Human Papillomavirus 16 E6 Induces FoxM1B in Oral Keratinocytes through GRHL2

High-risk human papillomavirus (HPV) is a major risk factor for oral and pharyngeal cancers (OPCs), yet the detailed mechanisms by which HPV promotes OPCs are not understood. Forkhead box M1B (FoxM1B) is an oncogene essential for cell cycle progression and tumorigenesis, and it is aberrantly overexpressed in many tumors. We previously showed that FoxM1B was the putative target of an epithelial-specific transcription factor, Grainyhead-like 2 (GRHL2). In the current study, we demonstrate that HPV type 16 (HPV-16) E6 induces FoxM1B in human oral keratinocytes (HOKs) and tonsillar epithelial cells (TECs) in part through GRHL2. FoxM1B was barely detectable in cultured normal human oral keratinocytes (NHOKs) and progressively increased in immortalized HOKs harboring HPV-16 genome (HOK-16B) and tumorigenic HOK-16B/BaP-T cells. Retroviral expression of HPV-16 E6 and/or E7 in NHOKs, TECs, and hypopharyngeal carcinoma cells (FaDu) revealed induction of FoxM1B and GRHL2 by the E6 protein but not E7. Both GRHL2 and FoxM1B were strongly induced in the epidermis of HPV-16 E6 transgenic mice and HPV+ oral squamous cell carcinomas. Ectopic expression of FoxM1B led to acquisition of transformed phenotype in HOK-16B cells. Loss of FoxM1B by lentiviral short hairpin RNA vector or chemical inhibitor led to elimination of tumorigenic characteristics of HOK-16B/BaP-T cells. Luciferase reporter assay revealed that GRHL2 directly bound and regulated the FoxM1B gene promoter activity. Using epithelial-specific Grhl2 conditional knockout mice, we exposed wild-type (WT) and Grhl2 KO mice to 4-nitroquinolin 1-oxide (4-NQO), which led to induction of FoxM1B in the tongue tissues and rampant oral tumor development in the WT mice. However, 4-NQO exposure failed to induce tongue tumors or induction of FoxM1B expression in Grhl2 KO mice. Collectively, these results indicate that HPV-16 induces FoxM1B in part through GRHL2 transcriptional activity and that elevated FoxM1B level is required for oropharyngeal cancer development.

[1]  Yifei Liu,et al.  FoxM1 overexpression promotes cell proliferation and migration and inhibits apoptosis in hypopharyngeal squamous cell carcinoma resulting in poor clinical prognosis , 2017, International journal of oncology.

[2]  Wei Chen,et al.  Grainyhead-like 2 regulates epithelial plasticity and stemness in oral cancer cells. , 2016, Carcinogenesis.

[3]  F. Kelleher,et al.  FOXM1 in sarcoma: role in cell cycle, pluripotency genes and stem cell pathways , 2016, Oncotarget.

[4]  L. Schumaker,et al.  Correlation of p16 expression and HPV type with survival in oropharyngeal squamous cell cancer. , 2015, Oral oncology.

[5]  B. Vanderhyden,et al.  Genetic determinants of FOXM1 overexpression in epithelial ovarian cancer and functional contribution to cell cycle progression , 2015, Oncotarget.

[6]  C. Dieterich,et al.  A Grhl2-dependent gene network controls trophoblast branching morphogenesis , 2015, Development.

[7]  Ya‐Wen Cheng,et al.  Up-Regulation of FOXM1 by E6 Oncoprotein through the MZF1/NKX2-1 Axis Is Required for Human Papillomavirus–Associated Tumorigenesis12 , 2014, Neoplasia.

[8]  Hongyan Chen,et al.  Down-regulation of FoxM1 by thiostrepton or small interfering RNA inhibits proliferation, transformation ability and angiogenesis, and induces apoptosis of nasopharyngeal carcinoma cells. , 2014, International journal of clinical and experimental pathology.

[9]  Alison M. Urvalek,et al.  The molecular features of tongue epithelium treated with the carcinogen 4-nitroquinoline-1-oxide and alcohol as a model for HNSCC. , 2013, Carcinogenesis.

[10]  J. Fujimoto,et al.  Comparable molecular alterations in 4-nitroquinoline 1-oxide-induced oral and esophageal cancer in mice and in human esophageal cancer, associated with poor prognosis of patients. , 2013, In vivo.

[11]  Udo Schumacher,et al.  Dual Roles of the Transcription Factor Grainyhead-like 2 (GRHL2) in Breast Cancer* , 2013, The Journal of Biological Chemistry.

[12]  M. Lingen,et al.  PTEN deficiency contributes to the development and progression of head and neck cancer. , 2013, Neoplasia.

