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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-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.
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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.

Mentions: In order to interrogate the individual and combined effect of RB loss and ErbB2 over expression, we utilized the immortalized mammary epithelial cell line MCF10A. Stable polyclonal RB-knockdown MCF10A cell line (MCF10A miRB) and scrambled control cell line (MCF10A miNS) are shown in Figure 1A. Biochemical analysis of the expression of downstream RB/E2F target genes (Cyclin A and MCM7) demonstrated minimal differences in protein expression from cells grown in culture as a monolayer (Figure 1A, top panel), as well as minimal change in proliferation as shown by BrdU incorporation (Figure 1A, bottom panel). To interrogate the phenotypic effects of pathway perturbation on the glandular organization of mammary epithelia, 3D cultures were employed (32, 33). RB-proficient MCF10A cells (miNS) generated normal acinar structures displaying growth arrest by 10 days as depicted by Ki67 staining. In contrast, RB-deficient cells showed approximately a 30% proliferation rate even after 10 days of culture (Figure 1B). To investigate the impact of RB loss in the context of ErbB2 over expression, we over expressed ErbB2 in RB-deficient MCF10A cells as shown in Figure 1C. These cultures exhibited similar levels of ErbB2 as observed in ErbB2 positive breast cancer cell lines, and demonstrated relatively uniform loss of RB throughout the culture as determined by immunofluorescence microscopy (Supplemental Figure 1). Similar to results displayed in Figure 1A, RB depletion resulted in minimal alterations in the expression of RB/E2F target genes (Cyclin A, MCM7) in ErbB2 over expressing MCF10A cells, and proliferation rates remained virtually unchanged compared to RB-proficient cells (Figure 1C). In the presence of ErbB2 over expression, RB-proficient and RB-deficient acinar structures exhibited similar proliferation rates as determined by Ki67 staining (Figure 1D). Thus, in the context of proliferation, loss of RB has little impact over that observed by ErbB2 over expression alone.


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-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.
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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.
Mentions: In order to interrogate the individual and combined effect of RB loss and ErbB2 over expression, we utilized the immortalized mammary epithelial cell line MCF10A. Stable polyclonal RB-knockdown MCF10A cell line (MCF10A miRB) and scrambled control cell line (MCF10A miNS) are shown in Figure 1A. Biochemical analysis of the expression of downstream RB/E2F target genes (Cyclin A and MCM7) demonstrated minimal differences in protein expression from cells grown in culture as a monolayer (Figure 1A, top panel), as well as minimal change in proliferation as shown by BrdU incorporation (Figure 1A, bottom panel). To interrogate the phenotypic effects of pathway perturbation on the glandular organization of mammary epithelia, 3D cultures were employed (32, 33). RB-proficient MCF10A cells (miNS) generated normal acinar structures displaying growth arrest by 10 days as depicted by Ki67 staining. In contrast, RB-deficient cells showed approximately a 30% proliferation rate even after 10 days of culture (Figure 1B). To investigate the impact of RB loss in the context of ErbB2 over expression, we over expressed ErbB2 in RB-deficient MCF10A cells as shown in Figure 1C. These cultures exhibited similar levels of ErbB2 as observed in ErbB2 positive breast cancer cell lines, and demonstrated relatively uniform loss of RB throughout the culture as determined by immunofluorescence microscopy (Supplemental Figure 1). Similar to results displayed in Figure 1A, RB depletion resulted in minimal alterations in the expression of RB/E2F target genes (Cyclin A, MCM7) in ErbB2 over expressing MCF10A cells, and proliferation rates remained virtually unchanged compared to RB-proficient cells (Figure 1C). In the presence of ErbB2 over expression, RB-proficient and RB-deficient acinar structures exhibited similar proliferation rates as determined by Ki67 staining (Figure 1D). Thus, in the context of proliferation, loss of RB has little impact over that observed by ErbB2 over expression alone.

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