A Pygopus 2-Histone Interaction Is Critical for Cancer Cell De-Differentiation and Progression in Malignant Breast Cancer

Author Manuscript Published OnlineFirst on June 25, 2020; DOI: 10.1158/0008-5472.CAN-19-2910 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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A Pygopus 2-histone interaction is critical for cancer cell de-differentiation and progression in malignant breast cancer

Meera Saxena1,*, Ravi K.R. Kalathur1,4,#, Natalia Rubinstein1,5,#, Andrea Vettiger1,6, Nami Sugiyama1, Melanie Neutzner1, Mairene Coto-Llerena2, Venkatesh Kancherla2, Caner Erkan2, Salvatore Piscuoglio2, Jonas Fischer1, Ernesta Fagiani1, Claudio Cantù3,7, Konrad Basler3 and Gerhard Christofori1,*

1Department of Biomedicine, University of Basel, Switzerland 2Institute of Pathology, University Hospital Basel, Switzerland 3Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland

Current affiliations: 4Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Australia 5Institute of Biosciences, Biotechnology and Translational Biology, Department of Physiology, Molecular and Cellular Biology, Faculty of Exact Science, University of Buenos Aires, Buenos Aires, Argentina 6Balvatnik Institute, Harvard Medical School, Boston, United States of America 7Wallenberg Centre for Molecular Medicine Linköping; Department of Biomedical and Clinical Sciences, Faculty of Health Science, Linköping University, Sweden

#Authors contributed equally *Corresponding authors: Meera Saxena and Gerhard Christofori Department of Biomedicine, University of Basel Mattenstrasse 28, CH-4058 Basel, Switzerland Tel. +41 61 207 35 64 Fax. +41 61 207 35 66 E-mail: [email protected]; [email protected]

Running Title: Pygo2 promotes breast cancer progression

The authors declare no potential conflicts of interest.

Word Count: 6640 words (main text); 7 Figures, 7 Supplementary Figures, 3 Supplementary Tables and 4 Supplementary Videos.

Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 25, 2020; DOI: 10.1158/0008-5472.CAN-19-2910 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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Abstract Pygopus 2 (Pygo2) is a co- -catenin signaling that can bind bi- or trimethylated lysine 4 of histone-3 (H3K4me2/3) and participate in chromatin reading and writing. It remains unknown whether the Pygo2- H3K4me2/3 association has a functional relevance in breast cancer progression in vivo. To investigate the functional relevance of histone binding activity of Pygo2 in malignant progression of breast cancer, we generated a knock-in mouse model where binding of Pygo2 to H3K4me2/3 was rendered ineffective. Loss of Pygo2-histone interaction resulted in smaller, -catenin signaling. RNA and ATAC sequencing analyses of tumor-derived cell lines revealed downregulation signaling and upregulation of differentiation pathways such as PDGFR signaling. Increased differentiation correlated with a luminal cell fate which could be reversed by inhibition of PDGFR activity. Mechanistically, the Pygo2-histone interaction p -catenin signaling in part -catenin regulated the expression of miR-29 family members which in turn repressed PDGFR expression to promote de- differentiation of wildtype Pygo2 mammary epithelial tumor cells. Collectively, these results demonstrate that the histone binding function of Pygo2 is important for driving de- differentiation and malignancy of breast tumors, and loss of this binding activates various differentiation pathways which attenuate primary tumor growth and metastasis formation. Interfering with the Pygo2- H3K4me2/3 interaction may therefore serve as an attractive therapeutic target for metastatic breast cancer.

Significance Pygo2 represents a potential therapeutic target in metastatic breast cancer, as its histone binding capability promotes beta-catenin-mediated Wnt signaling and transcriptional control in breast cancer cell de-differentiation, EMT, and metastasis.

Keywords Breast cancer, de-differentiation, Pygo2, Wnt/ -catenin, PDGFR

Introduction Breast cancer is the most frequent malignancy in women worldwide. Localized, differentiated breast cancers, as represented by the luminal subtypes, are responsive to therapeutic intervention (1). However, de-differentiated, aggressive subtypes, characterized as basal-like/triple-negative, in most cases represent therapy refractory primary tumors concomitant with the formation of distant metastasis (1). Inappropriate activation of the Wnt signaling pathway, for example by activating mutations in positively acting components or loss of function of negative regulators of the pathway,

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has been shown to drive several malignancies including breast cancer (2). The transcriptional activity of -catenin, the key transcriptional effector of Wnt signaling, is aided by binding to the co- transcriptional activators Pygopus 1 and 2 (Pygo1 and 2) via the Bcl9/9L adaptor proteins (3,4). Pygo proteins were first discovered in Drosophila melanogaster (5-7) and in Xenopus (8). Of the two mammalian Pygo homologues, Pygo2 has been shown to be more abundantly expressed and functionally relevant than Pygo1 (9,10). While deletion of Pygo2 affects the proper development of multiple tissues, additional deletion of Pygo1 does not appear to exacerbate the Pygo2 phenotype (9- 12). Furthermore -catenin signaling has been found to be context- -catenin-independent manner in lens development (12,13), in tooth enamel formation (14) and in spermatogenesis (11). However, during the activation of hair follicle stem/progenitor cells and the regeneration of skin (15) and also during the development of mammary glands and expansion of mammary stem/progenitor cells, Pygo2 functions at least in part by regulating Wnt/ -catenin signaling (16). The best-characterized function of the Pygo proteins is in cell proliferation. At actively transcribing gene loci, Pygo2 binds activating histone marks, such as bi- or trimethylated histones (H3K4me2/3), and recruits histone acetyltransferases (HAT) which in turn promote an open chromatin structure (17-19). Moreover, by acting as an epigenetic accessory protein, Pygo2 drives Myc- dependent activation of mitosis-related genes (20). Befitting its role in proliferation, an increased expression of Pygo2 has been observed in several malignancies, including ovarian cancer (21), glioma (22), lung cancer (23), hepatocellular carcinoma (24), and prostate cancer (25). Importantly, Pygo2 upregulation has also been observed in breast cancer (18,26). Interestingly, Pygo2 resides in the chromosomal region 1q21-q22, which is found to be amplified in more than half of breast cancer cases (27). In the breast, Pygo2 plays an important role in mammary lineage differentiation. Pygo2 maintains the fate of mammary stem cells/basal cells by suppressing their luminal/alveolar differentiation, at least in part through the suppression of Notch signaling (28). Finally, ablation of Pygo2 in the mammary gland was found to delay the onset of tumors in the Wnt signaling-driven MMTV-Wnt1 transgenic mouse model of breast cancer (29). The Pygo family proteins contain a highly conserved C-terminal plant homology domain (PHD) which mediates the direct binding to H3K4me2/3, a mark of active transcription (16,19,30). PHD- thus linking chromatin remodeling to changes in gene transcription (31) by interacting with and recruiting histone acetyltransferases (HAT), such as CBP [CREB (cAMP- responsive-element-binding protein)-binding protein] (17), or histone methyl transferases (HMT), such as mixed lineage leukemia-2 (MLL2) (18), and thus augments -catenin-mediated

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transcriptional activation. Hence, Pygo2 can function as an efficient chromatin effector participating (11,16). However, whether, the Pygo2-H3K4me2/3 association has a functional relevance in breast cancer progression in vivo has remained elusive. Here, we report that the interaction of Pygo2 with histones is critical for -catenin-driven de-differentiated invasive breast cancer and metastasis formation. In mammary tumors of knock-in mice carrying Pygo2 alleles which are unable to bind to H3K4me2/3, differentiation inducing pathways like PDGFR get activated and promote a luminal cell fate, thereby repressing primary tumor invasiveness and metastasis formation.

Materials and Methods Antibodies and reagents Axin-2 (Abcam, ab32197), -tubulin (Sigma, T-9026), -catenin (BD transduction labs, 610154; used for immunofluorescence staining of tumor spheroids), -catenin (Novus Biologicals, NBP1-32239; used for ChIP experiments), non-phospho (active) -catenin (Ser33/37/Thr41) (D13A1) (Cell signaling technology, 8814; used for immunoblotting), cleaved Caspase-3 (Cell Signaling Technology, 9664), c-Myc (D84C12) (Cell signaling technology, 5605), cyclin-D1 (EPR2241) (Abcam, ab134175), Cytokeratin-14 (ThermoFisher Scientific, RB-9020-P0), Cytokeratin-8/18 (Fitzgerald, 20R-CP004), E-cadherin (Zymed, 13-1900), fibronectin (Sigma-Aldrich, F3648), GAPDH (Abcam, ab9485), Notch 3 (Abcam, ab23426), phospho-Histone3 (Merck Millipore, 06- 570), phospho-PDGFR (Tyr751) (88H8) (Cell signaling technology, 3166), total-PDGFR Y-92-C- terminal (Abcam, ab32570), Pygopus 2 (Novus Biologicals, NBP1-46171; used for IHC on mouse tumor sections), Pygopus 2 (Abcam, ab155262; used for IHC on human breast tumors and also for ChIP experiments), total-AKT (Cell signaling technologies, 9272), Vimentin (Novus Biologicals, NB300-223), WIF1 (Abcam, ab155101), Alexa-Fluor 488 and 568 (Molecular Probes), secondary horse radish peroxidase (HRP)-conjugated antibodies against mouse and rabbit (Jackson ImmunoResearch). -diamidino-2-phenylindole (DAPI, Sigma-Aldrich, D9542), recombinant human TGF 1 (R&D Systems, 240-B), Wnt3a (Peprotech, 315-20), CHIR99021 (Abcam, ab120890), PDGF-BB (Peprotech, 315-18), Tyrphostin AG1296 (Selleckchem, S8024), DAPT (Selleckchem, S2215).

