Introduction
Breast cancer is a worldwide health concern with approximately 1,000,000 million new cases each year [
1]. Significant advances have been made in our understanding of this malignancy and several molecular subtypes of breast cancer have been characterized [
2‐
4]. This molecular understanding has paved the way for the development of new agents that target pathogenic molecular alterations that drive tumor cell growth while sparing patients many of the traditional toxicities associated with chemotherapy. Ubiquitous to all cancer types is abnormal proliferation with dysregulation of normal cell cycle control [
5]. For this reason, inhibitors of key cell cycle regulators are attractive targets for novel cancer therapeutics [
6]. Successful clinical development of this class of agents, however, will require some understanding of which subgroup of patients will be more likely to benefit from these targeted interventions.
Under normal control, the cell cycle functions as a tightly regulated and predictable process consisting of several distinct phases: G
0 (quiescence) followed by G
1 (pre-DNA synthesis), S (DNA synthesis), G
2 (pre-division), and M (cell division). The careful regulation of this system is of fundamental importance, and dysregulation can result in several disease processes including cancer. The progression from G
1 to S is a key checkpoint in protecting the cell from abnormal replication. Key to passage through this restriction point is the interaction between the cyclin-dependent kinases (CDKs) and cyclin proteins. CDKs are a subgroup of serine/threonine kinases that play a key role in regulating cell cycle progression by associating with cyclins. Hyperphosphorylation of the retinoblastoma (Rb) gene product pRb is mediated in early G
1 by CDK4 and CDK6 interacting with cyclin D
1. This results in pRB inactivation and release of transcription factors that allow progression to the S phase. Negative regulators of CDK4/6-cyclin activity include the INK4 family (p16, p15, p18, p19) [
7].
Several studies have identified alterations of cell cycle regulators in human breast cancer (reviewed in [
8,
9]) and provide a rationale for a potential therapeutic role for CDK4/6 inhibition in this tumor type. Amplification of the cyclin D
1 gene has been identified in approximately 15 to 20% of human breast cancers [
10,
11] while overexpression of the protein has been demonstrated in a higher percentage [
12,
13]. The prognostic significance of cyclin D
1 overexpression is not clear; some studies suggest it is a dominant oncogene associated with poor clinical outcomes [
11,
14‐
16], while other studies suggest it is associated with a more indolent, estrogen receptor (ER)-positive phenotype [
17,
18]. In addition, studies have associated cyclin D amplification with resistance to tamoxifen [
19,
20]. While the interaction between CDK4/6 and cyclin D
1 suggests their interdependence, cyclin D
1 has been found to function independently of CDK4/6 in supporting proliferation by directly activating ER [
21,
22]. Finally, loss of function of pRb has been described in 20 to 35% of breast cancers (reviewed in [
23]).
The majority of CDK targeted agents to date have not focused on CDK4/6 targeting but rather on CDK1/2 targeting. Consequently the most advanced agents in development are aimed at these targets [
24,
25]. Further, limited data exist regarding the preclinical activity of CDK4/6 inhibitors in breast cancer. PD 0332991 is an orally active potent and highly selective inhibitor of CDK4 and CDK6 kinases, which in low nanomolar concentrations blocks pRb phosphorylation - subsequently inducing G
1 arrest in sensitive cell lines [
26‐
29]. Preclinical studies have demonstrated that PD 0332991 induces G
1 arrest in primary bone marrow cells
ex vivo and prevents tumor growth in disseminated human myeloma xenografts [
30].
Based on the above biology, we hypothesized that there might be a molecular subgroup of human breast cancers that would be dependent on CDK4/6 function and would be likely to respond to this agent. Previous studies have demonstrated that
in vitro large-panel analyses of molecularly characterized breast cancer cell lines can offer insight into the molecular heterogeneity of the clinical disease [
31,
32]. To identify potential biomarkers of response to PD 0332991 and to assist in patient selection and clinical development, we therefore evaluated the effects of PD 0332991 in a panel of 47 human breast cancer and immortalized breast cell lines growing
in vitro.
