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Oncogenic transformation of mammary epithelial cells by transforming growth factor beta independent of mammary stem cell regulation.

Dunphy KA, Seo JH, Kim DJ, Roberts AL, Tao L, DiRenzo J, Balboni AL, Crisi GM, Hagen MJ, Chandrasekaran T, Gauger KJ, Schneider SS, Jerry DJ - Cancer Cell Int. (2013)

Bottom Line: The gene expression profiles of TGFβ-treated cells demonstrate de-differentiation with decreases in the expression of genes that define luminal, basal and stem cells.Additionally, the gene expression profiles demonstrate increases in markers of EMT, growth factor signalling, TGFβ2 and changes in extra cellular matrix.This model demonstrates full oncogenic EMT without an increase in stem cells, serving to separate EMT markers from stem cell markers.

View Article: PubMed Central - HTML - PubMed

Affiliation: Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA.

ABSTRACT

Background: Transforming growth factor beta (TGFβ) is transiently increased in the mammary gland during involution and by radiation. While TGFβ normally has a tumour suppressor role, prolonged exposure to TGFβ can induce an oncogenic epithelial to mesenchymal transition (EMT) program in permissive cells and initiate the generation of cancer stem cells. Our objective is to mimic the transient exposure to TGFβ during involution to determine the persistent effects on premalignant mammary epithelium.

Method: CDβGeo cells, a transplantable mouse mammary epithelial cell line, were treated in vitro for 14 days with TGFβ (5 ng/ml). The cells were passaged for an additional 14 days in media without TGFβ and then assessed for markers of EMT and transformation.

Results: The 14-day exposure to TGFβ induced EMT and transdifferentiation in vitro that persists after withdrawal of TGFβ. TGFβ-treated cells are highly tumorigenic in vivo, producing invasive solid de-differentiated tumours (100%; latency 6.7 weeks) compared to control (43%; latency 32.7 weeks). Although the TGFβ-treated cells have initiated a persistent EMT program, the stem cell population was unchanged relative to the controls. The gene expression profiles of TGFβ-treated cells demonstrate de-differentiation with decreases in the expression of genes that define luminal, basal and stem cells. Additionally, the gene expression profiles demonstrate increases in markers of EMT, growth factor signalling, TGFβ2 and changes in extra cellular matrix.

Conclusion: This model demonstrates full oncogenic EMT without an increase in stem cells, serving to separate EMT markers from stem cell markers.

No MeSH data available.


Related in: MedlinePlus

Persistently transdifferentiated pTD cells do not have an increase in the stem cell pool. (A) Initiation of primary or secondary mammospheres are not different between CDβGeo cells and pTD cells. (B) FACS for Aldefluor. (C) FACS for CD44 CD24. (D) FACS for Let7c sensor. (E) Limiting dilution for CDβGeo and pTD cells.
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Figure 4: Persistently transdifferentiated pTD cells do not have an increase in the stem cell pool. (A) Initiation of primary or secondary mammospheres are not different between CDβGeo cells and pTD cells. (B) FACS for Aldefluor. (C) FACS for CD44 CD24. (D) FACS for Let7c sensor. (E) Limiting dilution for CDβGeo and pTD cells.

Mentions: Previous reports suggest that the population of stem cells is increased during the implementation of a transient EMT program[20,25,26]. Because stem cell profiles and EMT profiles overlap, and because the pTD cells demonstrate persistent EMT-mediated changes in gene expression without increases in select stem cell genes, we compared stem cell ratios relative to the CDβGeo parental cells using three distinct assays. During the TGFβ exposure period, the mammosphere forming capability is transiently increased (Additional file4: Figure S3), but although EMT is persistent 14 days after withdrawal of TGFβ, on day 28 there was no increase in primary or secondary mammosphere formation (Figure 4A). FACs analysis also shows no difference in aldefluor positive cells or changes in the CD44High CD24Low population (Figure 4 B&C). The let-7c sensor assay, which utilizes the fact that stem cells express very low let7c microRNA[38], also demonstrates no differences in the proportion of stem cells between the CDβGeo and pTD cells (Figure 4D). A limiting dilution series also shows that the CDβGeo cells and the pTD cells have similar capacity to repopulate in the mammary gland (Figure 4E). Specifically, partial growth (<50% of fat pad filled) occurred equally between the two cell types when 5000 (2/5) or 1000 (1/5) cells were transplanted. Regardless of the number of cells transplanted, the successful pTD outgrowths always produced solid tumours, even when examined as early as three weeks after transplantation. We find no evidence that there is an increase in the stem cell population in the pTD cells.


