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Fbxw7 is a driver of uterine carcinosarcoma by promoting epithelial-mesenchymal transition

Significance Uterine carcinosarcoma (UCS) is an aggressive endometrial cancer variant distinguished from endometrial adenocarcinoma (EC) by admixed malignant epithelial and mesenchymal components (carcinoma and sarcoma). The molecular events underlying UCS are enigmatic, as cancer gene mutations are generally shared among UCS/EC. We take advantage of genetic approaches in mice to show that inactivation of Fbxw7 and Pten results in UCS through spontaneous acquisition of mutations in a third gene (Tp53), arguing for strong biological selection and synergism in UCS. We used this UCS model including tumor-derived cell lines to show that Fbxw7 loss drives epithelial–mesenchymal transition, explaining Fbxw7’s role in UCS. This model system argues that simultaneous genetic defects in 3 distinct pathways (Fbxw7, Pten/PI3K, Tp53) converge in UCS genesis.


Organoid culture
Adult uteri were fragmented into small pieces using scalpels and digested with 0.4 mg/ml Collagenase V (Sigma) and 1.25 U/ml Dispase II (Sigma) in Advanced DMEM/F12 (Invitrogen) medium containing pen/strep (1%; Gibco) at 37°C for 30 min with gentle shaking. Tituration disrupted epithelial glands into single cells and undigested tissue chunks were removed using forceps. Isolated cells were pelleted, resuspended in 100 µl FACS buffer (HBSS + 3%FBS) containing 2 µl FCR blocking buffer (Miltenyi Biotec) and incubated at 4°C for 10 min. After blocking, cells were pelleted and incubated at 4°C for 45 min in darkness with EpCAM antibody (1:40; BD Biosciences) to label epithelial cells. Cells were washed, pelleted, and resuspended in FACS buffer containing PI (50 µg/ml; BD Biosciences) and flow sorted for EpCAM +ve /PI -ve epithelial cells. Sorted epithelial cells were resuspended in ice-cold Matrigel (Corning). 20 µl matrigel-cell suspension was plated into individual wells of a 48-well plate and incubated at 37°C for 5 min to form a mini dome containing cells. To compare proliferation rate, organoids were harvested from Matrigel using Cell Recovery Solution (Corning) followed by pipetting and trypsinizing with TrypLE Express Enzyme (Gibco). 500 cells in 15 µl Matrigel were seeded per well of a 96-well plate in triplicate with 150 µl organoid expansion medium and incubated for 10 days at 37°C, 5% CO 2 . The proliferation of organoids was assayed on day 3, 5, 7 and 10. At the end of each incubation period, Cell Titer-Glo® 3D reagent (Promega) was added to each well and luminescence was recorded after 30 min using Tecan Spark 10M microplate reader.
Organotypic culture 2,000-2,500 single UCS1 or UCS2 cells were resuspended in 40l of a Matrigel (BD Biosciences) (3-5mg/ml) and Collagen I (BD Biosciences) (1.5-2.1 mg/ml) mix, and then plated on 25l of a basement layer of Matrigel/Collagen I. Cultures were incubated at 37ºC for 1 hr and then supplemented with a 2% Matrigel/growth medium mixture. Cultures were grown for 3-4 days and then analyzed by fluorescence microscopy by fixing in 2% formalin in PBS for 20 min, and permeabilizing with 0.5% Triton X-100 in PBS for 10 min at RT. Cells were stained with Hoechst and Phalloidin-Alexa Fluor 546nM (Life Technologies) and mounted using ProLong Gold antifade reagent (Life Technologies). Images were obtained a Zeiss LSM700 confocal microscope.

Tissue processing and immunohistochemistry (IHC)
Tissues were fixed in 10% buffered formalin overnight, embedded in paraffin and serially sectioned (5 m). For IHC, sections were rehydrated in a series of ethanol solutions after deparaffinization in xylene. Antigen retrieval was performed in parboiling 10mM sodium citrate (pH 6.0) for 20 min, cooled to RT, followed by peroxidase blocking in 3% H 2 O 2 and blocking in 1% BSA in PBS. ImmPRESS (Vector Labs) was used for detection. Color images were obtained using a Nikon Eclipse Ci microscope and analyzed with NIS-Elements BR software. L1CAM was scored in a blinded manner by Allred scoring with four categories (0,1, 2,3 corresponding to no, low, intermediate, and high expression).

