PD 0332991, a selective cyclin D kinase 4/6 inhibitor, preferentially inhibits proliferation of luminal estrogen receptor-positive human breast cancer cell lines in vitro

Abstract

Introduction: Alterations in cell cycle regulators have been implicated in human malignancies including breast cancer. PD 0332991 is an orally active, highly selective inhibitor of the cyclin D kinases (CDK)4 and CDK6 with ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. To identify predictors of response, we determined the in vitro sensitivity to PD 0332991 across a panel of molecularly characterized human breast cancer cell lines.

Methods: Forty-seven human breast cancer and immortalized cell lines representing the known molecular subgroups of breast cancer were treated with PD 0332991 to determine IC50 values. These data were analyzed against baseline gene expression data to identify genes associated with PD 0332991 response.

Results: Cell lines representing luminal estrogen receptor-positive (ER+) subtype (including those that are HER2 amplified) were most sensitive to growth inhibition by PD 0332991 while nonluminal/basal subtypes were most resistant. Analysis of variance identified 450 differentially expressed genes between sensitive and resistant cells. pRb and cyclin D1 were elevated and CDKN2A (p16) was decreased in the most sensitive lines. Cell cycle analysis showed G0/G1 arrest in sensitive cell lines and Western blot analysis demonstrated that Rb phosphorylation is blocked in sensitive lines but not resistant lines. PD 0332991 was synergistic with tamoxifen and trastuzumab in ER+ and HER2-amplified cell lines, respectively. PD 0332991 enhanced sensitivity to tamoxifen in cell lines with conditioned resistance to ER blockade.

Conclusions: These studies suggest a role for CDK4/6 inhibition in some breast cancers and identify criteria for patient selection in clinical studies of PD 0332991

Figure 1

Inhibitory concentration and cell type. Bar graph of IC₅₀ values (nM) and cell type. Cell lines are color coded by subtype: light blue, luminal; dark blue bars or stripes, HER2 amplified; yellow, nonluminal/undergone an epithelial-to-mesenchymal transition; red, nonluminal; turquoise, immortalized.

Figure 2

Differentially expressed genes between sensitive and resistant cell lines. Results of analysis of variance (ANOVA) identifying 450 differentially expressed genes between sensitive cell lines (IC₅₀ < 150 nM) and resistant cell lines (IC₅₀ > 1,000 nM). Retinoblastoma and cyclin D₁ expression were higher in, and CDKN2A (p16) was lower in, sensitive cell lines. Full results of the ANOVA are available in the Additional data files.

Figure 3

Effects of PD 0332991 on phosphorylation of retinoblastoma gene product. PD 0332991 blocks phosphorylation of retinoblastoma gene product pRb in sensitive cell lines but not in resistant cell lines. (a) In neither group does total pRb change significantly with treatment. (b) PD 0332991 significantly blocks phoshorylation of pRb (phospho-Rb) at serine 780 in sensitive cell lines (IC₅₀ < 150 nM), but not in resistant cell lines (IC₅₀ > 1,000 nM). All cell lines were treated with 100 nM PD 0332991 for the times specified and western blots were performed as described in Materials and methods.

Figure 4

Effects of PD 0332991 on cell cycle. (a) Sensitive cell lines (IC₅₀ < 150 nM) show marked G₀/G₁ arrest and a decrease in the S-phase fraction as compared with (b) resistant cell lines (IC₅₀ > 1,000 nM) after incubation with 100 nM PD 0332991 for 24 hours. Solid bars, control samples; hatched bars, treated samples. Error bars represent the standard error for two separate experiments.

Figure 5

Effects of PD 0332991, tamoxifen, and trastuzumab on growth of breast cancer cell lines. PD 0332991 (PD) acts synergistically with tamoxifen (Tam) and trastuzumab in inhibiting growth of (a) estrogen receptor-positive human breast cancer cell lines and (b) HER2-amplified cell lines in vitro, respectively. (a) Grey squares, PD 0332991 alone; open diamonds, tamoxifen alone; dark triangles, PD 0332991 and tamoxifen combination. (b) Grey squares, PD 0332991 alone; open diamonds, trastuzumab alone; dark triangles, PD 0332991 and trastuzumab combination. Error bars represent the standard error for two separate experiments. Mean combination index (CI_m) for the combination curves shown with standard error CI_m < 1, indicating synergy for the combinations.

Figure 6

PD 0332991 and tamoxifen in a tamoxifen-insensitive cell line. PD 0332991 (PD) enhances the effects of tamoxifen (Tam) in an MCF7 tamoxifen-insensitive cell line. Dark triangles, PD 0332991 and tamoxifen combination; open squares, PD 0332991 alone; grey diamonds, tamoxifen alone; grey circles, MCF7 parental with tamoxifen alone. Error bars represent the standard error for two separate experiments. The MCF7 parental line is 100% inhibited under these conditions in the presence of tamoxifen (grey circles).