Blockade of CDK7 Reverses Endocrine Therapy Resistance in Breast Cancer

Abstract

Cyclin-dependent kinase (CDK)-7 inhibitors are emerging as promising drugs for the treatment of different types of cancer that show chemotherapy resistance. Evaluation of the effects of CDK7 inhibitor, THZ1, alone and combined with tamoxifen is of paramount importance. Thus, in the current work, we assessed the effects of THZ1 and/or tamoxifen in two estrogen receptor-positive (ER+) breast cancer cell lines (MCF7) and its tamoxifen resistant counterpart (LCC2) in vitro and in xenograft mouse models of breast cancer. Furthermore, we evaluated the expression of CDK7 in clinical samples from breast cancer patients. Cell viability, apoptosis, and genes involved in cell cycle regulation and tamoxifen resistance were determined. Tumor volume and weight, proliferation marker (Ki67), angiogenic marker (CD31), and apoptotic markers were assayed. Bioinformatic data indicated CDK7 expression was associated with negative prognosis, enhanced pro-oncogenic pathways, and decreased response to tamoxifen. Treatment with THZ1 enhanced tamoxifen-induced cytotoxicity, while it inhibited genes involved in tumor progression in MCF-7 and LCC2 cells. In vivo, THZ1 boosted the effect of tamoxifen on tumor weight and tumor volume, reduced Ki67 and CD31 expression, and increased apoptotic cell death. Our findings identify CDK7 as a possible therapeutic target for breast cancer whether it is sensitive or resistant to tamoxifen therapy.

Keywords: breast cancer; c-Myc; cyclin-dependent kinase 7; estrogen receptor; resistance; tamoxifen.

Figure 1

The relationship between cyclin dependent kinase (CDK7) expression and survival in breast cancer patients. Scatter plots of the Spearman rank-order correlation between (a) CDK7 and estrogen receptor alpha or 1 (ESR1) expression in 981 patients with breast cancer and (b) 715 patients with ER+ breast cancer. Data are from TCGA samples (RNASEqv2 data type). (c) Kaplan–Meier curves comparing overall survival (OS) in patients with ER+ breast cancer stratified by CDK7 expression level. (d) Kaplan–Meier curves comparing OS in patients with ER+ breast cancer receiving tamoxifen (TAM) by CDK7 expression level. (e) Scatter plot showing correlation between CDK7 and MYC expression in breast cancer patients receiving tamoxifen. (f) Kaplan–Meier curves comparing OS in breast cancer patients receiving tamoxifen by MYC expression level. (g) Scatter plot showing the correlation between MYC and ESR1 expression in breast cancer patients receiving tamoxifen.

Figure 2

Expression of CDK7 in tamoxifen-sensitive and tamoxifen-resistant breast cancer cell lines. (a) Western blot showing CDK7 protein levels in tamoxifen-sensitive (MCF-7) and tamoxifen-resistant (LCC2) cells. (b) Western blots showing levels of ER-α and phosphorylated ER-α (at serine 118) (p-Ser118 ERα) in MCF-7 and LCC2 cells. (c,d) Western blots showing CDK7 protein levels in MCF-7 (c) and LCC2 (d) cells after transfection with 100 nM control siRNA (CT siR) or 50 or 100 nM siRNA-CDK7-1 (CDK7 siR-1) or siRNA-CDK7-2 (CDK7 siR-2) (72 h incubation) or treatment with 10 µM tamoxifen (TAM) or 13 nM THZ1 (48 h incubation). (e,f) Quantitative RT-PCR data showing CDK7 mRNA expression levels in MCF-7 (e) and LCC2 (f) cells after transfection or treatment as described above. The results are expressed as mean ± SD of five independent experiments performed in triplicate. Statistical significance was determined by one-way ANOVA using the Tukey multiple comparison test, ** p < 0.01, **** p < 0.001. (g,h) Western blots showing p-Ser118 ERα and ER-α levels in MCF-7 (g) and LCC2 (h) cells after transfection or treatment as described above. (i,j) Western blots showing levels of C-Myc, STAT3, β-catenin, and TFIIH in (i) MCF-7 and (j) LCC2 cells with no treatment after transfection with 100 nM CT siR, 50 or 100 nM siR-2, or 13 nM THZ1. For Western quantification imageJ software was used to measure the intensity and normalize each value to its corresponding β-actin or GAPDH.

