WNT inhibition creates a BRCA‐like state in Wnt‐addicted cancer
2021; Springer Nature; Volume: 13; Issue: 4 Linguagem: Inglês
10.15252/emmm.202013349
ISSN1757-4684
AutoresAmanpreet Kaur, Jun Yi Stanley Lim, Sugunavathi Sepramaniam, Siddhi Patnaik, Nathan Harmston, May Ann Lee, Enrico Petretto, David M. Virshup, Babita Madan,
Tópico(s)CRISPR and Genetic Engineering
ResumoArticle4 March 2021Open Access Transparent process WNT inhibition creates a BRCA-like state in Wnt-addicted cancer Amanpreet Kaur Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, Singapore Search for more papers by this author Jun Yi Stanley Lim Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, Singapore Search for more papers by this author Sugunavathi Sepramaniam Experimental Drug Development Centre, A*Star, Singapore, Singapore Search for more papers by this author Siddhi Patnaik Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, Singapore Search for more papers by this author Nathan Harmston Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, Singapore Science Division, Yale-NUS College, Singapore, Singapore Search for more papers by this author May Ann Lee Experimental Drug Development Centre, A*Star, Singapore, Singapore Search for more papers by this author Enrico Petretto Center for Computational Biology and Program in Cardiovascular and Metabolic Disorders, Duke-NUS Medical School, Singapore, Singapore Search for more papers by this author David M Virshup orcid.org/0000-0001-6976-850X Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, Singapore Department of Pediatrics, Duke University School of Medicine, Durham, NC, USA Search for more papers by this author Babita Madan Corresponding Author [email protected] orcid.org/0000-0003-1065-8589 Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, Singapore Search for more papers by this author Amanpreet Kaur Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, Singapore Search for more papers by this author Jun Yi Stanley Lim Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, Singapore Search for more papers by this author Sugunavathi Sepramaniam Experimental Drug Development Centre, A*Star, Singapore, Singapore Search for more papers by this author Siddhi Patnaik Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, Singapore Search for more papers by this author Nathan Harmston Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, Singapore Science Division, Yale-NUS College, Singapore, Singapore Search for more papers by this author May Ann Lee Experimental Drug Development Centre, A*Star, Singapore, Singapore Search for more papers by this author Enrico Petretto Center for Computational Biology and Program in Cardiovascular and Metabolic Disorders, Duke-NUS Medical School, Singapore, Singapore Search for more papers by this author David M Virshup orcid.org/0000-0001-6976-850X Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, Singapore Department of Pediatrics, Duke University School of Medicine, Durham, NC, USA Search for more papers by this author Babita Madan Corresponding Author [email protected] orcid.org/0000-0003-1065-8589 Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, Singapore Search for more papers by this author Author Information Amanpreet Kaur1, Jun Yi Stanley Lim1, Sugunavathi Sepramaniam2, Siddhi Patnaik1, Nathan Harmston1,3, May Ann Lee2, Enrico Petretto4, David M Virshup1,5 and Babita Madan *,1 1Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, Singapore 2Experimental Drug Development Centre, A*Star, Singapore, Singapore 3Science Division, Yale-NUS College, Singapore, Singapore 4Center for Computational Biology and Program in Cardiovascular and Metabolic Disorders, Duke-NUS Medical School, Singapore, Singapore 5Department of Pediatrics, Duke University School of Medicine, Durham, NC, USA *Corresponding author. Tel: +65 161790; E-mail: [email protected] EMBO Mol Med (2021)13:e13349https://doi.org/10.15252/emmm.202013349 See also: S Angers (April 2021) 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 Wnt signaling maintains diverse adult stem cell compartments and is implicated in chemotherapy resistance in cancer. PORCN inhibitors that block Wnt secretion have