RIT1 regulates mitotic processes in HCC cells

To investigate the underlying mechanism by which RIT1 promotes HCC progression, we conducted GSEA using the TCGA LIHC database. Our findings revealed a strong correlation between high RIT1 expression in HCC tissues and cell growth and mitotic processes, including sister chromatid segregation, spindle assembly, and nuclear division (Fig. 1A and Supplemental Table S5). We established stably RIT1-overexpressed HCC cells and performed RNA-Seq analysis to identify differential gene expression between RIT1 overexpressed and control groups. Genes showing at least a 1.5-fold difference in expression were selected for further gene enrichment analysis. Gene Ontology (GO) analysis demonstrated that RIT1 overexpression was associated with multiple GO terms related to cell growth and mitotic processes, such as mitotic cell cycle transition, mitotic nuclear division, spindle assembly, and sister chromatid segregation. In addition, KEGG pathway analysis along with hallmark analyses indicated that RIT1 was involved in various signaling pathways including MAPK signaling pathway, epithelial-mesenchymal transition, interferon alpha response, mTORC1 signaling, and hypoxia (Fig. 1B), which is consistent with previous reports [15, 20]. The above analyses of TCGA and RNA-Seq results support that RIT1 expression levels are closely associated with mitotic processes in HCC.

Fig. 1

RIT1 is closely associated with the mitotic processes in HCC. A Gene set enrichment analysis (GSEA) of a TCGA LIHC cohort with 374 tissues stratified by the mean cut-off value of RIT1 expression. B GO, KEGG, and HALLMARK enrichment analysis based on differential genes detected by RNA-Seq in the RIT1 overexpressing and control group of HCC cells. The terms are ordered by -log10 FDR. C Comparison of the representative time-lapse images of dynamic division at different time points between RIT1 knockdown and control groups in MHCC-97H and HCC-LY10 cells (upper section). The quantitation histogram showing the percentage of abnormal division cells (lower section). Data are presented as mean ± SD of three independent experiments. *** P < 0.001. The P values were calculated by unpaired Student’s t-test in (C)

To investigate the biological function of RIT1 in mitosis, we examined the expression of RIT1 protein in various HCC cell lines using western blot (Fig. S1A). MHCC-97H and HCC-LY10 cells were selected for lentiviral transfection to establish stably RIT1-knockdown cell lines. The knockdown efficiency of the RIT1 protein was assessed using qPCR and western blot analysis (Fig. S1B). Subsequently, we performed live cell imaging and dynamically observed the cell division process. The results demonstrated that depletion of RIT1 led to cell division failure, with abnormal phenomena observed in both MHCC-97H and HCC-LY10 cells including cell enlargement, cell vacuolation, binucleated cells, multinucleated cells, and cell death (Fig. 1C). These results suggest a pivotal role for RIT1 in mitosis in HCC.

Knockdown of RIT1 induces mitotic catastrophe and apoptosis in HCC cells

Rapid proliferation due to mitotic dysregulation is a significant feature of cancer, and precise cell cycle progression is essential for proper cell division and proliferation [25]. Given the involvement of RIT1 in mitotic processes and its promotion of HCC growth, we employed flow cytometry to investigate the effect of RIT1 on cell cycle progression. Cell cycle assays revealed an increased percentage of cells in the G2/M phase following RIT1 knockdown, indicating that RIT1 knockdown induced G2/M phase arrest (Fig. S2A). Precisely regulated mitosis ensures proper chromosome segregation, whereas abnormal mitosis can cause mitotic catastrophe, leading to cell death [26]. Subsequently, we examined whether silencing RIT1 affects spindle formation during mitosis in MHCC-97H and HCC-LY10 cells. The immunofluorescence (IF) staining results showed that silencing RIT1 caused a significantly increased multipolar spindle cells (Fig. 2A, B), which are hallmarks of mitotic catastrophe. To confirm the mitotic catastrophe caused by RIT1 interference, we further observed the morphological characteristics of cell nuclei. We found that RIT1 knockdown resulted in obvious multinucleated, heterogeneous nuclei in MHCC-97H and HCC-LY10 cells (Fig. 2C, D). Furthermore, according to the 7-ADD/propidium iodide staining analysis, RIT1 knockdown induced significant apoptosis in MHCC-97H and HCC-LY10 cells (Fig. 2E). These data suggest that knockdown of RIT1 causes cell cycle arrest, triggers mitotic catastrophe, and promotes apoptosis in HCC cells.