Figure 3

Viability of MCF-7 and LCC2 cells after tamoxifen, THZ1, or CDK7-targeting siRNA treatment individually and in combination. (a) Percentage of viable MCF-7 and LCC2 cells 48 h after treatment with the indicated concentrations of tamoxifen (TAM). (b) Percentage of viable MCF-7 and LCC2 cells after 48 h of incubation with THZ1 (2.5–50 nM). (c) (left) Viability of MCF-7 cells after treatment with the indicated concentrations of dimethyl sulfoxide (DMSO) (control), THZ1, tamoxifen, or THZ1 plus tamoxifen. (right) Viability of LCC2 cells after treatment with the indicated concentrations of dimethyl sulfoxide (DMSO), THZ1, tamoxifen, or THZ1 plus tamoxifen. (d) Cell viability after treatment with the indicated concentrations of control siRNA (CT siR) plus tamoxifen, siRNA-CDK7-2 (CDK7 siR), or siRNA-CDK7-2 plus tamoxifen in MCF-7 and LCC2 cells. The results are expressed as mean ± SD of five independent experiments performed in triplicate. Statistical significance was determined by one-way ANOVA using the Tukey multiple comparison test. *** p < 0.001. UT, untreated.

Figure 4

Effects of CDK7 inhibition combined with tamoxifen on the cell cycle and apoptosis. (a) Histograms showing the results of the flow cytometry analysis of the cell cycle and apoptosis assays of MCF-7 cells. (Left) Cell cycle analysis of MCF-7 cells treated with the indicated concentrations of tamoxifen, THZ1, and their combination. (Center) Cell cycle analysis of MCF-7 cells treated with the indicated concentrations of tamoxifen, CT siR, CDK7 siR, and the combination of tamoxifen plus CDK7 siR. (Right) Percentage of living and apoptotic MCF-7 cells treated with the indicated treatments. (b) Histograms showing results of flow cytometry analysis of the cell cycle and apoptosis assays of LCC2 cells. (Left) Cell cycle analysis of LCC2 cells treated with the indicated concentrations of tamoxifen, THZ1, and their combination. (Center) Cell cycle analysis of LCC2 cells treated with the indicated concentrations of tamoxifen CT siR, CDK7 siR, and the combination of tamoxifen plus CDK7 siR. (Right) Percentage of living and apoptotic LCC2 cells treated with the indicated treatments. (c) Western blots of cell cycle and apoptosis markers in cells exposed to tamoxifen, THZ1, and CDK7 siR, individually and in combination. For Western quantification imageJ software was used to measure the intensity and normalize each value to its corresponding β-actin. Results in the graphs are expressed as mean ± SD of two independent experiments performed in triplicate. Statistical significance was determined by one-way ANOVA using the Tukey multiple comparison test. * p < 0.05. UT, untreated.

Figure 5

In vivo therapeutic efficacy of CDK7 inhibition in combination with tamoxifen. (a) Tumor volume in mice bearing MCF-7 tumors treated with tamoxifen, THZ1, or their combination. (b) Tumor weight in mice bearing MCF-7 tumors in four treatment groups (10 mice per group): untreated (UT), tamoxifen (TAM), THZ1, and tamoxifen plus THZ1. (c) Western blot of c-MYC protein levels in tumor tissue from mice bearing MCF-7 tumors treated with tamoxifen, THZ1, or their combination, for quantification we used imageJ software to measure the intensity and normalize each value to its corresponding β-actin. (d) (Left) Representative micrographs (20×) showing immunohistochemical staining of CD31 and Ki67 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays in MCF-7 tumors from mice treated with tamoxifen, THZ1, or their combination. (Right) Quantification of microvessel density, cell proliferation, and apoptosis in the above groups. (e) Tumor volume in mice bearing LCC2 tumors treated with tamoxifen, THZ1, or their combination. (f) Tumor weight in mice bearing LCC2 tumors in four treatment groups (10 mice per group): UT, tamoxifen, THZ1, and tamoxifen plus THZ1. (g) Western blot of c-MYC protein levels in tumor tissue from mice bearing LCC2 tumors treated with tamoxifen, THZ1, or their combination. (h) (Left) Representative micrographs (20 ×) showing immunohistochemical staining of CD31 and Ki67 and TUNEL assays in LCC2 tumors from mice treated with tamoxifen, THZ1, or their combination. (Right) Quantification of microvessel density, cell proliferation, and apoptosis in the above groups. Statistical significance was determined by one-way ANOVA using the Tukey multiple comparison test. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Figure 6

Schematic illustration of the CDK7 signaling pathway and how it interacts with ER and ER-regulating genes including MYC. ① CDK7 activates ER phosphorylation at serine 118, which augments MYC transcription. ② Targeting CDK7 by siRNA or THZ1 blocks ER phosphorylation and MYC expression which could restore tamoxifen (TAM) toxicity in resistant breast cancers.