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The retinoblastoma tumor suppressor pathway modulates the invasiveness of ErbB2-positive breast cancer.

Witkiewicz AK, Cox DW, Rivadeneira D, Ertel AE, Fortina P, Schwartz GF, Knudsen ES - Oncogene (2013)

Bottom Line: Further, stable knockdown of RB resulted in invasive lesions in orthotopic xenograft assays, compared with DCIS-like lesions developing from RB-proficient cells.Conversely, the invasive phenotype observed in ErbB2-positive cancer models was inhibited through CDK4/6 inhibition in an RB-dependent manner.Taken together, these data demonstrate a key role for the RB pathway in invasion associated with breast tumor progression, and shed light on the key molecular events that promote the progression of DCIS to invasive disease.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pathology, UT Southwestern Medical Center, Dallas, TX, USA [2] Simmons Cancer Center, UT Southwestern Medical Center, Dallas, TX, USA.

ABSTRACT
The processes that control the progression of ductal carcinoma in situ (DCIS) to invasive breast cancer remain poorly understood. Epidermal growth factor receptor 2 (ErbB2) overexpression is common in DCIS, as is disruption of the retinoblastoma tumor suppressor (RB) pathway. Here, we examined the cooperative impact of ErbB2 and RB deregulation on facets of disease progression. Our studies demonstrate that RB deficiency altered the expression of key molecules needed for proper cellular organization and epithelial cell-cell adhesion as part of a program related to the epithelial-to-mesenchymal transition (EMT). An increase in the invasive potential of ErbB2-overexpressing cells was observed upon RB depletion. Further, stable knockdown of RB resulted in invasive lesions in orthotopic xenograft assays, compared with DCIS-like lesions developing from RB-proficient cells. Conversely, the invasive phenotype observed in ErbB2-positive cancer models was inhibited through CDK4/6 inhibition in an RB-dependent manner. Finally, in a cohort of DCIS cases, we show that, although elevated levels of ErbB2 are associated with increased risk of a subsequent DCIS recurrence, it is not associated with progression to invasive disease. In contrast, RB loss in ErbB2-positive DCIS cases was associated with increased risk for invasive breast cancer. Taken together, these data demonstrate a key role for the RB pathway in invasion associated with breast tumor progression, and shed light on the key molecular events that promote the progression of DCIS to invasive disease.

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RB loss promotes cell migration and invasion in ErbB2 over expressing cells(A) Cell migration and (B) cell invasion of MCF10A cells harboring ErbB2 over expression and/or RB-deficiency by Boyden Chamber assays; bars, SD. (***P < .0001) (C/D) SKBR3 and BT474 cells transduced with miRB or miNS retroviruses. Cells were harvested and cell lysates were analyzed by immunoblot for the indicated proteins. BrdU incorporation was analyzed by bivariate flow cytometry and data are average of at least three independent experiments; bars, SD. (E/F) Analysis of SKBR3 miNS/miRB and BT474 miNS/miRB cell migration by Boyden Chamber assays; bars, SD. (**P<0.01) (F) Analysis of SKBR3 miNS/miRB and BT474 miNS/miRB cell invasion by Boyden Chamber assays; bars, SD. (**P<0.01).
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Figure 4: RB loss promotes cell migration and invasion in ErbB2 over expressing cells(A) Cell migration and (B) cell invasion of MCF10A cells harboring ErbB2 over expression and/or RB-deficiency by Boyden Chamber assays; bars, SD. (***P < .0001) (C/D) SKBR3 and BT474 cells transduced with miRB or miNS retroviruses. Cells were harvested and cell lysates were analyzed by immunoblot for the indicated proteins. BrdU incorporation was analyzed by bivariate flow cytometry and data are average of at least three independent experiments; bars, SD. (E/F) Analysis of SKBR3 miNS/miRB and BT474 miNS/miRB cell migration by Boyden Chamber assays; bars, SD. (**P<0.01) (F) Analysis of SKBR3 miNS/miRB and BT474 miNS/miRB cell invasion by Boyden Chamber assays; bars, SD. (**P<0.01).

Mentions: To determine if RB-deficiency had an effect on cell motility, the migratory and invasive activity of RB-proficient and RB-deficient ErbB2 over expressing cells was assessed by Boyden chamber assays. While loss of RB or over expression of ErbB2 alone was observed to increase the migratory ability of MCF10A cells, the combined deregulation of the ErbB2- and RB-pathways demonstrated a cooperative enhancement of cell migration (Figure 4A). Upon examination of cell invasion, we found that while RB loss alone had minimal impact on the invasive potential of MCF10A cells, ErbB2 over expression significantly increased cell invasion (Figure 4B). Furthermore, combined deregulation of the ErbB2- and RB-pathways resulted in an ∼2-fold increase in cell invasion over ErbB2 over expression alone. These effects were dependent on downstream signaling through the ERK and AKT pathways. Specific pharmaceutical inhibition of AKT had a dramatic impact on invasion of all cells, while inhibition of ERK signaling had a particular impact on RB-deficient models (Supplemental Figure 2). These results suggest that RB-deficiency contributes to a more invasive phenotype in ErbB2 expressing mammary epithelial cells.


