SATB1 as oncogenic driver and potential therapeutic target in head & neck squamous cell carcinoma (HNSCC)

The Special AT-rich sequence binding protein 1 (SATB1) is a genome organizer protein that controls gene expression of numerous genes by regulating chromatin architecture and targeting chromatin-remodeling/-modifying enzymes onto specific chromatin regions. SATB1 is overexpressed in various tumors. In head and neck squamous cell carcinoma (HNSCC), SATB1 upregulation is correlated with TNM classification, metastasis, poor prognosis and reduced overall survival. In this paper, we comprehensively analyze cellular and molecular effects of SATB1 in a large set of primary cell lines from primary HNSCC or metastases, using RNAi-mediated knockdown in vitro and, therapeutically, in tumor xenograft mouse models in vivo. In a series of 15 cell lines, major differences in SATB1 levels are observed. In various 2-D and 3-D assays, growth inhibition upon efficient siRNA-mediated SATB1 knockdown depends on the cell line rather than initial SATB1 levels. Inhibitory effects are found to be based on cell cycle deceleration, apoptosis induction, decreased HER3 and Heregulin A&B expression, and effects on EMT genes. In vivo, systemic treatment of tumor xenograft-bearing mice with siRNAs formulated in polymeric nanoparticles inhibits tumor growth of two HNSCC xenograft models, resulting from therapeutic SATB1 reduction and concomitant decrease of proliferation and induction of apoptosis. In conclusion, SATB1 represents a promising target in HNSCC, affecting crucial cellular processes and molecular pathways.


Radiosensitivity assay
In the radiosensitivity assays conditions were 8% CO 2 and humidified atmosphere. Media / supplements were from Biotech, Aidenbach, Germany. Cells were cultured only for two months which accounted for 12-16 passages, after which a new culture was thawed from the stock.
Radioresponse was assessed in clonogenic survival assays. Single cell suspensions prepared from exponentially growing monolayer cultures pre-exposed for 72h to siLuc3 or siSATB1 in transfection reagent as described above were seeded into 6-well plates at defined densities (300 -4,800 cells/well) in supplemented standard DMEM. After a 10 hours incubation interval to allow cell adherence, culture plates were irradiated at room temperature using single doses of 0-10 Gy with a dose-rate of about 1.3 Gy/min (200 kV X-rays; Yxlon Y.TU 320, Yxlon, 0.5 mm Cu filter). UT-SCC-42B and FaDu cells were then cultured for a total of 8 and 10 days, respectively, to guarantee >5 doublings and the formation of colonies consisting of ≥50 cells. Colonies were fixed and stained followed by manual microscopic counting to determine plating efficiencies (PEs). Survival fractions (SFs) relative to control conditions at 0 Gy were calculated and clonogenic survival curves were fitted, employing the linear-quadratic model Clonogenic surviving fractions were statistically compared by independent Mann-Whitney-U tests including Bonferroni-Holm correction for multiple testing, while linear regression was used for statistical evaluation of the parameters A and B in the linear-quadratic functions modeling the irradiation dose dependent clonogenic survival curves. These tests were performed using SPSS Statistics 21 (IBM Corporation, NY) 1 .

Western Blotting
For protein isolation, identical amounts of cells in suspension (500,000 cells, harvested by trypsinization and counted in a Neubauer chamber) were lysed or, if cell numbers in the wells were substantially different, the protein concentration of the total cell lysates (generated from cells in monolayer) was measured using the BioRad DC protein quantification kit (BioRad Laboratories, Munich, Germany). RIPA-SDS buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 1% (v/v) TritonX-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mM EDTA, 10 mM NaF) was used for lysing the cells. Tumor homogenization was done with a tissue homogenizer (Ultra-turrax; IKA, Staufen, Germany). Cells in monolayer were scraped by cell scraper (Sarstedt; Nümbrecht, Germany) and cells in suspension were lysed only by pipetting. After homogenization (tumor/cell suspension/scrapped cell lysate), undissolved cell debris was eliminated by centrifugation at 5,000 rpm for 10 min at 4 °C. For sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), nitrocellulose membrane (Whatman, Dassel, Germany). Membranes were blocked with 5% (w/v) nonfat dry milk in TBST (10 mM Tris/HCl, pH 7.6, 150 mM NaCl, 0.1% Tween 20), washed with TBST and incubated overnight with primary antibodies at 4°C. For details on the specific antibodies used for immunodetection, see Suppl. Table 3. GAPDH or actin were used for loading control. After washing with TBST, membranes were incubated with horseradish peroxidase-coupled secondary antibodies conjugated with HRP for detection. Protein bands were visualized by using the chemiluminescence ECL kit from Thermo Fisher Scientific in a LAS-4000 chemiluminescence detection system (GE Healthcare, Munich, Germany). Immunoblots of SATB2 and its GAPDH loading controls were visualized using a secondary antibody conjugated with a NIR fluorophore. The detection was done in the near infrared region (700 and 800 nm) using an Odyssey Fc apparatus (LI-COR, Lincoln, NE).
Densitometric analysis of Western blots was performed using ImageJ software, and expression levels were normalized to GAPDH.