Oncogenic transformation of mammary epithelial cells by transforming growth factor beta independent of mammary stem cell regulation.

Dunphy KA, Seo JH, Kim DJ, Roberts AL, Tao L, DiRenzo J, Balboni AL, Crisi GM, Hagen MJ, Chandrasekaran T, Gauger KJ, Schneider SS, Jerry DJ - Cancer Cell Int. (2013)

Persistently transdifferentiated pTD cells do not have an increase in the stem cell pool. (A) Initiation of primary or secondary mammospheres are not different between CDβGeo cells and pTD cells. (B) FACS for Aldefluor. (C) FACS for CD44 CD24. (D) FACS for Let7c sensor. (E) Limiting dilution for CDβGeo and pTD cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3733955&req=5

Figure 4: Persistently transdifferentiated pTD cells do not have an increase in the stem cell pool. (A) Initiation of primary or secondary mammospheres are not different between CDβGeo cells and pTD cells. (B) FACS for Aldefluor. (C) FACS for CD44 CD24. (D) FACS for Let7c sensor. (E) Limiting dilution for CDβGeo and pTD cells.
Mentions: Previous reports suggest that the population of stem cells is increased during the implementation of a transient EMT program[20,25,26]. Because stem cell profiles and EMT profiles overlap, and because the pTD cells demonstrate persistent EMT-mediated changes in gene expression without increases in select stem cell genes, we compared stem cell ratios relative to the CDβGeo parental cells using three distinct assays. During the TGFβ exposure period, the mammosphere forming capability is transiently increased (Additional file4: Figure S3), but although EMT is persistent 14 days after withdrawal of TGFβ, on day 28 there was no increase in primary or secondary mammosphere formation (Figure 4A). FACs analysis also shows no difference in aldefluor positive cells or changes in the CD44High CD24Low population (Figure 4 B&C). The let-7c sensor assay, which utilizes the fact that stem cells express very low let7c microRNA[38], also demonstrates no differences in the proportion of stem cells between the CDβGeo and pTD cells (Figure 4D). A limiting dilution series also shows that the CDβGeo cells and the pTD cells have similar capacity to repopulate in the mammary gland (Figure 4E). Specifically, partial growth (<50% of fat pad filled) occurred equally between the two cell types when 5000 (2/5) or 1000 (1/5) cells were transplanted. Regardless of the number of cells transplanted, the successful pTD outgrowths always produced solid tumours, even when examined as early as three weeks after transplantation. We find no evidence that there is an increase in the stem cell population in the pTD cells.

Bottom Line: The gene expression profiles of TGFβ-treated cells demonstrate de-differentiation with decreases in the expression of genes that define luminal, basal and stem cells.Additionally, the gene expression profiles demonstrate increases in markers of EMT, growth factor signalling, TGFβ2 and changes in extra cellular matrix.This model demonstrates full oncogenic EMT without an increase in stem cells, serving to separate EMT markers from stem cell markers.

View Article: PubMed Central - HTML - PubMed

Affiliation: Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA.

ABSTRACT

Background: Transforming growth factor beta (TGFβ) is transiently increased in the mammary gland during involution and by radiation. While TGFβ normally has a tumour suppressor role, prolonged exposure to TGFβ can induce an oncogenic epithelial to mesenchymal transition (EMT) program in permissive cells and initiate the generation of cancer stem cells. Our objective is to mimic the transient exposure to TGFβ during involution to determine the persistent effects on premalignant mammary epithelium.

Method: CDβGeo cells, a transplantable mouse mammary epithelial cell line, were treated in vitro for 14 days with TGFβ (5 ng/ml). The cells were passaged for an additional 14 days in media without TGFβ and then assessed for markers of EMT and transformation.

Results: The 14-day exposure to TGFβ induced EMT and transdifferentiation in vitro that persists after withdrawal of TGFβ. TGFβ-treated cells are highly tumorigenic in vivo, producing invasive solid de-differentiated tumours (100%; latency 6.7 weeks) compared to control (43%; latency 32.7 weeks). Although the TGFβ-treated cells have initiated a persistent EMT program, the stem cell population was unchanged relative to the controls. The gene expression profiles of TGFβ-treated cells demonstrate de-differentiation with decreases in the expression of genes that define luminal, basal and stem cells. Additionally, the gene expression profiles demonstrate increases in markers of EMT, growth factor signalling, TGFβ2 and changes in extra cellular matrix.

Conclusion: This model demonstrates full oncogenic EMT without an increase in stem cells, serving to separate EMT markers from stem cell markers.

No MeSH data available.


Related in: MedlinePlus