SOX4 expression is associated with treatment failure and chemoradioresistance in oral squamous cell carcinoma

The HNSCC cell lines (PCI 50 and SNU 1041) were kindly provided by Dr. Sung MW (Seoul National University, Seoul, South Korea). The normal immortalized human keratinocyte cell line (HaCaT) was purchased from the American Type Culture Collection (Manassas, VA, USA). Cell lines were cultured in DMEM or RPMI1640 (Hyclone, Logan, UT, USA) supplemented with 10 % fetal bovine serum (Hyclone) in a humidified atmosphere of 5 % CO₂ at 37 °C. For transfection, cells were seeded on 6-well plates at 2 × 10⁵ cells per well at the time of transfection. Small interfering RNA (siRNA) was used to knock down endogenous SOX4 gene expression in HNSCC cells. Cells were transfected with SOX4-specific siRNA (Bioneer, Daejeon, Korea) or negative control siRNA (Qiagen, Valencia, CA, USA) using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA) for 48 h. SOX4 knockdown (SOX4-KO) was checked by reverse transcription-polymerase chain reaction (RT-PCR) and western blotting.

The total RNA from cells was extracted using Trizol reagent (Invitrogen), reverse transcribed, and amplified using specific primers for SOX4 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as previously described [15]. The extracted RNA loading was performed to verify the RNA integrity and double band (18S and 28S) was detected (Additional file 1). Primer sequences were: SOX4: 5′-GCA CAT GGC TGA CTA CCCC −3′/ 5′-GCC TTGTAC AGC GAG TGG TG-3′; and GAPDH: 5′-ACC ACA GTC CAT GCC ATC AC-3′/ 5′-TCC ACC ACC CTG TTG CTG TA-3′. PCR products were separated by electrophoresis on a 1 % agarose gel containing ethidium bromide. The signals were quantified by densitometric analysis using the Labworks Image Acquistion (UVP, Upland, CA).

Cells were lysed in RIPA buffer. Resolved proteins were electrophoretically transferred to polyvinylidene fluoride membranes. Specific proteins were sequentially blotted with primary antibodies: SOX4 (Catalogue# ab80261, Abcam, Cambridge, Mass, USA), cleaved caspase-3, cleaved caspase-7, cleaved poly-ADP ribose polymerase (PARP; Cell Signaling Technology, Danvers, MA, USA), X-linked inhibitor of apoptosis protein (XIAP) and polyclonal anti-GAPDH (Santa Cruz Biotechnology, CA, USA). Each membrane was incubated with anti-rabbit or anti-mouse HRP-conjugated secondary antibody (Santa Cruz Biotechnology). Immunoreactive proteins were visualized on the enhanced chemiluminescence detection system HRP substrate (Millipore, Billerica, MA, USA). The immunoreactive bands were quantified by densitometric analysis using the luminescent image analyzer LAS-4000.

Cells were seeded in a 96-well plate (5 × 10³ cells/well), and were transfected with SOX4 siRNA and negative control siRNA the next day. After incubation for 48 h, cell proliferation and viability were measured using the EZ-CyTox (tetrazolium salts, WST-1) cell viability assay kit (Daeil Lab Inc, Seoul, South Korea). After adding WST-1 reagent for 1–2 h at 37 °C, absorbance at 460 nm was determined using a microplate reader (Infinite M200; Tecan, Austria GmbH, Austria) with Magellan V6 data analysis software (Tecan). Triplicate wells were used for experimental conditions and all experiments were repeated at least three times.

Cells were treated with γ-irradiation at a single dose of 5 Gy (¹³⁷Cs, 2.875 Gy/min) using a Gammacell irradiator (Gammacell, Otawa, Canada) [16, 17]. Cells were treated with cisplatin at 10 μg/ml (Pharmachemie BV, New York, USA) for 24 h at 37 °C.

