Published OnlineFirst December 13, 2016; DOI: 10.1158/1078-0432.CCR-16-2811 Biology of Human Tumors Clinical Cancer Research Involvement of c-Fos in the Promotion of Cancer Stem-like Cell Properties in Head and Neck Squamous Cell Carcinoma Naoshad Muhammad1, Sourav Bhattacharya1, Robert Steele1, Nancy Phillips1, and Ratna B. Ray1,2 Abstract Purpose: Head and neck squamous cell carcinoma (HNSCC) is these cells tumorigenic in nude mice. Furthermore, subcutaneous the sixth most common cancer worldwide. Although improve- transplantation of c-Fos–overexpressing Cal27 cells (tumorigen- ments in surgical techniques, chemotherapy and radiation deliv- ic) into immunocompromised mice enhanced tumor growth as ery, and supportive care have improved quality of life for patients compared with parental cells. Mechanistic investigations dem- with HNSCC, regional and distant recurrence remain common. onstrated that c-Fos overexpression enhanced the epithelial– Recent evidence suggests that cancer stem-like cells (CSC) play a mesenchymal transition (EMT) state and expression of CSC significant role in recurrence and chemoresistance. We previously markers (Nanog, c-Myc, Sox2, and Notch1). Ectopic expression observed that c-Fos was highly upregulated in the HNSCC sphere– ofc-FosinHNSCCcellsalsodisplaysincreasedsphereforma- forming cells. Consequences of c-Fos upregulation for the biology tion. We further observed that overexpression of c-Fos increased of HNSCC-CSCs are poorly understood. In this study, we inves- the expression of pERK and cyclin D1 in HNSCC cells. tigated the role of c-Fos in renewal of stemness of HNSCC and Conclusions: Together, our results strongly suggest a novel tumor growth. role of c-Fos as a regulator of EMT and cancer stem cell repro- Experimental Design and Results: We generated stable gramming in HNSCC cells, which may hold potential as a CSC- HNSCC cell lines ectopically expressing the c-Fos gene. Exogenous directed therapeutic approach to improve HNSCC treatment. expression of c-Fos in nontumorigenic MDA1386Tu cells makes Clin Cancer Res; 1–9. Ó2016 AACR. Introduction of c-Jun and ultimately forms the transcription factor–activating protein 1 (AP-1). As a member of AP-1 family, c-Fos protein is Head and neck squamous cell carcinoma (HNSCC) is the sixth associated primarily in signal transduction, cell differentiation, most prevalent cancer in the world. Overall survival rate has not and proliferation (6). However, the downstream signaling path- improved in the past couple of decades, despite significant ways induced by c-Fos activation and their role in tumorigenesis improvements in surgical procedures, radiotherapy, and chemo- and metastasis remains to be understood. therapy. Thus, gaining insights into the signaling pathways that Transgenic mice overexpressing the c-Fos proto-oncogene post- control the high incidence of local recurrence and distant metas- natally develop osteosarcomas with 100% penetrance (7). In tases will be instrumental for developing new therapeutic modal- addition, overexpression of c-Fos has been shown to promote ities for the management of HNSCC. Solid tumors contain a small drug resistance phenotype (8). c-Fos can cause a loss of cell population of cancer stem-like cells (CSC) in several types of polarity and epithelial–mesenchymal transition, leading to inva- cancers including HNSCC, which plays a critical role in the sive and metastatic growth in mammary epithelial cells (9). AP-1 resistance to therapy, facilitating cell growth and metastasis. complex may play a crucial role in the maintenance of colon However, the pathogenesis and biological significance of CSCs cancer stem cells (10). Clinical data suggested that c-Fos may be in HNSCC has not been fully understood. associated with lymph node metastasis in oral cancer (11). Our We have recently observed that c-Fos was upregulated in previous results (1) and these observations in the literature HNSCC-sphere forming cells as compared with parental cells prompted us to investigate the role of c-Fos in cancer stemness (1). c-Fos is a proto-oncogene and present in various types of of HNSCC cells. In this study, we observed that c-Fos expression in cancers including HNSCC (2–5). c-Fos encodes a nuclear DNA- HNSCC cell lines augments cell proliferation, enhances CSC binding protein domain that forms a dimer with the gene product marker gene expression, and promotes tumor growth. Our results strongly demonstrated that c-Fos plays an important role in tumor 1Department of Pathology, Saint Louis University, St. Louis, Missouri. 