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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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Activation of the RB pathway prevents abnormal multi-acini growth and cell invasion(A) RB-proficient MCF10A/ErbB2 cells were treated with 0.5μM PD-0332991 for 24h. Cells were harvested and cell lysates were analyzed by immunoblot for the indicated proteins (left panel). BrdU incorporation was analyzed by bivariate flow cytometry and data are the average of at least three independent experiments; bars, SD (***P < .0001) (middle panel). Ki67 and phalloidin co-staining of MCF10A/ErbB2 acini treated with 0.5μM PD-0332991 for 72h (20X) (right panel). (B) RB-proficient MCF10A/ErbB2 cells grown in 3D culture and treated with 0.5μM PD0332991 for 72h, (20X) (left panel). Analysis of cell migration by Boyden Chamber assays supplemented with 0.5μM PD0332991 was performed; bars, SD. (**P=.003) (right panel). (C) RB-proficient BT474 cells were treated with PD-0332991 and analyzed by immunoblotting, BrdU incorporation and proliferation in 3D culture. BrdU incorporation data are the average of at least three independent experiments; bars, SD (***P < .0001). (D) RB-proficient BT474 cells were treated for growth in 3D culture and cell migration. Migration data are the average of at least three independent experiments; bars, SD (**P < 0.0037).
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Figure 5: Activation of the RB pathway prevents abnormal multi-acini growth and cell invasion(A) RB-proficient MCF10A/ErbB2 cells were treated with 0.5μM PD-0332991 for 24h. Cells were harvested and cell lysates were analyzed by immunoblot for the indicated proteins (left panel). BrdU incorporation was analyzed by bivariate flow cytometry and data are the average of at least three independent experiments; bars, SD (***P < .0001) (middle panel). Ki67 and phalloidin co-staining of MCF10A/ErbB2 acini treated with 0.5μM PD-0332991 for 72h (20X) (right panel). (B) RB-proficient MCF10A/ErbB2 cells grown in 3D culture and treated with 0.5μM PD0332991 for 72h, (20X) (left panel). Analysis of cell migration by Boyden Chamber assays supplemented with 0.5μM PD0332991 was performed; bars, SD. (**P=.003) (right panel). (C) RB-proficient BT474 cells were treated with PD-0332991 and analyzed by immunoblotting, BrdU incorporation and proliferation in 3D culture. BrdU incorporation data are the average of at least three independent experiments; bars, SD (***P < .0001). (D) RB-proficient BT474 cells were treated for growth in 3D culture and cell migration. Migration data are the average of at least three independent experiments; bars, SD (**P < 0.0037).

Mentions: Our data indicate that RB loss cooperates with ErbB2 in promoting invasive behavior. To investigate whether activation of the RB-pathway could have an inhibitory effect on the invasive phenotype observed with ErbB2 over expression, we used a highly specific CDK4/6 inhibitor PD-0332991 (PD) (37, 38). The ability of PD0332991 to decrease RB/E2F gene expression and inhibit cell proliferation in MCF10A cells has been previously established (39). As shown in Figure 5A, inhibition of CDK4/6 was exceedingly effective at repressing RB/E2F gene expression (MCM7, Cyclin A, PCNA) and cell proliferation even in cells harboring ErbB2 over expression. This inhibition of proliferation was observed in both 2D and 3D cell cultures (Figure 5A). Interestingly, CDK4/6 inhibition was also effective at preventing the formation of multi-acini structures in 3D culture (Figure 5B, left panel), and significantly inhibited cell invasion (Figure 5B, right panel). These effects were dependent on RB status as shown by the minimal effect of PD0332991 on invasive phenotype of RB-deficient cells (Figure 5B, right panel).


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)

Activation of the RB pathway prevents abnormal multi-acini growth and cell invasion(A) RB-proficient MCF10A/ErbB2 cells were treated with 0.5μM PD-0332991 for 24h. Cells were harvested and cell lysates were analyzed by immunoblot for the indicated proteins (left panel). BrdU incorporation was analyzed by bivariate flow cytometry and data are the average of at least three independent experiments; bars, SD (***P < .0001) (middle panel). Ki67 and phalloidin co-staining of MCF10A/ErbB2 acini treated with 0.5μM PD-0332991 for 72h (20X) (right panel). (B) RB-proficient MCF10A/ErbB2 cells grown in 3D culture and treated with 0.5μM PD0332991 for 72h, (20X) (left panel). Analysis of cell migration by Boyden Chamber assays supplemented with 0.5μM PD0332991 was performed; bars, SD. (**P=.003) (right panel). (C) RB-proficient BT474 cells were treated with PD-0332991 and analyzed by immunoblotting, BrdU incorporation and proliferation in 3D culture. BrdU incorporation data are the average of at least three independent experiments; bars, SD (***P < .0001). (D) RB-proficient BT474 cells were treated for growth in 3D culture and cell migration. Migration data are the average of at least three independent experiments; bars, SD (**P < 0.0037).
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Figure 5: Activation of the RB pathway prevents abnormal multi-acini growth and cell invasion(A) RB-proficient MCF10A/ErbB2 cells were treated with 0.5μM PD-0332991 for 24h. Cells were harvested and cell lysates were analyzed by immunoblot for the indicated proteins (left panel). BrdU incorporation was analyzed by bivariate flow cytometry and data are the average of at least three independent experiments; bars, SD (***P < .0001) (middle panel). Ki67 and phalloidin co-staining of MCF10A/ErbB2 acini treated with 0.5μM PD-0332991 for 72h (20X) (right panel). (B) RB-proficient MCF10A/ErbB2 cells grown in 3D culture and treated with 0.5μM PD0332991 for 72h, (20X) (left panel). Analysis of cell migration by Boyden Chamber assays supplemented with 0.5μM PD0332991 was performed; bars, SD. (**P=.003) (right panel). (C) RB-proficient BT474 cells were treated with PD-0332991 and analyzed by immunoblotting, BrdU incorporation and proliferation in 3D culture. BrdU incorporation data are the average of at least three independent experiments; bars, SD (***P < .0001). (D) RB-proficient BT474 cells were treated for growth in 3D culture and cell migration. Migration data are the average of at least three independent experiments; bars, SD (**P < 0.0037).
Mentions: Our data indicate that RB loss cooperates with ErbB2 in promoting invasive behavior. To investigate whether activation of the RB-pathway could have an inhibitory effect on the invasive phenotype observed with ErbB2 over expression, we used a highly specific CDK4/6 inhibitor PD-0332991 (PD) (37, 38). The ability of PD0332991 to decrease RB/E2F gene expression and inhibit cell proliferation in MCF10A cells has been previously established (39). As shown in Figure 5A, inhibition of CDK4/6 was exceedingly effective at repressing RB/E2F gene expression (MCM7, Cyclin A, PCNA) and cell proliferation even in cells harboring ErbB2 over expression. This inhibition of proliferation was observed in both 2D and 3D cell cultures (Figure 5A). Interestingly, CDK4/6 inhibition was also effective at preventing the formation of multi-acini structures in 3D culture (Figure 5B, left panel), and significantly inhibited cell invasion (Figure 5B, right panel). These effects were dependent on RB status as shown by the minimal effect of PD0332991 on invasive phenotype of RB-deficient cells (Figure 5B, right panel).

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