Acetylation suppresses breast cancer progression by sustaining CLYBL stability

Abstract

Background: The incidence of breast cancer remains high and it remains the leading cause of cancer-related deaths in women. A better understanding of the molecular mechanisms of breast cancer and identifying novel biomarkers will help improve therapeutic strategies. Citrate lyase beta like (CLYBL) is expressed at low levels in breast cancer tissues and is associated with low patient survival rates. In this study, we explored the regulatory mechanisms of CLYBL and its acetylation in breast cancer.

Methods: CLYBL expression patterns in breast cancer were assessed using a breast cancer tissue microarray, immunohistochemistry, and publicly available datasets. The acetylation patterns of CLYBL and the related regulatory functions were detected by high resolution mass spectrometry, immunoprecipitation assays, and western blot analysis. The potential effects of CLYBL and its acetylation on breast cancer were determined using both in vitro and in vivo assays.

Results: CLYBL was expressed at lower levels in breast cancer samples compared with normal tissues. This low CLYBL expression was associated with poor patient survival rates. Overexpressing CLYBL could inhibit breast cancer and reduce NRF2 pathway-mediated antioxidants. We identified two acetylated lysine sites in CLYBL, K57 and K82, using acetylated peptide affinity enrichment and high-resolution mass spectrometry. Our results suggest that K82 is the main acetylation site. Further work showed that the p300/CBP associated factor (PCAF) and histone deacetylase 3 (HDAC3) as the CLYBL acetyltransferase and deacetylase, respectively. Additionally, CLYBL acetylation facilitates its own protein stability by reducing it affinity for ubiquitin, thus enhancing the anti-breast cancer effects.

Conclusion: We revealed the role of CLYBL overexpression and its acetylation in breast cancer. Our study suggests that CLYBL is a potential molecular target for breast cancer therapy.

Keywords: Acetylation; Breast cancer (BC); CLYBL; HDAC3; PCAF.

Fig. 1

Breast cancer has low CLYBL expression level that is negatively associated with survival rates. (A-C) Kaplan–Meier survival analysis for OS and RFS of breast cancer patients according to CLYBL expression status. The P-value was determined using the log-rank test. (D) Box plots showing CLYBL mRNA expression patterns in breast cancer samples from the TCGA dataset

Fig. 2

CLYBL expression in breast cancer tissues. (A) Immunohistochemical staining of CLYBL protein in adjacent and matched breast cancer tissues. Scale bars: 200 µM. (B) CLYBL expression levels were quantified in both breast cancer tissues and adjacent tissues using the IRS score (P<0.001). Statistical analysis was performed using the unpaired t-test. (C) Kaplan-Meier survival curve showing a significant correlation between low CLYBL expression levels and decreased survival in human breast cancer. The P-value was determined using the log-rank test. (D) The correlations between CLYBL expression levels and various clinicopathological characteristics. (E) Univariate and multivariate analyses of the factors correlated with overall survival of breast cancer patients. The P-value was determined using the log-rank test

Fig. 3

Overexpression of CLYBL can inhibit breast cancer and reduce NRF2 pathway-mediated antioxidants. (A) Soft agar assays were performed using MDA-MB231 and Hs578T cells with or without CLYBL overexpression. Data are presented as the mean ± SD, n = 3, **P < 0.01. (B) MDA-MB231-Luc cells with or without CLYBL overexpression were injected into the second mammary fat pad of nude mice. The tumor volume was recorded every 2 days. After 19 days, the mice were sacrificed and the wet weight of each tumor was determined at autopsy. Data are presented as the mean ± SD of six mice, **P < 0.01 by Student’s t-test. (C) The nuclear proteins of MDA-MB231 and Hs578T cells were collected with or without CLYBL overexpression. (D) RT-qPCR was used to detect the mRNA expression levels of GCLM, GSS, HMOX, and NQO-1. Data are presented as the mean ± SD, n = 3, ∗P < 0.05, ∗∗P < 0.01

