Cyclin E overexpression as a biomarker for combination treatment strategies in inflammatory breast cancer

Inflammatory breast cancer (IBC) is a virulent form of breast cancer, and novel treatment strategies are urgently needed. Immunohistochemical analysis of tumors from women with a clinical diagnosis of IBC (n = 147) and those with non-IBC breast cancer (n = 2510) revealed that, whereas in non-IBC cases cytoplasmic cyclin E was highly correlated with poor prognosis (P < 0.001), in IBC cases both nuclear and cytoplasmic cyclin E were indicative of poor prognosis. These results underscored the utility of the cyclin E/CDK2 complex as a novel target for treatment. Because IBC cell lines were highly sensitive to the CDK2 inhibitors dinaciclib and meriolin 5, we developed a high-throughput survival assay (HTSA) to design novel sequential combination strategies based on the presence of cyclin E and CDK2. Using a 14-cell-line panel, we found that dinaciclib potentiated the activity of DNA-damaging chemotherapies treated in a sequence of dinaciclib followed by chemotherapy, whereas this was not true for paclitaxel. We also identified a signature of DNA repair–related genes that are downregulated by dinaciclib, suggesting that global DNA repair is inhibited and that prolonged DNA damage leads to apoptosis. Taken together, our findings argue that CDK2-targeted combinations may be viable strategies in IBC worthy of future clinical investigation.


Description of patient cohorts
We used four cohorts of breast cancer patients and two cohorts of IBC patients for our study comparing the expression of cyclin E. Some of these samples have been previously reported upon in our manuscript describing the validation of cyclin E antibodies and relationship with outcome [1]. In total, the non-IBC cohort of 2510 samples was derived from four sources: 1) The first set included 725 patients with early-stage breast cancer (stage I-II) who underwent surgery at MD Anderson Cancer Center between 1985 and 1999. These tissues were analyzed using tissue microarrays with 1-mm biopsy cores. 2) The second set was purchased from the National Cancer Institute (NCI) Cancer Diagnosis Program and was a collection of samples from 951 patients with stage I-II breast cancer who underwent surgery between 1985 and 1997 at four different hospitals around the United States (Kaiser Permanente in Portland, OR; Washington University in St Louis, MO; Fox Chase Cancer Center in Philadelphia, PA; and the University of Miami in Miami, FL). Tissue microarrays from these samples were generated at the University of Virginia using 0.6-mm cores.
3) The third set consisted of tissue microarrays from 515 older patients with stage I-II disease treated at Nottingham University Hospitals, Nottingham, England, between 1987 and 2005. 4) The fourth set of samples (totaling 319 samples) was collected prospectively from MD Anderson under an IRB-approved protocol to specifically examine the relationship between cyclin E and other clinical factors related to prognosis in patients treated between 2000 and the present. For all cohorts, formalin-fixed and paraffin-embedded tissues were collected, and clinical characteristics were collected by manual review of charts or from MD Anderson research databases in the Departments of Surgical Oncology and Breast Medical Oncology. All of the 147 IBC patients with cyclin E results and at least one item of follow-up post-initial consult were from MD Anderson, but not all patients received all therapy at MD Anderson. Charts and correspondence from outside physicians were reviewed to complete the clinical variables table.

Cell culture conditions
SUM149 and SUM185 cell lines were obtained from Asterand, KPL4 cells were obtained from Naoto Ueno (MD Anderson), and the remaining cell lines were purchased from American Type Culture Collection (ATCC). Cells were maintained in a humidified incubator at 37°C in a 6.5% CO 2 atmosphere, with the medium changed every 2-3 days to maintain cell health.

High-throughput survival assay development
The high-throughput survival assay was developed to increase the capacity for screening drug combinations in many cell lines versus classic clonogenic assays. HTSA and clonogenic assays in side-by-side experiments are highly concordant [2] .
Cell density was optimized by plating a range of densities (usually 500-5000, but some were at a lower density) and following cell growth by performing MTT assays every other day. MTT assays were performed by diluting the stock concentration by 5-fold in the medium in which the cells usually grew. Medium from the plates was aspirated, and then the diluted MTT was added for a 4-hour incubation back in the 37°C incubator. The diluted MTT was then aspirated and the converted MTT crystals were solubilized in an 86% isopropanol solution containing 1% sodium dodecyl sulfate (SDS) and 0.04 M www.impactjournals.com/oncotarget/

Cyclin E overexpression as a biomarker for combination treatment strategies in inflammatory breast cancer
Supplementary Materials hydrochloric acid. Characteristics of ideal densities are that they allow logarithmic growth throughout the experiment, and the absorbance of the wells remained within the linear range of the plate reader (Epoch reader, BioTek).
Prior to combination assays, the single drug doseresponse curves were used to derive IC 10 /IC 25 /IC 50 /IC 60 / IC 75 values for 24-hour or 48-hour treatments, performed in duplicate and averaged. Each plate in the combination treatment experiments contained two columns of cells that were left untreated, allowing internal controls to be set to 100% to derive the survival fraction. In addition, two columns contained cells just treated with drug A, and the other eight columns were different concentrations of drug A and drug B. The drug A concentrations were the IC 25 , IC 50 , or IC 75 values and the drug B concentrations ranged from IC 10 through IC 60 . After drug treatments, the old medium was replaced with fresh medium every 48 hours until the end of the experiment. CalcuSyn software was used to generate combination indexes from these data. The schematic for HTSA is provided in Supplementary Figure S1.

Quantitative real-time PCR primers
The sequences of the primers used were as follows:

Statistics and software
All experiments were performed in at least triplicate unless otherwise indicated in the figure legends or text. Bar graphs show the mean of all experiments pooled together, and error bars signify standard deviation from the mean. For comparisons, unpaired two-sided t-tests or ANOVAs (for more than two samples) were used, and P < 0.05 was the cut-off for significance.
Numerical data were processed using Excel for simple calculations and normalization. Final versions of graphs were made using GraphPad Prism 6 software, and t-tests and ANOVA were also performed using Prism. CalcuSyn software (Biosoft, Cambridge, UK) was used to generate combination indexes as described under HTSA. Heatmaps were generated using Tableau Desktop professional, version 9.3.4 (Tableau Software, Seattle, WA).   The cell numbers refer to cells plated in a 96-well plate.   The values are average combination indexes for two or three independent experiments for each direction. The values are average combination indexes for two or three independent experiments for each direction.  Figure 5A. The values are fold changes relative to the DMSO control for the same time point.  Figure 5B. The values are fold changes relative to the DMSO control for the same time point.  Figure 5C. The values are fold changes relative to the DMSO control for the same time point.