Journal: Cell Death & Disease

Article Title: Loss of function of BRCA1 promotes EMT in mammary tumors through activation of TGFβR2 signaling pathway

doi: 10.1038/s41419-022-04646-7

Figure Lengend Snippet: A Representative IHC analysis of mammary tumors with antibodies against Vim. Vim positive tumor cells (Black arrows) and stromal cells (Yellow arrows) are indicated. B Microarray analysis of mammary tumors. GSEA enrichment plot for a signature for Tgfβ signaling activity. NES: Normalized Enrichment Score (NES), Nominal p value (p), and False Discovery Rate q-value (FDR q) were detected comparing p18 −/− ; Brca1 +/− tumors ( n = 10) to p18 −/− and p18 +/− tumors including nine p18 −/− and one p18 +/− tumors. C Representative IHC analysis of mammary tumors from a p18 −/− and p18 −/− ; Brca1 +/− mice. To quantify the Tgfβr2 positive tumor cells, the intensity of Tgfβr2 antibody-specific staining by IHC in tumor cells were categorized into -, 1 + , 2 + , and 3 + . The representative images in the boxed area for each category were shown. D Summary of mammary tumors in mice with Balb/c background. EMT + tumors are tumors that are positive for at least two EMT markers (decreased E-Cad, increased Vim, Fn1, or CD29), and two EMT-TFs (Twist, Snail, Slug, Foxc2 or p-Fra1) in >2% tumor cells. Tgfβr2 + tumors are tumors that are positive for Tgfβr2 with 2+ or 3+ intensity in >20% tumor cells. The asterisk (*) denotes a significance from p18 −/− ; Brca1 +/− and p18 −/− tumors by two-tailed Fisher’s exact test. E Representative immunofluorescent staining of mammary tumors from a p18 −/− and p18 −/− ; Brca1 +/− mice. Note that the majority of Tgfβr2 positive p18 −/− ; Brca1 +/− tumor cells were co-stained with Ck14 (blue arrows). Ck14 singly positive basal epithelial cells (white arrows) in the normal gland are indicated.

Article Snippet: Anti-BRCA1 antibody (D-9, Santa Cruz) or control mouse IgG was used to precipitate chromatin associated with BRCA1.

Techniques: Microarray, Activity Assay, Staining, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Loss of function of BRCA1 promotes EMT in mammary tumors through activation of TGFβR2 signaling pathway

doi: 10.1038/s41419-022-04646-7

Figure Lengend Snippet: A, B Tumors (T) from p18 −/− and p18 −/− ; Brca1 MGKO mice were analyzed by qRT-PCR (A) and western blot ( B ). Tumor-free mammary glands (TF) from the same mouse were used as controls. Data in ( A ) represent the mean ± SD of three tumors in each group. The asterisk (*) denotes a significance from tumors and tumor-free tissues of the same genotype by a two-tailed, unpaired T test. C Four representative tumors from p18 −/− and p18 −/− ; Brca1 MGKO mice were analyzed by western blot. D Representative immunostaining of mammary tumors from a p18 −/− and p18 −/− ; Brca1 MGKO mice. E p18 −/− and p18 −/− ; Brca1 MGKO mammary tumors were analyzed by IHC, and H-scores for Tgfβr2 and p-Smad2 were calculated. The results represent the mean ± SD of five individual tumors per group for Tgfβr2 and four individual tumors per group for p-Smad2. F p18 −/− and p18 −/− ; Brca1 MGKO mammary tumor cells were transplanted into MFPs of NSG mice. Four weeks later, recipient mice with tumors generated (>0.5 cm 3 in size) were analyzed. # five tumor-bearing mice displayed lung metastasis by H.E. analysis. G 3 ×10 4 p18 −/− and p18 −/− ; Brca1 MGKO mammary tumor cells were cultured to generate primary tumorspheres in 10 days. The number of spheres large than 50 μm was quantified from triplicate experiments. The results represent the mean ± SD of three individual tumors per group. Statistical significance in ( E ) and ( G ) was determined by a two-tailed, unpaired T test.

Article Snippet: Anti-BRCA1 antibody (D-9, Santa Cruz) or control mouse IgG was used to precipitate chromatin associated with BRCA1.