proven effective in Wnt-addicted preclinical cancer models and are in clinical trials. In a survey for potential combination therapies, we found that Wnt inhibition synergizes with the PARP inhibitor olaparib in Wnt-addicted cancers. Mechanistically, we find that multiple genes in the homologous recombination and Fanconi anemia repair pathways, including BRCA1, FANCD2, and RAD51, are dependent on Wnt/β-catenin signaling in Wnt-high cancers, and treatment with a PORCN inhibitor creates a BRCA-like state. This coherent regulation of DNA repair genes occurs in part via a Wnt/β-catenin/MYBL2 axis. Importantly, this pathway also functions in intestinal crypts, where high expression of BRCA and Fanconi anemia genes is seen in intestinal stem cells, with further upregulation in Wnt-high APCmin mutant polyps. Our findings suggest a general paradigm that Wnt/β-catenin signaling enhances DNA repair in stem cells and cancers to maintain genomic integrity. Conversely, interventions that block Wnt signaling may sensitize cancers to radiation and other DNA damaging agents. Synopsis This study identifies that Wnt/β-catenin signaling regulates homologous recombination and Fanconi anaemia DNA repair pathways in Wnt-high cancers and intestinal stem cells. Wnt signaling inhibition induces a BRCA-like state; and Wnt and PARP inhibitors synergize to inhibit Wnt-addicted cancers. Wnt signaling regulates the expression of a broad set of genes involved in repairing DNA double-strand breaks. This is mediated in part via the transcription factor MYBL2. Wnt inhibition creates a BRCA-like state by inhibiting the expression of genes in the homologous recombination and Fanconi anemia DNA repair pathway. Wnt inhibition synergizes with PARP inhibitors to more effectively treat multiple Wnt-addicted cancers. Wnt inhibition causes homologous recombination deficiency that, when combined with blockage of PARP-mediated ssDNA repair leads to an accumulation of dsDNA breaks to enhance senescence. Wnt signaling also regulates Homologous recombination and Fanconi Anemia pathway genes in intestinal stem and transit amplifying cells. The paper explained Problem Resistance to DNA damage in both cancers and adult stem cells is often associated with activation of the Wnt signaling pathway. Understanding how Wnt signaling drives chemotherapy resistance is of major interest and may assist in developing novel anti-cancer therapies. Results We found that Wnt signaling turns on a broad set of genes involved in repairing DNA double-strand breaks and inhibiting Wnt signaling can reverse this. These DNA repair genes, including BRCA1, normally maintain genomic integrity in stem cells and cancers. This helps explain how activated Wnt signaling can drive chemotherapy resistance in both stem cells and cancer. Impact We find that Wnt pathway inhibition synergizes with PARP inhibition in multiple Wnt-addicted cancers. Wnt pathway inhibitors may be a useful intervention to reverse drug resistance in Wnt-high cancers. Introduction Stem cells in normal tissues protect the integrity of their genome by expression of diverse proteins that accurately detect and repair mutations introduced by DNA replication and environmental mutagens (Hua et al, 2012). Inherited defects in DNA repair pathways, such as germline mutations in BRCA1 or Fanconi anemia pathway genes that normally repair double-strand breaks and DNA cross-links, lead to the accumulation of mutations that impair stem cell function and promote tumorigenesis (Dietlein et al, 2014; Ma et al, 2018). The FA pathway proteins serve as interstrand cross-link (ICL)-sensors and promote DNA repair in conjunction with homologous recombination and other DNA repair pathways including BRCA proteins (Deans & West, 2011; Ceccaldi et al, 2016). Individuals with Fanconi anemia who have mutations in Fanconi anemia (FA) pathway genes are exquisitely sensitive to DNA ICL-generating agents. These DNA repair pathways are often co-opted in cancers during the development of drug resistance (Jackson & Bartek, 2009). Defects in specific DNA repair pathways also present an opportunity for targeted anti-cancer therapies (Dietlein et al, 2014; Ma et al, 2018; Ashworth & Lord, 2018). Breast