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Cigarette smoke induces epithelial to mesenchymal transition and increases the metastatic ability of breast cancer cells.

Di Cello F, Flowers VL, Li H, Vecchio-Pagán B, Gordon B, Harbom K, Shin J, Beaty R, Wang W, Brayton C, Baylin SB, Zahnow CA - Mol. Cancer (2013)

Bottom Line: Moreover, transplantation experiments in mice demonstrate that treatment with cigarette smoke extract renders MCF 10A cells more capable to survive and colonize the mammary ducts and MCF7 cells more prone to metastasize from a subcutaneous injection site, independent of cigarette smoke effects on the host and stromal environment.Analysis by flow cytometry showed that treatment with CSE leads to the emergence of a CD44(hi)/CD24(low) population in MCF 10A cells and of CD44+ and CD49f + MCF7 cells, indicating that cigarette smoke causes the emergence of cell populations bearing markers of self-renewing stem-like cells.The phenotypical alterations induced by cigarette smoke are accompanied by numerous changes in gene expression that are associated with epithelial to mesenchymal transition and tumorigenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD 21287, USA.

ABSTRACT

Background: Recent epidemiological studies demonstrate that both active and involuntary exposure to tobacco smoke increase the risk of breast cancer. Little is known, however, about the molecular mechanisms by which continuous, long term exposure to tobacco smoke contributes to breast carcinogenesis because most previous studies have focused on short term treatment models. In this work we have set out to investigate the progressive transforming effects of tobacco smoke on non-tumorigenic mammary epithelial cells and breast cancer cells using in vitro and in vivo models of chronic cigarette smoke exposure.

Results: We show that both non-tumorigenic (MCF 10A, MCF-12A) and tumorigenic (MCF7) breast epithelial cells exposed to cigarette smoke acquire mesenchymal properties such as fibroblastoid morphology, increased anchorage-independent growth, and increased motility and invasiveness. Moreover, transplantation experiments in mice demonstrate that treatment with cigarette smoke extract renders MCF 10A cells more capable to survive and colonize the mammary ducts and MCF7 cells more prone to metastasize from a subcutaneous injection site, independent of cigarette smoke effects on the host and stromal environment. The extent of transformation and the resulting phenotype thus appear to be associated with the differentiation state of the cells at the time of exposure. Analysis by flow cytometry showed that treatment with CSE leads to the emergence of a CD44(hi)/CD24(low) population in MCF 10A cells and of CD44+ and CD49f + MCF7 cells, indicating that cigarette smoke causes the emergence of cell populations bearing markers of self-renewing stem-like cells. The phenotypical alterations induced by cigarette smoke are accompanied by numerous changes in gene expression that are associated with epithelial to mesenchymal transition and tumorigenesis.

Conclusions: Our results indicate that exposure to cigarette smoke leads to a more aggressive and transformed phenotype in human mammary epithelial cells and that the differentiation state of the cell at the time of exposure may be an important determinant in the phenotype of the final transformed state.

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Exposure of mammary epithelial cells to CSE leads to gene expression changes indicative of EMT. (A, left panel) Western Blot showing upregulation of Vimentin and downregulation of E-cadherin in MCF10A and MCF7 cells exposed to CSE for 21 weeks. (A, right panel) qRT PCR analysis showing downregulation of e-cadherin mRNA and upregulation of Vimentin mRNA in two MCF10A subclones after 21 weeks of exposure to 0.5% CSE. (B) qRT PCR analysis showing downregulation of occludin and upregulation of N-cadherin and fibronectin in two MCF10A subclones at 21 and 40 weeks of exposure. (C-F) qRT PCR analysis of EMT genes in MCF 10A mammary epithelial cells exposed to 0.5% CSE for 21 weeks (clones SC1 and SC2) or 0.5-1.0% CSE for 40 weeks (without subcloning). Data in bar graphs are mean ± standard deviation of 3 replicates; *P<0.01, **P<0.001.
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Figure 5: Exposure of mammary epithelial cells to CSE leads to gene expression changes indicative of EMT. (A, left panel) Western Blot showing upregulation of Vimentin and downregulation of E-cadherin in MCF10A and MCF7 cells exposed to CSE for 21 weeks. (A, right panel) qRT PCR analysis showing downregulation of e-cadherin mRNA and upregulation of Vimentin mRNA in two MCF10A subclones after 21 weeks of exposure to 0.5% CSE. (B) qRT PCR analysis showing downregulation of occludin and upregulation of N-cadherin and fibronectin in two MCF10A subclones at 21 and 40 weeks of exposure. (C-F) qRT PCR analysis of EMT genes in MCF 10A mammary epithelial cells exposed to 0.5% CSE for 21 weeks (clones SC1 and SC2) or 0.5-1.0% CSE for 40 weeks (without subcloning). Data in bar graphs are mean ± standard deviation of 3 replicates; *P<0.01, **P<0.001.

