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PHF20 Inhibition Promotes Apoptosis and Cisplatin Chemosensitivity Via the OCT4‑P‑STAT3‑MCL1 Signaling Pathway in Hypopharyngeal Squamous Cell Carcinoma

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PHF20 Inhibition Promotes Apoptosis and Cisplatin Chemosensitivity Via the OCT4‑P‑STAT3‑MCL1 Signaling Pathway in Hypopharyngeal Squamous Cell Carcinoma INTERNATIONAL JOURNAL OF ONCOLOGY 59: 38, 2021 PHF20 inhibition promotes apoptosis and cisplatin chemosensitivity via the OCT4‑p‑STAT3‑MCL1 signaling pathway in hypopharyngeal squamous cell carcinoma XIUXIU LIU1,2*, ZHANCHENG ZHANG1,3*, SHIFENG KAN1,2, ZHENGHUA LV1,2, SHENGLI ZHOU1,2, XIANFANG LIU1,2, PEIHANG JING1 and WEI XU1,2 1Department of Otorhinolaryngology, Head and Neck Surgery, Shandong Provincial ENT Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong 250012; 2Shandong Provincial Key Laboratory of Otology, Jinan, Shandong 250022; 3Department of Otorhinolaryngology, The Fourth Hospital of Jinan, Jinan, Shandong 250031, P.R. China Received December 5, 2020; Accepted April 12, 2021 DOI: 10.3892/ijo.2021.5218 Abstract. Cisplatin is a widely used platinum‑based chemo‑ confirmed that PHF20 silencing induced tumor growth and therapeutic agent for hypopharyngeal squamous cell carcinoma increased apoptosis in HSCC cells compared with those (HSCC). However, resistance to cisplatin limits its use for the in the control cells. Thus, PHF20 inhibition may promote treatment of HSCC, and the underlying molecular mechanism apoptosis and improve cisplatin chemosensitivity via requires further investigation. The present study performed the OCT4‑p‑STAT3‑MCL1 signaling pathway in HSCC. functional assays to determine whether the expression of plant homeodomain finger protein 20 (PHF20) may be involved in Introduction the apoptosis and cisplatin resistance of HSCC. The expres‑ sion levels of PHF20 were higher in cisplatin‑resistant HSCC Hypopharyngeal squamous cell carcinoma (HSCC) is one of cells compared with those in cisplatin‑sensitive cells. The the most aggressive types of head and neck cancer; 70‑85% of inhibition of PHF20 suppressed cell viability but did not affect patients with HSCC are diagnosed at stage III or IV, and the the migratory and invasive abilities of HSCC cells compared 5‑year overall survival rates are 15‑45% based on studies in with those of negative control‑transfected cells. Furthermore, the USA and Australia between 1971 and 2002 (1‑3). In addi‑ PHF20 inhibition reduced cell viability by enhancing apoptosis tion to surgery, adjuvant chemotherapy is commonly used in compared with those in the control cells in vitro. Notably, the the multidisciplinary treatment of HSCC. Cisplatin is the most inhibition of PHF20 sensitized HSCC cells to cisplatin, thus widely used platinum‑based chemotherapeutic agent for solid increasing apoptosis via the signal transducer and activator tumors, such as HSCC (4). However, resistance to cisplatin of transcription 3 (STAT3)‑myeloid cell leukemia‑1 (MCL1) limits its clinical efficiency and the improvement of survival pathway. Octamer‑binding transcription factor 4 (OCT4) over‑ rate in patients with HSCC (5). expression restored phosphorylated STAT3‑MCL1‑mediated The anticancer mechanism of cisplatin involves the acti‑ apoptosis induced by PHF20 inhibition. In vivo experiments vation of DNA damage response and intrinsic apoptosis (6). Intrinsic apoptosis involves apoptotic executioners including caspase‑3, which cleave substrates such as poly(ADP‑ribose) polymerase 1 (PARP1), resulting in morphological and Correspondence to: Dr Wei Xu, Department of Otorhinolaryngology, biochemical alterations (7). In addition, cisplatin‑induced Head and Neck Surgery, Shandong Provincial ENT Hospital, Cheeloo apoptosis is regulated by the B‑cell lymphoma 2 (Bcl‑2) College of Medicine, Shandong University, 4 West Duanxing Road, protein family, which serves as an apoptotic switch (8). The Jinan, Shandong 250012, P.R. China Bcl‑2 protein family includes pro‑apoptotic and anti‑apoptotic E‑mail: [email protected] proteins, such as myeloid cell leukemia‑1 (MCL1), which regulates the intrinsic apoptosis pathway (9). Various MCL1 *Contributed equally inhibitors have been developed, providing effective approaches Abbreviations: HSCC, hypopharyngeal squamous cell carcinoma; Cis, for the chemosensitization of esophageal squamous carcinoma cisplatin; Bcl‑2, B‑cell lymphoma 2; MCL1, myeloid cell leukemia‑1; cells to cisplatin (10). Thus, understanding the detailed molec‑ PHF20, plant homeodomain finger protein 20 ular mechanisms through which cisplatin chemoresistance is developed may help in the development of effective treatments Key words: plant homeodomain finger protein 20, apoptosis, against HSCC. cisplatin resistance Plant homeodomain finger protein 20 (PHF20), also termed glioma‑expressed antigen 2, is a potent transcrip‑ tional activator (11). PHF20 expression is associated with 2 LIU et al: PHF20 INHIBITION PROMOTES APOPTOSIS AND CISPLATIN CHEMOSENSITIVITY OF HSCC CELLS tumorigenesis (12). PHF20 expression is also associated kit (cat. no. R2052; Zymo Research Corp.). The experiments with a good prognosis of non‑small‑cell lung cancer, and were performed in triplicate. Total RNA was qualified PHF20 