Fig 2: DRAM1 Interacts with SCAMP3 to Drive SLC1A5 and LAT1 to Lysosome Membranes(A) Saos TetOn-DRAM1-myc-his cells overexpressing V5-tagged SLC1A5 were grown for 24 h in the presence or absence of doxycycline (1 µg/mL) and then treated for 4 h with cycloheximide (100 µg/mL) or anisomycin (10 µg/mL). Cells were fixed in 4% paraformaldehyde and stained for Lamp2 and V5.(B) Saos2 TetOn-DRAM1-myc-his cells overexpressing V5-tagged SLC1A5 were, where indicated, treated with doxycycline for 24 h and then treated for 4 h with cycloheximide (100 µg/mL) or anisomycin (10 µg/mL). SLC1A5 expression was detected using an anti-V5 antibody: a short and long exposure is shown. DRAM-1 levels were detected using an anti-Myc tag antibody. Actin was used as a loading control(C) Saos2 TetOn-DRAM-1 cells treated with or without doxycycline for 24 h were lysed prior to immunoprecipitation of DRAM-1 using anti-Myc-tag antibody. Immunoblotting from total protein extracts (INPUT) or elutions (IP DRAM-1) was undertaken to detect SCAMP3, DRAM-1 (Myc tag), and ERK2.(D) Saos Tet-On-DRAM1-myc-his cells overexpressing V5-tagged SLC1A5 were grown for 24 h in the presence or absence of doxycycline (1 µg/mL). Cells were fixed in 4% paraformaldehyde and stained for SCAMP3, DRAM1 (Myc-tag), and SLC1A5 (V5-tag).(E) SCAMP3 was downregulated using siRNA in Saos-2 TetOn-DRAM1-myc-his cells overexpressing V5-tagged SLC1A5, grown for 24 h in the presence or absence of doxycycline (1 µg/mL). Cells were fixed in 4% paraformaldehyde and stained for SLC1A5(V5-tag) and LAMP2.(F) Colocalization coefficients were determined using Zeiss Zen Black software (average of at least 25 cells). Data are mean ± SEM.(G) SCAMP3 was downregulated using siRNA in Saos TetOn-DRAM1-myc-his cells overexpressing V5-tagged SLC1A5, then, where indicated, treated with Dox for 24 h, and then starved for 3 h in EBSS before treatment with EBSS containing leucine for 20 min. mTORC1 activation was evaluated by measuring phospho-S6 kinase and phospho-S6 levels by western blot. Levels of total S6 kinase, S6, and actin were used as loading controls. DRAM1 expression was detected using an anti-Myc-tag antibody, and SCAMP3 knockdown was measured using an anti- SCAMP3 antibody.

Fig 3: SCAMP3 promotes glioma growth in vivo. (A) The growth curve of implanted xenografts was plotted, which showed that U251-SCAMP3 cells possessed the highest growth rate. In contrast, U251 cells with SCAMP3-shRNA transfection showed impaired growth capacity compared with control cells. (B) The final tumor weight was tested after xenograft isolation and revealed consistent results with growth curves. (C) The photographed images of isolated xenografts. (D) The proliferation index was determined using the percentage of Ki-67 positive cells in isolated xenografts by IHC staining. As a result, SCAMP3-overexpression groups showed highest proliferation index while SCAMP3-shRNA groups showed lowest proliferation index. *P<0.05 by Student’s t-test compared with control group.

Fig 4: SCAMP3 promotes glioma cell proliferation. (A) The proliferation curves of transfected U251 cells were plotted based on MTS data, which indicated that overexpressing SCAMP3 enhanced while silencing SCAMP3 inhibited cell proliferation. (B) Colony formation assays were conducted for transfected U251 cells. Consistent with the proliferation results, SCAMP3 transfection resulted in more colonies while SCAMP3-knockdown attenuated colony formation. (C) Immunoblotting results confirmed the transfection efficiencies of SCAMP3 overexpression and knockdown. Besides, silencing SCAMP3 leads to impaired EGFR recycling, as well as the activation of S6K and 4E-BP1 proteins. (D) U251 cells transfected with SCAMP3 plasmids were further treated with rapamycin or gefitinib and then subjected to MTS assay. Either inhibiting mTOR or EGFR can significantly attenuate the proliferation-promoting effect of SCAMP3 on glioma cells. (E) The alteration of migration capacity by overexpressing or silencing SCAMP3 was tested and revealed no statistical significance. (F) Matrigel-transwell assay demonstrated that SCAMP3 has no significant effect on U251 cell invasion. Data were analyzed by Student’s t-test compared with control group. *P<0.05, **P<0.01. NS, not significant.

Fig 5: SCAMP3 is upregulated in glioma tissues. (A) RT-qPCR results demonstrated that SCAMP3-mRNA is higher in gliomas compared with normal brain tissues. (B) Representative IHC images of SCAMP3 staining in gliomas and brain tissues. Magnification: 400×. (C) The IHC score of SCAMP3 was significantly higher in gliomas than that in nontumorous tissues. (D) ROC curve was plotted to identify the cutoff value to divide patients into high-SCAMP3 group and low-SCAMP3 group based on the IHC scores. The area under curve is 0.650 and IHC score=5.0 was identified as the cutoff score. **P<0.01 by Student’s t-test, ***P<0.001 by Student’s t-test.