ATM suppresses SATB1-induced malignant progression in breast epithelial cells.

SATB1 drives metastasis when expressed in breast tumor cells by radically reprogramming gene expression. Here, we show that SATB1 also has an oncogenic activity to transform certain non-malignant breast epithelial cell lines. We studied the non-malignant MCF10A cell line, which is used widely in the literature. We obtained aliquots from two different sources (here we refer to them as MCF10A-1 and MCF10A-2), but found them to be surprisingly dissimilar in their responses to oncogenic activity of SATB1. Ectopic expression of SATB1 in MCF10A-1 induced tumor-like morphology in three-dimensional cultures, led to tumor formation in immunocompromised mice, and when injected into tail veins, led to lung metastasis. The number of metastases correlated positively with the level of SATB1 expression. In contrast, SATB1 expression in MCF10A-2 did not lead to any of these outcomes. Yet DNA copy-number analysis revealed that MCF10A-1 is indistinguishable genetically from MCF10A-2. However, gene expression profiling analysis revealed that these cell lines have significantly divergent signatures for the expression of genes involved in oncogenesis, including cell cycle regulation and signal transduction. Above all, the early DNA damage-response kinase, ATM, was greatly reduced in MCF10A-1 cells compared to MCF10A-2 cells. We found the reason for reduction to be phenotypic drift due to long-term cultivation of MCF10A. ATM knockdown in MCF10A-2 and two other non-malignant breast epithelial cell lines, 184A1 and 184B4, enabled SATB1 to induce malignant phenotypes similar to that observed for MCF10A-1. These data indicate a novel role for ATM as a suppressor of SATB1-induced malignancy in breast epithelial cells, but also raise a cautionary note that phenotypic drift could lead to dramatically different functional outcomes

ATM Suppresses SATB1-Induced Malignant Progression in Breast Epithelial Cells

<div><p>SATB1 drives metastasis when expressed in breast tumor cells by radically reprogramming gene expression. Here, we show that SATB1 also has an oncogenic activity to transform certain non-malignant breast epithelial cell lines. We studied the non-malignant MCF10A cell line, which is used widely in the literature. We obtained aliquots from two different sources (here we refer to them as MCF10A-1 and MCF10A-2), but found them to be surprisingly dissimilar in their responses to oncogenic activity of SATB1. Ectopic expression of SATB1 in MCF10A-1 induced tumor-like morphology in three-dimensional cultures, led to tumor formation in immunocompromised mice, and when injected into tail veins, led to lung metastasis. The number of metastases correlated positively with the level of SATB1 expression. In contrast, SATB1 expression in MCF10A-2 did not lead to any of these outcomes. Yet DNA copy-number analysis revealed that MCF10A-1 is indistinguishable genetically from MCF10A-2. However, gene expression profiling analysis revealed that these cell lines have significantly divergent signatures for the expression of genes involved in oncogenesis, including cell cycle regulation and signal transduction. Above all, the early DNA damage-response kinase, ATM, was greatly reduced in MCF10A-1 cells compared to MCF10A-2 cells. We found the reason for reduction to be phenotypic drift due to long-term cultivation of MCF10A. ATM knockdown in MCF10A-2 and two other non-malignant breast epithelial cell lines, 184A1 and 184B4, enabled SATB1 to induce malignant phenotypes similar to that observed for MCF10A-1. These data indicate a novel role for ATM as a suppressor of SATB1-induced malignancy in breast epithelial cells, but also raise a cautionary note that phenotypic drift could lead to dramatically different functional outcomes.</p> </div

Ectopic expression of SATB1 in MCF10A-1 cells induces tumor growth and lung colonization.

