Journal: American Journal of Cancer Research

Article Title: Novel mutations in BRCA2 intron 11 and overexpression of COX-2 and BIRC3 mediate cellular resistance to PARP inhibitors

doi:

Figure Lengend Snippet: BRCA2-deficient Capan-1 cells develop drug resistance to PARPi, independent of PARP activation and PARP1-DNA trapping. (A) Schematic representation of the generation of PARPi-resistant cells. BRCA2-deficient pancreatic cancer Capan-1 cells were separately exposed to increasing concentrations of simmiparib (SP), olaparib (OP) and talazoparib (TP), and the resistant monoclones were denoted Capan-1/SP (/SP), Capan-1/OP (/OP), and Capan-1/TP (/TP), respectively. (B and C) Cells were treated with simmiparib (B) or olaparib (C) for 7 days and subjected to SRB assays. (D) Cross-resistance of PARPi-resistant variants. Cells were exposed to olaparib (OP), talazoparib (TP), simmiparib (SP), niraparib (NP) or rucaparib (RP) for 7 days and assessed by SRB assays. The resistance factor was calculated as the ratio of the averaged IC50 value of the indicated PARPi in the given resistant cells to that of the same PARPi in the parental Capan-1 cells. (E and F) Protein levels of the drug transporters P-gp, BCRP (E) and PARP1, PARP2, PARP3 (F) detected by Western blotting in PARPi-resistant variants and parental Capan-1 cells. A stable vincristine (VCR)- resistant variant (KB/VCR) was a positive control of P-gp. (G and H) Cell lysates were analyzed by Western blotting to determine PAR formation (G) and chromatin-bound PARP1 (H) triggered by MMS. Histone 3 was used as the positive control for the chromatin-bound fractions. TP, talazoparib. MMS, methyl methanesulfonate. (I and J) Simmiparib reduced H2O2-triggered PAR formation in both Capan-1/SP and Capan-1 cells. (I) Representative images of PAR formation. Nuclei were stained with DAPI. Scale bar, 100 μm. (J) Inhibitory curves and IC50 values of PAR formation inhibited by simmiparib in Capan-1/SP and Capan-1 cells. Data are presented as the mean ± SD from three independent experiments.

Article Snippet: Antibodies against KU80 (#2753), NF-κB p65 (#4764), SMC1 (#4802), ATR (#13934), p-Chk1-Ser345 (#2348), Caspase 3 (#9662), Caspase 9 (#95022), COX-2 (#12282), BIRC3 (#3130), and C-BRCA2 (#10741) were from Cell Signaling Technology (Danvers, MA).

Techniques: Activation Assay, Western Blot, Variant Assay, Positive Control, Staining

Journal: American Journal of Cancer Research

Article Title: Novel mutations in BRCA2 intron 11 and overexpression of COX-2 and BIRC3 mediate cellular resistance to PARP inhibitors

doi:

Figure Lengend Snippet: PARPi-resistant variants harbor secondary mutations in intron 11 of BRCA2. (A) Cell lysates from indicated cells were immunoblotted with anti-BRCA2 antibodies recognizing N-terminal BRCA2 (residues 450-500 (Ab-1)) or C-terminal BRCA2 (residues 3319-3418 (Ab-2)) epitopes. FL, full length. BxPC3 and SKOV-3 cells are shown as a positive control of full length BRCA2. (B) Representative validation of NGS data by Sanger sequencing and two BRCA2 transcripts in Capan-1/TP cells. In genomic DNA (upper), a homozygous exon mutation (c.6174delT) and a heterozygous intron mutation (c.6841+1G>A) were identified. In complimentary DNA (cDNA, lower), a homozygous mutation (6174delT) in exon11 and a homozygous 4013-bp deletion from exon 11 to exon 22 were identified. P = 0.5 means that mutant allele frequencies are 0.5. (C) Schematic presentation of BRCA2 full-length protein and new isoforms. BRCA2 isoforms were predicted from the cDNA and genomic sequences. Open boxes represent the cDNA of BRCA2. The filled boxes represent the BRC Repeats (1-8) region in the cDNA of BRCA2. And dotted lines represent missing regions predicted based on sequencing results. (D) Knockdown of BRCA2. Cells were transfected with siRNA, and BRCA2 expression was evaluated by Western blotting. Upper panel: regions that the BRCA2 antibodies (Ab-1 and Ab-2) recognize and the siRNA (siN-BRCA2 and siM-BRCA2) targets are depicted. (E, F) Olaparib sensitivity assessed by the SRB assay after cells were treated with the indicated siRNAs. Cells were transfected with siRNA specific to C-terminus of BRCA2, and knockdown of BRCA2 isoform by siC-BRCA2 was confirmed by western blotting (F). Depletion of BRCA2 with siM-BRCA2, siN-BRCA2 (E) and siC-BRCA2 (F) sensitized cells to Olaparib. Data are presented as the mean ± SD from three independent experiments. **P<0.01; ***P<0.001.

