The retinoblastoma tumor suppressor modifies the therapeutic response of breast cancer

Abstract

The retinoblastoma tumor suppressor (RB) protein is functionally inactivated in the majority of human cancers and is aberrant in one-third of all breast cancers. RB regulates G(1)/S-phase cell-cycle progression and is a critical mediator of antiproliferative signaling. Here the specific impact of RB deficiency on E2F-regulated gene expression, tumorigenic proliferation, and the response to 2 distinct lines of therapy was investigated in breast cancer cells. RB knockdown resulted in RB/E2F target gene deregulation and accelerated tumorigenic proliferation, thereby demonstrating that even in the context of a complex tumor cell genome, RB status exerts significant control over proliferation. Furthermore, the RB deficiency compromised the short-term cell-cycle inhibition following cisplatin, ionizing radiation, and antiestrogen therapy. In the context of DNA-damaging agents, this bypass resulted in increased sensitivity to these agents in cell culture and xenograft models. In contrast, the bypass of antiestrogen signaling resulted in continued proliferation and xenograft tumor growth in the presence of tamoxifen. These effects of aberrations in RB function were recapitulated by ectopic E2F expression, indicating that control of downstream target genes was an important determinant of the observed responses. Specific analyses of an RB gene expression signature in 60 human patients indicated that deregulation of this pathway was associated with early recurrence following tamoxifen monotherapy. Thus, because the RB pathway is a critical determinant of tumorigenic proliferation and differential therapeutic response, it may represent a critical basis for directing therapy in the treatment of breast cancer.

Figure 1. Efficient RB knockdown in breast cancer cells causes deregulation of RB/E2F target genes and increased proliferation kinetics.

(A) MCF7 cells transfected with MSCV donor or MSCV siRb plasmids were selected with puromycin to isolate stable clones. Clones were screened by RB immunofluorescence as shown for MCF7 donor 1 and siRb28. Images were captured at equal exposures. Original magnification, ×20. (B) Lysates from MCF7 donor 1 and siRb28 clones were immunoblotted for expression levels of RB, PCNA, MCM7, cyclin E, cyclin A, cyclin D1, and p16INK4a. Cdk4 served as a loading control. (C) Cells represented in A were BrdU labeled for 10 hours, and BrdU immunofluorescence was performed and scored. (D) Cells represented in A were seeded at 3 × 10⁵, cell growth assays were carried out for 9 days, and cells were counted every 3 days. (E) Lysates represented in B along with lysates from polyclonal populations of T47D and Zr-75-1 cells infected with retrovirus encoding donor or siRb88 plasmids were immunoblotted for expression levels of RB and cyclin D1. Lamin B served as a loading control. (F) Retrovirally infected T47D and Zr-75-1 cells represented in E were seeded at 3 × 10⁵, and growth assays were carried out as described for D.

Figure 2. Tumor growth in nude mouse xenografts is accelerated in RB-knockdown cells.

(A) MCF7 donor 1 or siRb28 cells were harvested and resuspended in 3:1 PBS/Matrigel mixture. Cells (2 × 10⁶) in 150 μl of mixture were injected subcutaneously in a contralateral manner in flanks of ovariectomized nude mice. Mice were implanted with E2 pellets, and tumors were measured every 4 days. (B) Excised tumors were weighed 30 days after implantation. Tumor weights were plotted, and a 2-tailed Student’s t test assuming unequal variances was used to determine significance. Below: relative tumor size after excision 30 days following implantation. Scale bar: 1 cm. (C) Nude mice represented in A were injected with BrdU 1 hour prior to sacrifice. Sectioned tumors were immunohistochemically stained and scored for BrdU incorporation, and statistical analyses were carried out as described for B.

Figure 3. RB deficiency enables bypass of the DNA damage checkpoint, resulting in increased sensitivity.

