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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 plays a role in preventing invasive tumor growth in vivo(A) Dual staining for ErbB2 (red) and SMA (green) in SKBR3 miNS and SKBR3 miRB injected mammary glands. (B) Representative H&E, Smooth muscle actin (SMA) and Ki67 staining in SKBR3 miNS and SKBR3 miRB lesions are shown (left panels). Table depicting percentage of DCIS and invasive lesions from SKBR3 miNS and SKBR3 miRB orthotopic injections (n=15) (right panel)
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Figure 6: RB plays a role in preventing invasive tumor growth in vivo(A) Dual staining for ErbB2 (red) and SMA (green) in SKBR3 miNS and SKBR3 miRB injected mammary glands. (B) Representative H&E, Smooth muscle actin (SMA) and Ki67 staining in SKBR3 miNS and SKBR3 miRB lesions are shown (left panels). Table depicting percentage of DCIS and invasive lesions from SKBR3 miNS and SKBR3 miRB orthotopic injections (n=15) (right panel)

Mentions: Several commonly utilized breast cancer cell lines have been classified as weakly invasive, such as ZR75-1, MDA-MB-453, and SKBR3 cells (40). To examine the ability of RB loss to enhance the invasive potential of ErbB2 over expressing breast cancer cells in vivo, we injected RB-proficient and RB-deficient ErbB2-positive SKBR3 cells into the mammary fat pad of ten-week-old NOD/SCID mice. This model system gave rise to ErbB2 over expressing DCIS lesions after six months (Figure 6A). Smooth muscle actin (SMA) and ErbB2 staining was used to demonstrate ErbB2 over expressing DCIS lesions confined by a continuous myoepithelial layer (Figure 6A). Importantly, this confined structure was dependent on the presence of RB, as RB-deficient SKBR3 lesions displayed an invasive phenotype as indicated by the poorly defined SMA expression (Figure 6A and 6B) and invasion of proliferative cells into the surrounding stroma (Ki67, Figure 6B). Consistent with our in vitro analyses, no difference in proliferation was observed between the RB-proficient and RB-deficient lesions (Ki67, Figure 6B), indicating that the impact of RB loss in the context of ErbB2 over expression is predominantly associated with cell adhesion and epithelial cell motility.


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 plays a role in preventing invasive tumor growth in vivo(A) Dual staining for ErbB2 (red) and SMA (green) in SKBR3 miNS and SKBR3 miRB injected mammary glands. (B) Representative H&E, Smooth muscle actin (SMA) and Ki67 staining in SKBR3 miNS and SKBR3 miRB lesions are shown (left panels). Table depicting percentage of DCIS and invasive lesions from SKBR3 miNS and SKBR3 miRB orthotopic injections (n=15) (right panel)
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Related In: Results  -  Collection

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Show All Figures
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Figure 6: RB plays a role in preventing invasive tumor growth in vivo(A) Dual staining for ErbB2 (red) and SMA (green) in SKBR3 miNS and SKBR3 miRB injected mammary glands. (B) Representative H&E, Smooth muscle actin (SMA) and Ki67 staining in SKBR3 miNS and SKBR3 miRB lesions are shown (left panels). Table depicting percentage of DCIS and invasive lesions from SKBR3 miNS and SKBR3 miRB orthotopic injections (n=15) (right panel)
Mentions: Several commonly utilized breast cancer cell lines have been classified as weakly invasive, such as ZR75-1, MDA-MB-453, and SKBR3 cells (40). To examine the ability of RB loss to enhance the invasive potential of ErbB2 over expressing breast cancer cells in vivo, we injected RB-proficient and RB-deficient ErbB2-positive SKBR3 cells into the mammary fat pad of ten-week-old NOD/SCID mice. This model system gave rise to ErbB2 over expressing DCIS lesions after six months (Figure 6A). Smooth muscle actin (SMA) and ErbB2 staining was used to demonstrate ErbB2 over expressing DCIS lesions confined by a continuous myoepithelial layer (Figure 6A). Importantly, this confined structure was dependent on the presence of RB, as RB-deficient SKBR3 lesions displayed an invasive phenotype as indicated by the poorly defined SMA expression (Figure 6A and 6B) and invasion of proliferative cells into the surrounding stroma (Ki67, Figure 6B). Consistent with our in vitro analyses, no difference in proliferation was observed between the RB-proficient and RB-deficient lesions (Ki67, Figure 6B), indicating that the impact of RB loss in the context of ErbB2 over expression is predominantly associated with cell adhesion and epithelial cell motility.

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