STARD10 promotes progression of HER2+ breast cancer and intracellular lipid metabolism via the cAMP/PKA/CREB1 signaling axis

Figures

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  Figure 1.. Analysis of the Clinical Relevance of STARD10 Expression with Breast Cancer. (A, B) STARD10 expression levels in breast cancer, HER2+ , and normal breast tissues from in TCGA database. (C) Correlation analysis between STARD10 expression and ERBB2 from TCGA database. (D) Overall survival (OS) curve of HER2+ breast cancer patients with low and high STARD10 expression from TCGA cohort. (E) STARD10 expression levels in HER2- breast cancer and HER2+ breast cancer tissues analyzed from GEO database. (F-M) Correlation analysis between STARD10 and progesterone receptor status, gender, age, maximum tumor diameter, distant metastasis, lymph node metastasis, tumor-node-metastasis stage, and estrogen receptor in HER2+ breast cancer samples from the GEO database. *P < 0.05; ns, not significant.

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  Figure 2.. STARD10 overexpression promotes progression of HER2+ Breast Cancer and intracellular lipid droplets synthesis. (A, B) Western blot and q-PCR analysis for STARD10 expression in SKBR3 and HCC1954 cell lines transduced with lentiviral vectors. (C-E) CCK-8, colony formation and EdU assays were performed to evaluate the proliferation capacity of STARD10-overexpressing SKBR3 and HCC1954 cells. Scale bar = 100 μm. (F) Transwell assays were performed to evaluate cell migration and invasion capabilities of STARD10-overexpressing SKBR3 and HCC1954 cells. Scale bar = 50 μm. (G) Representative images of intracellular lipid droplets in STARD10-overexpressing SKBR3 and HCC1954 cells stained with LD540. The corresponding quantification shows the fluorescence intensity of lipid droplets. Scale bar = 100 μm. (H) Western blot analysis of FASN and DGAT1 expression levels in STARD10-overexpressing SKBR3 and HCC1954 cells. n = 3. *P < 0.05, **P < 0.01.

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  Figure 3.. STARD10 knockout inhibits the progression of HER2+ Breast Cancer and intracellular lipid droplets synthesis. (A, B) Western blot and q-PCR analysis for STARD10 expression in SKBR3 and HCC1954 cell lines edited using CRISPR/Cas9. (C-E) CCK-8, colony formation and EdU assays were performed to evaluate the proliferation capacity of stable STARD10-knockout SKBR3 and HCC1954 cells. Scale bar = 100 μm. (F) Transwell assays were performed to evaluate cell migration and invasion capabilities of stable STARD10-knockout SKBR3 and HCC1954 cells. Scale bar = 50 μm. (G) Representative images of intracellular lipid droplets in stable STARD10-knockout SKBR3 and HCC1954 cells stained with LD540. The corresponding quantification shows the fluorescence intensity of lipid droplets. Scale bar = 100 μm. (H) Western blot analysis of FASN and DGAT1 expression in STARD10-knockout SKBR3 and HCC1954 cells. n = 3. *P < 0.05, **P < 0.01.

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  Figure 4.. STARD10 promotes the growth and metastasis of HER2+ Breast Cancer cells in vivo. (A) Schematic diagram of the mouse xenograft tumor model. (B) Representative images of subcutaneous xenograft tumors from the Vector and STARD10-OE groups. (C, D) The volumes and weights of subcutaneous xenografts from the Vector and STARD10-OE groups. (E) Representative images of intracellular lipid droplets in tumor tissues from the Vector and STARD10-OE groups. Scale bar = 100 μm. (F) H&E staining images of tumor tissues from the Vector and STARD10-OE groups. Scale bar = 200 μm. (G) Schematic diagram of the mouse lung metastasis model. (H) Representative images of lung metastatic nodules on the surface. (I) The metastatic nodules were stained by H&E for histological analysis (left) and the number of metastatic nodules was statistically analyzed and was shown in the bar graph (right). n = 4. **P < 0.01.

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  Figure 5.. STARD10 activates the cAMP/PKA/CREB1 signaling pathway in HER2+ breast cancer. (A) Volcano plot comparing STARD10 overexpression and control SKBR3 cells. (B) KEGG pathway analysis of RNA-seq from STARD10 overexpression and control SKBR3 cells. (C) Correlation analysis between STARD10 expression and the levels of PRKAR1A, cAMP using TIMER2.0. (D) Western blot analysis of p-CREB1, CREB1, p-PKA, and PKA expression levels in STARD10-overexpressing SKBR3 and HCC1954 cells compared to control cells. n = 3. *P < 0.05, **P < 0.01. (E) Western blot analysis of p-CREB1, CREB1, p-PKA, and PKA expression levels in STARD10-knockout SKBR3 and HCC1954 cells compared to control cells. n = 3. *P < 0.05, **P < 0.01.

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  Figure 6.. The cAMP/PKA/CREB1 signaling axis mediates STARD10-driven malignant phenotypes and lipid droplets accumulation. (A) Western blot was performed to evaluate the expression of p-CREB1, CREB1, p-PKA, and PKA in SKBR3 and HCC1954 cell lines treated with or without H-89. (B–D) CCK-8, colony formation and EdU assays were performed to evaluate the proliferation capacity of SKBR3 and HCC1954 cells treated with or without H-89. Scale bar = 100 μm. (E) Transwell assays were performed to evaluate migration and invasion capabilities of SKBR3 and HCC1954 cells treated with or without H-89. Scale bar = 50 μm. (F) Representative images of intracellular lipid droplets in SKBR3 and HCC1954 cells stained with LD540 following treatment with H-89. The corresponding quantification shows the fluorescence intensity of lipid droplets. Scale bar = 100 μm. (G) Western blot was performed to evaluate the expression of FASN and DGAT1 in SKBR3 and HCC1954 cell lines treated with or without H-89. n = 3. *P < 0.05, **P < 0.01.