Journal: Cancers

Article Title: SCAMP3 Regulates EGFR and Promotes Proliferation and Migration of Triple-Negative Breast Cancer Cells through the Modulation of AKT, ERK, and STAT3 Signaling Pathways.

doi: 10.3390/cancers14112807

Figure Lengend Snippet: Figure 1. The proliferation of TNBC cells was suppressed after SCAMP3 knockout. (A) Immunoblot and proliferation of MDA-MB-231 cells after transfected with SCAMP3 targeting siRNA (siSC3) and nontargeting sequences (siNC). (B,C) Immunoblotting and proliferation of wild-type (WT) MDA-MB- 468 and SUM-149 cells after SCAMP3 gene knockout (SC3KO) using CRISPR/Cas9. WT and SC3KO (D) SUM-149 and (E) MDA-MB-468 cells were incubated for ten days. The colonies were stained with crystal violet and the relative number of colonies with >50 cells was graphed. Student’s t-test; *** p ≤0.001. WT and SC3KO SUM-149 were allowed to grow in a low-attachment plate for three days to allow them to form tumorspheres. (F) Illustrated micrographs represent 5 photos per condition.

Article Snippet: SCAMP3 siRNA (sc-41294) and scrambled siRNA (sc-37007) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Knock-Out, Western Blot, Transfection, Gene Knockout, CRISPR, Incubation, Staining

Journal: Cancers

Article Title: SCAMP3 Regulates EGFR and Promotes Proliferation and Migration of Triple-Negative Breast Cancer Cells through the Modulation of AKT, ERK, and STAT3 Signaling Pathways.

doi: 10.3390/cancers14112807

Figure Lengend Snippet: Figure 2. SCAMP3 modulates the EGFR signaling pathway. Immunoblots and densitometry quantification of (A,B) MDA-MB-231 cells transfected with SCAMP3 targeting siRNA and non- targeting sequence (siNC). (C,D) MDA-MB-468 WT and SC3 knockout cells. (E,F) MCF-10A con- trol cells and SC3 overexpressing cells (SC3OE). Lysates were probed using the indicated anti- bodies. Two-way ANOVA with Bonferroni’s multiple comparison test; * p ≤0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Data are represented as mean ± SEM. The experiments were carried out at least three times.

Article Snippet: SCAMP3 siRNA (sc-41294) and scrambled siRNA (sc-37007) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Western Blot, Transfection, Sequencing, Knock-Out, Comparison

Journal: Cancers

Article Title: SCAMP3 Regulates EGFR and Promotes Proliferation and Migration of Triple-Negative Breast Cancer Cells through the Modulation of AKT, ERK, and STAT3 Signaling Pathways.

doi: 10.3390/cancers14112807

Figure Lengend Snippet: Figure 3. SCAMP3 delayed tumor cell proliferation and decreased p-EGFR. Orthotopic models of breast cancer were generated using SUM-149 cells. (A) Body weight of mice injected with wild-type (WT) (n = 9) and SCAMP3 knockout cells (SC3KO) (n = 9). Two-way ANOVA: non-significant. (B) Tumor volume (mm3) was monitored for 10 weeks. Comparisons of the average tumor size by group and week were evaluated by unpaired t-test assuming unequal variances; * p ≤0.05. (C) Tumor weights at the end of the study. t-test; ns: not significant. (D) Immunoblots of SUM-149 tumors. Each lane represents a different animal. Samples are representative of n = 8/group. Lysates were probed using indicated antibodies. (E) Densitometry quantification analyses of (n = 8/group). Two-way ANOVA; * p ≤0.05, *** p < 0.001 and **** p < 0.0001; t-test. Data are represented as mean ± SEM.

