SOX2 boosts major tumor progression genes in prostate cancer and is a functional biomarker of lymph node metastasis

Critical issues in prostate cancer (PC) are a. identification of molecular drivers of the highly aggressive neuroendocrine differentiation (NED) in adenocarcinoma, and b. early assessment of disease progression. The SRY (sex determining region Y)-box 2 gene, SOX2, is an essential embryonic stem cell gene involved in prostate tumorigenesis. Here we assessed its implications in NED and progression of PC and its diagnostic and prognostic value. Laser microdissection, qRT-PCR, quantitative Methylation-Specific PCR and immunohistochemistry were used to analyze SOX2 gene expression and regulation in 206 PC samples. Results were examined according to the patient's clinical pathological profile and follow-ups. Functional studies were performed using PC cells transfected to overexpress or silence SOX2. SOX2 was consistently downregulated in PC, except in cell clusters lying within lymph node (LN)-positive PC. Multivariate analysis revealed that SOX2 mRNA expression in the primary tumor was significantly associated with LN metastasis. When SOX2 mRNA levels were ≥1.00, relative to (XpressRef) Universal Total RNA, adjusted Odds Ratio was 24.4 (95% CI: 7.54–79.0), sensitivity 0.81 (95% CI: 0.61–0.93) and specificity 0.87 (95% CI: 0.81–0.91). Patients experiencing biochemical recurrence had high median levels of SOX2 mRNA. In both PC and LN metastasis, SOX2 and NED marker, Chromogranin-A, were primarily co-expressed. In PC cells, NED genes were upregulated by SOX2 overexpression and downregulated by its silencing, which also abolished SNAI2/Slug dependent NED. Moreover, SOX2 upregulated neural CAMs, neurotrophins/neurotrophin receptors, pluripotency and epithelial-mesenchymal transition transcription factors, growth, angiogenic and lymphangiogenic factors, and promoted PC cell invasiveness and motility. This study discloses novel SOX2 target genes driving NED and spread of PC and proposes SOX2 as a functional biomarker of LN metastasization for PC.


Patients and samples
Patients entered in the study had not received hormone or immunosuppressive treatments or radiotherapy and were free from immune system diseases. Clinical and pathological stages were determined according to the 7 th edition of the TNM Classification of Malignant Tumours [1]. Tumor grade, from the prostate biopsies, was assessed according to the Gleason scoring system [2]. The PSA values were divided into 3 classes: 1: less than 10 ng/ml; 2: 10 to 20 ng/ml; 3: more than 20 ng/ml. The biological samples were cancer and normal prostate specimens (formalin-fixed and frozen) and draining lymph nodes (formalin-fixed) from each PCa patient who underwent radical prostatectomy. Half of each normal or neoplastic tissue sample was fixed in 4% formalin and embedded in paraffin. The other half was embedded in Killik frozen section medium (Bio-Optica, Milano, Italy), snap frozen in liquid nitrogen and preserved at −80°C. For histology, paraffin embedded samples were sectioned at 4 μm and stained with H&E. Single and double immunohistochemistry was done on paraffin-embedded or frozen sections, depending on the antibody (Ab) used.

Immunohistochemistry
Immunohistochemistry on formalin-fixed, paraffinembedded samples was performed using the primary Antibodies SOX2 (57CT23.3.4, Abcam, Cambridge, UK), CHGA (DAK-A3, Dako, Glostrup, DK) and SYP (Leica Biosystems, Newcastle Upon Tyne, UK) in a fully automated Leica Bond-Max instrument (Leica Biosystems). The immune complexes were detected using the Bond Polymer Refine Detection Kit (Leica Biosystems) according to the manufacturer's protocol. Negative controls were performed by replacing the primary Ab with 10% non-immune serum. Further controls were performed by omitting the secondary Ab. Controls were always negative.

Immunofluorescences and confocal microscopy
For double immunofluorescent staining on formalinfixed, paraffin-embedded samples, after deparaffination, the sections were subjected to antigen retrieval in ethylenediaminetetracetic acid (EDTA) buffer at pH 9. The slides were then incubated with the first primary antibody, SOX2 (Abcam), for 30 minutes, followed by incubation with the biotinylated secondary antiboby (Vector Laboratories, Burlingame, CA, USA) and subsequently with the Alexa Fluor 594 Streptavidin conjugated antibody (Molecular Probes, Eugene, OR, USA). Next, the sections were incubated with the second primary Ab, CHGA (Dako), for 30 minutes, followed by incubation with the biotinylated secondary Ab and subsequently with Alexa Fluor 488 Streptavidin conjugated antibody (Molecular Probes). Cross-reaction between the first secondary Ab and Alexa Fluor 488 Streptavidin conjugated was prevented by saturating all of its binding sites with the Alexa Fluor 594 Streptavidin conjugated antibody. Slides were mounted with vectashield medium (Vector Laboratories) and examined with a Zeiss LSM 510 Meta laser scanning confocal microscope (Zeiss, Oberkochen, D).

