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

Journal: NPJ Precision Oncology

Article Title: Vertical pathway inhibition of receptor tyrosine kinases and BAD with synergistic efficacy in triple negative breast cancer

doi: 10.1038/s41698-023-00489-3

Figure Lengend Snippet: a pBADSer99 levels and BAD expression were determined using immunohistochemistry (IHC) in adjacent normal (AD) and TNBC tissue specimens. Representative IHC images of pBADSer99 in TNBC and AD tissues (up). Scale bar, 20 μm. Analysis of the pBADSer99/BAD ratio and pBADSer99 staining (%) in AD and TNBC tissue specimens (down). For pBADSer99, the immunoreactive score (IRS) 0 to 4 was categorized as negative and IRS 5 to 12 as positive. For the pBADSer99/BAD ratio, the IRS ratio higher than 0.75 was regarded as positive . Cell survival of MDA-MB-231 ( b ) and BT549 ( c ) cells after transfection with pBADS99A knock in plasmid or vector control. Data represent means ± SD ( n = 3). * P < 0.05, ** P < 0.01, and *** P < 0.001. Corresponding immunoblots displaying levels of pBADSer99 and BAD. The sizes of detected bands in kDa are shown on the left. d Transwell analysis was performed to determine the effect of pBADS99A knock in on cell migration of MDA-MB-231 and BT549 cells. The TNBC cells were transfected with pBADS99A plasmid or vector control. Scale bar, 50 μm. Data represent means ± SD ( n = 5). * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The TNBC tissue microarray (ZL-Brc3N961) was obtained from Zhuoli Biotech Co., Ltd. (Shanghai, China).

Techniques: Expressing, Immunohistochemistry, Staining, Transfection, Knock-In, Plasmid Preparation, Control, Western Blot, Migration

Journal: NPJ Precision Oncology

Article Title: Vertical pathway inhibition of receptor tyrosine kinases and BAD with synergistic efficacy in triple negative breast cancer

doi: 10.1038/s41698-023-00489-3

Figure Lengend Snippet: a Petasis reaction, a three component boronic Mannich-type reaction which utilizes boronic acids as a potential nucleophilic species, salicylaldehyde, and substituted piperazines to form the new C–C bond of the formula I compound, was utilized to synthesize NCK (C 22 H 21 Cl 2 N 3 O). b 3D surface and enlarged view of the docked compounds NPB (red) & NCK (black) with the BAD protein (dim grey). The yellow color indicates the site of the Serine 99 residue. c 2D structure representation of NCK interacting with BAD protein residues. d Sensorgrams obtained by SPR analysis of NCK with the BAD protein. BAD protein was immobilized on the surface of a CM5 sensor chip. A solution of NCK at variable concentrations (1.25–160 μM) was injected to generate the binding responses (RU) recorded as a function of time (s). The results were analyzed using BIA evaluation 4.1. e Western Blot analysis was used to assess the level of BAD phosphorylation at Ser99, Ser75 and Ser118 in TNBC cells after treatment with NCK and NPB. β-ACTIN was used as input control for cell lysate. The sizes of detected bands in kDa are shown on the left. f Dose-dependent effect of NCK and NPB in 2D and 3D culture on MDA-MB-231 and BT549 TNBC cells measured by using total cell number and AlamarBlue assay respectively ( n = 3). g Western Blot analysis was used to assess the level of BAD phosphorylation at Ser99 in TNBC cells after transfection with siRNA-BAD. β-ACTIN was used as input control for cell lysate. The sizes of detected bands in kDa are shown on the left. h CASPASE3/7 activities of MDA-MB-231 cells after transfecting with siRNA targeting BAD transcript or scrambled control and treated with 5 μM NCK were evaluated using the Biovision Caspase 3/7 DEVD Assay Kit. Data represent means ± SD ( n = 3). * P < 0.05, ** P < 0.01, and *** P < 0.001. i Cell survival of MDA-MB-231 cells after transfecting with siRNA targeting BAD transcript or scrambled control and treated with 5 μM NCK. Data represent means ± SD ( n = 3). * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The TNBC tissue microarray (ZL-Brc3N961) was obtained from Zhuoli Biotech Co., Ltd. (Shanghai, China).

Techniques: Residue, Injection, Binding Assay, Western Blot, Phospho-proteomics, Control, Alamar Blue Assay, Transfection

Journal: NPJ Precision Oncology

Article Title: Vertical pathway inhibition of receptor tyrosine kinases and BAD with synergistic efficacy in triple negative breast cancer

doi: 10.1038/s41698-023-00489-3

Figure Lengend Snippet: a The survival fraction of NCK, OSI-930 (OSI) and Crizotinib (CRI) or combination treatments were evaluated with total cell number assay ( n = 3). b CI was measured with Chou-Talalay, where CI < 1 denotes synergy, CI = 1 denotes additivity, CI > 1 denotes antagonism. Synergy score was measured with HSA and bliss synergy analysis ( www.synergyfinder.com ), where CI > 0 denotes synergy, CI < 0 denotes antagonism. c Dose-response analysis of the shift in IC 50 of OSI-930 (OSI) and Crizotinib (CRI) in TNBC cells after co-treatment with NCK (2 μM) was evaluated with total cell number assay. Fold difference was calculated. Data represent means ± SD ( n = 3).