Article Snippet: SCAMP3 siRNA (sc-41294) and scrambled siRNA (sc-37007) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Generated, Injection, Knock-Out, Western Blot

Journal: Cancers

Article Title: SCAMP3 Regulates EGFR and Promotes Proliferation and Migration of Triple-Negative Breast Cancer Cells through the Modulation of AKT, ERK, and STAT3 Signaling Pathways.

doi: 10.3390/cancers14112807

Figure Lengend Snippet: Figure 4. SCAMP3 colocalizes with EGFR after receptor internalization. Cells were stimulated with 10 ng/mL EGF at the indicated time points to evaluate the location of SCAMP3 and EGFR using confocal microscopy. Representative images of the internalization assay and fluorescence quantification of SCAMP3, EGFR, and their colocalization in (A–C) SUM-149 and (D–F) MDA-MB- 468 cells. The nuclei were stained with DAPI (blue). Secondary antibodies conjugated to Alexa 488 (green) and Alexa 594 (red) were used to detect SCAMP3 and EGFR, respectively. The micrographs were taken at a magnification of 60× using confocal microscopy. The white arrows indicate the colocalization of EGFR and SCAMP3 in the zoom images. The zoom-in of each image is shown in white squares and each has equal dimensions. Scale bar = 20 µm. Total cell fluorescence and colocalization area analyses were performed in 20 cells from three experiments using Image J. Colocalization was calculated as the ratio of the colocalization fluorescence area to the total cell fluorescence area. One way or two-way ANOVA; * p ≤0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data are represented as mean ± SEM.

Article Snippet: SCAMP3 siRNA (sc-41294) and scrambled siRNA (sc-37007) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Confocal Microscopy, Staining

Journal: Cancers

Article Title: SCAMP3 Regulates EGFR and Promotes Proliferation and Migration of Triple-Negative Breast Cancer Cells through the Modulation of AKT, ERK, and STAT3 Signaling Pathways.

doi: 10.3390/cancers14112807

Figure Lengend Snippet: Figure 5. SCAMP3 modulates TNBC cell proliferation and motility through EGFR. (A) SUM-149 WT and SC3KO cells were stimulated with 10ng/mL of EGF for 30 min and proliferation was measured after 72 h using the Cell Proliferation Kit I (MTT). Data are expressed relative to WT without (EGF-) or after stimulation (EGF+). One-way ANOVA; *** p < 0.001 and **** p < 0.0001. EGF-stimulated SUM-149 WT stimulated with EGF and SC3KO were incubated in migration and Matrigel invasion chambers for 24 h to assess (B) migration and (C) invasion ability of cells. The nuclei of the migrating and invading cells were stained with PI. Illustrations represent 14 micrographs per condition at a magnification of 200×; scale = 100 µm. Cells were counted using ImageJ. Two-way ANOVA; * p ≤0.05, ** p < 0.01, *** p ≤0.001. (D) Wound healing assay of MCF-10A control and SC3OE cells using silicone insert Ibidi® plates after 24 h. The width of the wound was determined by measuring the distance between the edges of the wound. The nuclei were stained with DAPI (blue) and the actin cytoskeleton was stained with Rhodamine Phalloidin (red). Micrographs were taken at a magnification of 400×; scale = 200 µm. Data are expressed as percent relative to 0 h and analyzed relative to control cells. t-test: **** p ≤0.0001. Data are represented as mean ± SEM. The experiments were carried out at least three times.

Article Snippet: SCAMP3 siRNA (sc-41294) and scrambled siRNA (sc-37007) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Incubation, Migration, Staining, Wound Healing Assay, Control

Journal: Cancers

Article Title: SCAMP3 Regulates EGFR and Promotes Proliferation and Migration of Triple-Negative Breast Cancer Cells through the Modulation of AKT, ERK, and STAT3 Signaling Pathways.

