USP19 modulates cancer cell migration and invasion and acts as a novel prognostic marker in patients with early breast cancer

Tumor cell dissemination in cancer patients is associated with a significant reduction in their survival and quality of life. The ubiquitination pathway plays a fundamental role in the maintenance of protein homeostasis both in normal and stressed conditions and its dysregulation has been associated with malignant transformation and invasive potential of tumor cells, thus highlighting its value as a potential therapeutic target. In order to identify novel molecular targets of tumor cell migration and invasion we performed a genetic screen with an shRNA library against ubiquitination pathway-related genes. To this end, we set up a protocol to specifically enrich positive migration regulator candidates. We identified the deubiquitinase USP19 and demonstrated that its silencing reduces the migratory and invasive potential of highly invasive breast cancer cell lines. We extended our investigation in vivo and confirmed that mice injected with USP19 depleted cells display increased tumor-free survival, as well as a delay in the onset of the tumor formation and a significant reduction in the appearance of metastatic foci, indicating that tumor cell invasion and dissemination is impaired. In contrast, overexpression of USP19 increased cell invasiveness both in vitro and in vivo, further validating our findings. More importantly, we demonstrated that USP19 catalytic activity is important for the control of tumor cell migration and invasion, and that its molecular mechanism of action involves LRP6, a Wnt co-receptor. Finally, we showed that USP19 overexpression is a surrogate prognostic marker of distant relapse in patients with early breast cancer. Altogether, these findings demonstrate that USP19 might represent a novel therapeutic target in breast cancer.


Figure S2| Candidate regulators of migration.
We performed a loss-of-function screen using shRNAs targeting ubiquitination pathway-related genes in MDAMB231 cells. We implemented a cyclic functional selection and focused our analysis in cells that presented impaired migration. By sequencing DNA of selected cells, we calculated the frequency of each shRNA relative to the whole population of shRNAs after every cycle of selection. We then calculated the fold enrichment of each shRNA as their relative abundance in the fourth cycle compared to their abundance in the non-selected population of cells. The graph shows the mean fold enrichment (log2 of relative abundance [r.a.]) of at least two shRNAs targeting each candidate gene. Values above zero represent putative positive regulators of migration, and values below zero belong to shRNAs that were lost in the screen and could represent negative regulators of migration.  An area-based analysis of proliferation rate was used to determine cell growth over time. Cells were seeded onto wells, allowed to attach and imaged at the indicated time points. Left: relative mean occupied area at each time point ± S.E., and right: doubling time calculated for control and USP19 silenced cell lines (n= 9, one-way ANOVA, Dunnett's multiple comparison test. shRNA#1 p= 0.6284 and shRNA#2 p= 0.2123). We generated MDAMB231 cells expressing GFP tagged-H2B and then stably transduced them with control vector or shRNAs # 1 and # 2 directed against USP19. For the wound healing assays, a scratch was made with a pipette tip on a monolayer of the different cell cultures, and time-lapse imaging monitored the number of migrating cells across the border. (A) Representative images of a wound healing assay at time= 0 hours. Cells circled in magenta were considered for the analysis. Data corresponding to circled cells in the gap area were manually excluded from the analysis. Data was collected from three independent experiments for each cell line. Scale bar= 100 μm. shows the gap covered area (mm 2 ) at the indicated time. (C) Cells mean speed from each woundedge was calculated throughout the length of the experiment. (n=6, one-way ANOVA, Dunnett's multiple comparison test. shRNA#1 p< 0.0001 and shRNA#2 p= 0.0028). (D) Mean accumulated track displacement (total displacement) for cells in each wound-edge was calculated until the end of the experiment (n=6, one-way ANOVA, Dunnett's multiple comparison test. shRNA#1 p< 0.0001 and shRNA#2 p= 0.0059). (E) Persistence was calculated as the Euclidean/Accumulated track displacement ratio for control or USP19 silenced MDAMB231 cells at each wound-edge. (n=6, oneway ANOVA, Dunnett's multiple comparison test. shRNA#1 p= 0.0003 and shRNA#2 p= 0.0527).   There are a variety of USP19 mRNA transcripts generated by alternative splicing, and not all of them show the same functional properties. In this regard, changes in the last exon coding sequence generate soluble isoforms and endoplasmic reticulum (ER) membrane-bound isoforms. In order to analyze whether USP19's transmembrane domain was required for migration and 3D growth, we generated a GFP-tagged USP19 construct (wild type for its catalytic activity) with a point mutation that generates a premature stop codon. This mutation prevented the transmembrane domain to be translated, therefore generating a protein that mimicked USP19's soluble isoform (named WTDTM). We analyzed overexpressed-USP19 proteins subcellular localization by transiently transfecting U2OS cells with wild type (WT), catalytically dead mutant (C506S) or cytoplasmic (WTDTM) GFP-tagged USP19 constructs. Cells were fixed with 4% paraformaldehyde, stained with DAPI and mounted. Images were captured using an inverted fluorescence microscopy. Scale bar= 100 µm. Figure S8| Tumor growth analysis.