Cell invasion ability was measured by the number of cells that invaded through a transwell invasion apparatus with 8.0-μm pores (Costar, Cambridge, UK). Living cells transfected with SOX4 siRNA or negative control siRNA were seeded at 3 × 10⁵ cells in 120 μl of a 0.2 % bovine serum albumin (BSA) suspension in the upper chamber. We then loaded 400 μl of 0.2 % BSA containing 7-μg/ml fibronectin (Calbiochem, La Jolla, CA, USA) into the lower chamber as the chemoattractant. After incubation for 24 h, cells that had moved to the bottom Transwell surface were stained with Diff Quik solution (Sysmex, Kobe, Japan) and calculated in five random squares in the microscopic field of view. Results are shown as mean ± standard error of the number of cells/field in three individual experiments.

Cells transfected with SOX4 siRNA or negative control siRNA were seeded in each well of Culture-Inserts (Ibidi, Bonn, Germany) at 1.5 × 10⁵ cells/well. After incubation for 24 h, each insert was detached and the progression of cell migration was ascertained by photography at 0, 4, 8, 12, and 24 h, using an inverted microscope. Distances between gaps were normalized to 1 cm after capture of three random sites.

To evaluate SOX4 protein expression, paraffin-embedded tissue sections were collected from 95 patients who had undergone diagnostic biopsy or definitive surgery for OSCC at Chonnam National University Hwasun Hospital (Jeonnam, Korea) between May 2004 and June 2013. None of the collected tissues were obtained after radiotherapy and/or chemotherapy. Ten patients were excluded, because of follow-up loss or palliative treatment intent. Of the 85 remaining patients, 82 patients were treated with definitive surgery with/without adjuvant radiotherapy or cisplatin-based concurrent chemoradiotherapy (CRT). Three patients, who refused surgery, were treated with induction chemotherapy, followed by cisplatin-based concurrent CRT with curative intent. Patients with locoregional recurrence after primary treatment underwent salvage surgery or CRT. Of 85 patients in our study, 50 (58.8 %) underwent chemotherapy and/or radiotherapy. Treatment failure was defined as disease with inoperable locoregional progression or distant metastasis, even through salvage treatment. Patients provided the written informed consents for the surgical procedures, as well as for the use of resected tissue specimens. Patients’ clinicopathologic characteristics were reviewed in hospital records. Tumors were staged according to the seventh edition of the American Joint Committee on Cancer staging system [18]. Survival was measured from the date of starting treatment to the date of death or date last seen. This study was approved by the Institutional Review Board of Chonnam National University Hwasun Hospital (CNUHH-2015-028).

Tissue processing and immunohistochemical analysis were performed as previously described [15]. The tissues were incubated with polyclonal rabbit anti-human SOX4 (Abcam). Immunohistochemsitry was performed in five batches, averaging 18 samples, with one positive and one negative control per batch. Negative controls were treated similarly, except that primary antibodies were omitted.

Two independent observers interpreted SOX4 staining of specimens with no knowledge of the clinical information. Intensity was scored as follows: 0, no staning of tumor cells; 1+, weak to comparable staining in cytoplasm and/or the nucleus compared to that of non-tumoral cells; 2+, readily appreciable or dark brown staining distinctly marking the tumor cell cytoplasm and/or nucleus [10]. Percentages of stained cells were scored as follows: 0: 0 %; 1: 1–25 %; 2: 26–50 %; 3: 51–75 %; and 4, >75 % [6, 7]. Final staining scores were the product of the intensity and percentage scores, with ≤4 defined as low SOX4 expression and >4 defined as high SOX4 expression. Staining scores were discordant between the two pathologists (KHL and JHL) in five cases (5/85, κ = 0.875), which were re-evaluated by the two pathologists, who then reached an agreement for each inconclusive sample.

Relationships between SOX4 expression and various clinicopathologic parameters were compared using the χ² test and Fisher’s exact test. Survival curves were calculated by the Kaplan–Meier method, and compared using the log-rank test. Experimental differences between the SOX4-KO group and control group were tested with the Mann–Whitney U test. Analyses used Statistical Package for the Social Sciences (SPSS) version 21.0 (Microcal Software Inc, Chicago, IL, USA). P < 0.05 was considered significant.