The rearranged during transfection/papillary thyroid carcinoma tyrosine kinase is an estrogen-dependent gene required for the growth of estrogen receptor positive breast cancer cells

Abstract

The rearranged during transfection/papillary thyroid carcinoma (RET/PTC) tyrosine kinase is an oncogene implicated in the tumorigenesis of thyroid cancer. Recent studies by us and others have shown that RET/PTC kinase expression is induced by estrogen in breast cancer cells. Due to the critical involvement of estrogen-regulated genes in the pathogenesis of breast cancer, we investigated the expression, regulation, and function of RET/PTC kinase in breast cancer cells. We found that RET/PTC kinase expression correlates with estrogen receptor (ER) expression in breast cancer cells and tumor specimens, and that RET/PTC kinase expression is associated with a poor prognosis in ER-positive breast cancer patients. We found that estrogen rapidly induces RET/PTC kinase expression in an ER-dependent manner in breast cancer cells and that this induction is through a transcriptional regulatory mechanism. Using reporter assays, small interfering RNA (siRNA) assays, and chromatin immunoprecipitation (ChIP) assays, we demonstrated the necessity of crosstalk between ER and the forkhead box A1 (FOXA1) transcription factor in regulating RET/PTC kinase expression. In functional studies, increased expression of RET/PTC kinase induced by estrogen stimulation resulted in elevated phosphorylation of multiple downstream kinase signaling pathways. Conversely, knockdown of RET/PTC expression was associated with the inhibition of these same kinase signaling pathways, and, in fact, decreased the stimulatory effect of estrogen on the proliferation of ER-positive breast cancer cells. These results demonstrate a novel pathway of ER and FOXA1 transcription factor crosstalk in regulating RET/PTC kinase expression, and demonstrate that RET/PTC kinase is a critical regulator for the proliferation of ER-positive breast cancer cells. Taken together, our study suggests that RET/PTC kinase may serve as a novel prognostic biomarker and therapeutic target for prevention and treatment of ER-positive breast cancer.

Figure 1

RET/PTC kinase gene expression and its significance in breast cancer. (A) RET/PTC kinase mRNA expression in breast cancer cell lines as determined by qRT-PCR. (B) RET/PTC kinase protein expression in breast cancer cell lines as determined by Western blot. (C) Comparison of RET/PTC kinase mRNA expression in all ER-positive and ER-negative cell lines included in this study. (D) RET/PTC kinase mRNA expression in breast tumor tissue specimens from the three largest independent studies including a total of 625 patients. Data are from the clinical microarray datasets [Bittner et al IGC dataset, Not Published, 2005; Desmedt et al TRANSBIG dataset [25]; Wang et al Veridex dataset [26]]. (E) The correlation between ER and RET/PTC kinase expression in breast tumor tissue specimens [25]. (F) Kaplan-Meier analysis showing the overall survival of those ER-positive patients whose tumors had either significantly high or significantly low RET/PTC kinase expression [25].

Figure 2

Effect of estrogen on RET/PTC kinase gene expression. (A) E2 regulation of RET/PTC kinase expression in a panel of breast cancer cell lines. Hormone-depleted cells were treated with vehicle (V) or estrogen (E) for 6h and mRNA expression was analyzed by qRT-PCR. (B) Time course of E2 induction of RET/PTC kinase expression in MCF-7 cells. Inset: Western blot showing E2 induction of RET/PTC kinase expression in MCF-7 cells at 24h of E2 treatment. (C) Effects of Actinomycin D (ActD) and Cycloheximide (CHX) on E2 induction of RET/PTC kinase expression. Hormone depleted MCF-7 cells were treated with ActD (1µg/ml) or CHX (10µg/ml) for 1h before the addition of vehicle or E2. RNA was harvested 3h after treatment with vehicle or E2. (D) Effect of ICI 182.780 (ICI) on E2 induction of RET/PTC kinase expression. Hormone-depleted MCF-7 cells were treated with ICI (1µmol/l) for 1h before the addition of vehicle or E2, and RNA was harvested 3h after treatment with vehicle or E2. (E) Effects of siRNA against ER on RET/PTC kinase expression. Hormone-depleted MCF-7 cells were transfected with siRNA against luciferase (Luc) or estrogen receptor (ER) for 36h before the addition of vehicle or E2. RNA was harvested at 3h after treatment with vehicle or E2. Inset: verification of ER knockdown by Western blot.

Figure 3

Luciferase assay of RET/PTC kinase gene enhancer/promoter luciferase constructs. (A) Effect of E2 on the reporter activity of various lengths of RET/PTC kinase gene promoter- luciferase constructs. Hormone-depleted MCF-7 cells were transfected with RET/PTC kinase gene promoter luciferase plasmid. Sixteen hours later, cells were treated with vehicle or E2 for 6h and lysed for luciferase analysis. (B) More complete map of RET/PTC kinase gene structure in the genomic context, showing the ER binding sites that were previously identified by eight independent studies (chromosome 10: 42841683 ~ 42842495). (C) Effect of E2 on the reporter activity of RET/PTC kinase promoter luciferase construct with or without the upstream enhancer.

