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The KMT1A-GATA3-STAT3 Circuit Is a Novel Self-Renewal Signaling of Human Bladder Cancer Stem Cells Zhao Yang1, Luyun He2,3, Kais

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The KMT1A-GATA3-STAT3 Circuit Is a Novel Self-Renewal Signaling of Human Bladder Cancer Stem Cells Zhao Yang1, Luyun He2,3, Kais The KMT1A-GATA3-STAT3 circuit is a novel self-renewal signaling of human bladder cancer stem cells Zhao Yang1, Luyun He2,3, Kaisu Lin4, Yun Zhang1, Aihua Deng1, Yong Liang1, Chong Li2, 5, & Tingyi Wen1, 6, 1CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China 2Core Facility for Protein Research, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 3CAS Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 4Department of Oncology, the Second Affiliated Hospital of Soochow University, Suzhou 215000, China 5Beijing Jianlan Institute of Medicine, Beijing 100190, China 6Savaid Medical School, University of Chinese Academy of Sciences, Beijing 100049, China Correspondence author: Tingyi Wen, e-mail: [email protected] Chong Li, e-mail: [email protected] Supplementary Figure S1. Isolation of human bladder cancer stem cells. BCMab1 and CD44 were used to isolate bladder cancer stem cells (BCSCs: BCMab1+CD44+) and bladder cancer non-stem cells (BCNSCs: BCMab1-CD44-) from EJ, samples #1 and #2 by flow cytometry. Supplementary Figure S2. Gene ontology analysis of downregulated genes of human BCSCs. (A) Pathway enrichment of 103 downregulated genes in BCSCs. (B) The seven downregulated genes in BCSCs participating in centromeric heterochromatin, mRNA-3’-UTR binding and translation regulator activity signaling pathways were validated by qRT-PCR. Data are presented as mean ± SD. P < 0.05; P < 0.01. Supplementary Figure S3. The expression of KMT1A is higher in human BC than that in peri-tumor tissues. (A) The expression of KMT1A was higher in BC samples than that in peri-tumors as assessed by immunohistochemistry, Scale bar = 50 m. (B-C) The expression of KMT1A was analyzed according to the data from Bae’s cohort (GSE13507) and Kim’s cohort (GSE37815). Supplementary Figure S4. KMT1A is highly expressed in basal subtype of bladder cancer. (A and C). Clustering analysis of GSE48075 and GSE48276 according to the basal and luminal biomarkers. (B and D). The expression of KMT1A was analyzed in the basal and luminal subtypes (GSE48075 and GSE48276). Supplementary Figure S5. The expression of KMT1A and NSD1 in normal and tumor cell lines of different origin, and TCGA database. (A and B) The mRNA and protein expression levels of KMT1A in normal and tumor cell lines of different origin. (C and D) The analysis of the expression of KMT1A (C) and NSD1 (D) in tumor samples from TCGA database. KMT1A was highly expressed in ESCA, STES, STAD, LUSC, HNSC, BLCA and LIHC. NSD1 was only highly expressed in LIHC. BLCA, Bladder Urothelial Carcinoma; BRCA, Breast invasive carcinoma; COAD, Colon adenocarcinoma; COADREAD, Colorectal adenocarcinoma; ESCA, Esophageal carcinoma; HNSC, Head and Neck squamous cell carcinoma; KIPAN, Pan-kidney cohort (KICH+KIRC+KIRP); KIRC, Kidney renal clear cell carcinoma; KIRP, Kidney renal papillary cell carcinoma; LAML, Acute Myeloid Leukemia; LIHC, Liver hepatocellular carcinoma; LUAD, Lung adenocarcinoma; LUSC, Lung squamous cell carcinoma; OV, Ovarian serous cystadenocarcinoma; READ, Rectum adenocarcinoma; STAD; Stomach adenocarcinoma, STES, Stomach and Esophageal carcinoma; THCA, Thyroid carcinoma; UCEC, Uterine Corpus Endometrial Carcinoma. Data are presented as mean ± SD. P < 0.05. Supplementary Figure S6. The expression of KMT1A in human bladder. (A) The expression of KMT1A in bladder cancer stem cells (BCSCs: BCMab1+CD44+), bladder cancer non-stem cells (BCNSCs: BCMab1-CD44-), normal bladder stem cells (NBSCs: pan-CK+CD44+) and normal bladder non-stem cells (NBNSCs: pan-CK+CD44-) from primary bladder cancer samples, n = 5. (B) KMT1A was highly expressed in BCSCs and tumorspheres derived from bladder cancer cell lines compared to that in BCNSCs and non- sphere tumor cells, as assessed by qRT-PCR. Non-sphere: bladder cancer cell lines or primary bladder cancer cells that failed to form tumorspheres. (C) The expression levels of CD44 and KMT1A in BC cell lines and primary BC samples were examined by qRT-PCR and then subjected to a correlation analysis, n = 10. Data are presented as mean ± SD. P < 0.05; P < 0.01. Supplementary Figure S7. Depletion of KMT1A abrogates the self-renewal and tumorigenicity of human BCSCs. (A and B) The qRT-PCR (A) and WB (B) analysis of KMT1A in shCtrl and shKMT1A BCSCs. -actin served as a loading control. (C) Representative photographs of tumorspheres formed by shCtrl and shKMT1A BCSCs. The number of tumorspheres was counted in five independent fields/well after two weeks of cultivation. Scale bar = 100 m. (D) shKMT1A BCSCs consisted of fewer CD44+ cells than shCtrl BCSCs. (E) Kaplan-Meier curves comparing the overall survival between bladder carcinomas patients