Reactivation of p53 by a Cytoskeletal Sensor to Control the Balance Between DNA Damage and Tumor Dissemination

Background: Abnormal cell migration and invasion underlie metastasis, and actomyosin contractility is a key regulator of tumor invasion. The links between cancer migratory behavior and DNA damage are poorly understood. Methods: Using 3D collagen systems to recapitulate melanoma extracellular matrix, we analyzed the relationship between the actomyosin cytoskeleton of migrating cells and DNA damage. We used multiple melanoma cell lines and microarray analysis to study changes in gene expression and in vivo intravital imaging (n = 7 mice per condition) to understand how DNA damage impacts invasive behavior. We used Protein Tissue Microarrays (n = 164 melanomas) and patient databases (n = 354 melanoma samples) to investigate the associations between markers of DNA damage and actomyosin cytoskeletal features. Data were analyzed with Student’s and multiple t tests, Mann-Whitney’s test, one-way analysis of variance, and Pearson correlation. All statistical tests were two-sided. Results: Melanoma cells with low levels of Rho-ROCK–driven actomyosin are subjected to oxidative stress-dependent DNA damage and ATM-mediated p53 protein stabilization. This results in a specific transcriptional signature enriched in DNA damage/oxidative stress responsive genes, including Tumor Protein p53 Inducible Protein 3 (TP53I3 or PIG3). PIG3, which functions in DNA damage repair, uses an unexpected catalytic mechanism to suppress Rho-ROCK activity and impair tumor invasion in vivo. This regulation was suppressed by antioxidants. Furthermore, PIG3 levels decreased while ROCK1/2 levels increased in human metastatic melanomas (ROCK1 vs PIG3; r = -0.2261, P < .0001; ROCK2 vs PIG3: r = -0.1381, P = .0093). Conclusions: The results suggest using Rho-kinase inhibitors to reactivate the p53-PIG3 axis as a novel therapeutic strategy; we suggest that the use of antioxidants in melanoma should be very carefully evaluated.

PIG3 WT and PIG3 S151V plasmids were kindly provided by Dr Xavier Pares (Universitat Autònoma de Barcelona).
Flag-ARHGAP5 and myc-RhoA plasmids were kindly provided by Prof. Anne Ridley (King´s College London).

Transfection and RNAi
Melanoma cells (2x10 5 ) cells were seeded in a 6-well plate and transfected the next day with 20-40 nM SmartPool or individual OTs (On Target) siRNA oligonucleotides, using Optimem-I and Lipofectamine 2000 (Invitrogen). Forty eight h after transfection, cells were seeded on collagen in 10% FBS, the next day media was changed to 1% FCS (if specified) and cells analysed 24h later. Stable cell lines were selected with 100g/ml G418 (Sigma). GIPZ Lentiviral shRNA constructs were from Thermo Scientific and stable cells were selected with 1g/ml puromycin (Life Technologies).

Time-lapse microscopy
Multi-site bright-field microscopy imaging was performed in a humidified chamber at 37°C and 5% CO 2 using a 10×/0.3 NA Plan Fluor ELWD objective lens on a fully motorized (Prior Scientific) multi-field Nikon TE2000 microscope with an ORCA camera (Hamamatsu) controlled by MetaMorph (Molecular Devices) and Volocity (Perkin Elmer) software.

Tracking Assays
Tracking of cell migration within collagen matrices was performed as described (1)

Quantitative Real Time one step PCR
Trizol (200l/T12 well) was added to cells seeded on top of thick collagen and collected.
RNA was then precipitated and purified using iso-propanol and ethanol precipitation. RNA pellet was resuspended in RNase free water and further purified using spin-easy column purification kit (Sigma). QuantiTect Primer Assays (Qiagen) and Brilliant II SYBR Green QRT-PCR 1-step system (Agilent Technologies) and Stratagene MX 3005p qPCR system were used following the manufacturer's instructions. GAPDH was used as loading control. For 8-oxodG staining, cells were fixed with methanol followed by acetone, treated with 0.05N

Immunoblotting
HCl (5min on ice) and with 100µg/ml RNAse for 1h. DNA was denatured in situ with 0.15N NaOH in 70% EtOH. After DNA denaturation, treatment with proteinase K was performed. 6 Coverslips were blocked with 1% BSA for 1h at room temperature and then incubated with primary antibody (8-oxodG, Trevigen, 4354-MC-050), followed by followed by an Alexa 4888-conjugated secondary antibody and with DAPI to stain DNA. Samples were mounted with a medium from DakoCytomation (Carpinteria, CA, USA) and examined with a Leica scanning confocal microscope with 63x objective lense and Leica software. Nuclear 8-oxodG fluorescence signal was quantified calculating the pixel intensity in single cell nuclei relative to the nucleus area.

Annexin V apoptotic assay
To detect apoptotic cells, we used the Annexin V-FITC kit (Miltenyi Biotec) following the manufacture's instructions. Briefly, cells were incubated with Annexin V-FITC for 15 min in the dark at room temperature, then washed and incubated with propidium iodide (1 g/ml) prior to analysis by flow cytometry using BD FACSCanto™ II systems (BRC Flow Cytometry Core, Guy´s Hospital, London) and FlowJo software.

3D migration assays
Cells were suspended in serum-free bovine collagen I or rat tail collagen I at 2.3 mg/ml to a final concentration of 10x10 3 cells/100 μl in a 96-well plate and processed as previously described (3). Cells were mixed with collagen, seeded on a 96-well plate and spun down. 5% FBS-containing media was added on top of the gel after 4h and cells were allowed to invade upwards. After 24h incubation at 37ºC, cells were fixed in 4% formaldehyde for 16 h and

Immunofluorescence in xenografts
Six μm sections of formalin-fixed, paraffin-embedded material were used. Slides were dewaxed and antigen retrieval was performed using citrate buffer pH 6 followed by blocking in PBS-Tween 0.1% + 1% BSA for 15 min and overnight incubation with mouse monoclonal PIG3 antibody (1:100 in PBS+1% BSA). Antibody detection was performed using the Liquid Permanent Red Chromogen (Dako). Hematoxylin was used to counterstain. Quantification of cell morphology (roundness using ImageJ) was performed on H&E stainings. Each mouse xenograft was imaged for 6 separate fields and 10 representative cells were scored for roundness index. The average cell rounded index per field was plotted in Figure 7C (right panel).

RNAi sequences
Sh plasmid sequences