Roscovitine confers tumor suppressive effect on therapy-resistant breast tumor cells

Abstract

Introduction: Current clinical strategies for treating hormonal breast cancer involve the use of anti-estrogens that block estrogen receptor (ER)α functions and aromatase inhibitors that decrease local and systemic estrogen production. Both of these strategies improve outcomes for ERα-positive breast cancer patients, however, development of therapy resistance remains a major clinical problem. Divergent molecular pathways have been described for this resistant phenotype and interestingly, the majority of downstream events in these resistance pathways converge upon the modulation of cell cycle regulatory proteins including aberrant activation of cyclin dependent kinase 2 (CDK2). In this study, we examined whether the CDK inhibitor roscovitine confers a tumor suppressive effect on therapy-resistant breast epithelial cells.

Methods: Using various in vitro and in vivo assays, we tested the effect of roscovitine on three hormonal therapy-resistant model cells: (a) MCF-7-TamR (acquired tamoxifen resistance model); (b) MCF-7-LTLTca (acquired letrozole resistance model); and (c) MCF-7-HER2 that exhibit tamoxifen resistance (ER-growth factor signaling cross talk model).

Results: Hormonal therapy-resistant cells exhibited aberrant activation of the CDK2 pathway. Roscovitine at a dose of 20 μM significantly inhibited the cell proliferation rate and foci formation potential of all three therapy-resistant cells. The drug treatment substantially increased the proportion of cells in G2/M cell cycle phase with decreased CDK2 activity and promoted low cyclin D1 levels. Interestingly, roscovitine also preferentially down regulated the ERα isoform and ER-coregulators including AIB1 and PELP1. Results from xenograft studies further showed that roscovitine can attenuate growth of therapy-resistant tumors in vivo.

Conclusions: Roscovitine can reduce cell proliferation and survival of hormone therapy-resistant breast cancer cells. Our results support the emerging concept that inhibition of CDK2 activity has the potential to abrogate growth of hormonal therapy-resistant cells.

Figure 1

Effect of roscovitine on cell proliferation and survival of endocrine-resistant cancer cells. (a) Total cellular lysates from control and therapy-resistant model cells were subjected to western blotting using phosphor cell cycle dependent kinase (CDK) 2 antibody. Graph shows densitometry measurements of phosCDK2 protein bands relative to total CDK2 in each cell line. (b) Model cells were treated with or without roscovitine and cell proliferation was determined at indicated time points by using the Cell Titer Glo assay. (c) Model cells were treated with or without roscovitine and clonogenic survival was determined. Representative figures and quantitative analysis of survival was shown. Statistical significance was determined by student's t-test. P, P value; * P < 0.05; ** P < 0.01. Con, control; Phos, phospho; ROS, roscovitine.

Figure 2

Roscovitine modulates cell cycle status of endocrine-resistant cancer cells. (a, c, e, g) Control cells and/or (b, d, f, h) cells that were treated with roscovitine were subjected to flow cytometry. The percentage of cells in each cell cycle phase is depicted on the top of corresponding G1, S, and G2/M peaks. ROS, roscovitine.

Figure 3

Roscovitine treatment suppresses expression of key regulators of cell cycle and the ER α-signaling axis. (a) The model cells MCF7, MCF7-TamR, MCF7-HER2, and MCF7-LTLTca were treated with roscovitine and the status of cell cycle regulators and the estrogen receptor (ERα) signaling proteins was analyzed by western blotting. (b) MCF7, MCF7-Tam, MCF7-LTLTca, and MCF7-HER2 were treated with roscovitine and the levels of ERα transcripts were determined by quantitative RT-PCR. (c) Model cells were pre-treated with the proteosomal inhibitor MG132 and then subjected to roscovitine treatment. ERα status was determined by western blotting of total lysates. Actin was used as loading control. Con, control; ROS, roscovitine.

Figure 4

Roscovitine exerts a tumor suppressive effect on endocrine-resistant breast cancer xenografts. Nude mice were subcutaneously injected with the endocrine-resistant model cells (a) MCF7-LTLTca (n = 8), (b) MCF7-HER2 (n = 8), and (c) MCF7-TamR (n = 6). After three weeks, roscovitine treatment was given for 10 consecutive days and tumor volumes were recorded on the 5^th, 10^th, 16^th, and 25^th day after roscovitine treatment. P, P value; ** P < 0.01, * P < 0.05. (d) Representative images of tumors depicting the size of tumors derived from both control and roscovitine-treated groups.

Figure 5

Roscovitine treatment reduces proliferation and increases apoptosis in endocrine-resistant xenografts. (a) Immunohistochemical analysis of Proliferating Cell Nuclear Antigen (PCNA) on tumors treated with or without roscovitine. (b) Quantitation of PCNA staining using the PCNA index. P, P value; ** P < 0.01, * P < 0.05. (c) TUNEL staining for apoptosis in control and roscovitine-treated tumors. Representative images are depicted (upper panel). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) labeling was quantified as the mean TUNEL labeling percentage based on at least three randomly selected high-power microscope fields per group (lower panel). P, P value; ** P < 0.01, * P < 0.05. (d) Immunohistochemical analysis for estrogen receptor (ERα) status was performed in groups with or without roscovitine treatment. Con, control; ROS, roscovitine.