Immunofluorescence
Endometrial organoids were harvested from Matrigel by scraping with a 1 ml pipette tip on day 12 of culture and fixed in 4% PFA (Electron Microscopy Sciences) for 20 min followed by washing in PBS. Organoids were centrifuged, overlaid with 200 µl low melting agarose (Lonza) to form agarose plugs, with tissue processing and paraffin embedding as above. Sections were deparaffinized in xylene followed by rehydration through a series of graded ethanol. Antigen retrieval was performed using sodium citrate buffer (Vector Labs). Slides were washed with TBS containing 0.1% Tween 20 and incubated with primary antibody overnight, then washed and incubated with Alexa Fluor secondary antibodies (1:250; Life Technologies) and Hoechst (Fisher Scientific) for 1 hr at RT. For frozen sections, fresh tissue was fixed for 4 hrs in 4% paraformaldehyde,10% sucrose/PBS, followed by 12 hrs in 18% sucrose/PBS and 12 hrs in 30% sucrose/PBS. Tissues were frozen in OCT at -20ºC for several hrs. 5 m sections were air dried and mounted in Vibrance Antifade Mounting media with DAPI (Vector Labs). Fluorescent images were obtained on a fluorescence microscope (Nikon ECLIPSE Ni) using NIS-Elements BR software.

X-gal staining
Whole mount X-gal staining was performed by fixing tissues in 4% paraformaldehyde/PBS (Electron Microscopy Sciences) for 1 hr at 4ºC. After rinsing twice for 20 min in buffer I (100 mM sodium phosphate, pH 7.3, 2 mM MgCl 2 , 0.01% sodium deoxycholate, 0.02% NP40) at RT, tissues were incubated at 50ºC in buffer II (100 mM HEPES, 5 mM DTT, 1 mM MgSO 4 , 2% Triton X-100) for 1h. Tissues were rinsed in buffer I and incubated overnight in staining solution (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 1 mg/ml X-gal and brought up to volume in buffer I).

RNA extraction, library preparation and real-time PCR
Total RNA was isolated with the RNeasy Mini Kit (Qiagen #74104). RNA was treated with RNAsefree DNAse (Qiagen #79254) and Qubit quantified for library preparation at the McDermott Next Generation Sequencing Core at UTSW with quality control per Agilent 2100 Bioanalyzer. 4 g of total DNAse-treated RNA was prepared with TruSeq Stranded Total RNA LT Sample Prep kit (Illumina). Poly-A-RNA was purified and fragmented before strand specific cDNA synthesis. cDNA was poly A-tailed and indexed adapters were ligated. After adapter ligation, samples were PCR amplified and purified with Ampure XP beads, then validated again on the Agilent 2100 Bioanalyzer. Samples were quantified by Qubit before normalization and pooling. Samples were sequenced on the Illumina NextSeq 500 with read configuration as 75 bp, single end reads. 25-40 million reads were generated per sample.
For real-time PCR, 1 g of total RNA was used for cDNA synthesis using SuperScript VILO (Life Technologies) according to manufacturer's instruction and with an extension time of 90 min. The total reaction was diluted 1:2 and used in the PCR reactions. Real-time PCR was carried out using Taqman Fast Advanced Master Mix (Life Technologies) using QuantStudio5 (Applied Biosystems). Reactions were run in triplicate in at least 3 independent experiments. The geometric mean of actin was used as an internal control for normalization. The following FAM/MGB assays were purchased from Thermo Fisher: Mm00516558 (HeyL), Mm00556148 (Olfml2a), Mm00488601 (Fibulin 5), Mm01336227 (Fibulin 7), Mm00439498 (Mmp2), and Mm00485009 (Mgp). Expression data was analyzed with QuantStudio software using the 2 -CT method.

DNA and RNA sequencing analysis
A custom capture panel for the 76 mouse genes was designed using the Agilent SureDesign software suite based on UCSC mm9, NCBI Build 37, July 2007. The resulting design contained 4930 probes representing 366 kb of mouse genomic sequence. DNA capture, library preparation, and sequencing was performed using standard protocols per manufacturer's instructions. Reads were mapped to the mouse reference genome (GRCm38) using BWA 0.7.17 (1). PCR duplicates were marked using Picard and base quality score recalibration was applied using GATK 4.0 (2). SNP and INDEL discovery across all samples were performed using FreeBayes. A mutation was considered as a somatic mutation only if the alternative allele frequency was greater than 5% in tumor sample but less than 5% in normal sample. All mutations were annotated using SnpEff (3), based on GENCODE M16 annotation (4). The Sequence Read Archive (NCBI) accession number for the data is SUV5613208 For RNASeq, Fastq files were subjected to quality check using fastqc (version 0.11.2, https://www.bioinformatics.babraham.ac.uk/projects/fastqc) and fastqScreen (version 0.4.4, https://www.bioinformatics.babraham.ac.uk/projects/ fastq_screen), and trimmed using fastq-mcf (eautils/version 1.1.2-806, https://github.com/ExpressionAnalysis/ea-utils). Trimmed fastq files were mapped to Mus musculus reference genome (mm10, UCSC version from igenomes) using Tophat (5). Duplicates were marked using picard-tools (version 1.127, https://broadinstitute.github.io/picard/). Read counts were generated using featureCounts (6) and the differential expression analysis was performed using edgeR (7). The Geo (NCBI) accession number for the data is GSE138490.
Statistical cutoffs of FDR<0.001, log 2 CPM>0 were used to identify statistically significant differentially expressed genes. The filtered number of statistically significant genes in PLVX (empty vector) vs. Fbxw7 for UCS1 and UCS2, were 295 and 59 respectively. Pathway and network analysis were performed using DAVID v.6.8 software (8). The threshold in the DAVID gene ontology biological process analysis was set as modified Fisher Exact p-value (EASE score) ≤ 0.05. Heat maps were generated using ComplexHeatmap package (9). The dot plots were generated using ggplot2 (https://ggplot2.tidyverse.org/reference/geom_dotplot.html). Venn diagrams were plotted with Intervene (version 0.5.8) (9).

