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.

附加文件1：图S1。他莫昔芬（TMX）治疗后复发患者中SULT1A1的mRNA与蛋白表达上调。A、B：对患者A-F的配对原发与转移肿瘤进行微阵列分析的热图展示（表S2），比较了TMX治疗后SULT1A1（A）与SULT1A2（B）的表达水平，以原发肿瘤为参照。C：通过RNA测序分析乳腺癌患者配对原发肿瘤与肝转移样本中差异表达的磺基转移酶（SULTs）家族基因热图。D：自发获得他莫昔芬抗性（TMXR）的MCF7细胞中SULT1A1敲除（KO）克隆的代表性蛋白免疫印迹（WB）实验，以β-肌动蛋白（β-actin）作为上样对照，星号代表抗体产生的非特异性条带。E：MCF7细胞的自发TMXR克隆（红线）中SULT1A1缺失（蓝线，#10）在经不同浓度4OH-TMX处理4天后恢复药物敏感性，通过刃天青（resazurin）实验检测（***p < 0.001，采用Bonferroni多重比较校正的单因素方差分析（one-way ANOVA））。F：代表性免疫荧光图像显示，乳腺癌患者#K4的体外培养细胞经载体或1μM 4OH-TMX处理6天后，SULT1A1蛋白（红色，使用ab124011抗体）表达上调。G：如F中所述，经TMX处理的患者样本中SULT1A1 mRNA的定量实时聚合酶链反应（qRT-PCR）结果，患者信息详见表S1。

图S2。SULT1A1是癌细胞对RITA、AF及ONC-1产生敏感性所必需的。A：结晶紫染色检测高表达SULT1A1的细胞系（MCF7和T-47D）与低表达SULT1A1的细胞系（A375和SJSA）经指定浓度化合物处理72小时后的细胞活力。B：各癌细胞系的SULT1A1蛋白表达情况：乳腺癌细胞包括MCF7 TMXR、MDAMB-231；结肠癌细胞包括GP5d、HCT116；肺癌细胞包括H1299；黑色素瘤细胞包括A375、KADA、SKMEL28、SKMEL2、ESTDAB-37；神经母细胞瘤细胞包括SHSY-5Y；骨肉瘤细胞包括U2OS；皮肤癌细胞包括A431。以β-actin作为上样对照，使用SULT1A1抗体（ab124011）进行WB实验。

图S3。癌细胞中SULT1A1敲除克隆的构建与验证。A-D：利用CRISPR/Cas9基因编辑技术在MCF7和HCT116细胞中构建SULT1A1敲除（KO）克隆。A、C、D：分别展示MCF7野生型（WT）细胞（A）、HCT116细胞（D）以及使用SULT1A1抗体（ab124011）检测的HCT116细胞（C）的免疫印迹图像，均以β-actin作为上样对照，星号代表抗体ab191069与ab124011产生的非特异性条带。B：使用SULT1A1抗体（ab124011，红色荧光）对转染空载体的MCF7细胞与SULT1A1 KO克隆进行免疫荧光分析。E：结晶紫染色检测MCF7-WT与SULT1A1 KO克隆1、3在化合物处理第6天的长期细胞活力。

图S4。RITA、AF及ONC-1对硫氧还蛋白还原酶1（TrxR1）活性的抑制作用依赖于SULT1A1。A：HCT116细胞经1μM RITA（绿色）、3μM AF（红色）和5μM ONC-1（蓝色）处理24小时后氧化应激增强，联合使用1μM白藜芦醇（填充图案）可逆转该现象。B：MCF7-WT细胞裂解液中TrxR1与β-actin（上样对照）的免疫印迹结果。C、D：在SULT1A1过表达细胞中可诱导共价结合的TrxR1二聚体（通过TrxR1抗体检测）、细胞凋亡（切割型PARP的表达）与DNA损伤（磷酸化组蛋白H2A.X（p-S139，γH2A.X））。展示了低表达SULT1A1的A375（C）、SJSA（D）细胞与SULT1A1过表达细胞的细胞提取物中SULT1A1、p53、TrxR1、PARP及γH2A.X的免疫印迹结果，以β-actin作为上样对照。E：免疫印迹检测显示，使用氧化还原活性化合物白藜芦醇（1μM）处理可逆转γH2A.X（S139）与PARP的表达上调，二者分别为DNA损伤与细胞凋亡的标志物。