HIC1 regulates tumor cell responses to endocrine therapies

Abstract

An intractable problem impeding breast cancer treatment by the most frequently prescribed endocrine therapy tamoxifen is the inevitable development of resistance, and the molecular mechanisms underlying this loss of responsiveness by breast cancers have been under intense investigation but are not yet fully elucidated. Our recent reports demonstrated that the tumor suppressor heavily methylated in cancers 1 (HIC1) plays an essential role in growth suppression mediated by external stimuli. We report here that novel tumor suppressor HIC1 is required for growth suppression by estrogen antagonists in breast cancer cells. We also find that HIC1 expression is dramatically induced by exposure to estrogen antagonists in sensitive cells, via a c-Jun N-terminal kinase 1 (JNK1) and prohibitin-mediated signaling pathway. This induction is lost in spontaneously antagonist-resistant breast cancer cells. Furthermore, reintroducing HIC1 into resistant breast cancer cells restored their sensitivity to the estrogen antagonists, indicating the existence of a novel regulatory mechanism for growth control of breast cancer cells.

Figure 1

A, Induction of HIC1 protein expression by estrogen antagonists is lost in antagonist-resistant breast cancer cells. A, Wild-type MCF7 cells (WT) or MCF7 resistant to estrogen antagonists (4HT or ICI) (established by long-term culture/selection in estrogen antagonists) were treated with ethanol (control) or estrogen antagonists for 4 h. The cell extracts were analyzed by immunoblot using HIC1 antibody or tubulin antibody (control). B, The cell extracts were analyzed by quantitative RT-PCR for transcripts of the indicated genes.

Figure 2

HIC1 represses E2F-mediated transcription. A, Wild-type MCF7 cells or the tamoxifen-resistant MCF7 cells (MCF7-res) were transfected with the indicated plasmids (HI-Luc, HIC1 promoter driving a luciferase reporter; HIC1, HIC1 expression vector; E2FLuc, E2F-responsive promoter-driven luciferase reporter). Cell extracts were collected and analyzed by luciferase assay, using standard protocols. Results showed the luciferase activities as mean ± sem from four independent experiments in which each cohort of transfected cells was assayed in triplicate. The results in lane 3 are significantly different from those in lane 1, and the results in lanes 2, 4, and 5 are significantly different from lanes 1 and 3 (P ≤ 0.05). Results in lane C are significantly different from those in lane A, and the results in lanes B and D are significantly different from lanes A and C (P ≤ 0.05). Error bars show sem. B, Wild-type MCF7 cells or the tamoxifen-resistant MCF7 cells (MCF7-res) were transfected with the indicated plasmids. Cell extracts were analyzed by quantitative RT-PCR for the gene transcripts indicated. TK, Thymidine kinase.

Figure 3

Estrogen antagonists induce HIC1 recruitment to E2F1 promoter. A, Wild-type (WT) MCF7 cells, MCF7 cells resistant to estrogen antagonists, or the resistant cells transfected with HIC1 were treated with ethanol (control), estrogen antagonists (4HT or ICI) for 4 h. The cell extracts were analyzed by ChIP analysis with quantitative PCR at the endogenous E2F1 promoter, using the indicated antibodies. An adjacent region on the promoter was analyzed as control. Amplification of input DNA (total) produced similar levels of product in all lanes. B, MCF7 cells resistant to antagonists were transfected with control or HIC1 vector (0.5 μg/10-cm dish) followed by the treatment with 4HT or ICI as indicated. The transcript levels of the indicated genes were then analyzed by quantitative RT-PCR (qRT-PCR). C, Antagonist-resistant cells and the cells transfected with HIC1 were treated with the reagents indicated. Cell lysates were then analyzed by immunoblot (IB) analysis using the indicated antibodies.

Figure 4

HIC1 is required for the E2F repression induced by estrogen antagonists. A, MCF7 cells transfected with control or HIC1 siRNA were treated with ethanol (control) or tamoxifen for 4 h. The cell extracts were analyzed by immunoblotting (IB) using the indicated antibodies to demonstrate repression by siRNA. B, The same cells were assayed for transcript levels of the endogenous E2F-responsive E2F1 gene or the tubulin gene (control) by real-time RT-PCR. C, Antagonist-resistant MCF7 cells were transfected with HIC1 with or without control siRNA, Brg1 siRNA, or Brm siRNA. The cells were treated with vehicle control of 4HT, and collected cells were analyzed for transcription of E2F1 and tubulin genes by quantitative RT-PCR. Experiments were repeated four times and normalized to internal control gene β-actin, with average values shown.

Figure 5

Induction of HIC1 expression and promoter activity by prohibitin. A, MCF7 cells were transfected with empty vector (control) or prohibitin expression vector (PH). The cell extracts were analyzed by immunoblotting (IB), using the indicated antibodies. Tubulin antibody served as a negative control. B, Wild-type MCF7 cells were transfected with the indicated plasmids (HIC Pro-Luc, the HIC1 promoter driving a luciferase reporter gene; HIC Mut Pro-Luc, the HIC1 promoter mutated at the p53 binding site). The cells were collected and analyzed by luciferase assay, using standard protocols. Results showed the luciferase activities as mean ± sem from four independent experiments in which each experimental group was assayed in triplicate. The results in lanes 2 and 3 are significantly different from lane 1 (P ≤ 0.05), and the results in lanes 4–6 are significantly different from the results in lanes 2 and 3 (P ≤ 0.05); Error bars show sem. C, Prohibitin is required for the induction of HIC1 expression by estrogen antagonists. MCF7 cells stably transfected with control siRNA or prohibitin (PH) siRNA were treated with ethanol (control) or tamoxifen for 18 h. The cell extracts were analyzed by immunoblotting using the indicated antibodies.

Figure 6

Prohibitin associates with the HIC1 promoter. A, MCF7 cells were treated with vehicle control or 4HT for 2 h, followed by ChIP assay by quantitative PCR on the HIC1 promoter using the antibodies indicated. Amplification of input DNA (total) produced similar levels of product in all lanes. B, Prohibitin loses its effect on HIC1 induction in antagonist-resistant cells. Wild-type (WT) MCF7 cells or the cells resistant to antagonists were transfected with control or prohibitin followed by quantitative RT-PCR analysis for the gene transcripts indicated.

Figure 7

JNK1 is required for estrogen antagonist-induced HIC1 expression and promoter recruitment of prohibitin. A, MCF7 cells were transfected with a JNK1 expression vector, followed by immunoblot analysis using the antibodies indicated. B, MCF7 cells were transfected with JNK1 siRNA or control siRNA, followed by immunoblot (IB) analysis by the indicated antibodies. C, MCF7 cells with JNK1 depleted by siRNA were treated with vehicle control of 4HT for 4 h, followed by ChIP assay using the indicated antibodies and real-time PCR. Amplification of input DNA (total) produced similar levels of product in all lanes.