Animal experimentation Generation and validation of Pygo2 knock-in mouse strain in C57BL/6 background has been described previously (32). These mice were backcrossed into an FVB/N background using speed congenics (Taconic Inc. New York). Thereafter, the heterozygous mutant mice bearing the A342E point mutation (Pygo2A342E/+) were crossed with MMTV-PyMT (33) transgenic males. MMTV- PyMT-Pygo2+/+ (WT), MMTV-PyMT-Pygo2A342E/+ and MMTV-PyMT-Pygo2A342E/A342E female

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littermates were used for subsequent analysis. Animal experiments have been approved by the Cantonal Veterinary Office of Basel-Stadt (license numbers 1878 and 1907). In animal experiments, all efforts were made to minimize suffering, and mice were kept and bred under specific pathogen- free (SPF) conditions.

Primary tumor-derived cell line generation and treatments Pygo2+/+ and Pygo2AE/AE cell lines were isolated from mammary gland tumors (mammary gland 2/3) of littermate MMTV-PyMT transgenic female mice (FVB/N background). In brief, a small piece of a tumor was minced and subjected to enzymatic digestion with 0.1mg/ml DNaseI (Roche, 11284932001) and 1mg/ml Collagenase D (Roche, 11088858001) supplemented with 50 g/ml gentamycin (Sigma, G1397) and 1X antibiotic-antimycotic (ThermoFisher, 15240062) for 30 minutes in sterile conditions. The cell mixture was passed through a 70µm cell strainer (BD Falcon, 352350) and the single cells obtained were plated as a polyclonal population in a 10cm dish in DMEM supplemented with 10% FBS (Sigma), 10% horse serum (Amimed), 100U penicillin (Sigma-Aldrich) and 0.1mg/ml streptomycin (Sigma-Aldrich). Medium was changed regularly and any fibroblasts in culture were removed by several passages of differential trypsinization until only epithelial cells - Aldrich, D5671) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich, F7524), 2mM glutamine (Sigma-Aldrich, G7513), 100U penicillin (Sigma-Aldrich) and 0.1mg/ml streptomycin

(Sigma-Aldrich). All cell lines were grown at 37°C, 5% CO2, 95% humidity. Isolation of these cell lines was done with approval, and according to the rules and guidelines of the Swiss Federal Veterinary Office (SFVO) and the local ethics committee (Cantonal Veterinary Office, Basel-Stadt, Switzerland; license 1878). All the cell lines in this study were regularly confirmed for the absence of Mycoplasma contamination, especially after thawing, and prior to use in any experiment. Cells were used up to 15 passages after which fresh cells of an early passage were thawed and used. Py2T cells (34) was used as a second WT cell line (indicated in the figures as +/+ #2) in some experiments. For EMT experiments, cells were treated with 2ng/ml TGF 1 for the time points indicated. Cells were treated with 100ng/ml Wnt3a for time points specified in appropriate experiments. Cells were treated with 3µM CHIR99021 for three days.

Tumor spheroid generation Following mechanical and enzymatic digestion of primary tumors (described above under cell line generation), primary cells were washed and resuspended in serum free DME-F12 medium containing 10ng/ml hEGF, 1mg/ml hydrocortisone, 10mg/ml insulin, 20ng/ml bFGF, 4ng/ml heparin (Sigma Aldrich), B27 (Invitrogen) supplemented with 100U penicillin (Sigma-Aldrich) and 0.1mg/ml streptomycin (Sigma-Aldrich). 2x10^5 primary tumor cells/1.5 ml were then seeded in 6 well ultra-

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low attachment plates (Corning, CLS3471). 200µl medium was supplemented every third day and spheroids were harvested 1 week after culture. Similar seeding methodology was followed for generating tumor spheroids from primary tumor-derived cell lines.

EdU/PI cell cycle analysis For proliferation/cell cycle analysis, primary tumor-derived cell lines were incubated with 10µM EdU reagent for 2 hours in the incubator. The cells were then harvested and subjected to fixation, permeabilization and click reaction using Base Click EdU flow cytometry kit (Base Click, BCK- FC647-100) were resuspended in Propidium Iodide (PI) (50µg/ml; Sigma, P4170) + RNAse (10µg/ml; Roche, 11119915001) for 2 hours at 37°C and then taken to BD FACS Canto II Analyzer for flow cytometric analysis of proliferation and cell cycle stages. Analysis was performed using FlowJo software.

Transwell migration assay 50,000 untreated or long-term (>20d) TGF -treated cells were suspended in 500µl of DMEM+0.2% FBS and seeded into 24 trans-well migration inserts (Corning, 353097) in duplicates. The bottom chambers were filled with 700µl of DMEM+20% FBS to create a chemo-attractant gradient. The cells

were incubated in a tissue culture incubator at 37°C with 5% CO2. After 18 hours, inserts were fixed with 4% paraformaldehyde for 10 minutes. Cells that had not crossed the membrane were removed with a cotton swab, and cells on the bottom of the membrane were stained with DAPI. Images of five fields per insert were taken with a Leica DMI 4000 microscope and stained cells were counted using an ImageJ software plugin developed in-house.

Promoter reporter assay For TOP/FOP-Flash promoter reporter assays, cells were seeded in triplicates in 24-well plates and next day were co-transfected with 500ng of TOP- or FOP-Flash Firefly luciferase construct and 10ng of Renilla luciferase construct using lipofectamine 3000 (Thermofisher, L3000015). 24 hours post- transfection, medium was changed and 100ng/ml Wnt3a was added for 48 hours. Cells were then prepared for passive lysis and reading using the Dual luciferase Reporter Assay system (Promega, E1960 Technologies; Centro LB 960). Firefly luciferase activity was normalized to the Renilla luciferase activity and expressed as relative light units. For Smad4 promoter reporter assays, cells were co-transfected with 500ng of Smad4 Firefly reporter and 100ng of Renilla luciferase construct using lipofectamine 3000. pGL3-empty vector was used as a control. 24 hours post transfection, cells were treated with 2 ng/ml TGF for another 48 hours following which they were harvested and measured as above.

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Chromatin Immunoprecipitation ChIP experiments were performed as described previously (35). Briefly, crosslinked protein-bound DNA of WT or AE/AE mutant cells was sonicated (Bioruptor, Diagenode) to achieve chromatin fragments of sizes between 100-500 bps. For ChIP of endogenous Pygopus 2 or -catenin, at least 150 s incubated with 10 -catenin antibody (Novus Biologicals, NBP-32239), and immunocomplexes were precipitated with 40 pre-blocked Sepharose Protein A beads (Affi-Prep Protein A Support, Bio-Rad; 1560006). Immunocomplexes were eluted from the beads, de-crosslinked, and genomic DNA was purified by phenol/chloroform extraction and precipitated with sodium acetate. One out of forty of the ChIP sample and 1% of input DNA were used for quantitative RT- PCR. Fold enrichments for specific miR-29a/b1, PDGFR or WIF1 promoter regions were calculated by IP over input samples and normalized to isotype-specific IgG as negative control. Primers targeting different genomic regions of the miR-29a/b1, PDGFR or WIF1 promoters are listed in Suppl. Table I.

Flow cytometry Cells from a 15 cm dish each were trypsinized, washed with FACS buffer (2 % FCS, 2 mM EDTA/PBS) followed by blocking with anti-CD16/CD32 Fcg III/II receptor antibody (Biolegend, 101302) for 5 min at room temperature. Following washing, cells were divided for staining with specific antibodies (CD24-PE, CD29-APC, CD61-BV510) at 1:100 dilution for 20 min on ice. After washing, cells were resuspended in FACS buffer and filtered with a 40 m mesh before applying to flow cytometry. DRAQ7 was added just prior to putting cells on the FACS to mark dead cells. For flow cytometry-based stem cell analysis in mouse tumor tissues, primary tumors from WT or AE/AE mutant mice were collected, minced and subjected to enzymatic digestion first with 1mg/ml Collagenase D (Roche, 11088858001) and then with 0.1mg/ml DNaseI (Roche, 11284932001) under sterile conditions. After blocking with anti-CD16/CD32 antibody, lineage positive cells were removed by staining with biotinylated antibodies against CD45, CD31 and Ter119. Dead cells were then removed using EasySep Dead cell removal (Annexin V) Kit (StemCell Technologies, #17899) on a magnet. The resulting cell suspension was washed with FACS buffer and subjected to staining for stem cell markers using specific antibodies (CD24-PE, CD29-APC, CD61- BV510) as described above. Cells were analyzed using Cytoflex (Beckman Coulter) and data analysis was performed by Flowjo 10.6.2.

