KAT6A Acetylation of SMAD3 Regulates Myeloid‐Derived Suppressor Cell Recruitment, Metastasis, and Immunotherapy in Triple‐Negative Breast Cancer

Abstract Aberrant SMAD3 activation has been implicated as a driving event in cancer metastasis, yet the underlying mechanisms are still elusive. Here, SMAD3 is identified as a nonhistone substrate of lysine acetyltransferase 6A (KAT6A). The acetylation of SMAD3 at K20 and K117 by KAT6A promotes SMAD3 association with oncogenic chromatin modifier tripartite motif‐containing 24 (TRIM24) and disrupts SMAD3 interaction with tumor suppressor TRIM33. This event in turn promotes KAT6A‐acetylated H3K23‐mediated recruitment of TRIM24–SMAD3 complex to chromatin and thereby increases SMAD3 activation and immune response‐related cytokine expression, leading to enhanced breast cancer stem‐like cell stemness, myeloid‐derived suppressor cell (MDSC) recruitment, and triple‐negative breast cancer (TNBC) metastasis. Inhibiting KAT6A in combination with anti‐PD‐L1 therapy in treating TNBC xenograft‐bearing animals markedly attenuates metastasis and provides a significant survival benefit. Thus, the work presents a KAT6A acetylation‐dependent regulatory mechanism governing SMAD3 oncogenic function and provides insight into how targeting an epigenetic factor with immunotherapies enhances the antimetastasis efficacy.

Validating the specificity of the anti-SMAD3 K117 and K20 acetylation antibodies and identifying the importance of MH2 domain (D2 mutant) of SMAD3 for SMAD3 acetylation by KAT6A. A, The amino acid sequences around K20 and K117 in SMAD3 among multiple species. B, IHC assays of a clinical breast cancer tumor tissue with the specific anti-K20ac or anti-117ac antibody in the presence of a control peptide or the specific acetylated peptide containing K20ac or K117ac. IHC was performed two times on the sample with the blocking peptide with similar results. Scale bar, 50 μm. C and D, IP and WB of K20ac (C) or K117ac (D) in MDA-MB-231 cells. A rabbit K20ac or K117ac was generated against a specific acetylated peptide containing K20ac or K117ac. Before IP, agarose beads were pre-incubated with a control peptide or the specific acetylated peptide containing K20ac or K117ac.

Animal Xenograft Studies
For the orthotopic xenograft model, 4T1 cells (5 × 10 5 ) were suspended in 50 μl of PBS and mixed with matrigel (1:1), and then were injected into the fourth mammary fat pad of BALB/c mice following our established protocol [1]

immunoprecipitation (IP) and Western blotting (WB) assays
IP and WB assays were performed as previously described. [2] In brief, cells were lysed in IP lysis buffer (20 mM Tris-HCl pH 7.5, 1% Triton X-100, 150 mM NaCl, 2 mM Na 3 VO 4 , 5 mM NaF, and 1 mM EDTA) supplemented with complete protease inhibitor cocktail (Roche) at 4°C for 30 min. The lysates were cleared by centrifugation and immunoprecipitated with specific antibodies and protein G-G-agarose beads (Invitrogen).
Proteins were separated by SDS-PAGE gels and visualized by enhanced chemiluminescence (ECL, Bio-Rad) reaction according to the manufacturer's instructions.
Cell fractionation was conducted using Subcellular Protein Fractionation Kit (Pierce).

Purification of recombinant proteins and GST pull-down assay
Recombinant GST-conjugated KAT6A or SMAD3 was generated by transforming the E.

Proteomics Analysis
Proteomics analyses for KAT6A-associated proteins and SMAD3 acetylation were Then, the immunoprecipitants were washed three times with wash buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% NP40, 10% glycerin) and bead-bound proteins were eluted with wash buffer plus 500 µg /ml FLAG peptides. The samples were sent to Jiyun Biotech.Inc and analyzed as previous described. [3] Unique peptides that were detected only in Flag-KAT6A immunoprecipitants, or displayed at least 2-fold higher abundance than the empty vector control groups, were selected. From all these peptides, only the ones that emerged in all of the three replicates were considered as KAT6A associated proteins.

RNA-Seq analysis and gene set enrichment analysis
RNA-Seq and differentially expressed gene analysis were performed as previously described [2] . Gene set enrichment analysis (GSEA) was conducted using GSEA2. Dual-Luciferase® Reporter System following the manufacturer's recommendation.

Chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR)
ChIP analysis was performed using a SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads, Cell Signaling Technology, 9005S). Cells were harvested and cross-linked with 1% formaldehyde. Cell nuclei were prepared and chromatin was incubated with Micrococcal Nuclease 37°C for 20 min, followed by appropriate sonication.
The supernatants were immune-precipitated using 3 μg anti-SMAD3 antibody or the relevant non-specific IgG at 4°C for 16 h. ChIP DNA was purified and subsequently quantified by qPCR. following the manufacturer's recommendation.

Flow cytometry analysis (FACS) and sorting
Single-cell suspensions were prepared and incubated on the ice with a combination of antibodies for 30 min in the dark. FACS analysis was performed using the LSRII Flow Cytometer (BD Biosciences), and data were analyzed using the FlowJo software (Tree Star Inc.). For FACS sorting, Aria II or FACS Jazz instruments were used.

ALDH + cell staining
The ALDH + cell staining was performed by using a ALDEFLUOR assay kit according to the manufacturer's guidelines (STEMCELL Technologies
For ELDA, cells were seeded into 96-well ultralow attachment plates with sphere medium at density of 5, 10, 20, 50, 100 cells/well (12 wells per cell density). After 10 days, each well was examined for the formation of tumor spheres. Stem cell frequency was calculated using extreme limiting dilution analysis (http://bioinf.wehi.edu.au/software/elda/).