Materials and methods
Cell lines, cell culture, and reagents
The cell lines used in the analysis include MDA-MB-415, MDA-MB-134, HCC-1500, ZR-75-30, HCC-202, HCC-1419, HCC-38, HCC-70, HCC-1187, HCC-1806, HCC-1937, HCC-1954, MDA-MB-436, HCC-1569, Hs578t, HCC-1143, MDA-MB-175, BT-474, SK-BR-3, MDA-MB-361, UACC-893, UACC-812, UACC-732, T-47D, MDA-MB-453, MDA-MB-468, CAMA-1, MDA-MB-157, MCF-7, MDA-MB-435, ZR-75-1, BT-20, MDA-MB-231, BT-549, DU4475, HCC-1395, HCC-2218, 184A1, 184B5 and MCF-10A, and were obtained from American Type Culture Collection (Rockville, MD, USA). The cell lines EFM-192A, KPL-1, EFM-19, COLO-824 and CAL-51 were obtained from the German Tissue Repository DSMZ (Braunschweig, Germany), and the cell lines SUM-190 and SUM-225 were obtained from the University of Michigan (Ann Arbor, MI, USA).
MDA-MB-134, MDA-MB-415, MDA-MB-436, MDA-MB-175, UACC-893, UACC-812, and MDA-MB-157 cells were cultured in L15 medium supplemented with 10% heat-inactivated FBS, 2 mmol/l glutamine and 1% penicillin G-streptomycin-fungizone solution (PSF) (Irvine Scientific, Santa Ana, CA, USA). CAL-51, KPL-1, and Hs578t cells were grown in DMEM (Cellgro, Manassas, VA, USA) supplemented with 10% heat-inactivated FBS and PSF, as above. SUM-190 and SUM-225 cells were cultured in HAM's F12 supplemented with 5% heat-inactivated FBS, PSF, 5 mg/ml insulin and 1 mg/ml hydrocortisone. 184A1, 184B5, and MCF 10A cells were grown in a 50/50 mix of mammary epithelial basal medium (MCDB 170) (US Biological, Swampscott, MA, USA) supplemented with 1.5 ml/l bovine pituitary extract (Invitrogen, Carlsbad, CA, USA), 20 μl/l epidermal growth factor (Invitrogen), 10 ml insulin (Sigma, Saint Louis, MO, USA), 1 ng/ml cholera toxin (Calbiochem, San Diego, CA, USA), 0.5 mg/l hydrocortisone (Sigma), and RPMI 1640 supplemented with 10% heat-inactivated FBS, 2 mmol/l glutamine, and 1% PSF. The remaining cell lines were cultured in RPMI 1640 (Cellgro) supplemented with 10% heat-inactivated FBS, 2 mmol/l glutamine, and 1% PSF. A tamoxifen-resistant MCF7 cell line was developed after serial passage in RPMI 1640 without phenol red (Invitrogen) supplemented with 10% charcoal-stripped FBS (Fisher Scientific, Pittsburgh, PA, USA) and 2 mmol/l glutamine, and PSF.
Transcript microarray analyses
Briefly, cells were grown to log phase and then RNA was extracted using the RNeasy Kit (Qiagen, Valencia, CA, USA). The purified RNA was eluted in 30 to 60 μl diethylpyrocarbonate (DEPC) water and the quantity of RNA measured by spectral analysis using the Nanodrop Spectrophotometer (NanoDrop Products, Wilmington, DE, USA). RNA quality was determined by separation of the RNA via capillary electrophoresis using the Agilent 2000 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Microarray hybridizations of 51 breast cell lines were performed using the Agilent Human 1A V1 array.
Characterization of individual breast cancer cell line transcripts was performed by comparison with a breast cell line mixed reference pool of RNA and was conducted on a single slide in which the cell line mixture RNA was labeled with cyanine-3 and RNA from the individual cell line was labeled with cyanine-5. The mixed reference cRNA pool consisted of equal amounts of cRNA from nine breast cancer cell lines and one immortalized breast cell line selected to be representative of the full spectrum of breast cancer subtypes based on their expression of specific molecular markers - for example, ESR1, HER2, epidermal growth factor receptor, cytokeratins, and so forth - as well as growth characteristics. The reference cRNA pool includes RNA from 184B5, MDA-MB-468, MDA-MB-157, MDA-MB-231, MDA-MB-175, CAMA-1, MCF-7, MDA-MB-361, SK-BR-3, and DU4475 cell lines.