2Cancer progression and promotes cancer stemness in HNSCC. Center, Saint Louis University, St. Louis, Missouri. Corresponding Author: Ratna B. Ray, Saint Louis University, DRC 207, 1100 Materials and Methods South Grand Boulevard, St. Louis, MO 63104. Phone: 314-977-7822; Fax: 314-771- RNA-seq analysis 3816; E-mail: [email protected] þ À RNA was isolated from CD133 and CD133 cells and RNA- doi: 10.1158/1078-0432.CCR-16-2811 seq was done using Washington University ICTS Core Services. Ó2016 American Association for Cancer Research. Briefly, ribosomal RNA was removed by a hybridization method www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on October 4, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst December 13, 2016; DOI: 10.1158/1078-0432.CCR-16-2811 Muhammad et al. nonreplicating lentiviruswas purchasedfrom Origene.A-Fos(dom- Translational Relevance inant negative) plasmid DNA was obtained from Addgene. Head and neck squamous cell carcinoma (HNSCC) has a high mortality rate despite advancement in chemotherapy, Cell proliferation assay. Cells (Cal27 control, Cal27-c-Fos, radiotherapy, surgical procedure, and targeted therapy. Emerg- MDA1386Tu control, and MDA1386Tu-c-Fos) were seeded in ing evidence implicated that recurrence and metastasis from triplicates, and cells were trypsinized at 24, 48, and 72 hours. All these tumors occur from cancer-like stem cells, although the cells were counted by Trypan blue exclusion method using a mechanism remains unknown. Our study revealed the role of hemocytometer. c-Fos in the promotion of cancer stem-like cell properties in head and neck cancer. Overexpression of c-Fos in nontumori- Sphere (oralspheres) formation assay genic HNSCC cells exhibits tumorigenic phenotype, increases For sphere formation, Cal27 control, Cal27-c-Fos, stemness markers, and increases oralsphere formation. Taken MDA1386Tu control, and MDA1386Tu-c-Fos cells (5,000 cells/ together, our data indicate that c-Fos plays a crucial role in well) were seeded in single-cell suspensions on ultralow attach- driving the stemness properties in HNSCC and may be a good ment plates (Corning) as described previously (1). After 10 days therapeutic target. of incubation, numbers of oralspheres (>75 mm diameter) were counted using inverted microscope (Leica). Western blot analysis and antibodies using Ribo-ZERO kits (EpiCentre). mRNA was then fragmented Cell lysates were analyzed by SDS-PAGE and transferred onto and reverse transcribed to yield double-stranded cDNA. cDNA 0.45 mm nitrocellulose membrane (Bio-Rad). Membranes were was blunt ended, had an A base added to the 3' ends, and then had blocked using 5% low-fat dry milk in TBST and probed with the Illumina sequencing adapters ligated to the ends. Ligated frag- respective primary antibodies. Proteins were detected using ECL ments were then amplified for 12 cycles using primers incorpo- Western Blotting Substrate (Thermo Scientific) and autoradiogra- rating unique index tags. Fragments were sequenced on an Illu- phy. The protein loading was normalized using antibody to mina HiSeq-2500 using single reads extending 50 bases. b-actin or GAPDH. The following antibodies were used in this study: Nanog, Vimentin, c-Met c-Myc, Sox2, Notch 1, pERK, ERK, Cell lines and plasmid DNAs c-Fos (Cell Signaling Technology), cytokeratin19, cyclin D1, and HNSCC cell line (Cal27) were purchased from the ATCC and b-actin (Santa Cruz Biotechnology). nontumorigenic HNSCC MDA1386Tu cells were obtained from Jeff Myers (MD Anderson Cancer Center, Houston, TX), and main- Immunohistochemistry. The paraffin-embedded sections (5-mm tained in gDMEM or RPMI1640 (Sigma) medium supplemented thick) were deparaffinized and stained using antibody against with 10% FBS, and 1% penicillin/streptomycin in a humidified cytokeratin 19 and vimentin, followed by 3,30-diaminobenzidine CO2 incubator. c-Fos-Myc-DDK plasmid DNA or control plasmid staining in clinical histology laboratory. We used mouse mAbs for DNA in mammalian expression vector (Origene) was transfected Cytokeratin 19 (A53-B/A2.26) from Cell Marque (CA) and into Cal27 and MDA1386Tu cell lines, selected with puromycin, Vimentin (V9) from Ventana (AR). The slides were evaluated by pooled for establishment of a stable cell line. c-Fos shRNA in a pathologist (N. Phillips). Table 1. Differentially expressed genes Gene ID Symbol Fold change P FDR Highly upregulated genes 125740 FOSB 382.842 2.99EÀ85 1.73EÀ80 118503 TNFAIP3 195.041 252EÀ44 5.83EÀ40 169429 CXCL8 178.842 8.67EÀ41 1.67EÀ36 114315 HES1 168.761 1.38EÀ38 2.28EÀ34 128422 KRT17 157.761 3.49EÀ36 5.04EÀ32 143537 ADAM15 141.341 1.35EÀ32 1.74EÀ28 143398 PIP5K1A 126.338 2.59EÀ29 2.99EÀ25 137497 NUMA1 104.068 1.95EÀ24 1.88EÀ24 118515 SGK1 102.12 5.23EÀ24 4.64EÀ20 124788 ATXN1 99.408 2.05EÀ23 1.69EÀ19 Highly downregulated genes 150779 TIMM8B 11.899 5.62EÀ04 5.00EÀ02 168036 CTNNB1 11.911 5.58EÀ04 4.97EÀ02 176095 IP6K1 12.046 5.19EÀ04 4.72EÀ02 8710 PKD1 12.358 4.39EÀ04 4.15EÀ02 146678 IGFBP1 13.156 2.87EÀ04 3.00EÀ02 143514 TP53BP2 13.509 2.37EÀ04 2.62EÀ02 198001 IRAK4 14.154 1.68EÀ04 2.04EÀ02 99875 MKNK2 14.175 1.67EÀ04 2.02EÀ02 184545 DUSP8 15.185 9.75EÀ05 1.41EÀ02 33327 GAB2 15.261 9.37EÀ05 1.39EÀ02 OF2 Clin Cancer Res; 2017 Clinical Cancer Research Downloaded from clincancerres.aacrjournals.org on October 4, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst December 13, 2016; DOI: 10.1158/1078-0432.CCR-16-2811 c-Fos in the Enhancement of Cancer Stem-like Properties Figure 1.