KDELR2 promotes breast cancer proliferation via HDAC3-mediated cell cycle progression

Abstract

Background: Histone deacetylases (HDACs) engage in the regulation of various cellular processes by controlling global gene expression. The dysregulation of HDACs leads to carcinogenesis, making HDACs ideal targets for cancer therapy. However, the use of HDAC inhibitors (HDACi) as single agents has been shown to have limited success in treating solid tumors in clinical studies. This study aimed to identify a novel downstream effector of HDACs to provide a potential target for combination therapy.

Methods: Transcriptome sequencing and bioinformatics analysis were performed to screen for genes responsive to HDACi in breast cancer cells. The effects of HDACi on cell viability were detected using the MTT assay. The mRNA and protein levels of genes were determined by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting. Cell cycle distribution and apoptosis were analyzed by flow cytometry. The binding of CREB1 (cAMP-response element binding protein 1) to the promoter of the KDELR (The KDEL (Lys-Asp-Glu-Leu) receptor) gene was validated by the ChIP (chromatin immunoprecipitation assay). The association between KDELR2 and protein of centriole 5 (POC5) was detected by immunoprecipitation. A breast cancer-bearing mouse model was employed to analyze the effect of the HDAC3-KDELR2 axis on tumor growth.

Results: KDELR2 was identified as a novel target of HDAC3, and its aberrant expression indicated the poor prognosis of breast cancer patients. We found a strong correlation between the protein expression patterns of HADC3 and KDELR2 in tumor tissues from breast cancer patients. The results of the ChIP assay and qRT-PCR analysis validated that HDAC3 transactivated KDELR2 via CREB1. The HDAC3-KDELR2 axis accelerated the cell cycle progression of cancer cells by protecting the centrosomal protein POC5 from proteasomal degradation. Moreover, the HDAC3-KDELR2 axis promoted breast cancer cell proliferation and tumorigenesis in vitro and in vivo.

Conclusion: Our results uncovered a previously unappreciated function of KDELR2 in tumorigenesis, linking a critical Golgi-the endoplasmic reticulum traffic transport protein to HDAC-controlled cell cycle progression on the path of cancer development and thus revealing a potential therapeutical target for breast cancer.

Keywords: CREB1; HADC3; KDELR2; breast cancer; histone deacetylase inhibitor; protein of centriole 5.

FIGURE 1

Aberrant expression of KDELR2, a novel target of HDACi, correlated with the breast cancer process. A. Crystal violet staining was used to analyze the cell proliferation ability of MDA‐MB‐231 cells after treatment with TSA (10 μM), TDPA (10 nM) and FK228 (10 nM) for 48 h. B. MDA‐MB‐231 cells were treatment with TSA (10 μM), TDPA (10 nM) and FK228 (10 nM) for 48 h. Statistical analysis of relative mean fluorescence intensities (top) and the histrogram graph (bottom) of Ki67 expression were analyzed by flow cytometry. The data are presented as the mean ± SD of three independent experiments. *, P < 0.05. C. MDA‐MB‐231 cells (5× 10⁶ cells per mouse) were subcutaneously injected into female nude mice (n = 5 for each group). Mice were intraperitoneally injected with 3 mg/kg TDPA or 0.75 mg/kg FK228 every three days after inoculation. Tumor sizes were measured starting from 21 days after inoculation (top). At the end of the experiment, the tumors were extracted and compared (bottom). The data are presented as the mean ± SEM. *, P < 0.05 compared between the indicated groups. *, P < 0.05. D. The heat map from RNA‐Seq analysis showed alterations in the expression of genes in breast cancer cells treated with HDAC inhibitors relative to the expression of these genes in the control group. The colors indicate the ln‐transformed transcripts per kilobase of exon model per million mapped reads (TPM) values. E. Volcano plot showing the gene expression differences between breast tumor tissues and normal tissues and the statistical significance of genes related to patient survival in the TCGA database. Each point represents the gene that was consistently downregulated by 3 HDAC inhibitors from Figure 1D. The X axis represents the fold change in gene expression between breast tumor tissues and normal tissues. The Y axis represents the statistical significance of genes related to patient survival in TCGA. F. The growth of MDA‐MB‐231 cells with knockdown of the indicated genes identified by RNA‐Seq was calculated. The data are presented as the mean ± SEM of three independent experiments. *, P < 0.05; ns, not significant. G‐H. qRT‐PCR analysis of the mRNA levels of KDELR2 in MDA‐MB‐231 (G) and T47D (H) breast cancer cells treated with HDAC inhibitors TSA (10 μM), TDPA (10 nM) and FK228 (10 nM) for 48 h. The data are presented as the mean ± SD of three independent experiments. *, P < 0.05. I. Western blotting analysis of the protein levels of KDELR2 in MDA‐MB‐231 cells treated with HDAC inhibitors TSA (10 μM), TDPA (10 nM) and FK228 (10 nM) for 48 h. J. qRT‐PCR analysis of the mRNA levels of KDELR2 in 16 pairs of clinically matched tumor‐adjacent noncancerous breast tissues (Normal) and human breast cancer tissues (Tumor). The data are presented as the mean ± SD. Group differences were analyzed by the two‐tailed Student's t‐test. *, P < 0.05; ns, not significant. K. Kaplan‐Meier curves from univariate analysis for patients with low versus high KDELR2 expression. The data were obtained from the website (http://firebrowse.org). Patients were grouped according to the optimized cutoff

