YAP silencing by RB1 mutation is essential for small-cell lung cancer metastasis

Abstract

Small cell lung cancer (SCLC) is highly lethal due to its prevalent metastasis. Most SCLCs have inactivating mutations in TP53 and RB1. We find that loss of YAP expression is key for SCLC cells to acquire rapid ameboid migration and high metastatic potential. YAP functions through its target genes CCN1/CCN2 to inhibit SCLC ameboid migration. RB1 mutation contributes to YAP transcriptional silencing via E2F7, which recruits the RCOR co-repressor complex to YAP promoter. We discover that benzamide family HDAC inhibitors stimulate YAP expression by inhibiting the RCOR-HDAC complex, thereby suppressing SCLC metastasis and improving survival in a mouse model. Our study unveils the molecular and cellular basis underlying SCLC's high metastatic potential, the previously unrecognized role of YAP in suppressing ameboid migration and tumor metastasis, and the mechanism of YAP transcription regulation involving E2F7, RCOR, and Sin3 HDAC. This study reveals a therapeutic potential of benzamides for SCLC treatment.

Fig. 1. YAP is silenced in SCLC and re-expression inhibits tumor metastasis.

a YAP is expressed in lung adenocarcinoma (LUAD) and squamous carcinoma (LUSC) but not in SCLC tumors. YAP protein in tumors was detected by a specific antibody, and the dot plot on the right shows the IHC quantification of 10 samples for each cancer subtype. b YAP induction does not affect the growth of H209 in vivo and tumor formation in vitro. DOX denotes the presence of 100 ng/mL Dox in culture. Viable cells were determined by the CCK8 assay. c YAP induction inhibits H209 liver metastasis. Liver from vehicle and Dox-treated mice were stained with HE (left panels). Quantification of liver metastasis foci per mouse is shown in the right panel. d YAP induction at the early but not late stage inhibits liver metastasis. Liver from control and early/late Dox-treated mice were stained with HE (left panels). Quantification of metastatic sites per mouse is shown in the right panel. e The survival curve of nude mice after orthotopic injection of control or YAP inducible H209 cells. f YAP decreases CTCs. Equal amounts of EGFP-labeled control and mSCARLET-labeled YAP inducible cells were injected into mouse lungs. Blood cells were collected and analyzed for EGFP (x axis) and mSCALET (y axis) to quantify CTCs (green or red boxes). g YAP re-expression inhibits CTCs. Relative CTCs were determined by human GAPDH mRNA normalized against mouse GAPDH mRNA. h EGFP⁺/YAP⁻ cells but not mSCARLET⁺/YAP⁺ form liver metastasis. Mice were orthotopically injected with a mixture of both cells. Source data are provided as a Source data file.

Fig. 2. YAP inhibits SCLC ameboid migration through CCN1/2.

a YAP expression inhibits the fast ameboid-like migration of H209 cells under confinement. Ameboid migration speed were quantified and shown on the right. b TEAD-dependent YAP co-transcriptional activity is required to suppress H209 ameboid migration. c YAP expression alters genes in the cytoskeleton and Rho GTPase pathways. GO pathway enrichment scatterplot shows the enriched DEG pathways in control and YAP re-expressed H526 cells. Dot size is proportional to the number of DEGs. d The effect of YAP and CCN1/2 on F-actin polarization. Fluorescence images of Lifeact-TagGFP are shown for H209 cells under confinement. e YAP re-expression decreases F-actin intensity in H209 xenografted tumors. Quantification is shown in the right panel. f Purified CCN1 or CCN2 protein inhibits ameboid migration in H209 cells. g CCN1/2 are required for YAP to inhibit H209 cells ameboid migration. Source data are provided as a Source data file.

Fig. 3. RB1 loss results in YAP downregulation.

a Overlap of genes upregulated or downregulated by YAP re-expression or RB1 rescue in H526 cells. Only differentially regulated genes (>2-fold and p < 0.05 compared to the control H526 cells) were included in the analysis. b E2F7/8 KO induces YAP mRNA and protein in H209 cells. c Ectopic E2F7 expression blocks YAP induction by RB1 in H209 and H69 cells. d Volcano plot showing the upregulation and downregulation of YAP and E2F7/8 in H526 cells, respectively. e Effect of RB1 on chromatin accessibility in the promoters of YAP, E2F7, and E2F8. Control and RB1 rescue H526 cells were compared. TSS and TES denote transcription start and end site, respectively. All genes have been normalized to a 1 kb length. f RB1 re-expression abolishes E2F7 binding on the YAP promoter. Chromatin immunoprecipitation (ChIP) and RT-qPCR show E2F7 occupancy on the YAP promoter in control and RB1 rescued H209 and H69 cells. Source data are provided as a Source data file.

