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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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CSE causes changes in stem cell markers in mammary epithelial cells and breast cancer cells. FACS analysis of MCF 10A cells treated with CSE for 30 weeks and MCF7 cells treated with CSE for 17 weeks. Cells labeled with isotype control antibodies, or incubated with DEAB (negative control) are shown in grey. Cells labeled with specific antibodies, or by aldefluor reaction are in shown in black and the percentage of cells in each quadrant is shown. MCF 10A: quadrants were established to include 99.9% of CD24+/CD44+, or ALDEFLUOR+/CD44+ mock treated cells; cells with increased CD44 positivity (circled) are concentrating in the lower right quadrants after CSE treatment, indicating loss of CD24 positivity and ALDEFLUOR signal. MCF7: quadrants were established to include 99% of signal from isotype antibody (negative); mock treated MCF7 cells consist of a mixed population of CD44+ and CD44- cells, uniformly CD24+; CSE treatment caused a shift to the CD24+/CD44+ quadrant, and an increase in CD49f+ cells.
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Figure 4: CSE causes changes in stem cell markers in mammary epithelial cells and breast cancer cells. FACS analysis of MCF 10A cells treated with CSE for 30 weeks and MCF7 cells treated with CSE for 17 weeks. Cells labeled with isotype control antibodies, or incubated with DEAB (negative control) are shown in grey. Cells labeled with specific antibodies, or by aldefluor reaction are in shown in black and the percentage of cells in each quadrant is shown. MCF 10A: quadrants were established to include 99.9% of CD24+/CD44+, or ALDEFLUOR+/CD44+ mock treated cells; cells with increased CD44 positivity (circled) are concentrating in the lower right quadrants after CSE treatment, indicating loss of CD24 positivity and ALDEFLUOR signal. MCF7: quadrants were established to include 99% of signal from isotype antibody (negative); mock treated MCF7 cells consist of a mixed population of CD44+ and CD44- cells, uniformly CD24+; CSE treatment caused a shift to the CD24+/CD44+ quadrant, and an increase in CD49f+ cells.

Mentions: Self renewal is a critical component of tumorigenesis [11]. Thus, we analyzed how CSE affects the distribution of specific cell surface markers that are associated with tumor initiation and self renewal, specifically ALDH1 activity, high CD44/low CD24, CD49f and CD133 [19-21]. FACS analysis showed a sharp change in the distribution of CD44 and CD24 in CSE-treated MCF 10A and MCF7 cells. Most MCF 10A cells are CD44+/CD24+, but after exposure to 0.5% CSE, at least two cell populations with substantially increased CD44 and lower ALDH activity emerged. In one of these populations the expression of CD24 was particularly low (Figure 4, left panels). In MCF10A cells, the distribution of CD49f was virtually unaffected by treatment with CSE, and the cells appeared uniformly CD49f+ (not shown). A small number of CD133+ cells that were entirely distinguishable from the CD44hi cells were present in MCF 10A cells treated with 0.5% CSE, while untreated cells were CD133- (not shown). MCF7 cells are a mixed population of CD44+ and CD44- cells, but are uniformly CD24+ (Figure 4, right panels). CSE treatment caused a shift to the CD24+/CD44+ quadrant, (76.8% to 93.1%) and an increase of CD49f positivity in a portion of the CD44+ cells (18.4% to 29.9%). These results indicate that chronic low-dose exposure to cigarette smoke can alter cellular distributions of markers associated with self-renewal and stem-like properties.


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)

CSE causes changes in stem cell markers in mammary epithelial cells and breast cancer cells. FACS analysis of MCF 10A cells treated with CSE for 30 weeks and MCF7 cells treated with CSE for 17 weeks. Cells labeled with isotype control antibodies, or incubated with DEAB (negative control) are shown in grey. Cells labeled with specific antibodies, or by aldefluor reaction are in shown in black and the percentage of cells in each quadrant is shown. MCF 10A: quadrants were established to include 99.9% of CD24+/CD44+, or ALDEFLUOR+/CD44+ mock treated cells; cells with increased CD44 positivity (circled) are concentrating in the lower right quadrants after CSE treatment, indicating loss of CD24 positivity and ALDEFLUOR signal. MCF7: quadrants were established to include 99% of signal from isotype antibody (negative); mock treated MCF7 cells consist of a mixed population of CD44+ and CD44- cells, uniformly CD24+; CSE treatment caused a shift to the CD24+/CD44+ quadrant, and an increase in CD49f+ cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750372&req=5

Figure 4: CSE causes changes in stem cell markers in mammary epithelial cells and breast cancer cells. FACS analysis of MCF 10A cells treated with CSE for 30 weeks and MCF7 cells treated with CSE for 17 weeks. Cells labeled with isotype control antibodies, or incubated with DEAB (negative control) are shown in grey. Cells labeled with specific antibodies, or by aldefluor reaction are in shown in black and the percentage of cells in each quadrant is shown. MCF 10A: quadrants were established to include 99.9% of CD24+/CD44+, or ALDEFLUOR+/CD44+ mock treated cells; cells with increased CD44 positivity (circled) are concentrating in the lower right quadrants after CSE treatment, indicating loss of CD24 positivity and ALDEFLUOR signal. MCF7: quadrants were established to include 99% of signal from isotype antibody (negative); mock treated MCF7 cells consist of a mixed population of CD44+ and CD44- cells, uniformly CD24+; CSE treatment caused a shift to the CD24+/CD44+ quadrant, and an increase in CD49f+ cells.
Mentions: Self renewal is a critical component of tumorigenesis [11]. Thus, we analyzed how CSE affects the distribution of specific cell surface markers that are associated with tumor initiation and self renewal, specifically ALDH1 activity, high CD44/low CD24, CD49f and CD133 [19-21]. FACS analysis showed a sharp change in the distribution of CD44 and CD24 in CSE-treated MCF 10A and MCF7 cells. Most MCF 10A cells are CD44+/CD24+, but after exposure to 0.5% CSE, at least two cell populations with substantially increased CD44 and lower ALDH activity emerged. In one of these populations the expression of CD24 was particularly low (Figure 4, left panels). In MCF10A cells, the distribution of CD49f was virtually unaffected by treatment with CSE, and the cells appeared uniformly CD49f+ (not shown). A small number of CD133+ cells that were entirely distinguishable from the CD44hi cells were present in MCF 10A cells treated with 0.5% CSE, while untreated cells were CD133- (not shown). MCF7 cells are a mixed population of CD44+ and CD44- cells, but are uniformly CD24+ (Figure 4, right panels). CSE treatment caused a shift to the CD24+/CD44+ quadrant, (76.8% to 93.1%) and an increase of CD49f positivity in a portion of the CD44+ cells (18.4% to 29.9%). These results indicate that chronic low-dose exposure to cigarette smoke can alter cellular distributions of markers associated with self-renewal and stem-like properties.

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