Figure 4

Crosstalk between ER and FOXA1 in regulating RET/PTC kinase gene expression. (A) The predicted potential transcription factor binding sites in the upstream RET/PTC kinase gene enhancer, showing the two estrogen responsive elements (EREs), the single ERE half site (hERE), and the two forkhead responsive elements (FHREs). (B). Effect of E2 on the reporter activity of RET/PTC kinase gene enhancer-promoter luciferase constructs with mutations in the ERE and FHRE sites. (C) Effect of siRNA knockdown of FOXA1 on estrogen-induced RET/PTC kinase gene expression. Hormone-depleted MCF-7 cells were transfected with siRNA against FOXA1. Thirty-six hours later, cells were treated with vehicle or E2 for 3h and harvested for qRT-PCR. Inset: Validation of FOXA1 siRNA on ER and FOXA1 expression by Western Blot. (D). Effect of FOXA1 overexpression on estrogen-induced RET/PTC kinase gene expression. Hormone-depleted MCF-7 cells were transfected with vector (vec) or CMV-FOXA1 plasmid for 36h and then treated with vehicle or E2 for 3h. RNA was harvested and analyzed by qRT-PCR. Inset: Validation of FOXA1 overexpression by Western blot.

Figure 4

Crosstalk between ER and FOXA1 in regulating RET/PTC kinase gene expression. (A) The predicted potential transcription factor binding sites in the upstream RET/PTC kinase gene enhancer, showing the two estrogen responsive elements (EREs), the single ERE half site (hERE), and the two forkhead responsive elements (FHREs). (B). Effect of E2 on the reporter activity of RET/PTC kinase gene enhancer-promoter luciferase constructs with mutations in the ERE and FHRE sites. (C) Effect of siRNA knockdown of FOXA1 on estrogen-induced RET/PTC kinase gene expression. Hormone-depleted MCF-7 cells were transfected with siRNA against FOXA1. Thirty-six hours later, cells were treated with vehicle or E2 for 3h and harvested for qRT-PCR. Inset: Validation of FOXA1 siRNA on ER and FOXA1 expression by Western Blot. (D). Effect of FOXA1 overexpression on estrogen-induced RET/PTC kinase gene expression. Hormone-depleted MCF-7 cells were transfected with vector (vec) or CMV-FOXA1 plasmid for 36h and then treated with vehicle or E2 for 3h. RNA was harvested and analyzed by qRT-PCR. Inset: Validation of FOXA1 overexpression by Western blot.

Figure 5

ChIP assay showing recruitment of transcription factor proteins to the RET/PTC kinase gene enhancer. (A). Schematic representation of the ChIP primers used to amplify the RET/PTC kinase enhancer and promoter regions. (B-D). Recruitment of ER, FOXA1, and Pol II proteins, respectively, to the RET/PTC enhancer and promoter regions. Hormone-depleted MCF-7 cells were treated with vehicle or E2 for 30 min and then fixed with formaldehyde. Data were presented as relative amount of immunoprecipitated DNA normalized to input as measured by qPCR assay.

Figure 6

Effect of estrogen upregulation and siRNA knockdown of RET/PTC kinase expression on downstream kinase signaling pathways. Hormone-depleted MCF-7 cells were transfected with siRNA against RET/PTC kinase or siRNA against luciferase for 36h. Then cells were switched into serum free IMEM with E2 or vehicle for 24h. Before harvesting, cells were treated with physiological level (1ng/ml) of the RET/PTC kinase ligand glial cell line-derived neurotrophic factor (GDNF) for 10min to stimulate the RET/PTC kinase signaling pathway. Western blot analysis of both phosphorylated and unphosphorylated kinase signaling pathway components is shown.

Figure 7

Effect of estrogen upregulation and siRNA knockdown of RET/PTC kinase expression on breast cancer cell proliferation. (A) Upper Panel: MCF-7 cell proliferation as examined by MTS assay. Lower Panel: verification of RET/PTC kinase knockdown by qRT-PCR. (B) Upper Panel: MDA-MB-453 cell proliferation as examined by MTS assay. Lower Panel: verification of RET/PTC kinase knockdown by qRT-PCR. (C) Upper Panel: MCF-7 cell proliferation as examined by soft agar colony formation assay. Bar: 200µm. Lower Panel: A depiction of the colony counts.

Figure 8

Schematic representation of the ER and FOXA1 transcription factors regulating RET/PTC kinase expression, the associated downstream kinase signaling pathways, and ultimately, breast cancer cell proliferation.