<p><b>A</b>) (Left) Representative photographs of tumors formed by vector control and SATB1 overexpressing (pooled population and clone-1) MCF10A-1 cells injected into the mammary fat pad of nude mice. (Right) Corresponding tumor sections stained with haematoxylin and eosin. T, tumor; N, normal breast tissue. Scale bar, 80 µm. <b>B</b>) (Top) Mean tumor volumes as formed in (<b>A</b>). Note that the tumors derived from clone-1 grew at the fastest rate. Each data point is shown as the mean value (±s.e.m.) of 8–10 primary tumors. (Bottom) The number of metastatic nodules per lung formed by MCF10A-1 vector control (n = 7) and clone-1 (n = 5) 9 weeks after injection into the tail vein. The mean values for each group are underlined and shown below plot. <b>C</b>) (Top) The growth of tumor by vector control and SATB1 overexpressing (pLXSN-SATB1) MCF10A-2 cells after injection into mammary fat pads of nude mice. Note that MCF10A-2 cells did not form tumors upon SATB1 expression. (Bottom) The number of metastatic nodules per lung formed by MCF10A-2 vector control (n = 7) and SATB1 pool (n = 9) 9 weeks after injection into the tail vein. The mean values for each group are underlined and shown below plot. <b>D</b>) Schematic diagram for tumor formation and metastasis experiments. Primary tumor cell lines clone-1-TUM derived from xenografts of SATB1 overexpressing MCF10A-1 clone-1 were transfected with SATB1-shRNA and used for <i>in vivo</i> tumor formation and experimental metastasis experiments. <b>E</b>) Quantitative RT-PCR analysis for SATB1 transcript levels in parental MCF10A-1, clone-1, clone-1-TUM, clone-1-TUM (vector control), and clone-1-TUM (SATB1-shRNA) cells relative to GAPDH. <b>F</b>) (Top) The number of metastatic nodules per lung by the pooled population of clone-1-TUM (n = 7) and clone-1-TUM (SATB1-shRNA) (n = 6) 9 weeks after the injection into the tail vein of nude mice. The mean nodule per lung is underlined and shown above plot. (Bottom) The level of human SATB1 expression in mouse lungs relative to actin. RNAs were prepared from the metastatic lung of each injected nude mouse as described in (Top). Quantitative RT-PCR analysis was performed to evaluate the human specific SATB1 expression originating from the injected cells. The relative levels of SATB1 expression against sample #1 of clone-1-TUM (indicated as one) are shown. <b>G</b>) The growth of tumor formed by parental or SATB1-shRNA clone-1-TUM cells injected into mammary fat pads of nude mice. Mean volumes (n = 6 per group) of tumors formed in fat pads of mice are shown.</p

SATB1 overexpression induces EMT in MCF10A-1 cells.

<p><b>A</b>) Cell proliferation assay to compare the growth over time (days, d) of the parental MCF10A-1 and vector control cells with single-cell-derived SATB1-overexpressing MCF10A-1 clones clone-1 and clone-2 on plastic dishes (2D) or on Matrigel (3D). Error bars indicate ±s.e.m. from three independent experiments. <b>B</b>) Representative photographs of soft agar colonies formed by control and SATB1 overexpressing (clone-1 and clone-2) MCF10A-1 cells after 25 days of culture. The mean colony counts from three replicates are shown. <b>C</b>) Immunoblot analyses for the expression of mesenchymal markers (fibronectin and vimentin), epithelial markers (E-cadherin and ß-catenin) as well as SATB1 target ERBB2 in MDA-MB-231 (parental, control and SATB1-depleted by shRNA1 or shRNA2) and MCF10A-1 (parental, control, clone-1 and clone-2). Cell lysates were prepared from cells cultured on plastic dishes (2D). GAPDH was used as a loading control.</p

The combination of ectopic SATB1 expression and ATM depletion induces aggressive phenotype in non-malignant mammary epithelial cells.

<p><b>A</b>) Immunoblot showing ATM and SATB1 levels before and after SATB1 overexpression (SATB1) and ATM depletion (shATM) in non-malignant MCF10A-1, MCF10A-2, 184A1 and 184B5 cells. α-tubulin was used as an internal loading control. <b>B</b>) Colony morphologies of vector control, ATM depleted (shATM), SATB1 overexpressing (SATB1) and ATM depleted/SATB1 overexpressing (shATM+SATB1) cells generated as in (<b>C</b>). Cells were grown in 3D Matrix on-top cultures. The images were captured with phase 1 at 20X magnification on five days after plating. Scale bars, 100 µm. <b>D</b>) Invasion assay of ATM depleted (shATM), SATB1 overexpressing (SATB1) and ATM depleted/SATB1 overexpressing (shATM+SATB1) cells generated as in (<b>a</b>). Error bars indicate ±s.e.m., n = 3 experiments.</p

SATB1 is endogenously expressed in aggressive breast cancer cell lines and its ectopic expression induces malignant phenotype in non-malignant MCF10A-1, but not in MCF10A-2 cells.