Statistical analysis

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Statistical analysis was performed using GraphPad Prism 7.0 software. All data are presented as mean ± S.E.M. p-values < 0.05 were considered statistically significant. All experiments were repeated thrice, unless otherwise stated.

Data and Material Availability The datasets generated and analyzed within the current study are deposited at Gene Expression Omnibus (GEO; RNA sequencing data: accession number GSE130330; ATAC sequencing data: accession number GSE133066). RNA and ATAC-Sequencing data can also be accessed together under super series GSE133068. The cell lines generated within this work will be made available upon contacting the corresponding authors.

Results Pygo2 binding to histones is required for primary tumor growth and distant metastasis formation To assess the biological relevance of Pygo2 binding to H3K4me2/3 histone marks in mammary gland tumor formation, we crossed knock-in mice bearing an Ala-to-Glu point mutation at position 342 (A342E) in the PHD domain of Pygo2 (previously described in (32)) to MMTV-PyMT mice, a well- established mouse model of metastatic breast cancer (33). It has previously been shown that the A342E mutation effectively abolishes the binding of Pygo2 to histones (32). Interestingly, compared to Pygo2+/+ wild-type (WT) mice, Pygo2A342E/+ (AE/+) heterozygous and Pygo2A342E/A342E (AE/AE) homozygous mutant mice had significantly smaller tumors in the mammary glands at 12 weeks of age (Figure 1A, i and ii). This was due to a decrease in the number of proliferating cells in the mutants as assessed by immunofluorescence for phospho-histone 3 (Figure 1B, i and ii). Cell death by apoptosis was unaffected in the mutants as assessed by immunofluorescence for cleaved Caspase-3, an apoptosis marker (Figure S1A, i and ii). Immunohistochemical analysis of tumor sections confirmed that Pygo2 expression levels and localization remained unchanged between Pygo2 wild-type and mutant mice (Figure S1B), indicating that the inability of Pygo2 to bind histones did not affect its nuclear localization. To assess whether the tumors of mutant Pygo2 mice exhibit a proliferation block or reduced cell cycle progression, tumor bearing AE/+ heterozygous and AE/AE homozygous mutant mice were sacrificed at 13, 14 and 15 weeks of age before they reached the legally imposed termination endpoint. The tumor size consistently increased in both hetero- and homozygous mutants at 13, 14 and 15 weeks compared to 12 weeks of age (Figure S1C), suggesting that the mutant tumor cells still proliferated, albeit slower than WT. This proliferation defect in Pygo2 AE/+ and AE/AE mutants was also corroborated in primary tumor-derived WT, AE/+ and AE/AE cell lines by EdU/PI labeling and flow cytometry-based cell cycle analysis. End-point PCR using specific primers on genomic DNA isolated from the tumor-derived cell lines confirmed their genotype (Figure S1D). While 70-80% WT

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cells were found in S phase, only 10-20% AE-mutant cells were found in S phase and 60-70% in G0/G1 phase (Figure S1E). To determine whether Pygo1 could compensate for the loss of Pygo2 histone binding, we analyzed the effect of the Pygo2 AE mutation on tumor growth in PyMT mice carrying a genetic deletion of Pygo1 in mammary tumor cells (Figure S1F, i). Knockout of Pygo1 did not affect tumor growth, neither in WT mice nor in Pygo2 AE/+ and AE/AE mutant mice (Figure S1F, ii), suggesting that Pygo1 does not play an additive or compensatory role to Pygo2 in PyMT mammary tumors. To assess the differentiation status of tumors between Pygo2 WT and AE-mutant mice, we analyzed the expression of several differentiation markers such as ER , ER , Pgr, Gata3, Wap and Fabp4. Increased mRNA expression of these markers indicated that loss of Pygo2-histone binding leads to increased differentiation of tumors (Figure 1C). Tumor staging by quantifying the area fractions of hyperplasia, adenoma and carcinoma on Hematoxylin and Eosin-stained histological sections also confirmed the differentiated status of AE-mutant tumors (Figure 1D, i). Interestingly, WT mice developed significantly more carcinoma and less hyperplasia as compared to AE-mutant mice (Figure 1D, ii). This change in malignant phenotype was further corroborated by the observation that WT mice formed significantly higher numbers of metastases in the lungs as compared to AE-mutant mice (Figure 1E, i). The metastasis index (number of metastases in relation to primary tumor mass) and the incidence of metastasis was also significantly higher in the WT mice as compared to the AE-mutant mice (Figure 1E, ii and iii). Collectively, these results suggest that the Pygo2-histone interaction is important for tumor growth, de-differentiation, malignant tumor progression and for metastasis formation. Notably, the presence of one AE mutant allele was sufficient to induce the mutant phenotypes, indicating a dominant-negative function of the AE mutant protein. Interestingly, in support of previous findings (26), analysis of the TCGA dataset for human breast cancer patients revealed that PYGO2 expression was significantly higher in primary tumors and metastatic lesions as compared to normal breast tissue in both, matched as well as unmatched samples (Figure S1G), suggesting that PYGO2 might also be important in malignant progression of breast tumors in humans. A significant difference in PYGO2 levels across breast cancer subtypes (Figure S1H, i), TNM stages 1-3 (Figure S1H, ii), BRE grades 1-3 (Figure S1H, iii), or in patient samples with lymph node metastasis positivity (Figure S1H, iv) was, however, not observed.

Pygo2 binding to histones is required for -catenin-dependent Wnt signaling Since Pygo2 is a component of the nuclear -catenin transcriptional complex, we asked whether the inability of Pygo2 to bind histones would affect the output of Wnt/ -catenin signaling. Tumor lysates from primary tumors of WT and AE/+ and AE/AE mutant mice were analyzed by immunoblotting for the expression of several known Wnt target genes. Interestingly, the expression of well-established

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Wnt targets, such as Axin-2, c-Myc, and cyclin-D1, was found significantly reduced in AE/AE mutant tumors as compared to WT tumors (Figure 2A, i and ii). Further, non-phosphorylated, active - catenin levels were also found to be significantly higher in the WT than in the AE/AE mutant tumors (Figure 2A, i and ii). Immunohistochemical analysis of histological sections for c-Myc confirmed its higher expression in WT tumors as compared to AE/AE mutant tumors (Figure 2B). These results suggest that Pygo2 binding to histones contributes to the signaling output of Wnt/ -catenin in mammary tumors. To corroborate these findings, primary tumor-derived cell lines were treated with Wnt3a to activate canonical Wnt signaling. RT-qPCR analysis revealed a significantly higher upregulation of Wnt target genes, such as Axin2 and cyclin-D1 (Ccnd1), in the WT cells as compared to the AE- mutant cells (Figure 2C). Similar results were observed for Wnt3a-treated cells by immunoblotting analysis for c-Myc, cyclin-D1 and active -catenin (Figure S2A). Treatment with the Wnt agonist, CHIR99021, which activates the Wnt pathway by selectively inhibiting GSK3 , also led to an upregulation of the Wnt target genes Axin2 and Ccnd1 at a much higher extent in WT cells as compared to the mutant cells (Figure S2B). Additionally, TOP/FOP-Flash reporter assays also revealed an increased TCF/LEF reporter activity upon Wnt3a treatment in WT cells compared with mutant cells (Figure 2D). Finally, active -catenin could be detected only in nuclei of tumor spheroids formed by the WT primary tumor cells as compared to mutant tumor spheroids (Figure S2C; Suppl. Videos 1 and 2). Likewise, Axin-2 was expressed only in tumor spheroids formed by WT primary cells as compared to spheroids formed by mutant primary cells (Figure S2D; Suppl. Videos 3 and 4). RNA sequencing of the primary tumor-derived cell lines and subsequent gene ontology (GO) analysis revealed a significant upregulation of pathways involved in the negative regulation of canonical Wnt signaling in the mutant cell line compared to the WT cell line (Figure S2E). This is due to a significant upregulation of several Wnt signaling antagonists, including WIF1, sFRP-2, Dkk- 3, Kremen-1, Wnt-5a and APCDD1, upon loss of Pygo2-histone binding in the mutant cells (Suppl. Table II). Analysis of ATAC sequencing further corroborated the above findings, where open chromatin was observed near the transcriptional start sites (TSS) of several genes encoding for Wnt signaling antagonists, including Wif1, Sfrp2, Wnt5a and Apcdd1 (Figure S2F). These findings could also be extended to human cell lines with different driver mutations, where an siRNA mediated knockdown of Pygo2 in BT-474, BT-549 and MDA-MB-231 cells prevented a Wnt3a-mediated increase in Wnt target genes AXIN-2 and CYCLIN-D1 (Figure 2E). Further, analysis of the expression of several Wnt pathway genes in the TCGA database of breast cancer patients revealed an elevated expression of PORCN, NOTUM, LEF1, AXIN-1 and CYCLIN- D1 in patients with high PYGO2 expression compared to patients with low PYGO2 expression. Interestingly, patients with low levels of PYGO2, which implicitly would also have less interaction

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with histones, were found to have elevated levels of several WNT antagonist genes, such as APCDD1, DKK-2, DKK-3, DKK-4, sFRP1, sFRP4, sFRP5 and WIF1 corroborating our observations in the Pygo2 AE/AE mutant mice (Figure S2G). Collectively, these results suggest that the Pygo2-histone interaction contributes to the signaling output of -catenin in mammary tumors of MMTV-PyMT mice. Further, the changes in can be explained, at least in part, by a decrease in Wnt/ -catenin signaling in tumors.

