High Sugar Induced RCC2 Lactylation Drives Breast Cancer Tumorigenicity Through Upregulating MAD2L1

Abstract

Lactylation is a novel post-translational modification mediated by lactate, widely present in the lysine residues of both histone and non-histone proteins. However, the specific regulatory mechanisms and downstream target proteins remain unclear. Herein, it is demonstrated that the RCC2 protein may serve as a critical link between material metabolism and cell division, promoting the rapid proliferation of breast cancer under high glucose conditions. Mechanistically, the activation of glycolysis leads to an increase in lactate. Then, acyltransferase KAT2A mediates RCC2 lactylation at K124, which assists RCC2 in recruiting free SERBP1, thereby stabilizing MAD2L1 mRNA. The lactylation of RCC2 mediates the activation of the cellular MAD2L1 signaling pathway and contributes to the progression of breast cancer. A small molecule inhibitor slows down cell proliferation by binding to the RCC2 active pocket and specifically blocking RCC2 lactylation. The findings elucidate the mechanism behind the upregulation of MAD2L1 in murine tumors associated with a high-sugar diet as reported in prior study and suggest a novel therapeutic strategy of targeting RCC2 lactylation to restrict the rapid proliferation of breast cancer cell in a high-lactate microenvironment.

Keywords: MAD2L1; RCC2; SERBP1; cell division; high sugar diet.

Figure 1

Lactate upregulates MAD2L1 expression in breast cancer. A) MAD2L1 expression level under high‐glucose (25 mm) or low‐glucose (5 mm) culture conditions in HS578T cell in GSE202923. B) Western blot analysis of global lactylation level in seven pairs of normal tissue (N) and breast cancer tissue (T). C) IHC analysis of global lactylation level in 40 breast cancer, and divide them into two groups, high‐Kla and low‐Kla. D) Western blot analysis of global lactylation level in normal mammary epithelial MCF‐10A cell and breast cancer cells with different molecular subtypes. E) IF analysis after 24 h 10 mm 2‐DG treatment. Scale bars, 40 µm. F) PCR assays of MAD2L1 expression level after NaLa and 2‐DG treatment. G) Western blot analysis of MAD2L1 expression level after NaLa and 2‐DG treatment. H,I) MTT and colony formation assays were used to assess the proliferation capacity of the MDA‐MB‐231 cell when MAD2L1 was knocked down with or without 50 mm NaLa treatment. J) MDA‐MB‐231 cells stably expressing sh‐NC and sh‐MAD2L1 were subcutaneously injected into the nude mice. These mice were treated with or without NaLa (120 mg kg⁻¹ once a day). Tumor images and Ki‐67 staining were shown. Scale bars, 100 µm. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2

RCC2 serves as a lactylation substrate to upregulate MAD2L1. A) Correlation analysis of the expression of RCC2 and MAD2L1 in breast cancer tissues from the TCGA database. (http://gepia.cancer‐pku.cn/) B) PCR assays of MAD2L1 expression level when RCC2 was overexpressed. C) Western blot analysis of MAD2L1 protein level when RCC2 was overexpressed or knocked down. D) Detection of endogenous lactylation of RCC2 protein in MDA‐MB‐231 and HEK293T cells. E) Detection of lactylation of exogenous RCC2 protein in MDA‐MB‐231 and HEK293T cells after transfection plasmids. F) Lactylation level of exogenous RCC2 with or without 50 mm NaLa treatment. G,H) MTT and colony formation assays were used to assess the proliferation capacity of the MDA‐MB‐231 cell when RCC2 was knocked down with or without 50 mm NaLa treatment. I) MDA‐MB‐231 cells stably expressing sh‐NC and sh‐RCC2 were subcutaneously injected into the nude mice. These mice were treated with or without NaLa (120 mg kg⁻¹ once a day). Tumor images and Ki‐67 staining were shown. Scale bars, 100 µm. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3

KAT2A mediates RCC2 lactylation at K124. A) Plasmid schematic. B) Lysine lactylation of various peptide segments of the RCC2 protein was detected in HEK293T. C) Western blot analysis of exogenous RCC2 lactylation when transfected specific mutation plasmids. D) Exogenous RCC2 lactylation level when transfected RCC2 K124R plasmids with or without 50 mm NaLa treatment. E) Conservation analysis of RCC2 protein sequences in different species. F) Exogenous RCC2 lactylation level when a series of acyltransferases were overexpressed in MDA‐MB‐231. G) Exogenous RCC2 lactylation level when KAT2A was knocked down in MDA‐MB‐231. H,I) CoIP and WB analysis of RCC2 and KAT2A. J) Exogenous RCC2 lactylation level when KAT2A was overexpressed with or without RCC2 K124R mutation in MDA‐MB‐231. K) In vitro lactylation assay.