and ovarian cancers with defects in the homologous recombination (HR) repair pathway, especially BRCA1 and BRCA2 mutant tumors, are sensitive to poly ADP-ribose polymerase (PARP) inhibitors such as olaparib (Farmer et al, 2005; Bryant et al, 2005; Ashworth & Lord, 2018). These inhibitors work by blocking the function of PARP proteins essential for single-strand break (SSB) repair. Upon PARP inhibitor treatment, the unrepaired SSBs can progress to double-strand breaks that are then repaired by homologous recombination. In HR-deficient cells, treatment with PARP inhibitors results in growth arrest by apoptosis or senescence due to the accumulation of DNA damage (Ashworth & Lord, 2018). Wnt signaling is important for the maintenance of stem cell state, adult tissue homeostasis, and the prevention of differentiation (Clevers et al, 2014). Wnt signaling is also associated with the development of radioresistance in many cancers, but the underlying mechanisms are not well understood (Metcalfe et al, 2014; Jun et al, 2016; Emons et al, 2017; Zhao et al, 2018; Luo et al, 2019; Karimaian et al, 2017). Wnts are a family of 19 secreted palmitoleated glycoproteins that signal by binding to Frizzled (FZD) and additional co-receptors on the cell surface. The interaction of Wnts with their receptors stabilizes β-catenin, which then translocates to the nucleus to regulate gene expression. Aberrant stabilization of β-catenin can be caused by mutation of components of the Wnt pathway such as APC, AXIN and CTNNB1 (β-catenin), as is frequently seen in colorectal, gastric and liver cancers (Nusse & Clevers, 2017). Additionally, a subset of pancreatic, mucinous ovarian, colorectal, gastric, adrenocortical, and endometrial cancers harbor loss-of-function mutations in E3-ubiquitin ligases RNF43 or its paralog ZNRF3 or gene fusions leading to activation of Wnt agonists RSPO2/3 (R-spondin 2/3) (Wu et al, 2011; Bailey et al, 2016; Ryland et al, 2013; Wang et al, 2014; Giannakis et al, 2014; Assié et al, 2014; Seshagiri et al, 2012). These mutations enhance the abundance of Frizzleds and cause cancers to be dependent on activated Wnt signaling. This subset of cancers is highly sensitive to upstream inhibitors of Wnt signaling pathway such as anti-FZD and anti-R-spondin antibodies as well as PORCN inhibitors such as ETC-159 (Gurney et al, 2012; Jiang et al, 2013; Madan et al, 2016). Several of these agents are currently in clinical trials. These inhibitors are also powerful tools to investigate the pathways that are regulated by Wnt signaling in cancer (Madan et al, 2016, 2018). During a screen to identify approved drugs that synergize with PORCN inhibitors, we made the unexpected observation that the PARP inhibitor olaparib synergized with ETC-159 in Wnt-addicted cancers. Mechanistically, we found that inhibition of Wnt signaling in multiple cancer cell lines and normal intestinal crypts results in the suppression of multiple HR pathway genes. Wnt signaling through a β-catenin/MYBL2 pathway regulates the expression of BRCA1, BRCA2, RAD51, and FANCD2. This study uncovers a role for Wnt/β-catenin signaling in the regulation of homologous recombination DNA repair pathway in intestinal stem cells and in cancer and demonstrates that inhibition of Wnt signaling confers a BRCA-like phenotype, providing a novel therapeutic opportunity for Wnt high cancers. Results Wnt inhibition synergizes with PARP inhibitor Olaparib The PORCN inhibitor ETC-159 has shown efficacy as a monotherapy in preclinical models of Wnt-addicted cancers (Madan et al, 2016). To identify potential combinatorial therapeutic options, we performed a synergy screen using the Chou-Talalay method with selected drugs that are either FDA approved or in clinical trials (Chou, 2010). Since Wnt-addicted cells are substantially more sensitive to PORCN inhibitors when grown in suspension, the screen was performed in the Wnt-addicted RNF43-mutant pancreatic cancer cell line HPAF-II using soft agar colony formation as the readout (Madan et al, 2016; Zhong et al, 2019). We observed that the combination of olaparib and ETC-159 was significantly more effective in inhibiting colony formation of HPAF-II cells than treatment