Article Snippet: The TNBC tissue microarray (ZL-Brc3N961) was obtained from Zhuoli Biotech Co., Ltd. (Shanghai, China).

Techniques:

Journal: NPJ Precision Oncology

Article Title: Vertical pathway inhibition of receptor tyrosine kinases and BAD with synergistic efficacy in triple negative breast cancer

doi: 10.1038/s41698-023-00489-3

Figure Lengend Snippet: a Representative flow cytometry plots using Annexin V FITC/PI staining for apoptotic cell death of MDA-MB-231 and BT549 cells measured after treatment with 5 μM NCK, 5 μM OSI-930 (OSI), 5 μM Crizotinib (CRI) or combinations using flow cytometry analysis at 72 hours as described in materials and methods ( n = 3). b CASPASE 3/7 activities were evaluated in MDA-MB-231 and BT549 cells using the Biovision Caspase 3/7 DEVD Assay Kit after treatment with 5 μM NCK, 5 μM OSI-930 (OSI), 5 μM Crizotinib (CRI) or combinations. Data represent means ± SD ( n = 3). * P < 0.05, ** P < 0.01, and *** P < 0.001. c Crystal violet staining of foci in colonies generated by MDA-MB-231 cells and BT549 cells after exposure to 5 μM NCK, 5 μM OSI-930 (OSI), 5 μM Crizotinib (CRI) or combinations. d Representative images of MDA-MB-231 cells and BT549 cells cultured in 3D Matrigel after exposure to 5 μM NCK, 5 μM OSI-930 (OSI), 5 μM Crizotinib (CRI) or combinations. Scale bar, 100μm. e Western blot analysis was used to assess the level of various apoptotic proteins in TNBC cells after treatment with 1 μM NCK, 1 μM OSI-930 (OSI), 1 μM Crizotinib (CRI) or combinations. β-ACTIN was used as input control for cell lysate. The sizes of detected bands in kDa are shown on the left. f Western blot analysis was used to assess the expression/phosphorylation of various proteins of the PI3K/AKT and MAPK pathways after treatment with 1 μM NCK, 1 μM OSI-930 (OSI), 1 μM Crizotinib (CRI) or combinations. β-ACTIN was used as input control for cell lysate. The sizes of detected bands in kDa are shown on the left.

Article Snippet: The TNBC tissue microarray (ZL-Brc3N961) was obtained from Zhuoli Biotech Co., Ltd. (Shanghai, China).

Techniques: Flow Cytometry, Staining, Generated, Cell Culture, Western Blot, Control, Expressing, Phospho-proteomics

Journal: Frontiers in Immunology

Article Title: An exosome-based specific transcriptomic signature for profiling regulation patterns and modifying tumor immune microenvironment infiltration in triple-negative breast cancer

doi: 10.3389/fimmu.2023.1295558

Figure Lengend Snippet: The role of the hub gene CLDN7 in TNBC. (A, B) Random forest tree indicating the importance of exosome-related signatures. (C) Different expression levels of CLDN7 between normal and tumor tissues. (D) The impact of CLDN7 on OS and RFS in TNBC using Kaplan−Meier analysis.

Article Snippet: IHC staining of the CLDN7 protein in the TNBC tissue microarray was performed by incubation with human CLDN7 antibody (10118-1-AP, 1:400, Proteintech) overnight.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: An exosome-based specific transcriptomic signature for profiling regulation patterns and modifying tumor immune microenvironment infiltration in triple-negative breast cancer

doi: 10.3389/fimmu.2023.1295558

Figure Lengend Snippet: Tumor-promoting effects of CLDN7. (A) Western blot of TNBC cell-derived exosomes. (B) Wound healing of TNBC cells treated with exosomes. (C) Transwell assay of TNBC cells treated with exosomes. (D) RT−PCR of TNBC cells after treatment with exosomes. (E) Immunohistochemical staining of CLDN7 in TNBC tumor tissue (upper for high-expression group, lower for low-expression group) and survival analysis of different groups of TNBC patients based on IRS. *** P < 0.001, ** P < 0.01.

Article Snippet: IHC staining of the CLDN7 protein in the TNBC tissue microarray was performed by incubation with human CLDN7 antibody (10118-1-AP, 1:400, Proteintech) overnight.