Figure 4

RCC2 interacts with SERBP1 to regulate MAD2L1. A) Schematic diagram of the job screening process. B) GO molecular function enrichment analysis of RCC2's potential interacting proteins. C,D) PCR assays of MAD2L1 expression level when SERBP1 was overexpressed or knocked down. E,F) Western blot analysis of MAD2L1 expression level when SERBP1 was overexpressed or knocked down. G,H) CoIP and WB analysis of RCC2 and SERBP1. I) CoIP and Western blot analysis of exogenous SERBP1 and segmented RCC2. J,K) MTT and colony formation assays were used to assess the proliferation capacity of the SERBP1‐silenced MDA‐MB‐231 cell with or without RCC2 overexpression. L) Western blot analysis of MAD2L1 expression level in SERBP1‐silenced cell with or without RCC2 overexpression. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5

SERBP1 binds to and stabilizes MAD2L1 mRNA. A) RIP assay followed by nucleic acid gel electrophoresis analysis. B) RIP assays followed by PCR analysis. C,D) PCR assay showing the expression of MAD2L1 when SERBP1 was overexpressed or knocked down in MDA‐MB‐231 and BT‐549 cells at 0, 3, 6, 9, and 12 h Act D treatment. E) PCR assay of the level of MAD2L1 mRNA enriched by the same Flag antibodies when RCC2 was knocked down in MDA‐MB‐231. F) PCR assays of RCC2 expression level when SERBP1 was overexpressed in MDA‐MB‐231. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 6

RCC2 lactylation helps recruit free SERBP1. A,B) Cell proliferation of control (sh‐NC) or RCC2‐silenced MDA‐MB‐231 cell overexpressed with control vector or wt‐RCC2 (RCC2 wt.) or mut‐RCC2 (RCC2 K124R) in the presence of NaLa were measured in MTT and colony formation assays. C) Xenograft tumor images of control (sh‐NC) or RCC2‐silenced MDA‐MB‐231 cell overexpressed with control vector or wt‐RCC2 (RCC2 wt.) or mut‐RCC2 (RCC2 K124R) under Intermittent NaLa treatment. D) PCR assays of MAD2L1 expression level when overexpressed with control vector or wt‐RCC2 or mut‐RCC2 in MDA‐MB‐231. E) Molecular docking of RCC2 and SERBP1 using autodock software when incorporation or absence of a lactyl group at RCC2 site 124 lysine. F,G) Endogenous and exogenous IP and WB analysis of the interaction intensity between RCC2 and SERBP1 when overexpressed with wt‐RCC2 or mut‐RCC2. H,I) Endogenous and exogenous IP and WB analysis of the interaction intensity between RCC2 and SERBP1 when with or without 50 mm NaLa treatment. J) Immunofluorescence imaging and statistical analysis of cellular localization of RCC2 and SERBP1 proteins with or without 50 mm NaLa treatment. Scale bars, 20 µm. K) IP and WB analysis of the interaction intensity between RCC2 and SERBP1 when overexpressed with the control vector or KAT2A in MDA‐MB‐231. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 7

SBDA inhibits breast cancer cell RCC2 lactylation. A) The expression of RCC2, SERBP1, and MAD2L1 genes is correlated with the survival prognosis of breast cancer patients in the K‐M database (https://www.kmplot.com). B) The binding free energy of 7 candidate compounds docked to the RCC2 K124 active pocket. C) The chemical structure of SBDA. D) A docking visualization of SBDA with the active pocket of RCC2. E) MTT assays were used to assess the proliferation capacity of MDA‐MB‐231 cells after 24 h gradient concentration SBDA treatment (IC50 value was shown). F) Colony formation assays were used to assess the proliferation capacity of MDA‐MB‐231 cells after 24 h gradient concentration SBDA treatment under 50 mm NaLa. G) Exogenous RCC2 lactylation level was detected after 24 h SBDA treatment. H) The effect of 500 µm SBDA treatment on the growth of organoid cell spheres from breast cancer patients was observed by light microscopy (Shows images on day 1 and 5). Scale bars, 200 µm. I) The schematic diagram of the potential mechanism in this study. We created the images using the Biorender (https://www.biorender.com). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.