Collateral-resistance to estrogen and HER-activated growth is associated with modified AKT, ERα, and cell-cycle signaling in a breast cancer model

Abstract

Aim: A model of progressively endocrine-resistant breast cancer was investigated to identify changes that can occur in signaling pathways after endocrine manipulation.

Methods: The MCF7 breast cancer model is sensitive to estrogens and anti-estrogens while variant lines previously derived from wild-type MCF7 are either relatively 17β-estradiol (E₂)-insensitive (LCC1) or fully resistant to estrogen and anti-estrogens (LCC9).

Results: In LCC1 and LCC9 cell lines, loss of estrogen sensitivity was accompanied by loss of growth response to transforming growth factor alpha (TGFα), heregulin-beta and pertuzumab. LCC1 and LCC9 cells had enhanced AKT phosphorylation relative to MCF7 which was reflected in downstream activation of phospho-mechanistic target of rapamycin (mTOR), phospho-S6, and phospho-estrogen receptor alpha Ser167 [ERα(Ser167)]. Both AKT2 and AKT3 were phosphorylated in the resistant cell lines, but small interfering RNA (siRNA) knockdown suggested that all three AKT isoforms contributed to growth response. ERα(Ser118) phosphorylation was increased by E₂ and TGFα in MCF7, by E₂ only in LCC1, but by neither in LCC9 cells. Multiple alterations in E₂-mediated cell cycle control were identified in the endocrine-resistant cell lines including increased expression of MYC, cyclin A1, cyclin D1, cyclin-dependent kinase 1 (CDK1), CDK2, and hyperphosphorylated retinoblastoma protein (ppRb), whereas p21 and p27 were reduced. Estrogen modulated expression of these regulators in MCF7 and LCC1 cells but not in LCC9 cells. Seliciclib inhibited CDK2 activation in MCF7 cells but not in resistant variants; in all lines, it reduced ppRb, increased p53 associated responses including p21, p53 up-regulated modulator of apoptosis (PUMA), and p53 apoptosis-inducing protein 1 (p53AIP1), inhibited growth, and produced G2/M block and apoptosis.

Conclusions: Multiple changes occur with progression of endocrine resistance in this model with AKT activation contributing to E₂ insensitivity and loss of ERα(Ser118) phosphorylation being associated with full resistance. Cell cycle regulation is modified in endocrine-resistant breast cancer cells, and seliciclib is effective in both endocrine-sensitive and resistant diseases.

Keywords: Breast cancer; endocrine resistance; erbB receptor; estrogen; seliciclib.

Figure 1.

Effects of E₂, TGFα, and HRGβ on cell growth and cell cycle distribution in MCF7, LCC1, and LCC9 cell lines. A. Cell growth response to E₂, TGFα and HRGβ. Cells were grown for 24 h prior to treatment, then treated with 1 nmol/L growth factors or hormone. Cells were counted on the days indicated. Data shown are mean values of quadruplicates and are representative of three independent experiments. B. Cell cycle analysis after 72 h treatment with growth factors or hormone. Results are representative of three independent experiments. * statistically significant changes between control and treatment (P < 0.05)

Figure 2.

Expression of the HER receptors in the cell lines. Western analysis of the 3 cell lines and MDA-MB-231 (positive control for EGFR) as described in “Materials and methods”

Figure 3.

Comparison of the effect of E₂, TGFα and HRGβ stimulation on expression levels of P-AKT(Ser473) in the wild-type and resistant cell lines. A. Western blot of P-AKT [P-AKT(Ser473)] and T-AKT levels in cell lines after 15 min treatment with E₂, TGFα or HRGβ (1 nmol/L). B. Histogram of relative P-AKT expression levels (relative to T-AKT) in the cell lines after C, E, T or H. ANOVA test, n = 3. C. Effect of P in the absence or presence of growth factor T or H on growth over 72 h in the cell lines. Cell number was assessed by SRB assay as described in “Materials and methods”. C: untreated control; E: 1 nmol/L E₂; H: 1 nmol/L HRGβ; P: 100 nmol/L pertuzumab; T: 1 nmol/L TGFα; * P < 0.05; ** P < 0.01; *** P < 0.001

Figure 4.

AKT expression and associated signaling in the cell lines. A. Expression of AKT isoform mRNA measured by RT-PCR. Each column represents mean of quadruplicate values relative to actin expression. Error bar = standard deviation (SD). Statistical comparison of resistant line with MCF7; * P < 0.05; ** P < 0.01; *** P < 0.001 (ANOVA). B. Western blot of AKT isoforms, P-AKT(Ser743) and total AKT in the cell lines. C. P-AKT isoforms. Samples were immunoprecipitated with AKT isoform specific antibodies and then probed for P-AKT. The non-immunoprecipitated (IP) control was incubated with immunoglobulin G (IgG) rather than isoform specific antibody. D. Western blots of upstream (PI3K, PTEN) and downstream (mTOR, S6) signaling molecules of AKT in the cell lines. E. Western blot of phospho-ERα(Ser167) in the cell lines

Figure 5.

