Figure 1.
Status of pRb protein in human triple negative breast cancer (TNBC) cell lines.
Western blot analysis of pRb and phospho-ppRb-Ser795 in TNBC derived lines. HCC1937 expresses mutant pRb. MDA-MB-231, MDA-MB-435, MDA-MB-436, MDA-MB-157, Bt549, Du4475, Hs578T and HCC38 are claudin-low (BaB – depicted in orange). HCC70, HCC1937, HCC1954, HCC1187, HCC1569 and MDA-MB-468 are basal-like (BaA – depicted in green). MCF7, a luminal BC line, and 293T, a transformed kidney epithelium line, were used as control. Tubulin served as a loading control.
Figure 2.
Status of RB1 gene in human triple negative breast cancer (TNBC) cell lines.
(A) RB1 RNA expression correlates with pRb protein expression. Top, ratio of pRb to Tubulin from Figure 1. Botton, RNA expression of RB1 in indicated cell lines in 4 different studies and Pearson's correlation (r) relative to pRb protein expression. (B) Microarray analysis of cyclins and Cdks on the RB-pathway in human derived breast cancer cell lines, clustered according to subtype, from the 4 data sets. Cyclin E1 was consistently elevated in basal-A but not in basal-B tumors.
Figure 3.
Effect of RB1 status on response to radiation and anti-neoplastic drugs of TNBC lines.
Treatment with (A) gamma-irradiation, (B–C) CDK4/6 inhibitor PD-0332991, (D) doxorubicin, (E) 5-fluorouracil, (F) CDDP, and (G) methotrexate. RB+ lines: MDA-MB-231, HCC38, Hs578t and HCC70. RB− lines: MDA-MB-468, MDA-MB-436, Bt549 and Du4475. Values represent the average of 3–4 assays, each performed in triplicate. p-values for gamma-irradiation by two-tailed t-test: 5 Gy, p = 0.0610; 10 Gy, p = 0.0024. p-values for curve sets calculated using nonlinear regression analysis: doxorubicin, p = 0.0140; CDDP, p = 0.0515; 5-fluorouracil, p = 0.0187; methotrexate, p = 0.0043.
Figure 4.
Similar drug sensitivity observed following high-throughput screen of ∼3400 drugs against human RB+ vs. RB− TNBC lines.
RB+ lines (red): HCC70 and MDA-MB-231. RB− lines (blue): MDA-MB-436 and Bt549. (A) Top 25 hits from Sequoia library (1 µM, 268 drugs) ranked by average efficacy. Shaded values represent viability ≤50%. (B–D) Sensitivity of RB− cells relative to RB+ cells following screens with the Sequoia, Spectrum (1 µM, 2000 drugs) and Prestwick (0.8 µM, 1185 drugs) libraries. Arrow points to phenylmercuric acetate (see text).
Figure 5.
Effect of RB1 status on response of 14 human derived TNBC lines to idarubicin or epirubicin.
(A) Response of each individual line. Values represent the average of 3–5 assays, each performed in triplicate. (B) Average response based on RB1 status. RB+ lines: MDA-MB-231, HCC38, Hs578t, MDA-MB-157, HCC1954, HCC1569, HCC3153, SUM149 and HCC70. RB− lines: MDA-MB-436, MDA-MB-468, Bt549, Du4475 and HCC1937. Idarubicin, p = 0.3837; epirubicin, p = 0.1083. (C) Average response for hyper- and hypo-phosphorylated pRb states. epirubicin, p = 0.7905; idarubicin, p = 0.0353. (D) Average response for basal A (BaA) and basal B (BaB) TNBC subtypes. epirubicin, p = 0.6579. idarubicin, p = 0.5775. p-values calculated using nonlinear regression analysis.
Figure 6.
Effect of RB1 status on response to radiation or doxorubicin of CD44+/CD24−/low/ESA+ cancer stem cell (CSC)-enriched fraction in human TNBC lines.
Treatment with 5 Gy radiation or IC50 doses of CDDP or doxorubicin. (A) Viable cell population of 7-AAD control MDA-MB-231 cells. (B) ESA positive cell fraction of untreated MDA-MB-231 cells. (C–D) MDA-MB-231 CSC fraction in untreated and irradiated cells. (E–F) HCC1937 CSC fraction in untreated and irradiated cells. (G) Relative change in the CSC fraction for each line treated with CDDP, doxorubicin or radiation. (H) Average relative ratio of the CSC fraction for RB+ and RB− TNBC cell lines treated as indicated. RB+ lines (red): MDA-MB-231, MDA-MB-157 and HCC38. RB− lines (blue): MDA-MB-436, MDA-MB-468, and HCC1937. p-value calculated using a two-tailed t-test. n.s. = not significant.