Pygo2 binding to histones is required for TGF signaling and EMT Pathway analysis of the RNA sequencing results also suggested a significant downregulation of TGF signaling pathway in the AE/AE mutant cells as compared to the WT cells at the basal level (Figure S2E). Interestingly, the transcriptomic analysis revealed a decrease in the expression of TGF receptor-1 (TGF R1), Smad1, 2, 3, 4, and 5 and an upregulation of inhibitory Smad6 in the mutant cells as compared to WT cells (Suppl. Table II). Moreover, pathway analysis of RNA sequencing results performed on TGF -treated WT or AE/AE mutant cells revealed a significant upregulation of the TGF signaling pathway only in the WT cells (Figure S3A). To validate the functional defect in TGF signaling upon loss of Pygo2-histone binding, we performed a promoter reporter assay for Smad4 activity. TGF treatment led to a significant increase in Smad4-induced promoter reporter activity only in WT cells as compared to AE/AE mutant cells (Figure 3A). To assess whether the defect in TGF signaling in Pygo2 mutant cells could affect the induction of an epithelial-mesenchymal transition (EMT), we compared TGF -treated WT, AE/+ and AE/AE mutant cells. While, TGF treatment of Pygo2 WT cells induced a complete EMT, as observed by a clear change in cell morphology from epithelial to fully mesenchymal over 20 days, such a morphological change was not appreciable in the AE/AE mutant cells (Figure S3B). Immunocytochemical analysis revealed the loss of E-cadherin expression, a prototype epithelial marker, and an upregulation of Vimentin, a mesenchymal marker, only in WT cells and not in AE- mutant cells (Figure 3B). Interestingly, the level of Vimentin expression in the absence of TGF is higher in AE-mutant cells, as compared to WT cells, which could not be further increased by TGF treatment. RNA expression analysis by RT-qPCR also revealed the increased expression of the mesenchymal markers N-cadherin, NCAM-1, Fibronectin, Vimentin, Zeb1, and Zeb2 in 4 and 20 days TGF -treated WT cells (Figure 3C; Figure S3C). Further, compared to the AE/AE mutant cells, the fold-increase in the expression of EMT markers was significantly higher in the WT cells (Figure S3C). These results were also supported by performing a Transwell Boyden chamber migration assay with WT and AE-mutant cells treated with TGF for 20 days. While the WT cells

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increased their migratory ability by ~7 fold compared to untreated (UT) cells, this increase in migration was strongly reduced in TGF -treated AE/+ and AE/AE mutant cells (Figure 3D). These findings were validated in human cell lines where siRNA-mediated ablation of PYGO2 expression prevented the TGF -induced increase in the EMT-related markers Fibronectin and Vimentin in BT-474 and BT-549 cell lines (Figure 3E, i and ii). Similar results were observed in Hs- 578T and MDA-MB-231 cells (Figure S3D, i and ii). Notably, knockdown of PYGO2 also led to a decrease in the basal expression of EMT-related markers in BT-474, BT-549 and MDA-MB-231 (Figure 3E, i and ii; Figure S3D, ii). Further, we performed Gene Set Enrichment Analysis (GSEA) on TCGA gene expression data from human breast cancer patients with low or high levels of PYGO2 expression. As expected, the signature for genes that are downregulated during EMT, such as epithelial genes, was enriched in PYGO2 high-expressing patient tumors (Figure S3Ei). However, the genes that are typically upregulated in EMT, such as the mesenchymal genes, were found enriched in the PYGO2 low-expressing group (Figure S3Eii). Collectively, these results suggest that Pygo2-histone interaction is critical for the activation of TGF signaling, which in turn induces EMT and drives malignant tumor progression. Hence, a defect in the TGF signaling pathway can, at least in part, explain the observation of reduced tumor malignancy and fewer metastases in mice carrying the Pygo2-histone binding mutation.

Pygo2 binding to histones is required to suppress differentiation pathways Pathway analysis of the RNA and ATAC sequencing data described above suggested that the Pygo2- histone interaction was required to suppress pathways involved in cellular differentiation, such as PDGF receptor signaling (Figure S2E and S4A). Since histopathological analysis had revealed a more differentiated phenotype in AE/AE mutant tumors (Figure 1C and 1D ii), we investigated the activation of differentiation pathways in tumors with defective Pygo2-histone interaction. Notably, RNA and ATAC sequencing revealed increased mRNA expression (Figure S4B) and open chromatin at the transcriptional start sites or gene bodies (Figure S4C) of genes encoding for most PDGF ligands and their receptors PDGFR and PDGFR in AE/AE mutant cells. Further, RT-qPCR analysis confirmed the significant upregulation of PDGF ligands and PDGF receptors in tumors of AE-mutant compared to WT mice (Figure 4A). Immunohistochemical analysis also revealed that PDGFR expression was specifically upregulated in the differentiated cells of AE-mutant tumors, while it was expressed in the stromal cells of both AE-mutant and WT tumors (Figure 4B). Stimulation with PDGF-BB ligand revealed a phosphorylation and hence activation of PDGFR only in AE/AE mutant cells (Figure 4C; Figure S4D). This phosphorylation decreased in a dose- dependent manner upon treatment with the PDGFR inhibitor AG1296 (Figure 4C). PDGFR signaling has previously been shown to drive fibrotic disease by increasing the production of collagens, glycosaminoglycans and by ECM remodeling, and its activity has been

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shown to be promoted by binding to heparan sulfate (36). In fact, amongst the top significant pathways regulated in AE mutant cells, pathways involved in ECM organization and metabolism of carbohydrates were enriched (Figure S2E and S4A). Indeed, RNA sequencing revealed that the expression of genes involved in ECM and collagen formation, such as Col2a1, Col4a1, Col4a2, Col9a2, Itga8, 6, 11 and Itgb3, Laminins (Lama4, Lamb3) and fibulins (Fbln2, Fbln5), and genes involved in glycosaminoglycan metabolism, such as Hpse2, Hexa, Hs6st1, B4galt4, Hs3st3a1, Hs3st3b1, Gpc4 and Gpc6, was upregulated in AE mutant cells as compared to WT cells (Suppl. Table II). This observation was also confirmed by an increased production of Fibronectin, N- cadherin (Figure S4E, i and ii) and collagen deposition (Figures S4F, i and ii) in the stromal compartment of AE/AE mutant tumors, suggesting that they were indeed more fibrotic. In fact, many mutant tumors at the time of resection were found to have fluid-filled cysts, a phenotype reminiscent of benign fibrocystic disease of the breast in human patients (37). RNA and ATAC sequencing also revealed a significant upregulation of the Notch pathway in the AE-mutant cells (Figure S2E and S4A), in support of an earlier study, where the genetic deletion of Pygo2 in mammary glands was found to activate Notch signaling (28). In keeping with these findings, qRT-PCR analysis confirmed a higher expression of Notch receptors and their ligands in tumor lysates of mutant mice as compared to WT mice (Figure S4G). In contrast, expression of the canonical Wnt target gene Axin2 was found reduced in AE-mutant tumors as compared to WT tumors (Figure S4G). Immunohistochemical analysis confirmed the higher expression of Notch 3 in the homozygous AE/AE mutant tumors compared to WT tumors (Figure S4H). Notably, while Notch 3 was localized at the cell membranes in tumor spheroids formed by WT tumor cells, it was cytoplasmic or nuclear in the tumor spheroids formed by primary cells derived from the AE-mutant tumors (Figure S4I), suggesting an activation of Notch signaling in the Pygo2-mutant tumor cells. Analysis of the expression of PDGF receptors and ligands in the TCGA database of breast cancer revealed their elevated expression in patients with low PYGO2 expression compared to patients with high PYGO2 expression (Figure 4D). These results were also corroborated by performing a GSEA for PDGFR pathways in human breast cancer patients, where it was found elevated in the group with low PYGO2 expression (Figure S4J). Further supporting these results, PYGO2 expression was found to be inversely correlated to the expression of PDGFR and NOTCH 3 across 825 breast cancer patients of the Cancer Genome Atlas Network using cBioPortal (38) (Figure S4K). Collectively, these results strongly suggest that Pygo2 interaction with histones is required to suppress differentiation drivers like PDGFR and Notch signaling pathways.

