Fig 2: SMARCD1 is an “essential expression-restricted” metastasis modifier.Analysis of primary tumor (PT) weight (a), number of lung nodules (b), and number of lung nodules per gram of PT per mouse (c) 28 days after orthotopic injection of 6DT1 EV control and Smarcd1 OE lines (2 combined experiments, 10 mice per experiment). Analysis of PT weight (d), number of lung nodules (e), and number of lung nodules per gram of PT per mouse (f) 28 days after orthotopic injection of 6DT1 shScr control and shSmarcd1 lines (2 combined experiments, 10 mice per experiment). Number of lung metastases per mouse 28 days after tail vein injection (TV inj) of 6DT1 EV control and Smarcd1 OE lines (g) or 6DT1 shScr control and shSmarcd1 lines (h) (2 experiments combined, 10 mice per experiment). GOBO database distant metastasis-free survival (DMFS) Kaplan–Meier analysis stratified by SMARCD1 expression level for all breast cancer patients (i), Basal subtype patients (j), or ER+/HER2-enriched subtype patients (k).

Fig 3: Smarcd1 expression regulates tumorsphere formation and chromatin accessibility.a Western blot showing Smarcd1 and Actin protein levels after transduction of 6DT1 cells with empty vector (EV), Smarcd1 overexpression vector (OE), shScramble (shScr), and shSmarcd1 shRNA-1 and shRNA-2. Tumorsphere number grouped by size 10 days after seeding 6DT1 EV control and Smarcd1 OE lines (b), 6DT1 shScr control and shSmarcd1 lines (c), or 6DT1 cells transfected with Smarcd1-targeted siRNA or non-targeting control (d), representative data from 1 of 3 replicates, n = 6 wells per cell line. *p < 0.05. e–h Volcano plots showing differential gene expression analysis with false discovery rate (FDR) < 0.1 indicated by red intensity in Smarcd1-altered 6DT1 cell lines vs controls, in monolayer culture (e, f) and sphere culture (g, h). Histogram showing number of regions of open chromatin (i) detected by ATAC-seq analysis and their average length (j). k Venn diagram analysis of the genes associated with open regions of chromatin in Smarcd1-altered cells vs controls and in monolayer culture and in sphere culture. l Fold change in the enrichment of specific gene sections (Intergenic, Transcription start site or TSS, Intron, 5 prime untranslated region or 5’UTR, Transcription termination sequence or TTS, coding sequence or CDS, and 3’UTR) in open chromatin for Smarcd1-altered cells vs control lines grown in monolayer. m, Venn diagram analysis of genes from Smarcd1-altered cells and controls grown in monolayer for which 3’ UTR, 5’ UTR, and TTS were enriched in open chromatin.

Fig 4: Nanos1, Pum2, and Cpsf4 regulate Smarcd1 mRNA half-life.a Diagram illustrating a hypothetical metastatic gene expression program regulated by RNA-binding protein (RNA-BP) abundance and mRNA stability, amended from Ross et al.37. b Venn-diagram analysis of RNA-seq differential gene expression (≥1.5 fold change ≤−1.5) and false discovery rate (FDR) less than 0.05 from 4T1 shNanos1 vs shScr, shPum2 vs shScr, and shCpsf4 vs shScr. Venn-diagram analysis of RNA-seq differential gene expression for genes with ≥1.5 fold change (c) and ≤−1.5 fold change (d) with FDR of 0.05. e Kaplan–Meier analysis of distant metastasis-free survival (DMFS) in all breast cancer patients stratified by 21 commonly differentially expressed genes (used as a non-weighted signature) in the GOBO database. f–h Nine genes with consistent expression changes by qRT-PCR analysis of 21 differentially expressed genes in 6DT1 shPum2 (f), shNanos1 (g), and shCpsf4 (h) vs shScr (n = 3). Representative actinomycin D (actD) time courses showing Smarcd1 transcript decay in 6DT1 shPum2 (i), shNanos1 (j), and shCpsf4 (k) vs shScr lines (n = 3).