GLI3 knockdown decreases stemness, cell proliferation and invasion in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is an extremely aggressive disease associated with a poor prognosis. Previous studies have established that cancer stem cells (CSCs) actively participate in OSCC development, progression and resistance to conventional treatments. Furthermore, CSCs frequently exhibit a deregulated expression of normal stem cell signalling pathways, thereby acquiring their distinctive abilities, of which self-renewal is an example. In this study, we examined the effects of GLI3 knockdown in OSCC, as well as the differentially expressed genes in CSC-like cells (CSCLCs) expressing high (CD44high) or low (CD44low) levels of CD44. The prognostic value of GLI3 in OSCC was also evaluated. The OSCC cell lines were sorted based on CD44 expression; gene expression was evaluated using a PCR array. Following this, we examined the effects of GLI3 knockdown on CD44 and ESA expression, colony and sphere formation capability, stem-related gene expression, proliferation and invasion. The overexpression of genes related to the Notch, transforming growth factor (TGF)β, FGF, Hedgehog, Wnt and pluripotency maintenance pathways was observed in the CD44high cells. GLI3 knockdown was associated with a significant decrease in different CSCLC fractions, spheres and colonies in addition to the downregulation of the CD44, Octamer-binding transcription factor 4 (OCT4; also known as POU5F1) and BMI1 genes. This downregulation was accompanied by an increase in the expression of the Involucrin (IVL) and S100A9 genes. Cellular proliferation and invasion were inhibited following GLI3 knockdown. In OSCC samples, a high GLI3 expression was associated with tumour size but not with prognosis. On the whole, the findings of this study demonstrate for the first time, at least to the best of our knowledge, that GLI3 contributes to OSCC stemness and malignant behaviour. These findings suggest the potential for the development of novel therapies, either in isolation or in combination with other drugs, based on CSCs in OSCC.

[1]  A. Ruiz i Altaba,et al.  Human colon cancer epithelial cells harbour active HEDGEHOG-GLI signalling that is essential for tumour growth, recurrence, metastasis and stem cell survival and expansion , 2009, EMBO molecular medicine.

[2]  R. Segal,et al.  Hedgehog Signal Transduction: Key Players, Oncogenic Drivers, and Cancer Therapy. , 2016, Developmental cell.

[3]  M. Sugiyama,et al.  CD44(high) /ALDH1(high) head and neck squamous cell carcinoma cells exhibit mesenchymal characteristics and GSK3β-dependent cancer stem cell properties. , 2016, Journal of oral pathology & medicine : official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology.

[4]  A. van Oudenaarden,et al.  A predictive model of bifunctional transcription factor signaling during embryonic tissue patterning. , 2014, Developmental cell.

[5]  J. Nör,et al.  The biology of head and neck cancer stem cells. , 2012, Oral oncology.

[6]  Hua Tian,et al.  A paracrine requirement for hedgehog signalling in cancer , 2008, Nature.

[7]  O. Abdel-Rahman.  Hedgehog pathway aberrations and gastric cancer; evaluation of prognostic impact and exploration of therapeutic potentials , 2015, Tumor Biology.

[8]  S. Sell.  Cancer Stem Cells and Differentiation Therapy , 2006, Tumor Biology.

[9]  Takuji Tanaka,et al.  The pathological significance of Notch1 in oral squamous cell carcinoma , 2013, Laboratory Investigation.

[10]  F. Nunes,et al.  Cancer stem cell, cytokeratins and epithelial to mesenchymal transition markers expression in oral squamous cell carcinoma derived from ortothopic xenoimplantation of CD44high cells. , 2017, Pathology, research and practice.

[11]  Maria Kasper,et al.  Selective Modulation of Hedgehog/GLI Target Gene Expression by Epidermal Growth Factor Signaling in Human Keratinocytes , 2006, Molecular and Cellular Biology.

[12]  Cun-Yu Wang,et al.  Characterization of Side Populations in HNSCC: Highly Invasive, Chemoresistant and Abnormal Wnt Signaling , 2010, PloS one.

[13]  Chu-zhi Pan,et al.  The SHH/Gli axis regulates CD90‐mediated liver cancer stem cell function by activating the IL6/JAK2 pathway , 2018, Journal of cellular and molecular medicine.