Fig. 2

Knockdown of RIT1 induces mitotic catastrophe and apoptosis in HCC cells. A, B Representative immunofluorescence staining images of α-tubulin (red) and DAPI (blue) show spindle morphology during mitosis in MHCC-97H (A) and HCC-LY10 cells (B) with RIT1 knockdown and control (left). The quantitation histogram showing the percentage of cells with multipolar spindle (right). C, D Representative immunofluorescence staining images of α-tubulin (red) and DAPI (blue) show nucleus morphology in MHCC-97H (C) and HCC-LY10 cells (D) with RIT1 knockdown and controls (left). The quantitation histogram showing the percentage of cells with multinuclear (right). Cells were enriched in mitosis utilizing nocodazole. Scale bars, 20 μm. E Flow cytometry analysis of apoptosis in MHCC-97H and HCC-LY10 cells with RIT1 knockdown and control group (left). The quantitation histogram showing the percentage apoptotic cells (right). Data are presented as mean ± SD of three independent experiments. *** P < 0.001. The P values were calculated by unpaired Student’s t-test

RIT1 interacts with SMC3 in HCC cells

Previous studies on the oncogenic mechanism of RIT1 have focused mainly on its regulation of the RAS/MAPK pathway. A recent study revealed that the RIT1 is diffusely distributed in the cytoplasm during mitosis and interacts with MAD2, a component of the spindle assembly checkpoint (SAC), to negatively regulate SAC activity [27]. To explore the underlying molecular mechanism by which RIT1 regulates mitosis in HCC, we transfected Myc-tagged RIT1 plasmids into Huh7, Hep3B, and HCC-LY10 cells and detected RIT1 distribution during different cell cycle stages by immunofluorescence staining with an anti-Myc antibody. The results demonstrated that RIT1 exhibited diffuse cytoplasmic localization during interphase and gradually accumulated around the chromosomes during mitosis (Fig. 3A and Fig. S3A). These results further suggested that RIT1 is closely associated with mitotic processes in HCC.

Fig. 3

RIT1 interacts with SMC3 in HCC cells. A Immunofluorescence staining of Myc-tag (red) and DAPI (blue) shows the distribution of RIT1 at different cell cycle phases in HCC-LY10 and Huh7 cells transfected with Myc-RIT1 plasmid. Scale bars, 20 μm. B Heat map of mitotic chromosome segregation-related proteins across Myc-RIT1-precipitated compared to IgG-precipitated group in HCC-LY10, Huh7, and Hep3B cells transfected with Myc-RIT1 plasmid. C Co-IP analysis of RIT1 and SMC3 in HCC-LY10, Huh7 and Hep3B cells transfected with Myc-RIT1 plasmid. Cell lysates from indicated cells were immunoprecipitated using an anti-Myc antibody (upper section) and an anti-SMC3 antibody (lower section). D Interaction of RIT1 and SMC3 was analyzed by Co-IP analysis in 293T cells co-transfected with Myc-RIT1 and Flag-SMC3 plasmids. Cell lysates were immunoprecipitated using an anti-Myc antibody (upper section) and an anti-Flag antibody (lower section). E Schematic presentation of structural domain of RIT1. F Co-IP analysis of the indicated RIT1 truncation and wild-type with SMC3 for their interaction position in 293T cells co-transfected with Myc-RIT1 (WT, ΔN or ΔC) and Flag-SMC3 plasmids. G Co-IP analysis of RIT1 and SMC3 in Huh7 and HCC-LY10 cells with RIT1 overexpression during asynchronous and synchronized to mitosis (Full indicates asynchronization treatment; Mitosis indicates cells were enriched in mitosis). H Colocalization of RIT1 and SMC3 during interphase and different phases of mitosis was analyzed by co-immunofluorescence staining (Myc-RIT1: red, SMC3: green, DAPI: blue) in HCC-LY10 (left) and Huh7 (right) cells transfected with Myc-RIT1 plasmid. Scale bars, 20 μm

