Expression of the aryl hydrocarbon receptor is not required for the proliferation, migration, invasion, or estrogen-dependent tumorigenesis of MCF-7 breast cancer cells

Abstract

The AhR was initially identified as a ligand-activated transcription factor mediating effects of chlorinated dioxins and polycyclic aromatic hydrocarbons on cytochrome P450 1 (CYP1) expression. Recently, evidence supporting involvement of the AhR in cell-cycle regulation and tumorigenesis has been presented. To further define the roles of the AhR in cancer, we investigated the effects of AhR expression on cell proliferation, migration, invasion, and tumorigenesis of MCF-7 human breast cancer cells. In these studies, the properties of MCF-7 cells were compared with those of two MCF-7-derived sublines: AH(R100) , which express minimal AhR, and AhR(exp) , which overexpress AhR. Quantitative PCR, Western immunoblots, 17β-estradiol (E2 ) metabolism assays, and ethoxyresorufin O-deethylase assays showed the lack of AhR expression and AhR-regulated CYP1 expression in AH(R100) cells, and enhanced AhR and CYP1 expression in AhR(exp) cells. In the presence of 1 nM E2 , rates of cell proliferation of the three cell lines showed an inverse correlation with the levels of AhR mRNA. In comparison with MCF-7 and AhR(exp) cells, AH(R100) cells produced more colonies in soft agar and showed enhanced migration and invasion in chamber assays with E2 as the chemoattractant. Despite the lack of significant AhR expression, AH(R100) cells retained the ability to form tumors in severe combined immunodeficient mice when supplemented with E2 , producing mean tumor volumes comparable to those observed with MCF-7 cells. These studies indicate that, while CYP1 expression and inducibility are highly dependent on AhR expression, the proliferation, invasion, migration, anchorage-independent growth, and estrogen-stimulated tumor formation of MCF-7 cells do not require the AhR.

Figure 1

Expression of AhR and ERα in MCF-7 and MCF-7-derived cell lines. (A) Heterologous AhR mRNA expression in AhR^exp cells. Total RNA from AhR^exp (lanes 1-3) and MCF-7 cells (lanes 4-5) was isolated, and PCR was performed with primers specific for the coding sequence of AhR mRNA representing total AhR mRNA (377-bp product) or specific for the vector-derived mRNA (614-bp product). Lane 1, reverse transcriptase-negative control using vector-specific primers; lanes 2 and 5, PCR with vector-specific primers; lanes 3, 4, and 6, PCR with primers specific for the AhR coding sequence; lane 6, minus-RNA control using primers specific for the AhR coding sequence; lane 7, 100-bp ladder. (B) AhR and ERα protein levels in AH^R100, MCF-7, and AhR^exp cells. Western blots of whole cell lysates from MCF-7, AhR^exp, and AH^R100 cells were probed with anti-AhR and -ERα antibodies and detected with enhanced chemiluminescence. Lysates from TMX2-28 cells are included as an ERα-negative control. GAPDH was probed as a loading control for each blot. (C) The loss of TCDD-inducible EROD activity in AH^R100 cells is persistent. BaP was withdrawn from AH^R100 cultures for 10 or 20 weeks, and compared with cells without BaP withdrawal (designated as 0 weeks without BaP), after which cells were exposed for 72 h to 10 nM TCDD (gray bars) or the vehicle, 0.1% (v/v) DMSO (black bars). CYP1 activity was then measured by EROD assay. EROD activity of MCF-7 cells is shown for comparison; note the difference in scale. Data are presented as mean + SE; n = 5. Significant differences for TCDD-treated cultures in comparison with the TCDD-treated control group (AH^R100 cells without BaP withdrawal) are indicated (***P<0.001).

Figure 2

Ah-responsiveness in AH^R100, MCF-7, and AhR^exp cells as measured by the EROD assay. Confluent cultures of AH^R100, MCF-7, and AhR^exp cells were exposed for 48 h to 10 nM TCDD (gray bars), or the vehicle, 0.16% (v/v) DMSO (black bars), with or without 1 nM E₂ and 100 nM ICI, as indicated. CYP1 activity was then measured by the EROD assay. Data are represented as the mean + SE; n = 8. Significant differences between TCDD-exposed AH^R100 or AhR^exp cultures and MCF-7 cultures in which all other treatments were identical (***P<0.001); between groups differing only in E₂ exposure (⁺⁺⁺P<0.001); and between groups differing only in ICI exposure (⁺⁺⁺P<0.001) are indicated.

