Tumor-associated myoepithelial cells promote the invasive progression of ductal carcinoma in situ through activation of TGFβ signaling

Abstract

The normal myoepithelium has a tumor-suppressing nature and inhibits the progression of ductal carcinoma in situ (DCIS) into invasive ductal carcinoma (IDC). Conversely, a growing number of studies have shown that tumor-associated myoepithelial cells have a tumor-promoting effect. Moreover, the exact role of tumor-associated myoepithelial cells in the DCIS-to-IDC development remains undefined. To address this, we explored the role of tumor-associated myoepithelial cells in the DCIS-to-IDC progression. We developed a direct coculture system to study the cell-cell interactions between DCIS cells and tumor-associated myoepithelial cells. Coculture studies indicated that tumor-associated myoepithelial cells promoted the invasive progression of a DCIS cell model in vitro, and mechanistic studies revealed that the interaction with DCIS cells stimulated tumor-associated myoepithelial cells to secrete TGFβ1, which subsequently contributed to activating the TGFβ/Smads pathway in DCIS cells. We noted that activation of the TGFβ signaling pathway promoted the epithelial-mesenchymal transition, basal-like phenotypes, stemness, and invasiveness of DCIS cells. Importantly, xenograft studies further demonstrated that tumor-associated myoepithelial cells enhanced the DCIS-to-IDC progression in vivo Furthermore, we found that TGFβ-mediated induction of oncogenic miR-10b-5p expression and down-regulation of RB1CC1, a miR-10b-5p-targeted tumor-suppressor gene, contributed to the invasive progression of DCIS. Our findings provide the first experimental evidence to directly support the paradigm that altered DCIS-associated myoepithelial cells promote the invasive progression of DCIS into IDC via TGFβ signaling activation.

Keywords: breast cancer; epithelial-mesenchymal transition (EMT); microRNA (miRNA); transforming growth factor beta (TGFβ); tumor microenvironment.

Figure 1.

Coculture with tumor-associated Hs578bst myoepithelial cells induces the EMT/basal-like phenotypes in MCF10DCIS cells and enhances tumor cell migration, invasion, and stemness. A, coculture with Hs578bst cells leads to the increased expression of EMT-programming genes and down-regulation of E-cadherin expression in MCF10DCIS cells. qRT-PCR analysis of FOXC2, SNAIL, vimentin, ZEB1, ZEB2, and E-cadherin mRNA expression was performed on sorted non-cocultured control MCF10DCIS-GFP cells and on sorted MCF10DCIS-GFP cells cocultured with Hs578bst cells for 4 days. The quantitative bar graph was plotted based on triplicate experiments. B, myoepithelial coculture enhances basal-like phenotypes in MCF10DCIS cells. FACS analysis of CD44, CD49f, and EpCAM was performed on non-cocultured and cocultured MCF10DCIS-GFP cells. Dot-plot profiling analysis of CD44CD49f and histogram analysis of EpCAM were performed on gated GFP-positive MCF10DCIS cells. C, myoepithelial coculture enhances migration and invasion of MCF10DCIS cells. Migration and invasion assays were performed on sorted MCF10DCIS-GFP cells with or without coculture with Hs578bst cells. Migrated or invaded GFP-positive MCF10DCIS cells from six randomly selected fields were counted for each assay. The representative migrated and invaded cell pictures are shown at the top, and quantitative bar graphs (n = 3) are shown at the bottom. D, coculture with Hs578bst myoepithelial cells promotes CSC self-renewal of MCF10DCIS cells. Stem-cell sphere formation assays were performed on sorted GFP-positive cells as described in C. The representative CSC sphere pictures are shown at the top, and the scale bar indicates 100 μm. The quantitative bar graph for the CSC sphere data (n = 3) is shown at the bottom. The error bars in all bar graphs here indicate S.D. *, p < 0.05; **, p < 0.01.

Figure 2.

