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

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.

Figures

Figure 1. RB-deficient or ErbB2 over expression promotes bypass of acini growth arrest

(A) Cells stably expressing miRB or miNS were analyzed by immunoblot for the indicated proteins (top panel). BrdU incorporation was analyzed by bivariate flow cytometry and data are the average of at least three independent experiments; bars, SD (bottom panel). (B) Ki67 staining (green) on 10 day acini, (40X) and Ki67 quantification from at least three independent experiments scoring a minimum of 20 acinus; bars, SD (***P < .0001). (C) Cells were infected with ErbB2 lentivirus and subsequently infected with miRB or miNS retroviruses. Cells were harvested and cell lysates were analyzed by immunoblot for the indicated proteins (top panel). BrdU incorporation was analyzed by bivariate flow cytometry and data are the average of at least three independent experiments; bars, SD (bottom panel). (D) Ki67 staining (green) on 10 day acini (40X) and Ki67 quantification from at least three independent experiments scoring a minimum of 20 acinus; bars, SD.

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).

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).

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.

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).

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).

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)

Figure 7. RB deficiency is associated with increased disease progression in DCIS

(A) ErbB2 high (3+) status was compared against all other ErbB2 staining (0, 1+, 2+) for any recurrent disease (ipsilateral breast event) or invasive progression using Kaplan-Meier analysis. ErbB2 high was associated with risk of a subsequent ipsilateral breast event, but not progression to invasive disease. (B) Representative images of RB staining in DCIS lesions cases with high ErbB2. (C) ErbB2 high (3+) cases were stratified by RB status (positive vs. negative). Cases with RB loss in this subtype were associated with both risk of a subsequent ipsilateral breast event and progression to invasive disease.