Fig 2: Nuclear TYRO3 phosphorylates BRD3 to promote CRC malignancy.(A) Schematic drawing shows the flow chart of identifying BRD3 as ICD-TYRO3 nuclear partner by mass spectrometry from CCD-841CoN cells. (B) The interaction between TYRO3 (1rhf) and BRD3 (2nxb) and binding energies predicted by Structural Matching software. (C and D) Representative Western blots show the binding of endogenous TYRO3 and BRD3 in the nucleus of HCT116 cells (C) and ICD-TYRO3 and BRD3 in CCD-841CoN cells transiently transfected with GFP or ICD-TYRO3-GFP (D). Lamin A/C, loading control; WCL, whole cell lysate; Cy, cytosolic fraction; Nu, nuclear fraction; IgG, control. (E) Representative images show the binding of TYRO3 and BRD3 by in situ PLA. Scale bar, 5 µm. (F) Representative images and quantified data of nuclear TYRO3+/BRD3+ cells in orthotopic colon cancers (GFP, n = 9; TYRO3-GFP, n = 7; ARP100 + GFP, n = 10; ARP100 + TYRO3-GFP, n = 10). Scale bar, 10 µm. (G) The kinase activity of TYRO3 in phosphorylating BRD3. RFU, relative fluorescence units. (H) Representative Western blots show phosphorylated BRD3 (pBRD3) and unphosphorylated BRD3. The proteins were separated by 6% Zn2+-Phos-tag SDS–polyacrylamide gel electrophoresis (PAGE). (I) Biological processes associated with genes bound by BRD3 in nuclear TYRO3-overexpressing cells. (J) Results of ChIP-qPCR show the occupancies of BRD3 at the promoters of targeted genes. Distal primers were used as a quality control. (K) Results of ChIP-qPCR show the occupancies of TYRO3 at the promoters of SNAI1 and CDC27. Lysates from HCT-116 cells stably transfected with different constructs were immunoprecipitated using anti-GFP antibody and subjected for PCR amplification. (L) Heatmap shows the correlation of TYRO3 level with BRD3, SNAI1, and CDC27 by analyzing TCGA colon cancer dataset. **P < 0.01 and ***P < 0.001. m/z, mass/charge ratio.

Fig 3: Inhibition of BRD3 eradicates TYRO3-induced CRC metastasis in mice.(A) Representative Western blots and quantified result show SNAI1 in cells transfected with indicated plasmids in the presence (+) or absence (-) of MMP-2 inhibitor, ARP100 (1 µM). (B) Representative Western blots and quantified result show the levels of SNAI1 in HCT116 cells treated with (+) or without (-) BRD3 inhibitor, RVX-208 (10 µM). (C and D) Representative images and quantified results show BrdU+ (C) and annexin V+/PI+ (D) cells treated with or without BRD3 inhibitor. Scale bar, 50 µm. (E) Real-time reverse transcription–qRCR shows the expression of SNAI1. (F) Representative Western blot and quantified result show SNAI1 in ICD-TYRO3–overexpressed HCT116 cells with or without BRD3 knockout. (G) Representative Western blot and quantified result show global acetylation of histone H3 in HCT116 cells. (H) Potential target genes of BRD3 were analyzed by ChIP-qPCR. (I) Representative images show location of C-TYRO3, E-cadherin, and DAPI in colon organoids. Scale bar, 10 µm. (J) Representative images and quantified result of organoids (n = 3, 30 organoids per subject) treated with or without inhibitors were shown. Scale bar, 100 µm. (K) Representative images show local invasion and metastasis of cancer cells with or without different drug treatments. Arrows indicate the invasive cancer. Scale bar: 100 µm (left column); 50 µm (middle and right columns). (L) Representative images show local invasion and metastasis of cancer cells with or without BRD3 (sgBRD3) or BRD4 (sgBRD4) knockout. Arrows indicate the invasive cancer. Scale bars, 100 µm (left) and 50 µm (right). *P < 0.05, ##P < 0.01, ### and ***P < 0.001.

Fig 4: Model of nuclear TYRO3 contributing to CRC malignancy.We identified that MMP-2 releases ICD-TYRO3 to reduce survival rate and promote CRC migration, invasion, and cell growth through BRD3. TYRO3-induced CRC progression and metastasis are reversed by MMP-2 selective inhibitor in orthotopic mouse models.

Fig 5: ICD-TYRO3 promotes normal colon cell transformation and CRC malignancy.(A) Schematic drawing shows the sequence of TYRO3 and ICD-TYRO3. The ICD-TYRO3 starts from the MMP-2 cleavage site. Ig, immunoglobulin; TM, transmembrane domain. (B) Representative confocal images showed anti-N terminus of TYRO3 (red) and GFP signal detection in HCT116 cells after transfection. Scale bar, 10 µm. (C) Lengths and speed of nascent replication tracts labeled with CldU (red) and IdU (green) were measured (n = 150) after treatment. Scale bar, 10 µm. Asterisks indicate statistical differences in intragroup comparison and pound signs indicate statistical differences in intergroup comparison. (D) Representative Western blots and quantified data showed the levels of caspase 3, TYRO3, and ß-actin in HCT116 cells transfected with the indicated plasmids. (E) Representative Western blots showed the expression of ICD-TYRO3 (ICD) in the nucleus of CCD-841CoN cells after transfection. Cyto, cytosolic fraction; Nu, nuclear fraction; Ctrl, control IgG; red arrowhead, ICD-TYRO3-GFP; black arrowhead, GFP. (F) Colony formation assay was performed for 21 days after transfection of CCD-841CoN with control (Ctrl) or ICD-TYRO3 plasmid. Left, representative images; right, quantification analysis. Scale bar, 100 µm. (G) Representative Western blots and quantified data showed the levels of SNAI1, E-cadherin, TYRO3, and ß-actin in HCT116 cells transfected with the indicated plasmids. (H) Representative confocal images and quantified data showed the HCT116 transendothelial migration. CD31, endothelial cells (red); Yellow dashed lines, transwell filters; arrowheads, top and bottom sides of the filters. Scale bar, 10 µm. **P < 0.01; ### and ***P < 0.001.