Mentions: Two clones, designated SC1 and SC2 were isolated from MCF 10A cells exposed to 0.5% CSE for 13 weeks and expanded in the same concentration of CSE for 8 additional weeks, at which time total RNA was isolated for microarray and qPCR analysis. In addition, protein lysates and genomic DNA were prepared from independent samples treated with CSE for 21 weeks. The microarray analysis identified 186 unique genes that were upregulated and 308 unique genes that were downregulated at least two fold in both MCF 10A CSE clones (Additional file 1: Table S1). The expression of selected genes was verified by qRT-PCR or Western blot analysis (Figure 5), focusing on those genes associated with the phenotypes that we had observed, namely EMT, invasion and metastasis. E-cadherin and vimentin, which are associated with an epithelial state [22,23], were downregulated and upregulated respectively in MCF10As as determined by Western blot analysis (left panel) and PCR (right panel) at 21 weeks (Figure 5A). Similar changes were observed by Western blot analysis for MCF7 cells; however, vimentin data is only shown for a 0.25% CSE treatment (Figure 5A, left panel). Decreases in occludin, and increases in N-cadherin and fibronectin, which are also associated with a mesenchymal state [22], were observed in MCF10A cells treated with CSE for 21 weeks (Figure 5B left panel). In addition, we observed dysregulation of occludin and N-cadherin in an independent RNA sample of MCF 10A cells treated with CSE for 40 weeks (Figure 5B, right panel). We also observed a general downregulation of keratins (Additional file 1: Table S1), which is another hallmark of EMT [22]. Several members of the claudin family of tight-junction proteins were downregulated (Figure 5C), which fits with the observed increased motility induced by CSE treatment. The EMT-promoting transcription factors TWIST1, TWIST2, ZEB1, ZEB2, and FOXC2 were upregulated, while FOXC1 and SNAI1 (Snail) were downregulated by CSE in MCF 10A cells (Figure 5D). These transcription factors can be induced through TGF-β signaling [24,25], and we observed that TGF-β receptor I and III (TGFBR1, TGFBR3), and TGF-β2 (TGFB2) were upregulated in MCF 10A cells treated with CSE (Figure 5E). Some of these gene expression changes were significant in only one of the two CSE-treated MCF 10A clones indicating variability in the response to smoke exposure. Upregulation of TWIST1 and TWIST2, as well as of TGFBR3 was also observed in MCF 10A treated with CSE for 40 weeks, together with TGFB1, but not TGFB2 (Figure 5F). Since exposure to cigarette smoke has been previously linked to epigenetic silencing in human cancer [26,27], we investigated if promoter methylation could be responsible for gene downregulation in our model. We used a DNA methylation array to estimate the proportion of methylated loci (beta-value) in MCF 10A cells treated with CSE for 21 weeks (Additional file 2: Table S2), focusing on sites located within promoter CpG islands. The beta value of one occludin probe increased from 0.11 to 0.50 after treatment with CSE, indicating a substantial increase in methylation. Similarly, the beta-value of one claudin 1 site increased from 0.06 to 0.55. None of the other downregulated genes that we had validated up to this point were affected according to this analysis. However, we observed increased methylation of estrogen receptor beta (ERβ; 0.17 to 0.72), which can act as a tumor suppressor in the mammary epithelium [28]. Western blot analysis showed that the expression of ERβ was reduced in MCF 10A and MCF7 cells treated with CSE (Figure 6). Although our data are in agreement with other published reports and suggest that this receptor may be epigenetically repressed by cigarette smoke, we cannot discount alternative transcriptional mechanisms and processes related to protein degradation or decreased stability.