Coculture induces increased TGFβ1 secretion from tumor-associated myoepithelial cells and activation of the TGFβ/Smads pathway in MCF10DCIS cells. A, coculture of MCF10DCIS and Hs578bst cells results in an increase in secreted TGFβ1. ELISA analysis of TGFβ1 was performed on the culture medium and conditioned media from Hs578bst (MyoE-CM), MCF10DCIS (DCIS-CM), and cocultures of both cell lines (MyoE+DCIS-CM). The quantitative bar graph was plotted based on triplicate experiments, and error bars indicate S.D. B, coculture with DCIS cells results in increased TGFβ1 expression in tumor-associated myoepithelial cells. qRT-PCR analysis of TGFβ1 expression was performed on sorted non-cocultured control MCF10DCIS-GFP (DCIS-Ctrl) and Hs578bst (MyoE-Ctrl) cells and their respectively sorted cocultured cells (DCIS-Co and MyoE-Co). Experiments were performed in triplicate. C, analysis of TGFβ1 knockdown by siRNA in cocultured Hs578bst myoepithelial cells. qRT-PCR analysis of TGFβ1 expression was performed on sorted non-cocultured and cocultured Hs578bst cells with or without TGFβ1 knockdown by siRNA. Two distinct TGFβ1 siRNAs (siTGFβ1-1 and siTGFβ1-2) that target two different sequence regions of TGFβ1 mRNA and control siRNA (siControl) were included in knockdown experiments. Experiments were performed in triplicate. D, ELISA analysis of TGFβ1 in conditioned media from three different cultures, including Hs578bst cultures (the non-cocultured control) and cocultures of MCF10DCIS cells with siControl-, siTGFβ1–1- or siTGFβ1-2-transfected Hs578bst cells. Experiments were performed in triplicate. E, TGFβ1 knockdown in Hs578bst myoepithelial cells impairs coculture-induced activation of TGFβ signaling in MCF10DCIS cells. Western blot analysis of phospho-Smad2 (p-Smad2), total Smad2, and α-tubulin was performed on MCF10DCIS-GFP cells cocultured with or without siControl-, siTGFβ1-1-, or siTGFβ1-2-transfected Hs578bst cells. Non-cocultured and cocultured MCF10DCIS-GFP cells were sorted based on their GFP positivity before they were subjected to Western blot experiments. F, Western blot analysis of phospho-Smad2, total Smad2, and α-tubulin in mock-treated (control) and TGFβ1-treated MCF10DCIS-GFP cells. G, TGFβ receptors are responsible for coculture-induced activation of TGFβ signaling in MCF10DCIS cells. Western blot analysis of phospho-Smad2, total Smad2, and α-tubulin was performed on sorted non-cocultured (control), cocultured, and cocultured + SB431542-treated MCF10DCIS-GFP cells. H, the TGFβ/Smads pathway is activated in MCF10DCIS cells cocultured with Hs578bst cells under a transwell-based coculture setting. Western blot analysis of phospho-Smad2, total Smad2, and α-tubulin was performed on non-cocultured and cocultured MCF10DCIS cells with or without SB431542 treatment. I, coculture with Hs578bst myoepithelial cells activated TGFβ signaling in MCF10A, but not in MDA-MB-231 cells. MCF10A and MDA-MB-231 cells were cocultured with Hs578bst cells for 4 days under a transwell setting before they were subjected to Western blot analysis of phospho-Smad2, total Smad2, and α-tubulin. Their respective non-cocultured cells were included as controls. The arrow indicates total Smad2. Western blot data were quantified as described under “Experimental Procedures.” The quantitative phospho-Smad2 and total Smad2 data were normalized to their respective α-tubulin. **, p < 0.01; ***, p < 0.001; ns, not significant.

Figure 3.

