Prolactin enhances insulin-like growth factor I receptor phosphorylation by decreasing its association with the tyrosine phosphatase SHP-2 in MCF-7 breast cancer cells

Abstract

Normal mammary development requires coordinated interactions of numerous factors, including prolactin (PRL) and insulin-like growth factor I (IGF-I), both of which have also been implicated in breast cancer pathogenesis and progression. We previously reported that PRL and IGF-I synergize in breast cancer cells to activate ERK1/2 and AKT, leading to increased proliferation, survival, and invasion. Intriguingly, PRL co-treatment with IGF-I augments IGF-I receptor (IGF-IR) phosphorylation 2-fold higher than IGF-I alone. Here, we showed the importance of the tyrosine phosphatase SHP-2 in this cross-talk using pharmacological inhibition and small interfering RNA. SHP-2 recruitment to IGF-IR was significantly attenuated by PRL co-treatment. Src family kinase activity was required for IGF-IR association with SHP-2, ligand-induced IGF-IR internalization, and PRL-enhanced IGF-IR phosphorylation. Inhibition of internalization, via knockdown of the GTPase, dynamin-2, prevented not only IGF-IR dephosphorylation, but also PRL-enhanced IGF-IR phosphorylation. Consistently, PRL diminished IGF-I-induced IGF-IR internalization, which may result from reduced SHP-2 association with IGF-IR, because we demonstrated an essential role for SHP-2 in IGF-IR internalization. Together, these findings describe a novel mechanism of cross-talk between PRL and IGF-I in breast cancer cells, with implications for our understanding of tumor progression and potential therapeutic strategies.

FIGURE 1.

PRL enhances IGF-I-induced IGF-IR phosphorylation and activation of downstream signaling pathways. Serum-starved MCF-7 cells were treated with vehicle, IGF-I, PRL, or IGF-I/PRL for the indicated times. The immunoblots were performed using cell lysates and the indicated antibodies. A, representative experiment. B, levels of p-IGF-IR/total IGF-IR in response to IGF-I alone, compared with IGF-I + PRL, quantified as described under “Experimental Procedures” (means ± S.D., n = 3). The asterisks denote significant differences compared with IGF-I treatment. ** p < 0.01 (two-way ANOVA, Bonferroni post-test). C and D, levels of phosphorylated ERK1/2 and AKT, respectively, compared with total kinase levels, quantified as described under “Experimental Procedures” (means ± S.D., n = 3). The asterisks denote significant differences compared with IGF-I treatment. *, p < 0.05 (paired t test).

FIGURE 2.

SHP-2 mediates PRL enhancement of IGF-IR phosphorylation. A–C, serum-starved MCF-7 cells were pretreated with dimethyl sulfoxide (DMSO) and either 200 μm vanadate (A), 10 μm PTP-1B inhibitor (B, inh.), or 50 μm NSC-87877 (C) for 1 h prior to treatment with vehicle, IGF-I, PRL, or IGF-I/PRL for 15 min. D, MCF-7 cells were transfected with nontargeting (NT) or SHP-2-specific siRNA duplexes and treated with vehicle, IGF-I, PRL, or IGF-I/PRL for 15 min. Cell lysates were immunoblotted with the indicated antibodies. Top panels, representative experiment; bottom panels, quantification of p-IGF-IR signal/total IGF-IR in response to IGF-I alone, compared with IGF-I + PRL, as described under “Experimental Procedures” (means ± S.D., n = 3 (A–C) or n = 4 (D)). The asterisks denote significant differences compared with IGF-I treatment. *, p < 0.05; **, p < 0.01 (two-way ANOVA, Bonferroni post-test (A, C, and D) or paired t test (B)).

FIGURE 3.

PRL reduces association of SHP-2 with IGF-IR. A and B, serum-starved MCF-7 cells were treated with IGF-I, PRL, or IGF-I/PRL for 15 min. 1 mg of protein from cell lysates was immunoprecipitated (IP) with IGF-IRβ (A) or PRLR (B) antibodies and subjected to immunoblotting as shown. Top panel, immunoblot of representative immunoprecipitation results; middle panel, preimmunoprecipitation lysates; bottom panel, quantification of levels of SHP-2 in immunoprecipitates, compared with IGF-IRβ and PRLR in A and B, respectively, in response to IGF-I/PRL co-treatment compared with those induced by IGF-I alone (see “Experimental Procedures”; means ± S.D., n = 5 (A) or n = 4 (B)). The asterisks denote significant differences compared with IGF-I treatment (*p < 0.05, **p < 0.01, paired t test). C, serum-starved MCF-7 cells were pretreated with vehicle or PRL for 5 min, at which point medium containing vehicle or PRL was removed, and cells were washed with serum-free media. The cells were then exposed to medium containing IGF-I for an additional 15 min. The immunoblots were performed using cell lysates and the indicated antibodies (representative experiment shown). WB, Western blot.

