Journal: Cancer Communications

Article Title: NAT10‐mediated ac 4 C‐modified ANKZF1 promotes tumor progression and lymphangiogenesis in clear‐cell renal cell carcinoma by attenuating YWHAE‐driven cytoplasmic retention of YAP1

doi: 10.1002/cac2.12523

Figure Lengend Snippet: NAT10 catalyzed ac 4 C modifications and was upregulated in ccRCC. (A) Anti‐ac 4 C dot blotting of total mRNA from ccRCC tissues and adjacent normal tissues. The loading amount of each sample was fixed to 50 ng mRNA ( n = 12) ( t ‐test for statistics). (B) Anti‐ac 4 C dot blotting of total mRNA from cell lines. The loading amount of each sample was fixed to 50 ng mRNA ( n = 3) (ANOVA for statistics). The quantification results were normalized to the control cells. (C) The mRNA levels of NAT10 in ccRCC tissues and paired adjacent normal tissues based on TCGA‐KIRC and ICGC‐RECA (Wilcoxon test for statistics). (D) The protein level of NAT10 in ccRCC tissues and paired adjacent normal tissues based on the CPTAC3‐ccRCC database ( t ‐test for statistics). (E‐F) Protein and mRNA levels of NAT10 in cell lines ( n = 3) (ANOVA for statistics). (G‐H) Representative IHC staining images for NAT10 in ccRCC tissues and adjacent normal tissues ( n = 6) (Mann‐Whitney U test for statistics). (I‐J) Anti‐ac 4 C dot blotting of total mRNA from A498 and 786‐O cells with NAT10 overexpression or knockdown ( n = 3) ( t ‐test and ANOVA for statistics). The quantification results were normalized to the control cells. Results represented at least three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001). Abbreviations: NAT10, N‐acetyltransferase 10; ccRCC, clear‐cell renal cell carcinoma; ac 4 C, N4‐acetylcytidine; TCGA, the cancer genome atlas; KIRC, kidney renal clear‐cell carcinoma; ICGC‐RECA, the international cancer genome consortium‐renal cancer; CPTAC3, clinical proteomic tumor analysis consortium 3; IHC, immunohistochemical; FPKM, fragments per kilobase of exon model per million mapped fragments; RPKM, reads per kilobase per million mapped reads; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase.

Article Snippet: Human embryonic kidney 293T (HEK293T) cells, renal proximal tubule epithelial cells (RPTEC), ccRCC cells (A498, 786‐O, CAKI‐1, and OSRC‐2), and lymphatic endothelial cells (HLECs) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Control, Immunohistochemistry, MANN-WHITNEY, Over Expression, Knockdown, Immunohistochemical staining

Journal: Cancer Communications

Article Title: NAT10‐mediated ac 4 C‐modified ANKZF1 promotes tumor progression and lymphangiogenesis in clear‐cell renal cell carcinoma by attenuating YWHAE‐driven cytoplasmic retention of YAP1

doi: 10.1002/cac2.12523

Figure Lengend Snippet: NAT10 promoted tumor progression in ccRCC. (A) Colony formation assays for the NAT10‐knockdown ccRCC cells and the control cells ( n = 3) (ANOVA for statistics). (B) Transwell assays for the NAT10‐overexpressed ccRCC cells or the control cells ( n = 3). (C) Flow cytometry assays showed the proportion of apoptotic cells in NAT10‐knockdown ccRCC cells and the control cells ( n = 3). Comp‐PE‐A means Annexin V was compensated by negative control and single positive control. Comp‐Percp‐7‐AAD means 7‐AAD was compensated by negative control and single positive control. (D) CAKI‐1 cells with NAT10 knockdown were injected into nude mice. Tumors were extracted after mice were euthanized ( n = 5) ( t ‐test for statistics). (E‐F) IHC of NAT10 and Ki67 in subcutaneous tumors from the NAT10‐knockdown group and the control group ( n = 5) (Mann‐Whitney U test for statistics). (G) Live small animal fluorescent images of the metastasis model in the NAT10‐knockdown group and control group. The images depict one mouse from each group in four distinct body positions. Nude mice were imaged eight weeks after performing tail vein injections of A498 cells ( n = 5) ( t ‐test for statistics). Results represented at least three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001). Abbreviations: NAT10, N‐acetyltransferase 10; ccRCC, clear‐cell renal cell carcinoma; PE, phycoerythrin; 7‐AAD, 7‐amino‐actinomycin D; IHC, immunohistochemical; Ki‐67, marker of proliferation ki‐67.

