Mammary cancer chemoprevention by withaferin A is accompanied by in vivo suppression of self-renewal of cancer stem cells.
Current dogma favors elimination of therapy-resistant cancer stem cells for chemoprevention of breast cancer. We showed recently that mammary cancer development in a transgenic mouse model (mouse mammary tumor virus-neu; MMTV-neu) was inhibited significantly upon treatment with withaferin A (WA), a steroidal lactone derived from a medicinal plant. Herein, we demonstrate that the mammary cancer prevention by WA is accompanied by in vivo suppression of breast cancer stem cells (bCSC). In vitro mammosphere formation was dose-dependently inhibited by WA treatment in MCF-7 and SUM159 human breast cancer cells. Other markers of bCSC, including aldehyde dehydrogenase 1 (ALDH1) activity and CD44(high)/CD24(low)/epithelial-specific antigen-positive (ESA+) fraction, were also decreased significantly in the presence of plasma achievable doses of WA. However, WA exposure resulted in cell line-specific changes in Oct4, SOX-2, and Nanog mRNA expression. WA administration to MMTV-neu mice (0.1 mg/mouse, 3 times/week for 28 weeks) resulted in inhibition of mammosphere number and ALDH1 activity in vivo. Mechanistic studies revealed that although urokinase-type plasminogen activator receptor overexpression conferred partial protection against bCSC inhibition by WA, Notch4 was largely dispensable for this response. WA treatment also resulted in sustained (MCF-7) or transient (SUM159) downregulation of Bmi-1 (B-cell-specific Moloney murine leukemia virus insertion region-1) protein. Ectopic expression of Bmi-1 conferred partial but significant protection against ALDH1 activity inhibition by WA. Interestingly, WA treatment caused induction of Kruppel-like factor 4 (KLF4) and its knockdown augmented bCSC inhibition by WA. In conclusion, this study shows in vivo effectiveness of WA against bCSC.
Authors' Affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Su-Hyeong
SH
Kim
Authors' Affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania singhs@upmc.edu.
Shivendra V
SV
Singh
D000818:Animals; D014408:Biomarkers, Tumor / Q000032:analysis; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D014411:Neoplastic Stem Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology*; D063150:Polycomb Repressive Complex 1 / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D000074562:Receptor, Notch4; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D054358:Withanolides / Q000627:therapeutic use*
C069077:BMI1 protein, human; D014408:Biomarkers, Tumor; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C106075:NOTCH4 protein, human; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D000074562:Receptor, Notch4; D051880:Receptors, Notch; D055293:Receptors, Urokinase Plasminogen Activator; D054358:Withanolides; D063150:Polycomb Repressive Complex 1; C009684:withaferin A