Techniques: Western Blot, Derivative Assay, Transwell Assay, Reverse Transcription Polymerase Chain Reaction, Immunohistochemical staining, Staining, Expressing

Journal: Journal of Biomedical Science

Article Title: SCEL regulates switches between pro-survival and apoptosis of the TNF-α/TNFR1/NF-κB/c-FLIP axis to control lung colonization of triple negative breast cancer

doi: 10.1186/s12929-023-00986-4

Figure Lengend Snippet: SCEL expression is significantly upregulated in TNBC tumors and correlates with the poor outcome of patients with TNBC. A Analysis of the survival correlation of the candidate proteins in TCGA breast cancer dataset using UCSC Xena platform. B Analysis of mRNA expression levels of top seven candidates, NT5E, RAP1GAP2, SDPR, SCEL, LPCAT2, SMURF2, and CTGF, in TNBC tumors versus non-TNBC tumors using UCSC Xena. * P < 0.01. C The Kaplan–Meier survival analysis of BC patients and TNBC patients based on SCEL expression. The Kaplan–Meier survival curves of overall survival (OS) and progression-free survival (PFS) of breast cancer patients stratified by SCEL expression were generated by UCSC Xena using TCGA breast cancer dataset. The Kaplan–Meier survival curves of overall survival (OS) and distant metastasis-free survival (DMFS) of breast cancer patients stratified by SCEL expression were generated by KM plotter using 48 GSE datasets. The Kaplan–Meier survival curves of overall survival (OS), distant metastasis-free survival (DMFS), and relapse-free survival (RFS) of TNBC patients stratified by SCEL expression were similarly generated by KM plotter using 48 GSE datasets. D Immunohistochemistry (IHC) staining of SCEL protein expression in TNBC tissue microarrays (n = 191). Pearson Chi’s square test of the observed values and the expected values of the early/late-stage tumors with low and high SCEL expression ( Χ 2 = 22.32, P < 0.00001). Pearson Chi’s square test was similarly performed in the lower and higher-grade tumors with low and high SCEL expression ( Χ 2 = 0.38, P < 0.536). E H score of SCEL IHC staining of the early-stage and late-stage TNBC tumors. * P < 0.001. F H score of SCEL IHC staining of the lower and higher grade TNBC tumors. G H scores of SCEL IHC staining of TNBC (n = 191) and non-TNBC tumors (n = 82). * P < 0.001

Article Snippet: For the analysis of SCEL protein expression in clinical TNBC tumor samples, TNBC tissue microarrays were purchased from US Biomax, Inc. (CA, USA) (Additional file : Table S5).

Techniques: Expressing, Generated, Immunohistochemistry

Journal: Cell & Bioscience

Article Title: MiR-181a targets STING to drive PARP inhibitor resistance in BRCA - mutated triple-negative breast cancer and ovarian cancer

doi: 10.1186/s13578-023-01151-y

Figure Lengend Snippet: MiR-181a overexpression leads to Olaparib resistance and downregulates STING. A – C Quantification by RT-qPCR of miR-181a levels in miR-181a-OV and empty vector (CTRL) MDA-MB-436 ( A ), HCC1395 ( B ), and HCC1937 ( C ) cell lines (Student’s t-test). D Western blotting analysis for STING and β-actin (loading control) comparing miR-181a-OV and empty vector (CTRL) in MDA-MB-436, HCC1395, and HCC1937 cell lines. E – G Drug sensitivity assays comparing miR-181a-OV and empty vector (CTRL) in MDA-MB-436 ( E ), HCC1395 ( F ), and HCC1937 ( G ) cell lines, treated with different concentrations of olaparib (Two-way ANOVA and Sidak’s multiple comparisons test). H Western blotting analysis for STING, TBK1, cGAS, and β-actin (loading control), comparing miR-181a-OV and empty vector (CTRL) HCC1937 cell lines with or without olaparib treatment. I Western blotting analysis for STING and β-actin (loading control) comparing parental and OlaR in MDA-MB-436, HCC1395, and HCC1937 cell lines. J Western blotting analysis for STING and β-actin (loading control) comparing parental and OlaR HCC1937 cell line in resting and olaparib-treated (24 and 48 h) conditions. K Western blotting analysis for STING and β-actin (loading control) comparing parental and OlaR HCC1937 cell line in resting and cisplatin-treated (12 and 24 h) conditions. L , M Representative images of STING IHC ( L ) and miR-181a ISH ( M ) for TNBC tumor tissues in the TMA. Images of cases 1 and 2 (STING low, miR-181a high) and cases 3 and 4 (STING high, miR-181a low). N Comparison of STING protein levels in low versus high miR-181a TNBC tumor tissues in the TMA (Student’s t-test). * p < 0.05, ** p < 0.01, *** p < 0.001. Cell viability assays were performed in triplicates

Article Snippet: Furthermore, a clinically annotated tissue microarray (TMA) for TNBC (#BR1301) was obtained from US Biomax (Derwood, MD).

Techniques: Over Expression, Quantitative RT-PCR, Plasmid Preparation, Western Blot, Comparison