Techniques: Quantitative RT-PCR, Western Blot, Two Tailed Test, Immunostaining, Generated, Cell Culture

Journal: Cell Death & Disease

Article Title: Loss of function of BRCA1 promotes EMT in mammary tumors through activation of TGFβR2 signaling pathway

doi: 10.1038/s41419-022-04646-7

Figure Lengend Snippet: A , B Representative p18 −/− and p18 −/− ; Brca1 MGKO mammary tumor cells were analyzed by western blot ( A ) or treated with vehicle or TGFβ for different time periods, and then analyzed by qRT-PCR ( B ). Data in ( B ) represent the mean ± SD from duplicates of two independent experiments from two different pairs of primary tumor cell lines. C , E MCF7 ( C ) and MDA-MB-231 ( E ) cells were infected with either pGIPZ-empty (sh-Ctrl.) or pGIPZ-shBRCA1 targeting different sequences of human BRCA1 (sh-BRCA1-B7 and sh-BRCA1-G6). Cells stably expressing sh-Ctrl or shBRCA1 were analyzed by qRT-PCR. Data represent the mean ± SD from triplicates of each of the two independent experiments. D , F mRNA levels in MCF7-sh-Ctrl and MCF7-sh-BRCA1-G6 ( D ), or MDA-MB-231-sh-Ctrl and MDA-MB-231-sh-BRCA1-G6 ( F ) cells treated with TGFβ for 10 ( D ) or 24 ( F ) hours were analyzed. Data represent the mean ± SD from duplicates of two independent experiments. The asterisk (*) in ( B ) ( D ) and ( F ) denotes a statistical significance from vehicle- and TGFβ-treated samples determined by a two-tailed, paired T test. The asterisk (*) in ( C ) and ( E ) denotes a statistical significance from sh-BRCA1 and sh-Ctrl samples determined by a two-tailed, paired T test.

Article Snippet: Anti-BRCA1 antibody (D-9, Santa Cruz) or control mouse IgG was used to precipitate chromatin associated with BRCA1.

Techniques: Western Blot, Quantitative RT-PCR, Infection, Stable Transfection, Expressing, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Loss of function of BRCA1 promotes EMT in mammary tumors through activation of TGFβR2 signaling pathway

doi: 10.1038/s41419-022-04646-7

Figure Lengend Snippet: A SUM149 cells were transfected with pBabe-empty (Empty) or pBabe-HA-BRCA1 (BRCA1). The expression of genes indicated was determined by western blot and/or qRT-PCR 72 h after transfection. Data represent the mean ± SD from triplicates of two independent experiments. The asterisk (*) denotes a statistical significance from empty- and BRCA1-expressing samples determined by a two-tailed, paired T test. B Diagram showing the location of putative BRCA1 binding sites (dark black bars) in the human TGFβR2 gene, and that of primers (P1 - P8) used for ChIP analysis. +1, transcription start site. C ChIP analysis of endogenous BRCA1 binding to putative BRCA1 sites on the TGFβR2 locus in T47D cells. Normal IgG was used as a negative control. The ratio of binding signal to input was compared. Data represent the mean ± SD from triplicates of two independent experiments. The asterisk (*) denotes a statistical significance from IgG and anti-BRCA1 immunoprecipitated samples by a two-tailed, unpaired T test. D ChIP analysis of exogenous BRCA1 binding to the TGFβR2 locus in HCC1937 cells transfected with pBabe-empty (Empty) or pBabe-HA-BRCA1 (BRCA1). Normal IgG was used as a negative control. The ratio of binding signal to input was compared. Data represent the mean ± SD from triplicates of two independent experiments. The asterisk (*) denotes a statistical significance from Empty- and BRCA1-expressing samples immunoprecipitated by anti-BRCA1 by a two-tailed, unpaired T test. E A schematic representation of WT and Mut TGFβR2 promoter-reporter constructs. Numbers represent a position relative to the transcription start site and a letter (X) denotes a mutated GATA3 binding site. The location of primers used for ChIP analysis is shown. F T47D-sh-Ctrl and T47D-sh-BRCA1 cells were transfected with Renilla and pGL3-TGFβR2-WT or pGL3-TGFβR2-Mut (right panel), and SUM149 cells were transfected with Renilla and pGL3-TGFβR2-WT or pGL3-TGFβR2-Mut, as well as pBabe-empty or pBabe-HA-BRCA1 (left panel), which were then collected after 48 h and assayed for luciferase activity. Data represent the mean ± SD from triplicates of two independent experiments. The asterisk (*) denotes a statistical significance from BRCA1 and empty (left panel), or sh-BRCA1 and sh-Ctrl (right panel) samples determined by a two-tailed, paired T test. SUM149, BRCA1 mutant (Mut); T47D, BRCA1 wild type (Wt).

Article Snippet: Anti-BRCA1 antibody (D-9, Santa Cruz) or control mouse IgG was used to precipitate chromatin associated with BRCA1.