(A) Wild-type MCF7, T47D, and Zr-75-1 cells were treated with 0, 8, or 16 μM CDDP for 18 hours, washed, and labeled with BrdU for 10 hours in culture. Cells were then fixed, and BrdU immunofluorescence was performed and scored. (B) Retrovirally infected T47D (left) and Zr-75-1 (right) donor and siRb88 cells were treated with 0, 8, or 16 μM CDDP and BrdU labeled as described for A. (C) MCF7 donor 1 and siRb28 clones were treated with 0, 8, or 16 μM CDDP for 18 hours prior to washing (left) or with 0, 2.5, or 5 Gy IR (right) and BrdU labeled as described above. (D) MCF7 donor 1 or siRb28 cells were seeded at 3 × 10⁵ and treated with 2.5 (left) or 5 Gy IR (right), and cell growth assays were performed for 12 days and cells counted every 3 days. (E) Harvested MCF7 donor 1 and siRb28 cells were resuspended 3:1 in PBS/Matrigel and injected subcutaneously into the flanks of mice implanted with E2 pellets. When xenograft tumors reached approximately 110 mm³ during tumor development, mice were treated with CDDP (the E2 pellet was retained, and 5 mg/kg CDDP was injected i.p. every 4 days for 5 courses), and tumor size was monitored by caliper measurement. Tumor measurements are plotted, and a 2-tailed Student’s t test assuming unequal variances was used to determine significance of curves. (F) Tumors represented in E were weighed upon excision.

Figure 4. RB is necessary for sensitivity to antiestrogen therapy and long-term growth arrest.

(A) Lysates from wild-type MCF7, T47D, and Zr-75-1 cells were immunoblotted for the expression levels of RB, RB phospho-Ser780, cyclin D1, and p16INK4a. Lamin B served as a loading control, while lysates from U2OS and SaOS2 cells were included as controls for RB and p16INK4a expression, respectively. (B) MCF7 donor 1 and siRb28 clones were cultured in media containing FBS, CDT, CDT/Tam, or CDT/ICI for 3 days and were BrdU labeled for the final 10 hours of culture. Cells were then fixed, and BrdU immunofluorescence was performed and scored. A 2-tailed Student’s t test assuming unequal variances was utilized to determine significance. (C) T47D (left) and Zr-75-1 (right) donor and siRb88 cells were cultured, BrdU labeled, and scored as described for B. Statistical tests were performed as described for B. (D) MCF7 donor 1 or siRb28 cells were seeded at 3 × 10⁵, and cell growth assays were performed for 9 days while cells were cultured in CDT/Tam and counted every 3 days. (E) T47D (left) and Zr-75-1 (right) donor and siRb88 cells were seeded at 3 × 10⁵, and cell growth assays were performed as described for D. (F) When xenograft tumors (as in Figure 3E) reached 100–120 mm³, mice were treated with Tam (E2 pellet was removed and Tam pellet was added). Tumor size of the Tam-treated animals was monitored by calipers. (G) Final tumor weights of all tumors represented in F upon excision.

Figure 5. E2F3 overexpression in MCF7 cells allows bypass of antimitogenic checkpoints.

(A) MCF7 cells infected with adenoviral vectors encoding either lacZ or E2F3 were harvested 3 days after infection, lysed, separated by SDS-PAGE, and immunoblotted for determination of E2F3, RB, MCM7, and PCNA expression levels. Cdk4 served as a loading control. (B) The adenovirus-infected cells represented in A were cultured in media containing FBS, CDT, CDT/Tam, or CDT/ICI for 3 days or were treated as described above with 16 μM CDDP or 5 Gy IR prior to BrdU labeling for 10 hours. Cells were then fixed, and BrdU immunofluorescence and scoring were performed.

Figure 6. RB/E2F downstream target deregulation correlates with poor prognosis in human breast cancers treated with Tam monotherapy.

(A) Gene expression data from 60 ER-positive human breast tumors that were both micro- and macrodissected were analyzed for RB/E2F target gene expression using GeneSpring. The expression patterns of 59 known RB/E2F target genes and the expression levels of Rb, p16, cyclin D1, and cyclin E are displayed in a condition tree for each of the 2 tissue samples from each patient (120 samples). The average RB/E2F target gene expression levels of all 59 genes were categorized into 3 groups: low, medium, and high. Three HER2/neu-positive tumors (a, b, and c) were present in this tumor set. (B) The RB/E2F target gene expression levels in each group represented in C were averaged and displayed as a box-and-whisker plot. A 2-tailed Student’s t test assuming unequal variances was utilized to determine significance (P = 7.2 × 10^–12 low/medium; P = 1.3 × 10^–14 medium/high). (C) The survival data for each of the 60 patients from the low/medium and high gene expression groups represented in C was compiled into a disease-free survival curve. Statistical tests were performed as described for B.