Article Snippet: Human embryonic kidney 293T (HEK293T) cells, renal proximal tubule epithelial cells (RPTEC), ccRCC cells (A498, 786‐O, CAKI‐1, and OSRC‐2), and lymphatic endothelial cells (HLECs) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Knockdown, Control, Flow Cytometry, Negative Control, Positive Control, Injection, MANN-WHITNEY, Immunohistochemical staining, Marker

Journal: Cancer Communications

Article Title: NAT10‐mediated ac 4 C‐modified ANKZF1 promotes tumor progression and lymphangiogenesis in clear‐cell renal cell carcinoma by attenuating YWHAE‐driven cytoplasmic retention of YAP1

doi: 10.1002/cac2.12523

Figure Lengend Snippet: NAT10 promoted tumor lymphangiogenesis in ccRCC. (A) GSEA showed the associations between the VEGF signaling and the NAT10 mRNA levels in ccRCC. FDR q < 25% was considered statistically significant. Patients were categorized into low and high subgroups using median expression (50%) as the cut‐off. (B) Cell proliferation curves of HLECs treated with CM from the NAT10‐overexpressed ccRCC cells and the control cells ( n = 4) ( t ‐test for statistics). (C) Cell proliferation curves of HLECs treated with CM from the NAT10‐knockdown ccRCC cells and the control cells ( n = 4) ( t ‐test for statistics). (D) Transwell assays for HLECs treated with CM from the NAT10‐overexpressed ccRCC cells and the control cells ( n = 3) ( t ‐test for statistics). (E) Transwell assays for HLECs treated with CM from the NAT10‐knockdown ccRCC cells and the control cells ( n = 3) ( t ‐test for statistics). (F) Tube formation assays for HLECs treated with CM from the NAT10‐overexpressed ccRCC cells and the control cells ( n = 3) ( t ‐test for statistics). (G) Tube formation assays for HLECs treated with CM from the NAT10‐knockdown ccRCC cells and the control cells ( n = 3) ( t ‐test for statistics). (H) ELISAs were used to detect the VEGFD concentrations in CM from ccRCC cells with NAT10 overexpression or knockdown ( n = 3) ( t ‐test for statistics). (I) IHC of NAT10, VEGFC/D, and LYVE1 in subcutaneous tumors from the NAT10‐knockdown group and the control group ( n = 5) (Mann‐Whitney U for statistics). Results represented at least three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001). Abbreviations: NAT10, N‐acetyltransferase 10; ccRCC, clear‐cell renal cell carcinoma; GSEA, gene set enrichment analysis; FDR, false discovery rate; NES, normalized enrichment score; VEGF, vascular endothelial growth factor; HLEC, human lymphatic endothelial cell; CM, conditioned medium; ELISA, Enzyme‐linked immunosorbent assay; VEGFC/D, vascular endothelial growth factor‐C/D; LYVE1, lymphatic vessel endothelial hyaluronan receptor 1; KEGG, kyoto encyclopedia of genes and genomes.

