HMGA2/TET1/HOXA9 signaling pathway regulates breast cancer growth and metastasis - PubMed (original) (raw)

HMGA2/TET1/HOXA9 signaling pathway regulates breast cancer growth and metastasis

Miao Sun et al. Proc Natl Acad Sci U S A. 2013.

Abstract

The ten-eleven translocation (TET) family of methylcytosine dioxygenases initiates demethylation of DNA and is associated with tumorigenesis in many cancers; however, the mechanism is mostly unknown. Here we identify upstream activators and downstream effectors of TET1 in breast cancer using human breast cancer cells and a genetically engineered mouse model. We show that depleting the architectural transcription factor high mobility group AT-hook 2 (HMGA2) induces TET1. TET1 binds and demethylates its own promoter and the promoter of homeobox A (HOXA) genes, enhancing its own expression and stimulating expression of HOXA genes including HOXA7 and HOXA9. Both TET1 and HOXA9 suppress breast tumor growth and metastasis in mouse xenografts. The genes comprising the HMGA2-TET1-HOXA9 pathway are coordinately regulated in breast cancer and together encompass a prognostic signature for patient survival. These results implicate the HMGA2-TET1-HOX signaling pathway in the epigenetic regulation of human breast cancer and highlight the importance of targeting methylation in specific subpopulations as a potential therapeutic strategy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Induction of TET1 and HOX gene expression upon depletion of HMGA2 in 1833 cells, a bone-tropic derivative of human breast cancer cell line MDA-MB-231, or in MMTV_–_Wnt1 transgenic mouse breast tumors. (A, B, and D_–_H) We stably transduced 1833 cells with HMGA2 shRNA (shHMGA2) or control SCR sh. (A) Gene expression array analysis showing up-regulation of TET1 and 20 of 39 HOX genes in _HMGA2_-depleted cells. (B) The expression levels of HOXA genes are shown. *Fold change (fc) < 2; **fc > 2 based on the signal intensity of gene expression arrays. (C) Genomic transcription units of human HOXA genes on chromosome 7 viewed using the UCSC Genome Browser (40). HOXA genes are transcribed from right to left with the following order: 5′UTR (thin blue bar), Coding Sequence (thick blue bar), and 3′UTR (thin blue bar). Bar length is proportional to length of DNA sequence. (D_–_H) qRT-PCR and immunoblotting analyses validated induction of TET1 and HOXA gene expression in _HMGA2_-depleted cells. (D_–_F) HMGA2 (D), TET1 (E), or HOXA4/5/6/7/9/11 (F) mRNA analyzed by qRT-PCR (GAPDH as normalization control). (G) HMGA2, TET1, and HOXA9/7 protein analyzed by immunoblotting (GAPDH as control). (H) Genome-wide 5hmC levels analyzed by dot blot assay. (I and J) Loss of Hmga2 in MMTV_–_Wnt1 transgenic mouse breast tumors induced Tet1 and Hoxa9/7 expression. Wnt1 transgenic mice were crossed with _Hmga2_-specific knockout mice. Mouse primary breast tumors were obtained from Hmga2 wild-type (Hmga2+/+), heterozygous (Hmga2+/−), or null (H_mga2_−/−) mice. (I) Murine Hmga2, Tet1, and Hoxa9/7 mRNA analyzed by qRT-PCR (with mouse Gapdh as normalization control). (J) Murine Tet1 and Hoxa9 protein and 5hmC levels analyzed by immunostaining. (D_–_F, H, and I) Data are means ± SEM; n = 3. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2.

Fig. 2.