Pygo2 binding to histones is required to repress luminal cell fate It has previously been demonstrated that differentiation drivers like Notch and PDGFR signaling promote the luminal cell fate associated with mammary gland differentiation (28,39). Since, these pathways were activated upon loss of Pygo2 interaction with histones, we asked whether this loss of

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function mutation also promoted breast tumor cells to assume a luminal cell fate. Indeed, immunofluorescence analysis of WT and AE/AE tumors revealed a significant upregulation in the expression of the luminal cell markers cytokeratin-8 and 18 (CK-8/18) in the AE/AE mutant tumors as compared with the WT tumors, while the expression of the basal cell marker cytokeratin-14 (CK- 14) was comparable between the different genotypes (Figure 5A). Interestingly, while tumor spheroids formed by WT primary cells expressed both CK-14 and CK-8/18, spheroids formed by AE/AE mutant primary cells consisted almost exclusively of CK-8/18 expressing luminal cells (Figure 5B). Moreover, tumor spheroids formed by AE/AE mutant cells were primarily hollow (empty lumen) in contrast to solid spheroids formed by WT primary cells (Figure 5C; Figure S5A, i). Yet, no differences were observed in the sphere formation efficiency between spheroids formed by the WT and the AE/AE mutants over three passages (Figure S5A, ii). GSEA further revealed an upregulation of a luminal gene expression signature in mutant cell lines compared with WT tumor- derived cell lines (Figure S5B), lending further support to a luminal fate of cells upon loss of Pygo2- histone interaction. Interestingly, RNA sequencing also revealed a significant decrease in the expression of several stemness markers, such as CD24a, Prom1 (CD133), CD1d and Klf4 in AE/AE mutant cells compared to WT cells (Suppl. Table II). In contrast, expression of Itgb3 (CD61), a marker for luminal progenitors was found significantly higher in mutant as compared to the WT cells (Suppl. Table II). To further assess the effect of loss of Pygo2-histone binding on cell stemness, we performed flow cytometry analysis for the expression of basal (CD24+CD29hi), luminal progenitor (CD24+CD29loCD61+) and mature luminal (CD24+CD29lo) cell markers (40). While all cells were CD24+ in the WT cell line, a small CD24- population existed in the Pygo2-mutant cell line (Figure 5D, i). Further, WT cells were enriched in CD29hiCD61lo expression, while 92.9% of the AE/AE mutant cells were CD29+CD61hi, suggesting a strong enrichment in luminal progenitors (Figure 5D, ii and iii). In addition, flow cytometry analysis for the stem cell markers CD24, CD29, and CD61 in the lineage-negative (Lin-) population of primary tumors revealed a ~ two times larger CD24loCD29+ population in AE/AE mutant tumors as compared to WT tumors (Figure S5C), again suggesting an enrichment for luminal cells. In contrast, no difference in the expression of CD61was apparent between mutant and WT primary tumors. Collectively, and in keeping with previous studies (28), our results suggest that Pygo2-histone binding is important for expansion of basal mammary stem cells, and a loss in this interaction promotes the expansion of luminal cells. In line with a role of PDGFR signaling in luminal cell differentiation, the specific inhibition of PDGFR signaling with AG1296 led to a decrease in the number of hollow spheroids formed by AE/AE mutant cell lines, while it did not affect the phenotype of the solid spheroids formed by WT tumor cell lines (Figure 5E, i and ii; Figure S5D, i and ii). In fact, PDGFR signaling inhibition also led to a significant decrease in the expression of the differentiation markers Gata3 and Fabp4 in tumor spheroids generated by AE/AE mutant cells (Figure S5E). Inhibition of Notch signaling with the - secretase inhibitor DAPT, also resulted in the formation of solid spheroids by AE/AE mutant cells,

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however, this was observed only in one of the two AE/AE mutant cell lines (Figure S5F). Finally, siRNA-mediated ablation of PDGFR expression in AE/AE mutant cells also led to a significant decrease in the expression of genes involved in ECM organization, such as Col2a1, Col4a1, Col9a2, Bcan, Sdc3 and Chst11, indicating that the desmoplastic/ fibrotic phenotype observed upon loss of Pygo2-histone binding was at least in part regulated by PDGFR signaling (Figure S5G). Collectively, these results suggest that in mammary tumor cells, Pygo2-histone interaction suppresses differentiation-inducing pathways driven by PDGFR and Notch signaling. Upon activation, they drive breast cancer cells to assume a differentiated luminal, less aggressive and less metastatic phenotype.

Pygo2 suppresses cell differentiation through miR-29 expression To mechanistically explain how Pygo2-histone interaction suppressed pathways which induce differentiation, we performed Integrated Motif Activity Response Analysis (ISMARA) on the RNA sequencing transcriptomic data generated with the WT and AE-mutant cell lines. Though motifs for Snai1 and Tfdp1 transcription factors were found to be highly enriched (Figure S6A), their knockdown was found not to affect the expression of PDGFR or PDGFR in AE-mutant cells (Figure S6B, i and ii). Yet, ISMARA analysis also revealed an enrichment for motifs of miR-29-3p family members (Figure S6A). Notably, in addition to PDGFR , miR-29 family members were predicted to target several genes regulated in our data set, including many collagens (involved in ECM remodeling), Wisp1 (involved in fibrosis), and Atp1b1 (inhibits breast cancer invasion) (Figure S6C). Hence, we probed the functional involvement of miR-29 in defining the phenotypic changes observed in the Pygo2 mutant cells. Indeed, all three members of the miR-29 family (miR-29a-3p, miR-29b-3p, miR-29c-3p) were found to be significantly downregulated in two independent AE/AE mutant cell lines as compared to WT cells (Figure 6A). Interestingly, the forced transient expression of all the three miR-29-3p family members in AE-mutant cells led to a significant decrease in the levels of PDGFR and PDGFR (Figure 6B; Figure S6D, i). Moreover, the forced expression of miR-29 family members also led to a significant decrease in the expression of the Wnt antagonist WIF1 (Figure S6D, ii), yet likely due to an indirect effect, since WIF1 does not have a seed sequence for miR- . In contrast, expression of another Wnt antagonist, sFRP-2, was unaffected by the expression of any of the miR-29 family members (Figure S6D, ii). Immunoblotting analysis for PDGFR and WIF1 confirmed their decrease in the mutant cells upon overexpression of miR-29 family members (Figure S6E). The above findings were also confirmed in an independently generated mutant cell line (Figure S6F). Transient overexpression of miR-29 family members in long-term TGF -treated AE/AE mutant cells was able to significantly increase their migratory potential (Figure 6C), further highlighting the differentiation-repressing function of miR-

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29 family members. In contrast, cell proliferation was increased only marginally by miR-29 expression (Figure S7A). To further assess the effect of miR-29 overexpression on tumor growth, differentiation and metastasis formation, we generated AE/AE mutant cell lines stably overexpressing either control miRNA or miR-29 a/b/c family members (Figure S7B, i). As with the transient overexpression, stable overexpression of miR-29 family also led to a decrease in PDGFR and WIF1 levels, though the decrease in WIF1 was not statistically significant (Figure S7B, ii). AE/AE mutant cells stably expressing either miR-Ctrl or miR-29 family members were injected orthotopically into mammary gland 9 of female NSG mice and assessed for tumor growth weekly. Interestingly, the mice overexpressing miR-29a-3p and miR-29b-3p exhibited a significantly faster tumor growth rate (Figure 6D). The tumor weights at the time of termination were also higher in the mice overexpressing miR-29a and b (Figure S7C). Histopathological analysis of the lungs in these mice indicated a trend to higher numbers of metastatic lesions in the group overexpressing miR-29a, however without statistical significance (Figure S7D). Interestingly, the tumors of all the 3 groups overexpressing miR-29 family members formed de-differentiated tumors with cells invading into the surrounding stroma, while the control group developed highly differentiated tumors (Figure 6E). To assess whether miR-29 expression could also affect metastatic outgrowth, Pygo2 AE/AE cells stably expressing either miR-Ctrl or miR-29 family members were injected into the tail veins of NSG mice. After 4 weeks, mice were sacrificed, and histological sections of their lungs were analyzed for metastatic lesions. This analysis revealed a significantly higher number of metastases in the mice expressing miR-29b and c, as compared to the miR-Ctrl group (Figure 6F). Further, we assessed the expression of miR-29 family members in human breast cancer patients using the TCGA dataset. Low expression of miR-29a and miR-29b seemed to correlate with luminal/ low BRE grade of breast cancer patients however, this association was not statistically significant (Figure S7E). On the contrary, low expression of miR-29c was found to correlate significantly with basal/ high BRE grade of breast cancer patients (Figure S7E). Collectively, these results suggest that, Pygo2-histone interaction suppresses differentiation inducing pathways, such as PDGFR signaling, at least in part through induction of miR-29 expression. As a result, Pygo2-expressing tumors are proliferative, de-differentiated and metastatic.

