Fig 2: ß-Trcp and CK1d-mediated degradation of LZTS2 promotes cell proliferation and metastasis by activating PI3K/AKT signaling in HCC.a LZTS2-depleted SMMC-7721 cells were reconstituted with either LZTS2-WT or the LZTS2-M3 mutant as indicated (n = 3). b The growth curve of SMMC-7721 cells is shown. The cells were transfected with the indicated siRNAs and plasmids and then seeded at a low density in plates. The cell numbers were calculated every other day. ***P < 0.001 (n = 3). c SMMC-7721 cells were transfected with the indicated siRNAs and plasmids to form colonies. Left panel: Representative violet staining images are shown. Right panel: The numbers of colonies were counted and are shown with histograms. ***P < 0.001 (n = 3). d Upper panel: cell migration was determined using transwell assays. Lower panel: quantification of the migration of SMMC-7721 cells is shown. The data are shown as the mean ± SD. ***P < 0.001 (n = 3). e Upper panel: cell invasion was measured using transwell assays. Lower panel: quantification of the invasion of SMMC-7721 cells is shown. The data are shown as the mean ± SD. ***P < 0.001 (n = 3). f A proposed model mechanistically shows how LZTS2 is regulated by the E3 ligase ß-Trcp and the protein kinase CK1d in HCC. CK1d binds to and phosphorylates LZTS2, which is recognized by ß-Trcp via its phosphodegron sequences for subsequent ubiquitination and degradation, leading to the activation of the PI3K/AKT signaling pathway and ultimately driving HCC progression and metastasis.

Fig 3: LZTS2 silencing activates the PI3K/ATK signaling pathway to promote HCC progression and metastasis in a p85-dependent manner.a SMMC-7721 and MHCC-97H cells were collected and subjected to immunoprecipitation (IP) using an anti-IgG or anti-LZTS2 antibody and then analyzed by western blotting (n = 3). b SMMC-7721 and MHCC-97H cells were transfected with indicated plasmids for 24 h. Immunoprecipitation was performed with Myc beads, and western blotting was carried out with the indicated antibodies (n = 3). c LZTS2 deficiency increased the protein level of p-AKT in SMMC-7721 and MHCC-97H cells (n = 3). d SMMC-7721 cells were transfected with the indicated siRNAs. Forty-eight hours later, the cells were harvested and analyzed by western blotting (n = 3). e SMMC-7721 cells were transfected with the indicated siRNAs or plasmids. The cell numbers were calculated every other day. The results are presented as the mean ± SD. ***P < 0.001 (n = 3). f SMMC-7721 cells transfected with the indicated siRNAs or plasmids were grown for two weeks. The colonies were examined, and the results are shown with histograms (upper panel). Representative violet staining images are shown (lower panel). n.s. indicates no statistically significant difference (P > 0.05), ***P < 0.001 (n = 3). g SMMC-7721 cell migration (upper panel) and invasion (lower panel) were determined using transwell assays. ***P < 0.001 (n = 3).

Fig 4: The two conserved DSGXXS phosphodegrons are responsible for the degradation of LZTS2 by ß-Trcp and CK1d.a Upper panel: ß-Trcp reported substrates and their conserved DSGXXS (X represents any amino acid) phosphodegron. Lower panel: alignment of the candidate phosphodegron sequences in LZTS2 from different species. b HEK293T cells were transfected with Myc-ß-Trcp and LZTS2-WT or the LZTS2-M1 (S220/224A), LZTS2-M2 (S273/277A), or LZTS2-M3 (S220/224/273/277A) mutant constructs. Then, IP was performed with S-protein beads, and western blotting was performed with the indicated antibodies (n = 3). c HeLa cells were transfected with plasmids as indicated. At 24 h post transfection, the cells were treated with MG132 (10 µM) for 4 h before harvesting. The lysates were incubated with S beads and then subjected to immunoblotting (n = 3). d Upper panel: HeLa cells transfected with the indicated plasmids for 24 h were treated with CHX (20 µg/mL) for the indicated times and then analyzed by western blotting. Lower panel: quantification of the LZTS2 band intensity is shown. ***P < 0.001 (n = 3).

Fig 5: Knockdown of LZTS2 enhances HCC cell metastasis in vitro and in vivo.a Depletion of LZTS2 led to increased expression of Snail and Slug in HCC cells (n = 3). b The expression of Snail and Slug was detected by immunofluorescence staining in control and LZTS2-downregulated SMMC-7721 and MHCC-97H cells (n = 3). Scale bars, 50 µm. c LZTS2-depleted SMMC-7721 (upper panel) or MHCC-97H (lower panel) cells had a higher migratory capacity than the control cells. Scale bars, 100 µm. ***P < 0.001 compared with control cells (n = 3). d Transwell invasion analysis showed that LZTS2 silencing increased cell invasion ability. Scale bars, 100 µm. ***P < 0.001 compared with control cells (n = 3). e LZTS2 knockdown significantly enhanced HCC cell metastasis in vivo. MHCC-97H cells carrying the Sh-control or ShRNA-1 against LZTS2 were injected into nude mice via the tail vein. After 8 weeks, the mice were sacrificed, and lung tissues were stained with haematoxylin and eosin and photographed. Scale bars, 1 mm. f The number of lung metastatic nodules from each mouse was counted and statistically analyzed. n = 5 for each group, **P < 0.01 compared with the control group.