with either drug individually (Fig 1A and Table EV1). Consistent with this, the drug combination index values for ETC-159 and olaparib, as determined using the Chou-Talalay CompuSyn algorithm, showed a synergistic effect (Combination Index < 1) for each of the dosages tested (Fig 1B). To test whether this combination was also efficacious in vivo, we used the HPAF-II pancreatic cancer xenograft model. Treatment with olaparib alone or low dose of ETC-159 alone was less effective compared to the combination of olaparib and ETC-159 in preventing HPAF-II tumor growth in mice. This was shown by changes in tumor volumes during the course of treatment (Fig 1C) as well as the tumor weights at the end of 21 days treatment (Fig 1D). Figure 1. ETC-159 synergizes with the PARP inhibitor Olaparib A, B. PORCN inhibitor ETC-159 synergizes with PARP inhibitor olaparib in suppressing proliferation of HPAF-II pancreatic cancer cells in soft agar. The ED50 (dose that reduced colony formation by 50% of the maximal inhibition) was determined (Table EV1) and cells were treated with ETC-159, olaparib or the combination at the indicated dose (for example 0.25 x ED50 of Olaparib or ETC-159, or 0.25 x ED50 of Olaparib + 0.25 × ED50 of ETC-159, respectively). (A) The data is representative of two independent experiments with each point representing an average colony count ± SD of duplicates. (B) The Combination Index (CI) values of olaparib and ETC-159 calculated for the two independent experiments using the Chou-Talalay CompuSyn software. CI < 1, = 1, and > 1 indicate synergism, additive effect, and antagonism, respectively. The lower the CI, the stronger the synergism. C, D. Olaparib and ETC-159 synergize to prevent the growth of HPAF-II xenografts in mice. NSG mice with established HPAF-II subcutaneous xenografts were randomized into four groups. Mice were gavaged daily with ETC-159 (10 mg/kg), Olaparib (50 mg/kg) or a combination of ETC-159 (10 mg/kg) and Olaparib (50 mg/kg). Treatment was initiated after HPAF-II tumors were established. (C) Tumor volumes were measured starting from day 0 and during the course of treatment as shown. Data points represent the mean ± SD. n = 7–8 tumors/group. P-values indicate significant difference compared to the vehicle group. (D) Tumor weights in the respective groups at the end of 21 days treatment are shown, each dot represents an individual tumor and line represents mean. P-values were calculated with Mann–Whitney U-test. E, F. Olaparib and ETC-159 synergize in multiple Wnt-addicted cancer cells. Soft agar colony formation assays were performed as in Fig 1A with the indicated cell lines treated with varying concentrations of ETC-159, olaparib or a combination of both as indicated and the combination index calculated from two independent experiments using Chou-Talalay method are shown. PARP inhibitor olaparib and ETC-159 synergistically prevent colony formation of EGI-1, MCAS and CFPAC-1 cells in soft agar. (F) Representative image of soft agar colonies of EGI-1 cells is shown. Download figure Download PowerPoint Next, we assessed the combination of PORCN inhibitor and olaparib in three additional Wnt-addicted cell lines from diverse cancer types with distinct Wnt pathway mutations. The cholangiocarcinoma cell line EGI-1 (Wnt-addicted due to an R-spondin translocation), the ovarian cancer cell line MCAS (Wnt-addicted due to an RNF43 mutation), and the pancreatic cancer cell line CFPAC-1 (sensitive to Wnt inhibition, mechanism unknown) were used. Similar to what was observed with HPAF-II cells, the combination of ETC-159 and olaparib synergistically inhibited colony formation in all three cell lines in soft agar assay at all the doses tested (Figs 1E and F, and EV1A and B and Table EV1). Thus, the synergy of ETC-159 and olaparib is a general phenomenon. Taken together, the data indicate that blocking Wnt activity with a PORCN inhibitor sensitizes Wnt-addicted cells to a PARP inhibitor. Click here to expand this figure. Figure EV1. (accompanying Figs 1 and 2) A, B. Olaparib and ETC-159 synergize in multiple Wnt-addicted cancer cells. Soft agar colony formation assays were performed as in Fig 1A with the indicated cell lines treated with