Additional file 1 of Identification and targeting of selective vulnerability rendered by tamoxifen resistance

Additional file 1: Figure S1. Increased SULT1A1 mRNA and protein expression in relapsed patients after TMX treatment. A, B, Heat map representation of microarray analysis of matched primary and metastatic tumors from patients A – F (Table S2). The levels of SULT1A1 (A) and SULT1A2 (B) expression is compared with primary tumor after TMX treatment. C, Heatmap of SULTs family genes differentially expressed in matched primary tumor and liver metastasis samples from breast cancer patients as assessed by RNA-seq. D, Representative WB for SULT1A1 KO clones in spontaneous TMXR MCF7 cells. β-actin used as loading control. The asterisk indicates non-specific band produced by antibody. E, SULT1A1 deletion (blue line, #10) in spontaneous TMXR clone of MCF7 cells (red line) confers sensitivity after 4 days post treatment with different concentrations of 4OH-TMX, as determined by resazurin assay (***p < 0.001, one-way ANOVA with Bonferroni’s multiple comparison test). F, Representative immunofluorescence images showing increased SULT1A1 protein (in red, using ab124011 antibody) after treatment of breast cancer patient #K4 ex vivo cultured cells with either vehicle or 1 μM of 4OH-TMX for 6 days. G, qRT-PCR of SULT1A1 mRNA in patient samples treated with TMX as in F. Patient information is given in Table S1. Figure S2. SULT1A1 is required for RITA, AF and ONC-1 sensitivity in cancer cells. A, Crystal violet staining detecting cell viability of high (MCF7 and T-47D) and low SULT1A1 (A375 and SJSA) after 72 h treatment with the indicated concentrations of compounds. B, SULT1A1 protein expression in cancer cell lines. Breast cancer cells: MCF7 TMXR, MDAMB-231; colon cancer: GP5d, HCT116; lung cancer: H1299; melanoma: A375, KADA, SKMEL28, SKMEL2, ESTDAB-37; neuroblastoma: SHSY-5Y; osteosarcoma: U2OS; skin cancer: A431. β-actin used as loading control. SULT1A1 (ab124011) antibody was used to perform the WB. Figure S3. Generation and validation of SULT1A1 KO in cancer cells. A - D, Generation of SULT1A1 knock-out clones in MCF7 and HCT116 cells using CRISPR/Cas9 gene editing. A, C and D, shown is immunoblot images using SULT1A1 (ab191069) antibody in MCF7-WT (A), HCT116 (D) and in HCT116 using SULT1A1 (ab124011) antibody (C). β-actin used as loading control. The asterisk indicates non-specific band produced by the antibody ab191069 and ab124011 and B, Immunofluorescence analysis of SULT1A1 expression in MCF7-vector transduced and SULT1A1 KO clone using ab124011 antibody (in red). E, Crystal violet staining to detect cell viability in long-term viability assay on day 6 after compounds treatment of MCF7-WT and SULT1A1 KO clones 1 and 3. Figure S4. Inhibition of TrxR1 activity by RITA, AF, and ONC-1 is SULT1A1 dependent. A, Increased oxidative stress upon 24 h treatment by 1 μM RITA (green), 3 μM AF (red) and 5 μM ONC-1 (blue) in HCT116 cells was rescued by co-treatment with 1 μM resveratrol (filled pattern). B, Immunoblot of TrxR1 and β-actin as loading control in the lysates from MCF7-WT cells. C, D, Induction of covalent TrxR1 dimer (detected by TrxR1 Antibody), apoptosis (induction of cleaved PARP) and DNA damage (yH2A.X (p-S139)) in SULT1A1-overexpressing cells. Shown Immunoblot of SULT1A1, p53, TrxR1, PARP and ϒH2A.X in cell extracts from low SULT1A1-expressing A375 (C), SJSA (D) compared to SULT1A1 overexpressing cells. β-Actin was used as loading control. E, Induction of γH2A.X (S139) and PARP, indicators of DNA damage and apoptosis, respectively, was rescued by the treatment with redox active compound resveratrol (1 μM), as assessed by immunoblot.