Pygo2 and -catenin directly induce miR-29 expression To assess if Pygo2 and -catenin could directly regulate the expression of miR-29, PDGFR , and Wnt antagonists like WIF1, we assessed their differential binding at the TCF/LEF motifs in the promoter regions of miR-29a/b1, PDGFR and WIF1 in WT and AE/AE mutant tumor-derived cell lines using chromatin immunoprecipitation (ChIP) analysis. Interestingly, a significant decrease in binding of -catenin was observed at 3 different regions of the miR-29a/b1 promoter (region 1, 2 and

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7) in AE/AE mutant cells as compared to the WT cells (Figure 6G i). A similar trend was also observed with Pygo2 binding at 2 of these 3 sites (region 2 and 7), although the decrease was not statistically significant. These results suggest, that Pygo2 and -catenin can directly bind specific regions of the miR-29a/b1 promoter, thereby regulating its expression (Figure 6G i). In a comparable manner, both Pygo2 and -catenin occupancy decreased at regions 3 and 4 of the PDGFR gene promoter in AE/AE mutant cells as compared to WT cells, yet with statistical significance only at region 4 (Figure 6G ii). A reduced binding of Pygo2 occupancy (but not -catenin) was also observed at region 1 of the PDGFR gene promoter. While Pygo2 occupancy was higher at region 2 of the WIF1 gene promoter, binding of - catenin was significantly decreased at region 3 of the WIF1 promoter, suggesting that some regions are regulated independently between Pygo2 or -catenin (Figure S7F). An increase in Pygo2 occupancy at region 2 of the WIF1 promoter suggests a mechanism of how Wnt/ -catenin signaling output is decreased in a feedback loop on loss of Pygo2-histone interaction. The decreased occupancy of both Pygo2 and -catenin at the Notch3 gene promoter in AE/AE mutant cells as compared to WT cells (28) was used as a positive control for the ChIP experiment (Figure S7F). Collectively, these results imply, that Pygo2 and -catenin can directly regulate the expression of PDGFR , WIF1 and possibly other relevant genes whose expression is also affected on loss of Pygo2-histone binding, while miR-29 can in part regulate the expression of PDGFR and other target genes.

Discussion Pygopus proteins, initially discovered as co-transcriptional activators for -catenin in canonical Wnt signaling, have recently been shown to also function as chromatin effectors in a Wnt-dependent and independent fashion (11-16). Yet, whether Pygo2 interaction with chromatin, by binding histone marks, plays only an auxiliary role in potentiating Wnt/ -catenin signaling or has a more direct functional relevance in aiding malignant tumor progression has remained elusive. In our study, using a knock-in mouse model, where a point mutation in the PHD domain prevents Pygo2 to effectively bind histones (32), we demonstrate that the interaction of Pygo2 with histones is critical for breast primary tumor growth, malignant progression and metastasis formation by activating canonical Wnt/ -catenin signaling and by repressing tumor cell differentiation. Consistent with a role of Pygo2 in proliferation (20), our results demonstrate that loss of Pygo2-histone binding results in smaller tumors due to a proliferation defect rather than an increase in apoptosis. Interestingly, the phenotype was not accentuated by an additional loss of Pygo1 confirming previous observations that Pygo1 is functionally not as relevant as Pygo2 (9-12). Importantly, loss of Pygo2-histone binding decreased Wnt/ -catenin signaling output, as observed by a decrease in Wnt target gene expression in mutant tumors, the inability of mutant tumor cell lines to increase Wnt target gene expression or by TCF/LEF promoter-reporter activity upon stimulation with Wnt3a. Hence, the

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results suggest that the reduced tumor cell proliferation, malignant progression and metastasis formation observed in Pygo2 mutant tumors are due to a decrease in Wnt/ -catenin signaling output. The results also indicate that the loss of Pygo2-histone binding phenocopies the reported knockout of Pygo2 to a large extent, suggesting that the observations of decreased proliferation and differentiation to a luminal breast cancer subtype were primarily due to a loss of Pygo2 binding to histones. We find that Pygo2-histone binding is important for malignant tumor progression, i.e. the generation of de-differentiated, aggressive carcinomas and formation of distant lung metastasis. Further, Pygo2-histone binding also seems to be important for cells to undergo a complete EMT, an important pathological response in cancer cells that enables them to eventually invade and metastasize (41,42). RNA sequencing data and functional analysis reveals that AE/AE mutant tumor cells exert decreased TGF /Smad signaling. This defect is probably caused by diminished expression of TGF R-1 and several regulatory Smads and an increase in the expression of inhibitory Smad6 in the mutant cells. The lack of efficient TGF signaling may thus explain the reduced malignant tumor progression and metastasis formation of the Pygo2 mutant tumors. These results are in support of a recent study where overexpression of Pygo2 in prostate cancer cell lines was found to increase regional lymph node invasion in transplantation experiments (25). Collectively, the data suggest that Pygo2 in addition to proliferation, can also affect the malignant phenotype of a cancer cell. The complete ablation of Pygo2 expression in the normal mammary gland has been previously shown to promote luminal differentiation of the mammary cells in part due to activation of the Notch 3 pathway (28). In corroboration of these findings, we find that the binding of Pygo2 to histones is required to suppress the Notch pathway also in the MMTV-PyMT mouse model of breast cancer. Yet, in addition, our results reveal that Pygo2-histone binding is also critical to suppress other pathways known to induce cellular differentiation, importantly here, the PDGFR signaling pathway. These findings are strongly supported by the observation that in human breast cancer patients, Pygo2 expression correlates inversely with the expression of Notch 3 or several PDGF receptors and ligands. Functionally important, the inhibition of PDGFR could repress the luminal and differentiated cell fate of the Pygo2 mutant tumor cells. Also, the effect of PDGFR inhibition is stronger than Notch inhibition in our experimental systems, as the luminal fate of only one mutant cell line was repressed by Notch inhibition, while PDGFR inhibition was effective in two independent Pygo2 mutant cell lines. In support of these results, PDGFR signaling has previously been shown to promote proliferation of luminal breast cancer cells in the absence of estrogens (39). In another study, a significant association between high PDGF signature score and poor survival has been observed in LN+ (Lymph node +), ER+ (Estrogen Receptor +), luminal A, and grade 1 and 2 breast cancers (43). While autocrine PDGFR signaling has been implicated in glioblastomas and sarcomas (44), PDGFR signaling seems to require the cooperation of other genetic alterations to induce a fully malignant phenotype (45). For instance, autocrine PDGFR signaling, in conjunction with oncogenic Ras,

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appears to hyperactivate PI3K and drive malignant tumor progression of breast cancer in an MMTV- Neu mouse model (46). All these studies support our findings that PDGFR signaling promotes the growth of luminal breast cells and that additional pathways, such as activated Wnt signaling or additional genetic alterations, are required to suppress cancer cell differentiation and promote the development of de-differentiated, aggressive breast cancers. Amongst the various phenotypic changes, loss of Pygo2-histone binding also resulted in the formation of fibrotic/desmoplastic tumors as observed by a significant increase in fibronectin, N- cadherin and collagen deposition in the stromal compartment. RNA and ATAC sequencing analysis revealed an enrichment of biological processes involving ECM reorganization and metabolism of carbohydrates, including glycosaminoglycans, heparan sulfate, chondroitin sulfate and keratan sulfate. In fact, significant upregulation of several collagens (Col2a1, Col4a1, Col4a2, Col9a2), integrins (Itgb3, Itga11), laminins (Lama4, Lamb3, Lamc1), Adamts2 metalloproteinase, heparanase Hpse2, proteoglycans (Sdc3, Bcan), and proteoglycan sulfotransferases (Chst11, Chst12, Hs3st3a1, Hs3st3b1, Hs6st1) suggest active ECM remodeling contributing to the fibrotic phenotype in the mutants (Suppl. Table II and III). Moreover, Wnt1-inducible signaling pathway protein 1 (WISP1), a protein involved in fibrosis, is also found to be highly upregulated in Pygo2 mutant cells (Suppl. Table II). Activation of autocrine and paracrine PDGFR signaling, due to an elevated level of PDGFR in both, tumor and stromal cells might be responsible for the desmoplastic reaction by recruiting stromal fibroblasts and promoting stromal depositions and ECM remodeling in the mutant tumors as has been described previously (47). However, while tumor fibrosis has often been shown to foster tumor development by itself, it is not a driver of carcinogenesis (48). Fibrocystic tumors of benign nature are in fact often found in breast cancer patients (49). Interestingly, a study using pancreatic ductal adenocarcinoma (PDAC) has reported that at both early and late stages of pancreatic cancer, fibrosis- associated tumor stroma constitutes a protective response from the host rather than exerting an oncogenic supportive role (50). In our study, the Pygo2 mutant tumors are desmoplastic in their stroma, but the tumor cells themselves are not de-differentiated. Notably, treatment of Pygo2 AE/AE mutant cells with Wnt3a significantly downregulated the expression of several ECM remodeling genes (Suppl. Table III). In addition, knockdown of PDGFR in AE/AE mutant cells caused a significant decrease in the expression of genes involved in ECM reorganization, indicating that, upon loss of Pygo2-histone interaction, decreased Wnt activity and increased PDGFR signaling are at least in part responsible for the fibrotic stroma phenotype. Finally, we found that the upregulation of PDGFR signaling and a decrease in tumor growth and metastases formation caused by the loss of Pygo2-histone binding is at least in part due to a decrease in the levels of miR-29-3p family members. Notably, the expression of miR-29 family members is directly regulated by canonical Pygo2-dependent Wnt signaling. On the other hand, miR- 29 family members directly repress the expression of PDGFR , thus explaining the upregulated

