Hypoxia potentiates Notch signaling in breast cancer leading to decreased E-cadherin expression and increased cell migration and invasion.
BACKGROUND
Epithelial-to-mesenchymal transition (EMT) is associated with decreased adhesion and acquisition of metastatic potential of breast cancer cells. Epithelial-to-mesenchymal transition is mediated, in part, by two transcription repressors, Snail and Slug, that are known to be targets of the Notch signaling pathway, and JAGGED1-induced Notch activation increases EMT. However, the events that lead to increased Notch activity during EMT of breast cancer cells are unknown.


METHODS
The accumulation of hypoxia inducible factors (HIFs) under hypoxia was detected by western blot analysis, and their effects on Notch signaling were measured by an in vitro Notch reporter assay. The expression of Notch target genes under hypoxia was tested by real-time PCR. The knockdown of HIF-1alpha was mediated by retroviral delivery of shRNA. The expression of Slug and Snail under hypoxia was measured by real-time PCR. Breast cancer cell migration and invasion under hypoxia were tested with cell migration and invasion kits.


RESULTS
Hypoxia increased the expression of Notch target genes such as HES1 and HEY1 in breast cancer cells, as was expression of Notch receptors and ligands. The mechanism is likely to involve the accumulation of HIF-1alpha and HIF-2alpha in these cells by hypoxia, which synergised with the Notch co-activator MAML1 in potentiating Notch activity. Hypoxia inducible factor-1alpha was found to bind to HES1 promoter under hypoxia. Knockdown of HIF-1alpha with shRNA inhibited both HES1 and HEY1 expression under hypoxia. Hypoxia increased the expression of Slug and Snail, and decreased the expression of E-cadherin, hallmarks of EMT. Notch pathway inhibition abrogated the hypoxia-mediated increase in Slug and Snail expression, as well as decreased breast cancer cell migration and invasion.


CONCLUSIONS
Hypoxia-mediated Notch signaling may have an important role in the initiation of EMT and subsequent potential for breast cancer metastasis.
Chen
J
J
Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
Imanaka
N
N
Chen
J
J
Griffin
J D
JD
D051792:Basic Helix-Loop-Helix Proteins / Q000096:biosynthesis; D001943:Breast Neoplasms / Q000378:metabolism* / Q000503:physiopathology; D015820:Cadherins / Q000096:biosynthesis*; D018797:Cell Cycle Proteins / Q000096:biosynthesis; D002465:Cell Movement / Q000502:physiology*; D005260:Female; D018398:Homeodomain Proteins / Q000096:biosynthesis; D006801:Humans; D000860:Hypoxia / Q000503:physiopathology*; D009361:Neoplasm Invasiveness / Q000503:physiopathology; D051880:Receptors, Notch / Q000096:biosynthesis*; D015398:Signal Transduction; D000072056:Transcription Factor HES-1
D051792:Basic Helix-Loop-Helix Proteins; D015820:Cadherins; D018797:Cell Cycle Proteins; D018398:Homeodomain Proteins; D051880:Receptors, Notch; D000072056:Transcription Factor HES-1; C485631:HEY1 protein, human; C078493:HES1 protein, human