Laser capture microdissection (LCM)
LCM was performed using the P.A.L.M. Micro Beam System (P.A.L.M. Microlaser Technologies, Bernried, D). For LCM, 10 μm frozen sections (two sections per sample) from cancer and normal prostate specimens (of both control and PCa patients) were mounted on polyethylene naphthalate membrane-covered slides (P.A.L.M. Microlaser Technologies), thawed at room temperature, and immersed in cold acetone (5 min). Immediately after H&E staining, the sections were used for LCM. From 1000 to 1500 selected cells were cut and catapulted intact into the cap of an LPC-Microfuge Tube (P.A.L.M.), and RNA was immediately isolated with the RNeasy Plus Micro Kit (Qiagen, Hilden, D). Two sections per sample were analyzed. Epithelial components were dissected from cancer and normal prostate specimens (of both control and PCa patients) discriminating between neoplastic foci with low (well differentiated) versus high (poorly differentiated) Gleason grade (≤3 versus >3). Since the Gleason score is the sum of the grade assigned to the most common tumor pattern and the grade assigned to the next most common tumor pattern, to analyze homogeneous cell populations, only cells of the tumor foci with the highest Gleason grade were microdissected from each patient's tissue specimen. Foci with the same grade were microdissected from each specimen. Foci with low Gleason grade (≤3) were microdissected from PC samples of 75/206 patients, whereas those with high-grade (>3) were obtained from the other 131 patients.
All reagents used for LCM were prepared with Ultrapure DNase/RNase-free distilled water (Invitrogen, Paisley, UK).

Methylation analysis by quantitative Methylation-Specific PCR (qMSP)
The methylation levels of the SOX2 gene promoter, was performed by qMSP with primers and TaqMan probes specific for the methylated and the unmethylated converted DNA, for amplification and detection of a 128bp sequence located in the last portion of the promoter, 540bp upstream from the transcription start site. To detect insufficient bisulfite conversion, primers and probe for the housekeeping gene HPRT, specific for the unconverted DNA, were used. All primers and probes were designed with Beacon Designer software (Premier Biosoft, Palo Alto, CA, USA) in our laboratory and synthesized by Sigma-Aldrich Corporation (Sigma- LNCaP cells were cultured in the presence or absence of 5-Aza-2ʹ-deoxycytidine (5-Aza-dC) (AZA, 5-10 μM, Sigma-Aldrich) for 24-72 hours. Every 24 hours, RNA was extracted using RNeasy Mini Kit (Qiagen) and subjected to real time RT-PCR using the Quantifast SYBR Green PCR Kit (Qiagen).
Twenty-four, 48, and 72 hours after cell transfection, RNA was extracted and subjected to real time RT-PCR and the cell pellets were collected for Western Blotting.

Migration and invasion assay
CytoSelect TM Cell Migration and Invasion Assay Kit (Cell Biolabs, San Diego, CA, USA) was used for migration and invasion assays, according to the manufacturer′s protocol. Briefly, 24 hours after transfection, cells were counted and placed on polycarbonate membrane or matrigel inserts at 1 × 10 6 cells/mL in serum-free medium and were incubated for 24 hours (migration assay) or 48 hours (invasion assay) at 37°C. Cells were removed from the top of the inserts and cells that migrated and invaded through the inserts were fixed, stained, and quantified by optical density at 560 nm after extraction, using a SpectraMax 190 microplate reader (Molecular Devices, CA, USA).

Western blotting (WB)
For WB, cells were collected following standard procedures. Then, 1.0 mL of ice cold RIPA Lysis buffer (Thermo Scientific, Waltham, MA, USA) was added (supplemented with freshly added Protease and Phosphatase Inhibitors Cocktails (Thermo Scientific). Total proteins were measured in the extract by the Bradford assay. The whole cell lysates were examined on Mini-PROTEAN TGX Gels 4-20% (Bio-Rad). Proteins were transferred from the gels on Immun-Blot PVDF Membranes (Bio-Rad) in the transfer buffer (glycine, tris [pH 8.4] and methanol) using Mini Trans-Blot Cell apparatus (Bio-Rad). Membranes containing the proteins were blocked with milk 5% (Sigma-Aldrich) in TBST and subsequently, probed with primary and horseradish peroxidase conjugated secondary antibodies following standard procedures. Proteins transferred membranes were then washed with TBST and developed with Pierce ECL Western Blotting Substrate (Thermo Scientific).