Article22 May 2018Open Access Source DataTransparent process FAM35A associates with REV7 and modulates DNA damage responses of normal and BRCA1-defective cells Junya Tomida Corresponding Author Junya Tomida [email protected] orcid.org/0000-0002-0813-5757 Department of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX, USA Search for more papers by this author Kei-ichi Takata Kei-ichi Takata Department of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX, USA Search for more papers by this author Sarita Bhetawal Sarita Bhetawal Department of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX, USA Search for more papers by this author Maria D Person Maria D Person Proteomics Facility, University of Texas at Austin, Austin, TX, USA Search for more papers by this author Hsueh-Ping Chao Hsueh-Ping Chao Department of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX, USA Search for more papers by this author Dean G Tang Dean G Tang Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY, USA Search for more papers by this author Richard D Wood Corresponding Author Richard D Wood [email protected] orcid.org/0000-0002-9495-6892 Department of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX, USA Search for more papers by this author Junya Tomida Corresponding Author Junya Tomida [email protected] orcid.org/0000-0002-0813-5757 Department of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX, USA Search for more papers by this author Kei-ichi Takata Kei-ichi Takata Department of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX, USA Search for more papers by this author Sarita Bhetawal Sarita Bhetawal Department of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX, USA Search for more papers by this author Maria D Person Maria D Person Proteomics Facility, University of Texas at Austin, Austin, TX, USA Search for more papers by this author Hsueh-Ping Chao Hsueh-Ping Chao Department of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX, USA Search for more papers by this author Dean G Tang Dean G Tang Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY, USA Search for more papers by this author Richard D Wood Corresponding Author Richard D Wood [email protected] orcid.org/0000-0002-9495-6892 Department of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX, USA Search for more papers by this author Author Information Junya Tomida *,1, Kei-ichi Takata1, Sarita Bhetawal1, Maria D Person2, Hsueh-Ping Chao1, Dean G Tang3 and Richard D Wood *,1 1Department of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX, USA 2Proteomics Facility, University of Texas at Austin, Austin, TX, USA 3Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY, USA *Corresponding author. Tel: +1 512 237 6433; E-mail: [email protected] *Corresponding author. Tel: +1 512 237 9431; E-mail: [email protected] The EMBO Journal (2018)37:e99543https://doi.org/10.15252/embj.201899543 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract To exploit vulnerabilities of tumors, it is urgent to identify associated defects in genome maintenance. One unsolved problem is the mechanism of regulation of DNA double-strand break repair by REV7 in complex with 53BP1 and RIF1, and its influence on repair pathway choice between homologous recombination and non-homologous end-joining. We searched for REV7-associated factors in human cells and found FAM35A, a previously unstudied protein with an unstructured N-terminal region and a C-terminal region harboring three OB-fold domains similar to single-stranded DNA-binding protein RPA, as novel interactor of REV7/RIF1/53BP1. FAM35A re-localized in damaged cell nuclei, and its knockdown caused sensitivity to DNA-damaging agents. In a BRCA1-mutant cell line, however, depletion of FAM35A increased resistance to camptothecin, suggesting that FAM35A participates in processing of DNA ends to allow more efficient DNA repair. We found FAM35A absent in one widely used BRCA1-mutant cancer cell line (HCC1937) with anomalous resistance to PARP inhibitors. A survey of FAM35A alterations revealed that the gene is altered at the highest frequency in prostate cancers (up to 13%) and significantly less expressed in metastatic cases, revealing promise for FAM35A as a therapeutically relevant cancer marker. Synopsis Suppression of DNA double strand break resection