Mitosis is a process involving multiple protein interactions. Based on the subcellular localization pattern of RIT1 during mitosis, we hypothesized that RIT1 may affect the mitotic process of HCC cells by interacting with regulatory proteins involved in mitosis. Co-immunoprecipitation (Co-IP) combined with mass spectrometry (MS) was performed to screen for potential RIT1 interacting proteins in HCC-LY10, Huh7, and Hep3B cells transfected with Myc-tagged RIT1 plasmid. The screening for interacting proteins was based on their abundance in the Myc-RIT1 group, which was three times higher than that in the control IgG group. By intersecting the results from all three cell lines (Fig. S4A), we observed that MAD2 protein was not detected in our MS analysis; however, several proteins associated with regulation of mitotic chromosome separation, including structural maintenance of chromosome 3 (SMC3), structural maintenance of chromosome 2 (SMC2), polo like kinase 1 (PLK1), and protein kinase, DNA-activated, catalytic subunit (PRKDC) proteins, were identified in the intersection of three cell lines. In addition, we found that PDS5A and PDS5B, chromosomal cohesin regulatory subunits, were present at two cell intersections (Fig. 3B).

We selected SMC3, SMC2, PLK1, and PRKDC, which were present at the three-cell intersections, for further validation. Co-IP and western blotting analyses revealed that only SMC3 interacted with RIT1 in HCC-LY10, Huh7, and Hep3B cells (Fig. 3C and Fig. S4B). The interaction between RIT1 and SMC3 was further verified in 293T cells co-transfected with Myc-RIT1 and Flag-SMC3 plasmids (Fig. 3D). To determine which domain of RIT1 interacts with SMC3, wild-type or truncated Myc-RIT1 was transfected with Flag-SMC3 plasmids in 293T cells and immunoprecipitation and western blot were performed using anti-Myc and anti- Flag antibodies, respectively. The results showed that the wild-type and N-terminal deletion constructs of Myc-RIT1 could bind to SMC3, whereas the C-terminal deletion construct could not, indicating that the binding sites were present in the C-terminal domain of RIT1 (Fig. 3E, F). Moreover, Co-IP analyses with cells asynchronized or synchronized to mitosis showed that the interaction between RIT1 and SMC3 was significantly enhanced during mitosis (Fig. 3G). IF assays showed apparent colocalization of RIT1 with SMC3 proteins during mitosis in HCC-LY10, Huh7, and Hep3B cells (Fig. 3H and Fig. S5A). These results suggest that RIT1 interacts with SMC3, which is essential for mitosis in HCC.

SMC3 is essential for RIT1-mediated cell proliferation in HCC

Due to the interaction between RIT1 and SMC3, we hypothesized that RIT1 might play a regulatory role in mitosis through SMC3 in HCC cells. To investigate the role of SMC3 in RIT1-mediated HCC progression, we analyzed the mRNA expression of SMC3 in the TCGA LIHC database. As shown in Fig. 4A, SMC3 expression was significantly higher in HCC tissues than in non-cancerous tissues. We further examined the protein expression of SMC3 in 36 pairs of HCC tissues and matched non-cancerous tissues from our lab using western blot analysis. Consistent with the results of TCGA dataset analysis, SMC3 was highly expressed in HCC tissues compared with that in non-cancerous tissues (Fig. S6A, B). Immunohistochemistry (IHC) staining for SMC3 was performed on tissue samples from 201 patients with HCC, and patients were divided into high-SMC3 expression group (110 cases) and low-SMC3 expression group (91 cases) according to their IHC staining scores (Fig. 4B). Kaplan-Meier survival analysis showed that high SMC3 expression in patients with HCC was associated with a short survival time (Fig. 4C). These results indicate that SMC3 is highly expressed in HCC and is associated with a poor prognosis.