Techniques: Transfection, Expressing, Western Blot, Quantitative RT-PCR, Two Tailed Test, Binding Assay, Negative Control, Immunoprecipitation, Construct, Luciferase, Activity Assay, Mutagenesis

Journal: Cell Death & Disease

Article Title: Loss of function of BRCA1 promotes EMT in mammary tumors through activation of TGFβR2 signaling pathway

doi: 10.1038/s41419-022-04646-7

Figure Lengend Snippet: A – C p18 −/− ; Brca1 MGKO tumor cells were transfected with Tgfβr2 (Tgfβr2 CRISPR) and Control (Ctrl CRISPR) Double Nickase plasmids then selected with puromycin for 3 days. Tgfβr2- and control-knockout cells were then analyzed by western blot ( A ), Q-RT-PCR (B), and microscope for cell morphology ( C ). The areas with epithelial-like cells are indicated in ( C ). D Tgfβr2- and Ctrl-knockout p18 −/− ; Brca1 MGKO mammary tumor cells were treated with vehicle or TGFβ for different time periods, and the expression of Fn was then determined. Data in ( B ) and ( D ) represent the mean ± SD from duplicates of two independent experiments. The asterisk (*) in ( B ) denotes a statistical significance from Tgfβr2- and Ctrl-knockout samples, and in ( D ) denotes a statistical significance from TGFβ treated and vehicle treated samples determined by a two-tailed, paired T test. E , F 6 ×10 5 Tgfβr2- and Ctrl-knockout p18 −/− ; Brca1 MGKO mammary tumor cells were inoculated into the left and right inguinal MFPs of NSG mice, respectively, in a pairwise manner. Two weeks after transplantation, mice were dissected. The volume ( E ) and expression of Vim ( F ) of the regenerated tumors were determined by measurement and IHC. Data in ( E ) represent the mean ± SD of four tumors in each group. The asterisks (*) denote a statistical significance from Tgfβr2- and Ctrl-knockout tumors determined by a two-tailed, paired T test. G–I 6 ×10 5 Tgfβr2- or Ctrl-knockout p18 −/− ; Brca1 MGKO mammary tumor cells were inoculated into the MFPs of NSG mice. When newly generated tumors reached the maximum size allowed by IACUC in 4–7 weeks, or the mice became moribund, lungs were examined for gross appearance ( G ), H.E. staining ( H ), and quantification of the number of metastatic nodules ( I ). M, metastatic nodules. Data in ( I ) represent the mean ± SD for the numbers of metastatic nodules detected in all lobes of the lungs in each group ( n = 4). Statistical significance was determined by a two-tailed, unpaired T test.

Article Snippet: Anti-BRCA1 antibody (D-9, Santa Cruz) or control mouse IgG was used to precipitate chromatin associated with BRCA1.

Techniques: Transfection, CRISPR, Control, Knock-Out, Western Blot, Reverse Transcription Polymerase Chain Reaction, Microscopy, Expressing, Two Tailed Test, Transplantation Assay, Generated, Staining

Journal: Cell Death & Disease

Article Title: Loss of function of BRCA1 promotes EMT in mammary tumors through activation of TGFβR2 signaling pathway

doi: 10.1038/s41419-022-04646-7

Figure Lengend Snippet: A 18 −/− ; Brca1 MGKO tumor cells treated with ITD1 at 4 μm for 24 h were analyzed by western blot. B 10 4 cells dissociated from 18 −/− ; Brca1 MGKO primary tumorspheres were treated with ITD1. Secondary spheres formed after 6 days of treatment were counted from quadruplicate experiments. Data represent the mean ± SD from two independent primary tumorspheres. C 1000 p18 −/− ; Brca1 MGKO tumorsphere-dissociated cells pretreated with DMSO or ITD1 for 6 days were transplanted into MFPs of NSG mice. Four weeks later, mice were dissected and tumor volumes were measured. Values represent the average tumor volumes ± SD of four tumors. The asterisk (*) in ( B ) and ( C ) denotes a statistical significance from ITD1 treated and DMSO treated samples determined by a two-tailed, unpaired T test. D , E Representative tumors generated by DMSO- or ITD1-pretreated p18 −/− ; Brca1 MGKO cells were analyzed by Western blot ( D ) and IHC ( E ). The H-scores for p-Smad2, Twist, and Tgfβr2 in IHC were calculated (E, right panel). The results represent the mean ± SD of four individual tumors per group. Statistical significance was determined by a two-tailed, unpaired T test.

Article Snippet: Anti-BRCA1 antibody (D-9, Santa Cruz) or control mouse IgG was used to precipitate chromatin associated with BRCA1.