favors non-homologous end-joining over homologous recombination repair, and is mediated by 53BP1-RIF1-REV7 factors. Identification of the OB-fold protein FAM35A as additionally required REV7 interactor suggests a possible link to single-stranded DNA recognition in this process. The previously uncharacterized protein FAM35A interacts with REV7, 53BP1 and RIF1. FAM35A contains OB-fold domains that may mediate binding to single-stranded DNA. FAM35A depletion sensitizes cells to DNA-damaging agents. FAM35A loss suppresses non-homologous end joining and enhances homologous recombination markers. FAM35A is frequently deleted in prostate cancers and absent in a BRCA1-mutant cancer cell line with anomalous resistance to PARP inhibitors. Introduction REV7 is a multifunctional protein encoded by the MAD2L2 gene in human cells. REV7 acts as an interaction module in several cellular pathways. One of its functions is as a component of DNA polymerase ζ, where it serves as bridge between the Pol ζ catalytic subunit REV3L and the REV1 protein. A dimer of REV7 binds to two adjacent sites in REV3L by grasping a peptide of REV3L with a "safety-belt" loop (Hara et al, 2010; Tomida et al, 2015). REV7 protein is an order of magnitude more abundant than REV3L (Tomida et al, 2015) and has additional functions and protein partners including chromatin-associated and post-translational modification proteins (Medendorp et al, 2009; Vermeulen et al, 2010; Itoh et al, 2011; Listovsky & Sale, 2013; Pirouz et al, 2013; Hara et al, 2017). Complete ablation of REV7 gives rise to mice with defects in primary germ cells (Pirouz et al, 2013; Watanabe et al, 2013). Recently, studies uncovered a function of REV7 as a DNA resection inhibitor, limiting genomic repair by an unknown mechanism (Boersma et al, 2015; Xu et al, 2015). Although BRCA1-mutant cells are defective in homologous recombination, these studies found that one mode to partially restore recombination activity is by inactivation of REV7. It was proposed that REV7, together with 53BP1 and RIF1, inhibits 5′ DNA end resection to promote non-homologous end-joining at the expense of homologous recombination. To investigate novel functions and pathways involving REV7, we identified proteins associated with REV7 in vivo. We report here an analysis of a previously uncharacterized REV7-interacting protein, FAM35A. We discovered that FAM35A is a novel factor that modulates the DNA damage sensitivity of normal and BRCA1-defective cells. Our analysis reveals that the C-terminal half of FAM35A contains three OB-fold domains similar to those in the single-stranded DNA-binding protein RPA large subunit. FAM35A has a disordered N-terminal portion, containing sites of DNA damage-dependent post-translational modification. Moreover, the FAM35A gene is deleted at an unusually high rate in prostate cancers, and in cells from at least one well-studied BRCA1-defective breast cancer case. FAM35A is more weakly expressed in metastatic prostate cancers, suggesting it as an important marker for outcome and therapeutic decisions. Results and Discussion FAM35A interacts with REV7, 53BP1, and RIF1 in vivo To isolate proteins associated with REV7, we engineered HeLa S3 cells that stably express REV7 with a C-terminal FLAG–HA epitope tag (REV7-FH). REV7-FH was sequentially immunoprecipitated from nuclear extract using FLAG and HA antibody beads (Ikura et al, 2007). This purified complex was separated by gradient gel electrophoresis, and associated proteins from gel sections were identified by LC-MS/MS. We confirmed association with previously identified REV7-binding proteins including GLP (Nakatani & Ogryzko, 2003; Pirouz et al, 2013), G9A (Pirouz et al, 2013), CAMP (Itoh et al, 2011), GTF2I (Fattah et al, 2014), POGZ (Vermeulen et al, 2010), and HP1α (Vermeulen et al, 2010) (Fig 1A and Table 1). The highest-ranking previously unstudied association was with the uncharacterized FAM35A protein (Fig 1A). Figure 1. Identification of REV7- or FAM35A-associated proteinsProtein complexes were sequentially immunoprecipitated (using FLAG and HA antibody beads) from nuclear extracts of HeLa S3 cell lines stably expressing C-terminally FLAG–HA-tagged REV7 (REV7-FH) or (N-terminally FLAG–HA-tagged-FAM35A (FH-FAM35A). A, B. REV7 complex (A) and FAM35A complex (B); associated proteins were identified by mass spectrometry. The FH-FAM35A complex was purified from HeLa S3 nuclear extracts after 18 h exposure to MMC (100 ng/ml). Proteins labeled in blue are previously published REV7-binding partners. Proteins labeled in red are involved in end-joining pathways of DSB repair. 