An unanticipated tumor-suppressive role of the SUMO pathway in the intestine unveiled by Ubc9 haploinsufficiency

Sumoylation is an essential posttranslational modification in eukaryotes that has emerged as an important pathway in oncogenic processes. Most human cancers display hyperactivated sumoylation and many cancer cells are remarkably sensitive to its inhibition, thus supporting application of chemical sumoylation inhibitors in cancer treatment. Here we show, first, that transformed embryonic fibroblasts derived from mice haploinsufficient for Ubc9, the essential and unique gene encoding the SUMO E2 conjugating enzyme, exhibit enhanced proliferation and transformed phenotypes in vitro and as xenografts ex vivo. To then evaluate the possible impact of loss of one Ubc9 allele in vivo, we used a mouse model of intestinal tumorigenesis. We crossed Ubc9+/− mice with mice harboring a conditional ablation of Apc either all along the crypt–villus axis or only in Lgr5+ crypt-based columnar (CBC) cells, the cell compartment that includes the intestinal stem cells proposed as cells-of-origin of intestinal cancer. While Ubc9+/− mice display no overt phenotypes and no globally visible hyposumoylation in cells of the small intestine, we found, strikingly, that, upon loss of Apc in both models, Ubc9+/− mice develop more (>2-fold) intestinal adenomas and show significantly shortened survival. This is accompanied by reduced global sumoylation levels in the polyps, indicating that Ubc9 levels become critical upon oncogenic stress. Moreover, we found that, in normal conditions, Ubc9+/− mice show a moderate but robust (15%) increase in the number of Lgr5+ CBC cells when compared to their wild-type littermates, and further, that these cells display higher degree of stemness and cancer-related and inflammatory gene expression signatures that, altogether, may contribute to enhanced intestinal tumorigenesis. The phenotypes of Ubc9 haploinsufficiency discovered here indicate an unanticipated tumor-suppressive role of sumoylation, one that may have important implications for optimal use of sumoylation inhibitors in the clinic.


Mice
Deletion of the floxed exon 14 of one Apc allele was induced by intraperitoneal injections of 4-OHT (Sigma-Aldrich) prepared at 10 mg/mL in corn oil with 4% ethanol (total of 0.1 mg per g of body weight) given three times at 12-hour intervals to [8][9][10][11][12] week-old male and female mice that were housed all together independently of their genotype. Polyp development was analyzed in blinded fashion 12 and 16 weeks after 4-OHT treatment for Villin-and Lgr5-IRES-EGFP-Cre ERT2 mice, respectively, by sampling the entire small intestine that was washed, longitudinally opened and scored and measured under the stereoscope. Normal tissue and polyp samples were excised and frozen in liquid nitrogen and kept at -80ºC until processing. For survival studies, mice were followed daily (in blinded fashion) and sacrificed when presenting disease symptoms or weight loss exceeded 20%.

Histology
Sampled intestines were prepared by the "Swiss Roll" technique (without longitudinal incision) and were incubated overnight in 10% formalin solution at pH 7 (VWR, Radnor PA, USA), then rinsed with water and stored in 70% ethanol (Sigma-Aldrich, St. Louis MO, USA) at 4°C until embedding in paraffin.
Cells, regularly tested for Mycoplasma contamination, were incubated in a humidified atmosphere at 37°C and 5% CO2.

Viral production and infection of MEFs
4.5x10 5 Phoenix-Eco cells (HEK293T cells modified for the production of ecotropic retroviruses; ThermoFisher) were seeded in 10 cm dishes and incubated for 24 h. Medium was then changed for DMEM supplemented with 25 µM chloroquine (Sigma-Aldrich), and cells were transfected with calcium phosphate with 10 µg of retroviral plasmid construct DNA in 100 mM CaCl2 in HBS (Hepes-buffered saline; Fluka) for 30 min at RT. Transfected cells were then incubated overnight at 37°C. The same day, 4.5x10 5 target cells to be infected were seeded in 10 cm dishes. The following two days, supernatant of Phoenix cells was harvested 4 times every 3 h, filtered through 0.45 μm, supplemented with Polybrene (8 μg/mL; Sigma-Aldrich) and applied to the target cells. Finally, puromycin (4 μg/mL; InvivoGen, San Diego, CA) or hygromycin (100 μg/mL; InvivoGen) were added for selection until complete mortality of non-infected control cells was observed. Retroviral constructs used for expression of dominant-negative p53 (Trp53, pBABE-hygro p53 DD; Addgene #9058), and active HRas V12 (pBabe-puro Ras V12 ; Addgene #1768), were a kind gift from Bob Weinberg.