varying concentrations of ETC-159, olaparib, or a combination of both. Representative image of soft agar colonies of (A) MCAS and (B) CFPAC-1 cells is shown. C. Timeseries analysis clusters genes into distinct patterns based on their transcriptional response to PORCN inhibition. Reanalysis of data from (Madan et al, 2018), where HPAF-II cells were orthotopically injected into the tail of the pancreas. Tumors were established over a period of 28 days, and mice were treated with ETC-159 (37.5 mg/kg bid). RNA was isolated from the tumors at the indicated time points and analyzed by RNA-seq. The heatmap shows all genes that were differentially expressed over time (FDR < 10%) following PORCN inhibition, clustered into 64 clusters based on their pattern of transcriptional response. The clusters of genes that are robustly downregulated following Wnt inhibition are highlighted with colors and indicated as Wnt-activated genes. D. ETC-159 treatment of HPAF-II tumors downregulates protein levels of BRCA1. Tumor lysates from HPAF-II xenografts treated with vehicle or ETC-159 for 56 h were analyzed by SDS–PAGE and immunoblotted with the anti-BRCA1 antibody. Each lane represents an individual tumor. E. Wnt inhibition does not alter the cell cycle phases in HPAF-II cells. HPAF-II cells were treated with DMSO or ETC-159 for 48 h. After treatment, cells were stained with propidium iodide and analyzed using flow cytometry to determine the number of cells in G1, S, or G2/M phase of the cell cycle. Each bar represents mean ± SD of two replicates. F. Wnt inhibition reduces the expression of HR and FA pathway genes in HPAF-II cells. HPAF-II cells were treated with DMSO or ETC-159 (100 nM) for 48 h. Total RNA was isolated, and the normalized expression of DNA repair genes as measured by RNA-seq is shown. The horizontal lines represent mean of replicates. G. Wnt inhibition reduces the expression of HR and FA pathway genes in Wnt high EGI-1 cells. EGI-1 cells were cultured in low adherence plates and treated with DMSO or ETC-159 (100 nM) for 72 h. Total RNA was isolated, and the expression of AXIN2 and DNA repair genes was measured by qRT–PCR. The horizontal lines represent mean of replicates. H. Wnt inhibition does not alter the cell cycle phases in AsPC-1 cells. AsPC-1 cells were treated with DMSO or ETC-159 for 48 h. After treatment cells were stained with propidium iodide and analyzed using flow cytometry to determine the number of cells in G1, S, or G2/M phase of the cell cycle. Each bar represents the mean ± SD of two replicates. Download figure Download PowerPoint Wnt inhibition reduces expression of homologous recombination (HR) and Fanconi anemia (FA) repair pathway genes Olaparib and related PARP inhibitors are uniquely effective in BRCA-mutant and BRCA-like cancers that have dysfunctional homologous recombination (Armstrong & Clay, 2019). Diverse mechanisms can cause defective BRCA-like behavior, including inherited mutations in BRCA1, BRCA2, and Fanconi anemia complementation (FANC) group of genes (Lord & Ashworth, 2016). Epigenetic and transcriptional mechanisms that silence HR pathway genes can also cause a BRCA-like state (Ibrahim et al, 2012). To test whether Wnt inhibition induces a BRCA-like state, we examined the expression of genes involved in DNA repair using our transcriptome dataset from ETC-159-treated Wnt-addicted HPAF-II pancreatic cancer orthotopic xenografts (Fig EV1C) (Madan et al, 2018). Remarkably, among the genes downregulated following PORCN inhibition, i.e. Wnt-activated genes, there were three clusters of genes (C1, C5, and C12) that were significantly enriched for Gene Ontology (GO) annotated processes and pathways related to multiple components of the DNA damage repair pathway (Figs 2A and B and EV1C). Figure 2. Homologous recombination (HR) and Fanconi anemia (FA) repair pathway genes are regulated by Wnt signaling A. Heatmap of selected temporal clusters containing the Wnt-activated genes that are enriched for DNA repair pathways. Transcriptomic data from HPAF-II orthotopic pancreatic tumors (dataset originally reported in Madan et al, 2018) was assessed at multiple time points during treatment with the PORCN inhibitor 37.5 mg/kg