Identification of a gene expression driven progression pathway in myxoid liposarcoma.

AIM
to investigate the events involved in the progression of myxoid liposarcoma (MLS). Gene expression profiling and immunohistochemical/biochemical analyses were applied to specimens representative of the opposite ends of the MLS spectrum: pure myxoid (ML) and pure round cell (RC) liposarcomas. The analyses revealed the involvement of both coding and non coding RNAs (SNORDs located in DLK1-DIO3 region) and support a model of stepwise progression mainly driven by epigenetic changes involving tumour vascular supply and tumoral cellular component. In this model, a switch in the vascular landscape from a normal to a pro-angiogenic signature and the silencing of DLK1-DIO3 region mark the progression from ML to RC in concert with the acquisition by the latter of the over-expression of YYI/C-MYC/HDAC2, together with over-expression of genes involved in cell proliferation and stemness: MKNK2, MSX1 and TRIM71. Taken together, these findings strongly suggest that to progress from ML to RC liposarcoma the cells have to overcome the epigenetic silencing restriction point in order to reset their new stem-like differentiation signature. Our findings provide a first attempt at identifying the missing links between ML and RC liposarcomas, that may also have broader applications in other clinico-pathological settings characterised by a spectrum of progression.


Treatment and follow-up
All the data are detailed in Tables S1 and S2.
Myxoid tumours. All but three of the patients with pure ML underwent surgery alone. RT was administered to three patients (INT-B) in whom the surgical margins were contaminated. At the time of the last contact, all of the patients were alive, and 8/12 were NED (no evidence of disease) (follow-up duration: 32-312 months).
Round cell tumours. All of the patients underwent surgery and RT and/or chemotherapy but two (one only surgery and one only RT). Three patients of the INT-A series and three of the INT-B developed metastases. The most frequent site of metastasis was lung, followed by bone and liver. All the patients of the INT-B series (but one lost) died within a range of 26 to 144 mos.

Molecular and molecular/cytogenetic characterisation
The diagnosis of MLS was confirmed by FISH and/or RT-PCR in all cases. Sanger sequencing showed 3/24 had TP53 mutations (12.5%; two functional, and one non-functional), and two had TP53 polymorphisms; 5/24 had PIK3CA mutations (20.8%); and 1/23 had a PTEN mutation (4.35%). Case AU28 (see Table S1) had both a non-functional TP53 and a PIK3CA mutation. The percentages are in line with previously published data; there was no significant segregation of mutations with the ML or RC variants. For details, see Tables S1 and S2.

RNA extraction and gene expression microarray analysis
The microarrays were run at the Functional Genomics and Bioinformatics Core Facility of IRCCS Istituto Nazionale dei Tumori (Milan).
Total RNA was isolated using the Qiagen RNeasy FFPE kit (INT-A case material) or RNAeasy (INT-B case material) kit (Qiagen, Valencia, CA, USA). INT-A was profiled on Illumina whole-genome DASL HumanHT-12 v4 BeadChips containing probes for 29,285 transcripts (Illumina Inc., San Diego, CA). The DASL assay is a validated bead-based method that allows the expression profiling of RNA in FFPE samples. The experimental procedures, including labelling and hybridisation, were carried out following the manufacturer's protocol. Total RNA from the INT-B series was profiled on HumanHT-12_v4 BeadChips using the direct hybridisation assay, which allows the detection more than 47,000 transcripts. Briefly, 300 ng of total RNA was reverse transcribed, labelled with biotin, and amplified overnight using the Illumina RNA TotalPrep Amplification kit (Ambion); 1 ug of the biotinylated cRNA sample was hybridised to the BeadChips at 58°C overnight. After washing, the array were stained with 1 ug/mL Cy3-streptavidine and scanned with Illumina BeadArray Reader.
In both cases, the images were analysed and the primary data were collected using the supplied scanner software. The average intensity of all of the beads belonging to a single probe was calculated with its P value using BeadStudio software v3 and BeadStudio expression analysis module v3.3.8; the data were quantile normalised and exported without correcting for background or scale. The data matrices were filtered in order to exclude signals with detection P values of >0.05 and allow 50% of missing values.