Microarray slides were read using an Agilent Scanner, and Agilent Feature Extraction software version 7.5 was used to calculate gene expression values. The feature extracted files were imported into the Rosetta Resolver® system version 7.1 for gene expression data analysis (Rosetta Biosoftware, Seattle, WA, USA). The intensity ratios between the cell line sample and mixed reference calculated for each sequence were computed according to the Agilent error model. A particular sequence was considered differentially expressed if the calculated p-value of change was P ≤ 0.01. These data are available with accession number [GEO:GSE18496].
Proliferation assays
Cells were seeded in duplicate at 5,000 to 10,000 cells per well in 24-well plates. The day after plating, PD 0332991 was added at 1 μM and twofold dilutions over six concentrations were performed to generate a dose-response curve. Control wells without drug were also seeded. Cells were counted on day 1 when the drug was added as well as after 6 days when the experiment ended. After trypsinization, cells were placed in Isotone solution and counted immediately using a Coulter Z2 particle counter (Beckman Coulter Inc., Fullerton, CA, USA). Suspension lines were counted using a Coulter Vi-Cell counter (Beckman Coulter Inc.).
Growth inhibition was calculated as a function of the number of generations inhibited in the presence of PD 0332991 versus the number of generations over the same time course in the absence of PD 0332991. In addition, lethality was defined as any decrease in cell number in treated wells versus the baseline number of cells pre-treatment at day 1 of exposure.
For tamoxifen studies with the MCF7 tamoxifen-resistant cell line, proliferation studies were performed as above except cells were plated without FBS and were supplemented with 0.5 nM β-estradiol (Sigma). Proliferation assays were then performed as above.
Multiple drug effects analysis
Similar to above, the ER-positive cell lines MCF-7, T47-D, and EFM-19 were plated and treated with PD 0332991 alone, with 4-hydroxytamoxifen (Sigma) alone, or with the combination, in duplicate, over six twofold dilutions at a fixed molar ratio. For combination studies with trastuzumab, BT-474, EFM-192A, and MDA-361 lines were plated as above and treated with PD 0332991 alone, with trastuzumab (Genentech, South San Francisco, CA, USA) alone, or with the combination, in duplicate, over six twofold dilutions at a fixed molar ratio.
For each assay, the log of the fraction growth inhibition was plotted against the log of drug concentration, and the linear regression curve fit correlation coefficient (
r value) was calculated. Multiple drug effect analysis was performed using computer software as previously described [
33].
Combination index (CI) values were derived from parameters of the median effects plots, and statistical tests were applied (unpaired, two-tail Student t test) to determine whether the mean CI values at multiple concentrations were significantly different from CI = 1. In this analysis, synergy is defined as CI values significantly lower than 1.0, antagonism as CI values significantly higher than 1.0, and additivity as CI values equal to 1.0. All CI values were calculated using the conservative assumption of mutually nonexclusive drug interactions. All experiments were carried out at least twice. Combination studies were performed as above with the MCF7 tamoxifen-resistant clones with addition of estrogen back to the media at the time of the experiment (as described in cell culture above).
Western blot analysis
Cells in log-phase growth were treated with 100 nM PD 0332991 and were harvested at various timepoints by washing in PBS and lysis at 4°C in RIPA lysis buffer. Insoluble material was cleared by centrifugation at 10,000 × g for 10 minutes and protein was quantitated using bicinchoninic (BCA) (Pierce Biochemicals, Rockford, IL, USA). Protein content was resolved by SDS-PAGE electrophoresis, and was transferred to nitrocellulose membranes (Invitrogen). Total pRb expression was detected using a rabbit polyclonal antibody to pRb (Abcam, Cambridge, MA, USA). Rb phosphorylation was detected using rabbit polyclonal antibody to phospho-serine 780 (Cell-signaling, Danvers, MA, USA). Blots were washed and incubated with a goat-anti-rabbit IgG horseradish peroxidase conjugate (Upstate, Bellerica, MA, USA), developed using ECL Plus chemifluorescent reagent (Amersham Biosciences, Pistcataway, NJ), and imaged using chemifluorescence.