bid ETC-159 (Fig EV1C). Genes that were differentially expressed over time (FDR < 10%) following PORCN inhibition were clustered based on their pattern of transcriptional response. Clusters 1, 5 and 12 refer to temporal clusters defined in (Madan et al, 2018) and TI50 is the time (in hours) after start of therapy to achieve 50% inhibition in gene expression for each cluster. B. Gene Ontology (GO) biological process enrichment of each cluster of Wnt-activated genes. Analysis of Wnt-activated genes in clusters 1, 5 and 12 from HPAF-II orthotopic pancreatic tumors (Fig 2A) and colorectal cancer (CRC) patient-derived xenograft (PDX) highlights enrichment of genes involved in multiple DNA repair pathways including interstrand cross-link repair and double strand break (DSB) repair via homologous recombination (HR). C. DNA repair genes involved in HR and FA pathways were differentially expressed over time in response to PORCN inhibition in HPAF-II orthotopic xenografts. Comparison of log2 fold change (FDR < 10%) in the expression of Wnt-activated genes over time across multiple DNA repair pathways shows that genes regulating HR and FA pathways have higher fold changes compared to genes regulating BER, NER or other pathways. BER: base excision repair; HR: homologous recombination; FA: Fanconi anemia; MMR: mismatch repair; NER: nucleotide excision repair; NHEJ: non-homologous end joining; TLS: translesion DNA synthesis. The horizontal line represents median of 4–6 replicates with the upper and the lower edges of the box representing the 75th and 25th percentile of the data respectively and the whiskers representing 1.5× interquartile range. D, E. ETC-159 treatment of HPAF-II orthotopic xenografts reduces the expression and protein levels of multiple HR and FA pathway genes. (D) Temporal expression of selected Wnt-activated HR and FA pathway genes are shown. n = 7–10 tumors/group. (E) Tumor lysates from HPAF-II xenografts treated with vehicle or ETC-159 for 8 or 56 h were analyzed by SDS–PAGE and immunoblotted with the indicated antibodies. Each lane represents an individual tumor. F, G. Wnt inhibition reduces the expression and protein levels of HR and FA pathway genes in a Wnt-addicted colorectal cancer patient-derived xenograft. Mice with CRC PDX driven by a RSPO3 translocation were treated with vehicle or ETC-159 for 56 h before harvesting. (F) Gene expression was analyzed by RNA-seq (dataset reported in (Madan et al, 2016)). The graph shows the normalized expression of the indicated genes. The horizontal line represents mean of 4 tumors/group. (G) Tumor lysates were analyzed by SDS–PAGE and immunoblotted with the indicated antibodies. Each lane represents an individual tumor. H, I. Wnt inhibition reduces the expression of HR and FA pathway genes in a RNF43-mutant pancreatic cancer patient-derived xenograft and AsPC-1 cells. (H) Pancreatic cancer PDX with G371fs RNF43 mutation treated with vehicle or ETC-159 (30 mg/kg) for 21 days were analyzed for changes in the expression of the indicated genes measured by qRT–PCR. Each data point represents an individual tumor. The horizontal lines represent mean of 3 tumors/group. (I) AsPC-1 cells were seeded in low adherence plates, treated with DMSO or ETC-159 (100 nM) for 72 h and the expression of indicated genes was measured by qRT–PCR. J. Wnt inhibition reduces homologous recombination. AsPC-1 or Panc 08.13 cells with a stable integration of HR reporter construct DR-GFP (encoding a modified GFP gene with the I-SceI site and a donor GFP sequence that restores GFP sequence upon HR-mediated DNA repair) were transfected with a plasmid expressing I-SceI endonuclease, followed by treatment with DMSO or ETC-159 (100 nM) for 24 h. The percentage of GFP-positive cells as measured by flow cytometry indicates that the extent of repair via HR pathway in the presence of ETC-159 treatment is reduced in Wnt high AsPC-1 cells or but not in Wnt-insensitive Panc 08.13 cells. Data show the values from three independent experiments with two to three replicates each. P-values were calculated using Mann–Whitney U-test. Download figure Download PowerPoint Further analysis of the genes involved in regulating each of the DNA repair