TOX3 is expressed in mammary ER(+) epithelial cells and regulates ER target genes in luminal breast cancer

Abstract

Background: A breast cancer susceptibility locus has been mapped to the gene encoding TOX3. Little is known regarding the expression pattern or biological role of TOX3 in breast cancer or in the mammary gland. Here we analyzed TOX3 expression in murine and human mammary glands and in molecular subtypes of breast cancer, and assessed its ability to alter the biology of breast cancer cells.

Methods: We used a cell sorting strategy, followed by quantitative real-time PCR, to study TOX3 gene expression in the mouse mammary gland. To study the expression of this nuclear protein in human mammary glands and breast tumors, we generated a rabbit monoclonal antibody specific for human TOX3. In vitro studies were performed on MCF7, BT474 and MDA-MB-231 cell lines to study the effects of TOX3 modulation on gene expression in the context of breast cancer cells.

Results: We found TOX3 expression in estrogen receptor-positive mammary epithelial cells, including progenitor cells. A subset of breast tumors also highly expresses TOX3, with poor outcome associated with high expression of TOX3 in luminal B breast cancers. We also demonstrate the ability of TOX3 to alter gene expression in MCF7 luminal breast cancer cells, including cancer relevant genes TFF1 and CXCR4. Knockdown of TOX3 in a luminal B breast cancer cell line that highly expresses TOX3 is associated with slower growth. Surprisingly, TOX3 is also shown to regulate TFF1 in an estrogen-independent and tamoxifen-insensitive manner.

Conclusions: These results demonstrate that high expression of this protein likely plays a crucial role in breast cancer progression. This is in sharp contrast to previous studies that indicated breast cancer susceptibility is associated with lower expression of TOX3. Together, these results suggest two different roles for TOX3, one in the initiation of breast cancer, potentially related to expression of TOX3 in mammary epithelial cell progenitors, and another role for this nuclear protein in the progression of cancer. In addition, these results can begin to shed light on the reported association of TOX3 expression and breast cancer metastasis to the bone, and point to TOX3 as a novel regulator of estrogen receptor-mediated gene expression.

Figure 1

TOX3 is expressed in mouse and human mammary epithelium. A. Gating strategy used to sort mouse mammary epithelial cell populations is shown. Contour plot on the right is gated on the luminal epithelial population as shown. B. qRT-PCR for mouse Tox3 in the indicated sorted cell populations. Data are expressed as mean ± SD of three independent sorts. C. Immunohistochemical staining of TOX3 in reduction mammoplasty samples showing areas of high and low TOX3 protein expression within an individual (magnification, 40X). D. Immunohistochemical staining of serial sections with antibodies specific for TOX3, ER, and PR (magnification 20X). E. Immunohistochemical staining of serial sections for TOX3 and FOXA1 at indicated magnification. Examples of FOXA1⁺ and FOXA1⁻ nuclei are indicated by red and green arrows, respectively.

Figure 2

TOX3mRNA is expressed in multiple molecular subtypes of breast cancer. A. Expression of TOX3 was determined by qRT-PCR in breast cancer (red) and normal breast (blue) samples, relative to housekeeping gene MRPL. Relative expression was then normalized to ‘Normal 1’. B. RT-PCR analysis of the long (Var1) and short (Var2) variants of TOX3, and GAPDH, in breast cancer cell lines (BT474, MCF-7), two normal breast samples, and two primary breast cancer samples BC6 and BC1, as in (A). C. Relative TOX3 mRNA expression in six intrinsic molecular subtypes of breast cancer.

Figure 3

TOX3 is highly expressed in a subset of LumB tumors and is associated with poor prognosis. A. Examples of TOX3 protein expression in six primary breast cancer samples. B. Proportion of TOX3^hi (red) tumors among histologically defined breast cancer subtypes on tissue array of 188 breast tumors. C. Kaplan-Meier plots showing overall survival (OS) and recurrence free survival (RFS) of patients bearing TOX3^hi tumors of the LumB and LumA subtypes. Hazard ratios, confidence intervals, and logrank P values are shown.

Figure 4

Expression of TOX3 in MCF-7 cells leads to upregulation of genes implicated in cancer progression. A. Two-way hierarchical clustering of gene expression changes in MCF-7 cells upon transient expression of TOX3. B. qRT-PCR validation of selected genes from microarray result. Data are expressed as mean ± SD of three independent experiments. C. TOX3 mRNA and protein expression (inset) in MCF-7 stable cell lines. D. Cell survival of stably transfected MCF-7 cell lines at indicated days, grown in estrogen-depleted medium. Data are expressed as mean ± SD of three cultures. E. BT474 cell growth upon siRNA-mediated knockdown of TOX3. Data are expressed as mean ± SD of five independent experiments.

Figure 5

TOX3 enhances MCF-7 migration and is upregulated by IGF-1. A. qRT-PCR and B. FACS analysis, for CXCR4 expression in two independently generated vector or TOX3 stably transfected MCF-7 cell lines. C. Representative images of migratory cells in vector control (V) or TOX3-expressing (TOX3) cell lines. D. Quantification of migratory cells in vector and TOX3 transfected cell lines using FBS as chemo-attractant. Data are expressed as mean ± SD of three independent experiments. E. qRT-PCR analysis of TOX3 and GAPDH (a positive control for IGF-1 response), relative to ACTB, in MCF-7 cells treated with IGF-1. Data are expressed as mean ± SD of three independent experiments.

Figure 6

TOX3 regulates estrogen-responsive genes in a ligand-independent manner. A. Overlap of genes upregulated by TOX3 (yellow) in MCF-7 cells as shown here, and those previously reported to be bound by ER and/or be regulated by estrogen [31] (blue). B. TFF1 mRNA expression in transiently transfected MCF-7 cells under estrogen depleted conditions. Data are expressed as mean ± SD of three independent experiments. C. TFF1 gene expression in stably transfected MCF-7 cell lines. D. TFF1 pre-mRNA expression in indicated cell lines treated with estrogen (E2) or vehicle (EtOH). E. eRNA expression in stably transfected MCF-7 cells. Data are expressed as mean ± SD of three independent experiments. F. Estrogen-induced TFF1 mRNA expression following siRNA-mediated TOX3 knockdown as indicated. Data are expressed as mean ± SD of six independent experiments. G. TFF1 expression following siRNA mediated ER knockdown in MCF-7 stable cells lines as indicated in presence of estrogen (E2) or vehicle (EtOH). Data are expressed as mean ± SD of four independent experiments. H. TFF1 expression after ER and/or TOX3 transfection of MDA-MB-231 cells and with indicated additions. Data are expressed as mean ± SD of four independent experiments.