Limits...
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.

Show MeSH

Related in: MedlinePlus

Cigarette smoke induces anchorage-independent cell growth, migration, and invasion in mammary epithelial cell lines. (A) Treatment of MCF10A cells with CSE or CSC leads to soft agar colony formation that increases, as compared to mock, with increased weeks of exposure. (B) Treatment of MCF10A cells with CSE for 37 and 72 weeks leads to increased migration through uncoated transwell inserts as compared to mock treated cells. (E) Treatment of MCF 7 cells with CSE for 9 weeks leads to soft agar colony formation as compared to mock. (F) Treatment of MCF 7 cells with CSE for 9 weeks leads to increased invasion through matrigel-coated transwell inserts as compared to mock. (C) Treatment of MCF12A cells with CSC for 18 weeks leads to increased colony formation as compared to mock. (D) Treatment of MCF12 A cells with CSC leads to increased migration through uncoated transwell inserts as compared to DMSO control The increase in MCF-12A migration (right) was not quantified numerically because the cells became partially confluent after migration and could not be accurately counted. The dark patches are cells stained with crystal violet that have migrated though the filter pores (light grey dots); the size bar represents 100 μm. Data in bar graphs are mean ± standard deviation of 3–5 replicates; *P<0.05, **P<0.01, ***P<0.001 by Student’s t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750372&req=5

Figure 1: Cigarette smoke induces anchorage-independent cell growth, migration, and invasion in mammary epithelial cell lines. (A) Treatment of MCF10A cells with CSE or CSC leads to soft agar colony formation that increases, as compared to mock, with increased weeks of exposure. (B) Treatment of MCF10A cells with CSE for 37 and 72 weeks leads to increased migration through uncoated transwell inserts as compared to mock treated cells. (E) Treatment of MCF 7 cells with CSE for 9 weeks leads to soft agar colony formation as compared to mock. (F) Treatment of MCF 7 cells with CSE for 9 weeks leads to increased invasion through matrigel-coated transwell inserts as compared to mock. (C) Treatment of MCF12A cells with CSC for 18 weeks leads to increased colony formation as compared to mock. (D) Treatment of MCF12 A cells with CSC leads to increased migration through uncoated transwell inserts as compared to DMSO control The increase in MCF-12A migration (right) was not quantified numerically because the cells became partially confluent after migration and could not be accurately counted. The dark patches are cells stained with crystal violet that have migrated though the filter pores (light grey dots); the size bar represents 100 μm. Data in bar graphs are mean ± standard deviation of 3–5 replicates; *P<0.05, **P<0.01, ***P<0.001 by Student’s t-test.

