Clinical samples

Formalin-fixed paraffin-embedded tissues were collected from 49 Chilean patients with a diagnosis of OPC. The specimens were obtained from the Service of Pathological Anatomy from Clinical Hospital José Joaquín Aguirre, between 2010 and 2020. Additionally, 31/49 (63.3%) of the patients were men and 18/49 (36.7%) were women, with a mean age of 62.6 years. HPV presence/genotyping and p16 detection by IHC were previously determined in these specimens [30]. This study was approved by the Ethics Committee of the Clinical Hospital of the University of Chile (approval code 47/19). The epidemiological data contained in the anatomical-pathological report were obtained and those missing were looked up in the electronic clinical record. The clinical specimens were labeled with an internal number for their management. Each sample was analyzed by an experienced pathologist who selected the areas of the biopsy specimen in which neoplastic involvement was observed, from which material was obtained both to make tissue microarrays (tissue arrays) and for nucleic acid extraction (2 cuts of 10 microns, each).

Tissue arrays and immunohistochemistry (IHC)

Four tissue microarrays were manufactured, in which 13 5 mm samples of oropharynx, a control of non-neoplastic spleen tissue, and a control of neoplastic breast carcinoma tissue were included. The Benchmark Ultra equipment (Ventana, Medical Systems Inc., Tucson, AZ, USA) was used in which the tissue microarrays were introduced into it and Tris–Borate-EDTA (TBE) pH 8.0 buffer was added for 36 min to antigen recovery. Subsequently, nonspecific proteins were blocked by incubation with 3% albumin for 1 h. For cIAP2 IHC, the microarrays were incubated at 4 °C in a humid chamber overnight with the cIAP2 monoclonal antibody (E40 clone; 1:500 dilution). The p16 IHC data was obtained from a previous study by us [30]. Briefly, for p16 IHC, antigenic recovery was carried out in TBE buffer pH 8.0 for 92 min, incubation with the Cintec p16 antibody clone E6H4 (Ventana, Medical Systems Inc., Tucson, AZ, EE. USA) for 1 h at 37°. For cIAP2 and p16, revealing was performed using the UltraView Dab Kit (Ventana, Medical Systems Inc., Tucson, AZ, USA). For cIAP2 analysis, samples were classified as negative when the staining was negative or very slight, and positive when the staining was moderate to intense, which was evaluated by an experienced pathologist. The results for p16 IHC were expressed as negative and positive according to the standard of the Pathological Anatomy Service according to the indications provided by the American College of Pathologists (positive with ≥ 70% nuclear and cytoplasmic staining) (de C Ferreira et al., 2021).

Cell cultures and transfections

SCC-143 cells (squamous cell carcinoma of the floor of the mouth) were obtained from the University of Pittsburgh (White et al., 2007). Cells were incubated in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Fremont, CA, USA) and antibiotics (100 U/mL penicillin and 100 g/mL streptomycin) and were maintained at 37ºC in a 5% CO2 atmosphere incubator. For subculture, cells were incubated with trypsin for 3–5 min and maintained with fresh medium containing Fetal Bovine Serum (FBS) (Hyclone, Fremont, CA, USA). The cells were tested against mycoplasma contamination. The pLXSN and pLXSNVPH16E6/E7 plasmids were donated by Dr. Massimo Tommasino†, from the International Agency for Research on Cancer (IARC), Lyon, France. Retroviral transduction was performed with GP + envAM-12 packaging cells previously transfected with the pLXSN or pLXSNHPV16E6/E7 plasmids with lipofectamine 2000 and maintained for 24 h at 37 °C under conditions of 5% CO2 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. SCC-143 cells were stably transduced with retroviruses and then selected with 0.3 mg/mL geneticin (GIBCO, Carlsbad, CA, USA). Knocking down of HPV16 E6, HPV16 E7 and cIAP2 was performed in SCC-143 E6/E7 cells using small interfering RNA (siRNA) (SC-156008 for E6, SC-270423 for E7, SC-29850, Santa Cruz). A scramble sequence was used as the negative control (SC-37007, Santa Cruz). Transfection of SCC-143 cells with the siRNAs was performed using FuGENE® 6 (Promega, Madison, WI, USA), according to the manufacturer's instructions.

RNA extraction, reverse-transcriptase PCR, and reverse-transcriptase quantitative PCR

