
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T47D human breast cancer cells express undetectable levels of IGFBP-3. IGFBP-3 transfectants secrete similar levels of IGFBP-3 (
20–25 ng/ml 3 d post seeding; Fig. 1
) to the levels observed in other breast cancer-derived cell lines (e.g. Hs578T) and a phenotypically normal mammary epithelial cell line MCF-10A (14), emphasizing the relevance of this model in determining the effects of IGFBP-3 on breast cell growth. The growth rates of T47D cells stably transfected with human IGFBP-3 cDNA (T47D/BP-3) or vector control (T47D/VEC) were determined over a 13-d period with or without IGFBP-3 antiserum. Figure 1A demonstrates that stable expression of IGFBP-3 significantly inhibits the growth of early passage T47D cells compared with vector controls (P < 0.0005 by repeated-measures ANOVA). This inhibitory effect was ablated in the presence of anti-IGFBP-3 antibody R100 (Fig. 1A), which suggests it is mediated by IGFBP-3.
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, P < 0.05 by repeated-measures ANOVA). Furthermore, this enhancement of growth seen in T47D/BP-3 cells was not ablated in the presence of the IGFBP-3-neutralizing antibody. This suggests that the proliferative response seen at late passage is via a mechanism distinct from the antiproliferative effects. IGFBP-3 expression enhances the growth of human breast tumors in vivo
Stable transfectants (passage 4) were injected into groups of 12 female, nu/nu mice, and tumor size was measured weekly up to 10 wk post injection. Results from three independent experiments are summarized in Fig. 2A. Tumors derived from T47D/BP-3 cells had a significantly enhanced growth rate compared with vector control tumors (P < 0.002 by repeated-measures ANOVA). In addition, there was a significant increase in the wet tumor weight of IGFBP-3-expressing tumors compared with vector tumors (Fig. 2B; P < 0.01). The percentage of animals that developed visible tumors was not significantly different in the T47D/VEC (91.7%) and T47D/BP-3 groups (83.3%; P = 0.54 by 
2 test).
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). RNA from T47D/BP-3 cells was used as a positive control for IGFBP-3 cDNA expression (Fig. 2C, lane C). Endogenous mouse IGFBP-3 mRNA is 2.6 kb, and a band of this size was also detected in some of the tumor-derived RNA samples. Because this was not detected in T47D/BP-3 cells, this band may be due to contamination of tumor samples by murine cells. Serum samples were also analyzed for IGFBP-3 expression using a human IGFBP-3-specific RIA (21), but levels were undetectable by this method. Up-regulation of EGFR in IGFBP-3-expressing breast tumors
Human IGFBP-3 expression was examined in excised tumors by immunohistochemical staining of paraffin-embedded tumor sections (Fig. 3). Interestingly, IGFBP-3 expression was mainly cytoplasmic in T47D/BP-3-derived tumors but was strongly expressed in the nucleus of cells undergoing mitosis (Fig. 3, arrows), which has been previously reported in mitotic keratinocytes (23). Staining of sections with an anti-EGFR antibody demonstrated increased expression of EGFR in the IGFBP-3-expressing tumors compared with vector control tumors (Fig. 3).
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We have previously demonstrated that exogenous IGFBP-3 stimulates the growth of normal mammary epithelial cells in the presence of EGF (15). We determined whether the enhanced growth rate of late passage T47D/BP-3 cells was due to altered responsiveness to EGF by assessing the incorporation of [3H]thymidine as a measure of DNA synthesis. As illustrated in Fig. 4A, both sensitivity and maximal response to the growth-stimulatory effects of EGF were enhanced in late passage T47D/BP-3 cells by approximately 15–30% compared with either early passage cells or late passage T47D/VEC. Furthermore, similar to the growth stimulation observed in Fig. 1, this enhanced sensitivity to EGF in late passage T47D/BP-3 cells was not blocked by IGFBP-3 antiserum (data not shown).
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demonstrates a significant enhancement of EGFR phosphorylation in response to EGF in late passage T47D/BP-3 cells compared with vector controls. No significant difference was observed in the response of early passage transfectants to EGF. We then examined whether increased responsiveness to EGF was associated with increased binding of ligand to cells. As shown in Fig. 4C, binding of radiolabeled EGF was significantly higher in late passage T47D/BP-3 cells compared with early passage T47D/BP-3 or vector controls. Collectively, these data suggest that late passage T47D/BP-3 cells that are growth stimulated are more responsive to the mitogenic effects of EGF compared with vector controls, and this is associated with increased binding of EGF to cell surfaces.