TGFβ1 knockdown in Hs578bst myoepithelial cells impairs the coculture-promoted EMT and basal-like/stem-cell-like phenotypes of MCF10DCIS cells. A, expression profiling of EMT-programming genes in MCF10DCIS-GFP cells cocultured with or without siControl-, siTGFβ1-1-, or siTGFβ1-2-transfected Hs578bst cells. qRT-PCR experiments (n = 3) were performed on sorted non-cocultured and cocultured MCF10DCIS-GFP cells. *, p < 0.01 versus the non-cocultured control MCF10DCIS data set; #, p < 0.05 (or ¥, p < 0.05) versus the data set of MCF10DCIS cells cocultured with siControl-transfected Hs578bst cells (MyoE-siControl). B, FACS analysis of the basal-like/stemlike (CD44⁺CD49f⁺) cell population in MCF10DCIS-GFP cells cocultured with or without siControl-, siTGFβ1-1-, or siTGFβ1-2-transfected Hs578bst cells. GFP-positive MCF10DCIS cells were gated for analysis of the CD44/CD49f profile. C, FACS analysis of the luminal cell property of MCF10DCIS-GFP cells cocultured with or without siControl-, siTGFβ1-1-, or siTGFβ1-2-transfected Hs578bst cells. GFP-positive MCF10DCIS cells were gated for analysis of luminal (EpCAM-positive) cells in MCF10DCIS cells.

Figure 4.

Inactivation of the TGFβ pathway by the inhibitor of TGFβ receptors abolishes the coculture-induced effect that enhances basal-like/EMT phenotypes in MCF10DCIS cells. A, treatment with the TGFβ receptor inhibitor eradicates enhanced basal-like phenotypes in cocultured MCF10DCIS cells. FACS analysis of CD44, CD49f, and EpCAM was performed on non-cocultured (control) and cocultured MCF10DCIS-GFP cells with or without SB431542 treatment. Dot-plot profiling analysis of CD44CD49f and histogram analysis of EpCAM were performed on gated GFP-positive MCF10DCIS-GFP cells. B, treatment with TGFβ1 promotes basal-like phenotypes in MCF10DCIS cells. FACS analysis of CD44, CD49f, and EpCAM was performed on mock-treated (control) and TGFβ1-treated MCF10DCIS-GFP cells as described in A. Cells were treated with 5 nm recombinant TGFβ1 for 3 days before FACS analysis. C and D, the enhanced EMT in cocultured MCF10DCIS cells is attributable to activation of the TGFβ pathway. C, qRT-PCR analysis of five EMT-programming genes was performed on non-cocultured (control), cocultured, cocultured + SB431542-treated, and TGFβ1-treated MCF10DCIS-GFP cells after GFP-positive cells were sorted. Experiments were performed in triplicate. *, p < 0.05 versus the non-cocultured control data set; #, p < 0.05 versus the cocultured data set. D, Western blot analysis of vimentin, E-cadherin, and α-tubulin was performed on non-cocultured (control), cocultured, cocultured + SB431542-treated, mock-treated (control), and TGFβ1-treated MCF10DCIS-GFP cells. In the cocultured Western blot data (left), non-cocultured and cocultured MCF10DCIS-GFP cells were sorted based on their GFP positivity before they were subjected to Western blot experiments. The quantitative vimentin and E-cadherin data were normalized by their respective α-tubulin.

Figure 5.

The effect of coculture with tumor-associated myoepithelial cells on enhancing migration, invasion, and stemness of MCF10DCIS cells results from activation of TGFβ signaling. A and B, transwell-based migration (A) and invasion (B) assays were performed on non-cocultured (control), cocultured, cocultured + SB431542-treated, mock-treated (control), and TGFβ1-treated MCF10DCIS-GFP cells. Non-cocultured and cocultured MCF10DCIS-GFP cells were sorted based on their GFP positivity before they were subjected to experiments. The quantitative bar graphs were plotted based on triplicate experiments. C, stem-cell sphere formation assays were performed on non-cocultured and cocultured MCF10DCIS-GFP cells with or without SB431542 treatment after they were sorted based on their GFP positivity. D, stem-cell sphere formation assays were performed on mock-treated and TGFβ1-treated MCF10DCIS-GFP cells. Scale bars in C and D, 100 μm. The quantitative bar graphs for sphere data shown in C and D were plotted based on triplicate experiments. The error bars in all bar graphs shown here indicate S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 6.