expressing high or low levels of KMT1A, log-rank test. n, patient number. Data are presented as mean ± SD. P < 0.05; P < 0.01. Supplementary Figure S8. STAT3 is highly expressed and activated in human BCSCs. (A) The qRT-PCR analysis of the expression levels of GLI1, STAT3, BMI1, HES1, CTNNB1, NANOG, POU5F1, SOX2 and CD44 in shCtrl and shKMT1A BCSCs. (B) The WB analysis of pY-STAT3, STAT3, GLI1 and SOX2 in shCtrl and shKMT1A BCSCs. -actin served as a loading control. (C) The expression of STAT3 was higher in KMT1Ahigh samples than that in KMT1Alow samples based on an analysis of McConkey’s cohort (GSE48276). (D) The expression of STAT3 along with KMT1A were analyzed using the data from McConkey’s cohort (GSE48276). (E) STAT3 is highly expressed in BCSCs and tumorspheres derived from bladder cancer cell lines compared to that in BCNSCs and non-sphere tumor cells, as assessed by qRT-PCR. Non-sphere: bladder cancer cell lines or primary bladder cancer cells that failed to form tumorspheres. Data are presented as mean ± SD. P < 0.05; P < 0.01. Supplementary Figure S9. STAT3 activation is indispensable for the self-renewal maintenance of human BCSCs. (A) The qRT-PCR analysis of STAT3 in shCtrl and shSTAT3 BCSCs. (B) The WB analysis of pY-STAT3 and STAT3 in shCtrl and shSTAT3 BCSCs. -actin served as a loading control. (C) Representative photographs of tumorspheres formed by shCtrl and shSTAT3 BCSCs. The number of tumorspheres was counted in five independent fields/well after two weeks of cultivation. Scale bar = 100 m. (D) shSTAT3 BCSCs consisted of fewer CD44+ cells than shCtrl BCSCs. (E) Kaplan-Meier curves comparing the overall survival between bladder carcinomas patients expressing high or low levels of STAT3. n, patient number. Data are presented as mean ± SD. P < 0.05; **P < 0.01. Supplementary Figure S10. KMT1A is not recruited on the promoters of GATA1, GATA2, NFB, c-Myc, SOCS3, P53 and STAT3 in human BCSCs. (A-F) ChIP analysis of the promoters of GATA1, GATA2, NFB, c-Myc, SOCS3, P53 and STAT3 promoters with IgG and KMT1A antibodies in BCSCs. The enrichment of different regions of the promoter was detected by qRT-PCR. Data are presented as mean ± SD. Supplementary Figure S11. GATA3 was downregulated in BCSCs compared with that in BCNSCs. Supplementary Figure S12. The expression of GATA3 was downregulated in BCSCs. (A) GATA3 was downregulated in CD44high samples compared to that in CD44low samples based on an analysis of Michor’s cohort (GSE48276) and Michor’s cohort (GSE31684). (B) The expression of GATA3 along with CD44 were analyzed using the data from Michor’s cohort (GSE48276) and Michor’s cohort (GSE31684). (C) Kaplan-Meier curves comparing the overall survival between bladder carcinomas patients expressing high or low levels of GATA3. n, patient number. Data are presented as mean ± SD. P < 0.05; P < 0.01. Supplementary Figure S13. The destruction of STAT3 promoter by Cas9 in human BCNSCs. (A) Recognition sequence of GATA3 in the STAT3 promoter. (B) Sanger sequencing of PCR product including the STAT3 promoter in WT and Mut BCNSCs. Red bases indicated the abrogation of the binding motif of GATA3. Supplementary Fig. S14. Depletion of GATA3 promotes the transcription of STAT3 in BCNSCs. (A) The qRT-PCR analysis of GATA3 in shCtrl and shGATA3 BCNSCs, Student’s t test. (B) The WB analysis of pY-STAT3, STAT3 and GATA3 in shCtrl and shGATA3 BCNSCs. -actin served as a loading control. (C) Representative photographs of tumorspheres formed by shCtrl and shGATA3 BCNSCs. The number of tumorspheres was calculated in five independent fields/well after two weeks of cultivation, Student’s t test. Scale bar = 100 m. Data are presented as mean ± SD. P < 0.05; P < 0.01. Supplementary Figure S15. The KMT1A-GATA3-STAT3 circuit triggers the self- renewal and tumorigenicity of human BCSCs. (A) The qRT-PCR analysis of GATA3 and STAT3 in vec, oeGATA3 and oeGATA3/STAT3 BCSCs. (B) The WB analysis of pY-STAT3, STAT3 and GATA3 in vec, oeGATA3 and oeGATA3/STAT3 BCSCs. -actin served as a loading control. (C) oeGATA3 BCSCs consisted of fewer CD44+ cells than vec BCSCs. The overexpression of STAT3 in oeGATA3 BCSCs rescued the percentage of CD44+ cells, Student’s t test. Data are presented as mean ± SD. P < 0.05; P < 0.01. Supplementary Figure S16. The mutation of STAT3 promoter abolished the negative regulation of GATA3 on the expression of STAT3 in BCSCs. (A) Sanger sequencing of PCR product including the STAT3 promoter in vec and oeGATA3 Mut BCSCs. Red bases indicated the abrogation of the binding motif of GATA3. (B) The qRT-PCR analysis of GATA3 in vec and oeGATA3 Mut BCSCs (#3, T2a, high grade and #6, T2a, high grade), Student’s t test. (C) The WB analysis of pY-STAT3, STAT3 and GATA3 in vec and oeGATA3 Mut BCSCs. -actin served as a loading control. (D) The percentage of tumor-free mice four months after the subcutaneous injection of the different dilutions of vec or oeGATA3 Mut BCSCs into immunodeficient mice, n=6.
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