1 Oncogene 2003 Vol: 22(22):3441-3451. DOI: 10.1038/sj.onc.1206410

A deletion mutant of heregulin increases the sensitivity of breast cancer cells to chemotherapy without promoting tumorigenicity

Heregulin (HRG) is an activator of the erbB2-, erbB3- and erbB4-(erbB-2/3/4) signaling pathway. Transfection of full-length HRG cDNA into the estrogen (E2)-dependent cell line MCF-7 promoted an invasive E2-independent phenotype, as well as persistent activation of the erbB-2/3/4 receptors. Moreover, HRG expression in MCF-7 cells renders the cells sensitive to the topoisomerase II inhibitor doxorubicin (Doxo). In an attempt to dissociate the tumorigenic effect of HRG from the sensitizing effect to chemotherapy, we constructed a structural deletion mutant of HRG. Transfection of the deletion mutant of HRG described in this study (HRG/M) into MCF-7 cells resulted in the dissociation of the tumor-promoting activity of HRG from the sensitization to Doxo, that is, although the cells did not become more aggressive or E2-independent they became more sensitive to Doxo. HRG/M was unable to autophosphorylate the erbB receptors and did not affect the level of MAPK phosphorylation. Furthermore, the intracellular localization of the protein was different from that of the full-length protein. Our data show that the HRG/M sequences are sufficient to sensitize MCF-7 cells to Doxo, and provide evidence that this sensitization is independent of erbB2 activation.

