title: Evaluation of STAT3 signaling in ALDH+ and ALDH+/CD44+/CD24- subpopulations of breast cancer cells.
abstract: BACKGROUND
  STAT3 activation is frequently detected in breast cancer and this pathway has emerged as an attractive molecular target for cancer treatment. Recent experimental evidence suggests ALDH-positive (ALDH(+)), or cell surface molecule CD44-positive (CD44(+)) but CD24-negative (CD24(-)) breast cancer cells have cancer stem cell properties. However, the role of STAT3 signaling in ALDH(+) and ALDH(+)/CD44(+)/CD24(-) subpopulations of breast cancer cells is unknown.
  
  
  RESULTS
  We examined STAT3 activation in ALDH(+) and ALDH(+)/CD44(+)/CD24(-) subpopulations of breast cancer cells by sorting with flow cytometer. We observed ALDH-positive (ALDH(+)) cells expressed higher levels of phosphorylated STAT3 compared to ALDH-negative (ALDH(-)) cells. There was a significant correlation between the nuclear staining of phosphorylated STAT3 and the expression of ALDH1 in breast cancer tissues. These results suggest that STAT3 is activated in ALDH(+) subpopulations of breast cancer cells. STAT3 inhibitors Stattic and LLL12 inhibited STAT3 phosphorylation, reduced the ALDH(+) subpopulation, inhibited breast cancer stem-like cell viability, and retarded tumorisphere-forming capacity in vitro. Similar inhibition of STAT3 phosphorylation, and breast cancer stem cell viability were observed using STAT3 ShRNA. In addition, LLL12 inhibited STAT3 downstream target gene expression and induced apoptosis in ALDH(+) subpopulations of breast cancer cells. Furthermore, LLL12 inhibited STAT3 phosphorylation and tumor cell proliferation, induced apoptosis, and suppressed tumor growth in xenograft and mammary fat pad mouse models from ALDH(+) breast cancer cells. Similar in vitro and tumor growth in vivo results were obtained when ALDH(+) cells were further selected for the stem cell markers CD44(+) and CD24(-).
  
  
  CONCLUSIONS
  These studies demonstrate an important role for STAT3 signaling in ALDH(+) and ALDH(+)/CD44(+)/CD24(-) subpopulations of breast cancer cells which may have cancer stem cell properties and suggest that pharmacologic inhibition of STAT3 represents an effective strategy to selectively target the cancer stem cell-like subpopulation.
authors.affiliation: Divison of Cardiology, Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People's Republic of China ; Center for Childhood Cancer, The Research Institute at Nationwide Children's Hospital, Department of Pediatrics, Columbus, Ohio, United States of America.
authors.forename: Li
authors.initials: L
authors.lastname: Lin
authors.affiliation: Center for Childhood Cancer, The Research Institute at Nationwide Children's Hospital, Department of Pediatrics, Columbus, Ohio, United States of America ; Molecular, Cellular, and Developmental Biology Program, The Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Brian
authors.initials: B
authors.lastname: Hutzen
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, The University of Michigan, Ann Arbor, Michigan, United States of America.
authors.forename: Hsiu-Fang
authors.initials: HF
authors.lastname: Lee
authors.affiliation: Medical Technology Division, School of Allied Medical Professions, Columbus, Ohio, United States of America.
authors.forename: Zhengang
authors.initials: Z
authors.lastname: Peng
authors.affiliation: Divison of Cardiology, Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People's Republic of China.
authors.forename: Wenlong
authors.initials: W
authors.lastname: Wang
authors.affiliation: Divison of Cardiology, Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People's Republic of China.
authors.forename: Chongqiang
authors.initials: C
authors.lastname: Zhao
authors.affiliation: Medical Technology Division, School of Allied Medical Professions, Columbus, Ohio, United States of America ; Department of Medical Laboratory Sciences, College of Health Sciences, University of Delaware, Newark, Delaware, United States of America.
authors.forename: Huey-Jen
authors.initials: HJ
authors.lastname: Lin
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, The University of Michigan, Ann Arbor, Michigan, United States of America.
authors.forename: Duxin
authors.initials: D
authors.lastname: Sun
authors.affiliation: Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Pui-Kai
authors.initials: PK
authors.lastname: Li
authors.affiliation: Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Chenglong
authors.initials: C
authors.lastname: Li
authors.affiliation: Department of Internal Medicine, The University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan, United States of America.
authors.forename: Hasan
authors.initials: H
authors.lastname: Korkaya
authors.affiliation: Department of Internal Medicine, The University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan, United States of America.
authors.forename: Max S
authors.initials: MS
authors.lastname: Wicha
authors.affiliation: Center for Childhood Cancer, The Research Institute at Nationwide Children's Hospital, Department of Pediatrics, Columbus, Ohio, United States of America ; Molecular, Cellular, and Developmental Biology Program, The Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Jiayuh
authors.initials: J
authors.lastname: Lin
mesh_terms: D000080924:Aldehyde Dehydrogenase 1 Family; D000818:Animals; D000880:Anthraquinones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D051927:CD24 Antigen / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation; D002470:Cell Survival / Q000187:drug effects; D003499:Cyclic S-Oxides / Q000494:pharmacology; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D016513:Mice, SCID; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000378:metabolism; D050697:Retinal Dehydrogenase / Q000378:metabolism*; D050796:STAT3 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D054852:Small Molecule Libraries / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D023041:Xenograft Model Antitumor Assays
chemical_list: D000880:Anthraquinones; D051927:CD24 Antigen; D003499:Cyclic S-Oxides; D018960:Hyaluronan Receptors; D007527:Isoenzymes; C549232:LLL12 compound; D034741:RNA, Small Interfering; D050796:STAT3 Transcription Factor; D054852:Small Molecule Libraries; D013449:Sulfonamides; C517409:stattic; D000080924:Aldehyde Dehydrogenase 1 Family; C510223:ALDH1A1 protein, human; C515028:ALDH1A1 protein, mouse; D050697:Retinal Dehydrogenase