Cell-state transitions regulated by SLUG are critical for tissue regeneration and tumor initiation

Perturbations in stem cell activity and differentiation can lead to developmental defects and cancer. We use an approach involving a quantitative model of cell-state transitions in vitro to gain insights into how SLUG/SNAI2, a key developmental transcription factor, modulates mammary epithelial stem cell activity and differentiation in vivo. In the absence of SLUG, stem cells fail to transition into basal progenitor cells, while existing basal progenitor cells undergo luminal differentiation; together, these changes result in abnormal mammary architecture and defects in tissue function. Furthermore, we show that in the absence of SLUG, mammary stem cell activity necessary for tissue regeneration and cancer initiation is lost. Mechanistically, SLUG regulates differentiation and cellular plasticity by recruiting the chromatin modifier lysine-specific demethylase 1 (LSD1) to promoters of lineage-specific genes to repress transcription. Together, these results demonstrate that SLUG plays a dual role in repressing luminal epithelial differentiation while unlocking stem cell transitions necessary for tumorigenesis

CoREST1 Promotes Tumor Formation and Tumor Stroma Interactions in a Mouse Model of Breast Cancer

<div><p>Regulators of chromatin structure and gene expression contribute to tumor formation and progression. The co-repressor CoREST1 regulates the localization and activity of associated histone modifying enzymes including lysine specific demethylase 1 (LSD1) and histone deacetylase 1 (HDAC1). Although several CoREST1 associated proteins have been reported to enhance breast cancer progression, the role of CoREST1 in breast cancer is currently unclear. Here we report that knockdown of CoREST1 in the basal-type breast cancer cell line, MDA-MB-231, led to significantly reduced incidence and diminished size of tumors compared to controls in mouse xenograft studies. Notably, CoREST1-depleted cells gave rise to tumors with a marked decrease in angiogenesis. CoREST1 knockdown led to a decrease in secreted angiogenic and inflammatory factors, and mRNA analysis suggests that CoREST1 promotes expression of genes related to angiogenesis and inflammation including VEGF-A and CCL2. CoREST1 knockdown decreased the ability of MDA-MB-231 conditioned media to promote endothelial cell tube formation and migration. Further, tumors derived from CoREST1-depleted cells had reduced macrophage infiltration and the secretome of CoREST1 knockdown cells was deficient in promoting macrophage migration and macrophage-mediated angiogenesis. Taken together, these findings reveal that the epigenetic regulator CoREST1 promotes tumorigenesis in a breast cancer model at least in part through regulation of gene expression patterns in tumor cells that have profound non-cell autonomous effects on endothelial and inflammatory cells in the tumor microenvironment.</p></div

Decreased CoREST1 expression reduced tumor angiogenesis.

<p><b>(A)</b> Ki67 expression, a marker of proliferation, was detected in control (shCtrl) and shCoREST1 (shCoR #1) tumors. Percent Ki67 positive nuclei per high power field (HPF) were quantified in three images from each tumor using ImageJ. In this image, Ki67 immunostaining is red and DAPI (to detect nuclei) is blue. <b>(B)</b> Necrosis was quantified on hematoxylin and eosin (H&E) stained slides in tumors that formed from either shCoR or shCtrl cells. Differences were determined using Student’s t-test (n = 6 tumors/group). <b>(C)</b> CD31 expression was detected using immunofluorescence in shCoR or shCtrl tumors. CD31 expression (red) was quantified using five high power fields of DAPI positive nuclei (blue) from each tumor. Differences were determined using Student’s t-test (n = 3 tumors/group). Scale bar = 100μm.</p

Knockdown of CoREST1 decreased tumor cell-mediated stimulation of endothelial cells <i>in vitro</i>.

<p><b>(A)</b> HUVECs were grown in conditioned media (CM) from shCoREST1 (shCoR) or control (shCtrl) cells on Matrigel for 6 hr to assess changes in tube forming ability. <b>(B)</b> Quantification of tube formation of HUVEC treated with shCtrl or shCoR CM. Tubes from 5 high power fields (HPF) were averaged for each condition tested (n = 3 experiments). <b>(C)</b> HUVEC cells were exposed to CM from shCtrl cells or shCoR cells and wound closure was measured 6 hr after scratching confluent cells as described in Materials and Methods. Data is expressed as % of wound closure as determined from an average of 10 replicates per condition (n = 3 experiments). <b>(D)</b> Proliferation of HUVEC cells after exposure to shCtrl or shCoR CM was determined by counting cells after 72 hours (n = 3 experiments). Differences were determined by Student’s t-test (mean±s.d.). Scale bar = 100μm.</p

Depletion of CoREST1 altered the tumor cell secretome.