4–20% gradient gels were stained with SYPRO Ruby. C. FH-FAM35A was co-transfected into human 293T cells with GFP empty vector (control), GFP-53BP1 or GFP-RIF1. Forty-eight hours after transfection, cell lysates were made and used for immunoprecipitation with GFP antibody beads. After electrophoretic transfer of proteins, the membrane was cut into three sections to separate proteins > 250 kDa (GFP as GFP-53BP1 and GFP-RIF1), 37–250 kDa (α-tubulin), and < 37 kDa (GFP as control) and immunoblotted with the indicated antibodies. Results for the input and immunoprecipitation (IP) product after gel electrophoresis are shown. The asterisk (*) in the IP lane marks degraded or truncated forms of 53BP1 and RIF1. Source data are available online for this figure. Source Data for Figure 1 [embj201899543-sup-0002-SDataFig1.tif] Download figure Download PowerPoint Table 1. Identification of FAM35A with previously reported proteins in a REV7-associated complex Protein Accession number (UniProtKB) Molecular weight Spectral counts Unique peptides REV7 Q9UI95|MD2L2_HUMAN 24 kDa 453 15 FAM35A Q86V20-2|FA35A_HUMAN 92 kDa 74 23 GLP Q9H9B1|EHMT1_HUMAN 141 kDa 9 6 G9A Q96KQ7|EHMT2_HUMAN 135 kDa 7 5 CAMP Q96JM3|ZN828_HUMAN 89 kDa 127 29 GTF2I B4DH52|B4DH52_HUMAN 112 kDa 1780 88 POGZ Q7Z3K3| POGZ_HUMAN 154 kDa 147 31 HP1α P45973|CBX5_HUMAN 22 kDa 8 3 The immunoprecipitated sample was separated on a denaturing polyacrylamide gel, and proteins from gel sections of approximately equal size were identified by LC-MS/MS. FAM35A was a top specific hit, together with previously identified REV7-associated proteins. GTF2I (TFII-I) is likely a non-significant association as it is an abundant protein frequently found in control experiments with agarose supports and Flag-His tags (www.crapome.org), but it is included here for reference, as it was previously reported to interact with REV7 (Fattah et al, 2014). To validate the association, a reciprocal experiment was performed by constructing a HeLa S3 cell line stably expressing FAM35A with an N-terminal FLAG–HA tag (FH-FAM35A). Cells were exposed to mitomycin C (MMC, 100 ng/ml) for 18 h or mock-exposed. Following sequential immunoprecipitation with FLAG and HA antibody beads, proteins were separated and identified by mass spectrometry (Fig 1B). DNA repair proteins associating with FAM35A included REV7, RIF1, BLM, and TOP3A (Table 2), with relatively more RIF1 peptides and 53BP1 identified following MMC exposure (Table 3). Table 2. DNA repair proteins identified in the FAM35A complex Protein Accession number (UniProtKB) Molecular weight Spectral counts Unique peptides FAM35A Q86V20-2|FA35A_HUMAN 92 kDa 170 37 REV7 Q9UI95|MD2L2_HUMAN 24 kDa 5 2 RIF1 Q5UIP0-2|RIF1_HUMAN 272 kDa 6 4 BLM H0YNU5|H0YNU5_HUMAN 144 kDa 6 4 TOP3A Q13472|TOP3A_HUMAN 112 kDa 5 3 Proteins immunoprecipitating with exogenously expressed FAM35A were separated on a denaturing polyacrylamide gel, and proteins from gel sections of approximately equal size were identified by LC-MS/MS. Table shows the significant DNA repair-related proteins that were detected. Table 3. DNA repair proteins identified in the FAM35A complex after MMC exposure Protein Accession number (UniProtKB) Molecular weight Spectral counts Unique peptides FAM35A Q86V20-2|FA35A_HUMAN 92 kDa 623 69 RIF1 Q5UIP0-2|RIF1_HUMAN 272 kDa 315 112 REV7 Q9UI95|MD2L2_HUMAN 24 kDa 21 4 Ku80 P13010|XRCC5_HUMAN 83 kDa 6 4 BLM H0YNU5|H0YNU5_HUMAN 144 kDa 6 4 53BP1 Q12888-2|TP53B_HUMAN 214 kDa 3 3 Ku70 P12956|XRCC6_HUMAN 70 kDa 11 7 Proteins from gel sections were identified by LC-MS/MS. The gel sections were approximately equal size with the exception of a wider segment for the FAM35A bait. The top associated hits were RIF1 and REV7. All other known DNA repair-related proteins that were detected are shown. Ku70 and Ku80 may be non-significant associations as they are found frequently in control experiments with agarose supports and Flag-His tags (www.crapome.org), but they are included here as they were not detected with significance in the complex from non-damaged