Article Snippet: Antibodies against KU80 (#2753), NF-κB p65 (#4764), SMC1 (#4802), ATR (#13934), p-Chk1-Ser345 (#2348), Caspase 3 (#9662), Caspase 9 (#95022), COX-2 (#12282), BIRC3 (#3130), and C-BRCA2 (#10741) were from Cell Signaling Technology (Danvers, MA).

Techniques: Positive Control, Biomarker Discovery, Sequencing, Mutagenesis, Genomic Sequencing, Knockdown, Transfection, Expressing, Western Blot, Sulforhodamine B Assay

Journal: American Journal of Cancer Research

Article Title: Novel mutations in BRCA2 intron 11 and overexpression of COX-2 and BIRC3 mediate cellular resistance to PARP inhibitors

doi:

Figure Lengend Snippet: PARPi or PARP1 depletion induces upregulation of COX-2/BIRC3 expression. (A) Capan-1 cells were treated with olaparib (OP), simmiparib (SP) and talazoparib (TP) for 48 h, and then relative mRNA levels of COX-2 and BIRC3 were determined by qPCR. (B) Protein levels of COX-2 and BIRC3 were detected by western blotting after 72 h olaparib (OP) and talazoparib (TP) treatment in Capan-1 cells. (C and D) PARP1 was stably knocked down by CRISPR/Cas9 or TALEN in Capan-1 or RD-ES cells [31], and the mRNA levels (C) and protein levels (D) of COX-2 and BIRC3 were determined by qPCR and Western blotting. (E and F) PARPi-resistant variants of RD-ES or U251 [29], and the mRNA levels (E) and protein levels (F) of COX-2 and BIRC3 were determined by qPCR and Western blotting. Data are presented as the mean ± SD from three independent experiments. *P<0.05; **P<0.01; ***P<0.001. (G) Schematic model to show formation of PARPi resistance in pancreatic cancer Capan-1 cells. When chronically treated with increasing concentration of PARPi, cancer cells develop two distinct pathways to overcome PARPi cytotoxicity. Under selective pressure of PARPi exposure, BRCA2-mutated cancer cells acquired new mutations in intron 11 of BRCA2, which restored expression of the functional novel BRCA2 protein. Meanwhile, PARPi treatment significantly increased anti-apoptotic gene (COX-2 and BIRC3) expression by inhibiting PARP-1, thereby suppressing apoptosis by COX-2/PGE2/BIRC3 and promoting cell survival under DSBs. As a result, cells with secondary mutations and apoptotic resistance were significantly resistant to PARPi.

Article Snippet: Antibodies against KU80 (#2753), NF-κB p65 (#4764), SMC1 (#4802), ATR (#13934), p-Chk1-Ser345 (#2348), Caspase 3 (#9662), Caspase 9 (#95022), COX-2 (#12282), BIRC3 (#3130), and C-BRCA2 (#10741) were from Cell Signaling Technology (Danvers, MA).

Techniques: Expressing, Western Blot, Stable Transfection, CRISPR, Concentration Assay, Functional Assay