TRIM59 promotes breast cancer motility by suppressing p62-selective autophagic degradation of PDCD10
Fig 4
TRIM59 regulates breast cancer cell actomyosin contractility and metastasis.
(A) Representative images of cell morphology changes upon TRIM59 deletion (T59 KO) in MCF7 cells (upper, left) or overexpression (T59 OE) in MDA-MB-231 cells (bottom, left). Scale bars: 20 μm. Quantification of the cell area of MCF7 (upper, right) and MDA-MB-231 cells (bottom, right). n = 6. (B) Representative confocal images of MCF7 cells depleted of TRIM59 and stained for F-actin, pSer19-MLC, and E-cadherin. Scale bars: 20 μm. (C) IB analysis of E-cadherin, pSer19-MLC expression in WT and TRIM59-KO MCF7 cells. (D) Representative confocal images of TRIM59 overexpressing (T59 OE) MDA-MB-231 cells with IRES-GFP stained for pSer19-MLC (red). Scale bars: 10 μm. (E) Representative confocal images of p-ERM expression (green) in WT and TRIM59-deleted MCF7 cells. Scale bars: 10 μm. (F) IB analysis of p-ERM and total ERM levels in WT, TRIM59 KD, or TRIM59 KO MCF7 cells (lanes 1–3) and WT or TRIM59 OE MDA-MB-231 cells (lanes 4–6). (G) Representative IHC staining images for p-ERM in tissue sections from xenograft tumors of TRIM59 KO MCF7 cells or TRIM59 OE MDA-MB-231 cells compared with control cells. Scale bars: 200 μm. (H) Spearman correlation between TRIM59 expression and ezrin phosphorylation (p-S148, p-S366) levels. (I) Representative images showing HE and CK8 staining of lung micro-metastases of xenograft tumors in the indicated groups (WT versus TRIM59 OE or TRIM59 KO in MCF7 or MDA-MB-231 cells). Scale bars: 100 μm (HE); 20 μm (CK8). Red arrow: pulmonary metastatic foci. Statistical analysis of the number of lung metastases was shown in the right panel. n = 6. (J and K) Representative luminescent images of the primary and metastases acquired with an IVIS imaging system. Luminescence imaging in NSG mice was followed after inoculation with breast cancer cells into the mammary fat-pad at the indicated time points (n = 3). Note, 10-week-old NSG mice inoculated with TRIM59 OE MDA-MB-231-luc died without bioluminescent imaging. (L) Gene expression analysis of TRIM59 in primary and lymph node metastases (GSE30480 dataset). (M) Representative TRIM59 IHC staining images in paired primary breast cancer and metastatic lymph nodes from breast cancer patients (n = 19). Statistical analysis of paired patient samples (primary versus metastatic tissues in the same patient) were shown on the right. Data in A, I, L, and M are presented as means ± SD. *P < 0.05, **P < 0.01 versus controls. The underlying data can be found in S1 Data. CK8, cytokeratin 8; ERM, Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558); HE, hematoxylin–eosin; IB, immunoblot; IHC, immunohistochemistry; IRES, internal ribosome entry site; GFP, green fluorescent protein; IVIS, in vivo imaging system; KD, knockdown; KO, knockout; Min, minimum; Max, maximum; NSG, NOD scid gamma; OE, overexpressed; p-ERM, phosphorylation of ERM; pSer19-MLC, phosphorylation of MLC at serine 19; TRIM59, tripartite motif 59; WT, wild-type.