Cell cycle analysis and apoptosis studies
Effects of PD 0332991 on the cell cycle were assessed using Nim-DAPI staining (NPE Systems, Pembroke Pines, FL, USA). Cells were plated evenly in control and experimental wells, were allowed to grow to log phase and were then treated with 100 nM PD 0332991 for the defined times. To perform cell cycle analysis, cells were washed with PBS; then trypsin was applied to release cells, which were then centrifuged at 10,000 × g for 5 minutes. Supernatant was aspirated and cells were then resuspended in 100 μl Nim-DAPI (NPE Systems) and gently vortexed.
Cells were analyzed with UV using a Cell Lab Quanta SC flow cytometer (Beckman-Coulter Inc.). Apoptosis assays were performed using an Annexin V-FITC apoptosis detection kit (MBL, Woburn, MA, USA) and flow cytometry. Cells were plated and treated as for cell cycle studies and were exposed to 100 nM PD 0332991 for 5 days. After incubation, cells were processed as directed in the kit and were analyzed using a FITC signal detector and propidium iodide detector using a Cell Lab Quanta SC flow cytometer.
Statistical methods
Growth response to PD 0332991 and molecular subtype classification data were entered into the Statistica data analysis system version 8.0 (StatSoft Inc., Tulsa, OK, USA). The Pearson chi-square test was used to assess the relationship between response and subtype.
Breast cell lines were profiled on the Agilent Human 1A V1 platform that contains 17,086 probes including known genes and ESTs. The Resolver system analysis of variance (ANOVA) and hierarchical cluster analysis of the breast cell line expression profiles were used to compare the most sensitive cell lines (n = 21, IC50 < 150 nM) and the most resistant cell lines (n = 12, IC50 > 1,000 nM). All ANOVAs were performed using the Benjamini-Hochberg False Discovery Rate (FDR) multiple test correction and a statistical cutoff value for sequences of a twofold change in at least three experiments. The criteria used to determine differentially expressed genes were P < 0.05 with a difference between average expression for each group of at least |0.2|. Sequence sets were compared using the Venn Diagram tool in the Resolver system. The two-dimensional cluster analysis was performed using an agglomerative hierarchical clustering algorithm based on the cosine correlation similarity metric.
Discussion
The critical role of CDK-cyclin interactions in controlling cell growth has been an attractive target in cancer therapy for sometime. These data represent the most comprehensive preclinical evaluation of a CDK4/6 inhibitor in breast cancer cell lines to date, and build the case for its clinical development in specific molecular subgroups of breast cancer.
Using baseline Agilent gene expression profiles, we first demonstrated that luminal ER-positive and HER2-amplified breast cancer cell lines were more sensitive to CDK4/6 inhibition of proliferation and cell cycle arrest. ANOVA analysis of these data identified a set of genes that were associated with response to PD 0332991. While the majority of these genes were associated with the luminal subtype, increased RB1 and cyclin D1 as well as decreased CDKN2A (p16) were associated with sensitivity to the effects of PD 0332991 on the cell cycle and growth inhibition. Western blot analysis confirmed that Rb phosphorylation is decreased in sensitive cell lines after PD 0332991 treatment, while resistant lines that had detectable pRb were relatively resistant to the effects of PD 0332991 on Rb phosphorylation. In this case, the presence of pRb alone is not predictive of response to PD 0332991. pRb presence in the background of luminal ER-positive breast cancer, however, is predictive of response to the compound. pRb is present in some nonluminal breast cancer cell lines but these lines are resistant to both the antiproliferative effects of PD 0332991 and its ability to block Rb hyperphosphorylation.
Further studies will be required to determine why PD 0332991 cannot block hyperphosphorylation in cell lines that do contain pRb. One can speculate that potentially CDK4/6 is mutated in these cell lines and does not allow PD 0332991 binding and kinase inhibition, as is the case in resistance to BCR-ABL inhibitors in chronic myelogenous leukemia [
35]. Alternatively, there may be another mechanism driving Rb hyperphosphorylation in resistant cell lines, such as a greater dependence on CDK1/2-cyclin E interactions or loss of negative regulators of this pathway in these cell lines.