Tumor-associated myoepithelial cells activate TGFβ signaling and the EMT in DCIS and promote DCIS progression into invasive mammary tumors. A, co-transplantation of Hs578bst cells promotes the in vivo tumor growth of transplanted MCF10DCIS cells. The tumor growth curve (n = 4 for each experimental group) was plotted based on tumor size measurements from 4-week monitoring. The tumor size difference between these two experimental groups (MCF10DCIS only versus MCF10DCIS + Hs578bst) is statistically significant (***, p < 0.001). B, immunohistochemistry analysis of α-smooth muscle actin (α-SMA) was performed on paraffin-embedded tissue sections prepared from isolated MCF10DCIS only and MCF10DCIS + Hs578bst xenograft tumors for detecting myoepithelial and stromal cells in tissues. Representative staining pictures are shown. Scale bars, 50 μm. DCIS tumor areas are depicted by red dotted lines. Left (MCF10DCIS-only xenograft), red arrows indicate the α-SMA-positive myoepithelial cell layer, and blue arrows indicate α-SMA-positive stromal cells. Right (MCF10DCIS + Hs578bst xenograft), α-SMA-positive tissue areas (marked by brown dotted lines) indicate myoepithelium, stroma, or the mix of both tissue cell types. C, immunohistochemistry analysis of phospho-Smad2 was performed on paraffin-embedded tissue sections as described in B. Representative staining pictures are shown. Scale bars, 50 μm. DCIS tumor areas are depicted by red dotted lines. Ten randomly selected fields for each stained tissue section were used to count Smad2-positive and total tumor cells. Smad2 positivity was expressed as the Smad2-positive cell number per 1000 counted tumor cells. The quantitative bar graph was plotted based on the counting results from three different stained tumor tissue sections resected from three transplanted nude mice for each xenograft group. Tissue samples of each xenograft group were confirmed to have the same histological feature as shown in B before they were subjected to phospho-Smad2 IHC analysis. D, qRT-PCR analysis of five EMT-programming genes (n = 3) was performed on the RNA sample isolated from GFP-negative tumor cells that were purified either from MCF10DCIS only or from MCF10DCIS + Hs578bst xenograft tumors as described under “Experimental Procedures.” Error bars, S.D. **, p < 0.01; ***, p < 0.001.

Figure 7.

Tumor-associated myoepithelial cells activate the TGFβ/miR-10b-5p axis in DCIS cells and up-regulation of miR-10b-5p expression contributes to the coculture-enhanced EMT, invasiveness, and stemness of DCIS cells. A, PCR array profiling of miRNA expression in control and cocultured MCF10DCIS cells. miRNA expression data were plotted into a two-dimensional dot plot. Up-regulated (≥ 2-fold) and down-regulated (≤ −2-fold) miRNAs in sorted cocultured MCF10DCIS-GFP cells compared with the non-cocultured control are indicated. TGFβ-regulated miRNAs are depicted in the dot plot. B, qRT-PCR analysis of miR-10b-5p expression was performed on the sorted cell set as described in the legend to Fig. 4C. C, qRT-PCR analysis of miR-10b-5p expression was performed on RNA samples as described in the legend to Fig. 6D. D, qRT-PCR analysis of four EMT-programming genes was performed on MCF10DCIS cells stably overexpressing the control scramble, miR-10b, or miR-10b sponge RNA. Expression bar graph data shown in B–D were plotted based on triplicate experiments. E, inhibition of miR-10b by the sponge RNA partially suppresses coculture-promoted migratory and invasive activities of MCF10DCIS cells. Migration and invasion assays were performed on cocultured MCF10DCIS-GFP cells overexpressing the control scramble or miR-10b sponge RNA. Non-cocultured MCF10DCIS-GFP cells overexpressing the control scramble RNA served as a control. Non-cocultured and cocultured cells were sorted based on their GFP positivity before they were subjected to migration and invasion assays. Quantitative bar graph data (n = 3) were plotted as described in the legend to Fig. 1C. F, inhibition of miR-10b by the sponge RNA partially suppresses coculture-promoted CSC self-renewal of MCF10DCIS cells. Stem-cell sphere formation assays were performed on non-cocultured and cocultured cells as described in E. Quantitative CSC sphere formation data were plotted based on triplicate experiments. Error bars, S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 8.

