Additional file 1 of SALL1 functions as a tumor suppressor in breast cancer by regulating cancer cell senescence and metastasis through the NuRD complex
Creators: name: Ma, Chunling
name: Wang, Fang
name: Han, Bing
name: Zhong, Xiaoli
name: Si, Fusheng
name: Ye, Jian
name: Hsueh, Eddy C.
name: Robbins, Lynn
name: Kiefer, Susan M.
name: Zhang, Yanping
name: Hunborg, Pamela
name: Varvares, Mark A.
name: Rauchman, Michael
name: Peng, Guangyong
Publisher: name: figshare
Year: 2022
Subjects: subject: Biophysics
subject: Space Science
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: Plant Biology

description: Figure S1. SALL1 and SALL4 expression levels in different types of human cancers. (A) SALL1 expression in tumor cells in breast cancer and melanoma tissues was determined using the immunohistochemical staining. Numbers of SALL1+ tumor cells in melanoma tissues were much higher than those in breast cancer tissues. Expression level of each dot shown in the right panel is the average numbers of SALL1+ cells per high field (400 x) in each tissue sample. The median number of SALL1+ cells in each group is shown as a horizontal line. Significance was determined by unpaired T test. (B) The COSMIC analysis shows the mutations and the promoter methylation status of SALL1 gene in breast cancer tissues. (C) SALL1 promoter genes are highly methylated in the primary breast cancer tissues comparison with solid normal tissues. The 2 specific molecular probes which span the proximal promoter region of SALL1 gene were selected based on the information in COSMIC. The box plots showed that the methylation β value of particular promoter regions in the tissues. N indicated the number of sample size. **P &lt; 0.01 between 2 groups with the Mann-Whitney U test. (D) and (E) Gene expression levels of SALL4 in different cancer cell lines (in D) and in tumor tissues (in E) using Real-time PCR analyses. Tumor cell lines include breast cancer (human MDA, MCF7, BC80, 31, 30, 29, 16, and murine 4 T1 and E0771), melanoma (human Mel1938, Mel1586, Mel1860, Mel1363, Mel1526 and Mel1628, and murine B16F0), prostate cancer (PC3 and DU145), colon cancer (SW480), and lymphoma (L428 and L504). Normal breast cell lines (BN6 and BN16), fibroblasts (F163, F160, F158 and F112), 293 T cells, and normal breast tissues were included as controls. mRNA levels in each cancer cell line and tumor tissue were normalized to the relative quantity of GAPDH expression and then adjusted to the express levels in 293 T cells (set as 1). Results shown in the histogram are mean ± SD from three independent experiments. Figure S2. The inhibition of breast cancer cell proliferation and growth mediated by SALL1 expression is not due to the induction of apoptosis. (A) and (B) Transfection of SALL1 but not SALL4 significantly inhibited prostate cancer PC3 cell growth and proliferation. PC3 tumor cells transfected with or without plasmids pcDNA3.1-SALL1, pcDNA3.1-SALL4, and pcDNA3.1, were cultured at a starting number of 2 × 104/well in 24 wells (A), or 1 × 104/well in 96-well plates (B). Cell growth was evaluated at different time points by counting cell number (in A), and cell proliferation was determined using [3H]-thymidine assays (in B). Data are mean ± SD from three independent experiments with similar results. **p &lt; 0.01 compared with the vector control group. (C) Transfected breast cancer and melanoma cells were cultured for additional 24 h. Apoptosis in transfected tumor cells was analyzed after staining with PE-labeled Annexin V and 7-AAD. Data shown are representative of three independent experiments with similar results. Figure S3. Real-time PCR analyses of mRNA expression levels of the cell cycle regulatory genes in breast cancer MDA-MB-231 cells after transfection with SALL1. MDA-MB-231 tumor cells were transfected with SALL1 for 72 h and then RT-PCR for gene expressions was performed. The expression level of each gene was normalized to GAPDH expression and adjusted to the levels in vector-transfected tumor cells. Data show mean ± SD from three independent experiments with similar results. Figure S4. SALL1 over-expression in breast cancer cells induces tumor cell senescence. (A) Transfection of SALL1, but not SALL4 in MCF-7 and E0771 breast cancer cells significantly induced the increased SA-β-Gal+ cell populations. In contrast, over-expression of SALL1 in B16F0 cells did not induce senescent cells. Transfected tumor cells were cultured for additional 3 days, and senescent cells were analyzed using the SA-β-Gal activity assay. Data shown are mean ± SD from three independent experiments with similar results. **p &lt; 0.01 compared with the vector group. (B) SALL1 expression in breast cancer cells induced expression of phosphorylated active of ATM in the transfected cells. Transfected tumor cells were analyzed for the expression of p-ATM expression after culture for 24 h using the FACS analysis. Figure S5. SALL1 proteins expressed in transfected breast cancer cells. Wild type SALL1 is shown at the top. Red represents the 12 amino acid region that mediates NuRD complex recruitment. Blue ovals are C2H2 zinc fingers. In mutant mSALL1, the 12 amino acid NuRD recruitment domain is deleted. In SALL1-S2E, the serine at position 2 is mutated to a glutamic acid. This phosphomimetic mutation disrupts NuRD recruitment, similar to the deletion mutant. In SALL1-S2A, the serine at position 2 is mutated to an alanine. This substitution prevents the inactivating serine phosphorylation, thereby enhancing SALL1 mediated NuRD recruitment and its effects on gene transcription. Figure S6. Real-time PCR analyses of mRNA expression levels of the other SALL family members in breast cancer cells after transfection with SALL1. MCF7 and E0771 tumor cells were transfected with SALL1 for 24 h and then RT-PCR was performed. The expression level of each gene was normalized to GAPDH expression and adjusted to the levels in vector-transfected tumor cells. Data show mean ± SD from three independent experiments with similar results. Figure S7. SALL1 over-expression in breast cancer cells inhibited tumor metastasis in vivo. (A) Over-expression of SALL1 in MCF-7 breast cancer cells significantly inhibited the migration of tumor cells compared with the control mSALL1 and vector-transfected tumor cells in the wound closure assays. Data shown are from three independent experiments with similar results. (B) Over-expression of SALL1 in E0771 breast cancer cells markedly suppressed the tumor cell migration and metastasis in NSG mice. Lentivirus-transfected E0771 tumor cells were stained with VivoTag®680 XL and then injected tail intravenously (5 × 104/mouse) into NSG mice. Mice were imaged with the IVIS Spectrum at different time points following the tumor cell adoptive transfer. Data shown are the dorsal, ventral, and right lateral images of representative of 5 mice per group at day 3 and day 10. (C) H &amp; E staining on sections from embedded liver tissues showed that high amount of tumor cells infiltrated into livers obtained from control groups (mSALL1 and vector), but not from the SALL1 transfection group. (PDF 11758 kb)