shRNA library sequencing
The library preparation strategy uses genomic DNA and two rounds of PCR in order to isolate the shRNA cassette and prepare a single strand of the hairpin for sequencing by means of an XhoI restriction digest in the stem-loop region. We used barcoded half-hairpin sequences for multiplexing purposes. The procedure was performed as described previously (1). A HiSEQ 2500 HT Mode V4 Chemistry Illumina instrument was used with a simple 1x50 run and on average 1.2x10 6 reads were obtained per sample.
shRNA data were analyzed in a similar fashion to RNA-seq data. Briefly, quality control was performed with FastQC, reads were trimmed to include only shRNA sequences using FASTQ trimmer and filtered with the FASTQ Quality Filter. Reads were then aligned to a custom reference library of shRNA sequences using TopHat2.

Mutagenesis
Mutagenesis PCR amplification was performed with the KAPA HiFi System (Kapa Biosystems).
Mutations that generated a stop codon at amino acid 1290 were corroborated by sequencing and by checking expression in U2OS cells under a fluorescence microscopy (Supp. Fig. 7). Mutagenesis primers sequences are as follows:

Primers list
For mRNA expression analysis:

Primer name Primer sequence
Human GAPDH sense 5′-TACTAGCGGTTTTACGGGCG-3′ Human GAPDH antisense 5′-TCGAACAGGAGGAGCAGAGAGCGA-3′ Crystal violet proliferation assay 1x10 4 cells were seeded in 96-well plates at time= 0 hours in quadruple. Every 24 hours, medium was removed, cells were rinsed with 1X PBS and fixed with methanol. After 4 days, wells were stained with 150 μl of a 0.05% crystal violet solution. Plates were then rinsed with water, dried out and the remaining crystal violet was solubilized in 150 μl methanol. The absorbance was measured at 595 nm.

Area-based analysis of proliferation rate
This experiment was performed as described previously (2), with minor modifications. Briefly, 1x10 3 cells were seeded in 96-well plates and incubated overnight to allow cell attachment. Images were then acquired under bright field illumination every 6 hours for 3 days using a Zeiss Axio Observer Z1 Inverted Epi-fluorescence microscope equipped with an AxioCam HRm3digital CCD camera, a Stage Controller XY-STEP-SMC-2009 scanning stage and an Incubator XLmulti-S1 and Heating Unit XL-S1 for live imaging incubation. A 10X air objective and Zeiss Zen Blue 2011 software was used for image acquisition. Image analysis was performed with Fiji software, using an automated analysis macro to measure the occupied area by cells.

Wound healing assays
For wound-edge cells analysis, GFP-tagged H2B expressing MDAMB231 cells were used. Images were acquired every 30 minutes for 8 hours for three independent experiments, and calculations were performed using cell tracks from each wound edge separately. The number and position of cells were determined using, ImageJ/Fiji -'Trackmate' plug-in (3), and trajectory analysis was performed using the 'Chemotaxis and Migration Tool' 1.01 ImageJ plug-in (Ibidi).

Agar invasion assay
5 μL spots of a 1% noble agar melted solution were pipetted onto 96 well cell culture plates and allowed to cool for 20 min at RT. At this point, 5x103 cells were plated in culture media supplemented with 1 μg/ml Hoechst 33258 (Thermo Fisher Scientific) and allowed to adhere for 1 hour. Fluorescent images of the edges of each spot were taken every 20 minutes for 18 hours using a 10X magnification air objective, using the Fluorescent Microscope mentioned before. The number and position of cells were determined using ImageJ/Fiji -'Trackmate' plug-in (3).

Noble agar assay
A 2 ml mixture of 5,000 cells in assay medium and 0.3% noble agar was seeded onto a 4 ml-solidified bed of 0.6% noble agar on six-well plates. The plates were incubated at 37°C. and the cultures were fed once a week with assay medium, for 6 weeks.
Matrigel three-dimensional cell culture 1,000 cells were cultured embedded in 100 μl basement membrane gels composed of 9.2 μg/μl Matrigel (BD Bioscience, Cat#356231) in 96 well plates. 50 μl of fresh medium were added on top of each gel every three days.

Metastatic load quantification of lung Hematoxylin & Eosin (H&E) stained slides
The presence of tumor nodules was identified by scanning individual lung H&E-stained slides with an optical microscope. Digital image files were acquired for each specimen, and Adobe Photoshop software was used to determine the percentage of the lung section that was occupied by the tumor.

Patients
We retrospectively extracted the eligible patients for the study from a consecutive cohort of cases