doi: 10.3390/cancers14112807

Figure Lengend Snippet: Figure 6. SCAMP3 regulates EGFR signaling by degradation of the receptor. (A,B) Immunoblots and densitometry quantification of SUM-149 WT or SC3KO cells after stimulation with 10 ng/mL EGF for 30 min. Lysates were probed using the indicated antibodies. Two-way ANOVA; * p ≤0.05, ** p < 0.01, **** p < 0.0001. (C,D) Immunoblots and densitometry quantification analyses of SUM-149 WT or SC3KO lysates after cells treated with 1 µM erlotinib for 72 h. One-way ANOVA; * p ≤0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant. (E) Serum starved WT and SC3KO SUM-149 cells were treated with 100µM cycloheximide for 1 h before stimulation with 10 ng/mL of EGF. Lysates were obtained after each time point shown and immunoblotted using SCAMP3 and EGFR antibodies. After densitometry analysis, the EGFR intensities data were normalized to β-tubulin and correlated to 0 h. The plotted data represent the residual EGFR. t-test, * p ≤0.05, *** p < 0.001 and **** p < 0.0001. Data are represented as mean ± SEM. The experiments were carried out at least three times.

Article Snippet: SCAMP3 siRNA (sc-41294) and scrambled siRNA (sc-37007) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Western Blot

Journal: Cancers

Article Title: SCAMP3 Regulates EGFR and Promotes Proliferation and Migration of Triple-Negative Breast Cancer Cells through the Modulation of AKT, ERK, and STAT3 Signaling Pathways.

doi: 10.3390/cancers14112807

Figure Lengend Snippet: Figure 7. SCAMP3 expression analyses from the TCGA database. (A) UALCAN portal analysis comparing SCAMP3 expression between normal and breast cancer tumors. (B) Probability of survival between breast cancer patients with high and low SCAMP3 expression using The Human Protein Atlas portal. (C) Expression of SCAMP3 in different subtypes of breast cancer. (D) Probability of survival between breast cancer patients with different subtypes and low to high levels of SCAMP3 expression. Green line = SCAMP3 high expression (TNBC). (E) Probability of relapse-free survival (RFS) between TNBC patients with high and low SCAMP3 expression using Kaplan–Meier Plotter portal. (F) Probability of distant metastasis-free survival (DMFS) between patients with TNBC with high and low SCAMP3 expression using Kaplan–Meier Plotter portal.

Article Snippet: SCAMP3 siRNA (sc-41294) and scrambled siRNA (sc-37007) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing

Journal: Cancers

Article Title: SCAMP3 Regulates EGFR and Promotes Proliferation and Migration of Triple-Negative Breast Cancer Cells through the Modulation of AKT, ERK, and STAT3 Signaling Pathways.

doi: 10.3390/cancers14112807

Figure Lengend Snippet: Figure 8. Regulation of gene expression by SCAMP3 in breast cancer. (A) RT2 PCR arrays were performed to profile the expression of EGF/PDGF signaling pathway-specific genes in three different tumors per group. ACTB, B2M, GAPDH, HPRT1, and RPLP0 were used as reference genes. The Volcano plot shows the effects on gene expression analyzed at −2.0 ≥2.0 log2-fold change (vertical lines). Statistically significant (p ≤0.05) genes are above the horizontal black line. Correlation of the expression of the SCAMP3 and (B) EGFR, (C) STAT3, or (D) PDGF genes in breast cancer. BioPortal web server: Breast Invasive Carcinoma (TCGA, Firehouse Legacy). (E) Summary of our findings. The left panel shows the interaction of EGFR and SCAMP3 within the cell after activation of EGFR in WT cells and its effects on cell proliferation, motility, and modulation of EGFR, AKT, and ERK. The right panel shows our findings in our SCAMP3 depletion models. EGFR depletion decreased cell proliferation, migration, and invasion. Moreover, accelerated EGFR degradation and modulated EGFR, AKT, ERK, and STAT3.

Article Snippet: SCAMP3 siRNA (sc-41294) and scrambled siRNA (sc-37007) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Gene Expression, Expressing, Activation Assay, Migration