Mentions: It has been shown that the risk of developing cancer increases with the number of years a person has smoked or been exposed to second hand smoke [12,13]. For this reason we developed a model to study the progressive, chronic effects of cigarette smoke exposure. Cells were continuously cultured for 72 weeks with an aqueous cigarette smoke extract (CSE) from main stream smoke prepared in our laboratory (0.25%, 0.5% or 1% CSE) or for approximately 40 weeks with cigarette smoke condensate (CSC) a commercial product based on condensate from second-hand-like smoke (10 μg/ml or 25 μg/ml CSC). A concentration of 0.5% CSE, or 25 μg/ml CSC in the media corresponds to approximately 0.001 cigarettes/ml, which is an amount comparable to, or lower than those used in other studies [9,10,14-16]. The corresponding amount of nicotine in the media (1.3±0.1 μg/ml) approximates the upper limit of the concentrations of cotinine found in the plasma or breast milk of smokers, which has been reported as high as 300–800 ng/ml and 200–500 ng/ml, respectively [17]. Non-tumorigenic MCF 10A cells cultured with either CSE or CSC were transferred to soft agar to assess anchorage-independent growth after 15, 21, 27 and 39 or 37 weeks of treatment. Both CSE and CSC caused a significant increase in colony formation in soft agar (up to 42 fold; Figure 1A) which is a feature typical of cancer cells. Linear regression analysis indicated that the effect was both dose and time dependent as the number of colonies increased in parallel with the duration of treatment (r2>0.9; P<0.05 by F-test), and the concentration of CSE or CSC (P<0.01 by F-test). The vehicle control for CSC, which contains DMSO, led to the development of slightly more colonies than the saline control for CSE. Treatment with CSE also increased the migratory ability in MCF10As. After treatment with 0.5% or 1% CSE for 37 weeks MCF 10A cells showed a 3.1 and 3.6 fold increase in migration, respectively (Figure 1B). The experiment was replicated after 72 weeks of treatment with similar results, suggesting that the initial increase in motility is maintained during prolonged exposure to CSE (Figure 1B). To test whether colony formation and migration were unique to MCF10As or would also occur in other breast cell lines we treated non-tumorigenic, MCF-12A cells with CSC and the breast cancer cell line MCF7 with CSE. For MCF12A, we observed a 4 to 5-fold increase in colony formation after 18 weeks of treatment (Figure 1C, P<0.01 by Student’s t-test) and a significant increase in migration when exposed to CSC for 18 weeks (Figure 1D). MCF7 cells are capable of forming colonies without CSE treatment; however, a significant increase in colony formation was observed after only nine weeks of treatment with 0.5% CSE (Figure 1E). Moreover MCF7 cells, which are more motile then MCF 10A and -12A, and have the ability to migrate through matrigel coated filters, showed a marked increase of their invasive capability when exposed to 0.25% or 0.5% CSE for 9 weeks (Figure 1F). All cell lines tested altered their morphology when exposed to CSE or CSC. Untreated MCF 10A and MCF-12A cells normally display a typical cobblestone epithelial morphology in culture. Treatment with CSE or CSC caused them to adopt a more spindle shape and fibroblast-like morphology (shown clearly in the inserts, upper and middle panels, Figure 2), which is consistent with the increased motility that we observed in the migration assays shown in Figure 1. Similarly, MCF7 cells also became more elongated and spindle-shaped after exposure to cigarette smoke (Figure 2, bottom panel). The observed changes in morphology and motility are consistent with phenotypical changes associated with EMT, and suggest that chronic exposure to cigarette smoke may cause breast epithelial cells to acquire mesenchymal properties, which would render them more motile [11]. For cells that are already tumorigenic, such as MCF7, our observations suggest that the phenotype has become more invasive. Similar results were observed using either CSE or CSC, indicating that both mainstream smoke and second hand cigarette smoke contain compounds that can significantly alter the phenotype of these diverse cell lines.


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)