Techniques: Western Blot, Two Tailed Test, Generated

Journal: Cell Death & Disease

Article Title: Loss of function of BRCA1 promotes EMT in mammary tumors through activation of TGFβR2 signaling pathway

doi: 10.1038/s41419-022-04646-7

Figure Lengend Snippet: A Correlation analysis of the expression of BRCA1 and TGFβR2 for MetaBric breast cancer patients. B Analysis of gene expression in NKI295 breast cancer patients according to tumor subtype. BL basal-like, CL claudin-low, H2 Her2-enriched, LA luminal A, LB luminal B, NBL normal breast-like. C Representative immunostaining of ER positive and negative invasive human breast cancers with antibodies against TGFβR2. D, E Summary of expression of TGFβR2 by IHC and BRCA1 by Q-RT-PCR ( D ). The expression levels of TGFβR2 were quantified by H-scores. The expression of BRCA1 was determined by Q-RT-PCR. BRCA1 mRNA levels relative to that of T47D cells were determined as we previously reported . Analysis of the expression of TGFβR2 by IHC and BRCA1 by Q-RT-PCR in ER + and ER-invasive breast cancers ( E ). Statistical significance was determined by a two-tailed, unpaired T test. F Correlation analysis of BRCA1 mRNA levels and H scores of TGFβR2 expression for breast cancer samples.

Article Snippet: Anti-BRCA1 antibody (D-9, Santa Cruz) or control mouse IgG was used to precipitate chromatin associated with BRCA1.

Techniques: Expressing, Gene Expression, Immunostaining, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Nature structural & molecular biology

Article Title: Acetylation Limits 53BP1 Association with Damaged Chromatin to Promote Homologous Recombination

doi: 10.1038/nsmb.2499

Figure Lengend Snippet: (a) Schematic of inducible DSB generation by mCherry-LacI-FokI at an integrated reporter transgene in U2OS cells (U2OS-DSB-reporter). ChIP-qPCR primer sets are shown as p1–p4. (b) Immunofluorescence (IF) was performed at 5 hours after induction of DSBs by mCherry-LacI-FokI in the U2OS-DSB-reporter cells transfected with the indicated siRNA. Scale bar, 10 μm. ( c ) ChIP-qPCR was performed with an antibody to 53BP1 or BRCA1 in the U2OS-DSB-reporter cell line with (Damage) or without (Control) induction of DSBs by mCherry-LacI-FokI. Error bars represent S.E.M. ** p<0.005. ( d ) ChIP-qPCR was performed with antibody to H4ac in the U2OS-DSB-reporter cell line after treatment with DMSO (Control) or 0.5 μM TSA. Induction of DSBs by mCherry-LacI-FokI was included in all conditions tested. Error bars represent S.E.M. ** p<0.005. ( e) ChIP-qPCR was performed with antibody to BRCA1. Cells were treated with DMSO (Control) or 0.5 μM TSA for 5 hours and DSBs were induced by mCherry-LacI-FokI. Error bars represent S.E.M. * indicates p<0.05, ** p<0.005. (f) ChIP-qPCR was performed with antibody to 53BP1 in the U2OS-DSB-reporter cells (locus p4). DSBs were induced at 48–72 hours after siRNA transfection or 5 hours after TSA (0.5 μM) administration. Error bars represent S.E.M. * indicates p<0.05.

Article Snippet: Antibodies against 53BP1 and RPA were from Novus; rabbit polyclonal anti-BRCA1 antibodies from Millipore, mouse monoclonal anti-BRCA1 antibody D9 from Santa Cruz; anti-H4ac, anti-H4K16ac, anti-γH2AX, anti-His-tag and Streptavidin-HRP from Millipore; anti-Rad51 was from Santa Cruz; anti-GFP antibody was from Roche and anti-H4K20me2 was from Active Motif; and anti-TRRAP was purchased from Cell Signaling.

Techniques: ChIP-qPCR, Immunofluorescence, Transfection, Control

Journal: Nature structural & molecular biology

Article Title: Acetylation Limits 53BP1 Association with Damaged Chromatin to Promote Homologous Recombination