Immunofluorescence microscopy
5,000 cells in 500 µl of complete medium were seeded per compartment on Superfrost excel slides (Thermo Fisher Scientific). Compartments for several independent samples were performed by using flexiPERM ® reusable silicone inserts for compartmentalizing the growth surface (Sarstedt, Nümbrecht, Germany). 24 h after seeding, cells were transfected with 5 nM siRNA in 600 µl media as described above. Cells were fixed 96 h later with 100% ice cold methanol for 30 min at -20 °C.
Antigen retrieval was done by boiling the slides for 10 min in 1x citrate buffer, pH 6.0 (Dako/Agilent, Santa Clara, CA), prior to washing in PBS. Unspecific binding was blocked by exposure to 5% (v/v) goat serum, 1% (w/v) BSA in PBS followed by incubating the slides with antibodies against SATB1 (EPR 3951, Abcam) at 4 °C overnight. In parallel, slides without primary antibody were run as negative control. After washing with PBS 3 times for 5 min, Cy3-labelled anti-rabbit antibodies (Jackson Immunoresearch, Ely, UK) were added and incubated at room temperature for 1 h, again followed by washing. To stain cell nuclei, the slides were incubated with 40 µg/ml Hoechst 33342 in TBS (Molecular Probes, Leiden, The Netherlands) for 10 min at room temperature. Images were taken with a confocal microscope (Leica SP8 confocal microsope; Leica, Wetzlar, Germany).

Immunohistochemistry
Tissues were fixed in 4% paraformaldehyde, prior to dehydration and paraffin embedding. Paraffin sections were fixed on the slide by baking at 56 °C overnight and microwaving for 5 min. For deparaffination, slides were incubated 2 x 5 min in Neo-Clear (Merck, Darmstadt, Germany), followed by a series of decreasing ethanol concentrations (100%, 96%, 90%, 70%) and a final short washing step in distilled water. Antigen retrieval was done by microwave pre-treatment in 10mM sodium citrate buffer, pH 6.0, at 90 °C for 15 min. After cooling at room temperature for 10 min, slides were washed 2 x 5 min in PBST (PBS + 1% Tween-20). After washing 2 x 5 min in PBST, slides were treated for 15 min at 4°C with 0.3% H 2 O 2 in PBST, to block endogenous peroxidase activity. For blocking of nonspecific binding sites, slides were treated with blocking solution (PBST containing 2% bovine serum albumin, 10% normal serum from the secondary antibody source) at room temperature for 30 min, followed by incubation with the primary antibodies (mouse anti-Ki67 (1:100) or mouse anti-cleaved caspase-3 (1:50; see Suppl. Table 3

Colony formation and spheroid assay
For assessing the ability of the cells to form colonies on plastic, cells were trypsinized 72 h after transfection, counted as described above, and 1,000-2,000 cells were seeded in a 6 well plate. Cells were allowed to grow for 5 -8 days until each colony contained ~ 30 -50 cells, prior to staining with 1% (w/v) methylene blue in 70% (v/v) ethanol solution for 45 to 60 min. Stained colonies were washed with distilled water, air dried, photographed and counted by image J.
For spheroid assays, transfected cells were trypsinized, counted, and 5,000 cells per well in 250 µl medium were seeded in Nexcelom 3D 96-well round bottom Ultra-low attachment plates (Nexcelom Bioscience, Lawrence, MA). Spheroids were allowed to grow for two weeks prior to taking photos.

Apoptosis assay
Apoptosis was determined by detection of active Caspase 3/7 using a bioluminescent Caspase 3/7 Glo® assay (Promega, Mannheim, Germany). For this purpose, cells were seeded in 96 well plates at an initial density of 1,000 cells/well and transfected as described above. Detection of Caspase 3/7 activation was performed 72 h post transfection according to the manufacturer's protocol.
Luminescence was measured using a POLAR star Omega reader (BMG Labtec, Jena, Germany) after 1 h of incubation at room temperature in the dark. In parallel, cell numbers were determined using a WST-1 colorimetric assay (Roche, Mannheim, Germany), and used for the normalization of caspase luminescence results.