Resistance to PD 0332991 in many of the nonluminal breast cancer cell lines may be explained by the absence of pRb. Recent publications highlighted the lack of pRb in basal-like breast cancer tissue [
36] and observed that pRb depletion can result in the characteristic epithelial-to-mesenchymal transition changes seen in some breast cancer specimens [
37], recapitulating our
in vitro observations. The lack of activity of a CDK4/6 inhibitor in cell lines and tumors that lack pRb can be explained by the fact that cyclin D
1 does not offer G
1 control in the absence of pRb [
38].
Published studies evaluating the role of cyclin D
1 in breast cancer support the current observations of the activity of a CDK4/6 inhibitor in luminal ER-positive breast cancer, its synergism with tamoxifen in cell lines that are sensitive to hormone manipulation, as well as the reversal of resistance of those that have acquired a resistant phenotype in the face of anti-estrogen therapy. Estrogen effects on cell cycle progression are tightly linked to expression of cyclin D
1 [
39]. Cyclin D
1 amplification and/or overexpression has been more commonly associated with an ER-positive breast cancer subtype [
40] and, as mentioned previously, is associated with tamoxifen resistance [
19,
20]. Some studies suggest that overexpression of cyclin D
1 can directly activate ER in a hormonally independent manner that is also independent of CDK and pRb function [
21,
22]. The data supporting this concept were reviewed recently [
41]. In the large panel of human breast cancer lines we evaluated, however, 9/10 ER-positive lines were sensitive to PD 0332991 inhibition. Of interest, Wang and colleagues recently described a cyclin D
1 splice variant - named cyclin D
1b - that occurs in breast cancer tissue and cell lines, and whose expression can overcome cell cycle arrest induced by anti-estrogens via a CDK4 interaction [
42].
In addition, a pivotal role for cyclin D
1 function in HER2-mediated transformation has been described using transgenic mouse models [
43]. More recently, the same authors defined that the ability of cyclin D
1 to activate CDK4 is critical for driving tumorigenesis in these models. Moreover, CDK4-associated kinase activity is required to maintain breast tumorigenesis in this system [
44,
45] and a subset (~25%) of HER2-amplified breast cancers also have high cyclin D
1 levels. The authors hypothesized that this 'subset may benefit from inhibiting CDK4 kinase' [
44]. Our data would suggest that the benefit with PD 0332991 might be extended beyond that 25%, since the benefit was not dependent on elevated cyclin D
1 alone in the HER2-amplified cell lines. In addition, synergistic efficacy was observed between trastuzumab and PD 0332991 that may also be independent of cyclin D
1 measurement. Earlier work evaluating the nonspecific cyclin-CDK inhibitor flavopiridol in combination with trastuzumab also demonstrated similar activity in HER2-amplified cell lines [
46]. Finally, a recent study investigating gene expression profiles of women with HER2-amplified breast cancer who develop early brain metastasis identified CDK4 expression as part of a 13-gene profile that predicted for early brain metastasis and death [
47].
Acquired and
de novo resistance to trastuzumab remains a management challenge in clinical oncology. While limited data exist about the role of CDK4-cyclin D
1 interactions and trastuzumab resistance, these data suggest a role for dual targeting of the HER2 pathway and CDK4/6. Molecular profiling of the JIMT-1 human breast cancer cell line derived from a woman with progressive HER2-amplified disease while receiving trastuzumab did identify a small amplicon on 12q14.1, which contains the CDK4 gene [
48].
Competing interests
RSF and DJS research funding from Pfizer, Inc. IC, CF, and GL are employees of Pfizer, Inc. The other authors declare that they have no competing interests.
Authors' contributions
RSF and DJS designed and supervised the study, analyzed data, and drafted the manuscript. JD performed data analysis. DC, OK, DJC, and AD performed in vitro experiments and performed molecular biology. CG performed all microarrays and analyzed data. MA created the tamoxifen-resistant cell line and designed experiments. IC, CF, and GL collaborated in experimental design, data analysis, and manuscript writing. All authors read and approved the final manuscript.