pathways highlighted that Wnt inhibition robustly downregulated genes involved in homologous recombination (HR) and Fanconi anemia (FA) pathways (Fig 2C). By comparison, genes annotated to be involved in mismatch repair and in the repair of single strand breaks such as base excision repair (BER) or nucleotide excision repair (NER) were much less affected by changes in Wnt signaling. Most notably, the expression of BRCA1, BRCA2, RAD51, and multiple FANC genes was decreased by 70–80% compared to the vehicle group by 32 h after the start of treatment with the PORCN inhibitor (Fig 2C and D). Immunoblotting for BRCA1, BRCA2, FANCA, and FANCD2 in HPAF-II tumors harvested from ETC-159 treated mice confirmed the downregulation of these proteins by 56 h (Figs 2E and EV1D). This downregulation of genes involved in the HR and FA pathways following Wnt inhibition is consistent with the observed increase in sensitivity of Wnt-addicted tumors and cell lines to olaparib (Fig 1). While ETC-159 treatment of HPAF-II cells in vitro for 48 h significantly reduced the expression of HR pathway and FA genes, we did not observe a significant change in the percentage of cells in S-phase as compared to control (Fig EV1E and F). These results suggest that the suppression of HR and FA pathway gene expression by ETC-159 treatment is not due to its effect on cell proliferation (Saleh-Gohari, 2004; Davidson & Niehrs, 2010). Click here to expand this figure. Figure EV2. (accompanying Fig 5) Temporal regulation of FOXM1 in HPAF-II orthotopic xenografts treated with ETC-159. Each data point represents an individual tumor. The horizontal line represents the median with the lower and upper edges of the boxes representing 25th and the 75th percentile of the data, respectively, and the whiskers representing 1.5× interquartile range. Expression of DNA repair genes in Wnt-addicted cells is minimally regulated by FOXM1. HPAF-II cells were transfected with two independent siRNAs against FOXM1 or treated with ETC-159 (100 nM) for 48 h. Total RNA was isolated and expression of FOXM1 and DNA repair genes was measured by qRT–PCR. The horizontal lines represent mean of replicates. Wnt-regulated HR and FA pathway gene expression is MYC-independent. Mice bearing HPAF-II xenografts without or with stabilized MYC (T58A) were treated with ETC-159 for 56 h. Tumors from the control and treated groups were harvested and the expression of DNA repair genes was measured. ChIP-seq data from K562 cells from ENCODE shows the binding of MYBL2 on the promoters of DNA repair genes but not on AXIN2. The putative MYBL2 motif locations identified using FIMO analysis are indicated (ENCODE Project Consortium, 2012). Enrichment of MYBL2 binding on the promoters of DNA repair genes. Chromatin immunoprecipitated from HPAF-II cells with IgG or MYBL2 antibody was analyzed using primers specific for the DNA binding sites in the indicated DNA repair genes. Representative images of the PCR products resolved on a 12% acrylamide gel are shown. The arrows indicate specific bands. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. (accompanying Fig 6) Representative images showing 53BP1 foci and senescence associated heterochromatin foci in HPAF-II treated with DMSO, ETC-159 (50 nM), olaparib (20 µM), or both for 7 days. 53BP1 foci (red) and nuclei counterstained with DAPI (blue). Wnt inhibition induces senescence associated heterochromatin foci, that is further enhanced by co-treatment with olaparib. HPAF-II cells were treated with DMSO, ETC-159 (50 nM), olaparib (20 µM) or both for 7 days and the number of SAHF per cell was assessed by DAPI fluorescent staining. The horizontal lines represent mean of replicates. P-values were calculated by Mann–Whitney U-test. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. (accompanying Fig 7) HR and FA pathway genes are expressed in the Wnt high compartment of the small intestine: Immunohistochemical staining of sections of small intestine from C57BL/6J mice shows that the expression of Brca1, Fancd2, and Rad51 is high in the crypts (Wnt high compartment) but not the villi. Download figure Download PowerPoint Click here to exp
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