Article Snippet: Human embryonic kidney 293T (HEK293T) cells, renal proximal tubule epithelial cells (RPTEC), ccRCC cells (A498, 786‐O, CAKI‐1, and OSRC‐2), and lymphatic endothelial cells (HLECs) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Control, Knockdown, Over Expression, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay

Journal: Cancer Communications

Article Title: NAT10‐mediated ac 4 C‐modified ANKZF1 promotes tumor progression and lymphangiogenesis in clear‐cell renal cell carcinoma by attenuating YWHAE‐driven cytoplasmic retention of YAP1

doi: 10.1002/cac2.12523

Figure Lengend Snippet: NAT10 promoted tumor progression and lymphangiogenesis in ccRCC by enhancing the nuclear localization of YAP1. (A) Western blotting showed the subcellular localization of YAP1 after NAT10 knockdown ( n = 3) (ANOVA for statistics). (B) Immunofluorescence assays displayed subcellular localization of YAP1 after NAT10 overexpression. (C) Cell proliferation curves of cell viability assays for the indicated ccRCC cells ( n = 4) (ANOVA for statistics). (D‐E) Tube formation assays for HLECs treated with CM from the indicated ccRCC cells ( n = 3) (ANOVA for statistics). (F) The protein levels of VEGFC/D in ccRCC cells with NAT10 overexpression and the control cells. (G) The protein levels of NAT10, YAP1, and VEGFC/D in the indicated ccRCC cells. Results represented at least three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001). Abbreviations: NAT10, N‐acetyltransferase 10; ccRCC, clear‐cell renal cell carcinoma; YAP1, yes1‐associated transcriptional regulator; HLEC, human lymphatic endothelial cell; CM, conditioned medium; VEGFC/D, vascular endothelial growth factor‐C/D; pYAP1, phosphorylated YAP1; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; DAPI, 4',6‐diamidino‐2‐phenylindole.

Article Snippet: Human embryonic kidney 293T (HEK293T) cells, renal proximal tubule epithelial cells (RPTEC), ccRCC cells (A498, 786‐O, CAKI‐1, and OSRC‐2), and lymphatic endothelial cells (HLECs) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Knockdown, Immunofluorescence, Over Expression, Control

Journal: Cancer Communications

Article Title: NAT10‐mediated ac 4 C‐modified ANKZF1 promotes tumor progression and lymphangiogenesis in clear‐cell renal cell carcinoma by attenuating YWHAE‐driven cytoplasmic retention of YAP1

doi: 10.1002/cac2.12523

Figure Lengend Snippet: ANKZF1 was identified as a functional target of NAT10 in ccRCC. (A) Venn diagram of the downstream genes regulated by NAT10 in A498 ccRCC cells. “NAT10‐RIP‐seq” means that these genes were identified by NAT10‐RIP‐seq. “shNAT10‐RNA‐seq” means DEGs between the shNC and shNAT10 group. “NAT10‐|R| > 0.35” means that the correlation between this gene set and NAT10 is greater than 0.35. The list of candidates in the Venn diagram has been deposited in the Science Data Bank ( www.scidb.cn ). (B) The ac 4 C modification levels of ANKZF1 in ccRCC tissues and adjacent normal tissues ccRCC ( n = 12) (Wilcoxon test for statistics). (C) The ac 4 C modification levels of ANKZF1 in ccRCC cell lines and the normal cell line ( n = 3) (ANOVA for statistics). (D) The ac 4 C modification levels of ANKZF1 in NAT10‐overexpressed ccRCC cells or the control cells ( n = 3) ( t ‐test for statistics). (E) The decay rate of NAT10 mRNA after treatment with actinomycin D in A498 and 786‐O cells with NAT10 knockdown or overexpression ( n = 3) ( t ‐test for statistics). (F) The protein expression of ANKZF1 in ccRCC cells with NAT10 overexpression ( n = 3) ( t ‐test for statistics). (G) The apoptosis rates of the indicated ccRCC cells from flow cytometry assays ( n = 3) (ANOVA for statistics). (H) Tube formation assays for HLECs treated with CM from the indicated ccRCC cells ( n = 3) (ANOVA for statistics). (I) Cell proliferation curves of HLECs treated with CM from the indicated ccRCC cells ( n = 4) (ANOVA for statistics). (J) The protein expression of NAT10, ANKZF1, and VEGFC/D in the indicated ccRCC cells ( n = 3). Results represented at least three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001). Abbreviations: ANKZF1, ankyrin repeat and zinc finger peptidyl tRNA hydrolase 1; NAT10, N‐acetyltransferase 10; ccRCC, clear‐cell renal cell carcinoma; RIP‐seq, RNA immunoprecipitation‐sequencing; DEGs, differentially expressed genes; ac 4 C, N4‐acetylcytidine; HLEC, human lymphatic endothelial cell; CM, conditioned medium; VEGFC/D, vascular endothelial growth factor‐C/D; CRYBG1 , crystallin beta‐gamma domain containing 1; CDIPT , cdp‐diacylglycerol‐inositol 3‐phosphatidyltransferase; ATG16L2 , autophagy‐related 16 like 2; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase.

