title: Regulation of Head and Neck Squamous Cancer Stem Cells by PI3K and SOX2.
abstract: We have an incomplete understanding of the differences between cancer stem cells (CSCs) in human papillomavirus-positive (HPV-positive) and -negative (HPV-negative) head and neck squamous cell cancer (HNSCC). The PI3K pathway has the most frequent activating genetic events in HNSCC (especially HPV-positive driven), but the differential signaling between CSCs and non-CSCs is also unknown.
  
  
  
  We addressed these unresolved questions using CSCs identified from 10 HNSCC patient-derived xenografts (PDXs). Sored populations were serially passaged in nude mice to evaluate tumorigenicity and tumor recapitulation. The transcription profile of HNSCC CSCs was characterized by mRNA sequencing, and the susceptibility of CSCs to therapy was investigated using an in vivo model. SOX2 transcriptional activity was used to follow the asymmetric division of PDX-derived CSCs. All statistical tests were two-sided.
  
  
  
  CSCs were enriched by high aldehyde dehydrogenase (ALDH) activity and CD44 expression and were similar between HPV-positive and HPV-negative cases (percent tumor formation injecting ≤ 1x10(3) cells: ALDH(+)CD44(high) = 65.8%, ALDH(-)CD44(high) = 33.1%, ALDH(+)CD44(high) = 20.0%; and injecting 1x10(5) cells: ALDH(-)CD44(low) = 4.4%). CSCs were resistant to conventional therapy and had PI3K/mTOR pathway overexpression (GSEA pathway enrichment, P < .001), and PI3K inhibition in vivo decreased their tumorigenicity (40.0%-100.0% across cases). PI3K/mTOR directly regulated SOX2 protein levels, and SOX2 in turn activated ALDH1A1 (P < .001 013C and 067C) expression and ALDH activity (ALDH(+) [%] empty-control vs SOX2, 0.4% ± 0.4% vs 14.5% ± 9.8%, P = .03 for 013C and 1.7% ± 1.3% vs 3.6% ± 3.4%, P = .04 for 067C) in 013C and 067 cells. SOX2 enhanced sphere and tumor growth (spheres/well, 013C P < .001 and 067C P = .04) and therapy resistance. SOX2 expression prompted mesenchymal-to-epithelial transition (MET) by inducing CDH1 (013C P = .002, 067C P = .01), followed by asymmetric division and proliferation, which contributed to tumor formation.
  
  
  
  The molecular link between PI3K activation and CSC properties found in this study provides insights into therapeutic strategies for HNSCC. Constitutive expression of SOX2 in HNSCC cells generates a CSC-like population that enables CSC studies.
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Keysar
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: Phuong N
authors.initials: PN
authors.lastname: Le
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: Bettina
authors.initials: B
authors.lastname: Miller
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: Brian C
authors.initials: BC
authors.lastname: Jackson
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: Justin R
authors.initials: JR
authors.lastname: Eagles
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: Cera
authors.initials: C
authors.lastname: Nieto
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: Jihye
authors.initials: J
authors.lastname: Kim
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: Binwu
authors.initials: B
authors.lastname: Tang
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: Magdalena J
authors.initials: MJ
authors.lastname: Glogowska
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: J Jason
authors.initials: JJ
authors.lastname: Morton
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: Nuria
authors.initials: N
authors.lastname: Padilla-Just
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: Karina
authors.initials: K
authors.lastname: Gomez
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: Emily
authors.initials: E
authors.lastname: Warnock
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: Julie
authors.initials: J
authors.lastname: Reisinger
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: John J
authors.initials: JJ
authors.lastname: Arcaroli
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: Wells A
authors.initials: WA
authors.lastname: Messersmith
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: Lalage M
authors.initials: LM
authors.lastname: Wakefield
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: Dexiang
authors.initials: D
authors.lastname: Gao
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: Aik-Choon
authors.initials: AC
authors.lastname: Tan
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: Hilary
authors.initials: H
authors.lastname: Serracino
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: Vasilis
authors.initials: V
authors.lastname: Vasiliou
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Roop
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: Xiao-Jing
authors.initials: XJ
authors.lastname: Wang
authors.affiliation: Affiliations of authors: Division of Medical Oncology, Department of Medicine (SBK, PNL, BM, BCJ, JRE, CN, JK, MJG, JJM, NPJ, KG, EW, JR, JJA, WAM, ACT, AJ), Department of Biostatistics and Informatics (JK, DG, ACT), Department of Pathology (HS, XJW), Department of Dermatology (DRR), and Gates Center for Regenerative Medicine (DRR, XJW, AJ), University of Colorado Denver School of Medicine, Denver, CO; Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD (BT, LMW); Department of Environmental Health Sciences, Yale School of Public Health, Yale School of Medicine, New Haven, CT (VV).
authors.forename: Antonio
authors.initials: A
authors.lastname: Jimeno
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000378:metabolism; D000080924:Aldehyde Dehydrogenase 1 Family; D000818:Animals; D015703:Antigens, CD; D000970:Antineoplastic Agents / Q000494:pharmacology; D015820:Cadherins / Q000378:metabolism; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D002455:Cell Division; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000187:drug effects; D066246:ErbB Receptors / Q000378:metabolism; D005260:Female; D006258:Head and Neck Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D027383:Papillomaviridae / Q000302:isolation & purification; D058539:Phosphatidylinositol 3-Kinase / Q000235:genetics* / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012333:RNA, Messenger / Q000032:analysis*; D050697:Retinal Dehydrogenase; D055748:SOXB1 Transcription Factors / Q000235:genetics* / Q000378:metabolism; D017423:Sequence Analysis, RNA; D015398:Signal Transduction; D018874:Spheroids, Cellular; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D059467:Transcriptome; D014407:Tumor Cells, Cultured
chemical_list: D015703:Antigens, CD; D000970:Antineoplastic Agents; C513384:CDH1 protein, human; D015820:Cadherins; D018960:Hyaluronan Receptors; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012333:RNA, Messenger; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; D000080924:Aldehyde Dehydrogenase 1 Family; D000444:Aldehyde Dehydrogenase; C510223:ALDH1A1 protein, human; D050697:Retinal Dehydrogenase; C546842:MTOR protein, human; D058539:Phosphatidylinositol 3-Kinase; D066246:ErbB Receptors; D058570:TOR Serine-Threonine Kinases