Cigarette smoke induces epithelial to mesenchymal transition and increases the metastatic ability of breast cancer cells.

Di Cello F, Flowers VL, Li H, Vecchio-Pagán B, Gordon B, Harbom K, Shin J, Beaty R, Wang W, Brayton C, Baylin SB, Zahnow CA - Mol. Cancer (2013)

Exposure of mammary epithelial cells to CSE leads to gene expression changes indicative of EMT. (A, left panel) Western Blot showing upregulation of Vimentin and downregulation of E-cadherin in MCF10A and MCF7 cells exposed to CSE for 21 weeks. (A, right panel) qRT PCR analysis showing downregulation of e-cadherin mRNA and upregulation of Vimentin mRNA in two MCF10A subclones after 21 weeks of exposure to 0.5% CSE. (B) qRT PCR analysis showing downregulation of occludin and upregulation of N-cadherin and fibronectin in two MCF10A subclones at 21 and 40 weeks of exposure. (C-F) qRT PCR analysis of EMT genes in MCF 10A mammary epithelial cells exposed to 0.5% CSE for 21 weeks (clones SC1 and SC2) or 0.5-1.0% CSE for 40 weeks (without subcloning). Data in bar graphs are mean ± standard deviation of 3 replicates; *P<0.01, **P<0.001.
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Figure 5: Exposure of mammary epithelial cells to CSE leads to gene expression changes indicative of EMT. (A, left panel) Western Blot showing upregulation of Vimentin and downregulation of E-cadherin in MCF10A and MCF7 cells exposed to CSE for 21 weeks. (A, right panel) qRT PCR analysis showing downregulation of e-cadherin mRNA and upregulation of Vimentin mRNA in two MCF10A subclones after 21 weeks of exposure to 0.5% CSE. (B) qRT PCR analysis showing downregulation of occludin and upregulation of N-cadherin and fibronectin in two MCF10A subclones at 21 and 40 weeks of exposure. (C-F) qRT PCR analysis of EMT genes in MCF 10A mammary epithelial cells exposed to 0.5% CSE for 21 weeks (clones SC1 and SC2) or 0.5-1.0% CSE for 40 weeks (without subcloning). Data in bar graphs are mean ± standard deviation of 3 replicates; *P<0.01, **P<0.001.
Mentions: Two clones, designated SC1 and SC2 were isolated from MCF 10A cells exposed to 0.5% CSE for 13 weeks and expanded in the same concentration of CSE for 8 additional weeks, at which time total RNA was isolated for microarray and qPCR analysis. In addition, protein lysates and genomic DNA were prepared from independent samples treated with CSE for 21 weeks. The microarray analysis identified 186 unique genes that were upregulated and 308 unique genes that were downregulated at least two fold in both MCF 10A CSE clones (Additional file 1: Table S1). The expression of selected genes was verified by qRT-PCR or Western blot analysis (Figure 5), focusing on those genes associated with the phenotypes that we had observed, namely EMT, invasion and metastasis. E-cadherin and vimentin, which are associated with an epithelial state [22,23], were downregulated and upregulated respectively in MCF10As as determined by Western blot analysis (left panel) and PCR (right panel) at 21 weeks (Figure 5A). Similar changes were observed by Western blot analysis for MCF7 cells; however, vimentin data is only shown for a 0.25% CSE treatment (Figure 5A, left panel). Decreases in occludin, and increases in N-cadherin and fibronectin, which are also associated with a mesenchymal state [22], were observed in MCF10A cells treated with CSE for 21 weeks (Figure 5B left panel). In addition, we observed dysregulation of occludin and N-cadherin in an independent RNA sample of MCF 10A cells treated with CSE for 40 weeks (Figure 5B, right panel). We also observed a general downregulation of keratins (Additional file 1: Table S1), which is another hallmark of EMT [22]. Several members of the claudin family of tight-junction proteins were downregulated (Figure 5C), which fits with the observed increased motility induced by CSE treatment. The EMT-promoting transcription factors TWIST1, TWIST2, ZEB1, ZEB2, and FOXC2 were upregulated, while FOXC1 and SNAI1 (Snail) were downregulated by CSE in MCF 10A cells (Figure 5D). These transcription factors can be induced through TGF-β signaling [24,25], and we observed that TGF-β receptor I and III (TGFBR1, TGFBR3), and TGF-β2 (TGFB2) were upregulated in MCF 10A cells treated with CSE (Figure 5E). Some of these gene expression changes were significant in only one of the two CSE-treated MCF 10A clones indicating variability in the response to smoke exposure. Upregulation of TWIST1 and TWIST2, as well as of TGFBR3 was also observed in MCF 10A treated with CSE for 40 weeks, together with TGFB1, but not TGFB2 (Figure 5F). Since exposure to cigarette smoke has been previously linked to epigenetic silencing in human cancer [26,27], we investigated if promoter methylation could be responsible for gene downregulation in our model. We used a DNA methylation array to estimate the proportion of methylated loci (beta-value) in MCF 10A cells treated with CSE for 21 weeks (Additional file 2: Table S2), focusing on sites located within promoter CpG islands. The beta value of one occludin probe increased from 0.11 to 0.50 after treatment with CSE, indicating a substantial increase in methylation. Similarly, the beta-value of one claudin 1 site increased from 0.06 to 0.55. None of the other downregulated genes that we had validated up to this point were affected according to this analysis. However, we observed increased methylation of estrogen receptor beta (ERβ; 0.17 to 0.72), which can act as a tumor suppressor in the mammary epithelium [28]. Western blot analysis showed that the expression of ERβ was reduced in MCF 10A and MCF7 cells treated with CSE (Figure 6). Although our data are in agreement with other published reports and suggest that this receptor may be epigenetically repressed by cigarette smoke, we cannot discount alternative transcriptional mechanisms and processes related to protein degradation or decreased stability.