FIGURE 2

HDAC3 was responsible for HDACi‐mediated KDELR2 expression. A. qRT‐PCR analysis of the mRNA levels of KDELR2 in MDA‐MB‐231 cells transfected with pooled shHDACs and NTC cells. The data are presented as the mean ± SD of three independent experiments. *, P < 0.05. B. qRT‐PCR analysis of the mRNA levels of HDAC8 and KDELR2 in HDAC8 knockdown MDA‐MB‐231 cells and NTC cells. The data are presented as the mean ± SD of three independent experiments. *, P < 0.05; ns, not significant. C. qRT‐PCR analysis of the mRNA levels of HDAC3 and KDELR2 in HDAC3 knockdown MDA‐MB‐231 cells and NTC cells. The data are presented as the mean ± SD of three independent experiments. *, P < 0.05. D. Western blotting analysis of the protein levels of HDAC3 and KDELR2 in MDA‐MB‐231 cells stably expressing shHDAC3 and NTC cells. β‐Actin served as the loading control. E. qRT‐PCR analysis of the mRNA levels of HDAC3 and KDELR2 in MDA‐MB‐231 cells stably overexpressing 3×Flag‐HDAC3 and empty vector cells. The overexpression efficiency was analyzed by Western blotting. The data are presented as the mean ± SD of three independent experiments. *, P < 0.05. F. qRT‐PCR analysis of the mRNA levels of HDAC3 in 16 pairs of tumor‐adjacent noncancerous breast tissues (Normal) and breast cancer tissues (Tumor). The data are presented as the mean ±SD. Group differences were analyzed by the two‐tailed Student's t‐test. G. Kaplan‐Meier curves from univariate analysis for patients with low versus high HDAC3 expression. The data were obtained from the website (http://firebrowse.org). Patients were grouped according to the optimized cutoff. H. Western blotting analysis of HDAC3 and KDELR2 protein levels in 16 pairs of clinically matched adjacent noncancerous breast tissues (normal) and human breast cancer tissues (tumor). Ponceau staining is shown at the bottom as the loading control.

FIGURE 3

HDAC3 promoted KDELR2 expression via CREB1. A. Pie graph showing the predicted transcription factors of KDELR2 analyzed by the Genecards and Promo websites. B. qRT‐PCR analysis of the mRNA levels of KDELR2 in MDA‐MB‐231 cells with CREB1, SP1 or CEBP‐β knockdown. The data are presented as the mean ± SD of three independent experiments. *, P < 0.05; ns, not significant. C. A diagram shows the potential binding sites and sequences of CREB1 at the KDELR2 gene promoter. D. Endogenous ChIP was performed to identify the binding sites of CREB1 in the KDELR2 gene. The data are presented as the mean ± SD of three independent experiments. *, P < 0.05. E‐F. qRT‐PCR analysis and Western blotting analysis of the mRNA and protein levels of CREB1 in MDA‐MB‐231 cells with stable HDAC3 knockdown. The data are presented as the mean ± SD of three independent experiments. *, P < 0.05; β‐Actin served as the loading control. G. Western blotting analysis of the expression of CREB1 in MDA‐MB‐231 cells treated with HDAC inhibitors TSA (10 μM), TDPA (10 nM) and FK228 (10 nM) for 48 h. β‐Actin served as the loading control. H. qRT‐PCR analysis of KDELR2 and CREB1 mRNA in MDA‐MB‐231 cells treated with HDAC inhibitors (TDPA or FK228) for 48 h. The data are presented as the mean ± SD of three independent experiments. *, P < 0.05. I. qRT‐PCR analysis of the mRNA levels of KDELR2 in MDA‐MB‐231 cells with stable HDAC overexpression and CREB1 knockdown. The data are presented as the mean ± SD of three independent experiments. *, P < 0.05; ns, not significant. J. Western blotting analysis of the protein levels of KDELR2, CREB1 and HDAC3 in MDA‐MB‐231 cells with stable HDAC3 overexpression and CREB1 knockdown. β‐Actin served as the loading control.