Cigarette smoke induces anchorage-independent cell growth, migration, and invasion in mammary epithelial cell lines. (A) Treatment of MCF10A cells with CSE or CSC leads to soft agar colony formation that increases, as compared to mock, with increased weeks of exposure. (B) Treatment of MCF10A cells with CSE for 37 and 72 weeks leads to increased migration through uncoated transwell inserts as compared to mock treated cells. (E) Treatment of MCF 7 cells with CSE for 9 weeks leads to soft agar colony formation as compared to mock. (F) Treatment of MCF 7 cells with CSE for 9 weeks leads to increased invasion through matrigel-coated transwell inserts as compared to mock. (C) Treatment of MCF12A cells with CSC for 18 weeks leads to increased colony formation as compared to mock. (D) Treatment of MCF12 A cells with CSC leads to increased migration through uncoated transwell inserts as compared to DMSO control The increase in MCF-12A migration (right) was not quantified numerically because the cells became partially confluent after migration and could not be accurately counted. The dark patches are cells stained with crystal violet that have migrated though the filter pores (light grey dots); the size bar represents 100 μm. Data in bar graphs are mean ± standard deviation of 3–5 replicates; *P<0.05, **P<0.01, ***P<0.001 by Student’s t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Cigarette smoke induces anchorage-independent cell growth, migration, and invasion in mammary epithelial cell lines. (A) Treatment of MCF10A cells with CSE or CSC leads to soft agar colony formation that increases, as compared to mock, with increased weeks of exposure. (B) Treatment of MCF10A cells with CSE for 37 and 72 weeks leads to increased migration through uncoated transwell inserts as compared to mock treated cells. (E) Treatment of MCF 7 cells with CSE for 9 weeks leads to soft agar colony formation as compared to mock. (F) Treatment of MCF 7 cells with CSE for 9 weeks leads to increased invasion through matrigel-coated transwell inserts as compared to mock. (C) Treatment of MCF12A cells with CSC for 18 weeks leads to increased colony formation as compared to mock. (D) Treatment of MCF12 A cells with CSC leads to increased migration through uncoated transwell inserts as compared to DMSO control The increase in MCF-12A migration (right) was not quantified numerically because the cells became partially confluent after migration and could not be accurately counted. The dark patches are cells stained with crystal violet that have migrated though the filter pores (light grey dots); the size bar represents 100 μm. Data in bar graphs are mean ± standard deviation of 3–5 replicates; *P<0.05, **P<0.01, ***P<0.001 by Student’s t-test.
Mentions: It has been shown that the risk of developing cancer increases with the number of years a person has smoked or been exposed to second hand smoke [12,13]. For this reason we developed a model to study the progressive, chronic effects of cigarette smoke exposure. Cells were continuously cultured for 72 weeks with an aqueous cigarette smoke extract (CSE) from main stream smoke prepared in our laboratory (0.25%, 0.5% or 1% CSE) or for approximately 40 weeks with cigarette smoke condensate (CSC) a commercial product based on condensate from second-hand-like smoke (10 μg/ml or 25 μg/ml CSC). A concentration of 0.5% CSE, or 25 μg/ml CSC in the media corresponds to approximately 0.001 cigarettes/ml, which is an amount comparable to, or lower than those used in other studies [9,10,14-16]. The corresponding amount of nicotine in the media (1.3±0.1 μg/ml) approximates the upper limit of the concentrations of cotinine found in the plasma or breast milk of smokers, which has been reported as high as 300–800 ng/ml and 200–500 ng/ml, respectively [17]. Non-tumorigenic MCF 10A cells cultured with either CSE or CSC were transferred to soft agar to assess anchorage-independent growth after 15, 21, 27 and 39 or 37 weeks of treatment. Both CSE and CSC caused a significant increase in colony formation in soft agar (up to 42 fold; Figure 1A) which is a feature typical of cancer cells. Linear regression analysis indicated that the effect was both dose and time dependent as the number of colonies increased in parallel with the duration of treatment (r2>0.9; P<0.05 by F-test), and the concentration of CSE or CSC (P<0.01 by F-test). The vehicle control for CSC, which contains DMSO, led to the development of slightly more colonies than the saline control for CSE. Treatment with CSE also increased the migratory ability in MCF10As. After treatment with 0.5% or 1% CSE for 37 weeks MCF 10A cells showed a 3.1 and 3.6 fold increase in migration, respectively (Figure 1B). The experiment was replicated after 72 weeks of treatment with similar results, suggesting that the initial increase in motility is maintained during prolonged exposure to CSE (Figure 1B). To test whether colony formation and migration were unique to MCF10As or would also occur in other breast cell lines we treated non-tumorigenic, MCF-12A cells with CSC and the breast cancer cell line MCF7 with CSE. For MCF12A, we observed a 4 to 5-fold increase in colony formation after 18 weeks of treatment (Figure 1C, P<0.01 by Student’s t-test) and a significant increase in migration when exposed to CSC for 18 weeks (Figure 1D). MCF7 cells are capable of forming colonies without CSE treatment; however, a significant increase in colony formation was observed after only nine weeks of treatment with 0.5% CSE (Figure 1E). Moreover MCF7 cells, which are more motile then MCF 10A and -12A, and have the ability to migrate through matrigel coated filters, showed a marked increase of their invasive capability when exposed to 0.25% or 0.5% CSE for 9 weeks (Figure 1F). All cell lines tested altered their morphology when exposed to CSE or CSC. Untreated MCF 10A and MCF-12A cells normally display a typical cobblestone epithelial morphology in culture. Treatment with CSE or CSC caused them to adopt a more spindle shape and fibroblast-like morphology (shown clearly in the inserts, upper and middle panels, Figure 2), which is consistent with the increased motility that we observed in the migration assays shown in Figure 1. Similarly, MCF7 cells also became more elongated and spindle-shaped after exposure to cigarette smoke (Figure 2, bottom panel). The observed changes in morphology and motility are consistent with phenotypical changes associated with EMT, and suggest that chronic exposure to cigarette smoke may cause breast epithelial cells to acquire mesenchymal properties, which would render them more motile [11]. For cells that are already tumorigenic, such as MCF7, our observations suggest that the phenotype has become more invasive. Similar results were observed using either CSE or CSC, indicating that both mainstream smoke and second hand cigarette smoke contain compounds that can significantly alter the phenotype of these diverse cell lines.

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