Bottom Line: Moreover, transplantation experiments in mice demonstrate that treatment with cigarette smoke extract renders MCF 10A cells more capable to survive and colonize the mammary ducts and MCF7 cells more prone to metastasize from a subcutaneous injection site, independent of cigarette smoke effects on the host and stromal environment.Analysis by flow cytometry showed that treatment with CSE leads to the emergence of a CD44(hi)/CD24(low) population in MCF 10A cells and of CD44+ and CD49f + MCF7 cells, indicating that cigarette smoke causes the emergence of cell populations bearing markers of self-renewing stem-like cells.The phenotypical alterations induced by cigarette smoke are accompanied by numerous changes in gene expression that are associated with epithelial to mesenchymal transition and tumorigenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD 21287, USA.

ABSTRACT

Background: Recent epidemiological studies demonstrate that both active and involuntary exposure to tobacco smoke increase the risk of breast cancer. Little is known, however, about the molecular mechanisms by which continuous, long term exposure to tobacco smoke contributes to breast carcinogenesis because most previous studies have focused on short term treatment models. In this work we have set out to investigate the progressive transforming effects of tobacco smoke on non-tumorigenic mammary epithelial cells and breast cancer cells using in vitro and in vivo models of chronic cigarette smoke exposure.

Results: We show that both non-tumorigenic (MCF 10A, MCF-12A) and tumorigenic (MCF7) breast epithelial cells exposed to cigarette smoke acquire mesenchymal properties such as fibroblastoid morphology, increased anchorage-independent growth, and increased motility and invasiveness. Moreover, transplantation experiments in mice demonstrate that treatment with cigarette smoke extract renders MCF 10A cells more capable to survive and colonize the mammary ducts and MCF7 cells more prone to metastasize from a subcutaneous injection site, independent of cigarette smoke effects on the host and stromal environment. The extent of transformation and the resulting phenotype thus appear to be associated with the differentiation state of the cells at the time of exposure. Analysis by flow cytometry showed that treatment with CSE leads to the emergence of a CD44(hi)/CD24(low) population in MCF 10A cells and of CD44+ and CD49f + MCF7 cells, indicating that cigarette smoke causes the emergence of cell populations bearing markers of self-renewing stem-like cells. The phenotypical alterations induced by cigarette smoke are accompanied by numerous changes in gene expression that are associated with epithelial to mesenchymal transition and tumorigenesis.

Conclusions: Our results indicate that exposure to cigarette smoke leads to a more aggressive and transformed phenotype in human mammary epithelial cells and that the differentiation state of the cell at the time of exposure may be an important determinant in the phenotype of the final transformed state.

Show MeSH
Related in: MedlinePlus