Fig. 4

SMC3 is upregulated in HCC and contributes to HCC proliferation. A mRNA expression of SMC3 in 374 HCC tissues and 50 non-cancerous tissues in the TCGA LIHC database (left). mRNA expression of SMC3 in 50 pairs of HCC tissues and adjacent non-cancerous tissues in the TCGA LIHC database (right). B Representative IHC staining images of high and low SMC3 expression in 201 HCC tissue samples, including 110 cases of high expression and 91 cases of low expression based on the IHC staining score; scale bars, 200 μm. C Kaplan-Meier analysis showing the overall survival of 186 patients with HCC with high-SMC3 and low-SMC3 expression; HR, Hazard Ratio, P = 0.00016. D CCK8 assay for MHCC-97H and HCC-LY10 cells with SMC3 knockdown and control. E Colony formation assay of MHCC-97H and HCC-LY10 cells with SMC3 knockdown and control. F Flow cytometry analysis of the cell cycle of MHCC-97H and HCC-LY10 cells with SMC3 knockdown and control (left). Quantitation histograms showing the percentage of cells in different phases of the cell cycle (right panel). G Flow cytometry analysis of apoptosis in MHCC-97H cells with SMC3-knockdown and control cells (left). Quantitation histogram showing the percentage of apoptotic cells (right). H Representative immunofluorescence staining images of α-tubulin (red) and DAPI (blue) showing spindle morphology during mitosis in MHCC-97H cells with SMC3 knockdown and control(left). The quantitation histogram showing the percentage of cells with multispindle polar (right). Cells were enriched in mitosis using nocodazole. Scale bars, 20 μm. I Representative immunofluorescence staining images of α-tubulin (red) and DAPI (blue) show nucleus morphology in MHCC-97H cells with SMC3 knockdown and control (left). The quantitation histogram showing the percentage of cells with multinuclear (right). Scale bars, 20 μm. Data are presented as the mean ± SD of three independent experiments. ** P < 0.01, *** P < 0.001. The P values were calculated by paired Student’s t-test in (A), log-rank test in (C), and unpaired Student’s t-test in (D-I)

To further investigate the role of SMC3 in HCC cells, we examined the expression of SMC3 protein in various HCC cell lines and knocked down SMC3 via shRNA in MHCC-97H and HCC-LY10 cells and verified the silencing efficiency using western blotting (Fig. S6C, D). Similar effects were observed after SMC3- and RIT1-knockdown in HCC cells. SMC3 knockdown significantly inhibited proliferation and colony formation of MHCC-97H and HCC-LY10 cells (Fig. 4D, E and Fig. S6E) and led to G2/M phase arrest and apoptosis (Fig. 4F, G). Moreover, the knockdown of SMC3 induced mitotic catastrophe, multinucleated and heterogeneous nuclei, and multipolar spindles (Fig. 4H, I and Fig. S6F, G).

Given that RIT1 interacts with SMC3 and is co-localized during mitosis, it is reasonable to speculate that RIT1 might promote HCC progression by regulating SMC3. We silenced SMC3 in RIT1-overexpressed Huh7 and Hep3B cells (Fig. 5A). CCK8 and colony formation assays showed that silencing SMC3 attenuated the proliferative effect caused by RIT1 overexpression (Fig. 5B, C). SMC3 knockdown also reversed the tumor-promoting effects of RIT1 in vivo in mouse subcutaneous tumor experiments (Fig. 5D, E). Overall, these data suggest that RIT1 promotes HCC cell proliferation by interacting with SMC3.