To further investigate the involvement of EGF in the stimulatory effects of IGFBP-3, we examined the growth of T47D/BP-3 and vector controls in the presence or absence of the EGFR-specific inhibitor, PD153035 (20). Figure 5 demonstrates that the growth of T47D/VEC and early passage T47D/BP-3 cells was unaffected by treatment with PD153035. However, the stimulatory growth effects observed in late passage T47D/BP-3 cells compared with T47D/VEC (P < 0.03 by repeated-measures ANOVA) were ablated in the presence of the EGFR inhibitor, and treated T47D/BP-3 cells were significantly growth inhibited compared with T47D/VEC (Fig. 5B, P < 0.007 by repeated-measures ANOVA).
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Discussion |
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These breast cancer models of the stimulatory effects of IGFBP-3 appear to correlate with clinical data demonstrating that high IGFBP-3 breast tissue levels are associated with an unfavorable prognosis, namely large tumor size, low levels of steroid receptors, and a high proliferative fraction (18, 19). IGFBP-3 expression in primary tumors as well as breast cancer cell lines also appears to correlate inversely with the ER status of the cells, with ER-negative cells expressing high levels of IGFBP-3 (25). These breast tumors also grow more rapidly than ER-positive tumors and usually have a worse prognosis with a greater propensity to metastasize (26).
T47D breast cancer cells do not express detectable levels of endogenous IGFBP-3, and expression of IGFBP-3 in these cells is growth inhibitory (8, 12) and proapoptotic (7, 8). However, these cells eventually acquire resistance to the inhibitory effects of IGFBP-3 after long-term growth in vitro and have a higher growth rate compared with controls (12). In this study, we examined the effects of IGFBP-3 on the growth of T47D cells in vitro and in vivo. Expression of IGFBP-3 was clearly growth inhibitory up to 13 d in vitro. However, as we have previously observed, at later passages after transfection, IGFBP-3-expressing cells switch their response to IGFBP-3 and become growth stimulated compared with vector controls. The antiproliferative effects observed in early passage cells were ablated in the presence of an IGFBP-3-neutralizing antibody. In contrast, the stimulatory effects of IGFBP-3 were not, suggesting either that these effects are mediated by a distinct epitope of IGFBP-3 that is not neutralized by this antibody or that growth stimulation is not initiated directly by IGFBP-3. Our data showing the enhanced responsiveness of IGFBP-3-expressing cells to EGF support the latter hypothesis.
When examined in vivo, expression of IGFBP-3 was associated with enhanced growth of T47D-derived tumors compared with vector controls. Although this is in contrast to the growth-inhibitory and proapoptotic effects of IGFBP-3 in vitro, it appears to support data from clinical studies that have shown that a high level of IGFBP-3 in breast tumor tissue is associated with a more malignant phenotype (18, 19). It is also consistent with our observation that, after numerous passages in vitro, T47D/BP-3 cells acquire resistance to the inhibitory effects of IGFBP-3 and exhibit a higher proliferative rate than vector controls without any changes in expression levels (12). A similar in vitro phenomenon was observed by Hochscheid et al. (27) in non-small-cell lung cancer cells stably transfected with IGFBP-3. After approximately 9 d of growth in culture, the IGFBP-3-expressing cells appeared to lose their sensitivity to the growth-inhibitory effects of IGFBP-3 and exhibited an enhanced growth rate compared with vector controls (27). However, IGFBP-3 expression decreased lung tumor growth in vivo, which is contrary to our observations in breast tumors.
Although the mechanism underlying this apparent switch to IGFBP-3-mediated growth stimulation is unclear, a number of lines of evidence implicate an interaction between IGFBP-3 and the EGFR system, which is similar to that recently described by us in phenotypically normal breast epithelial cells (15). The enhanced growth of IGFBP-3-expressing tumors was associated with an up-regulation of EGFR protein expression, and late passage, growth-stimulated T47D/BP-3 cells showed increased sensitivity to EGF as demonstrated by a 15–30% increase in DNA synthesis in response to EGF, which would be expected to have a highly significant influence on mitogenesis and cell growth when amplified over many cell cycles. More specifically, we demonstrated enhanced signaling by the EGFR in late passage IGFBP-3-expressing cells in response to EGF. Significantly, blocking EGFR kinase activity with a specific inhibitor (20) specifically reversed the stimulatory effects of IGFBP-3, and restored IGFBP-3-mediated growth inhibition. Stimulation of IGF-dependent DNA synthesis by IGFBP-3 has been reported to involve signaling through the PI 3-kinase pathway (13, 28). Although EGFR activation can lead to enhanced signaling through this pathway, previous data from our laboratory, showing that IGFBP-3 potentiates both ERK and p38 MAPK activation but not Akt activation by EGF (15), suggests that an EGFR-dependent system not involving PI 3-kinase signaling may be involved in the phenomenon described here.
