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Regulation of epithelial-mesenchymal transition in breast cancer cells by cell contact and adhesion.

Cichon MA, Nelson CM, Radisky DC - Cancer Inform (2015)

Bottom Line: We show here that EMT-related processes are linked to a broad and conserved program of transcriptional alterations that are influenced by cell contact and adhesion.We further find that treatment of cells with matrix metalloproteinase-3 (MMP-3), an inducer of EMT, interrupts a defined subset of cell contact-regulated genes, including genes encoding a variety of RNA splicing proteins known to regulate the expression of Rac1b, an activated splice isoform of Rac1 known to be a key mediator of MMP-3-induced EMT in breast, lung, and pancreas.These results provide new insights into how MMPs act in cancer progression and how loss of cell-cell interactions is a key step in the earliest stages of cancer development.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Mayo Clinic Cancer Center, Jacksonville, FL USA.

ABSTRACT
Epithelial-mesenchymal transition (EMT) is a physiological program that is activated during cancer cell invasion and metastasis. We show here that EMT-related processes are linked to a broad and conserved program of transcriptional alterations that are influenced by cell contact and adhesion. Using cultured human breast cancer and mouse mammary epithelial cells, we find that reduced cell density, conditions under which cell contact is reduced, leads to reduced expression of genes associated with mammary epithelial cell differentiation and increased expression of genes associated with breast cancer. We further find that treatment of cells with matrix metalloproteinase-3 (MMP-3), an inducer of EMT, interrupts a defined subset of cell contact-regulated genes, including genes encoding a variety of RNA splicing proteins known to regulate the expression of Rac1b, an activated splice isoform of Rac1 known to be a key mediator of MMP-3-induced EMT in breast, lung, and pancreas. These results provide new insights into how MMPs act in cancer progression and how loss of cell-cell interactions is a key step in the earliest stages of cancer development.

No MeSH data available.


Related in: MedlinePlus

Density-dependent differences in expression of gene splicing factors and Rac1b in SCp2 and MCF10A cells. (A) Expression in SCp2 cells of the gene encoding hnRNPA1, known to inhibit expression of Rac1b, according to cell density and MMP-3 treatment (gene expression from normalized microarray data, scaled to 50K control, and displayed as means ± SEM; ANOVA P = 0.0575 for trend in control; P = ns for trend in MMP-3 treated). (B) Expression in SCp2 cells of Rac1b according to cell density and MMP-3 treatment (gene expression from QPCR, normalized to GAPDH and scaled to 50K control, and displayed as means ± SEM; ANOVA P = 0.0475 for trend in control; P = ns for trend in MMP-3 treated). (C) Expression in MCF10A cells of genes encoding ESRP1, known to inhibit expression of Rac1b and ESRP2, according to cell density (gene expression from normalized microarray data, scaled to 50K control). (D) Expression in MCF10A cells of Rac1b according to cell density treatment (gene expression from QPCR, normalized to GAPDH and scaled to 50K control, and displayed as means ± SEM; ANOVA P < 0.001 for trend).
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f6-cin-suppl.3-2015-001: Density-dependent differences in expression of gene splicing factors and Rac1b in SCp2 and MCF10A cells. (A) Expression in SCp2 cells of the gene encoding hnRNPA1, known to inhibit expression of Rac1b, according to cell density and MMP-3 treatment (gene expression from normalized microarray data, scaled to 50K control, and displayed as means ± SEM; ANOVA P = 0.0575 for trend in control; P = ns for trend in MMP-3 treated). (B) Expression in SCp2 cells of Rac1b according to cell density and MMP-3 treatment (gene expression from QPCR, normalized to GAPDH and scaled to 50K control, and displayed as means ± SEM; ANOVA P = 0.0475 for trend in control; P = ns for trend in MMP-3 treated). (C) Expression in MCF10A cells of genes encoding ESRP1, known to inhibit expression of Rac1b and ESRP2, according to cell density (gene expression from normalized microarray data, scaled to 50K control). (D) Expression in MCF10A cells of Rac1b according to cell density treatment (gene expression from QPCR, normalized to GAPDH and scaled to 50K control, and displayed as means ± SEM; ANOVA P < 0.001 for trend).

