Targeting Signal Transducer and Activator of Transcription 3 Pathway by Cucurbitacin I Diminishes Self-Renewing and Radiochemoresistant Abilities in Thyroid Cancer-Derived CD133 + Cells Ling-Ming Tseng, Pin-I Huang, Yu-Rung Chen, Yu-Chih Chen, Yueh-Ching Chou, Yi-Wei Chen, Yuh-Lih Chang, Han-Shui Hsu, Yuan-Tzu Lan, Kuan-Hsuan Chen, Chin-Wen Chi, Shih-Hwa Chiou, De-Ming Yang, and Chen-Hsen Lee Institutes of Clinical Medicine (L.-M.T., P.-I.H., Y.-C.Che., Y.-W.C., Y.-T.L., K.-H.C., S.-H.C.), Pharmacology (Y.-R.C., Y.-C.Cho., Y.-L.C., C.-W.C., S.-H.C.), and Emergency and Critical Care Medicine (H.-S.H., C.-H.L.), and Department of Surgery (L.-M.T., Y.-C.Che., H.-S.H., Y.-T.L., D.-M.Y., C.-H.L.), School of Medicine, National Yang-Ming University, Taipei, Taiwan; and Departments of Education and Research (Y.-C.Che., C.-W.C., S.-H.C., D.-M.Y.), Surgery (L.-M.T., Y.-R.C., H.-S.H., Y.-T.L., C.-H.L.), and Pharmacy (Y.-C.Cho., Y.-L.C., K.-H.C.), Cancer Center (P.-I.H., Y.-W.C., C.-W.C.), and Department of Emergency (C.-H.L.), Taipei Veterans General Hospital, Taipei, Taiwan Received October 7, 2011; accepted February 9, 2012 ABSTRACT Anaplastic thyroid cancer (ATC) is a lethal solid tumor with poor prognosis because of its invasiveness and its resistance to current therapies. Recently, ATC-CD133 + cells were found to have cancer stem cell (CSC) properties and were suggested to be important contributors to tumorigenicity and cancer me- tastasis. However, the molecular pathways and therapeutic targets in thyroid cancer-related CSCs remain undetermined. In this study, ATC-CD133 + cells were isolated and found to have increased tumorigenicity, radioresistance, and higher ex- pression of both embryonic stem cell-related and drug resis- tance-related genes compared with ATC-CD133 - cells. Mi- croarray bioinformatics analysis suggested that the signal transducer and activator of transcription 3 (STAT3) pathway could be important in regulating the stemness signature in ATC-CD133 + cells; therefore, the effect of the potent STAT3 inhibitor cucurbitacin I in ATC-CD133 + cells was evaluated in this study. Treatment of ATC-CD133 + cells with cucurbitacin I diminished their CSC-like abilities, inhibited their stemness gene signature, and facilitated their differentiation into ATC- CD133 - cells. Of note, treatment of ATC-CD133 + cells with cucurbitacin I up-regulated the expression of thyroid-specific genes and significantly enhanced radioiodine uptake. Further- more, cucurbitacin I treatment increased the sensitivity of ATC- CD133 + cells to radiation and chemotherapeutic drugs through apoptosis. Finally, xenotransplantation experiments revealed that cucurbitacin I plus radiochemotherapy significantly suppressed tu- morigenesis and improved survival in immunocompromised mice into which ATC-CD133 + cells were transplanted. In summary, these results show that the STAT3 pathway plays a key role in mediating CSC properties in ATC-CD133 + cells. Targeting STAT3 with cucur- bitacin I in ATC may provide a new approach for therapeutic treat- ment in the future. Introduction Thyroid cancer is a common endocrine tumor. Approxi- mately 85 to 90% of patients with thyroid cancer are diag- nosed with papillary thyroid cancer (PTC), which originates from thyroid follicular cells (Sipos and Mazzaferri, 2010). Patients with PTC are expected to survive for 10 years or more after diagnosis (Sipos and Mazzaferri, 2010). However, This work was supported by the Division of Experimental Surgery of the Department of Surgery, Taipei Veterans General Hospital, the National Sci- ence Council [Grants 97-3111-B-075-001-MY3, 98-2314-B-341-001-MY3]; Tai- pei Veterans General Hospital [Grants V97B1-006, E1-008, ER2-018, ER3- 005, F-001]; the Joint Projects of the University System of Taiwan and Taipei Veterans General Hospital [VGHUST 98-G6-6]; Yen-Tjing-Ling Medical Foun- dation; and National Yang-Ming University (Ministry of Education, Aim for the Top University Plan). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. http://dx.doi.org/10.1124/jpet.111.188730. ABBREVIATIONS: PTC, papillary thyroid cancer; ATC, anaplastic thyroid cancer; CSC, cancer stem cell; STAT3, signal transducer and activator of transcription 3; MMP-9, matrix metalloproteinase 9; RET/PTC, rearranged in transformation/papillary thyroid carcinoma; p-STAT3, phosphor- ylated STAT3; DMEM, Dulbecco’s modified Eagle’s medium; bFGF, basic fibroblast growth factor; EGF, epidermal growth factor; IR, ionizing radiation; RT, reverse transcriptase; PCR, polymerase chain reaction; NIS, sodium/iodide symporter; GFP, green fluorescent protein; JAK, Janus tyrosine kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; TPO, thyroperoxidase; Tg, thyroglobulin; PPAR, peroxisome prolif- erator-activated receptor-; 5-FU, 5-fluorouracil. 1521-0103/12/3412-410–423$25.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 341, No. 2 Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics 188730/3763966 JPET 341:410–423, 2012 410 at National Yang-Ming University on June 23, 2012 jpet.aspetjournals.org Downloaded from DC1.html http://jpet.aspetjournals.org/content/suppl/2012/02/10/jpet.111.188730. 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