Article Snippet: Human embryonic kidney 293T (HEK293T) cells, renal proximal tubule epithelial cells (RPTEC), ccRCC cells (A498, 786‐O, CAKI‐1, and OSRC‐2), and lymphatic endothelial cells (HLECs) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Functional Assay, RNA Sequencing, Modification, Control, Knockdown, Over Expression, Expressing, Flow Cytometry, RNA Immunoprecipitation, Sequencing

Journal: Cancer Communications

Article Title: NAT10‐mediated ac 4 C‐modified ANKZF1 promotes tumor progression and lymphangiogenesis in clear‐cell renal cell carcinoma by attenuating YWHAE‐driven cytoplasmic retention of YAP1

doi: 10.1002/cac2.12523

Figure Lengend Snippet: NAT10 inactivated the non‐canonical Hippo signaling by enhancing the ANKZF1‐YWHAE interaction. (A) Western blotting showed the subcellular localization of YAP1 after ANKZF1 knockdown ( n = 3) ( t ‐test for statistics). (B) Venn diagram of proteins pulled down through Flag‐ANKZF1, proteins pulled down through Vector, and proteins in the Hippo pathway from the KEGG database. Peptides with an unused score > 1.3 (a credibility of more than 95%) were considered credible peptides, and proteins containing at least one unique peptide were retained. The list of candidates in the Venn diagram has been deposited in the Science Data Bank ( www.scidb.cn ). (C) The endogenous ANKZF1‐YWHAE interaction was determined by co‐IP assays in HEK293T cells. (D) The exogenous ANKZF1‐YWHAE interaction was determined by co‐IP assays in HEK293T cells overexpressed Flag‐ANKZF1 and/or Myc‐YWHAE. (E) The exogenous ANKZF1‐YWHAE interaction was determined by co‐IP assays in ccRCC cells overexpressed Flag‐ANKZF1 and/or Myc‐YWHAE. (F) The YWHAE‐YAP1 interaction was determined by co‐IP assays in A498 and 786‐O cells with Flag‐ANKZF1 overexpression. (G) The YWHAE‐YAP1 interaction was determined by co‐IP assays in A498 and 786‐O cells with ANKZF1 knockdown. (H) The diagrams show wild‐type ANKZF1 (full length, 1‐726) and its five truncations. (I) HEK293T cells were transfected with the truncated plasmids, followed by co‐IP assays and Western blotting to examine the interaction between truncations of ANKZF1 and YWHAE. (J) Western blotting showed the subcellular localization of YAP1 in the indicated ccRCC cells. Results represented at least three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001). Abbreviations: ANKZF1, ankyrin repeat and zinc finger peptidyl tRNA hydrolase 1; NAT10, N‐acetyltransferase 10; YWHAE, tyrosine 3‐monooxygenase/tryptophan 5‐monooxygenase activation protein epsilon; YAP1, yes1‐associated transcriptional regulator; KEGG, kyoto encyclopedia of genes and genomes; co‐IP, co‐immunoprecipitation; pYAP1, phosphorylated YAP1; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; PPP1/2CB, protein phosphatase 1/2 catalytic subunit beta; PPP2R1A/B, protein phosphatase 2 scaffold subunit Aalpha/Abeta.