The RNA from clinical specimens was purified using the High Pure RNA Paraffin Kit (Roche, Pleasanton, CA, USA), following the manufacturer’s instructions. The RNA from SCC-143 cells was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After chloroform purification and isopropanol precipitation, the RNA was suspended in diethylpyrocarbonate (DEPC)-treated water and stored at − 80 °C. Next, the RNA was incubated with RQ1 RNase-free DNAse (Promega, Madison, WI, USA) at 37 °C for 60 min and then treated with RQ1 DNAse Stop Solution for 10 min. cDNA was prepared using a 20 µL-reaction volume containing DNAse treated RNA (1 µg), 1 U RNAse inhibitor (Promega, Madison, WI, USA), 0.04 µg/µL random primers (Promega, Madison, WI, USA), 2 mM dNTPs (Promega, Madison, WI, USA) and 10 U Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (Promega, Madison, WI, USA). The reaction mixture was incubated for 1 h at 37 °C. The obtained cDNA was stored at − 20 °C until use. For cIAP2 transcript detection in clinical specimens, a semiquantitative RT-PCR was carried out, using the following primers: cIAP2 F 5′-AGCTACCTCTCAGCCTACTTT-3′ and cIAP2 R 5′-CCACTGTTTTCTGTACCCGGA3′. The conditions of amplification were: 94 °C for 5 min, followed by 33 cycles that included 95 °C for 45 s, 56 °C for 40 s and 72 °C for 45 s, with a final extension at 72 °C for 5 min. The PCR products were characterized by 2.5% agarose gel electrophoresis and stained with SafeView PlusTM (abm, Vancouver, BC, Canada) and visualized by UV exposure in a transilluminator (Vilber-Lourmat, Marne La Vallée, France). The ImageJ software version 1.52a was used for semiquantitative analysis. For experiments in the SCC-143 cell line, RT-qPCR was performed on the AriaMx Real-Time machine (Agilent, Santa Clara, CA, USA) in a final volume of 25 μL. The reaction components were as follows: 12.5 μL of 2X SYBR Green Mastermix (Bioline, London, United Kingdom), 7.5 μL of nuclease-free water, and 1 μL of cDNA. The amplification conditions were as follows: initial denaturation at 94 °C for 30 s, 58 °C for 20 s, and 72 °C for 20 s, for a total of 40 cycles. The relative copy number of each sample was calculated using the 2 − ∆∆Ct method. A melting curve was carried to determine the specific melting temperature of each amplicon. Endogenous β-actin mRNA levels were used for the normalization of RNA expression. All reactions were performed in triplicate.

Western blotting

Protein lysate was obtained from SCC-143 cells transduced with either an empty vector or HPV16 E6/E7 vector, using 1X RIPA lysis buffer (Abcam, Cambridge, United Kingdom) containing protease and phosphatase inhibitors (Roche, Basel, Switzerland). The suspensions were centrifuged at 14,000 rpm for 15 min at 4 °C. Protein concentration was determined using the PierceTM BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Next, 15 μg of total protein was loaded per well and separated by SDS-PAGE on 12% gels. Proteins were then transferred by semidry electrotransfer to Hybond-P ECL membranes (Amersham, Piscataway, NJ, USA), using a Tris–glycine transfer buffer pH 8.3 (20 mM Tris, 150 mM glycine and 20% methanol) and the Trans-Blot SD® kit (Bio-Rad, Hercules, CA, USA). Membranes were blocked in 5% bovine serum albumin/0.5% Tween-20 in Tris-buffered saline pH 7.6 (TBS) for 1 h at room temperature and then incubated overnight at 4 ◦C with primary antibodies against cIAP2 (ab32059), p53 (BD554294) (BD PharmigenTM, San Diego, CA, USA), pRb (ab24) or β-actin (SC47778) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) diluted 1:1000 in TBS/Tween 20 (TBS–T20). After three washes in TBS-T20, the membranes were incubated with Anti-Mouse IgG (BD Pharmingen; BD Biosciences, Heidelberg, Germany) or Anti-Rabbit IgG (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). USA) conjugated with HRP, diluted 1:1000 in 5% BSA blocking buffer for 1 h at room temperature. Membranes were washed three times for 15 min and revealed with ClarityTM Western ECL detection reagent (Bio-Rad, Hercules, CA, USA) in a ChemiDoc™ Imaging System (Bio-Rad, Hercules, CA, USA), according to the manufacturer's instructions.

Radiation exposure and apoptosis analysis by flow cytometry

The cellular radiation assays were performed at the National Cancer Institute (INC) under the supervision of a radiation oncologist and a medical physicist. After 24 h post-transfection, SCC143 E6/E7 control and SCC143 E6/E7 si-cIAP2 cell cultures were irradiated in 6-well plates (200,000 cells per well), using the Trilogy clinical electron linear accelerator (Varian) at doses of 0, 2, 4, 6 and 8 Gy. SCC143 E6/E7 and SCC143 E6/E7 si-cIAP2 cells were trypsinized and harvested 8 h post-irradiation. The flow cytometry assay for the evaluation of apoptosis was performed using Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (ThermoFisher, Waltham, MA, USA) according to the manufacturer's instructions. The tests were carried out in triplicate.

In silico analysis

The UCSC Xena web source (https://xenabrowser.net) allowed us to explore functional genomic datasets and correlational genomic and phenotypic variables. We selected 612 HNSSC samples from GDC TCGA Head and Neck Cancer Database—first, BIRC3 gene expression concerning the HPV status (ISH information). Next, only 33 HNSSCs met the following criteria: (1) HPV status information, (2) BIRC3 expression, and (4) tumor primary site (tonsils and base of tongue cancers).

Statistical analysis

Statistical analysis was performed using GraphPad Prism 9 (Version 9.3.1) and Stata 17.0 (Stata Corp, Texas, USA) softwares. The results were considered statistically significant when p was less than or equal to 0.05.