TET1 is involved in an autoregulation in human breast cancer cells. (A) TET1 binds to its own promoter. We analyzed 1833 cells expressing TET1 or control by ChIP assay with anti-TET1 or H3K4Me3 antibody followed by qPCR analysis: TET1 and H3K4Me3 binding to the CpG island proximal to the TSS of TET1 (see site-1 and -2 in

SI Materials and Methods

). Site-3 is a negative control. (B and C) HMGA2 depletion causes demethylation of CpG islands at the TET1 promoter region. We analyzed 1833 cells stably expressing shHMGA2 or SCR sh for CpG island methylation status by multiple approaches. (B) TET1 promoter region was analyzed within ±1 kb from the TSS. Methylation-specific digestions followed by qPCR distinguished between methylated CpGs vs. unmethylated or other modified (e.g., 5hmC) CpGs. The percentage of methylation vs. unmethylation (includes unmethylated or other modified C) is indicated. (C) Bisulfite sequencing of specific CpGs (see

SI Materials and Methods

for primers) at the TET1 promoter proximal to the TSS. Results show unmethylated CpGs (open circles) vs. methylated or modified CpGs (filled circles) in 10 or more independent clones encompassing the region of interest. (D) We treated 1833 cells with 5-azacytidine followed by qRT-PCR analysis for TET1 mRNA expression (GAPDH as normalization control). (A, B, and D) Data are means ± SEM; n = 3. *P < 0.05; **P < 0.01.

Fig. 3.

Fig. 3.

TET1 induces HOXA gene expression. (A and B) Depletion of TET1 by siRNA partially countered induction of HOXA genes. We transfected 1833 cells stably expressing HMGA2 shRNA with control or TET1 siRNA. (A) Analysis of TET1 and HOXA gene mRNA by qRT-PCR. (B, Upper) Analysis of TET1 and HOXA9/7 protein by immunoblotting. (Lower) Analysis of 5hmC levels by dot-blot assay. (C and D) Expression of TET1 dramatically induced HOXA9 expression. We analyzed 1833 cells expressing constitutive Tet1 (Flag–Tet1) by qRT-PCR for HOXA9 mRNA (C) and by immunoblotting for Tet1 (Flag–M1) and HOXA9 protein (D, Upper) and dot-blot assay for 5hmC levels (D, Lower). (E and F) Induced expression of TET1 in breast xenograft tumors significantly induced HOXA9 expression. The 1833 cells stably expressing an inducible Tet1 expression vector were orthotopically injected into the mammary fat pad of nude mice. Tumor tissues were collected and analyzed after 6 wk with (+DOX) or without (−DOX) doxycycline treatment. (E) Tet1 and HOXA9 mRNA analyzed by qRT-PCR. (F) Tet1 and HOXA9 protein and 5hmC levels analyzed by immunostaining. (G) Significant positive correlation between TET1 and HOXA9/7 expression in breast cancer patients (see

Table S3

for patient information). Correlations were determined by Pearson’s correlation coefficient. P value was determined by Student t test. (A_–_E) GAPDH served as normalization control. Data are means ± SEM; n = 3. **P < 0.01; ***P < 0.001.

Fig. 4.

Fig. 4.

TET1 induces HOXA gene expression through binding to the promoter regions of HOXA genes and contributing to local demethylation in human breast cancer cells. (A and B) TET1 binds to the HOXA gene promoters. We analyzed 1833 cells expressing TET1 or control by ChIP assay with anti-TET1 or H3K4Me3 antibody followed by qPCR analysis: TET1 and H3K4Me3 binding to the CpG islands proximal to the TSS of HOXA7 (see site-1 and -2 in

SI Materials and Methods

) where site-3 is a negative control (A); or HOXA9 (see site-1 in

SI Materials and Methods

) where site-2 is a negative control (B). (C_–_E) HMGA2 depletion causes demethylation of CpG islands at HOXA gene promoter regions. We analyzed 1833 cells stably expressing shHMGA2 or SCR sh for CpG island methylation status by multiple approaches (see Fig. 2 B and C for the specificity of each method). (C) HOXA promoter regions were analyzed within −5 to +3 kb from the TSS. The percentage of methylation vs. unmethylation is indicated. (D and E) Bisulfite sequencing of specific CpGs (see

SI Materials and Methods

for primers) at HOXA7 (D) and HOXA9 (E) promoters proximal to the TSS. Results show unmethylated CpGs (open circles) vs. methylated or modified CpGs (filled circles) in 10 independent clones encompassing the region of interest. (F and G) We treated 1833 cells with 5-azacytidine followed by qRT-PCR analysis for expression of HOXA7 (F) or HOXA9 (G) mRNA (GAPDH as normalization control). (A_–_C, F, and G) Data are means ± SEM; n = 3. **P < 0.01; ***P < 0.001.