Mentions: The final two gene groups identified by the K-means clustering contained genes that were regulated by density in the absence of MMP-3, but less regulated by density in the presence of MMP-3 (Fig. 4G and H), an effect that could be induced if the protein(s) acting as sensors of cellular density for regulation of these genes was cleaved or blocked by MMP-3. These groups were enriched with genes encoding splicing factors (Table 1, Supplementary Tables 1–3). Evaluation of genes with these characteristics and annotated as associated with RNA processing revealed 56 gene features (mapped to 37 genes) that were upregulated by cell density in the absence of MMP-3 (Fig. 5A–C) and 80 gene features (mapped to 56 genes) that were downregulated by cell density in the absence of MMP-3 (Fig. 5D–F). Strikingly, these genes included HNRNPA1, which encodes a splicing factor that inhibits EMT86,87 and that is known to regulate the induction of Rac1b in mouse mammary and lung epithelial cells exposed to MMP-3.32,88 Analysis of HNRNPA1 expression data in the SCp2 cell experiments revealed substantial density-dependent differences in untreated cells, but relatively similar levels in MMP-3-treated cells (Fig. 6A). Consistent with the role of hnRNPA1 as a repressor of exon 3b inclusion,88 expression of Rac1b in the same samples decreased in the untreated samples as a function of density, while the levels of MMP-3-induced Rac1b remained constant (Fig. 6B). Other splicing factors contained in the density-regulated/MMP-inhibited gene clusters have been implicated in other studies to influence Rac1b inclusion, including SRSF1 (ASF/SF2), which acts to increase inclusion of exon 3b89 and which was downregulated by density in the absence of MMP-3, but maintained higher expression levels in the presence of MMP-3 (Fig. 5E). Furthermore, while expression levels of HNRNPA1 were undetectable in the MCF10A cells (data not shown), we did find that density-dependent differences in the expression of ESRP1 and ESRP2 (Fig. 6C), which encode splicing factors that have been shown to regulate exon 3b inclusion in human oral squamous carcinoma cells,90 were also associated with density-dependent differences in the expression of Rac1b (Fig. 6D).


Regulation of epithelial-mesenchymal transition in breast cancer cells by cell contact and adhesion.

Cichon MA, Nelson CM, Radisky DC - Cancer Inform (2015)

Density-dependent differences in expression of gene splicing factors and Rac1b in SCp2 and MCF10A cells. (A) Expression in SCp2 cells of the gene encoding hnRNPA1, known to inhibit expression of Rac1b, according to cell density and MMP-3 treatment (gene expression from normalized microarray data, scaled to 50K control, and displayed as means ± SEM; ANOVA P = 0.0575 for trend in control; P = ns for trend in MMP-3 treated). (B) Expression in SCp2 cells of Rac1b according to cell density and MMP-3 treatment (gene expression from QPCR, normalized to GAPDH and scaled to 50K control, and displayed as means ± SEM; ANOVA P = 0.0475 for trend in control; P = ns for trend in MMP-3 treated). (C) Expression in MCF10A cells of genes encoding ESRP1, known to inhibit expression of Rac1b and ESRP2, according to cell density (gene expression from normalized microarray data, scaled to 50K control). (D) Expression in MCF10A cells of Rac1b according to cell density treatment (gene expression from QPCR, normalized to GAPDH and scaled to 50K control, and displayed as means ± SEM; ANOVA P < 0.001 for trend).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4325704&req=5