cells (Table 2). Although REV7 is known to cooperate functionally with 53BP1 to limit resection at DNA breaks, REV7 was not detected in 53BP1 immunocomplexes, and it is unknown how REV7 connects with 53BP1 in vivo (Xu et al, 2015). To verify an association of FAM35A with 53BP1 and the additional DNA end resection control factor RIF1, FH-FAM35A was co-transfected with GFP-RIF1 or GFP-53BP1 and expressed in 293T cells. All proteins were expressed at the predicted molecular weights (Fig 1C, "input" lanes). Following immunoprecipitation with GFP antibody beads, FAM35A co-immunoprecipitated with recombinant RIF1 or 53BP1 (but not with the control vector, Fig 1C). These interactions suggest that FAM35A may functionally bridge 53BP1 and REV7 in human cells, directly or via interactions with other proteins. FAM35A is an OB-fold protein that changes localization following DNA damage We found FAM35A orthologs are present in vertebrate genomes, but not in invertebrates or plants. Multiple protein isoforms arising from alternative splicing are annotated in genomics databases for human (UniProt accession number Q86V20) and mouse FAM35A. Isoforms 1 and 2 are the most common, encoding 904 and 835 amino acid proteins, respectively. They arise by differential splicing of one in-frame exon (Fig 2A). Both mRNA isoforms of FAM35A are ubiquitously expressed in different cell and tissue types (www.gtexportal.org). Figure 2. FAM35A is an OB-fold protein with an N-terminal disordered region Domain schematic of human FAM35A derived from sequence prediction modeling. An N-terminal disordered region includes post-translational modification sites. Locations of the three OB-fold domains A, B, and C are shown, with a Zn-ribbon containing conserved Cys residues. One exon is absent in isoform 2 compared to isoform 1, deleting 69 aa from OB domain B. Multi-species alignment of a segment of FAM35A protein in the predicted Zn-ribbon. The four Zn-coordinating Cys residues (CxxC, CxxC), homologous to those in human RPA1, are evolutionarily conserved. Download figure Download PowerPoint BLAST searches for sequence homologs did not reveal significant primary sequence similarity to gene products other than FAM35A. We therefore analyzed the FAM35A protein sequence using structure prediction servers based on PSI-BLAST. The N-terminal half of the protein is predicted to be disordered up until about residue 420 (Fig 2A), and this region is likely to interact with other proteins, as found commonly for disordered regions of polypeptides (Receveur-Brechot et al, 2006). The N-terminal region contains previously identified post-translational modification sites, including a conserved ubiquitin modification (Kim et al, 2011) and a conserved SQ site (Matsuoka et al, 2007) in which residue S339 is phosphorylated after exposure to ionizing radiation or UV radiation (Matsuoka et al, 2007). With high significance, the C-terminal portion of FAM35A is predicted to contain three OB-fold domains structurally homologous to those in the 70 kDa subunit (RPA1) of the single-stranded DNA-binding protein RPA (Figs 2A and EV1). The three OB folds are similar to DNA-binding domain folds A, B, and C of RPA (Bochkareva et al, 2002; Fan & Pavletich, 2012). OB-fold domain C is predicted to include four conserved cysteine residues (Fig 2B) at the core of a zinc-binding ribbon, homologous to a loop in the same position in RPA (Fig EV2). Together, the OB folds and Zn-ribbon form the elements of DNA-binding and orientation-enabling RPA1 to simultaneously bind to protein partners and to single-stranded DNA in an 8–10 nt binding mode (Lin et al, 1998; Bochkareva et al, 2002; Arunkumar et al, 2003). The region including the 4-Cys Zn-ribbon identifies the domain of unknown function (PF15793) that is conserved in FAM35A homologs as annotated by the Pfam database. C-terminal helices are predicted present following OB domains A and B, in positions corresponding to helices involved in multimerization of the OB folds in RPA subunits (Bochkareva et al, 2002; Fan & Pavletich, 2012; Fig EV2). Click here to expand this figure. Figure EV1. Partial model of the OB-fold domains of FAM35AA view of a model generated by Phyre2 is shown for FAM35A isoform 1 residues 402–904, using template 4GOP (chain C, the RPA 70 kDa subunit from Ustilago maydis). The model includes OB folds with five-stranded beta barrels for OB domain A and OB domain B. The first three beta strands of OB domain C are modeled, with the Zn-ribbon that occurs in a loop between OB fold C beta strands 1 and 2 (Fig EV2). The Phyre2 coordinates were rendered in PyMOL using spectrum coloring. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Secondary structure element assignment for the ordered region of human FAM35AThe secondary structure assignment is derived from the Phyre2 model of human FAM35A isoform 1 (Fig EV1) and largely coincides with features in the three functional DNA-binding domains (dbdA, B, and C) of RPA1 (PDB: 4GOP, 1FGU), with a few differences. In FAM35A, a C-terminal helix is predicted to be present at the end of both OB folds A and B, unlike in RPA1 where this helix only follows OB domain B. Beta strands 4 and 5 of OB domain C were not modeled by Phyre2 but candidate beta strands are present in appropriate positions, as noted. The exon that is absent in FAM35A isoform 2 results in 69 fewer residues (red box), deleting structural elements of OB domain B. Download figure Download PowerPoint We constructed a human U2OS cell line stably expressing GFP-FAM35A. Cells were exposed to MMC (100 ng/ml, 24 h) or mock-exposed and then fixed and stained with DAPI and anti-GFP. In cells exposed to DNA-damaging agent, GFP-FAM35A was concentrated into foci in the nucleus (Fig 3A), suggesting a direct involvement in DNA repair. Figure 3. FAM35A is a DNA damage response gene A. GFP-FAM35A forms nuclear foci upon DNA damage. A U2OS cell line stably expressing GFP-FAM35A was exposed to MMC (100 ng/ml, 24 h) (right) or to mock treatment (left). The following day, cells were fixed and stained for DAPI and anti-GFP. Scale bars: 6 μm. B. Two pseudogenes (FAM35DP and FAM35BP) with > 98% identity to FAM35A are located on chromosome 10q22. Both FAM35DP and FAM35BP are present in genomes of apes and old-world monkeys, but not in other mammalian genomes. By inference, these pseudogenes arose by whole gene duplication in the common ancestor of the catarrhines about 25–30 million years ago. A third pseudogene (not shown) is FAM35CP, an inactive spliced product of reverse transcription (> 95% identity) that was integrated into an intron of the galactosylceramidase (GALC) gene on chromosome 14q31.3. FAM35CP is present in apes but not old-world monkeys, indicating a more recent evolutionary origin. C. Acute depletion of FAM35A causes hypersensitivity to several DNA-damaging agents but not to olaparib. The survival of HEK293 cells, FAM35A acutely depleted and control, was monitored following exposure to MMC, etoposide, and olaparib. siControl (circle symbol, green line). siFAM35A (square symbol, blue line). siFANCD2 (triangle symbol, red line). siFANCA (triangle symbol, black line). siRNA-treated cells were plated and exposed to indicate dose of agent for 48 h. Cellular viability was measured 48 h later. Data represent mean ± SEM. n = 3. *P < 0.05 and **P < 0.01 by unpaired t-test. D–F. Quantification analysis of > 5 nuclear γH2AX foci (D), > 5 FANCD2 foci (E), and > 5 RAD51 foci (F) in HEK293 cells acutely depleted for FAM35A. Cells with > 5 foci/nucleus were counted after MMC (100 ng/ml, 24 h) or control treatment. Data shown are means ± SE of more than 250 nuclei from two independent experiments. ns: not significant, **P < 0.01 and ***P < 0.001 by unpaired t-test. Source data are available online for this figure. Source Data for Figure 3 [embj201899543-sup-0003-SDataFig3.tif] Download figure Download PowerPoint FAM35A depletion sensitizes cell lines to DNA damage The human FAM35A gene is located on chromosome 10q23.2. Three pseudogenes are also present in the human genome, two of them on 10q22 (Fig 3B) with high (> 98%) sequence identity to FAM35A. Precise nuclease-mediated knockout of human FAM35A is therefore challenging, as simultaneous targeting of pseudogenes would likely cause chromosome rearrangements and deletion. siRNA was used to acutely deplete FAM35A from human HEK293 cells and investigate its role in DNA repair. FAM35A-depleted HEK293 cultures were hypersensitive to MMC and etoposide, with sensitivity comparable to that conferred by depletion of Fanconi anemia FANCA and FANCD2 gene expression (Fig 3C). In HEK293 cells, FAM35A-siRNA did not sensitize to the PARP