The retinoblastoma tumor suppressor pathway modulates the invasiveness of ErbB2-positive breast cancer.

Witkiewicz AK, Cox DW, Rivadeneira D, Ertel AE, Fortina P, Schwartz GF, Knudsen ES - Oncogene (2013)

RB loss promotes cell migration and invasion in ErbB2 over expressing cells(A) Cell migration and (B) cell invasion of MCF10A cells harboring ErbB2 over expression and/or RB-deficiency by Boyden Chamber assays; bars, SD. (***P < .0001) (C/D) SKBR3 and BT474 cells transduced with miRB or miNS retroviruses. Cells were harvested and cell lysates were analyzed by immunoblot for the indicated proteins. BrdU incorporation was analyzed by bivariate flow cytometry and data are average of at least three independent experiments; bars, SD. (E/F) Analysis of SKBR3 miNS/miRB and BT474 miNS/miRB cell migration by Boyden Chamber assays; bars, SD. (**P<0.01) (F) Analysis of SKBR3 miNS/miRB and BT474 miNS/miRB cell invasion by Boyden Chamber assays; bars, SD. (**P<0.01).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4150690&req=5

Figure 4: RB loss promotes cell migration and invasion in ErbB2 over expressing cells(A) Cell migration and (B) cell invasion of MCF10A cells harboring ErbB2 over expression and/or RB-deficiency by Boyden Chamber assays; bars, SD. (***P < .0001) (C/D) SKBR3 and BT474 cells transduced with miRB or miNS retroviruses. Cells were harvested and cell lysates were analyzed by immunoblot for the indicated proteins. BrdU incorporation was analyzed by bivariate flow cytometry and data are average of at least three independent experiments; bars, SD. (E/F) Analysis of SKBR3 miNS/miRB and BT474 miNS/miRB cell migration by Boyden Chamber assays; bars, SD. (**P<0.01) (F) Analysis of SKBR3 miNS/miRB and BT474 miNS/miRB cell invasion by Boyden Chamber assays; bars, SD. (**P<0.01).
Mentions: To determine if RB-deficiency had an effect on cell motility, the migratory and invasive activity of RB-proficient and RB-deficient ErbB2 over expressing cells was assessed by Boyden chamber assays. While loss of RB or over expression of ErbB2 alone was observed to increase the migratory ability of MCF10A cells, the combined deregulation of the ErbB2- and RB-pathways demonstrated a cooperative enhancement of cell migration (Figure 4A). Upon examination of cell invasion, we found that while RB loss alone had minimal impact on the invasive potential of MCF10A cells, ErbB2 over expression significantly increased cell invasion (Figure 4B). Furthermore, combined deregulation of the ErbB2- and RB-pathways resulted in an ∼2-fold increase in cell invasion over ErbB2 over expression alone. These effects were dependent on downstream signaling through the ERK and AKT pathways. Specific pharmaceutical inhibition of AKT had a dramatic impact on invasion of all cells, while inhibition of ERK signaling had a particular impact on RB-deficient models (Supplemental Figure 2). These results suggest that RB-deficiency contributes to a more invasive phenotype in ErbB2 expressing mammary epithelial cells.

Bottom Line: Further, stable knockdown of RB resulted in invasive lesions in orthotopic xenograft assays, compared with DCIS-like lesions developing from RB-proficient cells.Conversely, the invasive phenotype observed in ErbB2-positive cancer models was inhibited through CDK4/6 inhibition in an RB-dependent manner.Taken together, these data demonstrate a key role for the RB pathway in invasion associated with breast tumor progression, and shed light on the key molecular events that promote the progression of DCIS to invasive disease.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pathology, UT Southwestern Medical Center, Dallas, TX, USA [2] Simmons Cancer Center, UT Southwestern Medical Center, Dallas, TX, USA.

ABSTRACT
The processes that control the progression of ductal carcinoma in situ (DCIS) to invasive breast cancer remain poorly understood. Epidermal growth factor receptor 2 (ErbB2) overexpression is common in DCIS, as is disruption of the retinoblastoma tumor suppressor (RB) pathway. Here, we examined the cooperative impact of ErbB2 and RB deregulation on facets of disease progression. Our studies demonstrate that RB deficiency altered the expression of key molecules needed for proper cellular organization and epithelial cell-cell adhesion as part of a program related to the epithelial-to-mesenchymal transition (EMT). An increase in the invasive potential of ErbB2-overexpressing cells was observed upon RB depletion. Further, stable knockdown of RB resulted in invasive lesions in orthotopic xenograft assays, compared with DCIS-like lesions developing from RB-proficient cells. Conversely, the invasive phenotype observed in ErbB2-positive cancer models was inhibited through CDK4/6 inhibition in an RB-dependent manner. Finally, in a cohort of DCIS cases, we show that, although elevated levels of ErbB2 are associated with increased risk of a subsequent DCIS recurrence, it is not associated with progression to invasive disease. In contrast, RB loss in ErbB2-positive DCIS cases was associated with increased risk for invasive breast cancer. Taken together, these data demonstrate a key role for the RB pathway in invasion associated with breast tumor progression, and shed light on the key molecular events that promote the progression of DCIS to invasive disease.

Show MeSH
Related in: MedlinePlus