Fig. 4

CLYBL is acetylated at its lysine 82 site. (A) Acetylation of exogenous CLYBL-Flag in MDA-MB231 and Hs578T cells. Immunoprecipitation of whole cell lysates with an anti-Flag antibody or IgG was followed by western blot analysis with the anti-acetyl-lysine and anti-Flag antibodies. (B) Acetylation of exogenous CLYBL-Flag in HEK293T cells treated with deacetylase inhibitors CHI (3.2 µM) for 24 h. CLYBL-Flag was immunoprecipitated with the anti-Flag antibody, and then the precipitates were analyzed using an anti-acetyl-lysine antibody (Ace-lys). 4T1 cells were injected into the second mammary fat pad of BALB/C mice and tumor tissues were collected 14 days after CHI treatment, and then sent for profiling of the lysine acetylation proteome. (C) The bar diagram shows the subcellular localization of proteins modified by acetylation determined with WolF PSORT software. (D) The pie chart shows the distributions of the number of sites in the modified proteins. (E) The left and right sides of the bar chart represent the GO terms enriched in the proteins with upregulated or downregulated acetylation levels. (F) The tissues lysates and isotopically-labeled lysates were mixed and immunoprecipitated with anti-acetyl-lysine antibody-conjugated beads, and then were analyzed by mass spectrometry (MS). (G) Alignment of the CLYBL amino acid sequences from various species. Red shading indicates the conserved K57 and K82 residues. (H) CLYBL-Flag (K57R), CLYBL-Flag (K82R), CLYBL-Flag (K57R/K82R), or CLYBL-Flag (WT) were transfected into MDA-MB231 and Hs578T cells. The lysates were extracted, and then CLYBL acetylation was detected by co-IP and western blot analysis with the indicated antibodies

Fig. 5

CLYBL is acetylated by PCAF and deacetylated by HDAC3. (A) HEK293T cells were transfected with CLYBL-Flag and a serious of acetyltransferases, GCN5-Myc, P300-Myc, PCAF-Myc, TIP60-HA, and CREBP-HA, to detect CLYBL acetylation levels by immunoprecipitation. (B) Exogenous co-IP was used to detect the interactions between PCAF and CLYBL in HEK293T cells. (C) HEK293T cells co-transfected with CLYBL-HA and HDAC3-Flag were used to detect CLYBL acetylation levels by immunoprecipitation. (D, E) Co-immunoprecipitation of HDAC3 with CLYBL was performed using HEK293T cells co-transfected with CLYBL-HA and HDAC3-Flag or CLYBL-Flag and HDAC3-HA

Fig. 6

K82 acetylation inhibits CLYBL degradation and further repress breast cancer. (A) HEK293T cells were transfected with CLYBL-Flag and/or Ub-HA vectors, followed by treatment with MG132 (100 µM). The lysates were extracted and CLYBL ubiquitination was detected by co-IP and western blot analysis with the indicated antibodies. (B) CLYBL-Flag (K82Q), CLYBL-Flag (K82R), CLYBL-Flag (WT), or Ub-HA were transfected in HEK293T cells. The lysates were extracted and CLYBL ubiquitination was detected by co-IP and western blot analysis with the indicated antibodies. (C) CLYBL-Flag, Ub-K48-HA, or Ub-HA were transfected in HEK293T cells. The lysates were extracted and K48-linked ubiquitination of CLYBL was detected by co-IP and western blot analysis with the indicated antibodies. (D) HEK293T cells were transfected with CLYBL-Flag (WT) and CLYBL-Flag (K82Q) for 36 h, then incubated with 20 µg/mL cycloheximide (CHX) for the indicated times and analyzed by western blot analysis. (E) MDA-MB231 cells with CLYBL (WT) or CLYBL (K82Q) overexpression were injected into the second mammary fat pad of nude mice. The tumor volume was recorded every 2 days. After 23 days, the mice were sacrificed and the wet weight of each tumor was determined at autopsy. Data are presented as the mean ± SD of six mice, *P < 0.05 by Student’s t-test

Fig. 7

Schematic depicting CLYBL and its acetylation regulation in BC. CLYBL is modified by acetylation at K82. PCAF contributes to its acetylation and HDAC3 promotes its deacetylation. Acetylation of CLYBL facilitates its own stability by reducing the affinity of CLYBL for K48-linked ubiquitin, which could inhibit breast cancer and reduce NRF2 pathway-mediated antioxidants