Although increased EGFR expression in vivo is in contrast to our observations in MCF-10A cells in which 24-h preincubation with exogenous IGFBP-3 did not alter EGF binding (15), it is possible that chronic exposure to IGFBP-3 alters EGF responsiveness by conferring a growth advantage on cells expressing high levels of EGFR. Because EGFR protein levels were too low to be detected in late passage T47D cells by immunoprecipitation and immunoblotting (data not shown), we were unable to demonstrate this directly in our in vitro model of growth stimulation by IGFBP-3. However, the increased binding of radiolabeled EGF and enhanced phosphorylation of EGFR in response to EGF observed in late passage T47D/BP-3 suggests some modulation of EGFR levels after chronic exposure to IGFBP-3.
Interestingly, the effects of IGFBP-3 on the growth of human breast cancer cells differ from those of IGFBP-5, which is potently growth inhibitory and proapoptotic in breast cancer cells both in vitro and in vivo (29). This suggests that, although they share much structural and functional homology, IGFBP-3 and IGFBP-5 may elicit their growth effects through distinct regulatory pathways.
Collectively, clinical data and our current results support the hypothesis that increased tumorigenicity is associated with a change in the response of breast cancer cells to IGFBP-3, with interaction between IGFBP-3 and the EGFR system central to the switch in response from growth inhibited to growth stimulated. High expression of EGFR and IGFBP-3 is positively correlated in human breast tumor tissue (19), and we found that blockade of EGFR kinase activity restored the inhibitory effects of IGFBP-3 in vitro. The clinical implication of this is that breast (and other) cancer therapies that target EGFR may have increased efficacy in tumors in which IGFBP-3 expression is also high. Understanding mechanistically the interaction between IGFBP-3 and the EGFR system will provide increased opportunities to exploit the potential of IGFBP-3 as an antiproliferative and proapoptotic agent in a variety of cancers.
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Acknowledgments |
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Footnotes |
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Abbreviations: EGF, Epidermal growth factor; EGFR, epidermal growth factor receptor; ER, estrogen receptor; IGFBP, IGF binding protein; PI, phosphatidylinositol; RT, room temperature; SF, serum-free/insulin-free.
Received May 27, 2003.
Accepted January 12, 2004.
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) and T47D/BP-3 (•) cells at passage 4 (early passage) or passage 12 (late passage) post transfection were grown in media containing 10% fetal calf serum with normal rabbit serum IgG. Data are also shown for T47D/BP-3 cells grown in the presence of 10 µg/ml anti-IGFBP-3 antibody (
). At the indicated time points, cells were trypsinized, and viable cell numbers were determined by trypan blue exclusion and cell counting (lines, right-hand axis). Values shown are means of triplicate wells ± SE. Conditioned medium was collected from T47D/BP-3 cells at each time point and analyzed for levels of secreted IGFBP-3 by RIA (bars, left-hand axis).
and 
and •, respectively) cells under SF conditions in the presence of EGF at the indicated concentrations. Values shown are means of quadruple wells ± SE. B, Flow cytometric analysis of phospho-EGFR (Tyr1068) after 30-min incubation with EGF at the indicated concentrations. Histograms show an increase in fluorescence intensity of the late passage T47D/BP-3 population (white peak) compared with the late passage T47D/VEC population (black peak) after treatment with 10 ng/ml EGF. No change is seen in early passage cells. Values shown are means of triplicate wells from at least three independent experiments ± SE. *, P < 0.05; **, P < 0.01 for IGFBP-3 expressing cells vs. vector controls. C, Binding of radiolabeled EGF to early and late passage T47D/BP-3 (
and 
, respectively) or absence of the EGFR-specific inhibitor, PD153035 (0.5 µM). At each time point, cells were trypsinized, and viable cell numbers were determined by trypan blue exclusion and cell counting. Values shown are means of triplicate wells ± SE. *, P < 0.01 for treated cells vs. untreated controls.