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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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Combined effect of ErbB2- and RB-pathway deregulation on acinar morphology(A) Phase contrast images of acini (10X) were used to measure acini diameters and > 50 acini were scored for each genotype; bars, SD (***P < .0001) Phalloidin staining (red) was performed on acini grown for 10days (40x). (B) Phase contrast images of acini (20X) were used to visualize stellate acinar structures. Representative images are shown. (C) Gene expression data comparing MCF10A/ErbB2 miNS and MCF10A/ErbB2 miRB grown in 3D cultures. Changes in gene expression are displayed as a heat map (left panel). GSEA associated with the Taube-EMT signature (middle panel) and representative genes with corresponding fold change and P-values are highlighted (right panel).
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Figure 2: Combined effect of ErbB2- and RB-pathway deregulation on acinar morphology(A) Phase contrast images of acini (10X) were used to measure acini diameters and > 50 acini were scored for each genotype; bars, SD (***P < .0001) Phalloidin staining (red) was performed on acini grown for 10days (40x). (B) Phase contrast images of acini (20X) were used to visualize stellate acinar structures. Representative images are shown. (C) Gene expression data comparing MCF10A/ErbB2 miNS and MCF10A/ErbB2 miRB grown in 3D cultures. Changes in gene expression are displayed as a heat map (left panel). GSEA associated with the Taube-EMT signature (middle panel) and representative genes with corresponding fold change and P-values are highlighted (right panel).

Mentions: While RB-deficiency had little impact on the proliferation of ErbB2 over expressing mammary cells, a noticeable impact on the morphology of the acinar structures was observed (Figure 2A). RB-proficient and RB-deficient cells generated spherical acini with a hollow lumen, and well-organized actin expression throughout the epithelial cells as determined utilizing phalloidin staining (Figure 2A, top left panels). Consistent with the enhanced proliferation observed with RB loss in MCF10A acini, an increase in acini volume was observed (Figure 2A, bottom left panel). However, as previously shown (22), ErbB2 over expressing MCF10A cells displayed a multi-acinar phenotype and absence of a hollow lumen (Figure 2A, top right panels). Consistent with the minimum difference in proliferation rates, minimal difference in acinar volume was observed with RB-deficiency in cells expressing ErbB2 (Figure 2A, bottom right panel). However, the combined perturbation of the ErbB2- and RB-pathways resulted in further disorganization of the structures, with the appearance of “stellate” acinar morphology as shown by phase contrast images (34), with cellular protrusions escaping from the acinar structure into the surrounding matrigel (Figure 2A, right panels). The appearance of aberrant invasion into the surrounding matrix could be observed as early as 3 days post-plating (not shown), and was clearly apparent within 6 days (Figure 2B).


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)

Combined effect of ErbB2- and RB-pathway deregulation on acinar morphology(A) Phase contrast images of acini (10X) were used to measure acini diameters and > 50 acini were scored for each genotype; bars, SD (***P < .0001) Phalloidin staining (red) was performed on acini grown for 10days (40x). (B) Phase contrast images of acini (20X) were used to visualize stellate acinar structures. Representative images are shown. (C) Gene expression data comparing MCF10A/ErbB2 miNS and MCF10A/ErbB2 miRB grown in 3D cultures. Changes in gene expression are displayed as a heat map (left panel). GSEA associated with the Taube-EMT signature (middle panel) and representative genes with corresponding fold change and P-values are highlighted (right panel).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Combined effect of ErbB2- and RB-pathway deregulation on acinar morphology(A) Phase contrast images of acini (10X) were used to measure acini diameters and > 50 acini were scored for each genotype; bars, SD (***P < .0001) Phalloidin staining (red) was performed on acini grown for 10days (40x). (B) Phase contrast images of acini (20X) were used to visualize stellate acinar structures. Representative images are shown. (C) Gene expression data comparing MCF10A/ErbB2 miNS and MCF10A/ErbB2 miRB grown in 3D cultures. Changes in gene expression are displayed as a heat map (left panel). GSEA associated with the Taube-EMT signature (middle panel) and representative genes with corresponding fold change and P-values are highlighted (right panel).
Mentions: While RB-deficiency had little impact on the proliferation of ErbB2 over expressing mammary cells, a noticeable impact on the morphology of the acinar structures was observed (Figure 2A). RB-proficient and RB-deficient cells generated spherical acini with a hollow lumen, and well-organized actin expression throughout the epithelial cells as determined utilizing phalloidin staining (Figure 2A, top left panels). Consistent with the enhanced proliferation observed with RB loss in MCF10A acini, an increase in acini volume was observed (Figure 2A, bottom left panel). However, as previously shown (22), ErbB2 over expressing MCF10A cells displayed a multi-acinar phenotype and absence of a hollow lumen (Figure 2A, top right panels). Consistent with the minimum difference in proliferation rates, minimal difference in acinar volume was observed with RB-deficiency in cells expressing ErbB2 (Figure 2A, bottom right panel). However, the combined perturbation of the ErbB2- and RB-pathways resulted in further disorganization of the structures, with the appearance of “stellate” acinar morphology as shown by phase contrast images (34), with cellular protrusions escaping from the acinar structure into the surrounding matrigel (Figure 2A, right panels). The appearance of aberrant invasion into the surrounding matrix could be observed as early as 3 days post-plating (not shown), and was clearly apparent within 6 days (Figure 2B).

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