[13]  Hong Jiang,et al.  Grhl2 Determines the Epithelial Phenotype of Breast Cancers and Promotes Tumor Progression , 2012, PloS one.

[14]  M. Kang,et al.  Grainyhead-like 2 (GRHL2) inhibits keratinocyte differentiation through epigenetic mechanism , 2012, Cell Death and Disease.

[15]  J. Tagne,et al.  The Transcription Factors Grainyhead-like 2 and NK2-Homeobox 1 Form a Regulatory Loop That Coordinates Lung Epithelial Cell Morphogenesis and Differentiation* , 2012, The Journal of Biological Chemistry.

[16]  David P. Davis,et al.  A whole-genome RNAi screen identifies an 8q22 gene cluster that inhibits death receptor-mediated apoptosis , 2011, Proceedings of the National Academy of Sciences.

[17]  C. Buck,et al.  Glycosaminoglycans and Sialylated Glycans Sequentially Facilitate Merkel Cell Polyomavirus Infectious Entry , 2011, PLoS pathogens.

[18]  Hans Clevers,et al.  The cancer stem cell: premises, promises and challenges , 2011, Nature Medicine.

[19]  J. Hocking,et al.  Head and neck cancer in Australia between 1982 and 2005 show increasing incidence of potentially HPV-associated oropharyngeal cancers , 2011, British Journal of Cancer.

[20]  N. Park,et al.  Grainyhead-like 2 Enhances the Human Telomerase Reverse Transcriptase Gene Expression by Inhibiting DNA Methylation at the 5′-CpG Island in Normal Human Keratinocytes* , 2010, The Journal of Biological Chemistry.

[21]  A. Barsotti,et al.  Pro-proliferative FoxM1 is a target of p53-mediated repression , 2009, Oncogene.

[22]  S. Teo,et al.  FOXM1 Upregulation Is an Early Event in Human Squamous Cell Carcinoma and it Is Enhanced by Nicotine during Malignant Transformation , 2009, PloS one.

[23]  V. Patel,et al.  Targeting Mammalian Target of Rapamycin by Rapamycin Prevents Tumor Progression in an Oral-Specific Chemical Carcinogenesis Model , 2009, Cancer Prevention Research.

[24]  Wei Chen,et al.  Regulation of the hTERT promoter activity by MSH2, the hnRNPs K and D, and GRHL2 in human oral squamous cell carcinoma cells , 2008, Oncogene.

[25]  Takao Kawabe,et al.  Gain of GRHL2 is associated with early recurrence of hepatocellular carcinoma. , 2008, Journal of hepatology.

[26]  K. Yao,et al.  Over‐expression of FOXM1 transcription factor is associated with cervical cancer progression and pathogenesis , 2008, The Journal of pathology.

[27]  Goberdhan P Dimri,et al.  Elevated Bmi-1 expression is associated with dysplastic cell transformation during oral carcinogenesis and is required for cancer cell replication and survival , 2006, British Journal of Cancer.

[28]  Paul Martin,et al.  Cell Biology: Master Regulators of Sealing and Healing , 2005, Current Biology.

[29]  K. Kelsey,et al.  Human papillomavirus type 16 and squamous cell carcinoma of the head and neck. , 2002, Clinical cancer research : an official journal of the American Association for Cancer Research.

[30]  Chong Sze Tong,et al.  Over‐expression of FoxM1 stimulates cyclin B1 expression , 2001, FEBS letters.

[31]  M. Kang,et al.  Conversion of normal to malignant phenotype: telomere shortening, telomerase activation, and genomic instability during immortalization of human oral keratinocytes. , 2001, Critical reviews in oral biology and medicine : an official publication of the American Association of Oral Biologists.

[32]  M. Kang,et al.  In vitro replication and differentiation of normal human oral keratinocytes. , 2000, Experimental cell research.

[33]  H. Pitot,et al.  The Human Papillomavirus Type 16 E6 Gene Alone Is Sufficient To Induce Carcinomas in Transgenic Animals , 1999, Journal of Virology.

[34]  M. Bobrow,et al.  High sensitivity detection of HPV-16 in SiHa and CaSki cells utilizing FISH enhanced by TSA , 1997, Histochemistry and Cell Biology.

[35]  H. Pitot,et al.  Squamous epithelial hyperplasia and carcinoma in mice transgenic for the human papillomavirus type 16 E7 oncogene , 1996, Journal of virology.

[36]  N. Park,et al.  Combined oral carcinogenicity of HPV-16 and benzo(a)pyrene: an in vitro multistep carcinogenesis model. , 1995, Oncogene.

[37]  N. Park,et al.  Immortalization of normal human oral keratinocytes with type 16 human papillomavirus. , 1991, Carcinogenesis.