<p><b>A</b>) (Top) Quantitative RT-PCR analysis for SATB1 expression relative to GAPDH in MCF10A cell lines from two different sources (MCF10A-1 and MCF10A-2), 184A1, 184B5 and MCF10A progression series (MCF10A-neoT and CA1d), BT549 and MDA-MB-231. (Bottom) Immunoblot for SATB1 expression using the same cell lines as in quantitative RT-PCR analysis. α-tubulin was used as an internal loading control. <b>B</b>) Immunoblot showing the expression level of SATB1 before and after SATB1 overexpression in MCF10A-1 and MCF10A-2 cells. ß-actin was used as an internal loading control. <b>C</b>) Colony morphologies of MCF10A-1 and MCF10A-2 control and SATB1 overexpressing cells (pLXSN-SATB1: pooled populations), and MDA-MB-231 control and SATB1 depleted (SATB1 shRNA) cells at six days of 3D matrix on-top culturing. Images were captured with Phase 1 at 20X magnification. Scale bars, 100 µm. Note that SATB1 overexpression caused MCF10A-1 to form a mixture of large spheroid and spindle structures, indicated by white and blue arrows, respectively, while it caused MCF10A-2 cells to form larger spheroid structures. The aggressive breast cancer cell line, MDA-MB-231, formed network-like spindle structures, which was inhibited by knockdown of SATB1. <b>D</b>) Invasion assay of MCF10A-1, MCF10A-2, MCF10A-neoT and CA1d cell lines before and after SATB1 overexpression. Parental cells lines are shown in blue; SATB1 overexpressing cells are shown in pink. Error bars indicate s.e.m., n = 3 experiments. <b>E</b>) Vector control (top row) and SATB1-expressing MCF10A-1 (pLXN-SATB1) (bottom two rows) grown on Matrigel were stained for F-actin (red), ß-catenin (green), integrin α6 (green), SATB1 (green) and DAPI (blue). Note that control cells showed the typical acinar structure whereas SATB1-expressing cells showed large spheroid (i) and spindle (ii) structures. No acinar structures were detected in SATB1-expressing MCF10A-1. Scale bars, 15 µm.</p

Differential response to SATB1 overexpression by MCF10A-1 and MCF10A-2 cells is attributed to their disparate gene expression patterns involved in cell cycle regulation.

<p><b>A</b>) (Left) Gene copy number profiles for MCF10A-1 and MCF10A-2. Each dot represents the copy number for a given SNP that is ordered by genomic position, from chromosome 1 to chromosomes X and Y. Vertical lines represent chromosome boundaries for the autosomes. Data for MCF10A-1 and MCF10A-2 are plotted in green and blue, respectively. The copy number profiles for both cell lines were largely overlapping, indicating that the cell lines are genomically the same. (Right) Copy number correlation between MCF10A-1 and MCF10A-2. Each dot represents copy number for a single SNP, where copy number for the MCF10A-1 cell line is plotted along the x-axis and that for the MCF10A-2 cell line is plotted along the y-axis. The Pearson correlation is 0.96 (p<2.2e-16), which supports the idea that the two cell lines are identical. <b>B</b>) Expression analysis of genes associated with cancer progression in MCF10A-1 and MCF10A-2. Genes are categorized based on their specific biochemical functions. Fold gene expression in MCF10A-1 (blue bar) relative to MCF10A-2 (red dotted line at 1 fold) is shown. Each gene expression level was normalized to the level of GAPDH. <b>C</b>) Expression analysis of genes associated with cell cycle regulation in MCF10A-1 and MCF10A-2. Genes are categorized based on their roles in specific cell cycle phases. The result is shown as in (<b>B</b>).</p