[14]  P. Das,et al.  CD44(high)CD24(low) molecular signature determines the Cancer Stem Cell and EMT phenotype in Oral Squamous Cell Carcinoma. , 2016, Stem cell research.

[15]  M. Katoh,et al.  Hedgehog signaling, epithelial-to-mesenchymal transition and miRNA (review). , 2008, International journal of molecular medicine.

[16]  F. Soares,et al.  Activation of sonic hedgehog signaling in oral squamous cell carcinomas: a preliminary study. , 2011, Human pathology.

[17]  I. Ganly,et al.  Ten years of progress in head and neck cancers. , 2012, Journal of the National Comprehensive Cancer Network : JNCCN.

[18]  K. Watabe,et al.  The gain-of-function GLI1 transcription factor TGLI1 enhances expression of VEGF-C and TEM7 to promote glioblastoma angiogenesis , 2015, Oncotarget.

[19]  R. Myers,et al.  Human Homolog of patched, a Candidate Gene for the Basal Cell Nevus Syndrome , 1996, Science.

[20]  R. Agarwal,et al.  Implications of cancer stem cells in developing therapeutic resistance in oral cancer. , 2016, Oral oncology.

[21]  I. Mackenzie,et al.  Cancer stem cells in squamous cell carcinoma switch between two distinct phenotypes that are preferentially migratory or proliferative. , 2011, Cancer research.

[22]  V. Ramos-Mejia,et al.  Gli3-mediated hedgehog inhibition in human pluripotent stem cells initiates and augments developmental programming of adult hematopoiesis. , 2013, Blood.

[23]  Minhong Yan,et al.  Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis , 2006, Nature.

[24]  D. Rowitch,et al.  Sonic hedgehog Promotes G1 Cyclin Expression and Sustained Cell Cycle Progression in Mammalian Neuronal Precursors , 2000, Molecular and Cellular Biology.

[25]  R. Weinberg,et al.  EMT, CSCs, and drug resistance: the mechanistic link and clinical implications , 2017, Nature Reviews Clinical Oncology.

[26]  L. Chen,et al.  EZH2-mediated upregulation of ROS1 oncogene promotes oral cancer metastasis , 2017, Oncogene.

[27]  Y. Yen,et al.  A subpopulation of CD133(+) cancer stem-like cells characterized in human oral squamous cell carcinoma confer resistance to chemotherapy. , 2010, Cancer letters.

[28]  S. Akira,et al.  An epigenetic switch induced by Shh signalling regulates gene activation during development and medulloblastoma growth , 2014, Nature Communications.

[29]  G. Trigiante,et al.  GLI2 Is a Regulator of β-Catenin and Is Associated with Loss of E-Cadherin, Cell Invasiveness, and Long-Term Epidermal Regeneration. , 2017, The Journal of investigative dermatology.

[30]  Huiqing Zeng,et al.  The loss of Hh responsiveness by a non-ciliary Gli2 variant , 2015, Development.

[31]  I. Mackenzie,et al.  Phenotypic Plasticity Determines Cancer Stem Cell Therapeutic Resistance in Oral Squamous Cell Carcinoma , 2016, EBioMedicine.

[32]  E. Rosenthal,et al.  Notch Signaling Activation Is Associated with Patient Mortality and Increased FGF1-Mediated Invasion in Squamous Cell Carcinoma of the Oral Cavity , 2016, Molecular Cancer Research.

[33]  Premkumar Vummidi Giridhar,et al.  CD44 integrates signaling in normal stem cell, cancer stem cell and (pre)metastatic niches , 2013, Experimental biology and medicine.

[34]  A. Johansson,et al.  A CD44high/EGFRlow Subpopulation within Head and Neck Cancer Cell Lines Shows an Epithelial-Mesenchymal Transition Phenotype and Resistance to Treatment , 2012, PloS one.

[35]  T. McDonnell,et al.  Sonic hedgehog induces epidermal growth factor dependent matrix infiltration in HaCaT keratinocytes. , 2005, The Journal of investigative dermatology.

