Distinct pathways regulated by RET and estrogen receptor in luminal breast cancer demonstrate the biological basis for combination therapy

Abstract

Objective: We investigated directed therapy based on TFAP2C-regulated pathways to inform new therapeutic approaches for treatment of luminal breast cancer.

Background: TFAP2C regulates the expression of genes characterizing the luminal phenotype including ESR1 and RET, but pathway cross talk and potential for distinct elements have not been characterized.

Methods: Activation of extracellular signal-regulated kinases (ERK) and AKT was assessed using phosphorylation-specific Western blot. Cell proliferation was measured with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] after siRNA (small interfering RNA) gene knockdown or drug treatment. Cell cycle, Ki-67, and cleaved caspase 3 were measured by fluorescence-activated cell sorting. Tumorigenesis was assessed in mice xenografts.

Results: Knockdown of TFAP2C or RET inhibited GDNF (glial cell line-derived neurotrophic factor)-mediated activation of ERK and AKT in MCF-7 cells. Similarly, sunitinib, a small-molecule inhibitor of RET, blocked GDNF-mediated activation of ERK and AKT. Inhibition of RET either by gene knockdown or by treatment with sunitinib or vandetanib reduced RET-dependent growth of luminal breast cancer cells. Interestingly, knockdown of TFAP2C, which controls both ER (estrogen receptor) and RET, demonstrated a greater effect on cell growth than either RET or ER alone. Parallel experiments using treatment with tamoxifen and sunitinib confirmed the increased effectiveness of dual inhibition of the ER and RET pathways in regulating cell growth. Whereas targeting the ER pathway altered cell proliferation, as measured by Ki-67 and S-phase, anti-RET primarily increased apoptosis, as demonstrated by cleaved caspase 3 and increased TUNEL (terminal deoxyneucleotidyl transferase dUTP nick end labeling) expression in xenografts.

Conclusions: ER and RET primarily function through distinct pathways regulating proliferation and cell survival, respectively. The findings inform a therapeutic approach based on combination therapy with antiestrogen and anti-RET in luminal breast cancer.

Figures

FIGURE 1

TFAP2C regulates ERK and AKT activation via RET. (A), Treatment of MCF-7 with GDNF results in induction of ERK and AKT phosphorylation. This effect is prevented by gene knockdown of TFAP2C, with a similar effect noted with direct RET knockdown showing that GDNF-mediated activation of ERK and AKT is dependent on TFAP2C regulation of RET in MCF-7. (B), GDNF-mediated activation of ERK and AKT is prevented by treatment with the RET inhibitor sunitinib in MCF-7. (C), There is no effect of GDNF treatment or RET or TFAP2C knockout in MDA-MB-231, which do not express RET or TFAP2C. (D), There is no effect of sunitinib treatment on ERK or AKT activation in MDA-MB-231. *P < 0.001.

FIGURE 2

TFAP2C regulates cell growth through the ER and RET pathways. (A), Knockdown of RET or ER results in significantly reduced growth of MCF-7 cells. Knockout of TFAP2C results in a larger reduction in cell number than either RET or ER knockdown alone. (B), Knockdown of RET, ER, or TFAP2C does not affect growth of MDA-MB-231 cells. (C), Treatment with GDNF increases growth of MCF-7 cells and treatment with tamoxifen (TAM) or sunitinib (SUN) reduce cell growth, whereas treatment with both tamoxifen and sunitinib has a more significant effect on growth compared with either drug alone. (D), Treatment with GDNF, sunitinib, tamoxifen, or sunitinib and tamoxifen did not alter proliferation in MDA-MB-231 cells.

FIGURE 3

The effects of sunitinib (SUN) in luminal breast cancer are mediated by the RET receptor. (A), Western blot demonstrating RET expression in the luminal breast cancer cell line BT-474 and confirming effectiveness of RET siRNA to eliminate RET expression. (B), Treatment with sunitinib reduces cell growth in BT-474 with additive results when combined with tamoxifen (TAM) treatment. (C), Treatment with sunitinib or vandetanib reduced viability in MCF-7 and BT-474. Knockdown of RET expression by siRNA eliminated the antiproliferative effects of treatment of both sunitinib and vandetanib in both cell lines. For both cell lines, data were normalized to NT transfection without drug treatment.

FIGURE 4

ER controls proliferation and RET controls cell survival. (A), FACS showed a significant reduction in Ki-67 proliferative index with knockdown of ER or TFAP2C with nonsignificant effect with knockdown of RET in MCF-7. (B), Example of FACS of MCF-7 cells with GDNF stimulation and treatment with tamoxifen (TAM) and/or sunitinib (SUN). Histogram representation of the effect on Ki-67 positivity (C) and expression of CC3 (D) after GDNF stimulation with tamoxifen and/or sunitinib treatment. (E), Experimental data from FACS showing summary of S-phase, Ki-67, and CC3 with GDNF stimulation and treatment with tamoxifen and/or sunitinib treatment. *P < 0.05.

FIGURE 5

Treatment of mice with tumors confirms apoptotic effects of sunitinib (SUN). (A), Micrographs from MCF-7 xenograft tumors showing hematoxylin-eosin (H&E) staining and immunohistochemistry for Ki-67 and TUNEL. (B), Animals treated with sunitinib were significantly less likely to develop tumors than control animals. (C), Ki-67 positivity was significantly reduced by sunitinib treatment. (D), TUNEL-positive apoptotic cells were significantly increased in sunitinib-treated animals. *P < 0.05.