Growth of transformed MEFs
MEFs were seeded at 10 5 cells per well into 6-well plates in 2 mL of DMEM, treated with 100 μg/mL of 4-hydroxytamoxifen (4-OHT; Sigma-Aldrich) prepared in 100% ethanol or ethanol (EtOH) as control, grown for the indicated time, fixed for 10 min in 10% formalin pH 7, washed and then stained with 0.01% crystal violet solution (Sigma-Aldrich) for 10 min at RT.
After extensive washes, the crystal violet trapped in the cell membranes was solubilized with 1 mL of 10% acetic acid (Sigma-Aldrich) and its absorbance was measured at 590 nm to estimate the relative number of cells. Alternatively, MEFs were counted using Z1 Coulter Particle Counter (Beckman Coulter) according to manufacturer's instructions.
In 2D plate colony formation (low density) assays, 300 transformed MEFs were seeded in 10-cm dishes with DMEM that was changed every 3 days and were incubated for 14 days. MEFs were fixed with ice-cold methanol (Sigma-Aldrich) and then stained with 0.5% crystal violet. In foci formation (high density) assays, 10 6 transformed MEFs were seeded in 10-cm dishes with DMEM that was changed every 3 days and were incubated for 14 days.
MEFs were fixed with 4% PFA (Sigma-Aldrich) and stained with 0.5% crystal violet. In 3D soft-agar colony formation assays, 5x10 4 transformed MEFs were seeded per well of a 6-well plate in 2 mL of 0.5% agar (Sigma-Aldrich) in DMEM medium on top of 2 mL layer of 1% agar in medium previously poured and solidified and were incubated for 4 weeks. 1 mL of medium was added on top and was changed every 3 days. Colonies were stained with 0.005% crystal violet. Écully, France). Tumors were measured externally with a caliper every day and were allowed to grow until reaching the limit point set at 1000 mm 3 . Tumor volume was determined using the equation V = LxW 2 x0.5, where L is length and W is width.

Homologous recombination reporter assay
Homologous recombination was determined as described previously [1]
Extracts were later on sonicated and centrifuged at maximum speed for 5 min. Protein concentration in supernatants was measured using Pierce 660 nm protein assay supplemented with ionic detergent compatibility reagent (IDCR, ThermoFisher) following manufacturer's instructions. Equal amounts of proteins were loaded on 4-12% Bis-Tris Criterion (Bio-Rad) or NuPAGE 4-12% Bis-Tris (Invitrogen, Waltham, MA) gels for electrophoresis. Proteins were transferred to nitrocellulose membranes using the Trans-blot Turbo transfer pack and system (Bio-Rad). Equal loading of samples was assessed by Ponceau S staining (Sigma-Aldrich) after transfer or by staining with anti--actin antibody.
When necessary to detect multiple protein species, identical sample amounts were redeposited on the same or a separate gel, to be run, blotted and processed in parallel, each with loading controls (Ponceau S), as indicated. Membranes were blocked with either 5% low-fat milk or 3% BSA in PBS-Tween, incubated with primary antibodies overnight at 4ºC, followed by 1 h incubation with secondary antibodies coupled to DyLight-680 or -800 nm fluorophores (ThermoFisher) and scanned using the Odyssey infrared system (LI-COR, Lincoln NE, USA). Original TIFF files were transferred to ImageJ software and the entire image was inverted. Images were adjusted via Image>Adjust>Brightness/Contrast with no gamma adjustment to visualize differences and relevant parts were then transferred to Normalized log-scaled data were further considered for differential analysis with limma [3].
Moderated F-statistics that combines the empirical Bayes moderated t-statistics for all contrasts into an overall test of significance for each probe were used to assess the 7 significance of the observed expression changes for each contrast. P-values were corrected for multiple testing using the FDR approach. Functional characterization was performed via Gene Ontology (GO) and gene set over-representation analysis using curated databases GO-Biological Process, GO-Cellular Component, GO-Molecular Function [4,5], and pathways in REACTOME [6] and KEGG [7,8], and MSigDB via the ToppGene Suite portal [9]. GSEA analysis was performed using the GSEA software from the Broad Institute (http://software.broadinstitute.org/gsea/index.jsp) [10], with 1000 gene_set permutations, No_Collapse of data, and the previously published intestinal stem cell signature [11].      Unpaired two-tailed Mann-Whitney test was used. middle and distal regions, respectively, from n=3 Ubc9 +/+ mice. n=46, 26, 55 crypts in proximal, middle and distal regions, respectively, from n=4 Ubc9 +/-mice. b As in a for colon crypts. n=45 and 54 crypts for Ubc9 +/+ (n=3 mice) and Ubc9 +/-(n=4), respectively. Unpaired