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SNAI2/Slug gene is silenced in prostate cancer and regulates neuroendocrine differentiation, metastasis-suppressor and pluripotency gene expression.

Prostate Cancer (PCa)-related deaths are mostly due to metastasization of poorly differentiated adenocarcinomas often endowed with neuroendocrine differentiation (NED) areas. The SNAI2/Slug gene is a major regulator of cell migration and tumor metastasization. We here assessed its biological significance in NED, and metastatic potential of PCa. SNAI2 expression was down-regulated in most PCa epithelia, in association with gene promoter methylation, except for cell clusters forming: a. the expansion/invasion front of high-grade PCa, b. NED areas, or c. lymph node metastasis. Knockdown of SNAI2 in PC3 cells down-regulated the expression of neural-tissue-associated adhesion molecules, Neural-Cadherin, Neural-Cadherin-2, Neuronal-Cell-Adhesion-Molecule, and of the NED marker Neuron-Specific Enolase, whereas it abolished Chromogranin-A expression. The metastasis-suppressor genes, Nm23-H1 and KISS1, were up-regulated, while the pluripotency genes SOX2, NOTCH1, CD44v6, WWTR1/TAZ and YAP1 were dramatically down-regulated. Over-expression of SNAI2 in DU145 cells substantiated its ability to regulate metastasis-suppressor, NED and pluripotency genes. In PCa and lymph node metastasis, expression of SOX2 and NOTCH1 was highly related to that of SNAI2. In conclusion, I. SNAI2 silencing in PCa may turn-off the expression of NED markers and pluripotency genes, while turning-on that of specific metastasis-suppressors, II. SNAI2 expression in selected PCa cells, by regulating their self-renewal, NED and metastatic potential, endows them with highly malignant properties. SNAI2 may thus constitute a key target for modern approaches to PCa progression.


Patients and samples
Patients entering 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 7th edition of the TNM Classification of Malignant Tumors. Tumor grade was assessed according to the Gleason scoring system from the prostate biopsies.
The biological samples were cancer and normal prostate specimens (formalin-fixed and frozen) and draining lymph nodes (formalin-fixed) from PCa patient who underwent radical prostatectomy between 2009 and 2013 at the "S.S. Annunziata" Hospital, Chieti, Italy. Onehalf of each tissue sample was fixed in 4% formalin and embedded in paraffin. The other 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 were done on paraffin-embedded or frozen sections, depending on the antibody (Ab) used.

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 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, 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 and stromal components were dissected from cancer and normal prostate specimens (of both control and PCa patients) with discrimination between neoplastic foci with low (well differentiated) versus high (poorly differentiated) Gleason grade (≤ 3 versus > 3). Foci with the same grade were microdissected from each specimen.
Foci with low grades were obtained from 35/102 PCa patients, whereas foci with high grades were obtained from the other 67/102 PCa patients. The stroma was isolated among the glands of both low-grade and high-grade PCa, or in the histologically normal zones far from the PCa.
All reagents used for LCM were prepared with Ultrapure DNase/RNase-free distilled water (Invitrogen, Paisley, UK).

Methylation analysis by bisulfite genomic sequencing
Bisulfite-sequencing PCR primers, SNAI2 Forward (5′-TTGGTTTAAAATGGGTTGTTT-3′) and SNAI2 Reverse (5′-TATACAAAACAAACCTCTCCAAAT-3′) were designed, using the Methyl Primer Express program (Applied Biosystems, Foster City, CA, USA), for specific amplification of successfully converted DNA strands only and did not contain any CpG sites within their sequences. They were synthesized by the Sigma-Aldrich Corporation (St. Louis, MO, USA) and used for amplification of a 309 bp region of the SNAI2 gene promoter, located 130 bp upstream from the transcription start site and containing 9 sparsely spaced CpGs in its central portion. Thermal reactions were performed on a MJ Mini Gradient Thermal Cycler (Bio-Rad, Hercules, CA, USA). PCR conditions were as follows: 95°C for 2 min, 44 cycles of 95°C for 1 min., 55°C for 30 sec.,72°C for 1 min., followed by 72°C for 7 min. The PCR was carried out with the AmpliTaq Gold ® 360 Master Mix (Applied Biosystems). The sequence reaction was performed with the primers used for the PCR reaction. Cycle sequencing conditions were as follows: 96°C for 1 min., 25 cycles of 96°C for 10 sec., 55°C for 5 sec., 60°C for 4 min.

SUPPLEMENTARY DATA
www.impactjournals.com/oncotarget/ Oncotarget, Supplementary Materials 2015 treated with H 2 O 2 /3% for 5 min to inhibit endogenous peroxidase, and then washed in H 2 O. The slices were then incubated for 30 min with the first primary antibody (Ab) (mouse anti-SNAI2 Ab) followed by detection with the Bond Polymer Refine Detection Kit (Leica Biosystems) according to the manufacturer's protocol. Then, sections were incubated for 30 min with the second primary Ab (rabbit anti-AMACR, mouse anti-Cytokeratin 5.6, mouse anti-Cytokeratin 18) followed by detection with the Bond Polymer Refine Red Detection Kit (Leica Biosystems) according to the manufacturer's protocol.

Western blotting
Approximately 2.0 × 10 6 cells were collected by low-speed centrifugation (1200 rpm), at room temperature, for 5 minutes. Next, pellets were washed with PBS, at room temperature, and cells were collected by low-speed centrifugation. Then, 1.0 mL of ice cold RIPA Lysis buffer (Thermo Scientific, Waltham, MA, USA) was added, with freshly added Protease and Phosphatase Inhibitors Cocktails (Thermo Scientific), and the cells were incubated on ice for 30 minutes. Then, cells were disrupted and homogenized, transferred to microcentrifuge tubes and centrifuged at 13000 rpm for 10 minutes at 4°C. The supernatants represented the total cell lysates and were transferred to new microfuge tubes.
Membranes containing proteins were blocked with milk 5X (Sigma-Aldrich) in TBST for 1 hour and, subsequently, probed with primary antibodies and incubated overnight. The membranes were then washed for 3 x 15 minutes with TBST and incubated in diluted (1:5000) horseradish peroxidase conjugated secondary antibodies. The following secondary antibodies were used: rabbit anti-mouse IgG (whole molecule)-Peroxidase and rabbit anti-goat IgG (whole molecule)-Peroxidase (both from Sigma-Aldrich); goat anti-rabbit IgG (H + L)-HRP Conjugate (Bio-Rad). Proteins transferred membranes were washed with TBST for 3 x 15 min and developed with Pierce ECL Western Blotting Substrate (Thermo Scientific).