Fig. 5

Knockdown of SMC3 inhibits the pro-proliferative effect of RIT1 in HCC cells. A Western blot analysis of RIT1 and SMC3 protein expression after silencing SMC3 in RIT1-overexpressing Huh7 and Hep3B cells. B CCK8 assay was performed to examine proliferation after silencing SMC3 in RIT1-overexpressing Huh7 and Hep3B cells. C Representative images of colony formation assays for RIT1-overexpressing Huh7 and Hep3B cells with SMC3 knockdown (left panel). Quantitation histogram showing colony numbers (right). D Images of xenograft tumors of RIT1-overexpressing Huh7 cells or control cells with SMC3 knockdown. E Quantification of tumor weight (g) (n = 7 per group). Data are presented as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001. The P values were calculated by unpaired Student’s t-test for (B, C, E)

RIT1 affects SMC3 acetylation during mitosis

SMC3 is a member of the cohesin core subunit that ensures accurate chromosomal segregation. Establishing chromosome adhesion depends on the acetylation of K105 and K106 in the SMC3 head domain [28]. To further investigate how RIT1 regulates SMC3 and thus influences HCC cell mitosis, we detected the effect of RIT1 on SMC3 mRNA expression by qPCR and found that RIT1 did not affect the mRNA expression of SMC3 (Fig. S7A, B). Then, we examined the protein levels of total SMC3 and SMC3 acetylation in HCC cells with altered RIT1 expression. Protein samples were collected at different stages of the cell cycle after release from double thymidine block for western blot analysis. The results showed that RIT1 significantly affected the acetylation level of SMC3 during mitosis but not in the S and G2 phases. Knockdown of RIT1 decreased the acetylation level of SMC3, whereas overexpression of RIT1 increased the acetylation level of SMC3. RIT1 did not affect the total protein expression of SMC3 (Fig. 6A-D). These results suggest that RIT1 may regulate mitotic progression in HCC cells by influencing the acetylation level of SMC3 during mitosis. To further validate this result, we knocked down ESCO1, an SMC3 acetyltransferase, in RIT1 overexpressed HCC cells (Fig. 6E) and observed changes in their proliferative capacity. CCK8 experiments demonstrated that ESCO1 knockdown reversed the enhanced proliferative capacity induced by RIT1 overexpression (Fig. 6F). This evidence reveals that RIT1 exerts a pro-oncogenic effect by affecting the acetylation level of SMC3 during mitosis.

Fig. 6

RIT1 affects the acetylation of SMC3 during mitosis. A, B Western blot analysis of acetylated-SMC3, total SMC3, and RIT1 protein levels during different cell phases in MHCC-97H (A) and HCC-LY10 cells (B) with RIT1 knockdown. C, D Western blot analysis of acetylated-SMC3, total SMC3, and RIT1 protein levels during different cell phases in Huh7 (C) and Hep3B (D) cells with RIT1 overexpression. Cells were enriched in different cycle phases by releasing from the TT block for the indicated time. E qPCR assays of ESCO1 mRNA expression following si-ESCO1 transfection in RIT1-overexpressed Huh7 and Hep3B cells. F The cell proliferation ability was examined using CCK8 assay in RIT1-overexpressing Huh7 and Hep3B cells with ESCO1 knockdown. Data are presented as mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001. The P values were calculated by unpaired Student’s t-test in (E, F)

RIT1 protects the acetylation of SMC3 by binding to PDS5

Next, we investigated the regulatory mechanism by which RIT1 affects the acetylation of SMC3. Considering that RIT1 concentrates around the chromosome during mitosis and interacts with SMC3, we hypothesized that RIT1 might play a regulatory role in SMC3 acetylation during mitosis by influencing proteins or kinases involved in SMC3 acetylation regulation. qPCR analysis showed that RIT1 did not affect ESCO1 expression (Fig. S8A, B), and no SMC3 acetyltransferase was detected in the MS results for the Co-IP products mentioned above. However, our MS results indicated an interaction between RIT1 and PDS5 (Fig. 3B). PDS5 is an essential regulatory subunit of cohesin that maintains a dynamic balance between cohesin loading and unloading by binding to sororin and wings apart-like protein (WAPL) homolog proteins [29]. In addition, PDS5 can protect and maintain SMC3 acetylation during mitosis [30]. Therefore, we speculated that RIT1 might regulate the acetylation of SMC3 via PDS5. PDS5 has two subunits: PDS5A and PDS5B. Co-IP and western blot analyses revealed an interaction between RIT1 and PDS5A/B (Fig. 7A), while overexpression or knockdown of RIT1 had no effect on PDS5 expression (Fig. S9A, B). We silenced both PDS5A/B in RIT1 overexpressed HCC cells and examined the total SMC3 and acetylated protein expression. The results showed that the upregulation of SMC3 acetylation induced by RIT1 overexpression was suppressed by PDS5A/B knockdown during mitosis, whereas total levels of SMC3 protein remained unchanged (Fig. 7B). The PDS5A/B knockdown attenuated the enhanced growth capacity induced by RIT1 overexpression (Fig. 7C).

