Fig 2: Promoter methylation silences RASA4 expression, conversely activating Ras-MAPK signaling in SCLC.A Western blot analysis of RASA4 expression in SCLC cell lines (H82, H69, H526, H446, SBC-2, DMS114, SHP77, and DMS273) and the normal bronchial alveolar epithelial cell line (BEAS-2B). B qRT-PCR shows that the treatment of 5-Aza increases the transcription of RASA4 mRNA in H82, H526, SHP77, and DMS114 SCLC cells. Data are shown as mean ± SD from three independent biological replicates (n = 3, unpaired Student’s t-test). C Western blot shows that the treatment of 5-Aza increases the expression of RASA4 protein in H82, H526, SHP77, and DMS114 SCLC cells. D Targeted demethylation of the DMR (chr7:102254100-102254400) within the RASA4 promoter by dCas9-Tet1 and sgRNA. BSP analysis indicates a significant decrease in methylation levels at the target CpG sites in both DMS114 and SHP77 cells following co-transfection with vectors expressing dCas9-Tet1 and the sgRNA targeting the specific DMR (Chr7: 102254100-102254400). qRT-PCR indicates that RASA4 mRNA expression is significantly increased in DMS114 and SHP77 cells following targeted demethylation of the DMR (Chr7: 102254100-102254400) by dCas9-Tet1 and sgRNA. For the BSP analysis, data are shown as mean ± SD from six independent DNA clones (n = 6, unpaired Student’s t-test). For the qRT-PCR analysis, data are shown as mean ± SD from three independent biological replicates (n = 3, unpaired Student’s t-test). E Western blot shows that the treatment of 5-Aza inhibits the phosphorylation of MEK and ERK in H526 SCLC cells. F Ras activation assay shows that knockdown of RASA4 increases Ras activity in SBC-2 SCLC cells. The overexpression of RASA4 decreases Ras activity in SHP77 SCLC cells. G Western blot shows that knockdown of RASA4 increases the phosphorylation of MEK and ERK in SBC-2 SCLC cells. The overexpression of RASA4 decreases the phosphorylation of MEK and ERK in SHP77 SCLC cells.

Fig 3: RASA4 inhibits the proliferation and invasion of SCLC in vitro and in vivo.A Cell viability assay shows that the stable over-expression of RASA4 inhibits the proliferation in SHP77 and DMS114 cells. Data are shown as mean ± SD from three independent biological replicates (n = 3, unpaired Student’s t-test). B Colony formation assay shows that the ability of colony formation of SHP77 and DMS114 cells with stable over-expression of RASA4 is reduced. The colonies were quantified and shown by mean ± SD from three biologically independent experiments (n = 3, unpaired Student’s t-test). C Matri-gel invasion assay shows that the stable over-expression of RASA4 inhibits the invasion in DMS114 cells. The stable knockdown of RASA4 increases the invasion in SBC-2 cells. The invaded SCLC cells were quantified and shown by mean ± SD from three biologically independent experiments (n = 3, unpaired Student’s t-test). (scale bar = 200 μm) (D) Wound healing assay shows that the stable over-expression of RASA4 inhibits the migration in DMS114 cells. The stable knockdown of RASA4 increases the migration in SBC-2 cells. Wound closure was quantified as a percentage of the initial wound area. Data are shown as mean ± SD from three biologically independent experiments (n = 3, unpaired Student’s t-test). (scale bar = 200 μm) (E) The growth inhibition curves demonstrate that stable RASA4 over-expression sensitizes SHP77 cells to etoposide and carboplatin, respectively, as evidenced by decreased IC50 values, whereas RASA4 knock-down conversely increases the IC50 values in SBC-2 cells. Data are shown as mean ± SD from three independent biological replicates. F Subcutaneous tumorigenesis assay in nude mice. SHP77 cells with stable transfection of the empty vector and the RASA4-expressing vector were subcutaneously injected into nude mice. The growth curves show the tumor volume over a period of 6 weeks. Upon euthanasia at the end of the experiment, tumors were dissected and weighed. Images of tumors from each group were taken. Data are shown as mean ± SD from 5 mice for each group (n = 5, unpaired Student’s t-test). G H&E and Ki67 IHC staining in SHP77 xenografts stably transfected with empty vector or RASA4-expressing vector. (scale bar = 40 μm for 20×, scale bar = 200 μm for 4×) (H) The knockdown of RASA4 promotes the metastasis of SCLC cells. SBC-2 cells stably expressing scramble and RASA4 shRNA were intravenously injected into nude mice. After 6 weeks, the lungs of mice were dissected and photographed using an animal imaging system. Green fluorescence-expressing metastatic nodules were indicated. Each group comprised five mice injected with SBC-2 cells stably expressing scramble or RASA4 shRNA. Three mice in each group developed metastatic lung nodules, as observed during dissection. Metastatic lung nodules are quantified as mean ± SD from three mice (n = 3, unpaired Student’s t-test).

Fig 4: Ca2+ Influx and RASA4 expression in HPV-infected human cervical epithelial cells upon inhibition of TRPC3.H8 cell line and HCECs were infected with HPV16 or 18 for 72 hours prior to treatment with ATP in the presence of a TRPC3 inhibitor (Pyr3, 10 μM) or an equal volume of the DMSO solvent control. A Calcium influx was measured using Rhod-3 Calcium Imaging Kit and fluorescence microscopy. B Immunofluorescence images showing RASA4 expression on H8 and HCECs treated with Pyr3 or the DMSO solvent control following HPV16/18 infection.

Fig 5: RASA4 is down-regulated in SCLC tumor tissues and correlates with poor clinical prognosis.A Representative images of H&E staining in tumor and adjacent normal tissues from FFPE specimens of three surgical SCLC patients. B IHC staining of RASA4 in tumor and adjacent normal tissues from FFPE specimens of the same three SCLC patients. The results show significantly reduced RASA4 expression in the tumor tissues compared to the adjacent normal tissues. C IHC staining of SERPINE2 in tumor and adjacent normal tissues from FFPE specimens of the same three SCLC patient samples. The results show significantly increased SERPINE2 expression in the tumor tissues compared to the adjacent normal tissues. (scale bar = 20 μm for 40×, scale bar = 100 μm for 8×) (D) IHC staining shows that SERPINE2 expression is elevated in the invasive front of the SCLC tumor. (scale bar = 40 μm for 20×, scale bar = 200 μm for 5×) (E) IHC scores of RASA4 and SERPINE2 in tumor and adjacent normal tissues from 29 SCLC patients who underwent surgery (n = 29, Mann–Whitney test). F Statistical analysis indicates the negative association between RASA4 and SERPINE2 expression in SCLC tumor and adjacent normal tissues (n = 29, Chi-square test for R × C contingency tables). G Kaplan–Meier survival analysis of 29 SCLC patients who underwent surgery, stratified by RASA4 IHC scores in tumor tissues. The results show that SCLC patients with moderate scores of RASA4 expression have significantly better survival compared to those with weak scores. The statistical significance of the difference in survival was determined by the log-rank test.