Fig 2: OTUD7B-mediated removal of K63-linked poly-Ub chains on LSD1 determines LSD1/CoREST/HDACs integrity. A) MDA-MB-231 cells stably expressing the indicated lentiviruses were subjected to IB analysis. B) LSD1 reconstituting cells were infected with the indicated lentiviruses, followed by IB analysis. C) LSD1 reconstituting cells expressing CoREST shRNA were transfected with K63-only Ub plasmid, followed by IP and IB analysis as indicated. D) MDA-MB-231 cells expressing OTUD7B sgRNAs were treated with BTZ for 5 h, followed by IP and IB analysis as indicated. E) IP and IB analysis of LSD1 reconstituting cells expressing OTUD7B sgRNA as indicated.

Fig 3: OTUD7B binds LSD1 and regulates its stability. A) HA-tagged DUBs were transfected into HEK293T cells, followed by immunoprecipitation (IP) and immunoblotting (IB) as indicated. B) Co-IP of OTUD7B with LSD1 in MDA-MB-231 cells. The immunoprecipitated materials by the indicated antibodies were analyzed by western blotting. C) Colocalization of the indicated proteins by IF staining with the indicated antibodies was visualized by structured illumination microscopy (SIM). Scale bars: 10 µm. D) Flag-tagged LSD1 was co-transfected into HEK293T cells with the indicated constructs. Whole cell lysates (WCLs) were subjected to IB analysis. E) Cells were infected with the indicated lentiviruses. Cell lysates and RNA extracts were subjected to IB (left panel) and quantitative real-time PCR (qRT-PCR) analysis as indicated (right panel). F) OTUD7B reconstituting MDA-MB-231 cells were subjected to IB analysis as indicated. G) MDA-MB-231 cells stably expressing the indicated sgRNAs were treated with BTZ (100 × 10-9 m) for 5 h, followed by IB analysis. H) MDA-MB-231 cells stably expressing OTUD7B sgRNAs were treated with cycloheximide (CHX, 50 µg mL-1), followed by IB analysis as indicated (left panel). The graph shows the quantification of protein levels (right panel). I) IB analysis of WCL derived from 20 different cancer cells expressing the indicated lentiviral constructs, as shown in (E) and Figures S1J and S2A in the Supporting Information. The heatmap shows the fold change of quantified LSD1 protein levels. Data were presented as mean ± SEM of three independent experiments.

Fig 4: OTUD7B is a bona fide LSD1 deubiquitinase. A) IB analysis of WCL and Flag immunoprecipitate derived from HEK293T cells transfected with the indicated constructs and treated with BTZ for 5 h before harvesting. B) HEK293T cells stably expressing OTUD7B sgRNAs were transfected with Myc-tagged Ub, followed by IP and IB analysis as indicated. C) HEK293T cells stably expressing OTUD7B sgRNAs were co-transfected with the indicated Ub constructs, followed by IP and IB analysis as indicated. D) HEK293T cells were co-transfected with Flag-tagged LSD1, HA-tagged ubiquitin, and Myc-tagged OTUD7B. Cell lysates were subjected to IP and mass spectrometry analysis to identify ubiquitination sites. The recovered LSD1 peptide and the ubiquitination sites were highlighted in red (upper panel). Lower panel: A schematic diagram showing two evolutionarily conserved lysine residues (K226 and K277) within the SWIRM domain of LSD1. E) HEK293T cells were co-transfected with the indicated plasmids, followed by IP and IB analysis as indicated. F) LSD1WT- or LSD12KR-reconstituted MDA-MB-231 cells were treated with CHX for the indicated times. Lysates were subjected to IB analysis as indicated (top panel). The graph shows the quantification of protein levels (bottom panel). G) IB analysis of WCL derived from the indicated LSD1 reconstituting cells expressing OTUD7B sgRNA lentiviruses. H) LSD1-reconstituted cells stably expressing the indicated sgRNAs were transfected with K63-only Ub plasmid, followed by IP and IB analysis as indicated. Data were presented as mean ± SEM of three independent experiments.

Fig 5: OTUD7B promotes metastasis via deubiquitinating LSD1. A) Parental or C) LSD1 reconstituting LM2 cells infected with the indicated lentiviruses were injected into the mammary fat pads of nude mice. Representative bioluminescent images of mice with spontaneous lung metastasis (left panel), quantification (right panel), and (B) representative H&E staining analysis of lung metastasis is shown. Results represent mean ± SEM. n = 8 mice per group. Scale bars: 40 µm. ***P < 0.001. P values were calculated by two-tailed unpaired t test. D) Representative IHC staining of LSD1 and OTUD7B in normal breast tissues (n = 26) and breast carcinomas (n = 379) (histological I, II, and III). Scale bars: 100 µm for low magnification (10×) and 25 µm for high magnification (40×). E) Quantification of LSD1 and OTUD7B intensity in (D). P values were calculated by two-tailed unpaired t test. * P < 0.05, ** P < 0.01, and *** P < 0.001. F) Representative IHC staining of LSD1 and OTUD7B in normal tissue (n = 22) and the indicated breast carcinoma subtypes (luminal = 76, HER2+ = 49, and basal-like = 130). Scale bars: 100 µm. G) Quantification of OTUD7B and LSD1 intensity in (F). *** P < 0.001. P values were calculated by two-tailed unpaired t test. H) TCGA DNA sequencing results showing that the OTUD7B gene is amplified at higher frequencies in basal-like subtype (27%) compared to other subtypes (19%). I) Kaplan–Meier plots analysis of distant metastasis free survival rates (DMFS) in basal-like breast cancer patient with high or low OTUD7B mRNA levels. Patient number at risk at different times of analysis is shown at the bottom of the plots. J) The working model of OTUD7B-mediated LSD1 deubiquitination in coordinating LSD1 turnover, LSD1/CoREST corepressor complex assembly, and the subsequent impact on cell cycle and cancer metastasis.