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expression of PDGFR and a luminal tumor cell fate in addition to desmoplasia upon loss of Pygo2 function. Interestingly, miR-29 is since it has been shown to repress genes involved in ECM remodeling and collagen biogenesis. Moreover, Col4a2 and Atp1b1, known targets of miR-29 and previously shown to inhibit breast cancer invasion, are found to be highly upregulated in the Pygo2 AE-mutant cells (Suppl. Table II). Importantly, miR-29 has previously been shown to be transcriptionally activated by -catenin and in turn to target Wnt signaling antagonists, such as Dkk-1, sFRP-2 and Kremen-2, thereby augmenting Wnt/ -catenin signaling (51). Therefore, we propose that Pygo2 binding to histones potentiates Wnt/ -catenin signaling and drives the expression of miR-29 which in turn suppresses differentiation-inducing pathways (Figure 7). Further, the loss of Pygo2-histone binding also facilitates an increase in the expression of several Wnt antagonist genes including WIF1 and potentially others. While we show this could be in part due to differential binding of Pygo2 and -catenin at their promoter regions, we speculate that this could also be due to relieving the repression of an activator for Wnt antagonist genes explaining how this inhibitory feedback loop further decreases the Wnt/ -catenin pathway output. This mechanistic hierarchy also explains how the AE knock-in mutation of Pygo2 regulates Wnt/ -catenin signaling (Figure 7). Hence, we envisage an attractive therapeutic opportunity by specifically targeting the binding site in the PHD of Pygo2 to prevent Pygo2-histone interaction. Our results predict that such therapeutic treatment will repress primary tumor growth, malignant tumor progression and metastasis formation. Moreover, the remaining smaller, differentiated tumors may be more readily and successfully treated with conventional chemotherapy or targeted therapy, possibly by PDGFR inhibition.

Acknowledgements We thank P. Lorentz and the DBM microscopy facility (DBM, University of Basel) for support with microscopy, E. Panoussis, the DBM Animal Facility and D. Büchel for technical support in animal experimentation, U. Schmieder for mice genotyping, I. Galm for technical support with sectioning and staining of lung sections and T. Bürglin for help with FACS-related experiments. We are grateful to C. Beisel, K. Eschbach and the Genomics Facility Basel for library preparation and next-generation RNA and ATAC sequencing. Financial support: We acknowledge funding for this work by the SystemsX.ch MTD project MetastasiX (2014/268), a project grant by the Swiss National Science Foundation (310030B_163471), a Sinergia Grant by the Swiss National Science Foundation (CRSII3_136274), the Swiss Cancer League (KFS-3479-08-2014), and the Krebsliga Beider Basel (KlbB-4469-03- 2018).

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Figure Legends

Figure 1. Pygo2 binding to histones is required for primary tumor growth and distant metastasis formation. (A) (i) Photomicrographs of tumors formed in mammary glands (MG) 2/3 and 4 of Pygo2+/+ (+/+), Pygo2A342E/+ (AE/+) and Pygo2A342E/A342E (AE/AE) mice. (ii) Graph represents the total weight of all mammary gland tumors formed by the +/+, AE/+ and AE/AE mice at the time of sacrifice at 12 weeks of age. N=30 for +/+ mice, N=35 for AE/+ mice, and N=23 for AE/AE mice. Data are represented as mean ± SEM. (B) (i) Immunofluorescence microscopy analysis for phospho-histone 3 (pH3; green) in tumor sections of +/+, AE/+ and AE/AE mice. Nuclei are counterstained blue with DAPI. Scale bar=50µm. (ii) Graph represents the quantification of pH3-positive cells in (i). Each dot represents the average number of pH3-positive cells in each mouse. N=10 mice per genotype (1 tumor per mouse).

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(C) Quantitative RT-PCR analysis of RNA isolated from whole tumor lysates of +/+, AE/+ and AE/AE mice to assess expression levels of the indicated differentiation markers. Ct values in the +/+ group were averaged and normalized to 1. Fold-change was calculated over WT (+/+) mice. N=10 mice for +/+ and AE/AE groups and N=6 for AE/+ mice. Data are represented as mean ± SEM. (D) (i) Photomicrograph represents a hematoxylin and eosin stained tumor section of the mammary gland from an MMTV-PyMT mouse showing different stages of breast cancer progression from hyperplasia to adenoma to carcinoma. (ii) Graphs represent the quantification of the areas of hyperplasia, adenoma or carcinoma as a fraction of the total tumor area in the mammary gland 2/3 (left panel) and 4 (right panel). N=10 mice per genotype. Data are represented as mean ± SEM. (E) (i) Quantification of the number of metastases formed in the lungs of +/+, AE/+ and AE/AE tumor bearing mice. Each dot represents the total number of lung metastases per mouse. N=30 for +/+ mice, N=35 for AE/+ mice and N=23 for AE/AE mice. Data are represented as mean ± SEM. Significance analysis was performed using non-parametric, multiple comparisons Kruskal-Wallis test. *, p-value < 0.05. (ii) Graph represents the metastasis index calculated as the total number of lung metastases divided by the total primary tumor weight of that mouse. Data are represented as mean ± SEM. (iii) Graph shows the incidence of metastasis calculated as the number of mice with metastasis divided by total number of mice analyzed per genotype. See also Figure S1.

Figure 2. Pygo2 binding to histones is required for -catenin-dependent Wnt signaling. (A)(i and ii) Protein lysates from primary tumors of +/+, AE/+ and AE/AE mice were subjected to immunoblotting analysis for non-phosphorylated, active -catenin and indicated Wnt signaling targeted proteins. Representative immunoblots for 4 mice per genotype are shown in (i). The numbers on top of each lane, represent the in house mouse number. Graphs represent the densitometric quantification of Wnt target levels normalized to loading control GAPDH. Densitometric analysis of immunoblots was done using ImageJ. N=8 mice per genotype. Each dot in (ii) represents 1 mouse. Data are represented as mean ± SEM. (B) Immunohistochemical analysis for c-Myc in tumor sections of +/+ and AE/AE mice. Scale bar=50µm. N=4 mice per genotype (1 tumor per mouse). (C) Primary tumor-derived +/+, AE/+ and AE/AE cell lines were treated with Wnt3a for 3 days followed by RNA isolation and RT-qPCR for canonical Wnt signaling target genes Axin-2 and cyclin-D1. N=3. One-way ANOVA was performed to assess statistical differences across genotypes in Wnt3a treated samples. Performing a two- -test between untreated and Wnt3a- treated samples in each genotype revealed the increase in Axin-2 mRNA levels in AE/+ and AE/AE cell lines to also be significant. Data are represented as mean ± SEM.

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(D) The graph represents the fold-change in luciferase activity (in arbitrary relative luciferase units; RLU) in primary tumor-derived +/+ and AE/AE cell lines treated with Wnt3a for 2 days and subjected to TOP/FOP-flash reporter assay. N=3. Data are represented as mean ± SEM. (E) Human cell lines BT-474, BT-549 and MDA-MB-231 transiently transfected with control or Pygo2 siRNA were treated with Wnt3a for 2 days followed by RNA isolation and qRT-PCR for mRNAs encoding for Pygo2 and canonical Wnt signaling target genes Axin-2 and cyclin-D1. N=3. Data are represented as mean ± SEM. See also Figure S2 and Suppl. Videos 1 4.