inhibitor olaparib (Fig 3C), suggesting that homologous recombination repair is still active in the absence of FAM35A, as it is in cells with suppressed REV7 activity (Boersma et al, 2015; Xu et al, 2015). We investigated the impact of acute depletion of FAM35A on other markers of DNA damage responses in HEK293 cells. Cells with > 5 γH2AX foci, > 5 FANCD2 foci, or > 5 RAD51 foci were quantified after MMC exposure (100 ng/ml, 24 h) and in control cells. MMC exposure induced cellular γH2AX foci as expected; there was no significant change in this pattern in cells depleted for FAM35A, indicating intact signaling leading to γH2AX formation (Fig 3D). The Fanconi anemia signaling pathway (FANCD2 foci) was also functional in FAM35A-depleted cells, showing about 1.5-fold more foci after MMC exposure (Fig 3E). RAD51 foci, a readout of homologous recombination pairing, were formed in FAM35A-defective cells, with a twofold elevated frequency in non-damaged cells (Fig 3F). Depletion of REV7 reduces the efficiency of non-homologous end-joining (NHEJ; Boersma et al, 2015; Xu et al, 2015) by promoting resection that channels repair of DNA double-strand breaks into homologous recombination and other pathways. Because of the association between FAM35A and the resection control factors REV7, RIF1, and 53BP1 (Fig 1A–C), we investigated whether FAM35A depletion affects end joining, using a plasmid integration assay (Boersma et al, 2015). REV7, 53BP1, and RIF1 depletion decreased integration ratio in this assay. We confirmed efficient knockdown of FAM35A mRNA and protein in 293T cells using qPCR (Fig 4A) and immunoblot analysis (Fig 4B). The plasmid integration ratio decreased significantly after FAM35A depletion (Fig 4C), suggesting that FAM35A is involved in modulating double-strand break repair pathway choice. Expression of FAM35A isoform 1 in 293T cells (Fig 4D) restored NHEJ to normal levels (Fig 4E). This is consistent with increased resection in the absence of FAM35A, causing NHEJ to be less effective, which may account for the increased sensitivity of FAM35A-depleted cells to MMC and etoposide. Figure 4. Suppression of FAM35A reduces random DNA integration by non-homologous end-joining A, B. Depletion of endogenous FAM35A mRNA (A) and protein (B) from 293T cell lines carrying shRNA to FAM35A. Cell lysates were made from 293T cell lines stably depleted for FAM35A and controls. After electrophoretic transfer of proteins, the membrane was immunoblotted with anti-FAM35A or anti-α-tubulin. C. Plasmid integration assay. pDsRed-Monomer plasmid with antibiotic resistance (Hygromycin B) was linearized by restriction enzyme. The linearized plasmid was transfected into stably FAM35A-depleted 293T cell lines and control. Colonies were counted after antibiotic selection (Hygromycin B 300 μg/ml). D. Expression of FAM35A isoform 1 (mouse) in human 293T cells with stable suppression of human FAM35A. The immunoblot of cell lysates used an anti-HA antibody to recognize epitope-tagged mouse FAM35A. E. Plasmid integration frequency is restored by expression of FAM35A in cells. Data information: Data represent mean ± SEM. n = 4. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 by unpaired t-test. Source data are available online for this figure. Source Data for Figure 4 [embj201899543-sup-0004-SDataFig4.tif] Download figure Download PowerPoint FAM35A deficiency in BRCA1-deficient cells and resistance to camptothecin and PARP inhibitors In BRCA1-mutant cells, REV7 depletion restores homologous recombination (HR) by restoring 5′ end resection (Boersma et al, 2015; Xu et al, 2015). We therefore hypothesized that FAM35A depletion from BRCA1-mutant cells would increase resistance to a DNA strand-breaking agent. We engineered a BRCA1-mutant cell line (MDA-MB-436) expressing FAM35A shRNA. Efficient knockdown was verified by qPCR (Fig 5A). The FAM35A-depleted BRCA1-mutant cells and controls were assayed for sensitivity to camptothecin using a colony-forming assay. FAM35A depletion from the BRCA1-mutant cell line significantly alleviated the sensitivity to camptothecin (Fig 5A). Following exposure to ionizing radiation, a BRCA1-mutant cell line transfected with control siRNA did not form RAD51 foci, as expected (Johnson et al, 2013). However, the BRCA1-mutant cells formed damage-dependent nuclear RAD51 foci