Bioinformatics analysis
The differentially expressed genes were defined by imposing an FDR of <10%. The significance of the individual genes was tested by means of the univariate permutation test using BrB ArrayTools_v4.1.0-stable release (Simon, R. and Lam, AP; http://linus.nci.nih.gov/BRB-ArrayTools.html).
The sub-class mapping algorithm (Gene Pattern Software, Version 3.0; Broad Institute) [9] was used to ascertain whether the pattern of the modulated genes in the training set molecularly corresponded to that observed in the validation set. This algorithm computes the enrichment of each predefined phenotype between the first and second dataset, and also provides a p-value. Sub-class mapping has the advantage of being an unsupervised method for identifying molecular similarity in groups of samples from independent datasets beyond technical differences.

Quantitative reverse-transcription PCR (qRT-PCR) of gene and miRNA expression
The gene and miRNA primers were respectively purchased from Applied Biosystems (Foster City, CA, USA), and Exiqon (Vedbaek, Denmark), except for the primers for MSX1, which were custom designed on the basis of the MSX1 refseq (NM_002448) and purchased from IDT (Integrated DNA Technologies, Inc, CA, USA). The assay IDs are listed in Table S2.
Gene expression qRT-PCRs were performed using a TaqMan transcription kit (Applied Biosystem, Foster City, CA), and real-time PCRs using the TaqMan Gene Expression Master Mix, TaqMan MicroRNA assays (Applied Biosystems) or TaqMan gene expression assays (Applied Biosystems). The reactions were incubated for 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and one minute at 60°C. The qRT-PCR data shown in Table1 were normalised using the RPL13a housekeeping gene; similar results were obtained testing a second housekeeping gene (β-actin).
The miRNA qRT-PCRs were performed using the miRCURY LNA Universal RT microRNA PCR system (Exiqon) in accordance with the manufacturer's instructions. Total RNA (20 ng) was polyadenylated and reverse-transcribed at 42°C (60 min), and then heat-inactivated at 85°C (5 min) using a poly-T primer containing a 5′ universal tag. The resulting cDNA was diluted 80-fold, and 8 µL was used in 20 µL PCR amplification reactions at 95°C for 10 min, 40 cycles of 95°C for 10 sec, and 60°C for 60 sec. The results were normalised using snord48 (Assay ID:203903).
The expression levels were quantified using a sequence detection system (QuantStudio; LifeTechnologies) in triplicate, and the threshold cycle (Ct) was determined for each sample. Relative miRNA and mRNA expression was analysed using the ΔΔCT method.

Vorinostat treatment of the myxoid liposarcoma cell line
Vorinostat (Cat NoS1047-SAHA MK0683 Selleck Chemical) was resuspended at 150 mM in DMSO, and diluted to final concentrations ranging from 500 nM to 15 μM) in RPMI cell culture medium (maximum DMSO concentration 0.0005%).

Proliferation assay (MTT)
1.5x10 3 cells were seeded in a flat-bottomed 96-well plates in a volume of 200 μl of RPMI cell medium. After 24 hours, cells were treated with vorinostat at final concentration ranging from 1.87 μM to 15 μM. After 72 hours of culture, 20 μl of 5mg/L MTT (Cat M5655, Sigma Aldrich, St. Luis, MO) were added directly to the cells followed by an additional 4 hours incubation, and then 100 μl DMSO were added. The adsorbance of individual wells was read at a wavelength of 550nm.

Trypan blue assay (growth curve)
3x10 5 cells were seeded in 10x20 mm tissue culture dish (cat. 35503, Becton Dikinson). Vorinostat was added at final concentration ranging from 500 nM to 1.5 μM in RPMI cell culture medium. After 3 and 6 days, the cells were detached using a 1% trypsin-EDTA solution (Cat. No. 15400, Invitrogen) and counted using a Burker chamber.

c-MYC WB
Proteins were extracted from vorinostat-treated cells and immunoblotted with c-MYC antibody as described above.