f6-cin-suppl.3-2015-001: Density-dependent differences in expression of gene splicing factors and Rac1b in SCp2 and MCF10A cells. (A) Expression in SCp2 cells of the gene encoding hnRNPA1, known to inhibit expression of Rac1b, according to cell density and MMP-3 treatment (gene expression from normalized microarray data, scaled to 50K control, and displayed as means ± SEM; ANOVA P = 0.0575 for trend in control; P = ns for trend in MMP-3 treated). (B) Expression in SCp2 cells of Rac1b according to cell density and MMP-3 treatment (gene expression from QPCR, normalized to GAPDH and scaled to 50K control, and displayed as means ± SEM; ANOVA P = 0.0475 for trend in control; P = ns for trend in MMP-3 treated). (C) Expression in MCF10A cells of genes encoding ESRP1, known to inhibit expression of Rac1b and ESRP2, according to cell density (gene expression from normalized microarray data, scaled to 50K control). (D) Expression in MCF10A cells of Rac1b according to cell density treatment (gene expression from QPCR, normalized to GAPDH and scaled to 50K control, and displayed as means ± SEM; ANOVA P < 0.001 for trend).
Mentions: The final two gene groups identified by the K-means clustering contained genes that were regulated by density in the absence of MMP-3, but less regulated by density in the presence of MMP-3 (Fig. 4G and H), an effect that could be induced if the protein(s) acting as sensors of cellular density for regulation of these genes was cleaved or blocked by MMP-3. These groups were enriched with genes encoding splicing factors (Table 1, Supplementary Tables 1–3). Evaluation of genes with these characteristics and annotated as associated with RNA processing revealed 56 gene features (mapped to 37 genes) that were upregulated by cell density in the absence of MMP-3 (Fig. 5A–C) and 80 gene features (mapped to 56 genes) that were downregulated by cell density in the absence of MMP-3 (Fig. 5D–F). Strikingly, these genes included HNRNPA1, which encodes a splicing factor that inhibits EMT86,87 and that is known to regulate the induction of Rac1b in mouse mammary and lung epithelial cells exposed to MMP-3.32,88 Analysis of HNRNPA1 expression data in the SCp2 cell experiments revealed substantial density-dependent differences in untreated cells, but relatively similar levels in MMP-3-treated cells (Fig. 6A). Consistent with the role of hnRNPA1 as a repressor of exon 3b inclusion,88 expression of Rac1b in the same samples decreased in the untreated samples as a function of density, while the levels of MMP-3-induced Rac1b remained constant (Fig. 6B). Other splicing factors contained in the density-regulated/MMP-inhibited gene clusters have been implicated in other studies to influence Rac1b inclusion, including SRSF1 (ASF/SF2), which acts to increase inclusion of exon 3b89 and which was downregulated by density in the absence of MMP-3, but maintained higher expression levels in the presence of MMP-3 (Fig. 5E). Furthermore, while expression levels of HNRNPA1 were undetectable in the MCF10A cells (data not shown), we did find that density-dependent differences in the expression of ESRP1 and ESRP2 (Fig. 6C), which encode splicing factors that have been shown to regulate exon 3b inclusion in human oral squamous carcinoma cells,90 were also associated with density-dependent differences in the expression of Rac1b (Fig. 6D).

Bottom Line: We show here that EMT-related processes are linked to a broad and conserved program of transcriptional alterations that are influenced by cell contact and adhesion.We further find that treatment of cells with matrix metalloproteinase-3 (MMP-3), an inducer of EMT, interrupts a defined subset of cell contact-regulated genes, including genes encoding a variety of RNA splicing proteins known to regulate the expression of Rac1b, an activated splice isoform of Rac1 known to be a key mediator of MMP-3-induced EMT in breast, lung, and pancreas.These results provide new insights into how MMPs act in cancer progression and how loss of cell-cell interactions is a key step in the earliest stages of cancer development.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Mayo Clinic Cancer Center, Jacksonville, FL USA.

ABSTRACT
Epithelial-mesenchymal transition (EMT) is a physiological program that is activated during cancer cell invasion and metastasis. We show here that EMT-related processes are linked to a broad and conserved program of transcriptional alterations that are influenced by cell contact and adhesion. Using cultured human breast cancer and mouse mammary epithelial cells, we find that reduced cell density, conditions under which cell contact is reduced, leads to reduced expression of genes associated with mammary epithelial cell differentiation and increased expression of genes associated with breast cancer. We further find that treatment of cells with matrix metalloproteinase-3 (MMP-3), an inducer of EMT, interrupts a defined subset of cell contact-regulated genes, including genes encoding a variety of RNA splicing proteins known to regulate the expression of Rac1b, an activated splice isoform of Rac1 known to be a key mediator of MMP-3-induced EMT in breast, lung, and pancreas. These results provide new insights into how MMPs act in cancer progression and how loss of cell-cell interactions is a key step in the earliest stages of cancer development.

No MeSH data available.


Related in: MedlinePlus