<p><b>(A)</b> Conditioned media from shcontrol (shCtrl) and shCoREST1 (shCoR #1) MDA-MB-231 cells was incubated with a human angiogenesis antibody array as described in Materials and Methods. Immunoblot images from this screen, performed one time, are shown. <b>(B)</b> Quantification of the relative pixel density on the array for the indicated pro-angiogenic, pro-inflammatory and anti-angiogenic factors secreted by shCtrl and shCoR cell lines (n = 1 experiment). <b>(C)</b> VEGF-A mRNA expression was measured in shCoR cells compared to shCtrl cells in MDA-MB-231 and SUM159 cell lines. <b>(D)</b> CCL2 mRNA expression was measured in the indicated cell lines. Expression levels were detected by RT-qPCR and represented as fold change compared to shCtrl cells. Differences were determined by Student’s t-test (mean±s.d.; n = 3 experiments). <b>(E)</b> Luciferase activity from shCtrl or shCoR #1 MDA-MB-231 cells transfected with VEGF-luc or pMCP-luc and pRL-CMV-Renilla. Luciferase expression was normalized to Renilla, then expressed as fold change compared to shCtrl cells. Differences were determined by Student’s t-test (mean±s.d.; n = 3 experiments).</p

Knockdown of CoREST1 decreased tumor cell-mediated macrophage migration and activation.

<p><b>(A)</b> F4/80 immunostaining, a marker of macrophage infiltration, was performed in tumors that grew from shCtrl and shCoREST (shCoR) cells. F4/80 expression (red) was quantified using ImageJ using five high power fields of DAPI positive nuclei (blue) from each tumor. Differences were determined using Student’s t-test (n = 3 tumors/group). <b>(B)</b> Migration of HL-60 macrophages was measured in response to conditioned media (CM) from shCoR cells compared to shCtrl cells. HL-60 cells were differentiated into macrophages as described in Materials and Methods. Transwell migration of macrophages was quantified after 4 hr, and differences were determined by ANOVA analysis (n = 3 experiments in triplicate). <b>(C)</b> Migration of HL-60 macrophages was examined in response to CM from shCtrl cells supplemented with vehicle, a blocking antibody to CCL2, or RS504393, an inhibitor for the CCR2 receptor. Transwell migration of macrophages was quantified after 4 hr, and differences were determined by ANOVA analysis (n = 3 experiments in triplicate). <b>(D)</b> HUVEC tube formation was examined in response to CM collected from HL-60 macrophages activated with CM from either shCoR or shCtrl cells. Tubes from 3 high power fields (HPF) were averaged for each condition tested, and differences were determined by ANOVA analysis (n = 3 experiments). <b>(E)</b> HUVEC cell migration was measured following treatment with CM from macrophages activated with CM isolated from either shCoR or shCtrl cells. Wound closure was measured using ImageJ software 6 hr after scratching confluent cells as described in Materials and Methods. Data is expressed as % of wound closure as determined from an average of 10 replicates per condition (n = 3 experiments). <b>(F)</b> Proliferation of HUVEC cells was not altered in response to treatment with CM from macrophages activated with either shCtrl or shCoR CM. HUVEC were counted after 72 hr (n = 3 experiments). Differences were determined by ANOVA analysis (mean±s.d.). Scale bar = 100μm.</p

Knockdown of CoREST1 reduced MDA-MB-231 tumor formation.

<p>MDA-MB-231 cells were transduced with lentivirus encoding a control (shCtrl) or either of 2 shRNA constructs targeting CoREST1 (shCoR). <b>(A)</b> CoREST1 expression in MDA-MB-231 cell lines was quantified using RT-qPCR relative to β-actin expression. Differences were determined using Student’s t-test (n = 6 experiments; mean±s.e.m.). <b>(B)</b> Representative image of CoREST1 expression measured by immunoblotting (n>3 experiments). <b>(C)</b> NOD/SCID females were injected with shCoR #1 or shCtrl cells into the fourth mammary glands. Masses greater than 3 mm in diameter were defined as tumors (*p<0.001, Fischer’s exact test). <b>(D)</b> Tumor growth curve from mice injected with shCoR #1 cells compared to shCtrl controls (*p<0.005, Mann-Whitney test). <b>(E)</b> At end stage, tumor weights were measured from mice injected with either shCtrl or shCoR #1 cells. Differences were determined by Mann-Whitney test.</p

Cell-State Transitions Regulated by SLUG Are Critical for Tissue Regeneration and Tumor Initiation

Perturbations in stem cell activity and differentiation can lead to developmental defects and cancer. We use an approach involving a quantitative model of cell-state transitions in vitro to gain insights into how SLUG/SNAI2, a key developmental transcription factor, modulates mammary epithelial stem cell activity and differentiation in vivo. In the absence of SLUG, stem cells fail to transition into basal progenitor cells, while existing basal progenitor cells undergo luminal differentiation; together, these changes result in abnormal mammary architecture and defects in tissue function. Furthermore, we show that in the absence of SLUG, mammary stem cell activity necessary for tissue regeneration and cancer initiation is lost. Mechanistically, SLUG regulates differentiation and cellular plasticity by recruiting the chromatin modifier lysine-specific demethylase 1 (LSD1) to promoters of lineage-specific genes to repress transcription. Together, these results demonstrate that SLUG plays a dual role in repressing luminal epithelial differentiation while unlocking stem cell transitions necessary for tumorigenesis