Journal: bioRxiv

Article Title: USP7 inhibition perturbs proteostasis and tumorigenesis in triple negative breast cancer

doi: 10.1101/2025.01.28.635372

Figure Lengend Snippet: (A) cBioportal data for breast cancer tumors shows USP7 is recurrently amplified and overexpressed in breast cancer. Red=amplification; pink=increased expression; blue=deletion; light blue=decreased expression. (B) TCGA breast cancer database shows USP7 mRNA expression is elevated across all breast cancer tumors (n=1085, red) compared to normal breast tissue (n=291, grey). (C) Analysis of DepMap data on the effect of USP7 CRISPR KO on breast cancer cell fitness/proliferation separated by pathologic breast cancer subtypes. (D) Overall survival analysis from the METABRIC breast cancer database shows high USP7 expression correlates with poorer survival (n=1904). Patients grouped into quartiles based on USP7 expression: Q1 (yellow)=lowest USP7 expression; Q4 (magenta)=highest USP7 expression. Log-rank test p=0.0031, HR=1.34. (E) Representative IHC images of core human tissues from a TNBC tissue microarray stained for USP7. (F) Heat map showing percentage of core tissues with low, medium, or high USP7 protein levels, subdivided into TNBC stage I, II, or III.

Article Snippet: Tissue microarray (TMA) slides containing 110 TNBC cases were obtained from US Biomax (BRE1201; TissueArray.com ).

Techniques: Amplification, Expressing, CRISPR, Microarray, Staining

Journal: bioRxiv

Article Title: USP7 inhibition perturbs proteostasis and tumorigenesis in triple negative breast cancer

doi: 10.1101/2025.01.28.635372

Figure Lengend Snippet: (A) Western Blot analysis showing USP7 protein expression levels in malignant breast cancer cells, including TNBC, compared to the non-malignant MCF10A mammary epithelial cell line used as control. Quantification relative to MCF10A expression shown at the bottom after β-actin normalization. β-actin was used as loading control. (B) Representative Western blot analysis of USP7 in CRISPR/Cas9-mediated USP7 knockout (USP7 KO) clones of TNBC cell lines in comparison to non-targeting vector control (Ctrl). β-actin was used as loading control. (C) Bar graph of colony counts from clonogenic assays comparing USP7 KO to Ctrl. (D) Chemical structures of USP7 inhibitors that were used for in vitro assays. (E) Representative images of colonies stained with crystal violet comparing SUM159 treated with XL177A or FT671. DMSO used as vehicle control. (F) Bar graph of colony counts from clonogenic assays comparing TNBC cells treated with XL177A or FT671. DMSO as control. (G) Representative image from scratch-wound assay comparing wound closure of SUM159 non-targeting vector control (Ctrl) and USP7 KO clone over 8 hours. White lines represent the wound edge. (H) Quantification of the distance cells migrated in scratch-wound assays for each TNBC cell line. (I) Representative images from Transwell invasion assays with cells stained with crystal violet comparing SUM159 Ctrl and USP7 KO clone. (J) Quantification of the number of cells invaded across a Transwell membrane for each of the TNBC cell lines. (K) Quantification of the distance cells migrated for TNBC cells treated with XL177A or FT671. DMSO used as vehicle control. (L) Quantification of cell invasion for TNBC cells treated with XL177A or FT671. DMSO used as vehicle control. (M) Quantification of the distance cells migrated normalized to DMSO for MDA-MB-231 Ctrl and USP7 KO. (N) Quantification of cell invasion normalized to DMSO for MDA-MB-231 Ctrl and USP7 KO. For all bar graphs, values were normalized to control. Each bar is shown as mean ± SD, with each dot representing an independent replicate, n= 3. For all graphs: ns=not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by unpaired two-tailed t test.

Article Snippet: Tissue microarray (TMA) slides containing 110 TNBC cases were obtained from US Biomax (BRE1201; TissueArray.com ).

Techniques: Western Blot, Expressing, Control, CRISPR, Knock-Out, Clone Assay, Comparison, Plasmid Preparation, In Vitro, Staining, Scratch Wound Assay Assay, Membrane, Two Tailed Test

Journal: bioRxiv

Article Title: USP7 inhibition perturbs proteostasis and tumorigenesis in triple negative breast cancer

doi: 10.1101/2025.01.28.635372

Figure Lengend Snippet: (A) Schematic describing the quantitative proteomics workflow for the USP7 inhibitor (USP7i) analysis. SUM149, SUM159, and MDA-MB-231 TNBC cell lines were grown and treated with DMSO, 1 μM FT671, or 1 μM XL177A for 8 h. Lysates are digested using trypsin and LysC peptides are TMT labeled, mixed, and fractionated. Peptides are analyzed by mass spectrometry (MS) and protein abundances determined. (B) Volcano plot showing the log2 fold-change (x axis) and -log10 p-value (y axis) of all proteins identified by MS when comparing samples from USP7i-treated (FT671 or XL1774A) cells to samples from DMSO-treated cells. Each dot represents an individual protein. (C) Plot showing the log2 fold-change ratio of all proteins identified by MS when comparing samples from FT671-treated to DMSO-treated cells (x axis) vs the log2 fold-change ratio of all proteins identified by MS when comparing samples from XL177A-treated to DMSO-treated cells (y axis).

Article Snippet: Tissue microarray (TMA) slides containing 110 TNBC cases were obtained from US Biomax (BRE1201; TissueArray.com ).

Techniques: Labeling, Mass Spectrometry