[36]  I. Weissman,et al.  Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma , 2007, Proceedings of the National Academy of Sciences.

[37]  Alexander van Oudenaarden,et al.  Neural-specific Sox2 input and differential Gli-binding affinity provide context and positional information in Shh-directed neural patterning. , 2012, Genes & development.

[38]  R. Weinberg,et al.  Targeting the Epithelial-to-Mesenchymal Transition: The Case for Differentiation-Based Therapy , 2017, Cold Spring Harbor symposia on quantitative biology.

[39]  K. Nagata,et al.  GLI-mediated Keratin 17 expression promotes tumor cell growth through the anti-apoptotic function in oral squamous cell carcinomas , 2017, Journal of Cancer Research and Clinical Oncology.

[40]  C. Mathers,et al.  Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012 , 2015, International journal of cancer.

[41]  J. Pammer,et al.  Expression of the Sonic hedgehog pathway in squamous cell carcinoma of the skin and the mucosa of the head and neck , 2011, Head & neck.

[42]  Ming Yan,et al.  HH/GLI signalling as a new therapeutic target for patients with oral squamous cell carcinoma. , 2011, Oral oncology.

[43]  Walter Birchmeier,et al.  Deciphering the function of canonical Wnt signals in development and disease: conditional loss- and gain-of-function mutations of beta-catenin in mice. , 2008, Genes & development.

[44]  Miao Sun,et al.  Sonic hedgehog signaling may promote invasion and metastasis of oral squamous cell carcinoma by activating MMP-9 and E-cadherin expression , 2014, Medical Oncology.

[45]  A. Ruiz i Altaba,et al.  Context-dependent signal integration by the GLI code: The oncogenic load, pathways, modifiers and implications for cancer therapy , 2014, Seminars in cell & developmental biology.

[46]  F. D. de Sauvage,et al.  Mechanisms of Hedgehog pathway activation in cancer and implications for therapy. , 2009, Trends in pharmacological sciences.

[47]  Michael Kahn,et al.  Targeting Notch, Hedgehog, and Wnt pathways in cancer stem cells: clinical update , 2015, Nature Reviews Clinical Oncology.

[48]  A. Jemal,et al.  Global cancer statistics , 2011, CA: a cancer journal for clinicians.

[49]  Sharmila Shankar,et al.  PI3K/AKT/mTOR and sonic hedgehog pathways cooperate together to inhibit human pancreatic cancer stem cell characteristics and tumor growth , 2015, Oncotarget.

[50]  Barbara Stecca,et al.  Context-dependent regulation of the GLI code in cancer by HEDGEHOG and non-HEDGEHOG signals. , 2010, Journal of molecular cell biology.

[51]  N. Nakamura,et al.  Notch signaling induces EMT in OSCC cell lines in a hypoxic environment , 2013, Oncology letters.

[52]  A. Chinnaiyan,et al.  Cisplatin Induces Bmi­1 and Enhances the Stem Cell Fraction in Head and Neck Cancer , 2015 .

[53]  T. Shimo,et al.  Sonic hedgehog signaling promotes growth of oral squamous cell carcinoma cells associated with bone destruction. , 2012, Oral Oncology.

[54]  Quentin Liu,et al.  Inhibition of Sonic Hedgehog Signaling Pathway by Thiazole Antibiotic Thiostrepton Attenuates the CD44+/CD24-Stem-Like Population and Sphere-Forming Capacity in Triple-Negative Breast Cancer , 2016, Cellular Physiology and Biochemistry.

[55]  K. Plate,et al.  Sonic hedgehog acts as a negative regulator of {beta}-catenin signaling in the adult tongue epithelium. , 2010, The American journal of pathology.

[56]  Bhuvanesh Singh,et al.  Targeting cellular and molecular drivers of head and neck squamous cell carcinoma: current options and emerging perspectives , 2016, Cancer and Metastasis Reviews.

[57]  W. Birchmeier,et al.  Wnt signaling in stem and cancer stem cells. , 2013, Current opinion in cell biology.

[58]  Philip A Beachy,et al.  Hedgehog-Regulated Processing of Gli3 Produces an Anterior/Posterior Repressor Gradient in the Developing Vertebrate Limb , 2000, Cell.