Article Snippet: Human embryonic kidney 293T (HEK293T) cells, renal proximal tubule epithelial cells (RPTEC), ccRCC cells (A498, 786‐O, CAKI‐1, and OSRC‐2), and lymphatic endothelial cells (HLECs) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Knockdown, Plasmid Preparation, Co-Immunoprecipitation Assay, Over Expression, Transfection, Activation Assay, Immunoprecipitation

Journal: Cancer Communications

Article Title: NAT10‐mediated ac 4 C‐modified ANKZF1 promotes tumor progression and lymphangiogenesis in clear‐cell renal cell carcinoma by attenuating YWHAE‐driven cytoplasmic retention of YAP1

doi: 10.1002/cac2.12523

Figure Lengend Snippet: NAT10‐ANKZF1 axis inactivated the non‐canonical Hippo signaling and suppressed tumor progression in vivo. (A‐B) Representative images of isolated subcutaneous tumors from nude mice in the indicated groups. Tumors were extracted and weighed after mice were euthanized ( n = 5) (ANOVA for statistics). (C) Tumor size was measured every 4 days and the last measurement was performed on day 44 ( n = 5) (ANOVA for statistics). (D) IHC staining for NAT10, ANKZF1, Ki67, VEGFC/D, YAP1/TAZ, and LYVE1 in the subcutaneous tumors ( n = 5). The bottom left image is a 4 times magnified view of the original image's top‐left corner. (E) Live small animal fluorescent images of the indicated groups in the metastasis model. The images depict one mouse from each group in four distinct body positions. Nude mice were imaged eight weeks after performing tail vein injections of A498 cells ( n = 5) (ANOVA for statistics). (F) The Kaplan‐Meier analyses showed the association between the overall survival rates of ccRCC patients and the expression levels of NAT10 and ANKZF1. Patients were categorized into low and high subgroups using median expression (50%) as the cut‐off (log‐rank for statistics). Results represented at least three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001). Abbreviations: ANKZF1, ankyrin repeat and zinc finger peptidyl tRNA hydrolase 1; NAT10, N‐acetyltransferase 10; IHC, immunohistochemistry; VEGFC/D, vascular endothelial growth factor‐C/D; YAP1, yes1‐associated transcriptional regulator; TAZ, tafazzin; LYVE1, lymphatic vessel endothelial hyaluronan receptor 1; Ki‐67, marker of proliferation ki‐67; TAZ, tafazzin.

Article Snippet: Human embryonic kidney 293T (HEK293T) cells, renal proximal tubule epithelial cells (RPTEC), ccRCC cells (A498, 786‐O, CAKI‐1, and OSRC‐2), and lymphatic endothelial cells (HLECs) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: In Vivo, Isolation, Immunohistochemistry, Expressing, Marker

Journal: Cancer Communications

Article Title: NAT10‐mediated ac 4 C‐modified ANKZF1 promotes tumor progression and lymphangiogenesis in clear‐cell renal cell carcinoma by attenuating YWHAE‐driven cytoplasmic retention of YAP1

doi: 10.1002/cac2.12523

Figure Lengend Snippet: The mechanism scheme of NAT10 in ccRCC. NAT10‐mediated ac 4 C raised the expression of ANKZF1 in ccRCC; ANKZF1 interacted with YWHAE to promote the nuclear localization of YAP1, thereby activating the transcription of VEGFC/D. Then the NAT10/ANKZF1 axis‐regulated non‐canonical Hippo pathway promoted tumor progression and lymphangiogenesis in ccRCC. The mechanism scheme was drawn by using Figdraw ( www.figdraw.com ). Abbreviations: ANKZF1, ankyrin repeat and zinc finger peptidyl tRNA hydrolase 1; NAT10, N‐acetyltransferase 10; VEGFC/D, vascular endothelial growth factor‐C/D; YAP1, yes1‐associated transcriptional regulator; YWHAE, tyrosine 3‐monooxygenase/tryptophan 5‐monooxygenase activation protein epsilon; ac4C, N4‐acetylcytidine; TEAD, tea domain transcription factor.

Article Snippet: Human embryonic kidney 293T (HEK293T) cells, renal proximal tubule epithelial cells (RPTEC), ccRCC cells (A498, 786‐O, CAKI‐1, and OSRC‐2), and lymphatic endothelial cells (HLECs) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Activation Assay