Fig. 4. E2F7 and RCOR mediate the effect of RB1 loss on YAP downregulation.

a Mass spectrometry showing the Flag-E2F7 interacting proteins in H526 cells. The background of proteins recovered from the Flag vector control expressing cells has been subtracted. The 10 top enriched proteins are listed. b E2F7 interacts with RCOR1. Co-immunoprecipitation (Co-IP) experiment confirmed the interaction between Flag-tagged E2F7 and endogenous RCOR1 in H209 and H69 cells. c RCOR1 interacts with E2F7 but not E2F1. Co-IP showed the interaction between Flag-RCOR1 and endogenous E2F family proteins in H209 and H69 cells. d RCOR1/2/3 KO induces YAP mRNA and protein in H209 and H69 cells. e RCOR1/2/3 and E2F7/8 KO increases H3K4me3 YAP promoter of H209 and H69 cells. ChIP and RT-qPCR show YAP promoter H3K4 trimethylation. f YAP is required for RB1 to inhibit ameboid migration. Cell morphology (left) and migration (right) of control, RB1 rescued, YAP KO, and YAP KO and RB1 rescued H209 cells under confinement. g E2F7/8 KO or RCOR1/2/3 KO inhibits ameboid migration. Control, E2F7/8 KO, and RCOR1/2/3 KO H209 cells under confinement. h REST expression of SCLC cell lines and others in CCLE database. i Ectopic expression of REST in H209 and H69 cells induces YAP expression and abolishes the effect of RB. Source data are provided as a Source data file.

Fig. 5. Benzamide family HDAC inhibitors induce YAP transcription via the SIN3A-HDAC complex.

a Entinostat induces YAP and CCN2 expression. H209 and H69 cells were treated with 1 µM entinostat, 0.3 µM TSA, 1 µM GSK-LSD1, 1 µM DZNep, and 10 µM decitabine for 24 hours. b 1 µM entinostat treatment for 24 hours induces YAP protein in H209 and H69 cells. c TSA prevents entinostat from inducing YAP expression in H209 and H69 cells. d SIN3A/B KO prevents entinostat from inducing YAP expression in H209 and H69 cells. e TSA treatment blocks YAP induction by RCOR1/2/3 KO in H209 and H69 cells. f SIN3A/B KO suppresses YAP mRNA and protein induction by RCOR1/2/3 KO in H209 and H69 cells. g A proposed mechanism of YAP transcription regulation. RB1 acts through the E2F7-RCOR axis, while SIN3A/B is required for YAP expression in SCLC. h KO of SIN3A/B but not RCOR1/2/3 blocks YAP induction by RB1 re-expression in H209 and H69 cells. i SIN3A/B KO but not RCOR1/2/3 KO blocks YAP induction by E2F7/8 KO in H209 and H69 cells. j TSA suppresses RB1 rescue-induced YAP expression in H209 and H69 cells. k TSA suppresses E2F7/8 KO-induced YAP expression in H209 and H69 cells. l Entinostat but not TSA increases SIN3A occupancy on the YAP promoter. m E2F7 expression blocks RB1-induced SIN3A occupancy on the YAP promoter. n Benzamide HDAC inhibitors induce YAP protein in H209 and H69 cells. Source data are provided as a Source data file.

Fig. 6. Entinostat inhibits SCLC metastasis.

a Entinostat (1 µM, 24 hours) inhibits H209 cell ameboid migration under confinement. b Entinostat decreases H209 cell F-actin intensity in vitro. The fluorescence image and quantification of Lifeact-TagGFP are shown in the left and right panel, respectively. c Entinostat inhibits liver metastasis of orthotopically grafted H209 cells. HE staining shows liver metastasis. Quantification of metastasis sites per mouse is shown in the right panel. d Entinostat treatment decreases F-actin intensity in H209 xenografted tumors. Quantification is shown in the right panel. e Entinostat reduces CTCs in mice orthotopically inoculated with EGFP-labeled H209 or H526 cells. EGFP-positive CTCs in blood were detected by flow cytometry. f Entinostat reduces CTCs in mice orthotopically inoculated with either H209 or H526 cells. Experiments were similar to d, except CTCs were quantified by human GAPDH mRNA normalized against mouse GAPDH mRNA. g Entinostat prolongs survival of H209 xenografted mice. Nude mice were orthotopically inoculated with 5 × 10⁶ H209 cells. Seven days later, mice were orally treated with a control vehicle or 12.3 mg/kg entinostat once daily, five days per week, until the terminal event. h Entinostat acts through YAP to decrease H209 cell F-actin intensity in vitro. The fluorescence image and quantification of Lifeact-TagGFP are shown in the left and right panel, respectively. i Entinostat acts through YAP to inhibit H209 cell ameboid cell migration. j Entinostat acts through YAP to inhibit F-actin in the H209 orthotopic model. Quantification is shown in the right panel. k YAP KO blocks the inhibitory effect of entinostat on CTCs in H209 cells. l YAP is required for entinostat to inhibit H209 liver metastasis. Source data are provided as a Source data file.