Figure 3

Effect of AhR expression on ERα mRNA levels and the inducibility of CYP1 mRNAs. Confluent cultures of AH^R100, MCF-7, and AhR^exp cells were exposed to 10 nM TCDD, 1 nM E₂, or the vehicle, 0.16% (v/v) DMSO, for 48 h, and the RNA isolated. qPCR was performed with primers specific for the transcripts as indicated. Data are represented as the average +/- SEM; n=3, and are normalized to total RNA. Significant differences between TCDD-exposed AH^R100 or AhR^exp cultures in comparison with MCF-7 cultures in which all other treatments were identical (*P<0.05; ***P<0.001); and between E₂ exposure and the respective group differing only in the absence of E₂ exposure (⁺⁺P<0.01; ⁺⁺⁺P<0.001) are indicated.

Figure 4

Basal and TCDD-induced E₂-metabolite formation in AH^R100, MCF-7, and AhR^exp cells. Confluent cultures of AH^R100, MCF-7, and AhR^exp cells were exposed for 48 h to 10 nM TCDD or the solvent vehicle, 0.1% (v/v) DMSO, as indicated. Metabolite formation was determined with (+SULF) and without (-SULF) prior hydrolysis of conjugates as described in Materials and Methods. Data are represented as the mean + SE and are normalized to total cellular protein; n = 3. Significant differences between TCDD-exposed AH^R100 or AhR^exp cultures and MCF-7 cultures, where all other treatments were identical (***P<0.001) are indicated; ND denotes that the metabolites were not detected.

Figure 5

Proliferation, migration, and invasion of AH^R100, MCF-7, and AhR^exp cells. (A) Proliferation: AH^R100, MCF-7, and AhR^exp cells were seeded in 96-well plates at 6000 cells per well, and the media were replenished every 3 or 4 days with DC₅ (closed circles) or DC₅ containing 1 nM E₂ (open circles). Proliferation was measured at the indicated times using the Sulforhodamine B assay. Data are presented as mean + SE; n = 8; however, for all points the error bar falls within the symbol. Curves were obtained by fitting data to the four parameter Chapman model. (B) Anchorage-independent growth: AH^R100, MCF-7, and AhR^exp cells were suspended in 500 μL 0.3% soft agar in 24-well plates at 1500 cells per well. The cells were cultured for 2 weeks in DC₅ or DC₅ containing 1 nM E₂ as indicted. Anchorage-independent growth was assessed under 10X magnification by counting colonies larger than 50 μm (black bars) or larger than 100 μm (gray bars) in diameter. Significant differences between AH^R100 or AhR^exp cultures in comparison with MCF-7 cultures when all other treatments were identical (**P<0.01; ***P<0.001) are indicated. Data are represented as mean colonies per field + SE; n = 6. Significant differences for colonies ≥ 50 μm are shown. (C) Migration and invasion: AH^R100, MCF-7, and AhR^exp cells were suspended in DMEM containing 0.1% bovine serum albumin, and 10⁵ cells per well were seeded in Matrigel-coated chambers for measurement of invasion (black bars) or in control inserts for measurement of migration (gray bars). The lower chamber contained 1 nM E₂ in DC₁₀ as a chemoattractant. After 40 h, inserts were fixed and stained with crystal violet. Data are represented as mean cells per insert + SE; n = 3. Significant differences between AH^R100 or AhR^exp cultures in comparison with MCF-7 cultures (**P<0.01; ***P<0.001) are indicated.

Figure 6

Tumorigenicity of the MCF-7-derived cell lines with differential expression of AhR and ERα. AH^R100, MCF-7, AhR^exp cells, or TMX2 cells, as indicated, were implanted into the mammary glands of SCID mice, with (closed circles) or without (open circles) E₂ supplementation as a Silastic implant. Tumor growth was monitored by palpation at the times indicated. The latency of tumor formation is indicated in the plots by denotation of the day at which the maximal incidence of tumor formation was observed, with the number of mice with palpable tumors/total number of mice. Data are presented as mean tumor volume + SE of the mice with measurable tumors.