Western Blot
Cells from 10 cm plates were scraped in 5 ml cold D-PBS, pelleted by centrifugation at 1000g for 5 min and resuspended in 200 l ice cold RIPA buffer (Thermo Scientific) supplemented with Protease Inhibitor Cocktail (Sigma) and Phosphatase Inhibitor Cocktail 2 (Sigma). Following gentle mixing at 4ºC for 30 min, extracts were centrifuged at 10,000g for 10 min. Supernatants were collected, mixed with 4X Nupage LDS Sample Buffer (Life Technologies) and heated at 70ºC for 10 min. Equal amounts of protein were loaded in 4-12% NuPAGE Bis-Tris Mini Gels and run at 200V. Gels were transferred using Trans Blot Turbo Transfer System (BioRad) and PVDF membrane (BioRad). Membranes were blocked with 5% dry milk in TBST-T (BB), and antibodies incubated in BB overnight at 4ºC. Membranes were stripped with Restore Stripping Buffer (Thermo Scientific), blocked and probed again with GAPDH in BB for 1 hr RT. Blots were developed with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) and digital images acquired by ChemiDoc (BioRad).

Live cell imaging
Imaging for cell growth, scratch wound, and chemotaxis assays was performed using the IncuCyte S3 Live Cell Imaging System (Essen Bioscience). For proliferation assays, 2,000 cells/well were plated in 96-well plates and scanned every 6 hrs until confluence. Cell confluence was calculated using Incucyte software by phase contrast images and confluence at a given time was normalized to confluence at time 0. For scratch wound assays, 15,000 cells were plated/well in a 96-well plate and allowed to reach confluency overnight. Wounds were generated with WoundMaker™ (Essen Bioscience). Pictures were taken every 3 hrs, and wound width (cell migration) determined with IncuCyte software. For transwell migration assays, we used Incucyte Clearview 96-well cell migration plates (#4582). Top and bottom inserts were coated with matrigel (following manufacturer's instructions) and 1,000 cells grown overnight in DMEM/Insulin/Transferrin/Selenium seeded per well in DMEM/0.5% FBS. The bottom reservoir contained DMEM/10% FBS as chemoattractant. Top and bottom scanning were performed every 3 hrs. Figure S1. Confirmation of Pten protein loss in Fbxw7/Pten uteri. A) Pten IHC shows loss in some glands at 6 weeks of age.
Step sections show that Pten loss results in Akt hyperphosphorylation (Ser473) and Foxo1 relocalization from nucleus to cytoplasm. By 12 weeks, Pten-null clones expanded to take over entire endometrium, demonstrating selective growth advantage relative to non-recombined cells. The strongly Pten-positive cells within otherwise Pten-null glands are infiltrating leukocytes. Black dashed lines=Pten-null clones; blue-dashed line=Pten wt clone. Bar=50 m, all panels at same magnification. B) Percentage of unrecombined cells across genotypes at 12 weeks of age. Since efficiency of Cremediated recombination should be equivalent across genotypes, the results indicate that Fbxw7 loss confers an additional growth advantage to Pten deficient cells (p=0.027, t-test).      to left in order of mutation incidence in UCS. Red=more frequent in UCS relative to EC; black=less frequent in UCS relative to EC. C) L1CAM expression by IHC on human EC cases (not UCS) determined by Allred scoring. Filled red circles indicate cases also expressing p53; see also Table S3. D) L1CAM and p53 expression by IHC on human ECs with mutant or wild-type Fbxw7.