following FAM35A depletion, suggesting that 5′ end resection was more active in the absence of FAM35A (Fig 5B). Figure 5. FAM35A depletion/KO in BRCA1-deficient cells enhances markers of homologous recombination Quantification of depletion of endogenous FAM35A mRNA and protein from 293T cell lines carrying shRNA to FAM35A. Colony-forming assay: MDA-MB-436 cells infected with non-targeting control or FAM35A shRNAs were treated for 24 h with 20 nM camptothecin, with medium changed and incubated until colonies appear; cells were then fixed and stained. Top row is untreated; second row is treated with camptothecin. Colony and cell numbers were counted. Bottom left is the number of cells 24 h after seeding 1.5 × 105 cells. Bottom right is the colony count. Data represent mean ± SEM. n = 3. ns: not significant, *P < 0.05, **P < 0.01 and ***P < 0.001 by unpaired t-test. FAM35A-depleted BRCA1-mutant cells form nuclear foci of RAD51 following DNA damage. MDA-MB-436 cells infected with non-targeting control (second row) or FAM35A shRNAs (top row) were exposed to X-rays (10 Gy). Cells were fixed after 6 h and stained with DAPI and anti-RAD51. Scale bars: 10 μm. Genes related to PARP inhibitor resistance in BRCA1-mutant cells and alterations of these genes in the HCC1937 cell line according to the CCLE. Quantification of endogenous FAM35A mRNA expression. Endogenous FAM35A mRNA from HCC1937 (BRCA1 mutant), HCC1937BL, and MDA-MB-436 (BRCA1 mutant) cell lines was quantified using qPCR. Data represent mean ± SEM. n = 3. Detection of FAM35A exons 1 and 2 deletion in genomic DNA using PCR. Deletion of FAM35A exons 1 and 2 is confirmed in the HCC1937 cell line. In HCC1937 BL, endogenous FAM35A mRNA expression level was detectable. PCR primers for amplification of genomic DNA were designed in exon 1 (forward; primer 1) and exon 2 (reverse; primer 2). The predicted PCR product size is 1,794 bp. Source data are available online for this figure. Source Data for Figure 5 [embj201899543-sup-0005-SDataFig5.tif] Download figure Download PowerPoint We investigated a further widely used cell line, HCC1937, which has a known inactivati
Andrographolide, a major constituent of Andrographis paniculata, was previously shown to exhibit anti-inflammatory, antiviral, and anticancer activities. The anticancer activity of andrographolide includes growth suppression, apoptosis promotion, antiangiogenesis, and antitransformation. However, the effect of andrographolide on cancer metastasis, the most malignant feature of cancer, has not been elucidated extensively. In the present study, we demonstrated that andrographolide at nontoxic to subtoxic concentrations (0.3–3 µM) suppressed the invasion ability of CT26 cells in Matrigel-based invasion assays. In addition, the expression of cell adhesion regulators (β-catenin and ILK) was not altered by andrographolide treatment. However, andrographolide indeed inhibited matrix metalloproteinase 2 (MMP2) activity without affecting its expression. Furthermore, the activation of ERK, but not Akt, was attenuated by andrographolide treatment. Notably, a similar inhibitory effect of andrographolide on the invasion and MMP2 activity of the human colon cancer cell line HT29 was also observed. In summary, our results indicate that andrographolide exhibits anti-invasive activity against colon cancer cells via inhibition of MMP2 activity.
Abstract Dysregulation of mRNA alternative splicing (AS) has been implicated in development and progression of hematological malignancies. How the global AS dysregulation contributes to the development and progression of solid tumors is under-studied and remains generally unclear. Here we describe the first comprehensive AS landscape in the spectrum of human prostate cancer (PCa) development, progression and therapy resistance. We find that the severity of splicing dysregulation correlates with disease progression and establish intron retention (IR) as a hallmark of PCa stemness and aggressiveness. Systematic interrogation of 274 splicing-regulatory genes (SRGs) uncovers prevalent SRG mutations associated with, mainly, copy number variations leading to mis-expression of ~68% of SRGs during PCa evolution. Consequently, we identify many SRGs as prognostic markers associated with splicing disruption and patient outcome. Interestingly, androgen receptor (AR) controls a splicing program distinct from its transcriptional regulation. The spliceosome modulator, E7107, reverses cancer aggressiveness and inhibits the growth of castration-resistant PCa (CRPC) xenograft models and of the autochthonous Hi-Myc tumors. Altogether, our studies establish aberrant AS landscape caused by dysregulated SRGs as a hallmark of PCa aggressiveness and a novel therapeutic vulnerability for CRPC. Citation Format: Dingxiao Zhang, qiang Hu, Xiaozhuo Liu, Yibing Ji, Hsueh-Ping Chao, Amanda Tracz, Jason Kirk, Silvia Buonamici, Ping Zhu, Jianmin Wang, song liu, Dean Tang. Dysregulated alternative splicing landscape identifies intron retention as a hallmark and spliceosome as a therapeutic vulnerability in aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2197.
BACKGROUND: GP.Mur (Mi.III) is a glycophorin B‐A‐B hybrid sialoglycoprotein expressing several potent immunogens, including Mi a , Mur, and Hil. GP.Mur is considered one of the most important red blood cell (RBC) phenotypes in blood banking in Southeast Asia. However, there are no antibodies commercially available for the screening of GP.Mur RBCs. STUDY DESIGN AND METHODS: To develop a direct blood polymerase chain reaction (PCR) approach for the screening of GP.Mur cells, we first confirmed the genomic sequence differences among four GP.Mur and three Mi(a–) samples by sequencing their GYP.Mur and GYPB genes. With these data, we designed PCR primers that best discriminate GYPB and GYP.Mur . Our primer design also allows the detection of other Hil+ glycophorin variants. We also constructed two plasmids—pGBi2i3 and pMiIIIi2i3—which serve as the negative and positive control DNA, respectively, for the PCR procedure. Additionally, we designed a control PCR to be run side by side with the typing PCR. RESULTS: Because of the high specificity of our primers, we found it unnecessary to extract DNA from blood samples for PCR. We have tested this PCR method on 379 fresh and frozen blood samples. The results were further validated by serology and DNA sequencing and were shown to be completely accurate in our hand. We also found that the rapid genotyping method—high‐resolution melting—can be a timesaving alternative for DNA sequencing. CONCLUSION: This direct blood PCR approach for determination of GP.Mur and related Hil+ phenotypes is reliable and economical and is expected to be useful for blood banking in Southeast Asia.
Autophagy is critical for maintaining cellular homeostasis during times of stress, and is thought to play important roles in both tumorigenesis and tumor cell survival. Formation of autophagosomes, which mediate delivery of cytoplasmic cargo to lysosomes, requires multiple autophagy-related (ATG) protein complexes, including the ATG12-ATG5-ATG16L1 complex. Herein, we report that a molecular ATG5 "conjugation switch", comprised of competing ATG12 and ubiquitin conjugation reactions, integrates ATG12-ATG5-ATG16L1 complex assembly with protein quality control of its otherwise highly unstable subunits. This conjugation switch is tightly regulated by ATG16L1, which binds to free ATG5 and mutually protects both proteins from ubiquitin conjugation and proteasomal degradation, thereby instead promoting the irreversible conjugation of ATG12 to ATG5. The resulting ATG12-ATG5 conjugate, in turn, displays enhanced affinity for ATG16L1 and thus fully stabilizes the ATG12-ATG5-ATG16L1 complex. Most importantly, we find in multiple tumor types that ATG5 somatic mutations and alternative mRNA splicing specifically disrupt the ATG16L1-binding pocket in ATG5 and impair the essential ATG5-ATG16L1 interactions that are initially required for ATG12-ATG5 conjugation. Finally, we provide evidence that ATG16L2, which is overexpressed in several cancers relative to ATG16L1, hijacks the conjugation switch by competing with ATG16L1 for binding to ATG5. While ATG16L2 stabilizes ATG5 and enables ATG12-ATG5 conjugation, this endogenous dominant-negative inhibitor simultaneously displaces ATG16L1, resulting in its proteasomal degradation and a block in autophagy. Thus, collectively, our findings provide novel insights into ATG12-ATG5-ATG16L1 complex assembly and reveal multiple mechanisms wherein dysregulation of the ATG5 conjugation switch inhibits autophagy.
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