FIGURE 4.

SFKs mediate PRL enhancement of IGF-IR phosphorylation and SHP-2 association with IGF-IR. A–D, serum-starved MCF-7 cells were pretreated with DMSO and either 10 μm SU6656 (A), 10 μm PP1 (B), 10 μm U0126 (C), or 10 μm LY294002 (D) for 1 h prior to treatment with vehicle, IGF-I, PRL, or IGF-I/PRL for 15 min. The immunoblots were performed using cell lysates and the indicated antibodies (representative experiments shown). p-IGF-IR signals induced by IGF-I/PRL co-treatment were quantified and compared with those induced by IGF-I alone as described under “Experimental Procedures” (means ± S.D., n = 5 (A) or n = 3 (B)). The asterisks denote significant differences compared with IGF-I treatment. *, p < 0.05; **, p < 0.01) two-way ANOVA, Bonferroni post-test). E, serum-starved MCF-7 cells were pretreated with dimethyl sulfoxide (DMSO) or 10 μm SU6656 for 1 h prior to hormone treatment for 15 min. 1 mg of protein from cell lysates was immunoprecipitated (IP) with 1 μg of IGF-IRβ antibody and subjected to immunoblotting as indicated. Top panel, immunoblots of representative immunoprecipitation results; bottom panel, preimmunoprecipitation lysates. WB, Western blot.

FIGURE 5.

IGF-IR internalization is required for dephosphorylation and PRL-enhanced phosphorylation. MCF-7 cells were transfected with nontargeting (NT) or Dnm-2-specific siRNA. Following transfection, the cells were treated with IGF-I for the times indicated (A) or with vehicle, IGF-I, PRL, or IGF-I/PRL for 15 min (B). Immunoblots were performed using cell lysates and the indicated antibodies (representative experiments shown). p-IGF-IR signals induced by IGF-I/PRL co-treatment were quantified and compared with those induced by IGF-I alone as described under “Experimental Procedures” (means ± S.D., n = 5). The asterisks denote significant differences compared with IGF-I treatment. *, p < 0.05 (two-way ANOVA, Bonferroni post-test).

FIGURE 6.

PRL decreases IGF-I-induced IGF-IR internalization. Serum-starved MCF-7 cells were labeled with 0.25 mg/ml sulfo-NHS-biotin at 4 °C for 2 h. The cells were then treated for the times indicated with vehicle (B), IGF-I (A–C), PRL (B), or IGF-I/PRL (C) at 37 °C to allow for internalization of IGF-IR. Remaining plasma membrane-associated biotin was then cleaved with dithiothreitol (DTT). Biotinylated proteins that had internalized were immunoprecipitated using NeutrAvidin-conjugated beads and subjected to Western analysis (representative experiments shown). Internalized biotinylated IGF-IR was quantified using densitometry. A, means ± S.D. (n = 3). The asterisk denotes a significant difference in internalized biotinylated IGF-IR between 5 and 30 min. *, p < 0.05 (paired t test). C, means ± S.D. (n = 3). The asterisks denote significant differences in internalized biotinylated IGF-IR compared with IGF-I treatment. *, p < 0.05; **, p < 0.01 (one-way ANOVA, Neuman-Keuls post-test).

FIGURE 7.

SFKs and SHP-2 are required for IGF-IR internalization. Serum-starved MCF-7 cells were pretreated with dimethyl sulfoxide (DMSO) and either 10 μm SU6656 (A) or 50 μm NSC-87877 (B) or were transfected with nontargeting (NT) or SHP-2-specific siRNA duplexes (C) and labeled with 0.25 mg/ml sulfo-NHS-biotin at 4 °C for 2 h. The cells were then treated with IGF-I at 37 °C for 5 min to allow for internalization of IGF-IR. Plasma membrane-associated biotin was then cleaved with dithiothreitol (DTT). Biotinylated proteins that had internalized were immunoprecipitated using NeutrAvidin-conjugated beads and subjected to Western analysis. Top panels, representative experiment; bottom panels, quantitated internalized IGF-IRβ. The asterisks denote significant differences in internalized biotinylated IGF-IRβ compared with IGF-I treatment. *, p < 0.05; **, p < 0.01 (two-way ANOVA, Bonferroni post-test).