doi: 10.1038/nsmb.2499

Figure Lengend Snippet: (a) TRF1-FokI colocalizes with telomeres and 53BP1 foci are observed adjacent to telomeric signals as shown by immuno-FISH using peptide nucleic acid probes against telomeres (Telomere PNA). Scale bar, 10 μm. (b) ChIP using antibodies to BRCA1 and 53BP1 at different subtelomeric regions in 293T cells with (Damage) or without (Control) expression of TRF1-FokI. Treatment with TSA is indicated. The chromosome location and distance from the telomere is indicated at each qPCR primer set. Error bars represent standard error of the mean (S.E.M). * indicates p<0.05. (c) Schematic of the subtelomeric region of 16p denoting histone H4ac pattern (UCSC genome browser) and positions of ChIP-qPCR primer sets. (d) ChIP was performed at regions of high H4ac as indicated in c in 293T cells expressing TRF1-FokI following transfection of control or TIP60 siRNA. (e) Schematic of DSB induction in transcriptionally inactive or active chromatin. Transcription is induced with doxycycline prior to DSB induction. (f) ChIP was performed in the presence or absence of doxycycline as described in e . Induction of DSBs by mCherry-LacI-FokI was included in all conditions tested following the addition of doxycycline. Error bars represent S.E.M. * indicates p<0.05.

Article Snippet: Antibodies against 53BP1 and RPA were from Novus; rabbit polyclonal anti-BRCA1 antibodies from Millipore, mouse monoclonal anti-BRCA1 antibody D9 from Santa Cruz; anti-H4ac, anti-H4K16ac, anti-γH2AX, anti-His-tag and Streptavidin-HRP from Millipore; anti-Rad51 was from Santa Cruz; anti-GFP antibody was from Roche and anti-H4K20me2 was from Active Motif; and anti-TRRAP was purchased from Cell Signaling.

Techniques: Control, Expressing, ChIP-qPCR, Transfection

Journal: Nature structural & molecular biology

Article Title: Acetylation Limits 53BP1 Association with Damaged Chromatin to Promote Homologous Recombination

doi: 10.1038/nsmb.2499

Figure Lengend Snippet: ( a ) IB with cell lysates prepared from cells treated with siRNAs as indicated. (b) Representative images of 53BP1 (upper panel) or BRCA1 (lower panel) at mCherry-LacI-FokI-induced DSBs in cells transfected with the indicated siRNA. Scale bar, 10 μm. (c), Quantification of relative mean fluorescence intensity (RMFI) from b . RMFI was calculated from 200 cells and 3 independent experiments. Error bars represent S.E.M. * indicates p<0.05, ** p<0.005. ( d ), IB with cell lysates prepared from TIP60-GFP expressing cells treated with siRNAs as indicated. (e) Representative images of BRCA1 and 53BP1 co-staining following fixation at 5 hours after 2 Gy IR treatment of U2OS cells that were transfected with the indicated siRNA. Scale bar, 10 μm. (f) Quantification of percentage of cells possessing greater than 5 BRCA1 IRIF per cell from e . Error bars represent S.E.M. ** indicates p<0.005.

Article Snippet: Antibodies against 53BP1 and RPA were from Novus; rabbit polyclonal anti-BRCA1 antibodies from Millipore, mouse monoclonal anti-BRCA1 antibody D9 from Santa Cruz; anti-H4ac, anti-H4K16ac, anti-γH2AX, anti-His-tag and Streptavidin-HRP from Millipore; anti-Rad51 was from Santa Cruz; anti-GFP antibody was from Roche and anti-H4K20me2 was from Active Motif; and anti-TRRAP was purchased from Cell Signaling.

Techniques: Transfection, Fluorescence, Expressing, Staining

Journal: Nature structural & molecular biology

Article Title: Acetylation Limits 53BP1 Association with Damaged Chromatin to Promote Homologous Recombination

doi: 10.1038/nsmb.2499

Figure Lengend Snippet: (a) Representative metaphase images from 53BP1+/+ and 53BP1−/− MEFs following knockdown of TIP60 and Olaparib treatment. Significance was calculated using Fisher’s exact test. (b) Percentage survival following Olaparib administration in U2OS cells that had been transfected with the indicated siRNAs. (c) Model depicting that TIP60 -dependent acetylation limits 53BP1 binding to H4K20Me2 when present on the same histone H4 tail. TIP60 deficiency would result in hypoacetylated H4K16 and a 53BP1-dependent block of BRCA1 DSB chromatin association.

Article Snippet: Antibodies against 53BP1 and RPA were from Novus; rabbit polyclonal anti-BRCA1 antibodies from Millipore, mouse monoclonal anti-BRCA1 antibody D9 from Santa Cruz; anti-H4ac, anti-H4K16ac, anti-γH2AX, anti-His-tag and Streptavidin-HRP from Millipore; anti-Rad51 was from Santa Cruz; anti-GFP antibody was from Roche and anti-H4K20me2 was from Active Motif; and anti-TRRAP was purchased from Cell Signaling.

Techniques: Knockdown, Transfection, Binding Assay, Blocking Assay