Metalloprotease-disintegrin ADAM12 actively promotes the stem cell-like phenotype in claudin-low breast cancer.
ADAM12 is upregulated in human breast cancers and is a predictor of chemoresistance in estrogen receptor-negative tumors. ADAM12 is induced during epithelial-to-mesenchymal transition, a feature associated with claudin-low breast tumors, which are enriched in cancer stem cell (CSC) markers. It is currently unknown whether ADAM12 plays an active role in promoting the CSC phenotype in breast cancer cells.



ADAM12 expression was downregulated in representative claudin-low breast cancer cell lines, SUM159PT and Hs578T, using siRNA transfection or inducible shRNA expression. Cell characteristics commonly associated with the CSC phenotype in vitro (cell migration, invasion, anoikis resistance, mammosphere formation, ALDH activity, and expression of the CD44 and CD24 cell surface markers) and in vivo (tumor formation in mice using limiting dilution transplantation assays) were evaluated. RNA sequencing was performed to identify global gene expression changes after ADAM12 knockdown.



We found that sorted SUM159PT cell populations with high ADAM12 levels had elevated expression of CSC markers and an increased ability to form mammospheres. ADAM12 knockdown reduced cell migration and invasion, decreased anoikis resistance, and compromised mammosphere formation. ADAM12 knockdown also diminished ALDEFLUOR+and CD44hi/CD24-/loCSC-enriched populations in vitro and reduced tumorigenesis in mice in vivo. RNA sequencing identified a significant overlap between ADAM12- and Epidermal Growth Factor Receptor (EGFR)-regulated genes. Consequently, ADAM12 knockdown lowered the basal activation level of EGFR, and this effect was abolished by batimastat, a metalloproteinase inhibitor. Furthermore, incubation of cells with exogenously added EGF prevented the downregulation of CD44hi/CD24-/locell population by ADAM12 knockdown.



These results indicate that ADAM12 actively supports the CSC phenotype in claudin-low breast cancer cells via modulation of the EGFR pathway.
Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS, 66506, USA.|Current address: Department of Radiation Oncology, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA.
Sara
S
Duhachek-Muggy
Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS, 66506, USA.|Current address: Department of Diabetes Complications and Metabolism, Beckman Research Institute, City of Hope, Duarte, CA, USA.
Yue
Y
Qi
Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS, 66506, USA.
Randi
R
Wise
Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS, 66506, USA.
Linda
L
Alyahya
Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS, 66506, USA.|Current address: Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Hui
H
Li
Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS, 66506, USA.
Jacob
J
Hodge
Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS, 66506, USA. zolkiea@ksu.edu.
Anna
0000-0003-3632-9337
A
Zolkiewska
D000072199:ADAM12 Protein / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015415:Biomarkers; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000401:mortality / Q000473:pathology; D045744:Cell Line, Tumor; D057167:Claudins / Q000378:metabolism*; D016000:Cluster Analysis; D004195:Disease Models, Animal; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D064593:Heterografts; D006801:Humans; D016130:Immunophenotyping; D051379:Mice; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D010641:Phenotype; D011379:Prognosis; D015398:Signal Transduction; D047368:Tumor Burden
D015415:Biomarkers; D057167:Claudins; D066246:ErbB Receptors; D000072199:ADAM12 Protein
ADAM metalloprotease; Breast cancer; Cancer stem cells; Epidermal growth factor receptor; Epithelial-to-mesenchymal transition