Effects of AKT siRNAs on the expression and growth of the cell lines. A. Effect of AKT siRNAs on specific AKT isoform expression. B–D. Effect of AKT siRNAs (50 nmol/L) on proliferation of (B) MCF7 cells, (C) LCC1 cells, and (D) LCC9 cells. Each point represents the mean of 6 values. Neg: negative control

Figure 6.

The effects of E₂ and TGFα on the expression of P-ERα(Ser118) in the cell lines. A. Western blot example of P-ERα(Ser118) expression for MCF7 cells treated with E₂ or TGFα. B–D. Time courses of the effect of E₂ or TGFα on P-ERα(Ser118) expression for (B) MCF7 cells, (C) LCC1 cells, or (D) LCC9 cells. Legend for B–D is shown in panel C

Figure 7.

A. MYC mRNA expression as determined by RT-PCR in cell lines grown +/– E₂ (0.1 nmol/L). Data shown are expressed as the ratio of MYC: β-actin expression. Mean values +/– SDs are shown. Values were then normalized against the MCF7 control. Groups were compared by ANOVA followed by the Tukey-Kramer multiple comparison test. * P < 0.05. B. Western blot of MYC protein expression in cell lines grown +/– E₂ (0.1 nmol/L) and +/– seliciclib. C. Data shown are expressed as a ratio of MYC: tubulin expression. +: modulator shown present; −: modulator shown absent

Figure 8.

A. Expression of cell cycle regulatory gene mRNA levels in cell lines grown +/– E₂ (0.1 nmol/L) for 24 h. Data shown are expressed as the ratio of gene expression relative to that of β-actin. Mean of quadruplicate values is shown. Statistically significant values are indicated by either * (comparison with E₂-treatment, P < 0.05) or ** (comparison with MCF7 in absence of E₂, P < 0.05). Groups were compared by ANOVA followed by the Tukey-Kramer multiple comparison test. B. Western blot showing total and phosphorylated CDK2 expression in cell lines exposed to E₂ (0.1 nmol/L) +/– seliciclib (20 μmol/L) for 24 h. C. Western blot showing Rb phosphorylation in cell lines exposed to E₂ (0.1 nmol/L) +/– seliciclib (20 μmol/L) for 24 h. +: modulator shown present; −: modulator shown absent

Figure 9.

Effects of seliclib on cell growth, cell cycle and apoptosis in the cell lines. A. Inhibition of cell growth in cell lines as determined by seliciclib. The cell number is shown after 3 days treatment and the day 0 value is indicated for comparison. B. Cell cycle distribution of cell lines grown in E₂ (0.1 nmol/L) +/– seliciclib (20 μmol/L) for 24 h. Groups were compared by ANOVA followed by the Tukey-Kramer multiple comparison test. * P < 0.05 (comparison with same cell line control); ** P < 0.05 (comparison with MCF7 control). C. Proportion of apoptotic cells as determined by annexin V assay in E₂ (0.1 nmol/L) +/– seliciclib (20 μmol/L) for 24 h. Mean values +/– SDs are shown. D. Induction of apoptosis in cell lines exposed to seliciclib (20 μmol/L) +/– E₂ (0.1 nmol/L) for 24 h. PARP cleavage is represented by the lower row of bands (cleaved PARP) with full-length PARP above. PARP: polyadenosine-diphosphate-ribose polymerase; +: modulator shown present; −: modulator shown absent

Figure 10.

A. Western blots showing total expression and phosphorylation of p53, along with p21 and PUMA expression in cell lines grown +/– E₂ (0.1 nmol/L), +/– seliciclib (20 μmol/L). Lysates were harvested after 24 h. B. Expression of p53, MDM2 and downstream regulators of apoptosis in cell lines grown +/– E₂ (0.1 nmol/L), +/– seliciclib (20 μmol/L). Data shown are the expression of the named gene relative to that of β-actin. Mean values +/– SDs are shown. Groups were compared by ANOVA followed by the Tukey-Kramer multiple comparison test. * P < 0.05

Figure 11.

A. Western blots showing expression and phosphorylation of p53, MDM2 and CDK2 together with p21 and PUMA in MCF7-parent and MCF7-p53-KD cell lines in +/– seliciclib (20 μmol/L) for 24 h. Cleaved PARP expression illustrates the levels of apoptosis under these conditions. B. Expression of p53 mRNA in cell lines. Data shown are the ratio of expression of p53 relative to that of β-actin. C. Effect of seliciclib on the expression of p53 and associated genes in cell lines. The data shown is a ratio of expression of the named gene in seliciclib-treated relative to untreated cells (after correction of target mRNA relative to actin). * comparison with MCF7 parent cell line control, P < 0.05. D. Growth inhibitory effect of seliciclib in cell lines as determined using the SRB growth assay. +: modulator shown present; −: modulator shown absent

Figure 12.

Summary of the constitutive expression changes between the cell lines and estrogen-modulated expression changes. A. Changes between the cell lines. B. Estrogen-modulated expression changes. ↑: increased expression; ↓: reduced expression