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Figures
Figure 1: Schematic diagram of the HRG and HRG-M proteins Figure 2: Expression of HRG and HRG/M in MCF-7-derived clones. MCF-7/V, MCF-7/HRG1-HRG2, and MCF-7/M-M3 clones were grown to 80% confluency. Total RNA was extracted and 20 g of RNA was analysed in an RNAse protection assay as described in the 'Materials and methods'. The GAPDH probe was used as an internal control for RNA loading. Protected fragments of HRG2 (480 bp) HRG/M (360 bp) and GAPDH (100 bp) were visualized by autoradiography Figure 3: Assessment of sensitivity to Doxo of MCF-7-derived clones. MCF-7/V, MCF-7/HRG1-HRG2, and MCF-7/M1-M2 clones were plated at 1000 cells/well in IMEM 5% FCS in a 96-well plate. The cells were treated with increasing concentrations of Doxo. Viability of the cells was assessed on day 7 by a MTS assay. Cytotoxicity is expressed as the percentage of control absorbance at 490 nm. The results are mean of quadruplicate wells. The experiments were carried at least three times with similar results Figure 4: Anchorage-dependent growth in response to E2. MCF-7, MCF-7/V, MCF-7/HRG, and MCF-7/M clones were depleted of E2 for 5 days in phenol red-free IMEM with 5% CCS as described in 'Materials methods' prior to plating in 24-well plates at 10 000 cells/well. After 24 h, E2, ICI 182 780 (ICI) or ICI+E2 were added and the cells were continuously treated with the compounds, or untreated as controls. Cell growth was assessed by cell counting using the coulter counter. Results are mean of triplicate wells. Experiments were performed at least three times Figure 5: Anchorage-independent growth in response to E2. Growth in soft agar assay was performed to assess the ability of the cells to form colonies in the presence or absence of E2. The cells were depleted from steroid-like compounds by growing the cells in phenol red-free IMEM containing 5% CCS. Cells were then plated in six-well plates with a soft agar in the presence or absence of E2 or treated with ICI and ICI+E2. Cells were allowed to grow and colonies were counted after 14 days; results are mean of triplicate wells. Experiments were performed at least three times Figure 6: Tyrosine phosphorylation of erbB2/3 receptors in MCF-7-derived clones and MDA-MB-453 cells treated with CM or recombinant proteins. Cell lysates from MCF-7/V, MCF-7/HRG, and MCF-7/M cells were subjected to SDS–PAGE (a). The erbB2-overexpressing cell line, MDA-MB-453, was treated with recombinant HRG1, MBP, or MBP-HRG/M prior to lysis (b), or treated with concentrated CM obtained from the MCF-7-derived clones (c). Blots were probed with an antiphosphotyrosine antibody. Cell lysates from MCF-7/V, MCF-7/HRG, and MCF-7/M cells, treated with HRG1 or untreated as controls, were subjected to SDS–PAGE. Total erbB2 levels were assayed by blotting with an anti-erbB2 antibody (d). Actin levels were determined for equal protein loading (e). Experiments were performed at least three times Figure 7: Detection of full-length HRG and its mutant in cell-culture media. MCF-7, MCF-7/V, MCF-7/HRG, and MCF-7/M cells were grown to subconfluency. The media were changed to serum-free medium and CM were collected and concentrated prior to electrophoresis. The presence of the HRG protein (a) or the HRG/M (b) proteins was assayed by Western blot using an anti-HRG antibody specific to the EGF-like domain. Experiments were performed at least three times Figure 8: Activation of MAPK p44/42 in MCF-7 derived clones. MCF-7/V cells and MCF-7/HRG cells were serum starved overnight. MCF-7/V, were treated with HRG1 for 30 min at 37°C or untreated. Cell lysates were separated on SDS–PAGE gels and blotted with phospho-MAPK and MAPK antibodies (a). MCF-7/M cells were serum starved overnight, treated with HRG1 or untreated as controls, lysed and 50 g total protein was loaded on SDS–PAGE gels. Activated MAPK was measured by Western blot with an antibody specific for the activated form of the protein. Total MAPK protein was assessed using an anti-MAPK antibody (b). Experiments were performed at least three times Figure 9: Localization of the GFP-HRG and GFP-HRG/M proteins in MCF-7 cells. MCF-7 cells were plated in Lab-Teck chambers and transiently transfected with constructs containing GFP-HRG or GFP-HRG/M sequences as described in 'Materials and methods'. Intracellular localization of the GFP control, GFP-HRG, and GFP-HRG/M proteins was visualized using confocal microscopy
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References
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    • . . . This increase in sensitivity was correlated with an increase in the expression of Topo II at the mRNA and the protein level (Harris et al., 1998) . . .
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    • . . . We and others have demonstrated that HRG is localized mainly in the nucleus when added to SKBr-3 cells (Li et al., 1996) and unpublished data . . .
    • . . . HRG is a growth factor that is involved in tumorigenicity and tumor progression (Li et al., 1996; Lupu et al., 1996; Hijazi et al., 2000; Tsai et al., 2000) . . .
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    • . . . Cells were then transfected with HRG2 cDNA (Liu et al., 1999), with the deletion mutant of HRG containing construct (HRG/M) in pRC-CMV, or with the mammalian expression vector pRC-CMV as control, using FuGENE as per the manufacturer's instructions (Roche) . . .
    • . . . The antisense riboprobe plasmids for HRG and GAPDH were constructed as described previously (Liu et al., 1999) . . .
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    • . . . The activation of the erbB2 receptor, which has been correlated with poor prognosis, can occur via upregulation of expression and dimerization of the receptors, or via formation of heterodimers with erbB3 or erbB4 (Tang et al., 1996) . . .
    • . . . We concluded that the transfection of HRG was sufficient to promote the progression of MCF-7 cells to a more aggressive phenotype, E2-independent phenotype (Tang et al., 1996). . . .
    • . . . We have previously shown constitutive activation of the erbB receptors in MCF-7/HRG cells (Tang et al., 1996) . . .
    • . . . Our studies have shown that the introduction of HRG cDNA into MCF-7 cells provides the cells with a growth advantage in E2-depleted medium, as well as the ability to form tumors in vivo in the absence of E2 stimulation (Tang et al., 1996) . . .
    • . . . We have shown that the expression and autocrine function of HRG are sufficient to confer E2 independence in vivo and in vitro (Tang et al., 1996) . . .
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    • . . . It has been previously reported that the transmembrane (TM) domain of the protein has an important role for the secretion of the protein and its prior localization into the membrane as a TM protein (Wang et al., 2001) . . .
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    • . . . It has also been shown that the extracellular domain of HRG containing the EGF-like domain is sufficient for the tumorigenic effect, while the intracellular domain is sufficient for the apoptotic effect that HRG can have in some systems (Weinstein et al., 1998; Weinstein and Leder, 2000) . . .
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    • . . . Such studies have shown that the cytoplasmic domain of the protein is sufficient for the apoptotic effect that this protein may have, and that the EGF-like domain is sufficient for the proliferation and transformation of mammary epithelial cells (Weinstein and Leder, 2000) . . .
    • . . . These results are in concordance with previously published data, which demonstrate that the EGF-like domain of HRG is sufficient to induce activation of the erbB receptors (Weinstein and Leder, 2000) . . .
    • . . . It has also been shown that the extracellular domain of HRG containing the EGF-like domain is sufficient for the tumorigenic effect, while the intracellular domain is sufficient for the apoptotic effect that HRG can have in some systems (Weinstein et al., 1998; Weinstein and Leder, 2000) . . .
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