Figure 3. Pygo2 binding to histones is required for TGF signaling and EMT. (A) Primary tumor-derived +/+ and AE/AE cell lines were treated with TGF and subjected to a Smad4 promoter-luciferase reporter assay. Relative Luciferase Units (RLU) of Smad4 promoter containing plasmid was first normalized to the empty vector control. Fold change in Smad4 reporter activity on TGF treatment was then calculated over the untreated control. N=3. Data are represented as mean ± SEM. (B) Primary tumor-derived +/+, AE/+ and AE/AE cell lines were treated with TGF for 4 days followed by immunofluorescence analysis for mesenchymal marker Vimentin and epithelial marker E-cadherin. Nuclei were counterstained blue with DAPI. Pictures for all cell lines were acquired at the same magnification. Scale bar=50µm. N=3. (C) Primary tumor-derived +/+, AE/+ and AE/AE cell lines were treated with TGF for 4 days followed by RNA isolation and RT-qPCR for EMT-related genes. N=4. Data are represented as mean ± SEM. (D) Primary tumor-derived +/+, AE/+ and AE/AE cell lines were treated long-term with TGF (>20 days) and then subjected to Boyden chamber Transwell migration assay. Graph represents the number of cells migrated across the membrane per field. N=3. Data are represented as mean ± SEM. (E) Human breast cancer cell lines (i) BT-474 and (ii) BT-549 transiently transfected with control siRNA or siRNA against Pygo2 were treated with TGF for 2 days followed by RNA isolation and qRT-PCR for the indicated genes. N=3. Data are represented as mean ± SEM. See also Figure S3 and Suppl. Table II.

Figure 4. Pygo2 binding to histones is required to suppress differentiation pathways. (A) Quantitative RT-PCR on RNA isolated from whole tumor lysates of +/+, AE/+ and AE/AE mice to assess expression levels of PDGF ligands and PDGF receptors. Ct values in the +/+ group were averaged and normalized to 1. Fold-change was calculated over WT (+/+) mice. N=10 mice per genotype. Data are represented as mean ± SEM.

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(B) Immunohistochemical analysis for PDGFR in tumor sections of +/+, AE/+ and AE/AE mice. Scale bar=50µm. N=5 mice per genotype, 1 tumor per mouse. (C) Primary tumor-derived +/+ and AE/AE cell lines were treated with increasing concentrations of PDGFR inhibitor (AG1296) for 24 hours followed by ligand stimulation with PDGF-BB for 10 minutes. Immunoblots show the levels of phospho-PDGFR (p-PDGFR ) and total-PDGFR (t-PDGFR ) levels. Total-AKT (t-AKT) was used as a loading control. N=3. (D) Heatmap represents expression of PDGF receptors and ligands in the TCGA database of human breast cancer patients with high or low Pygo2 expression. See also Figure S4 and Suppl. Table II.

Figure 5. Pygo2 binding to histones is required to repress luminal cell fate. (A) Immunofluorescence microscopy analysis for Cytokeratin-14 (CK-14) (red) and Cytokeratin-8/18 (CK-8/18) (green) in tumor sections of +/+ and AE/AE mice. Nuclei were counterstained blue with DAPI. Scale bar=100µm. Graphs represent the quantification of fluorescence intensity per mm2 of the tumor section. Each dot represents average fluorescence intensity per mouse analyzed. N=10 mice per genotype (1 tumor per mouse). Data are represented as mean ± SEM. (B) Primary cells isolated from +/+ and AE/AE tumors were subjected to an anchorage independent tumor spheroid formation assay. The primary cell-derived tumor spheroids harvested after 7 days were immunostained for Cytokeratin-8/18 (CK-8/18) (green) and Cytokeratin-14 (CK-14) (red). Nuclei were counterstained blue with DAPI. Scale bar=50µm. N=2 mice per genotype were analyzed. (C) DAPI stained primary cell-derived tumor spheroids from +/+ and AE/AE mice were analyzed for solid/ hollow morphology under a confocal microscope. Scalebar=50µm. Graph represents quantification of solid or hollow tumor spheroids formed by +/+ or AE/AE-derived primary cells. N=2 mice per genotype and 10 spheroids per mouse were analyzed. Data are represented as mean ± SEM. (D) (i) Flow cytometry dot plot of the distribution of CD24+ cells plotted against side scatter (SSC-A) in Pygo2 +/+ versus AE/AE mutant cells. (ii) Flow cytometry dot plot of the expression of CD29 and CD61 in the CD24+ population from (i) in Pygo2 +/+ versus AE/AE mutant cells. (iii) Histograms depict the expression of CD29, CD24 or CD61 in Pygo2 +/+ versus AE/AE mutant cells. N=2. (E) (i) Immunofluoresence microscopy analysis by DAPI staining of the effect of PDGFR inhibition with AG1296 (Pi) on morphology of tumor spheroids formed by +/+ and AE/AE tumor-derived cell lines. (ii) Quantification of solid and hollow tumor spheroids formed by +/+ or AE/AE-derived cell lines with or without PDGFR inhibition on stimulation with PDGF-BB. DMSO served as the vehicle control for Pi. Scale bar=50µm. N=2. Data are represented as mean ± SEM. See also Figure S5.

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Figure 6. Pygo2 binding to histones drives canonical Wnt signaling and suppresses PDGFR expression through miR-29 expression. (A) Quantitative RT-PCR analysis of the expression of miR-29a-3p, miR-29b-3p and miR-29c-3p family members in 2 independent WT and AE/AE mutant cell lines (#1 and #2). N=3. Data are represented as mean ± SEM. (B) Quantitative RT-PCR analysis of the expression of PDGFR and PDGFR upon 3 days of the forced expression of miR-29-3p family members in primary tumor-derived AE/AE mutant cell line #1. N=3. Data are represented as mean ± SEM. (C) Long-term TGF -treated AE/AE cells transiently expressing miR-29a/b/c were subjected to a Boyden Transwell chamber migration assay. Graph represents the number of cells migrated across the membrane per field. N=3. Data are represented as mean ± SEM. (D) Pygo2 AE/AE mutant cells stably expressing control miRNA or miR-29 family members were injected orthotopically into mammary fat pads of NSG mice. Graph represents tumor growth monitored weekly. N=6 mice per cohort. (E) Representative photomicrographs showing histological tumor sections from tumors of AE/AE mutant cells overexpressing control or miR-29 family members described in (D). Scale bar =100µm (F) Quantification of the number of lung metastases formed in NSG mice injected in the tail vein with with AE/AE mutant cells overexpressing control or miR-29 family members. Mice were sacrificed 4 weeks post-injection, and lungs were resected for the analysis of cancer cell colonization/ metastases formation. N=6 mice per cohort. (G) (i) Top: Scheme depicting the promoter region (2500bp upstream and 200bp downstream of TSS) of the miR-29a/b1 gene. Red vertical lines mark the positions for TCF/LEF motifs, and red arrows indicate the primer pairs (1-8) used to amplify by PCR the regions containing the TCF/LEF motif. Bottom: Quantification of the fold-enrichment after ChIP with antibodies against -catenin and Pygo2, respectively, and PCR of the indicated miR-29a/ b1 promoter regions in +/+ versus AE/AE mutant cells. Data were normalized to control IgG and are presented as mean fold-enrichment above background ± SEM. N=3. (ii) Top: Scheme depicting the promoter region (2500bp upstream and 200bp downstream of TSS) of the Pdgfr gene. Red vertical lines mark the positions for TCF/LEF motifs, and red arrows indicate the primer pairs (1-4) that were designed to amplify the regions containing the TCF/LEF motif. Bottom: Quantification of the fold-enrichment after ChIP with antibodies against -catenin and Pygo2, respectively, and PCR of the indicated Pdgfr promoter regions in +/+ versus AE/AE mutant cell line. Data were normalized to control IgG and are presented as mean fold enrichment above background ± SEM. N=3. See also Figures S6 and S7.

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Figure 7. A working model of the functional requirement of the Pygo2-histone interaction during primary tumor growth, cell de-differentiation, EMT and metastasis formation. Scheme depicting the working model for how in wildtype Pygo2+/+ cells (left, black box highlighted) Pygo2-histone interaction potentiates Wnt signaling through -catenin. Active Wnt/ -catenin signaling drives the expression of one of its known target genes, miR-29. miR-29 family members in turn bind and target PDGFR mRNA for degradation, thereby suppressing cell differentiation. Both Pygo2 and -catenin can however also bind at TCF/LEF motifs in PDGFR and other genes (X), thereby directly regulating their expression. In Pygo2AE/AE mutant cells (right, red box highlighted), loss of Pygo2-histone binding decreases transcriptional activation of the Wnt/ -catenin target gene miR-29. The resulting decrease in expression of miR-29 family members relieves the post- transcriptional repression on PDGFR and potentially on other mRNAs. The increase in PDGFR protein (and PDGF ligands) activates downstream signaling to induce tumor cell differentiation. The increased expression of Wnt antagonists, such as WIF1, Dkk family members and others, in a negative feedback loop further inhibit the activity of Wnt/ -catenin signaling. Together, these changes in signaling pathways in Pygo2-deficient tumor cells results into delayed primary tumor growth and reduced metastasis formation.

A Pygopus 2-histone interaction is critical for cancer cell de-differentiation and progression in malignant breast cancer

Meera Saxena, Ravi K.R. Kalathur, Natalia Rubinstein, et al.

Cancer Res Published OnlineFirst June 25, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-2910

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