Disruption of insulin receptor function inhibits proliferation in endocrine-resistant breast cancer cells

Abstract

The insulin-like growth factor (IGF) system is a well-studied growth regulatory pathway implicated in breast cancer biology. Clinical trials testing monoclonal antibodies directed against the type I IGF receptor (IGF1R) in combination with estrogen receptor-α (ER) targeting have been completed, but failed to show benefits in patients with endocrine-resistant tumors compared to ER targeting alone. We have previously shown that the closely related insulin receptor (InsR) is expressed in tamoxifen-resistant (TamR) breast cancer cells. Here we examined if inhibition of InsR affected TamR breast cancer cells. InsR function was inhibited by three different mechanisms: InsR short hairpin RNA, a small InsR-blocking peptide, S961 and an InsR monoclonal antibody (mAb). Suppression of InsR function by these methods in TamR cells successfully blocked insulin-mediated signaling, monolayer proliferation, cell cycle progression and anchorage-independent growth. This strategy was not effective in parental cells likely because of the presence of IGFR /InsR hybrid receptors. Downregulation of IGF1R in conjunction with InsR inhibition was more effective in blocking IGF- and insulin-mediated signaling and growth in parental cells compared with single-receptor targeting alone. Our findings show TamR cells were stimulated by InsR and were not sensitive to IGF1R inhibition, whereas in tamoxifen-sensitive parental cancer cells, the presence of both receptors, especially hybrid receptors, allowed cross-reactivity of ligand-mediated activation and growth. To suppress the IGF system, targeting of both IGF1R and InsR is optimal in endocrine-sensitive and -resistant breast cancer.

Figure 1. TamR breast cancer cells were more sensitive to insulin treatment compared to their parental cells

(A) MCF-7L and T47D cells were plated, serum starved for 24 hours and treated with increasing concentrations of insulin for 15 minutes. Whole cell lysates were collected, separated by SDS-PAGE and subjected to the indicated immunoblotting analyses. (B) Cell monolayer growth of MCF-7L and T47D were measured using MTT proliferation assay. Cells were serum starved for 24 hours and then treated with increasing concentrations of insulin. Readings were taken 5 days later. The results were normalized to untreated group. (C) Anchorage-independent growth assay was carried out on MCF-7L and T47D cells. Colonies formed were counted 14 days and 20 days later, respectively. Values were normalized to untreated group and were presented as fold change (mean ± SD, n=3).

Figure 2. Insulin receptor knockdown reduced insulin-regulated signaling and growth in TamR cells, but not in parental cells

MCF-7L and T47D and their counter TamR cells were InsR knock-downed with lentiviral shRNA (shIR#2, shIR#6) or plasmid control (shSrcb or pLKO.1). InsR protein and mRNA expression levels were respectively determined using Western blot analyses as shown in lentiviral transduced (A) MCF-7L and T47D cells and qRT-PCR as shown in (B) MCF-7L and T47D cells. Cells were plated, serum started for 24 hours and treated with or without 10nM insulin for 15 minutes. Whole cell lysates were collected, separated by SDS-PAGE and subjected for indicated immunoblotting analyses. For qRT-PCR analysis, total RNA was collected from cells in full media. Data was normalized to housekeeping gene, GAPDH. Results represent mean ± SD of triplicates from three independent experiments. (C) Cell monolayer growth was determined using MTT assay. Transduced MCF-7L and TamR cells were serum starved for 24 hours and treated with or without insulin for 5 days. (D) Anchorage-independent growth of lentiviral transduced MCF-7L and T47D was measured after 19 days and 25 days, respectively. Values were normalized to untreated group and were represented in fold change (mean ± SD, n=3). Two-way ANOVA with Bonferroni comparison was performed to identify significance among untreated versus treated groups and shIR versus control groups. *, p<0.05; **, p<0.01.

Figure 3. S961 inhibited insulin-stimulated PI3K/MAPK signaling and growth in MCF-7L TamR cells, but not in parental cells

(A) MCF-7L and TamR cells were serum starved overnight and pre-treated with varying concentrations of S961 for 30 minutes before treating the cells with either 10nM insulin or 5nM IGF-I for 10 minutes. Whole cell lysates were separated by SDS-PAGE and immunoblotted for indicated antibodies. (B) Anchorage-independent growth assay was carried out on MCF-7L and TamR cells treated with varying concentrations of S961 and either 10nM insulin or 5nM IGF-I. Treatments were spiked in after 7 days. Colonies formed were counted 14 days later. Two-way ANOVA with Bonferroni comparison was performed to compare between treated and untreated group. *, p<0.05; **, p<0.01. (C) Cell cycle analysis was performed using flow cytometry. MCF-7L and TamR cells were plated, serum starved for 8 hours before treating with varying concentrations of S961 and either 10nM insulin or 5nM IGF-I overnight.

Figure 4. Monoclonal antibody clone 83-7 against insulin receptor (83-7) downregulated insulin receptor, inhibiting insulin-stimulated signaling and growth in TamR cells

(A) MCF-7L and T47D cells were serum starved and pre-treated with 2 μg/mL of 83-7 overnight before treating with either 5nM IGF-I, 10nM IGF-II or 10nM insulin for 15 minutes. Whole cell lysate were collected, separated by SDS-PAGE and subjected for indicated immunoblotting analyses. Anchorage-independent growth assay was carried out on (B) MCF-7L and T47D cells treated with 2 μg/mL of 83-7 without or with either 10nM IGF-I or 10nM insulin. Colonies formed were counted 14 days for MCF-7Ls and 21 days for T47D cells after first plated. Two-way ANOVA with Bonferroni comparison was performed to identify significance among untreated vs. treated groups. *, p<0.05; **, p<0.01.

Figure 5. Inhibition of InsR via S961 and shIR was not effective in parental cells due to the presence of IGF1R/InsR hybrid receptors

The presence of IGF1R/InsR hybrid receptors was identified via immunoprecipitation (IP). (A) Whole cell lysates were collected in full media and immunoprecipitated with either anti-InsR antibody or mouse IgG overnight. IP was then resolved with SDS-PAGE and subjected for IGF1R immunoblotting. (B) MCF-7L parental cells were serum starved overnight, pre-treated with 20 μg/mL HuEM164 for 3 hours and varying concentrations of S961 for 30 minutes before exposing toeither 10nM insulin or 5nM IGF-I for 10 minutes. Whole cell lysates were collected, separated by SDS-PAGE and subjected for immunoblotting analyses. (C) Lentiviral transduced MCF-7L cells were serum starved overnight and pre-treated with 20 μg/mL HuEM164 overnight before exposing to either 10nM insulin or 5nM IGF-I for 15 minutes. Whole cell lysates were collected, separated by SDS-PAGE and subjected for indicated immunoblotting analyses.