Fig. 5.

Fig. 5.

Both TET1 and its target, HOXA9, suppress breast tumor growth, invasion, and metastasis. (A_–_D) HMGA2/TET1/HOXA pathway regulates breast cancer cell invasion. (A) Inhibition of cell invasion in 1833 cells with HMGA2 depletion. (B) Transfection of TET1 siRNA into _HMGA2_-depleted 1833 cells increases invasion. (C) Transfection of HOXA7 or HOXA9 siRNA into _HMGA2_-depleted 1833 cells increases invasion. (D) Decitabine (5-aza-dC) treatment of 1833 cells decreases cell invasion, and transfection of HOXA9 siRNA into treated cells partially reversed cell invasion. (A_–_D) Data are means ± SEM; n = 3. (E_–_K) We orthotopically injected 1833 cells stably expressing an inducible control, Tet1, or HOXA9 expression vector into the mammary fat pad of nude mice. Mice were administered drinking water with (+DOX) or without (−DOX) addition of doxycycline. (E_–_G) Both TET1 and HOXA9 suppress xenograft breast tumor growth. (E) Representative bioluminescence images of mice bearing 1833 cells treated as indicated. (F) Photograph of representative xenograft breast tumors of 1833 cells treated as indicated. (G) Xenograft breast tumors of 1833 cells treated as indicated and analyzed for tumor weight. (F and G) Tumors were dissected at 6 wk after implantation. (H and I) Both TET1 and HOXA9 suppress the proliferation in xenograft breast tumors: immunostaining showing Ki67-positive cells in tumor sample of 1833 cells with induced (+DOX) vs. noninduced (−DOX) expression of Tet1 (H) or HOXA9 (I). (J and K) Both TET1 and HOXA9 inhibit intravasation of 1833 cells. Cells isolated from the blood after 6 wk were analyzed for GAPDH/Gapdh transcripts derived from human (tumor) or mouse (control) by qRT-PCR: intravasation of 1833 cells with induced (+DOX) vs. noninduced (−DOX) expression of Tet1 (J) or HOXA9 (K). Data are means ± SEM; n = 8 per group. (L_–_N) Both TET1 and HOXA9 suppress bone metastasis of 1833 cells. We injected 1833 cells stably expressing an inducible Tet1 or HOXA9 expression vector into the left ventricle of mice. Mice were administered drinking water with (+DOX) or without (−DOX) addition of doxycycline and imaged for luciferase activity after 3 wk. (L) Representative bioluminescence images of mice with bone metastasis. (M) Quantification of bone colonization by 1833 cells with induced (+DOX) vs. noninduced (−DOX) expression of Tet1 or HOXA9. Data are means ± SEM; n = 7–9 per group. (N) Kaplan–Meier survival analysis of mice over 8 wk after injection of the tumor cells.

Fig. 6.

Fig. 6.

The HMGA2/TET1/HOXA pathway regulates breast tumorigenesis. (A) Comparison of the genes regulated by HMGA2, TET1, or HOXA9 in 1833 cells (human breast cancer cells; hBrCa). (B) Scheme illustrating HMGA2/TET1/HOXA signaling pathway in breast tumorigenesis. (C) Kaplan–Meier analysis of gene expression data from 101 breast tumor patients (see

Table S3

for patient information). Patients were stratified for survival using HMGA2, TET1, HOXA9, HOXA7, or the complete pathway as indicated. (Right) Red line, high HMGA2 and low TET1/HOXAs (n = 34); blue line, low HMGA2 and high TET1/HOXAs (n = 35); P, χ2 P value.

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