FIGURE 4

HDAC3/KDELR2 axis promoted cell cycle progression by enhancing POC5 expression. A. Cell growth analysis of HDAC3‐knockdown MDA‐MB‐231 cells. The data are presented as the mean ± SEM of three independent experiments. *, P < 0.05. B. Cell growth analysis of KDELR2‐knockdown MDA‐MB‐231 cells. The data are presented as the mean ± SEM of three independent experiments. *, P < 0.05. C. Cell growth analysis of MDA‐MB‐231 cells with stable HDAC3 overexpression and KDELR2 knockdown. The data are presented as the mean ± SEM of three independent experiments. *, P < 0.05; ns, not significant. D. Analysis of the cell cycle distribution in MDA‐MB‐231 cells with KDELR2 knockdown by flow cytometry. Representative histogram data and statistical results are shown. The data are presented as the mean ± SEM of three independent experiments. *, P < 0.05. E. Thirty proteins were predicted to interact with KDELR2 in the the BioGRID database. F. Coimmunoprecipitation assay of the protein interaction between KDELR2 and POC5. MDA‐MB‐231 cells were cotransfected with Flag‐EV or Flag‐KDELR2 and HA‐POC5 plasmids. Cell lysates were immunoprecipitated with an anti‐Flag antibody, followed by Western blotting analysis with antibodies against Flag and HA tags. G. Western blotting analysis of the expression of POC5 and KDELR2 in MDA‐MB‐231 cells with stable KDELR2 knockdown. β‐Actin served as the loading control. H. Western blotting analysis of the expression of POC5 in MDA‐MB‐231 cells with HDAC3 knockdown. β‐Actin served as the loading control. I. Western blotting analysis of the expression of POC5 in MDA‐MB‐231 cells with HDAC3 overexpression. β‐Actin served as the loading control. J. Western blotting analysis of the expression of POC5 in MDA‐MB‐231 cells with stable HDAC3 overexpression and KDELR2 knockdown. β‐Actin served as the loading control. K. Western blotting analysis of the expression of POC5 in MDA‐MB‐231 cells with KDELR2 knockdown treated with lactacystin (5 μM) for 24 h. β‐Actin served as the loading control. L‐M. Ubiquitination analysis of POC5 protein in HEK293T cells with KDELR2 (L) or HDAC3 (M) knockdown. HEK293T cells were cotransfected with HA‐Ub, FLAG‐POC5, and shKDELR2 and treated with lactacystin (5 μM) for 24 h before lysis. Equal amounts of proteins were used for immunoprecipitation with an anti‐Flag antibody, followed by blotting with anti‐HA. N. Cell growth analysis of MDA‐MB‐231 cells with KDELR2 knockdown and stable POC5 overexpression. The data are presented as the mean ± SEM of three independent experiments. *, P < 0.05. O. Analysis of the cell cycle distribution of MDA‐MB‐231 cells with KDELR2 knockdown and stable POC5 overexpression by flow cytometry. The data are presented as the mean ± SEM of three independent experiments. *, P < 0.05; ns, not significant.

FIGURE 5

KDELR2 was critical for HDAC3‐regulated breast cancer progression in vivo. A‐C. MDA‐MB‐231 cells overexpressing empty vector or 3×Flag‐HDAC3 were further infected with lentivirus expressing NTC or shKDELR2. The cell lines above were subcutaneously injected into female nude mice (n = 5 for each group). Tumor sizes were measured starting from 14 days after inoculation (A). At the end of the experiment, the tumors were extracted and compared (B‐C). The data are presented as the mean ±SEM. *, P < 0.05; ns, not significant. D‐E. qRT‐PCR analysis of the mRNA levels of KDELR2 (D) and Western blotting analysis of the expression of HDAC3, KDELR2 and POC5 (E) in xenograft tumor tissues. The data are presented as the mean ±SEM. *, P < 0.05. β‐Actin served as the loading control.

FIGURE 6

KDELR2 is required for HDAC3‐regulated breast cancer progression via accelerating cell cycle. (Left) HDAC3 transactivates KDELR2 via CREB1 and activated KDELR2 in turn protects the centrosomal protein POC5 from proteasomal degradation to accelerate cell cycle progression and breast cancer progression. (Right) HDACI treatment inactivates HDAC3, leading to the decreased expression of KDELR2 and proteasomal degradation of POC5. Consequently, blockade of HDAC3/KDELR2 axis results in cell cycle arrest and retards tumor growth in vivo.