RB1CC1 is a miR-10b-5p target gene and functionally participates in cell migration regulation. A, qRT-PCR analysis of a panel of 15 predicted miR-10b-5p target genes was performed on biotin pull-down mRNA samples prepared from MCF10DCIS cells transfected with either the biotin-conjugated scramble or biotin-conjugated miR-10b-5p. The expression values were normalized to actin. The housekeeping gene GAPDH was included in the analysis as a negative control. B, overexpression of miR-10b-5p inhibits the luciferase expression of the wild-type, but not the mutated, RB1CC1 3′-UTR reporter. A map for the predicted miR-10b-5p targeting site in the 3′-UTR of the RB1CC1 mRNA is shown on the left. A DNA fragment with mutations in the miR-10b seeding site of RB1CC1 3′-UTR was used to construct the mutated reporter and its RNA sequence is shown under the map with its wild-type and miR-10b-5p sequences. HEK-293T cells were transfected with the wild-type or mutated RB1CC1 3′-UTR reporter plasmid DNA along with either the control scramble or the miR-10b expression plasmid. All cell samples were also co-transfected with the Renilla expression plasmid, which was used as a transfection efficiency control. Dual-Luciferase assays were performed on transfected cells 24 h after transfection. The measured luciferase activity values were normalized by Renilla activity values to make a quantitative bar graph (shown on the right). Error bars, S.D. of the data set (n = 3). C, miR-10b-5p negatively regulates endogenous RB1CC1 expression. Western blot analysis of RB1CC1 and α-tubulin was performed on MCF10DCIS-GFP cells overexpressing the control scramble, miR-10b, or miR-10b sponge RNA. The quantitative RB1CC1 data were normalized by their respective α-tubulin. D, knockdown of RB1CC1 enhances the migratory activity of MCF10DCIS cells. Wound-healing assays were performed for 16 h to measure the migratory activity of RB1CC1-knockdown cells compared with that of siControl-transfected cells. Two different RB1CC1 siRNAs (siRB1CC1-1 and siRB1CC1-2) were utilized in the knockdown experiment. Their knockdown efficiency is shown in supplemental Fig. S11. The representative pictures of scratched wounds for three different siRNA-transfected cells are shown on the left. The cell pictures were analyzed using ImageJ to measure wound closure percentage as described under “Experimental procedures.” The wound closure percentage data (n = 3) were plotted to make a bar graph (shown on the right). Error bars, S.D. *, p < 0.05; **, p < 0.01.

Figure 9.

RB1CC1 expression is down-regulated by activated TGFβ signaling in the invasive progression of DCIS in vitro and in vivo triggered by interaction with tumor-associated myoepithelial cells, and RB1CC1 down-regulation is clinically associated with TNBC. A, coculture with tumor-associated myoepithelial cells induces RB1CC1 down-regulation in DCIS cells through activation of the TGFβ signaling pathway. Western blot analysis of RB1CC1 and α-tubulin was performed on protein samples as described in the legend to Fig. 4D. The quantitative RB1CC1 data were normalized by their respective α-tubulin. B, immunohistochemistry analysis of RB1CC1 was performed on paraffin-embedded tissue sections prepared from isolated MCF10DCIS only and MCF10DCIS + Hs578bst tumors. Representative staining pictures with ×40 magnification are shown. Scale bars, 50 μm. C, in silico expression analysis of RB1CC1 in ductal breast carcinomas. RB1CC1 expression values in ductal breast carcinomas were plotted based on the previously published breast cancer microarray expression data sets of Esserman et al. (43) (n = 98) and Tabchy et al. (44) (n = 163) deposited in the Oncomine microarray database (http://www.oncomine.org).³ Ductal breast carcinomas subjected to in silico expression analysis were classified into TNBCs and non-TNBC according to estrogen receptor, progesterone receptor, and ERBB2 status. The median of each data set is indicated in the plot.