[59]  I. Mackenzie,et al.  Invasive oral cancer stem cells display resistance to ionising radiation , 2015, Oncotarget.

[60]  L. Sobin,et al.  TNM Classification of Malignant Tumours , 1987, UICC International Union Against Cancer.

[61]  X. Cui,et al.  LncRNA‐Hh Strengthen Cancer Stem Cells Generation in Twist‐Positive Breast Cancer via Activation of Hedgehog Signaling Pathway , 2015, Stem cells.

[62]  A. Olivi,et al.  Cyclopamine‐Mediated Hedgehog Pathway Inhibition Depletes Stem‐Like Cancer Cells in Glioblastoma , 2007, Stem cells.

[63]  A. Johansson,et al.  The relationship between EMT, CD44high /EGFRlow phenotype, and treatment response in head and neck cancer cell lines. , 2016, Journal of oral pathology & medicine : official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology.

[64]  R. Weinberg,et al.  EMT, cell plasticity and metastasis , 2016, Cancer and Metastasis Reviews.

[65]  M. Zöller.  CD44: can a cancer-initiating cell profit from an abundantly expressed molecule? , 2011, Nature Reviews Cancer.

[66]  Maryam K. Mohammed,et al.  The evolving roles of canonical WNT signaling in stem cells and tumorigenesis: Implications in targeted cancer therapies , 2015, Laboratory Investigation.

[67]  Yuanzhi Lu,et al.  Hedgehog signaling is a novel therapeutic target in tamoxifen-resistant breast cancer aberrantly activated by PI3K/AKT pathway. , 2012, Cancer research.

[68]  Yu-Chao Chang,et al.  Oct4 Mediates Tumor Initiating Properties in Oral Squamous Cell Carcinomas through the Regulation of Epithelial-Mesenchymal Transition , 2014, PloS one.

[69]  S. Oh,et al.  Sonic hedgehog pathway promotes metastasis and lymphangiogenesis via activation of Akt, EMT, and MMP-9 pathway in gastric cancer. , 2011, Cancer research.

[70]  Huan Yang,et al.  Identification of CD44+CD24+ gastric cancer stem cells , 2011, Journal of Cancer Research and Clinical Oncology.

[71]  E. Tajara,et al.  PROX1 Gene is Differentially Expressed in Oral Cancer and Reduces Cellular Proliferation , 2014, Medicine.

[72]  E. Antonarakis,et al.  Clinical Implications of Hedgehog Pathway Signaling in Prostate Cancer , 2015, Cancers.

[73]  T. Davis,et al.  BMI1 as a novel target for drug discovery in cancer , 2011, Journal of cellular biochemistry.

[74]  Changli Wang,et al.  Gli promotes epithelial-mesenchymal transition in human lung adenocarcinomas , 2016, Oncotarget.

[75]  Michael Dean,et al.  Mutations of the Human Homolog of Drosophila patched in the Nevoid Basal Cell Carcinoma Syndrome , 1996, Cell.

[76]  A. McMahon,et al.  The activity of Gli transcription factors is essential for Kras-induced pancreatic tumorigenesis , 2012, Proceedings of the National Academy of Sciences.

[77]  A. Lam,et al.  Regulation of tumorigenesis in oral epithelial cells by defined reprogramming factors Oct4 and Sox2 , 2016, Oncology reports.

[78]  L. Bourguignon,et al.  Hyaluronan-CD44 interaction promotes oncogenic signaling, microRNA functions, chemoresistance, and radiation resistance in cancer stem cells leading to tumor progression. , 2014, Advances in cancer research.

[79]  A. Kulkarni,et al.  NOTCH1 inhibition enhances the efficacy of conventional chemotherapeutic agents by targeting head neck cancer stem cell , 2016, Scientific Reports.

[80]  R. Carpenter,et al.  Targeting the Sonic Hedgehog Signaling Pathway: Review of Smoothened and GLI Inhibitors , 2016, Cancers.

[81]  W. Xiaojing,et al.  Sonic hedgehog–Gli1 signals promote epithelial–mesenchymal transition in ovarian cancer by mediating PI3K/AKT pathway , 2014, Medical Oncology.