Fig. 7

RIT1 promotes HCC proliferation by binding to PDS5 to protect the acetylation of SMC3. A Co-IP analysis of RIT1 and PDS5 in Huh7 and Hep3B cells with RIT1 overexpression. B Western blot analysis of acetylated-SMC3, total SMC3, PDS5A/B, and RIT1 protein upon knockdown of PDS5A/5B in RIT1-overexpressing Huh7 and Hep3B cells. C The cell proliferation ability was examined using CCK8 assay upon knockdown of PDS5A/5B in RIT1-overexpressed Huh7 and Hep3B cells. Data are presented as mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01. The P values were calculated by unpaired Student’s t-test in (C)

In addition, we further explored whether PDS5 expression affects the interaction between RIT1 and SMC3. Co-IP results demonstrated that knockdown of PDS5 did not affect the binding of RIT1 to SMC3 (Fig. S10A).

These results indicated that RIT1 may function as a molecular scaffold to protect SMC3 acetylation by binding to PDS5.

RIT1 expression positively correlates with PDS5 and SMC3 in HCC tissues

Given that RIT1 protects the acetylation of SMC3 by binding to PDS5, we analyzed the correlation between RIT1 expression and SMC3 as well as PDS5A/B in HCC tissues. The mRNA level of RIT1 was positively correlated with SMC3 and PDS5A/B expression in the TCGA LIHC dataset (Fig. 8A). Notably, the protein levels of acetylated SMC3, total SMC3, PDS5A/B, and RIT1 were also positively correlated in HCC tissues (Fig. 8B). IHC analysis further confirmed a positive correlation between RIT1 and SMC3 expression in HCC tissues (Fig. 8C). We then divided the patients with high RIT1 expression into two groups according to SMC3 expression levels and observed that patients with high expressions of both RIT1 and SMC3 had a worse prognosis compared to those with high RIT1 but low SMC3 expression (Fig. 8D). These results further conformed that RIT1 expression closely correlated to SMC3 and PDS5A/B expression, and RIT1 exerted its pro-proliferative function via SMC3 in HCC. Our data highlight the significance of the RIT1/PDS5/SMC3 axis in promoting HCC progression and suggest that RIT1 may serve as a biomarker and potential target for HCC diagnosis and therapy (Fig. 8E).

Fig. 8

RIT1 expression positively correlates with PDS5 and SMC3 in HCC tissues. A Correlation of mRNA level of RIT1 with PDS5A/B and SMC3 in TCGA LIHC dataset. B Western blot analysis of RIT1, PDS5A/B, acetylated-SMC3, and total SMC3 in HCC tissues from our lab (left). Spearman correlation analysis was performed between RIT1 and PDS5A/B, acetylated-SMC3, total SMC3 expression (right). C Representative IHC staining images of concurrent high or low RIT1/SMC3 expression in 201 HCC tissue samples. Spearman correlation analysis of RIT1 and SMC3 expression was performed (R = 0.319, P < 0.001). Scale bars, 200 μm. D Kaplan-Meier survival curves for 99 HCC patients with RIT1 high expression classified by high or low expression of SMC3 expression according to IHC results; HR, Hazard Ratio. E A schema showing that RIT1 regulates mitosis and promotes proliferation by interacting with SMC3 and PDS5 in HCC. With a high level of RIT1 in HCC, RIT1 binds with PDS5 and SMC3, which protects and maintains the acetylation of SMC3 during mitosis. HCC cells undergo successful and rapid mitosis, leading to tumor growth in HCC (left). A low level of RIT1 in HCC or knockdown of RIT1 leads to a reduction of the acetylation level of SMC3, resulting in mitotic catastrophe in HCC (right)