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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 compromises the integrity of cell-cell adhesion complexes(A) Immunoblot analysis of MCF10A ErbB2 cells either transduced with miNS or miRB are shown for the indicated proteins. (B) Representative images of cells in 2D cultured stained for the indicated cytokeratins. (C) Representative images of CK18 stained in 3D cultures. (D) Representative images of cells in 2D stained for junctional plakoglobin (JUP). (E) Representative images of cell in 2D cultured stained for E-cadherin (green) and Phalloidin (red) (top panel) and acini stained for E-cadherin (red) are shown. Leading edges of RB-deficient cells displaying decreased E-cadherin expression are highlighted (arrows, bottom panel). (F) Representative images for Laminin V in 3D cultures.
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Figure 3: RB loss compromises the integrity of cell-cell adhesion complexes(A) Immunoblot analysis of MCF10A ErbB2 cells either transduced with miNS or miRB are shown for the indicated proteins. (B) Representative images of cells in 2D cultured stained for the indicated cytokeratins. (C) Representative images of CK18 stained in 3D cultures. (D) Representative images of cells in 2D stained for junctional plakoglobin (JUP). (E) Representative images of cell in 2D cultured stained for E-cadherin (green) and Phalloidin (red) (top panel) and acini stained for E-cadherin (red) are shown. Leading edges of RB-deficient cells displaying decreased E-cadherin expression are highlighted (arrows, bottom panel). (F) Representative images for Laminin V in 3D cultures.

Mentions: To functionally validate the gene expression data, western blotting and immunofluorescence microscopy was performed. As shown, there was significant down-regulation of multiple cytokeratins at the protein level with RB-knockdown in ErbB2-expressing MCF10A cells (Figure 3A). There was a concordant reduction in cytokeratin expression as determined by fluorescence microscopy in 2D cultures (Figure 3B). These findings were also apparent in 3D cultures (Figure 3C). Correspondingly, junctional plakaglobin (JUP), which is a critical adhesion molecule, was significantly reduced consistent with the gene expression analyses (Figure 3D). While the levels of E-Cadherin were only modestly reduced as determined by microarray; there was a deficit in E-Cadherin expression/localization that was apparent in disorganized acinar structures at and the leading edge of ErbB2-positive and RB-deficient cells grown in 2D (Figure 3D). Together these data indicate that there is substantial decrease in the expression of key epithelial specific proteins that are important for tissue architecture and invasion. An important characteristic of normal acini is the deposition of basement membrane components such as Laminin V (36). While uniform basal Laminin V expression was displayed around RB-proficient ErbB2 over expressing acini, RB-deficient ErbB2 over expressing cells harbored diffuse Laminin V expression throughout the acini, indicating disruption of cellular polarization (Figure 3E). Taken together, these data indicate that upon RB loss in ErbB2 over expressing cells, there is a disruption of cell polarity and defective cell-cell junction complexes through both changes in gene expression and molecule localization that could lead to the deregulation of key cellular functions such as 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 loss compromises the integrity of cell-cell adhesion complexes(A) Immunoblot analysis of MCF10A ErbB2 cells either transduced with miNS or miRB are shown for the indicated proteins. (B) Representative images of cells in 2D cultured stained for the indicated cytokeratins. (C) Representative images of CK18 stained in 3D cultures. (D) Representative images of cells in 2D stained for junctional plakoglobin (JUP). (E) Representative images of cell in 2D cultured stained for E-cadherin (green) and Phalloidin (red) (top panel) and acini stained for E-cadherin (red) are shown. Leading edges of RB-deficient cells displaying decreased E-cadherin expression are highlighted (arrows, bottom panel). (F) Representative images for Laminin V in 3D cultures.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: RB loss compromises the integrity of cell-cell adhesion complexes(A) Immunoblot analysis of MCF10A ErbB2 cells either transduced with miNS or miRB are shown for the indicated proteins. (B) Representative images of cells in 2D cultured stained for the indicated cytokeratins. (C) Representative images of CK18 stained in 3D cultures. (D) Representative images of cells in 2D stained for junctional plakoglobin (JUP). (E) Representative images of cell in 2D cultured stained for E-cadherin (green) and Phalloidin (red) (top panel) and acini stained for E-cadherin (red) are shown. Leading edges of RB-deficient cells displaying decreased E-cadherin expression are highlighted (arrows, bottom panel). (F) Representative images for Laminin V in 3D cultures.
Mentions: To functionally validate the gene expression data, western blotting and immunofluorescence microscopy was performed. As shown, there was significant down-regulation of multiple cytokeratins at the protein level with RB-knockdown in ErbB2-expressing MCF10A cells (Figure 3A). There was a concordant reduction in cytokeratin expression as determined by fluorescence microscopy in 2D cultures (Figure 3B). These findings were also apparent in 3D cultures (Figure 3C). Correspondingly, junctional plakaglobin (JUP), which is a critical adhesion molecule, was significantly reduced consistent with the gene expression analyses (Figure 3D). While the levels of E-Cadherin were only modestly reduced as determined by microarray; there was a deficit in E-Cadherin expression/localization that was apparent in disorganized acinar structures at and the leading edge of ErbB2-positive and RB-deficient cells grown in 2D (Figure 3D). Together these data indicate that there is substantial decrease in the expression of key epithelial specific proteins that are important for tissue architecture and invasion. An important characteristic of normal acini is the deposition of basement membrane components such as Laminin V (36). While uniform basal Laminin V expression was displayed around RB-proficient ErbB2 over expressing acini, RB-deficient ErbB2 over expressing cells harbored diffuse Laminin V expression throughout the acini, indicating disruption of cellular polarization (Figure 3E). Taken together, these data indicate that upon RB loss in ErbB2 over expressing cells, there is a disruption of cell polarity and defective cell-cell junction complexes through both changes in gene expression and molecule localization that could lead to the deregulation of key cellular functions such as 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