activates NF‑κB at the transcriptional factor (13). and quantified using a NanoDrop 2000 and Agilent 2100 However, PHF20 is highly expressed in neuroblastoma Bioanalyzer (Thermo Fisher Scientific, Inc.). cDNA was and promotes the aggressiveness of neuroblastoma cells by generated according to the manufacturer's instructions. The directly binding to the promotor regions of octamer‑binding single‑stranded circular DNA was formatted as the final library, transcription factor 4 (OCT4) (14). OCT4 is a key nuclear which was amplified to produce a DNA nanoball (DNB) and transcriptional factor that maintains the pluripotency and loaded into the patterned nanoarray, and single‑end 50‑base self‑renewal properties of embryonic stem cells (15). OCT4 reads were generated on the MGISEQ2000 platform. The affects the tumorigenesis of a number of types of cancer concentration of the final library was detected by Nanodrop including pancreatic, cervical and ovarian cancer, as well as 2000. The final library was pooled in equimolar concentra‑ glioma (16‑18). OCT4 inhibits apoptosis in cervical cancer by tions (~9.4 nM) and sequenced on the MGISEQ2000 platform. inhibiting microRNA‑125b expression (19), whereas its knock‑ CoolMPS high‑through sequencing kit (cat. no. 1000018234; down suppresses the viability and mobility of hepatocellular MGISEQ‑2000RS FCL SE50; BGI Group) was used to deter‑ carcinoma cells and is associated with low expression levels mine the sequences of the libraries. Differential expression of survivin and phosphorylated STAT3 (20). Furthermore, analysis was performed using the DEGseq2 package with OCT4 expression promotes the differentiation of lung cancer R software (3.6.3) (26,27). Fold‑change >2 and P<0.001 were cells into small lung cancer cells and acquisition of drug used as the screening criteria. resistance (21,22), and its knockdown sensitizes cancer cells to cisplatin treatment in bladder and non‑small lung cancer Cell viability assay. Cell viability was detected using the cells (23,24). Thus, the present study aimed to determine Cell Counting Kit‑8 (CCK‑8) assay (Beyotime Institute of whether the expression of PHF20 may be associated with Biotechnology). A total of ~4x103 cells were seeded in each apoptosis and cisplatin resistance in HSCC cells. well of 96‑well plates containing 100 µl medium. A total of A previous study has demonstrated that PHF20 is involved 10 µl of CCK‑8 solution was added to each well at 24, 48, in tumorigenesis (25). However, the expression level and role 72 or 96 h after cell seeding and incubated for 2 h at 37˚C. of PHF20 in apoptosis and cisplatin sensitivity remain unclear. Subsequently, optical density was measured at 450 nm to The aim of the present study was to determine the expression determine the cell viability by using a microplate reader levels of PHF20 in cisplatin‑resistant and cisplatin‑sensitive (BioTek Instruments, Inc.). HSCC cells and to investigate whether PHF20 knockdown induced apoptosis and sensitized HSCC cells to cisplatin treat‑ RNA extraction and reverse transcription‑quantitative ment. These results may indicate whether PHF20 may be used PCR (RT‑qPCR). Total RNA was extracted from cells using as a potential therapeutic target in HSCC to improve cisplatin TRIzol® reagent (Thermo Fisher Scientific, Inc.) according sensitivity. to the manufacturer's instructions. cDNAs were synthesized using the RevertAid™ First Strand cDNA synthesis kit Materials and methods (Thermo Fisher Scientific, Inc.) as follows: The reaction mixture was prepared, the samples were incubated for 60 min Cell lines and culture. The FaDu cell line was used as the at 42˚C, following which the reaction was terminated for HSCC model and was obtained from the American Type 5 min at 70˚C. qPCR was performed using SYBR® Premix Culture Collection. FaDu cells were cultured in DMEM/F12 Ex Taq (Promega Corporation) on Mastercycler® RealPlex 2 (1:1; Gibco; Thermo Fisher Scientific, Inc.) supplemented (Eppendorf) under the following conditions: 95˚C for 2 min, with 10% fetal bovine serum (FBS; Biological Industries). followed by 40 cycles of 95˚C for 20 sec, 58˚C for 20 sec and The cells were incubated at 37˚C in a humidified incubator 72˚C for 20 sec, and a final dissolution curve. The primer with 5% CO2. Cisplatin was obtained from Sigma‑Aldrich; sequences were as follows: PHF20 forward, 5'‑GAA TGG TCA Merck KGaA (cat. no. P4394) and dissolved in 0.9% sodium ATG CGA GTG G‑3' and reverse, 5'‑TGG CAG CAA GGC TAC chloride solution to obtain a 2‑µM stock solution. TGT G‑3'; OCT4 forward, 5'‑TTG CCG CAA AGT GTG TAA CG‑3' and reverse, 5'‑GTC ACC CCT AAA TGC CAC CG‑3'; Establishment of a cisplatin‑resistant HSCC cell line. First, OCT4A forward, 5'‑CGT GAA GCT GGA GAA GGA GAA GCT FaDu cells were treated with various concentrations (0, 1, 2, 4, G‑3' and reverse, 5'‑CAA GGG CCG CAG CTT ACA CAT GTT 8, 16 and 32 µM) of cisplatin for 2 days, and the half maximal C‑3'; OCT4B forward, 5'‑ATG CAT GAG TCA GTG AAC AG‑3' inhibitory concentration (IC50) was determined. FaDu cells and reverse, 5'‑CCA CAT CGG CCT GTG TAT AT‑3'; OCT4B1 were subsequently treated with the culture medium containing forward, 5'‑GGG TTC TAT TTG GTG GGT TCC‑3' and reverse, 0.25 µM cisplatin for 48 h.
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